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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
ii
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
SUB-THEMES<br />
Structure Elucidation<br />
Chemotaxonomy<br />
Pharmacology and Bioassay on Natural Products<br />
Modern Chromatographic Techniques<br />
Herbal Remedies<br />
Role <strong>of</strong> Ethnobotany in Drug Discovery<br />
Bioassay screening<br />
Medicinal Chemistry<br />
Synthesis <strong>of</strong> Natural Products<br />
Drug Development<br />
Ethics and IPR <strong>of</strong> Drug Development<br />
Green Chemistry in Natural Product Research<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Local Organizing Committee<br />
NAPRECA/University <strong>of</strong> Nairobi<br />
Jacob O. Midiwo (Executive secretary, NAPRECA)<br />
Abiy Yenesew (Assistant Secretary/Treasurer, NAPRECA)<br />
Solomon Derese (Programme Officer, NAPRECA)<br />
Leonidah Kerubo (Member)<br />
Albert Ndakala (Member)<br />
Catherine Lukhoba (Member)<br />
Joe Mwaniki (Member)<br />
Charles Mirikau (Member)<br />
John Wanjohi (Member)<br />
John Onyari (Member)<br />
Milkyas Endale (Member)<br />
Ivan Gumula (Member)<br />
Martha Induli (Member)<br />
Aggrey Akimanya<br />
Nusrat Begum (Member)<br />
NAPRECA - Kenya<br />
Joseph M. Keriko (Chairman)<br />
Danstone Baraza ( Vice Chairman)<br />
John Onyari (Secretary)<br />
Josiah O. Omolo (Vice Secretary)<br />
Job .I. Jondiko (Treasurer)<br />
Catherine Lukhoba (Vice Treasurer)<br />
NAPRECA Advisory Panel<br />
Pr<strong>of</strong> B. Abegaz (Botswana)<br />
Pr<strong>of</strong> R. T. Majinda (Botswana)<br />
Dr. Bonaventure Ngadjui Cameroon)<br />
Pr<strong>of</strong>. Dibungi Kalenda (DR Congo)<br />
Pr<strong>of</strong> Ermias Dagne (Ethiopia)<br />
Pr<strong>of</strong> Jacob Midiwo (Kenya)<br />
Pr<strong>of</strong>. Philippe Rasoanaivo (Madagascar)<br />
Mr. Justin Kabera (Rwanda)<br />
Dr. Sakina Yagi (Sudan)<br />
Dr. Maud K. Mugisha (Uganda)<br />
Dr. Joseph J. Magadula (Tanzania)<br />
Dr. Stanley Mukanganyama (Zimbabwe)<br />
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FOREWORD<br />
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
The Natural Products Research Network for East and Central Africa (NAPRECA) was established in<br />
1984 and was subsequently affiliated to UNESCO as a network program in 1988. Since then it has<br />
held regular biennial Symposia which have continuously attracted top class research pr<strong>of</strong>essionals<br />
to its Symposia. The current Symposium is the 14 th edition and is attended by researchers from 22<br />
countries spread in four continents- Asia, America, Europe and Africa. It is being held in conjunction<br />
with the ethno-veterinary symposium organized by the Association for African Medicinal Plants<br />
Standards (AAMPS). This is the first time we are collaborating with AAMPS and I believe this makes<br />
the meeting even more exciting.<br />
The Symposium is going to have a total <strong>of</strong> 111 presentations- one keynote address, 25 plenary, 38<br />
short lectures, 26 young scientist presentations and 22 posters which is quite a heavy serving. The<br />
abstracts indicate that there will be a lot <strong>of</strong> substance in the presentations and we hope that<br />
delegates leave satisfied. The presentations cover the whole range <strong>of</strong> Natural Products fields with<br />
reports <strong>of</strong> quality results from East and Central African Institutions. There are papers discussing bioprospecting<br />
project strategies in Africa and elsewhere; due to the high throughput bioassay<br />
techniques that are now well established these presentations will be <strong>of</strong> particular interest to<br />
NAPRECA members because they <strong>of</strong>fer opportunity for wide collaboration across many institutions.<br />
For this reasons, NAPRECA members are particularly encouraged to attend the Pan African Natural<br />
Products Library meeting so as to determine how they would participate it in the future. The<br />
AAMPS Symposium also <strong>of</strong>fers a unique opportunity for researchers to interact with Industry and<br />
therefore reflect on how they could develop product prototypes from their research results and<br />
search for commercial outlets. Traditional medicine practitioners will also be participating in this<br />
ethno-veterinary function; we are lucky as NAPRECA members for this opportunity to interact with<br />
these people who are primary sources <strong>of</strong> information.<br />
Due to problems we have had with publication <strong>of</strong> books <strong>of</strong> proceedings for recent Symposia we<br />
have decided to produce only one publication for the symposium in the form <strong>of</strong> this extended book<br />
<strong>of</strong> abstracts; we believe that you will find it useful as a reference material. Enjoy the booklet and<br />
the symposium.<br />
Finally we wish you a nice stay in our beautiful, city in the sun as Nairobi is fondly referred to. You<br />
should take some time and enjoy tourist attraction spots in and around the city after the<br />
Symposium.<br />
Jacob O Midiwo, PhD<br />
Executive Secretary, NAPRECA.<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Table <strong>of</strong> Contents<br />
KEYNOTE ADDRESS<br />
Berhanu Abegaz<br />
Chemical Sciences in Africa Historical Insights and Major Milestones<br />
PLENARY LECTURE PRESENTATIONS<br />
PL 1 Ahmed Hassanali<br />
Natural Products: Evolution <strong>of</strong> Structural and Functional Diversity and Implication for R<br />
& D Targeting Useful Candidates<br />
PL 2<br />
Philippe Rasoanaivo, Sol<strong>of</strong>oniaina Razafimahefa, Emmanuel Randrianarivo<br />
Shifting the Paradigm to the Ethnobotany-based Drug Discovery<br />
PL 3 Kobus El<strong>of</strong>f<br />
Why Has There Been Hardly any Success in Developing Antimicrobial Products from<br />
Medicinal Plants?<br />
PL 4 Dulcie A Mulholland, Moses K Langat, Neil R Crouch and Jean-Marc Nuzillard<br />
Phytochemical Investigations <strong>of</strong> Leaves and Bark <strong>of</strong> Croton gratissimus (Euphorbiaceae)<br />
PL 5 Ivan Addae-Mensah<br />
Challenges and Opportunities <strong>of</strong> Traditional/Herbal Medicine<br />
PL 6 Raymond C F Jones, Abdul K Choudhury, Carole C M Law, Christopher Lumley, Terence A<br />
Pillainayagam and James P Bullous<br />
Approaches to the Synthesis <strong>of</strong> Tricarbonyl Metabolites: An Isoxazole Strategy<br />
PL 7 Gerhard Bringmann<br />
Absolute Stereostructures by LC-CD Coupling in Combination with Quantum-Chemical<br />
CD Calculations<br />
PL 8 Kelly Chibale<br />
Technology Platforms to Facilitate Natural Product-Based Drug Discovery from African<br />
Biodiversity<br />
PL 9 Ameenah Gurib-Fakim<br />
From Past Traditions to a Herbal Pharmacopoeia Africa s Green Gold<br />
PL 10 Máté Erdelyi<br />
NMR Analysis <strong>of</strong> the Molecular Structure <strong>of</strong> Flexible Molecules in Solution<br />
PL 11 Alain Meybeck<br />
Bioassay <strong>of</strong> Natural Products for Cosmetics<br />
PL 12 Rumbidzai Mangoyi, Theresa Chimponda, Elaine Chirisa, Tariro Chitemerere and Stanley<br />
Mukanganyama<br />
Multiple Anti-infective Properties <strong>of</strong> Selected Combretum species from Zimbabwe<br />
PL 13 Peter G. Ruminski<br />
The Center for World Health & Medicine at Saint Louis University: A New Translational<br />
Research Model to Develop Novel Therapies for Neglected Diseases and Other Unmet<br />
Medical Needs<br />
PL 14 Hulda Swai, Lonji Kalombo, Lebogang Katata, Rose Hayeshi, Yolandy Lemmer, Philip<br />
Labuschagne, Paula Melariri, and Belle Nyamboli<br />
Towards Gaining Recognition as an African Centre <strong>of</strong> Excellence in Applied<br />
Nanomedicine Research and Training for Poverty Related Diseases Focus on the<br />
DST/CSIR Nanomedicine Platform<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
PL 15 Ermias Dagne, Yehualashet Belete, Hanna Kassaye and Yadessa Melaku<br />
Application <strong>of</strong> UV-VIS Spectroscopy to Evaluate Quality <strong>of</strong> Medicinal and Edible Oils<br />
PL 16 Jacob O Midiwo, Francis Machumi, Abiy Yenesew, Leonida Kerubo, Solomon Derese<br />
Natural Products from Plant Diversity and their Potential in Management <strong>of</strong> Neglected<br />
Diseases<br />
PL 17 P. Okinda Owuor<br />
Changes in Plants Metabolites with Location <strong>of</strong> Growth and Agronomic Practices: Some<br />
Lessions from Black Tea Quality Studies<br />
PL 18 Vusumuzi Kalvin Ndhlovu, Simiso Dube, Namboole Moses Munkombwe and Mathew Muzi<br />
Nindi<br />
Challenges <strong>of</strong> Isolation, Characterization and Pr<strong>of</strong>iling <strong>of</strong> African Medicinal Plants:<br />
Analytical Prospective <strong>of</strong> Standardization and Quality Control Methods<br />
PL 19 M.A. Birkett, A.M. Hooper, Z.R. Khan C.A.O. Midega, J.A. Pickett and B. Torto<br />
Exploiting the Chemistry <strong>of</strong> African Biodiversity in Pest Management: from Extraction <strong>of</strong><br />
Plant chemicals to Expression in GMOs<br />
PL 20 Drissa DIALLO, Adiaratou TOGOLA, Rokia SANOGO, Chiaka DIAKITE<br />
Development <strong>of</strong> Medicines from African Medicinal Plants: Experiences in West Africa<br />
PL 21 Andrae-Marobela, K., Ghislain, F., Dube, M., Maher, F., Ntumy A.N.<br />
The pan-African Natural Product Library (p-ANPL): Giving Steam a Direction<br />
SHORT LECTURE PRESENTATIONS<br />
SL 1A Gihan O.M. ELhassan, Achyut Adhikari, Sammer Yousuf, Hafiz Ur Rahman, M. Ahmed Mesaik,<br />
Omer M. Abdalla, Asaad Khalid, Muhammad Iqbal Choudhary and Sakina Yagi<br />
Phytochemical and biological activity studies on Aloe sinkatana<br />
SL 1B DR Katerere & C. Rewerts<br />
Application <strong>of</strong> in vitro drug metabolism and disposition studies to assess risk <strong>of</strong> drug<br />
interactions with Sutherlandia frutescens extracts<br />
SL 2A Moses K. Langat, Dorota A. Nawrot and Dulcie A. Mulholland<br />
Chemical constituents <strong>of</strong> East European Species<br />
SL 2B Alice W. Njue, Dan O. Otaye, Peter K. Cheplogoi and Josiah O. Omolo<br />
In vitro inhibition <strong>of</strong> tomato Fusarium wilt causative agent by zearalenone from a soil<br />
inhabiting fungus<br />
SL 3A Robert Byamukama, George Ogweng, Angella Mbabazi, Irene Skaar, Monica Jordheim,<br />
Oyvind M. Andersen, and Bernard T. Kiremire<br />
Anthocyanins from selected plant species in Uganda<br />
SL 3B Hayder Abdelgader<br />
Side Effects <strong>of</strong> some Botanicals on the egg parasitoid Trichogramma spp.<br />
SL 4A Venkatesan Jayakumar, Nicholas Daniel, Kingsly Arachi.<br />
Bioactive constituents from Hyptis suaveolens<br />
SL 4B M. O. Omolo, B. Njiru, I. O. Ndiege, R. M. Musau, P. Njagi, A. Hassanali<br />
Blends <strong>of</strong> Chemicals in Smelly Feet Switch Malaria Mosquitoes on and <strong>of</strong>f.<br />
SL 5A Abdelhafeez M.A. Mohammed, Philip H. Coombes, Neil R. Crouch and Dulcie A. Mulholland<br />
Extractives from the genus Heteropyxis<br />
SL 5B Théoneste Muhizi, Stéphane Grelier, Véronique Coma<br />
Essential oil from some Rwandese plants and their antibacterial activity<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
SL 6A Osman N.A, Mohamed U.I, Ahmed N.E., and Elhussein S.A<br />
Chemical constituents <strong>of</strong> the essential oil <strong>of</strong> Cymbopogon proximus and their potential<br />
for the treatment <strong>of</strong> otomycosis<br />
SL 6B Machocho, A.K.<br />
Antimicrobial Activity and Phytochemical Studies <strong>of</strong> Some Selected Medicinal Kenyan<br />
Plants<br />
SL 7A Abiy Yenesew, Hoseah M Akala, Hannington Twinomuhwezi, Martha Induli, Beatrice Irungu,<br />
Fredrick L. Eyase, Solomon Derese, Bernard T. Kiremire, Jacob O. Midiwo , Norman C. Waters<br />
Antiplasmodial and Radical Scavenging Activities <strong>of</strong> Flavonoids from Kenyan Erythrina<br />
species<br />
SL 7B Brendler, T., Mortensen, D.J., Simon, J.E. 2 , Raskin, I., Feiter, U.<br />
Inventory and Pharmacological Screening <strong>of</strong> Selected Indigenous Plant Species <strong>of</strong><br />
Namibia for Development <strong>of</strong> New Natural Products.<br />
SL 8A C. Steinert, G. Bringmann<br />
Isolation and Structure Elucidation <strong>of</strong> Bioactive Compounds from the Tropical Liana<br />
Ancistrocladus congolensis<br />
SL 8B Elfahal I. A. , Abdelgader, H, , Elhussein, S. A. , Osman, N.A.<br />
The Efficacy <strong>of</strong> Extracts <strong>of</strong> the Plant Argemone Mexicana on Mosquito Species,<br />
Anopheles arabiensis.<br />
SL 9A Christian D. A. Fozing, Gilbert D.W.F. Kapche, P. Ambassa, Bonaventure T. Ngadju 1 ,<br />
Muhammad C. Iqbal, and Berhanu M. Abegaz<br />
Arylbenz<strong>of</strong>urans, Prenylated Flavonoids and Diels Ader Adducts with Biological Activities<br />
from Morus mesozygia<br />
SL 9B Danielle A. Doll RAKOTO, Ranjàna RANDRIANARIVO, Mounidati EL-YACHOUROUTUI 1 , Alain A.<br />
ARISOA, Noelinirina RAHARISOA, Noelitiana RAKOTONDRASOA, Pascaline RAONIHARISOA,<br />
Victor JEANNODA<br />
In vitro Effects <strong>of</strong> Extracts from five Malagasy Endemic Species <strong>of</strong> Albizia (Fabaceae) on<br />
Vegetable Seeds Germination<br />
SL 10A Stephen S. Nyandoro, Josiah O. Odalo, Mayunga H.H. Nkunya, Cosam C. Joseph<br />
Aristolactams, Indolidinoids and other Metabolites from Toussaintia orientalis - An<br />
Endangered Annonaceae Species Endemic to Tanzania<br />
SL 10B Innocent E, Hassanali A, Magesa SM and Kisinza NW<br />
From Laboratory to Field Application <strong>of</strong> Phyto-larvicides: An Outreach Community Based<br />
Experience in Bagamoyo District, Tanzania<br />
SL 11A Odalo JO, Joseph CC, Nkunya MHH, Sattler I, Lange C , Dahse H-M, Möllman, U<br />
Bioactive Furanoditerpenoids, a Dibenzopyranone, Nor-isoprenoid and Biflavonoids<br />
from Medicinal Stuhlmania moavi Verdc.<br />
SL 11B Job Isaac Jondiko, Dida Mathew, Orwa Jeniffer .Nyahanga Thomas, Manguro Lawrence<br />
Toddalia asiatica. Lin: A Potential Source and Model <strong>of</strong> Materials and Services for<br />
Control <strong>of</strong> Diseases and Implications for Herbal Medical Practice in Kenya.<br />
SL 12A Lilechi D. Baraza, Mayunga H.H. Nkunya, Nobert Arnold, Ludger Wessjohann, Cosam C.<br />
Joseph, Jürgen Schmidt, Andrea Porzel<br />
Fungitoxic C-18 hydroxy Unsaturated Fatty Acids from Fruiting Bodies <strong>of</strong> Cantharellus<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Species<br />
SL 12B Alvaro Viljoen<br />
Application <strong>of</strong> Vibrational Spectroscopy and Planar Chromatography in the Quality<br />
Control <strong>of</strong> South African Medicinal and Aromatic Plants<br />
SL 13A Benard Juma, Muhammad Adeel, Alexander Villinger, Helmut Reinke, Anke Spannenberg<br />
Christine Fische, Peter Langer<br />
Synthesis <strong>of</strong> 2,6-Dioxo-1,2,3,4,5,6-hexahydroindoles and their Transformation into<br />
5,8,9,10-Tetrahydro-6H-indolo[2,1-a]isoquinolin-9-ones<br />
SL 13B Najma Dharani<br />
Medicinal Plants <strong>of</strong> East Africa- Importance, Uses in Traditional Medicine, Challenges and<br />
Conservation Status<br />
SL 14A Maurice O. Okoth and Clare I. Muhanji<br />
Crystallization for Long Range Molecular Order Structure Elucidation<br />
SL 15A Elwaleed Elamin Hassan, Ahmed Mudawi Musa, Sara Hamad Hassab Elgawi, Tilal Elimam<br />
Elsammani, Waddah Gamal, Vanessa Yardley, Mahgoub Sherif Eltohami<br />
Antileishmanial Activity <strong>of</strong> Petroleum ether, n-hexane Crude Extract and (2E)-methyl 3-<br />
((1E, 4E)-7-methyl-4-(2-oxopropylidene) cyclohept-1-enyl) acrylate from Xanthium<br />
brasilicum Vell. leaves.<br />
SL 16A Ndze Ralph Kinyuy, Abubakar Abdulkadir, Wirkom Venansius Kihdze, Tanayen Grace Ghaife,<br />
Tanayen Julius Kihdze<br />
Assessment <strong>of</strong> Azadirachta Indica and Cassia Spectabilis for Some Immunomodulatory<br />
Properties<br />
SL 17A Maud Kamatenesi-Mugisha, Buyungo John Paul, Vudriko Patrick, Ogwal Patrick Arop Deng,<br />
Joshua Ogendo, J.M. Mihale<br />
Evaluation <strong>of</strong> the Biosafety <strong>of</strong> Selected Botanical Pesticide Plants Used by Subsistance<br />
Farmers Around the Lake Victoria Basin<br />
SL 18A Kirimuhuzya Claude, Bunalema Lydia, Tabuti John RS, Kakudidi K Esezah, Orodho, John<br />
Magadula Jangu Joseph, Otieno Nicholas and Paul Okemo<br />
The in vitro Antimycobacterial Activity <strong>of</strong> Medicinal Plants Used by Traditional Medicine<br />
Practitioners (TMPs) to Treat Tuberculosis in the Lake Victoria Basin in Uganda<br />
SL 19A Rehab E. H. Fadwal, Faiza E. E. Salah, Mohammed H. Z. Elabdeen and Elamin M. E.<br />
Effects <strong>of</strong> Aqueous Extracts <strong>of</strong> Basil, Ocimum basilicum L., Sodom s apple, Calotropis<br />
procera Ait and Coriander Coriandrum sativum L. on leaf miner, Liriomyza Spp., on okra<br />
Crop.<br />
SL 20A Nga ng a MM, Thoruwa-Langat C, Chhabra S<br />
Antiplasmodial Compounds from the leaves <strong>of</strong> Drypetes gerrardi<br />
YOUNG SCIENTIST PRESENTATIONS<br />
YS 1 Derek Tantoh Ndinteh, Joseph Tanyi Mbafor, Rui Werner Macedo Krause, Won Ken OH<br />
Flavonoids with Anti-Diabetic Activity from Erythrina Abyssinica<br />
YS 2 Elizabeth V.M. Kigondu, Ge<strong>of</strong>frey M. Rukunga, Joseph M. Keriko, Willy K. Tonui, Jeremiah W.<br />
Gathirwa, Peter G. Kirira, Beatrice Irungu, Johnstone M. Ingonga, Isaiah O. Ndiege.<br />
Antimalarial and Antileishmanial Activity and Cytotoxicity <strong>of</strong> Selected Medicinal Plants<br />
from Kenya<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
YS 3 Wanyama P. Juma, Hoseah M. Akala, Fredrick L. Eyase, Lois M. Muiva, Matthias Hydenreich,<br />
Faith A. Okalebo, Peter M. Gitu, Martin G. Peter, Douglas S. Walsh, Mabel Imbuga, Abiy<br />
Yenesew<br />
Terpurinflavone: Antiplasmodial Flavones from the Stem <strong>of</strong> Tephrosia purpurea<br />
YS 4 BN Irungu, GM Rukunga and CN Muthaura<br />
In vitro Antiplasmodial and Cytotoxicity Activities <strong>of</strong> Some Medicinal Plants from Kenya<br />
YS 5 Milkyas Endale, John Patrick Alao, Hoseah M. Akala, Nelson K. Rono, Fredrick L. Eyase,<br />
Solomon Derese, Albert Ndakala, Martin Mbugua, Douglas S. Walsh, Per Sunnerhagen, Mate<br />
Erdelyi, Abiy Yenesew<br />
Antiplasmodial Quinones from the Roots <strong>of</strong> two Pentas Species<br />
YS 6 C. Karangwa, J.N. Kabera, C. Mukayisenga, M.G. Ingabire<br />
Study <strong>of</strong> Verucidal Effect <strong>of</strong> the Crashed Leaves <strong>of</strong> Tetradenia riparia on the Warts<br />
YS 7 Ivan Gumula, Mathias Heydenreich, Solomon Derese 1 , Faith A. Okalebo, Isaiah O. Ndiege,<br />
Mate Erdelyi, Abiy Yenesew<br />
Is<strong>of</strong>lavanones and 3-Methoxyflavones from the Stem Bark <strong>of</strong> Platycelphium voënse<br />
YS 8 C. J. D. Obbo, B. Makanga, D. A Mulholland, P. H. Coombes, R. Brun, M. Kaiser, W. Olaho-<br />
Mukani<br />
Antitrypanosomal, Antileishmanial and Antiplasmodicidal Activities <strong>of</strong> Khaya<br />
anthotheca, a Plant used by Chimpanzees for Self Medication.<br />
YS 9 Francis Machumi, Abiy Yenesew, Jacob Midiwo, Larry Walker, Muhammad Illias, Matthias<br />
Heydenreich, Erich Kleinpeter<br />
Antiplasmodial and Antileishmanial Studies on Carvotacetone Derivatives from<br />
Sphaeranthus bullatus<br />
YS 10 Rechab, S.O., Kareru, P.G., Kutima, H.L, Gakuya, D.W., Nyagah, G.C., Njonge, F.K 4 , Waithaka,<br />
R.W.<br />
In vitro anthelmintic Effect <strong>of</strong> Prosopis juliflora (Sw.) DC (Fabaceae) on Haemonchus<br />
contortus, an Abomasal Nematode <strong>of</strong> Sheep<br />
YS 11 F. Ng ang a, A. Onditi, A. Gachanja, E. Ngumba<br />
Application <strong>of</strong> Solid Phase Extraction Gas Chromatography Mass Spectrometry in<br />
Geographical Pr<strong>of</strong>iling and Characterization <strong>of</strong> Volatile Organic Compounds in Kenyan<br />
Honey<br />
YS 12 Mokua G.N., Innocent E., Mbwambo Z., Lwande W., Ahmed Hassanali<br />
Evaluation <strong>of</strong> Larvicidal Activity and Phytoextract Induced Morhological Disruptions <strong>of</strong><br />
Vitex Schiliebenii Extracts Against Anopheles Gambiae Larvae<br />
YS 13 Gomotsang Bojase-Moleta, Alan D. Payne and Michael S. Sherburn<br />
The Relative Stabilities and Reactivities <strong>of</strong> the First Six Members <strong>of</strong> the Dendralene<br />
Family<br />
YS 14 A.L. Deng, J.O. Ogendo, P.K. Bett, M. Kamatenesi-Mughisha and J.M. Mihale<br />
Fumigant and Contact Toxicity <strong>of</strong> Cupressus lusitanica and Eucalyptus saligna Essential<br />
Oils against Insect Pests <strong>of</strong> Stored Cereals and Legumes<br />
YS 15 Mihigo, S.O., Mammo, W., Bezabih, M., Marobela, A-K., Abega, B.M.<br />
Rhuschalcone VI: Synthesis, Re-Isolation and Bioactivities in its Analogues<br />
YS 16 Robert Opiro, Anne M. Akol, Joseph Okello-Onen<br />
Acaricidal Effects <strong>of</strong> Four Plant Species on Rhipicephalus appendiculatus Neumann<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
(Acarina ixodidae) Ticks<br />
YS 17 Ruth A. Omole, Isaiah O. Ndiege and Alex K. Machocho<br />
Anti-Malarial Activity and Phytochemical Studies <strong>of</strong> Cissampelos Mucronata and<br />
Stephania Abyssinica<br />
YS 18 Anastasia N. Nandwa<br />
Effects <strong>of</strong> Sida Cuneifolia (A.Gray) Herbal Extracts on the Reproductive System<br />
Functioning in Male and Female Laboratory Rats<br />
YS 19 Odak Jenipher A., Manguro Lawrence O. and Ogur Joseph A<br />
Phytochemical Evaluation <strong>of</strong> Elaeodendron buchananii Stem Bark for Microbial Activities<br />
YS 20 Baluku Joward<br />
Evaluating Community Knowledge, Management and Economic Losses due to a<br />
Zoonotic Disease: A Case Study <strong>of</strong> Newcastle Disease in Kasese Municipal Council,<br />
Western Uganda<br />
YS 21 Sylvia A. Opiyo, Lawrence O.A. Manguro, Philip Okinda-Owuor, Elijah M. Ateka and Peter<br />
Lemmen<br />
Further Phytochemical and Antimicrobial Activity Studies <strong>of</strong> Warburgia Ugandensis<br />
against Sweet Potato Pathogens<br />
YS 22 Nalumansi Patricia, Kamatenesi Maud-Mugisha, John Robert Steven Tabuti<br />
Medicinal Plants used in Disease Management among Children in Namungalwe Sub<br />
County, Iganga District<br />
YS 23 Charles O Ochieng , P. Okinda Owuor, Lawrence A.O. Mang uro, Hosea Akala, Ismail O.<br />
Ishola.<br />
Antiplasmodial and Antinociceptive constituents from Caesalpinia volkensii Harms<br />
(Caesalpiniaceae) Root Bark<br />
YS 24 Kosgey Janet Cheruiyot<br />
Documentation <strong>of</strong> Medicinal Plants Found in Keiyo County Cherebes and Endo Village<br />
YS 25 Turibio K. Tabopda, Ghislain W. Fotso, Joseph Ngoupayo, Anne-Claire Mitaine-Offer,<br />
Bonaventure T. Ngadjui, Marie-Aleth Lacaille-Dubois<br />
Antimicrobial Dihydroisocoumarins from Crassocephalum biafrae<br />
YS 26 Chrian Marciale, Paul Erasto , Joseph N Otieno<br />
Antimycobacterial and Cytotoxicity Activity <strong>of</strong> Extracts from Zanthoxylum rhalybeum<br />
and Hallea rubrostipulata<br />
YS 27 Damien S. Tshibangu, Francis O. Shode, Neil Koorbanally, V. Mudogo, Pius T. Mpiana, Jean<br />
Paul K. Nbgolua<br />
Anti-Sickling Triterpenoids from Callistemon Viminalis, Melaleuca Bracteata Var.<br />
Revolution Gold Syzygium Guineense and Syzygium Cordatum<br />
POSTER PRESENTATIONS<br />
PS 1 John Onyango Adongo, Josiah O. Omolo, Peter K. Cheplogoi, Dan O. Otaye<br />
In vitro Inhibition <strong>of</strong> Botrytis cinerea - Causative Agent for Grey Mold by Crude Extracts<br />
<strong>of</strong> Basidiomycetes Fungi<br />
xi<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
PS 2 Daniel Bestina, Innocent E and Mbwambo Z.H.<br />
Biochemical Comparison <strong>of</strong> Annona Squamosa L. Leaves Growing In Different Eco-Zones<br />
In Tanzania For Mosquito Larvicidal Activity.<br />
PS 3 Bosire C. M., Kabaru J.M., Yenesew A., Kimata D. M<br />
A Toxicological Study <strong>of</strong> Millettia usaramensis Stem Bark Extract on Aedes aegypti<br />
(Mosquito), Schistocerca gregaria (Desert Locust) and Mus musculus (Mouse)<br />
PS 4 Ivan Gumula, Mathias Heydenreich, Solomon Derese, Faith A. Okalebo, Isaiah O. Ndiege,<br />
Mate Erdelyi, Abiy Yenesew<br />
Bioactivity <strong>of</strong> Flemingin A and other Natural Products from the Leaves <strong>of</strong> Flemingia<br />
grahamiana<br />
PS 5 Mohamed Said Hassani<br />
Seasonal Variation in the Chemical composition <strong>of</strong> the Bark Ocotea comoriensis<br />
Essential Oils<br />
PS 6 Khalid, I.I., Elhardallou, S.B. and Elkhalifa, E.A.<br />
Proximate and Amino Acid Composition <strong>of</strong> Cowpea (Vigna ungiculata L.walp) Flour and<br />
Protein Isolates<br />
PS 7 Kariuki S. M., D. J. Kim, Dossaji S. F., Kabaru J. M.<br />
Molecular Species Identification and the Respective Quantification <strong>of</strong> Dioscin: A Case <strong>of</strong><br />
Dioscorea spp<br />
PS 8 Kibrom Gebreheiwot, Dibaba Amenu and Nigist Asfaw<br />
Cuauthemone Sesquiterpenes from Laggera Tomentosa Endemic to Ethiopia<br />
PS 9 Joyce Jepkorir Kiplimo, Shahidul Md. Islam and Neil A. Koorbanally<br />
Novel Limonoids and Flavonoid from the Kenyan Vepris uguenensis Engl. and their<br />
Antioxidant Potential<br />
PS 10 Richard N. Mbithi<br />
Need to Regulate Herbal Remedies in Kenya<br />
PS 11 Mekonnen Abebayehu and Nigist Asfaw<br />
Phytochemical Investigation <strong>of</strong> Satureja abyssinica<br />
PS 12 Peninah Njoki, Hellen Kutima, Rebecca Waihenya, Dorcas Yole<br />
Determination <strong>of</strong> Efficacious Praziquantel Dose in Different Mouse Strains: BALB/c and<br />
Swiss Mice for Treatment <strong>of</strong> Schistosoma Mansoni<br />
PS 13 Jacqueline V. Ndlebe, Vinesh J. Maharaj , Gerda Fouche, Paul Steenkamp, Natasha<br />
Kolesnikova<br />
Cytotoxicity <strong>of</strong> a Novel Diterpenoid from Suregada zanzibariensis<br />
PS 14 Emmanuel Rubagumya, Emil K. Abotsi and Ignatious Ncube<br />
Solid-state Fermentation <strong>of</strong> Jatropha curcas Seed Meal Using Aspergillus niger<br />
Eliminates Molluscicidal Activity and Changes the Phorbol Ester Composition <strong>of</strong> the<br />
Seed Meal<br />
PS 15 Judith Agot Odhiambo, Catherine Wanjiru Lukhoba, Saifuddin Fidahussein Dossaji<br />
Ethnomedicinal Knowledge in the Traditional Management <strong>of</strong> Human Ailments in Lake<br />
Victoria Basin, Kenya<br />
PS 16 Danielle A. Doll RAKOTO, Clara RAJEMIARIMOELISOA, Ranjàna RANDRIANARIVO, Delphin<br />
RAMAMONJISON, Christian RAHERINIAINA, Noelinirina RAHARISOA, Victor JEANNODA<br />
In vitro Antimicrobial Activity <strong>of</strong> Extracts from five Malagasy Endemic Species <strong>of</strong> Albizia<br />
xii<br />
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298<br />
301<br />
305<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
(Fabaceae)<br />
PS 17 Sanogo R., Haïdara M., Dénou A., De Tommasi N. c , Occhiuto F.<br />
Phytochemical and Pharmacological Studies <strong>of</strong> Extracts <strong>of</strong> Trichilia emetica Used in the<br />
Treatment <strong>of</strong> Dysmenorrhoea in Mali<br />
PS 18 Sanogo R., Haïdara M., Dénou A., Diarra M., Kamaté B., Togola A., Bah S., Diallo, D.<br />
Hepatoprotective Activity <strong>of</strong> Aqueous Extracts <strong>of</strong> Leaves, Stem Bark and Roots <strong>of</strong> Entada<br />
africana Against Carbon Tetrachloride-Induced Hepatotoxicity in Rats.<br />
PS 19 Taiwo, B.J., Ogundaini, A.O. and Obuotor, E.M.<br />
The Radical Scavenging Activity <strong>of</strong> Flavonoids from Solenostemon monostachys (P.Beauv.)<br />
Briq (Lamiaceae).<br />
PS 20 Balogun S.O., Tanayen J.K. , Ajayi A.M., Ibrahim A., Ezeonwumelu J.O.C., Oyewale A.A., Loro J.<br />
O, Goji A.D.T., Kiplagat D.M., Adzu B.<br />
Preliminary Evaluation <strong>of</strong> Anti-Diarrheal, Ulcer-Protective and Acute Toxicity <strong>of</strong> Aqueous<br />
Ethanolic Stem Bark Extract <strong>of</strong> Ficus trichopoda in Experimental Rodents<br />
PS 21 James Ndia Muithya, Alex Kingori Machocho, Alphonse Wafula Wanyonyi and Paul Kipkosgei<br />
Tarus<br />
Phytochemical and In Vitro Antimicrobial Echinops Hispidus Fresen<br />
PS 22 E. Katuura, J.S.R.Tabuti, M. Kamatenesi-Mugisha & J. Ogwal Okeng<br />
Efficacy and safety pr<strong>of</strong>ile <strong>of</strong> some Ugandan antimalarial herbs used in Primary Health<br />
Care<br />
xiii<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Programme<br />
Programme for<br />
for<br />
the<br />
the<br />
14 th NAPRECA The 14 Symposium and AAMPS Ethnoveterinary Medicine<br />
Symposium<br />
th NAPRECA Symposium and AAMPS<br />
Ethnoveterinary Medicine Symposium<br />
08:00-09:00 Registration<br />
Chairperson: Pr<strong>of</strong>. Ahmed Hassanali<br />
MONDAY, AUGUST 8 TH 2011<br />
SESSION I: OPENING SESSION<br />
09:00 09:05 SPEECH 1 NAPRECA-K<br />
09:05 09:10 SPEECH 2 Pr<strong>of</strong>. J.O. Midiwo<br />
09:10 09:15 SPEECH 3 ICIPE<br />
09:15 09:20 SPEECH 4 DAAD<br />
09:20 09:25 SPEECH 5 ISP<br />
09:25 09:30 SPEECH 6 NCST<br />
09:30 - 10:15 KEYNOTE ADDRESS<br />
Chemical Sciences in Africa Historical<br />
Insights and Major Milestones<br />
10:15 10:30 Reaction to the Keynote Address<br />
10:30 11:00 HEALTH BREAK<br />
SESSION II - PLENARY LECTURES I<br />
Chairperson: Pr<strong>of</strong>. J. O. Midiwo<br />
PL 1 11:00 11:40 Natural Products: Evolution <strong>of</strong> Structural and<br />
Functional Diversity and Implication for R&D<br />
Targeting Useful Candidates<br />
PL 2 11:40 12:20 Shifting the paradigm to the ethnobotany-based<br />
drug discovery<br />
PL 3 12:20 13:00 Why has there been hardly any success in<br />
developing antimicrobial products from medicinal<br />
plants?<br />
13:00 14:00 LUNCH BREAK<br />
xiv<br />
Pr<strong>of</strong>. Berhanu Abegaz<br />
Executive Director<br />
African Academy <strong>of</strong> Sciences<br />
Pr<strong>of</strong>. Ahmed Hassanali<br />
Pr<strong>of</strong>. P. Rasoanaivo<br />
Pr<strong>of</strong>. Kobus El<strong>of</strong>f
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
SESSION III - PLENARY LECTURES II<br />
Chairperson: Pr<strong>of</strong>. Ermias Dagne<br />
PL 4 14:00 14:40 Phytochemical Investigations <strong>of</strong> leaves and<br />
bark <strong>of</strong> Croton gratissimus (Euphorbiaceae)<br />
PL 5 14:40 15:20 Challenges and opportunities <strong>of</strong><br />
traditional/herbal medicine<br />
PL 6 15:20 16:00 Approaches to the synthesis <strong>of</strong> tricarbonyl<br />
metabolites: An Isoxazole Strategy<br />
16:00 16:30 Health break<br />
SESSION IV - PARALLEL SESSIONS SHORT LECTURES I<br />
xv<br />
Pr<strong>of</strong>. D. Mullholland<br />
Pr<strong>of</strong>. Ivan Addae-Mensah<br />
Pr<strong>of</strong>. Ray Jones<br />
LECTURE ROOM A LECTURE ROOM B<br />
Chairperson: Pr<strong>of</strong>. I.O. Jondiko Chairperson: Dr. Cathrine Lukhoba<br />
16:30 16:50 [SL 1A] Sakina Yagi<br />
16:30 16:50 [SL-1B] David R. Katerere<br />
Phytochemical and biological activity<br />
Application <strong>of</strong> in vitro drug<br />
studies on Aloe sinkatana<br />
metabolism and disposition<br />
studies to assess risk <strong>of</strong> drug<br />
interactions with Sutherlandia<br />
16:50 -17:10 [SL 2A] Moses Langat<br />
16:50 -17:10<br />
frutescens extracts<br />
[SL 2B] Alice Njue<br />
Chemical constituents <strong>of</strong> East European<br />
In vitro inhibition <strong>of</strong> tomato<br />
Species<br />
Fusarium wilt causative agent by<br />
zearalenone from a soil inhabiting<br />
fungus<br />
17:10 17:30 [SL 3A] Robert Byamukama<br />
17:10 17:30 [SL 3B] Hayder Abdelgader<br />
Anthocyanins from selected plant species in<br />
Side Effects <strong>of</strong> some Botanicals on<br />
Uganda<br />
the egg parasitoid Trichogramma<br />
spp.<br />
17:30 17:50 [SL-4A] V. Jayakumar<br />
17:30 17:50 [SL-4B] M.M.O. Omolo<br />
Bioactive constituents from Hyptis<br />
Blends <strong>of</strong> Chemicals in Smelly Feet<br />
suaveolens<br />
Switch Malaria Mosquitoes on and<br />
<strong>of</strong>f.<br />
17:50 18:10 [SL 5A] A. Mohammed<br />
Extractives from the genus Heteropyxis<br />
18:10 18:30 [SL-6A] Nour A. Osman<br />
Chemical constituents <strong>of</strong> the essential oil <strong>of</strong><br />
Cymbopogon proximus and their potential<br />
for the treatment <strong>of</strong> otomycosis<br />
19:00 Mixer Cocktail<br />
17:50 18:10 [SL-5B] Theoneste Muhizi<br />
Essential oil from some Rwandese<br />
plants and their antibacterial<br />
activity<br />
18:10 18:30 [SL 6B] Machocho, A.K.<br />
Antimicrobial Activity and<br />
Phytochemical Studies <strong>of</strong> Some<br />
Selected Medicinal Kenyan Plants
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
TUESDAY, AUGUST 9 TH 2011<br />
SESSION V -PLENARY LECTURES II<br />
CHAIRPERSON: Pr<strong>of</strong>. Philippe Rasoanaivo<br />
PL 7 08:00 08:40 Absolute Stereostructures by LC-CD Coupling in<br />
PL 8 08:40 09:20<br />
Combination with Quantum-Chemical CD Calculations<br />
Technology Platforms to Facilitate Natural Product-<br />
Based Drug Discovery from African Biodiversity<br />
PL 9 09:20 10:00 From past traditions to a Herbal Pharmacopoeia<br />
Green Gold<br />
Africa s<br />
PL 21 10:00 10:20 The pan-African Natural Product Library (p-ANPL): Giving<br />
steam a direction<br />
10:20 10:40 Health break<br />
SESSION VI - PARALLEL SESSIONS SHORT LECTURES II<br />
IV A IVB<br />
LECTURE ROOM A LECTURE ROOM B<br />
Chairperson: Pr<strong>of</strong>. Joseph Keriko Chairperson: Pr<strong>of</strong>. Kobus El<strong>of</strong>f<br />
10:40 11:00 [SL-7A] Abiy Yenesew<br />
Antiplasmodial and Radical Scavenging<br />
Activities <strong>of</strong> Flavonoids from Kenyan<br />
Erythrina species<br />
11:00 11:20 [SL-8A] Claudia Steiner<br />
Isolation and structure elucidation <strong>of</strong><br />
bioactive compounds <strong>of</strong> the tropical liana<br />
Ancistrocladus congolensis<br />
11:20 - 11:40 [SL-9A] Bonaventure T. Ngadjui<br />
Arylbenz<strong>of</strong>urans, Prenylated Flavonoids<br />
and Diels Ader Adducts with biological<br />
activities from Morus mesozygia<br />
11:40 12:00 [SL-10A] Stephen S. Nyandoro<br />
Aristolactams, indolidinoids and other<br />
metabolites from Toussaintia orientalis -<br />
An endangered Annonaceae species<br />
endemic to Tanzania.<br />
12:00 12:20 [SL-11A] Josiah O. Joo<br />
Odalo<br />
Bioactive furanoditerpenoids, a<br />
dibenzopyranone, nor-isoprenoid and<br />
xvi<br />
Pr<strong>of</strong>. G. Bringmann<br />
Pr<strong>of</strong>. Kelly Chibale<br />
Pr<strong>of</strong>. Ameenah G-Fakim<br />
Andrae-Marobela K.<br />
10:40 11:00 [SL 20A] Margart Ng ang a<br />
Antiplasmodial Compounds from<br />
the leaves <strong>of</strong> Drypetes gerrardi<br />
11:00 11:20 [SL-8B] Inshirah. A. Elfahal<br />
The efficacy <strong>of</strong> extracts <strong>of</strong> the plant<br />
Argemone mexicana on mosquito<br />
species, Anopheles arabiensis<br />
11:20 - 11:40 [SL-9B] Danielle A. D. Rakoto<br />
In vitro effects <strong>of</strong> extracts from five<br />
Malagasy endemic species <strong>of</strong><br />
Albizia (Fabaceae) on vegetable<br />
seeds germination<br />
11:40 12:00 [SL-10B] Ester Innocent<br />
From laboratory to field application<br />
<strong>of</strong> phyto-larvicides: An outreach<br />
community based experience in<br />
Bagamoyo District, Tanzania.<br />
12:00 12:20 [SL-11B] I.O. Jondiko<br />
Toddalia Asiatica. Lin: A Potential<br />
Source and Model <strong>of</strong> Materials and<br />
Services for Control <strong>of</strong> Diseases and<br />
Implications for Herbal Medical
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
biflavonoids from medicinal Stuhlmania<br />
moavi verdc.<br />
12:20 12:40 [SL-12A] Danstone L. Baraza<br />
Fungitoxic C-18 hydroxy unsaturated fatty<br />
acids from fruiting bodies <strong>of</strong> Cantharellus<br />
species<br />
12:40 13:00 [SL-13A] Bernard F. Juma<br />
Synthesis <strong>of</strong> 2,6-Dioxo-1,2,3,4,5,6hexahydroindoles<br />
and their<br />
Transformation into 5,8,9,10-Tetrahydro-<br />
6H-indolo[2,1-a]isoquinolin-9-ones<br />
13:00 14:00 Lunch<br />
SESSION VII - PLENARY LECTURES III<br />
CHAIRPERSON: Pr<strong>of</strong>. Dulcie Mulholland<br />
PL 10 14:00 14:40 NMR Analysis <strong>of</strong> the Molecular Structure <strong>of</strong><br />
Flexible Molecules in Solution<br />
PL 11 14:40 15:20 Bioassay <strong>of</strong> natural products for cosmetics<br />
PL 12 15:00 15:40 Multiple anti-infective properties <strong>of</strong> selected<br />
Combretum species from Zimbabwe<br />
PL 13 15:40 16:20 The Center for World Health & Medicine at<br />
Saint Louis University: A New Translational<br />
Research Model to Develop Novel Therapies<br />
for Neglected Diseases and Other Unmet<br />
Medical Needs<br />
16:20 17:30 Health break/Poster Session<br />
SESSION VIII PLENARY LECTURES V<br />
PL 14 17:30 18:10 Towards Gaining Recognition as an African Centre <strong>of</strong><br />
Excellence in Applied Nanomedicine Research and<br />
Training for Poverty Related Diseases<br />
DST/CSIR Nanomedicine Platform.<br />
Focus on the<br />
PL 15 18:10 18:50 Application <strong>of</strong> UV-Vis Spectroscopy to Evaluate Quality <strong>of</strong><br />
Medicinal and Edible Oils<br />
xvii<br />
Practice in Kenya.<br />
12:20 12:40 [SL- 12B] Alvaro Viljoen<br />
Application <strong>of</strong> vibrational<br />
spectroscopy and planar<br />
chromatography in the quality<br />
control <strong>of</strong> South African medicinal<br />
and aromatic plants<br />
12:40 13:00 [SL-13B] Najma Dharani<br />
Medicinal Plants <strong>of</strong> East Africa-<br />
Importance, Uses in Traditiona<br />
Medicine, Challenges and<br />
Conservation Status<br />
Pr<strong>of</strong>. Máté Erdélyi<br />
Dr. Alain Meybeck<br />
Dr. S. Mukanganyama<br />
Pr<strong>of</strong>. Peter G. Ruminski<br />
Dr. Hulda Swai<br />
Pr<strong>of</strong>. Ermias Dagne
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Chairperson: Pr<strong>of</strong>. Berhanu Abegaz<br />
08.45<br />
08:55<br />
Welcome Thad Simons<br />
WEDNESDAY, AUGUST 10 TH 2011<br />
ETHNOVETERINARY MEDICINE SYMPOSIUM<br />
SESSION A<br />
Pr<strong>of</strong>. Ameenah Gurib Fakim<br />
Pr<strong>of</strong>. Jacob Midiwo<br />
PL I 9:00 9:40 From ethnoveterinary medicines to<br />
phytomedicines<br />
PL II 9:40 10:00 The role <strong>of</strong> botanical gardens in<br />
ethno-veterinary research<br />
SL I 10:00 - 10:15 Novel control strategies for the<br />
Southern Cattle Tick<br />
10:15 -10:45 Question and Answer<br />
Chairperson: Pr<strong>of</strong>. Jacob Midiwo<br />
10.45 11:15 HEALTH BREAK<br />
SESSION B<br />
PL III 11:15 11:50 Research on the use <strong>of</strong> plant extracts to<br />
enhance animal productivity in Southern<br />
Africa<br />
SL II 11:50 12:10 Validation <strong>of</strong> some plants used by Eastern<br />
Cape farmers in the control <strong>of</strong> internal and<br />
external parasites <strong>of</strong> livestock<br />
SL III 12:10 12:30 Evaluation <strong>of</strong> plants used traditionally to<br />
protect animals against myasis<br />
xviii<br />
President & CEO Novus International<br />
Chairman <strong>of</strong> AAMPS<br />
SL IV 12:30 12:50 Funding opportunities for research and the<br />
potential value <strong>of</strong> an African Herbal<br />
Pharmacopoeia for animal health and<br />
productivity<br />
12:50 13:00 Question and Answer<br />
Executive Secretary <strong>of</strong> NAPRECA<br />
Dr. David Katerere<br />
Medical Research Council, Cape<br />
Town, Editor <strong>of</strong> Ethno-veterinary<br />
Botanical Medicine: Herbal<br />
medicines for Animal Health<br />
Dr. Wendy Applequist<br />
Assistant Curator, Missouri<br />
Botanical Garden, USA<br />
Representative from ICIPE<br />
Pr<strong>of</strong> JN El<strong>of</strong>f<br />
Phytomedicine<br />
Programme, University <strong>of</strong><br />
Pretoria<br />
Pr<strong>of</strong>. Patrick Masika<br />
University <strong>of</strong> Fort Hare<br />
Zimbabwe<br />
Ms. Lilian Mukandiwe<br />
University <strong>of</strong> Harare,<br />
Zimbabwe<br />
Pr<strong>of</strong>. Ameenah Gurib-Fakim<br />
University <strong>of</strong> Mauritius
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
1300 1400 Lunch<br />
SESSION C<br />
Chairperson: Pr<strong>of</strong> Ameenah Gurib-Fakim<br />
PL IV 14:00 14:30 Highlights <strong>of</strong> East African ethnoveterinary<br />
medicine research<br />
SL V 14:30 15:00 Animal and human Trypanosomiasis:<br />
Challenges to medicinal plant research in<br />
Africa.<br />
SL VI 15:00 15:20 A brief review <strong>of</strong> the use <strong>of</strong> medicinal plants<br />
in veterinary medicine in Mali.<br />
15:20 16:00 General panel discussion and concluding<br />
remarks<br />
16:00 16:30 Health Break<br />
xix<br />
Dr. John Githiori<br />
16:30 Annual General Meeting <strong>of</strong> Association for African Medicinal Plant<br />
SESSION IX PARALLEL LECTURES- SHORT LECTURES III<br />
IV A IVB<br />
Pr<strong>of</strong>. Clement Adewunmi,<br />
Obafemi Awolowa<br />
African Journal <strong>of</strong><br />
Traditional,<br />
Complementary&<br />
Alternative Medicine<br />
Pr<strong>of</strong>. Drissa Diallo<br />
Dept. <strong>of</strong> Traditional<br />
Medicine<br />
Bamako, Mali<br />
Chairmen & speakers<br />
LECTURE ROOM A LECTURE ROOM B<br />
Chairperson: Chairperson:<br />
16:30 16:50 [SL 14A] Okoth, M.O.<br />
Crystallization for Long Range Molecular<br />
Order Structure Elucidation<br />
16:50 17:10 [SL 15A] Elwaleed E. Hassan<br />
Antileishmanial Activity <strong>of</strong> Petroleum<br />
ether, n-hexane Crude Extract and (2E)methyl<br />
3-((1E, 4E)-7-methyl-4-(2oxopropylidene)<br />
cyclohept-1-enyl)<br />
acrylate from Xanthium brasilicum Vell.<br />
leaves.<br />
17:10 17:30 [SL 18A] Claude Kirimuhuzya<br />
The in vitro antimycobacterial activity <strong>of</strong><br />
medicinal plants used by traditional<br />
medicine practitioners (TMPs) to treat<br />
tuberculosis in the Lake Victoria basin in<br />
Uganda<br />
16:30 16:50 [SL 16A] Tanayen, J.K.<br />
Assessment <strong>of</strong> Azadirachta Indica<br />
and Cassia Spectabilis for Some<br />
Immunomodulatory Properties<br />
16:50 17:10 [SL 17A] Maud K.-Mugisha<br />
Evaluation <strong>of</strong> the Biosafety <strong>of</strong><br />
Selected Botanical Pesticide Plants<br />
Used by Subsistence Farmers<br />
Around the Lake Victoria Basin<br />
17:10 17:30 [SL 19A] Faiza E. E. Salah<br />
Effects <strong>of</strong> Aqueous Extracts <strong>of</strong><br />
Basil, Ocimum basilicum L.,<br />
Sodom s apple, Calotropis procera<br />
Ait and Coriander Coriandrum<br />
sativum L. on leaf miner,<br />
Liriomyza Spp., on okra Crop.
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Chairperson: Pr<strong>of</strong>. D. Mullholland<br />
19.30 Novus International Sponsored Dinner<br />
Thursday 11 TH August 2011<br />
SESSION X - PLENARY LECTURES VI<br />
PL 16 08:00 08:40 Natural Products from Plant Diversity and their<br />
Potential in Management <strong>of</strong> Neglected Diseases.<br />
PL 17 08:40 09:20 Changes in Plants Metabolites with Location <strong>of</strong><br />
Growth and Agronomic Practices: Some Lessions<br />
from Black Tea Quality Studies<br />
PL 18 09:20 10:00 Challenges <strong>of</strong> Isolation, Characterization and<br />
Pr<strong>of</strong>iling <strong>of</strong> African Medicinal Plants: Analytical<br />
Prospective <strong>of</strong> Standardization and Quality<br />
Control Methods<br />
10:00 10:30 Tea break<br />
xx<br />
Pr<strong>of</strong>. J. O. Midiwo<br />
Pr<strong>of</strong>. P. Okinda Owuor<br />
Pr<strong>of</strong>. Mathew M Nindi<br />
SESSION XI PARALLEL SESSION- YOUNG SCIENTIST COMPETITION I/p-ANPL Meeting<br />
IV A IVB IV C<br />
LECTURE ROOM A LECTURE ROOM B LECTURE ROOM C<br />
Chairperson: Pr<strong>of</strong>. Gerhard Bringmann Chairperson: Pr<strong>of</strong>. Kelly Chibale Chairperson: Pr<strong>of</strong>. B. Abegaz<br />
10:30 10:50 [YS-1] Ndinteh D.T<br />
Derek Tantoh<br />
The Genus Erythrina a<br />
source <strong>of</strong> many useful<br />
phytochemicals.<br />
10:50 11:10 [YS-3] Lois Mwikali<br />
Mutisya<br />
Terpurinflavone:<br />
Antiplasmodial Flavones<br />
from the Stem <strong>of</strong><br />
Tephrosia purpurea<br />
11:10 11:30 [YS-5] Milkyas Endale<br />
Annisa<br />
Antiplasmodial<br />
quinones from selected<br />
Pentas species<br />
11:30 11:50 [YS-7] Ivan Gumula<br />
New Prenylated<br />
Is<strong>of</strong>lavanones From the<br />
Stem Bark <strong>of</strong><br />
Platycelphium Voense<br />
10:30 10:50 [YS-2] Elizabeth V.M.<br />
Kigondu<br />
Antimalarial and<br />
Antileishmanial Activity<br />
and Cytotoxicity <strong>of</strong><br />
Selected Medicinal Plants<br />
from Kenya<br />
10:50 11:10 [YS-4] Beatrice. N. Irungu<br />
In vitro antiplasmodial<br />
and cytotoxicity activities<br />
<strong>of</strong> selected medicinal<br />
plants from Kenya<br />
11:10 11:30 [YS-6] Justin N. Kabera<br />
Therapeutic (Verucidal)<br />
effect study <strong>of</strong> juice <strong>of</strong><br />
Tetradenia (Iboza) riparia<br />
leaves on the warts.<br />
11:30 11:50 [YS-8] Chris J.D. Obbo<br />
Antitrypanosomal,<br />
Antileishmanial and<br />
Antiplasmodicidal<br />
Activities <strong>of</strong> Khaya<br />
pan-African Natural<br />
Product Library (p-ANPL)<br />
General Meeting.
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
11:50 12:10 [YS-9] Francis Machumi<br />
Antiplasmodial and<br />
antileishmanial studies<br />
on carvotacetone<br />
derivatives from<br />
Sphaeranthus bullatus<br />
12:10 12:30 [YS-11] Fredrick Munga<br />
Ng ang a<br />
Application <strong>of</strong> Solid<br />
Phase Extraction Gas<br />
Chromatography Mass<br />
Spectrometry in<br />
Geographical Pr<strong>of</strong>iling<br />
and Characterization <strong>of</strong><br />
Volatile Organic<br />
Compounds in Kenyan<br />
Honey<br />
12:30 12:50 [YS-13] Gomotsang<br />
Bojase<br />
The Relative Stabilities<br />
and Reactivities <strong>of</strong> the<br />
First Six Members <strong>of</strong> the<br />
Dendralene Family<br />
12:50 13:10 [YS-15] Mihigo, S.O<br />
Rhuschalcone VI:<br />
Synthesis, Re-Isolation<br />
and Bioactivities in its<br />
Analogues<br />
xxi<br />
anthotheca, a Plant used<br />
by Chimpanzees for Self<br />
Medication.<br />
11:50 12:10 [YS-10] Rechab S.<br />
Odhiambo<br />
In vitro anthelmintic<br />
effect <strong>of</strong> Prosopis juliflora<br />
(Sw.) DC on Haemonchus<br />
contortus, an abomasal<br />
nematode <strong>of</strong> sheep<br />
12:10 12:20 [YS-12] Gladys Nyamoita<br />
Mokua<br />
Evaluation <strong>of</strong> larvicidal<br />
and phytoextract induced<br />
morphological activities<br />
<strong>of</strong> Vitex schiliebenii<br />
extracts against<br />
Anopheles gambiae<br />
larvae<br />
[YS-14] Philip K. Bett<br />
Fumigant and Contact<br />
Toxicity <strong>of</strong> Cupressus<br />
lusitanica and Eucalyptus<br />
saligna Essential Oils<br />
Against Insect Pests <strong>of</strong><br />
Stored Cereals and<br />
Legumes<br />
[YS-16] Robert Opiro<br />
Acaricidal Effects <strong>of</strong> Four<br />
Plant Species on<br />
Rhipicephalus<br />
appendiculatus Neumann<br />
(Acarina ixodidae) Ticks<br />
13:10 14:00 Lunch<br />
SESSION XII PLENARY LECTURES VII<br />
Chairperson:Pr<strong>of</strong>. Philip Owuor<br />
PL 19 14:00 14:40 Exploiting the chemistry <strong>of</strong> African biodiversity in Pr<strong>of</strong>. John Pickett<br />
pest management: from extraction <strong>of</strong> the<br />
PL 20 14:40 15:20<br />
chemicals to expression in GMOs<br />
Development <strong>of</strong> Medicines from African Medicinal<br />
Plants: Experiences in West Africa<br />
Pr<strong>of</strong>. Drissa Diallo
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
SESSION XII - YOUNG SCIENTIST COMPETITION III/SHORT LECTURES III<br />
IV A IVB<br />
LECTURE ROOM A LECTURE ROOM B<br />
Chairperson: Chairperson:<br />
15:20 15:40 [YS-17] Ruth A. Omole<br />
Anti-Malarial Activity and Phytochemical<br />
Studies <strong>of</strong> Cissampelos Mucronata And<br />
Stephania Abyssinica<br />
15:40 16:00 [YS-19] Jenipher Odak<br />
Phytochemical Evaluation <strong>of</strong><br />
Elaeodendron buchananii Stem Bark for<br />
Microbial Activities<br />
16:00 16:20 HEALTH BREAK<br />
SESSION X - YOUNG SCIENTIST COMPETITION III<br />
16:20 16:40 [YS-17] Ruth A. Omole<br />
Anti-Malarial Activity and Phytochemical<br />
Studies <strong>of</strong> Cissampelos Mucronata And<br />
Stephania Abyssinica<br />
16:40 17:00 [YS-19] Jenipher Odak<br />
Phytochemical Evaluation <strong>of</strong><br />
Elaeodendron buchananii Stem Bark for<br />
Microbial Activities<br />
17:00 17:20 [YS 21] Sylvia A. Opiyo<br />
Further Phytochemical and Antimicrobial<br />
Activity Studies <strong>of</strong> Warburgia Ugandensis<br />
Against Sweet Potato Pathogens<br />
17:20 17:40 [YS-23] Charles Ochieng<br />
Antiplasmodial and Antinociceptive<br />
constituents from Caesalpinia volkensii<br />
Harms (Caesalpiniaceae) Root Bark<br />
18:00 18:20 [YS -25] Fotso Ghislain Wabo<br />
Antimicrobial Dihydroisocoumarins from<br />
Crassocephalum Biafrae<br />
18:20 19:00 CLOSING CEREMONY<br />
xxii<br />
12:20 12:40 [YS-18] Anastasia Nandwa<br />
Effects <strong>of</strong> Sida Cuneifolia (A.Gray)<br />
herbal extracts on the reproductive<br />
system functioning in male and<br />
female laboratory rats<br />
12:40 13:00 [YS 20] Joward Baluku<br />
Risk-factors and the indigenous<br />
knowledge in the management <strong>of</strong><br />
Newcastle Disease. A case study <strong>of</strong><br />
Kasese District, Western Uganda.<br />
16:20 16:40 [YS-18] Anastasia Nandwa<br />
Effects <strong>of</strong> Sida Cuneifolia (A.Gray)<br />
herbal extracts on the reproductive<br />
system functioning in male and<br />
female laboratory rats<br />
16:40 17:00 [YS 20] Joward Baluku<br />
Risk-factors and the indigenous<br />
knowledge in the management <strong>of</strong><br />
Newcastle Disease. A case study <strong>of</strong><br />
Kasese District, Western Uganda.<br />
17:00 17:20 [YS-22 ] Nalumansi Patricia<br />
Medicinal Plants used in Disease<br />
Management Among Children in<br />
Namungalwe Sub County.<br />
17:20 17:40 [YS-24] Kosgey Janet Cheruiyot<br />
Documentation <strong>of</strong> Medicinal Plants<br />
Found in Keiyo County Cherebes<br />
and Endo Village<br />
18:00 18:20 [YS 26] Chrian Marciale<br />
Antimycobacterial and Cytotoxicity<br />
Activity <strong>of</strong> Extracts from<br />
Zanthoxylum rhalybeum and Hallea<br />
rubrostipulata
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
KEYNOTE ADDRESS<br />
Chemical Sciences in Africa Historical Insights and Major Milestones<br />
Berhanu Abegaz<br />
African Academy <strong>of</strong> Sciences, Nairobi, Kenya<br />
Key words: Chemistry in Africa; historical perspectives; natural products<br />
he idea to address this topic was prompted by the declaration <strong>of</strong> 2011 as the International Year<br />
<strong>of</strong> Chemistry. The intention is to outline the major contributions <strong>of</strong> those scientists and<br />
research groups that laid the foundation <strong>of</strong> chemistry in general and natural product sciences in<br />
particular in Africa. Historically the applications <strong>of</strong> natural products such as incense, opium, castor<br />
oil, etc can be traced through ancient Egyptian heliographic documents like the Ebers papyrus<br />
writings to 1500 BC. Despite these ancient beginnings, there is little documented on indigenous<br />
chemical science emerging during the post Lavoisier era up to the end <strong>of</strong> the 19 th T<br />
century.<br />
Information on Africans who may have trained in Europe or the US during this period is also very<br />
scanty, although one finds that a Ghanian by the name <strong>of</strong> Anthony William Amo was most probably<br />
the first African to study in Europe and to even become pr<strong>of</strong>essor in two German Universities (Halle<br />
and Jena) around 1730 (Abraham, 1996). Amo eventually returned to Ghana, but there is no record<br />
<strong>of</strong> him having started a modern school <strong>of</strong> learning and research. The development <strong>of</strong> African<br />
universities like Ibadan, Makerere and Khartoum during colonial times was linked to the University<br />
<strong>of</strong> London. The research and teaching <strong>of</strong> chemistry in these pioneering institutions dates back to<br />
nearly seventy years, and most <strong>of</strong> the research was focused on natural products (Abegaz and<br />
Davies-Coleman, 2009). The development <strong>of</strong> chemistry in South Africa dating back to nearly one<br />
hundred years is relatively well documented (Mulholland and Drewes, 2004). Key names that<br />
feature prominently in the early development <strong>of</strong> South African chemistry include: Theiler, du Toit,<br />
Marais, Warren, Enslin, Roux, etc. with others, like Steyn, Bull, Drewes, Ferreiera, Nyokong,<br />
Michael, etc., forming some <strong>of</strong> the notable contemporary chemists. Some <strong>of</strong> the outstanding<br />
research from their efforts led to the discovery <strong>of</strong> compounds like geigerin from Geigera aspera,<br />
mon<strong>of</strong>loroacetic acid from Dichapetalum cymosum, caespitin from Helichrysum caespititium,<br />
rooperol from Hypoxis hemerocallidea, ocholbullenone from Ocotea bullata. Pioneering Ghanian<br />
chemists include Torto and Quartey who returned from training in the UK and initiated research in<br />
Ghana. Torto is known for starting research on anti-sickeling agents in West Africa (Torto et al.,<br />
1973), while Quartey contributed on several fronts including the development <strong>of</strong> the Birch<br />
reduction, and became a life-long friend <strong>of</strong> AJ Birch (Birch et al., 1952). The development <strong>of</strong><br />
chemistry in Nigeria is a shared contribution <strong>of</strong> Nigerian as well as European chemists with the most<br />
prominent ones being: Bevan, Taylor, Akinsanya, Ekong, Ogan, Powell, Nwaji, Arene, Eshiet,<br />
Adesogan, Olagbemi, Okogun, etc. who began active research on natural products and discovered a<br />
whole range <strong>of</strong> natural compounds including the now well known substance Gedunin (Akinsanya et<br />
1
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
al., 1960). Looking for anti-sickling compounds has been a major trust for West African chemists,<br />
and this eventually led to the FDA approved drug (called Nicosan) based on a traditional<br />
phytomedicine preparation (Wambebe, 2008). The development <strong>of</strong> chemistry in Ethiopia was<br />
related to efforts to find applications to some widely used plants like Kosso (which engaged well<br />
known chemists like Birch and Todd and companies like Merck) (Abegaz et al., 1999) and the soap<br />
berry plant, Phytolacca dodecandra and the discovery made by Lemma <strong>of</strong> the properties <strong>of</strong> this<br />
bush plant in killing bilharzia-causing snails (Lemma, 1990). In Kenya, the establishment <strong>of</strong> the<br />
international Center for insect physiology (icipe) has made direct contributions to the study <strong>of</strong><br />
natural products that have insecticidal properties as well as the study <strong>of</strong> semiochemicals from<br />
insects. The lecture will also describe the roles <strong>of</strong> pioneer chemists in Cameroon, Malawi, Kenya,<br />
Tanzania, etc. Chemistry has developed relatively well during the last decades with several<br />
research groups actively discovering natural compounds, while others are undertaking synthesis<br />
and analysis. Green chemistry is also taking a foothold in many countries through the collaborative<br />
efforts <strong>of</strong> European and Ethiopian scientists. The celebration <strong>of</strong> the international year <strong>of</strong> chemistry<br />
should therefore allow reflections on the role <strong>of</strong> chemistry for development especially for Africa.<br />
Many countries in Africa will probably not meet the MDGs in 2015, and hence they will<br />
unquestionably remain as key drivers for policy and actions in subsequent years. Chemists should<br />
have significant roles in this regard since chemistry can make definite contributions to at least the<br />
six <strong>of</strong> the eight Millennium Development Goals.<br />
References<br />
Abegaz, B.M., Davies-Coleman, M. T., (2009); A Brief History <strong>of</strong> Natural Product Research in Africa, The American<br />
Society <strong>of</strong> Pharmacognosy, in 50 Years <strong>of</strong> Progress in Natural Products Research 1959-2009, In: G.C. Cragg, J.A.<br />
Beutler & W.P. Jones, (eds.) Chapter 5, Our partners abroad.<br />
Abegaz, B.M., Ngadjui, B.T. Bezabih, M. Mdee, L.K. (1999); Novel natural products from marketed plants <strong>of</strong> eastern and<br />
southern Africa. Pure and Applied Chemistry, 919-216.<br />
Abraham, W.E. (1996); The Life and Times <strong>of</strong> Anton Wilhelm Amo, the first African (black) Philosopher in Europe In:<br />
Asante, Molefi Kete, Abu S. Abarry (eds.), African Intellectual Heritage. A <strong>Book</strong> <strong>of</strong> Sources, Philadelphia: Temple<br />
University Press, 424-440.<br />
Akisanya, A., Bevan, C.W.L., Hirst, J., Halsall, T.G., Taylor, D.A.H. (1960); West African timbers. Part III. Petroleum<br />
extracts from the genus Entandrophragma. Journal <strong>of</strong> the Chemical Society, 3827-3835.<br />
Birch AJ, Quartey J.A.K., Smith H. (1952); Hydroaromatic Xteroid Hormones. Part 111." Some Angular-methylated<br />
Intermediates. Journal <strong>of</strong> the Chemical Society, 1768-1774.<br />
Lemma, A. (1990); Science from the Third World. Bulletin <strong>of</strong> the Chemical Society <strong>of</strong> Ethiopia, 4, 79-82.<br />
Mulholland, D and Drewes, S. (2004); Global phytochemistry: indigenous medicinal chemistry on track in southern<br />
Africa. Phytochemistry, 65, 769-782.<br />
Torto, Addae-Mensah, Baxter (1973); Alkaloids <strong>of</strong> Ghanaian Medicinal Plants III. Phytochemistry, 12, 2315.<br />
Wambebe, C. (2008); From Plants to Medicine: Research and Development <strong>of</strong> NIPRISAN, a Drug for the Treatment <strong>of</strong><br />
Sickle Cell Disease, The New Legon Observer, 2(15), 19-21.<br />
2
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
PLENARY LECTURES<br />
3
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[PL 1] Natural Products: Evolution <strong>of</strong> Structural and Functional Diversity and<br />
Implication for R & D Targeting Useful Candidates<br />
Ahmed Hassanali<br />
Chemistry Department, Kenyatta University, Nairobi, Kenya<br />
Key Words: Natural products & ecology, structural & functional diversity, blend effects, bio-rational prospecting,<br />
opportunities & challenges<br />
Introduction<br />
D<br />
espite their dominant role in human culture, in the past, natural products ( secondary<br />
metabolites ) were <strong>of</strong>ten dismissed as evolutionary relics or waste products <strong>of</strong> primary<br />
metabolism without any specific biological function. Earlier scientific interest in secondary<br />
metabolites was driven partly by search for bioactive agents from plants, as targets or as models for<br />
synthetic analogues useful in pharmacognosy, chemotherapy and pesticidal science. However, it<br />
was the fascination <strong>of</strong> chemists for structural diversity, synthetic challenges and biogenetic origin<br />
that saw rapid expansion <strong>of</strong> our knowledge <strong>of</strong> natural products. By 1070s, there was growing<br />
recognition that natural products play very important ecological functions in ecosystems in which<br />
they have evolved. An evolutionary view accounts for the origin, diversity, range <strong>of</strong> bioactivities,<br />
and long-term performance <strong>of</strong> natural products.<br />
Important consequences resulting from co-evolutionary interactions within and between organisms<br />
mediated by secondary metabolites include (i) structural and analogue diversity <strong>of</strong> compounds<br />
<strong>of</strong>ten acting as blends to provide effective protection against specific predators or invaders (Cates,<br />
1996) and to mitigate against speedy resistance development (Feng and Isman, 1995; Isman et<br />
al.,1996); (ii) functional diversity <strong>of</strong> individual constituents or blends to respond to diverse groups<br />
<strong>of</strong> predators/invaders (Berenbaum and Zangerl, 1996); and (iii) subtlety in the actions <strong>of</strong> secondary<br />
metabolites, <strong>of</strong>ten relying on repelling (or deterring) specific functions <strong>of</strong> predators/invaders,<br />
and/or inhibiting their normal physiology and development, and rarely on their acute toxicity<br />
(Romeo el., 1996). These have important implications on our approach to bioprospecting.<br />
Bio-prospecting: examples, highlights <strong>of</strong> results and challenges<br />
The presentation seeks to highlight the importance <strong>of</strong> a bio-rational approach to discovering<br />
useful natural products that build on chemo-ecological insights, and the different challenges they<br />
present in practical exploitations. These will be illustrated by the following on-going research<br />
activities.<br />
Two types <strong>of</strong> blend effects will be illustrated: (a) anti-Plasmodium effects <strong>of</strong> artemisinin and<br />
crude blends <strong>of</strong> Artemisia annua leaves constituents obtained by aqueous or solvent extraction,<br />
including performance <strong>of</strong> optimally dried whole-leaf A. annua tablets in a clinical trial; and (b)<br />
rate <strong>of</strong> resistance development in repeated cycles <strong>of</strong> exposures <strong>of</strong> malaria parasite cultures to<br />
4
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
pure artemisinin and a comprehensive blend <strong>of</strong> the dried A. annua leaves. A. annua<br />
constituents (Elford et al., 1987; 2005; Kangethe et al., in preparation) as well as potential<br />
resistance-mitigating effect <strong>of</strong> the full A. annua phytochemical blend relative to pure<br />
artemisinin (Kangethe et al., in preparation).<br />
Approaches to identifying constituents that contribute to a bioactive blend illustrated by<br />
research on herbal plants used traditionally in post-harvest crop protection (e.g. Ocimum<br />
kilimandcharicum) and in repelling mosquitoes and other disease vectors (e.g. Conyza newii);<br />
challenges encountered in downstream exploitation <strong>of</strong> the essential oils, particularly the<br />
problem <strong>of</strong> chemotypic differences in the composition and efficacy <strong>of</strong> the essential oils <strong>of</strong> plants<br />
collected from different agro-ecological sites (Bekele and Hassanali, 2001; Omolo et al., 2004,<br />
2005; Aswalam et al., 2008; Mayeku et al., submitted).<br />
Potential integration <strong>of</strong> chemo-ecological studies relating to semiochemical-mediated location<br />
<strong>of</strong> preferred feeding site by the vector (the Brown Ear Tick, Rhipicephalus appendiculatus) <strong>of</strong> the<br />
cattle disease East Coast Fever (and a related species, Rh. evertsi), research findings on ethnoveterinary<br />
practices based on the use <strong>of</strong> anti-tick products (Wanzala et al., 2004; Wanzala et al.,<br />
submitted; Wanzala et al., in preparation), toward the development <strong>of</strong> on-host push-pull<br />
strategy to control the Brown Ear Tick.<br />
References<br />
Aswalam, E.S., Emosairue, S.O. and Hassanali, A. (2008); Essential oil <strong>of</strong> Ocimum gratissimum (Labiatae) as Sitophilus<br />
zeamais (Coleoptera: Curculionidae) protectant African J. Biotech. 20: 3771-76.<br />
Bekele, J. and Hassanali, A. (2001); Blend effects in the toxicity <strong>of</strong> the essential oil constituents <strong>of</strong> Ocimum<br />
kilimandscharicum and Ocimum kenyense (Labiateae) on two post-harvest insect pests Phytochemistry, 57, 385-<br />
391.<br />
Berenbaum, M.R., Zangerl, A.R. (1996); Phytochemical diversity: adaptation or random variation? In: Romeo, J.T.,<br />
Saunders, J.A., Barbosa, P. (Eds.), Phytochemical Diversity and Redundancy in Ecological Interactions. Plenum Press,<br />
New York, pp. 1 24.<br />
Elford, B.C., Roberts, M.F., Phillipson, J.D., Wilsom, R.J.M. (1987); Potentiation <strong>of</strong> the anti-malarial activity <strong>of</strong> Qinghaosu<br />
by methoxylated flavones. Trans. Royal Soc. Trop. Med. & Hyg. 81: 434-436.<br />
Feng, R. and Isman, M.B. (1995); Selection for resistance to azadirachtin in the green peach aphid, Myzus persicae<br />
Experientia 51: 831-833.<br />
Isman, M.B., Matsuura, H. MacKinnon, S., Durst, T., Neil Towers, G.H. and Arnason, J.T. (1996); Phytochemistry <strong>of</strong> the<br />
meliaceae: so many terpenoids, so few insects In: Romeo Saunders, J.A., Barbosa, P. (Eds.), Phytochemical<br />
Diversity and Redundancy in Ecological Interactions, Plenum Press, New York, pp155-178.<br />
Mayeku, P.W., Hassanali, A., Ndiege, I.O., Odalo, J.O., Kiremire, B.T. and Swaleh, S. (2011); Variations in Chemical<br />
Composition and Mosquito Repellency <strong>of</strong> Essental oils <strong>of</strong> Conyza newii from different Locations <strong>of</strong> Kenya<br />
(Submitted).<br />
Omolo, M.O. Okinyo, D. Ndiege, I.O. Lwande, W., Hassanali A. (2004); Repellency <strong>of</strong> essential oils <strong>of</strong> some Kenyan<br />
plants against Anopheles gambiae . Phytochemistry 65, 2797-2802.<br />
Omolo, M. O., Okinyo, D., Ndiege, I. O., Lwande, W. and Hassanali A. (2005); Fumigant toxicity <strong>of</strong> the essential oils <strong>of</strong><br />
some African plants and constituents <strong>of</strong> Conyza newii (Compositeae) against Anopheles gambiae sensu stricto<br />
Phytomedicine Research 12, 241-6.<br />
Wanzala, W., Noel, S.F.K., Gule and Hassanali A. (2004); Attractive and repellent host odours guide ticks to their<br />
respective feeding sites Chemoecology Vol. 14: 229-232.<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Wanzala, W., Takken,W., Mukabana, W. R. and Hassanali, A. Time-course on-host performance <strong>of</strong> essential oils <strong>of</strong><br />
plants used traditionally to protect cattle from Rhipicephalus appendicualtus and other tick species in herds grazing<br />
in natural pastures in Kenya (To be submitted).<br />
6
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[PL 2] Shifting the Paradigm to the Ethnobotany-based Drug Discovery<br />
Philippe Rasoanaivo 1,2 , Sol<strong>of</strong>oniaina Razafimahefa 1 , Emmanuel Randrianarivo 1<br />
1 Ecole Supérieure Polytechnique, University <strong>of</strong> Antananarivo, BP 1500, Antananarivo, Madagascar<br />
2 Institut Malgache de Recherches Appliquées, BP 3833, 101-Antananarivo, Madagascar<br />
Key words: Ethnobotany; Drug discovery; Reverse pharmacology; Malaria; Brain-related disorders; Commercialisation.<br />
Introduction<br />
T<br />
here are approximately 60,000 plant species in African Union countries, which represent<br />
roughly a quarter <strong>of</strong> the world plants. Unfortunately, despite the wealth and endemicity <strong>of</strong> the<br />
African plant biodiversity and associated cultures, Africa has only contributed 83 <strong>of</strong> the world s<br />
1100 leading commercial medicinal plants.<br />
Investigation <strong>of</strong> medicinal plants for drug discovery has mainly followed the Western approach <strong>of</strong><br />
single active constituents using standard pharmacological methods. Unfortunately, there are<br />
several examples <strong>of</strong> herbal medicines with reputedly excellent therapeutic effects, which<br />
subsequently produce disappointing results when evaluated in the laboratory with the standard<br />
biological screenings. Conversely, there are several bioactive compounds isolated from plants with<br />
excellent biological activity in the laboratory, which are ineffective or too toxic to use in human<br />
patients.<br />
The advent <strong>of</strong> today-modern techniques, namely combinatorial chemistry, high throughput<br />
screening (HTS), bioinformatics, omics methods, etc., has revolutionised drug discovery.<br />
Unfortunately, biopharmaceutical companies attempting to increase productivity through these<br />
novel technologies have fallen short <strong>of</strong> achieving the desired results (1). Following the trend <strong>of</strong><br />
these novel technologies, there has been a paradigm shift in the investigation <strong>of</strong> plants, focusing<br />
more on chemical diversity than traditional uses. The exploration <strong>of</strong> biodiversity for new sources <strong>of</strong><br />
natural products termed bioprospecting is based on massive random collecting, systematic<br />
extraction and medium to high throughput biological screening. Disappointingly, the success in<br />
terms <strong>of</strong> new medicines reaching the market has not also increased with the application <strong>of</strong> the<br />
modern technologies (2).<br />
The World Health Organisation estimates that up to 80% <strong>of</strong> populations in Africa depend on<br />
traditional medicine for their primary health care requirements. This is attributed to cultural<br />
acceptability, efficacy against certain types <strong>of</strong> diseases and ailments, physical accessibility and<br />
economic affordability as compared to modern medicine. African scientists should therefore look at<br />
other possibilities, get outside the box , think otherwise on how to better harness traditional<br />
knowledge to drug discovery and formulation, taking into account the evolving equilibrium <strong>of</strong> living<br />
organisms including humans in ecosystems. We report here our recent results in malaria and brain-<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
related disorders, using the reverse pharmacology approach. Challenges in translating discovery<br />
into commercialisation will be also discussed.<br />
Material and methods<br />
Field work<br />
For the past six years, field works were conducted in two very different ecosystems: the rainforest<br />
<strong>of</strong> the Eastern region <strong>of</strong> Madagascar, and the dry, xerophytic forests in the South and South-west.<br />
The rural communities were involved in a complete contributively and participative manner. Before<br />
commencing the research, the field team used an informed consent protocol that he had<br />
developed. Communities were asked if they were willing to allow photographs or films to be taken.<br />
Focus was on learning from/with local people, listening to them and considering their ideas (using<br />
ears more than mouth). Emphasis was also put on understanding local health concepts and disease<br />
classifications in connection with the environment.<br />
Selection, collection and processing <strong>of</strong> plants<br />
Medicinal plants were selected and then collected on the basis <strong>of</strong> ethnobotanical outcomes and<br />
target diseases. Botanical identification was made the by the Department <strong>of</strong> Botany, Parc<br />
Botanique et Zoologique de Tsimbazaza. Voucher specimens were kept at the Institut Malgache de<br />
Recherches Appliquées (IMRA). Extraction was carried out according to the methods used by the<br />
local populations.<br />
Antimalarials tests<br />
In vivo antimalarials tests were based on the 4-day suppressive test. Parasitemia, number <strong>of</strong> mice<br />
survival, and appearance/proliferation <strong>of</strong> lymphocytes were respectively recorded from D-4 until<br />
mice death. In vitro antiplasmodial tests were based on the inhibition <strong>of</strong> tritiated hypoxanthine<br />
uptake by Plasmodium falciparum cultured in human blood.<br />
Brain-related tests<br />
Anticonvulsant activities were assessed chemically by the pentylenetetrazole- and picrotoxininduced<br />
seizure tests, and electrically by the maximal electroshock-induced seizure test. The Morris<br />
water maze test was used to evaluate cognitive behaviour.<br />
Isolation <strong>of</strong> bioactive constituents and structure elucidation<br />
Silica gel column chromatography was used to isolate bioactive constituents by bioassay-guided<br />
fractionation. Structure elucidation was mainly based on concerted interpretation <strong>of</strong> NMR data.<br />
Results and Discussion<br />
From a medicinal plant reputedly used to treat malaria, we found that the aqueous extract, in<br />
combination with sub-therapeutic doses <strong>of</strong> conventional drugs, exhibited strong immunostimulating<br />
effects. From a plant used to treat convulsions and related diseases, we showed that an<br />
extract obtained by specific plant processing had both anticonvulsant activities in different animal<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
models and positive effects on cognitive behaviour. Regarding the biological tests, we went beyond<br />
the boundary <strong>of</strong> classical test protocols, and devised modified protocols to demonstrate biological<br />
activities.<br />
Research on antimalarial drugs has been mainly focused on killing the parasites but rarely consider<br />
other mechanisms. Many anti-malarial herbal remedies may exert their anti-infective effects not<br />
only by directly affecting the parasite, but also by stimulating natural and adaptive defence<br />
mechanisms <strong>of</strong> the host. The immune system is the first line host defence, and it is always<br />
associated with a complex inflammatory process which is partly responsible for the disease<br />
symptoms. Unfortunately, little work has been devoted to the investigation <strong>of</strong> the immunostimulating<br />
and anti-inflammatory effects <strong>of</strong> these herbal remedies. Curcumin was reported to<br />
possess both immune-stimulating and anti-inflammatory effects, but its poor bioavailability has<br />
limited its clinical uses (3). Our preliminary results showed a promising approach for malaria<br />
treatment and protection.<br />
Epilepsy is the most common neurological disorder in young humans. Ion channels,<br />
neurotransmitters and second messenger systems constitute molecular targets <strong>of</strong> antiepileptic<br />
drugs (AEDs). The same targets regulate brain processes essential both for propagation <strong>of</strong> seizures<br />
and for learning, memory and emotional behaviour. Thus, AEDs which are used to treat seizures in<br />
infants, children and pregnant women can cause cognitive impairment, microcephaly and birth<br />
defects (4). All currently available AEDs are synthetic in nature. Our preliminary results showed that<br />
a clearly defined extract from a traditional antiepileptic plant had both anticonvulsant and<br />
behaviour-improving activities.<br />
As outlined in the figure below, drug discovery (DD) process has evolved following the evolution <strong>of</strong><br />
science and technology (S&T).<br />
Data intensive<br />
Computational<br />
Theorical &<br />
Laboratory<br />
Empirical<br />
S&T<br />
Bioinformatics<br />
In silico<br />
In enzymo<br />
In cellulo<br />
In vitro (isolated organs)<br />
In vivo<br />
In homo<br />
DD<br />
9<br />
Data intensive<br />
Reverse<br />
pharmacology<br />
Observational therapeutics
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Reverse pharmacology is defined as the combination <strong>of</strong> traditional knowledge and the application<br />
<strong>of</strong> modern technology and processes to provide better and safer drugs. Its aim is to reverse the<br />
routine laboratory-to-clinic pathway to clinics-to-laboratories (5).<br />
The today-dominating paradigm <strong>of</strong> drug discovery in biopharmaceutical companies is to find new<br />
single entity drugs acting selectively on individual drug targets. Natural product drug discovery<br />
based on traditional knowledge may also be considered as attractive strategic options.<br />
Nevertheless, it is important to bear in mind that most <strong>of</strong> pure natural products do not comply with<br />
the Lipinski Rule <strong>of</strong> Five . During evolution and natural selection, mammals, including humans, have<br />
developed biological systems for effluxing them, to prevent xenobiotics being absorbed (example <strong>of</strong><br />
curcumin). It may be therefore necessary to shift the paradigm from the notion that a single<br />
molecular abnormality is the cause <strong>of</strong> complex diseases, and focus on developing standardized<br />
extracts with multiple mechanisms <strong>of</strong> action. At this point, complex, pleiotropic diseases may<br />
require multi-component, multi-functional therapies, and complex molecular interactions produce<br />
effects that may not be achieved by single components. Treating malaria may involve killing the<br />
parasites, boosting the immune system and managing the inflammatory process. Treating epilepsy<br />
may involve the necessity <strong>of</strong> maintaining a clinical equilibrium between seizure control and<br />
potential behavioural and cognitive expressions that may implicate social and vocational life<br />
aspects. Standardized plant extracts may bring answers to these innovative treatments. And<br />
surprisingly, the next paradigm <strong>of</strong> drug discovery is reported to be network pharmacology,<br />
considering multi-target strategies over single-target approaches (6).<br />
Regarding the commercial aspect <strong>of</strong> discovery, there is a skill gap and a funding gap to translate<br />
research finding in academia into marketable products. It is now time to break the walls between<br />
science and commerce and to start building bridges instead (H.A.M. Dzinotyiweyi, Minister <strong>of</strong><br />
Science and Technology Development, Zimbawe).<br />
In conclusion, our results reinforce the therapeutic potential <strong>of</strong> the two species and point out to the<br />
biological and cultural value <strong>of</strong> studying traditional folk medicine as a source <strong>of</strong> innovative<br />
therapeutic treatment. Where there are no modern drugs, there could be a safe and effective<br />
local treatment.<br />
Acknowledgements<br />
We thank warmly the CNRS (France), the IFS/PRISM, TWAS, CYROI (La Réunion), for various<br />
supports.<br />
References<br />
1. R. Lahana. (2003); Who wants to be irrational? Drug Discovery Today 8(15), 655.<br />
2. D. J. Newman, and G. M. Cragg. (2007); Natural products as sources <strong>of</strong> new drugs over the last 25 Years, Journal <strong>of</strong><br />
Natural Products 70, 461.<br />
3. P. N. Mimche, D. Taramelli, L. Vivas. (2011); The plant-based immunomodulator curcumin as a potential candidate<br />
for the development <strong>of</strong> an adjunctive therapy for cerebral malaria, Malaria Journal, 10(Suppl 1):S10.<br />
10
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
4. A. E. Cavanna, F. Ali, H. E. Rickards, D. McCorry. (2010); Behavioral and cognitive effects <strong>of</strong> anti-epileptic drugs,<br />
Discovery Medicine 9(45), 138.<br />
5. B. Patwardhan, R. A. Mashelkar. (2009); Traditional medicine-inspired approaches to drug discovery: can Ayurveda<br />
show the way forward? Drug Discovery Today 14(15/16), 804.<br />
6. A. L Hopkins. (2008); Network pharmacology: the next paradigm in drug discovery, Nature Chemical Biology 4(11),<br />
682.<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[PL 3] Why Has There Been Hardly any Success in Developing<br />
Antimicrobial Products from Medicinal Plants?<br />
Kobus El<strong>of</strong>f<br />
Phytomedicine Programme, Faculty <strong>of</strong> Veterinary Science, University <strong>of</strong> Pretoria, Private Bag X04,<br />
Onderstepoort, Pretoria, 0110. kobus.el<strong>of</strong>f @up ac.za<br />
Key words: Extractant, serial dilution assay, synergism, tetrazolium violet<br />
Introduction<br />
I<br />
t is widely accepted that resistance <strong>of</strong> microorganisms to currently used antibiotics is growing at<br />
an alarming rate. Some authors have even stated that we are entering the post antibiotic era.<br />
Before the discovery <strong>of</strong> antibiotics infections <strong>of</strong> even simple wounds have led to the death <strong>of</strong> many<br />
people. Investigation <strong>of</strong> plants has led to the development <strong>of</strong> many pharmaceutical products in the<br />
therapy <strong>of</strong> many diseases. In the order <strong>of</strong> 25% <strong>of</strong> prescription drugs in the United States were based<br />
on products isolated from plants (Farnsworth, 1990). There have been thousands <strong>of</strong> publications <strong>of</strong><br />
authors investigating plants for antimicrobial activity, yet no commercially useful antibiotic has yet<br />
been developed from plants. Several aspects that could be responsible for this situation will be<br />
discussed.<br />
Material and Methods<br />
Many aspects that could have an influence on development <strong>of</strong> commercially useful antibiotics from<br />
plants were investigated to try to get an answer to the question. These include: selection <strong>of</strong> plant<br />
material, extraction <strong>of</strong> the plant material, determination <strong>of</strong> antimicrobial activity, isolation <strong>of</strong><br />
antimicrobial compounds, test organisms used, safety <strong>of</strong> compounds from plants or extracts,<br />
synergism <strong>of</strong> activity between different compounds.<br />
Results and Discussion<br />
Only a small proportion <strong>of</strong> the c. 250 000 plant species have been investigated in some detail to<br />
date. In selecting plant species to investigate most scientists examined plants that have been used<br />
traditionally to treat infections. Other possibilities are random screening or screening plants on a<br />
taxonomic basis (El<strong>of</strong>f, 1998a). Many parameters play a role in selecting the best extractants for<br />
plants and the best part <strong>of</strong> the plant to examine. We decided to focus on leaves and specifically tree<br />
leaves based on sustainable use considerations and the high diversity <strong>of</strong> compounds present in<br />
leaves. We have repeatedly found that acetone is the extractant <strong>of</strong> choice based on its ability to<br />
extract compounds with a wide range <strong>of</strong> polarity, ease <strong>of</strong> removal due to volatility and low toxicity<br />
to test organisms (El<strong>of</strong>f, 1998b).<br />
In examining methods to determine the antimicrobial activity <strong>of</strong> plant extracts we found that agar<br />
diffusion was not trustworthy because the polarity <strong>of</strong> the compounds in an extract had a major<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
effect on the diffusion within an agar matrix. We also found that serial dilution and measuring<br />
turbidity with a microplate reader gave variable results due to precipitation <strong>of</strong> compounds in more<br />
concentrated extracts. We developed a method based on measuring growth after serial dilution by<br />
the reduction <strong>of</strong> tetrazolium violet to a purple formazan due to respiratory enzymes (El<strong>of</strong>f, 1988c).<br />
This method provides reproducible results and has since then been widely used. We also decided<br />
to use four ATCC strains <strong>of</strong> the most important nosocomial bacterial pathogens as a standard to<br />
ensure that variation on strain susceptibility would not invalidate comparing data found in different<br />
laboratories.<br />
Traditional healers mainly have water as extractant available. We have repeatedly found that<br />
water extracts <strong>of</strong> plant leaves have a very low antimicrobial activity compared to other extracts<br />
(Kotze and El<strong>of</strong>f, 2002). By focusing on plant species used traditionally scientists may have missed<br />
plants with high antimicrobial activity. In comparing different plant species it also became clear<br />
that not only the MIC <strong>of</strong> the extract but also the quantity extracted from a plant plays a role. The<br />
concept <strong>of</strong> total activity was developed by dividing the mass in mg extracted from 1 g <strong>of</strong> plant<br />
material by the MIC in mg/ml. The result in ml/g provides an indication <strong>of</strong> the volume to which the<br />
antimicrobial compounds present in 1 g <strong>of</strong> the plant can be diluted and still inhibit the growth <strong>of</strong><br />
the microorganism (El<strong>of</strong>f, 2000). This approach is also useful in bioassay guided isolation <strong>of</strong><br />
bioactive compounds. If there is no loss <strong>of</strong> activity through deactivation <strong>of</strong> the bioactive compound<br />
there should not be a difference in the total activity <strong>of</strong> the crude and the sum <strong>of</strong> the total activities<br />
<strong>of</strong> the fractions (El<strong>of</strong>f, 2004).<br />
We determined the antimicrobial activity <strong>of</strong> acetone extracts <strong>of</strong> leaves <strong>of</strong> more than 600 tree<br />
species against eight important bacterial and fungal pathogens to establish if taxonomic<br />
relationships can be useful in predicting which taxa would be useful to investigate in depth. We<br />
discovered that many plant extracts had excellent activities. For example about 5% <strong>of</strong> extracts <strong>of</strong><br />
species examined had a minimum inhibitory concentration <strong>of</strong> 40 µg/ml or lower and 2% <strong>of</strong> 20 µg/ml<br />
or lower against the non pathogenic Mycobacterium smegmatis closely related to the species<br />
causing tuberculosis (Figure 1).<br />
Figure 1 Cumulative percentage <strong>of</strong> tree leaf extracts active against Mycobacterium smegmatis at<br />
different MIC values<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
By examining extracts <strong>of</strong> species with high activity many compounds were isolated. It became clear<br />
that even though bioautography indicated practically only one antimicrobial compound in some<br />
cases, if that compound was isolated the activity would be orders <strong>of</strong> magnitude lower than what<br />
would have been expected after removing the inactive compounds. This shows that in a crude<br />
extract there is a synergism not necessarily between different antimicrobial compounds, but<br />
between the antimicrobial compound and other compounds that may affect one or more <strong>of</strong> the<br />
absorption, distribution, metabolism or excretion <strong>of</strong> the antimicrobial compound in the<br />
microorganism.<br />
We could show that a simple manipulation <strong>of</strong> the crude extract could increase the activity per mass<br />
unit and this have led to several patents and in one case to a product in the market. We also have<br />
evidence that the development <strong>of</strong> resistance was much slower with a crude extract than with a<br />
single product antibiotic. Some examples will be discussed.<br />
Conclusion<br />
We conclude that the main reason why there have been no development <strong>of</strong> new antibiotics from<br />
plants are not the different methodological problems discussed above, but that plants appear to<br />
use a mixture <strong>of</strong> compounds to address microbial infection. The focus in developing effective<br />
antimicrobial products from plants should therefore move from a pure compound basis to an<br />
extract based product. In this way we may be able to use the biodiversity resources <strong>of</strong> Africa to<br />
address primary health needs <strong>of</strong> its people.<br />
Acknowledgements<br />
The National Research Foundation and the University <strong>of</strong> Pretoria provided funding and many<br />
students were involved with the work reported here. Several National Botanical Gardens <strong>of</strong> the<br />
National Biodiversity Institute allowed us to collect plant material.<br />
References<br />
ELOFF J N (1998a); Conservation <strong>of</strong> Medicinal Plants: Selecting Medicinal Plants for Research and Gene Banking.<br />
Monographs in Systematic Botany from the Missouri Botanical Garden 71, 209-222. [In: Conservation <strong>of</strong> Plant Genes III:<br />
Conservation and Utilization <strong>of</strong> African Plants. Robert P. Adams and Janice E. Adams, eds., Missouri Botanical Garden<br />
Press, St. Louis, USA..]<br />
ELOFF, J N (1998b); Which extractant should be used for the screening and isolation <strong>of</strong> antimicrobial components from<br />
plants? Journal <strong>of</strong> Ethnopharmacology 60, 1-8.<br />
ELOFF J N (1998c); A sensitive and quick microplate method to determine the minimal inhibitory concentration <strong>of</strong> plant<br />
extracts for bacteria. Planta Medica 64, 711-714.<br />
ELOFF J N (2000); A proposal on expressing the antibacterial activity <strong>of</strong> plant extracts - a small first step in applying scientific<br />
knowledge to rural primary health care in South Africa. South African Journal <strong>of</strong> Science 96,116-118.<br />
KOTZE M and ELOFF J N (2002); Extraction <strong>of</strong> antibacterial compounds from Combretum microphyllum (Combretaceae).<br />
South African Journal <strong>of</strong> Botany 68, 62-67.<br />
ELOFF J N (2004); Quantifying the bioactivity <strong>of</strong> plant extracts during screening and bioassay-guided fractionation.<br />
Phytomedicine 11, 370-371<br />
FARNSWORTH, N.R. (1990); The role <strong>of</strong> ethnopharmacology in drug development. In Chadwick, D. J. & Marsh, J. [eds.]<br />
Bioactive compounds from plants 2-21. John Wiley Chichester, England<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[PL 4] Phytochemical Investigations <strong>of</strong> Leaves and Bark <strong>of</strong> Croton gratissimus<br />
(Euphorbiaceae)<br />
Dulcie A Mulholland, 1,2 Moses K Langat 1 , Neil R Crouch 2,3 and Jean-Marc Nuzillard 4<br />
1<br />
Natural Products Research Group, Division <strong>of</strong> Chemical Sciences, University <strong>of</strong> Surrey, Guildford, GU2 7XH, United<br />
Kingdom. D.mulholland@surrey.ac.uk<br />
2<br />
School <strong>of</strong> Chemistry, University <strong>of</strong> KwaZulu-Natal, Westville Campus, Private Bag X54001, Durban 4000, South Africa<br />
3<br />
Ethnobotany Unit, South African National Biodiversity Institute, PO Box 52099, Berea Road 4007, South Africa<br />
4<br />
Institute <strong>of</strong> Molecular Chemistry, CNRS UMR 6229, University <strong>of</strong> Reims, BP 1039, 51687 Reims Cedex 2, France<br />
Key words: Euphorbiaceae, Croton gratissimus, diterpenoids, cembranolides.<br />
Introduction<br />
O<br />
f the approximately 750 species <strong>of</strong> Croton L. (Euphorbiaceae) distributed throughout the<br />
tropics, some 50 are found in Africa (Mabberley 2008), and only 10 species are native to the<br />
Flora <strong>of</strong> southern Africa region. Croton gratissimus Burch. (syn. C. zambesicus Müll. Arg.; C.<br />
microbotryus Pax.) is a semi-deciduous tree species, widespread in sub-Saharan Africa, occurring on<br />
stony or rocky hillsides throughout much <strong>of</strong> the warmer and drier regions, from South Africa<br />
northeastwards to the horn <strong>of</strong> Africa. The leaves produce a pleasant lavender-like scent when<br />
crushed, and are used dried and powdered for their perfume (Palmer and Pitman 1972). Across its<br />
range it is an important ethnomedicinal species: the Zulu use milk infusions <strong>of</strong> the bark as<br />
purgatives for stomach and intestinal disorders, despite its toxic reputation (Bryant 1966).<br />
Elsewhere several other crotons including the Asian C. tiglium L., and C. flavens L. from the<br />
Caribbean have been so employed, although diterpenes from both have been implicated in indirect<br />
carcinogenesis (oesophageal cancer) through activation <strong>of</strong> the Epstein-Barr virus (Hecker 1981;<br />
Bruneton 1995). The Zulu further treat unspecified uterine disorders with powdered bark blown<br />
into the womb. They also remedy pleurisy or pleurodynia by rubbing the powdered bark into chest<br />
skin incisions to act as a counter-irritant, given its cutaneous eruptive irritant and stimulant<br />
properties (Bryant 1966). As a cure for insomnia and restlessness, the leaves are ground with goat<br />
fat and those <strong>of</strong> two other Croton species, the paste heated on coals and the fumes inhaled (Palmer<br />
and Pitman 1972). Gerstner (1941) further recorded the purgative properties <strong>of</strong> the roots, and<br />
documented their application in treating fevers. Zimbabweans treat coughs with smoke from<br />
leaves, and take root infusions for abdominal pains and as an aphrodisiac (Gelfand et al. 1985). In<br />
Botswana a decoction prepared with leaves is taken for coughs (Hedberg and Staugård 1989). Watt<br />
and Breyer-Brandwijk (1962) documented the use <strong>of</strong> C. gratissimus bark in treating painful<br />
respiratory conditions (including intercostal neuralgia), unspecified plant parts as a remedy for<br />
fevers, charred, powdered bark for bleeding gums, and leaves to treat both eye disorders and<br />
rheumatism. In Venda, leaves are dried and smoked for influenza, colds and fevers (Mabogo 1990).<br />
Doubts about the toxicity <strong>of</strong> this species have been raised due to the esteem with which leaves<br />
have been held as a stock food in Namibia (Watt and Breyer-Brandwijk 1962). In Namibia roots and<br />
leaves <strong>of</strong> this taxon have found application as a treatment for colds and coughs, bark for ear<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
problems, and roots for chest ailments and fever (Von Koenen 2001). From the above usage pr<strong>of</strong>iles<br />
<strong>of</strong> C. gratissimus the following diverse bioactivities are indicated: analgesia, febrifugal, aphrodisiac,<br />
purgative, emetic, soporific, antibiotic and antiviral. The febrifugal activity <strong>of</strong> C. gratissimus (in the<br />
context <strong>of</strong> malaria) has earlier been demonstrated: Clarkson et al. (2004) found DCM extracts <strong>of</strong> the<br />
leaves to show a high antiplasmodial activity in vitro <strong>of</strong> 3.5 µg/ml, thus compounds 3 and the acetyl<br />
derivative <strong>of</strong> 12 were screened for antiplasmodial activity against the P. falciparum (CQS) D10<br />
strain. In the current study, congnisance has been taken <strong>of</strong> the use <strong>of</strong> this species for abdominal<br />
pains (Gelfand et al. 1985), an indication <strong>of</strong> potential antineoplastic applications (Charlson 1980),<br />
especially as the Zulu treat unspecified uterine disorders with powdered bark preparations (Bryant<br />
1966). Accordingly, isolates from the bark were screened against PEO1 and PEO1TaxR ovarian<br />
cancer cell lines.<br />
Previous investigations <strong>of</strong> the genus Croton have yielded pimarane (Block et al. 2004), kaurane (Kuo<br />
et al. 2007 ), labdane (Garcia et al. 2006 ), clerodane (Garcia et al. 2006) and cembrane (Pudhom et<br />
al. 2007) diterpenoids, isoquinoline alkaloids (New World species only) (Charris et al. 2000 ) and<br />
triterpenoids (Block et al. 2004).<br />
Materials and Methods<br />
Leaves and bark <strong>of</strong> Croton gratissimus Burch. var. gratissimus were collected from a mature tree<br />
cultivated on the campus <strong>of</strong> the University <strong>of</strong> KwaZulu-Natal, Durban, South Africa, and a voucher<br />
retained for verification purposes (Crouch 1051, NH).<br />
The ground stem bark <strong>of</strong> C. gratissimus was extracted using a Soxhlet apparatus for 24 h<br />
successively using hexane, methylene chloride, ethyl acetate and methanol. The hexane and<br />
methylene chloride extracts were separated using column chromatography over silica gel using a<br />
hexane/methylene chloride/methanol step gradient to yield the novel cembranolides, 1-4, lupeol,<br />
4(15)-eudesmene-1 ,6 -diol and -glutinol.<br />
The ground leaves <strong>of</strong> C. gratissimus were extracted and compounds purified similarly to give the<br />
following compounds: cembranolides 3 and 5-14, glutinol, 24-ethylcholesta-4, 22-dien-3-one,<br />
lupeol and eudesm-4(15)-ene-1 , 6 diol.<br />
Results and Discussion<br />
Fourteen novel cembranolides were isolated from the stem bark and leaves <strong>of</strong> C. gratissimus and<br />
are shown in Figure 1. Structures were determined using extensive 2D NMR spectroscopy and mass<br />
spectrometry, and the structure <strong>of</strong> 1 was confirmed by single crystal X ray analysis and using the<br />
LSD program (Nuzillard 2003). NMR data for these compounds are reported elsewhere.<br />
(Mulholland, Langat et al. 2010)<br />
Compounds 1 and 3 were screened against the PEO1 and PEO1TaxR ovarian cancer cell lines and<br />
were found to have IC50 values <strong>of</strong> 132 and 125nMolar (cf. paclitaxel 2.3) against PEO1 and 200 and<br />
16
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
135 respectively against PEO1TaxR (cf. paclitaxel 30.5). The acetate derivative <strong>of</strong> 12 was prepared<br />
in an attempt to produce a crystalline product for single crystal X-ray analysis, or a more stable<br />
compound for screening. Unfortunately, the derivative was not crystalline but was stable enough<br />
to undergo in vitro anti-plasmodial screening against P. falciparum (CQS) D10 strain. Both<br />
compounds 3 and 12 acetate showed moderate activity (IC50 values <strong>of</strong> 20.80 and 13.52 µg/ml<br />
respectively), compared to chloroquine (IC50 27.04 ng/ml), validating the traditional usage <strong>of</strong> this<br />
plant.<br />
Figure 1. Cembranolides from the leaves and stem bark <strong>of</strong> C.gratissimus<br />
Acknowledgements<br />
ML wishes to acknowledge a PhD studentship from the University <strong>of</strong> Surrey. This research was<br />
funded by the University <strong>of</strong> Surrey and South African National Research Foundation (NRF). We<br />
thank Dr Helen Coley <strong>of</strong> the University <strong>of</strong> Surrey for PEO1 and PEO1TaxR screening, Pr<strong>of</strong>essor Pete<br />
Smith <strong>of</strong> the University <strong>of</strong> Cape Town for antiplasmodial screening.<br />
References<br />
Block, S., Baccelli, C., Tinant, B., Meervelt, L.C., Rozenberg, R., Jiwan, H.J., Llabres, G., Pauw Gillet, D.M., Quetib<br />
Leclercq, J. (2004); Diterpenes from the leaves <strong>of</strong> Croton zambesicus. Phytochemistry 65, 1165 1171<br />
Bruneton, J. 1995. Pharmacognosy, phytochemistry, medicinal plants. Intercept Limited, Andover, UK.<br />
Bryant, A.T. (1966); Zulu medicine and medicine-men. Struik, Cape Town.<br />
Charris, J., Dominguez, J., De la Rosa, C., Caro, C. (2000); (-)-Amuronine from the leaves <strong>of</strong> Croton flavens L.<br />
(Euphorbiaceae). Biochem. Syst. Ecol. 28, 795 797<br />
Clarkson, C., Maharaj, V.J., Crouch, N.R., Grace, O.M., Pillay, P., Matsabisa, M.G., Bhagwandin, N., Smith, P.J., Folb, P.I.,<br />
(2004); In vitro antiplasmodial activity <strong>of</strong> medicinal plants native to or naturalised in South Africa. J.<br />
Ethnopharmacol. 92, 177 191.<br />
17
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Coley, M.H., Shotton, C.F., Ajose-Adeogun, A., Modjtahedi, H., Thomas, H. (2006); Receptor tyrosine kinase (RTK)<br />
inhibition is effective in chemosensitising EGFR-expressing drug resistant human ovarian cancer cell lines when<br />
used in combination with cytotoxic agents. Biochem. Pharmacol. 72, 941-948<br />
Crombie, L., King, R. W., Whiting, D.A. (1975); Carbon-13 magnetic resonance spectra. Synthetic presqualene esters,<br />
related cyclopropanes, and isoprenoids. J. Chem. Soc., Perkin Trans. 1. 913-915<br />
Garcia, A., Ramirez-Apan, T., Cogordan, J.A., Delgado, G. (2006); Absolute configuration assignments by experimental<br />
and theoretical approaches <strong>of</strong> ent-labdane and cis-ent-clerodane-type diterpenes isolated from Croton glabellus.<br />
Can. J. Chem. 84, 1593 1602<br />
Gelfand, M., Mavi, S., Drummond, R.B., Ndemera, B. (1985); The traditional medical practitioner in Zimbabwe. His<br />
principles <strong>of</strong> practice and pharmacopoeia. Mambo Press, Gweru.<br />
Gerstner, J. (1941); A preliminary check list <strong>of</strong> Zulu names <strong>of</strong> plants, with short notes. Bantu Studies 15, 277-301.<br />
Hecker, E., (1981); Cocarcinogenesis and tumor promoters <strong>of</strong> the diterpene ester type<br />
as possible carcinogenic risk factors. J. Can. Res. Clin. Oncol. 99, 103 124.<br />
Hedberg, I., Staugård, F. (1989); Traditional medicine in Botswana. Traditional medicinal plants. Ipeleng Publishers,<br />
Gaborone.<br />
Kuo, P.C., Shen, Y.C., Yang, M.L., Wang, S.H., Thang, T.D., Dung, W.X., Chiang, P.C., Lee, K.H., Lee, E.J., Wu, T.S. (2007);<br />
Crotonkinins A and B and related diterpenoids from Croton tonkinensis as anti-inflammatory and anti-tumor<br />
agents. J. Nat. Prod. 70, 1906 1909<br />
Mabberley, D.J. (1997); The Plant-book. A portable dictionary <strong>of</strong> the vascular plants. Cambridge University Press,<br />
Cambridge.<br />
Mabogo, D.E.N. (1990); The ethnobotany <strong>of</strong> the Vhavenda. Unpublished MSc, University <strong>of</strong> Pretoria.<br />
Mulholland, D.A. Langat, M.K., Crouch, N.R., Nuzillard, J.M., Coley, H.M. and Mutambi, E.M. (2010); Cembranolides from<br />
the stem bark <strong>of</strong> the southern African medicinal plant, Croton gratissimus (Euphorbiaceae). Phytochemistry 71,<br />
1381-1386<br />
Nuzillard, J.M. (2003); Automatic Structure Determination <strong>of</strong> Organic Molecules: Principles and Implementation <strong>of</strong> the<br />
LSD Program. Chin. J. Chem. 21, 1263-1267.<br />
Palmer, E., Pitman, N. (1972); Trees <strong>of</strong> southern Africa. Vol 2. A.A. Balkema, Cape Town.<br />
Pudhom, K., Vilaivan, T., Ngamrojanavanich, N., Dechangvipart, S., Sommit, D., Petsom, A. and Roengsumran, S. (2007);<br />
Furano-cembranoids from the stem bark <strong>of</strong> Croton oblongifolius. J. Nat. Prod. 70, 659-661<br />
Von Koenen, E. (2001); Medicinal, poisonous and edible plants in Namibia. Kluas Hess Publishers, Windhoek and<br />
Göttingen.<br />
Watt, J.M., Breyer-Brandwijk, M.G. (1962); The medicinal and poisonous plants <strong>of</strong> southern and eastern Africa. E. &. S.<br />
Livingstone Ltd., Edinburgh and London.<br />
18
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[PL 5] Challenges and Opportunities <strong>of</strong> Traditional/Herbal Medicine<br />
IVAN ADDAE-MENSAH<br />
Chemistry Department, University <strong>of</strong> Ghana Legon<br />
E-mail a-mensah@ug.edu.gh<br />
Key Words: Traditional Medicine; Herbal Medicine; WHO strategic plan; Research paradigm; Neglected tropical<br />
diseases; Biodiversity; Intellectual Property rights; Indigenous knowledge.<br />
H<br />
erbal medicine has become a multi-billion dollar enterprise worldwide. The world market for<br />
herbal medicines based on traditional knowledge is estimated at over US$60 billion, which is<br />
about 30 percent <strong>of</strong> total world pharmaceutical sales.Africa accounts for only 2.1 billion dollars<br />
(1.2%) <strong>of</strong> the world s total pharmaceutical sales. The developed world, which constitutes only 20<br />
percent <strong>of</strong> the world s population, consumes 80 percent <strong>of</strong> the total pharmaceutical output. So how<br />
do Africa s almost one billion people take care <strong>of</strong> themselves if they consume only 1.2% <strong>of</strong> the total<br />
output <strong>of</strong> pharmaceutical products? The answer lies in traditional/complementary alternative<br />
medicine (TM/CAM).<br />
A recent survey on treatment <strong>of</strong> fevers in children has shown that the cost <strong>of</strong> herbal treatment is<br />
only about 6 percent <strong>of</strong> the cost <strong>of</strong> clinical or hospital treatment (WHO 2002 p13). In a situation<br />
where it may be the only available treatment within a radius <strong>of</strong> more than 30 kilometres, it will be<br />
far better than nothing in circumstances where no treatment at all will mean certain death.The<br />
WHO s 3-year Strategic Plan for traditional medicine envisages that member countries should<br />
integrate Traditional Medicine (TM) with national health care systems, promote the safety, efficacy<br />
and quality <strong>of</strong> TM by expanding the knowledge base, increase the availability and affordability <strong>of</strong><br />
TM/CAM with emphasis on access for poor populations and promote therapeutically sound use <strong>of</strong><br />
appropriate TM/CAM by providers and consumers (WHO 2000; WHO 2002).<br />
The <strong>of</strong>ficial national norm for primary health care in Ghana is that no citizen should travel more<br />
than 8 kilometres to the nearest health facility, no matter the kind <strong>of</strong> facility. But currently in all the<br />
regions, whereas people in over 90 percent <strong>of</strong> all localities can reach a traditional healer within a<br />
radius <strong>of</strong> 5 kilometres, conventional hospitals are available in only 12 percent or less <strong>of</strong> localities.<br />
Over 60% <strong>of</strong> patients have to travel up to 30km or more to get to the nearest hospital. The national<br />
average allopathic doctor to patient ratio is about 1 to 11,000 with some districts having a ratio <strong>of</strong><br />
about 1 doctor to 256,000 population, whereas that <strong>of</strong> traditional healer to patient is about 1 to<br />
900. (Ghana Statistical Services 2005) The situation cannot be any different in other African<br />
countries.<br />
This situation makes research and development <strong>of</strong> traditional medicine a priority in the quest for<br />
adequate primary health. But research is extremely expensive. It can cost over $600 million to<br />
develop a major drug, but about $400 million <strong>of</strong> this goes into finding the best candidate, and<br />
19
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
discarding all other possibilities on the way. Rejection rate is very high. Hardly any African country<br />
can afford that sort <strong>of</strong> research, whether for the development <strong>of</strong> synthetic or plant-based natural<br />
medicines. There is therefore the need to adopt a completely different approach to research and<br />
development <strong>of</strong> traditional medicine.<br />
The continents <strong>of</strong> South America and Asia have both made major contributions from their<br />
biodiversity to the treatment <strong>of</strong> malaria, one <strong>of</strong> the most important tropical diseases. The discovery<br />
<strong>of</strong> quinine and quinidine from the South American cinchona bark, and compounds that emerged<br />
from this discovery were the mainstay <strong>of</strong> malaria chemotherapy for decades. The artemisinin-based<br />
anti-malarials which are now the mainstay <strong>of</strong> first-line malaria treatment are also <strong>of</strong> a Chinese plant<br />
origin. What has Africa got to <strong>of</strong>fer from its vast biodiversity resource? This is the challenge we face<br />
as African Researchers.<br />
This Plenary Lecture will focus on a selection <strong>of</strong> neglected tropical diseases such as malaria,<br />
helminthic diseases such as trypanosomiasis and leishmaniasis as well as cancer and HIV Aids and<br />
examine what research is being, or has been done in this area in the search for potential lead<br />
compounds for possible development into usable drug products for these diseases. The chemical<br />
constituents and biological activities <strong>of</strong> a selection <strong>of</strong> medicinal plants from various families<br />
including the Rutaceae, Chailetaceae, Meliaceae and Periplocaceae, will be discussed as illustrative<br />
examples. The challenges posed to African researchers in this quest will be discussed. The need for<br />
a major paradigm shift in traditional medicine research will be discussed. It will be suggested that a<br />
thorough re-examination <strong>of</strong> the constituents <strong>of</strong> plant species previously considered to be already<br />
thoroughly investigated, may reveal these as potential sources <strong>of</strong> compounds that could serve as<br />
new scaffolds for developing drugs for neglected tropical diseases.<br />
The issue <strong>of</strong> intellectual property rights and the protection <strong>of</strong> indigenous knowledge will also be<br />
critically examined. The need for regulatory measures for conservation and protection <strong>of</strong><br />
biodiversity as well as a rational and sustainable exploitation <strong>of</strong> this biodiversity will also be<br />
addressed. The pivotal role <strong>of</strong> African Governments in issues <strong>of</strong> scientific research and S&T<br />
governance structures will be dealt with. Our governments should realise that our science will<br />
never make any meaningful global impact if spending on S&T research continues to depend<br />
primarily on foreign donor funded programmes. Governments must take the lead in research<br />
funding, and devote a substantial portion <strong>of</strong> GDP to research.<br />
References<br />
1. Ghana Statistical Services (2005); Regional Reports <strong>of</strong> the 2000 Population and Housing Census Vols 1-10. (Ed. K.<br />
Twum-Baah, I Addae-Mensah and T. Kumekpor).<br />
2. World Health Organisation (2000); Promoting the Role <strong>of</strong> Traditional Medicine in Health Systems; A Strategy for the<br />
African Region, 2001-2010. Harare. WHO document ref. AFR/RC50/doc.9R<br />
3. World Health Organisation (2002 p12); WHO Traditional Medicine Strategy 2002-2005. pp.7, 12 and references<br />
therein. World Health Organisation, Geneva. (See also WHO Policy Perspectives on Medicines No.2. May 2002 p.1,<br />
Box 1.)<br />
4. World Health Organisation (2002); WHO Traditional Medicine Strategy 2002-2005. p. 13 and references therein.<br />
World Health Organisation, Geneva.<br />
20
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[PL 6] Approaches to the Synthesis <strong>of</strong> Tricarbonyl Metabolites:<br />
An Isoxazole Strategy<br />
Raymond C F Jones, Abdul K Choudhury, Carole C M Law, Christopher Lumley, Terence A<br />
Pillainayagam and James P Bullous<br />
Email: r.c.f.jones@lboro.ac.uk<br />
Department <strong>of</strong> Chemistry, Loughborough University, Loughborough, Leics. LE11 3TU, UK<br />
Keywords: Acyltetramic acid; acylpyridone; nitrile oxide; dipolar cycloaddition; pyrroloisoxazole; isoxazolopyridine<br />
Introduction<br />
atural products displaying the common acyltetramic acid motif 1 show a wealth <strong>of</strong> structural<br />
features, and significantly, a diversity <strong>of</strong> biological activity including antibiotic, antiviral,<br />
antitumour, antiulcerative, fungicidal, cytotoxic and mycotoxic properties. 1 N<br />
Examples are equisetin,<br />
a selective HIV integrase inhibitor, and the mycotoxin ikarugamycin. Acyltetramic acids are also<br />
implicated in a number <strong>of</strong> interesting biosynthetic processes: the 3-decalinoyl derivatives are<br />
believed to arise from intramolecular cycloaddition <strong>of</strong> a polyketide-derived side chain; acyltetramic<br />
acid ring expansion is believed to lead to bioactive 3-acyl-4-hydroxypyridone metabolites 2, e.g the<br />
protein tyrosine kinase inhibitor pyridovericin; and C12/14-TA formed from acylated homoserine<br />
lactones, are involved in bacterial quorum sensing. Recent renewed interest in these metabolites<br />
with reports <strong>of</strong> new natural products and synthetic work, confirms the relevance <strong>of</strong> our own<br />
approach.<br />
O<br />
R1 5<br />
N<br />
3<br />
R 3<br />
O<br />
OH<br />
1<br />
3-acyltetramic acids<br />
R 1<br />
R 2<br />
OH O<br />
R 3<br />
HO<br />
HO<br />
H<br />
H<br />
O<br />
Me<br />
N<br />
OH O<br />
O<br />
Equisetin<br />
21<br />
OH<br />
HN<br />
O<br />
H<br />
N<br />
H<br />
Et<br />
HO<br />
O<br />
O<br />
H H H<br />
H H H<br />
Ikarugamycin<br />
N O<br />
R<br />
3-acylpyridones<br />
2 N 2 N O<br />
O<br />
OH<br />
H<br />
HO<br />
H<br />
Pyridovericin C12/14-TA (R = (CH2) 8/10CH3) Results and Discussion<br />
We have developed a flexible synthetic strategy for this moiety, using dipolar cycloaddition <strong>of</strong><br />
alpha-amino acid-derived nitrile oxides with beta-ketoester enamines, via pyrroloisoxazole building<br />
blocks 3 2 . This is illustrated in Scheme 1 for the acyltetramic acids.<br />
O<br />
R<br />
OH
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
R1 CO 4 steps<br />
2Me<br />
NHBoc<br />
R 1<br />
R 1<br />
O<br />
O<br />
N<br />
NHBoc<br />
N<br />
H<br />
O<br />
OH<br />
Scheme 1<br />
22<br />
N<br />
CO 2Bu t<br />
i, H 2, Pd<br />
ii, aq. OH<br />
R 1<br />
BocHN<br />
R 1<br />
HN<br />
N O<br />
3<br />
CO 2Bu t<br />
N O<br />
O<br />
5<br />
i, TFA<br />
ii, EDCI<br />
A key principle <strong>of</strong> our strategy is to mask the highly polar, potentially reactive beta, beta -<br />
tricarbonyl (heterocyclic trione) moiety <strong>of</strong> the acyltetramic acids and acylpyridones as an isoxazole<br />
until required, allowing elaborations around the non-polar core pyrroloisoxazole structure 3. We<br />
will report elaboration at the C-3(methyl) <strong>of</strong> 3 using aldol-type chemistry, and subsequent<br />
unmasking <strong>of</strong> the tetramic acid moiety. 3 In a related approach to the acylpyridones (Scheme 2) we<br />
will report on the elaboration <strong>of</strong> isoxazolopyridones at C-3(methyl), again by aldol-type reaction,<br />
and at C-7 by palladium-mediated cross-couplings, followed by unmasking <strong>of</strong> the acylpyridone<br />
moiety. 4<br />
NOH O<br />
via<br />
nitrile oxide<br />
(PNH)<br />
H<br />
+ enamine (PNH)<br />
PNH<br />
CO 2 Et<br />
2,3-diaminopropanoic acid<br />
or<br />
-Alanine<br />
PNH<br />
N O<br />
CO 2Et<br />
[P = protecting gp.; e.g. Boc, Z]<br />
Scheme 2<br />
7<br />
3<br />
3<br />
N O<br />
3'<br />
N O<br />
H<br />
2nd 6<br />
generation<br />
building-block<br />
steps<br />
acylpyridones<br />
Acknowledgements:<br />
We acknowledge Loughborough University for studentships, Novartis & Syngenta for financial<br />
support, EPSRC Mass Spectrometry Service Centre for some high resolution MS data, & EPSRC<br />
National Crystallography Service, Pr<strong>of</strong> V McKee, Dr M R J Elsegood for X-ray crystal data.<br />
References:<br />
1. See: (a) Royles, B. J. L. (1995); Chem. Rev., 95, 1981; (b) Schobert, R.; Schlenk, A. (2008); Bioorg. Med. Chem. 16,<br />
4203; (c) Jeong, Y.-C.; Moloney, M. G. (2011); J. Org. Chem., 76, 1342.<br />
2. (a) Jones, R. C. F.; Bhalay, G.; Carter, P. A.; Duller K. A. M.; Dunn, S. H. (1999); J. Chem. Soc., Perkin Trans. 1 765; (b)<br />
Jones, R. C. F.; Dawson, C. E.; O Mahony, M. J.; Patel, P. (1999); Tetrahedron Lett., 40, 4085; (c) Jones, R. C. F.;<br />
Dawson, C. E.; O Mahony, M. J. (1999); Synlett, 873.<br />
3. (a) Jones, R. C. F.; Pillainayagam, T. A. (2004); Synlett, 2815; (b) Law, C. C. M., (2008); Ph.D. Thesis, Loughborough<br />
University.<br />
4. Jones, R. C. F.; Choudhury, A. K.; Iley, J. N.; Loizou, G.; Lumley, C.; McKee, V. (2010); Synlett, 654.
T<br />
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[PL 7] Absolute Stereostructures by LC-CD Coupling in Combination with<br />
Quantum-Chemical CD Calculations<br />
Gerhard Bringmann et al.<br />
Institute <strong>of</strong> Organic Chemistry, University <strong>of</strong> Würzburg,<br />
Am Hubland, D-97074 Würzburg, Germany<br />
bringman@chemie.uni-wuerzburg.de<br />
he search for novel bioactive compounds from nature is a rewarding, but also demanding task.<br />
It requires a set <strong>of</strong> modern methods to trace up these compounds, even from complex<br />
mixtures, to recognize their novelty and originality, and to assign their full absolute<br />
stereostructures reliably.<br />
By our analytical triad HPLC-MS/MS-NMR-CD (CD = circular dichroism), we can rapidly identify<br />
novel-type compounds and establish their full absolute stereostuctures online, right from the peak<br />
in the chromatogram. Of particular importance is the LC-CD option, which we have introduced into<br />
natural products chemistry (Bringmann et al. 1999). The interpretation <strong>of</strong> the CD spectra (whether<br />
measured online or <strong>of</strong>fline!) can be done empirically, by comparison with those <strong>of</strong> known<br />
compounds (but how related do they have to be?) or by applying empirical rules (but do they really<br />
apply in the present case?). But, much more reliably, the assignment can be achieved by quantumchemical<br />
CD calculations (Bringmann et al. 2008b, Bringmann et al. 2009, Bringmann et al. 2011).<br />
Scheme 1 outlines the strategy: For a new compound whose absolute configuration we want to<br />
assign, we simulate the CD spectrum for each <strong>of</strong> its possible enantiomers. The comparison <strong>of</strong> these<br />
two predicted CD spectra with the actual experimental CD curve will, if successful, give a good<br />
agreement for one enantiomer (top), and a mirror-like opposite situation for the other (bottom),<br />
which, thus, will permit a clear assignment <strong>of</strong> the absolute configuration.<br />
Scheme 1. General strategy for the assignment <strong>of</strong> absolute configurations by quantum-chemical CD calculations, here<br />
for the marine natural product sorbicillatone B (Bringmann et al. 2005).<br />
23
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
For the calculation <strong>of</strong> CD spectra one has to take into account that the CD <strong>of</strong> a molecule strongly<br />
depends on its conformational behavior - like for no other spectroscopic method! This requires a<br />
thorough conformational analysis beforehand, and that will be useful also for the interpretation <strong>of</strong><br />
NMR data.<br />
As an example, Scheme 2 below shows the stereochemical assignment <strong>of</strong> shuangancistrotectorine<br />
A (Xu et al. 2010), a demanding molecule with seven stereogenic elements: four stereocenters and<br />
three consecutive chiral axes, discovered in a tropical Ancistrocladus plant. Its constitution was<br />
established by NMR, like also the relative configurations at the centers and the outer axes. The<br />
absolute configuration at the central axis can be assigned by LC-CD in combination with quantumchemical<br />
CD calculations; only the elucidation <strong>of</strong> the absolute configurations <strong>of</strong> the centers has to<br />
be done <strong>of</strong>fline, in this case by chemical degradation.<br />
Scheme 2. Structural elucidation <strong>of</strong> the novel quateraryl alkaloid shuangancistrotectorine A.<br />
The lecture presents the strategy and illustrates examples out <strong>of</strong> most different classes <strong>of</strong><br />
structures configurationally assigned by the method, among them ancistrocladinium A and B,<br />
shuangancistrotectorine A and C, dioncophylline A, chloropupukeanolides C and D,<br />
cyclorocaglamide, xylogranatin I, sorbifuranone C, and -bisporphyrins (Bringmann et al. 2006,<br />
Xu et al. 2010, Bringmann et al. 2001, Liu et al. 2011, Bringmann et al. 2003, Wu et al. 2008,<br />
Bringmann et al. 2010b, Bringmann et al. 2008a) (see Scheme 3). Some <strong>of</strong> the structures have<br />
already been confirmed by total synthesis (Bringmann et al. 2010a).<br />
24
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Scheme 3. Some selected examples <strong>of</strong> chiral structures whose absolute configurations have been determined by the<br />
combination <strong>of</strong> experimental and calculated CD investigations.<br />
References:<br />
G. Bringmann, T. Bruhn, K. Maksimenka, Y. Hemberger (2009); The Assignment <strong>of</strong> Absolute Stereostructures by<br />
Quantum Chemical Circular Dichroism Calculations; Eur. J. Org. Chem. 2717-2727.<br />
G. Bringmann, D. Goetz T. Bruhn (2011); The Online Stereoanalysis <strong>of</strong> Chiral Compounds by HPLC-ECD Coupling in<br />
Combination with Quantum-Chemical Calculations; in Comprehensive Chiroptical Spectroscopy (N. Berova, P.<br />
Polavarapu, K. Nakanishi, R.W. Woody, Eds.), Vol. 2, John Wiley & Sons / Wiley-Blackwell, New York, in press.<br />
G. Bringmann, D.C.G. Götz, T.A.M. Gulder, T.H. Gehrke, T. Bruhn, T. Kupfer, K. Radacki, H. Braunschweig, A. Heckmann,<br />
C. Lambert (2008a); Axially Chiral , -Bisporphyrins: Synthesis and Configurational Stability Tuned by the Central<br />
Metals; J. Am. Chem. Soc. 130, 17812-17825.<br />
G. Bringmann, T. Gulder, B. Hertlein, Y. Hemberger, F. Meyer (2010a); Total Synthesis <strong>of</strong> the N,C-Coupled<br />
Naphthylisoquinoline Alkaloids Ancistrocladinium A and B, and Related Analogues; J. Am. Chem. Soc. 132, 1151-<br />
1158.<br />
G. Bringmann, T.A.M. Gulder, M. Reichert, T. Gulder (2008b); The Online Assignment <strong>of</strong> the Absolute Configuration <strong>of</strong><br />
Natural Products: HPLC-CD in Combination with Quantum Chemical CD Calculations; Chirality 20, 628-642.<br />
G. Bringmann, I. Kajahn, M. Reichert, S.E.H. Pedersen, J.H. Faber, T.Gulder, R. Brun, S.B. Christensen, A. Ponte-Sucre, H.<br />
Moll, G. Heubl, V. Mudogo (2006); Ancistrocladinium A and B, the First N,C-Coupled Naphthyldihydroisoquinoline<br />
Alkaloids, from a Congolese Ancistrocladus Species; J. Org. Chem. 71, 9348-9356.<br />
G. Bringmann, G. Lang, T. Bruhn, K. Schäffler, S. Steffens, R. Schmaljohann, J. Wiese, J.F. Imh<strong>of</strong>f (2010b); Sorbifuranones<br />
A-C, sorbicillinoid metabolies from Penicillium strains isolated from Mediterranean sponges; Tetrahedron 66, 9894-<br />
9901.<br />
G. Bringmann, G. Lang, T. A. M. Gulder, H. Tsuruta, J. Mühlbacher, K. Maksimenka, S. Steffens, K. Schaumann, R. Stohr,<br />
J. Wiese, J. F. Imh<strong>of</strong>f, S. Perovic-Ottstadt, O. Boreiko, W. E. G. Müller (2005); The first sorbicillinoid alkaloids, the<br />
antileukemic sorbicillactones A and B, from a sponge-derived Penicillium chrysogenum strain; Tetrahedron 61,<br />
7252-7265.<br />
25
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
G. Bringmann, J. Mühlbacher, K. Messer, M. Dreyer, R. Ebel, B.W. Nugroho, V. Wray, P. Proksch (2003);<br />
Cyclorocaglamide, the First Bridged Cyclopentatetrahydrobenz<strong>of</strong>uran, and a Related Open Chain Rocaglamide<br />
Derivative from Aglaia oligophylla; J. Nat. Prod. 66, 80-85.<br />
G. Bringmann, K. Messer, M. Wohlfarth, J. Kraus, K. Dumbuya, M. Rueckert (1999); HPLC-CD On-Line Coupling in<br />
Combination with HPLC-NMR and HPLC-MS/MS for the Determination <strong>of</strong> the Full Absolute Stereostructure <strong>of</strong> New<br />
Metabolites in Plant Extracts; Anal. Chem. 71, 2678-2686.<br />
G. Bringmann, J. Mühlbacher, C. Repges, J. Fleischhauer (2001); MD-Based CD Calculations on the Absolute Axial<br />
Configuration <strong>of</strong> the Naphthylisoquinoline Alkaloid Dioncophylline A; J. Comp. Chem. 22, 1273-1278.<br />
L. Du, J. Ai, D. Li, T. Zhu, Y. Wang, M. Knauer, T. Bruhn, H. Liu, M. Geng, Q. Gu, G. Bringmann (2011); Aspergiolides C and<br />
D, Spirocyclic Aromatic Polyketides with Potent Protein Kinase c-Met Inhibitory Effects; Chem. Eur. J. 17, 1319-<br />
1326.<br />
L. Liu, T. Bruhn, L. Guo, D.C.G. Götz, R. Brun, A. Stich, Y. Che, G. Bringmann (2011); Chloropupukeanolides C E, Cytotoxic<br />
Pupukeanane Chlorides with a Spiroketal Skeleton from Pestalotiopsis fici; Chem. Eur. J. 17, 2604-2613 (with cover<br />
picture).<br />
J. Wu, S. Zhang, Q. Xiao, H. Ding, T. Bruhn, G. Bringmann (2008); Xylogranatins F-R: Antifeedants from the Chinese<br />
Mangrove, Xylocarpus granatum, Suggesting a New Biogenetic Pathway to Tetranortriterpenoids; Chem. Eur. J. 14,<br />
1129-1144.<br />
M. Xu, T. Bruhn, B. Hertlein, R. Brun, A. Stich, J. Wu, G. Bringmann (2010); Shuangancistrotectorines A-E, Dimeric<br />
Naphthylisoquinoline Alkaloids with Three Chiral Biaryl Axes, from the Chinese Plant Ancistrocladus tectorius;<br />
Chem. Eur. J. 16, 4206-4216.<br />
26
I<br />
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[PL 8] Technology Platforms to Facilitate Natural Product-Based Drug<br />
Discovery from African Biodiversity<br />
Kelly Chibale<br />
Department <strong>of</strong> Chemistry and Institute <strong>of</strong> Infectious Disease and Molecular Medicine, University <strong>of</strong> Cape Town,<br />
Rondebosch 7701, South Africa; E-mail: Kelly.Chibale@uct.ac.za.<br />
n order for diseases that primarily affect the African population to receive worldwide scientific<br />
attention, African researchers must take a more active role in modern drug discovery approaches<br />
while making the most <strong>of</strong> the indigenous biodiversity available on the continent. 1 While African<br />
scientists do not have the luxury <strong>of</strong> access to large synthetic chemical libraries and other<br />
sophisticated technological platforms due, amongst other things, to limited financial and<br />
infrastructure resources, they have a powerful resource in uniquely endemic natural products<br />
and/or general biodiversity, which are yet to be exploited for health and economic benefits.<br />
In order for natural product-based drug discovery research in Africa to translate into tangible<br />
modern pharmaceuticals, or at the very least contribute more positively to the global drug<br />
discovery value chain, it is necessary for drug discovery in Africa to adopt a more integrated and<br />
multidisciplinary approach and take advantage <strong>of</strong> modern, available drug discovery technologies.<br />
Such a paradigm shift is likely to contribute immensely to the development <strong>of</strong> research<br />
infrastructure on the continent, providing not just the potential for exciting new discoveries, but<br />
also the opportunity to expose students and scientists to multi-disciplinary research even as a new<br />
generation <strong>of</strong> African scientists are trained in modern drug discovery approaches. We have<br />
recently 2 proposed an approach to the integration <strong>of</strong> African natural products in modern drug<br />
discovery, which is summarized in Figure 1.<br />
African scientists working in the area <strong>of</strong> drug discovery, to a large extent, must adopt<br />
pharmaceutical industry approaches to drug discovery through lead identification and optimization<br />
in typical hit to lead (H2L) and Lead Optimization (LO) campaigns. Increasingly integration <strong>of</strong> in<br />
silico, in vitro and in vivo drug absorption, distribution, metabolism and excretion (ADME) studies in<br />
the discovery and development <strong>of</strong> new chemical entities (NCE) has become a feature <strong>of</strong> medicinal<br />
chemistry programmes in the pharmaceutical industry and some academic and related<br />
institutions. 3<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Figure 1: Development <strong>of</strong> leads from natural products. 2<br />
This lecture will highlight some technology platforms that have been set up in our laboratories at<br />
the University <strong>of</strong> Cape Town (UCT) to facilitate the integration <strong>of</strong> African natural products into<br />
modern drug discovery paradigms.<br />
References<br />
[1] E. M. Guantai and K. Chibale( 2011); Malaria Journal 10(Suppl 1):S2 (15 March 2011).<br />
[2] E. M. Guantai. C. M. Masimirembwa, and K. Chibale (2011); Future Medicinal Chemistry 3(3) 257-261<br />
[3] E. M. Guantai, K. Ncokazi, T. J. Egan, J. Gut, P. J. Rosenthal, R. Bhampidipati, A. Kopinathan, P. J. Smith<br />
and K. Chibale (2011); Journal <strong>of</strong> Medicinal Chemistry, 54, in press,<br />
28
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[PL 9] From Past Traditions to a Herbal Pharmacopoeia Africa s Green Gold<br />
Ameenah Gurib-Fakim<br />
Centre for Phytotherapy Research, 7th Floor, Ebene, Mauritius afakim@cephyr-recherche.com<br />
Keywords: Medicinal plants, herbal medicine, validation, African Herbal Pharmacopoeia<br />
P<br />
lants have formed the basis <strong>of</strong> sophisticated traditional medicines that have existed for<br />
thousands <strong>of</strong> years and continue to provide Mankind with remedies. According to the WHO,<br />
over 80% <strong>of</strong> the world s population still depend on traditional medicine for their primary health<br />
care (WHO 1999). The interest in Nature continues not only as a potential source <strong>of</strong> herbal<br />
medicines, which is finding increasing acceptance in the developed world, but also as<br />
chemotherapeutic agents. It is a fact that natural products and their derivatives represent more<br />
than 50% <strong>of</strong> all drugs in clinical use in the world (Farnsworth et al, 1985).<br />
Whilst modern allopathic medicine usually aims to develop a patentable single compound or a<br />
magic bullet to treat specific condition, traditional medicine <strong>of</strong>ten aims to restore balance by using<br />
chemically complex plants, or by mixing together several different plants in order to maximise a<br />
synergistic effect or to improve the likelihood with a relevant molecular target. In most societies<br />
and increasingly in western societies, allopathic and traditional systems <strong>of</strong> medicine occur side by<br />
side in a complementary way. The former treats serious acute conditions while the latter is used for<br />
chronic illnesses, to reduce symptoms and improve the quality <strong>of</strong> life in a cost-effective way.<br />
The African continent is blessed with a unique biodiversity accounting for almost 25% <strong>of</strong> the global<br />
pool <strong>of</strong> genetic resources. Paradoxically, this continent is experiencing the highest rate <strong>of</strong><br />
destruction. The conservation <strong>of</strong> plant genetic resources, the documentation and validation <strong>of</strong> the<br />
traditional knowledge are key issues that will need to be addressed (Neuwinger 2000). The<br />
industrial potential <strong>of</strong> these plants is to be demonstrated especially as medicinal plants have not<br />
only the potential <strong>of</strong> addressing the Millennium Development Goals (MdGs) but also provide to<br />
Mankind cheap and efficacious remedies.<br />
African herbal medicine relies more on wild harvested plants than any continent on earth yet the<br />
sustainability <strong>of</strong> this indigenous resource is increasingly endangered with an average annual loss <strong>of</strong><br />
1% as opposed to 0.6% at the global level (Iwu, 1993). Fortunately the rate <strong>of</strong> this loss is reported to<br />
be slowing down. Loss <strong>of</strong> plants also means loss <strong>of</strong> accompanying traditional knowledge. The value<br />
<strong>of</strong> countless generations <strong>of</strong> observations <strong>of</strong> the application <strong>of</strong> certain plants on humans and animal<br />
disorders is impossible to value, especially in relation to present day global bioprospecting<br />
activities. The African Herbal Pharmacopoeia is one way <strong>of</strong> showcasing the important plants <strong>of</strong><br />
Africa (Brendler et al, 2010).<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
This presentation will show case not only the potential <strong>of</strong> important African medicinal plants but<br />
also the work being carried out on the validation <strong>of</strong> traditional herbal recipes against non<br />
communicable diseases (diabetes for example) and infectious diseases in general.<br />
References<br />
Brendler T et al (2010); African Herbal Pharmacopoeia. AAMPS, Mauritius<br />
Farnsworth N et al (1985); Bull. WHO, 63, 965-981<br />
Iwu M (1993). Handbook <strong>of</strong> African Medicinal Plants. CRC Press, USA<br />
Neuwinger HD (2000); African Traditional Medicine. MedPharm Scientific Publishers, Stuttgart, Germany<br />
WHO (1999); WHO Monographs on selected medicinal plants. World Health Organisation, Geneva, Switzerland.<br />
30
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[PL 10] NMR Analysis <strong>of</strong> the Molecular Structure <strong>of</strong> Flexible Molecules in<br />
Solution<br />
Máté Erdelyi<br />
Department <strong>of</strong> Chemistry, University <strong>of</strong> Gothenburg, SE-412 96 Gothenburg, Sweden, and the Swedish NMR Centre,<br />
SE-405 30 Gothenburg, Sweden. E-mail: mate@chem.gu.se<br />
Keywords: NMR, dynamics, ensemble, NAMFIS, residual dipolar coupling, pseudocontact shift<br />
Introduction<br />
L<br />
iving organisms biosynthesize a broad variety <strong>of</strong> substances from macromolecules to small<br />
molecular natural products <strong>of</strong> an astonishing chemical diversity. The identification and<br />
structural studies <strong>of</strong> both type <strong>of</strong> compounds is generally performed by NMR spectroscopy, which<br />
analysis provides different challenges for the two groups: the structure elucidation <strong>of</strong> biopolymers<br />
is <strong>of</strong>ten hampered by extensive signal overlaps, whereas that <strong>of</strong> small natural products may<br />
become difficult originating from their dynamic nature. In this presentation the focus will be on<br />
small flexible compounds that are commonly present in solution as rapidly equilibrating mixtures <strong>of</strong><br />
low-energy conformations and which cannot be accurately represented in form <strong>of</strong> a single structure<br />
as conventionally derived by experimentally-restrained structure calculations for biopolymers.<br />
(Nevins et al). The limitations <strong>of</strong> X-ray crystallography for reflecting highly dynamic solution<br />
structures are well-known, whereas computational efforts without experimental parameters can<br />
only provide predictions, but not description <strong>of</strong> the structure or dynamics <strong>of</strong> a molecule. Solution<br />
NMR is to date the method <strong>of</strong> choice for gaining an improved understanding <strong>of</strong> the behavior <strong>of</strong><br />
flexible molecules, however, should be applied with care as for example the simplified derivation <strong>of</strong><br />
an average structure from time averaged data, such as NOEs and scalar couplings, results in a non<br />
existing, erroneous structure Methods for the deconvolution <strong>of</strong> time-averaged NMR variables into<br />
structural families well-representing the solution ensemble and satisfactorily fulfilling the structural<br />
restraints are available, although still scarcely utilized.<br />
In this presentation solution NMR methods for the proper structural description <strong>of</strong> dynamic small<br />
molecules will be discussed on the examples <strong>of</strong> natural products and their synthetic analogues.<br />
Results and Discussion<br />
The method NAMFIS-NMR Analysis <strong>of</strong> Molecular Flexibility in Solution (Cicero et al)-was applied for<br />
determination <strong>of</strong> the relative configuration <strong>of</strong> Angiotensin IV analogues (Andersson et al) and for<br />
the elucidation <strong>of</strong> the conformational properties <strong>of</strong> natural as well as C3-modified epothilones<br />
(Erdelyi et al.2008 and 2010) and <strong>of</strong> chroman-4-one tetrahydropyrimidines (Fridén-Saxin et al.). For<br />
the above examples the experimental data (J couplings and NOEs) were fit to a priori computed set<br />
<strong>of</strong> theoretical structures generated by Monte Carlo conformational search.<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Determination <strong>of</strong> relative configuration was performed by identification <strong>of</strong> a conformationally welldefined,<br />
comparably rigid core <strong>of</strong> the studied 13- and 14-membered analogues followed by<br />
comparison <strong>of</strong> the quality <strong>of</strong> fit <strong>of</strong> the computed ensembles <strong>of</strong> all possible diastereomers to the<br />
experimental data. This procedure permitted the identification <strong>of</strong> the point <strong>of</strong> racemisation <strong>of</strong><br />
three sets <strong>of</strong> synthetic Angiotensin IV (Ang IV) analogues and gave insight in the tremendous<br />
importance <strong>of</strong> chirality on the insulin-regulated aminopeptidase (IRAP) inhibitory activity (50-500<br />
fold) <strong>of</strong> these substances (Figure 1.; Andersson et al).<br />
Figure 1. Overlaid backbones <strong>of</strong> the low energy conformations <strong>of</strong> Ang IV analogues possessing IRAP<br />
inhibitory activity, as identified by NAMFIS analysis.<br />
For conformational studies, the population distribution <strong>of</strong> the solution ensembles was evaluated by<br />
selection <strong>of</strong> feasible structures from a theoretically possible conformation pool and by estimation<br />
<strong>of</strong> their molar fractions. This procedure allowed the evaluation <strong>of</strong> the conformational directing role<br />
<strong>of</strong> the C3 substituents <strong>of</strong> the microtubule-stabilizing agent natural product epothilone A and<br />
provided a pro<strong>of</strong> for its previously determined NMR-derived tubulin-bound conformation<br />
(Carlomagno et al) over the one proposed by electron crystallography (Nettles et al). These findings<br />
were further confirmed by investigation <strong>of</strong> the binding mode <strong>of</strong> epothilone-tubulin complexes<br />
based on transferred NOE experiments (Erdelyi et al, 2010). The enormous potency <strong>of</strong> the NAMFISbased<br />
procedure is further shown by the elucidation <strong>of</strong> the conformational properties <strong>of</strong> novel<br />
synthetic chroman-4-one tetrahydropyrimidines that revealed their potential applicability as type<br />
VIII -turn peptidomimetics (Fridén-Saxin et al).<br />
A second methodology applying deconvolution <strong>of</strong> residual dipolar couplings (RDCs) and<br />
pseudocontact shifts (PCSs), as observed by NMR, was used to describe the dynamic properties <strong>of</strong><br />
oligosaccharides. A large series <strong>of</strong> distance and orientation dependent RDCs and PCSs was collected<br />
for two different oligosaccahrides, lactose (Erdelyi et al, 2011) and N,N -diacetylchitobiose<br />
(Yamamoto et al), by a paramagnetic tagging-based technology (Figure 2). The technology is shown<br />
to successfully distinguish dynamic linkages from rigid ones and estimate the probabilities <strong>of</strong><br />
conformations present in solution, even for compounds for which the NOE- and scalar couplingbased<br />
methods <strong>of</strong>ten are inadequate.<br />
32
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Figure 2. Overlaid conformations <strong>of</strong> lactose connected to a paramagnetic centre through a rigid<br />
biphenyl-EDTA-type paramagnetic tag that allows rotation only around a single axis and keeps the<br />
paramagnetic centre (green) at a well-defined distance from the saccharide to control<br />
paramagnetic relaxation-induced line broadening. Such utilization <strong>of</strong> the anisotropic magnetic<br />
susceptibility tensor <strong>of</strong> paramagnetic lanthanides <strong>of</strong>fers long distance information on conformation<br />
and dynamics by induction <strong>of</strong> residual dipolar couplings and pseudocontact shifts.<br />
Simultaneous fitting <strong>of</strong> the probabilities <strong>of</strong> computed conformations and the orientation <strong>of</strong> the<br />
magnetic susceptibility tensor <strong>of</strong> a series <strong>of</strong> lanthanide complexes <strong>of</strong> lactose shows that its<br />
glycosidic bond samples syn/syn, anti/syn and syn/anti regions <strong>of</strong> the conformational space in<br />
water. The obtained results demonstrate that the applied paramagnetic tagging-based technique<br />
allows for the description <strong>of</strong> the motion <strong>of</strong> the glycosidic linkage in saccharides. It permits the<br />
collection <strong>of</strong> several complementary series <strong>of</strong> RDC and PCS data <strong>of</strong> the same ensemble by simple<br />
variation <strong>of</strong> the complexed paramagnetic ion. The increased number <strong>of</strong> available experimental<br />
parameters provides greatly improved reliability for the investigation <strong>of</strong> dynamic processes. In a<br />
related study, we demonstrated on the example <strong>of</strong> N,N-diacetylchitobiose (Yamamoto et al) that<br />
the applied paramagnetic tagging technique is capable <strong>of</strong> identifying rigid glycosidic linkages <strong>of</strong><br />
sugars and thereby have proven that the approach is capable <strong>of</strong> differentiating between rigid and<br />
dynamic structures.<br />
The high importance <strong>of</strong> the proper description and the thorough understanding <strong>of</strong> the dynamic<br />
properties <strong>of</strong> small molecules with respect to their biological activity is demonstrated and the<br />
exceptional applicability <strong>of</strong> solution NMR techniques for the ensemble analysis <strong>of</strong> flexible, complex<br />
structures, such as natural products, is shown.<br />
Acknowledgements<br />
The Swedish Research Council (2004-3073, 2007-4407), the European Research Council (0411363,<br />
259638), the Royal Society <strong>of</strong> Arts and Sciences in Göteborg, the Carl Tryggers Foundation and the<br />
Åke Wibergs Foundation are gratefully acknowledged for their generous financial support. I am<br />
33
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
grateful for the contributions <strong>of</strong> all co-authors <strong>of</strong> the below cited original papers for their<br />
contributions to the presented work and for all stimulating discussions.<br />
References<br />
Andersson, H.; Demaegdt, H.; Vauquelin, G.; Lindeberg, G.; Karlén, A.; Hallberg, M.; Erdélyi, M.; Hallberg, A. (2010); J.<br />
Med. Chem. 53, 8059-8071.<br />
Carlomagno, T.; Sanches, V.M.; Blommers, M.J.J.; Griesinger, C. (2003); Angew. Chem. Int .Ed. 42, 2511.<br />
Cicero, D.O.; Barbato, G.; Bazzo, R. (1995); J. Am. Chem. Soc. 117, 1027-1033.<br />
Erdélyi, M.; Pfeiffer,B.; Hauenstein, K.;Fohrer, J.;Altmann, K.-H.; Carlomagno, T. (2008); J. Med. Chem., 51, 1469-1473.<br />
Erdélyi, M.; Navarro-Vázquez, A.; Pfeiffer, B.; Kuzniewski, C. N.; Felser, A.; Widmer, T.; Gertsch, J.; Pera, B.; Fernando-<br />
Díaz, J.; Altmann, K.H.; Carlomagno, T. (2010); ChemMedChem, 5, 911-920.<br />
Erdélyi, M.; D Auvergne, E.; Navarro-Vázquez, A.; Leonov, A.; Griesinger, C. (2011); Chem. Eur. J. in press.<br />
Fridén-Saxin, M.; Seifert, T.; Hansen, L.K.; Grøtli, M.; Erdélyi, M.; Luthman, K., (2011); submitted.<br />
Nettles, J.H.; Li, H.; Cornett, B.; Krahn, J.M.; Snyder, J.P.; Downing, K.H. (2005); Science, 305, 866.<br />
Nevins, N.; Cicero, D.; Snyder, J.P. (1999); A test <strong>of</strong> the single-conformation hypothesis in the analysis <strong>of</strong> NMR data for<br />
small polar molecules: A force field comparison. J. Org. Chem. 64, 3979-3986.<br />
Yamamoto, S.; Yamaguchi, T.; Erdélyi, M.; Griesinger, C.; Kato, K. (2011); Chem. Eur. J. in press.<br />
34
C<br />
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[PL 11] Bioassay <strong>of</strong> Natural Products for Cosmetics<br />
Alain Meybeck<br />
AM Phyto-Conseil, Courbevoie, France. ameybeck@club-internet.fr<br />
osmetic products are preparations intended to be placed in contact with the external parts <strong>of</strong><br />
the human body with a view to clean them, perfume them, change their appearance and/or<br />
protect them or keep them in good conditions.<br />
Anti-aging activity <strong>of</strong> 20-hydroxyecdysone (20-E). 20-E or beta-Ecdysone or Ecdysterone, is the<br />
most common member <strong>of</strong> the ecdysteroid family. It can be found in insects, in plants such as<br />
Cyanotis arachnoidea (C.a.), and even in edible plants like spinach. It has many types <strong>of</strong> activities in<br />
mammals [Lafont et al, 2003] such as improvement <strong>of</strong> skin healing [Meybeck et al, 1990], and<br />
stimulation <strong>of</strong> skin keratinocyte differentiation [Detmar et al, 1994]. It is known that many<br />
proliferative cell types like lung or skin human diploid fibroblasts (HDFs), exposed to subcytotoxic<br />
stress (UV, H2O2, etc.), undergo stress-induced premature senescence or SIPS which is closely<br />
related to replicative senescence. SIPS can be defined as the long term effect <strong>of</strong> subcytotoxic stress<br />
on proliferative cell types, including irreversible growth arrest <strong>of</strong> a majority <strong>of</strong> the cell population.<br />
The proportion <strong>of</strong> HDFs positive for senescence-associated ß-galactosidase (SA ß-gal) activity<br />
increases in SIPS [Toussaint et al, 2002].<br />
A study was undertaken in order to determine the potential anti-photoaging effects <strong>of</strong> 20-E<br />
extracted from C.a. in a model <strong>of</strong> dermal aging in vitro.<br />
The results show that 20-Hydroxyecdysone provides human BJ foreskin fibroblasts with some kind<br />
<strong>of</strong> protection against premature cellular senescence induced by repeated UV insults (UV-SIPS) as<br />
showed by the dramatic decrease by 20-E <strong>of</strong> the proportion <strong>of</strong> cells expressing SA -gal. Moreover<br />
it seems that this protection is due to a transient stimulation <strong>of</strong> p53, <strong>of</strong>ten called the guardian <strong>of</strong><br />
the genome, which probably prepares cells to face UVB injuries and induces an efficient repair<br />
process <strong>of</strong> the damages caused by UVB radiations. These findings reinforce the notion that UVBinduced<br />
premature senescence <strong>of</strong> HDFs can be used to screen potential anti-photoageing<br />
compounds, and allow the development <strong>of</strong> 20-E containing cosmetic formulations which will better<br />
protect the cells against chronic sunlight damage leading to premature ageing <strong>of</strong> exposed skin<br />
[Meybeck et al, 2006], characterized in particular by senescent fibroblasts expressing markers<br />
associated with the processes <strong>of</strong> inflammation and destruction <strong>of</strong> the dermal matrix [Funk et al,<br />
2000].<br />
Further studies have shown that the mechanism <strong>of</strong> action <strong>of</strong> 20E might pass through its binding to<br />
the alternative active site <strong>of</strong> the Vitamin D receptor responsible for the rapid effects.<br />
35
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Anti-melanogenesis activity <strong>of</strong> 5-hydroxy-tryptophan (5-HTP). There is a great demand worldwide<br />
but particularly in Asia for products aimed at controlling skin pigmentation. This is why Griffonia<br />
simplicifolia (G.s.) seed extracts were screened for an eventual inhibitory activity on melanogenesis<br />
in skin cells. (G.s.) is a tree growing in West African countries like Ivory Coast and Ghana, the seeds<br />
<strong>of</strong> which are the main industrial source <strong>of</strong> 5-HTP widely used as a food supplement to help reduce<br />
depression, migraines, insomnia, appetite [Lemaire et al, 2002] .<br />
An evaluation <strong>of</strong> the activity <strong>of</strong> G.s. extracts and 5-HTP on melanin pigment synthesis was carried<br />
out on normal human epidermal melanocytes (NHEM) and murine B16 melanoma cells. In order to<br />
have a better appreciation <strong>of</strong> 5-HTP activity, a test on B16 melanoma cells previously stimulated by<br />
the -MSH analog NP-MSH, has been carried out in comparison with Kojic acid and Arbutin. This<br />
protocol disconnects melanogenesis from the basic metabolism <strong>of</strong> melanocytes and allows to<br />
measure significant inhibitions at lower concentrations <strong>of</strong> active compounds.<br />
In this assay, 5-HTP induced 31 % melanogenesis inhibition already at 8 µg/ml, 70 % at 40 µg/ml,<br />
and 90 % or nearly complete inhibition at 100 µg/ml. This performance was better than that <strong>of</strong> Kojic<br />
acid (only 70 % inhibition at 200 µg/ml), and similar to that <strong>of</strong> Arbutin (97 % inhibition at 200<br />
µg/ml).<br />
This new protocol , together with more classical tests , has allowed to determine that G.s. seed<br />
extracts and 5-HTP inhibit melanin synthesis , thus confirming the results <strong>of</strong> Kim K.T. et al [2006]<br />
who found also that 5-HTP stimulates the ERK pathway therefore downregulating MITF and<br />
tyrosinase biosynthesis.<br />
The results obtained show that G.s. seed extracts and their active molecule 5-HTP might be efficient<br />
whitening or brightening ingredients in cosmetic formulations [Meybeck 2006].<br />
Protection by Notoginseng root saponins (NRS) against UV-induced immuno suppression. The<br />
root <strong>of</strong> Panax Notoginseng contains up to 10% saponins ( which are thought to be the major active<br />
components ) , flavonoids , polysaccharides , polyacetylenes , aminoacids The Panax Notoginseng<br />
Root Saponin Fraction (NRS) contains five major compounds ( content > 4% ) : ginsenosides Rg1 ,<br />
Rb1 , Rd and Re , and notoginsenoside R1 .<br />
In a recent study [Sene et al, 2007] , it was found that NRS have the effect in human skin fibroblasts<br />
, <strong>of</strong> up-modulating the m-RNA coding for Heme Oxygenase-1 (HO-1) , a very important protecting<br />
enzyme since it leads to the formation <strong>of</strong> biliverdin which is a very powerful natural antioxidant ,<br />
and <strong>of</strong> carbon monoxide which has been shown to protect Langerhans cells from photoimmunosuppression<br />
( Langerhans Cells located in the epidermis , are responsible for the immune<br />
protection <strong>of</strong> skin against aggressions . But when skin is exposed to UV light, they disappear from<br />
the epidermis, and as a result the skin becomes more vulnerable ) .<br />
36
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
In a first experiment on cultivated normal human dermal fibroblasts (pool <strong>of</strong> NHDF at 8 th passage),<br />
it was found through the use <strong>of</strong> a c-DNA array , that the expression <strong>of</strong> the m-RNA <strong>of</strong> HO-1 had been<br />
significantly enhanced (189%) by a 24 h treatment with Notoginseng Root Saponins at 0.2 mg/ml ,<br />
although it was not significantly affected by Ginseng Root Saponins. Then , in a second experiment<br />
on another culture (NHDF at 8 th passage) , a quantitative determination was achieved , after PCR<br />
amplification , and it was found that the expression <strong>of</strong> HO-1 m-RNA in the cells after 4h <strong>of</strong> contact<br />
with NRS at 0.2 mg/ml was increased to 313 % <strong>of</strong> the base level (277% at 8h ; 115% at 24h) .<br />
In a third experiment carried out on human skin ex-vivo, it has been put in evidence that NRS are<br />
able to protect the skin against Langerhans cells depletion by UV exposure . The observed<br />
protection was up to 63 % (at 1mg/ml <strong>of</strong> NRS), and almost as much as that <strong>of</strong> a UV filtering<br />
commercial formulation <strong>of</strong> SPF 20 .<br />
Finally, it was shown that NRS could provide a 40% protection (at 0.3mg/ml) against the<br />
appearance <strong>of</strong> SA- -Gal positive senescent fibroblasts induced by an H2O2 stress (senescent cells<br />
are unable to undergo mitosis and are characterized by the expression <strong>of</strong> SA- -Galactosidase) . This<br />
work has therefore established that Panax Notoginseng root saponins can protect skin cells against<br />
certain oxidative stresses and that this protection most probably results from stimulating the<br />
expression <strong>of</strong> the enzyme HO-1. And it is possible that HO-1 induction might as well be responsible<br />
for some known effects <strong>of</strong> Notoginseng on wound healing, in the treatment <strong>of</strong> cardio-vascular<br />
diseases, on memory improvement<br />
Anti-Wrinkle Effect <strong>of</strong> Extracts <strong>of</strong> Boswellia serrata<br />
Expression lines are produced by the mechanical stress exerted on the skin by facial muscles. So<br />
relaxing skin can help prevent there formation.Three extracts <strong>of</strong> Boswellia serrata were tested on a<br />
nerve-muscle coculture model which makes it possible to recreate a motor arc by innervations <strong>of</strong><br />
human striated muscle cells with explants <strong>of</strong> spinal cord and <strong>of</strong> spinal ganglia from rat embryos<br />
[Meybeck et al, 2004].<br />
This test predicts an anti-wrinkle effect, as was demonstrated in the case <strong>of</strong> diazepam, which<br />
inhibited muscle fibre contractions in this model, and showed an anti-wrinkle activity in vivo.<br />
Human muscle cells derived from human muscle samples from a healthy donor are seeded in 15<br />
mm-diameter wells (24-well culture dishes). After culturing for 10 days, these cells form a<br />
monolayer and fuse. At this stage, spinal cord explants from 13-day-old rat embryos, containing the<br />
spinal ganglion, are deposited onto the culture. The first muscle fibre contractions are observed<br />
after coculturing for one week. After coculturing for 3 weeks, the muscle fibres are striated and<br />
possess mature differentiated neuromuscular junctions.<br />
A muscle fibre having regular contractions (at least 60 contractions per minute) is then selected in<br />
three different culture wells and the number <strong>of</strong> contractions is counted over 30 seconds using<br />
image analysis s<strong>of</strong>tware. The extracts tested, diluted in ethanol, are then incubated for 60 seconds<br />
37
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
in these wells, at the concentrations C1 and C2 <strong>of</strong> 0.005% and 0.01%. At the end <strong>of</strong> the incubation,<br />
the number <strong>of</strong> contractions is again counted over 30 seconds.<br />
The percentage <strong>of</strong> inhibited contractions is then determined. The three Boswellia extracts tested<br />
induced inhibitions. The most active extract induced 72% <strong>of</strong> contraction inhibition at the<br />
concentration <strong>of</strong> 0.005%. The four pentacyclic triterpene acids found in extracts <strong>of</strong> Boswellia<br />
serrata, namely beta-boswellic acid, 3-O-acetylboswellic acid, 11-ketoboswellic acid and 3-O-acetyl-<br />
11-ketoboswellic acid, were tested in a model <strong>of</strong> calcium flux in order to evaluate their capacity for<br />
inhibiting calcium channels and therefore their ability to relax muscle fibers.<br />
The relaxing effect <strong>of</strong> 3-O-acetyl-11-ketoboswellic acid was found significantly greater than that<br />
obtained for the other three acids tested. This effect was confirmed with the muscle-nerve<br />
coculture test which showed a contraction-inhibiting effect <strong>of</strong> 74.7% at 5 microM and <strong>of</strong> 87% at 10<br />
microM for this compound. Anti-wrinkle cosmetic products can therefore be formulated with<br />
Boswellia extracts.<br />
References<br />
Detmar M., Dumas M., Bonte F., Meybeck A. and Orfanos C. (1994); Effects <strong>of</strong> ecdysterone on the differentiation <strong>of</strong><br />
normal human keratinocytes in vitro , Eur J Dermatol 4, 558-562<br />
Funk W.D., Wang C.K., Shelton D.N., Harley C.B., Pagon G.D., Hoeffler W.K. (2000); Telomerase expression restores<br />
dermal integrity to in vitro-aged fibroblasts in a reconstituted skin model , Exp Cell Res. 258, 270-8<br />
Kim K.T.,Lee B.Y.,Kim Y.H.,Kim K.S.,and Kim K.H. (2006); 5-hydroxytryptophan as a novel inhibitor <strong>of</strong> melanin<br />
synthesis , Poster PC-026, Preprints, 24 th IFSCC Congress, Osaka, Japan, pp.56-57<br />
Lafont R. and Dinan L. (2003); Practical uses for ecdysteroids in mammals including humans: an update , J Insect<br />
Science 3:7, 2003; http://www.insectscience.org/3.7,9/6<br />
Lemaire P. and Adosraku R. (2002); An HPLC method for the direct assay <strong>of</strong> the serotonin precursor, 5hydroxytryptophan,<br />
in the seeds <strong>of</strong> Griffonia simplicifolia .,Phytochem. Anal., 13, 333-337<br />
Meybeck A. and Bonte F. Hydrated lipidic lamellar phases or liposomes based on ecdysteroids . PCT Patent:<br />
WO90/03778<br />
Meybeck A., Zanvit A., 3-O-acetyl-11-ketoboswellic acid for relaxing the skin , Patent US2004166178 (A1)<br />
Meybeck A. « Utilisation d un extrait de Griffonia », Patent PCT/FR2006/001181<br />
Meybeck A. and Yang C.R. « Utilisation d une composition contenant un ecdysteroïde ». Patent PCT/FR2006/002407<br />
Sene G., Loiseau A., Meybeck A. and Yang C.R. Use <strong>of</strong> ginsenosides and extracts containing them . Patent WO<br />
2007131677 (A1)<br />
Toussaint O., Remacle J., Dierick J.F., Pascal T., Frippiat C., Zdanov S., Magalhaes J.P., Royer V.,and Chainiaux F. (2002);<br />
From the Hayflick mosaic to the mosaics <strong>of</strong> ageing. Role <strong>of</strong> stress-induced premature senescence in human<br />
ageing , Int J Biochem Cell Biol 34, 1415-1429<br />
38
The 14th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011<br />
[PL 12] Multiple anti-infective properties <strong>of</strong> selected Combretum species<br />
from Zimbabwe<br />
Rumbidzai Mangoyi, Theresa Chimponda, Elaine Chirisa, Tariro Chitemerere and Stanley Mukanganyama a<br />
Biomolecular Interactions Analyses Laboratory Department <strong>of</strong> Biochemistry, University <strong>of</strong> Zimbabwe<br />
smukanganyama@medic.uz.ac.zw.<br />
Key words: Antifungal, anti-inflammatory, antibacterial, Drug efflux, Mycobacterium aurum,<br />
Candida albicans, Rhodamine 6G, antimycobacterial, metabolism<br />
Introduction<br />
Medicinal plants are therapeutic resources used by traditional population specifically for<br />
health care and which may serve as precursors for the synthesis <strong>of</strong> useful drugs (Okigbo et<br />
al., 2009). Plants have served as a source <strong>of</strong> new pharmaceutical products and inexpensive<br />
starting materials for the synthesis <strong>of</strong> many known drugs. Natural products and their<br />
derivatives represent more than 50% <strong>of</strong> drugs in clinical use in the world (Cowan, 1999). A<br />
number <strong>of</strong> interesting outcomes have been found with the use <strong>of</strong> a mixture <strong>of</strong> natural<br />
products to treat diseases, most notably the synergistic effects and polypharmacological<br />
application <strong>of</strong> plant extracts (Ncube et al., 2008). The search for antimicrobial agents has<br />
mainly been concentrated on lower plants, fungi and bacteria as sources. Much less<br />
research has been conducted on antimicrobials from higher plants. Since the advent <strong>of</strong><br />
antibiotics, in the 1950s, the use <strong>of</strong> plant derivatives as antimicrobials has been virtually<br />
nonexistent. The interest in using plant extracts for treatment <strong>of</strong> microbial infections has<br />
increased in the late 1990s, as conventional antibiotics become ineffective (Cowan, 1999).<br />
Of interest in this study is the use <strong>of</strong> Combretum species in the treatment <strong>of</strong> diseases. At<br />
least twenty four species <strong>of</strong> Combretum are well known in African traditional medicine and<br />
are used for the treatment <strong>of</strong> a variety <strong>of</strong> ailments and diseases, ranging from scorpion and<br />
snake bites, mental problems, heart and worm remedies to fever and microbial infections.<br />
The fruits and seeds are, although, in general considered poisonous by traditional healers in<br />
various African countries and have been reported to give toxic effects on human (Fyhrquist,<br />
2007). Many species <strong>of</strong> Combretum have been found to possess powerful antibacterial and<br />
antifungal effects. Among antimicrobial active compounds isolated from Combretum<br />
species are combretastatins (bibenzyle compounds), acidic tetracyclic and pentacyclic<br />
triterpenes/triterpenoids, ellagitannins, phenanthrenes, flavonoids, saponins and<br />
cycloartane glycosides (El<strong>of</strong>f et al., 2005). The leaves, roots and stem bark <strong>of</strong> C. zeyheri are<br />
used medicinally. The leaves are used frequently and have a variety <strong>of</strong> uses in African<br />
traditional medicine. The smoke <strong>of</strong> burnt leaves is inhaled for treatment <strong>of</strong> coughs. The<br />
leaves <strong>of</strong> C. imberbe are used in the treatment <strong>of</strong> diarrhea and cough, symptoms that can be<br />
related to bacterial and fungal infection. The wide spread use <strong>of</strong> Combretum species in<br />
treatment <strong>of</strong> many ailments makes them potential sources <strong>of</strong> anti-infective target.<br />
Phytoconstituents from these plant species could prove to be more effective than the<br />
current available drugs. The study was undertaken to test the antimycobacterial activity <strong>of</strong><br />
the selected Combretum species plant species against Candida albicans, Mycobacterium<br />
aurum, E. coli, B .subtilis and human recombinant cyclooxygenase.<br />
39
The 14th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011<br />
Materials and Methods<br />
All the chemicals used were obtained were obtained from Sigma Aldrich (Darmstadt,<br />
Germany). Other chemicals used were <strong>of</strong> the highest grade available and were obtained<br />
from different sources. Mycobacterium aurum A+ were obtained were obtained from Pr<strong>of</strong>.<br />
Smith <strong>of</strong> the Department <strong>of</strong> Pharmacology, University <strong>of</strong> Cape Town and and<br />
Mycobacterium smegmatis, was obtained Pr<strong>of</strong>. Steenkamp, <strong>of</strong> the Department <strong>of</strong> Clinical<br />
Laboratory studies, University <strong>of</strong> Cape Town. Candida albicans strain ATCC 10231, B. subtilis<br />
and E.coli were obtained from Department <strong>of</strong> Biological Sciences, University <strong>of</strong> Botswana.<br />
The fungi were maintained on nutrient agar slants. The isolate was sub cultured regularly<br />
and stored at 4ºC as well as at -30ºC by making their suspension in 30% glycerol. The plants<br />
used in this work : Combretum imberbe, Combretum molle, Combretum apiculatum,<br />
Combretum elaegnoides, and Combretum krussaii, were collected from the National<br />
Botanical Gardens, Harare Province, Harare, Zimbabwe. Combretum platypetalum and<br />
Combretum zeyheri were obtained from Norton in Mashonaland West Provinces <strong>of</strong><br />
Zimbabwe. The plants were authenticated by Mr Christopher Chapano, a taxonomist at the<br />
National Botanical Gardens. The voucher specimens <strong>of</strong> the plants investigated were kept in<br />
the Department <strong>of</strong> Biochemistry, University <strong>of</strong> Zimbabwe.<br />
Plant leave material was dried in an oven at 50 o C and ground in a two speed blender (Cole<br />
Parmer Instrument Company, Connecticut, USA) to a fine powder. A volume <strong>of</strong> 10 ml <strong>of</strong><br />
absolute ethanol (or methanol) was added to 2 g <strong>of</strong> powder and shaken for 5 minutes on a<br />
vortex and left to sit for 24 hours. A syringe was prepared by inserting a piece <strong>of</strong> fine sieve.<br />
The plant suspension was then transferred into syringe and filtered into a small glass vial.<br />
The sterile suspension was filtered again using 0.45 µM Millipore® sterile filter (Sigma-<br />
Aldrich, Taufkirchen, Germany) into a labeled small glass vial. Ethanol was left to evaporate<br />
overnight in fume hood with air stream to quantify extraction. A constant dry weight <strong>of</strong><br />
each extract was obtained and the residues were stored at 4 o C until when required. To<br />
determine the activity <strong>of</strong> the plant extracts as antimicrobial, initial screening was carried out<br />
using the agar disc diffusion method. Once identified as being potent, growth inhibition<br />
parameters were determined using the broth microdilution method in a 96 wells microtitre<br />
plates. The S9 metabolites from C. zeyheri extract were prepared using fractions from rat<br />
livers <strong>of</strong> male Sprague-Dawley rats in which drug metabolizing enzyme induction had been<br />
done in vivo using phenobarbitone. The inhibitory effects <strong>of</strong> Combretum species on<br />
cycloxygenase I and 2 were also investigated using the Cox assay kit (Cayman, Estonia).<br />
2. Results and Discussion<br />
Table 3.1 shows the effects <strong>of</strong> selected Combretum species on the growth <strong>of</strong> Candida<br />
albicans. Combretum imberbe had the highest zone <strong>of</strong> inhibition followed by Combretum<br />
zeyheri. All the Combretum species used had some antifungal activity on C. albicans.<br />
40
The 14th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011<br />
Table 3.1:<br />
Plant name Zone MIC MFC<br />
<strong>of</strong> inhibition (mg/ml) (mg/ml)<br />
Combretum imberbe<br />
Combretum elaeagnoides<br />
Combretum apiculatum<br />
Combretum zeyheri<br />
Combretum molle<br />
Combretum kraussii<br />
Miconazole (+ve control)<br />
Dimethylsufoxide (DMSO)<br />
Ethanol(-ve control)<br />
(mm)<br />
20 0.31 0.52<br />
16 0.31 >10<br />
13 0.31 0.31<br />
19 0.31 >10<br />
10<br />
9<br />
20 1.25 1.25<br />
6<br />
6<br />
Antifungal activity <strong>of</strong> plant extracts as determined by the agar disc diffusion method<br />
(diameter in mm). These were the results obtained following 24 hours incubation at 37ºC on<br />
nutrient agar.<br />
It was also observed that 3 Combretum species, C. imberbe, C. apiculatum and C. zeyheri<br />
had efflux inhibitory activity on C. albicans when cipr<strong>of</strong>loxacin was used as the probe drug<br />
(Figure 3.1). This means that the antimicrobial action <strong>of</strong> these plants could also be due to<br />
efflux pump inhibition.<br />
% Cipr<strong>of</strong>loxacin<br />
accumulation<br />
150<br />
100<br />
KEY<br />
50<br />
0<br />
C. imberbe C. apiculatum verapamil<br />
% cipr<strong>of</strong>loxacin accumulation measured<br />
in the absence <strong>of</strong> either plant extract or verapamil<br />
% cipr<strong>of</strong>loxacin accumulation measured in the<br />
presence <strong>of</strong> 0.2 mg/ml <strong>of</strong> either plant extract or verapamil<br />
Figure 3.1: Accumulation <strong>of</strong> Cipr<strong>of</strong>loxacin in Candida albicans fungal cells at the final<br />
concentration <strong>of</strong> 0.2 mg/ml Combretum apiculatum and Combretum imberbe plant extracts.<br />
The effects <strong>of</strong> the Combretum species were also investigated in Mycobacterial species and it<br />
was found that only C. imberbe had inhibitory activity on the bacteria (Table 1.3). It was<br />
also observed that C. imberbe and C. hereroense had drug efflux inhibitory activity using<br />
cipr<strong>of</strong>loxacin as the probe drug.<br />
41
The 14th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011<br />
Table 3.2: Antimycobacterial activity <strong>of</strong> plant extracts as determined by the agar disc<br />
diffusion method (diameter in mm).<br />
Zone <strong>of</strong> inhibition (mm) MIC <strong>MB</strong>C<br />
Plant name M. smegmatis M. aurum µg/disc<br />
C. elaeagnoides na na<br />
C. hereroense na na<br />
C. imberbe 9.5 na 125 >1000<br />
C. zeyheri na na<br />
Rifampicin 50 µg/ml 33.0 21.75 0.2 50<br />
DMSO na na<br />
na - no activity.<br />
Metabolic studies using the liver S9 fractions from male Sprague-Dawley rats showed that<br />
the metabolites rather than the parent extracts were responsible for the antifungal effects<br />
observed in C. albicans (Figure 3.2) and that administration <strong>of</strong> the aqueous extract to the<br />
animals increased the activity <strong>of</strong> glutathione transferases (Figure 3.3)<br />
No <strong>of</strong> colonies<br />
No <strong>of</strong> colonies<br />
No <strong>of</strong> colonies<br />
150<br />
100<br />
50<br />
0<br />
150<br />
100<br />
50<br />
0<br />
150<br />
100<br />
50<br />
0<br />
(a) saline at conc o mg/ml<br />
0 30 60<br />
time/mins<br />
(b) Saline at conc. 0.5 mg /ml<br />
0 30 60<br />
time/mins<br />
(c) Saline at conc. 1 mg /ml<br />
0 30 60<br />
time/mins<br />
No <strong>of</strong> colonies<br />
No <strong>of</strong> colonies<br />
No <strong>of</strong> colonies<br />
150<br />
100<br />
50<br />
0<br />
150<br />
100<br />
50<br />
0<br />
150<br />
100<br />
50<br />
0<br />
(d) Pb at conc. 0 mg /ml<br />
0 30 60<br />
time/mins<br />
(e) Pb at conc. 0.5 mg /ml<br />
0 30 60<br />
time/mins<br />
(f) Pb at conc. 1 mg /ml<br />
0 30 60<br />
time/mins<br />
Specific Activity (units/min/mg)<br />
Specific Activity (units/min/mg)<br />
0.07<br />
0.06<br />
0.05<br />
0.04<br />
0.03<br />
0.02<br />
0.01<br />
0.00<br />
0.07<br />
0.06<br />
0.05<br />
0.04<br />
0.03<br />
0.02<br />
0.01<br />
0.00<br />
42<br />
Liver aqueous<br />
* P
The 14th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011<br />
Figure 3.4 Effects <strong>of</strong> C.zeyheri exyracts on Rhodamine 6G effelux in bacteria.<br />
Table 3.3 shows the activties <strong>of</strong> Combretum species on Cox activity. C.platypetalum showed<br />
potent inhibition <strong>of</strong> COX2 and had an IC50 <strong>of</strong> 571 µg/ml compared to 414 for indomethacin.<br />
Table 3.3: Summary <strong>of</strong> results obtained from COX Activity assay showing the prostaglandin<br />
concentrations for the various test extracts<br />
Enzyme Extract (2500 µg/ml) [PG] mg/ml % Inhibition Selectivity<br />
(concentration)<br />
COX-1 Combretum platypetalum 6.1240 41.5 0.5<br />
Combretum zeyheri 0.362 97.4 2.3<br />
Combretum molle 0.733 93.3 1.4<br />
Combretum molle (1250<br />
µg/ml)<br />
2.723 76.4 3.5<br />
Indomethacin 0.396 1.4<br />
COX-2 Combretum platypetalum 0.733 84.9 571<br />
Combretum zeyheri 2.76 42.2<br />
Combretum molle 0.509 67.6<br />
Combretum molle (1250<br />
µg/ml)<br />
1.556 22.0<br />
Indomethacin 0.280 414<br />
43<br />
IC50<br />
(µg/ml)<br />
The aqueous leaf extract and the methanolic leaf extract <strong>of</strong> C. zeyheri showed antimicrobial<br />
activity against E. coli and B subtilis. The plant extracts had an inhibition effect on drug<br />
efflux pumps with E. coli being inhibited the most. The water extract had a higher<br />
antibacterial and efflux pump inhibition effect as compared to the methanolic extract. The<br />
conclusion that can be drawn from this study is that S9 metabolites <strong>of</strong> an aqueous extract <strong>of</strong><br />
Combretum zeyheri inhibit growth <strong>of</strong> Candida albicans.<br />
Extracts from all the six plants investigated possessed inhibitory activity against Candida<br />
albicans. The Combretum imberbe plant extract showed almost comparable antifungal<br />
activity with miconazole, which was used as a positive control and reference antifungal.<br />
Combretum imberbe and Combretum apiculatum possessed fungicidal properties while<br />
Combretum elaeagnoides did not show such activities at the highest concentration tested.<br />
Combretum imberbe and Combretum apiculatum ethanolic plant extracts were found not to<br />
have an effect on the Candida albicans efflux pumps. Combretum platypetalum is likely<br />
source <strong>of</strong> compounds with COX 2 inhibitory activity and thus may serve as a source <strong>of</strong> antiinflammatory<br />
novel compounds. This study has identified Combretum species plants with
The 14th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011<br />
potential antifungal, antibacterial, antimycobacterial and anti-inflammatory activities that<br />
could be used as sources for the isolation <strong>of</strong> active compounds that may serve as lead<br />
compounds in development <strong>of</strong> phytomedicines. Further studies are being carried out to<br />
determine the cytoxicity <strong>of</strong> these compounds using cancer cell lines.<br />
Acknowledgments<br />
This work was supported by the International Program in the Chemical Sciences (IPICS<br />
ZIM01, Uppsala University, Sweden)<br />
References<br />
Cowan, M., (1999), Plant products as antimicrobial agents. Clinical Microbiology; 12(4): 564-582.<br />
El<strong>of</strong>f, J.N., Masoko, P., (2005), The diversity <strong>of</strong> antifungal compounds <strong>of</strong> six South African Terminalia species<br />
(Combretaceae) determined by bioautography. African Journal <strong>of</strong> Biotechnology 4: 1425–1431.<br />
Fyhrquist, P., Mwasumbi. L, Haeggstrom, C.A., Vuorela, H., Hiltunen, R., Vuorela. P. (2002). Ehnobotanical and<br />
antimicrobial investigation on some species <strong>of</strong> Terminalia and Combretum (Combretaceae) growing in<br />
Tanzania. Journal <strong>of</strong> Ethnopharmacology 79: 169-177<br />
Mangoyi, R., Mukanganyama, S. (2009). In vitro antifungal activities <strong>of</strong> crude extracts and purified compounds<br />
from selected plants in Zimbabwe against Candida albicans and Candida krusei. The S<strong>MB</strong>BM<br />
International Congress <strong>of</strong> Biochemistry, the IUB<strong>MB</strong> Special Meeting on Plant stresses and the Sixth<br />
FASB<strong>MB</strong> Congress, Marakech, Morocco, April 20-25<br />
McGaw L.J., Lall, N., Meyer, J.J.M., El<strong>of</strong>f, J.N., (2008), The potential <strong>of</strong> South African plants against<br />
Mycobacterium infections. Journal <strong>of</strong> Ethnopharmacology 119: 482-500.<br />
Ncube, N.S., Afolayan, A.J., and Okoh A.I., (2008), Assessment techniques <strong>of</strong> antimicrobial properties <strong>of</strong> natural<br />
compounds <strong>of</strong> plant origin: current methods and future trends. African Journal <strong>of</strong> Biotechnology,<br />
7(12):1797-1806<br />
Okigbo, R.N., Anuagasi, C.L., Amadi, J.E., Ukpabi, U.J. (2009). Potential inhibitory effects <strong>of</strong> some African<br />
tuberous plant extracs on Escherichia coli, Staphylococcus aureus and Candida albicans. International<br />
Journal <strong>of</strong> Intergrative Biology 6: 92<br />
44
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[PL 13] The Center for World Health & Medicine at Saint Louis University: A<br />
New Translational Research Model to Develop Novel Therapies for Neglected<br />
Diseases and Other Unmet Medical Needs<br />
Peter G. Ruminski<br />
Executive Director, Center for World Health and Medicine at Saint Louis University, 1100 S Grand Blvd., Suite 313; Saint<br />
Louis, Missouri 63104 pruminsk@slu.edu<br />
Key words: CWHM, ICCL, echistatin, RGDX, integrin<br />
T<br />
he Center for World Health & Medicine (CWHM) (www.cwhm.org) at Saint Louis University is a<br />
not-for-pr<strong>of</strong>it institution dedicated to the discovery and development <strong>of</strong> safe, effective and<br />
affordable therapies for neglected diseases <strong>of</strong> poverty in the developing world (especially infants<br />
and children), as well as rare and orphan diseases and other unmet medical needs. It evaluates<br />
promising drug candidates to find therapeutic solutions to debilitating and life threatening global<br />
health problems that pharmaceutical companies typically don t explore. Located in the Doisy<br />
Research Center at the Saint Louis University School <strong>of</strong> Medicine, the CWHM consists <strong>of</strong> a full team<br />
<strong>of</strong> former Pfizer pharmaceutical research and development scientists with specialized skills and<br />
over 200 years <strong>of</strong> combined experience in successful drug development. The CWHM team<br />
possesses the entire spectrum <strong>of</strong> the critical skill sets needed to translate basic science discoveries<br />
into clinically useful drug candidates. These skill sets range from medicinal chemistry and structure<br />
based drug design, in vitro assay development, in vivo pharmacology, development <strong>of</strong> animal<br />
models <strong>of</strong> human disease, PK/PD, and biomarker development and analysis. CWHM scientists are<br />
redirecting their specialized talents toward these global unmet medical needs. CWHM has<br />
established an efficient operating model that marries basic science with clinical opportunity and by<br />
forming international collaborations and partnerships with disease experts and institutions. A brief<br />
overview <strong>of</strong> the CWHM model, some <strong>of</strong> our disease targets, and how we are applying our expertise<br />
in advancing promising candidates to the clinic will be presented. It will also highlight our proposed<br />
partnerships in Africa, which includes a significant partnership opportunity as a member <strong>of</strong> the<br />
International Clinical Compound Library (ICCL) Consortium.<br />
The International Clinical Compound Library (ICCL), which is being coordinated by the CWHM, has<br />
the goal <strong>of</strong> accumulating compounds that have been discontinued (shelved) at clinical stages by<br />
pharmaceutical companies over the years to a central repository. There are more than 11,000 <strong>of</strong><br />
such compounds that never made it to market. This pool is comprised <strong>of</strong> late-stage pre-clinical<br />
through phase III clinical trial compounds. These compounds were discontinued for lack <strong>of</strong><br />
adequate efficacy for the original indication, lack <strong>of</strong> perceived commercial potential, company<br />
reorganization, companies exiting the therapeutic area, etc. Information about such discontinued<br />
compounds is <strong>of</strong>ten hard to find due to lack <strong>of</strong> public disclosures. And yet, these are high quality<br />
compounds with drug-like characteristics that would have tremendous value to the international<br />
research community. They represent a wide variety <strong>of</strong> structural diversity and mechanisms <strong>of</strong><br />
45
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
action. A lead generated from such a library would already be highly advanced, have passed safety<br />
assessments, and have either been in man or deemed appropriate for clinical trials. Such<br />
discontinued, or abandoned clinical compounds represent untapped potential.<br />
The ICCL would be made available for re-purposing or screening for new indications to academic<br />
and institutional investigators worldwide, researching a wide variety <strong>of</strong> diseases, including<br />
neglected diseases <strong>of</strong> the developing world, rare diseases and many additional diseases in need <strong>of</strong><br />
therapies or more effective therapies. Integration <strong>of</strong> researchers into collaborative repositioning<br />
efforts would substantially increase the knowledge base and the pool <strong>of</strong> methodologies available<br />
for pro<strong>of</strong>-<strong>of</strong>-concept studies. These matches will undoubtedly increase the number <strong>of</strong> approved<br />
drugs for new indications and considerable public benefit. Obtaining, managing and making<br />
available such a library to international research investigators has the potential to accelerate the<br />
advancement <strong>of</strong> new therapies for many unmet medical needs on a global scale.<br />
Adding global approved (marketed) drugs, active metabolites <strong>of</strong> approved drugs, veterinary drugs,<br />
and other appropriate high quality drug substances and diverse bioactive natural products to the<br />
ICCL would collectively constitute this world-class one stop shop screening and re-purposing<br />
library for the international research community.<br />
High quality bioactive natural products isolated from Africa s rich biodiversity would be a valuable<br />
component to the ICCL. Although not technically clinical compounds, the fact that a large<br />
majority <strong>of</strong> drugs used in clinical practice today are natural products or have their origins from a<br />
natural product lead, would allow for the screening <strong>of</strong> these novel compounds against a variety <strong>of</strong><br />
diseases on a global basis. The potential benefits <strong>of</strong> this will be discussed as well as a brief overview<br />
<strong>of</strong> the ICCL project, and Africa s proposed partnership in it.<br />
Pharmaceutical Companies abandoned natural products as a screening resource a number <strong>of</strong> years<br />
ago in favor <strong>of</strong> high throughput combinatorial chemistry, fragment based screening, in silico<br />
computational tools, etc. These were thought to be a more cost effective and imparted an<br />
attractive design tailored approach to new drug discovery as compared to the perceived costly<br />
and empirical approaches <strong>of</strong> natural product screening. In many regards this was ill-conceived,<br />
given the prior wealth <strong>of</strong> drugs that resulted from natural products. The view that combinatorial<br />
chemistry would provide millions <strong>of</strong> compounds with diverse structures to quickly and more<br />
cheaply (as opposed to natural products) provide leads for a new generation <strong>of</strong> drugs has not<br />
turned out to be the panacea envisioned. A shift back to a more balanced approach to new<br />
molecule drug discovery should be encouraged. Natural products can still provide a wealth <strong>of</strong><br />
structural diversity with which to provide novel substrates, ripe for optimization into novel drug<br />
candidates for a wide range <strong>of</strong> disease targets. The fact that there are still numerous untapped and<br />
untested natural products in existence (especially marine natural products) opens the door to those<br />
that want to bring such a balance back to new drug scaffold identification. With current analytical<br />
and separations technologies and advanced high throughput biological assays, the past perceived<br />
46
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
barriers to natural product screening should now be minimized. Controversy over ownership rights<br />
related to natural products identified in a native country is a separate topic, but one that should not<br />
preclude development <strong>of</strong> innovative new therapies for diseases in need. It can also present an<br />
opportunity for these countries to benefit from their natural riches.<br />
Several prime examples <strong>of</strong> extremely pr<strong>of</strong>itable and efficacious drugs that directly resulted from<br />
natural product leads (other than antibiotics and cancer agents) will be highlighted. A detailed case<br />
example <strong>of</strong> clinical compounds that were developed in our labs from a natural product lead will<br />
then be presented, as described below:<br />
The venom from the viper snake Echis carinatus produces dangerous systemic symptoms, with<br />
hemorrhage and coagulation defects being the most striking. These observations ultimately led to<br />
the isolation <strong>of</strong> the venom protein echistatin, and its subsequent clinical use as an anticoagulant.<br />
Isolated proteins from similar snake venoms, also with anti-coagulant activity, revealed a consensus<br />
RGD-X peptide sequence common to all <strong>of</strong> these venom proteins. It was further determined that<br />
this RGD-X sequence interacts with the integrin aIIbb3 (IIb/IIIa), a cell surface protein up-regulated<br />
on activated platelets, and thus interfering with platelet aggregation and clotting. Additional work<br />
demonstrated that the RGD-X peptide sequence itself was an antagonists <strong>of</strong> the IIb/IIIa receptor.<br />
Medicinal chemistry and in vitro and in vivo pharmacology efforts to transform this natural product<br />
lead into long acting, orally available, small molecule anti-platelet clinical compounds will be<br />
discussed. Additionally, and capitalizing on the work leading to these clinical anti-coagulant<br />
compounds, detailed drug development research leading to the discovery <strong>of</strong> potent, orally available<br />
clinical compounds as antagonists <strong>of</strong> the avb3 integrin receptor in my lab will also be shown. This<br />
will include the strategies employed that led to these avb3 antagonists which were designed to be<br />
selective against IIb/IIIa. These avb3 antagonists have potential utility in several therapeutic areas,<br />
such as oncology, osteoporosis, virology and angiogenesis.<br />
47
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[PL 14] Application <strong>of</strong> UV-VIS Spectroscopy to Evaluate Quality <strong>of</strong> Medicinal<br />
and Edible Oils<br />
Ermias Dagne * , Yehualashet Belete, Hanna Kassaye and Yadessa Melaku<br />
African Laboratory for Natural Products (ALNAP), Department <strong>of</strong> Chemistry, Addis Ababa University, P.O. Box 30270,<br />
Addis Ababa, Ethiopia. * E-mail: edagne@gmail.com<br />
Keywords: Nigella sativa, Brassica carinata, Sesamum indicum<br />
Introduction<br />
W<br />
e report here simplified methods for the isolation and quantitative determination <strong>of</strong><br />
thymoquinone (TQ), the main bioactive substance <strong>of</strong> the medicinal oil obtained from Nigella<br />
sativa, commonly known as black cumin or black seed. We also report a practical method <strong>of</strong><br />
determining the extent <strong>of</strong> adulteration <strong>of</strong> the edible oil <strong>of</strong> Sesame indica by the cheaper and less<br />
desirable edible oil Brassica carinata.<br />
In folk medicine <strong>of</strong> many countries black cumin seeds and its expressed oil are used as medicines.<br />
Biological activities <strong>of</strong> black cumin corroborated by scientific studies include: analgesic and antinflammatory<br />
(Hajhashemi, et al., 2004), antifungal (Khan, et al., 2003), antiasthmatic (Boskabady,<br />
et al., 2010), antiallergic (Kalus, et al., 2003) etc.<br />
Sesame (Sesamum indicum L.) is an ancient oilseed crop comprising <strong>of</strong> 50 60% oil (Arslan, et al.,<br />
2007). It is thought to have originated in Africa, although today India and China are the leading<br />
producers <strong>of</strong> sesame. In Ethiopia, three varieties are known: Humera, Gonder and Wellega. The oil<br />
extracted from sesame seeds is used mainly for cooking and in the production <strong>of</strong> margarine.<br />
Despite sesame oil's high proportion <strong>of</strong> polyunsaturated fatty acids, it is least prone, among cooking<br />
oils to turn rancid. This is believed to be due to the presence <strong>of</strong> endogenous antioxidants such as<br />
sesamol, sesamolin, sesamin etc.<br />
Brassica carinata, although now widely cultivated in different parts <strong>of</strong> the world, its origin is from<br />
the Ethiopian highlands, where its cultivation goes back to 4000 years B.C. In Amharic its leaves and<br />
seeds are known as Yabesha Gomen and Yegomen Zer respectively. The leaves are eaten cooked as<br />
vegetable whereas the seeds are used to oil the pottery baking pan (Mitad) <strong>of</strong> Injera.<br />
Materials and Methods<br />
NSPO (Nigella sativa pressed oil) was obtained by pressing black seeds or by purchasing oil from<br />
local commercial producers. Thymoquinone was isolated from powdered seeds <strong>of</strong> black cumin (10<br />
g) by first soaking in 80% aq. MeOH (70 ml), on a shaker for 4 h and filtering. Water was added to<br />
the filtrate to make the solution 50% aq. MeOH. This was then extracted twice using 50 ml CHCl3.<br />
The lower organic layer was separated, dried with anhyd Na2SO4, and concd to give dark brown<br />
gummy extract, that was subjected to CC on silica gel. Elution was carried out using hexane/CH2Cl2<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
(7:3), monitoring the separation <strong>of</strong> TQ by TLC (1:1, hexane/ CH2Cl2), which yielded pure TQ,<br />
confirmed by its 1 H, C-13, DEPT-135 NMR and UV spectra. TQ reference solutions were prepared<br />
(10 mg/10 ml).<br />
Relative comparison <strong>of</strong> TQ levels in different NSPO: Each sample <strong>of</strong> NSPO (10 mg) was dissolved in<br />
Solvent 1 [10 ml hexane: ethanol (1:9)]. For UV-Vis measuremnet 0.1 ml aliquotes from each<br />
sample was taken and diluted to 10 ml to give 0.01 mg/ml.<br />
Monitoring transformation <strong>of</strong> TQ in sunlight by UV-Vis: NSPO (200 mg) was placed in sunlight and<br />
about 10 mg was withdrawn after every 15 min, which was then dissolved in Solvent 1 and UV-Vis<br />
measured.<br />
Qualitative detection B. carinata oil: 5 mg and 20 mg <strong>of</strong> pure sesame oil were separately dissolved<br />
in 1 ml <strong>of</strong> Solvent 1 and their UV-Vis measured. Same procdure was repeated for B. carinata oil.<br />
Quantitative determination <strong>of</strong> B. carinata oil: Stock solutions <strong>of</strong> sesame (A) and B. carinata (B) oils<br />
were prepared by dissolving each time 100 mg <strong>of</strong> oil in 20 ml <strong>of</strong> Solvent 1. The volume ratio <strong>of</strong> A:B<br />
in each 5 mg was as follows: 5.0:0, 4.5:0.5, 4:1, 3.5:1.5, 3:2, 2.5:2.5, 2:3, 1.5:3.5, 1:4, 0.5:4.5, and<br />
0:5. In each case absorbance was measured at 287 nm.<br />
Instruments: 1 H and 13 C NMR spectral measurements were done on Bruker ACQ 400 AVANCE<br />
Spectrometer operating at 400 MHZ; UV-Vis on T 60 U spectrophotometer (PG instruments, UK)<br />
equipped with deuterium and tungsten lamps. The running parameters are controlled by UV-win<br />
s<strong>of</strong>tware.<br />
Results and Discussion<br />
It is well known that the substance which is most responsible for the diverse biological activity <strong>of</strong><br />
black cumin seeds and its pressed oil is thymoquinone (TQ).<br />
O<br />
CH 3<br />
O<br />
H 3C CH 3<br />
CH 3<br />
H 3C CH 3<br />
OH<br />
49<br />
H 3C<br />
H 3C<br />
O<br />
O<br />
O<br />
O<br />
CH 3<br />
Thymoquinone (TQ) Thymol Dithymoquinone (DTQ)<br />
The potential <strong>of</strong> using UV-Vis measurements for the detection and relative comparison <strong>of</strong> TQ levels<br />
in different oils is evident. In line with this, the UV-Vis spectra <strong>of</strong> different concentrations <strong>of</strong><br />
reference TQ (0.002, 0.004, 0.006 & 0.008 mg/ml), generated in ethanol ( max: 253 nm), are shown<br />
in Fig. 1. A similar linear curve was obtained when the spectra were generated using 0.1, 0.2, 0.3<br />
and 0.4 mg/ml <strong>of</strong> freshly prepared TQ dissolved in hexane/EtOH (1:9)/Solvent 1 (Fig. 2). Moreover,<br />
CH 3
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
the UV-Vis spectrum <strong>of</strong> NSPO after exposure to direct sunlight for 0 (C1), 30 (C2) and 45 (C3)<br />
minutes was obtained (Fig. 3). The result indicated that there is a gradual decline <strong>of</strong> the starting TQ<br />
(C1) and appearance <strong>of</strong> D1 and D2 peaks most likely due to its conversion products DTQ and<br />
thymol.<br />
Fig. 1: UV-Vis spectrum <strong>of</strong> different concentrations <strong>of</strong> TQ Fig. 2: UV-Vis spectrum <strong>of</strong> different NSPO<br />
samples<br />
C2<br />
C1<br />
C3<br />
D2<br />
D1<br />
Fig. 3: UV-Vis spectrum <strong>of</strong> TQ solutions after exposure to sunlight for 0 (C1), 30 (C2) and 45 (C3) minutes.<br />
Peaks D1 and D2 may be due to formation <strong>of</strong> DTQ and thymol.<br />
Sesame oil is widely believed to be adulterated by Brassica carinata oil. We attempt here to <strong>of</strong>fer a<br />
simple and quick method to detect this adulteration. The UV-Vis spectra <strong>of</strong> the two oil samples<br />
were obtained at 5 mg/ml (Fig. 4) and 20 mg/ml (Fig. 5) concentrations <strong>of</strong> the two pure oil samples.<br />
As shown in Figs. 4 and 5 their UV spectra are clearly different enough to enable one to distinguish<br />
the two oils. The spectrum <strong>of</strong> B. carinata (A) revealed three bands in the region between 400 - 500<br />
nm, insignificant in the spectrum <strong>of</strong> sesame oil (Fig. 5). On the other hand sesame oil displayed a<br />
typical band (B) with max absorption at 287 nm (Fig. 4). Therefore, these bands were used to<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
qualitatively distinguish between the two oil samples since addition <strong>of</strong> small amount <strong>of</strong> B. carinata<br />
to sesame oil changed the form <strong>of</strong> the spectrum <strong>of</strong> sesame oil.<br />
B<br />
A<br />
Fig. 4: Overlayed UV-Vis spectrum <strong>of</strong> oils <strong>of</strong> Fig. 5: Overlayed UV-Vis spectrum <strong>of</strong> oils <strong>of</strong><br />
B. carinata (A) and sesame (B), each 5 mg/ml B. carinata (A) and sesame (B), each 20 mg/ml<br />
In order to estimate sesame oil adulteration with B. carinata, different samples were prepared by<br />
mixing reference sesame oil with different volume ratios (10, 20, 30, 40, 50, 60, 80, and 90 %) <strong>of</strong> B.<br />
carinata oil. The absorbance for all samples including the pure and the blended oils were measured<br />
in triplicates. As observed from the spectrum, when the amount <strong>of</strong> B. carinata oil added to sesame<br />
oil increased, the absorbance <strong>of</strong> the blend at 287 nm decreased linearly while on the other hand<br />
the absorbance in the region 400 - 500 nm increased (Fig. 6).<br />
reference sesame oil<br />
51<br />
A<br />
reference B. carinata oil<br />
Fig. 6: UV-Vis spectrum <strong>of</strong> oils <strong>of</strong> B. carinata and sesame in different concentrations<br />
References<br />
1. Arslan, C., Uzun, B., Ulger, S., Cag�rgan, M., I. (2007); J. Am. Oil Chem. Soc. 84, 917-920<br />
2. Boskabady, M.H., Mohsenpoor, N., Takaloo, L. (2010); Phytomed. 17, 707-713<br />
3. Hajhashemi, V., Ghannadi, A., Jafarabadi, H. (2004); Phytother. Res. 18, 195-199<br />
4. Hunghton, P.J., Zarka, R., de las Heras, B., Hoult, J.R.S. (1995) Planta Med. 61, 33-36<br />
5. Kalus, U., Pruss, A., Bystron, J., Jurecka, M., Smekalova, A., Lichius, J.J., Kiesewetter, H. (2003); Phytother. Res. 17,<br />
1209-1214<br />
6. Khan, M.A.U., Ashfaq, M.K., Zuberi, H.S., Mahmood, M.S., Gilani, A.H. (2003); Phytother. Res. 17, 183-186.<br />
B
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[PL 15] Towards Gaining Recognition as an African Centre <strong>of</strong> Excellence in<br />
Applied Nanomedicine Research and Training for Poverty Related Diseases Focus<br />
on the DST/CSIR Nanomedicine Platform<br />
Hulda Swai, Lonji Kalombo, Lebogang Katata, Rose Hayeshi, Yolandy Lemmer, Philip Labuschagne,<br />
Paula Melariri, and Belle Nyamboli<br />
DST/CSIR nanomedicine platform Materials Science and Manufacturing<br />
Council for Scientific and Industrial Research<br />
PO Box 395, Pretoria, 0001, South Africa<br />
Hswai@csir.co.za Tel: +27 12 841 2366<br />
Keywords: Nanomedicine, nanoparticles, Tuberculosis, HIV/Aids, malaria, drug delivery, bioavailability, centre <strong>of</strong><br />
excellence<br />
Introduction<br />
S<br />
ub-Saharan Africa bears the brunt <strong>of</strong> Poverty Related Diseases (PRDs) such as Tuberculosis and<br />
malaria. Current therapies against PRDs are inadequate and warrant a quantum leap in drug<br />
development approaches. Nanomedicine is a rapidly advancing area <strong>of</strong> biomedical research with<br />
great potential to radically improve health, by enhancing shortfalls such as poor drug bioavailability,<br />
safety, efficacy, stability, and resistance, <strong>of</strong> new and existing therapeutic agents, used against<br />
diseases <strong>of</strong> poverty like TB and malaria.<br />
Technical Progress<br />
The DST/CSIR nanomedicine platform has made significant advances in TB drug delivery, and<br />
embarked on nanomedicine for HIV/Aids and malaria. We have successfully nanoencapsulated all<br />
four first line anti-TB drugs, in polymeric nanoparticles. In vitro release assays showed the drugs<br />
were released sustainably for up to 6 days. Intracellular drug delivery studies in two human cell<br />
lines demonstrated that the particles are taken up by the cells and delivered from the phagosomes<br />
into the cytoplasm. We also illustrated that the bacterial growth index in THP-1 cells treated with<br />
encapsulated rifampicin was reduced significantly, compared to cells treated with free rifampicin.<br />
Extracellular bacteria were also killed by the encapsulated drug over a period <strong>of</strong> time. Drug release<br />
was observed in vivo over a period <strong>of</strong> seven days. Further characterisation <strong>of</strong> the particles revealed<br />
that the particles did not elicit any inflammatory response when orally administered to both TB<br />
challenged and unchallenged mice. Preclinical studies on TB infected mice demonstrated that the<br />
encapsulated drugs, administered once weekly, over a period <strong>of</strong> 6 weeks, showed comparative<br />
efficacy against the TB bacterium, when compared to the free drugs that were administered once<br />
daily. Furthermore, we actively targeted TB infected macrophages with nanoparticles that are<br />
functionalised with aptamers against the target protein, and noted that intracellular delivery and<br />
slow release <strong>of</strong> the drugs is feasible.<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Focus on the Centre <strong>of</strong> Excellence<br />
The DST/CSIR nanomedicine platform is in a unique position in Africa; having built a substantial<br />
knowledge base in human capital, equipment/facilities and infrastructure, and is pioneering<br />
nanomedicine-based drug delivery research for PRDs. Given this advantage, the platform sees an<br />
urgent responsibility to advance nanomedicine research in Africa, while simultaneously generating<br />
highly qualified human capacity, in order to impact meaningfully at not only continental but also at<br />
a global level. In line with this goal, the platform is growing towards recognition as an African<br />
Centre <strong>of</strong> Excellence (CoE) in Applied Nanomedicine Research and Training . The proposed CoE will<br />
seek to deliver nanomedicine as an alternative therapy against PRDs through sharing <strong>of</strong> resources,<br />
know-how and technologies, which will avoid wasteful duplication <strong>of</strong> efforts and allow the most<br />
efficient use <strong>of</strong> pre-existing structures.<br />
Also geared at building and transforming human capital in Africa, the proposed CoE will concentrate<br />
existing capacity and resources to facilitate collaboration across disciplines and across organisations<br />
on long term programmes and projects <strong>of</strong> direct relevance to PRD drug development needs and<br />
aspirations. The proposed CoE will <strong>of</strong>fer researchers a stimulating and dynamic research<br />
environment by providing: Guidance/Support through mentoring, providing expertise, standards,<br />
methods, tools and knowledge repositories; Shared learning through training such as sensitisation<br />
seminars, workshops, summer schools, lab/researcher exchange programs, sabbaticals; Monitoring<br />
and Evaluation through conferences, publications, patents, technology transfer; Governance<br />
through coordinating <strong>of</strong> activities to enable valuable delivery.<br />
Acknowledgements<br />
DST/CSIR nanomedicine platform team<br />
The Council for Scientific and Industrial Research (CSIR), Polymers and Composites<br />
The Department <strong>of</strong> Science and Technology, South Africa<br />
The National Research Foundation, South Africa<br />
The Bill and Melinda Gates Foundation<br />
All collaborators<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[PL 16] Natural Products from Plant Diversity and their Potential in<br />
Management <strong>of</strong> Neglected Diseases<br />
Jacob O Midiwo, Francis Machumi, Abiy Yenesew, Leonida Kerubo, Solomon Derese<br />
Department <strong>of</strong> Chemistry, University <strong>of</strong> Nairobi, P.O. Box 30197, Nairobi<br />
Introduction<br />
C<br />
onventionally, neglected diseases are considered as a group <strong>of</strong> 13 infectious diseases that are<br />
endemic in the low income populations in the tropical developing world. They can be classified<br />
as those caused by trypanosomal parasites, helminthes, bacteria and viruses. They cause death to<br />
an estimated 0.5- 1m people annually. Trypanosomal diseases are represented by Kala-azar or<br />
visceral leshmaniasis, African sleeping sickness (African trypanosomiasis) and Chaga s disease<br />
(American trypanosomiasis); the current drugs for these diseases are relatively toxic even though<br />
the disease is not that lethal. Helminth infections include schistosomiasis treated with the<br />
inexpensive praziquantel but which cannot stop re-infection; onchocerciasis (river blindness), on<br />
which anthelmintic treatment is being tried; dracunculiasis (guinea worm), which should have been<br />
eradicated in 2009; lymphatic filiriasis (elephantiasis), managed by anthelmintic treatments. The<br />
others are soil transmitted worms such as ascariasis (round worms), trichuriasis (whipworms) and<br />
hookworms which are really best controlled by good hygienic practices. Leprosy, trachoma, Buruli<br />
ulcer and cholera represent the prevalent bacterial problems. Viral infections are yellow and<br />
dengue fevers caused by flavivirus transmitted by Aedes aegyptii and Japanese encephilitis caused<br />
by a flavivirus transmitted by Culex tritaeniorhynchus; the viral infections can be controlled through<br />
vaccination (WHO, 2008).<br />
However the WHO Innovative and Intensified Disease management (WHO-IDM) group considers<br />
NTDs to be only: Buruli ulcer, Chaga s diseases, cholera, sleeping sickness and leshmaniasis. This is<br />
because they get less funding than the big three HIV/AIDS, malaria and tuberculosis. However<br />
other groups led by Drugs for Neglected Diseases Initiative (DNDi) organization do not agree and<br />
consider African trypanosomiasis, leshmaniasis, Chaga s disease, and malaria as NTDs. This list<br />
leaves out Buruli ulcer but includes malaria with the argument that the disease does not receive<br />
adequate funding relative to its perilous effects on society. Their argument is that neglected<br />
diseases should be conditions that do not get enough funding relative to their impact on society<br />
(DNDi, 2003); in the presentation, this definition is considered to be the correct one.<br />
Clearly there is African folklore (mostly, medicinal herbal concoctions) about management <strong>of</strong> these<br />
neglected diseases even though such information is mostly scattered and may have run out <strong>of</strong><br />
vogue at the advent <strong>of</strong> civilization . It would be pertinent to explore the potential <strong>of</strong> certain local<br />
herbs for the development <strong>of</strong> phytomedicines for local populations for the diseases. Such practices<br />
have been recorded in monographs such as Medicinal Plants <strong>of</strong> East Africa (MPEA) (Kokwaro 2009)<br />
and Kenya Trees and Shrubs (Beentje 1994).<br />
Helminthiasis<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Helminthiasis as a disease group covers many <strong>of</strong> the neglected diseases. More than five hundred<br />
East African plants are listed by Kokwaro in MPEA as emetics/ purgatives, intestinal worm infections<br />
alleviators for hookworm, roundworm, tapeworm, threadworm, general anthelmintic, bilharzia and<br />
filarial infections. A few <strong>of</strong> these plants have been studied in our laboratory. They are the<br />
Myrsinaceae which are mentioned in several categories <strong>of</strong> anthelminthiasis and Rumex<br />
(Midiwo,2002) species listed as laxatives which can also be used to expel helminths from bowels.<br />
We have looked at the phytochemistry <strong>of</strong> the Kenyan Myrsinaceae reasonably and we find that it<br />
corresponds with that already reported in the literature. They are harbingers <strong>of</strong> long alkyl side chain<br />
2, 5-dihydroxybenzoquinones. We managed to chemotaxonomically group them into two subfamilies,<br />
the Myrsinodae and the Maesodae using these markers which coincides with their<br />
morphological delimitation. The Myrsinodae, which includes the species Myrsine africana, Rapanea<br />
melanphloes, Embelia schimperi and E. keniensis are chemically typified by the existence <strong>of</strong><br />
embelin/ rapanone while the Maesodae represented monotypically by Maesa lanceolata in Kenya<br />
is typified by the existence <strong>of</strong> maesaquinone. We have isolated several other benzoquinones from<br />
Kenyan Myrsinaceae and reported them in the literature (Midiwo, 2002). Rumex species are widely<br />
used locally for control <strong>of</strong> intestinal helminthic conditions. There are five Rumex species in Kenya -<br />
Rumex abyssinicus, R. usambarense, R. bequaertii, R. ruwenzoriensis and R. crispus, according to<br />
their grouping in the Key to Species . The compounds that are found in high concentration are the<br />
common anthraquinones, emodin, physcion and chrysophanol along with the polyketide<br />
naphthalenic compound, nepodin, whose distribution is in accordance with the Key (Midiwo, 2002).<br />
The anthraquinones have been reported to have purgative effect; no doubt this is the mechanism<br />
by which they exert their traditional anthelminthic activity.<br />
Considering the high concentration <strong>of</strong> bioactive principles in these Myrsinaceae, Rumex species and<br />
similar anthelmintic plants, it is suggested that they could be pursued for formulation <strong>of</strong> cheap<br />
phytomedicines for use by the local populations. Some <strong>of</strong> the herbs, like the berries <strong>of</strong> the<br />
Myrsinaceae, are usually sold in market places to be added food as medicinal spices for the desired<br />
effects for intestinal worm expulsion; they can be developed as food supplements to control<br />
intestinal worm proliferation.<br />
Malaria<br />
Malaria is a serious disease whose progression may lead to death. Its symptoms <strong>of</strong> high fever, chills,<br />
weakened joints, and flu-like illness are however well recognized by people in malaria endemic<br />
areas such as Kenya. Illness and death from malaria are largely preventable. Malaria is caused by<br />
Plasmodium parasites which are transmitted by mosquito vector. The most common parasite in<br />
Kenya which is the most virulent is Plasmodium falciparum; the others are P. vivax, P. ovale and P.<br />
malariae. The main mosquito vector in Africa and which, unfortunately, is also the most efficient is<br />
Anopheles gambiae. There are 300-500 million malaria infections world wide every year. About 80%<br />
<strong>of</strong> these are in Africa leading to 1.75- 2 m deaths annually, mostly children under five years old.<br />
Worldwide 3000 children die everyday from malaria. Approximately 90% <strong>of</strong> malaria deaths are in<br />
Africa. Malaria constitutes 10% <strong>of</strong> Africa s total disease burden; 40% health expenditure and 30-<br />
50% <strong>of</strong> in-patient cases (WHO 2001). Total African cost estimate is in the range <strong>of</strong> US$12b annually.<br />
55
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
In Kenya, 22 million people are at risk, 70% <strong>of</strong> them are in rural areas. About 34,000 Kenyan<br />
children die every year from malaria compared to a total estimate <strong>of</strong> 42,000 people dead (DMS<br />
Kenya 2006).<br />
Malaria can be controlled using three approaches- drug therapy, vector eradication and use <strong>of</strong><br />
vaccines (in the future). Drug therapy has been beset by development <strong>of</strong> resistance in virtually all<br />
the drugs developed by synthetic method or obtained from nature; the latter ones tend to take a<br />
longer period to experience resistance by the microorganisms. Malaria has been usefully been<br />
controlled using herbs such as Artemisia annua (Francois, 1993) and Cinchona which gave rise to<br />
quinine. These natural compounds have been noticed not to succumb to resistance as fast as the<br />
synthetic ones such as chloroquin, mefloquin and sulfadoxin/pyrimethamin. So it is attractive to<br />
look at new plant sources for these drugs. We have looked at several examples <strong>of</strong> these in our<br />
laboratory including Erythrina abysinica (Abiy, 2004 ) and Polygonum senegalense (Midiwo 2007)<br />
among others (Abiy 2003, Vlodomir Samolyenko, 2009).<br />
Conclusion<br />
Medicinal plants are useful sources <strong>of</strong> anti-plasmodial compounds that can be packaged as new<br />
anti-malarial drugs. It is worth pursuing the concept that mixtures <strong>of</strong> such compounds from plants<br />
can be formulated together for synergistic activity in which case the effective concentrations can be<br />
significantly reduced. It is the belief that this shift in paradigm from single or small number<br />
compound therapies may in the future lead to development <strong>of</strong> more efficient and cheaper drugs<br />
which also circumvent resistance development.<br />
References<br />
Abiy, Y, Induli, M., Derese, S., Midiwo, J.O., Heydenreich, M., Peter, M. G., Akala, H., Wangui, J., Liyala, P., Waters, N.C..<br />
Anti-plasmodial flavonoids from stem bark <strong>of</strong> Erythrina abysinica. Phytochemistry 2004, 65: 3029-3032.<br />
Abiy Y., Solomon D., Midiwo J.O., Oketch-Rabah, H.A., Lisgarten, J., Palmer, R., Heydenreich, M., Peter, M. G., Akala, H.,<br />
Wangui, J., Liyala, P., Waters, N.C. Anti-plasmodial activities and X-ray crystal structures <strong>of</strong> rotenoids from Millettia<br />
usaramensis subspecies usaramensis. Phytochemistry 64: 773-779.<br />
Beentje, H. (1994);The Kenya trees, shrubs and lianas. National Museums <strong>of</strong> Kenya<br />
DMS Kenya 2006. Figures provided by Director <strong>of</strong> Medical Services <strong>of</strong> Kenya as reported in the local press.<br />
Drugs for Neglected Diseases initiative (DNDi), Wikipedia 2003.<br />
Kokwaro J.O. Medicinal Plants <strong>of</strong> East Africa, 2 nd edition. Kenya Literature Bureau 1993.<br />
Midiwo J.O. A. Yenesew, B.F. Juma, S. Derese, J.A. Ayoo, A. O. Aluoch and S. Guchu, , Bioactive compounds from some<br />
Kenyan ethnomedicinal plants: Myrsinaceae, Polygonaceae and Psiadia punctulata. Phytochemical Reviews 2002,1:<br />
311-323.<br />
Francois G et al, Antiplasmodial activities <strong>of</strong> sesquiterpene lactones and other compounds in the organic extract <strong>of</strong><br />
Artemisia annua. Planta Medica 1993,59 (7), pp A677-A678.<br />
Midiwo Jacob O., Fidilia M. Omoto, Abiy Yenesew, Hosea M Akala, Julia Wangui, Pamela Liyala, Christine Wasuna,<br />
Norman C. Waters, The first 9-hydroxyhomois<strong>of</strong>lavanone, and Antiplasmodial chalcones, from the aerial exudates<br />
<strong>of</strong> Polygonum senegalense. Arkivoc 2007, (ix) 21-27.<br />
Volodymyr Samoylenko, Melissa R. Jacob, Shabana I. Khan, Jianping Zhao, Babu L. Tekwani, Jacob O Midiwo, Larry A.<br />
Walker and Ilias Muhammad( 2009) Anti- microbial, Antiparasitic, and Cytotoxic Spermine Alkaloids from Albizia<br />
schimperiana. Natural Products Communications. 4 (6) 791-796.<br />
WHO Publication January 2008.<br />
WHO Report 2001 pp 144-145, 150-151.<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[PL 17] Changes in Plants Metabolites with Location <strong>of</strong> Growth and Agronomic<br />
Practices: Some Lessions from Black Tea Quality Studies<br />
P. Okinda Owuor<br />
Department <strong>of</strong> Chemistry, Maseno University, P.O. Box 333 40105, Maseno, Kenya.<br />
Key words: Tea, Camellia sinensis, geographical location, yield, quality parameters<br />
Introduction<br />
ea (Camellia sinensis (L.) is an intensively cultivated perennial crop in many regions varying<br />
from 49ºN, outer Carpathians to 30ºS, Natal, South Africa 1 and from altitudes ranging from sea<br />
level in Japan and Sri Lanka to above 2700m above mean sea level (amsl) in Olenguruone, Kenya<br />
and Gisovu, Rwanda 2 . Despite the large variations in geographical locations <strong>of</strong> production, the<br />
recommended processing technologies and agronomic inputs are largely the same. For example,<br />
agronomic recommendations from Kenya 3 T<br />
are used throughout tea growing areas in East and<br />
Central Africa. Tea growers also have a desire to for high yielding and good quality panting<br />
materials in the hope that whatever identified good material in a location would replicate the good<br />
attributes in new areas <strong>of</strong> production. This paper examines the role <strong>of</strong> geographical location <strong>of</strong><br />
production on black tea yields, processing, and overall quality.<br />
Material and Methods<br />
In one trial 2 , clones TRFK 6/8 and TRFCA SFS 150 grown in at the Tea Research Foundation <strong>of</strong> Central<br />
Africa (TRFCA), Mulanje, Malawi, (altitude 650 m above mean sea level (amsl), latitude 16 o 05' S,<br />
longitude 35 o 37' E) and Tea Research Foundation <strong>of</strong> Kenya (TRFK), Kericho, Kenya, (altitude 2180 m<br />
amsl, latitude 0 o 22' S, longitude 35 o 21' E) for changes in quality parameters with fermentation time.<br />
The plants were grown under recommended agronomic practices. The cultivars were plucked both at<br />
the TRFCA and TRFK and processed by miniature CTC method under similar conditions at the<br />
respective institutions. Fermentation was varied at 30, 50, 70, 90 and 110 minutes at 28-30 o C before<br />
firing. The processing was done in replicate. The unsorted black teas were subjected to chemical<br />
analysis. The total theaflavins were analysed by the Flavognost method 4 while thearubigins,<br />
brightness and total colour were determined by the method <strong>of</strong> Robert and Smith 5 while the individual<br />
theaflavin ratios were determined using HPLC 6,7 as explained previously 8 . The astringency <strong>of</strong> the black<br />
teas was estimated using the modified theaflavins digallate equivalent factor 9 . Simultaneous steam<br />
distillation-extraction 10 was used to extract the volatile flavour compounds (VFC) with cumene as an<br />
internal standard. The dried (anhydrous Na2SO4), ether-VFC mixture was analysed using<br />
chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) 11 .<br />
In a second trial 12 20 widely cultivated (commercial) genotypes <strong>of</strong> tea were planted in Kangaita Tea<br />
Farm (latitude 0 o 30 S, longitude 37 o 16 E, altitude 2100 m amsl), TRFK, and Kipkebe Estate, Sotik<br />
(latitude 0 o 41 S longitude 35 o 5 E, altitude 1800 m amsl) were evaluated. At each site the plots<br />
were arranged in a randomised complete block design with three replicates 12 . Nitrogenous fertiliser<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
(NPKS 25:5:5:5) was applied at a single dose <strong>of</strong> 300 kg N ha _1 year _1 . Plucking was done at 10 14<br />
day intervals, depending on leaf availability. The plants were under uniform management and<br />
agronomic practices. One kilogram <strong>of</strong> leaf was plucked from each plot and processed by the<br />
miniature CTC method 2, 8 . The unsorted black teas were subjected to plain tea quality parameters<br />
chemical analysis 4, 5 and sensory evaluations.<br />
Another set <strong>of</strong> trials were laid out in five main tea growing regions <strong>of</strong> Kenya at Karirana (altitude<br />
2260 m amsl, latitude 1 0 6 S, longitude 36 0 39 E), TRFK, Changoi (altitude 1860 m amsl, latitude 0 0<br />
29 S, longitude 35 0 14 E), Sotik Highlands (altitude 1800 m amsl, latitude 0 0 35 S, longitude 35 0 5 E)<br />
and Kipkebe. Clone BBK 35 p uniformly managed and with known past cultivation history, were<br />
selected from each site. Fertilizer was applied at 0, 75, 150, 225 and 300 kg N ha -1 year -1 all in<br />
November, while plucking was done at 7, 14 and 21 days. The trials were laid out at each site as 5<br />
by 3 factorial design in randomized complete block arrangement. The data were analyzed as<br />
Randomised Complete Block in 5 by 3 factorial design at each site split for the five locations.<br />
Samples for miniature manufacture and fatty acids analyses were obtained when all plucking<br />
intervals coincided. Fatty acids 13 and plain black tea quality parameters 4, 5 were analysed according<br />
to published methods.<br />
Results and Discussion<br />
The optimal fermentation duration was reached much faster in Malawi tea leaves than tea leaves<br />
from Kenya 2 . Indeed, the quality parameters were lower in Malawi tea leaves than Kenyan tea<br />
leaves demonstrating that the plant metabolites responsible for making tea quality (polyphenols<br />
and volatile flavour compounds) and enzymes responsible for their transformation were not equal<br />
in the same clone produced in different regions. The individual theaflavins ratios in the same clone<br />
produced in different regions were not the same suggesting that the clones were not producing the<br />
flavan-3-ols in the same amounts and ratios. While black tea produced from Kenyan leaf was more<br />
aromatic, the same black tea from Malawi had high amounts <strong>of</strong> C6 alcohols and aldehydes which<br />
tend to lower black tea aroma quality. Thus production <strong>of</strong> the metabolites responsible for tea<br />
quality is influenced by the growing environment.<br />
The plain black tea quality parameters <strong>of</strong> the 20 clones varied with geographical area <strong>of</strong><br />
production 14 . The order <strong>of</strong> the variations and preferences <strong>of</strong> the clones also changed with location<br />
<strong>of</strong> production. However, some cultivars showed remarkable stability, with very little variations with<br />
geographical area <strong>of</strong> production. The results demonstrate need for assessing cultivars in new areas<br />
<strong>of</strong> intended cultivation before they are released to farmers for wide spread production, as it is not<br />
possible to predict the production rates and levels <strong>of</strong> the metabolites even in a single genotype<br />
when grown in different environment.<br />
Although the same clone was subjected to different rates <strong>of</strong> nitrogenous fertiliser and plucking<br />
intervals, the yields 15 , black tea quality parameters 15, 16 and fatty acids 17 levels changed with area <strong>of</strong><br />
production. These results demonstrate that same level <strong>of</strong> production or black tea quality cannot be<br />
obtained from the same cultivar when it is grown in different geographical regions even when<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
agronomic inputs are the same. The magnitudes and rates <strong>of</strong> changes in the individual parameters<br />
due to rates <strong>of</strong> nitrogenous fertiliser and plucking intervals changed with regions causing significant<br />
(P < 0.05) interactions effects. Thus the magnitude and rates <strong>of</strong> the changes in any parameter at<br />
one site cannot be used to predict responses at different locations.<br />
These results explain the general inability <strong>of</strong> tea growers to obtain same yields and/or quality <strong>of</strong><br />
black tea from same clone grown in different locations. There is therefore the need for<br />
development <strong>of</strong> region specific agronomic recommendations including cultivar selection to obtain<br />
high yields and quality. These results have further implications to phytochemical investigations.<br />
Levels <strong>of</strong> the metabolites detected in a plant from a given area may not be replicated in another<br />
area. Indeed in some cases, some metabolites detected in one area maybe absent altogether in<br />
another area. For, example nerolidol found in Kenya 2 and Longjing 18 tea has not been determined<br />
in Assam teas 11 .<br />
Acknowledgements<br />
The invitation by the organisers <strong>of</strong> the conference is gratefully acknowledged. I also thank Maseno<br />
University for granting me time <strong>of</strong>f to present this paper.<br />
References<br />
1 Shoubo, H. (1989); Meteorology <strong>of</strong> the tea plant in China. A review. Agriculture and Forestry Meteorology, 47, 19<br />
30.<br />
2 Owuor, P.O.; Obanda, M.; Nyirenda, H.E. & Mandala, W.L. (2008); Influence <strong>of</strong> region <strong>of</strong> production on clonal black<br />
tea chemical characteristics. Food Chemistry, 108, 363-271<br />
3 Othieno, C.O. (1988); Summary <strong>of</strong> recommendations and observations from TRFK. Tea, 9, 50-65.<br />
4 Hilton, P.J. Tea. (1973); In Encyclopedia <strong>of</strong> Industrial Chemical Analysis. Vol 18, Eds Snell, F.D. and Ettre, L.S., John<br />
Wiley, New York, USA, pp 453-516.<br />
5 Roberts, E.A.H. & Smith, R.F. (1963); Phenolic substances <strong>of</strong> manufactured tea. II, Spectroscopic evaluation <strong>of</strong> tea<br />
liquors. Journal <strong>of</strong> the Science <strong>of</strong> Food Agriculture, 14, 889-900<br />
6 Bailey, R.G.; McDowell, I. & Nurstein, H.E. (1990); Use <strong>of</strong> HPLC photodiode-array detector in a study <strong>of</strong> the nature <strong>of</strong><br />
black tea liquor. Journal <strong>of</strong> the Science <strong>of</strong> Food Agriculture, 52, 505-525<br />
7 Steinhaus, B. & Englehardt, U.H. (1989); Comparison <strong>of</strong> theaflavins Flavognost and HPLC analysis. Zeitschrift fur<br />
Lebensmittel-Unitersuchung und Forschchung, 188, 509-511.<br />
8 Owuor, P.O. & Obanda, M. (1997); The effects <strong>of</strong> some agronomic and processing practices and clone on the relative<br />
composition <strong>of</strong> the theaflavins in black tea. Food Science and Technology International, Tokyo, 3, 344-347.<br />
9 Owuor, P.O. & Obanda, M. (1995); Clonal variation in the individual theaflavins and their impact on astringency and<br />
sensory evaluation. Food Chemistry, 54, 273-277.<br />
10 Likens, S.T. & Nickerson, G.B. (1964); Detection <strong>of</strong> certain hop oil constituents in brewing products. Proceedings <strong>of</strong><br />
the American Society <strong>of</strong> Brewing Chemists, 5-13.<br />
11 Baruah, S.; Hazakira, M.; Mahanta, P.K.; Horita, H. & Murai, T. (1986); The effect <strong>of</strong> plucking intervals on the<br />
chemical constituents <strong>of</strong> CTC black teas. Agriculture and Biological Chemistry, 50, 1039-1041.<br />
12 Wachira, F.N.; Ng etich, W.; Omolo, J. & Mamati, G. (2002); Genotype x environment interactions for tea yields.<br />
Euphtica, 127, 289-296.<br />
13 Owuor, P.O.; Munavu, R.M. & Muritu, J.W. (1990); Plucking standards effects and the distribution <strong>of</strong> fatty acids in the<br />
tea (Camellia sinensis (L.O. Kuntze)) leaves. Food Chemistry, 37, 27-35.<br />
14 Owuor, P.O.; Wachira, F.N. & Ng etich, W.K. (2010); Influence <strong>of</strong> region <strong>of</strong> production on relative clonal plain tea<br />
quality parameters in Kenya, Food Chemistry, 119, 1168-1174.<br />
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15 Owuor, P. O.; Kamau, D. M. & Jondiko, E. O. (2009); Response <strong>of</strong> clonal plain black tea quality parameters and<br />
yields to geographical region <strong>of</strong> production and plucking frequencies. Food Chemistry, 115, 290-296.<br />
16 Owuor, P. O.; Kamau, D. M. & Jondiko, E. O. (2010); Responses <strong>of</strong> clonal tea to location <strong>of</strong> production and<br />
nitrogenous fertiliser rates. Journal <strong>of</strong> Food Agriculture and Environment, 8, 682-690.<br />
17 Okal, A.W. (2011); Influence <strong>of</strong> area <strong>of</strong> production, nitrogenous fertilizer rates and plucking intervals on the<br />
production <strong>of</strong> fatty acids in clonal tea (Camellia sinensis (L.O) Kuntze) leaves. MSc Thesis, Maseno University,<br />
18 Aisaka, H.; Kosuge, M.; Yamanishi, T. (1978); Comparison <strong>of</strong> the flavour <strong>of</strong> Chinese Kemmun Ceylon black tea.<br />
Agricultural and Biological Chemistry, 42, 2157-2159.<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[PL 18] Challenges <strong>of</strong> Isolation, Characterization and<br />
Pr<strong>of</strong>iling <strong>of</strong> African Medicinal Plants: Analytical Prospective <strong>of</strong><br />
Standardization and Quality Control Methods<br />
Vusumuzi Kalvin Ndhlovu 1 , Simiso Dube 1 and Namboole Moses Munkombwe 2 and Mathew Muzi<br />
Nindi 1<br />
1 Department <strong>of</strong> Chemistry, University <strong>of</strong> South Africa, Preller Street, Meckleneuk, City <strong>of</strong> Tshwane,<br />
0003 UNISA, RSA<br />
Nindimm@unisa.ac.za<br />
2 Department <strong>of</strong> Chemistry, University <strong>of</strong> Zambia, Lusaka, ZA<strong>MB</strong>IA<br />
Keywords: Harpagophytum procumbens, isolation, standards, pr<strong>of</strong>iling, quality control.<br />
P<br />
r<strong>of</strong>iling African medicinal plants is a challenge due to the lack <strong>of</strong> standards required for<br />
quantification. In this study a HPLC-DAD method is developed for quality control <strong>of</strong> Devil s Claw<br />
(Harpagophytum procumbens) products. Reference standards/compounds were isolated and<br />
purified using chromatographic methods. The standard compounds were used for pr<strong>of</strong>iling and<br />
quantification <strong>of</strong> Devil s Claw (Harpagophytum procumbens) species. Compounds such as<br />
harpagoside, acteoside, isoacteaoside, bioside and procumbide were isolated at high purity using<br />
chromatographic techniques. The purity and identification <strong>of</strong> the isolated compounds was<br />
determined using TLC, 1 H-NMR and HPLC.<br />
Introduction<br />
Harpagophytum procumbens is a plant found in dry and sandy regions <strong>of</strong> Namibia, Botswana, South<br />
Africa, Zambia and Zimbabwe [1]. Harpagophytum procumbens is commonly known as Devils Claw<br />
and has several vernacular names such as Sengaprile in Botswana. The root extracts <strong>of</strong><br />
Harpagophytum procumbens have been reported to be pharmacologically active. Clinical studies<br />
have shown that root extracts are effective in the treatment <strong>of</strong> degenerative rheumatoid arthritis,<br />
osteoarthritis and tendonitis, kidney inflammation and heart disease [2]. The tuber <strong>of</strong> this plants is<br />
commercially available and is sold on large quantities to European countries especially Germany [3].<br />
Currently there are no known quality assurance and standardization methods developed for Devil s<br />
Claw in supplying countries despite the fact that the importing countries require that the<br />
percentage <strong>of</strong> harpagoside should be at least 1 %. This work is an effort to develop suitable<br />
analytical procedures for quality control, standardization or pr<strong>of</strong>iling <strong>of</strong> Harpagophytum<br />
procumbens samples.<br />
Quantification and quality control studies require the availability <strong>of</strong> high purity standards. The<br />
major challenge in this work is that the reference standards for the analytical work for African<br />
traditional plants are either not available, limited or are very expensive in the market. For Devil s<br />
claw only one reference standard (harpagoside) is currently available in the market. Such a<br />
limitation necessitates the isolation <strong>of</strong> high purity standards (> 90 98 %.) from the plant. Thus the<br />
aim <strong>of</strong> this work is to isolate known compounds from the plant using chromatographic methods and<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
subsequently use the isolated compounds as reference standards for pr<strong>of</strong>iling Devil s Claw products<br />
using HPLC.<br />
Material and Methods<br />
The compounds were isolated from the Kalahari Devil s Claw tea packaged by Thusano Lefatsheng<br />
(Gaborone, Botswana). Pharma Tech Model 1000 High Speed Counter Chromatography (HSCCC),<br />
(Baltimore, Maryland USA) was used for chromatographic isolation <strong>of</strong> the tea. The isolated<br />
components were further cleaned-up by Sephadex LH-20 and Silica gel 60 (0.040-0.063) mm gravity<br />
column. In addition Dionex Ultimate 3000 HPLC (Germering, Germany) was used for semipreparative<br />
HPLC for the isolation and clean up <strong>of</strong> reference standards. Prep Xterra® MS C18 (3.5<br />
µm × 7.8 mm × 100 mm) column was used for semi-preparative HPLC work. Chlor<strong>of</strong>orm, ethyl<br />
acetate, n-butanol, methanol, dichloromethane and ethanol were <strong>of</strong> GPR grade and were used for<br />
the preparation <strong>of</strong> crude sample, gravity column and HSCCC separation. All the GPR grade solvents<br />
were distilled prior to use. In addition ultra high purity (UHP) water from a Milli-Q system<br />
(Millipore, Bedford, MA, USA) was used as part <strong>of</strong> the mobile phase. Silica GF254 Thin Layer<br />
Chromatography (TLC) plates from Merck (Darmstadt, Germany) were used for the isolated<br />
fractions. In addition Bruker Advance 300 MHz spectrometer (Karlsruhe, Germany) was used for 1 H<br />
proton structural elucidation. The identification was achieved by comparing <strong>of</strong> the obtained spectra<br />
with published spectra [4]. Devil s Claw (Harpagophytum procumbens) secondary roots (3.0 kg)<br />
were extracted using CH2Cl2/ MeOH (1:1 v/v) for 24 hrs followed by MeOH for 1 hr. The volume <strong>of</strong><br />
the extracts was reduced by evaporation before application to HSCCC and gravity column. The<br />
HSCCC solvent systems used in this study were prepared by mixing ethyl acetate n-butanol<br />
ethanol water (4:0.6:0.6:5, v/v). The upper organic phase was used as the mobile phase and the<br />
lower aqueous phase as the stationary phase. In gravity column, separations were achieved by<br />
using CHCl3/MeOH gradients saturated with H2O as the eluting solvent system. Further clean up<br />
was achieved on a column packed with Sephadex LH-20 using CHCl3/MeOH, 2:1 v/v as the eluting<br />
solvent.<br />
Result and Discussion<br />
A number <strong>of</strong> compounds are known to exist in Harpagophytum procumbens in appreciable<br />
quantities and these are 6-o-acetyl acteoside 1, iso-acteoside 2, bioside 3, acteoside 4, Harpagoside<br />
6, procumbens 9 and cinnamic acid 10 (Table 1). In this study 4 iridoid glycosides (5-6, 8-9), 3<br />
phenylethanoid 2-4 glycosides (2-4) and cinnamic acid 10 were isolated by means <strong>of</strong> HSCCC as well<br />
as column chromatography on Sephadex LH-20 and silica gel. The presence <strong>of</strong> compounds 1 and 7<br />
was detected in trace amounts on 1 H NMR spectra <strong>of</strong> other compounds.<br />
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Fig 1 Structures <strong>of</strong> Compounds isolated from Harpagophytum procumbens<br />
Table 1 Compounds isolated from Harpagophytum procumbens<br />
Reference compound Isolated Quantity (mg)<br />
Harpagoside 359<br />
Harpagide 67<br />
Procumbide 98<br />
8-0-p-coumaroylharpagide 50<br />
Acteoside 179<br />
Iso acteoside 194<br />
Bioside 4<br />
Cinnamic acid 10<br />
Preparative HPLC showed promising results for the isolation <strong>of</strong> known compounds at higher purity.<br />
Fig 2 shows a separation <strong>of</strong> the crude extract <strong>of</strong> Harpagophytum procumbens. From the<br />
chromatogram 16 peaks have been separated. Isolation <strong>of</strong> these peaks could result in an increased<br />
number <strong>of</strong> reference compounds needed for the pr<strong>of</strong>iling <strong>of</strong> the plant.<br />
Conclusions<br />
This study has shown that it is possible, although challenging, to isolate and purify compounds<br />
which can be used as reference standards. A HPLC-DAD method for the quality control <strong>of</strong> Devil s<br />
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Claw Harpagophytum procumbens products has been developed using the isolated reference<br />
standards.<br />
Fig 2. HPLC-DAD analysis <strong>of</strong> crude extract Prep Xterra® MS C18 3.5 µm × 7.8mm × 100 mm, CH3CN+0.1%<br />
HCOOH: 0.1% HCOOH, 20 min gradient, room temp<br />
Acknowledgements<br />
The authors would like to acknowledge SEANAC for the financial assistance <strong>of</strong> Mr Vusumuzi K.<br />
Ndhlovu through a six month student fellowship. We also acknowledge the Department <strong>of</strong><br />
Chemistry, University <strong>of</strong> Botswana for allowing the student to carry out some <strong>of</strong> the work in their<br />
laboratories. Finally, we are indebted to UNISA for funding this project.<br />
References<br />
1. Kristine M. Stewart, David Cole, (2005); The commercial harvest <strong>of</strong> Devil s claw (Harpagophytum spp.) in southern<br />
Africa: The devil s in the details, Journal <strong>of</strong> Ethnopharmacology 100 225 236<br />
2. Lanhers M.C, Fleurentin J, Mortier F, Vinche A, Younos C; Anti inflammatory and analgesic effects <strong>of</strong> an aqueous<br />
extract <strong>of</strong> Harpagophytum procumbens, Planta Medica, 58,117-123<br />
3. Bennett, B., (2006); Fair trade or foul: Using value chain analysis to understand power and governance on the<br />
South African Devil s claw industry, Natural Resources Institute.<br />
4. Namboole Moses Munkombwe, (2003); Acetylated phenolic glycosides from Harpagophytum procumbens,<br />
Phytochemistry 62(8) 1231-4<br />
5. Jin Qi, Ji-Jun Chen, Zhi-Hong Cheng, Jia-Hong Zhou, Bo-Yang Yu, Samuel X. Qiu, (2006); Iridoid glycosides from<br />
Harpagophytum procumbens D.C. (devil s claw), Phytochemistry 67 1372 1377<br />
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[PL 19] Exploiting the Chemistry <strong>of</strong> African Biodiversity in Pest Management:<br />
from Extraction <strong>of</strong> Plant chemicals to Expression in GMOs<br />
M.A. Birkett, 1 A.M. Hooper, 1 Z.R. Khan, 2 C.A.O. Midega 2 , J.A. Pickett 1 and B. Torto 2<br />
1 Department <strong>of</strong> Biological Chemistry, Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ, United Kingdom;<br />
john.pickett@rothamsted.ac.uk<br />
2 International Centre <strong>of</strong> Insect Physiology and Ecology (icipe), PO Box 30772, Nairobi, Kenya<br />
Key words: Africa, biodiversity, pest control, natural products, pheromones, semiochemicals, push-pull<br />
Introduction<br />
N<br />
atural products have long been viewed as providing leads for pest management, and<br />
particularly as insecticides. Indeed, many recently developed insecticides have followed<br />
natural product leads, such as the neonicotinoids, and some are indeed natural products<br />
themselves, for example spinosad. Perhaps the most well known are the synthetic pyrethroids,<br />
such as permethrin, cypermethrin and deltamethrin invented by Michael Elliott and his colleagues<br />
at Rothamsted from lead compounds, specifically pyrethrin 1, in the pyrethrum daisy,<br />
Chrysanthemum (= tanacetum) cinerariifolium (Asteraceae), currently grown commercially in Kenya<br />
for these natural insecticidal compounds.<br />
Here not only do we describe providing lead compounds and products directly for the international<br />
pest control market, but, by exploiting African biodiversity, compounds can be identified for<br />
exploitation locally in pest management. This can either be as plant products or their extracts,<br />
developed locally for feedstocks for pest control agents, or as pest management agents released<br />
from plants as wild-type cultivars or after genetic modification (GM). Although natural products do<br />
not represent intrinsically more benign agents than synthetic, by choosing those natural products<br />
that control pests, diseases and weeds, without the involvement <strong>of</strong> direct toxic modes <strong>of</strong> action<br />
then such intrinsically benign properties can be incorporated.<br />
Discussion<br />
Repellents against insect vectors <strong>of</strong> pathogens<br />
In global agricultural use, the neonicotinoids have now overtaken the pyrethroids, but for<br />
intervention against vectors <strong>of</strong> human pathogens, the pyrethroids still lead. In protecting against<br />
malaria, pyrethroids such as permethrin in long-lasting bednets act principally as toxicants, but<br />
there is evidence <strong>of</strong> repellency. The Sumitomo Chemical Company Ltd has introduced met<strong>of</strong>luthrin<br />
which acts aerially as a consequence <strong>of</strong> its very high vapour pressure, determined by a high level <strong>of</strong><br />
fluorine substitution, and which appears to show marked repellency. For natural products with non<br />
toxic repellency, there are many potential essential oil components, for example eucamalol from<br />
Eucalyptus camaldulensis (Myrtaceae), and a number <strong>of</strong> African herbs, e.g. Ocimum spp<br />
(Lamiaceae), yield essential oils with similarly useful properties. Although many, for various<br />
reasons, are not as useful repellents as the commercial gold standard DEET, the fact that they can<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
be produced locally could make these materials more useful. Also, African ethnobotany is able<br />
directly to <strong>of</strong>fer indicators <strong>of</strong> this type <strong>of</strong> biological activity and recently we showed that gum<br />
haggar from Commiphora holtziana (Burseraceae), growing in the arid regions <strong>of</strong> Kenya and to the<br />
East, contains volatile sesquiterpenes e.g. (R)-germacrene D, highly active against ticks and mites<br />
attacking cattle and camels, acting as repellents and which, although highly unstable, were<br />
protected by the gum matrix (Birkett et al. 2008). Recently, we and others have shown that DEET<br />
acts on the insect olfactory system in a similar way to the plant-derived volatiles (Pickett et al.,<br />
2008; Stanczyk et al. 2010) and this paves the way for more rapid electrophysiological screening <strong>of</strong><br />
putative repellents.<br />
Although many haematophagous insects feed on floral nectar, when foraging for blood meals<br />
haematophagous arthropods are mostly repelled by plant-derived volatile compounds. However,<br />
these can be overcome by the insect detecting host chemicals in spite <strong>of</strong> the presence <strong>of</strong> the plantderived<br />
signals. Exploiting mechanisms by which hosts are selected from within the host family, or<br />
even species via variation within the species, is likely to provide more durable and powerful<br />
repellents. Thus, although within the host family Bovidae, the water buck, Kobus defassa, releases<br />
volatile repellents against tsetse flies, which can mask the attractancy <strong>of</strong> hosts such as domestic<br />
cattle (Gikonyo et al. 2003). Even from cattle, and human subjects, repellents can also be identified<br />
from unattractive representatives <strong>of</strong> these animals, and can have potential commercial value as<br />
repellents in animal husbandry and against biting insects including the malaria mosquito Anopheles<br />
gambiae s.s. (Birkett et al. 2004, Logan et al. 2008, Logan et al. 2010).<br />
Antifeedants against crop pests<br />
Repellents have been largely unsuccessful as pest control agents in crop protection, but plant<br />
derived antifeedants, which interfere by non-toxic modes <strong>of</strong> action with normal feeding by<br />
herbivorous pests, have shown much greater promise. For these, East Africa has a range <strong>of</strong> plants<br />
yielding potentially valuable antifeedants, such as Ajuga spp. (Lamiaceae), particularly A. remota,<br />
yielding ajugarin 1, and the tree Warburgia ugandensis (Canellaaceae), yielding ugandensidial<br />
(Pickett et al. 1987). However, although these could be used locally and also form the basis <strong>of</strong><br />
exports to the north, such products have not yet been exploited.<br />
Direct production <strong>of</strong> crop protection agents by companion crops<br />
For generation directly by plants, it is possible to exploit the technique <strong>of</strong> companion cropping,<br />
referred to in kiSwahili as kilimo cha mchanganyiko , and the famous push-pull system, or vuta<br />
sukuma (pull-push), pioneered with the International Centre <strong>of</strong> Insect Physiology and Ecology<br />
(icipe) in Kenya www.push-pull.net, is an excellent example. Thus, by searching East African plant<br />
biodiversity for candidate repellent and attractant plants, a system has been developed for pushing<br />
away pests, and at the same time attracting beneficial enemies <strong>of</strong> these into the crop, so that the<br />
pests can be trapped in a peripherally grown trap crop (Khan et al. 1997; Hassanali et al. 2008).<br />
Each companion crop is used also as forage for cattle or dairy goats. Although knowledge intensive,<br />
once the technology has been acquired, this approach is extremely popular with farmers (Khan et<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
al. 2011). Of the intercrops one, which comprises forage legumes in the genus Desmodium<br />
(Fabaceae), also dramatically controls the African parasitic witchweeds in the genus Striga<br />
(Orobanchaceae), particularly S. hermonthica (Hassanali et al. 2008).<br />
Release from GM plants<br />
By identifying the chemistry <strong>of</strong> the companion crops that is responsible for repelling pests and<br />
attracting beneficial insects we have also created new targets for genetic modification, for example,<br />
increasing production <strong>of</strong> 4,8,12-trimethyl-(E,E)-trideca-1,3,7,11-tetraene which both repels pests<br />
and attracts parasitic wasps (Bruce et al. 2008; Matthes et al 2011). Insect-derived elicitors will<br />
provide a valuable tool by which to switch on defence based on this type <strong>of</strong> chemistry through<br />
breeding and also by GM approaches. Currently we are working on elicitors from the eggs <strong>of</strong> maize<br />
stem borers to identify elicitors that can have dramatic effects on the defence chemistry <strong>of</strong> African<br />
grasses (Bruce et al. 2010). The compounds from D. uncinatum that interfere with the<br />
development <strong>of</strong> the parasitic weeds Striga spp. comprise C-glycosylated flavonones and we have<br />
recently elucidated the mechanism by which these compounds are biosynthesised (Hamilton et al.<br />
2009). By heterologously expressing the C-glycosyltransferase enzymes involved into edible beans,<br />
we would create companion intercrop plants useful as human food but embodying the novel trait<br />
for controlling Striga spp. (Pickett et al. 2010; Khan et al. 2010).<br />
Acknowledgements<br />
This work was funded by the Gatsby Charitable Foundation (UK), Kilimo Trust (East Africa), the<br />
Rockefeller Foundation, and the Biovision Foundation (Switzerland). Rothamsted Research receives<br />
grant-aided support from the Biotechnology and Biological Sciences Research Council (BBSRC) and<br />
was funded through the BBSRC/DFID SARID initiative. The authors also acknowledge the assistance<br />
provided by icipe field staff, Ministry <strong>of</strong> Agriculture extension staff, and the farmers.<br />
References<br />
M.A. Birkett, N. Agelopoulos, K.-M.V. Jensen, J.B. Jespersen, J.A. Pickett, H.J. Prijs, G. Thomas, J.J. Trapman, L.J.<br />
Wadhams and C.M. Woodcock (2004); The role <strong>of</strong> volatile semiochemicals in mediating host location and selection<br />
by nuisance and disease-transmitting cattle flies. Medical and Veterinary Entomology 18, 313-322.<br />
M.A.Birkett, S. Al Abassi, T. Kröber, K. Chamberlain, A.M. Hooper, P.M. Guerin, J. Pettersson, J.A. Pickett, R. Slade, and L.J.<br />
Wadhams (2008); Antiectoparasitic activity <strong>of</strong> the gum resin, gum haggar, from the East African plant, Commiphora<br />
holtziana. Phytochemistry 69, 1710-1715.<br />
T.J.A. Bruce, M.C. Matthes, K. Chamberlain, C.M. Woodcock, A. Mohib, B. Webster. L.E. Smart, M.A. Birkett, J.A. Pickett<br />
and J.A. Napier (2008); cis-Jasmone induces Arabidopsis genes that affect the chemical ecology <strong>of</strong> multitrophic<br />
interactions with aphids and their parasitoids. Proceedings <strong>of</strong> the National Academy <strong>of</strong> Sciences USA 105, 4553-4558.<br />
T.J.A. Bruce, C.A.O. Midega, M.A. Birkett, J.A. Pickett and Z.R. Khan (2010); Is quality more important than quantity?<br />
Insect behavioural responses to changes in a volatile blend after stemborer oviposition on an African grass. Biol.<br />
Lett.6:314-317.<br />
N.K. Gikonyo, A. Hassanali, P.G.N. Njagi and R.K. Saini (2003); Responses <strong>of</strong> Glossina morsitans moristans to blends <strong>of</strong><br />
electroantennographically active compounds in the odors <strong>of</strong> its preferred (buffalo and ox) and non preferred<br />
(waterbuck) host. J. Chem. Ecol. 29:10:2331-2345.<br />
M.L. Hamilton, J.C. Caulfield, J.A. Pickett and A.M. Hooper (2009); C-Glucosylflavonoid biosynthesis from 2hydroxynaringenin<br />
by Desmodium uncinatum (Jacq.) (Fabaceae). Tetrahedron Letters 50: 5656-5659.<br />
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A. Hassanali, H. Herren, Z.R. Khan, J.A. Pickett and C.M. Woodcock (2008); Integrated pest management: the push-pull<br />
approach for controlling insect pests and weeds <strong>of</strong> cereals, and its potential for other agricultural systems including<br />
animal husbandry. Philosophical Transactions <strong>of</strong> the Royal Society London B 363, 611-621.<br />
Z.R. Khan, K. Ampong-Nyarko, P. Chiliswa, A. Hassanali, S. Kimani, W. Lwande, W.A. Overholt, J.A. Pickett, L.E. Smart, L.J.<br />
Wadhams and C.M. Woodcock (1997); Intercropping increases parasitism <strong>of</strong> pests. Nature 388, 631-632.<br />
Z.R. Khan, C.A.O. Midega, T.J.A. Bruce, A.M. Hooper and J.A. Pickett (2010); Exploiting phytochemicals for developing a<br />
push-pull crop protection strategy for cereal farmers in Africa. Journal <strong>of</strong> Experimental Botany 61:15 4185-4196.<br />
Z.R. Khan, C. Midega, J. Pittchar, J. Pickett and T Bruce (2011); Push-Pull technology: a conservation agriculture<br />
approach for integrated management <strong>of</strong> insect pests, weeds and soil health in Africa. UK government s Foresight<br />
Food and Farming Futures project. International Journal <strong>of</strong> Agricultural Sustainability 9:1: 162-170.<br />
J.G. Logan, M.A. Birkett, S.J. Clark, S. Powers, N.J. Seal, L.J. Wadhams, A.J. Mordue (Luntz) and J.A. Pickett (2008);<br />
Identification <strong>of</strong> human-derived volatile chemicals that interfere with attraction <strong>of</strong> Aedes aegypti mosquitoes.<br />
Journal <strong>of</strong> Chemical Ecology 34, 308-322.<br />
J.G.Logan, N.M. Stanczyk, A. Hassanali, J. Kerme, A.E.G. Santana, K.A.L. Ribeiro, J.A. Pickett and A.J. Mordue (Luntz)<br />
(2010); Arm-in-cage testing <strong>of</strong> natural human-derived mosquito repellents. Malaria Journal 9:239.<br />
M. Matthes, T. Bruce, K. Chamberlain, J. Pickett and J. Napier (2011); Emerging roles in plant defense for cis-jasmoneinduced<br />
cytochrome P450 CYP81D11. Plant Signaling & Behavior 6:4, 1-3.<br />
J.A. Pickett, G.W. Dawson, D.C. Griffiths, A. Hassanali, L.A. Merritt, A. Mudd, M.C. Smith, L.J. Wadhams, C.M. Woodcock<br />
and Z-n. Zhang (1987); Development <strong>of</strong> plant derived antifeedants for crop protection. In: Pesticide Science and<br />
Biotechnology, pp. 125 128. Editors R. Greenhalgh and T.R. Roberts. (Blackwell Scientific Publications).<br />
J.A. Pickett, M.A. Birkett and J.G. Logan (2008); DEET repels ORNery mosquitoes.<br />
Proceedings <strong>of</strong> the National Academy <strong>of</strong> Sciences USA 105, 36: 13195-13196.<br />
J.A. Pickett, M.L. Hamilton, A.M. Hooper, Z.R. Khan and C.A.O. Midega (2010); Companion cropping to manage parasitic<br />
plants. Annual Review <strong>of</strong> Phytopathology 48:161-177.<br />
N.M. Stanczyk, J.F.Y. Brookfield, R. Ignell, J.G. Logan, and L.M. Field (2010); Behavioral insensitivity to DEET in Aedes<br />
Aegypti is a genetically determined trait residing in changes in sensillum function. Proceedings <strong>of</strong> the National<br />
Academy <strong>of</strong> Sciences USA 107, 8575-8580.<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[PL 20] Development <strong>of</strong> Medicines from African Medicinal Plants: Experiences<br />
in West Africa<br />
Drissa DIALLO 1 . Adiaratou TOGOLA 1 Rokia SANOGO 1 Chiaka DIAKITE 1<br />
1. Department <strong>of</strong> Traditional Medicine BP 1746 Bamako.<br />
dri.diallo@yahoo.fr<br />
KEY WORDS: African pharmacopeia, Improved Traditional Medicines, Gastrosedal, Antianeamia, Dysenteral, Antitussif<br />
Introduction<br />
A<br />
frican flora contains many medicinal plants. Less than 10% <strong>of</strong> these plants have been<br />
investigated so far. Plants are sources <strong>of</strong> new molecules which can be developed as news<br />
medicines. From plants to medicines there are different steps. How Africans can improve the health<br />
state <strong>of</strong> their population by using natural products research?<br />
Material and Methods<br />
In West Africa many research institute are developing medicines from plants. Researches should<br />
use standardized methods and assure efficacy, safety and quality. Studies have been performed on<br />
different plants: Vernonia kostchyana (roots), Guiera senegalensis (leaves), Combretum micranthum<br />
(leaves), Euphorbia hirta (aerial part), Zanthoxylum zanthoxyloïdes (root bark), Crossopteryx<br />
febrifuga (fruits) Dissotis rotundifolia, Sclerocarya birrea, Cochlospermum tinctorium, and Cassia<br />
italica.<br />
Result and Discussion<br />
Vernonia kostchyana root powder has shown effect on gastric ulcer in Mali (Diallo et al., 1990).<br />
Coumarins, flavonoids, tannins, sugars, mucilage, sterols and triterpens were identified during the<br />
chemical screening (Diawara, 1989), more investigation by biological tests showed the activity on<br />
gastric ulcer <strong>of</strong> rats (Sanogo et al 1996); Saponins and polysaccharides were identified as<br />
responsible <strong>of</strong> the activity (Sanogo et al 1998; Sogn et al 2005). The Root powder <strong>of</strong> Vernonia<br />
kostchyana is being sold in Mali as Gastrosedal a phytomedicine used against gastric ulcer. Sirop<br />
Elooko is a phytomedicine prepared from the leaves <strong>of</strong> Guiera senegalensis and used against cough<br />
in Senegal . Dissotis rotundifolia is also used against cough in Guinea Conakry, a cough syrup has<br />
been prepared from this plant. After different researches, Euphorbia hirta was show to be<br />
efficacious against dysenteria, phytomedicines have then been prepare from it Dysenteral in Mali<br />
and sirup Amibex in Burkina Faso. The fruits <strong>of</strong> Crossopteryx febrifuga contain saponins, Balembo<br />
syrup has been made from their ethanol extract and being use in Mali against cough. Zanthoxylum<br />
zanthoxyloïdes is used in west Africa against sickle cell anemia in, Benin, Burkina Faso, Mali Nigeria<br />
and Togo. Chemical investigations showed that alkaloids, benzoic acid derivatives (phydroxybenzoic<br />
acid, 2-hydroxymethyl benzoic acid and vanillic acid); essential oil, tannins;<br />
flavonoïds and saponins are present in the roots bark (S<strong>of</strong>owora.E.A. Lloydia et al, 1971, Lamba S. et<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
al., 1990, Emerson et al., 2006, Pousset, 2007). Cassia italica is used against constipation, the<br />
phytomedicine Laxa cassia has been prepare from its leaves in Mali. Further studies are ongoing for<br />
plant against diabetes, hypertension and AIDS. The main aim <strong>of</strong> all these researches is to valorize<br />
traditional medicine and its products by making available to low income population quality<br />
medicines that are efficient safe and affordable.<br />
Acknowledgements<br />
Traditional Healers<br />
Partners (Ministry <strong>of</strong> Health, Université Of Oslo, Université de Marseille, Université de Messina,<br />
WHO, Antenna Technology, EU, DDC, HCNLS, Tradition et Médecine), DMT, DNS, UdB.<br />
References<br />
Cecilie Sogn Nergard,,* Tsukasa Matsumoto, Marit Inngjerdingen, Kari Inngjerdingen,Sanya<br />
Hokputsa, Stephen E. Harding, Terje E. Michaelsen, Drissa Diallo, Hiroaki Kiyohara Berit Smestad<br />
Paulsen and Haruki Yamada, Carbohydrate research, 2005, 340, 115-130<br />
Diallo, D, Koumare, A Koïta,N, Maïga A, Y, Cahier INRSP, 1990<br />
Diawara, D Thèse Pharmacie, 1989<br />
Emerson Queiroz, Anne-Emmanuelle Hay, Fatima Chaaib, Daphné van Diemen, Drissa Diallo, Kurt<br />
Hostettmann. Planta Med. 2006 72, 746-750.<br />
Lamba S.., Buch K., Lewis H., Planta Medica, 1990, 56, 681..<br />
Pousset J. CIPO, 2007<br />
Rokia Sanogo, Maria Paola Germano, Nunziatina de Tommasi, Cosimo Pizza and Rita Aquino ,<br />
Phytochemistry, 1998, 47, 73-78<br />
Sanogo, R.; Pasquale, R. d.; Germano, M. P.; Iauk, L.;Tommasi, N. d. Phytother. Res. 1996, 10, 169<br />
171.<br />
S<strong>of</strong>owora.E.A., Isaac-Sodeye W.A., Ogunkoya L.O., Lloydia, 38, 2, 169-171.<br />
70
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[PL 21] The pan-African Natural Product Library (p-ANPL): Giving Steam a<br />
Direction<br />
Andrae-Marobela, K. 1,2 , Ghislain, F. 3 , Dube, M. 1 , Maher, F. 1 , Ntumy, A.N. 1<br />
1 Department <strong>of</strong> Biological Sciences, University <strong>of</strong> Botswana, Gaborone, Botswana<br />
2 Center for Scientific Research, Indigenous Knowledge and Innovation (CesrIKi), Gaborone, Botswana<br />
3 Department <strong>of</strong> Chemistry, University <strong>of</strong> Botswana, Gaborone, Botswana<br />
frican scientists have in various ways shown immense creativity in efforts to take advantage <strong>of</strong><br />
natural product diversity on the African continent to identify lead compounds to use against<br />
locally relevant, but globally neglected diseases, including many parasitic infections.<br />
However, mainly due to a lack <strong>of</strong> resources many efforts remain uncoordinated and involve little<br />
cooperation between African natural product researchers. The exploitation <strong>of</strong> the African<br />
biodiversity for drug discovery has been largely confined to a model in which natural resources<br />
from Africa are extracted and evaluated in industrialized countries with minimal participation or<br />
direction from African collaborators. But to develop a more accurate global health perspective<br />
African scientists need to be in the forefront <strong>of</strong> drug research involving their unique resources in<br />
order to combat major health challenges effectively.<br />
To this end the pan-African Natural Product Library (p-ANPL) consortium was established in 2009,<br />
signed by representatives from seven African research institutions and a common declaration<br />
outlined the major goal <strong>of</strong> the consortium as bringing together biologically diverse natural<br />
compounds and extracts from the African continent and compounds originating from Africa that<br />
are held in other countries. This library will - in a first step - be subjected to screening for nonpeptide<br />
nematode G-protein coupled receptor agonists which are likely to act on specifically on<br />
multiple receptors preventing the development <strong>of</strong> anthelmintic resistance. However, other<br />
antibiotic or additional drug discovery programs can be included in the future.<br />
In this presentation we will discuss the current status <strong>of</strong> efforts to build p-ANPL, introduce our<br />
screening platforms and above all we would like to invite African natural product researchers to<br />
actively participate in the p-ANPL initiative.<br />
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SHORT LECTURES<br />
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[SL 1A] Phytochemical and Biological Studies on Aloe sinkatana Berger<br />
Gihan O.M. ELhassan 1 , Achyut Adhikari 2, Sammer Yousuf 2 , Hafiz Ur Rahman 3 , M. Ahmed Mesaik 3 ,<br />
Omer M. Abdalla 3 , Asaad Khalid 4 , Muhammad Iqbal Choudhary 2 and Sakina Yagi* 1<br />
1<br />
Department <strong>of</strong> Botany, Faculty <strong>of</strong> Science, University <strong>of</strong> Khartoum, Khartoum, Sudan.<br />
2<br />
H. E. J. Research Institute <strong>of</strong> Chemistry, International Center for Chemical and Biological Sciences, University <strong>of</strong><br />
Karachi, Karachi, 75270 Pakistan.<br />
3<br />
Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological<br />
Sciences, University <strong>of</strong> Karachi, Karachi-75270, Pakistan.<br />
4<br />
Medicinal and Aromatic Plants Research Institute, National Center for Research, Khartoum, Sudan.<br />
*Corresponding author: e-mail: sakinayagi@yahoo.com<br />
Kew wards: Aloe sinkatana, anthraquinones, antiglycation activity, T-cell proliferation, cytotoxicity.<br />
Introduction:<br />
A<br />
loe species (family Liliaceae) have enjoyed a wide folkloric usage and are also used in modern<br />
medicine and cosmetic products in many parts <strong>of</strong> the world. Carter (1994) and Lavranos (1995)<br />
reported the presence <strong>of</strong> 14 Aloe species in Sudan with 2 endemics namely; Aloe sinkatana Berger<br />
and A. macleayi Reynold. A. sinkatana is a shrub native to the Red Sea Hills in the Sudan. In folk<br />
medicine, the leaves and leaf exudates are used for the treatment <strong>of</strong> constipation, fever, skin<br />
disease and inflamed colon (MAPRI, 1997).<br />
Material and methods:<br />
The plant, A. sinkatana was collected from Arkawit, Sudan, in February 2009. Dry leaves (200g)<br />
were extracted with hexane, ethyl acetate and methanol respectively. Pure compounds were<br />
isolated from the ethyl acetate and methanol extracts using different chromatographic techniques.<br />
Their structures were identified on the basis <strong>of</strong> IR, UV, and 1D and 2D NMR and mass spectroscopic<br />
analysis. Evaluation <strong>of</strong> protein glycation was determined by -gluconolactone assay (Rahbar and<br />
Nadler, 1999). Antiglycation property <strong>of</strong> isolated compounds was evaluated by the method<br />
described by McPherson et al. (1988). Cell proliferation was evaluated by standard thymidine<br />
incorporation assay following a method reported by Nielsen et al. (1998). Cytotoxicity was<br />
performed according to method reported by Dariusz et al. (1993) with some modifications.<br />
Results and discussion<br />
From the leaves <strong>of</strong> A. sinkatana one new anthraquinone (2, 8 -dihydroxy -6-(hydroxymethyl)-1methoxyanthracene-9,10-dione)<br />
(1), aloe-emodin (2), aloin A & B, (3 & 4) chrysophanol (5),<br />
feralolide (6) microdontin (7), homoaloin (8) and -sitosterol (glycoside) (9) were isolated.<br />
Antiglycation activity <strong>of</strong> extracts and compounds 1 and 2 was carried out. The results obtained<br />
showed that the MeOH and EtOAc extracts as well as compound 1 showed inhibitory effect <strong>of</strong> early<br />
stage <strong>of</strong> protein glycation. Compound 1 also showed significant inhibitory effects against glucoseinduced<br />
advanced glycation end-products formation. The immunomodulatory and cytotoxic<br />
properties <strong>of</strong> some <strong>of</strong> the isolated compounds were also evaluated. Two <strong>of</strong> these compounds (2<br />
and 6) were found to exert significant suppressive effects on T-cell proliferation with an IC50 <strong>of</strong> 9.2<br />
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and 7.4 g/mL respectively and a suppressive IL-2 production activity with an IC50 <strong>of</strong> 1.1 and 1.9<br />
g/mL for 6 and 2 respectively. The cytotoxicity <strong>of</strong> these two compounds was evaluated using two<br />
cell lines and in vitro MTT assay, where none <strong>of</strong> them showed significant effect on the viability <strong>of</strong><br />
either <strong>of</strong> the two cell lines.<br />
Fig. 1: Structure <strong>of</strong> compounds 1 isolated from A. sinkatana.<br />
Acknowledgement<br />
Miss Gihan O. M. ELhassan gratefully acknowledges the financial support <strong>of</strong> TAWAS Organization.<br />
References<br />
Carter S. (1994); The Family Aloaceae. In: Polhill, R.M.(ed), Flora <strong>of</strong> Tropical East Africa. Balkema, Rotterdam and<br />
Brookfield.<br />
Dariusz S., Sarah J.S., Richard H.C., Michael B. (1993); An improved MTT assay. J. Immunol. Meth. 157: 203 207.<br />
Lavranos J. (1995). The Famiy Aloaceae. In: Thulin, M.(ed), Flora <strong>of</strong> Somalia. Royal Botanic Gardens, Kew.<br />
Mapri. (1997); Review <strong>of</strong> Trade in Widlife Medicinal Plant in Khartoum, Medicinal and Aromatic Plant Research<br />
Institute, Khartoum. Sudan.<br />
McPherson I.D., Shilton B. H., Walton P.J. (1988); Role <strong>of</strong> fructose in glycation and cross-linking <strong>of</strong> proteins.<br />
Biochemistry. 27: 1901 1907.<br />
Nielsen M., Gerwien J., Nielsen M., Geisler C., Ropke C., Svejgaard A., Odum N. (1998); MHC class II ligation induces<br />
CD58 (LFA-3)- mediated adhesion in human T-cells. Exp. Clin. Immunogenet. 15: 61 68.<br />
Rahbar S, Nadler J.L. (1999); A new rapid method to detect inhibition <strong>of</strong> Amadori product generated by<br />
deltagluconolactone. Clin. Chim. Acta. 287: 123 130.<br />
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[SL 1B] Application <strong>of</strong> in vitro Drug Metabolism and Disposition Studies to<br />
Assess Risk <strong>of</strong> Drug interactions with Sutherlandia frutescens Extracts<br />
DR Katerere 1 & C. Rewerts 2<br />
1 PROMEC Unit, SAMRC, Francie van Zijl Ave, Tygerberg, 7505, RSA. 2 Scynexis Inc, P.O. Box 12878, Research Triangle<br />
Park, N.C. 27709-2878, USA<br />
david.katerere@mrc.ac.za<br />
Key Words: Drug interactions, Sutherlandia frutescens, apparent permeability, CYP inhibition<br />
Introduction<br />
T<br />
he interactions <strong>of</strong> drugs with herbal drugs is an emerging area <strong>of</strong> interest in drug discovery<br />
studies and clinical use. Interactions may occur at luminal transporter level (e.g. pg-p), or by<br />
inhibition or induction <strong>of</strong> drug metabolizing enzymes (DME) impacting on the bioavailability <strong>of</strong> the<br />
victim drug(s). Most drug-herb interactions studies to date have used phytomedicines popular in<br />
industrialized countries but little attention has been paid to herbs which are used in Africa and Asia<br />
where co-medication with herbal medicine is rife (WHO, 2002; Okigbo et al, 2008). We investigated<br />
the potential for drug-herb interactions <strong>of</strong> extracts <strong>of</strong> ground leaves <strong>of</strong> Sutherlandia frutescens (socalled<br />
cancer-bush) which is widely marketed and used in Southern Africa for stress disorders,<br />
cancer, diabetes and more recently as an immunebooster in HIV / AIDS.<br />
Material and Methods<br />
The assay for Pg-p inhibition utilized the MDCK-MR1 cell line culture as a monolayer on Transwells ®<br />
(Corning) and dosed with sutherlandia extracts (SU) (6.8 mg / ml to 1.7 g /ml). Amprenavir was<br />
used as the permeability marker and GF120918 the standard Pg-p inhibitor. After pre-inculabtion<br />
the Transepithial Electrical Resistance (TEER) was measured, and then the concentration <strong>of</strong><br />
amprenavir analyzed by LC-MS in positive ion mode (API 4000 QTRAP, Applied Biosystems) postincubation.<br />
The effect <strong>of</strong> SU on cytochrome P450 was determined by the P450-Glo assay kit (Promega,<br />
Madison, WI, USA). Three controls were used viz. one with control membranes, one with vehicle<br />
(DMSO < 3.2%) and the third with standard inhibitors specific to the target enzyme i.e. -<br />
naphth<strong>of</strong>lavone (for 1A2), sulfaphenazole (2C9), troglitazone (2C19), sertraline (2B6) and<br />
ketoconazole (3A4). SU extract (25mM stock solution) was used to give final serial dilutions from<br />
100 to 1 µM. Luminescent activity was read using the Wallac® luminometer with Envision® s<strong>of</strong>tware<br />
(Perkin Elmer, Mass, USA). The data was processed by MS Excel and then transformed by GraphPad<br />
to give IC50 values.<br />
Result and Discussion<br />
Doses <strong>of</strong> SU >850 µg /ml reduced TEER readings implying possible damage to tight junctions.<br />
Amprenavir permeability increased in a dose-dependent manner in cells exposed to SU >53 µg /ml.<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
This suggests that the extract is inhibiting Pg-p and reducing amprenavir efflux. In the CYP inhibition<br />
assays, the IC50 values for sutherlandia extracts were above 10 µg / ml for all enzyme is<strong>of</strong>orms<br />
which implies a diminished potential for drug interactions at the tested concentrations.<br />
In conclusion this study suggests that persons self-medicating with sutherlandia may be prone to<br />
intoxication if they are also co-medicating with drugs which are pg-p substrates e.g. HIV protease<br />
inhibitors, digoxin, cyclosporine, macrolide antibiotics, anti-neoplastics and verapamil (Lee et al,<br />
1998; Huisman et al, 2002; Mills et al, 2005; Bauer et al, 2005). On the other hand sutherlandia<br />
does not cause clinically significant CYP enzyme inhibition.<br />
Acknowledgements<br />
This work was done as part <strong>of</strong> the NRF / Emory University / Scynexis Advanced Drug Discovery<br />
Programme for which DRK was a beneficiary<br />
References<br />
Bauer B, Hartz AM, Fricker G, Miller D. Modulation <strong>of</strong> p-Glycoprotein Transport Function at the Blood-Brain Barrier.<br />
Experimental Biology and Medicine 2005, 230:118-27<br />
Huisman, MT; Smit, JW; Crommentuyn, KML; Zelcer, N; Wiltshire, HR; Beijnen, JH; Schinkel, AH. Multidrug resistance<br />
protein 2 (MRP2) transports HIV protease inhibitors, and transport can be enhanced by other drugs. AIDS 2002, 16,<br />
2295-2301.<br />
Lee CGL., Gottesman MM., Cardarelli CO., Ramachandra M, Jeang K-T, Ambudkar S V., Pastan I, and Dey S. HIV-1<br />
Protease inhibitors are substrates for the MDR1 Multidrug Transporter. Biochemistry, 1998, 37, 3594 3601<br />
Mills E, Foster BC., van Heeswijk R, Phillips E, Wilson K, Leonard B, Kosuge K and Kanfer I. Impact <strong>of</strong> African herbal<br />
medicines on antiretroviral metabolism. AIDS 2005, 19, 95 97<br />
Okigbo, R N, Eme, U E. and Ogbogu S. Biodiversity and conservation <strong>of</strong> medicinal and aromatic plants in Africa.<br />
Biotechnology and Molecular Biology Reviews 2008, 3, 127-134.<br />
WHO. World Health Organization: 2002. Traditional Medicine Strategy 2002-2005.<br />
http://whqlibdoc.who.int/hq/2002/WHO_EDM_TRM_2002.1.pdf (accessed 8 April 2011)<br />
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[SL 2A] Chemical Constituents <strong>of</strong> East European Forest Species<br />
Moses K. Langat 1 , Dorota A. Nawrot 1 and Dulcie A. Mulholland 1<br />
1 Natural Products Research Group, Division <strong>of</strong> Chemical Sciences, University <strong>of</strong> Surrey, Guildford, GU2 7XH, United<br />
Kingdom. m.langat@surrey.ac.uk<br />
Key words: Pinus, Larix, Picea, Pinaceae, Salicaeae, diterpenoids, lignans and stilbenoids<br />
Introduction<br />
FORESTSPECS is an EC 7 th framework programme (FP7-KBBE-2008-2B-227239) consortium<br />
consisting <strong>of</strong> researchers from the UK (University <strong>of</strong> Surrey), Germany (Trifolio-M), Finland<br />
(Granula, VTT and University <strong>of</strong> Helsinki), Switzerland (FiBL) and Russia (FEFRI, NRIF and SPSMA).<br />
The overall aim <strong>of</strong> FORESTSPECS project is to utilize different types <strong>of</strong> wood residues from wood<br />
processing industry including Pinus sylvestris, Pinus pumila, Picea abies, P. Ajanensis Larix gmelinii,<br />
L. sibirica, L. sukaczewii, L. decidua, Abies nephrolepis (Pinaceae) and Populus tremula (Salicaceae)<br />
as raw materials to produce bioactive compounds and environmentally benign industrial chemicals<br />
and polymers as well as remediation chemicals.<br />
The Pinaceae (Pine) is an ancient and important family <strong>of</strong> trees which occur in the cooler parts <strong>of</strong><br />
the northern hemisphere, and mountains further south [Polunin, 1976]. The family consists <strong>of</strong><br />
about two hundred and sixty species worldwide, with about eighteen native to Europe [Polunin,<br />
1976]. The chemical composition <strong>of</strong> the wood varies from genus to genus and also between the<br />
species <strong>of</strong> the same genus. The compounds isolated from this family include, flavonoids, alkaloids,<br />
phenols, terpenoids, glucosides, phytosterols and lignans [Challen and Kucera, 1967; Slimestad,<br />
2003]. The Salicaceae or Willow family is widely distributed in the northern hemisphere and is a<br />
typical temperate family. This family is dispersed across the whole <strong>of</strong> Europe [Ding, 1995] and<br />
consists <strong>of</strong> over six hundred and fifty species worldwide with three genera, Chosenia Nakai, Populus<br />
L. and Salix L. [Ding, 1995]. Previous chemical investigations <strong>of</strong> the plants from the Salicaceae<br />
family have resulted in the isolation <strong>of</strong> a wide range <strong>of</strong> compounds including flavonoids, terpenoids,<br />
aromatic alcohols, glycerides and steroidal compounds [Hartonen, 2007].<br />
Chemical constituents <strong>of</strong> Larix gmelinii, L. sukaczewii, L. sibirica, Pinus sylvestris, P. Pumila and Picea<br />
abies will be presented.<br />
Materials and Methods<br />
The barks <strong>of</strong> ten east European forest species were collected from Arkhangelsk Territory, Northern<br />
Russia and Finland. The barks were air-dried and ground using a Glen Creston cross beater mill and<br />
extracted using a CEM Corporation, microwave assisted extraction system (MARS). Tests were<br />
performed to compare the performance <strong>of</strong> the MARS microwave extraction system against the two<br />
conventional extraction techniques, Soxhlet extraction and room temperature solvent extraction by<br />
shaking. Compounds were separated using silica gel (Merck Art. 9385)/sephadex (LH20100) packed<br />
column using different diameter-sized columns ranging from 2-6 cm depending on the amount <strong>of</strong><br />
sample available and thin layer chromatographic techniques (Merck Art. 9385). Final purification<br />
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was carried out using 1 cm diameter column, packed with silica gel (Merck Art. 9385) in<br />
dichloromethane, dichloromethane/ ethyl acetate or dichloromethane/ methanol systems.<br />
Structural determinations were done using 1D and 2D NMR, IR and CD spectroscopy and mass<br />
spectrometry.<br />
Results and discussion<br />
A comparative analysis on the performance <strong>of</strong> the MARS microwave extraction instrument against<br />
traditional extraction methods <strong>of</strong> Soxhlet extraction and room temperature solvent extraction using<br />
a shaker showed that the MARS microwave can be used instead <strong>of</strong> the conventional extraction<br />
techniques while retaining the yield and composition <strong>of</strong> the extract using considerably less solvent.<br />
Several compounds have been isolated and identified from Larix gmelinii, L. sukaczewii, L. sibirica,<br />
Pinus sylvestris, P. Pumila and Picea abies including the novel labdane diterpenoid, 6 ,13dihydroxy-14-oxo-8(17)-labdene<br />
(1) from L. gmelinii, the novel 4-acetyl-2,5-dihydroxy-3-methoxy-1methylbenzene<br />
(2) from L. sibirica, an unusual serratane triterpenoid, 3 -methoxyserrat-14-en-21one<br />
(3) and bornyl ferulate (4) (Figure 1) from P. pumila. Isolated pure compounds are being tested<br />
for the following activities: insect antifeedant activity (Helsinki University), herbicidal (Trifolio-M,<br />
Germany), fungicidal (FiBL, Switzerland), antiviral and antibacterial (Helsinki University) and antiinflammatory<br />
and antidiabetic (St. Petersburg State Medical Academy).<br />
OH<br />
1<br />
O<br />
OH<br />
HO<br />
O<br />
O<br />
2<br />
OH<br />
OM e<br />
MeO<br />
3<br />
Figure 1. Compounds from L. gmelinii, L. sibirica and P. pumila<br />
Acknowledgements<br />
The authors acknowledge funding from FP7-KBBE-2008-2B-227239. We thank FORESTSPECS<br />
partners, Claire Horner, Dani Shen, Dorota Nawrot, Christina Alexandrou, Qinmin Zhang, Judith<br />
Peters, Anuska Mann, Dan Drsicoll and other members <strong>of</strong> the Natural Products Group at the<br />
University <strong>of</strong> Surrey for their assistance.<br />
References<br />
Challen, S.B. and Kucera, M. (1967); Chromatographic studies on preservatives in the wood <strong>of</strong> some conifers, especially<br />
<strong>of</strong> the genus Abies, Picea and Pinus. Journal <strong>of</strong> Chromatography. 31, 345-353.<br />
Ding, T. (1995); Origin, divergence and geographical distribution <strong>of</strong> Salicaceae. Acta Botanica Yunnanica. 17, 277-290.<br />
Hartonen, K., Jevgeni, P., Sandberg, K., Bergelin, E., Nisula, L., Riekkola, M. L. (2007); Isolation <strong>of</strong> flavonoids from aspen<br />
knotwood by pressurized hot water extraction and comparison with other extraction techniques. Talanta. 74, 32<br />
38.<br />
Polunin, O. with drawings by Everard, B. (1976); Trees and Bushes <strong>of</strong> Europe . London, Oxford University Press,<br />
London.<br />
Slimestad, R. (2003); Flavonoids in buds and young needles <strong>of</strong> Picea, Pinus and Abies. Biochemical Systematics and<br />
Ecology. 31, 1247-1255.<br />
78<br />
O<br />
HO<br />
OCH3<br />
4<br />
O<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[SL 2B] In vitro Inhibitions <strong>of</strong> Tomato Fusarium Wilt by Zearalenone from a Soil<br />
Fungus<br />
Alice W. Njue a , Dan O. Otaye b , Peter K. Cheplogoi a and Josiah O. Omolo a<br />
a Chemistry Department, Egerton University, P. O. Box 536, 20115-Egerton, Kenya.<br />
b Biological Sciences Department, Egerton University, P. O. Box 536, 20115-Egerton, Kenya.<br />
To whom correspondence should be addressed: njuealice@yahoo.com<br />
KEYWORDS: Fusarium oxysporium f. sp. Lycosperci, Tomato, submerged cultures, column chromatography, zearalenone<br />
Introduction<br />
I<br />
n the course <strong>of</strong> continuous search for bioactive compounds from tropical forest which are a rich<br />
diversity <strong>of</strong> fungal genetic resources (Berdy, 2003), fungal strains were screened against Fusarium<br />
oxysporium f. sp. Lycosperci a disease <strong>of</strong> tomato in the farming fields. Synthetic chemical fungicides<br />
have been used for decades to control fungal diseases (Allen, 2004). However, the effectiveness <strong>of</strong><br />
fungicides is threatened by development <strong>of</strong> resistance by the pathogen and in some instances there<br />
are cases <strong>of</strong> efficacy concerns. In the last 2-3 decades efforts have been reported that involve<br />
control <strong>of</strong> Fusarium wilt using antagonistic fungi (Nel et al., 2006; Sabuquillo,et al., 2009). These<br />
anatagonistic interactions with other fungi typically have been classified as based on antibiosis,<br />
mycoparasitism and competition for nutrients (Hjeljord, and Tronsmo, 1998; Chet and Inbar, 1994).<br />
However, the spectrum <strong>of</strong> activity <strong>of</strong> microorganisms when they are used as biological control<br />
agents is usually narrower than that <strong>of</strong> synthetic pesticides (Janisiewicz, 1996; Copping, and Menn,<br />
2000)<br />
Methodology<br />
Aerial parts <strong>of</strong> infected tomato plants were collected from a greenhouse in Crops, Soils and<br />
Horticulture Department, Egerton University. The tomato fusarial causative agent was identified as<br />
F. oxysporum f. sp. Lycopersici from the cultures isolated from the infected plant and used as the<br />
test organism in the antifungal tests. The fungal strain was cultured in 35 replicates <strong>of</strong> liquid media<br />
which was prepared by dissolving 10.0g <strong>of</strong> molasses, 4.0g glucose, and 4.0g <strong>of</strong> yeast extract in 1.0L<br />
<strong>of</strong> tap water. The cultures were incubated at 25 o C and aerated by agitation for 21 days. The filtered<br />
fermentation broth gave 950 g <strong>of</strong> mycelium (Mex) and 30 liters <strong>of</strong> culture filtrate (Kex). Crude<br />
extract from culture filtrate were prepared using liquid-adsorption technique (Mitsubishi HP21<br />
DIAION) packed in a glass column. The column was eluted with acetone, followed in succession by<br />
methanol.<br />
Results and Discussion<br />
About 2.4 g <strong>of</strong> crude extract was obtained from acetone eluent while the methanol eluent afforded<br />
5.5 g <strong>of</strong> crude extract. However, only the acetone-eluted crude extract showed significant activity<br />
<strong>of</strong> 220.2 9.0 ppm as compared to the methanol-eluted crude extract, which had weak activity <strong>of</strong><br />
455 15.0 ppm, hence the latter, was not investigated. From the mycelium, 2.1 g <strong>of</strong> crude extract<br />
(Mex) was prepared using acetone, with an activity <strong>of</strong> 430 15.0 ppm, which was less active than<br />
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acetone-eluted crude. The crude extracts were subjected to silica gel chromatography and the<br />
purified compound established using two-dimensional experiments, COSY, HSQC and H<strong>MB</strong>C and<br />
zearalenone was found to be the main compound. The MIC for zearalenone was found to be<br />
550 10.5 ppm, which was less than the antifungal activity observed for the crude extracts. The<br />
diminution may be attributed concentration <strong>of</strong> zearalenone in the crude extract and partly to<br />
synergistic effects <strong>of</strong> other compounds present. The method <strong>of</strong> processing may be responsible for<br />
the reduction as well given that from the structure <strong>of</strong> zearalenone, stability factors are evident. The<br />
antifungal activity found for zearalenone is significant and can be <strong>of</strong> scientific value in the control<br />
Fusarium wilt in tomato farming.<br />
Acknowledgment<br />
Technical assistance was provided by Caleb Otieno and Nicholas Karubiu <strong>of</strong> Egerton University.<br />
Andy Foster <strong>of</strong> University <strong>of</strong> Kaiserslautern, Germany identified the producing organisms (Fusarium<br />
species) using molecular techniques.<br />
References<br />
Allen, T. W.; Enebak, S. A.; Carey, W. A. (2004); Evaluation <strong>of</strong> fungicides for control <strong>of</strong> species <strong>of</strong> Fusarium on long leaf<br />
pine seed Crop Protection, 23, 978-982.<br />
Berdy, J. (2003); Are actinomycetes exhausted as a source <strong>of</strong> secondary metabolites? In Biotechnologija (ISSN0234-<br />
2751) Prot. 9 th Int. Symp. On the Biology <strong>of</strong> Actinomycetes. Debanov VG. pp. 13-34.<br />
Chet, I. (1994); Inbar, J. Biological control <strong>of</strong> fungal pathogens. Applied Biochemistry and Biotechnology, 48, 37 43.<br />
Copping, L. G.; Menn, J. J. (2000); Biopesticides, their action application and efficacy. Pest Management Science, 56:<br />
651-676.<br />
Hjeljord, l.; Tronsmo, A. (1998); Trichoderma and Gliocladium in biological control: an overview. In: Kubicek, C. P.;<br />
Harman, G. E. (Eds.). Trichoderma and Gliocladium, vol. 2. Taylor and Francis, London, pp. 131-151,<br />
Janisiewicz, W. J. (1996); Ecological diversity, niche overlap, and coexistence <strong>of</strong> antagonists use in developing mixtures<br />
for biocontrol <strong>of</strong> postharvest diseases <strong>of</strong> apples. Phytopathology 86, 473 47<br />
Nel, B.; Stanberg, C.; Labuschagne, N.; Viljoen, A. (2006); Evaluation <strong>of</strong> fungicides and sterilants for potential application<br />
in management <strong>of</strong> Fusarium wilt. Crop Protection, 23, 1112-1114.<br />
Sabuquillo, P.; Sztejnberg, A.; De Cal, A.; Melgarejo, P. (2009); Relationship between number and type <strong>of</strong> adhesions <strong>of</strong><br />
Penicillium oxalicum conidia to tomato roots and biological control <strong>of</strong> tomato wilt. Biological Control, 48, 244 251.<br />
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[SL 3A] Anthocyanins from Selected Plant Species in Uganda<br />
Robert Byamukama a,* , George Ogweng a , Angella Mbabazi a , Irene Skaar b , Monica Jordheim b , Oyvind<br />
M. Andersen b , and Bernard T. Kiremire a<br />
a Chemistry Department, Makerere University, P.O. Box 7062, Kampala, Uganda<br />
b Department <strong>of</strong> Chemistry, University <strong>of</strong> Bergen, Allegt. 41, 5007, Bergen, Norway<br />
Corresponding author *rbyamukama@chemistry.mak.ac.ug<br />
Introduction<br />
A<br />
nthocyanins comprise a diverse group <strong>of</strong> intensely coloured pigments responsible for the<br />
appealing and <strong>of</strong>ten spectacular orange, red purple and blue colours <strong>of</strong> many fruits,<br />
vegetables, cereal grains, flowers, leaves, roots and other plant storage organs. The most common<br />
food colorants that have been used worldwide are synthetic ones some <strong>of</strong> which are deemed to be<br />
carcinogenic. Because <strong>of</strong> this, the safety <strong>of</strong> synthetic colorants has been questioned in the past<br />
years, and this has significantly increased the interest in natural colorants as food colour additives<br />
such as anthocyanins. Today, interest in anthocyanin pigments has also intensified because <strong>of</strong> their<br />
possible health benefits related to their antioxidant properties.<br />
Methods<br />
The anthocyanins were isolated from the plant materials by a combination <strong>of</strong> chromatographic<br />
techniques. Their structures were elucidated by online diode array detection chromatography and<br />
homo- and hetero-nuclear Nuclear Magnetic Resonance (NMR) spectroscopy and Mass<br />
spectrometry (LC-MS) techniques.<br />
Results and Discussion:<br />
This presentation will give the results <strong>of</strong> anthocyanin analysis (isolation and structure elucidation)<br />
from a number <strong>of</strong> plants plant species in Uganda including the novel compounds from Ricinus<br />
communis (caster plant) (Byamukama et al., 2008) Synadenium grantii (Andersen et al., 2010) and<br />
Plumbago auriculata (Jordheim et al., 2010), whose structures have been elucidated recently.<br />
In the sky-blue corollas <strong>of</strong> Plumbago auriculata, six new anthocyanins were isolated. All the six<br />
pigments in P. auriculata are based on three anthocyanidins, which for the first time in natural state<br />
are reported to have methylation <strong>of</strong> both <strong>of</strong> their A-ring hydroxyl groups. Four new together with<br />
two known anthocyanins pigments were isolated from S. grantii. The four were the first reported<br />
anthocyanins containing the monosaccharide apiose and the disaccharide 2 -( -apiosyl)- -xyloside.<br />
Cyanidin 3-xyloside-5-glucoside and cyanidin 3-O- -xylopyranoside-5-O-(6'''-O-methylmalonate- -<br />
glucopyranoside) were new anthocyanins isolated from Ricinus communis, and are relatively rare<br />
anthocyanins with monosaccharide xylose linked directly to the anthocyanidin 3-position.<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Acknowledgements:<br />
Authors acknowledge Carnegie Cooperation <strong>of</strong> New York and The Norwegian Programme for<br />
Development, Research and Education (NUFU) for funding the Research.<br />
References<br />
Andersen, M. Ø., Jordheim, M., Byamukama, R., Mbabazi, M., Ogweng, G., Skaar, I., Kiremire T. B. (2010); Anthocyanins<br />
with unusual furanose sugar (apiose) from leaves <strong>of</strong> Synadenium grantii (Euphorbiaceae). Phytochemistry 71 1558-<br />
1563.<br />
Byamukama, R., Jordheim, M., Andersen, M. Ø., Kiremire T. B. (2008); New anthocyanins from the stem bark <strong>of</strong> Castor,<br />
Ricinus communis. Natural Product Communications , 3 (9), 1497-1500.<br />
Jordheim, M., Byamukama, R., Andersen, M. Ø., Mbabazi, M., Skaar, I., Kiremire T. B. (2010); Natural anthocyanidins<br />
with methylated A-rings from Plumbago auriculata. Submitted<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[SL 3B] Effects <strong>of</strong> Botanical Insecticides on the Egg Parasitoid Trichogramma<br />
cacoeciae Marchal (Hym. Trichogrammatidae).<br />
Abdelgader, H.<br />
Agricutural Research Corporation, Crop Protection Research Center, Wadmedani P. O. Box 126, Sudan.<br />
Key words: Side effects; Trichogramm; Botanical Insecticides; Neemazal; Quassin.<br />
Introduction<br />
P<br />
arasitoids <strong>of</strong> the genus Trichogramma occure naturaly worldwide and play an important role as<br />
natural enemies <strong>of</strong> lepidopterous pests on a wide range <strong>of</strong> agricultural crops. Results <strong>of</strong><br />
augmentative releases <strong>of</strong> Trichogramma can be affected by the use <strong>of</strong> broad-spectrum insecticides<br />
in or near release plots (Stinners et al. 1974, Ables et al. 1979, King et al. 1984). The search for<br />
selective insecticides to be used with Trichogramma releases is <strong>of</strong> great importance. The recent<br />
laboratory studies were carried out to investigate the side effects on Trichogramma cacoeciae <strong>of</strong><br />
two formulated products <strong>of</strong> each <strong>of</strong> two botanical insecticides: Azadirachtine (Neemazal T/S Blank<br />
and Celaflor®) and Quassin (alcoholic or water extracts) to study there possible use with<br />
Trichogramma releases, since these insecticides are coming from plant origin they are believed also<br />
to have the advantage <strong>of</strong> having the lease impact on the environment.<br />
Materials and Methods<br />
Two formulations <strong>of</strong> the botanical active ingredient, azadrichtine (Neemazal T/s Blank and Celaflor)<br />
as well as two extracts <strong>of</strong> Quassin (Alcoholic and Water extracts) were included in the tests. The<br />
field recommended concentrations were prepared for the tests. The study included Exposing adults<br />
(susceptible life statgae) <strong>of</strong> Trichogramma to sprayed glass plates using the method described by<br />
Hassan et al. (2000). In other experiments adults <strong>of</strong> Trichogramma were exposed to sprayed host<br />
eggs. The treated host eggs were either <strong>of</strong>fered directly after drying <strong>of</strong> the spray or the eggs were<br />
hold at 15 °C and <strong>of</strong>fered to adults after 6 days. Less susceptible life stage (parasites within their<br />
hosts) were also exposed to tested treatments. Thhe method described by Hassan and Abdelgader<br />
(2001) was followed. The study included spraying <strong>of</strong> parasitised host eggs at different interval after<br />
parasitisation ranging from 1 8 days. The percentage <strong>of</strong> adult emergence and the reduction in<br />
emergence relative to the control were then determind and the pesticides were categorized<br />
accordingly.<br />
Results and Discussion<br />
Effects on adults<br />
Results <strong>of</strong> tested Botanicals on adults are presented in Table (1). The results showed that by<br />
exposing adults T. cacoeciae to residues <strong>of</strong> Neemazal formulations on glass plates (standard test<br />
method, Hassan et al. 2000), the preparations were either harmful (Neemazal-Blank) or moderately<br />
harmful (Celaflor). The two Quassin formulations tested were harmless. In another set <strong>of</strong><br />
experiments, where treated host eggs were <strong>of</strong>fered to adults T. cacoeciae, all tested chemicals<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
were almost harmless. By exposing adults to treated host eggs both Quassin formulations were<br />
harmless. Celaflor was slightly toxic for adults, both when freshly or 6-day old sprayed host eggs<br />
were <strong>of</strong>fered to adults <strong>of</strong> T. cacoeciae. Neemazal-Blank formulation was only slightly toxic when 6<br />
day old sprayed host eggs were <strong>of</strong>fered to the adults.<br />
Table 1. Effects <strong>of</strong> exposing adult Trichogramma cacoeciae to various treatments<br />
Treatment Parasitism rate<br />
eggs/female<br />
(glass plate test)<br />
Class Fresh residue<br />
host eggs<br />
spraying<br />
eggs/ female<br />
84<br />
Class 6 day residue<br />
host eggs<br />
spraying<br />
eggs/ female<br />
Control 18.9 abc 28.8 bc 36.0 b<br />
Quassin-Alcohol 21.2 bc 1 23.1 ab 1 31.6 ab 1<br />
Quassin-Water 22.0 c 1 33.0 c 1 33.9 b 1<br />
Neemazal-Blank 0.0 a 4 24.0 ab 1 24.0 a 2<br />
Celaflor 1.0 ab 3 20.3 a 1-2 23.2 a 2<br />
** = Figures followed by the same letter are not significantly different (Multiple Range Test , 5%); SE = Standard Error; %<br />
RC = Percentage Reduction relative to the control; Class = IOBC classification<br />
Effects on immature stages<br />
Spraying parasitized host eggs one day after parasitism resulted in a significantly lower number <strong>of</strong><br />
black eggs (i.e. lower pupation). All tested insecticides significantly reduced pupation, when host<br />
eggs where sprayed two days after parasitism, indicating that Trichogramma was very sensitive<br />
during this stage. This might have coincided with the hatching <strong>of</strong> the vulnerable neonate larvae <strong>of</strong><br />
Trichogramma from laid eggs. The pupation rate was not reduced as a result <strong>of</strong> treatment, when<br />
host eggs were sprayed on the third and subsequent days after parasitism (Table 2). This trend can<br />
also be seen clearly when the percentage reduction relative to the control and the categorisation<br />
according to the IOBC classification was determined (Table 3).<br />
Table 2. Developing Black eggs after treating parasitsed eggs at various days after parasitism<br />
Treatment 1 day 2 days 3 days 5 days 7 days 8 days<br />
Control 427.3 c 329.0 a 388.3 ab 465.2 ab 440.2 b 355.5 ab<br />
Quassin-Alcohol 400.8 c 189.8 b 441.7 bc 464.2 a 420.7 b 388.5 bc<br />
Quassin-Water 401.7 c 247.8 b 448.8 c 506.3 b 412.0 b 421.3 c<br />
Neemazal-Blank 219.0 a 219.8 b 357.5 a 437.5 a 340.3 b 325.3 a<br />
Celaflor 334.3 b 197.0 b 466.5 c 430.2 a 420.0 b 323.2 a<br />
SE 17.5 20.9 19.3 26.0 19.5 20.8<br />
Class<br />
** = Figures followed by the same letter are not significantly different (Multiple Range Test , 5%); SE =<br />
Standard Error
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Table 3. Developing Black eggs after treating parasitsed eggs at various days after parasitism (IOBC<br />
Classification)<br />
Treatment 1 day<br />
2<br />
days<br />
3<br />
days<br />
% RC Class % RC Class % RC Class<br />
85<br />
5<br />
days<br />
7<br />
days<br />
8<br />
days<br />
%<br />
RC Class % RC Class % RC Class<br />
Quassin-Alcohol 6.2 1 42.3 2 -13.7 1 0.2 1 4.4 1 -9.3 1<br />
Quassin-Water 6.0 1 24.8 1 -15.6 1 -8.9 1 6.4 1 -18.5 1<br />
Neemazal-Blank 48.8 2 33.2 2 7.9 1 6.0 1 22.7 1 8.5 1<br />
Celaflor 21.8 1 40.1 2 -20.1 1 7.5 1 4.6 1 <strong>9.1</strong> 1<br />
% RC = Percentage Reduction relative to the control; Class = IOBC classification<br />
Conclusion<br />
The results showed, in general, that both Azadirachtine and Quassin were relatively safe to the<br />
tested parasitoid and could therefore be used in combination with Trichogramma releases.<br />
References<br />
Ables, J. R., Johnes, R. K., Morrison, V. S., House, D. L., Bull, L. F., Bouse and Carlton, J. B. (1979); New developments in<br />
the use <strong>of</strong> Trichogramma to control lepidopteran pests <strong>of</strong> cotton, pp. 125-127. In: Proceedings, Beltwide Cotton<br />
Production Research Conference. National Cotton Council, Memphis, TN.<br />
Hassan, S. A., Halsall, N., Gray, A. P., Kuehner, C., Moll, M., Bakker, F.M., Roembke, J., Yousef, A., Nasr, F. and<br />
Abdelgader, H. (2000); A laboratory method to evaluate the side effects <strong>of</strong> plant protection products on<br />
Trichogramma cacocciae Marchal (Hym., Trichogrammtidae), 107 119. In: M.P. Candolfi, S. Blümel, R. Forster,<br />
F.M. Bakker, C. Grimm, S.A. Hassan, U. Heimbach, M.A. Mead-Briggs, B. Reber, R. Schmuck and H. Vogt (eds.)<br />
(2000); Guidelines to evaluate side-effects <strong>of</strong> plant protection products to non-target arthropods. IOBC/ WRPS,<br />
Gent.<br />
Hassan, S. and Abdelgader, H. (2001); A sequential testing program to assess the effects <strong>of</strong> pesticides on Trichogramma<br />
cacoeciae Marchal (Hym., Trichogrammatidae). Pesticides and beneficial Organisms. IOBC/ WRPS Bulletin, 24 (4):<br />
71-81.<br />
King, E. G., L. F. Bouse, D. L. Bull, W. A. Dickerson, W. J. Lewis, P. Liapis, J. D. Lopez, R. K. Morrison and Phillips, J. R.<br />
(1984); Potential <strong>of</strong> management <strong>of</strong> Heliothis spp. In: cotton by augmentative releases <strong>of</strong> Trichogramma pretiosum,<br />
pp. 232-236. In: Proceedings, Beltwide Cotton Production Research Conference. National Cotton Council, Memphis,<br />
TN.<br />
Stinners, R. E., R. L. Ridgway, J. R. Coppedge, R. K. Morrison and W. A. Dickerson, Jr. (1974); Parasitism <strong>of</strong> Heliothis eggs<br />
after field releases <strong>of</strong> Trichogramma pretiosum in cotton. Environ. Entmol., 3: 497-500.
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[SL 4A] Bioactive Constituents from Hyptis suaveolens<br />
Venkatesan Jayakumar 1 , Nicholas Daniel 2 , Kingsly Arachi 1<br />
1<br />
General Engineering Department, St.Joseph College <strong>of</strong> Engineering and Technology University, Post box no: 11007,<br />
Morogoro road, Mbezi Luguruni, Dar Es Salaam, Tanzania.<br />
2<br />
St.Joseph College <strong>of</strong> Engineering and Technology University, Post box no: 11007, Morogoro road, Mbezi Luguruni,<br />
Dar Es Salaam, Tanzania.<br />
Key words: Hyptis suaveolens, (2E)-1-(2-hydroxy phenyl) pent-2-en-1-one, 1-[(3-hydroxy-5, 5-dimethyl cyclohex-3-en-<br />
1yl) oxy] hexan-3-one, Antifeedant activity, Ovicidal activity.<br />
Introduction<br />
yptis suaveolens (L.) poit, a rigid sweetly aromatic herb belongs to the family Lamiaceae is a<br />
native <strong>of</strong> tropical America. The plant is used as green manure in India 1 , the edible shoot tips<br />
are used for flavoring the dishes. In Philippines, the leaves are used for antispasmodic,<br />
antirheumatic and antisoporific baths. A decoction <strong>of</strong> the roots is used as appetizer and the root is<br />
chewed with betel nuts as a stomachic 2, 3 . The leaves are used to treat cancer ailments 4 and antifertility<br />
causes 5 . This plant is used for ethano botanical applications in rural communities in African<br />
countries and shows the promising results to control the Sesamia calamistis on Maize 6 H<br />
.<br />
Previously isolated constituents<br />
The presence <strong>of</strong> ethereal oil, Monoterpenes, Diterpenes, Suaveolic acid, Suaveolol, Triterpenoid,<br />
Campesterol, Fucosterol, Sesquiterpene alcohols and essential oils have been reported to be<br />
present in this plant 7 .<br />
Experimental Procedure<br />
Shade dried, powdered leaf material (2 Kg) was subjected to sequential solvent extraction and the<br />
respective crude extract was then subjected to bio-activity studies. The crude extracts which shows<br />
promising activity alone further taken for column chromatographic isolation. Ethyl acetate crude<br />
extract (50g) showed promising activity was fractionated through flash column chromatography,<br />
using silica gel (230-400 mesh AR), column size (15cm X 100cm) using the gradient <strong>of</strong> solvent<br />
Hexane / Ethyl acetate ( 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 60:40, 50:50 and 100).Totally 20<br />
fractions were obtained, each fraction was tested for its bioactivity at various concentration.<br />
Promising fractions were further studied for their bioactivity at 100, 250, 500, 1000 and 2000 ppm.<br />
Purified promising fractions were subjected to FTIR, H NMR and C NMR for identification <strong>of</strong><br />
bioactive compounds.<br />
O<br />
OH<br />
CH 3<br />
HO<br />
86<br />
C<br />
H 3<br />
CH 3<br />
O<br />
O<br />
CH 3
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
(2E)-1-(2-hydroxyphenyl) pent-2-en-1-one (I) 1-[(3-hydroxy-5,5-dimethylcyclohex-3-en-1yl)oxy]hexan3-one(II)<br />
Table1. Bioactivity <strong>of</strong> ethyl acetate extract <strong>of</strong> Hyptis suaveolens at 1000 ppm concentration<br />
Tested insects<br />
Bioactivity Spodoptera litura Helicoverpa armigera<br />
Antifeedent (%) 65.3 ± 3.37 71.0 ± 1.90<br />
Oviposition detergent 39.0 ± 3.48 24.0 ± 4.21<br />
Ovicidal (%) 69.4 ± 2.99 65.7 ± 2.7<br />
Insecticidal (%) 19.4 ± 2.55 11.5 ± 2.28<br />
Values are expressed as percentage mean ± SD (n = 5).<br />
Maximum antifeedant and ovicidal activity were recorded in ethyl acetate extract <strong>of</strong> H. suaveolens<br />
and the results are presented in Table. 1. No antifeedent and ovicidal activity was recorded in<br />
positive and negative control. Among the 11 fractions tested, fraction II and IV showed maximum<br />
antifeedent and ovicidal activity. Statistically significant antifeedant and ovicidal activity were<br />
recorded at 1000 ppm concentrations. The bioactivity <strong>of</strong> fraction II seems to be due to the presence<br />
<strong>of</strong> long aliphatic chain group containing , -unsaturated keto-moiety, attached to phenolic<br />
nucleus. The presence <strong>of</strong> , -unsaturated ketone group seems to impart synergistic activity <strong>of</strong><br />
phenolic compound. Also, the presence <strong>of</strong> methyl residue seems to enhance the hydrophobic<br />
nature <strong>of</strong> the molecule, thereby indirectly enriching the bioactivity <strong>of</strong> the patent phenolic<br />
compound. Earlier bioactivity <strong>of</strong> polyphenolic rich fractions form the stem bark <strong>of</strong> Streblus asper<br />
against Dysdercus cingulatus has been reported 8 and several polyphenolic compounds have been<br />
reported to have insecticidal activity 9-11 .<br />
References:<br />
1. Yoganarasimhan, S. N. (2000); Medicinal Plants <strong>of</strong> India (eds Srinivasan, V. and KosalRam, N.), Cyber Media,<br />
Bangalore, vol. 2, p. 282.<br />
2. Wealth <strong>of</strong> India, (1959); CSIR, New Delhi, vol.V, p. 159.<br />
3. Dalziel, J. M. (1937); The Useful Plants <strong>of</strong> West Tropical Africa, Crown Agents, London, p. 461.<br />
4. Mabbereley, D. J., (1990); The Plant <strong>Book</strong>, Cambridge University Press, London, pp. 209, 289.<br />
5. Oliver-Bever, B. (1986); Medicinal Plants in Tropical West Africa, Cambridge University Press, London, p. 225.<br />
6. Adda.C., Atachi.P., Hell.K., Tamo.M., (2011); Journal <strong>of</strong> insect science., 11 (33): 1-13<br />
7. Peezada, N., (1997); Molecules, 2, 165-168.<br />
8. Hashim, M. S. and Devi, K. S. (2003); Fitoterapia, 74, 670-676.<br />
9. Khambay, B. P., Beddie, D. G. and Simonds, M. S. J., (1999); J. Natl. Prod., 62, 1423-1425.<br />
10. Schneider, C. et al., (2000); Phytochemistry, 54, 731-736.<br />
11. Kim, D. H. and Ahn, Y. (2001); J. Pest Manage. Sci., 57, 301-304.<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[SL 4B] Blends <strong>of</strong> Chemicals in Smelly Feet Switch Malaria Mosquitoes on and<br />
<strong>of</strong>f<br />
M. O. Omolo 1, *, B. Njiru 2 , I. O. Ndiege 3 , R. M. Musau 3 , P. Njagi 2 , A. Hassanali 3<br />
1<br />
Department <strong>of</strong> Pure & Applied Chemistry, Faculty <strong>of</strong> Science, Masinde Muliro University <strong>of</strong> Science and Technology<br />
(MMUST), P.O. Box 190, Kakamega, 00500, Kenya<br />
2<br />
Behavioural and Chemical Ecology Department (BCED), International Center <strong>of</strong> Insect Physiology and Ecology (ICIPE),<br />
P.O. Box 30772 Code 00100, Nairobi, Kenya.<br />
3<br />
Department <strong>of</strong> Chemistry, School <strong>of</strong> Pure and Applied Sciences, Kenyatta University, P.O. Box 43844 Code 00100<br />
Nairobi, Kenya.<br />
*Author to whom correspondence and reprint requests should be addressed.<br />
Key words: Human, foot, odours, mosquitoes, attractants, repellants<br />
Introduction<br />
M<br />
osquitoes are important vectors <strong>of</strong> several tropical diseases, including malaria, filariasis, and<br />
a series <strong>of</strong> viral diseases such as dengue, Japanese encephalitis, West Nile virus, and yellow<br />
fever. Of these, malaria-transmitting species are most important. Globally, an estimated 200-300<br />
million people are affected by malaria, <strong>of</strong> which 1.5-2.7 million die each year.<br />
Host location in nocturnal anthropophilic mosquitoes is mediated largely by volatile human odours<br />
associated with body and breath (Mukabana et al., 2004; Okumu et al., 2010, Verhulst et al., 2010).<br />
Some studies suggest that, some humans are more attractive than others to host seeking<br />
mosquitoes (Lindsay et al., 1993; Knols et al., 1995). Human foot odour is more attractive to An.<br />
gambiae Giles s.s. than cow leg odour (Pates et al., 2001). Washing <strong>of</strong> human feet substantially<br />
reduced their attraction to the mosquito (Knols et al., 1995). Moreover, placing recently worn<br />
socks next to a blood-feeding membrane device enhanced feeding, and in turn fecundity, in An.<br />
gambiae and An. stephensi (Andreasen, 2004). These observations suggest a two-step process in<br />
the orientation behaviour <strong>of</strong> anthropophilc mosquitoes: location <strong>of</strong> hosts from some distance<br />
mediated by gross odour emanating from the body and breath and closer range avoidance <strong>of</strong><br />
breath but attraction to preferred feeding sites that is mediated by site-specific volatiles (Suer,<br />
2011).<br />
In this study, we compared the attractiveness <strong>of</strong> foot odours collected on socks from 16 individuals<br />
and chemical compositions <strong>of</strong> the most and least attractive odours. Herein we report the results <strong>of</strong><br />
our study.<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Materials and methods<br />
Masking effect <strong>of</strong> the repellents<br />
The average catches <strong>of</strong> a counter flow geometry (CFG) trap (American Biophysics) baited with one<br />
<strong>of</strong> the following blends were compared with unbaited CFG trap under semi-field conditions in a<br />
screenhouse (11.5 x 7.1 x 3.0 m) between 20 hr in the night and 6 hr the following morning:<br />
(i) 11-component blend <strong>of</strong> EAG-active (electrophysiologically-active) components (<strong>of</strong><br />
isobutyric acid, isovaleric acid, 2-methylphenol, 4-ethylacetophenone, 4ethoxyacetophenone,<br />
n-octanal, n-nonanal, n-decanal, n-undecanal n-dodecanal and ntridecanal)<br />
in relative proportion present in typical human foot odour attractive to An.<br />
gambiae s.s.;<br />
(ii) (i) minus 4-ethylacetophenone<br />
(iii) (i) minus 4-ethoxyacetophenone<br />
(iv) (i) minus undecanal<br />
(v) 4-ethylacetophenone and 4-ethoxyacetophenone; and<br />
(vi) 4-ethylacetophenone, 4-ethoxyacetophenone and undecanal.<br />
The two traps were arranged diagonally in the screenhouse (Figure 1). The positions <strong>of</strong> the test and<br />
the control CFG traps were interchanged every following night. 200 starved female An. gambiae s.s.<br />
were released from a cup placed at the center between the baited trap and unbaited control trap<br />
(Figure 1) and the number <strong>of</strong> mosquitoes caught in each trap counted. Each test was replicated 8<br />
times on different nights. The relative catch size associated with each blend was computed and<br />
analysed statistically.<br />
Figure 1: Sketch showing the arrangement <strong>of</strong> baited and control CFG traps in a screenhouse (a and<br />
b represent test and control CFG traps at the 2 different locations with the cup c at equidistant from<br />
the traps. All the dimensions are in meters).<br />
Comparison <strong>of</strong> mosquito catches in traps baited with a synthetic blend and a human volunteer<br />
The average catches <strong>of</strong> a counter flow geometry (CFG) trap (American Biophysics Corporation)<br />
baited with a synthetic blend <strong>of</strong> eight constituents that make up the attractive mixture <strong>of</strong> human<br />
foot odours (i.e. isobutyric acid, isovaleric acid, octanal, nonanal, decanal, dodecanal, tridecanal,<br />
and 2-methylphenol) was compared with those <strong>of</strong> a bed-net trap (Mathenge et al. (2002). Each<br />
comparison was performed repeatedly between 20 hr in the night and 6 hr the following morning<br />
under semi-field conditions in a screenhouse (11.5 x 7.1 x 3.0 m) also at Mbita Point on the shores<br />
89
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
<strong>of</strong> Lake Victoria. The two types <strong>of</strong> traps in each test were located at the corners <strong>of</strong> the screenhouse<br />
and were interchanged before each replicate. 200 starved laboratory-reared female An. gambiae<br />
s.s. were released from a cup placed at the center between the two traps being compared and the<br />
number <strong>of</strong> mosquitoes caught in each trap counted. The relative catch sizes <strong>of</strong> each pair <strong>of</strong> traps<br />
were computed and analysed statistically.<br />
Results and Discussion<br />
The results <strong>of</strong> the experiment on masking effect <strong>of</strong> the repellants (Table 1) show that the presence<br />
<strong>of</strong> 4-ethylacetophenone, 4-ethoxyacetophenone and undecanal in the EAG-active 11-component<br />
foot odour blend in relative amounts found in the natural odour masks the attractiveness <strong>of</strong> the<br />
blend to the mosquito. This is reflected in higher catches in CFG traps baited with blends with one<br />
or more <strong>of</strong> these constituents missing.<br />
Table 1. Transformed mean mosquito catches <strong>of</strong> CFG traps baited with different blends <strong>of</strong> EAGactive<br />
human foot odour blends.<br />
Blend Transformed mean + SE<br />
Blend (i)<br />
Blend (ii)<br />
Blend (iii)<br />
Blend (iv)<br />
Blend (v)<br />
Blend (vi)<br />
15.86 + 2.26 d<br />
51.25 + 7.02 b<br />
27.13 + 5.85 c<br />
48.10 + 6.01 b<br />
73.50 + 2.91 a<br />
69.50 + 2.99 a<br />
The CFG trap baited with the synthetic odour blend caught significantly more (P < 0.01; t-Test)<br />
mosquitoes with an average (+ SE) <strong>of</strong> 105.3 + 14.5 compared with 45.5 + 5.6 in the bed-net trap.<br />
This study has demonstrated that there is differential attractiveness <strong>of</strong> An. gambiae s.s. to the<br />
human feet odours. The difference in attractiveness <strong>of</strong> the human feet odours is due to the<br />
variation in the composition <strong>of</strong> the skin micr<strong>of</strong>lora and fauna, which lead to variation in the<br />
chemical composition <strong>of</strong> the odours emanating from the feet. The repellents and attractants<br />
identified in the human foot odour can be exploited in reducing mosquito numbers in their natural<br />
habitats in a push-pull strategy wherein traps or targets attractive blends are used as baits in the<br />
pull mode and repellents or repellent blends are used in personal protection or space fumigation<br />
in the push mode.<br />
Acknowledegments<br />
This work was supported by funds from UNDP/World Bank/WHO/TDR (Grant No.) and NIH (Grant<br />
No. U19A14511-01). We thank Jactone Arija (ICIPE) for the supply <strong>of</strong> the insects, Moses Kimote<br />
(ICIPE) for his help during the bio-assays and all the sixteen human subjects for their voluntary<br />
participation in the experiments.<br />
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References<br />
Andreasen, M. H., (2004); Enhanced blood feeding <strong>of</strong> Anopheles mosquitoes (Diptera: Culicidae) through membranes<br />
with applied host odour. Bulletin <strong>of</strong> Entomological Research, 94, 291-295.<br />
Knols, B. G. J., De Jong, R., Takken, W., (1995); Differential attractiveness <strong>of</strong> isolated humans to mosquitoes in Tanzania.<br />
Trans. Royal Soc. Tropical Med. & Hyg., 89, 604-606.<br />
Lindsay, S. W., Adiamah, J. H., Miller, J. E., Pleass, R. J., Armstrong, J. R. M., (1993); Variation in attractiveness <strong>of</strong> human<br />
subjects to malaria mosquitoes (Diptera: Culicidae) in The Gambia. Journal <strong>of</strong> Medical Entomology, 30, 368-373.<br />
Mukabana, W. R., Takken, W., Killeen, F. G., Knols, B. G. J., (2004); Allomonal effect <strong>of</strong> breath contributes to differential<br />
attractiveness <strong>of</strong> humans to the African malaria vector Anopheles gambiae. Malaria Journal, 3: 1<br />
Okumu, F.O., Killeen, G.F., Ogoma, S., Biswaro, L., Smallegange R.C., Mbeyela, E., Titus, E., Munk, C., Ngonyani, H.,<br />
Takken, W., Mshinda, H., Mukabana, W.R., Moore, S.J., (2010); Development and field evaluation <strong>of</strong> a synthetic<br />
mosquito lure that is more attractive than humans. PLoS 5(1):e8951.<br />
Pates, H. V., Takken, W., Stake, K., Curtis, C. F., (2001); Differential behaviour <strong>of</strong> Anopheles gambiae sensu stricto<br />
(Diptera: Culicidae) to human and cow odours in the laboratory. Bulletin <strong>of</strong> Entomological Research, 91, 289-296.<br />
Suer, R., (2011); Malaria mosquitoes accurately find their way to smelly feet. Thesis,Wageningen University,<br />
Netherlands.<br />
Verhulst, N.O., Andriessen, R., Groenhagen, U., Bukovinszkine, K.G., Schulz S., Takken, W., Van Loon, J.J.A., Schraa, G.,<br />
Smallegange C.R., (2010); Differential attraction <strong>of</strong> malaria mosquitoes to volatile blends produced by human skin<br />
bacteria. PLoS ONE 5 (12): e15829.<br />
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[SL 5A] Non-Volatile Isolates from Two Members <strong>of</strong> the African Genus<br />
Heteropyxis (Myrtaceae)<br />
Abdelhafeez M.A. Mohammed 1,2 , Philip H. Coombes 1 , Neil R. Crouch 1,3 and Dulcie A. Mulholland 1,4<br />
1<br />
School <strong>of</strong> Chemistry, University <strong>of</strong> KwaZulu-Natal, Westville Campus, Private Bag X54001, Durban, 4000, South Africa<br />
2<br />
Department <strong>of</strong> Chemistry, Alzaiem Alazhari University, P O Box 1432, Khartoum North, 13311, Sudan,<br />
ahafeez61@yahoo.com<br />
3<br />
Ethnobotany Unit, South African National Biodiversity Institute, PO Box 52099, Berea Road 4007, South Africa<br />
4<br />
Division <strong>of</strong> Chemical Sciences, Faculty <strong>of</strong> Health and Medical Sciences, University <strong>of</strong> Surrey, Guildford, Surrey, GU2<br />
7XH, United Kingdom<br />
Keywords: Heteropyxis natalensis; Heteropyxis canescens; Psiloxyloideae; Myrtaceae<br />
Introduction<br />
H<br />
eteropyxis Harvey, one <strong>of</strong> two genera in the small subfamily Psiloxyloideae <strong>of</strong> the Myrtaceae<br />
(the other being Psiloxylon Thouars ex Tul.), comprises only three species: H. canescens<br />
Oliver, H. natalensis Harvey and H. dehniae Susseng (Heywood et al. 2007, Mohammed et al. 2009).<br />
A literature survey <strong>of</strong> Heteropyxis revealed that only the essential oil composition <strong>of</strong> H. natalensis<br />
has been studied, although it is used widely in Zulu traditional medicine as a tea, a drench for stock<br />
animals, an aphrodisiac, and to treat impotence (Hutchings et al. 1996, Sibanda et al. 2004). The<br />
Vhavenda <strong>of</strong> South Africa employ the plant in the treatment <strong>of</strong> bleeding gums and noses, and for<br />
menorrhagia (Hutchings et al. 1996). No ethnobotanical usage has been documented for H.<br />
canescens, nor has it previously been investigated phytochemically.<br />
The present study has considered the chemistry <strong>of</strong> two <strong>of</strong> the four subfamily members (tribe<br />
Heteropyxideae Harv.) in relation to the rest <strong>of</strong> the Myrtaceae (represented by the Myrtoideae) in<br />
view <strong>of</strong> the <strong>of</strong>times contentious placement <strong>of</strong> Heteropyxis and Psiloxylon in the Myrtaceae (Conti et<br />
al. 1997; refs within). Heteropyxis has, at various times, also been included in the Lythraceae,<br />
Rutaceae, and the monogeneric Heteropyxidaceae (Dahlgren and Van Wyk 1988), whilst the<br />
monotypic Psiloxylon has been placed in Bixaceae sensu lato, Flacourtiaceae, Myrtaceae, Guttiferae<br />
and the monogeneric Psiloxyloaceae (Schmid 1980). Darnley Gibbs (1974) earlier contemplated the<br />
familial position <strong>of</strong> Heteropyxis, remarking (p. 1495) that if we knew something <strong>of</strong> the chemistry <strong>of</strong><br />
the genus we might well be able to place it with confidence . Phytochemical data to assist in<br />
defining historical family relationships were lamented by Schmid (1980) to be meagre, and have<br />
remained so, thus providing the motivation for undertaking research on non-volatile elements in<br />
Heteropyxis.<br />
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Materials and Methods<br />
Plant materials:<br />
Heteropyxis natalensis Harvey (N. Crouch 1057, NH) was collected in Klo<strong>of</strong>, KwaZulu-Natal in<br />
October 2002 and Heteropyxis canescens Oliver (N. Crouch & J. Burrows 1007, NH) was collected in<br />
January 2004 at Buffelsklo<strong>of</strong> Private Nature Reserve in Mpumalanga Province, South Africa.<br />
Extraction, separation and isolation <strong>of</strong> compounds<br />
The air-dried, milled plant material was extracted separately and successively for 24 h each, in a<br />
Soxhlet apparatus with n-hexane, DCM, EtOAc and MeOH. Each crude plant extract was initially<br />
fractionated by either vacuum or gravity column chromatography, generating some 250-300<br />
fractions <strong>of</strong> 100 mL each. Fractions were pooled on the basis <strong>of</strong> their TLC pr<strong>of</strong>iles and further<br />
purified when necessary by further column chromatography. Separation <strong>of</strong> crude extracts was<br />
generally carried on a column using silica gel (Merck 9385). Both column and thin layer<br />
chromatographic techniques made use <strong>of</strong> varying ratios <strong>of</strong> n-hexane/dichloromethane, nhexane/EtOAc,<br />
dichloromethane/EtOAc or dichloromethane/methanol as eluting solvents. Thin<br />
layer chromatography was carried out on 0.2 mm silica gel, aluminium-backed plates (Merck 5554).<br />
The plates were first analysed under ultraviolet light (254 and 366 nm) and then sprayed with<br />
anisaldehyde: conc. H2SO4: methanol (1: 2: 97) spray reagent and heated.<br />
Characterization and identification <strong>of</strong> compounds<br />
Compounds were characterized and identified using NMR, IR, and UV spectroscopy and LRMS or<br />
HRMS and data were compared with literature data.<br />
Results and Discussion<br />
The chemical investigation <strong>of</strong> the leaves and stems <strong>of</strong> Heteropyxis canescens and the twigs and<br />
roots <strong>of</strong> H. natalensis afforded a total <strong>of</strong> twenty five known compounds. Eleven known<br />
compounds, comprising two cinnamic acid esters, eicosyl trans-7-hydroxycinnamateand eicosyl<br />
trans-7-hydroxy-6-methoxycinnamate, four lupane triterpenoids, lupeol, lupenone, betulinic acid,<br />
and 3 -hydroxylup-20(29)-en-28-al, and two phytosterols, sitost-4-en-3-one and sitosterol,<br />
O-methyl-3,4-methylenedioxyellagic acid and gallic acid were isolated from<br />
the twigs and roots <strong>of</strong> this species. Fourteen known compounds were isolated from the leaves,<br />
stems and roots <strong>of</strong> Heteropyxis canescens: two flavanoids, strobopinin and<br />
desmethoxymatteucinol, two flavones, quercetin and apigenin, four lupane triterpenoids, lupeol,<br />
lupenone, betulinic acid, and 3 -hydroxylup-20(29)-en-28-al, an oleanane triterpenoid, arjunolic<br />
acid, three phytosterols, sitosterol-3-O- -D-glucoside, stigmasterol and sitosterol, an ellagic acid<br />
O-methylellagic acid, and the phenolic acid, gallic acid (Table 1).<br />
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Table 1. Non-volatile isolates <strong>of</strong> Heteropyxis natalensis and H. canescens<br />
Compound class Heteropyxis natalensis Heteropyxis canescens<br />
Lupane triterpenoids Lupeol<br />
lupenone<br />
betulinic acid<br />
3 -hydroxylup-20(29)-en-28-al<br />
Phytosterols sitosterol<br />
sitost-4-en-3-one<br />
94<br />
lupeol<br />
lupenone<br />
betulinic acid<br />
3 -hydroxylup-20(29)-en-28al<br />
sitosterol<br />
Oleanane triterpenoids arjunolic acid<br />
C-methylated flavanoids 2 -methoxyand<br />
chalcones 3<br />
O-methyl-<br />
3,4-methylenedioxyellagic acid<br />
gallic acid<br />
Flavonols - apigenin<br />
quercetin<br />
Cinnamic acid esters eicosyl trans-7hydroxycinnamate<br />
eicosyl trans-7-hydroxy-6methoxycinnamate<br />
sitosterol-3-O- -D-glucoside<br />
stigmsterol<br />
strobopinin<br />
desmethoxymatteucinol<br />
3,3',4'-tri-O-methylellagic<br />
acid<br />
gallic acid<br />
The current phytochemical investigation <strong>of</strong> the Psiloxyloideae has not revealed a significant<br />
variance from the chemical pr<strong>of</strong>ile <strong>of</strong> the Myrtoideae (Myrtaceae). However, <strong>of</strong> the four hundred<br />
and seventy-one lupane triterpenoids reported to date from all natural sources, the Myrtoideae<br />
have yielded only four, from four species out <strong>of</strong> some one hundred and forty that have been<br />
investigated (DNP 2009). By comparison, two members <strong>of</strong> the Psiloxyloideae have currently yielded<br />
four lupane triterpenoids (Table 1). The phytochemical investigation <strong>of</strong> the Psiloxyloideae has not<br />
revealed a significant variance from the chemical pr<strong>of</strong>ile <strong>of</strong> the Myrtoideae, revealing that at least<br />
on the basis <strong>of</strong> non-volatile constituents, alternative family placements <strong>of</strong> Heteropyxis are not<br />
presently supported (Mohammed et al. 2009).<br />
Acknowledgements<br />
Thanks to Mr Dilip Jagjivan for NMR analysis, Mr Bret Parel for GC-MS analysis, and Dr Philip<br />
Bosh<strong>of</strong>f at the Cape Technikon and Dr Colin Sparrow at Oxford University for HRMS analysis.<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
References:<br />
Conti, E., Litt, A., Wilson, P.G., Graham, S.A., Briggs, B.G., Johnson, L.A.S., Sytsma, K.J., (1997); Interfamilial relationship<br />
in Myrtales: molecular phylogeny and patterns <strong>of</strong> morphological evolution. Systematic Botany 22, 629-647.<br />
Dahlgren, R. and Van Wyk, A.E., (1988); Structures and relationships <strong>of</strong> families endemic to or centered in southern<br />
Africa. Monographs in Systematic Botany from the Missouri Botanical Garden 25, 1-94.<br />
Darnley Gibbs, R., (1974); Chemotaxonomy <strong>of</strong> flowering plants. Volume III. Orders. McGill-Queen s University Press,<br />
Montreal and London.<br />
Dictionary <strong>of</strong> Natural Products on CD-ROM, (2009); Chapman and Hall/CRC<br />
Heywood, V. H., Brummitt, R. K., Culhan, A. and Seberg, O., (2007); Flowering Plant Families <strong>of</strong> the World, Firefly <strong>Book</strong>s,<br />
Ontario, Canada, 225-226.<br />
Hutchings A., Scott, A.H., Lewis, G. and Cunningham, A., (1996); Zulu Medicinal Plants. An inventory. University <strong>of</strong> Natal<br />
Press, Pietermaritzburg, South Africa, p. 219.<br />
Mohammed, A.M.A., Coombes, P.H., Crouch, N.R. and Mulholland, D.A., (2009); Non-volatile isolates <strong>of</strong> two<br />
Heteropyxis species: a first chemotaxonomic assessment <strong>of</strong> subfamily Psiloxyloideae (Myrtaceae). Biochemical<br />
Systematics and Ecology, 37, 241-243.<br />
Schmid, R., (1980); Comparative anatomy and morphology <strong>of</strong> Psiloxylon and Heteropyxis and the subfamilial and tribal<br />
classification <strong>of</strong> Myrtaceae. Taxon 29, 559-595.<br />
Sibanda, S., Chigwadda, G., Poole, M., Gwebu, E.T., Noletto, J.A., Schmidt, J.M., Rea, A.I. and Setzer, W.N., (2004);<br />
Composition and bioactivity <strong>of</strong> the leaf essential oil <strong>of</strong> Heteropyxis dehniae. Journal <strong>of</strong> Ethnopharmacology, 92,<br />
107-111.<br />
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[SL 5B] Essential Oils Extracted from Cymbopogon citratus Leaves, Citrus limon<br />
and Citrus sinensis Peels as an Alternative to Find New Friendly Environmental<br />
Biocides<br />
Théoneste Muhizi 1 , Stéphane Grelier 2 , Véronique Coma 2<br />
1. corresponding author: National University <strong>of</strong> Rwanda, Faculty <strong>of</strong> Science, Department <strong>of</strong> Chemistry, P.O.BOX 117<br />
Butare Rwanda, email: tmuhizi@nur.ac.rw<br />
2. Unité Science du bois et des biopolymères (US2B), Université de Bordeaux 1, France.<br />
Key words: Essential oils, Cymbopogon citratus, Citrus lemon, Citrus sinensis, antibacterial activity, Salmonella<br />
thyphimurium, Listeria innocua, Staphylococcus aureus, Escherchia coli<br />
Introduction<br />
iocides are substances or preparations containing one or several active substances which<br />
should kill or inhibit the action from pathogen microbes (CE, 1998). The use <strong>of</strong> biocides has<br />
been undertaken over many decades and by numerous people from over the world. For example<br />
sulphur and arsenic have been used as biocides in 1000 year before Christ (Muhizi, 2008).<br />
Furthermore, during the First World War, the bioactive properties <strong>of</strong> Pyrethrum were accidentally<br />
discovered while those from Tobacco, Derris and Lonchocarpus were reported in the end <strong>of</strong> 16 th<br />
century. On the beginning <strong>of</strong> 19 th B<br />
century, treatment <strong>of</strong> fungi was done using principally heavy<br />
metal salts containing copper, mercury and lead. Development <strong>of</strong> chemistry enabled to discover<br />
other type <strong>of</strong> biocides which are principally organic and their uses have been more intensified.<br />
According to Knight et al (2002), the world market <strong>of</strong> biocides cover approximately four milliards <strong>of</strong><br />
US dollars and increases for 4% each year. Among these substances figure disinfectants, antiinterferences,<br />
wood protection substances, conservators substances and so on.<br />
However, in this decade, the resistance <strong>of</strong> microorganisms towards conventional biocides is<br />
increasing (Corrégé, 2001; White et al, 2001; Prazak, 2004, Korsac, 2004, ) and constitutes public<br />
health problems. For example studies have shown that among 1001 isolates <strong>of</strong> Listeria genus from<br />
retail foods, about 10.9% was resistant to one or more antibiotics (Walsh et al, 2001). Furthermore,<br />
Prazak et al. (2002) showed that about 95% <strong>of</strong> L. monocytogenes isolated from cabbage, water and<br />
environment samples resisted to two or more antibiotics. At the present time these different<br />
microbial resistances remarked must be highly combated to protect humanity against various<br />
diseases.<br />
Not only the world is struggling to find solutions to resistant microorganisms but also those from<br />
the toxicity <strong>of</strong> many <strong>of</strong> the biocides used. In fact, depending on their higher toxicity some <strong>of</strong> the<br />
biocides have been removed from the market (Kamrin ,1998, GTIF, 2003, FAO/UNEP, 1996, Hanson,<br />
et al., 2007, Cooper, et al., 2008, Focant, 2002, INERIS, 2005 and 2007, CE. 2001, Gouv. F. 2004,<br />
Hughes, W. W. 1996,) and this resolution constitute a big challenge, principally to biocides users but<br />
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also to researchers. Nowadays, researchers should work hardly to discover new effective and<br />
friendly environmental biocides to replace the banned ones.<br />
Natural organic compounds isolated from plants should be a good candidate for achieving this goal.<br />
These natural products have shown various utilities in different domains which include food,<br />
cosmetic, perfume, pharmaceutical and pesticides industries and more others (Ibrahim et al, 2001,<br />
Cimanga et al, 2002). Some authors even described some <strong>of</strong> these natural compounds as safe and<br />
strong bactericidal (Takahashi, 2002). Essential oils extracted from plants are among these natural<br />
compounds and constitute inexhaustible resources in Rwanda and in over the world.<br />
In this study, the essential oils from C. citratus leaves and those obtained from the peels from C.<br />
lemon and C. sinensis were extracted, chemically characterized and evaluated on different<br />
microorganisms such us Salmonella thyphimirium, Escherchia coli, Staphylococcus aureus and<br />
Listeria innocua. We choose to work with C. citratus from Rwanda because <strong>of</strong> its uses as food<br />
additive and its reported biological activity (Onawunmi et al., 1984, Negrelle et al, 2007) while peels<br />
<strong>of</strong> C. lemon and C. sinensis were chosen because <strong>of</strong> their important amount found in Rwanda and<br />
their useless consideration. This study intended to valorise them by recuperating the essential oils<br />
they contain and verify their possible economic application. The biological activity study <strong>of</strong> essential<br />
oils was done for their possible application as biocides, especially in food chemistry. Note that the<br />
microbes used are generally contaminating foods and thus further studies on the obtained active<br />
essential oils should be done to verify if they can be used as food preservative compounds.<br />
Material and methods<br />
Plant materials<br />
Fresh leaves <strong>of</strong> Cymbopogon citratus were collected from the Garden <strong>of</strong> CURPHAMETRA (Centre<br />
Rwandaise de la Recherche sur la Pharmacopée et la Médécine Tradionnelle) in the Southern<br />
Province <strong>of</strong> Rwanda while peels <strong>of</strong> Citrus lemon and Citrus sinsensis were obtained from fruits<br />
collected in Tumba sector at Huye District, Southern Province <strong>of</strong> Rwanda. All these collections were<br />
done in the beginning <strong>of</strong> dry season, precisely in the month <strong>of</strong> July.<br />
Extraction process<br />
In this study the hydro-distillation <strong>of</strong> fresh peels or leaves was conducted using Clevenger-type<br />
apparatus. The essential oils obtained were dried on anhydrous sodium sulphate and kept into<br />
opaque vials at 0°C for further studies.<br />
Microbial strain and media<br />
Staphylococcus aureus (Institut Pasteur 25923), Escherichia coli (Institut Pasteur 25922), Salmonella<br />
typhimurium (Institut Pasteur 5858) and Listeria innocua (ISTAB, Université Bordeaux 1) were<br />
maintained at - 4°C in 20% <strong>of</strong> glycerol. Overnight pre-cultures were performed as follows:<br />
Staphylococcus aureus and Listeria innocua were grown in Tryptose broth (Difco 262200) while<br />
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Escherichia coli and Salmonella typhimurium were grown in nutrient broth (Difco 234000) at 37°C<br />
for 18 h.<br />
Antibacterial Activity Assessment<br />
The antibacterial assessment <strong>of</strong> pure essential oils was conducted using an agar plate method<br />
performed by a diffusion method. To do this, 20 mL <strong>of</strong> culture medium prepared by mixing tryptose<br />
broth (Difco 262200) or nutrient broth (Difco 234000) with 15%(w/w) agar (Difco 215530) for L.<br />
innocua or S. aureus and S. typhimurium or E. coli, respectively, was poured into each Petri dish.<br />
Then, one-hundred microliters <strong>of</strong> 10 -3 diluted inoculums from the microbial culture was gently<br />
spread on the surface <strong>of</strong> agar medium. Six-millimeter-diameter cellulosic disks were deposited on<br />
the agar medium surface and 10 L <strong>of</strong> essential oil were gently deposited on the disk. Control disks<br />
without any essential oil were concurrently tested. Thereafter plates were incubated at 37°C for 24<br />
h prior to determination <strong>of</strong> the diameters <strong>of</strong> inhibition zones surrounding the disks. Each test was<br />
performed three times and means <strong>of</strong> diameters <strong>of</strong> inhibition zones were calculated.<br />
Gas chromatography<br />
The essential oils were dissolved in ether prior to have a dilution <strong>of</strong> 100 times. The obtained<br />
samples were then analysed on Thermo Finnigan Gas Chromatography fitted with BP 21 SGE=FFAP<br />
column (25 m x 0.22 mm). The oven temperature was programmed from 50°C to 200°C at 4.5°C<br />
min -1 , the detector and injector temperatures were set at 240°C and 180°C respectively while<br />
Helium was used as carrier gas. Different internal references were used to identify essential oil<br />
components. Results obtained from GC were completed by those obtained from coupled Gas<br />
chromatography/mass spectrometry Ultra DSQ Thermo.<br />
Results and discussion<br />
From fresh leaves and peels, the essential oils were obtained with yields <strong>of</strong> 1.3, 0.19 and 0.16 % for<br />
C. citratus leaves, C. lemon and C. sinensis peels respectively. The yield from C. citratus was more<br />
than that found in previous reports (Onawunmi et al. (1984), 0.80-0.98% and Cimanga et al. (2002),<br />
0.3%), but is still being in the range reported by Negrelle et al., 2007. Essential oils from C. sinensis<br />
and C. limon have been used in different studies, but no yields <strong>of</strong> these oils have been reported (Hili<br />
et al; 1996; Caccioni et al., 1998; Steuer et al, 2001). All essential oils were then analysed on GC and<br />
GC/MS to determine their chemical composition. C. citratus essential oil is highly composed by<br />
geranial and neral with percentages <strong>of</strong> 33.0 and 49.7% respectively (Table 1 and figure 1). These<br />
two chemical components are mainly known as trans- and cis- citral respectively. The total amount<br />
<strong>of</strong> citral found in the Rwandese C. citratus essential oil, 82.7%, is quite similar on what reported<br />
(Negrelle et al, 2007 (70-80%)) but higher than that reported by Cimanga et al., 2002 (32.7%).<br />
Furthermore, this oil presented also -Myrcene and -phellandrene with percentages <strong>of</strong> 3.8 and<br />
2.5 respectively. The main component found from the essential oils <strong>of</strong> C. lemon and C. sinensis was<br />
limonene in the quantities <strong>of</strong> 77.5 and 83.3 respectively (Table 1 and Figures 2 and 3). These<br />
essential oils also contain -phellandrene with percentages <strong>of</strong> 8.1 and 10.8 respectively for C. lemon<br />
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and C. sinensis. In addition, the C. lemon oil indicates a remarkable percentage <strong>of</strong> (Z)-hex-3-enyl<br />
acetate (6.0%).<br />
Table 1: Chemical composition <strong>of</strong> essential oil <strong>of</strong> C. citratus, C. lemon and C. sinsensis<br />
Components C. citratus C. lemon C. sinensis<br />
-pinene tr 0.8 0.5<br />
Bicyoclo (3.1.0)hex-2-ene,4-methylene-1-(1methylethyl)tr<br />
tr tr<br />
Camphene tr<br />
-Phellandrene 8.1 10.8<br />
-Pinene 0.4 0.4 0.1<br />
(Z)-hex-3-enyl acetate 6.0 1.3<br />
-Myrcene 3.4 1.5 1.3<br />
-Phellandrene 2.5 0.2 0.1<br />
p-Cymene 0.5 0.1<br />
Limonene 0.5 77.5 83.3<br />
Eucalyptol 0.3<br />
3-Carene (chercher nom) 0.2<br />
-Terpinene 0.7<br />
Terpinolene 0.2 tr<br />
Citronellal 1.4<br />
Longifolene 1.2<br />
Caryophyllene 0,8 1.3<br />
Humulene 0.3<br />
Citronellyl acetate 0.5 0.8<br />
(cis )-p-(2-menthen)-1-ol 1.5<br />
Methyl geranate 0.5<br />
Cis-Verbenol 0.72<br />
Cis-Carveol 1.19<br />
Trans-Citral (=Geranial) 33.0<br />
Cis- Citral (=Neral) 49.7<br />
- Terpenyl acetate 2.4<br />
Total peak area 98.51 97.7 99.8<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Figure 1: Chromatogram <strong>of</strong> essential oil from C. citratus leaves<br />
Figure 2: Chromatogram <strong>of</strong> essential oil from C. lemon peels<br />
Figure 3: Chromatogram <strong>of</strong> essential oil from C. sinensis peels<br />
Biological activity <strong>of</strong> these three essential oils was evaluated on four food contaminating bacteria,<br />
E. coli, S. typhimurium, L. innocua and S. aureus. Disk-diffusion method was used to assess this<br />
activity. All essential oils tested showed the antibacterial activity but at different means. The oil<br />
from C. citratus was the more effectiveness compared to the remaining two others. This oil<br />
inhibited the bacteria with an inhibition diameter varying from 16 to 85 mm respectively for a Gram<br />
negative bacteria, S. typhimurium and a Gram positive bacteria, L. innocua (Table 2). The<br />
antibacterial activity <strong>of</strong> the oil extracted from C. lemon was observed with the inhibition diameters<br />
varying from 15 mm to 18 mm for E. coli, a gram positive bacteria and L. innocua. The less active<br />
essential oil is that obtained from C. sinensis with an inhibition diameter <strong>of</strong> 9 mm for L. innocua.<br />
According to Johnson and Case (1995), the activity <strong>of</strong> biocides can be considered as effective<br />
against bacteria when the inhibition diameter is over 16 mm. In this study, the essential oil from C.<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
citratus and Citrus lemon led to an inhibition diameter close or more than 16 mm and thus they<br />
might be considered as effective against all bacteria tested. This study showed that the Gram<br />
positive bacteria were less resistant to the effective essential oils than Gram negative ones. The<br />
mechanism <strong>of</strong> action <strong>of</strong> these essential oils is not known but different sensitivity between these<br />
bacteria can be explained by the difference noted in the chemical compositions <strong>of</strong> their cell walls<br />
(Muhizi et al., 2009). Differences remarked in the antibacterial activity <strong>of</strong> essential oils are closely in<br />
relationship with their chemical composition. The more effective essential oil from C. citratus is<br />
mainly composed by citral (trans and cis form). The antibacterial activity <strong>of</strong> these compounds was<br />
reported in previous works (Negrelle et al, 2007). According to these authors, it is not surprising<br />
that this oil become more active than those from C. lemon and C. sinensis which don t possess any<br />
trace <strong>of</strong> citral. The essential oil from C. lemon is more active than that from C. sinensis. In this study<br />
we didn t be able to determine the reason <strong>of</strong> this difference since the main component, Limonene,<br />
<strong>of</strong> these two oils is found in the quite same concentration (table 1). The more potent activity <strong>of</strong> C.<br />
lemon may be due to the other components found in small quantity.<br />
Table 2: Inhibition <strong>of</strong> E. coli, S. typhimurium, L. innocua and S. aureus by different essential oils<br />
Essential<br />
oils<br />
E. coli S. typhimurium L. innocua S. aureus<br />
C. citratus 1.9 0.1 1.6 0.2 8.5 0.0 6.8 0.3<br />
C. lemon 1.5 0.1 1.6 0.1 1.8 0.2 1.7 0.2<br />
C. sinensis 0.7 0.1 0.8 0.1 0.9 0.1 0.7 0.1<br />
In conclusion, essential oils from Cymbopogon citratus and Citrus lemon showed more pronounced<br />
antibacterial activity against bacteria contaminating food and can be further studied for their<br />
possible application as food preservative.<br />
Acknowledgements<br />
The authors thank the NUR-SIDA Project and NUR Research Commission for funding this work.<br />
Many thanks also to Ms Mélanie BOSQUET for technical assistance.<br />
References<br />
Caccioni, D. R.L., Guizzardi, M., Biondi, D. M., Renda, A., Ruberto, G. (1998); Relationship between volatile components <strong>of</strong> citrus fruit<br />
essential oils and antibacterial action on penicillium digitatum and Pencillium italicum, Int. J. <strong>of</strong> Food Microbiol. 43, 73-79<br />
CE. Directive 98/8/CE (1998); du Parlement Européen et Conseil du 16 février 1998 concernant la mise sur les marchés des produits<br />
biocides, J. Offic.Comm. Eur., L123/1-L123/63.<br />
CE. Directive 2001/90/CE (2001); de la Commission du 26 Octobre 2001 portant septième adaptation au progrès technique<br />
(Créosote) de l annexe I de la directive 76/769 CEE du Conseil concernant le rapprochement des dispositions législatives,<br />
réglementaires et administratives des Etats membres relatives à la limitation de la mise sur le marché et de l emploi de<br />
certaines substances et préparation dangereuses, J. Officiel Comm. Eur., , L283/41-L283/43.<br />
Cimanga, K., Kambu, K., Tona, L., Apers, S., De Bruyne, T., Hermans, N., Totté, J., Pieters, L., Vlietinck, A.J. (2002); Correlation<br />
between chemical composition and antibacterial activity <strong>of</strong> essential oils <strong>of</strong> some aromatic medicinal plants growing I the<br />
democratic republic <strong>of</strong> Congo, J. Ethnopharmac. 79, 213-220.<br />
Cooper, G. S.; Jones, S. (2008); Pentachlophenol and cancer risk: Fousing the lens on specific chlorophenols and contaminants-<br />
Review, Envir. Health Perspect., 116 (8), 1001-1008. Corrégé, I. La problématique salmonelles en filière porcine. Techni. Porc<br />
2001, 24 (2), 25-31.<br />
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FAO/UNEP, (1996); Pentachlorophenol and its salts and esters In Operation <strong>of</strong> the prior informed consent procedure for banned or<br />
severely restricted chemicals in international trade, Decision guidance documents (FAO, UNEP, Eds.), Rome- Geneva, 46-63.<br />
Focant, J. F.; Pirard, C.; Douny, C.; Scippo, M. L.; De Pauw, E.; Maghuin-Rogister, G. (2002); Le point, trois ans après, sur<br />
« la crise berge de la dioxine » , Impact probable sur la santé de la population belge, Ann. Méd. Vét., 146, 321-327.<br />
Gouv. F. Décret n o 2004-1227 du 17 novembre 2004 relatif aux conditions de mise sur le marché et d emploi de<br />
l arsenic et de ses composés, du colorant bleu,du pentacbromodiphényléther et de l octabromodiphényléther et<br />
modifiant le décret no 92-1074 du 2 octobre 1992, J. Officiel Rép. Française, texte 31 sur 83, 2004, 3 p.<br />
GTIF. Utilisation de Créosote dans le traitement du bois: point reglémentaire sur l arrêté du 2 juin 2003, Bulletin info<br />
sécurité, 2003, 3 p.<br />
Hanson, R. ; Dodoo, D. K. ; Essumang, D. K. ; Blay, J. ; Yankson, Jr. K. (2007); The effect <strong>of</strong> some selected pesticides on<br />
the growth and reproduction <strong>of</strong> fresh water Oreochromis niloticus, Chrysicthys nigrodigitatus and Clarias<br />
gariepinus, Bull. Environ. Contam. Toxicol., 79, 544-547.<br />
Hili , P. Evans, C.S., Veness, R.G. (1997); Antimicrobila action <strong>of</strong> essential oils: the effect <strong>of</strong> dimethylsulphoxide on the<br />
activity <strong>of</strong> cinnamon oil, Letters in applied microbiology, 24, 269-275<br />
Hughes, W. W. (1996); Essentials <strong>of</strong> environmental toxicology- The effects <strong>of</strong> environmentally hazardous substances on<br />
Human Health, Taylor & Francis, Bristol, 176 p.<br />
Ibrahim, M. A. Kainulainen, P., Aflatuni, A., Tiilikkala, K. Holopainen, (2001); Insecticidal, repellent, antimicrobial activity<br />
and phytotoxicity <strong>of</strong> essential oils: with special reference to limonene and its suitability for ontrol <strong>of</strong> insect pests,<br />
Agricultural and food science in Finland, 10, 243-259.<br />
INERIS. Données technico-économiques sur les substances chimiques en France : Hexachlorobenzène, Ineris, Paris,<br />
2005, 13 p.<br />
INERIS. Données technico-économiques sur les substances chimiques en France : Aldrine, Ineris, Paris, 2007, 11 p.<br />
Johnson and Case, (1995); Laboratory Experiments in Microbiology, 4th ed.; Benjamin Cummings Publishing Co:<br />
Redwood City, CA, 445 pp.<br />
Kamrin, M. (1998); Toxicity <strong>of</strong> arsenic, This Old House Magazine, 17, 118-125.Knight, D. J.;<br />
Cooke, M. (2002); The biocides business: regulation safety and applications, Wiley-VCH Verlag GmbH, Weinheim, 380 p.<br />
Korsak, N.; Clinquart, A.; Daube, G. (2004); Salmonella spp. dans les denrées alimentaires d origine animale : un réel<br />
problème de santé publique. Ann. Méd. Vét., 148, 174-193. 30-34)<br />
Muhizi, T. (2008); Synthèse d aminosucres conduisant à des biocides d origine naturelle, Thèse, Université Bordeaux 1,<br />
188 p.<br />
Muhizi, T., Grelier, S., Coma, V., (2009); Synthesis <strong>of</strong> N-Alkyl- -D-glucosylamines and Their<br />
Antimicrobial Activity against Fusarium proliferatum, Salmonella typhimurium, and Listeria innocua, J. Agric. Food<br />
Chem. 57(23), 11092 11099<br />
Negrelle R.R.B., Gomes, E.C. (2007); Cymbopogon citratus (DC) stapf: chemical Composition and biological activities.<br />
Rev. Bras. Pl. Med. 9(1), 80-92<br />
Onawunmi, G. O., Yisak, W., Ogunlana, E.O. (1984); Antibacterial constituents in essential oil <strong>of</strong> cymbopogon citratus<br />
(DC) stapf, J. Ethnopharm. 12, 279-286<br />
Prazak, M. A.; Murano,E. A.; Mercado, I.; Acuff, G. R. (2002); Antimicrobial resistance <strong>of</strong> Listeria monocytogenes<br />
isolated from various cabbage farms and packing sheds in Texas. J. Food. Prot. 65(11), 1796-1799.<br />
Steuer, B. Schulz, H., Lager, E. (2001); Classification and analysis <strong>of</strong> Citrus oils by NIR spectroscopy, Food chemistry, 72,<br />
113-117.<br />
Takahashi, T., (2002); Bactericides, US 6,352,727 B1, p. 30<br />
Walsh, D.; Duffy, G.; Sheridan, J. J.; Blair, I. S.; McDowell, D. A. (2001); Antibiotic resistance among Listeria, including<br />
Listeria monocytogenes, in retail foods. J. Appl. Microbiol. 90 (4), 517-522.<br />
White, D. G.; Zhao, S.; Sudler, R.; Ayers, S.; Friedman, S.; Chen, S.; McDermott, P. F.; McDermott, S. Wagner, D. D.;<br />
Meng, J. (2001); The isolation <strong>of</strong> antibiotic-resistant salmonella from retail ground meats. N. Engl. J. Med., 345 (16),<br />
1147-1154.<br />
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[SL 6A] Chemical constituents <strong>of</strong> the essential oil <strong>of</strong> Cymbopogon proximus and their<br />
potential for the treatment <strong>of</strong> Otomycosis<br />
Osman N.A 1* , Mohamed U.I. 1 , Ahmed N.E. 2 , and Elhussein S.A 3<br />
1<br />
Biotechnology Center, Faculty <strong>of</strong> Engineering and Technology, University <strong>of</strong> Gezira, Sudan.<br />
2<br />
Pathology Department, Agricultural Research Corporation,Wad-Medani. Sudan.<br />
3<br />
Faculty <strong>of</strong> Industrial Sciences and Technology, Unversiti Malaysia Pahang, Malaysia<br />
*Corresponding author: nourug@yahoo.com<br />
Keywords: Cymbopogon proximus, automycosis, essential oil, estragole PTLC-plate<br />
Introduction<br />
T<br />
he indigenous Cymbopogon proximus <strong>of</strong> the family poaeceae ( Graminae) growth as wild<br />
aromatic plant in Sudan, and used locally as carminatives, stimulatives, antiseptics, and for<br />
treatment <strong>of</strong> rheumatism, cholera ( Heiba, 1983). Fungal species associated with automycosis<br />
disease in Sudanese patients were Aspergillus niger, Aspergillus Flavus, and Aspergillus aculeatus<br />
(Elmustafa & Elmahi, 1999)<br />
Objective<br />
The main study objective was to test essential oil <strong>of</strong> C. proximus against main organisms responsible<br />
for otomycosis in Sudanese patients. Elucidating the nature <strong>of</strong> the active chemical ingredient was<br />
another target.<br />
Material and Methods<br />
The essential oil was prepared from the leaves <strong>of</strong> C. proximus by steam distillation method (<br />
Wagner, 1984). Fungal growth inhibition was obtained according to the agar plate-hole diffusion<br />
method and disc-diffusion method. Chemical analyses were carried out by FTIR, GLC, TLC, GC-MS<br />
and MS spectra.<br />
Result and Discussion<br />
The major fungal casual agent for automycosis in Sudan is : A. niger (60%), followed by A. flavus<br />
(30%). The extracted essential oil inhibited both <strong>of</strong> the test microorganisms in a dose-dependant<br />
manner. A dose <strong>of</strong> 20 l/ml essential gave 100% kill for both A. niger and A. flavus. The MIC was<br />
between 0.2 and 0.6 l/ml for A. niger and between 2 and 4 l/ml for A. flavus. The LC50 was 0.6<br />
l/ml for A. niger and 8.0 l/ml for A. flavus respectively. Only two out <strong>of</strong> ten components separated<br />
on PTLC-plate from leaves, <strong>of</strong> C. proximus essential oil, were found active against both tested<br />
microorganisms. The FTIR and MS spectra for the most active compound pointed to the presence <strong>of</strong><br />
alcoholic compound with M.W. 198 and chemical formula : C12H18O1. According to GC-MS<br />
chromatographic analysis, the major compound <strong>of</strong> indigenous C. proximus essential oil is estragole<br />
(43%).<br />
References<br />
1- Heiba, H. I. (983); "Chemical investigation <strong>of</strong> Cymbopogon proximus." Ph.D. Thesis, Cairo University.<br />
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2- Elmustafa, O. M. and Elmahi, N. A. (1999); "The causative organisms <strong>of</strong> automycosis in Sudanese patients." Journal <strong>of</strong><br />
Arab council for medical Specializion (Arabic), (4), pp 36-38.<br />
3- Wagner; H., Blaat; S., and Zgainski; E. M. (1984); "Plant Drug Analysis". Springer-Verlag, Berlin, New York, Tokyo.<br />
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[SL 6B] Antimicrobial Activity and Phytochemical Studies <strong>of</strong> Some Selected<br />
Medicinal Kenyan Plants<br />
Machocho A.K.<br />
Department <strong>of</strong> Chemistry; Kenyatta University<br />
C<br />
rude extracts <strong>of</strong> some plants species reported by herbalists to treat various bacterial and fungal<br />
complications were subjected to screening for the activity by use <strong>of</strong> various strains <strong>of</strong><br />
microbials in vitro. The plants are Tabernaemontana stapfiana, Echinops hispidus, Grewia similis,<br />
Ochna holtzii, Teclea nobilis among others. The extracts that showed positive or promising activity<br />
were subjected to fractionation using separation techniques including various chromatographic<br />
methods. Structures <strong>of</strong> the obtained compounds were elucidated by use <strong>of</strong> spectroscopic<br />
techniques which included IR, MS, proton and carbon-13 NMR. 2-D NMR (COSY, NOESY, HMQC and<br />
H<strong>MB</strong>C) was employed for complete elucidation. Some <strong>of</strong> the isolated compounds were equally<br />
tested antimicrobial activity some interesting results were reported. For example, methanolic<br />
extract <strong>of</strong> T. Stapfiana gave an average inhibition zone <strong>of</strong> 20 mm to various bacteria strains.<br />
Compounds responsible are bis-indole alkaloids like conodurine. Crude extract <strong>of</strong> E. hispidus<br />
showed strong antifungal activity and compounds responsible were polyacetylene thiophenes<br />
which gave an average inhibition <strong>of</strong> 23 mm. The results call for in vivo bioassay and toxicity<br />
evaluations.<br />
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[SL 7A] Antiplasmodial and Radical Scavenging Activities <strong>of</strong> Flavonoids from<br />
Kenyan Erythrina species<br />
Abiy Yenesew a* , Hoseah M Akala b,c Hannington Twinomuhwezi a , Martha Induli a,c , Beatrice Irungu d ,<br />
Fredrick L. Eyase b , Solomon Derese a , Bernard T. Kiremire c , Jacob O. Midiwo a , Norman C. Waters e<br />
a<br />
Department <strong>of</strong> Chemistry, University <strong>of</strong> Nairobi, P. O. Box 30197, Nairobi, Kenya<br />
b<br />
United States Army Medical Research Unit-Kenya, Walter Reed Project, Kisumu, MRU 64109, APO, AE 09831-4109,<br />
USA<br />
c<br />
Department <strong>of</strong> Chemistry, Makerere University, P. O. Box 7062 Kampala, Uganda<br />
d<br />
Center for Traditional Medicine and Drug Research, Kenya Medical Research Institute (KEMRI), P.O. Box 54840, Nairobi<br />
00200, Kenya<br />
e<br />
Department <strong>of</strong> Chemistry and Life Science, United States Military Academy, West Point, New York, NY 10996<br />
Abiy Yenesew: ayenesew@uonbi.ac.ke<br />
Keywords: Erythrina, antiplasmodial, radical scavenger, pterocarpan, flavanone, is<strong>of</strong>lav-3-ene, arylbenz<strong>of</strong>uran.<br />
T<br />
he success <strong>of</strong> quinine and artemisinin as potent natural antimalarial drugs demonstrates the<br />
importance <strong>of</strong> plants, especially those used in traditional medicine, as potential source <strong>of</strong><br />
antimalarial agents. Erythrina abyssinica (Leguminosae) is one <strong>of</strong> the most widely used plants to<br />
treat malaria in East Africa. The root bark <strong>of</strong> this plant showed antiplasmodial activity against the<br />
chloroquine sensitive (D6) and chloroquine resistant (W2) strains <strong>of</strong> Plasmodium falciparum, with<br />
IC50 values <strong>of</strong> 0.64 and 0.49 g/ml, respectively (Yenesew et al., 2003). Several compounds isolated<br />
from this plant (Kamat et al., 1981; Yenesew et al., 2003) were also tested (Yenesew et al., 2003;<br />
2004). Activity was observed among pterocarpans (e.g. erythrabyssin-II, IC50 8.1 and 6.5 M against<br />
the D6 and W2 strains, respectively), and flavanones (e.g. abyssinone-IV, IC50 9.0 and 7.7 M<br />
against D6 and W2 strains, respectively). However the activities <strong>of</strong> these compounds individually<br />
are much lower than that <strong>of</strong> the crude extract, indicating that these flavonoids and is<strong>of</strong>lavonoids<br />
may be more effective as mixtures.<br />
HO O<br />
O<br />
OH<br />
HO<br />
O<br />
Erythrabyssin II<br />
Abyssinone-IV<br />
Four additional Erythrina species <strong>of</strong> Kenya, namely E. burttii, E. melanacantha and E. sacleuxii, have<br />
been tested for antiplasmodial activities. Among these the root bark <strong>of</strong> E. burttii showed good<br />
antiplasmodial activity with IC50 value <strong>of</strong> 0.97 and 2.0 g/ml against the D6 and W2 strains <strong>of</strong><br />
106<br />
O<br />
OH
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Plasmodium falciparum, respectively. Flavonoids and is<strong>of</strong>lavonoids including is<strong>of</strong>lav-3-enes (eg<br />
burttinol A) and an aryl benz<strong>of</strong>uran burttinol D have been identified as the active principles. The<br />
root bark <strong>of</strong> E. sacleuxii was also active, with the most active compound being the is<strong>of</strong>lavone<br />
erusubin F (IC50 9.0+2.1 and 7.7+1.6 M against D6 and W2 strains respectively (Yenesew et al.,<br />
2006). In an in vivo assay, the extracts <strong>of</strong> the roots E. abyssinica, E. burttii and E. sacleuxii showed<br />
significant antimalarial activities against Plasmodium berghei.<br />
Oxidative stress normally follows malaria infection. This is due to elevated production <strong>of</strong> reactive<br />
oxygen species (Bahorun et al., 1996). It is therefore important that cells are protected from<br />
oxidative burden through the use <strong>of</strong> effective antioxidants. In a Radical Scavenging Activity (RSA)<br />
assay against 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical, using spectrophotometric method,<br />
the crude acetone extract <strong>of</strong> the root bark <strong>of</strong> E. abyssinica at 10 g/ml showed RSA <strong>of</strong> 82.2 %. In<br />
activity guided fractionation <strong>of</strong> the crude acetone extract, the pterocarpene erycristagallin, was<br />
identified as the most active principle with EC50 value <strong>of</strong> 8.2 M (Yenesew et al., 2009). The root<br />
bark <strong>of</strong> Erythrina burttii also showed high RSA activity (EC50 values <strong>of</strong> 10.8 g/ml) and the is<strong>of</strong>lav-3enes<br />
burttinol A and the aryl benz<strong>of</strong>uran burttinol D have been identified as the most active<br />
principles.<br />
HO<br />
O<br />
Burttinol A<br />
HO O<br />
O<br />
Erycristagallin<br />
OMe<br />
OH<br />
OH<br />
HO<br />
In conclusion, the wide traditional use <strong>of</strong> Erythrina abyssinica for treatment <strong>of</strong> malaria in East Africa<br />
has been justified. Antiplasmodial and radical scavenging activities have also been observed in<br />
other Erythrina species, E. burttii showing the highest activity. The activities <strong>of</strong> these plants are<br />
107<br />
HO<br />
O<br />
O<br />
Erysubin F<br />
O<br />
MeO<br />
Burttinol D<br />
OH<br />
OH
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
associated with flavonoids and is<strong>of</strong>lavonoids which appear to be more effective as mixtures rather<br />
than as pure compounds.<br />
References<br />
Andayi, A.W., Yenesew, A., Derese, S., Midiwo, J.O., Gitu, P.M., Jondiko, O.I., Waters, N., Liyala, P., Akala, H.,<br />
Heydenreich, M. and G Peter M. (2006); Anti-plasmodial activities <strong>of</strong> flavonoids from Erythrina sacleuxii Planta<br />
Medica 72, 187-189.<br />
Bahorun T., Gressier B., Trotin F., Brunet C., Dine T., Luycks M., Vasseur J., Cazin J., Pinkas M. (1996); Oxygen species<br />
scavenging activity <strong>of</strong> phenolic extracts from Hawthorn fresh plant organs and pharmaceutical preparations.<br />
Arzneimittel-Forschung / Drug Resarch 46, 1086-1089.<br />
Kamat, V.S., Chuo, F.Y., Kubo, I. and Nakanishi, K. (1981); Antimicrobial agents from an East African Medicinal Plant<br />
Erythrina abyssinica Heterocycles, 15, 1163-1170.<br />
Yenesew, A., Derese, S., Irungu, B., Midiwo, J.O., Waters, N.C., Liyala, P., Akala, H., Heydenreich, M. and Peter, M.G.<br />
(2003); Flavonoids and is<strong>of</strong>lavonoids with anti-plasmodial activities from the roots <strong>of</strong> Erythrina abyssinica Planta<br />
medica, 69, 658-661.<br />
Yenesew, A., Induli, M., Derese, S., Midiwo, J.O., Hedenreich, M., Peter, M.G., Akala, H., Wangui, J., Liyala, P., Waters,<br />
N.C. (2004). Anti-plasmodial Flavonoids from the roots <strong>of</strong> Erythrina abyssinica. Phytochemistry, 65, 3029-3032.<br />
Yenesew, A., Midiwo, J.O., Heydenreich, M. and Peter, M.G. (1998); Four is<strong>of</strong>lavones from stem bark <strong>of</strong> Erythrina<br />
sacleuxii . Phytochemistry, 49, 247-249.<br />
Yenesew, A., Midiwo, J.O., Miessner, M.,. Heydenreich, M. and Peter, M.G. (1998); Two prenylated flavanones from<br />
stem bark <strong>of</strong> Erythrina burttii. Phytochemistry, 48, 1439-1443<br />
Yenesew, A., Twinomuhwezi, H., Kiremire, B.T., Mbugua, M.N., Gitu, P.M. , Heydenreich M., Peter M.G. (2009); 8-<br />
Methoxyneorautenol and Radical Scavenging flavonoids from Erythrina abyssinica. Bull. Chem. Soc. Ethiopia 23,<br />
205-210.<br />
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[SL 7B] Inventory and Pharmacological Screening <strong>of</strong> Selected Indigenous Plant<br />
Species <strong>of</strong> Namibia for Development <strong>of</strong> New Natural Products.<br />
Brendler, T. 1 , Mortensen, D.J. 1 , Simon, J.E. 2 , Raskin, I. 3 , Feiter, U. 4<br />
1 Plantaphile, Belforter Strasse 20, Berlin, 10405, Germany, txb@plantaphile.eu<br />
2 Rutgers University, Department <strong>of</strong> Plant Biology and Plant Pathology, 59 Dudley Road, New Brunswick, NJ 08901, USA<br />
3 Rutgers University, School <strong>of</strong> Environmental and Biological Sciences, 59 Dudley Road, New Brunswick, NJ 08901, USA<br />
4 AAMPS, c/o Executive Services Ltd., 2nd Les Jamalacs Building, Vieux Conseil Street, Port Louis, Mauritius<br />
Keywords: Natural product development, Namibia, indigenous plant use, pharmacological screening, quality assurance<br />
W<br />
e present the outline and methodology <strong>of</strong> a project conducted in collaboration with Rutgers<br />
University, GIBEX, and AAMPS, which is funded by the Namibian Millennium Challenge<br />
account. This is a work in progress, having started in March 2011 and to be completed within<br />
approximately one year. We have surveyed and inventoried the utilized indigenous Namibian flora<br />
for the purpose <strong>of</strong> identifying potentially new ingredients for indigenous Namibian natural products<br />
in foods, flavors, health, nutrition, and cosmetic products in a context <strong>of</strong> local sustainable economic<br />
development. The survey and inventory has drawn from all available sources including early<br />
explorer's and other historical accounts in order to include traditional plant use which may have<br />
been lost over time (von Koenen 2008). From this inventory some 100 species are being selected<br />
for investigation based on a variety <strong>of</strong> criteria, with focus on local stakeholder's interests. Samples<br />
and herbarium specimen are being collected and uses will be verified in the field. Plant material is<br />
subjected to Screens-to-Nature technology (GIBEX 2011), where we introduce 11 portable, fielddeployable<br />
pharmacological screens, and facilitate into Namibia the training associated with their<br />
use. This approach does not remove any natural resources from its country <strong>of</strong> origin for the<br />
purpose <strong>of</strong> analysis. Based on the results <strong>of</strong> the screening process, select species will be subjected<br />
to further investigation including chemical pr<strong>of</strong>iling, development <strong>of</strong> quality assurance standards,<br />
regional and international market assessments, sustainability (wild-crafting vs. cultivation) and<br />
feasibility <strong>of</strong> creating a product development value chain benefitting Namibian PPOs. This work will<br />
lead to a shortlist <strong>of</strong> around 10 candidate species for product development employing sciencebased<br />
marketing strategies and establishing public-private sector partnerships.<br />
References<br />
Koenen, E v (2008). Namibias Heilkunde im Wandel. Hess, Windhoek.<br />
GIBEX (2011). Drug Discovery Paradigm. http://www.gibex.org/index.php?suj=91, accessed June 1 st , 2011<br />
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[SL 8A] Isolation and Structure Elucidation <strong>of</strong> Bioactive Compounds from the<br />
Tropical Liana Ancistrocladus congolensis<br />
C. Steinert, G. Bringmann *<br />
Institute <strong>of</strong> Organic Chemistry, University <strong>of</strong> Würzburg, Am Hubland, D-97074 Würzburg<br />
bringman@chemie.uni-wuerzburg.de<br />
Keywords: Naphthylisoquinoline alkaloids · Ancistrocladus congolensis · michellamine · 2D NMR experiments · CD<br />
spectroscopie · oxidative degradation<br />
Introduction<br />
T<br />
he small plant family <strong>of</strong> the Ancistrocladaceae comprises approximately 18 species in the<br />
palaeotropic regions and, up to now, one single genus named Ancistrocladus [Taylor et al.<br />
2005]. These lianas feature hooked branches as climbing implements, and are closely related to the<br />
Dioncophyllaceae, tropical lianas with hooked leaves. The two plant families are used in traditional<br />
African and Asian medicine against dysentery, malaria, African sleeping sickness and leishmaniasis<br />
[Franois et al. 1997]. The bioactivity results from naphthylisoquinoline alkaloids, an extraordinary<br />
class <strong>of</strong> biaryls, only found in Ancistrocladaceae and Dioncophyllaceae species. The<br />
naphthylisoquinolines consist <strong>of</strong> a naphthalene and an isoquinoline moiety, coupled via a biaryl<br />
axis. The axis joins the two molecule halves at various positions and usually is rotationally hindered.<br />
Several naphthylisoquinolines, C,C-coupled ones, like dioncophylline A and C and ancistrolikokine B,<br />
and N,C-coupled ones, like ancistrocladinium B, have shown in vitro and in vivo activities against<br />
pathogens <strong>of</strong> tropical diseases.<br />
Figure 1: selected naphtylisoquinoline alkaloids with excellent bioactivities against pathogens <strong>of</strong> the<br />
African sleeping sickness, malaria, and leishmaniasis.<br />
Previous work in our research group on Ancistrocladus congolensis showed the presence <strong>of</strong><br />
numerous interesting naphthylisoquinolines and also naphthylisoquinoline dimers, which are<br />
known for their anti-HIV activity [Boyd et al. 1994].<br />
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Materials and Methods<br />
Ground root bark <strong>of</strong> Ancistrocladus congolensis (collected in the Democratic Republic <strong>of</strong> the Congo<br />
in 2002) was extracted with methanol/dichloromethane (1:1, v/v) for several days. The solvents<br />
were evaporated in the vacuum, the resin was ground and mazerated in H2O (dest.), supported by<br />
ultrasonic sound. The water-resin-suspension was filtered, and the water phase was extracted with<br />
dichloromethane. The organic phase was evaporated to dryness, the water phase was frozen und<br />
lyophilized. The resin from the water phase was applied to the preparative HPLC. After several<br />
isolation circles, 3 pure fractions containing monomeric naphthylisoquinolines and 7 pure fractions<br />
containing dimeric naphthylisoquinolines were gained.<br />
Results and Discussion<br />
The 3 monomeric naphthylisoquinolines were identified with the aid <strong>of</strong> exact mass, 2D NMR<br />
experiments, CD spectroscopie, and oxidative degradation as korupensamine A, 5 -O-demethylhamatine<br />
and an ancistrocongoline derivative (figure 2).<br />
Figure 2: Korupensamine A, 5 -O-demethyl-hamatine, and ancistrocongoline derivative.<br />
The first dimeric napthylisoquinoline alkaloid was identified as michellamine A (figure 3), the<br />
corresponding dimer <strong>of</strong> korupensamine A. Three <strong>of</strong> the further dimers were found to have the<br />
same mass as michellamine A, meaning to be isomers, like michellamine B and C (figure 3).<br />
Figure 3: michellamines A, B, and C<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
The last two dimers corresponded to the mass m/z = 770, supposably being identical with or isomeric to<br />
michellamine D or F (figure 4).<br />
Figure 4: michellamines D and E<br />
Acknowledgements<br />
I thank Pr<strong>of</strong>. Virima Mudogo from the University <strong>of</strong> Kinshasa (DR Congo) for the collection <strong>of</strong><br />
Ancistrocladus congolensis plant material. From the University <strong>of</strong> Wuerzburg I thank Dr. Gruene and<br />
Dr. Buechner for the 2D NMR as well as the exact mass measurements. I thank B. Amslinger for CD<br />
acquisition, and M. Michel for the oxidative degradation.<br />
References<br />
C. M. Taylor, R. E. Gereau, G. M. Walters, (2005); Ann. Missouri Bot. Gard. 92, 360-399.<br />
G. François, G. Timperman, W. Eling, L. Aké Assi, J. Holenz, G. Bringmann, (1997); Antimicrob. Agents Chemother.<br />
41(11), 2533-2539.<br />
M. R. Boyd, Y. F. Hallock, J. H. Cardellina II, K. P. Manfredi, J. W. Blunt, J. B. McMahon, R. W. Buckheit, G. Bringmann, M.<br />
Schaeffer, G. M. Cragg, D. W. Thomas, J. G. Jato (1994); J. Med. Chem. 37, 1740-1745.<br />
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[SL 8B] The Efficacy <strong>of</strong> Extracts <strong>of</strong> the Plant Argemone Mexicana on Mosquito<br />
Species, Anopheles arabiensis.<br />
Elfahal I. A. 1, Abdelgader, H 1, Elhussein, S. A. 2 , Osman, N.A. 2 .<br />
1 Agricultural Research Corporation, P. O. Box 126, Wad Medani, Sudan; 2 University <strong>of</strong> Gezira,<br />
P. O. Box 20, Wad Medani, Sudan<br />
inshirahelfahal@yahoo.com<br />
Key words: Malaria vector; insecticide resistance; Botanical extract<br />
Introduction<br />
T<br />
he development <strong>of</strong> malaria control in irrigated Schemes <strong>of</strong> Central Sudan has gone through<br />
several phases. As a result <strong>of</strong> agricultural and irrigation practices in the Gezira, falciparum<br />
malaria transmission became perennial instead <strong>of</strong> seasonal and the mosquito vector developed<br />
resistance to several insecticides (Brown, 1986, Lee et.al., 2001, Wattal et.al.,1981). Subsequent<br />
failure to maintain control led to serious epidemics. A new control strategy <strong>of</strong> the vector is essential<br />
to help in insecticide resistance management.<br />
Objective<br />
In this study extracts <strong>of</strong> the plant Argemone mexicana were selected to investigate their larvicidal<br />
potential against mosquito.<br />
Methodology<br />
A known weight <strong>of</strong> leaf powder (range from 6 gm to 0.015 gm) was transferred to small permeable<br />
cloth cotton bags, knotted with cotton thread. The bags containing different weighs <strong>of</strong> leaf powder<br />
were placed in a fixed volume <strong>of</strong> distilled water to give different concentrations. Laboratory reared<br />
mosquito larvae species, Anopheline arabiensis were used for bioassay. Rearing was conducted<br />
under the standard conditions described by Busvine (1971) and WHO (1981). The numbers <strong>of</strong> dead<br />
larvae were assessed after 24 hours. The powder bags floated or remained close to the water<br />
surface throughout the bioassay period. The effect <strong>of</strong> pre incubation <strong>of</strong> Argemone leaf powder bags<br />
on mosquito larvicidal activity was assayed for 1, 2, 3, h etc up to 18.5 hours. The oil was extracted<br />
in n-hexane, hexane was evaporated completely and the oil was saponified according to Fadelmolla<br />
(2003) to give emulsion concentrate formulation(EC).Saponification was done by spotting 2N<br />
potassium hydroxide into freshly prepared Argemone oil.<br />
The results showed that the LC50 and LC90 <strong>of</strong> the leaf extract were 0.16% and 0.39%, respectively.<br />
The Slop <strong>of</strong> the Ld-p line was relatively steep (5.44) indicating a homogenous response. The LC50<br />
(0.006%) and LC90 (0.061%) <strong>of</strong> seed extract (EC formulated) were found to be lower than in leaf<br />
extract and the slope <strong>of</strong> the Ld-p line was 1.27. The mortality observed after exposing larvae <strong>of</strong><br />
mosquito to the concentration <strong>of</strong> 0.25% <strong>of</strong> seed extract were found to be 0, 25% and 100 % after ½<br />
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h, 1.5 h and 6.5 hours <strong>of</strong> exposure. In case <strong>of</strong> leaf extract the mortality <strong>of</strong> tested larvae were 0%,<br />
25% and 95% after 1 h, 6.5 h and 18.5 h <strong>of</strong> exposure.<br />
Table (1a) shows the effect <strong>of</strong> pre incubation <strong>of</strong> Argemone leaf powder bags (ALPB) on mosquito<br />
larvicidal activity as assayed for 1, 2, 3, h etc up to 18.5 hours. Significant mortality was observed<br />
between 5.5 and 6.5 hours from the time <strong>of</strong> incubation <strong>of</strong> the bags. This can be taken as the time<br />
required for release <strong>of</strong> larvicidal activity from the bag. By 16.5 h from bag introduction larvicidal<br />
activity was as high as (95%). These results were largely confirmed by the data <strong>of</strong> Table (1b) where<br />
the ALPB is more incubated for 0, 24, and 48 h before testing the larvicidal activity (at interval <strong>of</strong> ½,<br />
2, 4, 8, and 13 hours). In this separate experiment release <strong>of</strong> larvicidal active component was<br />
observed between 8 and 13 hours.<br />
Table (1a): Effect <strong>of</strong> exposure time on response <strong>of</strong> mosquito larvae to leaf powder. Tested larvae<br />
20. Bags containing 0.25 g each, <strong>of</strong> Argemone leaf powder/100 volume <strong>of</strong> water.<br />
ALPB<br />
incubation h.<br />
Table (1b): Effect <strong>of</strong> incubation time <strong>of</strong> Argemone leaf powder bags in bioassay aqueous medium<br />
114<br />
%Mortality<br />
1 0<br />
2 5<br />
3 10<br />
3.5 10<br />
4.5 10<br />
5.5 10<br />
6.5 25<br />
7.5 60<br />
16.5 95<br />
18.5 95
Powder bag<br />
incubation<br />
period<br />
48 hr<br />
24 hr<br />
0hr<br />
control<br />
SE +<br />
CV%<br />
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Bioassay observation time<br />
.5h 2h 4h 8h 13h<br />
14.5 b 16.5 b 17.0 b 18.5 b 19.0 c<br />
15 b 16 b 16 b 17 b 19.0 c<br />
0.0 a 0.0 a 0.0 a 0.0 a 13.0 b<br />
0.0 a 0.0 a 2.0 a 3.0 a 3.0 a<br />
2.76<br />
79<br />
3.17<br />
79<br />
3.29<br />
79<br />
Bags containing 0.75g/300 ml <strong>of</strong> Argemone leaf powder were introduced onto the aqueous<br />
bioassay medium & allowed to stand for 0, 24 & 48 h.20 larvae were introduced in each jar & larval<br />
mortality was observed at interval shown (1/2, 2, 4h etc )<br />
By 24 hours most <strong>of</strong> the ingredient was released to the aqueous medium, the 48 h incubation<br />
having little effect on the release. As for the exposure time necessary to kill larvae, it seems that the<br />
process is fast and can occurs in half an hour time (Table 2c, 24 and 48h pre incubation).<br />
Conclusion<br />
Larval mortality increased in a dose dependent manner. A100% mortality was reached by (0.25%)<br />
concentration .The minimum inhibitory dose was below (0.005%). Significant mortality was<br />
observed between 5.5 and 6.5 hours from the time <strong>of</strong> incubation <strong>of</strong> the bags. This can be taken as<br />
the time required for release <strong>of</strong> larvicidal activity from the bag. The fact that 48 h pre incubated<br />
medium was larvicidaly active for a further 13 hours implies good stability for more than 2 days<br />
application. Thus it seems that formulating Argemone leaf powder simply in permeable cloth bag<br />
release the larvicidaly active constituent in about 8 hours or just less. The activity remained highly<br />
effective for nearly 60 hours from introduction <strong>of</strong> bags. As for the exposure time necessary to kill<br />
larvae, it seems that the process is fast and can occur in.5 hours time. The Argemone plant<br />
produces a numerous number <strong>of</strong> seeds that separate easily during drying <strong>of</strong> shoots from the fruits.<br />
These seeds produce a fixed oil (30-40%) that was shown to have larvicidal activity when used as EC<br />
formulation.<br />
Acknowledgment: Thanks to the Agricultural Research Corporation for providing the financial<br />
support. The help <strong>of</strong> the staff <strong>of</strong> the Blue Nile Research and Training Institute in the bioassay is<br />
highly appreciated.<br />
115<br />
3.55<br />
79<br />
1.31<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
References<br />
Brown, A. W. A. (1986); Insecticide resistance in mosquitoes. Pragmatic review. J. Am. Mosq. Contr. 2: 123-140.<br />
Busvine, J.R.(1971); Critical review <strong>of</strong> the techniques for testing insecticides. Second Ed, London press.345.<br />
Fadalelmoula, R. A. (2003); Insecticidal activity <strong>of</strong> selected oils specially Agemone mexicana. Thesis submitted for the<br />
degree <strong>of</strong> Master <strong>of</strong> Science. University <strong>of</strong> Gezira.<br />
Lee S. E., Kim J. E. and Lee H. S.(2001); Insecticide resistance in increasing interest. Agric. Chem. Biotechnol.44: 105-<br />
112.<br />
Wattal, B. L., Joshi, G.C. and Das, M. (1981); Role <strong>of</strong> agricultural insecticides in precipitation vector resistance. Journal <strong>of</strong><br />
communicale disease 13: 71-73.<br />
WHO (1981); Instructions <strong>of</strong> determining the susceptibility or resistance <strong>of</strong> mosquito larvae to insecticides. World<br />
Health Organization VBC/81.807.<br />
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[SL 9A] Arylbenz<strong>of</strong>urans, Prenylated Flavonoids and Diels Ader Adducts with<br />
Biological Activities from Morus mesozygia<br />
Christian D. A. Fozing 1 , Gilbert D.W.F. Kapche 2 , P. Ambassa 1 , Bonaventure T. Ngadjui 1 , Muhammad<br />
C. Iqbal 3 , and Berhanu M. Abegaz 4<br />
1 Department <strong>of</strong> Organic Chemistry, Faculty <strong>of</strong> Science, University <strong>of</strong> Yaounde I, P.O. Box 812 Yaounde, Cameroon<br />
2 Department <strong>of</strong> Chemistry, Higher Teacher Training College, University <strong>of</strong> Yaounde I, P.O. Box 47 Yaounde, Cameroon<br />
3 Department <strong>of</strong> Chemistry, H.E.J. Research Institute <strong>of</strong> Chemistry, International Center for Chemical and Biological<br />
Sciences, University <strong>of</strong> Karachi, Karachi, Pakistan<br />
4 The African Academy <strong>of</strong> Sciences (AAS) Po Box 24916-00502, Nairobi, Kenyan<br />
Key words: Morus mesozygia, Moraceae, Arylbenz<strong>of</strong>urans, Diels Alder adducts, mesozygin<br />
Introduction<br />
M<br />
orus mesozygia Stapf. (Moraceae) is a small to medium-sized tree found in the tropical<br />
forests <strong>of</strong> Africa. The leaves and fruit provide food to the Mantled Guereza, a colobus<br />
monkey native to tropical Africa, and chimpanzee in West and Central Africa (Fashing, 2001).<br />
Traditionally, M. mesozygia is used to cure diabetes, arthritis, rheumatism, malnutrition, debility,<br />
stomach disorders, veneral diseases, and pain (Burkill, 1997). Recently, five new antioxidant and<br />
antimicrobial arylbenz<strong>of</strong>uran derivatives from the stem bark <strong>of</strong> this plant have been reported by us<br />
(Kapche et al. 2009; Kuete et al. 2009). Successive investigation on the bioactive constituents <strong>of</strong> M.<br />
mesozygia resulted in the isolation and identification <strong>of</strong> four new compounds from the leaves.<br />
Material and Methods<br />
General Experimental Procedures<br />
Optical rotations were measured on a JASCO P-2000 using a Glan-Taylor Prism as Polarizer. Melting<br />
points were determined on a Buchi 535 apparatus. IR and UV spectra were recorded on SHIMADZU<br />
8900 and Thermo-Evolution 300 spectrophotometers, respectively. EI-MS (ionization voltage 70 eV)<br />
and FAB-MS were measured on JEOL MS Route and JEOL HX 110 mass spectrometers, respectively.<br />
NMR spectra were run on Bruker AV- 400 and AV-500 MHz NMR spectrometers.<br />
Plant Material<br />
The leaves and trunk bark <strong>of</strong> Morus mesozygia Stapf. were collected in Yaoundé, Cameroon in<br />
January 2010 and identified by Mr. Nana, a botanist at the National Herbarium, Yaoundé<br />
(Cameroon), where a voucher specimen (No. 4228/SRFK) is deposited.<br />
Extraction and Isolation<br />
The air-dried leaves and trunk bark were ground into powder and extracted with MeOH at room<br />
temperature. Evaporation <strong>of</strong> the solvent under reduced pressure provided a MeOH extract. The<br />
EtOAc soluble part <strong>of</strong> the MeOH extract was subjected to consecutive column chromatography over<br />
silica gel and Sephadex LH-20 to give twenty compounds including six new stilbenoids: moracins O,<br />
Q- U(1 6) and three new Diels Alder adducts : mesozygins A- C. (7-9).<br />
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Phosphodiesterase I Inhibition Assay. Activity against snake venom was assayed by taking 33 mM<br />
Tris HC1 buffer pH 8.8, 30 mM Mg acetate with 0.000742 U/well final concentration <strong>of</strong> the<br />
enzyme using a micro titer plate assay and 0.33 mM bis-(p-nitropheny1) phosphate (Sigma N-3002)<br />
as a substrate. EDTA was used as a positive control (1C50 = 274 M 0.007).<br />
Results and discussion<br />
The MeOH extract <strong>of</strong> the trunk bark and leaves <strong>of</strong> M. mesozygia was subjected to consecutive<br />
column chromatography over silica gel and Sephadex LH-20 to give twenty compounds including six<br />
new stilbenoids: moracins O, Q- U(1 6) and three new Diels Alder adducts : mesozygins A- C. (7-9).<br />
Compounds 1-6 were characterized as 2-arylbenz<strong>of</strong>uran derivatives and were noted to have the<br />
following common features; their UV spectra displayed the characteristic two absorption bands in<br />
the regions 204- 237 and 290-320 nm (Nomura and Fukai, 1981). Compound 1-9 gave the expected<br />
colours upon reaction with methanolic ferric chloride confirming the presence <strong>of</strong> free phenolic<br />
hydroxyl substituent. Their IR spectra disclosed absorptions at max ca. 3400 (OH stretch) and typical<br />
aryl absorptions and overtones from 1610-1450 cm -1 . The 1 H-NMR spectra <strong>of</strong> 1-6 showed a sharp<br />
singlet at 6.88- 7.19 ppm characteristic <strong>of</strong> H-3 <strong>of</strong> the 2-arylbenz<strong>of</strong>uran derivatives and resonances<br />
for aryl A2X spin system [ 6.88- 7.05 (2H, d, J = 2.0 Hz, H-2'/6') and 6.39- 6.49 (1H, t, J = 2.0 Hz, H-<br />
4')]. and 2,5,6-trisubstituted benz<strong>of</strong>uran moiety [ 6.88 (1H, s, H-3), 7.26 (1H, s, H-4), and 6.91 (1H,<br />
s, H-7)] for compounds 1,2,4 and 6 (Kapche et al. 2009). Compounds 7-9 derived from Diels Alder<br />
reaction between the chalcone 10 and the flavone 11.<br />
Mesozygin A (7), + 343.2 (c 0.29, MeOH), displayed a quasi-molecular ion [M+H] + at m/z<br />
607.1640 in the HR-FAB-MS, indicating the molecular formula <strong>of</strong> C35H26O10 (calcd for C35H27O10,<br />
607.1604). The broadband-decoupled 13 C NMR spectrum <strong>of</strong> 7 displayed 35 resonances, which were<br />
differentiated via a DEPT spectrum as one methyl, one methylene, 15 methine, and 18 quaternary<br />
carbons. The 1 H and 13 C NMR spectra showed resonances assignable to a 6-C-substituted-5,7,2,4tetrahydroxyflavone<br />
moiety [ H/ c 7.23 (1H, s)/107.8 (CH-3), 6.67 (1H, s)/95.3 (CH-8), 6.61 (1H, d, J<br />
= 2.4 Hz)/104.5 (CH-3'), 6.51 (1H, dd, J = 8.7, 2.4 Hz)/108.7 (CH-5'), and 7.83 (1H, d, J = 8.7 Hz)/130.7<br />
(CH-6'); c 163.3 (C-2), 183.7 (C-4), 106.0 (C-4a), 161.6 (C-5), 108.9 (C-6), 159.2 (C-7), 156.7 (C-8a),<br />
110.2 (C-1'), 160.9 (C-2'), and 163.1 (C-4'); and H 13.97 (1H, s, 5-OH)]. The resonances observed in<br />
the 1 H and 13 C NMR spectra at H/ c 6.43 (1H, br. s)/122.2 (CH-2''), 3.42 (1H)/36.8 (CH-4''), 3.41<br />
(1H)/34.3 (CH-3''), 2.91 (1H)/28.3 (CH-5''), 2.70, 2.03 (1H each)/36.3 (CH2-6''), and 1.77 (3H, s)/23.8<br />
(CH3-7'') and c 134.1 (C-1'') indicated the presence <strong>of</strong> a methylcyclohexene ring (Zhang et al,<br />
2007). Consistent with the resonances <strong>of</strong> methylcyclohexene ring, a quaternary carbon in the 13 C<br />
NMR spectrum at c 103.3 (C-8'') revealed a ketal function. Additionally, two sets <strong>of</strong> ABX system<br />
observed in the 1 H NMR spectrum were ascribed to two 2,4-dihydroxyphenyl rings [ H 7.11 (1H, d, J<br />
= 8.4 Hz, H-14''), 6.21 (1H, dd, J = 8.4, 2.1 Hz, H-13''), and 6.43 (1H, d, J = 2.1 Hz, H-11'') and H 7.11<br />
(1H, d, J = 8.7 Hz, H-20''), 6.49 (1H, dd, J = 8.7, 2.1 Hz, H-19''), and 6.37 (1H, d, J = 2.1 Hz, H-17'')].<br />
The H<strong>MB</strong>C correlations between H-20''/C-5'' and H-2'', H-4''/C-6 revealed that the 2,4dihydroxyphenyl<br />
and the flavones moieties were attached to the methylcyclohexene ring through<br />
C3''-C6 and C5''-C15'' bonds. Also correlations <strong>of</strong> H-14'' and H-4'' with C-8'' showed that the remaining<br />
118
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
2,4-dihydroxyphenyl ring and the methylcyclohexene ring were connected to the ketal moiety. It is<br />
well established that structurally similar compounds possess absolute configuration <strong>of</strong> the chiral<br />
centers in the methylcyclohexene ring as R (C-3''), R (C-4''), and S (C-5'') for trans adducts, S (C-3''), R<br />
(C-4''), and S (C-5'') for cis-trans adducts, and S (C-3''), R (C-4''), S (C-5''), and R (C-8'') for ketal<br />
adducts; all trans adducts exhibit negative optical rotation values while the other two types show<br />
positive ones (Hano et al., 1988). Since (7) displayed a positive optical rotation, characteristic for<br />
cis-trans ketal adducts, the absolute configuration <strong>of</strong> the four chiral carbons were determined to be<br />
S (C-3''), R (C-4''), S (C-5''), and R (C-8''). The structure <strong>of</strong> (7) was established as mesozygin A.<br />
Mesozygin B (8), + 139.6 (c 0.16, MeOH), displayed a quasi-molecular ion at m/z 625 [M+H] + in<br />
the FAB-MS and its molecular formula, C35H28O11, was established by HR-FAB-MS (m/z 625.1688,<br />
calcd for C35H29O11, 625.1710). The 1 H and 13 C NMR spectral data <strong>of</strong> 8 were close to those <strong>of</strong> 7<br />
except that a carbonyl carbon resonance ( C 209.0, C-8'') in 8 replaced a ketal quaternary carbon ( C<br />
103.3) in 7. As a result, the resonance <strong>of</strong> an additional hydrogen-bonded hydroxyl proton was<br />
observed at H 12.49 (1H, s, OH-10''). The H<strong>MB</strong>C correlations between H-2'', H-4''/C-6 and H-20''/C-<br />
5'' suggested that the flavones and 2,4-dihydroxyphenyl moieties were connected to the<br />
methylcyclohexene ring at C-3'' and C-5'', respectively. In addition, the correlations <strong>of</strong> H-4'' and H-<br />
14'' with a carbonyl carbon at C 209.0 (C-8'') supported the existence <strong>of</strong> an oxo group between the<br />
2,4-dihydroxyphenyl and methylcyclohexene rings. The positive optical rotation observed for 8 is in<br />
favour for a cis-trans adduct as explained in discussion <strong>of</strong> 7 and supported the S, R, and S<br />
configuration at C-3'', C-4'', and C-5'', respectively. Thus, the structure <strong>of</strong> compound 8 was<br />
established as mesozygin B.<br />
The HR-FAB-MS <strong>of</strong> 9 provided a quasi-molecular ion [M+H] + at m/z 709.2315 corresponding to the<br />
molecular formula C40H36O12 (calcd for C40H37O12, m/z 709.2285). Compound 9 was also found to be<br />
an optically active Diels-Alder adduct ( + 85.7 (c 0.4, MeOH)); its 1 H and 13 C NMR spectra were<br />
very similar to those <strong>of</strong> 8, except for significant change in one <strong>of</strong> the 2,4-dihydroxyphenyl rings. This<br />
is reflected by the presence <strong>of</strong> an AB doublet [ H/ C 6.43 (1H, d, J = 9.0 Hz)/108.3 (C-13'') and 8.31<br />
(1H, d, J = 9.0 Hz)/132.0 (C-14'')] in the 1 H NMR <strong>of</strong> 9 instead <strong>of</strong> one <strong>of</strong> the ABX spin systems in 8. In<br />
addition, the resonances for an hydroxyisoprenyl moiety were observed in 9 The H<strong>MB</strong>C correlations<br />
<strong>of</strong> H2-21'' with C-10'' and C-12'', and H-22'' with C-11'' (Figure 1) revealed the position <strong>of</strong><br />
hydroxyisoprenyl at C-11'' in a 1,2,3,4-tetrasubstitutedphenyl ring. Based on the facts explained in<br />
8, the configuration at C-3'', C-4'', and C-5'' was determined to be S, R, and S, respectively, due to a<br />
positive optical rotation observed for 9. Finally, the structure <strong>of</strong> 9 was elucidated as mesozygin C.<br />
Compounds 7-9 were tested for phosphodiesterase I inhibitory activity against snake venom using<br />
EDTA as a positive control and found to be potent inhibitors compared to standard (Table 3).<br />
Compounds 8 showed the most potent phosphodiesterase I inhibitory activities [IC50 8.9 µM ± 1.26<br />
(IC50 for EDTA 274 µM ± 0.007)].<br />
119
H<br />
HO<br />
HO<br />
O<br />
HO<br />
HO<br />
HO<br />
7<br />
6'' O 6 7a O<br />
5''<br />
4''<br />
OH<br />
OH<br />
5<br />
HO<br />
4<br />
O<br />
O<br />
O<br />
3a<br />
1<br />
5<br />
OH<br />
HO<br />
O<br />
11<br />
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
8<br />
3<br />
OH<br />
2<br />
OH<br />
OH<br />
OH<br />
1'<br />
OH<br />
6'<br />
2'<br />
HO<br />
O<br />
O<br />
O<br />
O<br />
4'<br />
HO<br />
OH<br />
OH<br />
HO O<br />
O O<br />
HO<br />
OH<br />
HO<br />
6<br />
OH<br />
HO<br />
O<br />
2<br />
OH<br />
HO<br />
9<br />
O<br />
OH<br />
O<br />
120<br />
O<br />
O<br />
OH<br />
OH<br />
HO<br />
18''<br />
HO<br />
4'' 5''<br />
3"<br />
2''<br />
1''<br />
O<br />
HO<br />
12''<br />
3<br />
OH<br />
OH<br />
10''<br />
HO 9'' 14''<br />
17''<br />
O O 7<br />
8<br />
8a O<br />
8''<br />
20''<br />
OH<br />
15''<br />
4''<br />
5''<br />
OH<br />
6""<br />
6<br />
3'' 5<br />
4a 4<br />
OH O<br />
1''<br />
7'' 7<br />
HO<br />
HO<br />
O<br />
2<br />
3<br />
HO<br />
O<br />
6'<br />
1'<br />
2'<br />
OH<br />
HO<br />
10<br />
Acknowledgement<br />
This research program was supported by the Academy <strong>of</strong> Sciences for the Developing World<br />
(TWAS), the International Center <strong>of</strong> Chemical and Biological Sciences (ICCBS), University <strong>of</strong> Karachi,<br />
Pakistan and the Network <strong>of</strong> Analytical and Bioassay Services in Africa (NABSA).<br />
References<br />
Burkill, H.M. (1997); The useful plants <strong>of</strong> West Tropical Africa: Families M R; Royal Botanic Gardens, Kew: Richmond,<br />
Vol. 4, p 969.<br />
Fashing, P. (2001); J. Int. J. Primatol. 22, 579 609.<br />
Hano, Y.; Suzuki, S.; Kohno, H.; Nomura, T. (1988); Heterocycles 27, 75 81.<br />
Hano, Y.; Suzuki, S.; Nomura T.; Iitaka, Y. (1988); Heterocycles 27, 2315 2324.<br />
Kapche, G. D. W. F.; Fozing, D. C.; Donfack, J. H.; Amadou, D.; Tchana, A. N.; Bezabih, M.; Moundipa, P. F.; Ngadjui, B. T.;<br />
Abegaz, B. M. (2009); Phytochemistry 70, 216 221.<br />
Kuete, V.; Fozing, D. C.; Kapche, W. F. G. D.; Mbaveng, A. T.; kuiate, J. R.; Ngadjui, B. T.; Abegaz, B. M. (2009); J.<br />
Ethnopharmacol. 124, 551 555.<br />
Nomura, T. Fukai, T. (1981); Heterocycles, 15, 1531-1567<br />
Zhang, Q. J.; Tang, Y. B.; Chen R.Y.; Yu, D. Q. (2007); Chem. Biodivers. 4, 1533 1540<br />
OH<br />
4'<br />
O<br />
OH<br />
4<br />
OH<br />
OH
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[SL 9B] In vitro Effects <strong>of</strong> Extracts from five Malagasy Endemic Species <strong>of</strong><br />
Albizia (Fabaceae) on Vegetable Seeds Germination<br />
Danielle A. Doll RAKOTO 1 , Ranjàna RANDRIANARIVO 1 , Mounidati EL-YACHOUROUTUI 1 , Alain A.<br />
ARISOA 1 , Noelinirina RAHARISOA 1 , Noelitiana RAKOTONDRASOA 1 , Pascaline RAONIHARISOA 1 , Victor<br />
JEANNODA 1<br />
1: Laboratoire de Biochimie appliquée aux Sciences médicales, Département de Biochimie fondamentale et appliquée,<br />
Faculté des Sciences, BP 906, Université d Antananarivo, Madagascar<br />
Email: dad.rakoto@yahoo.fr<br />
Key-words: Albizia, crude extracts, purified extracts, saponins, alkaloids, inhibition, germination.<br />
Introduction<br />
T<br />
rees belonging to the genus Albizia (Fabaceae) grow in tropical areas such as Africa, Asia and<br />
South-America where they are widely used in traditional medicine (Kang et al., 2000; Zou et al.,<br />
2006; Rukunga et al., 2007). Chemical and pharmacological investigations on number <strong>of</strong> them have<br />
led to the isolation <strong>of</strong> novel structures with various properties, indicating the efficacy <strong>of</strong> the healers<br />
herb preparations.<br />
Thus, ethanolic extract from A. lebbeck exhibited anticonvulsive activity (Kasture et al., 2000). The<br />
structure <strong>of</strong> cytotoxic triterpenoidal saponins from Albizia julibrissin was established (Zou et al.,<br />
2006). Sedative activity <strong>of</strong> flavonol glycosides from this species was reported (Kang et al., 2000).<br />
Antiplasmodial spermin alkaloids were isolated from Albizia gummifera (Rukunga et al., 2007).<br />
Antimicrobial activity <strong>of</strong> several species such as A. ferruginea, A. gummifera, A. lebbeck, was<br />
reported (Agyare et al., 2005; Geyid et al., 2005; Sudharameshwari et al., 2007). It is also <strong>of</strong><br />
importance to note that some Albizia species are toxic (Gummow et al., 1992; Agyare et al., 2005)<br />
However, reports on the effects <strong>of</strong> these species on other plants are rare. Now, high application <strong>of</strong><br />
herbicides leads to resistance <strong>of</strong> many weeds.<br />
In Madagascar, Albizia is represented by 25 endemic species. No previous report on both the<br />
chemical constituents and the pharmacological activities <strong>of</strong> these plants could be found in the<br />
literature.<br />
The purpose <strong>of</strong> this study was to carry out assessment <strong>of</strong> the effects <strong>of</strong> extracts from five Malagasy<br />
species <strong>of</strong> Albizia: A2, A4, A5, A6 and A7 on Monocotyledons and Dicotyledons. Vegetables were used<br />
in germination assays since calibrated seeds were available.<br />
Materials and Methods<br />
Plant materials<br />
Collection and processing <strong>of</strong> plants<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Five species <strong>of</strong> Albizia encoded A2, A4, A5, A6 and A7 were used in this study. Plant parts were<br />
collected in western and southern regions <strong>of</strong> Madagascar. The organs used for each plant are<br />
shown in Table 1.<br />
Table 1: Organs used for the 5 species<br />
Species A2 A4 A5 A6 A7<br />
Organs Teguments Empty pods Seeds Leaves Seeds<br />
Seeds (for A2, A5, A7) were washed and all plant materials (seeds, pods and leaves) were sun-dried.<br />
Dried seeds (for A5, A7), empty pods (for A4) and leaves (for A6) were ground into a fine powder,<br />
using a microgrinder Culatti. For A2, teguments were separated from almond by several cycles <strong>of</strong><br />
grinding/sieving. Thereafter, teguments were washed to remove almond residues, sun-dried and<br />
ground into a fine powder.<br />
Tests-seeds<br />
Calibrated vegetable seeds used for germination tests came from the collection <strong>of</strong> Foibe Fikarohana<br />
momba ny Fambolena (F<strong>of</strong>ifa, Antananarivo) seed bank. For each test, experiments were carried<br />
out with one representative <strong>of</strong> Monocotyledons and one representative <strong>of</strong> Dicotyledons.<br />
Extracts preparation<br />
Crude extracts<br />
Cold extraction (A2, A4, A7)<br />
Powdered dried teguments (A2), pods (A4) and seeds (A7) were extracted with 75% ethanol, distilled<br />
water and 50% ethanol, respectively. Prior to extraction, A7 powdered dried seeds were defatted by<br />
Soxhlet extraction with hexan at 45°C for 18 h.<br />
Hot extraction (A5, A6)<br />
Powdered dried seeds (A5) were defatted by extraction with petroleum ether (60-80°C) in a<br />
Soxhlet s extractor. Using the same procedure, powdered dried leaves (A6) were depigmented with<br />
acetone. Both defatted powdered seeds and depigmented powdered leaves were extracted with<br />
absolute ethanol, using a reflux heating system.<br />
Purified extracts<br />
A2, A5, A6 and A7 crude extracts were purified using methods based on solubility, molecular weight<br />
or electric charge properties <strong>of</strong> active principles.<br />
Phytochemical screening<br />
Extracts were subjected to preliminary phytochemical testing for the major chemical groups<br />
(Fransworth, 1966; Marini-Bettolo et al., 1981).<br />
Assays on plants<br />
Assays on seedlings growth<br />
The effects <strong>of</strong> the crude (A2, A4, A7) or purified (A5, A6) extracts were studied on epicotyl and<br />
hypocotyl growth. Seven batches <strong>of</strong> 10 seeds were soaked for 48 h at 30°C in darkness, then<br />
transfered onto Petri dishes layered with cotton. Six batches among the seven ones were<br />
germinated and regularly watered with different concentration levels <strong>of</strong> the tested extract. One<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
batch was watered with distilled water as a control. Epicotyl and hypocotyl lengths were measured<br />
at 2 days intervals during 14 days.<br />
Assays on axillary bud growth<br />
Assays were carried out on 15-day-old pea seedlings previously sectionned above the second<br />
axillary bud. Effects <strong>of</strong> extracts were compared with those <strong>of</strong> the plant growth regulators giberellin<br />
and auxin. Tested solutions (1µl) were mixed with lanolin and deposited on the top <strong>of</strong> the<br />
sectionned part. Five groups <strong>of</strong> 5 plants each were studied: group 1received 50 µg giberellin; group<br />
2: 50 µg <strong>of</strong> auxin; groups 3 and 4: 50µg <strong>of</strong> extracts and group 5: 1µl distilled water. Axillary bud<br />
growth was measured at 2-days intervals during 10 days.<br />
Statistical analysis<br />
One-way analysis <strong>of</strong> variance (ANOVA) followed by Newman Keuls comparison test with Statitcf ®<br />
s<strong>of</strong>tware were used for statistical analysis. Statistical estimates were made at confidence interval <strong>of</strong><br />
95%.<br />
Results and discussion<br />
The extracts used, corresponding to the different species <strong>of</strong> Albizia, and the vegetable seeds tested in<br />
germination assays are shown in Table 2.<br />
Table 2: Extracts from for the 5 species and vegetable seeds used<br />
Plants A2 A4 A5 A6 A7<br />
Extracts<br />
CE = E23<br />
PE = E24<br />
CE = E41 PE = A5 PE = E6 CE = E71<br />
PE = E72<br />
Seeds Rice/Bean Rice/Bean Rice/Bean Maize/Pea<br />
Rice/White<br />
tissam<br />
CE = Crude extract; PE = Purified extract<br />
Vegetable seeds (from Monocotyledons and Dicotyledons) were chosen among those which did not<br />
germinate during preliminary tests with extracts at 1 mg/ml.<br />
All extracts inhibited the epicotyl and hypocotyl growth <strong>of</strong> seedlings tested at the used<br />
concentration levels. However, a slight stimulation effect was exhibited by some extracts at low<br />
concentrations, such as 0.23 mg/ml and 0.46 mg/ml for E23 or 0.035 mg/ml for E4. Above these<br />
concentrations, dose-effect was observed (p
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
E5 at 1.31 mg/ml was the most active extract as<br />
demonstrated by the total inhibition <strong>of</strong> growth<br />
noted for epicotyl <strong>of</strong> rice and beans seedlings<br />
(Figures 1 and 2).<br />
Crude extract E2 slightly inhibited axillary bud<br />
growth. This effect was lower than auxin at the<br />
same level. On the contrary, purified extract E24<br />
exhibited no effect. Active principles were probably<br />
removed by purification.<br />
The inhibition activity <strong>of</strong> the extracts from the various parts <strong>of</strong> the investigated plants appears to be<br />
due to saponins (A4, A5, A7) or alkaloids (A2, A6) identified by phytochemical screening (Table 3).<br />
In conclusion, these natural products were demonstrated to be toxic on seeds at the levels used in<br />
this study. On the other hand, these substances could be involved in plant-plant interactions,<br />
warranting studies <strong>of</strong> relationships between plants and their environment. Further investigations<br />
are necessary to determine their action mechanism, before undertaking research for the purpose <strong>of</strong><br />
their probable use as alternatives for herbicides.<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Table 3 : Phytochemical screening <strong>of</strong> extracts from species A2, A4, A5, A7, A6<br />
Phytochemical compounds Extracts<br />
E23 E24 E41 E5 E6 E71 E72<br />
Alkaloids + + +<br />
Flavonoids +<br />
Anthocyanins +<br />
Phenols<br />
Quinons<br />
Unsaturated sterols + + + + +<br />
Triterpenes + + + + + +<br />
Deoxysugars + + + +<br />
Saponins + + +<br />
Legend for extracts : see Table 2<br />
Acknowledgements<br />
The authors acknowledge the PER/AUF project for financial support and the center Foibe<br />
Fampandrosoana ny Fambolena (Antananarivo) for providing seeds.<br />
References<br />
1- Agyare, C., K<strong>of</strong>fuor, G. A., Mensah, A. Y., Agyemang, D. O. (2005); Antimicrobial and uterine smooth muscle<br />
activities <strong>of</strong> Albizia ferruginea extracts. BLACPMA, 5(2), 27-31.<br />
2- Fransworth, N. R. (1966); Biologica l and phytochemical screening <strong>of</strong> plants. J. Pharm. Sci., 55, 225-276.<br />
3- Geyid, A., Abebe, D., Debella, A., Makonnen, Z., Aberra, F., Teka, F., Kebede, T., Urga, K., Yersaw, K., Biza, T.,<br />
Mariam, B. H., Guta, M. (2005); Screening <strong>of</strong> some medicinal plants <strong>of</strong> Ethiopia for their anti-microbial properties<br />
and chemical pr<strong>of</strong>iles. J. Ethnopharmacol., 97, 421-427<br />
4- Gummow, B., Bastianello, S. S., Labuschagne, L., Erasmus, G. L. (1992); Experimental Albizia versicolor poisoning in<br />
sheep and its successful treatment with pyridoxine hydrochloride. Onderstepoort J. Vet. Res., 59, 111-118.<br />
5- Kang, T. H., Jeong, S. J., Kim, N. Y., Higuchi, R., Kim, Y. C. (2000); Sedative activity <strong>of</strong> two flavonol glycosides isolated<br />
from the flowers <strong>of</strong> Albizia julibrissin Durazz. J. Ethnopharmacol., 71, 321-323.<br />
6- Kasture V. S., Chopde, C. T., Deshmukh, V. K., (2000); Anticonvulsive activity <strong>of</strong> Albizia lebbeck, Hibiscus rosa<br />
sinensis and Butea monosperma in experimental animals. J. Ethnopharmacol., 71, 165-175.<br />
7- Marini-Bettolo, G. B., Nicoletti, M., Patamia, M. (1981); Plant screening by chemical and chromatographic<br />
procedure under field conditions. J. Chromatogr., 218,113-217.<br />
8- Rukunga, G/ M., Muregi, F. W., Tolo, F. M., Omar, S. A., Mwitari, P., Muthaura, C. N., Omlin, F., Lwande, W.,<br />
Hassanali, A., Githure, J., Iraqi, F. W., Mungai, G. M., Kraus, W., K<strong>of</strong>i-Tsekpo, W. M. (2007); The antiplasmodial<br />
activity <strong>of</strong> spermine alkaloids isolated from Albizia gummifera. Fitoterapia, 78, 455-459.<br />
9- Sudharameshwari, K., Radhika, J. (2007); Antibacterial screening <strong>of</strong> Aegle marmelos, Lawsonia inermis and Albizia<br />
lebbeck. Afr. J. Trad.CAM, 4(2), 199-204.<br />
10- Zou, K., Zhao, Y. Y., Zhang, R. Y. (2006); A cytotoxic saponin from Albizia julibrissin. Chem. Pharm. Bull., 54(8), 1211-<br />
1212.<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[SL 10A] Aristolactams, Indolidinoids and other Metabolites from Toussaintia<br />
orientalis - An Endangered Annonaceae Species Endemic to Tanzania<br />
Stephen S. Nyandoro a , Josiah O. Odalo, b Mayunga H.H. Nkunya, a Cosam C. Joseph a<br />
a Department <strong>of</strong> Chemistry, University <strong>of</strong> Dar es Salaam, P.O. Box 35061, Dar es Salaam, Tanzania;<br />
b Department <strong>of</strong> Pure and Applied Sciences, Mombasa Polytechnic University College, P.O. Box 90420-80100, Mombasa,<br />
Kenya<br />
samwel@chem.udsm.ac.tz<br />
Keywords: Toussaintia orientalis; Annonaceae; Aristolactams; Indolidinoids; Antimicrobial; Anti-inflammatory;<br />
Cytotoxicity.<br />
Introduction<br />
D<br />
uring our on-going investigations <strong>of</strong> Tanzanian Annonaceae species over the past two and half<br />
decades we have continued to focus particular interests on those species considered<br />
vulnerable to extinction (Nkunya, 2005). From our investigations as well as others reported<br />
elsewhere, it has been established that Annonaceae species are rich sources <strong>of</strong> structurally diverse<br />
natural products, some <strong>of</strong> which are also reported to exhibit wide spectra <strong>of</strong> biological activities<br />
(Leboeuf et al., 1982; Nkunya, 2005). In continuation with our studies it was noted that Toussaintia<br />
orientalis Verdc that has almost certainly gone extinct in Kenya and can still be found only in<br />
fragmented patches <strong>of</strong> coastal forests in Tanzania is under severe threat <strong>of</strong> extinction. For example<br />
the plant can no longer be found in Pugu Forest, and probably around Ifakara in the Kilombero river<br />
valley, localities which used to be its habitats (Verdcourt, 1971; Lovett and Clarke, 1998; IUCN,<br />
2010). Therefore, this prompted us to include T. orientalis (Odalo et al., 2010; Samwel et al., 2011)<br />
in our on-going investigations for chemical constituents <strong>of</strong> rarely occurring Tanzanian Annonaceae<br />
species (Nkunya, 2005). We hereby review the results from those investigations.<br />
Material and Methods<br />
The stem and root barks were collected in October 2004 while the leaves were collected in August<br />
2007. Both collections were made from Zaraninge Forest Reserve at the edge <strong>of</strong> Saadani National<br />
Park, Bagamoyo District in Tanzania. The identity <strong>of</strong> the plant species was confirmed at the<br />
Herbarium <strong>of</strong> the Department <strong>of</strong> Botany, University <strong>of</strong> Dar es Salaam, where a voucher specimen is<br />
preserved (specimen No. FMM 3330). Extraction, chromatographic and spectroscopic methods and<br />
instrumentations are reported in Odalo et al., 2010 and Samwel et al., 2011. Antimicrobial assays<br />
were done as described by Perez, et al., 1990 and Ell<strong>of</strong>f, 1998. Cytotoxicity, anti-inflammatory and<br />
antiproliferative effects were assessed as described by Meyer et al., 1982, Penning, 1985 and Dahse<br />
et al., 2001, respectively.<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Results and Discussion<br />
The cytotoxic stem and root bark extracts upon extraction and repeated chromatography (Odalo et<br />
al., 2010 and Samwel et al., 2011) yielded the hitherto new aristolactam alkaloid toussalactam (1),<br />
as well as the known ones, namely aristolactam AII (2), aristolactam BII (3), piperolactam C (4) and<br />
aristolactam FII (5); and 1-(2-C-methyl- -D-rib<strong>of</strong>uranosyl)-uracil (6), 3,4,5-trimethoxyphenyl- -Dglucopyranoside,<br />
3,4,5-trimethoxyphenyl- -D-glucopyranoside, 2-Hydroxy-3,4,6trimethoxychalcone,<br />
2-Hydroxy-3,4,6-trimethoxydihydrochalcone, (+)-Dependensin and Quercitin.<br />
The structures <strong>of</strong> the isolated metabolites were established based on extensive analysis <strong>of</strong><br />
spectroscopic data, aprticularly 2-D NMR (HSQC, H<strong>MB</strong>C and NOESY interactions, Odalo et al., 2010).<br />
The aristolactams exhibited antimicrobial, cytotoxic and antiinflammatory activities, aristolactam FII<br />
(5) showing almost the same level <strong>of</strong> activity as the standard anti-inflammatory agent<br />
Indomethacin. The compounds also exhibited either mild or no antiproliferative and cytotoxic<br />
activities, except aristolactam FII that showed the same level <strong>of</strong> cytotoxicity as the standard drug<br />
Camptothecin (Odalo et al., 2010). This was the first time for the isolation <strong>of</strong> the pseudo-nucleoside<br />
6 from a plant source.<br />
The unprecedented isolation <strong>of</strong> 6 that was previously known only as a synthetic product (Beigelman<br />
et al., 1987), prompted us to search for this type <strong>of</strong> compounds also from the leaves <strong>of</strong> T. orientalis,<br />
considered as renewable sources <strong>of</strong> that pharmacologically potent metabolite. However, the leaves<br />
yielded neither 6 nor its related compounds, nor those we obtained from the stem and root barks.<br />
Instead, the leaves yielded a series <strong>of</strong> variously cyclized aminocinnamoyl-tetraketide derivatives 7-<br />
11 (Samwel et al., 2011). These displayed similar spectroscopic features suggesting their structural<br />
similarities, consisting <strong>of</strong> a cinnamoyl moiety and indolidinoid or hydrobenz<strong>of</strong>uranoid, or<br />
cyclohexenoyl systems (1- and 2-D NMR, MS and IR). The compounds exhibited varying inhibitory<br />
potency and selectivity against the tested bacterial and fungal strains at 40 µg/mL concentration.<br />
The antibacterial compounds showed minimum inhibitory concentration (MIC) values that ranged<br />
between 5 and 20 µg/mL, the cynnamoylhydrobenz<strong>of</strong>uranoid 10 being the most potent (MIC = 5<br />
µg/mL), but showing less potency compared to the standard antibiotic Ampicillin (MIC = 2.5 µg/mL).<br />
127
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
R'<br />
R<br />
R'<br />
R'<br />
R''<br />
OH<br />
NH<br />
R'''<br />
O<br />
R R' R'' R'''<br />
1: OMe OH H OMe<br />
2: OMe OH H H<br />
3: OMe OMe H H<br />
4: OMe OMe OMe H<br />
5: OMe OH OMe H<br />
OH<br />
H<br />
N R'' R'<br />
H<br />
N R''<br />
7/8<br />
R'''<br />
9<br />
R'''<br />
OH<br />
OMe<br />
7:<br />
R' R'' R'''<br />
O O X<br />
H<br />
O<br />
NH<br />
R'''<br />
8:<br />
9:<br />
10:<br />
OH<br />
O<br />
O<br />
O<br />
H<br />
X<br />
X<br />
X<br />
Where, X =<br />
11 11: O X<br />
128<br />
R'<br />
HO<br />
OH<br />
O<br />
O<br />
OH HO<br />
6<br />
These results once gain demonstrate the significance <strong>of</strong> the less common Annonaceae species to be<br />
important sources <strong>of</strong> bioactive compounds having unprecedented chemical structures (Leboeuf et<br />
al., 1982; Nkunya, 2005), thus justifying the need for extended efforts for their continued<br />
conservation.<br />
Acknowledgement<br />
Financial support from Sida/SAREC and DAAD is gratefully acknowledged. We thank Mr. Frank M.<br />
Mbago, a curator at the Herbarium <strong>of</strong> the Department <strong>of</strong> Botany, University <strong>of</strong> Dar es Salaam for<br />
locating and identifying the investigated plant species.<br />
References<br />
Beigelman, L.N., Ermolinsky, B.S., Gurskaya, G.V., Tsapkina, E.N., Karpeisky, M.Y. and Mikhailov, S.N. (1987); New<br />
C-methylnucleosides starting from D-glucose and D-ribose. Carbohydrate Res., 166, 219-232.<br />
Dahse, H.M., Schlegel, B. and Gräfe, U. (2001); Differentiation between inducers <strong>of</strong> apoptosis and nonspecific cytotoxic<br />
drugs by means <strong>of</strong> cell analyzer and immunoassay using the cell lines K-562 (human chronic myeloid leukemia), and<br />
L-929 (mouse fibroblast) for antiproliferative effects and HeLa (human cervix carcinoma) for cytotoxic effects.<br />
Pharmazie, 56, 489-491.<br />
Ell<strong>of</strong>f, J.N. (1998); A sensitive and quick microplate method to determine the minimum inhibitory concentration <strong>of</strong> plant<br />
extracts for bacteria. Planta Med., 64, 711-714.<br />
IUCN, (2010); IUCN Red List <strong>of</strong> Threatened Species. Version 2010.4. . Downloaded on 26 May<br />
2011.<br />
Leboeuf, M., Cave, A., Bhaumik, P.K., Mukherjee, B. and Mukherjee, P. (1982); The phytochemistry <strong>of</strong> the Annonaceae.<br />
Phytochemistry, 21, 2783-2813.<br />
Lovett, J. and Clarke, G.P. (1998); Toussaintia orientalis. In: IUCN 2007. 2007 IUCN Red List <strong>of</strong> Threatened Species.<br />
. Downloaded on 12 July, 2008.<br />
10<br />
O<br />
O<br />
N<br />
Me<br />
NH<br />
R'''<br />
NH<br />
O
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Meyer, B.N., Ferrigini, R.N., Jacobsen, L.B., Nicholas, D.E., McLaughlin, J.L. (1982); Brine shrimp: A convenient general<br />
bioassay for active plant constituents. Planta Med., 45, 31-34.<br />
Nkunya, M.H.H. (2005); Unusual metabolites from some Tanzanian indigenous plant species. Pure Appl. Chem., 77,<br />
1943-1955.<br />
Odalo, J.O., Joseph, C.C., Nkunya, M.H.H., Sattler, I., Lange, C., Friedrich, G., Dahse, H.-M. and Möllman, U. (2010);<br />
-uracil and other bioactive constituents <strong>of</strong> Toussaintia orientalis.<br />
Nat. Prod. Comm., 5, 253-258.<br />
Penning, T.M. (1985); Inhibition <strong>of</strong> 5ß-dihydrocotisone reduction in rat liver cytosol: A rapid spectrophotometric screen<br />
for nonsteroidal anti-inflammatory drug potency. J. Pharma. Sci., 74, 651-654.<br />
Perez, C., Paul, M. and Bazerque, P. (1990); An antibiotic assay by the agar well diffusion method. Acta Biol. Med. Exp.,<br />
15, 113-115.<br />
Samwel, S., Odalo, J.O., Nkunya, M.H.H., Joseph, C.C., and Koorbanally, N.A. (2011); Toussaintines A E: antimicrobial<br />
indolidinoids, a cinnamoylhydrobenz<strong>of</strong>uranoid and a cinnamoylcyclohexenoid from Toussaintia orientalis leaves.<br />
Phytochemistry, accepted 19 th May, 2011.<br />
Verdcourt, B. (1971); Flora <strong>of</strong> Tropical East Africa: Annonaceae. Crown Agents, London, UK.<br />
129
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[SL 10B] From Laboratory to Field Application <strong>of</strong> Phyto-larvicides: An Outreach<br />
Community Based Experience in Bagamoyo District, Tanzania<br />
Innocent E 1 , Hassanali A 2 , Magesa SM 3 and Kisinza NW 3<br />
1<br />
Institute <strong>of</strong> Traditional Medicine, Muhimbili University <strong>of</strong> Health and Allied Sciences, P.O Box 65001, Dar es Salaam,<br />
Tanzania. Email: einnocent@muhas.ac.tz OR minza@talk21.com<br />
2<br />
Department <strong>of</strong> Chemistry, School <strong>of</strong> Pure & Applied Sciences, Kenyatta University, P.O Box: 43844-00100, Nairobi,<br />
Kenya<br />
3<br />
National Institute for Medical Research, Amani Research Centre, P.O. Box 81, Muheza-Tanga, Tanzania<br />
Keywords: Phyto-larvicide, Annona squamosa L., Traditional medicine, Mosquito-borne diseases, Ethno-knowledge,<br />
operational research, Livelihood improvement.<br />
Introduction<br />
T<br />
he burden <strong>of</strong> mosquito-borne diseases such as malaria, filariasis and yellow fever continue to<br />
lead in terms <strong>of</strong> morbidity and mortality. In the absence <strong>of</strong> promising vaccine for these<br />
diseases, high cost and detrimental effects caused by synthetic chemical insecticides, plants<br />
continue to play a very important role in traditional medicine and in protection against mosquito<br />
vectors especially in African communities. Since time immemorial, the use <strong>of</strong> plants in management<br />
<strong>of</strong> mosquitoes and other insects is well recognised in traditional (through ethno-knowledge<br />
passage) and academic (through publications) circles however, not given the deserving attention.<br />
Likewise, communities represent the greatest resource available for mosquito control, but least<br />
exploited. Although, communities have knowledge on the potential <strong>of</strong> plants growing in their<br />
environment, they lack proper processing technology. Therefore, simple and cheap-technological<br />
methods <strong>of</strong> harvesting and using bioactive agents that can be adopted by individuals and<br />
communities in order to improve their livelihood are encouraged.<br />
Objective<br />
To create awareness to the communities in Bagamoyo District <strong>of</strong> Tanzania on the potential <strong>of</strong><br />
Annona squamosa L. as accessible and affordable phyto-larvicide for mosquito control.<br />
Material and Methods<br />
Semi-structured questionnaires to assess knowledge, magnitude and attitude <strong>of</strong> using plants as an<br />
alternative to mosquito control was administered to individuals in the communities followed by<br />
participatory rural appraisal (PRA). Also, an outreach inspection <strong>of</strong> mosquito habitat and presence<br />
<strong>of</strong> Annona squamosa in selected villages was done by researchers in collaboration with village<br />
representatives.<br />
130
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Results and Discussion<br />
Initial results show that, most <strong>of</strong> the individuals in the rural communities do use plants in managing<br />
mosquito population. However, very few individuals were aware <strong>of</strong> the habitants <strong>of</strong> immature<br />
mosquitoes and Annona squamosa as an insecticide <strong>of</strong> which detailed results will be presented.<br />
Conclusion<br />
Annona squamosa is used as food, medicine and insecticide. The potential <strong>of</strong> this plant species<br />
continue to be extensively studied with an average <strong>of</strong> 12-20 publications been released annually<br />
since 1990 s hence its inclusion in operational research is justified.<br />
Acknowledgement<br />
This study was funded by DelPHE-British council. We are thankful to the support <strong>of</strong> the<br />
communities, The Office <strong>of</strong> District Medical Officer (DMO) and the District Community<br />
Development Officer (DCDO) in Bagamoyo District.<br />
References<br />
Gourevitch, A. (2003); Better Living Through Chemistry; DDT could save millions <strong>of</strong> Africans from dying <strong>of</strong> malaria- if<br />
only environmentalists would let it. Washington Monthly. WA.<br />
Kihampa, C., Joseph, C.C., Nkunya, M.H.H., Magesa, M.S., Hassanali, A., Hydenreich, M. and Kleinpeter, E. (2009);<br />
Larvicidal and IGR activity <strong>of</strong> extract <strong>of</strong> Tanzanian plants against malaria vector mosquitoes. J Vector Borne Diseases<br />
46, 145-152<br />
Magadula, J.J., Innocent, E. and Otieno, J.N. (2009); Mosquito larvicidal and cytotoxicity activities <strong>of</strong> three Annona<br />
species and isolation <strong>of</strong> active principles. Journal <strong>of</strong> Medicinal Plant Research, 3, 674-680<br />
Stephens, C., Masamu, E.T., Kiama, M.G., Keto, A.J., Kinenekejo, M., Ichimori, K., Lines, J. (1995); Knowledge <strong>of</strong><br />
mosquitoes in relation to public and domestic control activities in the cities <strong>of</strong> Dar es Salaam and Tanga. Bull World<br />
Health org, 73, 97-104<br />
131
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[SL 11A] Bioactive Furanoditerpenoids, a Dibenzopyranone, Nor-isoprenoid and<br />
Biflavonoids from Medicinal Stuhlmania moavi Verdc.<br />
Odalo JO 1 , Joseph CC 2 , Nkunya MHH 2 , Sattler I 3 , Lange C 3 , Dahse H-M 3 , Möllman, U 3<br />
1 Department <strong>of</strong> Pure and Applied Sciences, Mombasa Polytechnic University College, P.O. Box 90420-80100, Mombasa,<br />
Kenya; 2 Department <strong>of</strong> Chemistry, University <strong>of</strong> Dar es Salaam, P.O. Box 35061, Dar es Salaam, Tanzania; 3 Leibniz<br />
Institute for Natural Product Research and Infection Biology, Hans Knöll Institute, Beutenbergstrasse 11a, 07745 Jena,<br />
Germany<br />
Author for correspondence: Odalo JO, e-mail: odalo77@yahoo.com or joodalo@mombasapoly.ac.ke<br />
Keywords: Stuhlmania moavi Verdc., Ceasalpiniaceae, biflavonoids, phenolic lactone, glucopyranose, antimicrobial,<br />
anti-proliferative, cytotoxic.<br />
INTRODUCTION<br />
R<br />
ecent investigations have revealed bioactive and novel constituents from some Tanzanian<br />
Caesalpiniaceae species (Freiburghaus et al., 1998; Chin et al., 2006; Kihampa, 2008). The<br />
recent investigations on the roots and stem bark extracts <strong>of</strong> the Caesalpiniaceae species Stuhlmania<br />
moavi that grows in Tanzania and is used for the management <strong>of</strong> skin infections revealed the<br />
presence <strong>of</strong> cytotoxic, antiproliferative and antimicrobial furanoditerpenoids voucapane (1),<br />
voucapane-6 ,7 -diol (2), voucapane-18,19-diol (3) and 18-hydroxyvoucapan-19-al (4) (Odalo et al,<br />
2009).<br />
R'<br />
H<br />
R<br />
O<br />
H H<br />
R R'<br />
1: H H<br />
3: OH OH<br />
4: OH CH=O<br />
132<br />
2<br />
H<br />
O<br />
H H<br />
This prompted the investigation <strong>of</strong> the most redeemable part (leaves) <strong>of</strong> the plant for bioactive<br />
constituents. We thus report the isolation, structural determination, and antiproliferative, cytotoxic<br />
and antimicrobial activities <strong>of</strong> the 3-hydroxy-2,8-dimethoxydibenzo[b,d]pyran-6-one (5), (E)-4hydroxy-4-(3-hydroxybut-1-enyl)-3,5,5-trimethylcyclohex-2-enone<br />
(6), 4',4''',5,5'',7,7''-hexahydroxy-<br />
3',8''-biflavone (7), 4',4''',5,5'',7,7''-hexahydroxy-3'',6''-biflavone (8) and 1,2,3,4,6-penta-O-galloyl- -<br />
D-glucopyranose (9).<br />
MATERIALS AND METHODS<br />
Plant materials<br />
Leaves <strong>of</strong> S. moavi were collected from Kwedijela forest in Handeni District and Wami valley below<br />
Wami Bridge on the road to Segera-Tanga in Tanzania, respectively. The plant species was<br />
OH<br />
OH
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
authenticated at the Herbarium, Department <strong>of</strong> Botany, University <strong>of</strong> Dar es Salaam where a<br />
voucher specimen (No. FMM 3326) is preserved.<br />
Extraction and isolation<br />
The air-dried, powdered plant materials were extracted sequentially at room temperature with pet<br />
ether, CH2Cl2 and MeOH (each 2 x 72 h). The dichloromethane extract <strong>of</strong> the leaves <strong>of</strong> S. moavi was<br />
subjected to repeated pet ether/EtOAc gradient elution chromatography over silica gel coupled<br />
with Sephadex ® LH-20 chromatography (CHCl3/MeOH, 2:3 v/v) resulting into isolation <strong>of</strong><br />
compounds 5 and 6. The methanol extract <strong>of</strong> the leaves <strong>of</strong> S. moavi when fractionated by vacuum<br />
liquid chromatography (VLC, pet ether/EtOAc gradient elution) and then separation by repeated CC<br />
yielded compound 7. Further purification <strong>of</strong> the polar fractions <strong>of</strong> the CC by reversed phase (RP18)<br />
HPLC chromatography (H2O/MeOH) yielded compounds 8 and 9.<br />
Brine Shrimp Test<br />
The crude extracts were assayed in the brine shrimp test (BST) in artificial seawater and DMSO<br />
according to standard procedures (Meyer et al., 1982) and Cyclophosphamide was used as the<br />
standard toxic agent. LC 50 values (the concentration required to kill 50% <strong>of</strong> the shrimp larvae) were<br />
determined using Probit analysis (Finney, 1971).<br />
Antiproliferative and cytotoxicity assays<br />
The antiproliferative assay was carried out as described in the literature (Dahse et al., 2001) using<br />
the cell lines K-562 (human chronic myeloid leukaemia) and L-929 (mouse fibroblast) and the<br />
activity was expressed as GI50 values (concentration which inhibited cell growth by 50%).<br />
Cytotoxicity assay was carried out against HeLa cells and the activity expressed as GC50 values<br />
(concentration at which cells are destroyed by 50%; used partially in referring to the lysis <strong>of</strong> cells).<br />
For both the assays Taxol , Colchicine and Camptothecin were used as the standard anticancer<br />
agents.<br />
133
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Antibacterial and antifungal assay<br />
The agar diffusion method (Jorgensen et al., 1999) was used in the assays against microorganisms<br />
obtained from the Hans Knöll Institute for Natural Product Research and Infection Biology (HKI),<br />
Jena in Germany, Cipr<strong>of</strong>loxacin and Amphotericin being used as the standard antibacterial and<br />
antifungal agents, respectively. Antibacterial and antifungal activity was expressed as the average<br />
diameter <strong>of</strong> inhibition zones.<br />
The minimum inhibitory concentration (MIC) was determined for compound 9 in a serial microplate<br />
dilution assay against each test bacterial species (Ell<strong>of</strong>f, 1998), with two-fold serial dilution <strong>of</strong> the<br />
compound dissolved in DMSO, beyond the level where no inhibition <strong>of</strong> growth <strong>of</strong> the bacterial<br />
strains Bacillus subtilis [ATTC 6633 (IMET NA)], Staphylococcus aureus [SG511 (IMET 10760)],<br />
Mycobacterium smegmatis (SG987), M. aurum (SB66), M. vaccae (IMET 10670) and M. fortuitum<br />
was observed. Cipr<strong>of</strong>loxacin was used as the reference antibiotic.<br />
RESULTS AND DISCUSSION<br />
Repeated chromatography by silica gel and reverse phase chromatography (HPLC) <strong>of</strong> the<br />
dichloromethane and methanol extracts from the leaves <strong>of</strong> S. moavi resulted into isolation <strong>of</strong> the<br />
metabolites 5-9.<br />
The TLC pr<strong>of</strong>ile <strong>of</strong> the dichloromethane extract from the leaves <strong>of</strong> S. moavi indicated presence <strong>of</strong> a<br />
blue fluorescing compound 5, which was isolated upon repeated chromatography. The HR-ESIMS<br />
established a molecular formula C15H12O5 (MW 272.0685, calcd. for C15H12O5 272.2528) for the<br />
compound, indicating presence <strong>of</strong> 10 degrees <strong>of</strong> unsaturation. The IR spectrum showed strong<br />
absorptions at 3391, 1711, 1608, 1571 and 1491 cm -1 , this being consistent with the presence <strong>of</strong><br />
phenolic hydroxyl, carbonyl and benzenoid C=C groups, respectively (Kemp, 1991). The presence <strong>of</strong><br />
a conjugated carbonyl group was evident from the appearance <strong>of</strong> the corresponding 13 C NMR<br />
resonance at a particularly high field ( 161.7). The 1 H NMR spectrum exhibited signals due to two<br />
methoxyl groups at 4.00 and 3.91 (each s), two aromatic proton singlets at 6.93 and 7.24 and<br />
signals due to three other aromatic protons forming and ABX coupling pattern ( 7.35, dd, J = 8.8,<br />
2.7 Hz; 7.77, d, J = 2.7 Hz and 7.86, d, J = 8.8 Hz; Table 1). The aromatic proton coupling patterns<br />
suggested the presence <strong>of</strong> a tri- and a tetra-substituted benzene moiety in the compound. The<br />
presence <strong>of</strong> the methoxyl groups at C-2 and C-8, and a hydroxyl group at C-3 was indicated by the<br />
H/C H<strong>MB</strong>C correlations observed from H-1 and H-4 to C-2, C-3 and H-10 to C-8 as well as other H/C<br />
interactions (Fig. 1). Thus, based on these spectral data the structure <strong>of</strong> the isolated compound was<br />
established to correspond to 3-hydroxy-2,8-dimethoxy-dibenzo[b,d]pyran-6-one. The MS<br />
fragmentation pattern for 5 was typical <strong>of</strong> dibenzopyranone compounds (Concannon et al., 2000)<br />
and it consisted <strong>of</strong> fragment ion peaks at m/z 197, 213, 214 and 229 that were attributed to<br />
cleavage <strong>of</strong> CO2 and CO units from the parent skeleton.<br />
The spectroscopic data for the compounds 6-9 agrees with those already reported in the literature<br />
(Gonzalez et al., 1994; Markham et al., 1987; Chen and Hagerman, 2004).<br />
134
MeO<br />
HO<br />
H 3C<br />
O<br />
HO<br />
3<br />
3<br />
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
1<br />
5<br />
H<br />
1<br />
H<br />
10<br />
9<br />
6<br />
7<br />
O O<br />
5<br />
H<br />
10<br />
6<br />
H<br />
9<br />
7<br />
O O<br />
5<br />
OMe<br />
O<br />
H<br />
O<br />
CH 3<br />
11 12<br />
1<br />
5<br />
OH<br />
6<br />
3<br />
7<br />
135<br />
8<br />
OH<br />
GO<br />
10 GO<br />
H OG<br />
H<br />
H<br />
H O<br />
9<br />
OG<br />
H<br />
OG<br />
O<br />
OH<br />
OH<br />
OH<br />
G = Galloyl group<br />
Fig. 1. Important H/C H<strong>MB</strong>C correlations for 3-hydroxy, 2,8-dimethoxy-dibenzo[b,d]pyran-6-one (5)<br />
HO<br />
HO<br />
7<br />
7 9<br />
5<br />
OH<br />
5<br />
OH<br />
10<br />
9<br />
10<br />
O<br />
4<br />
O<br />
O<br />
4<br />
O<br />
2<br />
2<br />
3<br />
3<br />
1'<br />
7<br />
5'<br />
5'<br />
3'<br />
OH 2''<br />
O<br />
9''<br />
OH<br />
4'''<br />
1'''<br />
4''<br />
10''<br />
3''<br />
7''<br />
HO 5'' OH<br />
HO<br />
8<br />
4'<br />
1' 3'<br />
OH<br />
OH<br />
5''<br />
10''<br />
7'' 9''<br />
O<br />
4''<br />
O<br />
O<br />
3''<br />
2''<br />
1'''<br />
4'''<br />
OH<br />
Table 1. 1 H and 13 C-NMR spectral data for 3-hydroxy-2,8-dimethoxy-dibenzo[b,d]pyran-6-one 1.<br />
H/C H J (Hz) C H/C H J (Hz) C<br />
1 7.24 s 102.9 8 --- --- 159.2<br />
2 --- --- 144.3 9 7.35 dd, 8.8, 2.7 124.4<br />
3 --- --- 145.7 10 7.86 d,8.8 122.6<br />
4 6.9 s 103.8 10a --- --- 128.7<br />
4a --- --- 147.3 10b --- --- 110.3<br />
6 --- --- 161.7 2-OMe 4.00 s 56.4<br />
6a --- --- 121.3 8-OMe 3.91 s 55.8<br />
7 7.77 d, 2.7 111.1
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
The brine shrimp assay <strong>of</strong> the crude leaves extracts indicated potent levels <strong>of</strong> activity<br />
(Dichloromethane extract; LC50 13.01 µg/ml), being comparable with the efficacy shown by the<br />
standard cytotoxic agent Cyclophosphamide (LC50 16.33 µg/ml). Additionally, the isolated<br />
compounds from the crude extracts when assayed for in vitro antiproliferative and cytotoxic activity<br />
against L-929, K-562, and HeLa cell lines, respectively showed different levels <strong>of</strong> mild activity,<br />
compared with the standard anticancer agents Taxol , Colchicine and Camptothecin. The<br />
pyranolide 5, which is reported for the first time, demonstrated better activity as a cytotoxic agent<br />
as compared to antiproliferative activity (CC50 7.9 g/ml against HeLa cell line and GI50 16.4 and<br />
18.3 g/ml against L-929 and K-562, respectively).<br />
The compounds were evaluated for antibacterial and antifungal activities so as to establish possible<br />
activities that would corroborate the ethnomedicine application <strong>of</strong> the crude extracts <strong>of</strong> S. moavi in<br />
the treatment <strong>of</strong> skin infections. The compounds 5-9 were assayed for these activities in vitro<br />
against bacteria Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa<br />
and Mycobacterium vaccae, and against the fungal species Sporobolomyces salmonicolor, Candida<br />
albicans and Penicillium notatum. The galloylglucoside, 9 exhibited both antibacterial and<br />
antifungal activity. The compound was active against S. aureus (B3), E. coli (B4), M. vaccae (M4) and<br />
P. notatum (P1), displaying inhibition zones <strong>of</strong> 13, 18, 19 and 14 mm, respectively.<br />
The minimum (bacterial) inhibitory concentration (MIC) was determined for the more active<br />
compound 9 against S. aureus, M. smegmatis, M. aurum, M. vaccae, M. fortuitum and was found to<br />
be post potent against M. vaccae (MIC 12.5 g/ml).<br />
The demonstrated antibacterial and antifungal activities <strong>of</strong> the galloylglucoside 9 tend to further<br />
corroborate the traditional use <strong>of</strong> the crude extracts <strong>of</strong> S. moavi for the treatment <strong>of</strong> skin<br />
infections. It would be interesting to carry out further bioassays <strong>of</strong> combination formulations <strong>of</strong> the<br />
reported antimicrobial furanoditerpenoids from S. moavi and the galloylglucoside, so as to establish<br />
possible synergistic activities <strong>of</strong> the active plant metabolites.<br />
ACKNOWLEDGEMENTS<br />
Financial support from Germany Academic Exchange Services (DAAD) through NAPRECA and that<br />
Sida/SAREC is highly acknowledged. We thank Mr. F. Mbago <strong>of</strong> the Herbarium, Department <strong>of</strong><br />
Botany at the University <strong>of</strong> Dar es Salaam for identification <strong>of</strong> the investigated plant species.<br />
References<br />
Chen, Y., Hagerman, A.E. (2004); Characterization <strong>of</strong> soluble non-covalent complexes between bovine serum albumin<br />
and -1,2,3,4,6-Penta-O-galloyl-D-glucopyranose by MALDI-TOF MS. J. Agric. Food Chem., 52, 4008-4011.<br />
Chin, Y.W., Mdee, L.K., Mbwambo, Z.H., Mi, Q., Chai, H.B., Cragg, G.M., Swanson, S.M., Kinghorn, A.D., (2006);<br />
Prenylated flavonoids from the root bark <strong>of</strong> Berchemia discolor, a Tanzanian medicinal plant. J. Nat. Prod., 69,<br />
1649-1652.<br />
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Concannon, S., Ramachandran, V.N., Smyth, W.F., (2000); A Study <strong>of</strong> the electrospray ionization and ion trap<br />
fragmentation <strong>of</strong> hemiterpenoid and dimeric coumarin derivatives. Rapid Commun. Mass Spec., 14, 2260-2270.<br />
Dahse, H-M., Schlegel, B., Gräfe, U., (2001); Differentiation between inducers <strong>of</strong> apoptosis and nonspecific cytotoxic<br />
drugs by means <strong>of</strong> cell analyzer and immunoassay. Pharmazie, 56, 489-491.<br />
Ell<strong>of</strong>f, J.N., (1998); A sensitive and quick microplate method to determine the minimum inhibitory concentration <strong>of</strong><br />
plant extracts for bacteria. Planta Med., 64, 711-714.<br />
Finney, D.J., (1971); Probit Analysis: A statistical treatment <strong>of</strong> the sigmoid response curve, 3 rd Ed., Cambridge University<br />
Press, Cambridge, p. 318.<br />
Freiburghaus, F., Steck, A., Pfander, H., Brun, R., (1998); Bioassay-guided isolation <strong>of</strong> a diastereoisomer <strong>of</strong> kolavenol from<br />
Entada abyssinica active on Trypanosoma brucei rhodesiense. J. Ethnopharmacol., 61, 179-183.<br />
Gonzalez, A.G., Guillermo, J.A., Ravelo, A.G. and Jimenez, I.A., (1994); 4,5-Dihydroblumenol A, a new nor-isoprenoid from<br />
Perrottetia multiflora. J. Nat. Prod., 57, 400-402.<br />
Jorgensen, J.H., Turnidge, J.D., Washington, J.A., (1999); Antibacterial susceptibility tests: Dilution and disk diffusion<br />
methods. In: Murray, P.R., Pfaller, M.A., Tenover, F.C., Baron, E.J. and Yolken, R.H. (Eds.). Manual <strong>of</strong> clinical<br />
microbiology, 7 th Ed., ASM Press, Washington, DC, p. 1526-1543.<br />
Kemp, W., (1991); Organic Spectroscopy, 3 rd Ed. Macmillan, Hong Kong, p 59-83, 122-127.<br />
Kihampa, C., (2008); Mosquitocidal and antimicrobial nor-halimanoids, ent-clerodanoids, and other metabolites from<br />
some Tanzanian Tessmannia and Annonaceae species. Ph.D. Thesis, University <strong>of</strong> Dar es Salaam.<br />
Markham, K. R., Sheppard, C., Geiger, H., (1987); 13 C NMR Studies <strong>of</strong> Some Naturally Occurring Ament<strong>of</strong>lavone and<br />
Hinokiflavone Biflavonoids. Phytochemistry, 26, 3335-3337.<br />
Meyer, B.N., Ferrigini, R.N., Jacobsen, L.B., Nicholas, D.E., McLaughlin, J.L., (1982); Brine shrimp: A convenient general<br />
bioassay for active plant constituents. Planta Med., 45, 31-34.<br />
Odalo, J.O., Joseph, C.C., Nkunya, M.H.H., Sattler, I., Lange, C., Dahse, H-M., Möllman, U., (2009); Cytotoxic, antiproliferative<br />
and antimicrobial furanoditerpenoids from Stulhmania moavi. Phytochemistry, 70, 2047-2052.<br />
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[SL 11B] Toddalia asiatica. Lin: A Potential Source and Model <strong>of</strong> Materials and<br />
Services for Control <strong>of</strong> Diseases and Implications for Herbal Medical Practice in<br />
Kenya.<br />
Job Isaac Jondiko 1 , Dida Mathew 1 Orwa Jeniffer 2 .Nyahanga Thomas 1 , Manguro Lawrence 1<br />
1 Maseno University ,P.O.Box 333- 40100 Maseno Kenya. 2 Kenya Medical Research Institute, P. O. Box 54840- 00200,<br />
T<br />
Nairobi, Kenya<br />
he systematic diseases caused by physiological problems and infectious ones caused by<br />
helminthes, protozoans , viruses, bacteria and fungi and remain terrible killer diseases in<br />
tropical Africa due to development <strong>of</strong> drug resistance to disease germs and vectors and expensive<br />
insecticides which may be used for therapeutic and insect vector management respectively. The<br />
published ethno-botanical uses, phytochemical, pharmacological and biological evaluations <strong>of</strong><br />
Toddalia asiatica attracted us to investigate the potentials <strong>of</strong> this plant for the management <strong>of</strong><br />
these diseases (Orwa et al 2008).The ethno-botanical ( Kokwaro 1976, Beentje,1994)<br />
,phytochemical (Buckingham J. 1994 , Rashid et al 1995, Ishii et al 1991) and pharmacological<br />
(Okech-Rabbah H.A. et al 2000,Gakunju et al 1995, Ishi et 1991, Heather et al 2002, Hao et al 2004,<br />
Lu et al 2005, Iwasaki et al 2006 , Guo et al 1998, Kavimani et al 1996) and larvicidal (Korir ,2002)<br />
including repellency properties rejuvenated our interest in T. asiatica .<br />
This paper reports the approach that can be used as a model for the aim <strong>of</strong> validation <strong>of</strong> the<br />
traditional medicinal practices in Kenya. Thus the study guided by both ethno-botanical,<br />
pharmacological, agronomic, biological activity studies and chromatographic isolation <strong>of</strong> active<br />
principles led to identification and structural studies <strong>of</strong> many compounds which can be used as<br />
markers for efficacy, safety and quality in traditional medicinal practice by other researchers. In our<br />
own laboratory two compounds with invitro antiplasmodial activity against Plasmodium falciparum<br />
and essential oils with repellent activity against adults <strong>of</strong> mosquitoes as well as several essential oils<br />
with larvicidal activity against mosquito larvae <strong>of</strong> Anopheles gambiae were isolated and structurally<br />
elucidated. Further the extracts and isolated pure compounds <strong>of</strong> this plant exhibited interesting<br />
antibacterial and antifungal properties. This paper further discusses the implication <strong>of</strong> information<br />
being developed on T. asiatica that is required for purposes <strong>of</strong> implementation <strong>of</strong> the recently<br />
completed draft policy on traditional herbal medicinal practice in Kenya.<br />
References<br />
Heather, E.K., Suryanarayana, V.V., Matthew, F.S., Melissa, J.R. and John, D. (2002); Effects <strong>of</strong> isopimpinellin on<br />
blockage DNA adduct formation and skin tumour initiation. Carcinogenesis. 23(10): 1667-1675<br />
Hao, X.Y., Peng, L., Ye, L., Huang, N.H., Shen, Y.M. (2004); A study on anti-inflammatory and analgesic effects <strong>of</strong><br />
alkaloids <strong>of</strong> Toddalia asiatica. Journal <strong>of</strong> Chinese integrative Medicine. 2(6): 450-2.<br />
Guo, S., Li, S., Peng, Z.,Ren, X., (1998); Isolation and identification <strong>of</strong> active constituent <strong>of</strong> Toddalia Asiatica in<br />
cardiovascular system. Journal <strong>of</strong> Chinese Medicinal Material. 21(10):515-6<br />
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Gakunju, D.M.N., Mberu, E.K., Dossaji, S.F., Gray, A.I. ,Waigh, R. D., Waterman P. G. and Watkins W. M. (1995); Potent<br />
antimalarial activity <strong>of</strong> the alkaloid nitidine isolated from a Kenyan Herbal remedy. Antimicribiol. agents and<br />
Chemotherapy. 39(12): 2606-2609.<br />
Ishii, H., Kobayashi, J., Ishikawa, M., Haginiwa, J., Ishikawa, T. (1991); Studies on the chemical constituents <strong>of</strong><br />
Rutaceae++ plants. LXVI. The chemical constituents <strong>of</strong> Toddalia asiatica. Lam. (T. aculeate pers.). (1). Chemical<br />
constituents <strong>of</strong> the root bark. Yakugaku Zasshi, 111(7): 365-375<br />
Iwasaki, H.,Oku, H.,Takara, R.,Miyahira, H., Hanashiro, K., Yoshida Y., Kamada Y., Toyokawa T., Takara K. and Inakuju M.<br />
(2006); The tumour specific cytotoxicity <strong>of</strong> dihydronitidine from Toddalia asiatica. Cancer Chemother. Pharmacol.<br />
8: 1-9.<br />
Kavimani, S., Vetrichelvan, T., Ilango, R. and Jaykar, B., (1996); Anti-inflammatory activity <strong>of</strong> the volatile oil <strong>of</strong> Toddalia<br />
asiatica. Indian Journal <strong>of</strong> Pharmaceutical Sciences. 58(2): 67-70.<br />
Kokwaro, J. O. (1976); Medicinal Plants <strong>of</strong> East Africa, Kenya literature Bureau, Nairobi, Kampala, Dar-es-salaam. 2nd<br />
ed. pp 212.Lu ,S.Y., Qiao, Y.J., Xiao, P.G. and Tan X.H. (2005); Identification <strong>of</strong> antiviral activity <strong>of</strong> Toddalia asiatica<br />
against influenza type A virus. Zhongguo Zhong Yao Za Zhi. ;30(13):998-1001.<br />
Rashid, M.A., Gustafson, K.R., Kashman, Y., Cardellina, J.H, McMahon, J.B. and Boyd, M.R. (1995); Anti-HIV alkaloids<br />
from Toddalia asiatica. Nat. Prod. Lett. 6: 153-156.<br />
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[SL 12A] Fungitoxic C-18 hydroxy Unsaturated Fatty Acids from Fruiting Bodies<br />
<strong>of</strong> Cantharellus Species<br />
Lilechi D. Baraza a<br />
, Mayunga H.H. Nkunya b<br />
, Nobert Arnold c<br />
, Ludger Wessjohann c<br />
, Cosam C. Joseph b<br />
,<br />
Jürgen Schmidt c<br />
, Andrea Porzel c<br />
a<br />
Department <strong>of</strong> Pure and Applied chemistry, Masinde Muliro University <strong>of</strong> Science and Technology, 190-50100,<br />
Kakamega, Kenya.<br />
b<br />
Department <strong>of</strong> Chemistry, University <strong>of</strong> Dar es Salaam, P.O Box 35061,Dar es Salaam, Tanzania.<br />
c<br />
Department <strong>of</strong> Bioorganic Chemistry, Leibniz Institute <strong>of</strong> Plant Biochemistry,<br />
Weinberg, 3, D-06120 Halle (Saale), Germany.<br />
Key words: Cantharellus, fungitoxicity, unsaturated fatty acids<br />
Introduction<br />
The mushroom genus Cantharellus belongs to the fungi class Chanterellales that has been reported<br />
to consist <strong>of</strong> a number <strong>of</strong> species, some <strong>of</strong> which were only very recently described, growing wildly<br />
in the Miombo woodlands during the rainy season (HARKONEN et al. 2003, BUYCK et al. 2000).<br />
Although Cantharellus species are among the most popular edible mushrooms in East Africa and<br />
Northern Europe (HARKONEN et al. 2003, BUYCK et al. 2000), so far very few studies have been<br />
carried out to evaluate the constituents <strong>of</strong> Cantharellus and other mushroom species wildly<br />
occurring in East Africa. Recently, it was observed that there could be many mushroom species in<br />
East Africa that have not yet been scientifically documented (KARHULA et al. 1998, BUYCK et al.<br />
2000). These studies and the fact that previous chemical investigations <strong>of</strong> mushrooms had yielded<br />
compounds with interesting pharmacological properties (KAWAGISHI et al. 1994, SIMON et al.<br />
1995, KAWAGISHI et al. 1997, MEKKAWY et al. 1998) prompted us to analyze extracts <strong>of</strong> the<br />
Cantharellus species C. isabellinus, C. cibarius, C. platyphyllus, and C. tubaeformis with respect to<br />
their constituents, as part <strong>of</strong> our on-going investigations for nutritional and biologically active<br />
chemical substances from wildly occurring edible mushroom species, part <strong>of</strong> whose results on the<br />
analysis <strong>of</strong> amino acid constitution were recently published (MDACHI et al. 2004). We now report<br />
the isolation and structural determination <strong>of</strong> fungitoxic C18 unsaturated fatty acids from<br />
Cantharellus isabellinus and C. platyphyllus. 10-Hydroxy-8E,12Z-octadecadienoic acid (1), (9Z,14Z)octadecadien-12-ynoic<br />
acid (2), and 9-hydroxy-10E,14Z-octadecadien-12-ynoic acid (3), which was<br />
previously reported as an oxidation product <strong>of</strong> 2 (PANG & STERNER 1991, PANG et al. 1992), were<br />
obtained upon HPLC separation <strong>of</strong> the extracts <strong>of</strong> the dried fruiting bodies <strong>of</strong> C. isabellinus, C.<br />
cibarius, C. platyphyllus, and C. tubaeformis. Cibaric acid (4) also isolated from C. cibarius (PANG &<br />
STERNER 1991, PANG et al. 1992) could not be observed.<br />
140
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
OH<br />
1<br />
3<br />
COOH<br />
COOH<br />
HO<br />
HO<br />
Materials and methods<br />
1D-NMR spectra were recorded on a Varian 300 spectrometer (300 MHz for 1 H-NMR and 75 MHz<br />
for 13 C NMR); 2D-NMR on a Varian Inova 500 instrument at 500 MHz. Analytical HPLC was carried<br />
out on Lichrospher 100-RP 18 (5 µm) column (4 x 125 mm) using a Merck-Hitachi D-7000 system.<br />
Preparative HPLC was carried out on Lichrospher 100-RP 18 (10µm) column (10 x 250 mm) with a<br />
Merck-Hitachi L-6250 low-pressure gradient pump with L-4250 UV/Vis detector, and thin layer<br />
chromatography (TLC) on plastic or aluminium SiO2 plates (solvent system: n-hexane : EtOAc 10 : 3),<br />
visualization by anisaldehyde/sulphuric acid after spraying and heating at 120ºC (blue colour).<br />
Column chromatography was performed using silica gel <strong>of</strong> particle size 230 400 mesh ASTM<br />
(Merck), eluting with mixtures <strong>of</strong> n-hexane and ethyl acetate. High-resolution ESI mass spectra<br />
(HRMS) were obtained from a Bruker Apex III Fourier Transform ion cyclotron resonance (FT-ICR)<br />
mass spectrometer (Bruker Daltonics, Billerica, USA). IR spectra were measured on a Bruker IFS 28<br />
spectrophotometer; specific rotations were measured on a JASCO DIP-1000 polarimeter. The<br />
fungitoxicity assays for crude extracts and the isolated fatty acids 1 3 were carried out according to<br />
Gottstein et al. (1982). An aliquot <strong>of</strong> a CHCl3 solution <strong>of</strong> test sample (20, 50, 100 and 200 g) was<br />
spotted on a thin layer silica gel plate (0.5 mm layer thickness) and then the plate was sprayed with<br />
an aqueous nutritive suspension <strong>of</strong> the phytopathogen Cladosporium cucumerinum Ell. & Arth.<br />
Results and Discussion<br />
Compound 1 (C18H32O3 by HRMS) exhibited IR (( CO = 1710, C=C = 3025 and 1610 cm -1 ) and NMR<br />
absorptions due to carboxylic carbonyl and olefinic functionalities, the latter showing the presence<br />
<strong>of</strong> two double bonds whose positions, as well as that <strong>of</strong> the secondary alcohol function ( H = 4.08,<br />
and C = 72.5 ppm), were established from C/H correlations in the H<strong>MB</strong>C plot <strong>of</strong> 1, and from the<br />
ESI-CIDMS with key ions at m/z 183 (base peak), 155 (CO) and 139, characterizing the position <strong>of</strong><br />
the double bonds (MURPHY et al., 2001). The 8E and 12Z geometry <strong>of</strong> the double bond in 1 was<br />
deduced based on the magnitude <strong>of</strong> the J8,9 and J12,13 values (15.9 and 10.9 Hz respectively) as<br />
indicted in the 1 H-NMR spectrum. The rest <strong>of</strong> the 1 H- and 13 C-NMR spectral features were in<br />
agreement with those previously reported for unsaturated fatty acids similar to 1 (KOSHINO et al.<br />
1987). The structure <strong>of</strong> (9Z,14Z)-octadecadien-12-ynoic acid (2, C18H28O2, by HRMS) was established<br />
141<br />
O<br />
2<br />
4<br />
COOH<br />
COOH
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
upon comparison <strong>of</strong> its spectral features with those previously reported for similar compounds<br />
(HADACEK et al. 1987, PATIL et al. 1989). 1 H- and 13 C-NMR parameters, and the MS fragmentation<br />
pattern established the positions <strong>of</strong> the triple and double bonds, while the Z geometry at C-9 and C-<br />
14 was deduced from the magnitude <strong>of</strong> the corresponding 1 H-NMR coupling constants. 9-Hydroxy-<br />
10E,14Z-octadecadien-12-ynoic acid (3) displayed spectral features that indicated structural<br />
similarities <strong>of</strong> this compound with 1 and 2, whereby the 1 H- and 13 C-NMR, IR and UV spectra <strong>of</strong> 3<br />
exhibited absorptions due to a triple bond that is conjugated to two double bonds, one <strong>of</strong> which<br />
has a Z geometry and is vicinal to a CH2 group while the other double bond has an E configuration<br />
and is adjacent to a secondary alcohol function. The ESI-CIDMS <strong>of</strong> 3 exhibited characteristic<br />
fragment ion peaks at m/z 119 and 171. These spectral features, as well as the long range CH<br />
correlations observed in the H<strong>MB</strong>C spectrum, established the positions <strong>of</strong> the triple and double<br />
bonds, the secondary alcohol function, and hence structure 3. HPLC analysis <strong>of</strong> Cantharellus<br />
isabellinus, C. platyphyllus C. tubaeformis, and C. cibarius extracts indicated the presence <strong>of</strong><br />
compounds 1 3, and ergosterol. The fatty acids 1 - 3 exhibited mild antifungal activity against C.<br />
cucumerinum.<br />
The results reported herein have shown that Cantharellus species are a source <strong>of</strong> fungitoxic,<br />
unsaturated C18-fatty acids, which is in agreement with previous investigations that indicated<br />
Cantharellus mushrooms as producers <strong>of</strong> polyunsaturated free fatty acids as well as triglycerides,<br />
steroids and carotenoids (PANG & STERNER 1991, PANG et al. 1992).<br />
Acknowledgement<br />
Financial support through DAAD/NAPRECA and a Sida/SAREC grant, Mr. L.B. Mwasumbi, Herbarium<br />
<strong>of</strong> the Department <strong>of</strong> Botany at the University <strong>of</strong> Dar es Salaam for locating and identifying the<br />
investigated Cantharellus species and support <strong>of</strong> IPB staff, Hahn for HPLC analysis.<br />
References<br />
ANKE, H, MORALES, P, STERNER, O (1996); Planta Medica 62: 181-183.<br />
BUYCK, B, EYSSARTIER, G, KIVAISI, A (2000) Nova Hedwigia 71: 491-502.<br />
GOTTSTEIN, D, GROSS, D, LEHMANN, H (1982); Archiv für Phytopathologie und Pflanzenschutz 20: 111-116.<br />
HADACEK, F, GREGER, H, GRENZ, M, BOHLMANN, F (1987); Phytochemistry 26: 1529-1530.<br />
HARKONEN, M, NIEMELA, T, MWASU<strong>MB</strong>I, L (2003) Norrlinia 10: 52-54.<br />
KARHULA, P, HARKONEN, M, SAARIMAKI, T, VERBEKEN, A, MWASU<strong>MB</strong>I, LB (1998); Karstenia 38: 49-68.<br />
KAWAGISHI, H, ISHIYAMA, D, MORI, H, SAKAMOTO, H, ISHIGURO, Y, FURUKAWA, Y, LI, J (1997); Phytochemistry 45:<br />
1203-1205.<br />
KAWAGISHI, H, LI, H, TANNO, O, INOUE, S, IKEDA, S, KAMAYAMA, MO, NAGATA, T (1997); Phytochemistry 46: 959-961.<br />
KOSHINO, H, TOGIYA, S, YOSHIHARA, T, SAKAMURA, S (1987); Tetrahedron Letters 28: 73-76.<br />
MDACHI, SJM, NKUNYA, MHH, NYIGO, VA, URASA, IT (2004); Food Chemistry 86: 179-182.<br />
MEKKAWY, SE, MESELHY, RM, NAKAMURA, N, TEZUKA, Y, HATTORI, M, KAKIUCHI, N, SHIMOTOHNO, K, KAWAHATA, T,<br />
OTAKE, T (1998); Phytochemistry 49: 1651- 1657.<br />
MONTILLET, J, CACAS, J, GARNIERM, L, MONTANE, M, DOUKI, T, BESSOULE, J, POLKOWSKA-KOWALCZYK, L,<br />
MACIEJEWSKA, U, AGNEL, J, VIAL, A, TRIANTAPHYLIDESC, C (2004); The upstream oxylipin pr<strong>of</strong>ile <strong>of</strong> Arabidopsis<br />
thalania: A tool to scan for oxidative stresses. Plant Journal 40: 439-451.<br />
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MUI, D, FEIBLMAN, T, BENNETT, JW (1998); International Journal <strong>of</strong> Plant Science 159: 244-248.<br />
MURPHY, RC, FIEDLER, J, HEVKO, J (2001); Chemical Review 101: 479-526.<br />
PANG, Z, STERNER, O (1991); Journal <strong>of</strong> Organic Chemistry 56: 1233-1235.<br />
PANG, Z, STERNER, O, ANKE, H (1992); Acta Chemica Scandinavica 46: 301-303.<br />
PATIL, AD, CHAN, JA, LOIS-FLA<strong>MB</strong>ERG, P, MAYER, RJ, WESTLEY, JW (1989); Journal <strong>of</strong> Natural Products 52: 153-161.<br />
SIMON, B, ANKE, T, ANDERS, U, NEUHAUS, M, HANSSKE, F (1995); .Z. Naturforschung 50c: 173-180.<br />
STADLER, M, STERNER, O (1998); Phytochemistry 49:1013-1019.<br />
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[SL 12B] Application <strong>of</strong> Vibrational Spectroscopy and Planar Chromatography in<br />
the Quality Control <strong>of</strong> South African Medicinal and Aromatic Plants<br />
Alvaro Viljoen<br />
Departments <strong>of</strong> Pharmaceutical Sciences , Tshwane University <strong>of</strong> Technology, Private Bag X680, Pretoria, 0001, South<br />
Africa. Email: ViljoenAM@tut.ac.za<br />
I<br />
t is reported the approximately 80% <strong>of</strong> the population <strong>of</strong> African countries rely on medicinal<br />
plants for their primary health care while 70-80% <strong>of</strong> the population <strong>of</strong> developed countries use<br />
herbs as a form <strong>of</strong> alternative or complementary medicine. Quality control procedures are vital in<br />
plant-based medicines to guarantee the authenticity and quality <strong>of</strong> products. A major challenge in<br />
quality assurance <strong>of</strong> botanicals is their complex phytochemistry. It is now widely accepted that<br />
many molecules may work in an addictive / synergistic manner and that is it short-sighted to<br />
standardize plant extracts on a couple <strong>of</strong> molecules. Pr<strong>of</strong>iling a greater part <strong>of</strong> the plant<br />
metabolome is hence more desirable and vibrational spectroscopy is a useful tool in this regard.<br />
Furthermore, cost and efficiency remains a determining factor in the analysis <strong>of</strong> plant extracts. The<br />
equipment used in planar chromatography has greatly advanced in the past decade and remains an<br />
indispensible chromatographic technique in the quality assessment <strong>of</strong> botanicals. Various examples<br />
( Buchu , Pelargonium, Sutherlandia, Hoodia, etc) will be discussed to show the powerful<br />
application <strong>of</strong> these two techniques to assess the quality <strong>of</strong> both the raw material and commercial<br />
products derived from indigenous South African plant.<br />
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[SL 13A] Synthesis <strong>of</strong> 2,6-Dioxo-1,2,3,4,5,6-hexahydroindoles and their<br />
Transformation into 5,8,9,10-Tetrahydro-6H-indolo[2,1-a]isoquinolin-9-ones<br />
Benard Juma*, a Muhammad Adeel, b Alexander Villinger, b Helmut Reinke, c Anke Spannenberg, c<br />
Christine Fischer, c b, c<br />
Peter Langer<br />
a Masinde Muliro University <strong>of</strong> Science and technology, bfjuma@gmail.com<br />
b Institut für Chemie, Universität Rostock, Albert-Einstein-Str. 3a, 18059 Rostock, Germany<br />
c Leibniz-Institut fur Katalyse e. V. an der Universit_t Rostock, Albert-Einstein-Str. 29a, 18059 Rostock, Germany<br />
Keywords: Alkaloids; amides; C-C bond formation; cyclization; nitrogen heterocycles<br />
Introduction<br />
E<br />
rythrina alkaloids have been prepared through different ways but an important strategy relies<br />
on the acid-mediated domino reaction <strong>of</strong> (2-oxocyclohex-1-yl)acetic amides (Stanislawski, P. C.<br />
et. al. 2006, Tietze, L. F. et. al. 2008, Padwa, A. et. al. 2006).For example, spirocycle I has been<br />
directly prepared from the amide II under various conditions (Scheme 1). However, the preparative<br />
scope <strong>of</strong> this reaction is very narrow and its success strongly depends on the structure <strong>of</strong> the<br />
substrate.<br />
To address this problem, we planned to prepare the unknown erythrina derivative III, which<br />
contains an additional carbonyl group, from the corresponding amide IV (Scheme 1). The carbonyl<br />
group <strong>of</strong> III was expected to be a useful tool for the synthesis <strong>of</strong> erythrina-type natural products<br />
and their non-natural analogues.<br />
MeO<br />
N<br />
MeO O<br />
Ref.<br />
4-6<br />
MeO<br />
MeO<br />
O<br />
N<br />
O<br />
? MeO<br />
MeO<br />
HN<br />
O<br />
O<br />
O<br />
III IV<br />
Scheme 1. Strategy for the synthesis <strong>of</strong> the novel erythrina-type spiro-compound III containing an<br />
additional carbonyl group<br />
145<br />
MeO<br />
MeO<br />
HN<br />
I II<br />
Experimental Section<br />
General<br />
Chemical shifts <strong>of</strong> the 1 H and 13 C NMR are reported in parts per million using the solvent internal<br />
standard (chlor<strong>of</strong>orm, 7.26 and 77.0 ppm, respectively). Infrared spectra were recorded on a FTIR<br />
spectrometer. Mass spectrometric data (MS) were obtained by electron ionization (EI, 70 eV),<br />
O<br />
O
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
chemical ionization (CI, isobutane) or electrospray ionization (ESI). Column chromatography was<br />
performed using 60 A silica gel (60 200 mesh). Cyclization reactions were carried out in Schlenk<br />
tubes under an argon atmosphere. Crystallographic data were collected on a Bruker X8Apex with<br />
MoK radiation ( = 0.71073 Å).<br />
2-(1,4,8,11-Tetraoxadispiro[4.1.4.3]tetradec-12-yl)acetic acid (7a)<br />
To a stirred water solution (250 ml) <strong>of</strong> NaIO4 (15.00 g, 196.0 mmol) and KMnO4 (0.63 g, 3.9 mmol)<br />
was added an acetone solution (39 ml) <strong>of</strong> 6a (2.30 g, 11.7 mmol). The solution was stirred at room<br />
temperature until a colour change from violet to red was observed. The solution was then<br />
extracted with EtOAc (3 x 100 ml) and the combined organic layers were dried (MgSO4). The<br />
solution was filtered and the filtrate was concentrated in vacuo to give 7a (1.61 g, 65%) as a light<br />
brown gummy substance which required no further purification.<br />
Typical procedure for the synthesis <strong>of</strong> amides 10<br />
To a CH2Cl2 solution (20 mL) <strong>of</strong> 7a (200 mg, 0.8 mmol) was added N-hydroxysuccinimide (88 mg,<br />
0.78 mmol) and dicyclohexylcarbodiimide (162 mg, 0.8 mmol) at 0 °C and the mixture was stirred<br />
for 1 h at the same temperature. After stirring for 12 h, the mixture was filtered, 1-amino-2phenylethane<br />
(0.01 mL, 0.85 mmol) was added to the filtrate and the mixture was stirred for 2 h.<br />
The mixture was filtered and washed for several times with water (50 mL for each washing). The<br />
organic layer was dried (NaSO4), filtered and the filtrate was concentrated in vacuo. The residue<br />
was purified by column chromatography (silica gel, heptanes/EtOAc) to give 10k (180 mg, 64%) as a<br />
colourless solid.<br />
General procedure for the synthesis <strong>of</strong> 11a-z<br />
An acetone solution <strong>of</strong> amide 10 and <strong>of</strong> a catalytic amount <strong>of</strong> p-toluenesulfonic acid (PTSA) was<br />
heated under reflux for 6 h. The solution was cooled to 20 °C and concentrated in vacuo to give a<br />
solid residue which was purified by column chromatography (silica gel, heptanes/EtOAc).<br />
2,3-Dimethoxy-5,8,10,11-tetrahydroindolo[2,1-a]isoquinolin-9(6H)-one (14a)<br />
A CH2Cl2 solution (16 ml) <strong>of</strong> 11o (150 mg, 0.5 mmol) and <strong>of</strong> TfOH (0.7 mL) was heated under reflux<br />
for 4 h, cooled to room temperature, and quenched with water. The aqueous layer was extracted<br />
with CHCl3 (3 x 80 ml) and the combined organic layers were dried (MgSO4). The solution was<br />
filtered and the solvent <strong>of</strong> the filtrate was removed under reduced pressure. The residue was<br />
purified by flash chromatography (silica gel, heptanes/EtOAc) to give 14a (120 mg, 84%) as white<br />
crystals which proved to be unstable at room temperature.<br />
1,4,5,10,11,12,13,13a-Octahydro-7,8-dimethoxy-2H-indolo[7a,1-a]isoquinolin-2-one (12).<br />
The synthesis was carried out following the procedure as given for the synthesis <strong>of</strong> 14a. Starting<br />
with 10x (380 mg, 1.2 mmol), PTSA (5 mg, 0.02 mmol) and dry acetone (70 mL), 12 (309 mg, 86%)<br />
was isolated as a colourless viscous oil.<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Results and Discussion<br />
The synthesis <strong>of</strong> (2,4-dioxocyclohex-1-yl)acetic acid (4), despite its structural simplicity was done for<br />
the first time by our group. Accessing it through reactions <strong>of</strong> cyclohexane-1,3-dione (1) with 1bromo-2,2-diethoxyethane<br />
and epibromohydrin and similar bromides failed.<br />
Ozonolysis <strong>of</strong> 4-allylcyclohexane-1,3-dione (5a), following previous reports(Guay, B. et al. 2003),<br />
prepared by reaction <strong>of</strong> the dianion <strong>of</strong> 1a with allylbromide, (Zhang, W. et al 2003, 59) afforded the<br />
triacid 9 rather than the desired aldehyde 8. The formation <strong>of</strong> 9 can be explained by oxidative<br />
cleavage <strong>of</strong> the enolic double bond. The problem was solved by protection <strong>of</strong> the carbonyl groups<br />
<strong>of</strong> 5a to give the bis(acetal) 6a. The oxidation <strong>of</strong> 6a by KMnO4/NaIO4 (in acetone) afforded the acid<br />
7a. Likewise, derivative 7b was prepared in three steps from 1b. The bis(acetal) 7a can be<br />
deprotected to give the desired (2,4-dioxocyclohex-1-yl)acetic acid (4) which, however, proved to<br />
be unstable. Therefore, bis(acetals) 7a,b were directly used for all further transformations.<br />
HO<br />
HO<br />
i<br />
O<br />
1<br />
O<br />
Br<br />
Br<br />
OEt<br />
OEt<br />
O<br />
i<br />
Pb(OAc) 4<br />
HO<br />
O<br />
2 (8%)<br />
O<br />
HO<br />
3 (8%) 4<br />
O<br />
OEt<br />
O<br />
OEt<br />
CrO 3<br />
H 2SO 4<br />
OH<br />
147<br />
O<br />
R<br />
HO<br />
R<br />
1a (R = H)<br />
1b (R = Me)<br />
HO<br />
O<br />
i<br />
R<br />
R<br />
5a (R = H): 72%<br />
5b (R = Me): 95%<br />
O<br />
O<br />
iv<br />
O<br />
ii<br />
HO 2C<br />
8 9<br />
O<br />
O<br />
O<br />
O<br />
O<br />
O<br />
O<br />
O<br />
R<br />
R<br />
iii<br />
O<br />
R<br />
R<br />
OH<br />
7a (R = H): 65%<br />
7b (R = Me): 98%<br />
6a (R = H): 90%<br />
6b (R = Me): 40%<br />
CO2H CO2H The DCC-mediated reaction <strong>of</strong> 7a with various amines afforded the 10a-like amides. Reflux <strong>of</strong> an<br />
acetone solution <strong>of</strong> 10a in the presence <strong>of</strong> para-toluenesulfonic acid (PTSA) afforded the 2,6-dioxo-<br />
1,2,3,3a,4,5-tetrahydroindole 11a. The formation <strong>of</strong> an erythrina-type spiro-compound, such as III<br />
(see Scheme 1), was not observed.
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
O<br />
O<br />
O<br />
7a,b<br />
O<br />
O<br />
R1 R1 O<br />
OH<br />
R<br />
N<br />
2<br />
H 2NR 2<br />
O<br />
R<br />
11a-v<br />
1R1<br />
i<br />
ii<br />
O<br />
O<br />
O<br />
R<br />
O<br />
10a-v<br />
1<br />
R1 The structures <strong>of</strong> all products were established by spectroscopic methods. Some structures e.g.<br />
10o, 11o, and 11e were independently confirmed by X-ray crystal structure analyses (Figures 1-2). 6<br />
Figure 1. Ortep plot <strong>of</strong> 10o (50% probability level)<br />
probability level)<br />
148<br />
O<br />
NHR 2<br />
Figure 2. Ortep plot <strong>of</strong> 11o (50%<br />
For comparison, we studied the reaction <strong>of</strong> PTSA with amide 10x which contains one free carbonyl<br />
group. The reaction <strong>of</strong> 10x with PTSA afforded the erythrina-type spiro-compound 12 in excellent<br />
yield. The formation <strong>of</strong> 12 can be explained by acid-mediated reaction <strong>of</strong> the keto group with the<br />
electron-rich phenyl group to give intermediate A, protonation <strong>of</strong> the enamine moiety to give<br />
iminium salt B, and subsequent Pictet-Spengler reaction. It is important to be noted that this<br />
reaction is not general: The reaction <strong>of</strong> PTSA with amides 10y,z, again prepared from 7c in good<br />
yields, afforded the 2-oxo-1,2,3,4,5,6-hexahydroindoles 11y,z rather than the expected spirocyclic<br />
products. This can be explained by the higher strain <strong>of</strong> a 5,5,6- compared to a 5,6,6-spirocyclic<br />
system.<br />
Our next plan was to study the transformation <strong>of</strong> 2,6-dioxo-1,2,3,4,5,6-hexahydroindoles 11 into<br />
erythrina-type spirocycles, such as III, under more forcing conditions. Heating <strong>of</strong> 2,6-dioxo-<br />
1,2,3,4,5,6-hexahydroindole 11o in the presence <strong>of</strong> PTSA for an extended period <strong>of</strong> time (48 h) did<br />
not result in any conversion. The reaction <strong>of</strong> 11o with triflic acid (TfOH) afforded the 5,8,9,10tetrahydro-6H-indolo[2,1-a]isoquinolin-9-one<br />
14a (84% yield) rather than the erythrina-type<br />
spirocycle 13. The formation <strong>of</strong> 14a can be explained by protonation <strong>of</strong> the amide oxygen atom to<br />
give the cationic intermediate C, cyclization via the electron-rich aryl group (intermediate D), and<br />
subsequent extrusion <strong>of</strong> water and double bond migration.
MeO<br />
MeO<br />
HO<br />
O<br />
7c<br />
+ N<br />
O<br />
+<br />
B<br />
O<br />
NH 2<br />
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
OMe<br />
MeO<br />
i<br />
OMe<br />
MeO<br />
MeO<br />
O<br />
HN<br />
10x (75%) O<br />
N<br />
N<br />
MeO O<br />
12 (86%)<br />
A<br />
MeO<br />
MeO<br />
MeO<br />
MeO<br />
ii<br />
OMe<br />
O<br />
OMe<br />
O<br />
11o<br />
N<br />
N<br />
HO<br />
+<br />
C<br />
H +<br />
O<br />
O<br />
PTSA<br />
TfOH<br />
i<br />
H + _<br />
149<br />
MeO<br />
MeO<br />
MeO<br />
MeO<br />
MeO<br />
MeO<br />
R<br />
14a (84%)<br />
O<br />
MeO<br />
N<br />
MeO<br />
O<br />
N<br />
O<br />
13<br />
HO<br />
D<br />
NH 2<br />
R<br />
7c<br />
11y (R = OMe): 68%<br />
11z (R = H): 68%<br />
N<br />
N<br />
_<br />
H2O O<br />
O<br />
i<br />
ii<br />
R<br />
OMe<br />
HN<br />
O<br />
O<br />
10y (R = OMe): 86%<br />
10z (R = H): 79%<br />
In conclusion, we have reported the synthesis <strong>of</strong> the first (2,4-dioxocyclohex-1-yl)acetic amides.<br />
Their reaction with PTSA provides a general method for the synthesis <strong>of</strong> 2,6-dioxo-1,2,3,4,5,6hexahydroindoles.<br />
The reaction <strong>of</strong> the latter with triflic acid afforded 5,8,9,10-tetrahydro-6Hindolo[2,1-a]isoquinolin-9-ones<br />
rather than erythrina-type spirocycles.<br />
Acknowledgements:<br />
We are grateful for an experimental contribution by Mr. Olumide Fatunsin. Financial support from<br />
the Alexander-von-Humboldt foundation (Georg-Forster scholarship for B. J.) is gratefully<br />
acknowledged.<br />
References<br />
1. Stanislawski, P. C., Willis, A. C., Banwell, M. G., (2006); Org. Lett., 8:2143<br />
2. Tietze, L. F.; Tölle, N.; Noll, C. (2008); Synlett, 525.<br />
3. Padwa, A. and Wang, Q. (2006); J. Org. Chem. 71:7391.<br />
4. Guay, B. and Deslongchamps, P. (2003); J. Org. Chem. 68:6140.
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
5. Zhang, W. and Pugh, G. (2003); Tetrahedron 59:4237.<br />
6. CCDC-xxx contain the supplementary crystallographic data for this paper. These data can be obtained free <strong>of</strong> charge<br />
from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.<br />
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[SL 13B] Medicinal Plants <strong>of</strong> East Africa-<br />
Importance, Uses in Traditional Medicine, Challenges and Conservation Status<br />
M<br />
Najma Dharani<br />
International Centre for Research in Agr<strong>of</strong>orestry (ICRAF)<br />
United Nations Avenue<br />
P O Box 30677 00100, Nairobi, Kenya<br />
Email: ndharani@cgiar.org<br />
edicinal plants are important part <strong>of</strong> Traditional Health Systems or Primary healthcare<br />
systems in most <strong>of</strong> the East African Region. Where access to clinics dispensing modern<br />
medicine is either, limited or prohibitively costly; affordable traditional herbal remedies are still the<br />
primary means <strong>of</strong> meeting the health and medical needs <strong>of</strong> most rural communities. According to<br />
World Health Organization, <strong>of</strong> the 700 million population <strong>of</strong> sub Saharan Africa, fewer than 30%<br />
have access to modern health care and pharmaceuticals. Remaining 70% people still depend<br />
entirely on traditional herbal remedies. WHO has listed 10,000 medicinal plant species that people<br />
use regularly on the African continent.<br />
Common ailments such as stomach problems, diarrhoea, skin infections, infected wounds and<br />
sores, fevers, colds, coughs; parasitic diseases such as malaria, bilharzia and trypanosomiasis ;<br />
treatment <strong>of</strong> sexually transmitted diseases (STD s), tuberculosis, pneumonia and devastating<br />
afflictions associated with HIV/AIDS all can be prevent, treated and cured by using Medicinal<br />
Plants.Natural products and their derivatives represent more than 50% <strong>of</strong> all drugs in clinical use in<br />
the world. Medicinal Plants contribute no less than 25% to the total.<br />
Malaria, a deadliest - life threatening disease especially during pregnancy contributes to maternal<br />
anemia, premature delivery and low birth rate leading to increased child mortality. Due to high<br />
cost, limited availability <strong>of</strong> malaria drugs and remoteness <strong>of</strong> the area, medicinal plants have clearly<br />
played an important role in malarial treatment for centuries.<br />
Traditional herbal medicine in East Africa been prominently in existence for centuries remains<br />
almost wholly unregulated. Cut <strong>of</strong>f from the mainstream economy, traditional herbal medicine<br />
receives little or no development support and importance. Catastrophic habitat loss is threatening<br />
the survival <strong>of</strong> many important Medicinal plants. Steps must be taken to ensure use <strong>of</strong> local plants<br />
is sustainable and does not threaten biodiversity.<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
References<br />
1. World Health Organization (2009); Malaria Report 2009. The World Health Organization, Geneva, Switzerland.<br />
2. World Bank (2001); Medicinal Plants: Rescuing a Global Heritage. World Bank Technical Report No 355. The World<br />
Bank, Washington DC, USA.<br />
3. Dharrani, N. and Yenesew (2010); Medicinal plants <strong>of</strong> East Africa: An Illustrated guide. Publisher-Najma Dharani; in<br />
association with Dorongo Editing & Publishing. ISBN 978-9966- 167-8.<br />
4. Roll Back Malaria Action Plan for a Malaria free World. The Roll Back Partnership, The World Health Organization,<br />
Geneva, Switzerland.<br />
5. Dharani, N. et al; (2010); Common Antimalarial Trees and Shrubs <strong>of</strong> East Africa: a Description <strong>of</strong> Species and a<br />
Guide to Cultivation and Conservation through Use. World Agr<strong>of</strong>orestry Centre (ICRAF). ISBN 978-92-9059-238-9.<br />
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[SL 14A] Crystallization for Long Range Molecular Order Structure Elucidation<br />
Maurice O. Okoth* and Clare I. Muhanji<br />
Department <strong>of</strong> Chemistry & Biochemistry; Chepkoilel University College, Moi University<br />
P. O. Box 1125 30100; Eldoret, Kenya<br />
*Corresponding author -Email; okoth_mdo@hotmail.com<br />
Key words: Crystallization, Structure elucidation, X-ray crystallography, NMR<br />
Introduction<br />
N<br />
atural products are very important in the pharmaceutical industry. Within pharmaceutical<br />
analysis, there exist a hierarchy <strong>of</strong> activities related to molecular structure. Typically, structure<br />
characterization is a collection <strong>of</strong> primary spectroscopic data obtained from IR, UV, NMR, MS and<br />
clues from chromatography among others.<br />
However, more <strong>of</strong>ten than not, such structure characterization are neither conclusive nor involve<br />
interpretation <strong>of</strong> the data in a structural context. As such, structural confirmation methods i.e. the<br />
use <strong>of</strong> these data to confirm the proposed molecular structure <strong>of</strong> the chemical substance are still<br />
necessary. In effect the process <strong>of</strong> structure determination can be long, costly and without as much<br />
details.<br />
X-ray crystallography can be an invaluable tool in structure elucidation with which molecular<br />
structure can be worked out without preconception. Using this technique it is possible to solve<br />
molecular structures that are either related to known chemical structures (e.g. degradants and<br />
metabolites) or those from unknown origins (e.g. impurities) more cheaply, faster and more<br />
completely. However, the real bottleneck in this is the preparation <strong>of</strong> suitable crystals. For years<br />
crystallization has been a standard technique for purification <strong>of</strong> materials but in recent years it is a<br />
lost art that needs attention at all levels.<br />
Through rediscovery <strong>of</strong> the lost art <strong>of</strong> crystallization and utilization <strong>of</strong> recent advances in X-ray<br />
diffraction methods, elaborate structure elucidation has never been made easier for natural<br />
product chemistry<br />
Materials and Methods<br />
In the course <strong>of</strong> typical organic reactions, several intermediates were monitored using TLC and<br />
some isolated and characterized using NMR, IR etc. All synthetic reactions were performed under<br />
an atmosphere <strong>of</strong> nitrogen. Some <strong>of</strong> the intermediates were liquids but were crystallized using<br />
standard crystallization techniques. The resulting crystals were taken through a recrystallization<br />
process in appropriate solvents for purification and then subjected to the single crystal X-ray<br />
diffraction studies. Data collection was using a CrysAlis CCD and Bruker Nonius APEXII CCD<br />
diffractometers. Various s<strong>of</strong>twares were used; Data collection: CrysAlis CCD (Oxford Diffraction,<br />
2007) and COLLECT (Ho<strong>of</strong>t, 1998); cell refinement: CrysAlis CCD and DENZO (Otwinowski & Minor,<br />
153
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
1997) and COLLECT; data reduction: CrysAlis RED (Oxford Diffraction, 2007) and DENZO and<br />
COLLECT; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to refine<br />
structure: SHELXL97 (Sheldrick, 2008); molecular graphics: ORTEP-3 (Farrugia, 1997); s<strong>of</strong>tware used<br />
to prepare material for publications: SHELXL97.<br />
Results and Discussion<br />
Although some results were obtained using IR, NMR, MS and clues from chromatography, they<br />
were not as conclusive as those from X-ray crystallography. The X-ray diffraction revealed the<br />
structure <strong>of</strong> two compounds in more details including Hydrogen bonds.<br />
4-(Benzyloxy)benzaldehyde<br />
The compound, C14H12O2, has an essentially planar conformation with the two aromatic rings<br />
forming a dihedral angle <strong>of</strong> 5.23 (9) and the aldehyde group lying in the plane <strong>of</strong> its aromatic group<br />
[maximum deviation = 0.204 (3) Å]. Weak intermolecular C H...O contacts are found to be shortest<br />
between the aldehyde O-atom acceptor and the H atoms <strong>of</strong> the methylene group.<br />
1-Benzyloxy-4-(2-nitroethenyl)benzene<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
The compound, C15H13NO3, crystallizes with three independent molecules per asymmetric unit (Z =<br />
3). One <strong>of</strong> these molecules is found to have a configuration with a greater twist between its two<br />
aromatic rings than the other two [compare 70.26 (13) and 72.31 (12) with 84.22 (12) ]. There are<br />
also differences in the number and nature <strong>of</strong> the weak intermolecular C H .O contacts formed by<br />
each <strong>of</strong> the three molecules.<br />
The art <strong>of</strong> crystallization should be strengthened to enhance structure elucidation.<br />
Acknowledgements<br />
We are grateful to the National Crystallography Service, University <strong>of</strong> Southampton, for data<br />
collection. Dr Okoth also thanks the Commonwealth Scholarship Commission and the British<br />
Council for funding, Dr Alan Kennedy and the University <strong>of</strong> Strathclyde for hosting him as a visiting<br />
scholar and Moi University for sabbatical leave to do this work.<br />
REFERENCES<br />
Allwood, B. L., Kohnke, F. H., Slawin, A. M. Z., Stoddart, J. F. &Williams, D. J. (1985); Chem. Commun. pp. 311 314.<br />
Christer, S., Rolf, N., Per, E., Marita, H., Jusii, K., Lotta, V. & Hong, Z. (1998); Bioorg. Med. Chem. Lett. 8, 1511 1516.<br />
Farrugia, L. J. (1997); J. Appl. Cryst. 30, 565.<br />
Ho<strong>of</strong>t, R. (1998); COLLECT. Nonius BV, Delft, The Netherlands.<br />
Hunter, R., Younis, Y., Muhanji, C. I., Curtin, T.-L., Naidoo, K. J., Petersen, M., Bailey, C. M., Basavapathruni, A. &<br />
Anderson, K. S. (2008); Bioorg. Med. Chem. 16, 10270 10280.<br />
Kennedy, A. R., Kipkorir, Z. R., Muhanji, C. I. & Okoth, M. O. (2010); Acta Cryst. E66, o2110.<br />
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Otwinowski, Z. & Minor, W. (1997); Methods in Enzymology, Vol. 276,<br />
Oxford Diffraction (2007); CrysAlis CCD and CrysAlis RED. Oxford Diffraction Ltd, Abingdon, England.<br />
Macromolecular Crystallography, Part A, edited by C. W. Carter Jr & R. M. Sweet, pp 307-326. New York: Academic<br />
Press.<br />
Sheldrick, G. M. (2008); Acta Cryst. A64, 112-122.<br />
Stomberg, R. & Lundquist, K. (1994); Z. Kristallogr. 209, 835-836.<br />
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[SL 15A] Antileishmanial Activity <strong>of</strong> Petroleum ether, n-hexane Crude Extract<br />
and (2E)-methyl 3-((1E, 4E)-7-methyl-4-(2-oxopropylidene) cyclohept-1-enyl)<br />
acrylate from Xanthium brasilicum Vell. leaves.<br />
Elwaleed Elamin Hassan a , Ahmed Mudawi Musa b , Sara Hamad Hassab Elgawi b , Tilal Elimam<br />
Elsammani c , Waddah Gamal d , Vanessa Yardley e , Mahgoub Sherif Eltohami a .<br />
a Department <strong>of</strong> Pharmacognosy, Omdurman Islamic University, Khartoum, Sudan.<br />
b Department <strong>of</strong> Immunology and Clinical Pathology, University <strong>of</strong> Khartoum, Khartoum, Sudan.<br />
c Department <strong>of</strong> Pharmaceutical Chemistry, Faculty <strong>of</strong> Pharmacy, King Saud University, Elreydh, Saudi Arabia.<br />
d Department <strong>of</strong> Pharmacognosy, Faculty <strong>of</strong> Pharmacy, King Saud University, Elreydh, Saudi Arabia.<br />
e Department <strong>of</strong> Infectious & Tropical Diseases, London School <strong>of</strong> Hygiene & Tropical Medicine, London, UK.<br />
Introduction<br />
L<br />
eishmaniases are a group <strong>of</strong> parasitic diseases <strong>of</strong> multifaceted clinical manifestations, worldwide<br />
spread with epidemiological diversity. It is caused by at least 20 species <strong>of</strong> protozoan<br />
parasites <strong>of</strong> the genus Leishmania. The parasites are transmitted by the bite <strong>of</strong> sandflies <strong>of</strong> the<br />
genus Phlebotomous in the Old World and Lutzomia in the New World. About 30 species <strong>of</strong> the<br />
sandflies are proven vectors (Desjeux, 1996). Most leishmaniases are zoonosis and the reservoir<br />
hosts are mammals other than man. Man is secondarily infected, being an incidental host (Ashford,<br />
1996). Leishmaniasis is endemic in 88 countries. The disease ranges from self-healing disease <strong>of</strong> the<br />
skin (cutaneous leishmaniasis; CL), disfiguring disease <strong>of</strong> the mucous membranes (mucosal<br />
leishmaniasis; ML) to a fatal disease if not treated (visceral leishmaniasis; VL). More than 90% <strong>of</strong> VL<br />
is reported from Bangladish, Brazil, India and Sudan, and more than 90% <strong>of</strong> CL from Afaganistan,<br />
Iran, Saudi Arabia, Syria, Brazil and Peru (Desjeux, 1996; WHO, 2002; Guerin et al., 2002). WHO<br />
estimates the worldwide prevalence to be approximately 12 million cases, with annual mortality <strong>of</strong><br />
about 60 000. The size <strong>of</strong> the population at risk is about 350 million (WHO, 2010). Every year 1-1.5<br />
million new cases <strong>of</strong> CL, and 0.5 million <strong>of</strong> VL are reported, and the incidence is substantial when<br />
subclinical infections are included; asymptomatic infections outnumber symptomatic infections by<br />
a ratio ranging from 10:1 to 100:1 (Murray, 2002). Leishmaniasis is associated with about 2.4<br />
million disability adjusted life years (DALY). (Murray et al., 2005).<br />
Folk medicine is very <strong>of</strong>ten a good source for researchers looking for bioactive substances<br />
potentially useful against many diseases. Plants were used for leishmaniasis treatment by the<br />
people who live far from modern medicine; these plants <strong>of</strong>fered many lead substances for new<br />
antileishmanial drugs discovery (Carvalho and Ferreira, 2001). Natural products provided highly<br />
successful new drugs such as artemisinine. Further more screening natural products found in all<br />
environments such as the deep sea, rain forests and hot springs, and produced by all sorts <strong>of</strong><br />
organisms ranging from bacteria, fungi and plants to protozoa, sponges and invertebrates (Kayser<br />
et al., 2003). There are many problems associated with it's treatment, like development <strong>of</strong><br />
resistance to current treatment in many areas <strong>of</strong> the world where the disease is endemic, their high<br />
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costs, there are some serious side effects associated with many <strong>of</strong> them, and need for<br />
hospitalization (Cr<strong>of</strong>t et al., 2006).<br />
Methods<br />
Extraction<br />
X. brasilicum was collected from Gezira in August- 2008 and identified by Dr. Wail Elsadig and<br />
Hayder Abd el-Gadir M. Ahmed at Medicinal and Aromatic Plants Research Institute, National<br />
Center for Research, Khartoum. Extraction <strong>of</strong> X. brasilicum was done first using (50 gm) successively<br />
with six solvents in order <strong>of</strong> increasing polarity: petroleum ether, n-hexane, chlor<strong>of</strong>orm,<br />
ethylacetate, ethanol and methanol for at least 12 hrs. For isolation and characterization <strong>of</strong> the<br />
active compounds 1 kg <strong>of</strong> the powdered plant was used for extraction. Extracts were dried under<br />
reduced pressure using rotary evaporator. Stock solutions with a concentration <strong>of</strong> 10 mg/ml were<br />
made in DMSO and kept at 4�C.<br />
Antileishmanial activity (promastigotes)<br />
Antileishmanial activity screening was carried using Atta-ur-Rahman et al. (2005) method. In a 96<br />
well microtiter plate; 90 µl <strong>of</strong> the parasite culture with count <strong>of</strong> 2x10 6 parasites/ml were taken into<br />
each well. 10 µl <strong>of</strong> each concentration was taken and mixed well. Amphotericin B was set as the<br />
positive control with the concentrations up to 1µg/ml. The plate was incubated in the dark at 25 o C.<br />
Counting <strong>of</strong> the living parasites was done after 72 hours.<br />
Five concentrations 5, 10, 100, 500 and 1000 µg/ml were prepared <strong>of</strong> each extract <strong>of</strong> X. brasilicum.<br />
N-hexane extract was further assayed with concentrations <strong>of</strong> 5, 6.5, 8, 9.5 and 11µg/ml. The activity<br />
<strong>of</strong> the 14 chromatographic fractions were assayed using 5 and 10 µg/ml. Fraction 10 and 11 were<br />
further investigated using concentrations <strong>of</strong> 2.5, 5, 7.5 and 10 µg/ml. The test was performed using<br />
the above mentioned method.<br />
Anti-amastigotes<br />
Culture and preparation <strong>of</strong> human monocytes (THP-1)<br />
THP-1 cell lines were used for infection with the promastigotes. Cells were cultured in RPMI-1640<br />
complete medium at 37�C, 5% CO2/ 59% air mixture. THP-1 cells were transferred to 50 ml<br />
centrifuge tube and centrifuged at 4�C, 2000 rpm for 10 minutes. The supernatant was discarded<br />
and the pellet gently resuspended in a small volume <strong>of</strong> fresh culture medium, and then counted<br />
using Neubauer hemocytometer. Phorbol 12-myristate 13-acetate (PMA) 20 ng/ml was added to<br />
allow cells differentiation and adherence. The cells were seeded in 16-well tissue-culture slides at a<br />
density <strong>of</strong> 40,000 cells/well i.e. 4x10 5 /ml, 100µl/well and maintained at 37ºC, 5% CO2/ 95% air<br />
mixture for 48 hours. After that the cells were washed by replacing the overlay with 100 µl fresh<br />
culture medium without PMA and incubated for further 24 hours.<br />
Infection <strong>of</strong> the macrophages with Leishmania promastigotes<br />
Adherent macrophages were infected with late-stage promastigotes and incubated at 37�C, 5%<br />
CO2/ 95% air mixture, for 24 hours. The infected cells were washed with cold (4�C) culture medium.<br />
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The overlay was removed with sterile Pasteure pipette, and with a multi-channel pipette, 100 µl <strong>of</strong><br />
the cold medium gently dispensed and withdrawn 2-3 times. This dislodges the majority <strong>of</strong> any<br />
extracellular promastigotes. Finally, 100 µl <strong>of</strong> the complete medium was added prior administration<br />
<strong>of</strong> the drug. Sample was applied when the infection average reached 80<br />
Drug application<br />
Stock solution <strong>of</strong> the drug with concentration <strong>of</strong> 20 mg/ml was prepared; from this solution 2x<br />
dilution <strong>of</strong> 60µg/ml was made by taking 3µl <strong>of</strong> the stock solution + 997µl complete medium. After<br />
that serial dilutions were made in the wells as follows: the first well contained 75µl and the<br />
remaining wells each had 100µl fresh complete medium. 75µl <strong>of</strong> the 2x dilution <strong>of</strong> 60µg/ml was<br />
placed into the first well and mixed. Using 4 channels <strong>of</strong> a multichannel pipette, 50µl is removed,<br />
transferred and mixed (Conc 2) and so on, to produce a 3-fold dilution series and leaving 100µl in<br />
each well.<br />
Isolation <strong>of</strong> compound A & B<br />
After cooling the petroleum ether and n-hexane extracts at room temprature a pale yellow crystalline<br />
precipitate was formed on the bottom and the wall <strong>of</strong> the flask. The precipitate was collected by decantation<br />
<strong>of</strong> the extract, then subjected to further decolorization by dissolving it in chlor<strong>of</strong>orm and passing the<br />
chlor<strong>of</strong>orm solution through a small column (30 x 1.5 cm) containing activated charcoal loaded on a piece <strong>of</strong><br />
cotton wool and sand. The filtrate was collected in small vials and the solvent was evaporated to dryness to<br />
obtain a pale yellow crystals. Further more the compound was recrystallized.<br />
Recrystallization <strong>of</strong> compound A & B<br />
Compounds A & B were dissolved in a little amount <strong>of</strong> chlor<strong>of</strong>orm, and then petroleum ether was added<br />
gradually with stirring till complete solubility, and then allowed for sometime for recrystallization <strong>of</strong> the<br />
compound. The solvent was removed by decantation, and the crystals were dried on a filter paper. This<br />
process was repeated three times. Colourless crystals were obtained.<br />
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Results and discussion<br />
General Antileishmanial screening<br />
Table 1.<br />
No Plant Inhibition %<br />
Petroleum ether Chlor<strong>of</strong>orm Methanol<br />
1 Cassia obtusifolia + +++ -<br />
2 Indig<strong>of</strong>era oblongifolia +++ ++ +++<br />
3 Xanthium brasilicum +++ +++ ++<br />
4 Tinospora bakis +++ - -<br />
5 Striga hermonthica +++ +++ -<br />
6 Anogeissus leiocarpus + +++ +++<br />
7 Annona spp. - +++ +++<br />
8 Croton zambesicus +++ +++ +++<br />
9 Pulicaria crispa +++ +++ -<br />
10 Lwasonia innermis + +++ ++<br />
11 Argemone mexicana ND +++ -<br />
(-): No or very weak activity, (+) Inhibition % < 50%, (++): Inhibition %
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
1 2 3 4 5<br />
Conc µg/ml 5 6.5 8 9.5 11<br />
Inhibition % 56 69.25 76.57 100 100<br />
SD 0.5 1.5 2.1 0 0<br />
Fig. 1. Inhibition % for n-hexane crude extract obtained successively, after 72 hours incubation. Inhibition<br />
was dose dependent. The parasites morphology and motility were completely changes even at small doses.<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
1 2 3 4 5 6<br />
Conc µg/ml 3.5 5 6.5 8 9.5 11<br />
Inhibition % 70 73.35 82.5 86.65 100 100<br />
SD 2 1.5 1.7 2.5 0 0<br />
Fig. 2. Inhibition % for compound B, isolated from n-hexane successive extract, after 72 hours incubation<br />
period. The compound was more active than the original crude extract, reaching100% inhibition at a dose <strong>of</strong><br />
9.5µg/ml and 70 at 3.5µg/ml marked changes in the morphology <strong>of</strong> the remaining parasites. Inhibition<br />
occurred in a dose dependant manner.<br />
Table 3. Inhibition % for compound B against the intracellular amastigotes. The compound was toxic to both<br />
macrophages and the parasites at the concentration <strong>of</strong> 3.3µg/ml indicating its low selectivity towards the<br />
parasites.<br />
T: toxic to both macrophage and the parasite<br />
Conc µg/ml Inhibition %<br />
3.3 T<br />
1.1 29.8<br />
0.37 7.4<br />
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O<br />
O<br />
H<br />
H<br />
Chemical name: (2E)-methyl 3-((1E,4E)-7-methyl-4-(2oxopropylidene)cyclohept-1enyl)acrylat<br />
Molecular formula: C 15H 20O 3<br />
Molecular weight: 248.32<br />
Melting point: 94°C<br />
H<br />
Fig. 5. The proposed structure <strong>of</strong> compound (B). This structure was based on the above spectroscopic data.<br />
However it is not the final suggestion, further analysis like two dimensional NMR is needed to confirm this<br />
suggestion.<br />
Conclusion<br />
This study showed the importance <strong>of</strong> plants and plant-derived compounds as source for new<br />
molecules and important leads for drug discovery and development. X. brasilicum possesses very<br />
good antileishamial activity, in its nonpolar extracts mostly n-hexane extract. Bioactivity guided<br />
fractionation had led to isolation <strong>of</strong> an active compound which was identified with the means <strong>of</strong><br />
spectroscopic method as: (2E)-methyl 3-((1E,4E)-7-methyl-4-(2-oxopropylidene)cyclohept-1enyl)acrylate,<br />
beside this compound we have other active compounds. Intracellular amastigotes<br />
studies showed that the compound exhibited some toxicity against human macrophages.<br />
References<br />
Ashford, R. W. (1996); Leishmaniasis reservoirs and their significance in control. Clinics in dermatology, 14, 523-532.<br />
Carvalho, P. B., and, Ferreira, E. I. (2001; Leishmaniasis phytotherapy. Natures s leadership against an ancient disease.<br />
Fitoterapia, 72, 599-618.<br />
Cr<strong>of</strong>t, S. L., Sundar, S., Fairlamb, A. H., (2006); Drugs resistance in leishamanaisis. Clinical Microbiology Reviews, 19 (1),<br />
111-126.<br />
Desjeux, P. (1996); Leishmaniasis: public health aspects and control. Clincs in Dermatology, 14, 417-423.<br />
Guerin, P. J., Olliaro, P., Sundar, S., Boelaert, M., Cr<strong>of</strong>t, S., Desjeux, P., Wasunna, M. K. and Bryceson, D. M. (2002).<br />
Visceral lieshmaniasis: current status <strong>of</strong> control, diagnosis, and treatment and a proposed research and<br />
development agenda. The Lancet Infectious Diseases, 2, 494-501.<br />
Kayser, O., Kiderlen, A. F., Cr<strong>of</strong>t, S. L. (2003); Natural products as antiparasitic drugs. Parasitology Research, 90, 55-62.<br />
Murray, H., W. (2002); Kalaazar progress against a neglected disease. New England Journal <strong>of</strong> Medicine, 347 (22), 1793-<br />
1794.<br />
Murray, H. W, Berman, J. D, Davis, C. R, Saravia, N. G. (2005); Advances in leishmaniasis. The Lancet, 366, 1561-1577.<br />
World Health Organization. (2002); Leishmaniasis. Technical Report . 53. WHO, Genevava.<br />
World Health Organization. (2010); Geneva. http://www.who.int/leishmaniasis/disease_epidemiology.<br />
162<br />
O
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[SL 16A] Assessment <strong>of</strong> Azadirachta Indica and Cassia Spectabilis for Some<br />
Immunomodulatory Properties<br />
Ndze Ralph Kinyuy 1* , Abubakar Abdulkadir 2 , Wirkom Venansius Kihdze 1 , Tanayen Grace Ghaife 3 ,<br />
Tanayen Julius Kihdze 4<br />
1 Chemical Pathology Department, Federal College <strong>of</strong> Veterinary and Medical Laboratory Technology, N.V.R.I. Vom,<br />
Plateau State, Nigeria<br />
2 Biochemistry and Chemotherapy Division, Nigerian Institute for Trypanosomiasis Research, P.M.B. 03 Vom, Plateau<br />
State, Nigeria<br />
3 Diagnostics Department, Kampala International University Teaching Hospital, Ishaka, P.O. Box 71, Bushenyi, Uganda<br />
4 Department <strong>of</strong> Pharmacology and Toxicology, Kampala International University Western Campus. P.O. Box 71<br />
Bushenyi, Uganda.<br />
* Corresponding author (Phone: +2348061632025; E-mail ndzeraphael@yahoo.com)<br />
Key words: Azadirachta indica, Cassia spectabilis, immunomodulatory properties, medicinal plants, traditional<br />
medicine.<br />
Introduction<br />
I<br />
t has become a common practice to search for contemporary drugs among herbs used in<br />
traditional medicines partially because the plants contain bioactive substances. Also there is the<br />
general belief that the herbal medicines are safer. In developing countries herbal medicines are<br />
more available and affordable. It is thus necessary to explore this area to provide a scientific<br />
rationale for the use <strong>of</strong> herbal medicines on which 80% <strong>of</strong> our rural populations rely for primary<br />
healthcare (Kamatenesi-Mugisha et al, 2000).<br />
Methods<br />
Healthy male swiss albino mice were orally administered 2.0 and 4.0mg/kg bodyweight and, 100<br />
and 200mg/kg bodyweight <strong>of</strong> Azadirachta indica and Cassia spectabilis respectively, for 7days while<br />
the control group was given distilled water. Each group contained five animals (n=5). The animals<br />
were then sacrificed humanely and blood collected by cardiac puncture. After centrifugation, the<br />
blood was analysed for adenosine deaminase (ADA) activity (Matinek 1963), total protein, serum<br />
albumin and serum globulin.<br />
Results<br />
There was a statistically significant (p
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
GROUPS EXTRACT/DO<br />
SAGE<br />
A A. indica<br />
2mg/kg<br />
B A. indica<br />
4mg/kg<br />
C C. spectabilis<br />
100mg/kg<br />
D C. spectabilis<br />
200mg/kg<br />
ADA activity<br />
(IU/l)<br />
TOTAL<br />
PROTEIN<br />
(g/dl)<br />
164<br />
SERUM<br />
ALBUMIN (g/dl)<br />
SERUM<br />
GLOBULIN<br />
(g/dl)<br />
1.34±0.3 7.03± 0.9 4.31± 0.5 2.72 ±1.3<br />
6.65± 2.5* 5.76± 0.8 4.66± 2.5 1.10± 1.4<br />
5.15± 2.3 5.65± 0.6 4.59 ±2.3 1.06± 0.7<br />
19.34<br />
±3.2*<br />
7.60± 0.9 4.75 ±3.2 2.85 ±1.9<br />
E Control 6.85 ±2.6 7.12± 1.1 4.39 ±0.7 2.73± 0.4<br />
* Significant at p 0.05 and n=5<br />
Conclusion<br />
A. indica directly affects the pathogens while C. spectabilis modulates the immune system response.<br />
Acknowledgement<br />
We are deeply indebted to the following for their technical support; Dr. Bulus Adzu, Dr. Christian Ezeala and<br />
Mr. Claude Kirimuhuzya.<br />
References<br />
Kamatenesi-Mugisha M, HÖft R, Bukenya Ziraba R. (2000); Ethnomedical use <strong>of</strong> Rytigynia [Nyakibazi] in Bwindi<br />
Impenetrable National Park, SW Uganda. Norwegian Journal <strong>of</strong> Botany, LIDIA; 5 (4): 97-108.<br />
Martinek G. Robert (1963); Micromethod for the Estimation <strong>of</strong> Serum Adenosine Deaminase. Journal <strong>of</strong> Clinical<br />
Chemistry, vol 9, No 5, 620-642.<br />
http://www.clinchem.org/cgi/reprint/9/5/620.pdf
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[SL 17A] Evaluation <strong>of</strong> the Biosafety <strong>of</strong> Selected Botanical Pesticide Plants Used<br />
by Subsistance Farmers Around the Lake Victoria Basin<br />
Maud Kamatenesi-Mugisha 1 , Buyungo John Paul 1 , Vudriko Patrick 2 and Ogwal Patrick 3 Arop Deng 4 ,<br />
Joshua Ogendo 4 , J.M. Mihale 5<br />
1 Division <strong>of</strong> Ethnobotany, Department <strong>of</strong> Biology, Makerere University. P.O Box 7062, Kampala, Uganda.<br />
2 Division <strong>of</strong> Pharmacology, Therapeutics and Toxicology Department <strong>of</strong> Physiological Sciences, School <strong>of</strong> Veterinary<br />
Medicine. Makerere University. P.O Box 7062, Kampala, Uganda.<br />
3<br />
Natural Chemotherapeutics research Laboratory, Ministry <strong>of</strong> Health Uganda.<br />
4<br />
Egerton University, P.O Box 536 Egerton, Kenya<br />
5<br />
Open University <strong>of</strong> Tanzania, P.O Box 31608, Dar es salaam, Tanzania<br />
mkamatenesi@botany.mak.ac.ug,<br />
Key words: botanical pesticides, biosafety, oral acute toxicity, Lake Victoria Basin<br />
Introduction<br />
T<br />
here is a very long history <strong>of</strong> use <strong>of</strong> botanical extracts for human and veterinary medicines as<br />
well as for the protection <strong>of</strong> field and stored crops (Berger 1994). In the recent decades<br />
however, due to the introduction <strong>of</strong> synthetic pesticides, the adoption <strong>of</strong> these traditional<br />
approaches <strong>of</strong> crop and post harvest protection have not been improved (Berger 1994). Today, the<br />
use <strong>of</strong> plant extracts for controlling pests has been limited to small holder farmers, who in most<br />
cases have been supported by various Non Government Organizations (NGOs) and women groups (<br />
Mugisha-Kamatenesi et al. 2008). The use <strong>of</strong> synthetic pesticides has undoubtedly increased crop<br />
production. This has been possible through reduced losses caused by crop pests but many <strong>of</strong> these<br />
chemicals are hazardous to both humans and the environment at large. According to Blackman et<br />
al. (1999), it has been estimated that hardly 0.1% <strong>of</strong> the agrochemicals used for crop protection<br />
reach the target pest leaving the remaining 99.9% to enter the environment and cause hazards to<br />
non-target organisms including humans.<br />
Materials and methods<br />
Collection <strong>of</strong> plant materials<br />
Leaves <strong>of</strong> the selected pesticide plants were collected from Mukono district, Uganda.<br />
Preparation <strong>of</strong> plant materials<br />
Identification <strong>of</strong> the collected plants<br />
The plants were identified by taxonomists in the Herbarium Department <strong>of</strong> Botany, Makerere<br />
University. This was done to ensure that the plants are rightly identified to avoid any confusion.<br />
Drying <strong>of</strong> plant materials<br />
The collected plant materials were separately exposed under shade until they become dry.<br />
Extraction and concentration <strong>of</strong> Plant extracts<br />
Ethanol and water extracts <strong>of</strong> plant materials<br />
Leaves <strong>of</strong> Cupressus lusitanica, Ocimum suave, Tithonia diversifolia and Eucalyptus globulus were<br />
pounded. Known weights <strong>of</strong> the pounded material were separately soaked in ethanol (95 %) for<br />
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three days and then filtered. The solutions were filtered by use <strong>of</strong> filter papers and the extracts<br />
were thereafter concentrated to thick residues on a rotary evaporator and water bath maintained<br />
at 70 C. The concentrated ethanol extracts were put in well labeled, dry, clean bottles. In order to<br />
obtain the water extract, known amounts <strong>of</strong> the pounded materials were separately soaked in<br />
distilled water for two days to obtain the aqueous extract. The solutions were then filtered through<br />
a cheese cloth before further filtration using a Whitman No.A-1 filter paper. The filtrates were<br />
concentrated in a hot air oven maintained at 50 C for tow days and were subsequently air-dried to<br />
thicker residues.<br />
Extraction <strong>of</strong> Essential oils<br />
For Cupressus lusitanica, Ocimum suave and Eucalyptus globulus, essential oils were extracted by<br />
steam distillation from fresh leaves. The leaves will be cut into small pieces, put into a distillation<br />
flask. Steam was allowed to pass through each batch <strong>of</strong> leaves for two hours. Essential oils were<br />
trapped in collecting tubes and put in clean, dry and well labeled bottles, which were then kept in a<br />
fridge maintained at low temperatures to freeze the water which had been trapped together with<br />
the oil.<br />
Preparation <strong>of</strong> the test animals<br />
Test animals (wistar mice and rats) were purchased at 4 weeks <strong>of</strong> age from the faculty <strong>of</strong> veterinary<br />
medicine, Makerere University. They were randomly group housed in stainless wire cages living<br />
enough space for clear observation <strong>of</strong> each animal. The animals were kept on a 12 hour artificial<br />
light and dark cycle at 22 + 30 C. Conventional laboratory diets were used to feed the animals with<br />
unlimited supply <strong>of</strong> drinking water. This was done for 5 days prior to dosing so as to get them<br />
acclimatized to laboratory conditions. During this period, the mice were observed to asses their<br />
health conditions basing on their external appearance, nutritional conditions and general behavior.<br />
Determination <strong>of</strong> the acute and chronic toxicity <strong>of</strong> the plant extracts using mice<br />
Acute toxicity studies<br />
In order to determine the preliminary acute toxicity <strong>of</strong> the different plant extracts, 4 dose levels<br />
were prepared for each plant extract. Each dose level was assigned 2 test animals (mice). The<br />
administration <strong>of</strong> the test substances was done by use <strong>of</strong> intragastric plastic tubes to the different<br />
groups <strong>of</strong> test animals. The rough LD 50 was then used to determine the accurate LD50. For the<br />
accurate LD 50, five dose levels were set within the range <strong>of</strong> the rough LD50 for each extract. Each<br />
group was assigned 6 members (mice).<br />
Sub chronic toxicity studies using rats<br />
Sub chronic studies were done for two essential oils (Eucalyputus globulus and Cuppresus<br />
lusitanica). Test animals were divided into three groups <strong>of</strong> 10 members for each essential oil. A<br />
group <strong>of</strong> 10 members was kept as a control group and received 1 ml <strong>of</strong> the carrier substance<br />
(oil/emulsifier/water respectively) in the ratio 4:2:1 .Tween 80 2 % was used as the emulsifying<br />
agent.<br />
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Haematology<br />
Blood samples for bioassays were obtained from the tails <strong>of</strong> rats under anesthesia every after two<br />
weeks <strong>of</strong> administration. Heamatological parameters including haemoglobin (Hb), total leukocyte<br />
count (TLC), differential count including total lymphocyte, poly-morphonuclear leucocytes, and<br />
eosinnophil counts, platelet count, protrombin time and packed cell volume (PCV) were analyzed<br />
using standard techniques.<br />
Table <strong>of</strong> results showing the LD 50s <strong>of</strong> the different plant extracts<br />
NO. Plant name Extract LD 50 Value<br />
1. Ocimum suave Essential oil 4,677 mg/kg<br />
2. Ocimum suave Ethanol extract 13,182 mg/kg<br />
3. Ocimum suave Aqueous extract<br />
4. Cupressus lusitanica Essential oil 2,951.2 mg/kg<br />
5. Cupressus lusitanica Ethanol extract 14,791 mg/kg<br />
6. Cupressus lusitanica Aqeous extract<br />
7. Eucalyptus globulus Essential oil 2,290 mg/kg<br />
8. Eucalyptus globulus Ethanol extract 12,589.3 mg/kg<br />
9. Eucalyptus globulus Aqeous extract<br />
10. Tithonia diversifolia Ethanol extract 11,748 mg/kg<br />
11. Tithonia diversifolia Aqeous extract 11,885 mg/kg<br />
12. Tithonia diversifolia<br />
Acknowledgements<br />
We acknowledge VICRES for funding this research and the laboratories we used especially the<br />
Division <strong>of</strong> Pharmacology, Therapeutics and Toxicology Department <strong>of</strong> Physiological Sciences,<br />
School <strong>of</strong> Veterinary Medicine, Makerere University and Natural Chemotherapeutics Research<br />
Laboratory, Ministry <strong>of</strong> Health Uganda. The local people especially the farmers who provided the<br />
knowledge are appreciated.<br />
References<br />
1. Ajala, S.O, K, Kiling, J.G, Cardwell, K, (2001); Pests and Pesticides. International Institute <strong>of</strong> Tropical Agriculture,<br />
Ibadan, Nigeria.<br />
2. Akanbi, W.B, Adebayo, F. A, Togun, A.O, Adeyeye, A.J, Olaniran, O.A (2007); The use <strong>of</strong> Composst Extracts as Folia<br />
spray Nutrient source and botanical<br />
3. Insecticide in Telfairia Occidentalis. World Journal <strong>of</strong> Agricultural science 3(5) Pages 642-652. IDOSI publications.<br />
4. Attila Kis-Tamas (1990); Study on the Production Possibilities <strong>of</strong> Botanical Pesticides in developing African countries.<br />
United Nations Industrial development Organisation Vol.90-86421 Budapest, Hungary.<br />
5. Belmain, S, Neal S.R., Ray, G. E, Golob D. E. (2001); Ethnobotanicals in Ghana. Insecticidal and Vertebrate toxicity<br />
associated with ethno botanicals. Journal <strong>of</strong> toxicology, 39(3) 287-291.<br />
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[SL 18A] The in vitro Antimycobacterial Activity <strong>of</strong> Medicinal Plants Used by<br />
Traditional Medicine Practitioners (TMPs) to Treat Tuberculosis in the Lake Victoria<br />
Basin in Uganda<br />
*Kirimuhuzya Claude 1 , Bunalema Lydia 1 , Tabuti John RS 2 , Kakudidi K Esezah 2 , Orodho John 3 , Magadula Jangu<br />
Joseph 4 , Otieno Nicholas 4 and Paul Okemo 5<br />
1,2 Makerere University Department <strong>of</strong> Botany & Pharmacology and Therapeutics, respectively<br />
3,5 Kenyatta University, School <strong>of</strong> Education & Pure and Applied Sciences, respectively<br />
4 Muhimbili University, Institute <strong>of</strong> Traditional Medicine<br />
* Corresponding Author: Department <strong>of</strong> Pharmacology and Therapeutics, Makerere University College <strong>of</strong> Health<br />
Sciences, P.O.Box7062, Kampala. E-mail: claudekirim@yahoo.co.uk. Phone: +256 772 645 991<br />
Key words: In vitro activity; Anti-mycobacterial; Medicinal plant; Mycobacterium tuberculosis; Mycobacterium avium;<br />
rifampicin; isoniazid<br />
Introduction<br />
T<br />
uberculosis (TB) is one <strong>of</strong> the dreadful infectious diseases and the leading cause <strong>of</strong> mortality<br />
worldwide, with approximately 9 million people developing the disease and 2 million people<br />
dying annually (WHO, 2007; Sanjay 2004; Navin et al., 2002). Globally, more than one-third <strong>of</strong> the<br />
world s population (more than 2 billion) is infected with MTB (CDC Report, 2005; Navin et al., 2002).<br />
Outbreaks <strong>of</strong> multi-drug resistant (MDR) and extensively drug-resistant (XDR) tuberculosis have also<br />
compounded the problem. The emergence <strong>of</strong> XDR TB is cause for concern because it is widely<br />
distributed geographically (now in over 50 countries on all inhabited continents), and renders<br />
patients virtually untreatable with available drugs. New drugs have to be developed to deal with<br />
MDR and XDR TB strains, which have already become a problem especially where there is coinfection<br />
with HIV/AIDS. There is an urgent need to search for and develop new, comprehensive,<br />
safer and more effective quick acting and affordable anti-TB agents, and this also includes searching<br />
for leads from natural products <strong>of</strong> plant origin. This presentation is a report <strong>of</strong> results <strong>of</strong> a study<br />
that was carried out to identify the medicinal plants used by Traditional Medicine Practitioners in<br />
the Lake Victoria Region <strong>of</strong> Uganda, and their subsequent screening against rifampicin-resistant<br />
mycobacterium tuberculosis. The objectives <strong>of</strong> the study included documenting indigenous<br />
knowledge and the existing practices used by traditional medicine practitioners (TMPs) in the<br />
treatment <strong>of</strong> TB and to scientifically validate the TMPs claims.<br />
Materials and Methods<br />
The study area included three districts <strong>of</strong> Mayuge, Mbarara and Mukono as part <strong>of</strong> a wider East<br />
African regional survey in the Lake Victoria basin. Data was mainly gathered using key informant<br />
interviews, guided questionnaire interviews and direct observation techniques. A total <strong>of</strong> 31 TMPs<br />
were interviewed as well as 16 patients who had received TM treatment for TB about their health<br />
seeking behaviour and attitudes towards use <strong>of</strong> TM. After the survey, a list <strong>of</strong> the most frequently<br />
mentioned plant species was prepared and parts <strong>of</strong> 11selected plants were collected from various<br />
areas, their crude petroleum ether, chlor<strong>of</strong>orm and methanol extracts prepared and tested in a<br />
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bioassay on three strains <strong>of</strong> Mycobacterium. The antimycobacterial tests were done according to<br />
Parish and Stroker (1998). Susceptibility tests were carried out using the disc diffusion method on<br />
Middlebrook 7H10, while MIC and <strong>MB</strong>C tests for the active extracts were carried out using the<br />
Microtitre plate method where Middle brook 7H9 broth was used. Phytochemical screening<br />
(Edeoga et al., 2005) and acute toxicity tests (Gosh 1984) were also done for the most active<br />
extracts. Mycobacterium strains used were obtained from Joint Clinical Research Centre (JCRC)<br />
Mengo in Kampala, Uganda, where the mycobacteriology work was carried out, and included a<br />
rifampicin-resistant strain (TMC -331strain) to serve as an indicator <strong>of</strong> MDR, a fully susceptible<br />
strain (H37Rv) as a control, and Mycobacterium avium (MA) a wild strain from a Ugandan patient to<br />
represent the Mycobacterium other than tuberculosis (MOTT) group. Acute toxicity tests were<br />
done on the most active extracts according to Ghosh (1984).<br />
Results and Discussion<br />
Over 50 plant species were mentioned by the TMPs. Herbal drugs were prepared as mixtures <strong>of</strong><br />
four or more plants. The most frequently used plant parts were leaves, root wood, stem bark and<br />
fruit. Of the screened plants, four were found active against TB, two <strong>of</strong> which were active on all the<br />
three strains <strong>of</strong> Mycobacterium used, including the rifampicin-resistant. All the active extracts were<br />
bactericidal although their activity was lower compared with isoniazid and rifampicin. However,<br />
they had an advantage over rifampicin, one <strong>of</strong> the first-line anti TB drugs, by being active against<br />
rifampicin resistant TB. With regard to susceptibility tests the highest activity was registered with<br />
Erythrina abyssinica (VT8), Cryptolepis sanguinolenta (VT10), Warburgia ugandensis (VT2) (Wube et<br />
al., 2005), Mangifera indica (VT6) with zones <strong>of</strong> inhibition ranging between 10.7 and 23mm<br />
(including diameter <strong>of</strong> the disc which was 6mm). The concentrations <strong>of</strong> the extracts were at 50<br />
mg/ml (25 mg/ml for C.sanguinolenta). Rifampicin was not active on Mycobacterium avium<br />
complex and a rifampicin resistant strain TMC-331 but it showed a zone <strong>of</strong> inhibition <strong>of</strong> 26 mm for<br />
H37Rv (a pan sensitive strain) at a concentration <strong>of</strong> 0.1 mg. Isoniazid cleared the quadrant for two<br />
strains at a concentration <strong>of</strong> 0.05mg but it was also not effective on M.avium. The MICs <strong>of</strong> the<br />
active crude extracts ranged between 1.17 and 6.25 mg/ml while for rifampicin and isoniazid they<br />
were between 0.25 and 9.38µg/ml. The <strong>MB</strong>Cs for the active crude extracts were between 0.20 and<br />
6.25 mg/ml while for rifampicin and isoniazid they were between 0.25 and 1.0 µg/ml (but<br />
rifampicin was inactive on TMC-331). Alkaloids were found mainly in C. sanguinolenta (Gibbons et<br />
al., 2003) and flavones mainly in the extracts <strong>of</strong> E. abyssinica. Acute toxicity tests on E. abyssinica<br />
and C. sanguinolenta gave LD50 between 700 and 800mg/kg body weight which were in the<br />
relatively safe range. Phase III <strong>of</strong> the project involving isolation, characterization and identification<br />
<strong>of</strong> the compounds that are active on M. tuberculosis is under way.<br />
Conclusion<br />
The bioassays conducted on the selected plant species further vindicated some <strong>of</strong> the claims by the<br />
TMPs by showing activity against M. tuberculosis although more research is required especially in<br />
the area <strong>of</strong> standardization. However, isolation and screening active compounds and more in vitro<br />
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and in vivo studies on the toxicity <strong>of</strong> the plants are needed before declaring them completely safe<br />
for use in humans.<br />
Acknowledgements<br />
We are grateful to the following:<br />
1. Lake Victoria Research Initiative (VicRes) who provided the funding<br />
2. Uganda National Council for Science and Technology, for allowing the study to take place<br />
3. The Departments <strong>of</strong> Pharmacology and Therapeutics and Botany, Makerere University, for<br />
providing the equipment and some <strong>of</strong> the materials used during the study;<br />
3. Joint Clinical Research Centre (JCRC), Mengo, Kampala, Uganda for allowing us access to their<br />
Mycobacteriology laboratory.<br />
4. The herbalists, who provided the ethnobotanical information used as the basis for screening the<br />
plants.<br />
References<br />
Centre for Disease Control (2005); Worldwide emergence <strong>of</strong> Mycobacterium tuberculosis with extensive resistance to<br />
second-line drugs. Morbidity and Mortality Weekly Report, 55, 250-253<br />
Edeoga, HO, Okwu, DE, Mbaebie, BO (2005); Phytochemical constituents <strong>of</strong> some Nigerian medicinal plants. Afri. J.<br />
Biotechnol. 4: 685-688.<br />
Gibbons, S, Fallah, F., Wright, CW. (2003); Cryptolepine Hydrochloride: A Potent Antimycobacterial Alkaloid Derived<br />
from Cryptolepis sanguinolenta. Phytotherapy research, 17, 434-436<br />
Ghosh, MN. (1984); Fundamentals <strong>of</strong> Experimental Pharmacology.2 nd Edition;153-190; Scientific <strong>Book</strong> Agency , Culcutta.<br />
Navin, TR, Mc Nab, SJ, Crawford, JT (2002); The continued threat to tuberculosis. Emerg Infec Dis. 8: 1187.<br />
Parish, T., Stroker, NG. (1998); Mycobacteria Protocols: Methods in molecular Biology. (vol 101) (eds,) Humana Press,<br />
Totowa, NJ. 395-422.<br />
Sanjay, MJ. (2004); Natural Products: An important source for Anti-tubercular Drugs. CRISP. 5 :1.<br />
World Health organization (2007); Global Tuberculosis Database. [Online Tuberculosis Database as <strong>of</strong> 21 st March 2005].<br />
Wube, AA, Bucar, F, Gibbons, S, Asres, K. (2005); Sesquiterpenes from Warburgia ugandensis and their<br />
antimycobacterial activity. Phytochemistry 66(19): 2309-15.<br />
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[SL 19A] Effects <strong>of</strong> Aqueous Extracts <strong>of</strong> Basil, Ocimum basilicum L., Sodom s apple,<br />
Calotropis procera Ait and Coriander Coriandrum sativum L. on leaf miner,<br />
Liriomyza Spp., on okra Crop.<br />
Rehab E. H. Fadwal, Faiza E. E. Salah, Mohammed H. Z. Elabdeen and Elamin M. E.<br />
Dept. <strong>of</strong> Crop Protection, Faculty <strong>of</strong> Agricultural sciences, U. <strong>of</strong> Gezira, Sudan<br />
Introduction<br />
O<br />
kra, Ablemoschus (Hibiscus) esculentus (L.) (Moench), is ranking third <strong>of</strong> the major vegetable<br />
crops, and is one <strong>of</strong> the most popular and main Sudanese dishes. The most famous cultivars in<br />
the Sudan are Khartoumia, Momtaza, Karrari, Kassala, Dwarf long green and Clemson spineless<br />
(Bugstaler et al., 1984). Okra is grown under irrigation all year around, but partienlarly in summer. It<br />
is attacked by a number <strong>of</strong> insect pests one <strong>of</strong> which is the vegetable leafminers, Liriomyza spp.<br />
These are one <strong>of</strong> the largest groups or genera with over 300 species through out the world. Only<br />
about 15 different species are known to feed on cultivated plants and thus have some actual or<br />
potential economic significance. In the Sudan two species <strong>of</strong> liriomyza were reported: L. trifolii<br />
(Burgess) (Sharaf EL-Din et al., 1997) and L. sativae (Blanchard) recorded by Martinez and Brdat<br />
(1996).<br />
Materials and Methods<br />
An experiment was conducted at the U. Of Gezira Experimental farm to evaluate the effects <strong>of</strong> 10%<br />
aqueous leaf and fruit extracts <strong>of</strong> Basil, (Ocimum basilicum L.), leaf extracts <strong>of</strong> Sodom s apple,<br />
(Calotropis procera Ait ) and fruit extract <strong>of</strong> Coriander , (Coriandrum sativum L.) on the vegetable<br />
leaf miner, Liriomyza Spp .on okra. The experimental design comprised 16 plots assigned to 4<br />
treatments (replicated 4 times), arranged in a completely, randomized block design. Un dressed<br />
okra seeds were sown on 7/7/2008. Three natural products were used in the study Sodom s Apple<br />
(Usher), Calotropis procera Ait, Basil (Rehan), (Ocimum basilicum L.) and coriander (Kasbra),<br />
Coriandrum sativum L. The treatments consisted <strong>of</strong> spraying okra plants with either Basil, Sodom s<br />
apple and Coriander 10% aqueous extract or distilled water (control). The efficacy <strong>of</strong> the extracts<br />
was assessed in terms <strong>of</strong> active mines in okra leaves i.e., where leaf miner larvae were alive and<br />
feeding. Fresh leaves <strong>of</strong> Usher plant, leaves and fruits <strong>of</strong> Rehan plant and fruits <strong>of</strong> Kasbara plant,<br />
were dried in the laboratory at room temperature <strong>of</strong> 30 C. Dried plant materials were first crushed<br />
by hand then ground by an electric blender mixer, the powder was then stored in tightly covered<br />
glass jars and kept at room temperature in the laboratory ready for extraction.<br />
Results<br />
The results (Table 1) indicated that the three aqueous extracts significantly (P
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
treatment mean values for Usher, Kasbara and Rehan were significantly different from each other<br />
and the mean value recorded for the untreated okra.<br />
Table (1): Mean number <strong>of</strong> active mines <strong>of</strong> the leafminers Liriomyza spp. on okra leaves during the<br />
experimental period.<br />
Treatment Means *1<br />
Mean *2<br />
count 1 count2 count3 count4 count5 count6 Count7 <strong>of</strong> all<br />
counts<br />
Control 405 a 457 a 492 a 505 a 513 a 526 a 537 a 489 a<br />
Usher 348 b 117 b 113 b 69 c 33 b 33 b 32 b 106 d<br />
Rehnan 399 a 218 c 155 c 113 d 17 c 12 c 12 c 132 b<br />
Coriander 396 a 236 d 215 d 23 d 0 d 0 d 0 d 124 c<br />
S.E. 2.624 2.217 2.107 1.493 1.987 2.072 2.098 0.49<br />
CV% 14.22 14.43 14.54 10.00 22.40 24.01 24.19 16.30<br />
Means followed by the same letter (s) were not significantly different (P
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[SL 20A] Antiplasmodial Compounds from the leaves <strong>of</strong> Drypetes gerrardi<br />
Nga ng a MM, Thoruwa-Langat C, Chhabra S<br />
Department <strong>of</strong> Chemistry, Kenyatta University, Nairobi, KENYA.<br />
Mwihaki71@yahoo.com<br />
Key words: Drypetes gerrardi, Antimalarials, Antiplasmodial activity, Cytotoxicity<br />
Introduction<br />
M<br />
alaria is one <strong>of</strong> the most important parasitic infections <strong>of</strong> humans due to its high morbidity<br />
and mortality, a threat to over 2 billion people living in areas <strong>of</strong> high incidence (Andrade-<br />
Neto et al., 2004). Plasmodium falciparum, the causative agent <strong>of</strong> the malignant form <strong>of</strong> malaria,<br />
has high adaptability by mutation and is resistant to various types <strong>of</strong> antimalarial drugs, a serious<br />
setback to antimalarial programs, since it precludes the use <strong>of</strong> cheap and previously effective drugs<br />
like chloroquine. New families <strong>of</strong> active compounds are needed as well as poly chemotherapy<br />
associating molecules with independent mechanism <strong>of</strong> action, in order to decrease the risk <strong>of</strong><br />
resistance. In this paper we report the isolation <strong>of</strong> eight compounds (1-8) from Drypetes gerrardii<br />
(Euphorbiaceae) and there in vitro antiplasmodial activities against P. falciparum and their<br />
cyctotoxicity.<br />
Materials and Methods<br />
Extraction and Isolation<br />
The leaves <strong>of</strong> D. gerrardii were collected in Kilifi district Coast province in Kenya, in July 2004 and<br />
authenticated by Simon Mathenge, <strong>of</strong> Nairobi University, Kenya. A voucher specimen (MM/07/04)<br />
is deposited in Nairobi University herbarium, Chiromo Campus. The dried and powdered leaves (1<br />
kg) <strong>of</strong> D. gerrardii were exhaustively and sequentially extracted with petroleum ether, CH2Cl2,<br />
EtOAc and MeOH. The petroleum ether and the DCM crude extracts were combined based on their<br />
similarity on the TLC plate. The combined extract (32.6 g) was subjected to column chromatography<br />
on silica gel using petroleum ether, petroleum ether-EtOAc, EtOAc-MeOH and finally, pure MeOH<br />
as the mobile phase to yield 95 fractions (F1-95). Fractions 15-35 were combined and further<br />
separated by silica gel column chromatography eluting with petrol ether-EtOAc (3:1) to give white<br />
cotton needles <strong>of</strong> friedelin (1, 50 mg) and epifriedelanol (2, 10 mg). Similarly, repeated column<br />
chromatography <strong>of</strong> F54-67, which were eluted with petroleum ether: EtOAc (3:2) furnished<br />
friedelanol methyl ether (3, 12 mg). Further purification <strong>of</strong> F70-75 [petroleum ether-EtOAc (5.5:4.5)]<br />
and F78-85 [petroleum ether-EtOAc (3:7)] on a Sephadex LH-20 column with CH2Cl2: MeOH (7:3) as<br />
eluant combined with repeated crystallization using acetone afforded 5 ,24-cycl<strong>of</strong>riedelan-3-one<br />
(4, 8.6 mg).<br />
The crude ethyl acetate extract (15 g) was similarly chromatographed on a silica gel column and<br />
eluted with a gradient <strong>of</strong> petroleum ether, CH2Cl2, EtOAc, and MeOH yielding 65 fractions (F1-65).<br />
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Repeated column chromatography <strong>of</strong> F20-30 using a 5 % stepwise gradient <strong>of</strong> petroleum ether and<br />
ethyl acetate afforded 3-epimoretenol (5, 6.5 mg). Similarly repeated CC <strong>of</strong> fraction F31-35, eluted<br />
with a mixture <strong>of</strong> petroleum ether-EtOAc (8:2) and further purification in a Sephadex column using<br />
CH2Cl2: MeOH (1:1) gave resinone (6, 4.0 mg). Fraction F38-45 on CC using CH2Cl2-EtOAc (4:6), gave -<br />
sitosterol glucopyranoside (7, 50 mg). Preparative TLC <strong>of</strong> F50-58 eluted with ethyl acetate: MeOH<br />
(9.5:0.5) from the column, using CH2Cl2: MeOH (7:3) as the solvent system yielded 5 fractions. The<br />
polar fraction was further purified on a Sephadex LH-20 column using CH2Cl2:MeOH (1:1) and<br />
furnished ament<strong>of</strong>lavone (8, 6 mg) as a yellow powder.<br />
In Vitro drug sensitivity Protocol<br />
The semi automated micro dilution technique <strong>of</strong> Desjardins et al., (1979) for assessing the in vitro<br />
anti malarial activity as modified by le Bras and Deloron (1983) was adopted in the drug sensitivity<br />
studies for crude extracts, pure compounds and standard drugs against P. falciparum. This test<br />
evaluates the ability <strong>of</strong> the crude and pure compounds to inhibit growth <strong>of</strong> Plasmodium falciparum<br />
by preventing the uptake <strong>of</strong> [ 3 H]-hypoxanthine in vitro was carried out at the Kenya Medical<br />
Research Institute (KEMRI). For each assay chloroquine (Sigma C6628) and artemisinin (Arteannuin,<br />
Qinghaosu; Sigma 36,159-3) were used as the standard drugs with the highest concentration at 200<br />
ng/ml as positive control.<br />
Cytotoxicity assay<br />
In vitro cytotoxicity assay was carried out at KEMRI following a modified rapid calorimetric assay Of<br />
Mosmann, (1983) using Vero (199) cells.<br />
Results and Discussion<br />
The DCM and EtOAC extract <strong>of</strong> the leaves <strong>of</strong> D. gerrardii afforded one new flavone dimer, four<br />
friedelane-type triterpenoids namely friedelin (1) (Patra et al., 1990), epifriedelanol (2) (Bentacor et<br />
al., 1980), friedelanol methyl ether (3) (Samaraweera et al., 1983), and 5 ,24-cycl<strong>of</strong>riedelan-3-one<br />
(4) (Connolly et al., 1986) together with 3-epimoretenol (5) (hopane-type triterpenoid) (Khastgir et<br />
al., 1967), resinone (6) (lupane-type triterpenoid) (Pyrek and Baranowska, 1977), -sitosterol<br />
glucopyranoside (7) (Seo et al., 1978), and ament<strong>of</strong>lavone (8) (Goh et al., 1992; Lin et al., 2001) by<br />
comparison <strong>of</strong> 1 H and 13 C NMR data with reported data.<br />
In vitro antiplasmodial activity <strong>of</strong> pure isolated compounds from D. gerrardii<br />
Nine compounds isolated from D. gerrardii, were tested for in vitro anti-plasmodial activity against<br />
a K1 multidrug resistant strain <strong>of</strong> P. falciparum. The IC50 values are tabulated in Table 1 and their<br />
chemical structures are given in Figure 1. The antiplasmodial activity for the pure compounds (1-8)<br />
was considered high when IC50 < 1 µg/ml, moderate when between 1 and 5 and low when between<br />
5 and 10 µg/ml. Compounds with IC50 exceeding 10 µg/ml were considered to be inactive<br />
(Likhitwitayawuid et al., 1993). The definition <strong>of</strong> the cytotoxicity used: CC50 < 1.0 g/ml high<br />
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toxicity; CC50 1.0-10.0 g/ml moderate; CC50 10.0-30.0 g/ml mild toxicity; and CC50 > 30 g/ml<br />
non toxic. Podophyllotoxin (CC50 0.009+0.00003 g/ml) was used as standard toxin<br />
Selectivity index (SI) defined, as the ratio <strong>of</strong> IC50 (concentration required to cause visible alterations<br />
in 50% intact cells) <strong>of</strong> vero cells to IC50 P. falciparum was also determined. Selectivity index (SI) is<br />
used as a parameter <strong>of</strong> clinical significance <strong>of</strong> the test samples by comparing general toxins and<br />
selective inhibitory effect on P. falciparum (Wright and Phillipson, 1990). A pure compound is<br />
considered a hit if it is active in vitro against Plasmodium species with an IC50 <strong>of</strong> 1µg/ml and<br />
selective if it 10-fold more active against parasite than against a mammalian cell line<br />
(Likhitwitayawuid et al., 1993).<br />
Resinone (6) exhibited high antiplasmodial activity against K1 strain <strong>of</strong> P. falciparum with an IC50<br />
value <strong>of</strong> 0.09±0.01 µg/ml as well as satisfactory selectivity index. Ament<strong>of</strong>lavone (8) and 5 ,24cycl<strong>of</strong>riedelan-3-one<br />
(4) also exhibited moderate antiplasmodial activity <strong>of</strong> IC50 2.6+0.01 µg/ml and<br />
2.2±0.02 µg/ml respectively. Interestingly, ament<strong>of</strong>lavone (8) had high toxicity <strong>of</strong> CC50 0.34±0.00<br />
µg/ml as compared to 5 ,24-cycl<strong>of</strong>riedelan-3-one (4) that displayed mild toxicity This clearly<br />
indicated that the high antiplasmodial activity observed for ament<strong>of</strong>lavone (8) was probably due to<br />
cytotoxicity rather than the activity against the parasites The other compounds (1-3,5-7) gave<br />
antiplasmodial activity ranging from 4.8±0.11 µg/ml to >10 µg/ml. In addition 5 ,24-cycl<strong>of</strong>riedelan-<br />
3-one (4) that exhibited good antiplasmodial activity, did not demonstrate sufficient selectivity to<br />
kill the parasites without damaging mammalian cells. The selectivity index observed suggested that<br />
the antiplasmodia activity might be due to general toxicity. The antimalarial activity reported herein<br />
may explain the therapeutic efficacy claimed for these plants in traditional medicine and the<br />
compounds with appreciable activity may be used as scaffolds to generate leads with enhanced<br />
antiplasmodial activity, reduced cytotoxicity and improved bioavailability.<br />
R 2<br />
R 1<br />
1 R 1 , R 2 = O, R 3 = H<br />
R 3<br />
2 R 1 = H, R 2 = OH, R 3 = H<br />
O<br />
3 R 1 = OCH 3, R 2 = H, R 3 = H<br />
4<br />
HO<br />
175<br />
5
O<br />
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
6<br />
HO<br />
OH<br />
OH<br />
HO HO<br />
O<br />
O<br />
OH<br />
8<br />
176<br />
O<br />
OH<br />
HO<br />
O<br />
OH O<br />
OH<br />
OH<br />
Figure 1. Compounds 1 8 isolated from D. gerrardii<br />
Table 1. Antiplasmodial and cytotoxicity activity a <strong>of</strong> compounds 1-8 <strong>of</strong> D. gerrardii<br />
Compound IC50 (µg/ml) CC50 ± S.E<br />
( g/ml)<br />
1 4.8±0.11 >90 18.75<br />
2 >10 90.0±1.30 >9.00<br />
3 >10 32.8±0.90 3.28<br />
4 2.2±0.02 21.2±0.01 9.64<br />
5 >10 7.9±0.05 > 0.79<br />
6 0.09±0.01 84.8±2.34 942.2<br />
7 5.4±0.02 14.3±0.03 1.46<br />
8 2.6+0.01 0.34±0.00 0.13<br />
a P. falciparum K1 strain, Vero 199 cells, IC50<br />
inhibitory concentration for 50% <strong>of</strong> tested parasites,<br />
CC50 cytotoxic concentration for 50% <strong>of</strong> tested, cells,<br />
chloroquine IC50 0.063+0.03, artemisinin IC50<br />
0.002+0.000, Pdx - podophyllotoxin CC50 0.009+0.000<br />
O<br />
7<br />
5<br />
SI
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
References<br />
Andrade-Neto VF, Goulart MOF, Filho JFS, Silva MJ, Pinto MCFR, Pinto AV, Zalis MG Betancor C, Freire R, Gonzalez A,<br />
Salazar GJA, Pascard C, Prange T. 1980. Phytochem 19: 1989 1993.<br />
Carvalhoa LH, Krettli AU. 2004. Biorg Med Chem Lett 14: 1145 1149.<br />
Connolly, D.J., Freer, A.A., Anjaneyulu, V., Ravi, K., Sambasivarao, G., 1986. Acta Cryst C 42: 1352 1354.<br />
Desjardins, R.E., Canfield, R.E., Hayness, C.J., Chuby, J.D., (1979). Antimicrobial Agents and Chemotherapy 16, 710-718.<br />
Goh SH, Jantan I, Waterman PG. 1992. J Nat Prod 55: 1415 1420.<br />
Khastgir HN, Pradhan BP, Duffield AM, Durham LJ. 1967. J Chem Soc Chem Comm 1217 1218.<br />
Le Bras, J., Deloron, P., (1983). American Journal <strong>of</strong> Tropical Medicine and Hygiene 32, 447-51.<br />
Likhitwitayawuid K, Angerh<strong>of</strong>er CK, Chai H, Pezzuto JM, Cordell GA, Ruangrungsi N. 1993. J Nat Prod 56: 131-1338. J Nat<br />
Prod 64: 707-709.<br />
Mosmann, T., (1983). Journal <strong>of</strong> Immunology Methods 65, 55-63.<br />
Patra A, Chaudhuri SK, Acharyya AK. 1990. Magn Reson Chem 28: 85-89.<br />
Pyrek, J.S., Baranowska, E., 1977. Rocz Chem 51: 1141-1146.<br />
Samaraweera U, Sotheeswaran S, Sultanbawa MUS. 1983. Phytochem 22: 565-567.<br />
Seo S, Tomita Y, Tori K, Yoshimura Y. 1978.. J Amer Chem Soc 100: 3331 3339.<br />
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YOUNG SCIENTIST PRESENTATIONS<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[YS 1] Flavonoids with Anti-Diabetic Activiy from Erythrina Abyssinica<br />
Derek Tantoh Ndinteh 1 , Joseph Tanyi Mbafor 3 , Rui Werner Macedo Krause 1 , Won Ken OH 2<br />
1. Department <strong>of</strong> Chemical Technology, University <strong>of</strong> Johannesburg, Doorfontein, 2028, P.O. Box 17011.e.mail<br />
tantohtantoh@gmail.com<br />
2. College <strong>of</strong> Pharmacy, Chosun University, 375 Seosuk-dong, Gwangju 501-759, Republic <strong>of</strong> Korea<br />
3. Department <strong>of</strong> Organic Chemistry, Faculty <strong>of</strong> Sciences, University <strong>of</strong> Yaoundé I, P.O. box 812, Yaoundé Cameroon.<br />
Key words: Erythrina abyssinica, Flavonoids, PTP1B inhibitor.<br />
Introduction<br />
S<br />
ince the beginning <strong>of</strong> humankind people have relied primarily on plants for nourishment.<br />
Through trial and error they discovered that some plants are good for food, that some are<br />
poisonous, and that some produce bodily changes such as increased perspiration, bowel<br />
movement, urination, relief <strong>of</strong> pain, hallucination, and healing. Over the millennia these<br />
observations were passed orally from generation to generation, with each generation adding to and<br />
refining the body <strong>of</strong> knowledge. Every culture the world over has in this manner developed a body<br />
<strong>of</strong> herbal knowledge as part <strong>of</strong> its tradition. Some examples which have been historically proven as<br />
example are the India Aryuveda the Chinese Pen Tsao etc.<br />
Unlike many other genera <strong>of</strong> forage tree legumes, Erythrina is pan-tropical, consisting <strong>of</strong> some 112<br />
species, 70 neo-tropical, 31 African and 12 Asian. Only one species, Erythrina fusca, occurs in both<br />
the New and Old Worlds. The genus is probably <strong>of</strong> South American origin but the ability <strong>of</strong> the<br />
seeds to float and retain viability after prolonged immersion in salt water and the probable riverine,<br />
coastal or estuarine environments inhabited by the ancestral species have resulted in worldwide<br />
distribution. Pollination by birds and a marked ability to hybridize have resulted in a tremendous<br />
amount <strong>of</strong> ecological and morphological diversity, both within and between species, but with rather<br />
close cytological and phytochemical relationships. They have been known to possess predominantly<br />
flavonoids and have also been shown to have a considerable amount <strong>of</strong> alkaloids. The alkaloids <strong>of</strong><br />
Erythrina are distinct from those <strong>of</strong> other legumes and they all possess an unusual high activity, low<br />
affinity nitrate reductase system distinct from known nitrate reduction patterns in other<br />
angiosperms (Neill, 1988).<br />
Erythrina abyssinica is medium-sized tree, usually 5-15 m in height, deciduous, thickset, with a wellbranched,<br />
rounded, spreading crown; trunk short; bark yellow-buff when fresh, otherwise greybrown<br />
to creamy brown, deeply grooved, thickly corky and <strong>of</strong>ten spiny; when damaged the tree<br />
exudes a brown, gummy sap. Leaves are compound, trifoliolate, alternate; leaflets almost as broad<br />
as long, 5.5-15 x 6-14 cm, with the terminal leaflet being largest; lateral leaflets rather smaller than<br />
this, if 3 lobed then obscurely so, densely woolly when young, losing most <strong>of</strong> these hairs by<br />
maturity; midrib and main veins on the undersurface <strong>of</strong>ten bear scattered prickles. Flowers<br />
spectacular, in strong, sturdy racemes on the ends <strong>of</strong> branchlets, orange-red, up to 5 cm long; calyx<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
joined to form a tube, split along the under surface almost to the base and separating away into<br />
long, slender, distinctive lobes at the apex; calyx and standard petal striking scarlet to brick red.<br />
Fruit a cylindrical, woody pod, 4-16 cm long, deeply constricted between the seeds, densely furry,<br />
light brown in colour, opening to set free 1-10 shiny, red seeds with a grey-black patch.<br />
Diabetes is the body's failure to metabolize blood sugar properly. There are two widely known<br />
forms <strong>of</strong> diabetes. TYPE 1 is a failure <strong>of</strong> the pancreas to produce insulin. Daily injection <strong>of</strong> insulin<br />
replacement is the treatment, itself a triumph <strong>of</strong> twentieth century science. TYPE 2 is insulin<br />
resistance or impaired glucose tolerance. Insulin appears to be the key factor in developing Type 2<br />
diabetes. Some people have impaired glucose tolerance. What happens is that there is just a<br />
moderate rise in blood sugar--enough so that it silently triggers heart disease. In others, they<br />
develop insulin resistance. The insulin is produced in adequate quantity, but the body no longer<br />
responds effectively to it.<br />
Insulin resistance is one <strong>of</strong> the characteristic pathogenic signs <strong>of</strong> type-2 diabetes, and several drugs<br />
that increase the insulin sensitivity are currently in clinical use. However, these drugs have a<br />
number <strong>of</strong> limitations, which include adverse effects and high rates <strong>of</strong> secondary failure. Of the<br />
various potential drug targets for treatment <strong>of</strong> type-2 diabetes, protein tyrosine phosphathase-1B<br />
(PTP1B) has recently been considered as a major negative regulator in the insulin signaling<br />
pathway. It has been suggested that compounds reducing PTP1B activity or the genetic expression<br />
levels <strong>of</strong> PTP1B may be useful in the treatment <strong>of</strong> type-2 diabetes and possibly obesity as well.<br />
Although there have been a number <strong>of</strong> reports on the development <strong>of</strong> PTP1B inhibitors new types<br />
<strong>of</strong> PTP1B inhibitors having improved pharmacological properties remain to be discovered. It is with<br />
this in view that we under took the phytochemical and pharmacological study <strong>of</strong> Erythrina abysinica<br />
stem bark. (Rob Ho<strong>of</strong>t van Huijsduijnen et al, 2004).<br />
Materials and Methods<br />
The optical rotations were obtained in MeOH using a Rudolph Autopol IV polarimeter, whereas the<br />
IR spectra (KBr) were recorded on a Bruker Equinox 55 FT-IR spectrometer. The UV spectra were<br />
taken in MeOH using a Shimadzu spectrophotometer, with CD spectra obtained in MeOH using a<br />
JASCO J-710 spectrometer. The nuclear magnetic resonance (NMR) spectra were obtained on<br />
Varian Unity Inova 500 MHz spectrometer using TMS as the internal standard. Mass spectra were<br />
performed on a Micromass QTOF2 (Micromass, Wythenshawe, UK) mass spectrometer. Silica gel<br />
(Merck, 63 200 lm particle size) and C-18 silica gel (Merck, 75 µm particle size) were used for<br />
column chromatography (CC). TLC was carried out using Merck silica gel 60 F254 and RP-18 F254<br />
plates, whereas HPLC employed a Gilson system with a UV detector and an Optima Pak C18 column<br />
(10-250 mm, 10µm). Enzyme PTP1B (human, recombinant) was purchased from BIOMOL<br />
International LP. The solvents used for the bioassay were <strong>of</strong> analytical grade and obtained from<br />
Merck and Sigma Aldrich.<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Results and Discussion<br />
Erythrina abyssinica stem bark was collected from Mukono, Kampala, Uganda in June 2005 and was<br />
authentified and a voucher specimen No 0001 was deposited at the Department <strong>of</strong> Botany,<br />
Makerere University, Kampala, Uganda. The stem was air dried chopped and ground. It was then<br />
extracted repeatedly in Ethyl Actetate and about 500 grams <strong>of</strong> crude extract were obtained. The<br />
extract was tested positive for flavonoids using hydrochloric acid and Magnesium chips. The extract<br />
underwent a series <strong>of</strong> various chromatographic separations and pure compounds were isolated,<br />
their structures elucidated and their Protein Tyrosine Phosphate Inhibitory activity tested. Over<br />
seven known flavanones and twenty one novel flavanones were isolated and characterized using<br />
usual characteristic spectroscopic techniques. (Long Cui et al, 2007)<br />
The known flavanones were, Sigmoidin A, Sigmoidin B, Sigmoidin C, Sigmoidin D, Sigmoidin E,<br />
Sigmoidin F, Sigmoidin G. Abyssinin. The novel flavanones were given the trivial names<br />
Abyssin<strong>of</strong>lavanones V- XXV. Some <strong>of</strong> the structures are given below.<br />
HO<br />
HO<br />
HO<br />
O H<br />
O H<br />
O H<br />
O<br />
O<br />
O<br />
O<br />
O<br />
O<br />
OCH 3<br />
O H<br />
A34-32<br />
Abyssinin II<br />
O H<br />
O H<br />
OCH 3<br />
O<br />
HO<br />
HO<br />
HO<br />
O H<br />
O<br />
O<br />
A34-21<br />
Sigm oidin F<br />
OH<br />
A34-33<br />
Sigm oidniA A34-34<br />
A34-64(1)<br />
Abyssinin I<br />
O H<br />
O<br />
O<br />
O<br />
O<br />
O H<br />
O<br />
O H<br />
A34-65<br />
Licoagrochalcone A<br />
HO<br />
HO<br />
HO<br />
O H<br />
O<br />
O<br />
O<br />
OH<br />
O H<br />
O<br />
A34-35<br />
Abyssinon e IV<br />
O<br />
O<br />
A34-22<br />
Abyssinon e V<br />
OCH 3<br />
O H<br />
A34-62<br />
5-Deoxyabyssinin I I<br />
Figure 1: Some compounds isolated from Erythrina abyssinica.<br />
Table 1: Inhibitory Activity <strong>of</strong> some compounds against PTP1B<br />
compound inhibitory activity a<br />
abyssin<strong>of</strong>lavone V >60<br />
abyssin<strong>of</strong>lavone VI 18.9 ±1.9<br />
abyssin<strong>of</strong>lavone VII 15.7 ±0.4<br />
sigmoidin F 14.2± 1.7<br />
sigmoidin B 19.4± 2.3<br />
abyssinin II 17.3± 1.4<br />
181<br />
HO<br />
HO<br />
HO<br />
O H<br />
O H<br />
O H<br />
O<br />
O<br />
O<br />
O<br />
O<br />
OH<br />
OH<br />
OH<br />
O H<br />
A34-24<br />
5-Pren ylbutein<br />
A34-61<br />
Sigm oidin C<br />
A34-24(2)<br />
Sigm oidin B<br />
O<br />
OH<br />
HO<br />
HO<br />
HO<br />
O H<br />
O<br />
O<br />
A34-24(1)4<br />
O H<br />
O H<br />
H O<br />
O<br />
O<br />
O<br />
A34-25<br />
(-)Sigmoidin E<br />
O<br />
O<br />
A34-63<br />
3'-prenylnaringenin<br />
O H<br />
O<br />
O<br />
A34-24(1)2<br />
HO<br />
O H<br />
O H<br />
O
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
sigmoidin A 14.4 ± 0.8<br />
sigmoidin C >60<br />
5-deoxyabyssinin II 19.2 ±1.1<br />
3-prenylnaringenin 26.7 ± 1.2<br />
abyssinin I 18.2 ± 1.4<br />
abyssinone-VI 20.6 ± 2.1<br />
licoagrochalcone A 16.9± 0.7<br />
RK-682b 4.5 ± 0.5<br />
ursolic acid b 3.6 ± 0.2<br />
a Results are expressed as IC50 values (µM), determined by regression analyses and expressed as<br />
the mean ( SD <strong>of</strong> three replicates.<br />
b Positivecontrol. (Na M et al, 2006)<br />
References<br />
Liu, G. (2003); Protein tyrosine phosphatase 1B inhibition: opportunities and challenges. Curr. Med. Chem., 10, 1407-<br />
1421.<br />
Long Cui, D. T. Ndinteh, M. Na, J. S. Muruumu, D. Njamen, J.T. Mbafor, Z.T. Fomum, W. J. Yi, W. K. Oh, and J. S. Ahn.<br />
(2007); Isoprenylated Flavonoids from the stem bark Erythrina abyssinica J. Nat. Prod, 70, 1039-1042.<br />
Na, M.; Jang, J.; Njamen, D.; Mbafor, J. T.; Fomum, Z. T.; Kim, B. Y.; Oh, W. K.; Ahn, J. S. (2006); J. Nat. Prod. 2006, 69,<br />
1572-1576.<br />
Neill, D. (1988); Experimental studies on species relationships <strong>of</strong> Erythrina Leguminosae, Papilionoideae. Annals <strong>of</strong> the<br />
Missouri Botanical Gardens 75, 1988, 886-969.<br />
Rob Ho<strong>of</strong>t van Huijsduijnen, Wolfgang H. B. Sauer, Agnes Bombrun, and Dominique Swinnen Prospects for Inhibitors <strong>of</strong><br />
Protein Tyrosine Phosphatase 1B as Antidiabetics Drugs (2004); J. Med. Chem., 47, 4142-4146.<br />
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[YS 2] Antimalarial and Antileishmanial Activity and Cytotoxicity <strong>of</strong> Selected<br />
Medicinal Plants from Kenya<br />
Elizabeth V.M. Kigondu a , Ge<strong>of</strong>frey M. Rukunga a , Joseph M. Keriko b , Willy K. Tonui c , Jeremiah W.<br />
Gathirwa a , Peter G. Kirira a , Beatrice Irungu a , Johnstone M. Ingonga c , Isaiah O. Ndiege d .<br />
a<br />
Center for Traditional Medicine and Drug Research, Kenya Medical Research Institute (KEMRI), P.O. Box 54840, Nairobi<br />
00200, Kenya<br />
b<br />
Department <strong>of</strong> Chemistry, Jomo Kenyatta University <strong>of</strong> Agriculture and Technology (JKUAT), P.O. Box 62000, Nairobi<br />
00200, Kenya<br />
c<br />
Center for Biotechnology Research and Development, Kenya Medical Research Institute, P.O. Box 54840, Nairobi<br />
00200, Kenya<br />
d Department <strong>of</strong> Chemistry, Kenyatta University, P.O. Box 43844, Nairobi 00100, Kenya<br />
Key words: Plant extracts, Anti-plasmodial, Anti-leishmanial, Cytotoxicity, In vitro<br />
Introduction<br />
M<br />
alaria is perceived as the world's worst health problem. This burden <strong>of</strong> mortality is not<br />
equally shared, falling most heavily on sub-Saharan Africa. Global figures for deaths from<br />
malaria range from 1.5 to 2.7 million each year, most <strong>of</strong> whom are children under 5 years <strong>of</strong> age<br />
and pregnant women (WHO, 2006). In Kenya, more than half <strong>of</strong> the population is exposed to<br />
malaria transmission. Areas endemic are: Western, Coastal, parts <strong>of</strong> Rift Valley, Central and Eastern<br />
provinces (Ministry <strong>of</strong> Health, 2006). Indigenous rural communities in the tropics manage parasitic<br />
diseases, like malaria and leishmaniasis, using herbal drugs. The efficacy, dosage, safety and active<br />
principles <strong>of</strong> most <strong>of</strong> the herbal preparations are not known (Njoroge et al., 2004).<br />
Materials and methods<br />
Extraction<br />
The leaves, stem bark and root bark <strong>of</strong> the plants were air dried and ground using an electrical mill.<br />
Extraction <strong>of</strong> the various parts <strong>of</strong> the medicinal plants was carried out using organic solvents and<br />
water. Extracts were subjected to anti-leishmanial and anti-malarial bioassay (Harborne, 1994).<br />
Anti-malarial Plasmodium falciparum in vitro assay:<br />
Two strains <strong>of</strong> P. falciparum parasites (D6, chloroquine sensitive and W2 chloroquine resistant)<br />
were used. Parasite cultivation was done on in vitro technique described by Trager and Jensen<br />
(1976). In vitro assay semi-automated microdilution assay technique that measures the ability <strong>of</strong><br />
the extracts to inhibit the incorporation <strong>of</strong> [G- 3 H] hypoxanthine into the malaria parasite was<br />
adapted (Matile & Pink, 1990; Desjardins et al. 1979). Computation <strong>of</strong> the concentration <strong>of</strong> drug<br />
causing 50% inhibition <strong>of</strong> [G- 3 H] hypoxanthine uptake (IC50) was carried out by interpolation after<br />
logarithmic transformation <strong>of</strong> both concentration and cpm values using the formula,<br />
IC50 = antilog (logX1 + [(logY50 logY1)(logX2 log X1)]/(logY2 logY1)])<br />
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Where; Y50 is the cpm value midway between parasitized and non-parasitized control cultures and<br />
X1, Y1, X2, and Y2 are the concentrations and cpm values for the data points above and below the<br />
cpm midpoints (Sixsmith et al., 1984).<br />
Anti-leishmanial in vitro bioassay: Anti-promastigote assay<br />
Leishmania major promastigotes were cultured in NNN media overlaid with Schneider s Drosophila<br />
insect medium supplemented with 20% FBS, 100 g/ml streptomycin and 100 U/ml penicillin-G,<br />
and 5-fluorocytosine at 25 o C, in tissue culture flasks and the assay was done as described by<br />
Delornzi et al. (2001). Counting <strong>of</strong> the promastigotes using an improved neubauer chamber.<br />
Calculation <strong>of</strong> percentage mortality (PM).<br />
PM = Number <strong>of</strong> dead parasites x 100<br />
Total number <strong>of</strong> parasites<br />
Anti-amastigote (Macrophage) assay)<br />
Harvesting <strong>of</strong> mouse peritoneal macrophages BALB/c mice was carried out and the macrophages<br />
were cultured in RPMI-1640 medium, 37 o C, 5% CO2, in 24 microwell plates. Infection <strong>of</strong><br />
macrophages with Leishmania major (Strain IDU/KE/83 = NLB-144) amastigotes followed and then<br />
introduction <strong>of</strong> plant extracts. Determination <strong>of</strong> infection rate (IR) and multiplication index (MI).<br />
The assay was carried out as described by (Delorenzi et al., 2001).<br />
IR = No. <strong>of</strong> infected macrophages in 100 macrophages<br />
MI = (No. <strong>of</strong> amastigotes in experimental culture/100 macrophages) x 100 %<br />
No. <strong>of</strong> amastigotes in control culture/100 macrophages).<br />
Nitric oxide determination<br />
Nitric oxide release in supernatants <strong>of</strong> macrophage culture was measured by the Griess reaction for<br />
nitrites (Holzmuller et al., 2002).<br />
Results and Discussion<br />
After screening extracts from the six selected plant species, for in vitro anti-plasmodial and antileishmanial<br />
activity, against 2 laboratory-adapted Plasmodium falciparum isolates (D6, CQ-sensitive<br />
and W2, CQ-resistant) and Leishmania major (IDU/KE/83 = NLB-144 strain), respectively, the<br />
methanol extract <strong>of</strong> Suregada zanzibariensis leaves exhibited good anti-plasmodial activity (IC50<br />
4.66±0.22 and 1.82±0.07µg/ml for D6 and W2, respectively). Similarly, the methanol extracts <strong>of</strong><br />
Albizia coriaria (IC50 37.83±2.11µg/ml for D6) and Asparagus racemosus (32.63±2.68 and<br />
33.95±2.05µg/ml for D6 and W2, respectively) had moderate anti-plasmodial activity. Acacia tortilis<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
(IC50 85.73±3.36µg/ml for W2) and Albizia coriaria (IC50 71.17±3.58µg/ml for W2) methanol extracts<br />
and Aloe nyeriensis var kedongensis (IC50 87.70±2.98 and 67.84±2.12µg/ml for D6 and W2,<br />
respectively) water extract exhibited mild anti-plasmodial activity. The rest <strong>of</strong> the extracts did not<br />
exhibit any anti-plasmodial activity.<br />
Although the leishmanicidal activity <strong>of</strong> extracts were lower than for pentosam (80%), reasonable<br />
activity was observed for Aloe nyeriensis methanol (68.4±6.3%), Albizia coriara water (66.7±5.0%),<br />
Maytenus putterlickoides methanol (60.0±6.23%), Asparagus racemosus methanol and water<br />
(58.3±8.22 and 56.8±6.58%, respectively), Aloe nyeriensis water (53.3±5.1%) and Acacia tortilis<br />
water (52.9±6.55%) extracts at 1000µg/ml. Leishmania major infected macrophages treated with<br />
methanol extracts <strong>of</strong> Suregada zanzibariensis and Aloe nyeriensis var kedongensis and Pentostam ®<br />
had infection rates <strong>of</strong> 28±2.11, 30±1.22 and 40±3.69%, respectively at 1000µg/ml, indicating better<br />
anti-leishmanial activity for the extracts. The methanol extract <strong>of</strong> Albizia coriara (44.0±3.69%) and<br />
aqueous extracts <strong>of</strong> Asparagus racemosus (42±3.84%) and Acacia tortilis (44±5.59%) had similar<br />
activity to pentosam ® . Multiplication indices for Leishmania major amastigotes treated with<br />
methanol extracts <strong>of</strong> Albizia coriaria, Suregada zanzibariensis and Aloe nyeriensis var kedongensis,<br />
aqueous extract <strong>of</strong> Acacia tortilis and pentosam ® were 28.5±1.43, 29.4±2.15, 31.1±2.22,35.9±3.49<br />
and 44.0±3.27%, respectively, at 1000µg/ml, confirming better anti-leishmanial activity for the<br />
extracts. Aqueous extracts <strong>of</strong> Aloe nyeriensis (46.7±3.28%) and Albizia coriaria (47.5±3.21%) had<br />
similar activity level to pentosam ® . The plant extracts have better inhibitory activity while<br />
pentosam ® has better leishmanicidal activity. All extracts exhibited very low cytotoxicity (CC50<br />
>500µg/ml) against human embryonic lung fibroblast (HELF) cells. The investigations demonstrated<br />
the efficacy and safety <strong>of</strong> some extracts <strong>of</strong> plants that are used by rural indigenous communities for<br />
the treatment <strong>of</strong> parasitic diseases.<br />
Acknowledgement<br />
The authors acknowledge the research grants from World Health Organization (TDR-WHO A30707)<br />
and IFS that enabled this work. We are grateful to the Director, CBRD, KEMRI and the Head, Malaria<br />
Culture Laboratory, where part <strong>of</strong> the work was done. We thank Mr. Ge<strong>of</strong>frey M. Mungai, The<br />
National Museums <strong>of</strong> Kenya, for the identification <strong>of</strong> the plant species. The technical assistance <strong>of</strong><br />
Ms. Cecilia W. Kimani and Mr. Dennis M. Misigo during bioassays is appreciated.<br />
References<br />
Delorenzi, J. C.; Attias, M.; Gattass, C. R.; Andrade, M.; Rezende, C.; da Cunha Pinto, A.; Henriques, A. T.; Bou-Habib, D.<br />
C.; Saraiva, E. M. (2001); Anti-leishmanial activity <strong>of</strong> an indole alkaloid from Peschiera australis. Antimicrob. Agents<br />
Chemother. 45: 1349-1354.<br />
Desjardins, R. E.; Canfield, C. J.; Haynes, J. D.; Chulay, J. D. (1979); Quantitative assessment <strong>of</strong> anti-malarial activity in<br />
vitro by a semi-automated micro-dilution technique. Antimicrob. Agents Chemother. 16: 710-718.<br />
Harborne, J. B. (1994); Biochemistry <strong>of</strong> Phenolic Compounds. Academic Press, London.<br />
H<strong>of</strong>fman, S.L., Gardner, M.J., Doolan, D.L., Hedstrom, R.C., Wang, R., Sedegah, M., Gramzinski, R.A., Aguiar, J.C., Wang,<br />
H., Margalith, M., Hobart, P., 1996; DNA vaccines against malaria: immunogenicity and protection in a rodent<br />
model. Journal <strong>of</strong> Pharmaceutical Sciences 85, 1294 1300.<br />
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Matile, H.; Pink J. R. L. (1990); Plasmodium falciparum malaria parasite cultures and their use in immunology. In:<br />
Immunological Methods, Lefkovits, I. & Pernis, B. (eds). Academic Press, San Diego, p 221.<br />
Ministry <strong>of</strong> Health, 2006; National Guidelines for Diagnosis, Treatment and Prevention <strong>of</strong> Malaria for Health Workers in<br />
Kenya.<br />
Njoroge, N. G.; Bussmann, W. R.; Gemmill, B.; Newton, L. E.; Ngumi, V. W. (2004); Utilization <strong>of</strong> weed species as sources<br />
<strong>of</strong> traditional medicines in central Kenya. Lyoni 7: 72-87.<br />
Sixsmith, D. G.; Watkins, W. M.; Chuly, J. D.; Spencer, H. C. (1984); In vitro anti-malarial activity <strong>of</strong> tetrahydr<strong>of</strong>olate<br />
dehydrogenase inhibitors. Am. J. Trop. Med. Hyg. 33: 772-776.<br />
Trager, W.; Jensen, J. B. (1976); Human malaria parasites in continuos culture. Science 193: 673-675.<br />
WHO, 2006. The Africa Malaria Report. www.afro.who.int/malaria/publications/ annual_reports/africa<br />
malaria_report_2006.pdf 2006(Accessed 17th December 2008).<br />
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[YS 3] Terpurinflavone: Antiplasmodial Flavones from the Stem <strong>of</strong> Tephrosia<br />
purpurea<br />
Wanyama P. Juma, Hoseah M. Akala, Fredrick L. Eyase, Lois M. Muiva, Matthias Hydenreich, Faith<br />
A. Okalebo, Peter M. Gitu, Martin G. Peter, Douglas S. Walsh, Mabel Imbuga, Abiy Yenesew<br />
Introduction<br />
T<br />
ephrosia Pers (Leguminosae- Papilionoideae) is a large tropical and sub-tropical genus<br />
estimated to contain about three hundred species (Waterman and Khalid, 1980; Abou-Douh et<br />
al., 2005) out <strong>of</strong> which thirty are found in Kenya (Tarus et al., 2002). The extracts <strong>of</strong> the some<br />
Tephrosia species have shown various biological activities including antiplasmodial (Muiva et al.,<br />
2009), antibacterial (Abou-Douh et al., 2005). Various biological activities including antibacterial<br />
(Hegazy et al., 2009), antidiabetic and cancer chemoprevetive activities (Chang et al., 2000) have<br />
been reported for extracts and pure compounds from this plant. Tephrosia is rich in prenylated<br />
flavonoids including flavones (Hegazy et al., 2009; Pelter et al., 1981). Flavanones (Pelter et al.,<br />
1981); chalcones (Chang et al., 2000; Pelter et al., 1981) and rotenoids (Ahmad et al., 1999). In the<br />
search for compounds with antiplasmodial activity from Kenyan plants, the stem <strong>of</strong> T. purpurea has<br />
been investigated. This report is on the isolation and characterization <strong>of</strong> new prenylated flavones,<br />
named terpurinflavone (1), with antiplasmodial activity along with three known flavonoids.<br />
AcO<br />
O<br />
H<br />
O<br />
1'' O<br />
2''<br />
5''<br />
3<br />
4''<br />
8<br />
5<br />
1<br />
H<br />
H<br />
4a<br />
OAc<br />
8a<br />
O<br />
O<br />
H<br />
OAc<br />
O<br />
O<br />
4<br />
2<br />
1'<br />
5'<br />
3'<br />
Results and Discussion<br />
Compound 1 was obtained as a white amorphous powder with an Rf value <strong>of</strong> 0.45 in n-hexane/ethyl<br />
acetate (3:2). It showed [M+H] + peak at m/z 437.1593 in its positive electrospray ionization time <strong>of</strong><br />
flight mass spectrum (ESI-TOF-MS) constituting the molecular formula C25H2407. The presence <strong>of</strong> a<br />
flavone skeleton was deduced from the UV ( max 295, 325 nm), 1 H ( 6.79 s for H-3) and 13 C (163.9<br />
for C-2, 108.5 for C-3 and 177.6 for C-4) NMR spectroscopic data (Table 1).<br />
The presence <strong>of</strong> unsubstituted ring-B was clearly shown in 1 H ( 7.63 m for H-3'/4'/5', 8.18 m for<br />
H-2'/6') and 13 C ( 127.9 for C-2'/6', 130.7 for C-3'/5', 133.1 for C-4' and 133.6 for C-1') NMR<br />
187<br />
O<br />
MeO<br />
OH<br />
4<br />
2<br />
O<br />
O<br />
O<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
spectra. In ring-A, an AX protons which are ortho-coupled at 8.00 and 6.94 (J = 8.5 Hz) were<br />
assigned to H-5 and H-6, respectively, with C-7 and C-8 being substituted with an acetoxy (at C-7)<br />
and a tetrahydr<strong>of</strong>uran ring (at C-8) derived from a modified prenyl group as in tephrorin B (Chang et<br />
al., 2000). The H<strong>MB</strong>C spectrum showed correlation <strong>of</strong> H-4'' ( 4.44) with C-5'' (7<strong>9.1</strong>), C-3'' (78.7), C-<br />
2'' (84.0), C-7 (167.9), C-8 (116.0) and C-8a (155.9) confirming that the tetrahydr<strong>of</strong>uran ring is<br />
placed at C-8. The presence <strong>of</strong> a second acetate group was also evident from the NMR spectra<br />
(Table 1) and placed at C-3'' <strong>of</strong> the tetrahydr<strong>of</strong>uran group based on the H<strong>MB</strong>C spectrum which<br />
showed correlation <strong>of</strong> H-3'' ( 5.35) with acetoxy carbon ( 170.3) and the two methyl carbon<br />
atoms ( C 22.4 and 24.4) at C-2''). The 1 H and 13 C NMR chemical shift values <strong>of</strong> the tetrahydr<strong>of</strong>uran<br />
ring <strong>of</strong> 1 were quite similar to those reported for tephrorin B (Chang et al., 2000). The coupling<br />
constant (J = 8.5 Hz) between H-3'' and H-4'' indicated that the relative orientation <strong>of</strong> these two<br />
protons is cis as in tephrorin B. In the NOESY spectrum, NOE interaction <strong>of</strong> H-3'' with H-4''<br />
supported the cis geometry. The new compound was therefore characterized as 7-acetoxy-8-[3''acetoxy-2'',2''-dimethyltetrahydro-4''-furanylflavone<br />
(1) for which trivial name terpurinflavone was<br />
assigned. The isolation <strong>of</strong> this new compound once again demonstrated the unique capacity <strong>of</strong> T.<br />
purpurea to oxidize the C-7 methoxy group in compound 2 and cyclize it with the adjacent 2hydroxy-2-methylbut-1-enyl<br />
group into complex C-8 substituted flavonoids (Pelter et al., 1981).<br />
The EtOAc fraction <strong>of</strong> the CH2Cl2/MeOH (1:1) extract <strong>of</strong> T. purpurea showed moderate<br />
antiplasmodial activity against chloroquine-sensitive (D6) and chloroquine-resistant (W2), strains <strong>of</strong><br />
Plasmodium falciparum with IC50 values <strong>of</strong> 10.47 ± 2.22 µg/ml and 12.06 ± 2.54 µg/ml, respectively,<br />
respectively. The pure compounds isolated from this plant were also tested with the new<br />
compound terpurinflavone (1) exhibiting the highest activity with IC50 values <strong>of</strong> 3.12 ± 0.28 µM and<br />
6.26 ±2.66 µM against D6 and W2 stains <strong>of</strong> P. falciparum, respectively (Table 2). The activity <strong>of</strong> the<br />
crude extract could be due to these compounds, especially that <strong>of</strong> compound 1 which showed the<br />
highest activity.<br />
Table 1: 1 H (500 MHz) and 13 C (125 MHz) NMR data along with H<strong>MB</strong>C correlations for compound 1<br />
in acetone-d6<br />
Position H (in Hz) C H<strong>MB</strong>C ( 2 J, 3 J)<br />
2 163.9<br />
3 6.79 s 108.5 C-1', 2, 4, 4a<br />
4 177.6<br />
4a 119.9<br />
5 8.00 d (8.5) 129.3 C-4, 7, 8a<br />
6 6.94 d (8.5) 110.1 C-4a, 8<br />
7 167.9<br />
8 116.0<br />
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Position H (in Hz) C H<strong>MB</strong>C ( 2 J, 3 J)<br />
8a 155.9<br />
1' 133.6<br />
2'/6' 8.18 m 127.9 C-1', 2<br />
3'/5' 7.63 m 130.7 C-2'/6', 4<br />
4' 7.63 m 133.1<br />
2'' 84.0<br />
3'' 5.35 d (8.5) 78.7 C-2'', 5'', COMe-3''<br />
4'' 4.44 ddd (2.0, 8.0, 8.5) 42.3 C-2'', 3'', 5'', 7, 8, 8a<br />
5'' 4.84 dd (8.0, 9.5) 7<strong>9.1</strong> C-3''<br />
5.02 dd (2.0, 9.5) C-4'', 8<br />
COMe-3'' 170.3<br />
COMe-3'' 1-61 s 20.9 COMe-3''<br />
Me-2'' 1.76 s 22.4 C-2'', 3'', Me-2''<br />
Me-2'' 1.61 s 24.4 C-2'', 3'', Me-2''<br />
COMe-7 170.8<br />
COMe-7 2.00 s 23.0 COMe-7<br />
Table 2: In vitro IC50 values <strong>of</strong> pure compounds isolated form T. purpurea against the D6 and W2<br />
strains <strong>of</strong> P. falciparum.<br />
Sample IC50 (µM ± SD)<br />
(D6) (W2)<br />
Terpurinflavone (1) 3.12 ± 0.28 6.26 ± 2.66<br />
Lanceolatin A (2) 11.36 ± 2.97 14.97 ± 3.09<br />
Semiglabrin (3) 25.77 ± 6.08 35.58 ± 5.41<br />
Lanceolatin B (4) 27.02 ± 2.65 35.99 ± 4.24<br />
Mefloquine - 0.013 ± 0.002<br />
Chloroquine 0.035 ± 0.003 -<br />
Methods and Materials<br />
T. purpurea was collected from Kilifi District, Coast province, Kenya in August, 2007. The plant was<br />
identified by Mr. Patrick C. Mutiso <strong>of</strong> the the University Herbarium, Botany Department, University<br />
<strong>of</strong> Nairobi, where a voucher specimen (Mutiso-520-August 2007) is deposited.<br />
Extraction and Isolation<br />
Air dried and ground stems <strong>of</strong> T. purpurea (2 kg) were extracted with dichloromethane/methanol<br />
(1:1) by cold percolation at room temperature (3 x 1.5 L). The extract was filtered and the solvent<br />
removed under vacuum using a rotary evaporator at 35 o C. This gave dark oily extract that was<br />
partitioned between water and ethyl acetate. The organic layer (36 g) was subjected to CC on silica<br />
gel (400g) eluting with n-hexane containing increasing percentages (2%, 4%, 6%, 8%, 10%, 12.5%,<br />
15%, 17.5%, 20%, 25%, 30%, 40%, 50%, 75%, and 100%) <strong>of</strong> ethyl acetate and gave 15 fractions each<br />
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<strong>of</strong> 1.5L. The fraction eluted with 15% ethyl acetate in n-hexane was separated on Sephadex LH-20<br />
(CH2Cl2-MeOH; 1:1) to give terpurinflavone (1, 34 mg).<br />
In vitro antiplasmodial activity assay<br />
Antiplasmodial activities <strong>of</strong> crude extract and pure compounds against chloroquine-sensitive Sierra<br />
Leone 1 (D6) and chloroquine-resistant Indochina 1 (W2) strain <strong>of</strong> P. falciparum was tested using a<br />
non-radioactive assay technique (Smilkstein et al., 2004) with modifications. This method use the<br />
fluorochrome called SYBR Green 1 , a non-radioactive DNA dye that accurately depicts in vitro<br />
parasite replication.<br />
References<br />
Abou-Douh, A.M., Ito, C., Toscano, R.A., El-Baga, N.Y., El-Khrisy, E.A., Furukawa, H., (2005); Prenylated flavonoids from<br />
the roots <strong>of</strong> Egyptian T. apollinea- crystal structure analysis. Z, Naturforsch.60b, 458-470.<br />
Ahmad, V.U., Ali, Z., Hussaini, S.R., Iqbal, F., Zahid, M., Abbas, M., Saba, N., (1999). Flavonoids <strong>of</strong> T. purpurea.<br />
Fitoterapia 70, 443-445.<br />
Hegazy, M. E.F., Abd El-Razek, M.H., Asakawa, Y., Par e, Pw., (2009). Rare prenylated flavonoids from T. purpurea.<br />
Phytochemistry 70, 1474-1477.<br />
Johnson, J.D., Dennull, R.A., Gerena, L., Lopez-Sanchez, M., Roncal, N.E and Waters N.C (2007). Assessment and<br />
Continued Validation <strong>of</strong> Malaria SYBR Green I Based Fluorescence Assay for use in Malaria Drug Screening,<br />
Antimicrobial Agents and Chemotherapy 51: 1926-1933.<br />
Muiva, L.M; Yenesew, A; Derese, S; Matthias, H; Martin, G.P; Akala, M.H; Fredrick, E;Norman, C.W; Mutai, C; Keriko, M.J<br />
and Douglas, W. (2009); Antiplasmodial hydroxydihydrochalcone from seedpods <strong>of</strong> Tephrosia elata.<br />
Phytochemistry letters 2, 99-102.<br />
Peter, A., Ward, R.S., Rao, E.V., Raju, N,R., (1981); 8-substituted flavonoids and 3'-<br />
substituted 7-Oxygenated chalcones from T. purpurea. J. Chem. Soc. Perkin I 2491-2998.<br />
Smilkstein , M., Sriwilaijaroen, N., Kelly J.X., Wilairat, P., Riscoe, M., (2004); Simple and<br />
inexpensive fluorescence-based technique for high-throughput antimalarial drug screening. Antimicrob. Agents<br />
Chemother, 48, 375-379.<br />
Tarus, P.K., Machocho, A.K., Lang at-Thoruwa, C.., Chhabra,S.C., (2002); Flavonoids from T.<br />
aequilata. Phytochemistry 60, 375-379.<br />
Waterman, P.G and Khalid, A.S., (1980); The major flavonoids <strong>of</strong> the seeds <strong>of</strong> T. apollinea.<br />
Phytochemistry 19, 909-915.<br />
Chang, L.C., Chavez, D., Song. L.l., Farnsworth, N.R., Pezutto, J.M., Kinghorn, A.D., (2000); Absolute configuration <strong>of</strong><br />
novel bioactive flavonoids from T. purpurea. Org. Lett. 2, 515-518.<br />
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[YS 4] In vitro Antiplasmodial and Cytotoxicity Activities <strong>of</strong> Some Medicinal Plants<br />
from Kenya<br />
BN Irungu, GM Rukunga and CN Muthaura<br />
Centre for Traditional Medicine and Drug Research, Kenya Medical Research Institute, P.O. Box 54840-00200, Nairobi-<br />
Kenya. Tel 254-20-2722541<br />
Corresponding author: birungu@yahoo.com<br />
Key Words: Malaria, medicinal plants, Plasmodium falciparum, cytotoxicity<br />
Introduction<br />
M<br />
alaria still remains a major cause <strong>of</strong> morbidity and mortality especially in sub-Saharan Africa<br />
despite intensive efforts to control it. This pervasiveness is compounded by emergence and<br />
spread <strong>of</strong> drug resistant parasites combined with the absence <strong>of</strong> a vaccine and lack <strong>of</strong> systematic<br />
vector control strategies. As a result, efforts are being directed towards discovery and development<br />
<strong>of</strong> novel and affordable malaria control tools including the search for new drugs leads from plants.<br />
Materials and Methods<br />
Plants materials were collected from Meru and Mombasa regions <strong>of</strong> Kenya. They were air dried and<br />
ground. Cold extraction was done at room temperature ~ 25 o C successively using dichloromethane<br />
and methanol. They were dried using a rotary evaporator. Water extracts were extracted for 6<br />
hours in a waterbath at 70 0 C and dried in a freeze drier. Continuous in vitro cultures <strong>of</strong> asexual<br />
erythrocytic stages <strong>of</strong> Plasmodium strains {NF54 from unknown origin- chloroquine sensitive and K1<br />
from Thailand-CQ/pyrimethamine resistant} were maintained following a modified procedure<br />
described by Trager and Jensen (1976). Drug assay was done using a modification <strong>of</strong> the semi<br />
automated micro dilution technique <strong>of</strong> Desjardin et al. (1979) which measures the ability <strong>of</strong> the<br />
extracts to inhibit the incorporation <strong>of</strong> [ 3 H] hypoxanthine into the malaria parasite. Chloroquine<br />
was used as the reference drug.<br />
Cytotoxicity assay was done following the method <strong>of</strong> Page et al. (1993) and Ahmed et al. (1994)<br />
where rat skeletal myoblast L6 cell line was used. Podophyllotoxin (Polysciences Inc. USA) was used<br />
as a positive reference.<br />
Results and Discussion<br />
26 extracts from 14 plants were tested for their antiplasmodial properties in vitro (Table 1 & 2).<br />
Water extracts showed no activity with IC50 > 50 µg/ml in both strains except for S. heningsii, which<br />
had moderate activity. Methanol extracts had moderate activity except for T. robusta that was<br />
highly active with an IC50 <strong>of</strong> 3.5 and 2.4 µg/ml against K1 and NF54 strains respectively.<br />
Dichloromethane extracts showed the highest activity with IC50 s ranging from 35.2 - 1.4 µg/ml<br />
against the two strains except for S. heningsii which had dichloromethane extract as the least<br />
active. This phenomenon <strong>of</strong> high activity on dichloromethane extracts over water and methanol<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
extracts was also reported by Koch et al. (2005). A probable explanation could be due to lack <strong>of</strong><br />
tannins, polysaccharides and other water-soluble molecules that have no antiplasmodial properties.<br />
It was also noted that sensitivity <strong>of</strong> the extracts to both CQ-resistant and CQ-sensitive strains did<br />
not differ significantly.<br />
Table 1: In vitro antiplasmodial activities against K1 strain<br />
IC's 50 in µg/ml<br />
Type <strong>of</strong> extract<br />
Plant/part water MeOH CH2Cl2<br />
Caesalpinia volkensii 68.7 51.4 25.6<br />
Clerodendrum eriophyllum 82.7 47 2.7<br />
Clerodendrum myricoides 64 48.2 15.8<br />
Harrisonia abyssinica 91.1 52.3 4.4<br />
Strychnos heningsii 29.6 14.6 35.2<br />
Turraea robusta 91.5 3.5<br />
Vernonia auriculifera 84.5 53.8 32.7<br />
Vernonia lasiopus 52.2 31.2 4.7<br />
Warbugia ugandensis 31.8 17.8 1.4<br />
Chloroquine 0.091 0.05 0.061<br />
Table 2: In vitro antiplasmodial activity against NF54<br />
NF54 strain: IC's 50 in µg/ml<br />
Plant/part water M eOH CH2Cl2<br />
Caesalpinia volkensii 100 65.1 11.9<br />
Clerodendrum eriophyllum 100 79 5.3<br />
Clerodendrum myricoides 94.3 51.5 10.9<br />
Harrisonia abyssinica 100 55.4 5.6<br />
Strychnos heningsii 33.7 17.9 33.3<br />
Turraea robusta 100 2.4<br />
Vernonia auriculifera 100 60.8 27.3<br />
Vernonia lasiopus 100 50.5 4.9<br />
Warbugia ugandensis 64 24.3 2.2<br />
Chloroquine 0.003 0.003 0.004<br />
The dichloromethane extracts and methanol extract <strong>of</strong> T. robusta were tested for their cytotoxicity<br />
properties in vitro using L6, rat skeletal myoblast cells (Table 3). Most extracts showed low<br />
cytotoxicity with IC50 value <strong>of</strong> > 20 µg/ml (Zirihi et al., 2005). Selectivity index was also calculated<br />
with V. lasiopus having the highest selectivity <strong>of</strong> malaria parasite with an SI >10. Though W.<br />
ugandensis possessed a high antiplasmodial activity (1.4 µg/ml against K1), it also expressed the<br />
highest cytotoxicity with an IC50 <strong>of</strong> 0.34 µg/ml and a SI <strong>of</strong> 0.24. This indicates that the high<br />
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antiplasmodial activity noticed is probably due to cytotoxicity rather than activity against the<br />
parasite themselves.<br />
Table 3: Cytotoxicity results and SI <strong>of</strong> dichloromethane extracts<br />
Plant/ part L6 cells IC50's (µg/ml) K1 IC50's (µg/ml) SI<br />
Caesalpinia volkensii 82.4 25.6 3.2<br />
Clerodendrum eriophyllum 7.9 2.7 2.9<br />
*Clerodendrum myricoides 90 15.8 5<br />
Harrisonia abyssinica 32.8 4.4 7.5<br />
*Strychnos heningsii 90 35.2 2.6<br />
**Turraea robusta 14.3 3.5 4.1<br />
Vernonia auriculifera 84.8 32.7 2.6<br />
*Vernonia lasiopus 90 4.7 10.7<br />
Warbugia ugandensis 0.34 1.4 0.24<br />
Chloroquine 37 0.061 607<br />
Podophyllotoxin 0.01<br />
According to our classification, dichloromethane extract <strong>of</strong> V. lasiopus exhibited promising<br />
antimalarial activity with relatively minimal cytotoxicity. This plant is commonly used in traditional<br />
medicine in Kenya to manage malaria (Beentje, 1994). At least from ethnomedicinal use there is<br />
some evidence that the plant may be safe in humans. It is therefore planned to evaluate the activity<br />
<strong>of</strong> V. lasiopus in vivo using P. berghei mouse model then followed by isolation <strong>of</strong> the active<br />
compound (s). It s hoped that a lead compound may be identified that could be developed further<br />
as antimalarial agent.<br />
Acknowledgements<br />
This work received financial support from UNICEF/UNDP/World Bank/WHO special programme for<br />
Research and Training in Tropical Diseases.<br />
References<br />
Ahmed, S.A., Gogal, R.M., Walsh, J.E., 1994. A new rapid and simple non-radioactive assay to monitor and determine<br />
proliferation <strong>of</strong> lymphocytes. An alternative to [3H] thymidine incorporation assay. Journal <strong>of</strong> Immunology<br />
Methods 170, 211-224.<br />
Beentje, H. (1994);The Kenya trees, shrubs and lianas. National Museums <strong>of</strong> Kenya<br />
Desjardins, R.E., Canfield, C.J., Haynes, J.D., Chulay, J.D., (1979); Quantitative assessment <strong>of</strong> antimalarial activity in vitro<br />
by semi automated microdilution technique. Antimicrobial Agents in Chemotherapy 16, 710-718.<br />
Koch, A., Tamez, P., Pezzuto, J., Soejarto, D., (2005); Evaluation <strong>of</strong> plants used for antimalarial treatment by Maasai <strong>of</strong><br />
Kenya. Journal <strong>of</strong> Ethnophamacology 101, 95-99.<br />
Page, C., Page, M., Noel, C., (1993); A new flourimetric assay for cytotoxicity measurements in vitro. International<br />
Journal <strong>of</strong> Oncology 3, 473-476.<br />
Trager, W., Jensen, J.B., 1976. Human malaria parasites in continuous culture. Science 193, 673-675<br />
Zirihi, G.N., Mambu, L., Guede-Guina, F., Bodo, B., Grellier, P., (2005); In vitro antiplasmodial activity and cytotoxicity <strong>of</strong><br />
33 West African plants used for treatment <strong>of</strong> malaria. Journal <strong>of</strong> Ethnopharmacology 98, 281-285.<br />
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[YS 5] Antiplasmodial Quinones from the Roots <strong>of</strong> two Pentas Species<br />
Milkyas Endale, 1 John Patrick Alao, 2 Hoseah M. Akala, 3 Nelson K. Rono, 1 Fredrick L. Eyase, 3<br />
Solomon Derese, 1 Albert Ndakala, 1 Martin Mbugua, 1 Douglas S. Walsh, 3 Per Sunnerhagen, 2 Mate<br />
Erdelyi, 4* Abiy Yenesew 1*<br />
1<br />
Department <strong>of</strong> Chemistry, University <strong>of</strong> Nairobi, P.O. Box 30197-00100, Nairobi, Kenya.<br />
2<br />
Department <strong>of</strong> Cell- and Molecular Biology, University <strong>of</strong> Gothenburg, SE-405 30 Gothenburg, Sweden.<br />
3<br />
United States Army Medical Research Unit-Kenya, MRU 64109, APO, AE 09831-4109, USA.<br />
4<br />
Department <strong>of</strong> Chemistry, University <strong>of</strong> Gothenburg, SE-412 96 Gothenburg, Sweden and the Swedish NMR Centre,<br />
University <strong>of</strong> Gothenburg, P.O. Box 465, SE-40530 Gothenburg, Sweden<br />
Correspondence<br />
* (AbiyYenesew) Tel/Fax: +254-02-4446138, E-mail: ayenesew@uonbi.ac.ke. (Mate Erdelyi) Tel: +46-31-7869033, Email:<br />
mate@chem.gu.se<br />
Keywords: Pentas longiflora; Pentas lanceolata; Rubiaceae; anthraquinone; 5,6-dihydroxydamnacanthol; malaria<br />
Introduction<br />
M<br />
alaria, caused by the protozoan parasites <strong>of</strong> the genus Plasmodium, is a major disease in the<br />
tropical and subtropical regions <strong>of</strong> the world. Out <strong>of</strong> the yearly 300 to 500 million clinical<br />
episodes, 1.5-2.7 million cases are lethal [Snow et al., 2005]. The emergence <strong>of</strong> multidrug-resistant<br />
strains <strong>of</strong> the parasite P. falciparum and the rising resistance <strong>of</strong> the vector (Anopheles spp.) to<br />
insecticides on top <strong>of</strong> poverty and lack <strong>of</strong> a well-functioning healthcare system are the main causes<br />
for the relentless increase <strong>of</strong> malaria morbidity and mortality over the past decade. To date, over a<br />
thousand herbal species are employed in indigenous health care systems as a means <strong>of</strong> treating<br />
malaria and managing fever associated to the disease. However, neither the efficacy <strong>of</strong> most <strong>of</strong><br />
such plants established nor the active components responsible for activity identified.<br />
In Kenya, a decoction <strong>of</strong> Pentas longiflora mixed with milk and is taken as a cure for malaria<br />
[Kokwaro, 2010].We report here the isolation, structure elucidation, antiplasmodial and cytotoxicity<br />
studies <strong>of</strong> the secondary metabolites isolated from the roots <strong>of</strong> Pentas longiflora and Pentas<br />
lanceolata.<br />
Results and Discussion<br />
The roots <strong>of</strong> P. longiflora and P. lanceolata were extracted with CH2Cl2:MeOH (1:1) and tested for<br />
antiplasmodial activities and both extracts showed good activities (IC50
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[Van Puyvelde et al., 1985], and antiviral [Li-Kang et al., 1994] properties, their antiplasmodial<br />
activities are reported here for the first time. Unfortunately, compounds 1 and 2 showed high<br />
cytotoxicity (LD50 0.80 µg/mL for 1 and 0.89 µg/mL for 2).<br />
Compounds 4-13 were isolated from the root extract <strong>of</strong> P. lanceolata <strong>of</strong> which 5,6dihydroxydamnacanthol<br />
(11) is a new compound; while nordamnacanthal (7), lucidin- -methyl<br />
ether (9), and damnacanthol (10) are reported here for the first time from the genus Pentas.<br />
Compounds 4-11 (Fig 2, Table 2), especially 9 and 11, showed a valuable compromise between the<br />
antiplasmodial activity and cytotoxicity, revealing that by reaching better understanding <strong>of</strong> the<br />
factors governing their activities against P. falciparum and against mammalian cells,<br />
anthraquinones may become promising targets for further lead optimization.<br />
The anthraquinones isolated from the roots <strong>of</strong> Pentas lanceolata have a hydroxyl, and/or methoxyl<br />
and carbon (CH2, CHO, CH3, etc) substitution at C-2 <strong>of</strong> ring A [Kusamba et al., 1993; Han et al.,<br />
2001], and in the case <strong>of</strong> compound 11, additional hydroxyl groups at positions 5 and 6 <strong>of</strong> ring C are<br />
observed. These latter oxygens are introduced at a late stage <strong>of</strong> the biogenesis [Han et al., 2001].<br />
8<br />
5<br />
8a<br />
5a<br />
O<br />
O<br />
1a<br />
4a<br />
1<br />
4<br />
O<br />
O<br />
O<br />
195<br />
O<br />
OH<br />
1 2 3<br />
7<br />
10<br />
OH<br />
6<br />
O<br />
O<br />
4<br />
14 15<br />
OCH 3<br />
Figure 1. Compounds isolated from the roots <strong>of</strong> Pentas longiflora<br />
R 6<br />
Compound R 1 R 2<br />
4 H CH3 H H H<br />
5 OH CH3 OH H H<br />
6 OCH3 CH3 OH H H<br />
7 OH CH2OCH3 OH H H<br />
8 OH CHO OH H H<br />
9 OCH3 CHO OH H H<br />
10 OCH3 CH2OH OH H H<br />
11 OCH3 CH2OH OH OH OH<br />
12 OH CH3 OPrimveroside H H<br />
13 OCH3 CH3 OPrimveroside H H<br />
8<br />
5<br />
R 5<br />
O<br />
9<br />
10<br />
O<br />
1<br />
R 1<br />
4<br />
R 4<br />
R 2<br />
R 3<br />
Figure 2. Compounds isolated from the roots <strong>of</strong> Pentas lanceolata<br />
R 3<br />
R 4<br />
R 5
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Table 1. In vitro antiplasmodial activity and cytotoxicity <strong>of</strong> crude extracts and pure compounds<br />
Sample (purity in %) Antiplasmodial<br />
acitivty IC50*<br />
(µg/mL)<br />
W2 clone<br />
(CQ-R)<br />
196<br />
D6 clone<br />
(CQ-S)<br />
Cytotoxicity<br />
LD50 §<br />
(µg/mL)<br />
W2<br />
Selectivity<br />
Index<br />
Pentas longiflora (root extract) 0.93 ± 0.16 0.99 ± 0.09<br />
Pentalongin (1, 98%) 0.27 ± 0.09 0.23 ± 0.08 0.80 2.96 3.48<br />
Psychorubrin (2, 98%)) 0.91 ± 0.15 0.82 ± 0.24 0.89 0.98 1.09<br />
Mollugin (3, 95%) 10.22 ± 1.37 7.56 ±1.13 20.0 1.96 2.65<br />
Pentas lanceolata (root extract) 2.55 ± 0.30 1.33 ± 0.15<br />
Tectoquinone (4, 98%) 10.78 ± 1.33 6.74 ± 1.73 > 10 #<br />
D6<br />
> 0.93 >1.48<br />
Rubiadin (5, 98%) 8.36 ± 2.19 5.47 ± 0.70 53.0 6.34 9.69<br />
Rubiadin-1-methyl ether (6, 98%) 18.91± 0.39 12.08 ± 2.28 64.0 3.38 5.30<br />
Nordamnacanthal (7, 99%) 9.33 ± 2.98 9.29 ± 0.00 51.0 5.47 5.49<br />
Damnacanthal (8, 99%) 10.88 ± 2.09 7.67 ± 0.36 73.0 6.71 9.52<br />
Lucidin- -methyl ether (9, 98%) 13.19 ± 2.15 12.08 ± 3.69 > 100 >7.58 > 8.28<br />
Damnacanthol (10, 98%) 31.42 ± 2.32 16.07 ± 1.15 > 100 > 3.18 > 6.22<br />
5,6-Dihydroxydamnacanthol 11, > 99% 19.33 ± 6.36 15.02 ± 4.28 > 100 > 5.17 > 6.66<br />
Chloroquine 0.07 ± 0.01 0.01 ± 0.01<br />
Mefloquine 0.004 ± 0.38 0.06 ± 0.04<br />
* Data are the mean <strong>of</strong> at least 3 independent experiments. § The mean value <strong>of</strong> at least 6<br />
independent experiments are given; 95% confidence interval and dose-response curves are<br />
presented. # Reference [Costa et al., 2001].<br />
Acknowledgement<br />
M. Endale is thankful to the German Academic Exchange Service (DAAD) and the Natural Products<br />
Research Network for Eastern and Central Africa (NAPRECA) for a PhD. Scholarship. M. Erdelyi is<br />
thankful for the financial support <strong>of</strong> the Swedish Research Council (VR2007-4407) and the Royal<br />
Society <strong>of</strong> Arts and Sciences in Göteborg.<br />
References<br />
Costa, S.M.O, Lemos, T.L.G, Pessoa, O.D.L, Pessoa, C., Montenegro, R.C., Braz-Filho, R. (2001); J Nat Prod, 64792-795.<br />
De Kimpe, N., Van Puydele, L., Schripsema, J., Erkelius, C., Verpoorte, R. (1993); Magn Reson Chem; 31, 329-330.<br />
Han, Y.S., Van der Heijen, R., Verpoorte, R. (2001); Biosynthesis <strong>of</strong> Anthraquinones in Cell Cultures <strong>of</strong> Rubiaceae. Plant<br />
Cell, Tissue and Organ culture, 67, 201-220.<br />
Hari, L., Buyck, L.F, De Pooter, H.L. (1991); Naphthoquinoid Pigments from Pentas longiflora. Phytochemistry, 30, 1726-<br />
1727.<br />
Kokwaro, J.O. (2010); Medicinal Plants <strong>of</strong> East Africa; University <strong>of</strong> Nairobi press, Nairobi, pp. 247-248.<br />
Leistner, E. (1973); Biosynthesis <strong>of</strong> Morindone and Alizarin in Intact Plants and Cell Suspension Cultures <strong>of</strong> Morinda<br />
citrifolia. Phytochemistry, 12, 1669-1674.<br />
Kusamba, C., Federici, E., De Vicente, Y., Galeffi, C. (1993); The Anthraquinones <strong>of</strong> Pentas zanzibarica. Fitoterapia, 64,<br />
18-22.
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Snow, R.W., Guerra, C.A., Noor, A.M., Myint, H.Y , Hay, S.I., (2005); The Global Distribution <strong>of</strong> Clinical Episodes <strong>of</strong><br />
Plasmodium. falciparum Malaria, Nature, 434, 214-217.<br />
Tuyen Nguyen V, De Kimpe N. (2004); Synthesis <strong>of</strong> Pyranonaphthoquinone Antibiotics Involving the Ring Closing<br />
Metathesis <strong>of</strong> a Vinyl ether. Tetrahedron Lett. 45, 3443-3446.<br />
Van Puyvelde L., Geysen D., Ayobangira F.X., Hakizamungu E., Nshimyimana A., Kalise A. (1985); Screening <strong>of</strong> Medicinal<br />
Plants <strong>of</strong> Rwanda for Acaricidal Activity. J Ethnopharmacol, 13, 209-215.<br />
Wanyoike G.N., Chhabra S.C., Lang at-Thoruwa C.C., Omar S.A. (2004); Brine Shrimp Toxicity and Antiplasmodial Activity<br />
<strong>of</strong> Five Kenyan Medicinal Plants. J Ethnopharm, 90, 129-133.<br />
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[YS 6] Study <strong>of</strong> Verucidal Effect <strong>of</strong> the Crashed Leaves <strong>of</strong> Tetradenia riparia on the<br />
Warts<br />
C. Karangwa 1 , J.N. Kabera 2 , C. Mukayisenga 1 , M.G. Ingabire 2<br />
1 National University <strong>of</strong> Rwanda, Faculty <strong>of</strong> Medicine, Department <strong>of</strong> Pharmacy. (NUR)<br />
2 Institute <strong>of</strong> Scientific and Technological Research, Phytomedicine and Life sciences Program ( IRST)<br />
Corresponding author: kajust68@yahoo.fr<br />
Keywords: Tetradenia riparia leaves, warts, Juice, Nitrogene, Magnesium and Chrome.<br />
Introduction<br />
T<br />
etradenia riparia was previously classified under the genus Iboza, which was derived from its<br />
Zulu name and apparently this refers to the aromatic qualities <strong>of</strong> the plant. The Zulu people<br />
have many uses for the plant including the relief <strong>of</strong> chest complaints, stomach ache and malaria.<br />
Inhaling the scent <strong>of</strong> the crushed leaves apparently also relieves headaches. In Rwanda, the plant is<br />
used to treat mainly the fever, cough, you find also others medicinal uses such us respiratory<br />
problems, stomach ache, diarrhea, dropsy, angina pectoris, fever, malaria and dengue fever, yaws,<br />
headache, toothache and as an antibiotic. (Hakizamungu, E. et al, 1986).<br />
The natural habitat <strong>of</strong> Tetradenia riparia is along river banks, forest margins, dry wooded valleys<br />
and hillsides in areas where there is little frost. The natural distribution ranges from KwaZulu-Natal,<br />
Northern Province, Mpumalanga in South Africa to Swaziland, Namibia, Angola and northwards<br />
through tropical east Africa into Ethiopia (Codd, L.E, 1985).<br />
Figure 1: distribution map<br />
Botanic description<br />
Synonyms<br />
Iboza bainesii N. E. Br.<br />
Iboza galpinii N. E. Br.<br />
Iboza riparia (Hochst.) N. E. Br.<br />
Moschosma riparium Hochst.<br />
At Macroscopical level<br />
S<strong>of</strong>t much-branched dioecious shrub or small tree 1-3m in height, with brittle, semi-succulent<br />
stems and sticky-aromatic foliage; leaves petiolate, ovate-oblong to round, 35-80 × 35-70mm,<br />
sparsely to densely glandular-pubescent on both surfaces, margin coarsely crenate to dentate,<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
variable in size, shape and degree <strong>of</strong> hairiness; flowers (May-Aug) in large branched terminal<br />
panicles, the male flower-spikes longer than the female, small (corolla 3-3.5mm long), white to pale<br />
mauve ( Codd, L.E, 1985)<br />
At Microscopical Level<br />
Figure 2: line drawing (female flowers)<br />
Figure 3: Microscopical features<br />
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Characteristic features are: the cells <strong>of</strong> the lower leaf epidermis with sinuous walls and numerous<br />
anomocytic stomata (1); the polygonal cells <strong>of</strong> the upper leaf epidermis with occasional stomata<br />
and underlying palisade layer (4); the numerous glandular hairs <strong>of</strong> leaf and stem, <strong>of</strong> two types:<br />
those with 2-3 celled stalk and unicellular head, up to 650 in length, raised on papillae (5),<br />
particularly abundant on main leaf veins and those having a unicellular stalk and bicellular head up<br />
to 25 in diameter, filled with yellow-brown contents (3); the uniseriate clothing hairs <strong>of</strong> both leaf<br />
surfaces, up to 800 long, thin-walled, smooth, 2-3 cells long, with swollen base (2); the microrosettes<br />
<strong>of</strong> calcium oxalate, 10-12 in diameter, in cells <strong>of</strong> the leaf palisade and mesophyll. (Codd,<br />
L.E, 1985); ( Pyvelde, 1983), (Davies Coleman et al, 1995)<br />
Chemical constituents<br />
Previously, several new substances have been isolated from the leaves <strong>of</strong> this plant, including a new<br />
diterpene diol, i.e. 8(14), 15-sandaracopimaradiene-7 alpha, 18-diol. This new diterpene diol<br />
exhibits significant antimicrobial activity against several bacteria and funga. (Bodenstein, J. W.<br />
,1977) diterpenes e.g. ibozol ( Zelnik, R et al,1978), 7 -hydroxyroyleanone, 8 (14), 15sandaracopimaradiene-7<br />
,18-diol ( Puyvelde Van et al, 1987) -pyrones e.g. umuravumbolide 4, 5 ,<br />
tetradenolide 6<br />
Figure 4: Umuravumolide<br />
Essential oil (1.9%) <strong>of</strong> which the main components are: -terpineol (22.6%), fenchone (13.6%), -<br />
fenchyl alcohol (10.7%), -caryophyllene (7.9%) and perillyl alcohol (6.0%), ( Campbell, W.E, 1997).<br />
But the chemical composition <strong>of</strong> essential oil can vary depending on season and time <strong>of</strong> harvest<br />
(Gazim ZC et al, 2010).<br />
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Material and Methods<br />
Plant material<br />
The plant material consists <strong>of</strong> Tetradenia riparia leaves from which the juice has been extracted.<br />
Figure 1. Live plant<br />
In a primary school in Butare ( Ikibondo primary school) , 64 volunteers pupils aged between 12 and<br />
17 years old with warts have been selected for the treatment.<br />
Figure 3. Photos <strong>of</strong> some Pupils with warts.<br />
Methods<br />
Data collection procedure<br />
A form <strong>of</strong> structured questionnaires where convenience sampling was used. The focus was on<br />
crashed leaves <strong>of</strong> Tetradenia riparia used in treating the warts. Filling the form processing was done<br />
by patients or their parents. The forms were collected and analyzed after the period <strong>of</strong> treatment.<br />
Determination <strong>of</strong> chemical constituents: Nitrogen, Magnesium and Chrome.<br />
According the theory <strong>of</strong> DEGOS, recurrent warts are treated with magnesium salt, liquid nitrogen,<br />
and chromic acid instead <strong>of</strong> dry ice. We have previously investigated the presence <strong>of</strong> those<br />
constituents in the leaves <strong>of</strong> Tedradenia riparia to finally confirm or disprove the DEGOS theory,<br />
such as traditional healers had observed in treating warts. (Degos R., 1981)<br />
Determination <strong>of</strong> Magnesium and Crome (Digestion <strong>of</strong> organic matte).<br />
We used the method <strong>of</strong> wet digestion for the determination <strong>of</strong> magnesium (Mg) and Chromium (Cr)<br />
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or wet digestion method,( MOORS, 1967).<br />
Determination <strong>of</strong> total nitrogen.<br />
For determination <strong>of</strong> total nitrogen, we used the Kjeldahl digestion method, distillation and<br />
titration, (Blume, 1966).<br />
Application <strong>of</strong> crushed leaves <strong>of</strong> Iboza riparia on warts<br />
The crushed leaves <strong>of</strong> Tedradenia riparia were rubbed two to three times per day on warts .The<br />
application is distributed over a treatment period ranging between one and five weeks.<br />
Results and Discussions<br />
Determination <strong>of</strong> chemical constituents in the leaves <strong>of</strong> Tedradenia riparia<br />
The results <strong>of</strong> the analysis and determination <strong>of</strong> chemical constituents in the leaves are presented<br />
in the table below:<br />
Table No. 1: Concentration <strong>of</strong> chemical constituents in Tedradnia riparia leaves.<br />
No Chemical<br />
constituents<br />
1 Total nitrogen (N) 4850<br />
2 Magnesium (Mg) 6720<br />
3 Chromium (Cr) 1072<br />
202<br />
Concentration ( ppm)<br />
Application <strong>of</strong> crashed leaves <strong>of</strong> Tedradenia riparia on the warts<br />
The leaves were crushed and rubbed precisely on where the warts were observed in faces. Applying<br />
the juice <strong>of</strong> crushed leaves <strong>of</strong> Tedradenia riparia was regularly done three times a day (Morning-<br />
Noon-Night) and for a period ranging between one and five weeks.<br />
After regular application <strong>of</strong> the juice <strong>of</strong> the leaves <strong>of</strong> Tetradenia riparia on the warts, we observed<br />
the following results:<br />
Table No. 2: Distribution <strong>of</strong> patients according the number <strong>of</strong> treatment day.<br />
Number <strong>of</strong><br />
days<br />
Number <strong>of</strong><br />
patients<br />
Patients<br />
cured<br />
Resistant<br />
cases<br />
%<br />
healing<br />
1 14 12 2 85.7 14.3<br />
2 20 18 2 90 10<br />
3 30 27 3 90 10<br />
TOTAL 64 57 7 89 11<br />
.<br />
% Resistance
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Figure 4 : Photos showing some children cured after treatment.<br />
After carry out this study, in general 89% <strong>of</strong> cases were cured. These observations allow us to<br />
confirm the therapeutic effect <strong>of</strong> the crashed leaves <strong>of</strong> Tedrania riparia on warts. The plant<br />
contains essential oils, nitrogen, magnesium and chromium. All these chemical constituents have<br />
veridical activities acting on the warts. After the treatment with crashed leaves, non scars remains<br />
on the faces as you can see on the pictures above. This healing is due mainly to the presence in<br />
leaves <strong>of</strong> Nitrogen, Magnesium, and Chromium, as well as the essential oils. These results confirm<br />
the scientific work <strong>of</strong> (Degos, R., 1981), that the warts are treated by liquid nitrogen, nitric acid,<br />
chromic acid and magnesium salt.<br />
Acknowledgements<br />
Authors would like to thank the pupils, their teachers and parents for having accepted to carry out<br />
our research at Ikibondo primary school.<br />
References<br />
1. Blume (1966, USAID, 1972).Méthode de Kjeldahl pour la détermination de l azote totale ;<br />
2. Bodenstein, J. W. (1977); Toxicity <strong>of</strong> traditional herbal remedies. South African Medical Journal 52:790.<br />
3. Codd, L.E. (1985); The genus Tetradenia. Flora <strong>of</strong> Southern Africa 28(4): 113-116. ;<br />
4. Campbell, W.E., Gammon, D.W., Smith. P., Abrahams, M. and Purves, T. (1997); Composition and antimalarial<br />
activity in vitro <strong>of</strong> the essential oil <strong>of</strong> Tetradenia riparia. Planta Medica 63: 270-272.;<br />
5. Degos R, Delort J, Civatte J, Poiares Baptista A. (1962); Epidermal tumor with an unusual appearance: clear cell<br />
acanthoma. Ann Dermatol Syphiligr (Paris) 89:361 371.<br />
6. Davies-Coleman, M. and Rivett, D.E.A. (1995); Structure <strong>of</strong> the 5,6-dihydro- -pyrone umuravumbolide.<br />
Phytochemistry 38(3): 791-792.<br />
7. Gazim ZC, Amorim AC, Hovell AM, Rezende CM, Nascimento IA, Ferreira GA, Cortez DA. (2010); Seasonal variation,<br />
chemical composition, and analgesic and antimicrobial activities <strong>of</strong> the essential oil from leaves <strong>of</strong> Tetradenia<br />
riparia (Hochst.) Codd in southern Brazil. Molecules. Aug 10; 15(8):5509-24.<br />
8. Puyvelde Van L, de Kimpe N, Ayobangira FX, Costa J, Nshimyumukiza P, Boily Y, Hakizamungu E, Schamp N. (1988);<br />
Wheat rootlet growth inhibition test <strong>of</strong> Rwandese medicinal plants: active principles <strong>of</strong> Tetradenia riparia and<br />
Diplolophium africanum. J Ethnopharmacol. Dec; 24(2-3):233-46;<br />
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9. Puyvelde Van L, Nyirankuliza S, Panebianco R, Boily Y, Geizer I, Sebikali B, de Kimpe N, Schamp N. (1986); Active<br />
principles <strong>of</strong> Tetradenia riparia. I. Antimicrobial activity <strong>of</strong> 8(14),15-sandaracopimaradiene-7 alpha,18-diol. J<br />
Ethnopharmacol. Sep; 17(3):269-75.<br />
10. Puyvelde Van, L., Lefebvre, R., Mugabo, P., de Kimpe, N. and Schamp, N. (1987); Active principles <strong>of</strong> Tetradenia<br />
riparia.II. Antispasmodic activity <strong>of</strong> 8 (14), 15-sandaracopimaradiene-7 ,18-diol. Planta Medica 52: 156-158.<br />
11. Puyvelde Van, L. et al.(1979); New -pyrones from Iboza riparia. Phytochemistry 18: 1215-1218.<br />
12. Puyvelde Van, L. and de Kimpe, N. (1998); Tetradenolide, an -pyrone from Tetradenia riparia. Phytochemistry<br />
49(4): 1157-1158.<br />
13. Zelnik, R., Rabenhorst, E., Matida, A., Gottlieb, H.E., Lavie, D and Panizza, S. (1978); Ibozol, a new diterpenoid from<br />
Iboza riparia. Phytochemistry 17: 1795-1797.<br />
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[YS 7] Is<strong>of</strong>lavanones and 3-Methoxyflavones from the Stem Bark <strong>of</strong><br />
Platycelphium voënse<br />
Ivan Gumula 1,2 , Mathias Heydenreich 3 , Solomon Derese 1 , Faith A. Okalebo 4 , Isaiah O. Ndiege 2 ,<br />
Mate Erdelyi 5 , Abiy Yenesew 1 *<br />
1 Department <strong>of</strong> Chemistry, University <strong>of</strong> Nairobi, P.O. Box 30197-00100, Nairobi, Kenya<br />
2 Department <strong>of</strong> Chemistry, Kyambogo University, P.O. Box 1, Kyambogo-Kampala, Uganda<br />
3 Institut f r Chemie, Universität Potsdam, P.O. Box 60 15 53, D-14415, Potsdam, Germany<br />
4 School <strong>of</strong> Pharmacy , University <strong>of</strong> Nairobi, P.O. Box 30197-00100, Nairobi, Kenya<br />
5 Department <strong>of</strong> Chemistry, University <strong>of</strong> Gothenburg, SE-412 96 Gothenburg, Sweden and<br />
Swedish NMR Centre, University <strong>of</strong> Gothenburg, Box 465, SE-405 30 Gothenburg, Sweden<br />
* Corresponding Author E-mail Address: ayenesew@uonbi.ac.ke (A. Yenesew).<br />
Key Words: Platycelphium voënse; Stem bark; Leguminosae; Is<strong>of</strong>lavanones; 3-methoxyflavones; Glyasperin F;<br />
Sophorais<strong>of</strong>lavanone A; 5,7-dihydroxy-4'-methoxy-[2'',3'':2',3']-furanois<strong>of</strong>lavanone<br />
Introduction<br />
P<br />
latycelphium (Engl.) Wild (Leguminosae) is a monotypic genus that occurs in the drier parts <strong>of</strong><br />
Eastern Africa (Gillett et al., 1971). Prior to this report, the only phytochemical studies on<br />
Platycelphium voënse describe the identification <strong>of</strong> quinolizidine alkaloids through the GC-MS<br />
analysis (Asres et al., 1997; Van Wyk et al., 1993). As part <strong>of</strong> the ongoing study <strong>of</strong> some<br />
Leguminosae species <strong>of</strong> Kenya, we hereby discuss the isolation and characterization <strong>of</strong> eight<br />
compounds including three is<strong>of</strong>lavanones, one is<strong>of</strong>lavone, two 3-methoxyflavones and two<br />
triterpenes.<br />
Materials and Methods<br />
The stem <strong>of</strong> Platycelphium voënse was collected from Eastern Province, Kenya, in January 2009. The<br />
plant was identified at the University Herbarium, Botany Department, University <strong>of</strong> Nairobi, where<br />
a voucher specimen was deposited.<br />
The air-dried and pulverized stem bark (1.6 Kg) <strong>of</strong> P. voënse was extracted with CH2Cl2-MeOH (1:1)<br />
at room temperature to afforded 114 g <strong>of</strong> crude extract. The extract was subjected to CC on silica<br />
gel, using increasing amounts <strong>of</strong> EtOAc in n-hexane as the mobile phase. Purification was done by<br />
repeated chromatography on silica gel, prepTLC, and gel filtration over sephadex LH-20. The<br />
structures <strong>of</strong> isolated compounds were established using a combination <strong>of</strong> spectroscopic<br />
techniques and by comparing the results with published data.<br />
Results and Discussion<br />
Compounds 1 and 3 were obtained as white amorphous solids with 1 H (Table 1) NMR spectral<br />
features characteristic <strong>of</strong> 5-hydroxyis<strong>of</strong>lavanones each with a pair <strong>of</strong> meta-coupled protons<br />
between H 5.90 and 6.10 for H-6 and H-8 (in A-rings); and two ortho-coupled protons resulting in<br />
an AX spin system at ca. 6.40-6.70 ppm (H-5') and 6.80-7.20 ppm (H-6'). The 1 H, 13 C NMR and DEPT<br />
spectra further revealed the presence <strong>of</strong> 2,2-dimethylpyrano ( C 27.0, 27.5 for 2''-OCH3, C 76.4 for<br />
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C-2'', C 118.4 for C-4'', C 129.9 for C-3'') and isoprenyl ( C 23.7 for C-1'', C 121.4 for C-2'', C 135.6<br />
for C-3'', C 18.0 for C-4'', C 25.8.8 for C-5'') moieties in 1 and 3, respectively. The 13 C NMR<br />
spectrum for each <strong>of</strong> the two compounds 1 and 3 exhbited five peaks due to quaternary sp 2<br />
hybridized enolic carbons in the C 150-167 range, four <strong>of</strong> which (based on biogenesis) are<br />
attributed to C-5, C-7, C-8a and C-4'; and the fifth (based on the chemical shift range) is assignable<br />
to C-2' and not C-3'. The presence <strong>of</strong> peaks at H 3.74 (3H) and C 62.6 in the spectra for compound<br />
3 was indicative <strong>of</strong> a di-ortho substituted methoxyl group, hence, its placement at position 2' and<br />
the isoprenyl unit at 3'. The ESI-mass spectra gave [M+1] + peaks at m/z 355.5 for compound 1 and<br />
371.4 for 3, which is in agreement with the molecular formulae C20H18O6 and C21H22O6, respectively.<br />
In attempts to confirm the structure <strong>of</strong> 1, a portion <strong>of</strong> the compound was treated with<br />
dimethylsulphate and acetone in the presence <strong>of</strong> potassium carbonate resulting in compound 2<br />
with two methoxyl carbon peaks resonating at 55.8 ppm (7-OCH3 and 4'-OCH3) implying that the 2'enolic<br />
carbon (rather than the 4'-enolic carbon) was part <strong>of</strong> the pyrano ring in compound 1. Based<br />
on these data, HMQC and H<strong>MB</strong>C experiments, compound 1 was identified as glyasperin F,<br />
previously isolated from the roots <strong>of</strong> Glycrrhiza aspera (Zeng, et al., 1992). Compound 3 was<br />
identified as sophorais<strong>of</strong>lavanone A, first reported from the aerial parts <strong>of</strong> Sophora tomentosa<br />
(Komatsu et al., 1978).<br />
HO 7<br />
1<br />
O<br />
O 2'' 3'' H3CO O<br />
3<br />
1'<br />
2'<br />
4''<br />
5<br />
OH<br />
1<br />
O<br />
4' OH<br />
OH<br />
2<br />
O<br />
HO O<br />
4'' HO<br />
O<br />
HO<br />
OCH 3<br />
2'' 5''<br />
OH O<br />
3<br />
OH<br />
OH<br />
4<br />
O<br />
5<br />
O<br />
O<br />
1''<br />
3''<br />
OCH 3<br />
RO<br />
206<br />
OH<br />
O<br />
O<br />
O<br />
O<br />
OCH 3<br />
6: R = CH 3, 7: R = H<br />
OCH 3<br />
2''<br />
3''<br />
OCH 3<br />
OH
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Table 1. 1 H NMR Spectroscopic Data (200 MHz) for Compounds 1, 3 & 4<br />
H (J in Hz)<br />
Position 1 (acetone-d6) 3 (CDCl3) 4 (CDCl3)<br />
2 a 4.41, dd (11.0, 5.7) 4.46, m 4.75, m<br />
b 4.56, dd (11.0, 11.0) 4.46, m 4.75, m<br />
3 4.19, dd (11.0, 5.5) 4.35, dd (9.8, 6.4) 4.48, m<br />
6 5.97, d (2.2) 6.00, d (2.0) 6.00, d (2.0)<br />
8 5.94, d (2.2) 5.96, d (2.0) 5.97, d (2.0)<br />
5' 6.40, d (8.4) 6.63, d (8.0) 6.64, d (7.8)<br />
6' 6.87, d (8.4) 6.88, d (8.2) 7.06, d (8.2)<br />
5-OH 12.40, s 12.21, s 12.13, s<br />
1'' - 3.43, d (6.8) -<br />
2'' - 5.25, t (6.6) 7.52, d (2.2)<br />
3'' 5.63, d (10.1) - 6.87, d (2.2<br />
4'' 6.67, d (10.1) 1.77, s -<br />
5'' - 1.65, s -<br />
2'-OCH3 - 3.74, s -<br />
4'-OCH3 - - 3.93, s<br />
2''-OCH3 1.33, s<br />
1.34, s<br />
- -<br />
Compound 4, obtained as a yellow gum, exhibited 1 H NMR spectral features similar to those <strong>of</strong> 1<br />
and 3. However, the presence <strong>of</strong> a pair <strong>of</strong> doublets, in the 1 H NMR spectrum, at 6.87 (J = 2.4 Hz)<br />
and 7.52 (J = 2.0 Hz) was suggestive <strong>of</strong> a furano moiety. The 1 H NMR spectrum further revealed<br />
the presence <strong>of</strong> a methoxyl group ( 3.93, s, 3H) that exhibited an nOe difference interaction with a<br />
proton at 6.64 (d, J = 7.8 Hz, H-5'), hence allowing the placement <strong>of</strong> the methoxyl group at<br />
position 4' and the furano at 2' and 3'. Compound 4 was, therefore, identified as 5,7-dihydroxy-4'methoxy-[2'',3'':2',3']-furanois<strong>of</strong>lavanone.<br />
The other compounds identified included formononetin (5), kumatakenin (6), isokaempferide (7), -<br />
amyrin and betulin. All these compounds are reported from Platycelphium voënse for the first time.<br />
Acknowledgements<br />
One <strong>of</strong> the authors (I.G.) is grateful to the Natural Products Research network for Eastern and<br />
Central Africa (NAPRECA) for a Scholarship and the German Academic Exchange Services (DAAD) for<br />
financing the studies. Mr S.G. Mathenge is acknowledged for identification and collection <strong>of</strong> the<br />
species.<br />
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References<br />
Asres, K., Tei, A. and Wink, M., (1997); Quinolizidine Alkaloids <strong>of</strong> Platycelphium voense (Engl.) Wild (Leguminosae) Z<br />
Naturforsch Ser C 52, 129-131.<br />
Gillett, J.B., Polhill, R.M. and Verdcourt, B., (1971); Flora <strong>of</strong> Tropical East Africa-Subfamily Papilionoideae., In Brenan, J.<br />
P. M., (Ed.), Leguminosae (part 3), Royal Botanic Gardens, Kew, Richmond, UK, 31-60.<br />
Komatsu, M., Yokoe, I., Shirataki, Y. (1978); Studies on the constituents <strong>of</strong> Sophora species XIII. Constituents <strong>of</strong> the<br />
aerial parts <strong>of</strong> Sophora tomentosa. Chem. Pharm. Bull., 26, 3863-3870.<br />
Van Wyk, B.-E., Greinwald, R. and Witte, L., (1993); Alkaloids <strong>of</strong> the Genera Dicraeopetalum, Platycelyphium and<br />
Sakoanala. Biochem. Syst. & Ecol. 21, 711-714.<br />
Zeng, L.,. Fukai, T., Nomura, T., Zhang, R.-Y., Lou, Z.-C. (1992); Five New Isoprenoid-Substituted Flavonoids Glyasperins<br />
F, G, H, I and J from the roots <strong>of</strong> Glycrrhiza aspera. Hetercycles 34, 1813-1828.<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[YS 8] Antitrypanosomal, Antileishmanial and Antiplasmodicidal Activities <strong>of</strong><br />
Khaya anthotheca, a Plant used by Chimpanzees for Self Medication.<br />
C. J. D. Obbo 1 , B. Makanga 1 , D. A Mulholland 2,3 , P. H. Coombes 2 , R. Brun 4,5 , M. Kaiser 4,5 , W. Olaho-<br />
Mukani 6<br />
1<br />
Department <strong>of</strong> Zoology, Makerere University, Post Box 7062, Kampala Uganda<br />
2<br />
School <strong>of</strong> Pure and Applied Chemistry, University <strong>of</strong> Kwazulu-Natal, South Africa.<br />
3<br />
Divisional <strong>of</strong> Chemical Sciences, Faculty <strong>of</strong> Health and Medical Sciences, University <strong>of</strong> Surrey, GU2 7XH, Guildford,<br />
United Kingdom<br />
4<br />
Medical Biology and Infection Biology, Parasite Chemotherapy, Swiss Tropicaland Public Health Institute, Socinstrasse<br />
57, CH-4002 Basel, Switzerland.<br />
5 University <strong>of</strong> Basel, Petersplatz 1, CH4051 Basel, Switzeland<br />
6 Livestock Research Institute, Tororo, Uganda.<br />
Key words: Khaya anthotheca, chimpanzees, self-medication, antiprotozoal, drug-resistance, Trypanosoma,<br />
Leishmania, Plasmodium<br />
Introduction<br />
T<br />
rypanosomiasis, leishmaniasis and malaria are among the major devastating protozoan diseases<br />
<strong>of</strong> the underdeveloped tropical regions <strong>of</strong> the world, the first two being neglected tropical<br />
diseases that afflict extremely poor and disadvantaged rural settings (WHO 2002, Fevre et al.,<br />
2008). For each one <strong>of</strong> these diseases, there are no practical vaccines available yet to kill all<br />
parasites in infected communities and to block the transmission (Target and Greenwood 2008)<br />
Defence and protection against these diseases remains to be chemotherapy, but the number <strong>of</strong><br />
therapeutic drugs available to each <strong>of</strong> them is very limited and unsatisfactory (Falade et al., 2005).<br />
Variable and marginal efficacies, severe toxicities high costs, requirements for parenteral<br />
administration and for long course <strong>of</strong> treatment constitute some <strong>of</strong> the major drawbacks to current<br />
antitrypanosomal and antileishmanial drugs (Osorio et al., 2006). Resistance to the commonly used<br />
drugs against these pathogens poses the greatest challenge to their usefulness (Cr<strong>of</strong>t et al., 2006,<br />
Hyde 2007). The urgent need for newer, safer and more practical antiprotozoal drugs continues<br />
unabated (Bouteille et al., 2003) and their searches from plant sources are among the major<br />
priorities (Wilcox et al., 2004).<br />
Khaya anthotheca (Welw.) C.D.C. belongs to the family Meliaceae. Members <strong>of</strong> this family have<br />
important roles in the ethno-pharmacological practices <strong>of</strong> tropical and subtropical communities<br />
(Phillipson and O Neill 1986). Reports indicate that chimpanzees also ingest different parts <strong>of</strong> plants<br />
<strong>of</strong> this family in unusual feeding behaviour (Krief et al., 2004). Researchers have suggested<br />
medicinal benefits <strong>of</strong> such feeding behaviour for parasite control and provided chemical evidence<br />
to support the pharmacological roles <strong>of</strong> secondary metabolites in that respect (Huffman and Seifu<br />
1989). This report presents the antiprotozoal potency <strong>of</strong> the crude petroleum ether extract and two<br />
pure compounds, grandifolione and 7-deacetykhivorin, from K. anthotheca seed.<br />
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K. anthotheca was selected for this study on the basis <strong>of</strong> its common use in West African traditional<br />
medicine and from documented evidence <strong>of</strong> its use by primates in Budongo forest, western<br />
Uganda. Plant materials were collected, identified at Makerere University Herbarium and a voucher<br />
specimen retained. Mature healthy seeds were collected from a pure K. anthotheca tree plantation<br />
at the Budongo Conservation station.<br />
Extraction and purification Procedure<br />
The pericarp was removed and the dried seeds ground into smaller particles. Batches <strong>of</strong> 100<br />
grammes <strong>of</strong> the powder were packed in thimbles and successively extracted with hexane,<br />
petroleum ether, dichloromethane and methanol using a modified protocol <strong>of</strong> Rosanaivo and<br />
Ratsimamanga-Urveg (1993). The extracts were filtered and solvents removed in a Bucchi R rotary<br />
evaporator below 50 o C under reduced pressure. The crude extracts were freeze-dried in a shell<br />
freezer system (Labconco R /103 M BAR) at 0 o C. The hexane extract gave viscous colourless oil and<br />
the petroleum ether extract separated out into white crystals and a viscous oily portion. The<br />
solvent free extracts were placed in sample bottles, weighed, sealed with parafilm and stored at<br />
room temperature. The petroleum ether extract (3.22g) was purified by column chromatography<br />
eluted with dichloromethane : methanol (v/v 48: 2). Further purification was achieved with<br />
preparative thin layer chromatography. The pure compounds were identified as 7deacetoxykhivorin,<br />
(1), grandifolione, (2), 1, 3-diacetyldeoxyhavenensin (3) using 2D NMR<br />
spectroscopy and by comparison against literature data (Adesogan et al., 1971).<br />
In vitro drug sensitivity assays<br />
Activity against erythrocytic stages <strong>of</strong> P. falciparum was determined by a modified [3H]hypoxanthine<br />
incorporation assay (Matile and Pink, 1990) using the chloroquine- and<br />
pyrimethamine-resistant K1 strain and the standard drug chloroquine. Activity <strong>of</strong> all the extracts<br />
against Trypanosoma brucei rhodesiense was determined by the methods described (Räz et al.,<br />
1997). Activity against trypomastigote forms <strong>of</strong> T. cruzi, Tulahuen C2C4 strain containing the -<br />
galactosidase (Lac Z) gene, was determined by the methods <strong>of</strong> Buckner et al., (1996). Activity<br />
against amastigotes <strong>of</strong> L. donovani strain MHOM/ET/67/L82 was determined according to the<br />
method described by Al-Bashir et al., (1992). Cytotoxicity assays against L6-cells were performed<br />
according to the methods described by Ahmed et al., (1994). The IC50 values were calculated from<br />
the sigmoidal inhibition curves using S<strong>of</strong>tmaxPro s<strong>of</strong>tware. The selectivity index (SI), ratio <strong>of</strong> the IC50<br />
for the L-6 cells to the IC50 for the protozoan parasite was calculated for each compound.<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Results<br />
Table 1: Antiprotozoal activities <strong>of</strong> limonoids from K. anthotheca seed. IC50 shown are means <strong>of</strong> two<br />
independent assays. Selectivity index (SI ) in brackets<br />
Drug<br />
substance<br />
T. b.<br />
rhodesiense<br />
T. cruzi L.<br />
donovani<br />
P.<br />
falciparum<br />
Cytotoxicity<br />
L6<br />
IC50 IC50 IC50 IC50 IC50<br />
Crude Ka2-b 5.72 (17) 14.51 (6) 30 (3) 0.955 (95) 90<br />
Grandifolione 10.66 (4) 20.97 (2) 13.31 (3) 0.372 (121) 44.7<br />
7-deacetylkhivorin 16.88 (0) 31.82 (0.5) 36.71 (0.4) 1.370 (11) 14.9<br />
Melarsoprol 0.004 - - - -<br />
Benzinidazole - 0.296 - - -<br />
Miltefosine - - 0.156 - -<br />
Chloroquine - - - 0.058 -<br />
Podophillotoxin - - - - 0.005<br />
Discussion<br />
Sequential maceration <strong>of</strong> the seedcake <strong>of</strong> K. anthotheca with organic solvents <strong>of</strong> increasing<br />
polarities yielded crystals and gummy crude extracts with moderate antitrypanosomal,<br />
antileishmanial and antimalarial activities as compared to standard drugs. The crude petroleum<br />
ether extract demonstrated IC50 activities at 5.2 g/ml, 14.51 g/ml, >30 g/ml against T.b.<br />
rhodesiense, T.cruzi and L. donovani.<br />
Purified compounds, grandifolione and 7-deacetylkhivorin, showed IC50 activities at 10.66,<br />
16.88 g/ml against T. b. rhodesiense, 20.97, 31.82 g/ml against T. cruzi, and 13.31, 36.71 g/ml)<br />
against L. donovani respectively. Their selectivity indices were correspondingly low, at 4 and 0; 2<br />
and 0.5; 3 and 0.4 respectively for the three pathogens. The pure compounds exhibited lower<br />
activities than the crude form <strong>of</strong> the drug and weak antiprotozoal activities as compared to<br />
standards.<br />
However, P. falciparum (KI) was more sensitive to the crude form and pure compounds than the<br />
test trypanosomes and Leishmania. The crude form <strong>of</strong> the extract gave a mean antiplasmodial IC50<br />
value <strong>of</strong> 0.955 g/ml. Grandifolione gave an IC50 <strong>of</strong> 0.372 g/ml and 7-deacetoxykhivorin gave an<br />
IC50 <strong>of</strong> 1.37 g/ml. Selectivity indices for both compounds were 121 and 11. Grandifolione was 12x<br />
more selective than 7-deacetylkhivorin. The antiplasmodial activity <strong>of</strong> 7-deacetykhivorin was still<br />
moderate as compared to the activities <strong>of</strong> chloroquine and artemisinin. Cytotoxicity levels <strong>of</strong> the<br />
crude extracts and pure compounds were appreciably low, at 90, 44.7 and 14.9 g/ml, making the<br />
two compounds 8940x and 2980x less toxic than podophyllotoxin.<br />
From this study, it is evident that an ethnopharmacological use <strong>of</strong> Khaya seed to treat<br />
trypanosomiasis would be insufficient and perhaps that explains why there are no such anecdotal<br />
reports in ethnobotanic literature. The observed activity <strong>of</strong> grandifolione supports the claim, in<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
part, that chimpanzees might be using the seeds for self medication and in general, the use <strong>of</strong><br />
Khaya plant material by humans in disease endemic Tropical areas to treat fevers. Their low toxicity<br />
levels and high selectivity, especially for P. falciparum, make them attractive as possible<br />
antiplasmodial drug candidates. The antitrypanosomal and antileishmanial properties <strong>of</strong><br />
grandifolione and 7-deacetylkhivorin are here reported for the first time.<br />
Acknowledgements<br />
This study was co-funded by the DAAD, UNCST, Peoples and Plants Initiative UK. We thank the<br />
Swiss Tropical STIB Basel and LIR Tororo for the running the antiprotozoal assays. More gratitude to<br />
the Department <strong>of</strong> Pure and Applied Chrmistry UKZN S. Africa, for determining the molecular<br />
structures.<br />
References<br />
Adesogan et al., (1967); Extractives from the seed <strong>of</strong> Khaya anthotheca (Welv.) C.D.C. Chem. Com. 379-380.<br />
Ahmed et al., (1994); A new rapid and simple non-radioactive assay to monitor and determine the proliferation <strong>of</strong><br />
lymphocytes: an alternative to [3H] thymidine ncorporation assay. The Journal <strong>of</strong> Immunological Methods 170,<br />
211 224.<br />
Al-Bashir et al., (1992); Axenic cultivation <strong>of</strong> amastigotes <strong>of</strong> Leishmania donovani and Leishmania major and their<br />
infectivity. Annals <strong>of</strong> Tropical Medicine and Parasitology 86, 487-502.<br />
Page et al., (1993); A new fluorimetric assay for cytotoxicity measurements in vitro. International Journal <strong>of</strong> Oncology 3,<br />
473-476.<br />
Buckner et al., (1996). Buckner, F.S., Verlinde, C.L., La Flamme, A.C., van Voorhis, W.C., 1996. Efficient technique for<br />
screening drugs for activity against Trypanosoma cruzi using parasites expressing betagalactosidase. Antimicrobial<br />
Agents and Chemotherapy 40, 2592-2597.<br />
Bouteille et al., (2003). Fundam. Clin. Pharmacol. 17, 171.<br />
Cr<strong>of</strong>t and Coombs (2003). Leishmaniasis current chemotherapy and recent advances in the search for novel drugs.<br />
Trends in Parasitology Vol.19 No.11 p. 502-508.<br />
Cr<strong>of</strong>t et al., (2006). Drug Resistance in Leishmaniasis. Clinical Microbiology Reviews, January 2006, p. 111-126, Vol. 19,<br />
No. 1<br />
Falade et al., (2005). Efficacy and safety <strong>of</strong> artemether lumefantrine (Coartem®) tablets (six-dose regimen) in African<br />
infants and children with acute, uncomplicated falciparum malaria. Transactions <strong>of</strong> the Royal Society <strong>of</strong> Tropical<br />
Medicine and Hygiene (2005) 99, 459 467<br />
Fevre et al., (2008). The Burden <strong>of</strong> Human African Trypanosomiasis. PLoS Neglected Tropical Diseases. Vol. 2(12): e333.<br />
Hyde (2007). Drug-resistant malaria: an insight . FEBS J. 274: 4688-4698.<br />
Huffman and Seifu (1989). Observations <strong>of</strong> illness and consumption <strong>of</strong> a possibly medicinal plant Vernonia amygdalina<br />
(Del.), by a wild chimpanzee in the Mahale Mountains National Park, Tanzania. Primates 30: 51 63.<br />
Krief et al., (2004). Krief, B.; Martin, M. T.; Grellier, P.; Kasenene, J.; Sévenet, T. Antimicrob. Agents Chemother. 2004,<br />
48, 3196.<br />
Matile and Pink, (1990). Plasmodium falciparum malaria parasite cultures and their use in immunology. In:<br />
Immunological Methods. Lefkovits, I., Pernis, B., Eds., Academic Press, San Diego, CA, USA, 221-234.<br />
Osorio et al., (2006). Antileishmanial and Cytotoxic Activity <strong>of</strong> Synthetic Aromatic Monoterpens Acta Farm. Bonaerense<br />
25 (3): 405-13 (2006).<br />
Phillipson and O Neill (1986). Novel antimalarial drugs from plants? Parasitol. Today 2, 355 358.<br />
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[YS 9] Antiplasmodial and Antileishmanial Studies on Carvotacetone<br />
Derivatives from Sphaeranthus bullatus<br />
Francis Machumi 1 , Abiy Yenesew 2 , Jacob Midiwo 2 , Larry Walker 3 , Muhammad Illias 3 , Matthias<br />
Heydenreich 4 , Erich Kleinpeter 4<br />
1 Open University <strong>of</strong> Tanzania, Faculty <strong>of</strong> Science, P.O. Box 23409 Dar es Salaam, Tanzania<br />
2 Department <strong>of</strong> Chemistry, University <strong>of</strong> Nairobi, P.O. Box 30197 (00100), Nairobi, Kenya<br />
3 National Center for Natural Products Research, School <strong>of</strong> Pharmacy, University <strong>of</strong> Mississippi, University,<br />
Mississippi 38677, USA<br />
4 Institute für Chemie, Universität Potsdam, P.O. Box 60 15 53, D-14476 Potsdam, Germany<br />
Correspondence: francis.machumi@gmail.com<br />
Key words: Sphaeranthus bullatus, antiplasmodial, antileishmanial, carvotacetone derivatives<br />
Introduction<br />
M<br />
alaria and leishmaniasis are both parasitic diseases caused by protozoan parasites and<br />
transmitted by bite <strong>of</strong> infected insects, mosquitoes and sand flies respectively. Malaria,<br />
predominant in the tropics, is caused by blood parasites <strong>of</strong> the genus Plasmodium and transmitted<br />
to humans by female Anopheles mosquito. Clinical malaria is manifested by a range <strong>of</strong> symptoms<br />
such as fever, vomiting, joint pain and convulsions [Nkuo-Akenji and Menang, 2005]. Besides<br />
contributing to over a million deaths yearly, malaria is known to be a cause <strong>of</strong> anaemia and its<br />
various complications, miscarriages, brain damage, decreased cognition and irreversible disabilities<br />
[Rugemalila et al., 2006]. Leishmaniasis on the other hand is transmitted by a bite <strong>of</strong> some species<br />
<strong>of</strong> sand flies which infect the blood with parasites <strong>of</strong> the genus Leishmania [Tonui 2006]. Two<br />
common forms <strong>of</strong> leishmaniasis are known; cutaneous leishmaniasis (CL) which causes sore at the<br />
bite site and visceral leishmaniasis (VL) which affects vital organs. Leishmaniasis is spread in tropical<br />
and subtropical regions <strong>of</strong> the world, with estimated number <strong>of</strong> new cases <strong>of</strong> CL and VL at<br />
1.5million and 500,000 annually, respectively [Kigondu et al., 2009]. This study paper we report the<br />
first account <strong>of</strong> antiplasmodial and antileishmanial properties <strong>of</strong> abietane diterpenoids and<br />
carvotacetone derivatives from traditionally used medicinal plant Sphaeranthus bullatus.<br />
Material and Methods<br />
The aerial parts <strong>of</strong> Sphaeranthus bullatus were collected from Ngong forest Nairobi, in November<br />
2007. They were air dried in shade and pulverized to give 2.3 Kgs which were extracted by cold<br />
percolation at room temperature using 1:1 CH2Cl2/MeOH (3×5 L, 24 h each), followed by 100%<br />
methanol (1×4 L, 24 h) to give 168 g <strong>of</strong> black-brown gummy extract, <strong>of</strong> which 100 g were<br />
chromatographed over silica gel giving fractions which were further purified using to give five<br />
carvotacetone derivatives. The compounds were identified using spectroscopic method ( 1 H-NMR,<br />
13 C-NMR, DEPT, COSY, NOESY, H<strong>MB</strong>C, HSQC, EI-MS) and direct comparison with published spectral<br />
data and structures.<br />
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The antiplasmodial activity was measured in vitro by a colorimetric assay that determines the<br />
parasitic lactate dehydrogenase (pLDH) activity [Samoylenko et al., 2009]. The assay was performed<br />
in 96-well microplate and included two P. falciparum strains [Sierra Leone D6 (chloroquinesensitive)<br />
and Indochina W2 (chloroquine-resistant)]. DMSO, artemisinin and chloroquine were<br />
included in each assay as vehicle and drug controls, respectively. Antileishmanial activity <strong>of</strong> the<br />
compounds was tested in vitro on a culture <strong>of</strong> Leishmania donovani promastigotes. In a 96 well<br />
microplate assay compounds with appropriate dilution were added to the Leishmania<br />
promastigotes culture (2×106 cells/mL). The plates were incubated at 26°C for 72 hours and growth<br />
<strong>of</strong> Leishmania promastigotes was determined by Alamar blue assay [Mikus and Steverding 2000].<br />
Pentamidine and amphotericin B were used as standard antileishmanial agents. IC50 values for each<br />
compound were computed from the growth inhibition curve.<br />
Results and discussion<br />
Five known carvotacetone derivatives (Figure 1) were isolated from the aerial parts <strong>of</strong><br />
Sphaeranthus bullatus. These are 3-acetoxy-7-hydroxy-5-tigloyloxycarvotacetone (1), 3,7dihydroxy-5-tigloyloxycarvotacetone<br />
(2), 3-acetoxy-5,7-dihydroxycarvotacetone (3), 3,5,7trihydroxycarvotacetone<br />
(4), and 5-O- -glucopyranosylcarvotacetone (5) which is reported for the<br />
first time from the genus. With excerption <strong>of</strong> 5, the compounds showed antiplasmodial and<br />
antileishmanial activities as tabulated in Table 1. The activities seemed to be enhanced by presence<br />
<strong>of</strong> the acetyl or tiglyl substituents at 3-OH and 5-OH respectively, as witnessed by the weaker<br />
activity <strong>of</strong> 4 as compared to 1, 2 and 3. Compound 3 (3-acetoxy-5,7-dihydroxycarvotacetone) was<br />
the most active compound, having antiplasmodial activity <strong>of</strong> IC50 0.6 and 0.7 µg/ml against<br />
chloroquine sensitive and chloroquine resistant strains <strong>of</strong> P. falciparum respectively, as well as<br />
antileishmanial activity <strong>of</strong> IC50 0.7 against L. donovanii.<br />
Figure 1: Carvotacetone derivatives from the aerial parts <strong>of</strong> Sphaeranthus bullatus<br />
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Table 1: In-vitro antiplasmodial activity, antileishmanial activity and cytoxicity<br />
Antiplasmodial activity (µg/ml) Antileishmanial<br />
activity<br />
Cytotoxicity<br />
P. falciparum D6 P. falciparum W2 L. donovanii VERO<br />
IC50 SI IC50 SI IC50 SI<br />
Extract 9.7 NT 15.0 NT NT NT NT<br />
1 1.4 0.002 2.0 0.001 0.7 0.001 2.8<br />
2 0.8 0.006 0.9 0.005 3.0 0.007 NC<br />
3 0.6 0.022 0.7 0.019 0.7 0.001 0.013<br />
4 3.4 0.001 2.8 0.002 17.0 >0.04 NC<br />
5 NA NT NA NT 17.0 0.035 NC<br />
Chloroquin<br />
e<br />
0.01 NT 0.14 NT NT NT NT<br />
Artemisinin 0.004 NT 0.0048 NT NT NT NT<br />
Pentamidin<br />
e<br />
NT NT NT NT 0.1 NT NT<br />
NA = Not Active (up to the maximum dose tested 47.6 µg/ml); NC = Not Cytotoxic; NT = Not Tested,<br />
IC50 = concentration that affords 50% inhibition <strong>of</strong> growth, SI = Selectivity index<br />
References<br />
Kigondu E. V. M., Rukunga G. M., Keriko J. M., Tonui W. K., Gathirwa J. W., Kirira P. G., Irungu B., Ingonga J. M., Ndiege<br />
I. O. (2009); Anti-parasitic activity and cytotoxicity <strong>of</strong> selected medicinal plants from Kenya. Journal <strong>of</strong><br />
Ethnopharmacology 123, 504-509.<br />
Nkuo-Akenji, T.K., Menang, O.N. (2005); Prevelance <strong>of</strong> falciparum malaria together with acute diarrhoea in children<br />
residing in a malaria endemic zone. Africa Journal <strong>of</strong> Traditional, Complementary and Alternative Medicine 12, 26-<br />
30.<br />
Rugemalila, J.B., Wanga, C.L., Kilama W.L. (2006); Sixth Africa malaria day in 2006: how far have we come after Abuja<br />
declaration? Malaria Journal 12, 102-106.<br />
Tonui W. K. (2006; Situational Analysis <strong>of</strong> Leishmaniases Research in Kenya. African Journal <strong>of</strong> Health Sciences 13, 7-21.<br />
Samoylenko V., Jacob M. R., Khan S. I., Zhao J., Tekwani B. L., Midiwo J. O., Walker L. A., Muhammad I. (2009);<br />
Antimicrobial, antiparasitic and cytotoxic spermine alkaloids from Albizia schimperiana. Natural Product<br />
Communications, 4, 791-796.<br />
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[YS 10] In vitro anthelmintic Effect <strong>of</strong> Prosopis juliflora (Sw.) DC (Fabaceae) on<br />
Haemonchus contortus, an Abomasal Nematode <strong>of</strong> Sheep<br />
Rechab, S.O. 1* , Kareru, P.G. 1 , Kutima, H.L 2 , Gakuya, D.W. 3 , Nyagah, G.C. 1 , Njonge, F.K. 4 , Waithaka,<br />
R.W. 1<br />
1 Chemistry Department, Jomo Kenyatta University <strong>of</strong> Agriculture and Technology (JKUAT), P.O Box 62000, Nairobi,<br />
Kenya<br />
2 Zoology Department, JKUAT, P.O Box 62000, Nairobi, Kenya<br />
3 College <strong>of</strong> Agricultural and Veterinary Sciences, University <strong>of</strong> Nairobi, Kenya<br />
4 Land Resource, Planning & Management Department, JKUAT, P.O Box 62000, Nairobi, Kenya<br />
rechabsylvester@gmail.com<br />
Key Words: Haemonchus contortus, Tannins, saponins, anthelmintic activity, ruminants<br />
INTRODUCTION<br />
T<br />
he economic impact <strong>of</strong> parasitic gastroenteritis caused by mixed infection with several species<br />
<strong>of</strong> stomach and intestinal round worms, as a production disease in ruminants lies in direct<br />
losses involving mortality due to the clinical form <strong>of</strong> the disease and also indirect losses due to<br />
weaknesses, loss <strong>of</strong> appetite, decreased feed efficiency, reduced weight gain and decreased<br />
productivity. In Kenya, economic loss to the agricultural sector due to Haemonchus contortus<br />
parasite <strong>of</strong> small ruminants is estimated at over US$ 26 million per year (Githiori, 2004). Control<br />
programs based on the use <strong>of</strong> synthetic anthelmintics are no longer sustainable because <strong>of</strong> high<br />
prevalence <strong>of</strong> gastrointestinal nematode resistance, slow development <strong>of</strong> new anthelmintics, high<br />
costs to poor farmers and concerns regarding residue in food and the environment (Singh et al.,<br />
2002). Alternative methods <strong>of</strong> control such as use <strong>of</strong> tanniferous plants are thus required for<br />
introduction into farm production systems (Niezen et al., 2002). Prosopis juliflora (Sw.) DC<br />
(Fabaceae) is an evergreen tree native to South America, Central America and the Caribbean.<br />
Prosopis species are generally fast-growing, drought-resistant, nitrogen-fixing trees or shrubs<br />
adapted to poor and saline soils in arid and semi-arid zones. (Pasiecznik et al., 2001).<br />
MATERIALS AND METHODS<br />
Sample collection, preparation and extraction<br />
Leaves and root bark samples <strong>of</strong> P. juliflora, obtained from Endao, Marigat district, in Baringo<br />
county <strong>of</strong> Kenya were botanically identified and authenticated by field a <strong>of</strong>ficer from Kenya Forestry<br />
Research Institute, Marigat station and a taxonomist from Botany Department <strong>of</strong> Jomo Kenyatta<br />
University <strong>of</strong> Agriculture and Technology, where voucher specimens were also deposited. The<br />
collected materials were washed thoroughly in water, chopped; air dried for two week, pulverized<br />
in electric grinder and exhaustively extracted using ethanol. The extracts were concentrated in<br />
vacuo, dried and stored at 4°C until required for bioassay.<br />
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Phytochemical screening<br />
Phytochemical screening was performed using standard procedures (Harborne, 1998) and the<br />
extracts were tested for triterpenes, sterols, flavonoids, saponins, tannins and alkaloids.<br />
In vitro ovicidal activity:<br />
The egg hatch assay (EHA) was carried out using the World Association for the Advancement <strong>of</strong><br />
Veterinary Parasitology (W.A.A.V.P.) guidelines for determination <strong>of</strong> anthelmintic resistance (Coles<br />
et al., 1992) with modifications that allowed the testing <strong>of</strong> the natural compounds. The number <strong>of</strong><br />
eggs which had not hatched and number <strong>of</strong> hatched larvae were counted and percentage hatching<br />
calculated. There were three replicates for each concentration and controls.<br />
Results and Discussion<br />
Extraction yield<br />
Ethanolic extraction <strong>of</strong> the roots gave a higher yield <strong>of</strong> 16.78% as compared to that <strong>of</strong> the leaves<br />
which was 6.94%, an indication that there were more polar compounds in roots as compared to the<br />
leaves.<br />
Results for phytochemical screening<br />
Table 1: Phytochemical pr<strong>of</strong>ile <strong>of</strong> LEE and REE <strong>of</strong> P. juliflora<br />
Secondary metabolite LEE REE<br />
Alkaloids + +<br />
Tannins + +<br />
Saponins + ++<br />
Flavonoids + +<br />
Sterols/ Triterpenes + +<br />
+ Present, ++ Present in high concentration, LEE: Leaf Ethanolic extract;<br />
REE: Root Ethanolic Extract<br />
Phytochemical analysis showed that LEE and REE possess alkaloids, tannins, saponins, flavonoids,<br />
sterols and triterpenes. The root ethanolic extract had higher concentration <strong>of</strong> saponins as<br />
compared to the leaves ethanolic extract as exhibited by higher volume <strong>of</strong> persistent frothing.<br />
Results for in vitro anthelmintic activity<br />
In the search for natural anthelmintics, in vitro tests are used as preliminary studies <strong>of</strong> plants. In<br />
these tests, the plant extracts are directly placed in contact with the eggs larvae or adult parasites<br />
to evaluate the effect on egg hatching, larval development or motility and mortality <strong>of</strong> adult worms<br />
(Hammond et al., 1997).<br />
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120<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
EGG HATCH ASSAY FOR ALB, LEE AND REE<br />
0 0.03125 0.0625 0.125 0.25 0.5 1 2<br />
Concentration in mg/ml<br />
Fig1: Graph showing mean percentage egg hatching <strong>of</strong> various concentrations <strong>of</strong> LEE, REE and ALB<br />
after 48 hours<br />
Both LEE and REE showed anthelmintic activity in a concentration dependent manner. However,<br />
LEE had a higher activity (LD50=0.857 mg/ml) as compared to REE (LD50=1.782 mg/ml). Albendazole<br />
had significantly higher activity as compared to the ethanolic extracts. (LD50=0.046mg/ml). The<br />
anthelmintic activity <strong>of</strong> LEE and REE may be attributed to presence <strong>of</strong> phytochemicals such as<br />
saponins, tannins and alkaloids. Min et al. (2003) reported that Condensed tannins might diffuse<br />
through the external surfaces such as eggshells and bind to faecal egg proteins thus inhibiting egg<br />
hatching and larval development. Saponins destabilize membranes and increase cell permeability<br />
by combining with membrane-associated sterols (Gee and Johnson, 1988) while alkaloids may<br />
improve tonicity <strong>of</strong> the gastrointestinal tract and thus expel the worms or may have a direct effect<br />
on the nervous system <strong>of</strong> nematodes. Other phytochemicals like flavonoids and oleane type<br />
triterpenes may also have their independent or synergistic effects (Brantner et al., 1996). The use <strong>of</strong><br />
botanical anthelmintics has been proposed as an alternative strategy for the control <strong>of</strong><br />
gastrointestinal nematode infections in order to reduce the dependence on chemical anthelmintic<br />
treatments and to delay the selection and the transmission <strong>of</strong> anthelmintic resistances in worm<br />
populations (Hoste et al., 2006).<br />
Acknowledgement:<br />
The authors acknowledge Jomo Kenyatta University <strong>of</strong> Agriculture and Technology for funding the<br />
project.<br />
References<br />
Brantner, A., Males, Z., Pepeljak S. and Antolic A. (1996); Antibacterial activity <strong>of</strong> Paliurus spina-christi Mill (Christ s<br />
thorn). Journal <strong>of</strong> Ethnopharmacology, 52: 119 122.<br />
218<br />
ALB<br />
LEE<br />
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Coles, G.C., Bauer, C., Borgsteede, F.H., Geerts, S., Klei, T.R., Taylor, M.A., Waller, P.J., (1992); World Association for the<br />
Advancement <strong>of</strong> Veterinary Parasitology (W.A.A.V.P.) methods for the detection <strong>of</strong> anthelmintic resistance in<br />
nematodes <strong>of</strong> veterinary importance. Veterinary Parasitology, 44: 35 44.<br />
Gee, J.M. and Johnson, I.T. (1988); Interaction between haemolytic saponins, bile salts and small intestinal mucosa in<br />
rat. Journal <strong>of</strong> Nutrition,118: 1391 1397.<br />
Githiori, J.B. (2004). Evaluation <strong>of</strong> anthelmintic properties <strong>of</strong> ethnoveterinary plants preparations used as livestock<br />
dewormers by pastoralist and small holder farmer in Kenya. Doctoral dissertation, Department <strong>of</strong> Biomedical<br />
Sciences and Veterinary Public Health, SLU. Acta Universitatis Agricultural sulciae, p. 76.<br />
Hammond, J.A., Fielding, D. and Bishop, S.C. (1997); Prospects for plant anthelmintics in tropical veterinary medicine.<br />
Veterinary Research Communication,21: 213 228.<br />
Harborne, J.B (1998); Phytochemical Methods: A guide to Modern Techniques <strong>of</strong> Plant Analysis. 3rd edition. Chapman<br />
and Hall Ltd, London pp 279.<br />
Hoste, H., Jackson, F., Athanasiadou, S., Thamsborg, S.M. and Hoskin, S.O. (2006); The effects <strong>of</strong> tannin-rich plants on<br />
parasitic nematodes in ruminants. Trends Parasitology,22: 253 261.<br />
Min, B.R., Barry, T.N., Attwood, G.T. and McNabb, W.C. (2003); The effect <strong>of</strong> condensed tannins on the nutrition and<br />
health <strong>of</strong> ruminants fed fresh temperate forages: a review. Animal Feed Science and Technology, 106: 3-19.<br />
Niezen, J.H., Charleston,W.A.G., Robertson, H.A., Shelton, D., Whaghorn, G.C. and Green, R. (2002); The effect <strong>of</strong><br />
feeding sulla (Hedysarum coronarium) or lucerne (Medicago sativa) on lamb parasite burdens and development <strong>of</strong><br />
immunity to gastrointestinal nematodes. Veterinary Parasitology 105: 229 245.<br />
Pasiecznik, Nick, Peter Felker, P.J.C., Harris, L.N. Harsh, G. Cruz, J.C., Tewari, K. Cadoret and L.J. Maldonado (2001); The<br />
Prosopis juliflora-Prosopis pallida complex: A monograph. HDRA, Coventy, UK.<br />
Singh, D., Swarnkar, C.P. and Khan, F.A. (2002); Anthelmintic resistance in Gastrointestinal nematodes in livestock in<br />
India. Journal <strong>of</strong> Veterinary Parasitology 16: 115-130.<br />
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[YS 11] Application <strong>of</strong> Solid Phase Extraction Gas Chromatography Mass<br />
Spectrometry in Geographical Pr<strong>of</strong>iling and Characterization <strong>of</strong> Volatile Organic<br />
Compounds in Kenyan Honey<br />
F. Ng ang a 1 , A. Onditi 1 , A. Gachanja 1 , E. Ngumba 1<br />
1 Department <strong>of</strong> Chemistry, Jomo Kenyatta University <strong>of</strong> Agriculture and Technology, P.O. Box<br />
E-mail: mungaf@yahoo.com<br />
Key Words: Honey, Solid Phase Extraction (SPE); Gas Chromatography-Mass Spectrometry (GC-MS);<br />
Introduction<br />
H<br />
oney as a natural product is greatly appreciated by consumers, not only for its nutritive<br />
properties, but also for its characteristic aroma and sweet taste. Aroma is caused by the<br />
presence <strong>of</strong> many different volatile compounds in it (Soria et al., 2003). The composition and flavor<br />
<strong>of</strong> honey varies, this mainly depends on the source <strong>of</strong> the nectar(s) from which it originates and to a<br />
lesser extent on certain external factors - climatic conditions and beekeeping practices in removing<br />
and extracting honey (White, 1975). A large number <strong>of</strong> organic compounds have been described in<br />
different types <strong>of</strong> honey. Some <strong>of</strong> the compounds have been described as characteristic <strong>of</strong> the floral<br />
source whereas other compounds like some alcohols, branched aldehydes and furan derivatives may<br />
be related to microbial purity or processing and storage <strong>of</strong> honey (Bouseta et al., 1992).<br />
Because <strong>of</strong> the high price <strong>of</strong> certain honey types based on botanical and geographical origin,<br />
adulteration with low cost and nutritional value substances ( Cotte et al., 2007) or mislabeling<br />
regarding the botanical or geographical origin (Alissandrakis et al., 2007a) sometimes occurs.<br />
Nonetheless, the discrimination between different types <strong>of</strong> honey is important for honeys that<br />
possess discrete aroma, taste and special nutritional properties (Lusby et al 2005; Ma�rghitas et al.,<br />
2009) which make them preferable for consumers. Thus, the pr<strong>of</strong>iling and identification <strong>of</strong> Organic<br />
compounds which could be used as markers for the discrimination and classification <strong>of</strong> honey based<br />
on their geographical origin is <strong>of</strong> high importance.<br />
Aroma compounds are present in honey at very low concentrations as complex mixtures <strong>of</strong> volatile<br />
components <strong>of</strong> different functionality and relatively low molecular weight.<br />
Gas Chromatography Mass Spectrometry (GC-MS) is usually the technique <strong>of</strong> choice for the<br />
determination <strong>of</strong> volatile Organic compounds in honey this is due to its high separation efficiency<br />
and sensitivity and also it provides qualitative and quantitative data for these compounds. However,<br />
GC-MS requires the previous removal <strong>of</strong> sugars and water (Soria et al., 2003). Several fractionation<br />
techniques have been employed for the removal <strong>of</strong> sugars and water, they include: Solvent<br />
extraction, simultaneous steam distillation extraction Method, Static headspace analysis, Dynamic<br />
headspace (Purge and Trap), Solid Phase Extraction (SPE) and Solid phase micro extraction (SPME).<br />
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In this study solid-phase extraction followed by gas chromatography-mass spectrometry (GC-MS)<br />
was used to extract and identify volatile organic compounds in honey obtained from various<br />
geographical origin in Kenya. Solid phase Extraction technique was employed as it <strong>of</strong>fers the<br />
advantage <strong>of</strong> eliminating, by washing with water, some interfering substances such as sugars and<br />
acids thus making it possible to obtain the honey volatile fraction without the need <strong>of</strong> applying heat;<br />
however optimization <strong>of</strong> several parameters is necessary before applying this technique (V´azquez,<br />
et al 2006). Extraction conditions were optimized in order to obtain the highest yields <strong>of</strong> volatile<br />
substances.<br />
Material and methods<br />
Study <strong>of</strong> choice <strong>of</strong> eluting solvent<br />
A 30 % honey solution was prepared.10ml <strong>of</strong> these solution was passed through 2 precondition Sep-<br />
Pak (waters) C18 SPEcartridges , each <strong>of</strong> the catridge was washed with 5ml <strong>of</strong> distilled water . One<br />
was eluted with Hexane while the other one with Dichloromethane. Each <strong>of</strong> the eluent was spiked<br />
with 100µl <strong>of</strong> 100ppm Internal Standard (Benzophenone). Both eluent were concentrated to 1ml<br />
and analysed by GC-MS.<br />
Sample throughput (Volume) optimization<br />
5,10 15and 20ml <strong>of</strong> the 30 % honey solution prepared above was passed through a precondition<br />
C18 SPE catridge at a flow rate <strong>of</strong> approximatelty 1ml/min. 5 ml <strong>of</strong> distilled water were run into<br />
each <strong>of</strong> the catridge to wash the sugars. 5ml <strong>of</strong> DCM was used to elute the volatile organic<br />
compounds from each <strong>of</strong> the cartridges at a flow rate <strong>of</strong> 1ml/min. The eluent was spiked with 100µl<br />
<strong>of</strong> 100ppm <strong>of</strong> internal standard. The DCM eluent was further preconcentrated in a vacuum sample<br />
concentrator at 30 o c to a volume <strong>of</strong> 1ml and analyzed by GC-MS.<br />
Honey amount optimization<br />
4 honey solutions i.e 10%, 20%, 30% and 40% honey solutions were prepared, 10 ml <strong>of</strong> each <strong>of</strong> the<br />
solution (the optimum sample volume determined previously) was passed through a preconditioned<br />
C18 SPE catridge. 5 ml <strong>of</strong> distilled water were run into each <strong>of</strong> the catridge. 5ml <strong>of</strong> DCM was used to<br />
elute the volatile organic compounds from each <strong>of</strong> the cartridges at a flow rate <strong>of</strong> 1ml/min. the<br />
eluent was spiked with 100µl <strong>of</strong> 100ppm internal standard. The DCM eluent was further<br />
preconcentrated in a vacuum sample concentrator at 30 o c to a volume <strong>of</strong> 1ml and analysed by GC-<br />
MS.<br />
Blank preparation<br />
10ml <strong>of</strong> water was passed through a preconditioned cartridge at a rate <strong>of</strong> 1ml/min. The cartridge<br />
was eluted with 5ml <strong>of</strong> Dichloromethane. The eluent was further preconcentrated in a vacuum<br />
sample concentrator at 30 o c to a volume <strong>of</strong> 1ml and analyzed by GC-MS.<br />
Analysis <strong>of</strong> Volatile compounds in honey from various geographical regions in Kenya.<br />
20 grams <strong>of</strong> honey obtained were dissolved in 100ml <strong>of</strong> distilled water. 10ml were passed through a<br />
preconditioned cartridge at a rate <strong>of</strong> 1ml/min. the volatile fraction was eluted with 5ml <strong>of</strong><br />
Dichloromethane. The eluent was spiked with 100µl <strong>of</strong> 100ppm internal standard. The DCM eluent<br />
was further preconcentrated in a vacuum sample concentrator at 30 o c to a volume <strong>of</strong> 1ml and<br />
analysed by GC-MS.<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Chromatographic conditions<br />
GC-MS analyses were perfomed in a GC8000 Top series (CE instruments) coupled to a Voyager-<br />
Finnigan quadruople mass spectrometer detector. 1µl <strong>of</strong> each extracts were injected into the<br />
splitless mode in a DB5 (Crosslinked 5% Phenyl-95% Methyl Siloxane) capillary column (30m ×<br />
0.25mm i.d × 0.1µm film thickness) The injection temperature was maintained at 200 0 c, while the<br />
oven temperature was kept at 60 o c for 3min and programmed to rise at 3oc/min to 240 where it<br />
would be held for 5min. Helium was used as the carrier gas at flow rake <strong>of</strong> 1ml/min.Mass spectra<br />
were recorded in the Electron Ionization mode at 70 ev scanning the 40-450m/z range, the ion<br />
source and transfer line temperature were maintained at 200 o c and 250 o c respectively.<br />
Sampling<br />
33 Fresh unprocessed honey samples were obtained from farmers in different parts <strong>of</strong> the country<br />
geographical sampling was employed.<br />
Data analysis<br />
Peak identifications were performed by comparison <strong>of</strong> their mass spectra with spectral data from<br />
the NIST library. Peaks which were present in the blank were not considered. While quantitation was<br />
done by comparing the peak areas <strong>of</strong> individual compounds identified with that <strong>of</strong> the internal<br />
standard used (100µl <strong>of</strong> Benzophenone.)<br />
Results and Discussion<br />
Optimisation studies<br />
In the optimization study, 10 compounds identified to be present in both eluents were used so as to<br />
establish the trend. These 10 compounds identified were further used in subsequent optimization<br />
studies.<br />
It was established that the Dichloromethane eluent was found to have higher concentrations <strong>of</strong> the<br />
Volatile Organics as compared to the Hexane eluent, identified thus it was chosen as the best<br />
solvent for elution during the sample extraction phase with the C18 SPE cartridges as illustrated in<br />
Figure 1.<br />
In the sample optimization stage it was found that the optimum sample throughput volume was<br />
10ml. as shown in figure 2. Lastly in the e honey amount optimization stage, a 20 % honey solution<br />
was found to give the highest concentrations <strong>of</strong> the 10 compounds analyzed in these stage. As<br />
shown in figure 3.<br />
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Fig 1: Comparison <strong>of</strong> the concentration <strong>of</strong> compounds eluted from SPE cartridge by Hexane and<br />
DCM<br />
Fig 2: Comparison <strong>of</strong> concentration <strong>of</strong> compounds eluted with DCM with varying sample volume<br />
Fig 3: Comparison <strong>of</strong> concentration <strong>of</strong> compounds eluted with DCM with varying sample<br />
concentration.<br />
Volatile compounds present in honey from different geographical regions in Kenya.<br />
Chromatographic analysis <strong>of</strong> the extracts obtained by solid phase extraction enabled the<br />
identification <strong>of</strong> 57different compounds from samples collected in Kenya. A typical honey VOC<br />
chromatogram exhibited from 15 to 25 peaks. Differences in chromatographic pr<strong>of</strong>iles were<br />
observed when comparing honey samples from the different geographical origins. The volatile<br />
compounds identified were into 5 groups. Namely terpenes and derivatives, aldehydes, ketones,<br />
carboxylic acids and esters. These was in agreement with reports from researchers in other parts <strong>of</strong><br />
the world who have been pr<strong>of</strong>iling volatile compounds in honey from different floral sources (Soria<br />
et al., 2003, Baroni et. al 2006, Wolski, et al., 2006).<br />
Table 2 shows the pr<strong>of</strong>ile developed form these study.<br />
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Table 2: Concentration in ppm <strong>of</strong> compounds identified from the SPE extract <strong>of</strong> honey obtained<br />
from various geographical regions in Kenya.<br />
Compound Region<br />
Lower eastern Province Upper<br />
Eastern<br />
Province<br />
Carboxylic Acids Mwingi Kitui<br />
224<br />
Central<br />
Riftvalley<br />
Province<br />
South<br />
Riftvalley<br />
Province<br />
Nairobi &<br />
Central<br />
Province<br />
n-hexadecanoic acid 1970.667±351.269 176.67±55.13 35.12±9.85 230.96±11.95 46.71±2.94 556.93±114.89<br />
2-decanynoic acid __ __ __ 53.45±9.98 __<br />
14-methyl<br />
pentadecanoic acid<br />
Trans-e(sup 9)octadecenoic<br />
acid<br />
16.methylheptadecanoic<br />
acid<br />
Ketones and Aldehydes<br />
__ __ __ __ 10<strong>9.1</strong>5±23.45<br />
__ __ 11.78±4.35 __ 29.41±6.78<br />
__ __ __ __ 34.47±8.78<br />
2-pentadecanone __ 66.2±14.56 __ __ _<br />
E-14-Heptadecenal 35±12.34 14.94±2.43 __ 61.23±25.54 32.78±8.89<br />
3-heptadecenal __ __ __ 13.86±4.71 __<br />
Esters<br />
1-Methylethyl ester<br />
tetradecanoic acid<br />
__ 27.34±7.45 __ __ __<br />
Oleic acid,methyl ester __ __ 23.98±6.45 __ __<br />
Palmitic acid,methyl<br />
ester<br />
Stearic acid, methyl<br />
ester<br />
Terpenes and<br />
derivatives<br />
2,6,10,15tetramethylheptadecane<br />
45.62±8.834 __ 23.45±5.45 __ __<br />
__ __ 17.45±4.34 __ __<br />
__ 26.11±3.13 __ 216.98±39.7 __<br />
2,4-dimethyleicosane __ 44.30±7.70 __ __ __<br />
10-MethylEicosane __ __ __ 68.03±13.61 __<br />
n-nonadecane 38.67±6.34 15.65±5.63 __ __ __<br />
7-n-Hexyleicosane __ __ __ __ 12.89±3.56<br />
n-docosane __ __ __ 56.51±9.78 37.06±7.98<br />
Others Mwingi Kitui<br />
Sandoracopimar-15en8á-yl<br />
acetate<br />
2,2'-Methylenebis(4-<br />
Methyl-6-Tert-<br />
Butylphenol)<br />
__ 66.03±15.70 __ __ __ __<br />
582.46±106.98 4793.08±1205.95 __ 769.45±87.98 __ __
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
References<br />
Alissandrakis, E., Tarantilis, P. A., Harizanis, P. C., & Polissiou, M. (2007a); Comparison <strong>of</strong> the volatile composition in<br />
thyme honeys from several origins in Greece. Journal <strong>of</strong> Agricultural and Food Chemistry, 55, 8152 8157.<br />
Arvanitoyannis, I. S., Chalhoub, C., Gotsiou, P., Lydakis-Simantiris, N., & Kefalas, P. (2005); Novel quality control<br />
methods in conjunction with chemometrics (multivariate analysis) for detecting honey authenticity. Critical<br />
Reviews in Food Science and Nutrition, 45, 193 203.<br />
Baroni, M. V., Nores, M. L., Diaz, M. P., Chiabrando, G. A., Fassano, J. P., Costa, C., et al. (2006); Determination <strong>of</strong><br />
volatile organic compound patterns characteristic <strong>of</strong> five unifloral honey by solid-phase microextraction gas<br />
chromatography mass spectrometry coupled to chemometrics. Journal <strong>of</strong> Agricultural and Food Chemistry, 54,<br />
7235 7241.<br />
Bouseta, A., Collin, S. (1995); Optimized Likens-Nickerson Methodology for Quatifying Honey Flavours, Journal <strong>of</strong><br />
Agriculture. Food Chemistry. 43: 1890 1896.<br />
Cotte, J. F., Casabianca, H., Lheritier, J., Perrucchietti, C., Sanglar, C., Waton, H., et al. (2007); Study and validity <strong>of</strong> 13 C<br />
stable carbon isotopic ratio analysis by mass spectrometry and 2H site-specific natural isotopic fractionation by<br />
nuclear magnetic resonance isotopic measurements to characterize and control the authenticity <strong>of</strong> honey.<br />
Analytica Chimica Acta, 582, 125 136.<br />
Lusby, P. E., Coombes, A. L., & Wilkinson, J. M. (2005); Bactericidal activity <strong>of</strong> different honeys against pathogenic<br />
bacteria. Archives <strong>of</strong> Medical Research, 36, 464 467.<br />
Ma�rghitas, L. A., Dezmirean, D., Moise, A., Bobis, O., Laslo, L., & Bogdanov, S. (2009); Physico-chemical and bioactive<br />
properties <strong>of</strong> different floral origin honeys from Romania. Food Chemistry, 112, 863 867.<br />
V´azquez, L.C., D´�az-Maroto M. C., Guchu, E., & P´erez-Coello, M. S. (2006); Analysis <strong>of</strong> volatile compounds <strong>of</strong><br />
eucalyptus honey by solid phase extraction followed by gas chromatography coupled to mass spectrometry.Eur<br />
Food Res Technol 224: 27 31.<br />
White, J. (1975); Honey A Comprehensive Survey, Heinemann, London: 157-206.<br />
Wolski,T., Tambor, K., Rybak-Chmielewska, H., Kêdzia, B. (2006); Identification <strong>of</strong> Honey Volatile Components by Solid<br />
Phase Microextraction (SPME) and Gas chromatography/Mass Spectrometry (GC/MS). Journal <strong>of</strong> Apicultural<br />
Science, 50, (2): 34-49.<br />
225
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[YS 12] EVALUATION OF LARVICIDAL ACTIVITY AND PHYTOEXTRACT INDUCED<br />
MORHOLOGICAL DISRUPTIONS OF VITEX SCHILIEBENII EXTRACTS AGAINST<br />
ANOPHELES GA<strong>MB</strong>IAE LARVAE<br />
Mokua G.N. 1,2 , Innocent E. 1 , Mbwambo Z. 1 , Lwande W. 2 , Ahmed Hassanali. 3 ,<br />
1 Muhimbili University <strong>of</strong> Health and Allied Sciences, Institute <strong>of</strong> Traditional Medicine<br />
2 International Centre <strong>of</strong> Insect Physiology and Ecology, Department <strong>of</strong> Applied Bio-prospecting<br />
3 Kenyatta University, Department <strong>of</strong> Chemistry<br />
Key words: Phytochemicals, larvicidal, Vitex schiliebenii, Verbenaceae, Anopheles gambiae.<br />
Introduction<br />
M<br />
ost synthetic larvicides act on target and non-target organisms therefore representing a<br />
danger to beneficial insects, wildlife and human beings. Consequently, environmentally safe<br />
methods must be found to enhance or minimize the use <strong>of</strong> conventional chemical insecticides.<br />
Some plants are known to contain toxic principles that are useful in the control <strong>of</strong> vectors. Such<br />
plants exhibit insecticidal and biological activities. Botanical insecticides may provide a safe and<br />
effective short-term strategy for larval and adult mosquito control. Mosquito responses to<br />
phytochemicals from different plants or parts vary and have been studied. Among the plant families<br />
studied include Meliaceae, Rutaceae, Labiatae, Piperaceae, Verbenaceae, Asteraceae,<br />
Cladophoraceae, Oocystaceae, and Annonaceae and perhaps are the most promising (Akhatar &<br />
Isman, 2004).<br />
The genus Vitex in the family Verbenaceae and its species consists <strong>of</strong> shrubs or trees found mainly<br />
in tropical and sub-tropical regions although a few species may be found in temperate zones<br />
(Mokua et al., 2008). In Kenya, there are 12 different species <strong>of</strong> the genera Vitex found naturally<br />
from the Kenyan Coast through the dry woodlands to Mt. Kenya area and across the Rift Valley to<br />
the shores <strong>of</strong> Lake Victoria. (Kimondo et al., 2010). Vitex species besides their popular use as<br />
traditional medicines in many countries have been reported to exhibit insecticidal activities against<br />
a variety <strong>of</strong> insects (Karunamoorthi et al 2008; Yuan et al., 2006; Rodríguez-Lopez et al., 2007;<br />
Kannathasan et al., 2007; Rahman& Talukder. 2006).<br />
In this study, Anopheles gambiae were investigated using Vitex schiliebenii (Verbenaceae) a<br />
branched shrub with multiple stems and low height (4-8 m). The leaves have 3-leaflets with a<br />
smooth surface, about 10-12 cm long. In Kenya, it grows in the coastal region at Watamu which is a<br />
low and semi-arid land. The present investigation assessed the laboratory larvicidal effect <strong>of</strong> V.<br />
schiliebenii polar extracts against 3 rd and 4 th instar larvae <strong>of</strong> An. gambiae s.s. This is the first report<br />
on the evaluation <strong>of</strong> the acetone extracts <strong>of</strong> Vitex schiliebenii against An. gambiae through larvicidal<br />
bioassays.<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Materials and methods<br />
Plant materials<br />
The plant species was authenticated at the field by a botanist at the National Museum <strong>of</strong> Kenya<br />
(NMK). The leaf, stem and root barks <strong>of</strong> V. schiliebenii were collected from North Coast at Kenya<br />
Forest Research Institute (KEFRI) near Gede along Mombasa-Malindi road 18 km from Malindi<br />
town. A voucher specimen Ref. Nos. GMN/22 was deposited at the NMK herbarium. The plant<br />
materials were dried at room temperature (29 o C) for three weeks, ground into powder using an<br />
electric miller, from which the extracts were prepared.<br />
Acetone and methanol extracts <strong>of</strong> the plant materials were obtained sequentially by soaking 200 g,<br />
400 g and 800 g <strong>of</strong> the dried root bark, leaf and stem bark respectively in 1.0 L, 2.0 L and 4.0 L<br />
acetone in separate containers with occasional stirring. The mixture was kept for 24 h then filtered<br />
under gravity and concentrated to dryness using a rotary evaporator while maintaining the water<br />
temperature at 40 o C in order to avoid decomposition <strong>of</strong> thermally labile compounds. This<br />
procedure was repeated three times and the filtrates were then pooled and stored at 4 o C.<br />
Methanol extract <strong>of</strong> the plant material was prepared in a similar manner with that <strong>of</strong> acetone.<br />
Mosquito species<br />
The eggs <strong>of</strong> An. gambiae were procured from the research insectary at the International Center <strong>of</strong><br />
Insect Physiology and Ecology (ICIPE), Nairobi. The Kenya highland strain <strong>of</strong> An. gambiae s.s<br />
originated from ICIPE s Mbita Point Field Station in 2003 was used because it is the most efficient<br />
vector due to its high anthropophilic character. It is the one which transmits malaria mainly in the<br />
Sub-Saharan Africa. The eggs were obtained as rafts on a filter paper and kept in plastic tray<br />
containing distilled water at a temperature <strong>of</strong> 28±2 o C as culture medium and at laboratory<br />
conditions 30±2 o C. The larvae were fed on Tetramin ® fish food (Terta GmbH, Germany). The water<br />
temperature was maintained at 28±2 o C throughout the larval development period. The larvae at<br />
third and early fourth instar stages were used for larvicidal assay.<br />
Laboratory larvicidal assay<br />
Bioassays were conducted following the standard World Health Organization (WHO, 1981) larval<br />
susceptibility test method at ICIPE. The extracts were dissolved in 2 ml dimethyl sulfoxide and<br />
prepared into different concentrations viz 25, 50, 100, 250 and 500 ppm with distilled water.<br />
Twenty freshly molted late 3 rd and early 4 th instar larvae <strong>of</strong> Anopheles gambiae were tested in three<br />
replicates with two controls running simultaneously. During the experiment, the larvae were fed on<br />
Tetramin ® fish food (Terta GmbH, Germany) at about 1mg per beaker every 24h. The experiment<br />
room was kept at a temperature <strong>of</strong> 30 o C and an average humidity <strong>of</strong> 48 % and a photo period <strong>of</strong> 12<br />
hours <strong>of</strong> light and 12 hours <strong>of</strong> darkness. The lethal concentrations LC50, LC75 and LC90 were<br />
calculated using GenStat teaching edition s<strong>of</strong>t ware.<br />
Phytochemical screening<br />
Phytochemical tests were carried out on the acetone and methanol extracts using standard<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
procedures to identify the constituents as described by (Egwaikhide & Gumba, 2007) with some<br />
modifications.<br />
Results and discussion<br />
In this study, larval mortality <strong>of</strong> An. gambiae 3 rd and 4 th instar larvae under laboratory conditions<br />
were investigated after 24h, 48h, and 72h treatment with the extracts. The highest larval mortality<br />
was observed in the acetone leaf extract (VSL-221) with LC50 = 14.6 ppm followed by acetone stem<br />
bark (VSSB-221) extract (LC50 =17.4 ppm), methanol leaf (VSL-222) extract (LC50 = 136.3 ppm) and<br />
acetone root bark (VSRB-221) extract (LC50 = 252.1 ppm). Methanol root bark (VSRB-222) and stem<br />
bark (VSSB-222) extracts exhibited moderate larvicidal activity with LC50 = 444 and 522.6 ppm<br />
respectively (Table 2). In the case <strong>of</strong> control, no morality was observed within 24-h and the larvae<br />
developed<br />
Table 2: Bioefficacy <strong>of</strong> crude extracts <strong>of</strong> Vitex schiliebenii on 3 rd and early 4 th instar larvae <strong>of</strong><br />
Anopheles gambiae s.s after 24h, 48h and 72h <strong>of</strong> exposure<br />
Extract code Time<br />
(Hr)<br />
VSSB-221<br />
VSSB-222<br />
VSRB-221<br />
VSRB-222<br />
VSL-221<br />
VSL-222<br />
24<br />
48<br />
72<br />
24<br />
48<br />
72<br />
24<br />
48<br />
72<br />
24<br />
48<br />
72<br />
24<br />
48<br />
72<br />
24<br />
48<br />
72<br />
LC50<br />
16.3<br />
14.7<br />
12.4<br />
540.7<br />
104.3<br />
40.1<br />
251.1<br />
136.3<br />
38.7<br />
444.4<br />
295.9<br />
80.6<br />
13.5<br />
12.7<br />
11.1<br />
97.1<br />
63.1<br />
40.1<br />
95% CL<br />
13.6-19.3<br />
12.0-17.8<br />
9.8-15.5<br />
472.4-621.7<br />
92.2-118.1<br />
35.6-45.2<br />
222.6-283.0<br />
120.2-154.7<br />
34.3-43.6<br />
392.0-505.0<br />
260.8-335.7<br />
71.6-90.9<br />
10.9-16.6<br />
10.1-15.8<br />
8.5-14.39<br />
86.3-109.4<br />
56.0-71.1<br />
35.6-45.2<br />
228<br />
Lethal concentration values (ppm)<br />
LC75<br />
22.9<br />
21.2<br />
17.7<br />
758.9<br />
150.8<br />
57.5<br />
352.4<br />
197.1<br />
55.4<br />
623.7<br />
427.8<br />
115.4<br />
18.9<br />
18.4<br />
15.9<br />
136.3<br />
91.2<br />
57.4<br />
95% CL<br />
19.3-27.0<br />
17.5-25.6<br />
14.1-22.1<br />
65<strong>9.1</strong>-882.2<br />
133.0-171.6<br />
51.0-65.0<br />
312.3-399.4<br />
173.6-224.7<br />
4<strong>9.1</strong>-62.6<br />
547.9-715.5<br />
376.7-487.6<br />
102.2-130.9<br />
15.4-23.1<br />
14.7-22.8<br />
12.3-20.<br />
120.8-154.8<br />
80.8-103.3<br />
51.0-64.9<br />
LC90<br />
31.0<br />
29.6<br />
24.5<br />
1029.6<br />
210.1<br />
79.4<br />
478.0<br />
274.7<br />
76.5<br />
846.1<br />
596.1<br />
159.4<br />
25.7<br />
25.6<br />
21.9<br />
185.0<br />
127.1<br />
79.3<br />
95% CL<br />
26.2-36.8<br />
24.5-35.7<br />
19.6-30.6<br />
885.6-1214.2<br />
184.3-241.4<br />
70.1-90.5<br />
421.1-547.7<br />
240.7-315.9<br />
67.6-87.2<br />
736.8-983.9<br />
522.2-685.4<br />
140.3-182.5<br />
21.0-31.4<br />
20.6-31.8<br />
17.0-28.2<br />
162.7-212.6<br />
112.0-145.3<br />
70.0-90.4
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
VSRB-221- Acetone root bark extract <strong>of</strong> V.schiliebenii, VSRB-222-Methanol root bark <strong>of</strong><br />
V.schiliebenii VSSB-221- Acetone stem bark extract <strong>of</strong> V.schiliebenii, VSSB-222- Methanol root bark<br />
extract <strong>of</strong> V.schiliebenii, VSL-221-Acetone leaf extract <strong>of</strong> V.schiliebenii, VSL-222-Methanol leaf<br />
extract <strong>of</strong> V.schiliebenii<br />
The present findings are comparable with the methanol leaf extracts <strong>of</strong> V. negundo, V. trifolia, V.<br />
peduncularis and V. altissima against the early 4 th instar larvae <strong>of</strong> Culex quinquefasciatus with LC50<br />
212.57, 41.41, 76.28 and 128.04 respectively and the petroleum ether (60-80 o C) extracts <strong>of</strong> the<br />
leaves <strong>of</strong> V. negundo against larval stages <strong>of</strong> Culex tritaeniorhynchus in the laboratory (Kannathasan<br />
et al., 2007, Karunamoorthi et al., 2008).<br />
The extracts showed morphological deformation disruptions, prolongation <strong>of</strong> developmental period<br />
and highly significant reduction in adult emergence to the test larvae. The pupal-adult dead<br />
intermediates had question mark-like remains and the live larvae after 24-h could not move deep<br />
into the water.<br />
Dead deformed larva Pupal-adult dead<br />
intermediates<br />
Although there has been no phytochemical investigation <strong>of</strong> the polar extracts <strong>of</strong> V. schiliebenii<br />
reported, the plant has shown larvicidal activity against Anopheles gambiae larvae s.s in the current<br />
study. Phytochemical investigation <strong>of</strong> the extracts revealed the presence <strong>of</strong> flavonoids, terpenoids,<br />
steroids, alkaloids, saponins and tannins. The biological activity <strong>of</strong> the evaluated phytoextracts<br />
could therefore be attributed to the presence <strong>of</strong> these compounds which could synergistically,<br />
antagonistically or independently contribute to the activity <strong>of</strong> the crude extracts. Elango et al.<br />
(2010) reported the presence <strong>of</strong> these compounds in leaf extracts <strong>of</strong> four Andrographis species<br />
which exhibited adulticidal activity and adult emergence inhibition (EI) against a malarial vector<br />
Anopheles subpictus Grassi.<br />
Semi field experiments are in progress to enable the technology to be used in small water bodies<br />
around the homesteads. In addition, isolation, purification, characterization and bioassay <strong>of</strong> the<br />
pure compounds independently and in blends are in progress.<br />
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Acknowledgement<br />
The authors are grateful to Natural Product Research Network for Eastern and Central Africa<br />
(NAPRECA-DAAD) for financial assistance and the International Centre for Insect Physiology and<br />
Ecology (ICIPE) for providing the insects and necessary facilities to carry out this research work<br />
successfully. We also thank Mr. Simon Mathenge for identifying the plants under the study.<br />
Acknowledgement is extended to Muhimbili University <strong>of</strong> Health and Allied Sciences (MUHAS),<br />
Institute <strong>of</strong> Traditional Medicine (ITM) for its financial support in publishing this paper.<br />
References<br />
Akhatar Y. & Isman M.B. (2004); Comparative Growth <strong>of</strong> Inhibitory and Anti-feedant Effects <strong>of</strong> Plant Extracts and Pure<br />
Allelochemicals on Four Phytophagus Insect Species. J. Appl. Entomol. 128, 32-38.<br />
Egwaikhide P.A. & Gumba C.E. (2007); Analysis <strong>of</strong> the Phytochemical and Anti-microbial Activity <strong>of</strong> Plectranthus<br />
glandulosis Whole Plant Middle-East J. <strong>of</strong> Scientific Res. 2 (3-4), 135-138.<br />
Elango G., Rahuman A.A.m Kamaraj C., Bagavan A. &Zahir A.A. (2010); Efficacy <strong>of</strong> Medicinal Plant Extracts Against<br />
Malarial Vector, Anopheles subpictus Grassi. Parasitol Res.<br />
Kannathasan K., Senthilkumar A., Venkatesalu V. & Chandrasekaran M. (2008); Larvicidal Activity <strong>of</strong> Fatty Acid Methyl<br />
Esters <strong>of</strong> Vitex Species Against Culex quinquefasciatus. Parasitol. Res. 103 (4), 999-1001.<br />
Karunamoorthi K., Ramanujam S. & Rathinasamy R. (2008); Evaluation Of Leaf Extracts <strong>of</strong> Vitex Negundo L. (Family:<br />
Verbenaceae) Against Culex trataeniorhynchus and Repellent Activity on Adult Vector Mosquito. Parasitol. Res. 103<br />
(3): 545-550.<br />
Kimondo J.M., Agea J.G., Okia C.A., Abohassan R.A.A., Mulatya J. & Teklehaimanot Z. (2010); Vitex payos (Lour.) Merr.<br />
Fruit Trees in Dry Land Areas <strong>of</strong> Eastern Kenya: Use, Marketing and Management.<br />
Kuppusamy C., Murugan K., Arul N. & Yasodha P. (2009); Larvicidal and Insect Growth Regulatory Effect <strong>of</strong> -amyrin<br />
acetate from Catharanthus roseus Linn against the malaria vector An. stephensis Liston (Diptera:Culicidae). J.<br />
Entomological Res. 39, 78-83.<br />
Mokua G.N., Ndiege I.O., Hassanali A. & Tarus P.K. (2008); Anti-larval Activity <strong>of</strong> Crude Plant Extracts from Vitex payos<br />
and Vitex schilebenii. Aspects <strong>of</strong> African Biodiversity. In the Proceedings <strong>of</strong> the Pan African Chemistry Network<br />
Biodiversity Conference. 29-34.<br />
Rahman A. & Talukder F.A. (2006); Bio-Efficacy <strong>of</strong> Some Plant Derivative that Protect Grains against the Pulse Beetle,<br />
Callosobruchus maculates. J. Insect. Sci. 6 (3), 1-10.<br />
Rodríguez-Lopéz V., Figueroa-Suarez M.Z., Rodríguez T. & Aranda E. (2007); Insecticidal Activity <strong>of</strong> Vitex mollis.<br />
Fitoterapia, 78, 37-39.<br />
World Health Organization (1981); Instruction for Determining the Susceptibility or Resistance <strong>of</strong> Mosquito Larvae to<br />
Insecticides. WHO-VBC 81,807, 1-6<br />
Yuan L., Xue M., Liu Y., Wang H. (2006); Toxicity and Oviposition Deterrent <strong>of</strong> Vitex negundo Extracts to Plutella<br />
xylostella. Yingyong Shengtai Xuebao 1714, 695-698.<br />
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[YS 13] The Relative Stabilities and Reactivities <strong>of</strong> the First Six Members <strong>of</strong> the<br />
Dendralene Family<br />
Gomotsang Bojase-Moleta , Alan D. Payne and Michael S. Sherburn<br />
Department <strong>of</strong> Chemistry, University <strong>of</strong> Botswana, Private Bag 00704, Gaborone, Botswana.<br />
Research School <strong>of</strong> Chemistry, Australian National University, ACT 0200<br />
Introduction<br />
ith oligoalkene structures, four fundamental hydrocarbon families can be defined (Figure 1),<br />
with each differing in the type <strong>of</strong> atom connectivity (unbranched or branched; cyclic or<br />
acyclic). 1-5 W<br />
acyclic, unbranched acyclic, branched<br />
linear polyenes<br />
dendralenes<br />
cyclic, unbranched<br />
annulenes<br />
231<br />
cyclic, branched<br />
radialenes<br />
Figure 1 Fundamental classes <strong>of</strong> conjugated alkenic hydrocarbons.<br />
The unbranched acyclic and cyclic systems, namely the linear polyenes and annulenes, respectively,<br />
have been thoroughly studied. The alternation in behaviour <strong>of</strong> the annulenes (i.e. aromaticity and<br />
antiaromaticity for odd and even numbers <strong>of</strong> conjugated alkenes) played an important role in the<br />
development <strong>of</strong> modern theories <strong>of</strong> structure and reactivity. 3 On the other hand, the acyclic and<br />
cyclic branched systems, that is, the dendralenes and radialenes, are much less well investigated.<br />
The cross conjugated polyenes known as dendralenes are fascinating compounds with enormous<br />
untapped potential in chemical synthesis. 1-5 These hydrocarbons have remained unexplored<br />
because <strong>of</strong> limited accessibility and their reported instability. Contrary to earlier assertions that<br />
they are too prone to polymerisation to be synthetically useful, we have seen that these<br />
compounds are readily made and stored. 6-8 In this paper, it is demonstrated that these fundamental<br />
hydrocarbons exhibit alternation in their physical and chemical properties. Evidence is provided<br />
that this alternating behaviour stems from the conformational preferences <strong>of</strong> dendralenes with<br />
even- and odd numbers <strong>of</strong> alkene units. 9<br />
Materials and Methods<br />
NMR spectra were recorded at 298K using a Varian Unity INOVA 500 MHz or a Varian Unity INOVA<br />
300 MHz spectrometer. Residual chlor<strong>of</strong>orm ( 7.26 ppm) was used as an internal reference for 1 H<br />
NMR spectra measured in this solvent. Coupling constants (J) are quoted to the nearest 0.1 Hz.
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Residual chlor<strong>of</strong>orm ( 77.1 ppm) was used as an internal reference for 13 C NMR spectra.<br />
Assignment <strong>of</strong> carbon signals was assisted by DEPT experiments. IR spectra were recorded on a<br />
Perkin-Elmer 1600 F.T.I.R, spectrometer as neat films on NaCl plates for oils. Low resolution mass<br />
spectra were recorded on a Finnigan PolarisQ ion trap mass spectrometer using electron impact<br />
(EI + ) ionisation mode at 40 or 70 eV. High resolution mass spectra were recorded on a VG Autospec<br />
mass spectrometer operating at 70 eV. Analytical TLC was performed with Merck silica gel plates,<br />
precoated with silica gel 60 F254 (0.2 mm). Flash chromatography employed Merck Kiesegel 60<br />
(230-400 mesh) silica gel. Reactions were conducted under a positive pressure <strong>of</strong> dry argon or<br />
nitrogen in oven-dried glassware. Ether and THF were dried over sodium wire and distilled from<br />
sodium benzophenone ketyl before use. Dichloromethane was distilled from calcium hydride.<br />
Commercially available chemicals were purified by standard procedures or used as purchased.<br />
Results and Discussion<br />
During synthesis <strong>of</strong> the first six members <strong>of</strong> the dendralene family it was observed that some<br />
members were more stable than others. This observation led to investigations into the physical and<br />
chemical properties <strong>of</strong> the first six members <strong>of</strong> the dendralene family.<br />
It was established from the stability studies on the first six members <strong>of</strong> this class <strong>of</strong> hydrocarbons<br />
that the even numbered dendralenes are more stable than the odd numbered dendralenes. The<br />
major pathway <strong>of</strong> decomposition <strong>of</strong> these hydrocarbons is via Diels Alder dimerisation (Scheme 1).<br />
[n]dendralene heat<br />
Diels-Alder dimers<br />
Scheme 1<br />
For example, [5]dendralene (1) underwent DA dimerisation to give dimer which underwent further<br />
6 -electrocyclisation/[4+2] cycloaddition cascade to give a tetracyclic fenestrane (2) (Scheme 2)<br />
upon refluxing in chlorobenzene.<br />
2<br />
1<br />
1. [4+2] cycloaddition<br />
2. 6 -electrocyclization<br />
3. [4+2] cycloaddition<br />
232<br />
2<br />
X-ray<br />
Scheme 2 Intermolecular [4+2]/6 -electrocyclisation/intramolecular [4+2] cascade for [5]dendralene (7).<br />
Reaction conditions: (i) 5.0M 1.7, CH2Cl2, 72h, 16%, (ii) PhCl, BHT, reflux, 24h, 80%.<br />
Following work on the stabilities <strong>of</strong> these hydrocarbons the relative reactivities <strong>of</strong> the<br />
[n]dendralenes were also investigated. Thus, the Diels Alder reactivity <strong>of</strong> the dendralenes towards<br />
the electron deficient dienophile N-methylmaleimide (NMM) was examined. Odd numbered<br />
dendralenes underwent a rapid and clean conversion to (predominantly) the corresponding
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
terminal mono adduct at ambient temperature, even in the presence <strong>of</strong> a small excess <strong>of</strong> the<br />
dienophile (Scheme 3). In contrast even numbered dendralenes exhibit significantly lower reactivity<br />
towards NMM, producing mixtures <strong>of</strong> starting dendralenes, mono and bis adducts. Thus, there is<br />
clear difference in chemical reactivity between the odd and even dendralenes.<br />
odd dendralenes<br />
even dendralenes<br />
O<br />
O<br />
N<br />
Me<br />
N<br />
Me<br />
O<br />
( 1 mol equiv)<br />
233<br />
terminal mono adducts<br />
O<br />
( 1 mol equiv) mono adducts + bis adducts<br />
+ unreacted dendralene<br />
Scheme 3<br />
As is the case with the annulenes, the dendralenes exhibit alternating behaviour, with the physical<br />
and chemical properties <strong>of</strong> even members <strong>of</strong> the family being distinctly different from odd<br />
members. This alternating behaviour has been traced to conformational preferences in the<br />
dendralenes.<br />
Acknowledgements<br />
The authors gratefully acknowledge the Australian Research Council and The University <strong>of</strong> Botswana<br />
(scholarship for G. B.) for funding.<br />
References<br />
[1] M. Gholami, R. R. Tykwinski, Chem. Rev. 2006, 106, 4997 5027.<br />
[2] H. Hopf, in Organic Synthesis Highlights V (Eds: H.-G. Schmalz, T. Wirth), Wiley-VCH, Weinheim, 2003; pp. 419<br />
427.<br />
[3] H. Hopf, Classics in Hydrocarbon Chemistry: Syntheses, Concepts, Perspectives, Wiley-VCH, Weinheim, 2000; H.<br />
Hopf, G. Maas, Angew. Chem. 1992, 104, 953 977; Angew. Chem. Int. Ed.Engl. 1992, 31, 931 953; G. Maas, H.<br />
Hopf in Chemistry <strong>of</strong> Functional Groups: Dienes and Poplyenes, Vol. (Ed.: Z. Rappoport). Wiley, Chichester, 1997,<br />
pp. 927-977.<br />
[4] H. Hopf, Angew. Chem. 2001, 113, 727 729; Angew. Chem. Int. Ed. 2001, 40, 705 707.<br />
[5] H. Hopf, Angew. Chem. 1984, 96, 947 959; Angew. Chem. Int. Ed. Engl. 1984, 23, 948 960.<br />
[6] S. Fielder, D. D. Rowan, M. S. Sherburn, Angew. Chem. 2000, 112, 4501 4503; Angew. Chem. Int. Ed. 2000, 39,<br />
4331 4333.<br />
[7] A. D. Payne, A. C. Willis, M. S. Sherburn, J. Am. Chem. Soc. 2005, 127, 12188 12189.<br />
[8] G. Bojase, A. D. Payne, A. C. Willis, M. S. Sherburn, Angew. Chem. Int. Ed. 2008, 47, 910-912.<br />
[9] Payne, A. D.; Bojase, G.; Paddon-Row, M. N.; Sherburn, M. S. Angew. Chem. Int. Ed. 2009, 48, 4836-4839
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[YS 14] Fumigant and Contact Toxicity <strong>of</strong> Cupressus lusitanica and Eucalyptus<br />
saligna Essential Oils Against Insect Pests <strong>of</strong> Stored Cereals and Legumes<br />
A.L. Deng 1 , J.O. Ogendo 2 , P.K. Bett 1 , M. Kamatenesi-Mughisha 3 and J.M. Mihale 4<br />
1 Dept. <strong>of</strong> Biological Sciences, Egerton University, P.O. Box 536 Egerton, Kenya.<br />
2 Dept. <strong>of</strong> Crops, Horticulture and Soils, Egerton University, P.O. Box 536 Egerton, Kenya.<br />
3 Makerere University, P.O. Box 7062 Kampala, Uganda<br />
4 Open University <strong>of</strong> Tanzania, P.O. Box 31608 Dar es salaam, Tanzania.<br />
Corresponding author e-mail: pkkbett@yahoo.co.uk<br />
Key words: Botanical, extract, concentration, Callosobruchus chinensis, Tribolium castaneum, mortality<br />
Introduction<br />
I<br />
nsects cause substantial quantitative and qualitative pre- and post harvest losses varying in<br />
magnitude from 10 to 100% in tropical countries and in Kenya, 10-60 % losses <strong>of</strong> stored cereal<br />
and legume grains. These substantial losses are caused by Sitophilus spp., Sitotroga cerealella and<br />
Prostephanus truncatus on cereals, Acanthoscelides and Callosobruchus spp. on legumes (Mugisha-<br />
Kamatenesi et al., 2008). Current recommended control measures for insect pests rely on synthetic<br />
insecticides which pose health and environmental hazards. Research focus has now shifted to<br />
botanical pesticides, which are target-specific, relatively safe, affordable and readily available. The<br />
insecticidal activity <strong>of</strong> several plant essential oils, powders and other extracts have been evaluated<br />
against several insect pests <strong>of</strong> cereals and legumes and found to have contact toxic (Asawalam et<br />
al., 2006), repellence (Ogendo et al., 2008), fumigant toxicity and antifeedant (Rosman et al., 2007)<br />
effects. In the current study fumigant and contact toxicity <strong>of</strong> essential oils obtained from aerial<br />
parts <strong>of</strong> C. lusitanica and E. saligna were evaluated against C. chinensis and T. castaneum.<br />
Materials and Methods<br />
Essential oils extracted by hydro distillation using a modified clavenger apparatus. Experiments<br />
were carried out under controlled temperatures (28±2 o C) and relative humidity (65±5%) and laid<br />
out in completely randomized design with four replicates. The fumigant toxicity test was carried<br />
out in space fumigation chambers consisting <strong>of</strong> a 3.4L flask and suspended metallic cages carrying<br />
20 adult insects and food. Essential oils were separately applied to provide dosages <strong>of</strong> 0, 5, 10 and<br />
20 µl/L air on filter papers and suspended in the fumigation chamber. Insect mortality was recorded<br />
24, 72, 120 and 168 h post fumigation. Contact toxicity was evaluated in 100ml glass jars,<br />
containing 10-20g <strong>of</strong> grain depending on size <strong>of</strong> grain. Each test oil was separately applied to<br />
provide concentrations <strong>of</strong> 0, 0.05, 0.10,0.15 and 0.20 %w/w. Number <strong>of</strong> dead insects were recorded<br />
1, 3, 5, 7 and 10 days after treatment.<br />
Results and Discussion<br />
Results <strong>of</strong> fumigant toxicity bioassay reveal that C. lusitanica, and E. saligna plant parts(leaves,<br />
flowers and fruits) essential oils at the end <strong>of</strong> 168h was significantly(p
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
interactions. At the highest concentration (20µl/L) percent mortality for leaves, flowers and fruits<br />
was 75%, 92.5% and 32.5% for C. lusitanica (Fig.2a) and 60%, 20% and 22.5 % for E. saligna<br />
respectively (Fig.2b). Similarly analyzed results <strong>of</strong> leaf essential oils extracted from test plants and<br />
tested against T. castaneum were significantly (P
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Fig.2: Percent mortality (Mean±SD; n=4) <strong>of</strong> adult T. castaneum after 168 h exposure to C.<br />
lusitanica and E. saligna essential oils in space fumigation chambers.<br />
The observed differentail responses by test insect species to C. Lusitanica and E saligna essentail<br />
oils could be explained by individual/or synergistic fumigant and contact toxicity <strong>of</strong> their chemical<br />
constituents. Similarly, the toxicity differences <strong>of</strong> leaf, flower, and fruit essentail oils may be<br />
attributed to the existing intra-species variations in the quantitative chemical compositions. Results<br />
<strong>of</strong> this study point E. saligna, and C. lusitanic, essential oils as candidate substances for further<br />
bioactivity studies to determine their individual and combined effects on more insect pests as<br />
fumigants and possible integration into pest management options in subsistence agriculture.<br />
(a) C. chinensis<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
(b) T. castaneum<br />
Fig.3: Percent mortality (Mean±SD, n=4) <strong>of</strong> adult (a) C. chinensis and (b) T. castaneum after 10<br />
days contact with C. lusitanica and E. saligna essential oils<br />
Acknowledgements<br />
Authors are indebted to Inter-Universities Council <strong>of</strong> East Africa(IUCEA) thro VicRes, Egerton<br />
University and colleagues for financial, material and moral support.<br />
References<br />
Asawalam, E.F., Emosairue, S.O., Hassanali, A. (2006); Bioactivity <strong>of</strong> Xylopia aetiopica (Dunal) A. rich essential oil<br />
constituents on maize weevil Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae). Electronic J. Environ.,<br />
Agric. and Food Chem. 5(1): 1195-1204.<br />
Mugisha-Kamatenesi M., Deng A.L., Ogendo J.O., Omolo E.O., Mihale M.J, Otim, M., Buyungo, J.P. and Bett P.K. (2008);<br />
Indigenous knowledge <strong>of</strong> field insect pests and their management around Lake Victoria basin in Uganda. African<br />
Journal <strong>of</strong> Environmental Science and Technology. 2(8) 342-348.<br />
Ogendo, J.O., Kostyukovsky, M., Ravid, U., Matasyoh, J.C., Deng, A.L., Omolo, E.O., Kariuki, S.T., Shaaya, E. (2008);<br />
Bioactivity <strong>of</strong> Ocimum gratissimum oil and two constituents against five insect pests attacking stored food<br />
products. Journal .<strong>of</strong> Stored Products Research 44: 328-334.<br />
Rajendran S. and Sriranjini V. (2008);. Plant Products as Fumigants for Stored-Product Insect Control (Review). Journal<br />
<strong>of</strong> Stored Products Research 44: 126 135.<br />
Rosman, V.; Kalinovic, I & Korunic Z. (2007); Toxicity <strong>of</strong> naturally occurring compounds <strong>of</strong> Lamiaceae and Lauraceae to<br />
three stored-product insects. Journal <strong>of</strong> Stored Products Research 43: 349-355.<br />
237
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[YS 15] Rhuschalcone VI: Synthesis, Re-Isolation and Bioactivities in its<br />
Analogues<br />
Mihigo, S.O. 1 , Mammo, W. 2 , Bezabih, M. 1 , Marobela, A-K. 3 , Abegaz, B.M. 1<br />
1 Department <strong>of</strong> Chemistry, University <strong>of</strong> Botswana, P. Bag 00704, Gaborone, Botswana, Email: smihigo@yahoo.com;<br />
2 Department <strong>of</strong> Chemistry, Addis-Ababa University, PO Box 1176, Addis-Ababa, Ethiopia; 3 Department <strong>of</strong> Biological<br />
Sciences, University <strong>of</strong> Botswana, P. Bag 00704, Gaborone, Botswana.<br />
Key words: Suzuki-Miyaura reaction, Bichalcones, Rhuschalcones, Antiprotozoal activities, Bodo caudatus.<br />
Introduction<br />
R<br />
hus pyroides Burch. (Anacardiaceae) is a shrub to a medium-sized tree which is widely<br />
distributed in the eastern parts <strong>of</strong> Botswana, and South Africa where it is used against epilepsy<br />
in traditional medicine. Previous reports dealing with phytochemical investigations on R. pyroides<br />
indicate the isolation <strong>of</strong> a number <strong>of</strong> compounds belonging to various chemical classes and <strong>of</strong><br />
interesting biological activities, including Rhus bichalcones I-VI, but in small quantities. To date, as<br />
long as we are aware, rhuschalcone VI (1) is the first and unique example <strong>of</strong> a natural dimer in<br />
which two chalcones are linked by a C C bond. Furthermore, it is remarkable that whereas (a) the<br />
total syntheses <strong>of</strong> the bi-aryl ether-type bichalcones (Rhus bichalcones and verbenachalcone) have<br />
been reported by Mdee et al. (2003) employing a novel application <strong>of</strong> the microwave assisted<br />
Ullmann synthesis, and Xing et al. (2002) employing catalytic copper-mediated oxidation coupling<br />
and the Weinreb ketone synthesis as key steps, respectively; and (b) a number <strong>of</strong> flavonoids have<br />
been employed in Suzuki reactions (Parry et al., 2002), the use <strong>of</strong> chalcones and the synthesis <strong>of</strong><br />
any bi-aryl type rhus bichalcones have not yet been reported. In order to provide sufficient<br />
quantities <strong>of</strong> material for more complete biological studies, as well as a general route for the<br />
preparation <strong>of</strong> rhuschalcone VI and its structural analogues, we undertook and successfully<br />
achieved both the first-time total syntheses <strong>of</strong> rhuschalcone VI and analogues and the use <strong>of</strong><br />
Suzuki-Miyaura reaction for the synthesis <strong>of</strong> AB C-C linked bichalcones.<br />
The synthesis has been approached by constructing chalcone halides containing functionalities at<br />
using transition metal (Pd) catalysis. Within<br />
this context, a total <strong>of</strong> fourteen (14) bromochalcones, <strong>of</strong> which 13 are new compounds, have been<br />
synthesized using a solvent-free methodology (Toda et al., 1990). In addition, the first total<br />
syntheses <strong>of</strong> eight rhuschalcone VI-type bichalcones were achieved, indicating that the general<br />
methodology developed by our group is <strong>of</strong> practical use in the syntheses <strong>of</strong> more congeners<br />
carrying the same carbon-framework and the creation <strong>of</strong> biologically more potent substances.<br />
Material and Methods<br />
a) General: Commercially available reagents were used without further purification. Most<br />
solvents were purified by simple distillation, apart from THF which was distilled from<br />
sodium-benzophenone under nitrogen, immediately before use.<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
b) Chromatographic separations: Analytical thin layer chromatography was carried out using<br />
aluminium or glass-backed plates coated with Merck Kieselgel 60 GF254. Developed plates<br />
were visualized under ultra-violet light (254 nm) and/or sprayed with vanillin-sulphuric acid.<br />
Column chromatography was conducted on columns <strong>of</strong> different sizes using silica gel 60,<br />
particle size 0.040-0.063 mm, or Sephadex LH-20 (Merck). Fully characterized compounds<br />
were chromatographically homogeneous.<br />
c) Physical and spectroscopic measurements: Melting points were determined using a Stuart ®<br />
SMP3 5.0 or a Büchi mp B545 apparatus and are uncorrected. The ultraviolet and visible<br />
(UV-Vis) spectra were measured on a Shimadzu UV-2101PC UV-VIS scanning spectrometer.<br />
Infrared (IR) spectra were measured on PerkinElmer System 2000 FT-IR spectrometer as KBr<br />
pellets or on a PerkinElmer Spectrum 100 FT-IR Spectrometer. NMR spectra were recorded<br />
on Bruker Avance 300, 400 or 600 MHz spectrometers. For 1 H NMR, when complex spectra<br />
due to overlapping resonances were encountered, the range was recorded. High-resolution<br />
mass spectra were obtained on GCT Premier Instrument.<br />
d) Chemical reactions.<br />
1. Solvent-free Aldol condensation: For a typical experiment, equimolar quantities <strong>of</strong> the<br />
acetophenone and benzaldehyde derivatives and NaOH were ground in a porcelain<br />
mortar at room temperature. After a few minutes, the mixture turned to a yellow solid<br />
which was treated with water and filtered to give the desired bromochalcone.<br />
2. Suzuki-Miyaura cross-coupling reaction: A mixture <strong>of</strong> equimolar quantities <strong>of</strong> the<br />
boronate ester, the bromochalcone, and Pd(PPh3)4 (5 mol%, relative to the ester) in<br />
toluene was refluxed under nitrogen atmosphere for 10 min. Afterwards, a 20% aqueous<br />
solution <strong>of</strong> tetraethylammonium hydroxide (4.2 equiv. relative to the ester) was added<br />
and the resulting mixture was refluxed following the progress <strong>of</strong> reaction by TLC. After<br />
completion <strong>of</strong> the reation, the mixture was cooled to room temperature and water was<br />
added, and the mixture extracted with diethyl ether and dried (Na2SO4). Then the<br />
solvent was removed and the residue was chromatographed over silica gel (with<br />
appropriate mobile solvent systems). After evaporation <strong>of</strong> solvent under reduced<br />
pressure, the resulting solid was dried overnight in a vacuum oven to afford the ketals<br />
(coupling products) as solids.<br />
e) Biological activities: These were performed as reported by Mihigo et al. (2010).<br />
Results and Discussion<br />
In the last few years, special attention has been paid to the Suzuki-Miyaura reaction as one <strong>of</strong> the<br />
most popular and powerful methods for the coupling <strong>of</strong> aryl-aryl moieties. This methodology has<br />
gained prominence and found many applications (Braga et al., 2006) both in research laboratories<br />
and in large-scale industrial processes due to its compatibility with a variety <strong>of</strong> functional groups,<br />
the stability and the commercial availability <strong>of</strong> a wide range <strong>of</strong> organoboron starting materials, and<br />
the ease <strong>of</strong> working up the reaction mixtures. Another advantage <strong>of</strong> the Suzuki-Miyaura crosscoupling<br />
reaction over similar methods is its tolerance <strong>of</strong> water, which can be used as solvent or co-<br />
239
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
solvent (Kotha et al., 2002; Miyaura and Suzuki, 1995). These desirable features made us choose<br />
the Suzuki-Miyaura coupling reaction for the synthesis <strong>of</strong> Rhuschalcone VI and analogues.<br />
The conversion <strong>of</strong> bromochalcones to their corresponding chalconylboronate esters was envisaged<br />
via bromine-lithium exchange with n-BuLi followed by a reaction with 2-isopropoxy-4,4,5,5tetramethyl-1,3,2-dioxaborolane<br />
in THF. However, several attempts failed to yield the desired<br />
products (boronate esters); instead, n-BuLi (a highly reactive nucleophile) reacted with the<br />
electrophilic , -unsaturated carbonyl group (Wu and Huang, 2006) <strong>of</strong> the chalcone, and the<br />
resulting 1,2- and 1,4-addition products were isolated, and their structures confirmed from NMR<br />
data. In addition, attempts to protect the carbonyl groups <strong>of</strong> the chalcones in order to reduce the<br />
electrophilicity <strong>of</strong> the , -unsaturated carbonyl group, were not successful. At this point it was<br />
decided to form the boronate ester at an early stage <strong>of</strong> the ketal obtained from 5-bromo-2,4dimethoxyacetophenone<br />
to give boronate ester (11) and then perform the Suzuki-Miyaura coupling<br />
with various bromochalcones. This would then allow the synthesis <strong>of</strong> various bichalcones by aldol<br />
condensation <strong>of</strong> the acetophenone derivatives (resulting from the deketalization <strong>of</strong> the coupling<br />
products) with variously substituted benzaldehydes. This approach was found successful and the<br />
details are presented in Scheme 1 below, where p-anisaldehyde (12) is used for the aldol<br />
condensation reaction.<br />
R 2<br />
R 3<br />
Br<br />
O<br />
MeO<br />
H<br />
O<br />
2 R1 = H<br />
3 R 1 = OMe<br />
R 2<br />
Br<br />
R<br />
NaOH<br />
MeO<br />
O<br />
1 R 1<br />
4 R2 = R3 = OMe<br />
5 R 2 = R3 = H<br />
6 R2 = H, R3 +<br />
= Me<br />
7 R 1 = R 2 = R 3 = H<br />
8 R1 = R2 = H, R3 = Me<br />
9 R 1 = OMe, R 2 = R 3 = H<br />
10 R1 = R2 = R3 = OMe<br />
13 R1 = H, R2 = R3 (d)<br />
= OMe 1, 14 and 15<br />
16 R1 = R2 = R3 = H 17<br />
18 R1 = OMe, R2 = R3 = H<br />
19 R1 = R2 = H, R3 = Me<br />
20 R 1 = R 2 = R 3 (d)<br />
= OMe<br />
OMe O<br />
240<br />
R 3<br />
MeO R 3<br />
MeO<br />
OMe<br />
R 1<br />
O R 2<br />
MeO<br />
O<br />
Me<br />
B O<br />
O<br />
O<br />
11<br />
(c)<br />
O<br />
MeO R 3<br />
MeO<br />
OMe O<br />
Me<br />
O<br />
O<br />
(b)<br />
MeO R 3<br />
MeO<br />
Scheme 1. General synthetic strategy for Rhuschalcone VI (1) and analogues. Reagents and<br />
conditions: (a) Pd(PPh3)4, toluene, tetraethylammonium hydroxide, reflux; (b) I2, acetone, reflux; (c)<br />
p-anisaldehyde (12), solid NaOH, rt; (d) BBr3, dichloromethane, reflux.<br />
The chemical structures <strong>of</strong> the newly synthesized compounds were assigned by means <strong>of</strong> extensive<br />
1D and 2D NMR, and IR, UV, and MS analysis. For rhuschalcone VI, an authentic natural product<br />
was sought to make direct comparison possible. Such a sample was not available and hence the<br />
natural compound was re-isolated (using the synthetic material as reference) from the roots <strong>of</strong> the<br />
producing Rhus pyroides species. The Rf pattern, and the 1 H and 13 C NMR data generated for the<br />
natural product and the synthetic material were found to be in complete agreement.<br />
(a)<br />
R 1<br />
R 1<br />
O<br />
O<br />
R 2<br />
O<br />
R 2
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
The synthesized bichalcones 1, 13, 14, 15, 16, 17, 18, 19, and 20, together with isobavachalcone 21<br />
(a prenylated chalcone isolated from Dorstenia kameruniana and which has shown activity on other<br />
organisms (Mbaveng et al., 2008)), were preliminarily evaluated for their in-vitro anti-protozoal<br />
activities using a free-living, non-pathogenic protozoa Bodo caudatus (Bodonidae) as a model, and<br />
for the most active, for their cytotoxicity using the MTT viability assay (Mosmann, 1983). The results<br />
showed that compounds 1, 15, 17 and 21 were the most active and induced the largest reduction<br />
in viability <strong>of</strong> protozoa with an inhibition <strong>of</strong> 75-83 % compared to controls, CuSO4. LC50<br />
concentrations were determined for the four most active compounds. The most active compound<br />
(21) displayed LC50 concentration <strong>of</strong> 4.36 g/mL, which was about twice less active than CuSO4<br />
(2.30 g/mL). The other compounds (1, 15, and 17) killed protozoa with lesser efficacy at LC50<br />
concentrations <strong>of</strong> 53.32, 118.20, and 20.59 g/mL, respectively. The cytotocity test results showed<br />
that <strong>of</strong> the four most antiprotozoal active compounds, 1 and 21 induced significant cell death <strong>of</strong><br />
BHK cells after exposure for 48 hours at a concentration <strong>of</strong> 100 g/mL, which corresponded to CC50<br />
value <strong>of</strong> 97.59 g/mL and 86.88 g/mL, respectively. The CC50 concentration <strong>of</strong> compound 21 was<br />
found to be approximately 20 times higher than its antiprotozoal concentration (LC50 = 4.36 g/mL),<br />
while antiprotozoal activity <strong>of</strong> compound 1 is in the range as its cytotoxic concentration.<br />
Compounds 15 and 17 induced a small decrease in cell viability compared to non-treated cells, but<br />
did not exhibit significant cytotoxicity up to a concentration <strong>of</strong> 100 g/mL.<br />
Acknowledgements<br />
S.O.M. is gratefully indebted to DAAD-NAPRECA for a Ph.D. fellowship. Financial support from IPICS<br />
(Uppsala University, Sweden) to NABSA is gratefully acknowledged. We thank Gili Joseph at Gibex-<br />
USA who developed and provided the anti-protozoal assay protocols.<br />
References<br />
Mbaveng, A.T., Ngameni, B., Kuete, V., Simo, I.K., Ambassa, P., Roy, R., Bezabih, M., Etoa, F.-X., Ngadjui, B.T., Abegaz,<br />
B.M., Meyer, J.J.M., Lall, N. and Beng, P. (2008); Antimicrobial activity <strong>of</strong> the crude extracts and five flavonoids<br />
from the twigs <strong>of</strong> Dorstenia barteri (Moraceae). Journal <strong>of</strong> Ethnopharmacology, 116, 483-489.<br />
Mdee, L.K., Yeboah, S.O. and Abegaz, B.M. (2003); Rhuschalcone II-VI, five new bichalcones from the root bark <strong>of</strong> Rhus<br />
pyroides. Journal <strong>of</strong> Natural Products, 66, 599-604.<br />
Mihigo, S.O., Mammo, W., Bezabih, M., Andrae-Marobela, K. and Abegaz, B.M. (2010); Total synthesis, antiplasmodial<br />
and cytotoxicity activities <strong>of</strong> rhuschalcone VI and analogs. Bioorganic and Medicinal Chemistry, 18, 2464-2473.<br />
Miyaura, N. and Suzuki, A. (1995); Palladium-catalyzed cross-coupling reactions <strong>of</strong> organoboron compounds. Chemical<br />
Reviews, 95, 2457-2483.<br />
Mosmann, T. (1983); Rapid colorimetric assay for cellular growth and survival: Application to proliferation and<br />
cytotoxicity assays. Journal <strong>of</strong> Immunological Methods, 65, 55-63.<br />
Parry, P.R., Wang, C., Batsanov, A.S., Bryce, M.R. and Tarbit, B. (2002); Functionalized pyridylboronic acids and their<br />
Suzuki cross-coupling reactions to yield novel heteroarylpyridines. Journal <strong>of</strong> Organic Chemistry, 67, 7541-7543.<br />
Toda, F., Tanaka, K. and Hamai, K. (1990); Aldol condensation in the absence <strong>of</strong> solvent: Acceleration <strong>of</strong> the reaction<br />
and enhancement <strong>of</strong> the stereochemistry. Journal <strong>of</strong> Chemical Society: Perkin Trans 1, 3207-3209.<br />
Wu, G. and Huang, M. (2006); Organolithium reagents in asymmetric processes. Chemical Reviews, 106, 2596-2616.<br />
Xing, X., Padmanaban, D., Yeh, L.-A. and Cuny, G.D. (2002); Utilization <strong>of</strong> a copper-catalyzed diaryl ether synthesis for<br />
the preparation <strong>of</strong> verbenachalcone. Tetrahedron, 58, 7903-7910.<br />
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[YS 16] Acaricidal Effects <strong>of</strong> Four Plant Species on Rhipicephalus<br />
appendiculatus Neumann (Acarina ixodidae) Ticks<br />
Robert Opiro 1 , Anne M. Akol 2 , Joseph Okello-Onen 1<br />
1 Department <strong>of</strong> Biology, Gulu University, Box 166, Gulu, Uganda<br />
2 Department <strong>of</strong> Zoology, Makerere University, Box 7062, Kampala, Uganda<br />
Corresponding author: Robert Opiro (famousopiro@yahoo.com)<br />
Keywords: Tick mortality, lethal concentrations, probit analysis, Interaction effects<br />
Introduction<br />
T<br />
here is currently a worldwide trend towards reducing the use <strong>of</strong> chemical acaricides as much as<br />
possible in several parts <strong>of</strong> the world. This has been mainly due to the development <strong>of</strong><br />
resistance by ticks to acaricides, together with the well-documented damage these compounds<br />
cause to the environment and food chain. An alternative with proven efficacy at controlling ticks<br />
but with lower environmental impact is the use <strong>of</strong> plants with established acaricidal properties<br />
which possess numerous advantages (Liang et al., 2003).<br />
So far, promising results have been obtained from some plants screened for anti-tick properties<br />
(Wilson and Surthest, 1990; Nchu et al., 2005; Magano et al., 2007; Kaaya and Saxena, 1998; Kaaya<br />
et al., 1995). However, despite the promising results, many plants are still scientifically untested for<br />
anti-tick properties (Magano et al., 2007).<br />
It is against this background that this study evaluated the acaricidal properties <strong>of</strong> four plants<br />
species. These species had been cited as useful in the control <strong>of</strong> ticks in an ethnoveterinary survey<br />
amongst cattle keepers <strong>of</strong> Gulu and Amuru districts <strong>of</strong> Northern Uganda (Opiro, 2009, unpublished<br />
data).<br />
Materials and Methods<br />
Tick mortality following exposure to extracts <strong>of</strong> four plants species (Cassia didymobotrya, Kigelia<br />
africana, Euphorbia hirta and Cissus adenocucaulis) at five serial dilutions (10 -1 10 -5 ) and three<br />
periods <strong>of</strong> exposure (24, 48 and 72 h) was assessed. Ticks were exposed to filter paper discs<br />
impregnated with the extracts for various periods <strong>of</strong> time and the number dead were determined.<br />
Plant extracts were obtained using three different solvents (methanol, dichloromethane and<br />
hexane).<br />
Results<br />
Data analysis using ANOVA showed significant interactions among species, time and concentration.<br />
The highest mortality was observed at 72 h and the least was at 24 h. The difference in mortality<br />
was highest among plant species at the lowest concentration <strong>of</strong> extracts, but this gap decreased as<br />
the concentration increased. Additionally, tick mortality increased with increasing concentration <strong>of</strong><br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
extract and period <strong>of</strong> exposure to the plant extracts; C. didymobotrya was the most potent species<br />
followed by K. africana, E. hirta and C.adenocucaulis in order <strong>of</strong> decreasing potency. The solvents<br />
did not show significant differences in causing tick mortality. Probit analysis showed that the LC<br />
(lethal concentration) value <strong>of</strong> the extract decreased with longer periods <strong>of</strong> tick exposure to the<br />
extracts.<br />
Table 2: Table <strong>of</strong> means<br />
Grand mean 54.90<br />
Time (h) 24 48 72<br />
42.41 53.69 68.59<br />
Plant_spp C. didymobotrya K.africana E. hirta C. adenocucaulis<br />
68.81<br />
64.21 45.04 41.53<br />
Solvent Dichloromethane Hexane Methanol<br />
54.28 54.79 55.62<br />
Concn 100000 10000.0 1000.00 100.00 10.00<br />
75.41 66.96 58.12 42.34 31.6<br />
Time concn 100000 10000.0 1000.00 100.00 10.00<br />
24.00 66.93 56.20 46.21 26.61 16.10<br />
48.00 76.34 66.16 56.22 39.98 29.76<br />
72.00<br />
82.96 78.52 71.93 60.43 4<strong>9.1</strong>1<br />
Plant_spp concn 100000 10000.0 1000.00 100.00 10.00<br />
C.didy 88.84 82.34 74.18 55.91 42.81<br />
E.hirta 63.81 57.44 48.88 31.75 23.35<br />
K.africana 82.45 72.63 64.80 56.46 44.68<br />
S.adedacule<br />
66.55 55.42 44.62 25.25 15.79<br />
Solvent concn 100000.0 10000.0 1000.00 100.00 10.00<br />
Dichloromethane 75.25 67.36 58.03 40.73 30.04<br />
Hexane 75.11 66.41 57.81 43.12 31.46<br />
Methanol 75.88 67.10 58.52 43.17 33.46<br />
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Table 3: Estimated LC Values <strong>of</strong> extracts <strong>of</strong> different plants (values in mg/L=ppm)<br />
Cassia didymobotrya<br />
Time methanol dichloromethane hexane<br />
24h 48h 72h 24h 48h 72h 24h 48h 72h<br />
LC50 5610.01 2140.81 161.641 5841.14 2493.89 172.39 5785.91 2293.64 182.27<br />
LC99 9687.52 6891.34 1398.80 9737.35 6951.12 1927.02 9928.0 7243.31 2164.25<br />
Kigelia africana<br />
Time methanol dichloromethane hexane<br />
24h 48h 72h 24h 48h 72h 24h 48h 72h<br />
LC50 6216.39 3041.47 502.44 7286.1 3350.25 522.86 7107.59 3151.43 516.55<br />
LC99 14480.00 7714.00 1882.43 1192.65 7243.08 2242.12 11692.67 9235.12 3168.56<br />
Euphorbia hirta<br />
Time methanol dichloromethane hexane<br />
24h 48h 72h 24h 48h 72h 24h 48h 72h<br />
LC50 64886 41841 19478 68598 33631 18425 66363 43361 20624<br />
LC99 586628 274057 68487 424096 247713 68370 570361 267415 64590<br />
Symphostema adedacule<br />
Time methanol dichloromethane hexane<br />
24h 48h 72h 24h 48h 72h 24h 48h 72h<br />
LC50 74571 48272 21471 85050 53399 20118 75228 63182 21117<br />
LC99 451470 245831 79142 488885 251901 71476 581263 265850 68434<br />
Discussion and Conclusions<br />
This study indicates that all plants tested showed acaricidal activity. The analysis revealed that the<br />
interaction between plant species and concentration were significant, meaning the potencies <strong>of</strong> the<br />
former were influenced by the latter. The same applies to the effect <strong>of</strong> exposure duration. The<br />
interaction effects between plant species and concentration levels were probably caused by<br />
responses <strong>of</strong> any <strong>of</strong> the pairs <strong>of</strong> plant species reacting in the same manner i.e. mortality as<br />
described between C. didymobotrya and K. africana and also E. hirta and C. adenocucaulis.<br />
The good correlation between concentration and mean mortality is in line with the findings <strong>of</strong><br />
Magano et al., (2007) who investigated the anti-tick properties <strong>of</strong> the root extracts <strong>of</strong> Senna italica<br />
subsp. arachoides against adults <strong>of</strong> Hyalomma marginatum rufipes. These authors found that the<br />
higher the concentration <strong>of</strong> the extracts, the lesser the time needed for a certain proportion <strong>of</strong><br />
arthropod pests to die.<br />
There were significant differences in acaricidal activities at different exposure times though this<br />
effect depended on the concentration. This probably indicates that the active compounds bound to<br />
receptor sites would be expected to increase with period <strong>of</strong> exposure to the extracts (Lullmann and<br />
Bieger, 1993). The same could be true for the significant differences in activity <strong>of</strong> the extracts with<br />
increase in concentration. This finding agrees with the observation that time is a crucial factor that<br />
increases the amount and distribution <strong>of</strong> active compounds in the body (Lullman and Bieger, 1993).<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Solvent as a factor did not influence tick mortality and the observed mortality was solely due to<br />
species. This renders the discussion on effects <strong>of</strong> polarities on extraction <strong>of</strong> the plants ingredients<br />
irrelevant, implying that any could be used to make the extracts. The observation could be<br />
attributed to the nature <strong>of</strong> the chemical compounds in the plants. It is however important to note<br />
that sharp conclusions cannot be drawn at this stage unless chromatographic, GC-MS and NMR<br />
analysis are carried out to identify precisely the components.<br />
The LC values obtained by probit analysis decreased with an increase in the duration <strong>of</strong> exposure to<br />
the test extracts. The values obtained for the two best performing species therefore shows that<br />
they have great potential for development into commercial acaricides.<br />
On the whole, the results indicated that by considering the % mortality as a main index, C.<br />
didymobotrya extracts on average performed best, closely followed by K. africana, and this may be<br />
attributed to their high potencies as acaricides. The traditional uses and pharmacological properties<br />
<strong>of</strong> these two plants have already been highlighted by other authors (Tuwangye and Olila, 2006;<br />
Sangita et al. 2008).<br />
Conclusively, the study has demonstrated the acaricidal activity <strong>of</strong> these four plant species, as well<br />
as their potential to provide new compounds for tick control. The promising results therefore<br />
highlight the importance <strong>of</strong> these plants as a natural resource, providing a further impetus for<br />
measures to conserve them.<br />
Acknowledgements<br />
We acknowledge the financial support <strong>of</strong> Gulu University. The following persons are also greatly<br />
acknowledged: Mr. Oduru Ambrose, the Laboratory Technician Biology Department, Gulu<br />
University; Mr. Kimondo Mark <strong>of</strong> the ICIPE, Nairobi for providing the test stages used in the<br />
bioassays and Mr Ragama Phillips <strong>of</strong> Kawanda Agricultural Research Station for analyzing the data.<br />
References<br />
Fernández, F.F., Paula, E., Freitas, S., Anna, C. and Garcia, I. S (2005); Larvicidal potential <strong>of</strong> Sapindus saponaria to<br />
control the cattle tick Boophilus microplusPesq. agropec. bras.,Brasília, v.40, n.12, p.1243-1245.<br />
Kaaya, G.P., Mwangi, E.N., and Malonza, M.M. (1995); Acaricidal activity <strong>of</strong> Margaritria discoidea plant extracts against<br />
the ticks R.s appendiculatus and Amblyomma variegatum. International Journal <strong>of</strong> Acarology. 21, 123-129.<br />
Liang, G.M, Chen W, Liu T.X. (2003); Effects <strong>of</strong> three neem-based insecticides on diamondback moth<br />
(Lepidoptera: Plutellidae). Crop Protection. 22: 333-40.<br />
Lullman, H.K. Morh and Bieger, D. (1993); Colour atlas <strong>of</strong> pharmacology. Theme medical publishers. Inc. New York,<br />
pp52-98.<br />
Magano, S. R., Thembo, K. M., Ndlovu S. M., and Makhubela, N. F. H. (2007); The anti-tick property <strong>of</strong> the root extracts<br />
<strong>of</strong> Senna italica subsp. Arachoides. African Journal <strong>of</strong> Biotechnology Vol. 7 (4), pp. 476-48.<br />
Nchu, F., Magano, S.R, El<strong>of</strong>f, N. (2005); In vitro investigation <strong>of</strong> the toxic effects <strong>of</strong> extracts <strong>of</strong> Allium sativum bulbs on<br />
adults <strong>of</strong> Hyalomma marginatum rufipes and Rhipicephalus pulchellus. Journal <strong>of</strong> South African Veterinary<br />
Association. 76: 99-103.<br />
Sangita, S., Harmeet, K., Bharat, V., Ripudaman and Singh, S. K. (2009); Kigelia africana (Lam) Benth.-An oeverview.<br />
Natural product radiance. vol 8(2). Pp 190-197<br />
Tuwangye, I and Olila, D. (2006); The anthelmintic activity <strong>of</strong> selected indigenous medicinal plants used by the<br />
banyankole <strong>of</strong> western Uganda. Journal <strong>of</strong> animal and veterinary advances 5(8); 712-717.<br />
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[YS 17] Anti-Malarial Activity and Phytochemical Studies <strong>of</strong> Cissampelos<br />
Mucronata and Stephania Abyssinica<br />
Ruth A. Omole, Isaiah O. Ndiege and Alex K. Machocho<br />
Department <strong>of</strong> Chemistry, Kenyatta University<br />
Key words: Stephania abyssinica, Cissampelos mucronata hasubanan (-)-5-oxoaknadinine bisbenzylisoquinoline, (-)curine,<br />
(-)-isocurine and (-)-pseudocurine, anti-plasmodial activity.<br />
Introduction<br />
E<br />
ach year, there are about 500 million and 2.7 million reported malaria cases and deaths,<br />
respectively world wide (WHO 2002; Greenwood et al., 2005). The resurgence <strong>of</strong> malaria is<br />
partly attributed to development <strong>of</strong> drug resistance by the most common malaria parasite<br />
(Plasmodium falciparum). Plants are considered as important sources <strong>of</strong> lead compounds in drug<br />
development and discovery. Some <strong>of</strong> the anti-malarial in use today such as artemisinin and its<br />
derivatives were obtained directly from plants or developed using chemical structures <strong>of</strong> plants or<br />
developed using chemical structures <strong>of</strong> plant derived compounds as templates (Philipson et al.,<br />
1993). Previous research by Muregi et al. (2004) revealed that the crude extracts <strong>of</strong> Stephania<br />
abyssinica and Cissampelos mucronata exhibited strong anti-malarial activity. Isolation <strong>of</strong> the<br />
constituents that are responsible for anti-plasmodial activity has not been done. Phytochemical<br />
investigation <strong>of</strong> the crude alkaloid and dichloromethane extracts <strong>of</strong> S. abyssinica have resulted to<br />
characterization <strong>of</strong> two new bisbenzlyisoquinoline (BBIQ) and a new hasubanan alkaloid (-)-5oxoaknadinine,<br />
respectively. They were largely identified by interpretation <strong>of</strong> their 2D NMR spectral<br />
data. Hexane extract <strong>of</strong> S. abyssinica also gave (+)-nonacosan-10-ol which was previously isolated<br />
from Cocculus hirsutus (Ahmad et al., 1987). Investigation <strong>of</strong> DCM extracts <strong>of</strong> C. mucronata, (-)curine<br />
and stigmasterol were isolated. In this paper we wish to report the spectral characterization<br />
<strong>of</strong> these compounds and their biological evaluation in terms <strong>of</strong> anti-plasmodial activity.<br />
Materials and methods:<br />
Plant materials<br />
Stephania abyssinica was collected from Kisii highlands and Cissampelos mucronata was collected<br />
from Kabondo village Rachuonyo district, Nyanza province, Kenya in August 2006. The plants were<br />
authenticated by Mr. Simon Mathenge, Department <strong>of</strong> Botany University <strong>of</strong> Nairobi and voucher<br />
specimens (RO/01/2006 and RO/02/2006) deposited at the University <strong>of</strong> Nairobi Herbarium in the<br />
Department <strong>of</strong> Botany. The plant samples were air dried under shade and ground using a<br />
laboratory mill.<br />
Extraction<br />
The cold organic extractions were performed by soaking 1.57kg <strong>of</strong> the S. abyssinica and 0.9 kg <strong>of</strong> C.<br />
mucronata chaff for 48h in solvents <strong>of</strong> increasing polarity. The dichloromethane <strong>of</strong> S. abyssinica<br />
gave the alkaloid extract after acid hydrolysis. The extracts were preserved at -20°C until used.<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Instruments<br />
1 H and 13 C NMR spectra <strong>of</strong> the compounds were recorded at 400 MHz and 100 MHz, respectively on<br />
a Bruker DRX-500 spectrometer at 292.9k with a Bruker gradient unit. Optical rotation was<br />
measured using Polax-2L polarimeter at 25°C and IR on Hyper IR. Melting points were done using<br />
Gallen Kemp apparatus and were uncorrected.<br />
Isolation<br />
Column chromatography was done using silica gel and Sephadex LH-20 was used as the filter gel.<br />
UV was done using CECIL 2041 UV spectrometer. Repeated column chromatography <strong>of</strong> n-Hexane<br />
extract (6 g) and DCM (2 g) extract <strong>of</strong> S. abyssinica using hexane: ethylacetate followed by<br />
preparative column chromatography and recrystalization gave (-)-nonacosan-10-ol (1) and (-)-5oxoaknidinine<br />
(2). The alkaloid extract (266 mg) <strong>of</strong> the same plant was fractionated by Sephadex<br />
LH-20 eluting with DCM:MeOH (1:1) and then preparative thin layer chromatography (2%<br />
MeOH/DCM) with (-)-isocurine (3) and (-)-pseudocurine (4) being isolated. Repeated column<br />
chromatography <strong>of</strong> DCM extract <strong>of</strong> C. mucronata with n-Hexane:EtOAc, DCM:MeOH followed by<br />
preparative TLC gave stigmasterol (5) and (-)-curine (6).<br />
H 3C(H 2C) 5<br />
OH<br />
1<br />
(CH 2) 15CH 3<br />
247<br />
H 3CO<br />
HO<br />
O<br />
2<br />
OCH 3<br />
OCH3 OH<br />
N<br />
O<br />
OH<br />
O<br />
N<br />
H3CO 4 5<br />
4a OH<br />
3<br />
6<br />
N 1<br />
8a 7<br />
8 O<br />
OH<br />
9<br />
O<br />
N<br />
OCH3 H3CO 10 4<br />
4a<br />
5<br />
6<br />
3<br />
7<br />
1<br />
8a<br />
8<br />
9<br />
13'<br />
12'<br />
14'<br />
11'<br />
9'<br />
10' 15'<br />
15 11 7' 8a'<br />
8' 1'<br />
14<br />
6' 4a'<br />
12<br />
3'<br />
13<br />
5' 4'<br />
4<br />
10 15 11<br />
14<br />
7'<br />
1'<br />
13 12 6' 4a'<br />
3'<br />
5' 4'<br />
8' 12'<br />
9'<br />
11'<br />
10'<br />
8a'<br />
13'<br />
14'<br />
15'<br />
3<br />
4<br />
4a<br />
5<br />
6 OCH3 7 OH<br />
N 1<br />
8a<br />
8 O<br />
9<br />
O<br />
N<br />
OH<br />
H3CO 10 15 11<br />
14<br />
7'<br />
1'<br />
12 6' 4a'<br />
13<br />
3'<br />
5' 4'<br />
8' 12'<br />
9'<br />
11'<br />
10'<br />
8a'<br />
13'<br />
2<br />
HO 3<br />
19<br />
1<br />
10<br />
5<br />
4<br />
29<br />
28<br />
21 22<br />
20<br />
24 27<br />
18<br />
12 23 25<br />
17<br />
11 13<br />
26<br />
14 16<br />
9<br />
8 15<br />
7<br />
6<br />
5<br />
14'<br />
15'<br />
6<br />
Results and Discussion<br />
(+)-Nonacosan-10-ol (1) was isolated as white crystals. The IR spectrum showed the presence <strong>of</strong><br />
hydroxyl group at 3337.6 cm -1 . Important clue for the structure was obtained from mass spectrum.<br />
O<br />
OCH 3<br />
N
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
A molecular peak at m/z 424 corresponding to C29H60O.was revealed in EIMS. The peak at m/z 297<br />
indicated loss <strong>of</strong> CH3(CH2)18CH(OH) group while the one at m/z 157 indicated loss <strong>of</strong><br />
CH3(CH2)8CH(OH) group. This was consistent with the fact that OH group was located at 10 th<br />
position. The structure was further established through 1 H and 13 C NMR which were consistent with<br />
the reported data (Dragota & Riederer, 2008). (-)-5-Oxoaknadinine (2) was isolated as light yellow<br />
crystals. It gave appositive test for alkaloids with Dragend<strong>of</strong>f s reagent. UV (280 nm) spectra<br />
indicated the presence <strong>of</strong> , -unsaturated carbonyl moiety. The structure was determined on basis<br />
<strong>of</strong> 1D ( 1 H and 13 C NMR and DEPT) and 2D (HMQC, H<strong>MB</strong>C and COSY) NMR experiments.<br />
(-)-Isocurine (3) and (-)-pseudocurine (4) were isolated as brown amorphous solid and both gave a<br />
positive reaction for alkaloids with Dragend<strong>of</strong>f s reagents. The 1 H NMR spectrum <strong>of</strong> alkaloid 3 and 4<br />
presented two N-methyl groups and two methoxy groups. The electron impact mass spectrum<br />
presented a molecular peak ion at m/z 594 (C36H38N2O6) and a prominent peak at m/z at 298 for<br />
compound, indicative <strong>of</strong> a head to tail linked BBIQ (Baldas et al., 1972). Position <strong>of</strong> attachment <strong>of</strong><br />
the two diaryl ether bridges and location <strong>of</strong> hydroxyl groups were established through<br />
interpretation <strong>of</strong> the HMQC spectrum <strong>of</strong> the methylated derivatives. From the 1 H NMR spectrum <strong>of</strong><br />
alkaloid 3 it appeared related to compound 4 but the arrangement <strong>of</strong> the protons were totally<br />
different as revealed by COSY, HMQC and H<strong>MB</strong>C. Another difference between 3 and 4 one <strong>of</strong> the<br />
methoxy groups is at different position. Compound 3 the methoxy is attached to C-6 and 4 attached<br />
to C-12. The structures <strong>of</strong> the two compounds were established by careful analysis <strong>of</strong> the 1D-NMR<br />
and 2D-NMR (1H-1H correlation spectroscopy (COSY), heteronuclear multiple quantum correlation<br />
(HMQC) and heteronuclear multiple bond correlation (H<strong>MB</strong>C)<br />
A triterpene (+)-stigmasterol (5) was isolated as white crystals. Its IR spectrum revealed the<br />
presence <strong>of</strong> hydroxyl group at 3422cm -1 and double bond at 1653cm -1 . A molecular formula <strong>of</strong><br />
C29H48O was established by EIMS at m/z 412. All the spectroscopic data confirmed the structure and<br />
were consistent with literature values (Morale et al (6) was<br />
isolated as white amorphous solid. The IR spectrum showed the presence <strong>of</strong> hydroxyl group at 3384<br />
cm -1 and aromatic C=Cstr at 1507 cm -1 . The 1 H NMR spectra revealed 10 aromatic protons. Other<br />
peaks at 3.87 and 3.89 showed a presence <strong>of</strong> a methoxy group while signals at 2.28 and 2.49<br />
indicated presence <strong>of</strong> alkyl amine thus two N-methyl groups. EIMS for (-)-curine revealed a<br />
molecular ion peak at m/z 594 corresponding to C36H38N2O6 The spectral data was in agreement<br />
with those reported for (-)-curine (Koike et al., 1981; Lengo et al., 2000). The anti-plasmodial<br />
activity <strong>of</strong> the isolates against P. falciparum D6 and W2 strains in vitro are reported in table 1.<br />
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Table 1: Anti-plasmodial activity for the isolated compounds<br />
Compound P. falciparum (D6)<br />
IC50±SD (µg/ml)<br />
249<br />
P. falciparum (W2)<br />
IC50±SD (µg/ml)<br />
(+)-Nonacosan-10-ol (1) 13.79±1.02 4.35±2.45<br />
(-)-5-oxoaknadinine (2) 10.25±1.84 3.45±2.22<br />
(-)-Isocurine (3) 0.75±0.11 1.65±0.03<br />
(-)-Pseudocurine (4) 0.29±0.00 0.31±0.01<br />
(+)-Stigmasterol (5) >5 >5<br />
(-)-Curine (6) 0.24±0.03 0.22±0.06<br />
Chloroquine 1.16±0.00 1.69±0.14<br />
Artemisinin 8.34±0.14 56.87±1.27<br />
Apart from (-)-stigmasterol (5), (+)-nonacosan-10-ol (1) and (-)-oxoaknadinine (2) which were<br />
inactive, (-)-curine (6), (-)-isocurine (3) and (-)-pseudocurine (4) showed a strong anti-plasmodial<br />
activity. Chloroquine and artemisinin was used as positive controls. For D6 strain the IC50 range for<br />
compounds was 0.24±0.03-13.79±1.02 µg/ml while that for W2 the IC50 range was 0.22±0.06-<br />
4.35±2.45 µg/ml. (-)-Curine exhibited the strongest anti-plasmodial activity against P. falciparum D6<br />
and W2 strains as compared to other isolates.<br />
Acknowledgement<br />
RAO thank Kenyatta University through SPAS for the scholarship that enabled me to undertake the<br />
course. Thanks to Jeremiah Gathirwa and Hosea Akalla <strong>of</strong> KEMRI for evaluation <strong>of</strong> anti-plasmodial<br />
activity. SIDA/SAREC/IUCEA acknowledged for financial grants that made this research possible.<br />
References<br />
Ahmad V.U., Mohammad F.V., Rasheed T. (1987). Hirsudiol a titerpenoid from Cocculus hirsutus. Phytochemistry. 26:<br />
793-794.<br />
Baldas J., Bick I.R.C., Falco M.R., De Vries J.X., Porter Q.N. (1972). Mass spectrometry <strong>of</strong> bisbenzylisoquinoline alkaloids.<br />
J. Chem. Soc. 592-601.<br />
Dragota S., Rieder M. (2008). Comparative study on epicuticular leaf waxes <strong>of</strong> Araucacia araucana, Agadhis robusta and<br />
Wollemia nobilis (Araucaraceae). Austr. J. Bot. 56: 644-650.<br />
Greenwood B.M., Bojang K., Whitty C.J.M., Target G.A.T. (2005). Malaria. Lancet 365: 1487-1498.<br />
Forgo P., Köver K.E. (2004). Gradient enhanced selective experiments in the 1 H NMR chemical shift assignment <strong>of</strong><br />
skeleton and side-chain resonances <strong>of</strong> stigmasterol, a phytosterol derivative. Steroids. 69: 43-50.<br />
Lengo M., Marie-Therese M., Dorothee R., David R., Phillipe R., Francois F. (2000). Spectral characterization and<br />
antiplasmodial activity <strong>of</strong> bisbenzylisoquinolines form Isolona ghesquiereina. Planta Medica. 66: 537-540.<br />
Morale G., Sierra P., Mancilla A., Parexades A.N., Loyola L.A., Gallado O., Borquez J. (2003). Secondary metabolites from<br />
four medicinal plants from north Chile: antimicrobial activity and biotoxicity against Artemia salina. J. Chil. Chem.<br />
Soc. 48: 1-11.<br />
Muregi F.W., Chhabra S.C., Njagi E.N.M., Langat-Thoruwa C.C., Njue W.M., Orago A.S.S., Omar S.A., Ndiege I.O. (2003).<br />
Anti-plasmodial activity <strong>of</strong> some Kenyan plant extracts singly and in combination with chloroquine. Phytotherapy<br />
Research 18: 379-384.<br />
Philipson J.D., Wright W.C., Kirby G.C., Warhurst D.C. (1993). Tropical Plants as Sources <strong>of</strong> Antiprotozoal. In: Potential <strong>of</strong><br />
tropical trees. Recent advances in Phytochemistry. Plenum Press New York. 27: 1-40.<br />
WHO (2002). The world Health Report 2002: Reducing Risks Promoting Healthy Life. WHO Geneva
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[YS 18] Effects <strong>of</strong> Sida Cuneifolia (A.Gray) Herbal Extracts on the Reproductive<br />
System Functioning in Male and Female Laboratory Rats<br />
Anastasia N. Nandwa<br />
Etradiol, Polymorphnuclear cells, Gonadotropin Releasing Hormone, Leutinizing hormone, Follicle Stimulating<br />
Hormone, Epithelial cells<br />
INTRODUCTION<br />
S<br />
ida cuneifolia whose synonym is Billieturneria helleri is an ascendent to procumbent shrub. The<br />
leaves are small, being 0.5-1.5 cm long, and about as wide. It used by some communities in<br />
Kenya for contraception, however the mechanism <strong>of</strong> its action have not been documented.<br />
Methodology<br />
Herbs Preparation and Administration<br />
Fifty female albino rats <strong>of</strong> the species Rattus norvegicus, <strong>of</strong> average weight 200g were used in the<br />
investigation. The cages were kept at room temperature i.e. 20 o C and exposed to twelve hours<br />
daylight and twelve hours darkness.<br />
After a week, forty (40) rats were given root extracts from Sida cuneifolia at a concentration <strong>of</strong> 1g in<br />
100ml tap water making a 1% solution. This was given as drinking water twice at four day intervals.<br />
Ten rats continued to take plain tap water. These were the controls. Ten experimental and five<br />
control rats were mated with untreated males. The toxicity level <strong>of</strong> the drug was carried out at<br />
Kenya Medical Research Institute (KEMRI) laboratories.<br />
Results<br />
All female rats given the sida cuneifolia extract and mated with normal (untreated males) failed to<br />
conceive even after staying with the males for over three months. On the other hand control rats<br />
had litter normally, that is five to six young every three weeks. Female rats that had, had litter<br />
before also failed to conceive when treated with the extract and mated with normal males. Their<br />
counterparts that were kept as controls continued to have litter normally.Treated males also failed<br />
to sire <strong>of</strong>fspring while control males had <strong>of</strong>fspring.<br />
Day 1<br />
The smears showed equal numbers <strong>of</strong> cornified and epithelial cells. Leukocytes were either minimal<br />
or lacking.<br />
Day 2<br />
Polymophonuclear cells esp. many neutrophilis and monocytes observed in smears from<br />
experimental rats. The slide is clean and devoid <strong>of</strong> mucus. Smears from control rats show sheets <strong>of</strong><br />
ephilelial cells with mucous indicative <strong>of</strong> proestrus.<br />
Sheets <strong>of</strong> ephithelial cells seen.<br />
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Day 3<br />
Epithelial cells mostly <strong>of</strong> parabasal and intermediate type observed in smears from experimental<br />
rats. These are normally not observed during Estrus. Smears from control rats with superficial cells<br />
appearing in strings indicative <strong>of</strong> Estrus.<br />
Day 4<br />
High numbers <strong>of</strong> Leucocytes and total absence <strong>of</strong> superficial epithelial cells typical <strong>of</strong> metestrus and<br />
diestrus (infertile phases). Control rats showing almost equal numbers <strong>of</strong> ephithelial and cornified<br />
cells.<br />
Day 5<br />
A sharp rise in leukocytes and progressing decline in epithelial cells characteristic <strong>of</strong> diestrus in<br />
smears from experimental rats. The smear is devoid <strong>of</strong> mucus. The smear from control rat has<br />
increasing number <strong>of</strong> epithelial cells and declining numbers <strong>of</strong> cornified cells indicating a return to<br />
cyclicity.<br />
Histology <strong>of</strong> sections<br />
Uteri, Ovaries and vaginas were obtained from both experimental and control rats. Sections <strong>of</strong><br />
Testes from experimental and control rats were also studied.<br />
Uteri<br />
In some areas the lining was thin while in others it was thick. There were large epithelial cells with<br />
vacuolations.<br />
Ovaries<br />
Whole ovaries were shrunk and sections showed degenerative granulosa cells. Few follicles were<br />
observed and these had missing or degenerate ova.<br />
Vagina<br />
There was peeling <strong>of</strong>f <strong>of</strong> the Stratum Corneum into the lumen also few glands were observed in the<br />
Stratum Germinativum and the vagina had a narrow lumen. The vaginal walls were comparatively<br />
thinner than in control rats<br />
Testes<br />
Sections <strong>of</strong> testes from treated rats showed seminiferous tubules with sloughed <strong>of</strong>f epithelial lining,<br />
degenerated interstitial cells with immature spermatids in the lumens. The epithelial lining was<br />
thin, two-three cell thickness and even two cell thickness in some areas.<br />
Epididymis<br />
Those from control rats showed complete outlines with normal epithelial lining. Their lumens were<br />
full <strong>of</strong> spermatozoa. Sections from experimental rats showed scanty broken epithelial lining and<br />
very large empty or scantly filled lumens i.e aspermia.<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Acknowledgement<br />
My heartfelt thanks go to Jehovah God who always makes a way where none exists. I thank<br />
Chepkoilel University College for facilitating the research. I am deeply indebted to Janet Kosgey<br />
who worked tirelessly with me to accomplish this project. My sincere thanks go to my supervisors;<br />
Dr. A.G. M Ng wena and Pr<strong>of</strong>. R. Ochieng for their pr<strong>of</strong>essional guidance and superior advice during<br />
the course <strong>of</strong> the research. I also sincerely thank Dr. Ndiema <strong>of</strong> School <strong>of</strong> Medicine for always being<br />
ready to help.<br />
Refferences<br />
Ads by google (2009); htttp://pregnancy.lovetoknow.com/wiki/Male-contraception-options<br />
Anatomy and Physiology <strong>of</strong> Animals/Reproductive System Wikibooks (2010); Oct. 2010.<br />
en.wikibooks.org/wiki/anatomy../reproductivesystem<br />
BMJ 2004;329: 1156-1159 (13 November), doi: 10.1 136/bmj.329.7475. 1156 Van Wyk, 1t.E. &.Wikipedia August, 2009<br />
Channing C.P, Schaerf F.W, Anderson L.D, et al. (1980); Ovarian follicular and luteal physiology. In: Greep RO, editor.<br />
International Review <strong>of</strong> Physiology. Baltimore: University Park Press, I. I.<br />
F. Russel Westwood (2008); The female reproductive cycle: A practical Histological guide to staging. PP 1-10.<br />
Markiewicz L. Garey J. Aldecreutz H. et al. (1993); In vitro bioassays <strong>of</strong> non-steroidal phytoestrogens. J. Steroid Biochem<br />
Mol Biol 45:399-405.<br />
Shaunfang Li and Barbara Davis (2007); Evaluating rodent vaginal and uterine histology in Toxicity studies. Birth defects<br />
Research part B: Developmental and Reproductive Toxicology Vol.80. Issue 3. PP. 246-252.<br />
252
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[YS 19] Phytochemical Evaluation <strong>of</strong> Elaeodendron buchananii Stem Bark for<br />
Microbial Activities<br />
1 Odak Jenipher A., 1 Manguro Lawrence O. and 1 Ogur Joseph A<br />
1 Maseno University, Department <strong>of</strong> Chemistry, P.O.Box 333 Maseno, Kenya. odakjenipher@yahoo.com<br />
Key words: Elaeodendron buchananii (Loes), stem bark, coumarins, sterols, triterpenoids, and antimicrobial activities.<br />
Introduction<br />
D<br />
espite the wide availability <strong>of</strong> clinically useful antifungal and antibacterial drugs, there is need<br />
for search for effective antimicrobials with new strategies to replace those with limited<br />
antimicrobial spectrum. Plants that are used traditionally in the management <strong>of</strong> ailments could be<br />
sources <strong>of</strong> safe and effective drugs. Elaeodendron buchananii (Loes) belongs to the family<br />
Celastraceae and is a tree <strong>of</strong> tropical Africa that grows to 20m high with dense evergreen foliage<br />
found in wooded grassland and dry evergreen forest (Burkill, 1985). Many communities in Africa<br />
use E. buchananii stem bark to manage fungal (Vazquez, 2000) and bacterial (Kokwaro, 1976;<br />
Bekalo et al., 1996; Maundu and Tengnas, 2005) infections. Previous phytochemical analyses on its<br />
fruits and root bark revealed steroids and terpenoids (Kubo and Fukuhara, 1990; Tsanuo et al.,<br />
1993; Tsujino et al., 1995). The current investigation was undertaken because no phytochemical<br />
evaluations <strong>of</strong> the stem bark had been done despite its widespread use to manage both fungal and<br />
bacterial infections.<br />
Material and Methods<br />
Plant material<br />
The plant material was collected from Ngong Forest, Kenya (1°18'13.62"S; 36° 43' 07.11" E; Altitude<br />
5808ft above mean sea level) in April 2007. A voucher specimen <strong>of</strong> the plant was deposited in the<br />
Department <strong>of</strong> Botany, University <strong>of</strong> Nairobi following identification by the Taxonomist.<br />
Test organisms<br />
Gram-positive (Staphylococcus aureus, ATCC 25923, Diplococcus pneumoniae and Staphylococcus<br />
albus), gram-negative bacteria (Eschirichia coli, ATCC 25922, Vibrio cholerae, Shigella dysenterae<br />
and Neisseria meningitidis) and fungi (Candida albicans, ATCC 90028 and Cryptococcus<br />
ne<strong>of</strong>ormans) were obtained from the Microbiology Section <strong>of</strong> the New Nyanza Provincial General<br />
Hospital, Kisumu, Kenya.<br />
Isolation <strong>of</strong> compounds and characterization<br />
The shade-dried ground powdered stem bark (2kg) was exhaustively extracted sequentially using n-<br />
hexane, ethyl acetate and methanol at room temperature. The extracts were fractionated over<br />
silica gel packed as outlined in Figure 1. Structural elucidation was done by MS, IR and NMR.<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Disc diffusion assay<br />
The paper disc diffusion method was employed according to method described by Murray et al., 1999 with<br />
some modifications. The extracts and isolates were used at concentrations 1.5 and 5mg/ml respectively.<br />
Tetracyline (30 g/disk), gentamicin (10 g/ disk), clotrimazole (3 g/disk) and 5% DMSO ware used as<br />
control.<br />
Minimum Inhibitory Concentration (MIC) determination<br />
The MIC was determined by broth microdilution method according to Murray et al., 1999.<br />
Gentamycin and clotrimazole were used as positive control, 5% DMSO was used as negative<br />
control. The assays were done in replicate and analyzed statistically using MSTAT-C statistical<br />
package.<br />
Results and Discussions<br />
The n-hexane extract yielded 3 -acetylamyrin (5), stigmasterol (8) and 3-Ox<strong>of</strong>riedooleonane (2)<br />
while ethyl acetate extract gave coumarin (6), -sitosterol (9), 3 -hydroxyfriedooleonane (3),<br />
umbelliferone (7), carnophyllol (1) and ursolic acid (4) in addition to those obtained from n-hexane<br />
extract. Methanol extract gave negligible isolates and were not analyzed.<br />
R 1<br />
R 2<br />
. R 1O<br />
254<br />
R 2
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
3: R1=OH, R2=CH3<br />
1: R1=O, R2=CH2OH 4: R1= H R2 =CO2H<br />
2: R1=O, R2=CH3 5: R1=OCH3 R2 =CH3<br />
R O O<br />
HO 5<br />
6: R = H 8<br />
7: R =OH<br />
-Sitosterol (9) differed from compound 8 by the absence <strong>of</strong> C=C at position 22. These nine<br />
compounds have been isolated from this plant for the first time.<br />
The ethyl acetate extract exhibited strong antibacterial activities against most bacteria tested Table<br />
1; E. coli, (15.2 mm), D. pneumoniae (16.1 mm), S. albus (19.2 mm), S. aureus (22.2mm), N.<br />
meningititis (24.1 mm) and the best MIC <strong>of</strong> 15.62 g/ml against N. meningititis. The extract had<br />
better activity than the conventional gentamyacin used as positive control (zone and MIC <strong>of</strong> 21.13<br />
mm and 31.3 respectively against S. aureus). Methanol extract displayed strong antifungal activities<br />
especially against Candida albicans (zone 25.13mm and MIC 31.25 g/ml). The n-hexane extract<br />
showed mild antimicrobial activities.<br />
255<br />
6<br />
22<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Table 1: Antimicrobial activities <strong>of</strong> the extracts <strong>of</strong> E. buchananii stem bark<br />
Microorganisms<br />
Extract/standard SA DP SAL EC VC SD NM CN CA<br />
Sample<br />
(1.5mg/ml)<br />
n-hexane 12.1 10.1 11.3 9.3 5.2 5.2 14.2 3.2 5.1<br />
Ethyl<br />
acetate<br />
Tetracycline (30 g<br />
/disc)<br />
Gentamyacin (10 g<br />
/disc)<br />
Clotrimazole (30 g<br />
/disc)<br />
22.2 16.1 19.2 15.2 12.2 13.1 24.1 13.5 15.1<br />
methanol 14.2 12.2 13.2 13.1 <strong>9.1</strong> 10.2 16.2 16.1 25.1<br />
12.1 14.2 14.8 14.2 10.1 9.2 13.2 NT NT<br />
21.1 17.2 18.2 17.2 15.2 14.1 16.8 NT NT<br />
NT NT NT NT NT NT NT 15.1 22.2<br />
SA= S. aureus, DP= D. pneumoniae =SAL= S. albus, EC= E. coli, VC=V. cholerae SD=S. dysenterae, NM= N.<br />
meningititis, CN=C.ne<strong>of</strong>ormans,CA= C. albicans, NT=Not tested<br />
Coumarin (6) and umbelliferone (7) showed antifungal activities (11.2mm and 13.6mm respectively<br />
against C. albicans) while 3-Ox<strong>of</strong>riedooleonane/friedelin (2) and carnophyllol (1) exhibited<br />
antibacterial activities. Ursolic acid (4) displayed antibacterial (l13.2mm against C. albicans) and<br />
antifungal (12.2mm against C. albicans) activities.<br />
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Table 2: Antimicrobial activities <strong>of</strong> the isolates from E. buchananii stem bark<br />
Compound Microorganisms<br />
SA DP SAL EC VC SD NM CN CA<br />
Canophyllol (1) 11.2 10.2 12.2 11.2 8.1 5.2 15.1 3.1 3.2<br />
3 -acetyl amyrin<br />
(5)<br />
10.1 8.2 11.2 3.2 3.1 7.2 10.3 0.0 0.0<br />
Coumarin(6) 4.2 5.2 5.1 0.0 0.0 3.2 9.2 11.2 10.3<br />
Umbelliferone(7) 9.2 5.1 7.3 4.2 3.2 3.2 5.2 10.8 13.6<br />
Ursolic acid (4) 13.2 10.2 12.3 8.2 5.4 3.3 7.2 9.2 12.2<br />
Hydroxy friedelin<br />
(3)<br />
Friedelin (2)<br />
5.2 0.0 0.0 0.0 0.0 0.0 9.2 10.2 9.2<br />
14.2 13.2 10.2 12.2 10.3 <strong>9.1</strong> 14.2 3.2 7.4<br />
Sitosterol (9) 5.2 5.1 3.2 0.0 0.0 0.0 6.2 10.2 7.2<br />
Tetracycline 11.9 14.2 14.8 14.1 10.1 <strong>9.1</strong> 13.1 NT NT<br />
Gentamycin 21.1 17.2 17.6 17.1 15.1 14.1 16.5 NT NT<br />
Clotrimazole NT NT NT NT NT NT NT 15.2 21.9<br />
CV% 1.06 1.14 1.17 1.356 1.53 1.90 0.72 0.99 0.74<br />
LSD Comp<br />
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Acknowledgements<br />
The authors are grateful to Pr<strong>of</strong>. Johann Jauch, University <strong>of</strong> Saarlandes, Germany for analysing samples and<br />
availing the IR, MS and NMR spectra and Dr. Santana Odhiambo <strong>of</strong> New Nyanza Provincial General Hospital<br />
for his assistance in bioassay experiments.<br />
References<br />
Bekalo, I. M.; Keengwe, E.; Mathias, P.; Mundy, A. L. (1996). Ethnoveterinary Medicine in Kenya. A field manual <strong>of</strong><br />
traditional animal health care practice Intermediate Technology Development Group and International Institute <strong>of</strong><br />
Rural Reconstruction Nairobi, Kenya p 226.<br />
Burkill, H. M. (1985). Royal Botanic Gardens (Kew). The useful plants <strong>of</strong> West Tropical Africa, 1, 121-122.<br />
Kokwaro, J. O. (1976). Medicinal plants <strong>of</strong> East Africa, 3 rd Edition Kenya Literature Bureau, Kampala, Nairobi, Dar-essalaam.,<br />
pp 254-257.<br />
Maundu, P.; Tengnas, B. (2005). Useful trees and plants for Kenya. Technical Handbook, Nairobi Kenya, 35, p 216.<br />
Murray, P. R.; Baron, E. J.; Pfaller, M. A.; Tenover, F. C.; MYolken, R. H. (1999). National Committee for Clinical<br />
Laboratory Standards. Antibacterial Susceptibility Tests: Dilution and Disk Diffusion Methods. Manual <strong>of</strong> Clinical<br />
Microbiology Washington D.C.: American Society for Microbiology, pp 1526-1543.<br />
Tsanuo, M. K.; Hassanali, A.; Jondiko I. J.; Torto, B. (1993). Mutangin, a dihydroagar<strong>of</strong>uranoid sesquiterpene insect<br />
antifeedant from Elaeodendron buchananii. Phytochemistry, 34, 665-667.<br />
Tsujino, Y.; Ogoche, I. J.; Tazaki, H.; Fujimori, T.; Mori, K. (1995). Buchananioside, a steroidal glycoside from<br />
Elaeodendron buchananii. Phytochemistry, 40, 753-756.<br />
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[YS 20] Evaluating Community Knowledge, Management and Economic Losses<br />
due to a Zoonotic Disease: A Case Study <strong>of</strong> Newcastle Disease in Kasese Municipal<br />
Council, Western Uganda<br />
Baluku Joward<br />
Key Words: Poultry, Poultry farmers, Newcastle Disease (NCD), Newcastle Disease Virus (NCDV)<br />
Introduction<br />
P<br />
oultry production is recognised as an important activity in all developing countries.However,<br />
over the past few decades, the focus has been on the production <strong>of</strong> commercial poultry in rural<br />
areas, while traditional village poultry systems have been largely ignored.There are many<br />
constraints to poultry production (Sonaiya et al. 1999) including a range <strong>of</strong> bacterial and other viral<br />
diseases, internal and external parasites (Permin and Hansen 1998), poor nutrition and predation.<br />
Poultry farmers are disheartened by the loss <strong>of</strong> large numbers <strong>of</strong> their birds to NCD outbreaks that<br />
<strong>of</strong>ten occur on an annual basis.<br />
NCDV is wide spread among several different taxonomic groups <strong>of</strong> wild birds and appears capable<br />
<strong>of</strong> infecting all species <strong>of</strong> birds (especially the domestic chickens) and other vertebrates including<br />
humans.<br />
Collecting information on knowledge regarding folk beliefs, skills, methods and practices pertaining<br />
to the management <strong>of</strong> Newcastle Disease in poultry enables veterinarians to understand farmers<br />
knowledge <strong>of</strong> the disease transmission process, local remedies that may be worthy <strong>of</strong> further study<br />
and the type <strong>of</strong> animal husbandry currently being practiced.<br />
Objectives<br />
i. To establish peoples knowledge with respect to causes and risk factors <strong>of</strong> Newcastle<br />
Disease.<br />
ii. To evaluate the economic losses due to Newcastle Disease among households.<br />
iii. To establish the practices for management <strong>of</strong> Newcastle Disease.<br />
Materials and Methods<br />
The study was conducted on poultry farmers randomly sampled from the two parishes <strong>of</strong><br />
Nyakabingo II (far from QENP) and Railway (at the border with QENP), each with three villages in<br />
the central division <strong>of</strong> Kasese Municipality. The questionnaires were distributed randomly to 20<br />
poultry farmers from each <strong>of</strong> the 6 villages.Data analysis was performed using the descriptive<br />
method for the qualitative data from the two parishes, to capture information with regard to<br />
household size, flock structure, signs observed, causes and management <strong>of</strong> NCD, and economic loss<br />
suffered due NCD outbreak.<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Results and Discussion<br />
Seasonality <strong>of</strong> outbreaks <strong>of</strong> NCD<br />
Figure 1: The probability by month <strong>of</strong> an outbreak <strong>of</strong> NCD occurring according to the experience <strong>of</strong><br />
local farmers.<br />
Causes<br />
Table 6: Possible causes <strong>of</strong> NCD according to the opinion <strong>of</strong> the farmers<br />
CAUSES Percentage<br />
260<br />
Nyakabingo II Railway<br />
Too much sunshine(drought 30 31.7<br />
Introduction <strong>of</strong> new birds especially those from markets 18.3<br />
Poor hygiene especially in the poultry houses 13.3 5<br />
Congestion/overcrowding in the poultry house 5 10<br />
Rainy weather 3.3 3.3<br />
Wondering <strong>of</strong> the birds mainly due to the free ranging<br />
type <strong>of</strong> production system<br />
3.3 1.7
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Risks<br />
Table 7: Risk factors that may increase the chances <strong>of</strong> transmission and spread <strong>of</strong> NCD, according to<br />
farmers<br />
NYAKABINGO II RAILWAY<br />
Interaction with other birds<br />
Improper disposal <strong>of</strong> the remains <strong>of</strong><br />
birds after slaughter<br />
Free ranging type <strong>of</strong> production system<br />
Introduction <strong>of</strong> new birds either for restocking<br />
or reinforcement <strong>of</strong> an<br />
existing stock<br />
Rodents and other wild animals<br />
especially those that predate on the<br />
chickens<br />
Wild birds<br />
Poor housing<br />
Too much sunshine<br />
Congestion in the poultry houses<br />
Treatment <strong>of</strong> NCD<br />
Table 8: Forms <strong>of</strong> treatments used by the respondents<br />
261<br />
Wild birds <strong>of</strong> prey<br />
Free ranging type <strong>of</strong> production system<br />
Wondering<br />
Beatings by malicious neighbours<br />
especially children, which mostly affects<br />
ducks and ducklings<br />
Drought (too much sunshine)<br />
The sewage dumping pit (lagoon) from<br />
which some chickens drink when the day<br />
gets hot<br />
Introduction <strong>of</strong> new birds from anywhere<br />
The garbage/waste plant for the Clean<br />
Development Mechanism.<br />
Treatment NYAKABINGO II RAILWAY<br />
Frequency %age Frequency %age<br />
Conventional only 1 1.7 1 1.7<br />
Conventional (Name forgotten) 7 11.6 10 16.7<br />
Traditional only 33 55 32 53.3<br />
Both 9 15 9 15<br />
None 10 16.7 14 23.3<br />
Total 60 100 60 100
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
How the plants/items can be combined<br />
Tobacco 0 + + + + + + + +<br />
Cannabis + 0 + + + + + + + + + + + + +<br />
Red<br />
pepper<br />
+ + 0 + + + + + + + + + + + +<br />
Ash + + + 0 + + + + + + + + + + + + + +<br />
Pawpaw<br />
roots<br />
+ + + 0 + + + + + +<br />
Aloe + + + + 0 + + + + + +<br />
Palm oil + + + + + + 0 +<br />
Bitter leaf + +<br />
Tall<br />
fleabane<br />
Euphorbia + + + + 0<br />
Urine + + + + + +<br />
Soot + + + + + + + +<br />
Rock salt + + + + +<br />
Poke root + + + +<br />
Scales <strong>of</strong> a<br />
snake<br />
+ + +<br />
Garlic + + + +<br />
Esyantony<br />
era<br />
0<br />
Moringa + + + +<br />
Faeces <strong>of</strong><br />
ducks<br />
Black jack +<br />
vegetable<br />
oil<br />
+ + +<br />
Paraffin + + +<br />
Neem +<br />
Pluchea<br />
ovalis<br />
KEY<br />
+ is combined with<br />
0 can be used alone<br />
Birds lost in previous encounter<br />
262<br />
0<br />
+
4.4.2 Financial loss caused<br />
Poultry<br />
type<br />
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Number lost Av. Unit cost Total cost<br />
Nyakabingo II Railway Nyakabingo II Railway Nyakabingo II Railway<br />
Hens 219 267 10,000 10,000 2,190,000 2,670,000<br />
Cocks 90 83 20,000 25,000 1,800,000 2,075,000<br />
Chicks 205 393 - - - -<br />
Ducks 13 15 12,500 20,000 162,500 300,000<br />
Guinea<br />
fowl<br />
0 2 15,000 20,000 0 40,000<br />
Pigeons 0 0 4,000 4,000 0 0<br />
Turkeys 4 12 40,000 40,000 160,000 480,000<br />
TOTAL 531 772 4,312,500 5,565,000<br />
Conclusion<br />
A wide range <strong>of</strong> herbal medicines were used in poultry health (NCD) management. Although these<br />
herbal drugs were used in poultry disease management, there are information gaps related to<br />
efficacy, effectiveness, lethal doses and standardized doses and active ingredients <strong>of</strong> these plants.<br />
Therefore, this study recommends that further research on information gaps be identified and<br />
investigated.<br />
NCD causes a financial loss <strong>of</strong> more than 10million Uganda shillings annually, just in only the two<br />
parishes <strong>of</strong> Kasese Municipal council, which when extrapolated for the whole District and then<br />
country, is a lot <strong>of</strong> money. Therefore improved poultry productivity through improving disease<br />
(especially NCD) management is likely to contribute to poverty alleviation in the rural areas/villages.<br />
Reference<br />
1. Alders, R., and Spradbrow, P. (2001); Controlling Newcastle Disease in Village Chickens. A Field Manual<br />
2. Permin, A. and Hansen, J.W. (1998); Epidemiology, Diagnosis and Control <strong>of</strong> Poultry Parasites. FAO Animal Health<br />
Manual No. 4. Rome, FAO.<br />
3. Sonaiya, E.B., Branckaert, R.D.S. and Guèye, E.F. (1999); Research and development options for family poultry.<br />
First INFPD/FAO Electronic Conference on Family Poultry, Guèye, E.F., (Ed).<br />
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[YS 21] Further Phytochemical and Antimicrobial Activity Studies <strong>of</strong> Warburgia<br />
Ugandensis against Sweet Potato Pathogens<br />
Sylvia A. Opiyo a , Lawrence O.A. Manguro a , Philip Okinda-Owuor a , Elijah M. Ateka b and Peter<br />
Lemmen c<br />
a<br />
Department <strong>of</strong> Chemistry, Maseno University, P. O. Box 333, Maseno, Kenya;<br />
b<br />
Department <strong>of</strong> Horticulture, Jomo Kenyatta University <strong>of</strong> Agriculture and Technology, P.O. Box, 62000-00200, Nairobi,<br />
Kenya;<br />
c<br />
Department <strong>of</strong> Chemistry, Technical University <strong>of</strong> Muenchen, Lichtenbergstrasse 4, 85747 Garching, Germany<br />
E-mail address: sylvopiyo@yahoo.com<br />
Keywords: Warburgia ugandensis; Canellaceae; 7 -aceylugandensolide; antibacterial; antifungal.<br />
Introduction<br />
S<br />
weet potato is important potato crop worldwide since it is drought tolerant and acts as a<br />
famine relief crop (Gibson et al., 1997). However, its production is limited by viral, fungal and<br />
bacterial infections (Gibson et al., 1997). Use <strong>of</strong> synthetic chemicals to manage the infections cause<br />
adverse effects to the ecosystem (Cameron and Julian, 1984) and is unaffordable by most farmers.<br />
Moreover, resistance by pathogens has rendered some chemicals ineffective. There is a need to<br />
search for affordable, readily available, sustainable and environmentally friendly means <strong>of</strong><br />
managing the pathogens. This study evaluated the antimicrobial activity <strong>of</strong> W. ugandensis extracts<br />
and isolates, which are traditionally used as a remedy for fungal and bacterial infections (Kokwaro,<br />
2009), in the management <strong>of</strong> sweet potato infections.<br />
Materials and methods<br />
Powdered stem bark <strong>of</strong> W. ugandensis was sequentially extracted with n-hexane, EtOAc and<br />
MeOH. Crude extracts were subjected to repeated column chromatography over silica gel to give<br />
pure compounds. Extracts and isolates were tested for antimicrobial activity against sweet potato<br />
pathogens: Alternaria spp, Aspegillus niger, Fusarium oxysporum, F. solani, Rhizopus stolonifer<br />
(fungi), Ralstonia solanacearum and Steptomyces ipomoeae (bacteria) using disc diffusion method<br />
(Barry et al., 1979).<br />
Results and discussion<br />
Phytochemical studies afforded one new sesquiterpene, 7 -aceylugandensolide (1), together with<br />
thirteen known ones namely bemadienolide, drimenin, polygodial, warburganal, mukaadial,<br />
ugandensidial, muzigadial, 6 -hydroxymuzigadial, 9-deoxymuzigadial, ugandensolide,<br />
deacetoxyugandensolide, cinnamolide and 3 -acetoxycinnamolide. Their structures were<br />
determined using spectroscopic methods as well as comparison with literature data.<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
H 3b<br />
H 2a<br />
Me15 H1a H 3a<br />
H 2b<br />
Me 14 H 1b<br />
H 5<br />
1<br />
Me 14<br />
H6<br />
Antimicrobial activity<br />
All extracts were active against the tested pathogens (Table 1). Ethyl acetate extract exhibited the<br />
highest (P 0.05) inhibitory effects against the tested pathogens. A. niger and R. stolonifer were the<br />
most susceptible to the ethyl acetate extract.<br />
Table 1: Antimicrobial activity <strong>of</strong> crude extracts<br />
265<br />
OAc<br />
O<br />
OAc<br />
O<br />
H 7b<br />
H 12a<br />
Test microorganism *Zone <strong>of</strong> growth inhibition in mm<br />
EtOAc<br />
H 12b<br />
Extracts Standard drugs<br />
nhexane<br />
Methan<br />
ol Blitox Streptocycline<br />
Fungi Alternaria<br />
spp.<br />
22.1 1<strong>9.1</strong> 9.4 27.1 ND<br />
A. niger 26.1 16.5 8.1 33.0 ND<br />
F. oxysporum 18.4 13.1 6.0 21.9 ND<br />
F. solani 14.4 9.0 8.5 30.1 ND<br />
R. stolonifer 29.5 18.7 10.6 23.3 ND<br />
Bacteri<br />
a<br />
R.<br />
solanacearum<br />
21.2 17.3 10.0 ND 23.8<br />
S. ipomoeae 17.3 15.2 11.2 ND 19.4<br />
Mean 21.3 15.2 11.2 27.1 21.6<br />
*Values are means <strong>of</strong> three replicates and includes 5 mm diameter <strong>of</strong> disk; ND = Not done.<br />
Out <strong>of</strong> the 14 isolates, polygodial and mukaadial were the most effective against Alternaria spp<br />
(MIC = 25 µg/ml) while warbuganal and mukaadial showed strong activity against A. niger (MIC =<br />
12.5 µg/ml) (Table 2). Warbuganal and muzigadial exhibited fairly strong activity against F.<br />
oxysporum (MIC = 25 µg/ml) while polygodial and warburganal gave promising result with F. solani<br />
(MIC = 12.5 µg/ml). R. stolonifer was also found to be susceptible to compounds warbuganal and<br />
ugandensidial (MIC = 25 µg/ml).<br />
Table 2: Minimum inhibitory concentration (MIC, µg/ml) <strong>of</strong> isolated compounds<br />
Compound MIC, µg/ml <strong>of</strong> isolated compounds<br />
Test fungi Test bacteria
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Alter<br />
spp<br />
A.<br />
nig<br />
F.<br />
oxy<br />
266<br />
F. sol R. sto R. sola S. ipo<br />
7 -<br />
Acetoxyugandensolide<br />
(1)<br />
>200 >200 >200 >200 >200 200 >200<br />
Bemadienolide >200 >200 >200 >200 >200 >200 100<br />
Drimenin >200 >200 >200 >200 >200 >200 >200<br />
Polygodial 25 50 50 12.5 50 25 50<br />
Warburganal 50 12.5 25 12.5 25 50 50<br />
Mukaadial 25 12.5 100 25 50 25 50<br />
Ugandensidial 50 25 50 100 25 100 25<br />
Muzigadial 50 50 25 100 50 25 50<br />
6 -Hydroxymuzigadial 200 >200 100 >200 >200 100 >200<br />
9-Deoxymuzigadial >200 >200 >200 >200 >200 >200 >200<br />
Ugandensolide 50 50 100 200 100 >200 100<br />
Deacetoxyugandensolid<br />
e<br />
50 >200 >200 100 200 100 200<br />
Cinnamolide 100 100 >200 200 >200 >200 >200<br />
3 -Acetoxycinnamolide >200 >200 >200 >200 >200 >200 >200<br />
Blitox 50 6.25 12.5 6.25 12.5 ND ND<br />
Streptocycline ND ND ND ND ND 25 12.5<br />
ND = Not done; Alter spp = Alternaria spp, A. nig = Aspegillus niger, F. oxy = Fusarium oxysporum, F.<br />
sol = Fusarium solani, R.. sto = Rhizopus stolonifer, R.. sola = Ralstonia solanacearum, S. ipo =<br />
Steptomyces ipomoeae.<br />
This study revealed that extracts <strong>of</strong> W. ugandensis have antimicrobial activity against F. oxysporum,<br />
F. solani, Alternaria spp, R. stolonifer, A. niger R. solanacearum and S. ipomoeae which are soil<br />
pathogens associated with rotting <strong>of</strong> sweet potato and other root crops (Ristaino, 1993). This<br />
suggests that the pathogens can be managed using herbal extracts as had also been observed in<br />
other studies (Okigbo and Nmeka, 2005). The herbal extracts are more environmentally safe<br />
compared to the synthetic antimicrobial drugs currently used (Masuduzzaman et al., 2008; Siva et<br />
al., 2008). Extracts from W. ugandensis are not only active against fungi and bacteria that cause<br />
disease in animals/man (Kubo and Nakanishi, 1979; Mbwambo et al., 2009) but are also active<br />
against plant pathogens thus suggesting that the antimicrobial principles in the plant have broad<br />
spectrum activity.<br />
Acknowledgement<br />
The authors are thankful to Kenya Medical Research Institute (KEMRI), Kisumu, Kenya for the use <strong>of</strong><br />
their laboratory to perform the biological activity tests and Biosciences Eastern and Central Africa<br />
Network (BecANet) for financial support. Mr. Mutiso <strong>of</strong> Botany Department, Nairobi University is<br />
thanked for plant identification.
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
References<br />
Kokwaro, J.O., 2009. Medicinal Plants <strong>of</strong> East Africa. University <strong>of</strong> Nairobi Press, Nairobi, Kenya.<br />
Barry, A.L., Coyle, M.B., Gerlach, E.H., Haw-Kinson, R.W., Thornberry, C., 1979. Methods <strong>of</strong> measuring zones <strong>of</strong><br />
inhibition with the Baver-Kirby disc susceptibility test. Clin. Microbial. 10, 885-889.<br />
Cameron, H.J., Julian, G.R., 1984. The effects <strong>of</strong> four commonly used fungicides on the growth <strong>of</strong> cyanobacteria. Plant<br />
Soil, 78, 409-415.<br />
Gibson R.W., Mwanga R.O.M., Kasule S., Mpembe I., Carey E.E., 1997. Apparent absence <strong>of</strong> viruses in most<br />
symptomless field-grown sweet potato in Uganda. Ann. Appl. Biol. 130, 481-490.<br />
Okigbo, R.N., Nmeka, I.A., 2005. Control <strong>of</strong> Yam tuber rot with leaf extracts <strong>of</strong> Xylopia aethiopica and Zingiber <strong>of</strong>ficinale.<br />
Afr. J. Biotec. 4, 804-807.<br />
Masuduzzaman, S., Meah, M.B., Rashid, M.M., 2008. Determination <strong>of</strong> inhibitory action <strong>of</strong> Allamanda leaf extracts<br />
against some important plant pathogens. Agric. Dev. 6, 107-112.<br />
Mbwambo, Z.H., Erasto, P., Innocent, E., Masimba, P.J., 2009. Antimicrobial and cytotoxic activities <strong>of</strong> fresh leaf extracts<br />
<strong>of</strong> Warburgia ugandensis. Tanz. Health Res. 11, 75-78.<br />
Siva, N., Ganesan, S., Banumathy, N., Muthuchelian, J., 2008. Antifungal effect <strong>of</strong> leaf extract <strong>of</strong> some medicinal plants<br />
against Fusarium oxysporum causing wilt disease <strong>of</strong> Solanum melogena L. Ethnobot. Leafl. 12, 156-163.<br />
Ristaino, J.B., 1993. Infection <strong>of</strong> sweet potato fibrous roots by Streptomyces ipomoeae: Influence <strong>of</strong> soil water potential.<br />
Soil Biol. 25, 185-192.<br />
Kubo, I., Nakanish, I.K., 1979, Advances in pesticide Science (Geissbuhler, H, ed), p 284. Paragon Press, New York.<br />
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[YS 22] Medicinal Plants used in Disease Management among Children in<br />
Namungalwe Sub County, Iganga District<br />
Nalumansi Patricia, Kamatenesi Maud-Mugisha, John Robert Steven Tabuti<br />
Makerere University, College <strong>of</strong> Agricultural and Environmental Sciences, Department <strong>of</strong> Environmental Management,<br />
P.O. Box 7062, Kampala 1 nalumansip@yahoo.com<br />
Makerere University, College <strong>of</strong> Agricultural and Environmental Sciences, Department <strong>of</strong> Environmental Management,<br />
P.O. Box 7062, Kampala 2<br />
Makerere University, College <strong>of</strong> Natural Sciences, School <strong>of</strong> Biological sciences P.O. Box 7062, Kampala<br />
KEY WORDS: Children, traditional medicine, Medicinal plants, diseases<br />
Introduction<br />
T<br />
he national under-five mortality rate is 137 deaths per 1,000 live births and infant mortality<br />
rate is 75 deaths per 1,000 live births. Seventy percent <strong>of</strong> overall child mortality is due to<br />
malaria (32%), perinatal and neonatal conditions (18%), meningitis (10%), pneumonia (8%), HIV and<br />
AIDS (5.6%) and malnutrition (4.6%) (The Second NHP, 2010).<br />
Utilisation <strong>of</strong> the health facilities in Uganda is still a challenge due to poor infrastructure,<br />
inadequate medicines and other health supplies, the shortage and low motivation <strong>of</strong> human<br />
resource (the second NHP, 2010). And poverty aggravates the prevalence <strong>of</strong> diseases such as<br />
malaria, malnutrition and diarrhea (UBOS, 2007).<br />
Over 80% <strong>of</strong> the people World depend on medicinal plant species to meet their day today<br />
healthcare needs (WHO, 2002). The use <strong>of</strong> traditional medicine in rural Ugandan population for<br />
day-to-day health care needs is close to 90% (Kamatenesi and Oryem, 2006). The treatments <strong>of</strong><br />
most ailments that women and children suffer especially in rural areas depend on herbs first and in<br />
case the condition deteriorates, then they seek health facilities.<br />
Materials and Methods<br />
Ethno botanical data was collected by carried out household interviews, key informants traditional<br />
healers and traditional birth attendants. These techniques were complemented by direct<br />
observation, photography, collecting voucher specimens and making notes <strong>of</strong> relevant issues.<br />
Results<br />
A total <strong>of</strong> 67 species and one mushroom Termitomyces microcarpus were documented as medicinal<br />
plants used in the disease management among children. These species belonged to 38 families and<br />
61 genera. Faboideae (6) family had the most number <strong>of</strong> plant species. Herbs (37%) were the most<br />
used plant life forms in disease management among the children. Leaves (54%) were the most used<br />
plant parts. These plant species are mainly harvested from the wild (68%). The plants were mainly<br />
boiling. Vernonia amygdalina, Chenopodium opulifolium and Albizia corialia was the most<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
mentioned plant species.Twenty seven percent <strong>of</strong> the recorded plant species were reported for<br />
treating malaria. The commonest ailments were digestive system disorders.<br />
Discussion<br />
There is a diversity <strong>of</strong> knowledge on medicinal plants known to be used in disease management<br />
among children in Namungalwe Sub County. Majority <strong>of</strong> the respondents were women (54%) and<br />
these were using the plants in the treatment <strong>of</strong> diseases among children. Vernonia amygdalina,<br />
Chenopodium opulifolium and Albizia corialia are the commonly known to be used medicinal plant<br />
species. These have been reported by different researchers as medicinal plants used by local<br />
communities country wide. A lot <strong>of</strong> knowledge has existing on the plants used in the treatment <strong>of</strong><br />
malaria because in Uganda, it is highly prevalent and according to the (the second NHP, 2010),<br />
malaria (32%) is the leading cause <strong>of</strong> child mortality.<br />
Conclusion<br />
There is diversity <strong>of</strong> traditional knowledge on medicinal plants used in the management <strong>of</strong> ailments<br />
among children in Namungalwe Sub County.<br />
References<br />
Kamatenesi. M. M and O. H Oryem (2006): Medicinal plant species used to induce labour during childbirth in western<br />
Uganda. Journal <strong>of</strong> Ethnopharmacology 109:1-9.<br />
The Second National Health Policy (2010): Promoting People s Health to Enhance Socio-economic Development. The<br />
Republic <strong>of</strong> Uganda Ministry <strong>of</strong> Health<br />
WHO (2002): Mental health Global Action Program (MHGAP), Geneva, Switzerland.<br />
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[YS 23] Antiplasmodial and Antinociceptive constituents from Caesalpinia<br />
volkensii Harms (Caesalpiniaceae) Root Bark<br />
Charles O Ochieng 1 , P. Okinda Owuor 1 , Lawrence A.O. Mang uro 1 , Hosea Akala 2 , Ismail O. Ishola 3 .<br />
1<br />
Department <strong>of</strong> Chemistry, Maseno University, Private Bag, 40105, Maseno, Kenya.<br />
2<br />
United State Army Medical Research Unit-Kenya, MRU 64109, APO, AE 09831-4109, USA.<br />
3<br />
Department <strong>of</strong> Pharmacology, Faculty <strong>of</strong> Basic Medical Sciences, College <strong>of</strong> Medicine, University <strong>of</strong> Lagos, P.M.B.<br />
12003 Lagos, Nigeria<br />
Corresponding author: otieno.charles9@gmail.com<br />
KEY WORDS: Caesalpinia volkensii, voucapan-1, 5-diol, deoxycaesaldekarin D, antiplasmodial activity, antinociceptive<br />
activity<br />
Introduction<br />
D<br />
espite intense research, studies on more therapeutic options towards management <strong>of</strong> malaria<br />
are attractive due to the polymorphism <strong>of</strong> Plasmodium falciparum. Caesalpinia volkensii H.<br />
(Caesalpiniaceae) is used in East Africa for management <strong>of</strong> many diseases, including malaria, pain<br />
during pregnancy, aphrodisiac and retinoblastoma (Kokwaro, 2009). Medicinal values are ascribed<br />
to its root back, seed kernels and leaves (Beentje, 1994). However, pharmacogically active<br />
components responsible for the activities have not been identified. This study evaluated the<br />
antiplasmodial and antinociceptive actions <strong>of</strong> the organic extracts prepared from root bark <strong>of</strong> C.<br />
volkensii and characterized the active compounds.<br />
Materials and Methods<br />
The root bark <strong>of</strong> Caesalpinia volkensii was collected from Eldoret County <strong>of</strong> Kenya, identified and a<br />
voucher specimen (COO-CV- 2010-01) was deposited at the University <strong>of</strong> Nairobi Herbarium,<br />
Department <strong>of</strong> Botany. The ground-dried root bark (1.2 kg) was extracted with 95% Methanol:<br />
water, then partitioned between hexane, chlor<strong>of</strong>orm, ethyl acetate and n-butanol. The active<br />
fractions were further subjected to various chromatographic techniques (Column chromatography<br />
on silica gel and Sephadex, preparative TLC) to isolate their constituents. Antinociceptive effect <strong>of</strong><br />
the extracts and the compounds were assessed in mice using hot plate method (Eddy & Leimbach,<br />
1953) and acetic acid writhing tests (Koster et al., 1959) using Swiss albino mice (20-25 g) following<br />
the United States National Institutes <strong>of</strong> Health Guidelines for Care and Use <strong>of</strong> Laboratory Animals in<br />
Biomedical Research (NIH, 1985). In vitro antiplasmodial activity was performed using a non<br />
radioactive assay technique described by Smilkstein et al., (2004). The data were analysed using<br />
one way analysis <strong>of</strong> variance (ANOVA) followed by Bonferroni posttests and Dunnett s multiple<br />
comparison tests. Values were considered significant when P 0.05.<br />
Result and discussion<br />
On enteral administration <strong>of</strong> different extracts <strong>of</strong> Caesalpinia volkensii at doses <strong>of</strong> 100 mg/kg,<br />
chlor<strong>of</strong>orm and ethyl acetate extracts produced 40.6 and 40% inhibition <strong>of</strong> the writhing process in<br />
mice (p 0.05) (Table 1) while from the hot plate test similar trend was observed as presented in<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Table 1. On the other hand, the ethyl acetate extract (50 g/ml) showed significantly strong activity<br />
against D6 and W2 strains <strong>of</strong> Plasmodium falciparum with IC50 values <strong>of</strong> 0.23 ± 0.07 and 4.39 ± 2.49<br />
g/ml, respectively, compared to standard drug chloroquine (P 0.05) (Table 2). This prompted the<br />
chemical assessment <strong>of</strong> the components <strong>of</strong> active extracts by various chromatographic techniques<br />
leading to isolation and characterization seven furanoditerpene; voucapan-1, 5-diol (1),<br />
deoxycaesaldekarin D (2) voucapane (3), voucapan-5-ol (4), deoxycaesaldekarin C (5), caesaldekarin<br />
C (6), 5-hydroxy vinhaticoic acid (7) and three cinnamyl esters viz triacontanyl-(E)-ferulate (8),<br />
triacontanyl-(E)-caffaete (9) and 30 -hydroxytriacontanyl-(E)-ferulate (10).<br />
Table 1: Effects <strong>of</strong> Caesalpinia volkensii root bark extracts on hot plate-induced pain and acetic<br />
acid-induced writhing in mice<br />
Dose treatment (mg/kg) Pre- treat latency % inhibition <strong>of</strong> pain<br />
threshold (hot plate test)<br />
271<br />
Writhing response (%<br />
inhibition)<br />
30 min 60 min<br />
CV1 (100) 1.12 ± 0.37 1.25 6.92* 37.5<br />
CV2 (100) 1.26 ± 0.15 2.09 6.05* 40.62* a<br />
CV3 (100) 1.2 ± 0.45 3.13 8.13** 44.38** a<br />
CV4 (100) 1.34 ± 0.19 1.19 4.40 35.63<br />
Morphine (10ml/kg) 1.9 ± 0.1 10.07 14.70*** NT<br />
Ibupr<strong>of</strong>en (10) NT NT NT 81.88*** a<br />
N.saline water 1.27 ± 076 0.45 0.10 0.0<br />
Values are mean ± SEM (n = 6). Significant activities *P
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Table 2: In vitro antiplasmodial activity (50% growth inhibition) <strong>of</strong> a fractionated MeOH extract <strong>of</strong> C.<br />
volkensii and some isolates against D6 and W2 strains <strong>of</strong> Plasmodium falciparum.<br />
D6 Clone (IC50 g/ml) W2 Clone (IC50 g/ml)<br />
CVR A01 16.10±7.04 a<br />
10.84±3.85 a<br />
CVR-F02 >50 >50<br />
CVR-F03 15.68±2.42 a<br />
13.98±4.71 a<br />
CVR-F04 0.23±0.07 b<br />
4.39±2.49 b<br />
CVR-F05 >50 >50<br />
Voucapan-1, 5-diol 28.66±2.22 48.24±8.55<br />
Voucapan-5-ol >50 >50<br />
Deoxycaesaldekarin D 18.21±5.65 a<br />
15.38±3.13 a<br />
Deoxycaesaldekarin C 13.93±1.32 a<br />
13.57±1.78 a<br />
Caesaldekarin C 1<strong>9.1</strong>0±1.99 a<br />
32.58±2.87<br />
Triacontanyl-(E)-ferulate >50 >50<br />
Triacontanyl-(E)-caffaete >50 >50<br />
Chloroquine 0.005±0.002 b<br />
0.27±0.04 b<br />
Values with same superscript in the same column are statistically similar at P50 did not show activity in tested<br />
range (50 g/ml and below).<br />
The entereal administration <strong>of</strong> 3, 4, 5 and 6 (100 mg/kg) caused a significant reduction in the<br />
number <strong>of</strong> writhing episodes induced by acetic acid (Fig 1) and increased pain threshold on hot<br />
plate method (Fig 2) statistically similar to the ibobrufen and morphine, respectively (p
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Fig 1: The antinociceptive effect <strong>of</strong> the compounds <strong>of</strong><br />
C.volkensii root bark, ibobrufen, and 0.8% saline water<br />
as observed in acetic acid-induced writhing test. Values<br />
are presented as the mean ± SEM (n = 6). ***P
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[YS 24] Documentation <strong>of</strong> Medicinal Plants Found in Keiyo County Cherebes<br />
and Endo Village<br />
Kosgey Janet Cheruiyot<br />
Chepkoilel University College, Constituent College <strong>of</strong> Moi University, School <strong>of</strong> Science,<br />
Department <strong>of</strong> Biological Science<br />
Key words: Documentation, ethnobotany, traditional medicine, indigenous knowledge, herbalist<br />
Introduction<br />
T<br />
raditional herbal remedies are an important component in the provision <strong>of</strong> primary health care.<br />
They serve as an alternative to conventional medicines which are normally too expensive for<br />
most Kenyans, but the rate at which these herbal remedies are disappearing from their natural<br />
habitat is alarming. There is little or no documentation on the uses <strong>of</strong> these plants (Tildhun and<br />
Mirutse, 2007). Moreover, the indigenous knowledge on medicinal properties <strong>of</strong> these plants is<br />
secretly guarded by the herbalists and is not available to most Kenyans. The methods that were<br />
previously used to pass the information are presently not applicable. Traditional medicine has and<br />
still remains the main source for a large majority(80%) <strong>of</strong> people in Ethiopia for treating health<br />
problems and medicinal consultancy including consumption <strong>of</strong> the medicinal plants has a much<br />
lower cost than modern medicine attention (Tildhun and Mirutse, 2007). Traditional medicine is<br />
used throughout the world as it is dependent on locally available plants, which are easily accessible,<br />
and capitalizes on traditional wisdom-repository <strong>of</strong> knowledge, simple to use and affordable<br />
(Tesfaye and Sebsebe, 2009). Plants continue to be a rich source <strong>of</strong> therapeutic agents. The<br />
remarkable contribution <strong>of</strong> plants to the drug industry was possible because <strong>of</strong> the large number <strong>of</strong><br />
phytochemical and biological studies all over the world (Kesaran et al, 2007). The documentation <strong>of</strong><br />
medicinal plants in Kenya is very poor. Few <strong>of</strong> documentation have been done on plants from<br />
central Kenya as veterinary medicine, also few plants have been studied from kakamega rain forest;<br />
hence there is need to document medicinal plants found in Keiyo County. However, there is need to<br />
carry out proper identification <strong>of</strong> the medicinal plants (Tildhun and Mirutse, 2007). Equally<br />
threatened is the knowledge based on which the traditional system is based, as the ethno-botanical<br />
information is not documented and remains in the memory <strong>of</strong> the elderly practitioners (Tesfaye<br />
and Sebsebe, 2009). The purpose <strong>of</strong> documentation in ethno-botany is to try and find out how<br />
people have traditionally used plants, for whatever purpose and how is still doing so. Thus, ethnobotany<br />
tries to preserve valuable traditional knowledge for both future generations and other<br />
communities (Tesfaye and Sebsebe, 2009).<br />
Materials and methods<br />
An ethnobotanical study was done using a structured questionnaire to gather information on the<br />
plants used medicinally. This was administered randomly mainly targeting herbalists and adult<br />
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villagers. This information provides baseline data for collection <strong>of</strong> the medicinal plants. Plant<br />
Collection and identification was also done. Selection <strong>of</strong> the plants was based on available<br />
ethnobotanical information from traditional health practitioners consulted during the pilot study as<br />
well as available literature. The plant materials were photographed and collected in duplicates for<br />
identification, verification and storage at the herbarium in Chepkoilel University College.<br />
Results and Discussion<br />
A total <strong>of</strong> twenty five plants were documented. These plants are used for varied reasons ranging<br />
from stomachache, headache, dysmenorrhoea, chest infection, and increasing fertility in both<br />
males and females among others. Many plants are boiled together for better results. Some <strong>of</strong> the<br />
plants that were to be gathered were extinct as per the herbalists or are located in specific forested<br />
areas within the neighboring Kapnorok game reseave. Many other plants are endangered among<br />
them are Zanthoxylum usambarensis, Trichillia ometca, Terminaria spinosa, Senna siamea,<br />
Sanseveria conspicua, Sanseveria suffruticosa, Teclea nobilis, Salvadora persica, Landolphia<br />
swynnertonii, and Capparis tomentosa. From the statistics, the endangered species were ten out<br />
<strong>of</strong> twenty five collected. Some <strong>of</strong> the plants like Terminaria spinosa have become source <strong>of</strong> conflict<br />
among the people since in the past one would collect the plants from any region without<br />
quarrelling with the owner <strong>of</strong> the plot but presently some <strong>of</strong> the plants are jealously guarded by the<br />
plot owners.<br />
Plant Name Local name<br />
(Vernacular)<br />
BALANITES<br />
AEGYPTIACA<br />
Balanites<br />
pedicellaris<br />
Coccinea<br />
grandis<br />
Traditional uses<br />
Ng oswet The plant roots are boiled and taken for the purpose <strong>of</strong> opening blocked or<br />
narrow reproductive tubes, for treating typhoid and for the purpose <strong>of</strong><br />
increasing fertility in women<br />
MUIYENG WET THE PLANT ROOTS AND THE BACK ARE BOILED AND TAKEN FOR THE PURPOSE OF RECTIFYING<br />
DYSMENORRHOEA, FOR TREATS STOMACHACHE AND FOR STOPPING DIARRHEA<br />
SOTOP-CHEPTUGE THE PLANT LEAVES AND ROOTS ARE BOILED OR SOAKED AND TAKEN FOR THE PURPOSE OF<br />
SHRINKING OR TREATING FIBROIDS, DISSOLVING CLOT, TREATS PAINFUL SEXUAL<br />
INTERCOURSE AND FOR TREATING INTERNAL ORGANS, DISORDER AND INFECTION<br />
Acacia seyal LENG NET THE PLANT ROOT AND BACK ARE SOAKED OR BOILED AND TAKEN FOR THE PURPOSE OF<br />
Capparis<br />
tomentosa<br />
Landolphia<br />
swynnertonii<br />
Withania<br />
somnifera<br />
Salvadora<br />
persica<br />
KU<strong>MB</strong>OLWOP<br />
KIMAGET<br />
TREATING TYPHOID KIDNEY INFECTION AND AMOEBIASIS ESPECIALLY IN CHRONIC STAGES<br />
ESPECIALLY WHEN THE PATIENT HAS BLOOD STAINED STOOL.<br />
THE PLANT ROOTS AND BACK IS BOILED AND USED FOR THE PURPOSE OF TREATING<br />
GONORRHEA, INCREASING FERTILITY, FOR TREATING MASTITIS IN HUMANS AND FOR<br />
TREATING DYSMENORRHOEA<br />
MOKOKWET THE PLANT ROOTS ARE BOILED AND TAKEN FOR THE PURPOSE OF TREATING BACKACHE AND<br />
FOR INCREASING FERTILITY IN WOMEN<br />
KUMYAP CHEPKUK THE PLANT ROOTS ARE BOILED AND TAKEN FOR THE PURPOSE OF TREATING MALARIA AND<br />
YELLOW FEVER<br />
CHOGOWET THE PLANT BACK AND ROOTS ARE BOILED AND TAKEN FOR THE PURPOSE OF TREATING<br />
ALLERGY, COMMON COLD AND PAINFUL CHEST INFECTION<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Teclea nobilis KURYOT THE PLANT ROOTS ARE BOILED AND TAKEN FOR THE PURPOSE OF TREATING PERSISTENT<br />
Sanseveria<br />
suffruticosa<br />
Sanseveria<br />
conspicua<br />
BOSCIA<br />
ANGUSTIFOLIA<br />
HEADACHE AND COMMON COLD<br />
MOKOLATIET THE PLANT ROOTS ARE BOILED AND TAKEN FOR THE PURPOSE OF OPENING BLOCKED OR<br />
NARROWED REPRODUCTIVE TUBES AND FOR TREATING GONORRHEA<br />
Sagaratiet The plant roots and young leaves are either boiled or soaked and taken for<br />
the purpose <strong>of</strong> Removing placenta retained after birth in both humans and<br />
animals<br />
LIKWOT THE ROOTS ARE BOILED OR GROUND INTO POWDER AND USED FOR THE PURPOSE OF<br />
HEALING WOUNDS WHEN USED IN POWDER FORM, FOR TREATING TYPHOID AND THROAT<br />
INFECTION<br />
SENNA SIAMEA CHAKARANDAYAT ITS LEAVES IS BOILED AND TAKEN FOR THE PURPOSE OF TREATING MALARIA<br />
CISSUS<br />
VOTINDIFOLIA<br />
MAERUA<br />
SUBCORDATA<br />
INDIGOFERA<br />
HO<strong>MB</strong>EI<br />
TERMINARIA<br />
SPINOSA<br />
EUPHORBIA<br />
TIRUCALLI<br />
Trichillia<br />
ometca<br />
Commelina<br />
Africana<br />
Zanthoxylum<br />
usambarensis<br />
CHEROROWET ITS FLESHY ROOTS ARE BOILED AND TAKEN FOR THE PURPOSE OF TREATING AMOEBIASIS,<br />
TYPHOID, INCREASING FEMALE FERTILITY AND RECTIFYING INCONSISTENCY IN CHILDREN.<br />
CHEPYETABEI THE FLESHY ROOT IS EATEN RAW DRY OR FRESH FOR THE PURPOSES OF TREATING DIABETES,<br />
HIGH BLOOD PRESSURE, FOR IMPROVING APPETITE, PURIFIES WATER, FOR INDUCING SLEEP<br />
(INDUCES SLEEP AT HIGH DOSE)<br />
PARKELAT ITS ROOTS ARE EATEN RAW WHEN FRESH OR BOILED WHEN DRY FOR THE PURPOSES OF<br />
TREATING ALLERGY AND FOR RELIVING TOOTHACHE.<br />
TIKIT ITS LEAVES, BACK AND ROOTS ARE BOILED FOR THE PURPOSES OF TREATING PANCREAS<br />
DISORDER, TOOTHACHE, PERSISTENT HEADACHE ACCOMPANIED BY PAINFUL TEETH AND FOR<br />
TREATING NOSE AND EYES ALLERGY. IT IS ALSO USED TO INCREASE MALE LIBIDO.<br />
KIPNAGET THE LEAVES/STEM ARE BOILED AND TAKEN FOR THE PURPOSE FOR TREATING MALARIA AND<br />
STOMACHACHE.<br />
MOINET THE STEM AND ROOTS ARE BOILED AND TAKEN FOR THE PURPOSE OF TREATING ALLERGY<br />
AND FOR STOPPING DIARRHEA.<br />
CHESEPER THE LEAVES AND POUND FRESH AND USED TO TREAT WOUNDS AND FOR REMOVING OBJECTS<br />
THAT INJURED THE BODY LIKE THORNS.<br />
KOKCHAT THE ROOTS ARE BOILED AND TAKEN FOR THE PURPOSE OF TREATING RASHES ON THE<br />
TONGUE, FOR TREATING ULCERS, COUGH AND COMMON COLD.<br />
The residents also do not cultivate the plants and the herbalists depend 100% from the plants from<br />
the wild. This has posed danger since they are competing with the wild and domestic animals,<br />
including among themselves. The region is also being consumed by deep gulley erosion which<br />
frequently wipes away the plants. All the plants succumb to this since the gulley erosion are so<br />
deep such that not even a single plant survives the pressure. The villagers confirmed the heredity <strong>of</strong><br />
the knowledge and the danger <strong>of</strong> fast disappearance <strong>of</strong> the same.<br />
Acknowledgement<br />
I would wish to thank Mr. Wanjohi <strong>of</strong> Chepkoilel University College for naming the plants, and my<br />
supervisors Dr. Njenga, Dr. Mutai and Dr. Bii for their corrections, inspiration and guidance.<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Reference<br />
Kesavan Srinivasan, Devarajan Natarajan, Chokkalingam Mohanasundari, Chinthambi Venkatakrishnan And<br />
Nandakumar Nagamurugan(2007) Antibacterial, Preliminary Phytochemical And Pharmacognostical Screening<br />
On The Leaves Of Vicoa Indica (L.)Dc 1735-2657/07/61-109-113 Iranian Journal Of Pharmacology &<br />
Therapeutics<br />
Tilahun Teklehaymanot and Mirutse Giday(2007) Ethnobotanical study <strong>of</strong> medicinal plants used by people in Zegie<br />
Peninsula, Northwestern Ethiopia Journal <strong>of</strong> Ethnobiology and Ethnomedicine 2007, 3:12<br />
Tesfaye Awas and Sebsebe Demissew(2009) Ethnobotanical study <strong>of</strong> medicinal plants in Kafficho people, southwestern<br />
Ethiopia In: Proceedings <strong>of</strong> the 16th International Conference <strong>of</strong> Ethiopian Studies, ed. by Svein Ege, Harald<br />
Aspen, Birhanu Teferra and Shiferaw Bekele, Trondheim 2009<br />
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[YS 25] Antimicrobial Dihydroisocoumarins from Crassocephalum biafrae<br />
Turibio K. Tabopda 1 , Ghislain W. Fotso 1 , Joseph Ngoupayo 1 , Anne-Claire Mitaine-Offer 2 ,<br />
Bonaventure T. Ngadjui 1 , Marie-Aleth Lacaille-Dubois 2<br />
1<br />
Département de Chimie Organique, Université de Yaoundé I, Yaoundé, Cameroon<br />
2<br />
Université de Bourgogne, Faculté de Pharmacie, Laboratoire de Pharmacognosie, Dijon cedex, France<br />
KEY WORDS: Crassocephalum biafrae, Asteraceae, dihydroisocoumarins, antimicrobial activity<br />
Introduction<br />
C<br />
rassocephalum biafrae S. Moore (Asteraceae) and other Crassocephalum species are widely<br />
used as food additives and in traditional medicine in many Africa countries (Adebooye et al,<br />
2004). Aqueous extracts <strong>of</strong> C. biafrae rhizomes are used in Cameroon folk medicine to treat<br />
tuberculosis, epilepsy, respiratory infections, diarrhea, wounds, and cancer (Adjanohoun et al,<br />
1996). There is little phytochemical data on C. biafrae, although its essential oil has been<br />
characterized (Zollo et al, 2000).<br />
Materials and Methods<br />
The air-dried roots and rhizomes (2.5 kg) <strong>of</strong> C. biafrae were extracted with MeOH (1 L × 3) for 72 h<br />
at room temperature. A preliminary biological screening <strong>of</strong> this extract exhibited significant<br />
antimicrobial activity. The methanol extract (65 g) was evaporated to dryness, defatted with<br />
hexane, suspended in H2O, and partitioned between CHCl3 (400 mL × 3) and n-BuOH (400 mL× 3).<br />
The hexane (13 g), n-butanol (7 g), and chlor<strong>of</strong>orm (19 g) extracts were subjected to antibacterial<br />
and antifungal tests. The CHCl3 extract, which showed appreciable antibacterial activity, was then<br />
concentrated to a brown, viscous mass under reduced pressure. The residue was then dissolved in a<br />
small amount <strong>of</strong> MeOH. Purification <strong>of</strong> this extract using Sephadex LH-20 column (MeOH, 3 L, 9 ×<br />
7.3 cm), and silica gel column(20mg, 40 63 m, 5 × 48 cm) using n-hexane AcOEt (3: 2) and nhexane<br />
AcOEt (1: 1) solvent systems led to the isola on <strong>of</strong><br />
Three new dihydroisocoumarins (1) (67 mg), (2) (53 mg) and (3) ( 89 mg) along two know<br />
triterpenoids (4) (43 mg) and (5) (23 mg) and one known ceramide (6) (51 mg)<br />
In vitro antibacterial and antifungal activities were determined using the agar-well diffusion method<br />
(Atta-ur-Rahman et al, 2001) and the tube diffusion test (McLauglin et al, 1991).<br />
Results and Discussion<br />
As part <strong>of</strong> our ongoing research on Cameroonian medicinal plants, we report herein the isolation <strong>of</strong><br />
three new dihydroisocoumarins: Biafraecoumarin A (1), Biafraecoumarin B (2) and Biafraecoumarin<br />
C (3) along with three known compounds Fernenol (4) (Albrecht et al, 1969) Sorghumol acetate (5)<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
(Jain et al, 2002) and (2S, 2 R, 3S, 4R, 6E)-N-(2'-hydroxytetracosanoyl) -2-aminooctadec-6-ène-1, 3,<br />
4-triol (6) (Mendelsohn et al, 2000) from Crassocephalum biafrae. The structures <strong>of</strong> the new<br />
compounds wer established on the basis <strong>of</strong> the physical, chemical and spectroscopic data as shown<br />
in chart 1. Circular dichroism technique was used to determine the configuration <strong>of</strong> asymmetric<br />
carbons.<br />
Compounds 1-6 (1 mg/mL) have been tested in vitro for their antibacterial activity against Bacillus<br />
subtilis, Escherichia coli, Micrococcus luteus, Pseudomonas picketti and Staphylococcus aureus<br />
bacteria by the agar-well diffusion method [15]. DMSO was used as a control solvent, and cefixime<br />
was used as a standard drug. Minimum inhibitory concentration (MIC) values <strong>of</strong> all six isolated<br />
compounds were determined. The results showed that extract, fractions, and all compounds were<br />
active against E. coli and B. subtilis.<br />
Compounds 1-6 (200 g/mL) were also screened in vitro for their antifungal activity against six fungi<br />
species using the tube diffusion test [16]. Miconazole and Amphotericin (200 g/mL) were used as<br />
standard drugs. The linear growth <strong>of</strong> the fungus was obtained by measuring the diameter <strong>of</strong> the<br />
fungal colony after seven days. The amount <strong>of</strong> growth inhibition in each case was calculated as<br />
percentage inhibition. The screening results showed that compounds 1 and 2 exhibited significant<br />
activity (> 75%) against Candida albicans, Fusarium solani, and Trichphyton longifusus, whereas<br />
compound 3 showed significant activity (80%) against F. solani. The other compounds showed no to<br />
low activity. It is worthwhile to note that biafraecoumarins A (1), B (2), and C (3) exhibited<br />
maximum antibacterial and antifungal activities, possibly due to the presence <strong>of</strong> the<br />
dihydroisocoumarin moiety.<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
HO<br />
HO<br />
OH O<br />
HO<br />
1<br />
4<br />
O<br />
O NH<br />
HO<br />
OH<br />
OH<br />
HO<br />
OH<br />
Chart 1<br />
Acknowledgements<br />
This study was supported by a grant from the French government. We are grateful to the H.E. J.<br />
Research Institute <strong>of</strong> Chemistry and to the Dr. Panjwani Center for Molecular Medicine and Drug<br />
Research, University <strong>of</strong> Karachi, for spectra data and antimicrobial bioassays.<br />
References<br />
Adebooye, O.C., Jeffery, C., Solanecio biafrae (Olive and Heirne),. In: Grubben, G.J.H., Denton, O.A., (eds.) (2004); Plant<br />
resources <strong>of</strong> tropical Africa 2: Vegetables. Leiden, Netherlands: PROTA Foundation; 469 471.<br />
Adjanohoun, J.E., Aboubakar, N., Dramane, K., Ebot, M.E., Ekpere, J.A., Enow-Orock, E.G., Focho, D., Gbile, Z.O.,<br />
Kamanyi, A., Kamsu, K.J., Keita, A., Mbenkum, T., Mbi, C.N., Mbiele, A.L., Mbome, I.L., Mubiru, N.K., Nancy, W.L.,<br />
Nkongmeneck, B., Satabie, B., S<strong>of</strong>owora, A., Tamze, V., Wirmum, C.K. (1996); Traditional medicine and<br />
pharmacopoeia: contribution to ethnobotanical and floristic studies in Cameroon. Porto-Novo: Organisation <strong>of</strong><br />
African Unity Scientific, Technical and Research Commission. Centre National de Production de Manuels Scolaires;<br />
85.<br />
Albrecht, P., Ourisson, G. (1969); Triterpene alcohol isolation from oil shale. Science 163: 1192 1193.<br />
Atta-ur-Rahman, Choudhary, M.I., Thomsen, W.J. (1991); Bioassay techniques for drug development. Amsterdam, The<br />
Netherlands: Harwood Academic Elsevier Science; 383 409.<br />
Jain, R., Nagpal, S. (2002); Chemical constituents <strong>of</strong> the roots <strong>of</strong> Kirganelia reticulate. J Indian Chem Soc; 79: 776 777.<br />
O<br />
3<br />
6<br />
280<br />
O<br />
O<br />
O<br />
HO<br />
OH<br />
O<br />
2<br />
5<br />
O
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
McLauglin, J.L., Chang, C.J., Smith, D.L. (1991); Bench top bioassay for the discovery <strong>of</strong> bioactive natural products: an<br />
update. In: Atta-ur-Rahman, editor. Studies in natural products chemistry, Vol. 9. Amsterdam, The Netherlands:<br />
Elsevier Science;: 383 409.<br />
Mendelsohn, R., Rerek, M.E., Moore, D.J. (2000); Infrared spectroscopy and microscopic imaging <strong>of</strong> stratum corneum<br />
models and skin. Phys Chem 2: 4651 4657 <strong>of</strong> bioactive natural products: an update. In: Atta-ur-Rahman, editor.<br />
Publishers; 2001: 16 22.<br />
Zollo, P.H.A., Kuiate, J.R., Menut, C., Bessiere, J.M. (2000); Aromatic plants <strong>of</strong> tropical central Africa. XXXVI. Chemical<br />
composition <strong>of</strong> essential oils from seven Cameroonian Crassocephalum species. J Essent Oil Res 12: 533 536.<br />
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[YS 26] Antimycobacterial and Cytotoxicity Activity <strong>of</strong> Extracts from<br />
Zanthoxylum rhalybeum and Hallea rubrostipulata<br />
Chrian Marciale , Paul Erasto , Joseph N Otieno<br />
Institute <strong>of</strong> Tradition Medicine,<br />
Muhimbili University <strong>of</strong> Health and Allied Sciences; Dar es Salaam, Tanzania<br />
Key words: Hallea rubrostipulata, Zanthoxylum chalybeum, Antimycobacterial activity, Minimum<br />
Inhibition Concentration, Brine Shrimp Lethality Assay.<br />
Introduction<br />
The genus Mycobacterium (Mycobacteriaceae) is highly diverse with 85 species known in nature<br />
(Adewole et al., 2004). The common mycobacterial diseases are pulmonary infections, leprosy,<br />
buruli ulcers and tuberculosis. Tuberculosis (TB) is one <strong>of</strong> the most pervasive diseases caused by<br />
Mycobacterium tuberculosis (Mtb) and Mycobacterium africanum (Hugo et al., 2009). The current<br />
treatment <strong>of</strong> TB is facing a challenge <strong>of</strong> emerging multidrug resistant strains that refuse to respond<br />
to the available anti-TB drugs (Gemma et al., 2006). Therefore there is a need to search for novel<br />
compounds that can be used as effective lead compounds/extracts in the development <strong>of</strong> new anti-<br />
TB drugs. Medicinal plants are among the best sources <strong>of</strong> new chemical compounds that can<br />
facilitate discovery <strong>of</strong> new anti-TB chemotherapies. Examples <strong>of</strong> such medicinal plants are<br />
Zanthoxylum chalybeum and Hallea rubrostipulata.<br />
Zanthoxylum chalybeum (Rutaceae) and Hallea rubrostipulata (Rubiaceae) are medicinal plants that<br />
are used in the treatment <strong>of</strong> various diseases such as headaches, diarrhea, wound healing and<br />
respiratory tract infections. In Uganda and Kenya these plant species are used for treatment <strong>of</strong><br />
malaria, sickle cell diseases, measles, skin infections and severe coughs (Olila et al., 2002). The plant<br />
decoction <strong>of</strong> Hallea rubrostipulata is used for treatment <strong>of</strong> pre-hepatic jaundice, pregnancy related<br />
illness, back ache, salpingitis and diabetes (Ssegawa et al., 2007). In this study, the crude extracts<br />
from leaves and stem barks <strong>of</strong> Hallea rubrostipulata and stem barks <strong>of</strong> Zanthoxylum chalubeum<br />
were screened against for their antimycobacterial and cytotoxicity activity.<br />
Material and Methods<br />
Plant materials were collected from Karagwe District in Kagera Region, Tanzania. Identification was<br />
done at the site with the aid <strong>of</strong> the taxonomist. The sample specimens were deposited in the<br />
Herbarium <strong>of</strong> Institute <strong>of</strong> Traditional Medicine at Muhimbili University <strong>of</strong> Health and Allied<br />
Sciences. Plant materials were air dried and macerated into powdery form. Thereafter, powdered<br />
plant materials were extracted separately using dichloromethane (DCM), ethyl acetate (EtOAc) and<br />
finally with methanol (MeOH). Each extraction process took 24 hours before concentrating the<br />
extracts in vacuo using rotary evaporator.<br />
Antimycobacterial Activity<br />
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The crude extracts were screened against two fast growing non-pathogenic Mycobacterium strains<br />
namely: Mycobacterium madagascariense and Mycobacterium indicus pranii as markers for<br />
detection <strong>of</strong> potential anti-TB extracts using two folds broth microdilution method. The medium<br />
used was middlebrook 7H9 broth base containing tween 80 (0.1%) and the turbidity was adjusted<br />
to 0.5 McFarland units (approximately 1.2x10 8 CFU/ml) where the minimum inhibitory<br />
concentrations were determined according to McGaw et al. (2008). Isoniazid and cipr<strong>of</strong>loxacin were<br />
used as positive controls.<br />
Results and Discussion<br />
The dichloromethane extracts <strong>of</strong> the leaves <strong>of</strong> Hallea rubrostipulata exhibited higher activity<br />
against test organisms namely Mycobacterium madagascariense and Mycobacterium indicus pranii<br />
with the MIC values <strong>of</strong> 0.156 mg/ml and 0.625 mg/ml respectively. In the same assay conditions,<br />
the methanol extracts exhibited moderate to higher activities with the MIC values <strong>of</strong> 1.25 mg/ml<br />
and 0.3125 mg/ml respectively (Table 1). Furthermore, the dichloromethane and methanol extracts<br />
from Z. chalybeum showed moderate activities with MIC values <strong>of</strong> 1.25 mg/ml against<br />
Mycobacterium madagascariense and 2.5 mg/ml against Mycobacterium indicus pranii.<br />
Table 1: Antimycobacterial activity <strong>of</strong> the extracts <strong>of</strong> Hallea rubrostipulata and Zanthoxylum<br />
chalybeum<br />
Extracts Minimum Inhibitory Concentration (mg/ml)<br />
M. madagascariense M. indicus pranii<br />
HRSD 1.25 2.5<br />
HRSM 5 2.5<br />
HRSE 1.25 1.25<br />
HRLD 0.156 0.625<br />
HRLM 1.25 0.3125<br />
ZCSD 1.25 2.5<br />
ZCSM 1.25 2.5<br />
Isoniazid NA NA<br />
Cipr<strong>of</strong>loxacin
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
dichloromethane and methanol extracts <strong>of</strong> Z. chalybeum may partly be due to alkaloids present in<br />
the extracts. Further work to isolate and characterize the alkaloids and other natural products in<br />
the bioactive extracts <strong>of</strong> the screened plant species is in progress.<br />
The extracts from the two plant species were subjected on the brine shrimp lethality assay to test<br />
their potential cytotoxicity effect. All extracts with the exception <strong>of</strong> the dichloromethane extracts <strong>of</strong><br />
the stem bark <strong>of</strong> Z. chalybeum were less or not toxic against shrimps as compared to the standard<br />
anticancer agent cyclophosphamide (Table 2).<br />
Table 2: Cytotoxicity activity (BST) <strong>of</strong> the extracts <strong>of</strong> Hallea rubrostipulata and Zanthoxylum<br />
chalybeum<br />
Extracts LC50 (µg/ml) 95% Confidence Interval (µg/ml)<br />
HRSD 193.5 113 330.5<br />
HRSM 71.5 52.3 97.8<br />
HRSE 60.3 45.7 79.4<br />
HRLD 33.1 23.1 47.4<br />
HRLM 67.6 51.1 89.2<br />
ZCSD 5.7 3.1 10.5<br />
ZCSM 87.7 71.8 106.9<br />
Cyclophosphamide* 16.3 10.6 25.2<br />
Abbreviations<br />
MM Mycobacterium madagascariense<br />
MIP Mycobacterium indicus pranii<br />
HRLD Hallea rubrostipulata leaves, dichloromethane<br />
HRLE Hallea rubrostipulata leaves, ethyl acetate<br />
HRLM Hallea rubrostipulata leaves, methanol<br />
HRSD Hallea rubrostipulata stem bark, dichloromethane<br />
HRSM Hallea rubrostipulata stem barks, methanol<br />
ZCSD Zanthoxylum chalybeum stem barks, dichloromethane<br />
ZCSM Zanthoxylum chalybeum stem barks, methanol<br />
Acknowledgements<br />
MC would like to thank the Ministry <strong>of</strong> Health and Social Welfare, Tanzania for scholarship. PE and<br />
JNO appreciate the support <strong>of</strong> DelPHE-BC (Ref No 607).<br />
References<br />
Adesina SK. (2005); The Nigerian Zanthoxylum, Chemical and biological values. Afr. J. Trad. CAM. 2, 282.<br />
Adewole LO, Lewis PFE, Lewis HW. (2004); Natural antimycobacterial metabolites: current status. Phytochemistry, 65,<br />
1017-1032.<br />
Gemma OD, Franz B, Simon G. (2006); Phytochemistry and Mycobacterium activity <strong>of</strong> Chlorophytum inortum.<br />
Phytochemistry, , 67, 178-182.<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Hugo M, Turell L, Manta B, Botti H, Monteiro G, Netto LES, Alvarez B, Radi R, Trujillo M. (2009); Thiol and sulfenic Acid<br />
oxidation <strong>of</strong> AhpE, the one-cysteine peroxiredoxin from Mycobacterium tuberculosis: Kinetics, acidity constants<br />
and conformational dynamics. Boichem. 48, 9416-9426.<br />
Mcgaw LJ, Lall N, Hlokwe TM, Michel AL, Meyer JJM, Ell<strong>of</strong> NF (2008);. Purified compounds and extracts from Euclea<br />
species with antimycobacterial activity against Mycobacterium bovis and fast- Growing Mycobacteria. Biol. Pharm.<br />
Bull, 31, 1429-1433.<br />
Okunande AL, Elvin LMP, Lewis WH. (2004); Natural antimycobacterial metabolites: current status. Phytochemistry, 65,<br />
1017-1032.<br />
Olila D, Olwa O, Opunda A. Screening <strong>of</strong> extracts <strong>of</strong> Zanthoxylum chalybeum and Warbugia ugandensis for activity<br />
against measles virus in vitro. J. Afr. health science, 2002, 2, 1.<br />
Ssegawa P, Kasenene JM. Medicinal plant diversity and uses in Sango bay area, Southern Uganda. J. Ethnopharmacol.<br />
2007, 113, 521-540.<br />
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[YS 27] Anti-Sickling Triterpenoids from Callistemon Viminalis, Melaleuca<br />
Bracteata Var. Revolution Gold Syzygium Guineense and Syzygium Cordatum<br />
Damien S. Tshibangu 2 , Francis O. Shode 1 , Neil Koorbanally 1 , V. Mudogo 2 , Pius T. Mpiana 2 , Jean Paul<br />
K. Nbgolua 3<br />
1 School <strong>of</strong> Chemistry/Faculty <strong>of</strong> Science and Agriculture/ University <strong>of</strong> KwaZulu-Natal<br />
2 Department <strong>of</strong> Chemistry/ Faculty <strong>of</strong> Science/ University <strong>of</strong> Kinshasa<br />
3 Department <strong>of</strong> Biology/ Faculty <strong>of</strong> Science/ University <strong>of</strong> Kinshasa<br />
Corresponding author: tshibangud@yahoo.fr<br />
Key words: Anti-sickling, biological activity, maslinic, betulinic and oleanolic acids.<br />
Introduction<br />
A<br />
ll over Africa, traditional healers use medicinal plants to prepare medicines to treat a wide<br />
range <strong>of</strong> illnesses. One <strong>of</strong> these illnesses is sickle cell anaemia or drepanocytosis or sicklemia.<br />
This disease is particularly common among Sub-Saharan Africans with a clear predominance in<br />
equatorial Africa. However, it also exists in North Africa, Greece, Turkey, Saudi Arabia and India<br />
(Fleming, 1989). An estimated 50 million people are affected worldwide (Buchanan et al., 2004;<br />
Girot, 2003). A literature review on sickle cell anaemia revealed that a number <strong>of</strong> plants have antidrepanocytosis<br />
activity (Neuwinger, 2000; Elujoba et al., 2005, Ekeke et al., 1990, Mpiana et al.,<br />
2007; Mpiana et al., 2008). Callistemon viminalis, Melaleuca bracteata var. Revolution Gold,<br />
Syzygium guineense and Syzygium cordatum (from Democratic Republic <strong>of</strong> Congo and South Africa)<br />
were selected for investigation on the basis <strong>of</strong> their reported medicinal uses. The main aim <strong>of</strong> this<br />
study was to isolate and characterize anti-sickling (anti-drepanocytosis) compounds from the above<br />
mentioned medicinal plants.<br />
Materials and Methods<br />
Materials<br />
The leaves <strong>of</strong> Syzygium guineense were collected in Kinshasa, Democratic Republic <strong>of</strong> Congo (DRC)<br />
and on the campus <strong>of</strong> the University <strong>of</strong> KwaZulu Natal in South Africa, while the leaves <strong>of</strong> Syzygium<br />
cordatum, Callistemon viminalis and Melaleuca bracteata were collected in Durban, South Africa.<br />
The blood samples used to perform the antisickling activity <strong>of</strong> the crude extracts and the pure<br />
isolated compounds in this study were collected from a known drepanocitary center named<br />
Centre de Médecine Mixte et d`Anémie SS located in Kinshasa area, DRC. The blood samples were<br />
first characterized by Hb electrophoresis on cellulose acetate gel, in order to confirm their SS<br />
nature, as previously reported by (Mpiana, 2007). They were found to be SS blood and were then<br />
stored in a refrigerator. These SS blood samples were so used to perform biological tests.<br />
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Methods<br />
The selected plants were subjected to phytochemical extraction methods and crude extracts were<br />
separated using chromatographic techniques. The structures <strong>of</strong> the isolated compounds were<br />
determined by spectroscopic techniques (1D and 2D NMR, FT-IR and MS). The powdered plant<br />
materials were sequentially extracted with n-hexane, dichloromethane, ethyl acetate, methanol,<br />
and aqueous methanol 80%. Each extraction was optimized by repeating the maceration twice.<br />
Each solvent extraction was concentrated under reduced pressure and allowed to dry at room<br />
temperature and weighed to give hexane-, dichloromethane-, ethyl acetate-, methanol- and<br />
aqueous methanol-solubles, respectively. These extracts were subjected to chromatographic<br />
techniques over silica gel 60 F254 aluminum barking from Merck, Germany for thin layer<br />
chromatography (TLC) and over silica gel 60 (0.040 0.63mm [230-400 mesh]) for column<br />
chromatography. Collected fractions were monitored on TLC plate and similar fractions were<br />
combined according to their TLC pattern.<br />
The 1 H, 13 C and all 2D NMR spectra were recorded using a 400 MHz Bruker spectrometer at the<br />
University <strong>of</strong> KwaZulu Natal, Westville Campus. All the spectra were recorded at room temperature<br />
using deuteriochlor<strong>of</strong>orm (CDCl3) or the mixture <strong>of</strong> CDCl3 and deuteriomethyl sulfoxide (DMSO). All<br />
spectra were referenced according to the central line <strong>of</strong> deuteriorated chlor<strong>of</strong>orm at H 7.24 for 1 H-<br />
NMR spectra and C 77.20 for 13 C -NMR spectra.<br />
The FT-IR spectra were recorded using; Perkin-Elmer Spectrum 100 FT-IR spectrometer. Samples<br />
were calibrated against an air background. Crystalline samples were directly placed on the window<br />
and then analyzed. KBr was used as salt.<br />
The mass spectrometry <strong>of</strong> compounds was recorded using an Agilent 6890 series gas<br />
chromatography system. The compound was dissolved in HPLC grade methanol (1:100) and helium<br />
(at 0.8 ml/minute), the gas carrier was maintained at 200 °C for 4 minutes and then programmed to<br />
300 °C at 5 °C/minute.<br />
The mass spectrometry/liquid chromatography <strong>of</strong> compounds was recorded using an Agilent 1100<br />
series LC/MSD Trap system. Compounds were dissolved in HPLC grade methanol and the following<br />
parameters were observed for the run: Injection: 6 l; flow: 0.3 ml/min; time: 1.5 minutes;<br />
maximum pressure: 150 bar, minimum pressure: 0 bar, temperature 24.4 - 26.8 °C. Carrier solvents<br />
used were acetonitrile 95% and Millipore water 5%.<br />
Results and Discussion<br />
The methanol extract <strong>of</strong> the leaves <strong>of</strong> S. guineense <strong>of</strong> DRC gave the highest quantity <strong>of</strong> extract<br />
(25.65%), while ethyl acetate extract gave the lowest yield (0.59%) <strong>of</strong> crude extract.<br />
Dichloromethane extract <strong>of</strong> the leaves <strong>of</strong> S. guineense <strong>of</strong> South African origin gave the highest<br />
extract (1.05%), while ethyl acetate extract gave the lowest (0.08 %). Dichloromethane extract <strong>of</strong><br />
the leaves <strong>of</strong> C. viminalis gave the highest extract (4.63%), while ethyl acetate extract gave the<br />
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lowest (2.75%) <strong>of</strong> crude extract. Dichloromethane extract <strong>of</strong> M. bracteata gave the highest extract<br />
(9.33%), while ethyl acetate extract gave the lowest yield (0.80%) <strong>of</strong> crude extract.<br />
S. guineense from DRC yielded a major natural product which was a flavanone glycoside (1). S.<br />
guineense from South Africa afforded 4 compounds namely betulinic acid (2), sitosterol (3),<br />
friedelan-3-one (4) and a betulinic acid derivative. Callistemon viminalis afforded one compound,<br />
betulinic acid (2). Melaleuca bracteata afforded two compounds which were characterized as<br />
betulinic acid acetate (5) and ursolic acid acetate (6) and Syzygium cordatum afforded two<br />
compounds namely maslinic acid (7) and oleanolic acid (8).<br />
AcO<br />
O<br />
C<br />
H 3<br />
AcO<br />
O<br />
1``` O<br />
OAc<br />
6"<br />
AcO<br />
O<br />
4"<br />
O<br />
4<br />
2"<br />
OAc<br />
8<br />
OAc<br />
1<br />
5<br />
O<br />
OAc<br />
2'<br />
3<br />
O<br />
AcO<br />
4'<br />
6'<br />
OAc<br />
HO<br />
H<br />
H<br />
5<br />
H<br />
H<br />
H<br />
H<br />
2<br />
H<br />
288<br />
H<br />
CO 2 H<br />
CO 2 H<br />
AcO<br />
H<br />
H<br />
H H<br />
HO 3<br />
H<br />
H<br />
6<br />
H<br />
COOH<br />
7 8<br />
The investigation <strong>of</strong> the anti-drepanocytosis activities <strong>of</strong> the extractives and their crude extracts<br />
showed in vitro effective antisickling activity. Ethyl acetate crude extracts <strong>of</strong> Callistemon viminalis<br />
and Melaleuca bracteata; hexane, dichloromethane and ethyl acetate crude extracts <strong>of</strong> Syzygium<br />
guineense <strong>of</strong> D R Congo, betulinic acid, betulinic acid acetate and maslinic acid showed a high<br />
antisickling activity, more than 70% <strong>of</strong> normalization. The fatty acid from Melaleuca bracteata was<br />
found to have an activity, between 50 and 70% <strong>of</strong> normalization and oleanolic acid showed the<br />
weakest activity, between 10 and 50 % <strong>of</strong> normalization. The maslinic acid and oleanolic acid used<br />
in this study were extracted from Syzygium cordatum.
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
However, some crude extracts and pure isolated compounds were found to have no antisickling<br />
activity. These were crude dichloromethane extract <strong>of</strong> Callistemon viminalis; crude<br />
dichloromethane, methanol and aqueous methanol (80%) extracts <strong>of</strong> Melaleuca bracteata; crude<br />
hexane, dichloromethane, ethyl acetate and methanol extracts <strong>of</strong> Syzygium guineense <strong>of</strong> South<br />
Africa; ursolic acid from Melaleuca bracteata and flavanone glycoside from Syzygium guineense <strong>of</strong><br />
D R Congo.<br />
According to these results, it can be seen that the activity <strong>of</strong> ethyl acetate extract from Melaleuca<br />
braceta can be attributed to the betulinic acid and its acetate. Betulinic acid and maslinic acid were<br />
found to have the highest activities. We wish to recommend further investigations <strong>of</strong> the hexane,<br />
dichloromethane, ethyl acetate, and methanol extracts <strong>of</strong> S. guineense from DRC, in order to<br />
identify the active principles. Furthermore, different derivatives <strong>of</strong> betulinic acid, maslinic acid and<br />
oleanolic acid should be synthesized, in order to compare their anti-sickling activities with the<br />
starting materials.<br />
Acknowledgements<br />
We express our deepest thanks and pr<strong>of</strong>ound gratitude to Third World <strong>of</strong> Academy and Science<br />
(TWAS), for the 2008 Research and Advanced Training fellowship. We are also so thankful to the<br />
School <strong>of</strong> Chemistry <strong>of</strong> the Faculty <strong>of</strong> Science and Agriculture at the University <strong>of</strong> KwaZulu Natal for<br />
providing laboratory support for this study.<br />
We are also grateful to the International Foundation for Science (IFS, Sweden) and to the<br />
Organization for the Prohibition <strong>of</strong> Chemical Weapons (OPCW) for the Research Grant F/4921-1<br />
given to Mr Jean Paul NGBOLUA Koto-Te-Nyiwa.<br />
References<br />
1. Buchanan GR; De Baun MR; Quinn CT; Steinberg MH (2004); Sickle Cell Disease, Hematology 1: 35-47.<br />
2. Ekeke, GI and Shode, FO (1990); Phenylalanine is the predominant antisickling agent in Cajanus cajan seed extract.<br />
Planta Medica 56, 41 43.<br />
3. Elujoba, AA; Odeleye, OM and Ogunyemi, CM (2005a);. Traditional medicine development for medical and dental<br />
primary health care delivery system in Africa. Afr. J. Trad.Cam 2(1): 46 61.<br />
4. Fleming, AP (1989); The presentation management and prevention <strong>of</strong> crises in sickle cell disease in Africa. Blood<br />
Revue 3, 19-28.<br />
5. Girot R, Bégué P (2003); La Drépanocytose. John Libbey-Eurotext, Paris.<br />
6. Mpiana PT; Mudogo V; Tshibangu DST; Kitwa, EK; Kanangila, AB; Lumbu, JBS; Ngbolua, KN; Atibu, EK and Kakule,<br />
MK (2008);. Antisickling Activity <strong>of</strong> Anthocyanins from Bombax pentadrum, Ficus capensis, Ziziphus mucronata:<br />
Photodegradation effect. J. Ethnopharmacol; 120: 413-418.<br />
7. Mpiana PT; Tshibangu DST; Shetonde OM and Ngbolua KN (2007);. In vitro antidrepanocytary activity (anti-sickle<br />
cell anaemia) <strong>of</strong> some Congolese plants. Phytomedicine; 14: 192-195.<br />
8. Neuwinger, H.D (2000); African Traditional Medicine. Mephaim Scientific Publisher, Stuttgart.<br />
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POSTER PRESENTATION<br />
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[PS 1] In vitro Inhibition <strong>of</strong> Botrytis cinerea - Causative Agent for Grey Mold<br />
by Crude Extracts <strong>of</strong> Basidiomycetes Fungi<br />
John Onyango Adongo a , Josiah O. Omolo a , Peter K. Cheplogoi a , Dan O. Otaye b<br />
a Chemistry Department, Egerton University, P. O. Box 536, 20115-Egerton, Kenya.<br />
b Biological Sciences Department, Egerton University, P. O. Box 536, 20115-Egerton, Kenya.<br />
Key words: Botrytis cinerea, grey mold, basidiomycetes, submerged cultures, column chromatography.<br />
Introduction<br />
B<br />
otrytis cinerea the cause <strong>of</strong> grey mold is a well-known fungus with a wide host range that<br />
causes heavy economic losses <strong>of</strong> yield in more than 200 crop species including: onions, potato,<br />
strawberry, rose flowers, table grape and other ornamental plants (Guinebretiere et al., 2000).<br />
Current commercial synthetic fungicides used for its control such as Mancozeb have been shown<br />
to be carcinogenic (Marta et al, 2011). In addition, there have been documented evidences on<br />
traces <strong>of</strong> these fungicide residues persisting in vegetable crops and soil (Apladasarlis et al., 1994).<br />
Resistance <strong>of</strong> B. cinerea isolates from vegetable crops towards the major classes commercial antibotrytis<br />
fungicides: anilinopyrimidines, phenylpyrroles, hydroxyanilides, benzimidazoles and<br />
dicarboximides have also been recently confirmed (Myresiotis et al., 2007). It is imperative that<br />
alternative fungicides from naturally occurring compounds that are easily biodegradable and <strong>of</strong> low<br />
mammalian toxicity be explored for safe control <strong>of</strong> crop fungal pathogens since low mammalian<br />
toxicity, minimal environmental impact and novel modes <strong>of</strong> action are very important features <strong>of</strong><br />
natural antifungal compounds.<br />
The production <strong>of</strong> antimicrobial secondary metabolites has been reported in many fungal<br />
biocontrol agents (Turner, 2003); antibiosis mechanism best explains this phenomena, in which the<br />
antagonists produce a wide range <strong>of</strong> secondary metabolites such as antibiotics and toxins, which<br />
contribute to the antagonistic activity <strong>of</strong> fungal control agents against plant pathogens (Yazaki et<br />
al., 2008). Basidiomycetes fungi have been known to synthesize a vast array <strong>of</strong> secondary<br />
metabolites that possess beneficial biological activities, which can be exploited through research<br />
for crop protection purposes, in fact, current research on antifungal agents is based on the<br />
principle; that new generation fungicides should be practically non-toxic, except for the target<br />
organism (Komarek et al, 2009). Of relevance to this research, is the exploration <strong>of</strong> antagonistic<br />
strains belonging to the basidiomycete class <strong>of</strong> fungi, which are able to produce secondary<br />
metabolites that display antagonistic activity against B. cinerea. For this reason, armed with current<br />
methods in fungal biotechnology, there is high prospect <strong>of</strong> finding novel biologically active<br />
compounds that can be a potential fungicide for the control <strong>of</strong> grey mould disease.<br />
Materials and Methods<br />
All the glassware used in this work was standard quality and flasks as well as beakers were<br />
autoclaved before being used in the isolation <strong>of</strong> the test organism and cultivation <strong>of</strong> the submerged<br />
cultures. Crude extracts were then prepared using solvent extraction method using Acetone,<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Methanol and Ethyl acetate, which were recovered by concentrating using the Rotary evaporator<br />
apparatus. Silica gel 60 (0.063 0.2 mm/70-230 mesh) was used as stationery phase for column<br />
chromatography. Thin layer chromatography (TLC) was done with Macherey Nagel pre-coated<br />
silica gel 60 F254 plates (ALUGRAM ® SIL G/UV254 0.25 mm, Duren, Germany). The developed TLC<br />
plate was viewed under dual fixed wavelength UV lamp ( = 254 nm and 365 nm) and the spots<br />
visualized by spraying with freshly prepared p-anisaldehyde solution, then heated to 112°C. In vitro<br />
antifungal testing was done by impregnating filter paper disc (Rundfilter, 6 mm, Schleicher &<br />
Schuell) with known amounts <strong>of</strong> the crude extracts and enriched fractions. The glucose levels in the<br />
cultures was monitored using glucose testing strips (Diabur-test ® 5000 (Roche). The media and<br />
flasks were initially heat sterilized using an autoclave for 15 minutes at a temperature <strong>of</strong> 115 °C and<br />
pressure <strong>of</strong> 1.5 bars. The inoculation and monitoring <strong>of</strong> growth parameters were done under a<br />
lamina flow hood backed with a hot flame produced by a Bunsen burner. Bruker ARX300<br />
spectrometer will be used to perform NMR experiments upon successful isolation and purification.<br />
Results and Discussion<br />
From the initial screening crude extracts using agar diffusion assay, 22 out <strong>of</strong> 400 strains produced<br />
appreciable antifungal activities against B. cinerea (figure 1. below). The results significant since<br />
about 5% <strong>of</strong> the crude extracts screened showed significant activity against the B. cinerea, an<br />
accepted standard in microbial screening research (Rosa, 2003). The diameters <strong>of</strong> the inhibition<br />
zones were measured in millimetres and analyzed using SPSS 11.5 and all the 22 strains collectively<br />
had mean <strong>of</strong> 14.2mm, standard deviation <strong>of</strong> 1.8, the greatest inhibition zone being 17mm and the<br />
lowest being 11mm. The means were found to differ significantly at 95% confidence limit by<br />
running student t-test using the SPSS 11.5 s<strong>of</strong>tware. The strain that gave the greatest inhibition<br />
zone was consequently selected for further cultivation.<br />
Figure 1: Selected glass plates showing some <strong>of</strong> the bioactive basidiomycetes strains<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Figure 2: Comparison <strong>of</strong> "control" against selected fractions <strong>of</strong> an active crude extract<br />
A particular fraction produced by the selected active strain (labelled 2) showed reproducible<br />
antifungal activity in 5 replicates as was tested against a control - a commercial fungicide<br />
Dithane® (labelled 1) as shown in figure 2 above. A close examination <strong>of</strong> the result above concludes<br />
that the selected strain at least produces a compound that has potent and reproducible antifungal<br />
activity that can be compared to that <strong>of</strong> the control. Column chromatography and TLC techniques<br />
are being applied in an attempt to purify the responsible compound(s) after which minimum<br />
inhibitory concentrations (MIC) tests will be carried out and followed by structure elucidation<br />
experiments.<br />
References<br />
Apladasarlis P., Liapis K. S. and Miliadis, G. E. (1994); Study <strong>of</strong> procymidone and propargite residue levels resulting from<br />
application to greenhouse tomatoes. Journal Agricultural and Food Chemistry. 42: 1575-1577.<br />
Guinebretiere, M. H., Morrison, C., Reich, M. and Nicot, P. (2000); Isolation and characterization <strong>of</strong> antagonists for the<br />
biocontrol <strong>of</strong> the postharvest wound pathogen Botrytis cinerea on strawberry fruits. Journal <strong>of</strong> Food Protection.<br />
63: 386-394.<br />
Komarek, M., Cadkova, E., Chrastny, V., Bordas, F. and Bollinger, J. (2009); Contamination <strong>of</strong> vineyard soils with<br />
fungicides: A review <strong>of</strong> environmental and toxicological aspects. Environment International. 10: 11-12.<br />
Marta, A., Julie, B., Nelleman, C., Kiersgaard, M., Rosenkjold, P, J., Christiansen, S., Hongard, K, S. and Halla, U. (2011);<br />
Exposure to widely used Mancozeb causes thyroid hormone disruption in rat dams but no behavioral effects in<br />
the <strong>of</strong>fsprings. Journal <strong>of</strong> Toxicological Sciences. 10: 1093-1094.<br />
Myresiotis, C. K., Karaoglanidis, G. S. and Tzavella-Klonari, K. (2007); Resistance <strong>of</strong> Botrytis cinerea isolates from<br />
vegetable crops to anilinopyrimidine, phenylpyrrole, hydroxyanilide, benzimidazole and dicarboximide<br />
fungicides. Plant Diseases. 91: 407-413.<br />
Rosa, H. L., Machado, K. M. G., Jacob, C. C., Capaleri, M., Rosa, C. A. and Zani, L. C. (2003); Screening <strong>of</strong> Brazillian<br />
basidiomycetes for antimicrobial activity. Mem. Inst. Owswaldo Cruz, Rio de Janeiro. 98: 967-974.<br />
Turner, W. (2003); The effect <strong>of</strong> fungal secondary metabolites on bacterial and fungal pathogens. In Handbook <strong>of</strong><br />
Fungal Secondary Metabolites. Academic Press: New York - USA. pp. 252-259.<br />
Vettorazzi, G., Almeida, W. F., Burin, G. J., Jaeger, R. B., Puga,, F. R., Rahde, A. F., Reyes, F. G., Schvartsman, S. (1995);<br />
International safety assessment <strong>of</strong> pesticides: Dithiocarbamate pesticides, ETU, and PTU-A review and update.<br />
Teratogenesis, Carcinogenesis, and Mutagenesis. 15: 313-317.<br />
Yazaki, K., Sugiyama, A., Morita, M. and Shitan, N. (2008); Secondary transport as an efficient membrane transport<br />
mechanism for plant secondary metabolites. Photochemistry Review. 7: 513-524.<br />
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[PS 2] Biochemical Comparison <strong>of</strong> Annona Squamosa L. Leaves Growing In<br />
Different Eco-Zones In Tanzania For Mosquito Larvicidal Activity.<br />
Daniel Bestina, Innocent E and Mbwambo Z.H.<br />
Institute <strong>of</strong> Traditional Medicine, Muhimbili University <strong>of</strong> Health and Allied Science,<br />
P.0.BOX 65001,Dar-es salaam.Tanzania<br />
. bestinad@yahoo.com<br />
Key words - Anon squamosa L., HPTLC analysis, HPLC analysis, mosquito larvicidal assay<br />
Introduction<br />
A<br />
nnona squamosa L. (Annoneceae) is a medicinal plant used in treatment <strong>of</strong> different disorders<br />
such as constipation, fever, ulcer, cancer, and tumor (Saleem et al., 2009). This plant species is<br />
widely distributed in Tanzania mostly along the coastal area and Zanzibar (Nyambo et al., 2005).<br />
The leaves and root <strong>of</strong> Annona squamosa L have been reported to possess mosquito larvicidal<br />
activity against Culex quinqefasciatus (Magadula et al., 2009) and Anopheles gambiae (Kihampa et<br />
al., 2009) respectively. Therefore, the aim <strong>of</strong> this study was to investigate if geographical location<br />
had any effect on larvicidal activity <strong>of</strong> leaves <strong>of</strong> A.squamosa L.growing in different eco zones in<br />
Tanzania.<br />
Material & Methods<br />
The leaves <strong>of</strong> Annona squamosa L. were collected from five different eco zones in Tanzania.<br />
Identification were done with the aid <strong>of</strong> the taxonomist at the site. The voucher specimens were<br />
kept in the herbarium <strong>of</strong> Institute <strong>of</strong> Traditional Medicine (ITM), Muhimbili University <strong>of</strong> Health and<br />
Allied Science (MUHAS), Tanzania. The plant materials were soaked in ethanol and the crude<br />
extracts were screened for mosquito larvicidal activity against Culex quinqefasciatus based on WHO<br />
protocol (1996 and 2003). Mortality was recorded after 24h <strong>of</strong> exposure. Furthermore each extract<br />
was then partitioned using DCM, EtoAc and BtOH before being subjected to HPLC for analysis.<br />
Results<br />
The extract from Mbeya region exhibited the highest larvicidal activity with LC50 value <strong>of</strong> 6.60 g/ml<br />
and 5.20 g/ml while the extracts from Morogoro had a lowest larvicidal activity with LC50 value <strong>of</strong><br />
416.02 g/ml and 230.06 g/ml after 24 and 48h <strong>of</strong> exposure. The trend <strong>of</strong> mortality increased with<br />
increase in time <strong>of</strong> exposure.<br />
The HPTLC analysis <strong>of</strong> ethanol extracts indicated that this plant species contained compounds <strong>of</strong><br />
different polarity but most <strong>of</strong> compounds from each extract observed to be similar with others<br />
based on their Rf values ( fig. a & b).<br />
Figure a& b: HPTLC chromatograms <strong>of</strong> crude ethanol extract <strong>of</strong> Annona squamosa L. from different<br />
eco zones in Tanzania viewed in (a) fluorencence (366nm) (b) at low wavelength <strong>of</strong> (254nm)<br />
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(a) (b)<br />
In the chromatogram (Fig.a & b) from left was extracts from Kilimanjaro, Dar es salaam, Mwanza,<br />
Morogoro(A), Morogoro (B) and Mbeya region respectively.<br />
N.B: Mor(A)-The fresh leaves collected Morogoro region, Mor(B)-The dry leaves collected<br />
Morogoro region.<br />
Phytochemical screening <strong>of</strong> the leaf extracts shown positive results for alkaloids from butanol<br />
fraction and terpenoids from ethylacetate fraction as indicated in (Table 1) below.<br />
TABLE 1. Phytochemical screening <strong>of</strong> leaf extracts <strong>of</strong> A.squamosa L.<br />
Class Test used Dichlormethane<br />
fraction<br />
295<br />
Ethylacetate<br />
fraction<br />
Alkaloids Dragendorff reagent _ - +<br />
Flavanoids Aluminium chloride - - -<br />
Terpenoids Vannilin-sulphuric acid - + -<br />
Butanol fraction<br />
+: presence -: absence<br />
A comparative study <strong>of</strong> HPLC pr<strong>of</strong>iles showed that most <strong>of</strong> the compounds from different zones correlated<br />
in terms <strong>of</strong> peak number and retention time but differed in percentage area <strong>of</strong> the peak ( fig. c)
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Figure C. HPLC pr<strong>of</strong>ile <strong>of</strong> ethylacceteate fraction.<br />
BLANK<br />
5<br />
0<br />
DAD1 C, Sig=210,8 Ref=360,100 (BESTINA 2010-12-21 10-34-47\001-0501.D)<br />
0 2 4 6 8 10 12 14 16<br />
DAD1 C, Sig=210,8 Ref=360,100 (BESTINA 2010-12-21 10-34-47\1AD-0801.D)<br />
100<br />
50<br />
0<br />
0 2 4 6 8 10 12 14 16<br />
DAD1 DC, Si Sig=210,8 230 16Ref=360,100 R f 360 100 (BESTINA 2010-12-21 2010 12 2110-34-47\1AE-1001.D) 10 34 4 \1AD 0801 D)<br />
100<br />
50<br />
0<br />
0 2 4 6 8 10 12 14 16<br />
DAD1 DAD1 DC, Si Sig=210,8 230 16Ref=360,100 R f 360 100 (BESTINA 2010-12-21 2010 12 2110-34-47\1AF-1201.D) 10 34 4 \1AE 1001 D)<br />
50<br />
0<br />
0 2 4 6 8 10 12 14 16<br />
DAD1 C, Sig=210,8 S Ref=360,100 f (BESTINA ( S 2010-12-21 10-34-47\1AG-1401.D)<br />
\ )<br />
100<br />
50<br />
0<br />
0 2 4 6 8 10 12 14 16<br />
DAD1 D Si 230 16 R f 360 100 (BESTINA 2010 12 21 10 34 4 \1AG 1401 D)<br />
50<br />
25<br />
DAD1 C, Sig=210,8 Ref=360,100 (BESTINA 2010-12-21 10-34-47\1AH-1601.D)<br />
0<br />
0 2 4 6 8 10 12 14 16<br />
DAD1 DC, Sig=210,8 Si 230 16Ref=360,100 R f 360 100 (BESTINA 2010-12-21 2010 12 2110-34-47\1AI-1801.D) 10 34 4 \1AH 1601 D)<br />
100<br />
0<br />
0 2 4 6 8 10 12 14 16<br />
DAD1 D Si 230 16 R f 360 100 (BESTINA 2010 12 21 10 34 4 \1AI 1801 D)<br />
N.B;In the pr<strong>of</strong>iles, from top was blank, Kilimanjaro, Dar es salaam, Mwanza,Morogoro (A), Morogoro (B)<br />
and Mbeya extract.<br />
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Discussion & Conclusion<br />
The experimental results supported that the larvicidal activity <strong>of</strong> A.squamosa leaves varied from<br />
one eco zone to another. Phytochemical screening indicated the presence <strong>of</strong> alkaloids and<br />
terpenoids in each extract which was in agreement with Magadula et al., (2009) and Kihampa et<br />
al., (2009). Generally, the activity showed by extracts can be further used for validation <strong>of</strong> method<br />
for development <strong>of</strong> active botanical formulation for mosquito larvae control.<br />
Acknowledgement<br />
I would like to acknowledge the MoHSW-Tanzania, DelPHE British council, NAM S &T CENTRE-India<br />
and ICCBS center- Karachi-Pakistan for their supports in funds and experimental facilities.<br />
References<br />
Kihampa, C., Joseph, C.C., Nkunya, M.H.H., Magesa, S.M., Hassanali, A., Heydenrecich. M. and Kleinpeter, (2009);<br />
Larvacidal and IGR activity <strong>of</strong> extracts <strong>of</strong> Tanzanian plants against malaria vector mosquitoes. Journal <strong>of</strong> Vector<br />
Borne Disease, 46, 145-152.<br />
Magadula. J.J., Innocent, E. and Otieno, J.N. (2009); Mosquito larvacidal and cytotoxic activities <strong>of</strong> three Annona species<br />
and Isolation <strong>of</strong> active principles. Medicinal Plants Research, 3, 674-680.<br />
Nyambo, A., Nyamora, A., Ruffo, C.K. and Tengnas, B. (2005); Fruits and Nuts species with potential for Tanzania.<br />
Technical Hand <strong>Book</strong>, No.34, RELMA Nairobi, Kenya, pp.160.<br />
Saleem, M.T.S., Christina, A.J.M., Chidambaranathan, N., Ravi, V. and Gauthaman, K. (2008); Hepatoprotective activity<br />
<strong>of</strong> Annona squamosa Linn. on experimental animal model. International Journal <strong>of</strong> Applied Research in Natural<br />
Products., 1, 1-7.<br />
Saleem, M.T.S., Hema, B., Ravi, V., Bhupendra, S., Verma, N.K., Patel, S.S., Vijaya, K.S. and Gauthaman, K (2009); Phytopharmacological<br />
review <strong>of</strong> Annona squamosa Linn. Trade Science Inc. An Indian Journal. East Sikkim, India, 5, 85-<br />
88.<br />
WHO . Evaluation and Testing <strong>of</strong> Insecticides. Report <strong>of</strong> the WHO Informal Consultation WHO Geneva. 1996, pp 9.<br />
WHO. Malaria Vector Control Decision Making Criteria and Procedures for Judicious Use <strong>of</strong> Insecticides .WHO Geneva.<br />
2003.<br />
297
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[PS 3] A Toxicological Study <strong>of</strong> Millettia usaramensis Stem Bark Extract on<br />
Aedes aegypti (Mosquito), Schistocerca gregaria (Desert Locust) and<br />
Mus musculus (Mouse)<br />
Bosire C. M a ., Kabaru J.M b ., Yenesew A c ., Kimata D. M b<br />
a<br />
Department <strong>of</strong> Pure & Applied Sciences, Mombasa Polytechnic University College, P.O Box 90420-80100, Mombasa,<br />
Kenya.<br />
b<br />
Department <strong>of</strong> Zoology, School <strong>of</strong> Biological Sciences, University <strong>of</strong> Nairobi. P.O. Box 30197, Nairobi, Kenya.<br />
c<br />
Department <strong>of</strong> Chemistry, School <strong>of</strong> Physical Sciences, University <strong>of</strong> Nairobi.P.O. Box 30197, Nairobi, Kenya. Email:<br />
ayenesew@uonbi.ac.ke<br />
Key words: Millettia usaramensis, Aedes aegypti, Schistocerca gregaria, Mus musculus, toxicological study.<br />
Introduction<br />
S<br />
ome <strong>of</strong> the many insects that proliferate in tropical environments due to conduciveness <strong>of</strong> its<br />
weather conditions are crop pests both in the field and in storage. Others transmit diseases and<br />
affect the health <strong>of</strong> both man and livestock. Synthetic insecticides have been used to control them<br />
but these have shown pest resistance, they bio-accumulate and are non-biodegradable (Rembold,<br />
1984). This has led to a search for alternative insecticides and botanical insecticides are a promising<br />
source.<br />
The genus Millettia (Wight et Arn.) belongs to family Leguminosae (alternative name Fabaceae).<br />
The family has been reported to contain insecticidal rotenoids (Fukami and Nakajima, 1971).<br />
Millettia usaramensis stem bark extract is therefore a potential source <strong>of</strong> insecticidal rotenoids and<br />
it was interesting to investigate the activity <strong>of</strong> the extract on species <strong>of</strong> insects, and non-target<br />
organisms.<br />
The action <strong>of</strong> several insecticides is influenced by environmental factors, temperature being one <strong>of</strong><br />
them and is a critical factor in tropical areas (Narahashi and Chambers, 1989; Harris and Kinoshita,<br />
1977; Kabaru, 1996; Mwangi et al., 1997). It was therefore worthwhile to investigate posttreatment<br />
effect <strong>of</strong> temperature on the insecticidal activity <strong>of</strong> M. usaramensis stem bark extract on<br />
the locust Schistocerca gregaria. Mice Mus musculus were used in toxicity testing as non-target<br />
organisms.<br />
Materials and Methods<br />
M. usaramensis (stem bark) used in this study was collected from Diani along the Kenyan coast in<br />
2008. The sample was dried in the shade to constant weight and ground to fine powder in a mill,<br />
extracted using dichloromethane/ methanol at the ratio <strong>of</strong> 1: 1 (v/v) and stored desiccated at 4 o C.<br />
Aedes aegypti (L) colony, Schistocerca gregaria Forskal and male Mus musculus used in this study<br />
were obtained from the School <strong>of</strong> Biological Sciences, University <strong>of</strong> Nairobi.<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
A. aegypti larvae were exposed to 0-800mg/L M. usaramensis crude stem bark extract and larval<br />
mortality recorded after 24 h and 48 h exposure. S. gregaria 5 th instar nymphs were exposed to 0-<br />
800mg/L M. usaramensis crude stem bark extract through injection (10µl/g ), topical application<br />
(10µl/g ) and oral administration (6ml per 200 seedlings ) at 28 0 C. Mortality was recorded after 24<br />
h, 48 h, 72 h and 144 h post-exposure. The plant extract anti-feedant tests were conducted<br />
according to Butterworth and Morgan (1971) and Relative Anti-feedant Percentage (RAP)<br />
calculated. A. aegypti larvae were exposed to 0-100 mg/L <strong>of</strong> (+)-12a-epimilletosin, (+)usararotenoid-A<br />
and deguelin isolated from M. usaramensis subspecies usaramensis by Yenesew<br />
(1997). After 24 h and 48 h exposure, larval mortality was recorded.<br />
The toxicity <strong>of</strong> 10 µl/g injected M. usaramensis crude stem bark extract (0-1000 mg/L) to S. gregaria<br />
5 th instar nymphs was tested at 25 0 C and 40 0 C post-treatment temperatures. Mortality was<br />
observed and recorded after 24 and 48 hours. M. musculus were also exposed to M. usaramensis<br />
crude stem bark extract through intraperitoneal (50 µl <strong>of</strong> 0-1600 µg/g), oral (100 µl <strong>of</strong> 0-8000<br />
mg/kg) and topical (200 µl <strong>of</strong> 0-2000 mg/g) administration. Observations for signs <strong>of</strong> toxicity were<br />
made after every 24 hours for 2 weeks.<br />
Results and Discussion<br />
Log probit analysis <strong>of</strong> the larvicidal activity <strong>of</strong> M. usaramensis crude stem bark extract on the 4 th<br />
instar A. aegypti larvae showed a 48 hour activity with a median lethal dose <strong>of</strong> 50.82 mg/L. The<br />
crude extract administered to the locust S. gregaria elicited insecticidal activity <strong>of</strong> LD50 values <strong>of</strong><br />
445.65µg/g through injection at 48 hours, 569.77 µg/g through topical treatment at 72 hours and<br />
504.69 µg/g through oral treatment at 144 hours post exposure. The difference in duration taken<br />
for insecticidal activity to be manifested with method <strong>of</strong> administration could be due to relative<br />
proximity to internal organs, vehicle-solvent penetration ability (Kabaru, 1996; Ware, 1982) and<br />
losses during administration. The crude extract also showed an anti-feedant activity <strong>of</strong> ED50 660.71<br />
µg/ml. The pure compounds (+)-12a- epimilletosin, (+)-usararotenoid-A and deguelin elicited LC50<br />
activities <strong>of</strong> 2037 mg/L, 4.27 mg/L and 2.63 mg/L respectively at 48 hours post exposure. M.<br />
usaramensis crude stem bark extract is therefore larvicidal against A. aegypti and insecticidal as<br />
well as anti-feedant against S. gregaria. The moderate A. aegypti larvicidal activity and S. gregaria<br />
insecticidal activity observed in the crude stem bark extract can be attributed to (+)-usararotenoid-<br />
A, one <strong>of</strong> the major compounds in the extract.<br />
The activity <strong>of</strong> rotenoids against insects is associated with their chemical structure. The main<br />
structural unit <strong>of</strong> all the rotenoids and associated compounds is a fused four-ring system- a<br />
chromanochromanone known as 6a, 12a-dihydrorotoxen-12 (6H)-one . The B/C ring junction in all<br />
<strong>of</strong> the active rotenoids is also cis. Compounds with modified rings and a trans- B/C ring junction are<br />
less insecticidal (Fukami and Nakajima, 1971; Joseph and Casida, 1992). (+)-12a-epimilletosin has<br />
the B/C ring junction with a trans-stereochemistry. This explains its low insecticidal activity. Despite<br />
(+)-usararotenoid-A also having a trans-B/C ring junction, its activity is relatively high. It could be an<br />
exception to the rule or its mechanism <strong>of</strong> action could be different (Yenesew, 1997). However, the<br />
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structure-activity relationship is not entirely clear as less structurally complex is<strong>of</strong>lavonoids have<br />
shown some activity against insects (Bowers, 1983).<br />
The acute toxicity <strong>of</strong> M. usaramensis crude stem bark extract on S. gregaria has a positive<br />
temperature coefficient. Increase in temperature significantly increased toxicity <strong>of</strong> the extract in<br />
the locust S. gregaria by decreasing LD50 from 913.65 µg/g at 25 0 C to 323.59 µg/g at 40 0 C in a 48<br />
hour post-treatment exposure period. This could be due to high temperature accelerating catabolic<br />
reactions or disrupting cell membrane integrity (Kabaru, 1996). This would be <strong>of</strong> great advantage as<br />
locust-prone areas are generally hot.<br />
Neither mortality nor signs <strong>of</strong> toxicity were observed in 2 weeks in mice. This agrees with other<br />
studies (Brook and Price, 1961; Fukami and Nakajima, 1971) that rotenoids are non-toxic to<br />
mammals. This suggests that M. usaramensis stem bark extract is safe to mammalian non-target<br />
organisms.<br />
Acknowledgement<br />
We acknowledge the University <strong>of</strong> Nairobi for providing laboratory space to carry out<br />
investigations. We are grateful to Mr. Francis K. Kamau <strong>of</strong> the Animal House and Mr. Paul<br />
Ambundo <strong>of</strong> the Insectary, School <strong>of</strong> Biological Sciences, University <strong>of</strong> Nairobi, for rearing the test<br />
organisms used in this study.<br />
References<br />
Bowers, W.S. (1983); In Natural products for innovative pest management. Ed. D.L. Whitehead and W.S. Bowers. pp. 47-<br />
72, 313-322. Pergamon Press. New York.<br />
Brooks I.C and Price R.W. (1961); Studies on the chronic toxicity <strong>of</strong> Pro-Noxfish<br />
300<br />
® , a proprietary synergized rotenone fishtoxicant.<br />
Toxicology and Applied Pharmacology 3:49-56.<br />
Butterworth, J.B. and Morgan, E.D. (1971); Investigation <strong>of</strong> the locust feeding inhibition <strong>of</strong> the seeds <strong>of</strong> the neem tree<br />
(Azadirachta indica). J. Insect Physiol. 17: 969-977.<br />
Fukami H and Nakajima M. (1971); Rotenone and rotenoids, In Naturally Occurring Insecticides, Ed by Jacobson M and<br />
Crosby D.G, Marcel Dekker, New York, pp 71-79.<br />
Harris, C.R. and Kinoshita, G.B. (1977); Influence <strong>of</strong> post-treatment temperatures on the toxicity <strong>of</strong> pyrethroid<br />
insecticides. J. Economic Ent. 70:215-218.<br />
Joseph, J. L. and Casida, J.E. (1992); The rotenoid core structure: Modifications to define the requirements <strong>of</strong> the<br />
toxophore. Bio-Organic and Medicinal Chemistry Letters 2: 593.<br />
Kabaru, J.M (1996); A toxicological study <strong>of</strong> Melia volkensii (Gurke) extracts on Locusta migratoria migratoroides (R&F).<br />
PhD Thesis. University <strong>of</strong> Nairobi.<br />
Mwangi R.W, Kabaru J.M and Rembold H. (1997); In New strategies in locust control. Eds S. Krall and Wilps, H. GTZ.<br />
Eschborn pp 193-200.<br />
Narahashi, T. and Chambers, J.E. (1989); Insecticide action: from molecule to organism. pp 75-77. Plenum press, New<br />
York<br />
Rembold, H. (1984); Secondary plant compounds in insect control with special reference to azadirachtins. Advances in<br />
Invertebrate Reproduction 3: 481-491.<br />
Ware, G.W. (1982); Pesticide: Theory and application. Thompson publications, Fresno, California. pp 308.<br />
Yenesew A. (1997); Chemical investigation <strong>of</strong> two Millettia and two Erythrina species (Leguminosae) for bioactive<br />
constituents. PhD thesis. University <strong>of</strong> Nairobi.
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[PS 4] Bioactivity <strong>of</strong> Flemingin A and other Natural Products from the Leaves<br />
<strong>of</strong> Flemingia grahamiana<br />
Ivan Gumula 1,2 , Mathias Heydenreich 3 , Solomon Derese 1 , Faith A. Okalebo 4 , Isaiah O. Ndiege 2 ,<br />
Mate Erdelyi 5 , Abiy Yenesew 1 *<br />
1 Department <strong>of</strong> Chemistry, University <strong>of</strong> Nairobi, P.O. Box 30197-00100, Nairobi, Kenya<br />
2 Department <strong>of</strong> Chemistry, Kyambogo University, P.O. Box 1, Kyambogo-Kampala, Uganda<br />
3 Institut f r Chemie, Universität Potsdam, P.O. Box 60 15 53, D-14415, Potsdam, Germany<br />
4 School <strong>of</strong> Pharmacy, University <strong>of</strong> Nairobi, P.O. Box 30197-00100, Nairobi, Kenya<br />
5 Department <strong>of</strong> Chemistry, University <strong>of</strong> Gothenburg, SE-412 96 Gothenburg, Sweden and<br />
Swedish NMR Centre, University <strong>of</strong> Gothenburg, Box 465, SE-405 30 Gothenburg, Sweden<br />
*Corresponding Author E-mail Address: ayenesew@uonbi.ac.ke (A. Yenesew).<br />
Key Words: Flemingia grahamiana; Leaves; Flemingin A; Emodin; antioxidant activity<br />
Introduction<br />
F<br />
lemingia grahamiana (Wight & Arn.) is an erect herb or sub-shrub up to 1.8 m tall with deep<br />
(sometimes tuberous) roots and 3-foliate alternate leaves. It is distributed in Tropical Africa and<br />
occurs in open and wooded savanna, sometimes near water in riverine vegetation, on hillside,<br />
termite mounds and along roadsides (Gillett, et al., 1971; Jansen, 2005). The powder from the fruits<br />
and inflorescence <strong>of</strong> the plant is one <strong>of</strong> the principal sources <strong>of</strong> a dye and cosmetic called Waras<br />
(or Wurrus, or black kamala) sold in India and Arabia (Cardillo, et al., 1968; Jansen, 2005). The root<br />
decoction <strong>of</strong> the plant is used against diarrhoea and dysentery in Zimbabwe and Malawi. The plant<br />
is also used externally against skin diseases and internally as a purgative and against colds in India<br />
(Jansen, 2005).<br />
In our search for cancer chemopreventive agents from plants, we wish to report the antioxidant<br />
properties <strong>of</strong> a known chalcone, Flemingin A (1) and the characterization <strong>of</strong> a new chalcone with a<br />
3,4-disubstituted-1-methylcyclohexene moiety (2) from the leaves <strong>of</strong> F. grahamiana. Also reported,<br />
for the first time from the genus Flemingia, is the known anthraquinone, emodin (3).<br />
Materials and Methods<br />
The leaves <strong>of</strong> Flemingia grahamiana were collected from Kitale District, Western Province, Kenya,<br />
in October 2008. The plant was identified at the University Herbarium, Botany Department,<br />
University <strong>of</strong> Nairobi.<br />
The air-dried leaves (413.2 g) <strong>of</strong> F. grahamiana were pulverized and extracted with CH2Cl2-MeOH<br />
(1:1) at room temperature to yield 29.6 g <strong>of</strong> crude extract. The extract was subjected to CC on silica<br />
gel, using gradient elution <strong>of</strong> EtOAc in n-hexane as the solvent. Further fractionation and<br />
purification was done by repeated chromatography on silica gel, PTLC, and sephadex LH-20. The<br />
structures <strong>of</strong> isolated compounds were elucidated based on a combination <strong>of</strong> spectroscopic<br />
techniques and by comparing with the data in the literature. Antioxidant property test was done as<br />
described by Ohnishi, et al. (1994).<br />
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Results and Discussion<br />
Compound 1 was obtained as an orange powder with characteristic spectral features <strong>of</strong> a 2'hydroxyl<br />
chalcone and it was identified as Flemingin A (1) (Cardillo, et al., 1968, 1973). The<br />
compound exhibited strong in vitro antioxidant activity against DPPH with an EC50 value <strong>of</strong> 33.3 nM<br />
comparable to that <strong>of</strong> quercetin (21.5 nM, used as the standard), under the same experimental<br />
conditions.<br />
Compound 2 was obtained as a yellow gum and exhibited spectral features typical <strong>of</strong> 2'-hydroxyl<br />
chalcone [ H 13.91, s, 1H (2'-OH); 8.14, d, J= 15.6Hz, 1H (H- ; 7.75, d, J = 15.6Hz 1H (H- ); C 192.8<br />
(C=O); 121.9 (C- ; 139.4 (C- )] similar to those <strong>of</strong> compound 1. Compound 2 differed from 1 by the<br />
presence <strong>of</strong> a 3,4-disubstituted-1-methylcyclohexene moiety instead <strong>of</strong> a 2-methyl-2-(4'-methylpet-<br />
3'-enyl) chromene unit. The presence, in compound 2, <strong>of</strong> 3,4-disubstituted-1-methylcyclohexene<br />
was deduced from 1 H and 13 C NMR spectral data (Table 1): an allylic correlation observed between<br />
H-10'' and H-2'' in the 1 H- 1 H COSY experiment and a long range (H<strong>MB</strong>C) interaction between H-10''<br />
and C-4''. The closeness <strong>of</strong> Rf values, on TLC plate, for the two compounds implied that the number<br />
<strong>of</strong> hydroxyl groups is the same (three) rather than five (with extra OH groups at 4' and 7'').<br />
Therefore, it was concluded that the 3,4-disubstituted-1-methylcyclohexene, in this case, is fused<br />
with a pyrano ring as shown in structure 2 and therefore characterized as: (2E)-1-(6a,7,8,10atetrahydro-1,4-dihydroxy-6,6,9-trimethyl-6H-benzo[c]chromen-2-yl)-3-(2-hydroxyphenyl)prop-2-en-<br />
1-one. The relative stereochemistry <strong>of</strong> the pyrano-cyclohexene junction could not be established<br />
because the peaks (in 1 H NMR spectrum) for H-1'' and H-6'' appeared as multiplets and due to lack<br />
<strong>of</strong> NOE data. The cyclohexene moiety in 2 must have resulted, biogenetically, from the cyclisation<br />
<strong>of</strong> the geranyl group which is common in the chalcones <strong>of</strong> Flemingia species (Cardillo, et al., 1968,<br />
1973). Compounds <strong>of</strong> similar moieties have been reported by Simon-Levert, et al. (2005), Garrido,<br />
et al. (2002) [meroterpenoids]; Botta, et al. (2003) [is<strong>of</strong>lavanones with cannabinoid-like moieties]<br />
but not from the genus Flemingia. Compounds 2 and 3 were not tested for antioxidant properties<br />
due to paucity <strong>of</strong> the samples.<br />
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OH<br />
OH<br />
OH<br />
O<br />
1<br />
1'<br />
O<br />
1"<br />
9"<br />
7"<br />
6"<br />
O OH<br />
2 2"<br />
4"<br />
O<br />
3"<br />
3<br />
10"<br />
OH<br />
5'<br />
2'<br />
OH<br />
5"<br />
303<br />
8"<br />
O<br />
HO<br />
OH<br />
O OH<br />
Table 1. 1 H (300 MHz) & 13 C (75 MHz) NMR Data for the 3,4-disubstituted-1-methylcyclohexene<br />
moiety in Compound 2 (CDCl3)<br />
Position<br />
1<br />
H (J = Hz)<br />
13<br />
C<br />
1 1<br />
H- H COSY<br />
1" 3.65, m 31.5 2", 6"<br />
2" 6.40, d (3.6) 121.4 6", 10"<br />
3" - 134.4 -<br />
4" 2.07, m 29.5 -<br />
5" ax<br />
1.28, m<br />
20.8 -<br />
eq<br />
2.07, m<br />
8"/9"<br />
6" 1.88 m 39.9 1", 8"/9"<br />
7" - 79.7 -<br />
8" 1.51 s 25.5 6"<br />
9" 1.37 s 25.6 6"<br />
10" 1.72 s 23.6 2" (allylic)<br />
Acknowledgements<br />
I.G. is grateful to the Natural Products Research Network for Eastern and Central Africa (NAPRECA)<br />
and the German Academic Exchange Services (DAAD) for a PhD Scholarship and financing the<br />
research studies. Mr S.G. Mathenge is hereby acknowledged for identification <strong>of</strong> the species.<br />
References<br />
Cardillo, B., Genarro, Merlini, L., Nasini, G. and Servi, S. (1973); New Chromenochalcones from Flemingia.<br />
Phytochemistry, 12, 2027-2031.<br />
Cardillo, G., Merlini, L.,Mondelli, R. (1968); Natural Chromenes-III, Colouring Matters <strong>of</strong> Wars: The Structure <strong>of</strong><br />
Flemingins A, B, C and Hom<strong>of</strong>lemingin. Tetrahedron, 24, 497-510.
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Gillett, J.B., Polhill, R.M., Verdcourt, B., Schubert, B.G., Milne-Redhead, E., & Brummitt, R.K., (1971); Leguminosae (Parts<br />
3 4), subfamily Papilionoideae (1 2). In: Milne-Redhead, E. & Polhill, R.M. (Eds.). Flora <strong>of</strong> Tropical East Africa.<br />
Crown Agents for Overseas Governments and Administrations, London, UK. Pp. 1108.<br />
Ohnishi, M., Morishita, H., Iwahashi, H., Toda, S., Shirataki, Y., Kimura, M., Kido, R. (1994); Inhibitory In Vitro Linaleic<br />
Acid Peroxidation and Haemolysis by Caffeoyl Tryptophan. Phytochemistry 47, 1215-1218.<br />
Jansen, P.C.M., (2005); Flemingia grahamiana Wight & Arn. In: Jansen, P.C.M. & Cardon, D. (Eds.). PROTA 3: Dyes and<br />
tannins/Colorants et tanins. [CD-Rom]. PROTA, Wageningen, Netherlands<br />
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[PS 5] Seasonal Variation in the Chemical composition <strong>of</strong> the Bark Ocotea<br />
comoriensis Essential Oils<br />
Mohamed Said Hassani<br />
Faculté des Sciences et Techniques, Université des Comores, B.P. 2052, Moroni Comores<br />
Centre National de Documentation et de Recherche Scientifique (CNDRS), B.P. 169, Moroni Comores<br />
msaidhassani@gmail.com<br />
Key Words: Ocotea comoriensis; bark; essential oil; chemical composition; seasonal variation; Comoros archipelago<br />
Introduction<br />
O<br />
cotea comoriensis Kostermans (Lauraceae) is a tall beautiful and evergreen tree, 10 15 m tall,<br />
with a trunk 30-50 cm in diameter, endemic in the Comoros Islands (Kostermans, 1950).<br />
Localy, this plant is known under the names "Mnaliwa," "Ganja Mrihali" and "Mkanfure". The<br />
Ocotea comoriensis wood is particularly valued for woodworking (Said Hassani et al, 1995).<br />
Our work reports the results <strong>of</strong> the seasonal variation (fruition period and vegetative rest) in the<br />
chemical composition <strong>of</strong> the bark Ocotea comoriensis essential oils.<br />
Material and Methods<br />
Plant Material and Extraction<br />
Essential oils <strong>of</strong> Ocotea comoriensis were extracted by hydro distillation <strong>of</strong> the dry material <strong>of</strong> the<br />
bark collected in different periods (fruition and dormancy periods), using a Clevenger type<br />
arrangement with a 2 L flask. After 5 hours <strong>of</strong> extraction, the essential oils were recovered by<br />
decantation and dried over magnesium sulfate.<br />
Chemical Analysis<br />
Fruition period<br />
GC analyses were performed on a fused silica capillary column SPB-5 (60 m x 0.32 mm, inside<br />
ature was programmed from 60 °C to 200 °C at<br />
4 °C/min and to 230°C (60 min). Nitrogen was used as the carrier gas at a flow rate <strong>of</strong> 0.7 ml/min.<br />
Injector temperature, 250°C; splitless mode; FID temperature, 300°C.<br />
GC-MS analyses were conducted using a Hewlett-Packard chromatograph, Type 6890 and 6890N<br />
series, coupled to an HP 5972 and HP 5973N mass selective detector. The MS detector was used in<br />
the EI mode with an ionization voltage <strong>of</strong> 70 eV. Two capillary columns were used under the<br />
following conditions: (a) Supelcowax TM 10 (60 m x 0.32 mm i.d., film thickness 0.25 m); the oven<br />
temperature programme, 50°C rising at 4°C/min to 230 °C, held for 30 min; ion source temperature,<br />
280°C; injector temperature, 250°C; carrier gas, helium; flow rate, 0.8 ml/min. (b) SBP-5 (60 m x<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
0.32 mm i.d., film thickness 0.25 m); the oven temperature programme, 60°C rising at 4°C/min to<br />
230°C, held for 60 min; ion source temperature, 280°C; injector temperature, 250°C: carrier gas,<br />
helium; flow rate, 0.7 ml/min.<br />
a- Dormancy period<br />
GC analyses were performed on a fused silica capillary column (25 m x 0.32 mm, coated with OV-<br />
101); the oven temperature was programmed from 50 °C to 200 °C at 5 °C/min. GC MS analyses<br />
were carried out on a Hewlett-Packard capillary GC-quadrupole MS system (Model 5970), operating<br />
at 70 eV and fitted with a 25 m x 0.23 m i.d. fused-silica column with DB-5. The temperature was<br />
programmed as follows: 50 °C (3 min), 50 - 200°C at 3°C/min. Helium was used as the carrier gas at<br />
a flow rate <strong>of</strong> 0.9 ml/min.<br />
Identification and Quantification<br />
Retention indices <strong>of</strong> all the constituents were determined by the Kovats method; the oils were<br />
spiked on both phases with a standard mixture <strong>of</strong> n-alkanes series (C8-C22) and analyzed by GC-MS<br />
under the previous conditions. Constituents <strong>of</strong> the volatile oil were identified by comparison <strong>of</strong><br />
their retention indices and their mass spectral fragmentation patterns with those reported in the<br />
literature (Adams, 200l and Stenhagen et al., 1974) and those stored on MS Library (NBS75K).<br />
Results and Discussion<br />
The essential oil yields for fruition and dormancy periods were 0.20% and 0.35%, respectively.The<br />
bark essential oil collected during fruiting contains monoterpenes (0.4%), sesquiterpenes (84.7%)<br />
and aromatic compounds (5.8%). This oil is dominated by caryophyllene oxide (11.3%), -ylangene<br />
(8.2%), epi- -cadinol (6.1%), -muurolene and -muurolène (5.1% and 5.0%, respectively), -<br />
amorphene (4.4%), -cadinene (3.3%), -copaene (3.1%), -cadinene (2.8%) and -selinene (2.4%)<br />
(Said Hassani M., 2010).<br />
The bark essential oil collected in dormancy period exclusively consists <strong>of</strong> monoterpenes and<br />
sesquiterpenes. In fact, it includes: 67.9% monoterpenes and 27.4% sesquiterpenes. Its high<br />
content <strong>of</strong> monoterpenes is mainly owed to high percentages <strong>of</strong> camphene (18.1%), bornyl acetate<br />
(13.8%), -pinene (13.7%), -pinene (8.4%) and limonene (5.6%). Among the identified<br />
sesquiterpenes, there are -cubebene (4.5%) and -cadinol (3.0%) (Menut et al., 2002).<br />
The essential oil yield appears somewhat more important in vegetative rest period (0.35%) than in<br />
the fruiting period (0.20%).<br />
For the bark essential oil studied at two periods <strong>of</strong> the vegetative cycle (fruiting and vegetative rest<br />
periods) for a same specimen <strong>of</strong> Ocotea comoriensis, significant differences in chemical<br />
composition can be identified. Whereas in fruiting period, essential oil is largely dominated by<br />
sesquiterpenes (84.7%), in vegetative rest period, its proportion in monoterpenes becomes<br />
definitely more important, passing from 0.4% to 67.9%. The conducted study thus highlights a<br />
variation <strong>of</strong> the chemical composition <strong>of</strong> the bark essential oil according to the vegetative cycle.<br />
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Acknowledgements<br />
The author would like to thank Pr<strong>of</strong>essor Labat JN (MNHN-Paris), Mrs Ramadhoini Islame (TSF -<br />
Comoros University) and Mr Yahaya Ibrahim (CNDRS-Moroni) for the identification <strong>of</strong> the plant<br />
material. The author is also highly grateful to Pr<strong>of</strong>essor Smadja J., Dr-HDR Bialecki A. (Reunion<br />
University), Pr<strong>of</strong>essor Gurib Fakim A. (Mauritius University) and Menut C (Chemistry School-<br />
Montpellier University-France) for their guidance and assistance. Financial support Agence<br />
Universitaire de la Francophonie.<br />
References<br />
Adams, RP, (2001); In ldentification <strong>of</strong> Essential Oils by Gas Chromatography/Quadrupole Mass Spectroscopy. Allured:<br />
Carol Stream, IL.<br />
Kostermans, AJGH. (1950); In Flore de Madagascar et des Comores, 81 st Famille. Muséum National d Histoire Naturelle:<br />
Paris, France.<br />
Menut, C., Bessière, J.M., Said Hassani, M., Buchbauer, G. and Schopper, B., (2002); Chemical and Biological studies <strong>of</strong><br />
Ocotea comoriensis bark essential oil, Flavour Fragr. J., 17, 459-461.<br />
Said Hassani M. and Yahaya I., (1995); Programme Tramil-Comores. Rapport final. Moroni. Comores.<br />
Said Hassani, M., (2010); Valorisation de quatre plantes endémiques et indigènes des Comores. Thèse de doctorat, UFR<br />
Sciences et Technologies, Université de la Réunion, La Réunion-France.<br />
Stenhagen, E, Abrahamsson, S and McLafferty, FW, (1974); Registry <strong>of</strong> Mass Spectra Data. Wiley: New York<br />
307
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[PS 6] Proximate and Amino Acid Composition <strong>of</strong> Cowpea (Vigna ungiculata<br />
L.walp) Flour and Protein Isolates<br />
Khalid, I.I., Elhardallou, S.B. and Elkhalifa, E.A.<br />
Key Words: Cowpea, Protein isolates, Amino acid Composition<br />
Introduction<br />
T<br />
he cowpea (Vigna ungiculata L.walp) is a grain legumes believed to have originated in Africa<br />
and Asia (Taiwo, 1998), and is widely cultivated in the tropics (Chavan et al., 1989). As a<br />
legume, cowpeas are rich and low cost sources <strong>of</strong> proteins and nutrients (Egounlety and Aworth,<br />
2003) and they form part <strong>of</strong> staple diet in most African and Asian countries (Aykroyd and Doughty,<br />
1964).<br />
Material and Methods<br />
Proteins were isolated from dehulled cowpea flour by isoelectric and micellization precipitation<br />
techniques. Cowpea protein isolate- A (CPIA) was prepared from cowpea seed flour following the<br />
method described by Fernandez-quintela et al., (1997). Protein isolate-B (CPIB) was prepared using<br />
micella method as described by Lampart-Szczapa, (1996). Amino acids analysis was performed on<br />
(DDCF) and protein isolates (CPIA and CPIB) using amino acid analyzer according to the method<br />
described by Moore, et al., (1958), using the FAO/WHO (1973) reference pattern.<br />
Results and Discussion<br />
Chemical composition <strong>of</strong> dehulled defatted cowpea flour (DDCF) and protein isolates (CPIA and<br />
CPIB) are presented in Table 1. The CPIB showed significantly (P
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Table: 1 Proximate composition <strong>of</strong> whole cowpea flour (WCF), dehulled defatted cowpea flour<br />
(DDCF) and protein isolates (CPIA) and (CPIB) % dry basis<br />
Chemical constituents WCF DDCF CPIA CPIB LSD<br />
Crude protein (N x 6.25) 22.30 d 0.20 26.73 c 0.06 75.0 b 0.06 76.0 a 0.12 0.26<br />
Crude fat 2.10 a 0.10 2.30 a 0.10 Traces Traces 0.43<br />
Crude fibre 4.10 a 0.20 1.02 b 0.08 Traces Traces 0.35<br />
Total ash 3.77 a 0.06 3.87 a 0.06 2.63 b 0.15 2.3 b 0.20 0.55<br />
Carbohydrate (by<br />
difference)<br />
60.07 a 0.06 59.78 a 0.28 13.0 b 0.17 13.1 b 0.0 0.43<br />
Means in the same raw with different letters are significantly different (P < 0.05).<br />
Means ± standard deviation <strong>of</strong> triplicate analysis.<br />
LSD = Least significant differences.<br />
Table 2: Amino acid composition <strong>of</strong> dehulled defatted cowpea flour (DDCF) and cowpea protein isolates<br />
(CPIA and CPIB)<br />
Amino acid<br />
Isolucine<br />
Leucine<br />
Lysine<br />
Cystine<br />
Methionine<br />
Tyrosine<br />
Phenylalanine<br />
Threonine<br />
Tryptophan<br />
Valine<br />
Histidine<br />
Argnine<br />
Aspartic<br />
Glutamic acid<br />
Serine<br />
Proline<br />
Glysine<br />
Alanine<br />
DDCF<br />
0.98<br />
1.58<br />
4.28<br />
0.032<br />
-<br />
3.33<br />
2.0<br />
0.44<br />
ND<br />
0.72<br />
0.77<br />
2.66<br />
ND<br />
3.32<br />
0.89<br />
7.71<br />
0.68<br />
0.89<br />
CPIA<br />
309<br />
7.92<br />
8.81<br />
22.99<br />
0.06<br />
27.22<br />
16.31<br />
12.37<br />
7.18<br />
ND<br />
ND<br />
7.88<br />
17.09<br />
ND<br />
21.49<br />
8.09<br />
23.14<br />
3.32<br />
2.74<br />
CPIB<br />
8.20<br />
8.85<br />
15.78<br />
-<br />
30.60<br />
19.83<br />
11.96<br />
4.18<br />
ND<br />
6.61<br />
9.07<br />
19.26<br />
ND<br />
39.69<br />
11.19<br />
24.33<br />
3.97<br />
3.0<br />
FAO/WHO<br />
(1973) (g/16g<br />
nitrogen)<br />
4.0<br />
CPIA= Cowpea protein isolate by isoelectric point precipitation<br />
CPIB = Cowpea protein isolate by micellization precipitation<br />
Table 3: Classification <strong>of</strong> amino acids (g/ 16 g) <strong>of</strong> dehulled defatted cowpea flour (DDCF) and protein<br />
isolates (CPIA) and (CPIB).<br />
7.0<br />
5.50<br />
3.5<br />
3.5<br />
6.0<br />
6.0<br />
4.0<br />
1.0<br />
5.0<br />
-<br />
-<br />
-<br />
-<br />
-<br />
-<br />
-<br />
-
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Amino acid description<br />
Total amino acids (TAA)<br />
Total essential amino acids (TAA)<br />
with histidine<br />
Total essential amino acids (TAA)<br />
with out histidine<br />
Total non essential amino acids<br />
(TNAA)<br />
Essential aromatic amino acid<br />
(EArAA)<br />
Total acid amino acid (TAAA)<br />
Total basic amino acid (TBAA)<br />
Total sulphur amino acid (TSAA)<br />
310<br />
DDCF<br />
30.28<br />
14.13<br />
13.36<br />
16.92<br />
5.33<br />
3.32<br />
8.79<br />
0.032<br />
CPIA<br />
192.21<br />
156.34<br />
108.46<br />
83.75<br />
28.68<br />
21.49<br />
23.98<br />
27.28<br />
CPIB<br />
216.52<br />
115.08<br />
106.01<br />
110.51<br />
31.79<br />
39.69<br />
22.05<br />
30.60<br />
References<br />
Aykroyd, W.R. and Doughty. J. (1964); Legumes in human nutrition, Food and Agriculture Organization <strong>of</strong> the United<br />
Nation (FAO), Nutritional Studies No. 19, FAO, Rome.<br />
Chavan, J.K.; Kadam, S.S. and Salunkhe, D.K. (1989); Cowpea in: Hand book <strong>of</strong> world food legume: Nutritional chemistry,<br />
processing technology and utilization, volume 2. Editors Salunkhe and Kadam. CRC press, Florida.<br />
Egouniety, M. and Aworh, O.C. (2003); Effect <strong>of</strong> soaking, dehulling, cooking and fermentation on the oligosaccharides,<br />
trypsin inhibitors, phytic acid and tannins <strong>of</strong> soybean (Glysin max Merr.), cowpea (Vigna ungiculata Walp) and<br />
ground bean (Mccrotyloma geocarpa Harms). Journal <strong>of</strong> Engineering 56: 249 254.<br />
FAO/WHO (1973); Energy and protein requirements. Report <strong>of</strong> a joint FAO/WHO adhoc expert committee. FAO<br />
Nutritional Meeting Report Series No. 52, Technical Report Series No. 522 Food and Agriculture Organization<br />
<strong>of</strong> the United Nation, Rome (1973).<br />
Fernandez-Quintela, Q.A.; Macarulla, M.T.; Del-Barrio, A.S. and Martinez. J.A. (1997); Composition and functional<br />
properties <strong>of</strong> protein isolates obtained from commercial legumes grown in northern Spain.<br />
Plant Food Human Nutrition 51: 331 342.<br />
Lampart-Szczapa, E.; Obuchowski, W.; Czaczyk, K.; Pastuszewska, B. and Buraczewska, L. (1996); Effect <strong>of</strong> lupin flour on<br />
the quality and oligosaccharides <strong>of</strong> pasta and crisps. Nahrung/Food 41 (4): 219 223.<br />
Moore, S.; Spackman, D. H. and Stein, W. H. (1958); Chromatography <strong>of</strong> amino acid on sulphonated polystyrene resins.<br />
A analytical Chemistry 30: 1185- 1190.<br />
Taiwo, K.A. (1998); The potential <strong>of</strong> cowpea as human food in Nigeria Technovation 18: 469 481.
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[PS 7] Molecular Species Identification and the Respective Quantification <strong>of</strong><br />
Dioscin: A Case <strong>of</strong> Dioscorea spp<br />
Kariuki S. M. 1, 2 , D. J. Kim 2* , Dossaji S. F. 1 , Kabaru J. M. 1<br />
1 School <strong>of</strong> Biological Sciences, University <strong>of</strong> Nairobi Po Box 30197-00100 Nairobi<br />
2 International Institute <strong>of</strong> Tropical Agriculture (IITA), Po Box 30709-00100 Nairobi<br />
Key Words: Dioscorea, PCR, Species, dioscin, HPLC<br />
* Corresponding author: D. J. Kim: dj.kim@cgiar.org<br />
Introduction<br />
D<br />
ioscorea (Yams) in Kenya is a neglected crop, despite having a lot <strong>of</strong> potential as food source<br />
and as well playing a role in pharmaceutics (Mwirigi et al, 2009). Pharmacologically, Dioscorea<br />
species is known to have health promoting molecules among them the steroidal Saponin called<br />
dioscin. On acid hydrolysis <strong>of</strong> dioscin, diosgenin is derived. Diosgenin is used in the production <strong>of</strong><br />
steroidal hormones like progesterone (Sautour et al., 2004). Apart from being used in steroidal<br />
hormones production, dioscin has other health promoting effects such as antioxidant effects and<br />
reduction <strong>of</strong> postmenopausal symptoms in women (Wang et al., 2002). It is therefore essential to<br />
quantify the amounts <strong>of</strong> dioscin in Dioscorea species in Kenya. However, for dioscin quantification<br />
to be possible it was imperative to identify the taxonomic position <strong>of</strong> these yams. The taxonomic<br />
position <strong>of</strong> cultivated yams in Kenya has been a subject <strong>of</strong> speculation over the years (Mwirigi et al,<br />
2010). This project aimed at establishing the taxonomic position <strong>of</strong> Kenyan yams in relation to gene<br />
bank species and West African yams using a molecular approach and the respective identification<br />
and quantification <strong>of</strong> dioscin from the tubers.<br />
Materials and methods<br />
This study used three universal molecular markers (matK, rbcL, trnL_F) to investigate the species<br />
position <strong>of</strong> cultivated Kenyan yams and identify their relatedness to major African species and to<br />
species in the gene bank. The study also used reverse phase High performance Liquid<br />
Chromatography (RP-HPLC) to identify and quantify dioscin content in Kenyan yams. DNA was<br />
extracted from lyophilized tubers and leaf samples and Polymerase chain reaction (PCR) carried out<br />
on the DNA samples with direct sequencing <strong>of</strong> the PCR products. DNA sequences were assembled<br />
with sequencher ® and then a multiple sequence alignment through clustalW in MEGA 5 from which<br />
phylogenetic trees were drawn. Dioscin was extracted from 5g <strong>of</strong> freeze dried tubers for<br />
consequent identification and quantification. Identification and quantification <strong>of</strong> dioscin was<br />
carried out using a Reverse Phase High Pressure Liquid Chromatography (RP-HPLC).<br />
Results and discussion<br />
Results indicated that there is more to the species taxonomy in Kenyan Yams than currently known.<br />
Currently yams are said to be <strong>of</strong> the species D. minutiflora, D. dumetorum and D. bulbifera;<br />
however from our study, the species D. cayanensis, D. alata, D. rotundata, D. mangenotitana, D.<br />
schimperiana and D. bulbifera were also shown to be growing in Kenya. HPLC analysis indicated the<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
presence <strong>of</strong> dioscin in Kenyan yam samples albeit at low quantities. The quantities <strong>of</strong> dioscin were<br />
variable with the least being 0.884 Parts per billion (ppb) and the highest 5.12 ppb). The dioscin<br />
quantities were not species specific and a high dioscin variability was also noted in samples<br />
identified to be <strong>of</strong> the same species. Further sampling <strong>of</strong> Kenyan Dioscorea samples both wild and<br />
cultivated is essential to enhance detailed dioscin identification and quantification and as well<br />
check for more species. A tool that is robust enough to identify intra specific variability in<br />
Dioscorea species is also suggested for future comparative studies.<br />
Acknowledgements<br />
We would like to thank the Biosciences Eastern and Central Africa (BECA) for their Laboratory space<br />
and use <strong>of</strong> equipments. This research was funded by the International Institute <strong>of</strong> Tropical<br />
Agriculture (IITA) Ibadan Nigeria.<br />
References<br />
Mwirigi P. N, Kahangi, E. M., Nyende, A. B. and Mamati, E.G. (2009); Morphological variability within the Kenyan yam (<br />
Dioscorea spp .). Agriculture, 894 - 901.<br />
Sautour M, Mitaine-Offer AC, MiDioscoreaoto T, Dongmo A and Lacaille-Dubois MA (2004); Antifungal steroid saponins<br />
from Dioscorea cayenensis. Planta Med 70:90-2.<br />
Wang L. J, Wang Y, Chen SW, Ma JS, Fu Q, Wang BX. (2002); The antitumor activity <strong>of</strong> Diosgenin in vivo and in vitro].<br />
Zhongguo Zhong Yao Za Zhi. 2002 Oct; 27(10):777-9. Chinese. PubMed PMID: 12776562<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[PS 8] Cuauthemone Sesquiterpenes from Laggera Tomentosa Endemic to<br />
Ethiopia<br />
Kibrom Gebreheiwot 1 , Dibaba Amenu 2 and Nigist Asfaw 1*<br />
1 Department <strong>of</strong> Chemistry, Addis Ababa University, P.O. Box 1176. Addis Ababa, Ethiopia<br />
2 Gilgel Beles College <strong>of</strong> Teachers Education, P.O. Box 36. Gilgel Beles, Ethiopia<br />
*Corresponding author. E-mail: nigista@chem.aau.edu.et; nigistasfaw@hotmail.com<br />
KEY WORDS: Laggera tomentosa, Asteraceae, Cuauthemone sesquiterpenes, 3-O-(3 -acetoxy-2 -hydroxy-2 -<br />
methylbutyryl)cuauthemone<br />
Introduction<br />
L<br />
aggera tomentosa (Sch. Bip. ex A. Rich) Oliv & Hiern (Asteraceae) is a perennial fragment bushy<br />
herb (0.5-1.2 m high) endemic to Ethiopia. Traditionally, the juice <strong>of</strong> the crushed leaves is<br />
ingested as a treatment for stomachache, and is used against migraine. It is also used as a fumigant<br />
and for cleansing milk containers (Mesfin, 2004). Phytochemical studies on the essential oil <strong>of</strong> L.<br />
tomentosa have been reported before (Asfaw et al, 1999, 2003). However, there are no reports on<br />
the chemical investigation <strong>of</strong> the solvent extract <strong>of</strong> this species prior to this work.<br />
MATERIAL AND METHODS<br />
Plant material<br />
Laggera tomentosa was collected from Daletti, Western Shoa <strong>of</strong> Ethiopia in November 2005. A<br />
voucher specimen (SD 6487) is deposited at the National Herbarium (ETH), Department <strong>of</strong> Biology,<br />
Addis Ababa University.<br />
Methods<br />
The dried and milled aerial parts <strong>of</strong> L. tomentosa were extracted by maceration with petroleum<br />
ether (54-93 °C) at room temperature for 24 hours, and then evaporated in vacuo. The residue was<br />
then soaked with ethanol twice at room temperature for up to 24 hours each and then evaporated<br />
in vacuo. The dried petroleum ether and ethanol extract were fractionated on column<br />
chromatography using pet. ether and ethylacetate as solvent system and the fractions monitored<br />
with TLC. The structures <strong>of</strong> the pure compounds were elucidated by spectroscopic techniques<br />
including 1D and 2D NMR techniques as well as by chemical methods.<br />
Results and Discussion<br />
The three cuauthemone sesquiterpenes, 3-(3 -acetoxy-2 -hydroxy-2 -methylbutyryl)- cuauthemone<br />
(1), 4-O-acetylcuauthemone-3-O-angelate (2), 4-O-acetylcuauthemone-3-O-(2 -hydroxy-2 -methyl-<br />
3 - acetoxybutyrate) (3) are reported for the first time from L. tomentosa. The compounds, 4-Oacetylcuauthemone-3-O-angelate<br />
(2) (Guilhon and Muller, 1996), 4-O-acetylcuauthemone 3-O-(2 -<br />
hydroxy-2 -methyl-3 -acetoxybutyrate) (3) (Bohlmann et ak, 1985) were identified by comparison <strong>of</strong><br />
their spectroscopic data with reported values in the literature. The structure <strong>of</strong> compound 1 was<br />
fully characterized in this work based on 1D and 2DNMR data including DEPT-135, COSY, HSQC, and<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
H<strong>MB</strong>C. Dominguez et al, 1988 reported similar structure as 1 for a compound isolated from Pluchea<br />
purpurescens and the structure was elucidated from MS, IR and 1H NMR data. The MS and IR data<br />
reported are similar with those <strong>of</strong> compound 1 isolated in this study. However, the 1HNMR<br />
assignment by Dominguez et al. is different from some <strong>of</strong> the protons reported by us. This prompts<br />
us to do comprehensive NMR analysis <strong>of</strong> compound 1 (Table 1).<br />
4'<br />
3'<br />
AcO<br />
O<br />
1' O<br />
OH<br />
1<br />
HO<br />
1<br />
9<br />
3 5 7<br />
15<br />
H<br />
O<br />
13<br />
2<br />
314<br />
3<br />
R<br />
AcO<br />
1<br />
5<br />
H<br />
9<br />
7<br />
O<br />
13<br />
15<br />
O<br />
R= O<br />
3 R=<br />
AcO<br />
O<br />
O<br />
OH<br />
Figure 1: Cuauthemone sesquiterpenes isolated from L. tomentosa<br />
Table 1. 1 H, 13 C, and H<strong>MB</strong>C (H C) spectral data <strong>of</strong> compound 1 a (in CDCl3).<br />
no. C (ppm) H(ppm) H<strong>MB</strong>C H (ppm) Dominguez et al<br />
1 33.4 1.45 (m), 1.28 (m) H 1a C 10 , H 1a C 14<br />
H 1b C 10 , H 1b C 14<br />
no value<br />
2 23.9 1.79 (m), 1.75 (m) H 2 C 10 no value<br />
3 78.9 4.89 (br t) H 3 C 1 , C 4 , C 5 , C 15 , C 1 4.91(ht, J = 3 Hz)<br />
4 72.2 - -<br />
5 46.6 1.92 (dd, J = 4, 8 Hz) H 5 C 6 , C 7 , C 9 , C 14 , C 15 no value<br />
2.91 (dd, J = 4, 16 Hz), 2.17<br />
6 25.5 (dd, J = 12, 16 Hz) H 6 C 5 , C 7 , C 8 , C 11<br />
2.94 (ddbr, J = 4, 15 Hz), 2.09<br />
(ddbr, J = 13, 15 Hz)<br />
7 130.5 - -<br />
8 202.1 - -<br />
9 59.7 2.22 (s) H 9 C 1 , C 5 , C 7 , C 8 , C 14 1.93(dd, J = 13,5)<br />
10 35.8 - -<br />
11 145.9 - -<br />
12 23.6 2.03 (s) H 12 C 7 , C 8 , C 11 , C 13 2.23 (brs)<br />
13 22.9 1.82 (s) H 13 C 7 , C 8 , C 12 1.83 (brs)<br />
14 18.7 0.94 (s) H 14 C 1 , C 5 , C 9 , C 10 0.93 (s)<br />
15 21.5 1.26 (s) H 15 C 5 1.27 (s)<br />
1 174.7 - -<br />
2 76.4 - -<br />
3 74.4 5.12 (q, J = 4 Hz) H 3<br />
C 1 , C 4 , C 5 , COCH3 5.13(q, J = 6.5 Hz)<br />
4 13.3 1.28 (d, J = 4 Hz) 1.30(d, J = 6.5 Hz)<br />
5 22.4 1.40 (s) H 5<br />
C 1 , C 3 1.42(s)<br />
- OAc 169.8 -<br />
21.0 1.98 (s) COCH3 C 3 -<br />
a<br />
400 and 100 MHz, respectively.<br />
, COCH3<br />
1.99(s)<br />
Among the 20 Laggera species, L. pterodonta, L.alata, L. crispata and L. decurrens have been<br />
extensively investigated and 51 eudesmanes sesquiterpenes and five flavonoids have been<br />
reported from these species (Li et al, 2007). The cuauthemone eudesmanes isolated from<br />
L.tomentosa have not been reported from the above mentioned species. The cuauthemone<br />
eudesmanes are rather characteristic <strong>of</strong> the genus Pluchea (Mukhopadhyay et al, 1983), which<br />
belong to the same tribe, Plucheeae, as the genus Laggera. This study indicated that L. tomentosa is<br />
chemically related to some Pluchea species, due to the co-occurrence <strong>of</strong> cuauthemone
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
sesquiterpenes, rather than to the Laggera species studied so far. Further studies need to be<br />
carried out to establish the chemotaxonomic relationship <strong>of</strong> the species within the genus Laggera,<br />
and between L.tomentosa and Pluchea species.<br />
Acknowledgements<br />
Financial support from NAPRECA and Addis Ababa University for the participation <strong>of</strong> 14 th NAPRECA<br />
symposium is gratefully acknowledged. We thank ALNAP for the provision <strong>of</strong> NMR spectroscopic<br />
equipment used in this investigation. Pr<strong>of</strong>. Wendemagegn Mamo is gratefully acknowledged for his<br />
constructive suggestions in the interpretation <strong>of</strong> the 2D-NMR spectra. We are thankful to Dr.<br />
Haregewine Taddese and School <strong>of</strong> Chemistry, University <strong>of</strong> Nottingham for recording HRMS<br />
spectrum. NA acknowledges partial support from the ChemRAWN XIV International Green<br />
Chemistry Grants Program, USA. Pr<strong>of</strong>. Sebsebe Demissew is gratefully acknowledged for the<br />
collection and authentication <strong>of</strong> the plant material.<br />
References<br />
Asfaw, N., Storesund, H.J., Skattebol, L, Aasen, A.J. (2003); J. Essent. Oil Res. 15, 102.<br />
Asfaw, N., Storesund, H.J., Skattebol, L, Aasen, A.J. (1999); Phytochemistry, 52, 1491.<br />
Bohlmann, F., Wallmeyer, M., Jakupovic, J., Gerke, T., King, R.M., Robinsone, H. (1985); Phytochemistry, 24, 505<br />
Dominguez, X.A., Sanchez V.H., Vazquez, G., Grenz, M. (1988); Rev. Latinoamer. Quim.19, 91<br />
Guilhon, G.M.S.P., Muller, A.H. (1996); Phytochemistry, 43, 417.<br />
Li, X.C., Huo, C.H., Shi, Q.W., Kiyota, H. (2007); Chem. Biodiver. 4, 105.<br />
Mesfin, T. (2004); The Flora <strong>of</strong> Ethiopia and Eritrea, Vol. 4, Part 2, The National Herbarium, Addis Ababa University:<br />
Addis Ababa and Uppsala University: Uppsala; p 140.<br />
Mukhopadhyay, S., Cordell, G.A, Ruangrungsi, N., Rodkird, S., Tanyivatana, P., Hylands, P.J. (1983); J. Nat. Prod., 46, 671.<br />
315
A<br />
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[PS 9] Novel Limonoids and Flavonoid from the Kenyan Vepris uguenensis<br />
Engl. and their Antioxidant Potential<br />
Joyce Jepkorir Kiplimo a , Shahidul Md. Islam b and Neil A. Koorbanally a*<br />
a School <strong>of</strong> Chemistry, University <strong>of</strong> KwaZulu Natal, Westville, Durban 4000, South Africa<br />
b School <strong>of</strong> Biochemistry, Genetics and Microbiology, University <strong>of</strong> KwaZulu Natal, South Africa<br />
phytochemical investigation <strong>of</strong> Vepris uguenensis (Rutaceae) has led to the isolation <strong>of</strong> two<br />
new A, D-seco-limonoids that were accorded the trivial names, uguenensene (1) and<br />
uguenensone (2) and a new C-7 prenylated flavanoid, uguenenprenol (3). In addition, known<br />
compounds, kihadalactone A, tricoccin S13 acetate, limonyl acetate, methyl uguenenoate, niloticin,<br />
chisocheton A, skimianine, flindersiamine, 8 ,11-elemodiol, 7-O-methylaromadenrin, and lupeol<br />
were also isolated. The structures <strong>of</strong> the new compounds were elucidated and characterized by<br />
both 1D and 2D NMR and mass spectroscopy. The crude extracts were obtained by sequential<br />
extraction using a soxhlet apparatus and compounds were isolated and purified by repeated<br />
column chromatography. Antioxidant activity <strong>of</strong> the isolated compounds using the DPPH,<br />
Deoxyribose and Ferric Reducing Power assays showed that uguenenprenol (3) and 7-Omethylaromadenrin<br />
are good antioxidant agents. The isolated Limonoids displayed an interesting<br />
biogenetic relationship that might be expected for limonin biosynthesis. The current contribution<br />
adds uguenensene (1) and uguenensone (2) to the class <strong>of</strong> citrus limonoids common to Rutaceae<br />
[1].<br />
28<br />
O<br />
2<br />
1<br />
O<br />
3<br />
4<br />
O<br />
19<br />
5<br />
9<br />
10<br />
29<br />
11<br />
6<br />
12<br />
8<br />
30<br />
7<br />
18<br />
22<br />
13<br />
14<br />
O<br />
OCOCH3<br />
1. R = 2H<br />
2. R = O<br />
20<br />
17<br />
16<br />
15<br />
23<br />
O<br />
21<br />
Reference<br />
1. Moriguchi, T.; Kita, M.; Hasegawa, S.; Omura, M. J. Food Agri. Environ. 2003, 1 (1), 22-25.<br />
R<br />
5''<br />
3''<br />
316<br />
4''<br />
OH<br />
2''<br />
1''<br />
6<br />
7<br />
8<br />
5<br />
OH<br />
9<br />
10<br />
3<br />
O<br />
O<br />
4<br />
3<br />
2<br />
6'<br />
1'<br />
OH<br />
5'<br />
2'<br />
4'<br />
3'<br />
OH
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[PS 10] Need to Regulate Herbal Remedies in Kenya<br />
Richard N. Mbithi<br />
MITI MINGI CONSERVATION CENTRE; PROGRAM OFFICER; P.O. BOX 4500, ELDORET KENYA<br />
nyamaimbithi@gmail.com<br />
Introduction<br />
T<br />
raditional plant based medicines play an important role in African society; supplying accessible<br />
medicines, sustaining cultures and providing income earning and enterprise development<br />
opportunities. For Kenya, the new constitution, now, recognizes herbal Practitioners and reads:<br />
Every person has a right to health, which includes the right to Health care services, whether<br />
allopathic or complementary and alternative medicine including reproductive health care.<br />
According to WHO report estimates show that at least 80% <strong>of</strong> Kenyans have used herbal medicines<br />
at least once.<br />
Though herbal medicines are vital in improving the health <strong>of</strong> Kenyans there is urgent need to<br />
formulate safety and governance regulations.<br />
Herbal medicines are not regulated in Kenya. In Kenya, the only instrument available for protecting<br />
traditional knowledge is the trade secret. The Industrial Property Act Cap 509 <strong>of</strong> the Laws <strong>of</strong> Kenya,<br />
which could protect the intellectual integrity <strong>of</strong> traditional practitioners, disqualifies traditional<br />
knowledge. Medicinal plants are therefore collected and used without any regulation, opening<br />
them to indiscriminate exploitation and bio-piracy. Huge pr<strong>of</strong>its are made but never shared with<br />
the custodians <strong>of</strong> biological diversity.<br />
Observations<br />
In Kenya there is no registration system for herbal medicines and they are not included on the<br />
essential the essential drug list. These traditional medicines are sold without restriction. Due to<br />
lack <strong>of</strong> these guidelines, it has been discovered that the herbal remedies sector in Kenya is not<br />
harmony.<br />
It was discovered that some herbalists were mixing concoctions <strong>of</strong> conventional medicines and<br />
passing them as herbal medicines. Crook herbalists targeted immunity boosting cures, sexual<br />
enhancement and contraceptives.<br />
The ingredients currently used by most herbalists are a matter <strong>of</strong> guess since there are no machines<br />
to test the content <strong>of</strong> the herbs they recommend to patients. A draft policy formulated in 2008 lies<br />
unimplemented. Other consequences <strong>of</strong> the unregulated herbs were noted with a lot <strong>of</strong> concern by<br />
the pharmacy and poisons board <strong>of</strong> Kenya in July 2010.PPB drew guidelines on herbal and<br />
complimentary medicine but not yet in force .These guidelines are meant to harmonize the<br />
industry and also dismiss the issues as follows:<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
a) Misconception amongst herbalists that documentation requested for by PPB is intended<br />
to steal their indigenous knowledge and thus, there has been hesitation to submit<br />
applications.<br />
b) Lack <strong>of</strong> documented evidence on quality, safety & efficacy <strong>of</strong> Herbal and complementary<br />
products.<br />
c) Unethical practices that include:-<br />
Adulteration <strong>of</strong> herbal and complementary products with conventional medicines.<br />
Advertising <strong>of</strong> Herbal and complementary products in print media, electronic and bill<br />
boards.<br />
Peddling <strong>of</strong> products with no therapeutic benefits.<br />
Unsubstantiated medicinal claims by herbal practitioners.<br />
Dealing with herbal products whose toxicological pr<strong>of</strong>ile is not known.<br />
d) Poor standards <strong>of</strong> preparation / manufacture and sale <strong>of</strong> herbal and complementary<br />
products.<br />
This guideline will focus on the manufacture, registration and marketing <strong>of</strong> herbal and<br />
complementary medicines.<br />
Fake herbal contraceptives being sold caused serious side effects on the users. It was noted that the<br />
women who used the contraceptives developed serious hormonal alterations that made the<br />
children develop adolescent features, including the start <strong>of</strong> menstrual cycle in three year old girls.<br />
Several women and patients and children admitted at Kenyatta National Hospital had been<br />
diagnosed with the side effects <strong>of</strong> the drug.<br />
There were cases <strong>of</strong> cancer and HIV aids patients losing lives after using these drugs.<br />
With the few mentioned cases <strong>of</strong> mal -practices in the herbal industry noted in the country and<br />
many more unreported, there is urgent need for Kenya to enact guidelines on the use <strong>of</strong> herbal<br />
cures.<br />
Why regulate the industry?<br />
It is the duty <strong>of</strong> the nation to protect the lives <strong>of</strong> its citizens. Health is a human right and in the<br />
assurance <strong>of</strong> healthy nation, the subject <strong>of</strong> safety counts a great deal.<br />
Regulation will check the following:-<br />
a) Safety assessment <strong>of</strong> the traditional medicines<br />
b) Reduce the vice <strong>of</strong> counterfeit drugs<br />
c) The standards ranging from the contents, prescription and packaging.<br />
d) Help weed out rogue practitioners<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
e) Protection <strong>of</strong> intellectual property rights (patenting formulas and the ingredients used<br />
by specific herbalists)<br />
f) Protect the indigenous knowledge /culture preservation<br />
Recommendations<br />
Regulations on the use <strong>of</strong> herbal medicines in Kenya ought to be issued in order to harmonize the<br />
industry. Herbalists need to undergo training, registration and the medicines having to undergo<br />
laboratory analysis.<br />
In 2008 Kenya was singled out as one <strong>of</strong> the countries without intellectual property laws to<br />
encourage communities to share traditional knowledge. Development <strong>of</strong> laws and policies for<br />
protection <strong>of</strong> traditional knowledge like herbal medicines must be undertaken with the<br />
communities that sell them.<br />
There is need to draft an education capacity building initiative to Kenyans <strong>of</strong> the use <strong>of</strong> herbal<br />
medicines as an alternative source <strong>of</strong> treatment.<br />
There is need to intensify research on these remedies and establishment <strong>of</strong> large scale<br />
medicinal plant production.<br />
Conclusion<br />
When a traditional medicine policy will be place, Kenya has the potential <strong>of</strong> improving the<br />
healthcare <strong>of</strong> its citizens. The herbal industry can and is a tool <strong>of</strong> economic empowerment.<br />
While unregulated use <strong>of</strong> traditional medicine can have negative effects, a claim that<br />
Herbal medicine can cure every disease brings even good practice into disrepute. With<br />
increased prevalence the questions <strong>of</strong> safety, efficacy and quality are some <strong>of</strong> the challenges<br />
that need to be overcome. More work is also needed to raise public<br />
awareness <strong>of</strong> appropriate use <strong>of</strong> traditional medicine<br />
References<br />
1. .NCAPD Policy Brief No. 1 May 2008 Seeking Solutions for Traditional Herbal Medicine<br />
2. Pharmacy and poisons board <strong>of</strong> Kenya draft guidelines july 2010<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[PS 11] Phytochemical Investigation <strong>of</strong> Satureja abyssinica<br />
Mekonnen Abebayehu 1 and Dr. Nigist Asfaw 1*<br />
1 Department <strong>of</strong> Chemistry, Addis Ababa University, P.O. Box 1176, Addis Ababa, Ethiopia<br />
*Corresponding author. E-mail: nigista@chem.aau.edu.et; nigistasfaw@hotmail.com<br />
Keywords: Satureja abyssinica, Lamiaceae, Triterpene, Pulegone, Ursolic acid, Sucrose<br />
Introduction<br />
T<br />
he genus Satureja belongs to the family Lamiaceae (Labiatae). In Ethiopia, the genus is<br />
represented by eight species (1, 2). Satureja abyssinica ssp. abyssinica is an annual or perennial<br />
herb indigenous to Ethiopia where it is locally known as "Mutansa" (1). Previous work on the<br />
essential oil has been done and the result showed that it has a wide range <strong>of</strong> biological activities<br />
such as anti bacterial, anti fungal, anti-inflammatory and antioxidant activities (3). Here we report<br />
the preliminary phytochemical investigation on the solvent extract <strong>of</strong> Satureja abyssinica.<br />
Methods<br />
The aerial parts <strong>of</strong> S. abyssinica were collected in January 2011 from Endode-mariam near<br />
Debresina, 194 km away from Addis Ababa at an altitude <strong>of</strong> 1700 m. The air-dried leaves <strong>of</strong> S.<br />
abyssinica were successively extracted with hexane, chlor<strong>of</strong>orm and methanol. These extracts were<br />
used for further isolation on silica gel column chromatography. Isolated compounds were<br />
elucidated based on the spectral data <strong>of</strong> 1 H- NMR, 13 C-NMR, DEPT-135, 2D-NMR (H,H-COSY, HMQC,<br />
H<strong>MB</strong>C) experiments and mass spectroscopy.<br />
Result and Discussion<br />
Four compounds were isolated from the leaves <strong>of</strong> S, abyssinica, <strong>of</strong> which Pulegone and (1Z,5Z)-1,6dimethylcycloocta-1,5-diene<br />
were isolated from hexane extract and ursolic acid and Sucrose from<br />
methanol extract. The structure <strong>of</strong> the compounds was elucidated from 1D and 2D-NMR and in<br />
comparison the data reported in the literature for the compounds. It is noteworthy that the<br />
methanol extract was found to have high content <strong>of</strong> sugar. Future work on bioassay guided analysis<br />
will be done for a better understanding <strong>of</strong> the chemical composition and bioactivities <strong>of</strong> the plant.<br />
HO<br />
H<br />
ursolic acid<br />
H<br />
O<br />
OH<br />
(1Z,5Z)-1,6-dimethylcycloocta-1,5-diene<br />
Figure 1. Isolated compounds from Satureja abyssinica.<br />
320<br />
O<br />
pulegone<br />
HO<br />
HO<br />
HO<br />
HO<br />
O<br />
O<br />
OH<br />
O<br />
sucrose<br />
OH<br />
OH<br />
OH
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Acknowledgements<br />
Financial support from NAPRECA and Addis Ababa University for the participation on the 14 th<br />
NAPRECA symposium is gratefully acknowledged. We thank ALNAP for the provision <strong>of</strong> NMR<br />
spectroscopic equipment used in this investigation. We are grateful to Dr. Mick Cooper, University<br />
<strong>of</strong> Nottingham for mass analysis <strong>of</strong> the triterpenoid. We extend our gratitude to pr<strong>of</strong>essor Sebsebe<br />
demissew for the identification <strong>of</strong> the plant material, and to Ms. Senite dagne for her help and<br />
support in the lab work.<br />
References<br />
1. Sebsebe Demissew, Edwards S, Hedberg I, Ensermu Kelbessa, Persson E, (2006); Flora <strong>of</strong> Ethiopia and Eritrea. Addis<br />
Ababa University, Addis Ababa, 5, 516 517.<br />
2. Sebsebe Demissew, and Teshome Soromessa, (2002); Some uses <strong>of</strong> plants by the Benna Tsemay, and Zeyise<br />
people, southern Ethiopia. Ethiop. J. Nat. Resources, 4(1), 107-122.<br />
3. Kaleab Asres, Bucar Franz, El-Fiky Fathy K., Singab Abdel Nasser B, Ketema Tolossa, (2007); Journal <strong>of</strong> Essential Oil<br />
Research: JEOR, 19, 295-300.<br />
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[PS 12] Determination <strong>of</strong> Efficacious Praziquantel Dose in Different Mouse<br />
Strains: BALB/c and Swiss Mice for Treatment <strong>of</strong> Schistosoma Mansoni<br />
Peninah Njoki,¹ Hellen Kutima, 2 Rebecca Waihenya, 2 Dorcas Yole 3<br />
1<br />
Deparment <strong>of</strong> Medical Laboratory Science,School <strong>of</strong> Health Sciences,Mount Kenya University,(MKU) P.O. Box 342-<br />
01000 Thika,Kenya.<br />
2<br />
Department <strong>of</strong> Zoology, Faculty <strong>of</strong> Science, Jomo Kenyatta University <strong>of</strong> Agriculture and Technology,<br />
(J.K.U.A.T) P.O. Box 62000-00200 Nairobi, Kenya<br />
3 Institute <strong>of</strong> Primate Resarch(I.P.R) P.O. Box 24481-00200 Nairobi, Kenya<br />
Keywords: Schistosoma mansoni; Praziquantel; Mice; pathology; immunological<br />
Introduction<br />
F<br />
or 25 years Praziquantel has been the recommended treatment in mice (450mg/kg body<br />
weight) for schistosomiasis, a parasite transmitted by freshwater snails in Africa, Asia and Latin<br />
America, and infecting some 200 million people worldwide. Long experience with the WHOrecommended<br />
single dosage <strong>of</strong> 40 mg/kg has shown it to be safe and relatively efficacious. Murine<br />
models are used in S. mansoni studies because they could have different responses to S.mansoni<br />
infection hence vary in the effective dose <strong>of</strong> Praziquantel that can possibly eliminate the worms.<br />
Materials and Methods<br />
We infected BALB/c and Swiss mice with 250 S.mansoni cercariae.Serum were prepared and IgG<br />
ELISA carried out.Four weeks post infection mice were treated with PZQ 450, PZQ 900 and PZQ<br />
1350.Perfusion and adult worm recovery was done 2 weeks post infection. We also carried out<br />
histopathology on livers.<br />
Experimental Design<br />
Mouse<br />
strains<br />
Groups Doses<br />
(mg/kgbw)<br />
Week 0 Week 4 Week 6<br />
BALB/c Exp 450 I T S.(6)P(6)<br />
Swiss<br />
Exp 900 I T S.(6)P(6)<br />
Exp 1350 I T S.(6)P(6)<br />
IC - I S(6) S.(6)P(6)<br />
Exp 450 I T S.(6)P(6)<br />
Exp 900 I T S.(6)P(6)<br />
Exp 1350 I T S.(6)P(6)<br />
IC - I S(6) S.(6)P(6)<br />
Key: Exp- Experimental, IC Infected control, I- Infected, T-Treated, S-Sample, P- Perfusion<br />
mg/kgbw- milligram/ kilogram- body weight, -No activity, ( )-Number <strong>of</strong> mice per group<br />
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RESULTS<br />
Worm maturation<br />
Infected control BALB/c was 10% while that in Swiss mice was 14%. Swiss mice had more infecting<br />
parasites maturing into adult worms than BALB/c mice.<br />
Schistosome Worm recovery<br />
PZQ1350 had the highest worm reduction (69.70%) followed by PZQ900 (66.76%) and PZQ450<br />
(53.88%) had the lowest worm reduction in BALB/c..In Swiss mice PZQ1350 had the highest worm<br />
reduction (65.92%) followed by PZQ900 (52.48%) and PZQ450 (24.34%) had the lowest worm<br />
reduction (Fig 1&2).<br />
Ig G SPECIFIC ELISA<br />
8<br />
7<br />
6<br />
5<br />
4<br />
3<br />
2<br />
1<br />
0<br />
PZQ1350 PZQ900 PZQ450<br />
Doses <strong>of</strong> Praziquantel<br />
Figure 1: Percentage Schistosome worm recovery and worm reduction in BALB/c mouse strains in<br />
different treatment<br />
323<br />
% recovery<br />
%
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Figure 2: Percentage Schistosome worm recovery and worm reduction in Swiss mouse strain in<br />
different treatment<br />
PZQ1350 was statistically different when compared with PZQ900 and PZQ 450 (P
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
References<br />
Farah I O., Nyindo M., King C.I., Hau J. (2000); Hepatic granulomatous response to Schistosoma mansoni eggs in BALB/c<br />
mice and Olive baboons (Papio cynocephalus anubi). Journal <strong>of</strong> Comparative Pathology, 123: 7-14.<br />
Smithers S.R., and Terry R.J., (1965); The infection <strong>of</strong> laboratory hosts with the cercariae <strong>of</strong> mansoni and the recovery <strong>of</strong><br />
adults worms. Parasitology, 5: 695-700.<br />
World Health Organization, (2001); World Health Report 2001<br />
World Health Organization, (2002); Prevention and control <strong>of</strong> schistosomiasis and soil-transmitted helminthiasis. Report<br />
<strong>of</strong> a WHO Expert Committee. WHO Technical Report Series Geneva, No. 912.<br />
Yole, D.S., Pemberton R., Reid G.D. and Wilson R.A.,(1996); Protective immunity to Schistosoma mansoni induced in the<br />
Olive baboon, Papio anubis, by the irradiated cercariae vaccine. Parasitology, 12: 37-46.<br />
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[PS 13] Cytotoxicity <strong>of</strong> a Novel Diterpenoid from Suregada zanzibariensis<br />
Jacqueline V. Ndlebe 1 , Vinesh J. Maharaj 2, Gerda Fouche 2 , Paul Steenkamp 3 , Natasha Kolesnikova 4<br />
Bioprospecting, Biosciences, CSIR, P. O. Box 395, Pretoria 0001, South Africa<br />
Key words: Cytotoxicity, cancer cell lines, diterpenoid lactone, Suregada zanzibariensis, Euphorbiaceae, Bioassayguided<br />
fractionation<br />
Introduction<br />
A<br />
s part <strong>of</strong> the CSIR Bioprospecting platform research, 11 000 plants were collected throughout<br />
South Africa. Approximately 7 500 plant extracts were made from these plants and randomly<br />
screened for their anti-cancer properties against three cancer cell lines (melanoma UACC62, breast<br />
MCF7 and renal TK10). Based on screening results S. zanzibariensis (Euphorbiaceae) extract<br />
exhibited good anti-cancer activity against all the cell lines tested which prompted further research.<br />
A novel diterpenoid-lactone was isolated through the bioassay-guided fractionation <strong>of</strong> the organic<br />
extract <strong>of</strong> S. zanzibariensis. The compound exhibited potent anti-cancer activity (growth inhibition<br />
GI50 = 20ng/ml) against the melanoma cell line and found to be more potent that the corresponding<br />
plant extract. An accelerated 96 well microtitre plate semi preparatory HPLC purification method<br />
aimed at rapidly identifying actives in complex extracts also led to the identification <strong>of</strong> the same<br />
compound as the active ingredient. 1D, 2D NMR spectroscopy and UPLC TOF MS data were used to<br />
elucidate the structure <strong>of</strong> the active compound.<br />
Material and Methods<br />
The stem bark <strong>of</strong> the plant species S. zanzibariensis was collected from Matonela Sand forest in<br />
South Africa. The plant material was authenticated at the South African National Biodiversity<br />
Institute (SANBI), where voucher specimens were deposited.<br />
Different methods for purification <strong>of</strong> the extract was used, namely the classical method <strong>of</strong> bioassayguided<br />
fractionation and an accelerated 96-well microtitre semi preparatory HPLC purification<br />
method.<br />
Bioassay Guided Fractionation Method<br />
The organic extract <strong>of</strong> S. zanzibariensis was purified by silica gel column chromatography resulting<br />
in the generation <strong>of</strong> thirteen semi-pure fractions which were screened against the three cancer cell<br />
lines. The active refractions were further purified using silica gel column gravimetry to isolate the<br />
active compound(s).<br />
96 Well Microtitre Plate Semi-Preparatory HPLC Accelerated Method<br />
The organic extract <strong>of</strong> S. zanzibariensis was fractionated into 96 well microtitre plates with triplicate<br />
copies using Agilent 1200 semi preparative HPLC system. The microtitre plates were separately<br />
screened against three cancer cell lines, chemically analysed by UPLC MS TOF while the third plate<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
is stored as a retention sample. A correlation plot was developed between biological and chemical<br />
data to identify the active compound (s).<br />
Results and Discussion<br />
The needle-like crystals <strong>of</strong> compound 1 as shown in Figure 1 was isolated from the organic extract<br />
<strong>of</strong> S. zanzibariensis through bioassay-guided fractionation method.<br />
O<br />
327<br />
O<br />
O<br />
O<br />
O<br />
Figure 1: Compound 1<br />
The 1 H NMR and 13 C NMR spectra and 2D experiments were used to elucidate the structure <strong>of</strong> the<br />
compound. The two double bonds, acetate group, ketone and lactone carbonyl resonances seen in<br />
the 13 C NMR spectrum, accounted for the five double equivalence which in turn indicated that the<br />
molecule is tetracyclic. A search on the Dictionary <strong>of</strong> Natural Product database showed no hits,<br />
indicating that the compound could be novel. Abiatanes ditepenoids lactones, were previously<br />
isolated from the related species <strong>of</strong> Suregada, (I. Jahan et al, 2002 and I.A Jahan et al, 2004) and<br />
cytotoxicity studies <strong>of</strong> diterpenoids from the same genus. C. L Lee et al, 2008.<br />
Screening results <strong>of</strong> the 96 well microtitre plates showed that wells E3-E4 to be most active against<br />
the melanoma cells (AUCC). These active wells were analysed using UPLC MS TOF data and resulted<br />
in the confirmation that compound 1 was present in these wells. The TOF-MS + spectrum <strong>of</strong><br />
compound 1 showed a [M+Na+] ion peak at mz 395.1825, retention time (17.92 min) and UV max<br />
absorption at 263 nm. The MS spectrum indicated a mass fragmentation ion [M-59] + indicating loss<br />
<strong>of</strong> acetate group [-COOCH3], which corresponded to a molecular formula <strong>of</strong> C22H24O5.
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Figure 2: Histogram <strong>of</strong> UACC-62 results <strong>of</strong> most active fractions from the 96 well plate<br />
Conclusions<br />
Isolation <strong>of</strong> compound 1 was achieved through classical bioassay-guided fractionation approach.<br />
The 96 well plate accelerated approach confirmed the previously identified compound but also<br />
served as a validation <strong>of</strong> application the new accelerated method. The 96 well approach serve to<br />
tentatively identify compound/class responsible for activity in shorter time than that <strong>of</strong> classical<br />
bioassay guided fractionation, which took much longer. As the compound is novel, the 96 well plate<br />
approach and analysis through UPLC TOF MS analysis would not have been sufficient to elucidate<br />
the structure using only this technology.<br />
Acknowledgements<br />
The authors would like to thank the South African National Biodiversity Institute (SANBI) for the<br />
identification <strong>of</strong> plant specimens, NRF (YREF Young researcher Fund) and PG (Parliamentary Grant)<br />
for funding the project.<br />
References:<br />
1. I. Jahan, N. Nahar, M. Mosihuzzaman, F. Shaheen, Z. Parween, A. Rahman and M. I. Choudhary, (2002); Novel<br />
diterpenoids Lactones from Suregada multiflora. Journal <strong>of</strong> Natural Products, 65, 932-934<br />
2. I.A Jahan, N. Nahar, M. Mosihuzzaman, F.Shabeen, A. Rahman and M.I Choudhary, (2004); Six new diterpenoids<br />
from Suregada multiflora. Journal <strong>of</strong> Natural Products, 67, 1789-1795<br />
3. C. L Lee, F. R Chang, P. W Hsieh, M.Y. Chiang, C.C Wu, Z.Y Huang, Y.H Lan, M. Chen, K.H Lee, H.F Yen, W.C. Hung and<br />
Y.C Wu, (2008); Cytotoxic ent-abiatane diterpenoids from Gelonium aequoreum. Phytochemistry, 69, 276-287.<br />
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[PS 14] Solid-state Fermentation <strong>of</strong> Jatropha curcas Seed Meal Using<br />
Aspergillus niger Eliminates Molluscicidal Activity and Changes the Phorbol Ester<br />
Composition <strong>of</strong> the Seed Meal<br />
Emmanuel Rubagumya, Emil K. Abotsi and Ignatious Ncube*<br />
Department <strong>of</strong> Biochemistry, Microbiology and Biotechnology, School <strong>of</strong> Molecular and Life Sciences,<br />
University <strong>of</strong> Limpopo, P. Bag X1106, Sovenga 0727, South Africa<br />
Email: Ignatious.ncube@ul.ac.za<br />
Key words: Jatropha; solid-state fermentation; snails; Phorbol ester; Aspergills niger<br />
Introduction<br />
J<br />
atropha curcas, commonly referred to as physic nut, is a member <strong>of</strong> Euphorbiaceae family, and<br />
grows well in the tropical region (Aderibigbe et al., 1996). The genus name for Jatropha is<br />
derived from the Greek iotrós (doctor) and trophé (food) which implies medicinal uses (Makkar et<br />
al., 2009). Jatropha curcas seeds have high potential for utilization as food or feed <strong>of</strong> high<br />
nutritional value. Although the seed contains 40-60% oil which is similar to oil used for human<br />
consumption, this oil is not used for cooking purposes because it contains some toxic substances<br />
(Rakshit et al., 2008). The seed cake obtained after extraction <strong>of</strong> oil, has a protein content <strong>of</strong><br />
between 53 58% crude protein (Aregheore et al., 2003) and cannot be used as animal feed due to<br />
toxic elements such as lectins and phorbol esters. A critical obstacle in the establishment <strong>of</strong> J.<br />
curcas as a commercial crop could be overcome by detoxifying Jatropha seeds. Heat treatment<br />
followed by chemical (sodium hypochlorite (NaOCl) and sodium hydroxide (NaOH)) treatment has<br />
been used as one <strong>of</strong> the methods <strong>of</strong> detoxification (Waled and Jumat, 2009), but the method is<br />
found to be expensive and complicated. The aim <strong>of</strong> this study, therefore, was to detoxify J. curcas<br />
seed meal using solid state fermentation technology as a simple and an alternative method, so that<br />
seed cake can be used as animal feed or protein supplement in animals feed.<br />
Materials and Methods<br />
Materials<br />
Mature sun dried seeds <strong>of</strong> J. curcas were provided by the Harare Polytechnical College <strong>of</strong><br />
Zimbabwe. Snails were provided by Aquaculture Unit at University <strong>of</strong> Limpopo and Aspergillus niger<br />
FGSC A733 was obtained from Fungal Genetics Stock Centre (FGSC).<br />
Methods<br />
Seed kernels were ground to a meal using a pestle and mortar and sieved using 2 mm sieve to<br />
obtain J. curcas seed meal (JCSM). JCSM (10 g) was added into 500 mL Erlenmeyer flasks and the<br />
contents in the flasks were autoclaved at 120°C for 20 min and then cool to room temperature. Salt<br />
solution (0.2% K2PO4, 0.5% NH4NO3, 0.1% NaCl and 0.1% MgSO4) was also autoclaved separately<br />
and left to cool to room temperature. The seed meal was moistened with 4 mL <strong>of</strong> salt solution.. The<br />
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medium was inoculated with 1 mL <strong>of</strong> A. niger spore suspension(5 X 10 4 CFUs/ml). The flasks were<br />
incubated at 30°C for 10 days.<br />
After fermentation the fermented and unfermented J. curcas seed meal was extracted with<br />
acetone in ratio <strong>of</strong> 1:5 (w/v). The contents were stirred for 1 hr at room temperature. Acetone<br />
fraction was decanted and the extraction was repeated twice. The collected acetone fraction was<br />
filtered using Whatman No 1 filter paper. The acetone was evaporated from the extract using<br />
rotary evaporator at 40ºC. The Jatropha oil obtained was thoroughly mixed with 2 volumes <strong>of</strong><br />
methanol at room temperature in a separating funnel. The methanol fraction was collected. This<br />
procedure was repeated 4 times and methanol extractions were pooled together. Methanol was<br />
evaporated using rotary evaporator at 40°C.<br />
Snails were cultivated in an aquarium filled with fresh water, in controlled photoperiod <strong>of</strong> 12 hrs<br />
light and 12 hrs darkness. An air pump was used to supply air into the water tanks and the<br />
temperature was maintained at 24°C using heaters. The snails were fed on lettuce. After<br />
multiplication, snails were divided into separate tanks for further experiments involving exposure<br />
to Jatropha oil.<br />
Tanks <strong>of</strong> 10 L capacity were filled with 10 L <strong>of</strong> fresh water and 20 snails were introduced into each<br />
tank, fractions <strong>of</strong> oil extracted from unfermented Jatropha seed ranging between 0.1 1 mL was<br />
added to each tank and mortality rate <strong>of</strong> snails was recorded every 12 hrs over a period <strong>of</strong> 48 hrs.<br />
Sunflower oil was used as control. Different trials were performed by exposing snails to 0.7 mL <strong>of</strong><br />
Jatropha seed oil extracted from fermented or unfermented seed meal. Also snails were exposed to<br />
Jatropha oil hydrolysed using a lipase.<br />
Results and Discussion<br />
The lowest experimental dose where there is no measurable affect is known as the no observable<br />
adverse effects level (NOEL). Dosage range between 0.1 to 0.3 mL <strong>of</strong> Jatropha oil, show (NOEL)<br />
after 48 hrs <strong>of</strong> exposure. Dosage <strong>of</strong> 0.4 mL <strong>of</strong> Jatropha oil, 50% <strong>of</strong> snails were dead after 48 hrs <strong>of</strong><br />
exposure. While in dosage range from 0.6 to 1 mL, all snails are died after 36 hrs <strong>of</strong> exposure. The<br />
more mortality rate increases <strong>of</strong> snails exposed to the Jatropha oil was thus dose dependent (Figure<br />
1).<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
0.0 0.2 0.4 0.6 0.8 1.0<br />
Jatropha Oil Dose (mL/10 L)<br />
Figure: 1. Mortality <strong>of</strong> snails exposed to Jatropha oil at different doses <strong>of</strong> (0.1-1.0) mL per 10 L <strong>of</strong><br />
water. 12 hrs <strong>of</strong> exposure( ),24 hrs <strong>of</strong> exposure( ), 36 hrs <strong>of</strong> exposure( ) and 48 hrs <strong>of</strong><br />
exposure( ).<br />
After 12 hrs <strong>of</strong> exposure in all treatments the snails were normal, while after 36 hrs <strong>of</strong> exposure,<br />
only 5% <strong>of</strong> snails exposed to unfermented Jatropha oil survived (Figure 2). In fermented oil 90%<br />
survived. Sunflower oil had no lethal effect on the snails (Figure 2). These findings reveal that solid<br />
state fermentaion detoxifies Jatropha seed oil possibly through biochemical modification <strong>of</strong><br />
phorbol esters.<br />
120<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
0 20 40 60 80 100 120 140<br />
Time (hrs)<br />
Figure 2: Snails exposed to 0.7 ml <strong>of</strong> oil over 140 hrs. Unfermented Jatropha oil. ( ), fermented<br />
Jatropha oil( ) and sun�ower oil( ).<br />
There was no difference in survival rates <strong>of</strong> snails exposed to unfermented oil (25% survival) and<br />
those exposed to unfermented oil that had been hydrolysed with lipases. Oil extracted from<br />
fermented seed meal had similar survival rates <strong>of</strong> to sunflower oil (84%) (Figure 3). Lipolysis using<br />
the current lipase therefore does not detoxify the oils as the unfermented oil that was hydrolysed<br />
using a lipase had the same toxicity to snails as untreated oil (Figure 3).<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
120<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
0 20 40 60 80<br />
Tim e (hrs)<br />
Figure: 3. Snails exposed to 0.7 mL <strong>of</strong> oil treated in different ways. Oil extracted from unfermented<br />
Jatropha curcas seed( ), oil extracted from fermented Jatropha curcas seed( ),unfermented<br />
Jatropha curcas seed oil hydrolysed using lipase( ) and sunflower oil hydrolysed using lipase ( ).<br />
Acknowledgements<br />
The authors would are grateful to the Flemish Inter-University Council (VLIR-UOS) for funding the<br />
research and the Ministry <strong>of</strong> Education <strong>of</strong> Rwanda for providing a scholarship to Emmanuel<br />
Rubagumya.<br />
References<br />
ABBOTT, W. S. (1925); A method <strong>of</strong> computing the effectiveness <strong>of</strong> an insecticide. J. Econ. Entomol., 18 265-267.<br />
ADERIBIGBE, A. O., JOHNSON, C. O. L. E., MAKKAR, H. P. S., BECCKER, K. & FOIDL, N. (1996); Chemical composition and<br />
effect <strong>of</strong> heat on organic matter- and nitrigen-degradability and some antinutritional components <strong>of</strong> Jatropha<br />
meal. Animal Feed Sci. Technol., 67, 223-243.<br />
AREGHEORE, E. M., BECCKER, K. & MAKKAR, H. P. S. (2003); Detoxification <strong>of</strong> a toxic variety <strong>of</strong> Jatropha curcas using<br />
heat and chemical treatment, and preliminary nuttrional evaluation with rats. South Pacific J. Nat. Sci., 21, 50-56.<br />
MAKKAR, H. P. S., SIDDHURAJU, P. & BECKER, K. (2009); Plant secondary metabolites. Plant Sci., 393.<br />
RAKSHIT, K. D., DARUKESHWARA, J., NARASIMHAMURTHY, K., SAIBABA, P. & BHAGYA, S. (2008); Toxicity studies <strong>of</strong><br />
detoxified Jatropha meal (Jatropha curcas) in rats. Food Chem. Toxicol., 46, 3621-3625.<br />
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[PS 15] Ethnomedicinal Knowledge in the Traditional Management <strong>of</strong> Human<br />
Ailments in Lake Victoria Basin, Kenya<br />
Judith Agot Odhiambo 1 *, Catherine Wanjiru Lukhoba 1 , Saifuddin Fidahussein Dossaji 1<br />
1 School <strong>of</strong> Biological Sciences, University <strong>of</strong> Nairobi. P.O Box 30197-00100 Nairobi, Kenya<br />
*Corresponding author: judyodhis2003@yahoo.co.uk, judyaswani@uonbi.ac.ke ;<br />
Key words: Indigenous knowledge, human ailments, medicinal plants, ethnomedicine.<br />
Introduction<br />
U<br />
ses <strong>of</strong> plants in the indigenous cultures <strong>of</strong> developing countries are numerous and diverse.<br />
This indigenous knowledge evolved for a long time through trial and error. Though the<br />
majority <strong>of</strong> inhabitants in the Kenya rely on ethnomedicinal plant species to manage a wide range<br />
<strong>of</strong> human ailments, much the indigenous knowledge largely remains undocumented.<br />
Materials and methods<br />
An ethnomedicinal survey was conducted to document the plant species used medicinally in the<br />
Lake Basin. The ethnomedicinal data were based on structured interviews that sought answers to<br />
questions about the human ailments treated, local names <strong>of</strong> plant species, plant parts used,<br />
methods <strong>of</strong> preparation, and administration. In some cases, the interviews were facilitated by<br />
translators who were well conversant with the local language. This was done having first obtained<br />
verbal informed consent from each traditional healer.<br />
Results and discussion<br />
Traditional healers <strong>of</strong> the Lake Victoria Basin, Kenya were found to be rich in their indigenous<br />
knowledge on the use <strong>of</strong> ethnomedicinal plant species to manage various human ailments within<br />
the study area. This was evidenced by the result that a wide range <strong>of</strong> human ailments were<br />
reported to be treated using thirty four medicinal plant species distributed within twenty one<br />
botanical families.They were found to play a vital role in the primary healthcare <strong>of</strong> the local poor<br />
people as they were the main resource persons to their health problems. This may have been due<br />
to the inability by the locals to afford modern healthcare costs and the healers capability to handle<br />
most <strong>of</strong> their health problems. The plant family reported with the highest number <strong>of</strong> medicinal<br />
plant species was|Compositae followed by Leguminosae and Labiatae. This trend is in agreement<br />
with the findings by Yineger et al., (2008) who reported Compositae and Labiatae as the first and<br />
third families, respectively, with the highest number <strong>of</strong> medicinal plant species, but is contrary to<br />
those <strong>of</strong> Yineger & Yewhalaw (2007) who reported the most representive families as Leguminosae,<br />
Acanthaceae and Curcubitaceae successively. The discrepancy may be due to factors such as<br />
ecological, geographical and environmental (Runyoro et al., 2006) which favour the growth <strong>of</strong> some<br />
plant families and not others. There was evidence <strong>of</strong> high secrecy in the medicinal plant usage with<br />
a number <strong>of</strong> healers not ready to reveal full details <strong>of</strong> their knowledge about the medicinal plants<br />
to us. Many reported not having transferred the knowledge to the subsequent generation. The<br />
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secrecy surrounding the ethnomedicinal knowledge among the traditional healers could be<br />
attributed to the fact that traditional healers derive income from the treatments they provide. This<br />
is in agreement with findings done by Yineger et al., (2008) and Yineger & Yewhalaw, (2007) that<br />
apart from income, traditional healers get in-kind compensation and would therefore like the value<br />
<strong>of</strong> the indigenous knowledge maintained. Ng etich, (2005) reported that some traditional healers<br />
regard the knowledge as personal property, Kokwaro (1993) reported that in some cases oaths are<br />
taken during passing <strong>of</strong> the information so that it is not revealed to any one else. The degree <strong>of</strong><br />
agreement by traditional healers in dealing with ailments such as malaria which is managed using<br />
Tithonia diversifolia and Schkuria pinnata, ; sexually transmitted infections managed using Albizia<br />
coriaria and Harrisonia abyssinica and ringworms managed using Moringa sp. could give high<br />
validity to these species used to treat these ailments and could be due to the effective results on<br />
their usage from past experience, the species availability and existence <strong>of</strong> these ailments as the<br />
most commonly encountered ones. The use <strong>of</strong> traditional medicinal plants as mixtures by<br />
traditional healers to manage one or more human ailments was reported. In fact the majority <strong>of</strong> the<br />
ethnomedicinal plants collected were used as mixtures. This could be due to the additive effects<br />
that they could have during ailment treatment (Bussman and Sharon, 2006; Igoli et al., 2002). The<br />
traditional healer may not be sure <strong>of</strong> the specific ailment the patient could be suffering from and<br />
therefore gives a mixture <strong>of</strong> several herbal medicinal preparations as a remedy to potential<br />
ailments judging from the patients condition. The other reason could be due to the synergism<br />
action <strong>of</strong> the different preparations expected by the practitioner. Uses <strong>of</strong> traditional herbal<br />
remedies as mixtures <strong>of</strong> different herbs have also been reported in the Chinese traditional medicine<br />
by Xiao (1983). Some <strong>of</strong> the plants in the mixture could however be acting as antipyretics, immune<br />
stimulants to relieve the symptoms <strong>of</strong> the disease rather than having direct activity as reported by<br />
Philipson et al., (1993), and some could also be nutritive. Xiao (1983) explained that determination<br />
<strong>of</strong> the pharmacological effects and isolation <strong>of</strong> active principles from the herbal mixtures is much<br />
more difficult than in the case <strong>of</strong> single medicinal plants owing to the interaction <strong>of</strong> various<br />
constituents. In this study, it was observed that most <strong>of</strong> the medicinal plant species were used to<br />
treat more than one ailment. This could be due to the availability <strong>of</strong> the herbal plant or its<br />
effectiveness from past experience in the treatment <strong>of</strong> various human ailments. This is in<br />
agreement with reports by Lukhoba et al., (2006), Boer et al., (2005) Okemo et al., (2003) and<br />
Kokwaro, (1993). Results <strong>of</strong> this ethnomedicinal study revealed that traditional healers used<br />
additives such as the traditional ghee, Vaseline, oil during preparation. This could be attributed to<br />
the increase <strong>of</strong> potency <strong>of</strong> the medicinal plant. This result was in agreement with the findings <strong>of</strong><br />
Otieno et al., (2007) in Tanzania who reported that crude mineral Kadosero supplemented to other<br />
plants extracts by the herbal practitioners showed increased activity <strong>of</strong> the herbal medicine.<br />
Olembo et al., (1995) also reported a similar scenario about Dichondria repens (Convolvulaceae)<br />
whose leaves are crushed, mixed with oil to treat dermatological ailments. The same plant showed<br />
no activity when tested against a dermatological fungus by Kariba (2000).<br />
This study reported herbs to be the most used growth form used for remedy prepararation. The<br />
second and third being shrubs and trees respectively. High use <strong>of</strong> herbs could be attributed to the<br />
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fact that they tend to be most available in nearly all climatic conditions, have fast growth and tend<br />
to be available in conspicuous places like crop farms, disturbed areas, along the roadsides and along<br />
fences where they can easily be accessed by the practitioners. Leaves were the most cited plant<br />
parts used by the healers for the preparation <strong>of</strong> medicine followed by the roots. This finding is in<br />
line with the results <strong>of</strong> other ethnomedicinal studies such as those by Yineger et al., (2008) and<br />
Yineger & Yewhalaw, (2007). Most <strong>of</strong> the ethnomedicinal plant species were reported to be<br />
processed through concoction, decoction, powdering and administered mainly through oral and<br />
dermal routes. Remedies were mostly prescribed by the traditional healers, however the dosages<br />
lacked precision as they were given in cups, water glasses or in a basin. This report was found to be<br />
in agreement with that <strong>of</strong> Erasto et al ., (2008) , Boer et al., (2005) , Kokwaro, (1993) and Yineger et<br />
al., (2008) who in addition mentioned that there could a rise cases <strong>of</strong> over dose which could cause<br />
serious health problems due to toxicity <strong>of</strong> some species. Our report on remedy preparation is<br />
however in contrary to the findings by Yineger & Yewhalaw, (2007) in Ethiopia who reported the<br />
principal methods <strong>of</strong> remedy preparation as crushing and squeezing. Nature <strong>of</strong> ailments treated<br />
and healers past experience on results may have contributed to the observed difference.<br />
Acknowledgement<br />
The authors wish to thank the InterUniversity Council <strong>of</strong> East Africa (IUCEA) for sponsoring this<br />
research. Traditional healers <strong>of</strong> Lake Victoria Basin are genuinely acknowledged for their hospitality.<br />
Technical assistance by Mr. Simon Mathenge is greatly appreciated, and the University <strong>of</strong> Nairobi,<br />
School <strong>of</strong> Biological sciences for letting us use the available facilities.<br />
References<br />
Boer, J.H., Kool, A., Mziray, W.R., Herdberg I., Levenfors J.J., (2005); Antifungal and antibacterial activity <strong>of</strong> some herbal<br />
remedies from Tanzania. Journal <strong>of</strong> Ethnopharmacology 96 461-469.<br />
Bussmann, R.W., and Sharon, D.,(2006); Traditional plant use in Northern Peru: Tracking two thousand years <strong>of</strong> health<br />
culture. Journal <strong>of</strong> Ethnobiology and Ethnomedicine.2; 47.<br />
Erasto, P., Adebola, P. O., Grierson, D. S., Afolayan, A. J., (2005); An ethnobotanical study <strong>of</strong> plants used for the<br />
treatment <strong>of</strong> diabetes in the Eastern Cape Province, South Africa. African Journal <strong>of</strong> Biotechnology 4 (12), 1458-<br />
1460<br />
Igoli, J.O., Tor-Anyiin, TA., Usman, S.S., Oluma, H.O.A., Igoli, NP., (2002); Folk medicines <strong>of</strong> the lower Benue valley <strong>of</strong><br />
Nigeria. In: Recent Progress in Medicinal Plants, Vol.7 Ethnomedicine and Pharmacognosy II, (Eds. V.K Singh, J.N.<br />
Govil, S. Hashmi and G. Singh), Sci. Tech. Pub. , USA. 327-338.<br />
Kariba M. R., (2000); Antifungal activity <strong>of</strong> extracts from selected Kenyan medicinal plants. Ph.D thesis, University <strong>of</strong><br />
Nairobi, Nairobi, Kenya.<br />
Kokwaro J.O., (1993); Medicinal plants <strong>of</strong> East Africa. East Africa 2 nd .Ed. Literature Bureau, Nairobi.<br />
Lukhoba C.W., Simmonds M.S.J., Paton A.J. ( 2006); Plectranthus: A review <strong>of</strong> ethnobotanical uses. Journal <strong>of</strong><br />
Ethnopharmacology 103 (1):1-24.<br />
Ng etich K.A., (2005); Indegenious Knowledge, Alternative medicine and Interlectual Property Rights concerns Kenya.<br />
Paper presented in the 11 th General Assembly Maputo, Mozambique, 6-10.<br />
Okemo, P.O., Bais, P.H.,Vivanco, M.J., (2003); In vitro activities <strong>of</strong> Maesa lanceolata extracts against fungal plant<br />
pathogens. Fitoterapia 74 : 312-316.<br />
Olembo, N.K., Fedha, S.S., Ngaira, E.S., (1995); Medicinal and Agricultural plants <strong>of</strong> Ikolomani Division, Kakamega<br />
District. Signal press L.t.d Nairobi.<br />
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Otieno, J.N., Hosea, K.M.M., Lyaruu, H.V. (2007); The effect <strong>of</strong> a local mineral Kadosero towards the antimicrobial<br />
activity <strong>of</strong> medicinal plant s extract: case <strong>of</strong> Lake Victoria, Basin, Tarime Tanzania. African Journal <strong>of</strong> Traditional,<br />
Complimentary and Alternative medicines 4, (1): 1-6.<br />
Palombo, E.A (2006); Phytochemicals from Traditional Medicinal Plants used in the Treatment <strong>of</strong> Diarrhoea: Modes <strong>of</strong><br />
Action and Effects on Intestinal Function. Phytotherapy Research 20, 717 724<br />
Phillipson, J.D., Wright, C.W., Kirby, G.C., Warhurst, D.C., (1993); Tropical Plants as sources <strong>of</strong> antiprotozoal agents.<br />
Recent Advances in Phytochemistry, 27, 1 40.<br />
Runyoro D., Matee M., Ngassapa O., Joseph C., Mbwambo Z.,(2006); Screening <strong>of</strong> Tanzanian medicinal plants for anticandida<br />
activity.BMC Complementary and Alternative Medicines 30;6 (1).<br />
Xiao P.G., (1983); Medicinal plants: the Chinese approach. Journal <strong>of</strong> Ethnopharmacology, 7:95<br />
Yineger, H., Kelbessa , E., Bekele, T., Lulekal, E., (2008); Plants used in traditional management <strong>of</strong> human ailments at<br />
Bale Mountains National Park, Southeastern Ethiopia. Journal <strong>of</strong> medicinal plant research. 2 (6,) 132-153.<br />
Yineger, H., and Yewhalaw, D., (2007); Traditional medicinal plants knowledge and use by Ioc Sekoru District, Jimma<br />
Zone, Southwestern Ethiopia. Journal <strong>of</strong> Ethnobiology and Ethnomedicine 3; 24.<br />
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[PS 16] In vitro Antimicrobial Activity <strong>of</strong> Extracts from five Malagasy Endemic<br />
Species <strong>of</strong> Albizia (Fabaceae)<br />
Danielle A. Doll RAKOTO 1 , Clara RAJEMIARIMOELISOA 1<br />
Ranjàna RANDRIANARIVO 1 , Delphin RAMAMONJISON 1 , Christian RAHERINIAINA 1 , Noelinirina<br />
RAHARISOA 1 , Victor JEANNODA 1<br />
1 Laboratoire de Biochimie appliquée aux Sciences médicales, Département de Biochimie fondamentale et appliquée,<br />
Faculté des Sciences, BP 906, Université d Antananarivo, Madagascar<br />
Email: dad.rakoto@yahoo.fr<br />
Key-words: Albizia, seeds, extracts, antimicrobial, MIC, <strong>MB</strong>C.<br />
Introduction<br />
F<br />
or centuries, most <strong>of</strong> the population in many developing countries have relied on a system <strong>of</strong><br />
traditional medicine in which plants constitute the principal element <strong>of</strong> therapy. Plants<br />
belonging to the genus Albizia (Fabaceae) are trees distributed in African, Asian and South-<br />
American countries where they are widely used in indigenous pharmacopoeia (Agyare et al., 2005;<br />
Geyid et al., 2005; Murugan et al., 2007; Rukayadi, 2008). Albizia species have been the subject <strong>of</strong><br />
several chemical and pharmacological studies. Thus, many structures (heterosids, alkaloids, ) were<br />
elucidated (Zou et al., 2006; Rukunga et al., 2007) and various activities such as anthelmintic<br />
(Githiori et al., 2003), cytotoxic (Zou et al., 2006 ), larvicidal (Murugan et al., 2007) or antimicrobial<br />
(Agyare et al., 2005; Geyid et al., 2005; Sudharameshwari et al., 2007) were found.<br />
In Madagascar, Albizia is represented by 25 endemic and 2 introduced species. No previous report<br />
on both the chemical constituents and the pharmacological activities <strong>of</strong> these plants could be found<br />
in the literature. Since infectious diseases account for the significant proportion <strong>of</strong> health problems,<br />
antimicrobial principles from five Malagasy species <strong>of</strong> Albizia encoded A1, A2, A3, A4 and A5, were<br />
studied in this work. They were purified and the major secondary metabolites were identified by<br />
phytochemical screening. Extracts or pure compounds were tested in vitro against two Gram<br />
positive bacteria, three Gram negative bacteria and one yeast Candida albicans. Minimum<br />
inhibitory concentration (MIC) and Minimum bactericidal concentration (<strong>MB</strong>C) were determined on<br />
susceptible germs.<br />
Materials and methods<br />
1- Plant materials<br />
Seeds <strong>of</strong> plants A1, A2, A3, A4 and A5 were used in this study. Fruits were collected in western and<br />
southern regions <strong>of</strong> Madagascar.Seeds were washed, sun-dried and ground into a fine powder,<br />
using a microgrinder Culatti.<br />
2- Microorganisms<br />
The pathogenic microorganisms consisted <strong>of</strong> two Gram positive bacteria: Staphylococcus aureus,<br />
Bacillus subtilis, three Gram negative bacteria: Klebsiella pneumoniae, Escherichia coli Salmonella<br />
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typhi and one yeast Candida albicans. They were isolated and identified from heterogeneous<br />
cultures available in Institut Pasteur de Madagascar.<br />
3- Extracts preparation<br />
3.1- Extraction<br />
Powdered dried seeds were defatted by extraction with petroleum ether (60-80°C) in a Soxhlet s<br />
extractor, then extracted with distilled water, 50% ethanol or 75% ethanol.<br />
3.2- Purification<br />
Crude extracts were purified using methods based on solubility, molecular weight or electric charge<br />
properties <strong>of</strong> active principles.<br />
4- Phytochemical screening<br />
Extracts were subjected to preliminary phytochemical testing for the major chemical groups<br />
(Fransworth, 1966; Marini-Bettolo et al., 1981).<br />
5- Assays on microorganisms<br />
The antimicrobial tests were carried out by disc diffusion method in Mueller Hinton agar (Rios et al.,<br />
1988).MIC was determined by broth dilution method (Duval et Soussy, 1990; Ferron, 1994). Each<br />
medium showing no visible growth is subcultured on Mueller Hinton agar plates. After 24 hours at<br />
37°C, <strong>MB</strong>C was the corresponding concentration required to kill 99.9% <strong>of</strong> the cells (Duval et Soussy,<br />
1990; Ferron, 1994).<br />
6- Statistical analysis<br />
One-way analysis <strong>of</strong> variance (ANOVA) followed by Newman Keuls comparison test with Statitcf ®<br />
s<strong>of</strong>tware were used for statistical analysis. Statistical estimates were made at confidence interval <strong>of</strong><br />
95%.<br />
Results and Discussion<br />
1- Phytochemical screening<br />
The major secondary metabolites identified in extracts are shown in Table 1<br />
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Table 1: Phytochemical screening <strong>of</strong> extracts from 5 Malagasy species <strong>of</strong> Albizia (A1 to A5)<br />
Phytochemical compounds Extracts<br />
Alkaloids<br />
Flavonoids<br />
Anthocyanins<br />
Phenols<br />
Quinons<br />
E1 E21 E22 E3 E4 E5<br />
Unsaturated sterols + + + + + +<br />
Triterpenes + + + + +<br />
Deoxysugars + + + + + +<br />
Saponins + + + + + +<br />
: negative test +: positive test<br />
E1, E3, E4, E5 : purified extracts from plants A1, A3, A4 and A5 respectively<br />
E1, E21, E22 : pure compounds from plant A1, A2 respectively Except A1 which didn t contain<br />
triterpenes, all extracts showed the presence <strong>of</strong> unsaturated sterols, triterpenes and deoxysugars,<br />
indicating glycosidic nature <strong>of</strong> active principles. The presence <strong>of</strong> saponins, in addition with positive<br />
foam test and hemolytic effect (not shown) mean that antimicrobial compounds may be saponins.<br />
Saponins and other glycosides were isolated and identified from other species <strong>of</strong> Albizia (Pal et al.,<br />
1995; Debella et al., 2000; Zou et al., 2006).<br />
2- Antimicrobial activity<br />
According to these results, the extracts E3 and E5, respectively from A3 and A5, showed activity<br />
against all the tested germs. Bacillus subtilis seemed to be the most susceptible bacterium (13 mm<br />
inhibition zone for E3 and 16 mm for E5) to these extracts. On the other hand, all the extracts<br />
inhibited the growth <strong>of</strong> Staphylococcus aureus and Candida albicans at the tested concentrations.<br />
E21 (pure compound) exhibited the strongest activity against the fungus (20 mm). In a general<br />
manner, Gram positive germs, including Candida albicans, were more susceptible than Gram<br />
negative ones.<br />
Similar results were obtained with some other species <strong>of</strong> Albizia (Mbosso et al., 2010; Rukayadi,<br />
2008; Sudharameshwari et al., 2007).<br />
Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (<strong>MB</strong>C)<br />
determined on susceptible germs are given in Table 2.<br />
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Table 2: Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration<br />
(<strong>MB</strong>C) <strong>of</strong> extracts from 5 Malagasy species <strong>of</strong> Albizia<br />
Extracts Sensitive germs MIC (µg/ml) <strong>MB</strong>C (µg/ml)<br />
E1 Staphylococcus aureus 320 2500<br />
E22 Candida albicans 6.25 100<br />
E22 Klebsiella pneumoniae 50 800<br />
E3 Escherichia coli 2500 10 000<br />
E4 Staphylococcus aureus 625 10 000<br />
E4 Escherichia coli 1250 20 000<br />
E5 Escherichia coli 12 500 12 500<br />
Pure compound E22 from the plant A2, showed the lowest MIC (6.25 µg/ml) and <strong>MB</strong>C (100 µg/ml)<br />
against Candida albicans. With MIC values respectively corresponding to 100 µg/ml and 12.5 µg/ml,<br />
Albizia myriophylla and Albizia gummifera (Mbosso et al., 2010; Rukayadi, 2008) showed lower<br />
activity than A2 against this germ.<br />
Acknowledgements<br />
The authors acknowledge the PER/AUF project for financial support and the Institut Pasteur de<br />
Madagascar for providing microorganisms.<br />
References<br />
Agyare, C., K<strong>of</strong>fuor, G. A., Mensah, A. Y., Agyemang, D. O. (2005); Antimicrobial and uterine smooth muscle activities <strong>of</strong><br />
Albizia ferruginea extracts. BLACPMA, 5(2), 27-31.<br />
Debella, A., Haslinger, E., Schmid, M. G., Bucar, F., Michl, G., Abebe, D., Kunert, O. (2000); Triterpenoid saponins and<br />
sapogenin lactones from Albizia gummifera. Phytochemistry, 53, 885-892.<br />
Duval, J., Soussy, C. J. (1990); Antibiothérapie. Masson éd.<br />
Ferron, A. (1994). Bactériologie médicale. Edition C. et R.<br />
Fransworth, N. R. (1966); Biologica l and phytochemical screening <strong>of</strong> plants. J. Pharm. Sci., 55, 225-276.<br />
Geyid, A., Abebe, D., Debella, A., Makonnen, Z., Aberra, F., Teka, F., Kebede, T., Urga, K., Yersaw, K., Biza, T., Mariam, B.<br />
H., Guta, M. (2005); Screening <strong>of</strong> some medicinal plants <strong>of</strong> Ethiopia for their anti-microbial properties and chemical<br />
pr<strong>of</strong>iles. J. Ethnopharmacol., 97, 421-427.<br />
Githiori, J. B., Höglund, J., Waller, P. J., Baker, R.L. (2003); The anthelmintic efficacy <strong>of</strong> the plant, Albizia anthelmintica,<br />
against the nematode parasites Haemonchus contortus <strong>of</strong> sheep and Heligmosomoides polygyrus <strong>of</strong> mice. Vet.<br />
Parasitol., 116, 23-24.<br />
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Mbosso, E. J. T., Ngouela, S., Nguedia, J. C. A., Beng, V. P., Rohmer, M., Tsamo, E. (2010); In vitro antimicrobial activity <strong>of</strong><br />
extracts and compounds <strong>of</strong> some selected medicinal plants from Cameroon. J. Ethnopharmacol., 128, 476-481.<br />
Marini-Bettolo, G. B., Nicoletti, M., Patamia, M. (1981); Plant screening by chemical and chromatographic procedure<br />
under field conditions. J. Chromatogr., 218,113-217.<br />
Murugan, K, Murugan, P., Noortheen, A. (2007); Larvicidal and repellent potential <strong>of</strong> Albizia amara Boivin and Ocimum<br />
basilicum Linn against dengue vector, Aedes aegypti (Insecta: Diptera: Culicidae). Biores. Technol.,98, 198-201.<br />
Pal, B. C., Achari B., Yoshipppkawa, K., Arihara, S. (1995); Saponins from Albizia lebbeck. Phytochemistry, 38(5), 1287-<br />
1291.<br />
Rios, J. J., Recio, M. C., Villar, A. (1988); Screening methods for natural products with anti-microbial activity : a review <strong>of</strong><br />
literature. J. Ethnopharmacol., 23, 127-149.<br />
Rukayadi, Y., Shim, J. S., Hwang, J.K. (2008); Screening <strong>of</strong> Thai medicinal plants for anticandidal activity. Mycoses, 51,<br />
308-312.<br />
Rukunga, G/ M., Muregi, F. W., Tolo, F. M., Omar, S. A., Mwitari, P., Muthaura, C. N., Omlin, F., Lwande, W., Hassanali,<br />
A., Githure, J., Iraqi, F. W., Mungai, G. M., Kraus, W., K<strong>of</strong>i-Tsekpo, W. M. (2007); The antiplasmodial activity <strong>of</strong><br />
spermine alkaloids isolated from Albizia gummifera. Fitoterapia, 78, 455-459.<br />
Sudharameshwari, K., Radhika, J. (2007); Antibacterial screening <strong>of</strong> Aegle marmelos, Lawsonia inermis and Albizia<br />
lebbeck. Afr. J. Trad.CAM, 4(2), 199-204.<br />
Zou, K., Zhao, Y. Y., Zhang, R. Y. (2006); A cytotoxic saponin from Albizia julibrissin. Chem. Pharm. Bull., 54(8), 1211-<br />
1212.<br />
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[PS 17] Phytochemical and Pharmacological Studies <strong>of</strong> Extracts <strong>of</strong> Trichilia<br />
emetica Used in the Treatment <strong>of</strong> Dysmenorrhoea in Mali<br />
Sanogo R. a,b* , Haïdara M. a , Dénou A. a , De Tommasi N. c , Occhiuto F. d<br />
a<br />
Faculty <strong>of</strong> Medicine, Pharmacy, Odontostomatology, University <strong>of</strong> Bamako, Mali;<br />
b<br />
Département Médecine Traditionnelle, B.P. 1746 Bamako Mali;<br />
c<br />
Dipartimento di Scienze Farmaceutiche, Università di Salerno, Via Ponte Don Melillo, 84084 Fisciano, Italy.<br />
c<br />
Dipartimento Farmacobiologico, Università di Messina, Vill. SS Annunziata, 98168 Messina, Italy.<br />
*Email <strong>of</strong> corresponding author: rosanogo@yahoo.fr; aidemet@afribonemali.net<br />
Keywords: Dysmenorrhea; Trichilia emetica; anti-DPPH activity; Analgesic and anti-inflammatory activities.<br />
Introduction<br />
D<br />
ysmenorrhoea affects many women in reproductive age, and is a frequent cause <strong>of</strong> time lost<br />
from work or school as well as interfering with daily living. Treatment is usually done with<br />
NSAIDs and minor analgesics. Maytenus senegalensis Lam. (Celastraceae), Stereospermum<br />
kunthianum Cham. (Bignoniaceae) and Trichilia emetica Vahl. (Meliaceae) are traditionally used in<br />
Mali for the treatment <strong>of</strong> menstrual pains. Previous screening demonstrated the antiinflammatory,<br />
analgesic and antispasmodic activities <strong>of</strong> these plants (Sanogo et al., 2006 and 2007).<br />
The aim <strong>of</strong> the present project is to carry out further investigations on the extracts <strong>of</strong> the leaves<br />
and roots <strong>of</strong> T. emetica, in order to corroborate their use in the treatment <strong>of</strong> dysmenorrhoea.<br />
Material and Methods<br />
A qualitative phytochemical analyse was carried out using thin layer chromatography (TLC)<br />
methods. Some chemical components were isolated and their structures were elucidated by NMR.<br />
The toxicological study was performed on mice. Pharmacological investigations were carried out on<br />
acetic acid-induced writhing (pain) and hind paw oedema in mice. The Paracetamol and<br />
Indometacin are used as reference drugs, respectively in analgesic and anti-inflammatory test. The<br />
effects <strong>of</strong> the aqueous extract were studied on the isolated uterus <strong>of</strong> rat. The Nifedipin was used as<br />
inhibition drug <strong>of</strong> the uterus muscle contraction. The active extracts were studied by bio-guided<br />
fractionation, using the antiradical (1,1-diphenyl-2-picrylhydrazyl, DPPH) on chromatograms. The<br />
phenol content <strong>of</strong> active fractions was determined using Folin-Ciocolteu method.<br />
Results and Discussion<br />
Phytochemical analysis <strong>of</strong> the extract revealed the presence <strong>of</strong> coumarin, flavonoids, tannins,<br />
saponin glycosides and terpenoids. The polyphenolic compounds demonstrated the anti-DPPH<br />
activity. The phenol compounds content <strong>of</strong> aqueous extract <strong>of</strong> the leaves is high than those <strong>of</strong> the<br />
roots. Lignan structures were also isolated from root extract.The aqueous extract had an oral LD<br />
(50) more than 2000 mg in mice. Results <strong>of</strong> pharmacological investigations showed that the<br />
aqueous extracts possess significant (P
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
72% respectively for leaves and roots. Leaves and roots <strong>of</strong> T. emetica, also reduced the oedema<br />
with 67% and 76% 3h after carrageenan injection. The extract <strong>of</strong> the roots also exhibited the<br />
inhibition <strong>of</strong> spontaneous and acetylcholine-induced contractions in the uterus <strong>of</strong> rat. The best<br />
analgesic activity has been demonstrated by aqueous extracts <strong>of</strong> the leaves <strong>of</strong> T. emetica. In the<br />
anti-inflammatory test, the best activity was obtained with the extracts <strong>of</strong> leaves and roots bark <strong>of</strong><br />
T. emetica.<br />
These data tend to suggest that the aqueous extracts possess peripherally-mediated analgesical<br />
properties like Paracetamol. This peripheral analgesic and the anti-inflammatory effects <strong>of</strong> the<br />
decoctions may be mediated via inhibition <strong>of</strong> cyclooxygenases and/or lipoxygenases, like nonsteroidal<br />
anti-inflammatory drugs, commonly employed in the treatment <strong>of</strong> inflammation induced<br />
by prostaglandins (McGaw, et al., 1997). The aqueous extracts <strong>of</strong> T. emetica may contribute in<br />
some way in the prevention <strong>of</strong> synthesis <strong>of</strong> prostaglandins that caused menstrual pains and uterine<br />
hyper-contractility. The results <strong>of</strong> our studies can corroborate the use <strong>of</strong> the extracts <strong>of</strong> T. emetica<br />
in the treatment <strong>of</strong> dysmenorrhoea.<br />
Acknowledgement<br />
This project is supported by grants International Foundation for Science (IFS) N° F/3771-2 and<br />
WACP) (Dr Rokia SANOGO).<br />
References<br />
McGaw, L.J.; Jager, A.K. Van Staden, J, (1997). Prostaglandin synthesis inhibitory activity in Zulu, Xhosa and Otho<br />
medicinal plants. Phytotherapy Research 11: 113-117.<br />
Rokia Sanogo, Ababacar Maiga, Drissa Diallo (2006); Antalgesic and anti-inflammatory activities <strong>of</strong> Maytenus<br />
senegalensis, Stereospermum kunthianum et Trichilia emetica extracts used in the traditional treatment <strong>of</strong><br />
dysmenorrhoea in Mali. Pharmacopée et Médecine et Traditionnelles africaines Vol. XIV, pp.123-136.<br />
Sanogo R., D. Diallo A. Maiga, N. De Tommasi, R. and De Pasquale (2007); Analgesic and anti-inflammatory activities <strong>of</strong><br />
the aqueous extracts <strong>of</strong> Maytenus senegalensis, Stereospermum kunthianum and Trichilia emetica used in the<br />
treatment <strong>of</strong> dysmenorrhoea in Mali (YS -6, 12 th NAPRECA Symposium, Kampala, Uganda, July 2007).<br />
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[PS 18] Hepatoprotective Activity <strong>of</strong> Aqueous Extracts <strong>of</strong> Leaves, Stem Bark<br />
and Roots <strong>of</strong> Entada africana Against Carbon Tetrachloride-Induced Hepatotoxicity<br />
in Rats.<br />
Sanogo R. a,b* , Haïdara M. a , Dénou A. a , Diarra M. c , Kamaté B. d , Togola A. b , Bah S. a , Diallo D. a,b<br />
a Faculty <strong>of</strong> Medicine, Pharmacy, Odontostomatology, University <strong>of</strong> Bamako, Mali;<br />
b Département Médecine Traditionnelle, B.P. 1746 Bamako-Mali;<br />
c Service de Biochimie, Institut National de Recherche en Santé Publique<br />
d Service de Histopathologie, Institut National de Recherche en Santé Publique<br />
*Email <strong>of</strong> corresponding author: rosanogo@yahoo.fr ; rosanogo@gmail.com<br />
Keywords: Entada africana, carbon tetrachloride, Hepatoprotective activity, anti-DPPH activity.<br />
Introduction<br />
E<br />
ntada africana Guillet Perr. (Mimosaceae) is used in African traditional medicine for the<br />
treatment <strong>of</strong> many diseases including hepatic syndromes, jaundice, hepatitis and other hepatic<br />
disorders (Kerharo and Adam, 1974). E. africana, known in Mali with the local name <strong>of</strong> Samanéré<br />
in the Bambara language, is one <strong>of</strong> the phyto-medicines more prescribed for liver diseases.<br />
Preclinical and clinical studies <strong>of</strong> the aqueous extract <strong>of</strong> the roots <strong>of</strong> E. africana demonstrated it<br />
effectiveness in hepatoprotection (Douaré, 1991, Sanogo et al., 1998). Moreover, some<br />
antiproliferative triterpene saponins were isolated from the roots <strong>of</strong> E. africana (Ci<strong>of</strong>fi et al., 2006).<br />
The objective <strong>of</strong> our study was to compare the hepatoprotective activities <strong>of</strong> roots with leaves and<br />
stem bark ones, in order to use them for the production <strong>of</strong> phytomedicines. The aim was to avoid<br />
the excessive exploitation <strong>of</strong> the root <strong>of</strong> E. africana.<br />
Materials and Methods<br />
Our study was designed to evaluate the hepatoprotective activity <strong>of</strong> aqueous extracts <strong>of</strong> leaves,<br />
stem bark and roots <strong>of</strong> in experimental liver injury induced by carbon tetrachloride in rats. The<br />
animals were starved for 18h with water at libitum. E. africana leaf, stem bark and roots aqueous<br />
extracts were administrated orally to rats respectively at the doses <strong>of</strong> 190, 110 and 100 mg/kg. The<br />
extracts were administrated once a day for seven days. On the seventh day after the extract<br />
treatment, a single dose <strong>of</strong> the carbon tetrachloride (50% in olive oil) was injected by IP at the dose<br />
<strong>of</strong> 5 ml/kg.<br />
The levels <strong>of</strong> hepatic marker enzymes, alanine aminotransferase (ALT), aspartate aminotransferase<br />
(AST) and biluribin were used to assess the hepatoprotective activity against carbon tetrachloride<br />
(CCl4)-induced hepatotoxicity. The hepatoprotective activity was also assessed by histopathological<br />
studies <strong>of</strong> liver tissue. The ant-DPPH activity was determined on the chromatogram <strong>of</strong> extracts <strong>of</strong><br />
three parts <strong>of</strong> E africana by spraying plate with methanol solution <strong>of</strong> radical 1,1-diphényl 1-2-<br />
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picrylhydrazyle. Mains chemical constituents were analysed by colorimetric reactions, and by thin<br />
layer chromatography methods.<br />
Results and Discussion<br />
The highest hepatoprotective activity was observed for the aqueous extract <strong>of</strong> roots and stem bark<br />
<strong>of</strong> E. africana, when administrated once a day for seven days before intoxication. The protection<br />
according to the serum level was respectively for ALAT (63,84% and 61, 36%) for ASAT (63,04 and<br />
61,22) and for biluribin (40,00% and 28,57%). For the root decoction, the hepatoprotective activity<br />
was also supported by histopathological studies <strong>of</strong> liver tissue. The high content in polyphenolic<br />
components <strong>of</strong> aqueous extracts <strong>of</strong> roots and stem bark <strong>of</strong> E. africana can be contributed to the<br />
hepatoprotective activity. The presence <strong>of</strong> many polyphenolic compounds and polysaccharides in<br />
the aqueous extracts <strong>of</strong> the plant can also support a good hepatoprotective activity.<br />
The results <strong>of</strong> our researches showed the effectiveness in hepatoprotection <strong>of</strong> extract <strong>of</strong> stem bark<br />
more than leaves. Their possible use in the traditional herbal treatment in liver diseases, instead <strong>of</strong><br />
roots extracts, should be encouraged, in order to avoid the excessive exploitation <strong>of</strong> the root <strong>of</strong> E.<br />
africana.<br />
Acknowledgement<br />
This study was financed on National Budget via the National Istitute <strong>of</strong> Research in Public Health<br />
(Protocole N° 006/2010/INRSP/DG).<br />
References<br />
Douaré I. (1991); Contribution à l étude clinique de l Entada africana dans le traitement des hépatites virales. Thèse de<br />
Médicine, ENMP. Bamako (Mali).<br />
Kerharo J., Adam J. G. (1974); Pharmacopée Sénégalaise Traditionnelle: Plantes Médicinales et toxiques. Edit. Vigotfrères,<br />
Paris.<br />
Sanogo, R., Germanò, M.P., D Angelo, V., Guglielmo, M., De Pasquale, R. (1998); Anti-Hepatotoxic Properties <strong>of</strong> Entada<br />
africana Guil. et Perr. (Mimosaceae). Phytotherapy Research Vol. 12, S157- S159.<br />
Ci<strong>of</strong>fi, G., Dal Piaz, F., De Caprariis, P., Sanogo, R., Marzocco, S.,Autore, G, De Tommasi N., (2006); Antiproliferative<br />
triterpene saponins from Entada africana. Journal <strong>of</strong> Natural Products 69 (9), pp.1323-1329.<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[PS 19] The Radical Scavenging Activity <strong>of</strong> Flavonoids from Solenostemon<br />
monostachys (P.Beauv.) Briq (Lamiaceae).<br />
1.<br />
2.<br />
Taiwo, B.J 1 ., Ogundaini, A.O 1 . and Obuotor, E.M 2 .<br />
Department <strong>of</strong> Pharmaceutical Chemistry, Obafemi Awolowo University, Osun State, Nigeria.<br />
Department <strong>of</strong> Biochemistry, Obafemi Awolowo University, Osun State, Nigeria.<br />
Taiwobami2001@yahoo.com ; bamit@oauife.edu.ng.<br />
Key words:-Flavonoids, radical scavenging ability, Lamiaceae.<br />
Introduction<br />
F<br />
lavonoids are -benzo-pyrone derivatives, which resemble coumarin and are ubiquitous in<br />
photosynthesing cells. Their occurrence is therefore widespread in the plant kingdom and about<br />
500 varieties <strong>of</strong> flavonoids are known (Havsteen, 1983). Flavonoids are known to have antiinflammatory,<br />
anticancer, antimicrobial, antioxidant, antiretroviral etc activities. This made them<br />
subject <strong>of</strong> intense researches in the recent past. The different classes <strong>of</strong> flavonoid arise from the<br />
difference in the oxygenation pattern on the -benzo-pyrone skeleton. These compounds, which<br />
exist as aglycones and therefore lipophilic constituents, are not as universal in occurrence in higher<br />
plants as the more polar flavonoid glycosides, which occur in vacuoles in the plant cells. However,<br />
surface flavonoids are common in the Lamiaceae, especially in the subfamily Nepetoideae. Their<br />
presence is <strong>of</strong>ten correlated with the production <strong>of</strong> other lipophilic secondary products such as<br />
essential oil terpenoids (Wollenweber, 1982). Solenostemon monostachys (P.Beauv.) Briq<br />
(Lamiaceae) is in Ghana as a leafy vegetable while in South Western Nigeria, it is commonly<br />
employed as a recipe in herbal medicines for especially infectious and inflammatory diseases.<br />
Miyase et al. (1980) reported unusually rearranged diterpenoid from the plant. We now report the<br />
presence <strong>of</strong> three flavonoids from the aerial parts <strong>of</strong> the plants as well as the radical scavenging<br />
ability <strong>of</strong> the flavonoids.<br />
Materials and Methods<br />
General<br />
UV spectral was recorded on Cecil UV- Spectrophotometer. 1 H and 13 C NMR spectral were acquired<br />
in deuterated chlor<strong>of</strong>orm using 200 MHz Varian NMR Spectrometer with TMS as internal standard.<br />
Solvents used were general reagents solvents redistilled before used.<br />
Plant Material<br />
The aerial part <strong>of</strong> Solenostemon monostachys P. Beauv. (Briq.) (Lamiaceae) was collected on<br />
Obafemi Awolowo University campus premise on October, 2006. The plant was identified by Mr.<br />
Oladele A.T. by comparison <strong>of</strong> sample with voucher specimen deposited at the herbarium <strong>of</strong> the<br />
Obafemi Awolowo University, Ile Ife.<br />
DPPH Quantitative Spectrophotometic assay<br />
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This was carried out using the method described by Menzor et. al., (2001) with a slight modification.<br />
The reaction was carried out in a 96-well microtitre plates. Stock solutions <strong>of</strong> the isolated<br />
compounds and L-ascorbic acid (100 µM) were separately diluted to a concentration <strong>of</strong> 100, 50, 25,<br />
12.5, 6.25, 3.13, and 1.56 µM in methanol. Twenty microlitres <strong>of</strong> 0.25 µM DPPH solution in<br />
methanol was added to 50 µL <strong>of</strong> each concentration <strong>of</strong> sample to be tested and allowed to react at<br />
room temperature in the dark for thirty minutes. Blank solution was prepared with 50 µL <strong>of</strong> sample<br />
solution and 20 µL <strong>of</strong> methanol only while the negative control was DPPH solution (20 µL) plus 50 µL<br />
methanol. Methanol was used to blank the microplate reader and the decrease in absorbance was<br />
measured at 517 nm. Absorbance values were converted to percentage antioxidant activity (AA%)<br />
using the formula:<br />
AA% = 100 - {[( Abs sample - Abs blank) X 100]/Abs control}<br />
Abssample is the absorbance <strong>of</strong> the sample, Absblank is the absorbance <strong>of</strong> the blank and Abscontrol is the<br />
absorbance <strong>of</strong> L-ascorbic acid. The inhibitory concentration (IC50), is the concentration <strong>of</strong> the<br />
sample that brought about 50% inhibition <strong>of</strong> the DPPH free radicals. The values were obtained from<br />
the separate linear regression <strong>of</strong> plots <strong>of</strong> the mean percentage <strong>of</strong> the antioxidant activity, (AA%)<br />
against concentration <strong>of</strong> the test compounds from the replicate assays.<br />
Extraction and Isolation<br />
Ethyl acetate extract <strong>of</strong> the powdered dried aerial parts <strong>of</strong> Solenostemon monostachys (2.1 g) was<br />
adsorbed on silica gel mesh 200-400 and eluted on an open column with solvent mixtures <strong>of</strong><br />
increasing polarity from 100% hexane through 100% ethyl acetate to 100% methanol. The thin layer<br />
chromatography (TLC) analysis <strong>of</strong> the eluates on silica gel plate using 100% ethyl acetate as mobile<br />
phase gave fractions I-IV. Fraction I (0.49 g) with DPPH active spots in the DPPH autographic assay<br />
was dissolved in 2 ml <strong>of</strong> EtOAc: MeOH (9:1) and was layered on a column <strong>of</strong> Sephadex LH-20. The<br />
column was eluted with the following solvent systems: Hex : EtOAc (7:3, 8:2, 9:1), 100% EtOAc,<br />
MeOH : EtOAc (1:9, 2:8, 4:6 and 5:5). TLC analysis <strong>of</strong> eluates on silica with n-hexane: EtOAc (3:7) as<br />
mobile phase led to the isolation <strong>of</strong> compound 1 (0.027 g), compound 2 (0.021g) and three other<br />
fractions. The methanol extract (6 g) was eluted on silica gel on open column with 200 ml each, <strong>of</strong><br />
100% ethyl acetate, ethyl acetate: methanol (9:1, 8:2, 7:3, 5: 5) and 100% methanol to give fractions<br />
I-III. Fraction III (2.3 g) that tested positive in the DPPH autographic assay (Burits and Bucar, 2000)<br />
was eluted on a column <strong>of</strong> Sephadex LH-20 pre-swollen with 100% ethyl acetate and was eluted<br />
with the following solvent mixtures 100% EtOAc MeOH: EtOAc (1:9, 2:8,3:7, 5:5), 100% MeOH and<br />
20% water in MeOH. TLC analysis on reversed phase plate with water: methanol (6:4) led to three<br />
fractions; SMMa-SMMc. SMM3b (1.1 g extracted with 50-100% MeOH), was subjected to elution on<br />
a Lobar RP-18 column with the following solvent mixtures: MeOH: H2O (3:7, 4:6, 5:5, 6:4) and 100%<br />
MeOH. TLC analysis <strong>of</strong> the eluates using a RP-18 plate with MeOH: H2O (4:6) as mobile phase to give<br />
compound 3 (0.038g) and two other fractions. Compounds 1, 2 and 3 were subjected to quantitative<br />
evaluation <strong>of</strong> the antioxidant activity using the DPPH spectrophotometric assay as described above.<br />
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Table 1: Radical scavenging activities <strong>of</strong> isolated compounds compared with quercetin.<br />
Comp Regression equation R 2<br />
IC50<br />
1 Y=1.4682x+10.849 0.9994 26.67±0.21<br />
2 Y=8.4913x+4.596 0.9908 5.35 ± 0.31<br />
3 Y=0.258x+2.0396 0.9789 185.89 ± 1.02<br />
4 Y=11.487x+23.382 0.933 2.32 ± 0.08<br />
Compound 1: 1 H NMR (200 MHz, DMSO).<br />
d, J=0.977 Hz, H-8), 6.174 (1H, d, J=0.977 Hz, H-<br />
6). 13 C NMR (50 MHz, DMSO), : 164.82 (C-2), 103.52(C-3), 182.43(C-4), 161.85(C-5), 99.53(C-6),<br />
162.15(C-7), 94.65(C-8), 158.00(C-9), 104.39(C-10), 121.87(C-11), 116.64(C-12), 12<strong>9.1</strong>4(C-13),<br />
164.43(C-14), 12<strong>9.1</strong>4(C-15), 116.64(C-16).<br />
Compound 2: UV: 352, 280, 1 H NMR (200 MHz, DMSO).<br />
6.17 (1H, d, J=1.8Hz, H-6). 13 C NMR, (50 MHz, DMSO), : 164.3 (C-2), 103.3(C-3), 182.1(C-4),<br />
161.9(C-5), 99.3(C-6), 164.6(C-7), 94.3(C-8), 157.7(C-9), 104.1(C-10), 122.9(C-11), 113.8(C-12),<br />
146.2(C-13), 150.2 (C-14), 116.5 (C-15), 119.4 (C-16).<br />
Compound 3: UV: 365, 268 1 H NMR (200 MHz, DMSO),<br />
3), 6.766, (2H, d, J=1.8 Hz), 6.393 (1H, d, J= 1.8<br />
Hz). 13 C NMR (50 MHz, DMSO). : 164.74 (C-2), 103.17 (C-3), 182.37 (C-4), 161.39 (C-5), 100.00 (C-6),<br />
162.23 (C-7), 95.03 (C-8), 157.36(C-9), 105.18(C-10), 120.33(C-11), 116.54(C-12), 128.84(C-13),<br />
162.34(C-14), 128.84 (C-15), 116.54(C-16).<br />
Discussion<br />
Compound 1 was isolated as an amorphous powder. The UV spectrum <strong>of</strong> the methanolic solution<br />
gave two absorption bands at 266 and 365 characteristic <strong>of</strong> flavone UV absorption band II and<br />
band I respectively. The shift reagent studies gave a bathochromic shift <strong>of</strong> 36 nm in the NaOMe<br />
3 spectrum indicated no ortho-dihydroxyl<br />
substitution pattern on ring B while addition <strong>of</strong> NaOAc to the methanolic solution <strong>of</strong> compound 1<br />
gave a bathochromic shift <strong>of</strong> 36 nm indicating that 7-OH is free. The 1 HNMR spectrum showed 5<br />
signals in all; an AB coupling, a signal at<br />
with a signal at a monosubstituted pattern on ring B.<br />
The singlet signal at 6.93 ppm integrating for 1 proton was assigned to H-3. A doublet at 6.17<br />
ppm with coupling constant value <strong>of</strong> 0.977 Hz assigned to H-6 was observed to be coupling with the<br />
signal at 6.45 ppm (d, J= 0.977 Hz) assigned to H-8. The UV studies, NMR studies and comparison<br />
with literature values (Xiao et al, 2006) led to the characterization <strong>of</strong> compound 1 as apigenin.<br />
Compound 3 was isolated as an amorphous powder. The UV spectrum <strong>of</strong> the methanolic solution<br />
indicated a flavone with a substituted 7-OH group. The 1 H NMR and 13 CNMR spectra were similar<br />
to that <strong>of</strong> compound 1 apart from a doublet at 4.86 ppm, a doublet, (J=6.2 Hz) for an anomeric<br />
proton . The carbon signals showed additional four oxygenated methine carbon with one anomeric<br />
carbon and a carbonyl carbon at 172.52. Thus 3 was characterized as apigenin-7-O- -D-<br />
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glucuronide. The UV studies <strong>of</strong> compound 2 indicated a flavone with an ortho dihydroxy group and<br />
a free 7-OH. The proton NMR spectrum showed 6 protons all in the aromatic region. In the COSY<br />
spectrum, the signal at 6.42 correlated with the signal at 6.17, a doublet with coupling constant<br />
<strong>of</strong> 1.8 Hz which is assignable to H-8 and H-6 respectively. A singlet at 6.64 was assigned to H-3.The<br />
6.9, a doublet<br />
<strong>of</strong> doublet, (J=8 Hz and 1.5 Hz) as 6.7 (1H, d, J=1.5 Hz) is assignable<br />
is a correlation between the proton at 6.17 and the<br />
carbon at 99.29 for H-6 and the proton at 6.43 correlated with the carbon at 94.31 for H-8. The<br />
singlet at 6.64 correlated with the carbon at 103.28. On comparison with literature values<br />
(Markham, 1982), compound 2 was therefore identified as luteolin. The radical scavenging activity<br />
<strong>of</strong> the flavonoids was evaluated as shown above. Luteolin with an ortho dihydroxy group on ring B<br />
has the highest activity compared with apigenin wi<br />
hydroxyl group on apigenin drastically reduced the radical scavenging ability as indicated from the<br />
IC50. The presence <strong>of</strong> these compounds in Solenostemon monostachys may justify the<br />
ethnomedicinal uses <strong>of</strong> the plant.<br />
HO<br />
O<br />
OH O<br />
1<br />
H<br />
OH<br />
HO<br />
O<br />
OH O<br />
2<br />
H<br />
OH<br />
OH<br />
349<br />
O<br />
OH O<br />
References<br />
Burits, M. and Bucar, F. (2000); Antioxidant activity <strong>of</strong> Nigella sativa essential oil. Phytotherapy research. 14, 323-328.<br />
Havsteen, B. (1983); Flavonoid, a class <strong>of</strong> natural products <strong>of</strong> high pharmacological potency. Biochem Pharmacol<br />
32:1141-1148<br />
Markham K, K. (1982); The usefulness <strong>of</strong> NMR and MS data in flavonoid structure elucidation. In Techinques <strong>of</strong><br />
flavonoid identification. Accademic Press Inc, ltd. London. pp 24-28.<br />
Miyase, T., Ruedi, P. and Eugester, C.H. (1980); Structures <strong>of</strong> six coleons from Solenostemon monostachys (P.BEAUV.)<br />
BRIQ. (Labiateae). Helvetica Chimica Acta. 63, (.1). 95-101.<br />
Wollenweber, E., (1982); Flavones and flavonols. In: Harborne, J.B., Mabry, T.J. (Eds.), The Flavonoids, Advances in<br />
Research. Chapman and Hall, London, pp. 189 260.<br />
Xiao, J. B., Ren, F. L. and<br />
Xu, M. (2006); Flavones from Marchantia convolute: Isolation <strong>of</strong> Apigenin-7-O- -D-glucuronide<br />
and 5-Hydroxyl-7-methoxy-2-methylchromone. Journal <strong>of</strong> Pharmaceutical and Allied Sciences 3 (1), 310 313.<br />
HO<br />
HO<br />
COOH<br />
OH<br />
O<br />
O<br />
3<br />
H<br />
OH
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[PS 20] Preliminary Evaluation <strong>of</strong> Anti-Diarrheal, Ulcer-Protective and Acute<br />
Toxicity <strong>of</strong> Aqueous Ethanolic Stem Bark Extract <strong>of</strong> Ficus trichopoda in Experimental<br />
Rodents<br />
Balogun S.O. 1,6,7 , Tanayen J.K. 2,6 , Ajayi A.M. 2,6 , Ibrahim A. 1,6 , Ezeonwumelu J.O.C. 5,6 ,<br />
Oyewale A.A. 3,6 , Loro J. O 2,6 , Goji A.D.T. 4,6 , Kiplagat D.M. 5,6 , Adzu B. 5,6<br />
1<br />
Department <strong>of</strong> Biochemistry,<br />
2<br />
Department <strong>of</strong> Pharmacology and Toxicology,<br />
3<br />
Department <strong>of</strong> Anatomy,<br />
4<br />
Department <strong>of</strong> Physiology<br />
5<br />
Department <strong>of</strong> Clinical and Biopharmacy, School <strong>of</strong> Pharmacy,<br />
6<br />
Kampala International University Complementary and Alternative Medicine Research<br />
(KIUCAMRES), Kampala International University - W estern Campus,P.O. Box 71 Bushenyi, Uganda<br />
7<br />
Programa de pós-graduação em Ciências da Saúde,(Farmacologia), Faculdade de<br />
Ciencias Medicas, Universidade Federal de M ato Grosso, Mato Grosso, Cuiaba, Brazil<br />
Corresponding Author: tanayenjk@yahoo.com<br />
Key words: Acute toxicity, anti-diarrheal, Ficus trichopoda, gastroprotective, loperamide, Uganda<br />
Introduction<br />
G<br />
enus Ficus belongs to Family Moraceae commonly referred to as fig trees. Forty-four species<br />
are known from Uganda (Berg and Hijman, 1989; Verdcourt, 1998). A number <strong>of</strong> Ficus sp. are<br />
used as food and for medicinal properties (Lansky et al., 2008). Several ficus species are<br />
traditionally used in African folk medicine in the treatment <strong>of</strong> many illnesses such as convulsions<br />
and respiratory disorders (Wakeel et al., 2004). The decoction <strong>of</strong> Ficus rhynchocarpa, Ficus<br />
sycomorus, Ficus natalensis and Ficus vasta are used to treat various stomach disorders. Several<br />
other reports have demonstrated different biological activities <strong>of</strong> Ficus plants including peptic ulcer<br />
treatment (Kokwaro, 1993; Akah et al., 1997; Mandal et al., 2000; Chiang et al., 2005; Kuetea et al.,<br />
2008; Rao et al., 2008; Singh et al.,2009). The reported medicinal uses <strong>of</strong> these plants includes:<br />
treatment <strong>of</strong> various gastrointestinal disorders, infectious diseases, fertility treatment and<br />
induction <strong>of</strong> labor (Kamatenesi-Mugisha and Oryem-Origa, 2007; Ssegawa and Kasenene, 2007).<br />
Ficus trichopoda, Baker, is a medicinal plant belonging to the Moraceae family used popularly as a<br />
multi-purpose herb in Uganda. The aim <strong>of</strong> this study was to evaluate the anti-diarrheal, ulcer<br />
protective effects <strong>of</strong> 70% ethanolic extract <strong>of</strong> Ficus trichopoda stem bark (FTE) and its acute toxicity.<br />
Materials and Methods<br />
The laboratory animals used in these experiments were obtained from the laboratory animal facility<br />
<strong>of</strong> the department <strong>of</strong> Pharmacology and Toxicology <strong>of</strong> Kampala International University (KIU). The<br />
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stem bark and the leaves <strong>of</strong> F. trichopoda were collected in the month <strong>of</strong> June, 2009 in the<br />
morning. The plant was identified by a taxonomist at Makerere University Kampala and a voucher<br />
specimen deposited at Herbarium section <strong>of</strong> School <strong>of</strong> Pharmacy, Kampala International University.<br />
The stem bark was then ground into powder and was used for the subsequent experimentation.<br />
The powdered material was exhaustively extracted by cold aqueous ethanolic maceration for two<br />
days and the supernatant decanted. The entire process was repeated three times, and the extracts<br />
combined and filtered through Whatman No 1 filter paper. The crude extract was evaporated to dry<br />
powder at 40ºC in an oven.The anti-diarrheal effect was evaluated using castor-oil induced diarrhea<br />
model while anti-ulcer effect was evaluated using ethanol-induced ulcer model using rats.<br />
Loperamide and misoprostol were used as standard drugs for diarrhea and ulcer studies<br />
respectively. The extract was administered orally at three different doses <strong>of</strong> 125, 250 and<br />
500mg/kg. Acute toxicity was evaluated by oral administration <strong>of</strong> the extract at 1000, 2000 and<br />
4000 mg/kg body weight in mice. The extract exhibited a graded dose-dependent inhibition <strong>of</strong> the<br />
castor oil induced diarrhea.<br />
Results<br />
The onset-time and severity <strong>of</strong> diarrhea was significantly reduced (p4000 mg/kg in mice. Preliminary phytochemical screenings<br />
indicated the presence <strong>of</strong> reducing sugar, alkaloids, saponnins, pyrocathecolic tannins and free<br />
amino acids/amines.<br />
Table 1: Effect <strong>of</strong> aqueous ethanolic extract <strong>of</strong> FTE on ethanol-induced ulceration (125-500mg/kg,<br />
p.o)<br />
Treatment Dose Ulcer index (mm) 1<br />
% Inhibition <strong>of</strong><br />
ulceration 2<br />
Ficus trichopoda 125mg/kg 80.90±17.23 1.63<br />
Ficus trichopoda 250mg/kg 58.80±18.68 28.50<br />
Ficus trichopoda 500mg/kg 41.90±19.23 49.05<br />
Misoprostol 400µg/kg 16.00±6.78 80.54<br />
Distilled water 10ml/kg 82.24±13.3 -<br />
1 : Values expressed as Mean±S.E.M . (n = 5); p
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Table 2: Effect <strong>of</strong> ethanolic extract <strong>of</strong> FTE on castor oil-induced diarrhea and loss in body weight<br />
(125-500mg/kg, p.o)<br />
Loss in<br />
B Wt (g) 1<br />
Diarrhea Score Total<br />
Treatment Dose ++ + 0 Score<br />
352<br />
Percentage<br />
Inhibition 2<br />
Ficus<br />
trichopoda<br />
125mg/kg 11.22±3.14 3 1 1 7 22.22<br />
Ficus<br />
trichopoda<br />
250mg/kg 9.98 ±3.17 0 3 2 3 66.67 3<br />
Ficus<br />
trichopoda<br />
500mg/kg 7.88 ±4.13 0 1 4 1 88.89 3<br />
Loperamide 400µg/kg 6.84 ±4.67 0 0 5 0 100 3<br />
Distilled<br />
water<br />
10ml/kg 7.04 ±1.70 4 1 0 9 -<br />
1 2 3:<br />
: Values expressed as Mean±S.E.M. (n = 5); : Compared with saline control; Denotes statistical<br />
significance between treated groups and control.(One way ANO VA p
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
the anti-diarrheal effect observed may be due to the presence <strong>of</strong> these various phytochemicals in<br />
the extract. However, pretreatment <strong>of</strong> the rats with FTE did evoke a dose dependent inhibition <strong>of</strong><br />
ethanol-induced gastric erosion and hemorrhage (1.63, 28.5 and 49.05% for 125, 250 and 500<br />
mg/kg, respectively), but these observations were not statistically significant as compared to the<br />
control. It should be noted also that although the standard drug had an inhibition <strong>of</strong> 80.54% but<br />
this difference was not statistically significant either as compared to the control. The import <strong>of</strong><br />
these findings is the need to assess the anti-ulcer activity <strong>of</strong> FTE using other ulcer models.<br />
Conclusion<br />
This study confirmed the antidiarrheal properties <strong>of</strong> this plant as it is used in traditional medicine. It<br />
has exploitable ulcer protective effects and is considerably safe on oral administration.<br />
Acknowledgement<br />
We are grateful to KIU-CAMRES for the financial support provided. We also declare no conflict <strong>of</strong><br />
interest.<br />
References:<br />
Adzu, B., S. Amos, M.B. Amizan and K. Gamaniel, (2003); Evaluation <strong>of</strong> the antidiarrhoeal effects <strong>of</strong> Zizyphus spinachristi<br />
stem bark in rats. Acta Trop., 87: 245-250.<br />
Ahmadua, A.A., A.U. Zezib and A.H. Yaro, (2007); Antidiarrheal activity <strong>of</strong> the leaf extracts <strong>of</strong><br />
Daniellia oliveri spp. Hutch and Dalz (Fabaceae) and Ficus sycomorus Miq (Moraceae). Afr. J. Tradit. Complem., 4(4):<br />
524-528.<br />
Akah, P.A., K.S. Gamaniel, C.N. Wambebe, A . Shittu, S.D. Kapu and O.O. Kunle, (1997); Studies on gastrointestinal<br />
properties <strong>of</strong> Ficus exasperata. Fitoterapia, 68: 17-20.<br />
Bafor, E.E. and O. Igbinuwen, (2009); Acute toxicity studies <strong>of</strong> the leaf extract <strong>of</strong> Ficus exasperata on haematological<br />
parameters, body weight and body temperature. J. Ethnopharmacol., 123: 302-307.<br />
Berg, C.C. and M .E.E. Hijman, (1989); Ficus. In: Polhill, R.M. (Ed.), Flora <strong>of</strong> Tropical East Africa. A.A. Balkema, Rotterdam,<br />
Chap. 11, pp: 43-86.<br />
Kamatenesi-Mugisha, M. and H. Oryem-Origa, (2007); Medicinal plants used to induce labour during childbirth in<br />
western Uganda. J. Ethnopharmacol., 109: 1-9.<br />
Kinyi, H.W. and S.O. Balogun, (2009); Effects <strong>of</strong> Crude Ethanolic Extract <strong>of</strong> Ficus natalensis on liver function.<br />
(Unpublished).<br />
Kokwaro, J.O., (1993); Medicinal Plants <strong>of</strong> East Africa. Kenya Literature Bureau Publishers, 2nd Edn., pp: 174-176.<br />
Lansky, E.P., H.M. Paavilainen, A.D. Pawlus and R.A. Newman, (2008); Ficus spp. (fig): Ethnobotany and potential as<br />
anticancer and anti-inflammatory agents. J. Ethnopharmacol., 119: 195-213.<br />
Longanga, O.A., A. Vercruysse and A. Foriers, (2000); Contribution to the ethnobotanical, phytochemical and<br />
pharmacological studies <strong>of</strong> traditionally used medicinal plant in the treatment <strong>of</strong> dysentery and diarrhoea in<br />
Lomela area, Democratic Republic <strong>of</strong> Congo (DRC). J. Ethnopharmacol., 71(3): 411-423.<br />
Mandal, S.C., B.P. Saha and M. Pal, (2000); Study on antibacterial activity <strong>of</strong> Ficus racemosa Linn. Leaf extract.<br />
Phytother. Res., 14: 278-280.<br />
Mandal, S.C. and C.K. Kumar, (2002); Studies on antidiarrheal activity <strong>of</strong> Ficus luspida leaf extract in rats. Fitoterapia,<br />
73(7-8): 663-667.<br />
Mckeon, T.A., J.J. Lin and A.E. Stafford, (1999); Biosynthesis <strong>of</strong> ricinoleate in castor oil. Adv. Exp. Med. Biol., 464: 37-47.<br />
Palombo, E.A., (2006); Phytochemicals from traditional medicinal plants used in the treatment <strong>of</strong> diarrhoea: Modes <strong>of</strong><br />
action and effects on intestinal function. Phytother. Res., 20: 717-724.<br />
353
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Ssegawa, P. and J. Kasenene, (2007); Medicinal plant diversity and the uses in the Sango bay area, Southern Uganda. J.<br />
Ethnopharmacol., 113: 521-540.<br />
Tripathi, K.D., (1994); Essentials <strong>of</strong> Medical Pharmacology. Jay Pee Brothers Medical Publishers,<br />
New Delhi.<br />
Verdcourt, B., (1998); A new species <strong>of</strong> Ficus (Moraceae) from Uganda. Kew Bulletin, 53: 233-236.<br />
Wakeel, O.K., P.I. Aziba, R.B. Ashorobi, S. Umukoro, A.O. Aderibigbe and E.O. Awe, (2004); Neuropharmcological<br />
activities <strong>of</strong> Ficus platyphylla stem bark in mice. Afr. J. Biomed. Res., 7: 75-78.<br />
354
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[PS 21] Phytochemical and In Vitro Antimicrobial Echinops Hispidus<br />
Fresen<br />
James Ndia Muithya 1 , Alex Kingori Machocho 1* , Alphonse Wafula Wanyonyi 1 and Paul Kipkosgei<br />
Tarus 2<br />
1<br />
Department <strong>of</strong> Chemistry, School <strong>of</strong> Pure and Applied Sciences, Kenyatta University, P.O Box<br />
43844 - 00100, Nairobi-Kenya<br />
2<br />
Department <strong>of</strong> Chemistry and Biochemistry, Scool <strong>of</strong> Science, Moi University <strong>of</strong> Science and<br />
Technology, P.O Box 1125, Eldoret- Kenya<br />
Key words: Echinops hispidus, terpenoids, fur<strong>of</strong>uran lignans, thiophenes, antimicrobial<br />
The genus Echinops belongs to the family Asteraceae and comprises <strong>of</strong> 120 species. E. hispidus is<br />
widely distributed in Kenya and it is used by the Kipsigis in the treatment <strong>of</strong> bacterial infections.<br />
This work reports the isolation and anti-microbial studies <strong>of</strong> the sesquiterpenoid; cameroonan-7 -<br />
ol (1), the triterpenoid; acetyltaraxerol (2), fur<strong>of</strong>uran lignans; membrin-8 -ol (3) and membrin-8 -ol<br />
(4) and polyacetylene thiophenes; 2-Octyl-5-(3,4-dihydroxybut-1-enyl)thiophene (5), [4-[5-(penta-<br />
1,3-dieynyl) thien-2-yl] but-3-ynyl alcohol (6), 2-(Penta-1,3-diynyl)-5-(3,4-dihydroxybut-1ynyl)thiophene<br />
(7). Compounds 1, 2, 6 and 7 are being reported for the first time from this plant<br />
while 3, 4 and 5 have not been reported before. Compounds 5 showed moderate activity against<br />
Staphylococcus aureus while 6 and 7 exhibited high antibacterial activities against S. aureus.<br />
Compounds 5 - 7 showed no activity against Pseudomonas aeruginosa and Eschelicia colli.<br />
Compounds 1-4 exhibited no anti-microbial activities against S. aureus, E. coli, P. eruginosa and<br />
Cryptococcus ne<strong>of</strong>ormans. Compound 5 showed no antifungal activity but compound 6 and 7<br />
exhibited very strong antifungal activities against C. ne<strong>of</strong>ormans. The isolated compounds 5 7<br />
showed high antifungal activities against C. ne<strong>of</strong>ormans. These biologically active compounds are<br />
templates for synthesis <strong>of</strong> more potent and water soluble derivatives. These antimicrobial results<br />
support the use <strong>of</strong> E. hispidus for the treatment <strong>of</strong> antimicrobial related ailments by the Kipsigis in<br />
Kenya.<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
[PS 22] Efficacy and safety pr<strong>of</strong>ile <strong>of</strong> some Ugandan antimalarial herbs used in<br />
Primary Health Care<br />
E. Katuura 1,4 , J.S.R.Tabuti 2 , M. Kamatenesi-Mugisha 3 & J. Ogwal Okeng 4 .<br />
1 Natural Chemotherapeutic Research Laboratory, Ministry <strong>of</strong> Health, Kampala, Uganda. 2 Colledge <strong>of</strong> Natural Sciences,<br />
Department <strong>of</strong> Natural Resources Makerere University, Kampala, Uganda. 3 Colledge <strong>of</strong> life Sciences, Department <strong>of</strong><br />
Biological Sciences, and 4 College <strong>of</strong> Health Sciences, School <strong>of</strong> Biomedical Sciences, Department <strong>of</strong> Pharmacology &<br />
Therapeutics, Makerere University, Kampala, Uganda.<br />
Corresponding Author: ekatuura@yahoo.com<br />
Key words; efficacy, safety, antimalarial, plants,Uganda<br />
Introduction<br />
Plasmodium falciparum malaria is one <strong>of</strong> the most important parasitic diseases affecting Sub-<br />
Saharan Africa. It is the most prevalent infection in Uganda, despite the availability <strong>of</strong> interventions<br />
(MoH, 2007). There is widespread resistance to the available antimalarial agents, the effective <strong>of</strong><br />
which are too expensive for the great majority <strong>of</strong> patients, hence the need for new antimalarial<br />
drugs (MoH/MCU, 2003, 2007). In this study the efficacious and safe extracts <strong>of</strong> the commonly used<br />
medicinal plants that can be used as sources or templates in development <strong>of</strong> new antimalarial<br />
drugs were investigated.<br />
Materials and methods<br />
The petroleum ether, chlor<strong>of</strong>orm and ethanol plant crude extracts were subjected to in vitro<br />
screening against Plasmodium falciparum using the nitro-tetrazolium blue based plasmodium<br />
lactate dehydrogenase (pLDH) assay (Markler et al., 1993). Toxicity pr<strong>of</strong>ile was done on the<br />
antiplasmodialy active extracts (Lorke D. (1983). Selected extracts were orally administered in<br />
albino mice for acute toxicity and Wister rats for subacute toxicity tests.<br />
Results and Discussion<br />
In the antiplasmodial activity tests a number <strong>of</strong> plants showed high sensitivity towards P.<br />
falciparum parasite while others were not active. The chlor<strong>of</strong>orm extract <strong>of</strong> M. lanceolata (EC50 1.60<br />
µg ml), showed the highest antiplasmodial activity followed by R. natalensis (EC50 1.80 µg ml) as<br />
presented in Table 1 below.<br />
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The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Table 1: In vitro antiplasmodial activity (IC50) <strong>of</strong> extracts <strong>of</strong> selected plants used in<br />
the treatment <strong>of</strong> malaria in western Uganda<br />
Plant species IC50 median values (µg/ml) and percent yield <strong>of</strong> the extracts<br />
PE % yield CHCl3 02 % yield EtoH % yield<br />
M. Lanceolata >50.0 1.7 1.6 2.6 11.4 7.0<br />
E. suaveolens >50.0 2.8 >50.0 2.9 >50.0 4.2<br />
Conyza Sp. >50.0 0.8 <strong>9.1</strong> 1.5 20.5 5.8<br />
R. natalensis >50.0 1.2 1.8 2.8 6.6 3.7<br />
L. trifolia 13.2 0.8 >50.0 3.4 >50.0 4.4<br />
T. asiatica 6.6 1.0 22.4 2.5 >50.0 5.5<br />
B. longipes >50.0 2.4 3.7 2.7 50.0 3.9<br />
T. bakeri 3.9 0.6 >50.0 2.1 33.2 6.6<br />
I. emerginella 38.0 1.0 25.3 1.5 5.8 2.1<br />
V. lasiopus 43.9 0.8 >50.0 1.3 >50.0 1.9<br />
PE-Petroleum ether; CHCl3-Chlor<strong>of</strong>orm; EtoH-Ethanol; EC50-Effective concentration<br />
The study showed no acute toxicity with all the plants except B. longipes which showed slight<br />
toxicity (LD50 <strong>of</strong> 4.70 g/kg) (Table below) The biochemical parameters related to liver function tests,<br />
kidney function tests and haematological analysis <strong>of</strong> the treated animals showed no significant<br />
change compared to the control group. There was no significant change observed in organ<br />
weight/body weight ratios in treated groups as compared to the control group while only slight<br />
histological changes were observed in some organs <strong>of</strong> the high dose treated animals after 28 days<br />
<strong>of</strong> oral administration.<br />
Table 2: Summary <strong>of</strong> the effect <strong>of</strong> the different dose levels <strong>of</strong> the chlor<strong>of</strong>orm extract <strong>of</strong> B. longipes<br />
and chloroquine on the different haematological parameters <strong>of</strong> Wister rats after 28 days <strong>of</strong><br />
treatment<br />
GROUP WBC LYMP MONO GRAN Hb RBC PLT<br />
High<br />
dose<br />
Median<br />
dose<br />
9.83±2.5<br />
1 a<br />
6.30±1.2<br />
3 a<br />
Low dose 8.937±2.<br />
30 a<br />
48.0±11.<br />
41 a<br />
57.10±7.<br />
30 a<br />
54.5±10.<br />
83 a<br />
42.53±15<br />
.54 a<br />
26.87±4.<br />
64 a<br />
14.33±8.<br />
24<br />
9.433±4.<br />
36 a<br />
15.97±3.<br />
32 a<br />
16.8±3.4<br />
1 a<br />
357<br />
9.913±0.48<br />
a<br />
7.437±1.47<br />
a<br />
9.49±0.20 a<br />
15.80±0.<br />
32 a<br />
13.87±1.<br />
13 a<br />
15.53±0.<br />
46 a<br />
494±62.0<br />
8 a<br />
559±225 a<br />
633±133.<br />
9 a<br />
Chloroqui 13.57±1. 67.5±6.7 19.43±7. 13±1.13 9.053±0.53 15.07±0. 339±76.9<br />
ne<br />
28<br />
73<br />
62<br />
5<br />
a<br />
P > 0.05 at 95% confidence level. Normal values for the Wister Albino rats are: White Blood Cells, (6.325±0.0029);<br />
Lymphocytes, (70.95±0.3175); Granulocytes, (3.15±0.259); Haemoglobin, (8.024±0.321); Red Blood Cells, (15.88±0.266);<br />
Monocytes, (11.64±1.381).<br />
The toxicity studies suggest that these plants maybe safe in humans if used at controlled doses. The<br />
plants can also be further investigated for identification <strong>of</strong> simple antiplasmodial active molecules<br />
that can be synthesized in the laboratory. However it should be noted that experience in malaria
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
control programmes has shown that in vitro tests <strong>of</strong> parasite susceptibility to antimalarial drugs<br />
cannot substitute for in vivo observations on malaria therapy, some compounds that show in vitro<br />
activity may not possess in vivo activity due to pharmacokinetic and immunological factors (Waako<br />
et al., 2005; WHO, 2001). Hence the need to carry out clinical trials on these active extracts.<br />
Acknowledgements<br />
My sincere gratitude to the Norwegian Council <strong>of</strong> Universities Committee for Development<br />
Research and Education (NUFU), Swedish International Development Agency (SIDA) through VICRES<br />
project <strong>of</strong> the inter-University Council for East Africa and The Uganda Joint Clinical Research Centre.<br />
References<br />
Lorke D. (1983). A new approach to acute toxicity testing. Arch. Toxicol. 54: 275-287.<br />
Makler, M. T., Ries, J. M., Williams, J. A., Bancr<strong>of</strong>t, J. E., Piper, R. C., Gibbins, B. L. & Hinrichs, D. J. (1993). Parasite lactate<br />
dehydrogenase as an assay for P. falciparum drg sensitivity. Am. J. Trop. Hyg. 48(6): 739-741.<br />
MoH- Uganda. (2003). Financial year 2003/2004. District transfers for health services. Ministry <strong>of</strong> health Government <strong>of</strong><br />
Uganda.<br />
MoH-Uganda. (2007). Burden <strong>of</strong> Malaria (Accessed29/5/2007) www:health .go.ug /malaria.htm.<br />
Waako, P. J., Gumede, B., Smith, P. & Folb, P. I. (2005).The in vitro and in vivo antimalarial activity <strong>of</strong> Cardiospermum<br />
halicacabum L. and Momordica foetida Schumch. Et Thonn. J Ethnopharmacol. 99(1): 137-43.<br />
WHO. (2001). The use <strong>of</strong> antimalarial drugs. Report <strong>of</strong> a WHO informal consultation, Geneva. WHO/CDS/RBM/2001.33<br />
358
A<br />
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Abdalla .................................................................................. 73<br />
Abdelgader ................................................................... 83, 113<br />
Abdulkadir .......................................................................... 163<br />
Abebayehu ......................................................................... 320<br />
Abegaz .................................................................... 1, 117, 238<br />
Abotsi .................................................................................. 329<br />
Adeel .................................................................................. 145<br />
Adhikari................................................................................. 73<br />
Adongo ............................................................................... 291<br />
Adzu .................................................................................... 350<br />
Ahmed N ............................................................................. 103<br />
Ajayi .................................................................................... 350<br />
Akala ........................................................... 106, 187, 194, 270<br />
Akol .................................................................................... 242<br />
Alao ..................................................................................... 194<br />
Ambassa ............................................................................. 117<br />
Amenu ................................................................................ 313<br />
Andersen .............................................................................. 81<br />
Andrae-Marobela ................................................................. 71<br />
Arachi .................................................................................... 86<br />
ARISOA ................................................................................ 121<br />
Arnold ................................................................................. 140<br />
Asfaw .......................................................................... 313, 320<br />
Ateka ................................................................................... 264<br />
B<br />
Bah ...................................................................................... 344<br />
Balogun ............................................................................... 350<br />
Baluku ................................................................................. 259<br />
Baraza ................................................................................. 140<br />
Belete .................................................................................... 48<br />
Bestina ................................................................................ 294<br />
Bett ..................................................................................... 234<br />
Bezabih ............................................................................... 238<br />
Birkett ................................................................................... 65<br />
Bojase ................................................................................. 231<br />
Bosire .................................................................................. 298<br />
Brendler .............................................................................. 109<br />
Bringmann .................................................................... 23, 110<br />
Brun .................................................................................... 209<br />
Bullous .................................................................................. 21<br />
Bunalema ............................................................................ 168<br />
Byamukama .......................................................................... 81<br />
Author index<br />
359<br />
C<br />
Cheplogoi ...................................................................... 79, 291<br />
Cheruiyot ............................................................................ 274<br />
Chhabra ............................................................................... 173<br />
Chibale .................................................................................. 27<br />
Chimponda ............................................................................ 39<br />
Chirisa ................................................................................... 39<br />
Chitemerere .......................................................................... 39<br />
Choudhary............................................................................. 73<br />
Choudhury ............................................................................ 21<br />
Coma ..................................................................................... 96<br />
Coombes ....................................................................... 92, 209<br />
Cosam ................................................................................. 140<br />
Crouch ............................................................................. 15, 92<br />
D<br />
Dagne .................................................................................... 48<br />
Dahse .................................................................................. 132<br />
Daniel .................................................................................... 86<br />
Deng ............................................................................ 165, 234<br />
Dénou ......................................................................... 342, 344<br />
Derese ................................................... 54, 106, 194, 205, 301<br />
Dharani ............................................................................... 151<br />
DIAKITE ................................................................................. 69<br />
Diallo ................................................................................... 344<br />
DIALLO .................................................................................. 69<br />
Diarra .................................................................................. 344<br />
Dossaji ......................................................................... 311, 333<br />
Dube ............................................................................... 61, 71<br />
E<br />
Elabdeen ............................................................................. 171<br />
Elamin ................................................................................. 171<br />
Elfahal ................................................................................. 113<br />
Elgawi .................................................................................. 157<br />
Elhardallou .......................................................................... 308<br />
ELhassan ............................................................................... 73<br />
Elhussein ..................................................................... 103, 113<br />
Elkhalifa ............................................................................... 308<br />
El<strong>of</strong>f....................................................................................... 12<br />
Elsammani ........................................................................... 157<br />
Eltohami .............................................................................. 157<br />
EL-YACHOUROUTUI ............................................................. 121<br />
Endale ................................................................................. 194<br />
Erasto .................................................................................. 282
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Erdelyi ........................................................... 31, 194, 205, 301<br />
Eyase ................................................................... 106, 187, 194<br />
Ezeonwumelu ..................................................................... 350<br />
F<br />
Fadwal ................................................................................ 171<br />
Feiter .................................................................................. 109<br />
Fischer ................................................................................ 145<br />
Fotso ................................................................................... 278<br />
Fouche ................................................................................ 326<br />
Fozing .................................................................................. 117<br />
G<br />
Gachanja ............................................................................. 220<br />
Gakuya ................................................................................ 216<br />
Gamal ................................................................................. 157<br />
Gathirwa ............................................................................. 183<br />
Gebreheiwot ...................................................................... 313<br />
Ghaife ................................................................................. 163<br />
Ghislain ................................................................................. 71<br />
Gitu ..................................................................................... 187<br />
Goji ..................................................................................... 350<br />
Grelier ................................................................................... 96<br />
Gumula ....................................................................... 205, 301<br />
Gurib-Fakim .......................................................................... 29<br />
H<br />
Haïdara ....................................................................... 342, 344<br />
Hassan ................................................................................ 157<br />
Hassanali .......................................................... 4, 88, 130, 226<br />
Hassani ............................................................................... 305<br />
Hayeshi ................................................................................. 52<br />
Heydenreich........................................................ 205, 213, 301<br />
Hooper .................................................................................. 65<br />
Hydenreich ......................................................................... 187<br />
I<br />
Ibrahim ............................................................................... 350<br />
Illias ..................................................................................... 213<br />
Imbuga ................................................................................ 187<br />
Induli ................................................................................... 106<br />
Ingabire ............................................................................... 198<br />
Ingonga ............................................................................... 183<br />
Innocent ............................................................. 130, 226, 294<br />
Iqbal .................................................................................... 117<br />
Irungu ................................................................. 106, 183, 191<br />
360<br />
Ishola ................................................................................... 270<br />
Islam .................................................................................... 316<br />
J<br />
Jangu ................................................................................... 168<br />
Jayakumar ............................................................................ 86<br />
JEANNODA .................................................................. 121, 337<br />
Jeniffer ................................................................................ 138<br />
Jondiko ............................................................................... 138<br />
Jones ..................................................................................... 21<br />
Jordheim ............................................................................... 81<br />
Joseph ......................................................................... 126, 132<br />
Juma .................................................................................... 145<br />
K<br />
Kabaru ......................................................................... 298, 311<br />
Kabera ................................................................................ 198<br />
Kaiser .................................................................................. 209<br />
Kakudidi .............................................................................. 168<br />
Kalombo ................................................................................ 52<br />
Kamaté ................................................................................ 344<br />
Kapche ................................................................................ 117<br />
Karangwa ............................................................................ 198<br />
Kareru ................................................................................. 216<br />
Kariuki ................................................................................. 311<br />
Kassaye ................................................................................. 48<br />
Katata .................................................................................... 52<br />
Katerere ................................................................................ 75<br />
Katuura ............................................................................... 356<br />
Keriko .................................................................................. 183<br />
Kerubo .................................................................................. 54<br />
Khalid ............................................................................ 73, 308<br />
Khan ...................................................................................... 65<br />
Kigondu ............................................................................... 183<br />
Kihdze ................................................................................. 163<br />
Kim ...................................................................................... 311<br />
Kimata ................................................................................. 298<br />
Kinyuy ................................................................................. 163<br />
Kiplagat ............................................................................... 350<br />
Kiplimo................................................................................ 316<br />
Kiremire ........................................................................ 81, 106<br />
Kirimuhuzya ........................................................................ 168<br />
Kirira .................................................................................... 183<br />
Kisinza ................................................................................. 130<br />
Kleinpeter ........................................................................... 213<br />
Kolesnikova ......................................................................... 326<br />
Koorbanally ................................................................. 286, 316<br />
Krause ................................................................................. 179<br />
kutima ................................................................................. 322<br />
Kutima ................................................................................. 216
L<br />
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Labuschagne ......................................................................... 52<br />
Lacaille-Dubois 2 .................................................................. 278<br />
Langat ..................................................................... 15, 77, 173<br />
Lange .................................................................................. 132<br />
Langer ................................................................................. 145<br />
Law ....................................................................................... 21<br />
Lemmen .............................................................................. 264<br />
Lemmer ................................................................................. 52<br />
Loro ..................................................................................... 350<br />
Lukhoba .............................................................................. 333<br />
Lumley .................................................................................. 21<br />
Lwande ............................................................................... 226<br />
M<br />
Machocho ........................................................... 105, 246, 355<br />
Machumi ....................................................................... 54, 213<br />
Magadula ............................................................................ 168<br />
Magesa ............................................................................... 130<br />
Maharaj .............................................................................. 326<br />
Maher ................................................................................... 71<br />
Makanga ............................................................................. 209<br />
Mammo .............................................................................. 238<br />
Mang uro ............................................................................ 270<br />
Mangoyi ................................................................................ 39<br />
Manguro ............................................................. 138, 253, 264<br />
Marciale.............................................................................. 282<br />
Marobela ............................................................................ 238<br />
Mathew .............................................................................. 138<br />
Mbabazi ................................................................................ 81<br />
Mbafor ................................................................................ 179<br />
Mbithi ................................................................................. 317<br />
Mbugua .............................................................................. 194<br />
Mbwambo .................................................................. 226, 294<br />
Melaku .................................................................................. 48<br />
Melariri ................................................................................. 52<br />
MENSAH ............................................................................... 19<br />
Mesaik .................................................................................. 73<br />
Meybeck ............................................................................... 35<br />
Midega .................................................................................. 65<br />
Midiwo ................................................................. 54, 106, 213<br />
Mihale ......................................................................... 165, 234<br />
Mihigo ................................................................................ 238<br />
Mitaine-Offer ...................................................................... 278<br />
Mohamed U.I. ..................................................................... 103<br />
Mohammed .......................................................................... 92<br />
Mokua ................................................................................ 226<br />
Möllman ............................................................................. 132<br />
Mortensen .......................................................................... 109<br />
Mpiana ................................................................................ 286<br />
Mudogo .............................................................................. 286<br />
361<br />
Mughisha ............................................................................ 234<br />
Mugisha .............................................................. 165, 268, 356<br />
Muhanji ............................................................................... 153<br />
Muhizi ................................................................................... 96<br />
Muithya .............................................................................. 355<br />
Muiva .................................................................................. 187<br />
Mukanganyama .................................................................... 39<br />
Mukayisenga ....................................................................... 198<br />
Mulholland ....................................................... 15, 77, 92, 209<br />
Munkombwe ......................................................................... 61<br />
Musa ................................................................................... 157<br />
Musau ................................................................................... 88<br />
Muthaura ............................................................................ 191<br />
N<br />
Nalumansi ........................................................................... 268<br />
NANDWA ............................................................................ 250<br />
Nawrot .................................................................................. 77<br />
Nbgolua ............................................................................... 286<br />
Ncube .................................................................................. 329<br />
Ndakala ............................................................................... 194<br />
Ndhlovu ................................................................................ 61<br />
Ndiege ................................................... 88, 183, 205, 246, 301<br />
Ndinteh ............................................................................... 179<br />
Ndlebe ................................................................................ 326<br />
Ng ang a .............................................................................. 220<br />
Nga ng a .............................................................................. 173<br />
Ngadjui ....................................................................... 117, 278<br />
Ngoupayo ............................................................................ 278<br />
Ngumba .............................................................................. 220<br />
Nindi ..................................................................................... 61<br />
Njagi ...................................................................................... 88<br />
Njiru ...................................................................................... 88<br />
Njoki ................................................................................... 322<br />
Njonge ................................................................................. 216<br />
Njue ...................................................................................... 79<br />
Nkunya ................................................................ 126, 132, 140<br />
Ntumy ................................................................................... 71<br />
Nuzillard ................................................................................ 15<br />
Nyagah ................................................................................ 216<br />
Nyamboli ............................................................................... 52<br />
Nyandoro ............................................................................ 126<br />
O<br />
Obbo ................................................................................... 209<br />
Obuotor .............................................................................. 346<br />
Occhiuto .............................................................................. 342<br />
Ochieng .............................................................................. 270<br />
Odak ................................................................................... 253<br />
Odalo .......................................................................... 126, 132
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Odhiambo ........................................................................... 333<br />
Ogendo ....................................................................... 165, 234<br />
Ogundaini ........................................................................... 346<br />
Ogur .................................................................................... 253<br />
Ogweng ................................................................................ 81<br />
OH ....................................................................................... 179<br />
Okalebo............................................................... 187, 205, 301<br />
Okello-Onen 1 ...................................................................... 242<br />
Okemo ................................................................................ 168<br />
Okeng.................................................................................. 356<br />
Okoth .................................................................................. 153<br />
Olaho-Mukani ..................................................................... 209<br />
Omole ................................................................................. 246<br />
Omolo ..................................................................... 79, 88, 291<br />
Onditi .................................................................................. 220<br />
Opiro ................................................................................... 242<br />
Opiyo .................................................................................. 264<br />
Orodho ................................................................................ 168<br />
Osman ........................................................................ 103, 113<br />
Otaye ............................................................................ 79, 291<br />
Otieno ......................................................................... 168, 282<br />
Owuor ................................................................... 57, 264, 270<br />
Oyewale .............................................................................. 350<br />
P<br />
Patrick ................................................................................. 165<br />
Paul ..................................................................................... 165<br />
Payne .................................................................................. 231<br />
Peter ................................................................................... 187<br />
Pickett .................................................................................. 65<br />
Pillainayagam ........................................................................ 21<br />
Porzel .................................................................................. 140<br />
R<br />
RAHARISOA ................................................................. 121, 337<br />
RAHERINIAINA .................................................................... 337<br />
Rahman ................................................................................. 73<br />
RAJEMIARIMOELISOA ......................................................... 337<br />
RAKOTO ...................................................................... 121, 337<br />
RAKOTONDRASOA .............................................................. 121<br />
RAMAMONJISON ................................................................ 337<br />
Randrianarivo ......................................................................... 7<br />
RANDRIANARIVO ........................................................ 121, 337<br />
RAONIHARISOA ................................................................... 121<br />
Raskin ................................................................................. 109<br />
Rasoanaivo ............................................................................. 7<br />
Razafimahefa .......................................................................... 7<br />
Rechab ................................................................................ 216<br />
Reinke ................................................................................. 145<br />
Rewerts ................................................................................. 75<br />
362<br />
Rono .................................................................................... 194<br />
Rubagumya ......................................................................... 329<br />
Rukunga ...................................................................... 183, 191<br />
Ruminski ............................................................................... 45<br />
S<br />
Salah ................................................................................... 171<br />
Sanogo ........................................................................ 342, 344<br />
SANOGO ................................................................................ 69<br />
Sattler ................................................................................. 132<br />
Schmidt ............................................................................... 140<br />
Sherburn ............................................................................. 231<br />
Shode .................................................................................. 286<br />
Simon .................................................................................. 109<br />
Skaar ..................................................................................... 81<br />
Spannenberg ...................................................................... 145<br />
Steenkamp .......................................................................... 326<br />
Steinert ............................................................................... 110<br />
Sunnerhagen ....................................................................... 194<br />
Swai ...................................................................................... 52<br />
T<br />
Tabopda .............................................................................. 278<br />
Tabuti .................................................................. 168, 268, 356<br />
Taiwo .................................................................................. 346<br />
Tanayen ...................................................................... 163, 350<br />
Tarus ................................................................................... 355<br />
Thomas ............................................................................... 138<br />
Togola ................................................................................. 344<br />
TOGOLA ................................................................................ 69<br />
Tommasi ............................................................................. 342<br />
Tonui ................................................................................... 183<br />
Torto ..................................................................................... 65<br />
Tshibangu ........................................................................... 286<br />
Twinomuhwezi .................................................................... 106<br />
V<br />
Viljoen ................................................................................. 144<br />
Villinger .............................................................................. 145<br />
W<br />
Waihenya ............................................................................ 322<br />
Waithaka ............................................................................. 216<br />
Walker ................................................................................. 213<br />
Walsh .................................................................................. 194<br />
Wanyama ............................................................................ 187
The 14 th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium Nairobi, 2011.<br />
Wanyonyi ............................................................................ 355<br />
Waters ................................................................................ 106<br />
Wessjohann ........................................................................ 140<br />
363<br />
Y<br />
Yagi ....................................................................................... 73<br />
Yardley ................................................................................ 157<br />
Yenesew ......................... 54, 106, 187, 194, 205, 213, 298, 301<br />
Yole ..................................................................................... 322<br />
Yousuf ................................................................................... 73