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UNIVERSITY OF KWAZULU-NATALTHE PHYTOCHEMISTRY AND BIOLOGICALACTIVITY OF SECONDARY METABOLITES FROMKENYAN VERNONIA AND VEPRIS SPECIES2012JOYCE JEPKORIR KIPLIMO

P a g e | iiTHE PHYTOCHEMISTRY AND BIOLOGICALACTIVITY OF SECONDARY METABOLITES FROMKENYAN VERNONIA AND VEPRIS SPECIESJOYCE JEPKORIR KIPLIMO2012A thesis submitted to the school of Chemistry, Faculty of Science and Agriculture,University of KwaZulu-Natal, Westville, for the degree of Doctor of Philosophy.This Thesis has been prepared according to Format 4 as outlined in the guidelines from theFaculty of Science and Agriculture which states:This is a thesis in which chapters are written as a set of discrete research papers, with anOverall Introduction and Final Discussion. Where one (or all) of the chapters has alreadybeen published. Typically these chapters will have been published in internationallyrecognized,peer- reviewed journals.As the candidate’s supervisor, I have approved this thesis for submission.Supervisor:Signed: --------------------------------Name: ------------------------- Date: --------------

P a g e | iiiABSTRACTThis work is an account of the phytochemical analysis of two genera, Vernonia and Vepriswhich are used as remedies for illness by the Kalenjin community of Kenya. Species ofVernonia are known to yield sesquiterpene lactones, which typify the genus whereas Veprisis rich in alkaloids and limonoids which have a wide range of biological activities. Thespecies studied in this work were Vernonia auriculifera, Vernonia urticifolia, Veprisglomerata and Vepris uguenensis.Phytochemical studies revealed a range of compounds being present in the four species.From Vernonia, triterpenoids, a sesquiterpene amine, a carotenoid and a polyene wereisolated. This was the first account of a sesquiterpene amine from a plant species and thefirst account of the novel polyene. The triterpenoids showed moderate antibacterial activity,with β-amyrin acetate and oleanolic acid being effective at decreasing adhesion of selectedgram-negative and gram-positive bacteria.Lutein and urticifolene showed goodantibacterial activity against Enterococcus feacium and Pseudomonas aeruginosa.In Vepris, a range of compounds were isolated, belonging to the furoquinoline alkaloids,coumarins, flavonoids, cinnamic acid derivatives, lignins, cinnamaldehydes, triterpenoidsand limonoids.Five new compounds; a cinnamaldehyde derivative (glomeral), twoflavonoids (veprisinol, uguenenprenol) and two A, D-seco-limonoids (uguenensene anduguenensone) were amongst the compounds isolated. Antibacterial studies showed thatglomeral inhibited the growth of Staphylococcus aureus and Shigella dysentrieae at lowconcentrations (MIC of 2 µg mL -1 and 0.4 µg mL -1 respectively). Antioxidant assays ofseveral compounds revealed that, veprisinol, isohaplopine-3,3’-dimethylallyl ether,uguenenprenol and 7-O-methylaromadenrin are good antioxidant agents. The limonoidsisolated from Vepris uguenensis also make up an interesting biogenetic relationship.

P a g e | ivStructural elucidation was carried out by 1D and 2D NMR spectroscopy in conjuction withmass spectrometry, infrared, ultraviolet and circular dichroism analysis where applicable.Biological assays were carried out using standard methods at laboratories in the Universityof KwaZulu-Natal and Kenya Medical Research Institute (KEMRI-Nairobi).

P a g e | vSUMMARY OF COMPOUNDS ISOLATED1) Compounds Isolated from Vernonia species29H 3 COCO32 3123232511191054624P1-130122687181314271920151716212228HO1225112612910 83 4 75623 2430 2919 20 211817 2213COOH14161527P1-230293029HO1225112612910 83 475623 2419 1920 2112181817 221125261313142811416291510 815273 47 27H 3COCO5623 24P1-3 P1-420 211722281630HO22313 452511910246812267292719181314P1-51520 2117162228O21211251910 83 47624233019271813141526P1-62920 2117162228

P a g e | vi302921112591012871927181314152620 2117162228NH2P1-93 46H 3COCO2324P1-724 1 24 2121 22181220111719 13162324252726291081415HO34567P1-845OH321171OH52 3 4 67891011121314151617186 1 6207 89 11 1319 1 0 12 1 41515'14'12 1013 '11' 9'19 'HOO31OH30 28P2-12624192122P2-2208'17'16' 1'2'7'6'3'OH5'4'18'2) Compounds Isolated from Vepris species5''4''2''3''OH1''8O 765OHOCH 35'6' OCH 354a 43a34'69 O1' 3'8a2OH2'H 3 CO 7 NO8 9a1034OO1'4'P3-1 P3-22'3'25'

P a g e | vii5'3'4'4' 5'3'2'O1'OCH 3HO2'OH1'OCH 3O654a43a32O654a43a32H 3 CO788aN9aOH 3 CO788aNOP3-3P3-45'H 3 CO6754a8aOCH 3ON8OCH 343a9a3P3-524'3'2'O1'HO879 3456OCH 3O12P4-1HOH34O2 719 OH8652 7OH136OH 45P4-2 P4-38O9OHOH3OH 42 7165P4-48O9OCH 3OCH 3HO678O9105 4OH OP4-52'1'23OH6'3'5'OCH 3H 3 COH 3 CO67584109 OP4-632OHOH 3 CO4325617OP4-7989'O7'1'8'2'6'5'4'3'OCH 3OCH 3OH

P a g e | viiiOOOOOOOOCOCH 31234567891011121314151617181920212223282930OOOOOOOO1234567891011121314151617181920212223282930P4-8P4-9O1234567891011121314151617181928 2930OHO21 22202324252627HOH 3COCOOO1234568910121113141618192021 23222425262728 2930HHOH7P5-1 P5-2OOOOCOCH 3 OCOCH 32134567891011121315161718192022212328 2930P5-3OOOOOOCOCH 31234567891011121314151617181920212223282930P5-4

P a g e | ixOOOOOOCOCH 31234567891011121314151617181920212223282930P5-5OOOOOOOH 3COOOH1 2 34567891011121314151617181920212223282930P5-6OOHOHOOH23456789101'2'5'6'OOH1''2''3''4''5''3'4'P5-7OOOHOHH 3COOH23456789101'2'3'4'5'6'P5-8NOOOOCH 3OCH 3 233a44a56788a9aP5-9OHOH123456789141011121315P5-10OOOOCOCH 321345678910111213151617181920212328 2930O23P5-11HO123456789101112131415161718192021232425 262728293022P5-12

P a g e | xABBREVIATIONSANOVAanalysis of variance1 H NMR proton nuclear magnetic resonance spectroscopy13 C NMR C-13 nuclear magnetic resonance spectroscopyCOSYDEPTDPPHEIMSFRAPHMBCHSQCHRMSMSNOESYRSAUVAcbrmccccorrelated spectroscopydistortionless enhancement by polarization transfer2,2-diphenyl-1-picrylhydrazylelectron-impact mass spectroscopyferric reducing antioxidant potentialheteronuclear multiple bond coherenceheteronuclear single quantum coherencehigh-resolution mass spectrometrymass spectrometrynuclear overhauser effect spectroscopyradical scavenging activityultravioletacetatebroad resonancemultipletconcentrationcolumn chromatography

P a g e | xidddHzMestIRMptlcdoubletdouble doubletHertzmethylsinglettripletinfraredmelting pointthin-layer chromatography

P a g e | xiiDECLARATIONSDECLARATION 1 - PLAGIARISMI, Joyce Jepkorir Kiplimo declare that1. The research reported in this thesis, except where otherwise indicated, is my originalresearch.2. This thesis has not been submitted for any degree or examination at any otheruniversity.3. This thesis does not contain other persons’ data, pictures, graphs or other information,unless specifically acknowledged as being sourced from other persons.4. This thesis does not contain other persons' writing, unless specifically acknowledgedas being sourced from other researchers. Where other written sources have beenquoted, then:a. Their words have been re-written but the general information attributed to themhas been referencedb. Where their exact words have been used, then their writing has been placed initalics and inside quotation marks, and referenced.5. This thesis does not contain text, graphics or tables copied and pasted from theInternet, unless specifically acknowledged, and the source being detailed in the thesisand in the References sections.Signed …………………………………………………………

P a g e | xiiiDECLARATION 2-PUBLICATIONSDETAILS OF CONTRIBUTION TO PUBLICATIONS that form part and/or includeresearch presented in this thesis (include publications in preparation, submitted, in press andpublished and give details of the contributions of each author to the experimental work andwriting of each publication)Publication 1Kiplimo, J. J., Chenia, H., Koorbanally N. A. 2011. Triterpenoids from Vernoniaauriculifera Hiern exhibit antimicrobial activity, African Journal of Pharmacy andPharmacology, 5 (8), 1150 – 1156.Publication 2Kiplimo, J. J., Everia, C. A. and Koorbanally, N. A. 2011. A Novel polyene from VernoniaUrticifolia (Asteraceae), Journal of Medicinal Plants Research, 5 (17), 4202-4211.Publication 3Kiplimo, J. J., Islam, Md. S., Koorbanally, N. A. Novel flavonoid and furoquinolinealkaloids from Vepris glomerata and their antioxidant activity, Natural ProductsCommunication accepted for publication on 4 th October, 2011.Publication 4Kiplimo, J. J. and Koorbanally, N. A. Antibacterial activity of an epoxidised prenylatedcinnamaldehdye derivative from Vepris glomerata, manuscript submitted to PhytochemistryLetters.Publication 5Kiplimo, J. J., Islam, Md. S., Koorbanally, N. A. Ring A, D-seco Limonoids and Flavonoidfrom the Kenyan Vepris uguenensis Engl. and their antioxidant activity, manuscriptsubmitted to Journal of Natural Products in November 2011.

P a g e | xivFrom all the above publications, my role included carrying out all the experimental workand writing of the publications. The co-authors contribution was that of an editorial natureand checking on the scientific content and my correct interpretation.Signed: …………………………………..

P a g e | xvACKNOWLEDGEMENTSI would like to thank my supervisor, Dr Neil Anthony Koorbanally. Thank for your supportand encouragement throughout my three years of study. Your passion and humility hasalways encouraged me to give my best to my studies. These three years in your dynamicNatural Products Research Group (NPRG) have afforded me with a wealth of learningexperience and I want to thank you for your input into my academic career.My thanks also go to Dr Shahidul Islam for guiding me through the antioxidant assays andfor helping with statistical analyses of my data. Dr Hafizah Chenia is also acknowledgedfor guiding me through the antibacterial and antibiofilm assays.I would also like to thank my colleagues in the Natural Products Group Dr Nizam Shaik, DrHabila, Adele, Aliyu, Amaya, Asif, Damien, Dorothy, Edith, Erick, Hamisu, Kaalin,Shiksha, Sunayna, Thrineshen and Victoria for the good working relationship in the lab.My special thanks goes to my friend Mrs Roshila Moodley who has been very close to mehere in UKZN.I am grateful to Mr Dilip Jagjivan for guiding me in NMR analyses, Mathew Sibonelo forHREIMS analysis, Neal Broomhead and Anita Naidoo for GC-MS analysis and Dr Brand ofthe University of the Stellenbosch for CD analyses. My thanks also go out to all theacademic and technical staff in the School of Chemistry.My special thanks goes to the Kenyan community here at the University of KwaZulu-Natal,especially ‘Nduguzangu’ my brothers in the Analytical and Catalysis lab. They have beenmy family here in Durban ‘Asante Sana’.To my Patient Husband Mr Philip Bett I would like to say “you are the Best” you have keptme going. Thank you for your support, understanding and encouragement during my study

P a g e | xviiTABLE OF CONTENTSABSTRACT .............................................................................................................. iiiSUMMARY OF COMPOUNDS ISOLATED .......................................................... vABBREVIATIONS ................................................................................................... xDECLARATIONS................................................................................................... xiiACKNOWLEDGEMENTS .................................................................................... xvTABLE OF CONTENTS ...................................................................................... xviiCHAPTER ONE ....................................................................................................... 1INTRODUCTION ..................................................................................................... 11.1 Introduction to the genus Vernonia ........................................................................ 11.1.1 Phylogeny .......................................................................................................... 11.1.2 Ethnobotanical information of Vernonia species ................................................. 21.1.3 Biological activity of extracts from Vernonia species ......................................... 51.1.4 A phytochemical review of the triterpenoids from Vernonia species ................... 81.1.4.1 A Brief Introduction to Triterpenoids .............................................................. 91.1.4.2 Tetracyclic triterpenoids ................................................................................ 141.1.4.3 Lupane triterpenoids ...................................................................................... 151.1.4.4 Oleanane triterpenoids ................................................................................... 161.1.4.5 Taraxarane and Ursane triterpenoids .............................................................. 171.1.4.6 Friedoursane triterpenoids ............................................................................. 201.1.4.7 The Friedelane triterpernoids ......................................................................... 201.2 Introduction to the genus Vepris .......................................................................... 221.2.1 Phylogeny ........................................................................................................ 221.2.2. Ethnobotanical use of Vepris species ............................................................... 231.2.3. Biological activity of extracts from Vepris species .......................................... 25

P a g e | xviii1.2.4. A phytochemical review of Vepris species....................................................... 261.2.4.1. Quinoline alkaloids ...................................................................................... 271.2.4.2. Acridone alkaloids........................................................................................ 351.2.4.3. Limonoids .................................................................................................... 371.2.4.5. Cinnamic acid derivatives and simple aromatics ........................................... 421.2.4.6. Flavonoids .................................................................................................... 441.2.4.7. Miscellaneous compounds ............................................................................ 451.3 Aim of the study ................................................................................................. 461.3.1 Objectives ........................................................................................................ 47References ................................................................................................................ 48CHAPTER TWO .................................................................................................... 66TRITERPENOIDS FROM VERNONIA AURICULIFERA HIERN EXHIBITANTIMICROBIAL ACTIVITY ............................................................................. 66ABSTRACT ............................................................................................................. 66INTRODUCTION .................................................................................................... 67MATERIALS AND METHODS .............................................................................. 68RESULTS AND DISCUSSION ................................................................................ 73CONCLUSION ........................................................................................................ 79REFERENCES ......................................................................................................... 81CHAPTER THREE ................................................................................................ 85A NOVEL POLYENE FROM VERNONIA URTICIFOLIA (ASTERACEAE) ... 85ABSTRACT ............................................................................................................. 85INTRODUCTION .................................................................................................... 85MATERIALS AND METHODS .............................................................................. 86RESULTS AND DISCUSSION ................................................................................ 88

P a g e | xixCONCLUSION ........................................................................................................ 94References ................................................................................................................ 95CHAPTER FOUR ................................................................................................... 97A NOVEL FLAVONOID AND FUROQUINOLINE ALKALOIDS FROMVEPRIS GLOMERATA AND THEIR ANTIOXIDANT ACTIVITY ................... 97Abstract .................................................................................................................... 97Introduction .............................................................................................................. 97Results and Discussion ............................................................................................. 99Experimental .......................................................................................................... 104References .............................................................................................................. 107CHAPTER FIVE ................................................................................................... 111ANTIBACTERIAL ACTIVITY OF AN EPOXIDISED PRENYLATEDCINNAMALDEHDYE DERIVATIVE FROM VEPRIS GLOMERATA ............ 111Abstract .................................................................................................................. 111Introduction ............................................................................................................ 112Results and discussion ............................................................................................ 113Experimental .......................................................................................................... 117Conclusion .............................................................................................................. 121References .............................................................................................................. 123CHAPTER SIX ..................................................................................................... 127RING A, D-SECO LIMONOIDS AND FLAVONOID FROM THE KENYANVEPRIS UGUENENSIS ENGL. AND THEIR ANTIOXIDANT ACTIVITY .... 127Abstract .................................................................................................................. 127Introduction ............................................................................................................ 128Results and Discussion ........................................................................................... 130

P a g e | xxExperimental Section .............................................................................................. 142Associated Content ................................................................................................. 148References .............................................................................................................. 149CHAPTER SEVEN ............................................................................................... 152SUMMARY AND CONCLUSION ....................................................................... 1527.1 Summary .......................................................................................................... 1527.1.1 Recommendations for Vernonia auriculifera .................................................. 1527.1.2 Recommendations for Vernonia urticifolia ..................................................... 1537.1.3 Recommendations for Vepris glomerata ......................................................... 1547.1.4 Recommendations for Vepris uguenensis ....................................................... 1567.2 Conclusion ........................................................................................................ 157SUPPORTING INFORMATION ......................................................................... 158

P a g e | 1CHAPTER ONEINTRODUCTION1.1 Introduction to the genus Vernonia1.1.1 PhylogenyVernonia is one of the largest genera of flowering plants in the Asteraceae family, whichincludes more than 1500 species distributed widely in the tropical and sub-tropical region ofAfrica, Asia and America. It has two major centres of origin, South America and tropicalAfrica, with approximately five hundred species found in Africa and Asia, three hundred inMexico, Central and South America and sixteen in the USA. Of the five-hundred speciesfound in Africa, thirty are endemic to Kenya (Beentje, 1994; Oketch-Rabah et al., 1997).It has been difficult to establish relationships within the Vernonia species due to theiroverlapping characteristics.While the original family members (Barnadesioideae) arefound in South America, the tribe Vernonieae is believed to have originated in Madagascarand its sister tribe, the Liabeae is found in America. This difficulty in classifying thespecies gave rise to the nickname, the ‘evil tribe’ (Funk et al., 2005). As such there hasbeen no phylogeny proposed for the tribe Vernonieae and only a few relationships had beensuggested even among the best known species (Jones, 1977). The tribe Vernonieae has beentraditionally placed in the subfamily Cichorioideae (Funk et al., 2005). In a recent study itwas found that the species in America were derived from those of Europe, Asia and Africa(Keeley et al., 2007).The leaves of Vernonia are bright green, with a serrated edge and a light yellow central vein.They are alternate with each other along the stems, getting smaller towards the tip. The

P a g e | 2stems are upright and stiff and branch sparingly to about 1.2 m high. The flowers are lightto dark purple and are grouped in dense clusters at the tip of the stems. The seeds are verylight in weight and hairy (Olorode, 1984).The majority of these plants are used asornaments and vegetables, while others are considered as weeds in agriculture.Thevegetables have a bitter taste, hence the name “the bitter genus Vernonia” (Aliyu et al.,2011).1.1.2 Ethnobotanical information of Vernonia speciesAn ethnomedical survey of Vernonia species (Table 1) revealed widespread and diversemedical usage. Generally these species are used in the treatment of infectious and parasiticdiseases. The infectious diseases ranged from those affecting the skin to those of thestomach (gastrointestinal infections).Other major applications include treatment ofbacterial infections, gynaecological diseases and complications, respiratory diseases,diabetes, urinary tract infections and venereal diseases. Parasitic diseases include malaria,worm infection, amoebiasis and schistomiasis. Some species are also used as antivenomagainst snakebites and insect bites.Of the Vernonia species, V. amygdalina appears to be the most widely used. The leaves arethe most commonly used plant part and is prepared as a decoction. In some cases adecoction of the whole plant is prepared. The method of preparation depends on theapplication, for example, a mixture of the root and leaf decoction is drunk to treatgastrointestinal diseases and as an antipyretic and the leaves soaked in alcohol is drunk totreat diabetes mellitus. The traditional medicinal applications of members of the genus areillustrated in Table 1.

P a g e | 3Table 1 Species of Vernonia used in traditional medicinePlant species Plant Traditional useReferencespartV. ambigua Whole antimalarial Builders et al., 2011plantV. amygdalina leaves antimalarial, antidiabetic,antipyretic, gastrointestinaldiseases, appetite stimulant,dermatological infections,Akah and Okafor 1992;Akah and Ekekwe 1995;Yeap et al., 2010;Huffman et al., 1993anthelmintic, respiratorytract infections,gynaecological diseases andcomplications, infertility,antibacterial, antifungal andantivenom (snake bite)stems anti-HIV, antiviral and antiamoebicYeap et al., 2010leavesandfruitsantimalarial, anthelmintic,antibacterial and antiviralYeap et al., 2010;Erasto et al., 2006roots venereal diseases andgynaecologicalcomplicationsV. anthelmintica seeds anthelmintic, respiratorydiseases, gastrointestinaldiseases and complications,diuretic, anti-inflammatory,kidney protection and antiulcerV. branchycalyx roots gastrointestinalV. cinerea leavesandrootscomplicationanthelmintic, astringent,conjuctivitis, dermatologicaldiseases, diuretic,antipyretic,gastrointestinal diseases,gynaecological diseases,respiratory diseases, antidoteand urinary tract diseasesV. colorata leaves dermatological infections,respiratory diseases,antidiabetic, gastrointestinaldiseases, antipyretic,antiviral (hepatitis) andvenereal diseasesV. condensata leaves analgesic, anti-ulcer, antidiarrhoea,gastrointestinalYeap et al., 2010;Geissler et al., 2002Otari et al., 2010Oketch-Rabah et al., 1997Deborah et al., 1992;Khare, 2007;Marita et al., 1999;Misra et al., 1993Cioffi et al., 2004;Rabe et al., 2002;Sy et al., 2004Frutuoso et al., 1994;Pereira et al., 1994

P a g e | 4diseases, liver protectionand antivenom (snakebites)V. conferta bark anti-diarrhoea (bloody) Aliyu et al., 2011V. cumingiana ns antirheumatic arthritis,antiviral, bone and muscularinjury,respiratory diseases,antimalarial, and dentaldiseasesLin et al., 1985;Zhonghuabencao, 1999V. ferruginea ns anti-inflammatory remedies Malafronte et al., 2009V. galamensis leaves antidiabetic (mellitus) and Chhabra et al., 1989gastrointestinal diseasesV. guineensis ns antidote to poison,aphrodisiac, jaundice, antimalarial and prostatitisTchinda et al., 2002V. jugalis bark anti-diarrhoea (bloody) Aliyu et al., 2011V. kotschyana roots abdominal pains, respiratorydiseases including TB,antibacterial, dermatologicalinfections, gastrointestinaldisorder, analgesic,antiparasitic, antiprotozoaand anti-ulcersSanogo et al., 1998;Nergard et al., 2004V. mapirensis ns anti-inflammatory Morales-Escobar et al., 2007V. nigritiana root diuretic, gastrointestinalinfections, emetic,antipyretic and anthelminticAliyu et al., 2011V. nudicaulis whole venereal diseases Aliyu et al., 2011plantV. pachyclada ns dermatological injury Williams et al., 2005V. paltula wholeplantantimalarial, antirheumaticand gastrointestinalChiu and Chang, 1987leavesrootsflowersseedsinfectionsantiamoebic, anthelmintic,antiviral, respiratory tractinfection, gastrointestinalinfections and dermatitisgastrointestinal infections,respiratory tract infectionand coliceye problem, antipyreticand antirheumaticanthelmintic,gastrointestinal disorder,urinary tract infectionand dermatologicalinfectionV. polytricholepis ns antipyretic and respiratoryinfectionsGani, 1998Gani, 1998Mollik et al., 2010Gani, 1998Gani, 1998Aliyu et al., 2011

P a g e | 5V. potamophila leaves anticancer and skinBabady-bila et al., 2003infectionV. saligna ns respiratory tract infections Huang et al., 2003and gynaecologicalcomplicationsV. scorpioides leaves dermatological infection(diabetic lesions) and antiulcerPagno et al., 2006;Buskuhl et al., 2010V. trichoclada ns anti-inflammatory Morales-Escobar et al., 2007V. tweediana ns respiratory tract diseases Zanon et al., 2008Key: ns-not specified1.1.3 Biological activity of extracts from Vernonia speciesBiological activities of Vernonia extracts have been extensively studied as far back as 1969.The variety of secondary metabolites extracted from Vernonia species, explains the diversityof their biological activities. Among the diverse biological activities, antibacterial studiesare the most reported. Antibacterial activity was found to be common to all species in allextracts followed by conditions associated with pain, fever and inflammation andantiparasitic activity, including malaria, schistomiasis and leishmania. Antibacterialcompounds are mainly lipophilic and will partition from an aqueous phase into bacterialmembrane structures, causing expansion of the membranes, increased fluidity, disorderingof the membrane structure and inhibition of membrane embedded enzymes (Sikkema et al.,1995). Antifungal activity is also reported in five of the Vernonia species, while antiviral isnot commonly reported in the Vernonia with only one report in the ethanol extract of thefruit of V. amagdalina.Seven species have been associated with being active in assays against pain, fever orinflammation, which includes conditions such as arthritis and gastritis. In rural areas whereaccess to healthcare is not readily available, people rely on these extracts for common painrelief, inflammation and fever. Four of the nineteen species have also been reported to havesome form of antiparasitic activity, either antiplasmodial, antileishmanial, antischistomatic,

P a g e | 6anti-amoebic or antihelmintic. These are mainly reported for the polar extracts such as theaqueous and methanol extracts with few reports being in the chloroform or hexane extract.Beside the antibacterial, antiparasitic and conditions associated with pain, inflammation andfever, extracts of these plants have also shown other activities such as antidiabetic,antioxidant, anticancer, antiulcer, immunomodulatory, pesticidal and insecticidal activity.The most extensively studied plant among all the Vernonia species is V. amygdalina,reported to possess several pharmacological activities such as antidiabetic, antibacterial,antimalarial, antifungal, antioxidant, liver protection and cytotoxic effects (Table 2). Thebiological activities of Vernonia species that have been studied and documented are listed intable 2.Table 2: Biological activities of extracts from VernoniaPlant species Biological activity Extract Reference (s)V. ambigua antibacterial ethanol a and chloroform a Aliyu et al., 2011antiparasitic water wpBuilders et al.,2011(antiplasmodial)and antioxidantV. amagdalina antibacterial water r and ethanolic l Ogbulie et al., 2007bactericidal (oral cold water s, b, p Rotimi and Mosadomi, 1987bacterial)antifungal water sb,r and methanol sb,r Nduagu et al., 2008antiviral ethanol fr Vlietinck et al., 1995antiparasitic methanol l and water l Moundipa et al., 2005(antiamoebic)(antileishamanial) chloroform l and ethanol l Carvalho and Ferreira 2001Adedapo et al., 2007(antischistomiasis) petroleum ether land ethanolic l(anthelmintic) water l,s,r Adedapo et al., 2007(antiplasmodial) ethanolic l,r Abosi and Raseroka, 2003analgesic and water l and ethanol r Tekobo et al., 2002antipyreticanti-inflammatory water l Iroanya et al., 2010antioxidant ethanol r and methanol ns Yeap et al., 2010

P a g e | 7cytotoxic cold water wp Vlietinck et al., 1995anticancer cold water l Izevbigie et al., 2003antidiabetic water wp and isopropanol wp Erasto et al., 2006Taiwo et al., 2009liver protective methanol l and water l Adesanoye and Farombi, 2009pesticidal andinsecticidalmethanol l Ohigashi et al., 1991V. anthelmintica antibacterial water ns -ethanolic ns Parekh and Chanda 2008anti-arthritic ethanolic sd Otari et al., 2010anti-inflammatory water ns -ethanolic ns Parekh and Chanda 2008antidiabetic and EtOAc:isopropanol (1:1) sd Fatima et al., 2010antihyperglycemicV. blumeoides antibacterial ethanol a and chloroform a Aliyu et al., 2011V. branchycalyx antiparasitic CHCl 3 :EtOAc(1:1) l Oketch-Rabah et al., 1998(antileishmanial)(antiplasmodial)(antimalarial) water l Oketch-Rabah et al., 1997V. brasiliana (antiplasmodial) hexane l Alves et al., 1997V. cinerea antibacterial benzene l Gupta et al., 2003a;Latha et al., 2009antifungal methanol a Latha et al., 2009antioxidant methanol a Kumar et al., 2009;Latha et al., 2009anti-inflammatory alcoholic fl Latha et al., 1998antipyretic methanol wp Gupta et al., 2003banalgesic and chloroform l , methanol l Mazumder et al., 2003antipyretic and ether limmunomodulatory methanolic nsPratheeshkumar andKuttan, 2010V. colorota antibacterial,antiparasitic(antiplasmodial),antidiabetic andanti-inflammatoryCHCl 3 -MeOH (9:1) l Chukwujekwu et al., 2009;Cioffi et al., 2004V. condensata analgesic,antigastritis,antiulcer andantinociceptiveantidiabetic aqueous l Sy et al., 2004acetone-EtOH-EtOAc l Frutuoso et al., 1994;Sanogo et al., 1996;Risso et al., 2010V. glabra antifungal ns Gundidza, 1986V. hymenolepis tumor inhibitory alcoholic l Kupchan et al., 1969activity

P a g e | 8V. karaguensis antibacterial andantileukaemicNsMungarulire,1993;Taiwo et al., 1999V. kotchyana antibacterial,analgesic,antigastritis andaqueous/n-butanol rextract and acidic r Frutuoso et al., 1994;Germano et al., 1996;Sanogo et al., 1996, 1998antiulcerV. oocephala antibacterial ethanol/chloroform a Aliyu et al., 2011V. paltula antibacterial, ethanolic wp, fl, fr, t, l Chiu and Chang 1987antifungal,anti-inflammatoryand antipyreticV. polyanthes antiulcerogenic methanolic a andBarbastefano et al., 2007chloroform aV. pogosperma antibacterial Ns Tripathi et al., 1981V. scorpioides cytotoxic effects dichloromethane l Pagno et al., 2006(anti-tumor)bactericidal andfungicidalchloroform l and hexane l Buskuhl et al., 2010;Freire et al., 1996V. thomsoniana antibacterial andantileukaemicNs Mungarulire, 1993;Taiwo et al., 1999Key: superscripts, a = aerial parts, fr = fruits, fl = flowers l = leaves, ns = not specified,r = roots, s = stems, sb = stem bark, sd = seeds, wp = whole plant1.1.4 A phytochemical review of the triterpenoids from Vernonia speciesPhytochemical compounds previously isolated in Vernonia include sesquiterpene lactones,triterpenoids, flavonoids, coumarins, steroidal glycosides and carotenoids.Althoughsesquiterpene lactones are the main chemotaxonomic markers of this genus, none wereisolated in this work.To date, there are approximately twenty-six publications ontriterpenoids in Vernonia species (Scifinder, 2009). The isolation of tritepenoids from thisgenus is recorded to have begun as early as 1979, where the tetracyclic triterpenoid,fasciculatol was isolated from V. fasciculata.This remains to be the only tetracyclictriterpenoid isolated from Vernonia and all other phytochemical studies have reportedpentacyclic triterpenoids. In this study, triterpenoids were the main compounds isolatedfrom the Vernonia species investigated and as a result the literature review which followsfocuses on the triterpenoids in Vernonia.

P a g e | 91.1.4.1 A Brief Introduction to TriterpenoidsTriterpenoids are compounds based on a 30-carbon skeleton consisting of five sixmemberedrings or four six-membered rings and a five-membered ring. They are a widespreadgroup of natural terpenoids and are found in most plants. In Vernonia, seven classesof triterpenoids have been isolated and reported, of which five are closely related and possesfive six-membered rings in their basic skeleton (oleanane, ursane, taraxarene, friedelane andfriedoursane), the other two classes being lupane (four six membered rings and a fivememberedring) and a tetracyclic triterpenoid.Triterpenoids are produced by rearrangement of squalene epoxide (10) which is believed tohave been biosynthesized by the mevalonate pathway, but recently the route via thedeoxyxylulose phosphate pathway (DXP (4)) is reported to be more widely accepted(Dewick, 2006). The main precursor of terpenoids is the isoprene unit, which is formed viaisopentyl diphosphate (IPP) (1) and dimethylallyl diphosphate (DMAPP) (2), the latterbeing formed from IPP (1) with the isomerase enzyme (Scheme 1).O5HH13Isomerase2OPP4 OPPH R H SIPP (1) DMAPP (2)Scheme 1: Isomerisation of IPP to DMAPP (Dewick, 2006)These units may be derived from mevalonic acid (MVA) (3) or deoxyxylulose phosphate(DXP) (4).OHOHHOOCOHOP3O4OH

P a g e | 10In plants, the enzymes from the mevalonate pathway are found in the cytosol, thereforetriterpenoids and steroids (cytosolic products) are thought to be formed through thispathway, whereas in the deoxyxylulose phosphate (4), the enzymes are found mainly in thechloroplasts where the other terpenoids are derived (Dewick, 2006). IPP (1) isomerises toDMAPP (2) via the isomerase enzyme which stereospecifically removes the pro-R protonfrom C-2 and incorporates a proton from water onto C-4. Although the isomerisationreaction of IPP (1) to DMAPP (2) is reversible, the equilibrium favours the formation ofDMAPP (2) (Scheme 1).When the phosphate (a good leaving group) of DMAPP (2) leaves, it yields an allyliccarbocation that is stabilised by charge delocalisation, therefore making it electrophilic. IPP(1) on the other hand is a strong nucleophile due to the terminal double bond. This differingreactivity of DMAPP (2) and IPP (1) forms the basis of terpenoid biosynthesis in the linkageof isoprene units in a head-to-tail manner (Dewick, 2006; Zulak and Bohlmann, 2010).The combination of DMAPP (2) and IPP (1) through prenyl transferase mediation, results inthe formation of geranyl diphosphate (GPP) (5), the fundamental precursor formonoterpenoid (C 10 ) compounds. This involves ionisation of DMAPP (2) to the allyliccation to which the double bond of IPP (1) adds, followed by the stereochemical loss of thepro-R proton. GPP (5) possesses the reactive allyl diphosphate group and the reactionmediated by prenyl tranferase continues by addition of an IPP (1) unit to the geranyl cationwhich results in the formation of farnesyl diphosphate (FPP) (6), the sesquiterpenoid (C 15 )and geranyl geranyl diphosphate (GGPP) (7), the diterpenoid precursor (Scheme 2).

P a g e | 11Triterpenoids are formed by joining two FPP (6) units in a tail-to-tail manner to yield thehydrocarbon squalene (9), formed from the precursor presqualene diphosphate (8) by areaction that follows a different path from the previous condensation reactions. It wasdifficult to formulate the mechanism for the formation of squalene (9) however it wasresolved when presqualene diphosphate (8) was isolated from rat liver (Dewick, 2006). Theformation of presqualene diphosphate (8) (Scheme 3) is by an attack of the ∆ 2 double bondof one FPP (6) molecule on the farnesyl cation (formed by enzymatic ionisation of thesecond FPP (6) molecule). The resulting tertiary cation is discharged by loss of a protonwith the formation of a cyclopropane ring to yield presqualene diphosphate (8). This isfollowed by loss of diphosphate to form an unstable primary cation that undergoesrearrangement to generate a more favourable secondary carbocation and less strainedcyclobutane ring.Bond cleavage produces an allylic cation, stabilised by chargedelocalisation, which is quenched by attack of a hydride ion from NADPH to form squalene(9), an important precursor of cyclic triterpenoids (Scheme 3).

P a g e | 12prenyltransferaseOPPDMAPP (2)H RH SOPPOPPH R H SOPPstereospecificloss of protonMonoterpenoids (C 10 )GPP (5)IPP (1)Sesquiterpenoids (C 15 )Triterpenoids (C 30 )FPP (6)FPP (6)IPP (1)OPPDiterpenoids (C 20 )GGPP (7)OPPScheme 2: Formation of triterpenoid precursors GPP, FPP and GGPP (Dewick, 2006,Zulak and Bohlmann, 2010).

P a g e | 13FPP (6)SqualenesynthasePPO23FPP (6)allylic cationelectrophilic additiongiving tertiary cationHHOPPHloss of proton with formationof cyclopropane ringOPPHHPresqualene diphosphate (8)HW-M 1,3-alkylshiftbond cleavage yields alkeneand stable allylic cationHNADPHHcation quenchedby attack of hydrideHSqualene (9)Scheme 3: Formation of squalene (Dewick, 2006)Cyclisation of squalene (9) is possible through the intermediate squalene-2,3-oxide (10)which is produced in a reaction catalysed by a flavoprotein in the presence of O 2 andNADPH cofactors, resulting in the epoxidation of the terminal double bond in squalene(10).If squalene-2,3-oxide (10) is folded onto a chair-chair-chair-boat conformation on theenzyme surface, the transient dammarenyl cation (11) is produced through a series ofcyclisations followed by a sequence of Wagner-Meerwein rearrangements of 1,2-hydride

P a g e | 14and 1,2-methyl migrations to form the C-20 epimers, euphol (12) and tirucallol (13)(Scheme 4) (Dewick, 2006).HOH 2 Ochair-chair-chair-boatsqualene oxide (10)OHHHHHHOHDammarenyl cation (11)HODammarenediols-H + 20HLimonoids& tetracyclictriterpenoidsHOEuphol H-20β (12)Tirucallol H-20α (13)Scheme 4: Formation of limonoid and triterpenoid precursors (Dewick, 2006)1.1.4.2 Tetracyclic triterpenoids1213OHO 14Fasciculatol (14) is the only reported tetracyclic triterpenoid from Vernonia being isolated inV. fasciculata and is formed by the oxidation and then cyclisation of the side chain of

P a g e | 15euphol (12)/tirucallol (13). Reduction at position 12 leads to the formation of a double bondwith the concomitant loss of the methyl group at position 13.1.1.4.3 Lupane triterpenoidsThe lupane triterpenoids are characterised by four six-membered rings with a fifth fivememberedring to which an isopropyl group is attached. Five lupane triterpenoids havebeen isolated from Vernonia species, all with the same basic skeleton which is formed by a1,2-alkyl shift in the dammarenyl cation (11) resulting in the bacharenyl cation (15)followed by cyclisation onto ring D leading to a lupenyl cation (16) which ultimately leadsto lupeol (17) through the loss of a proton (Scheme 5). All Vernonia species except for V.cinerea, V. fasciciulata and V. saligna were found to contain lupeol (17). V. cinerea and V.saligna although not containing lupeol did contain derivatives of lupeol, lupenyl acetate (18)in V. cinerea and lupeol palmitate (19), a glycoside (20) and an acetate (21) in V. saligna.R23231430122511 26910 85 762417-2029202119H18 2213 17H 281416152723AcO23142511910562430122687292019H1821212213 172814161527R17 lupeol OH18 lupenyl acetate OAc19 lupenyl palmitate OCO(CH 2 ) 14 CH 320 lupenyl-20(29)en-3β-O-D-glucoside O-Glu21 18,19-dehydrolupenyl acetate OAc

P a g e | 16HH1,2-alkyl shiftfive membered ringformation gives atertiary cationHOHDammarenyl cation (11)HOHbaccharenyl cation (15)HOHlupenyl cation (16)-HHOHlupeol (17)Scheme 5: Formation of lupeol (Dewick, 2006)1.1.4.4 Oleanane triterpenoidsAnother widely distributed triterpenoid in Vernonia is the oleanane triterpenoid, β-amyrin(23), being found in all Vernonia species except V. arkansana, V. chunii, V. fasciculata andV. potamophilia, however V. chunii did contain oleanolic acid (27). V. mollissima and V.patula contained β-amyrin acetate (24), while V. saligna and V. patula contained β-amyrinpalmitate (25) and β-amyrin benzoate (26) respectively.The oleanane triterpenoids are characterised by five six-membered rings with a double bondat ∆ 12 and two methyl groups situated on the same carbon at position 20. They are formedby ring expansion in the lupenyl cation (16) by a Wagner-Meerwein rearrangement (a 1,2-alkyl shift) leading to the formation of the oleanyl cation (22) (Scheme 6), which is thendischarged by hydride migration and loss of a proton to form β-amyrin (23). Acetylation,aliphatic esterification and aromatic esterification has led to the other three β-amyrin

P a g e | 17derivatives (24-26). Oleanolic acid (27) is formed through the oxidation of the methylgroup at C-17.R 1 R 223 β-amyrin OH CH 324 β-amyrin acetate OAc CH 325 β-amyrin palmitate OCO(CH 2 ) 14 CH 3 CH 326 β-amyrin benzoate OCOPh CH 327 oleanolic acid OH COOH30 2919 20 2112181117 222526 131R142162910 8153 47 27R 51 623 243029HHHHOH1,2-alkylshiftHOLupenyl cation (18) oleanyl cation (22)HH1,2-hydride shiftR22313 4525119102468122672718131419 20 211517222816R = OH β−amyrin (23)R = OAc β−amyrin acetate (24)Scheme 6: Formation of oleanyl cation precursor (Dewick, 2006).1.1.4.5 Taraxarane and Ursane triterpenoidsThe difference between the oleanane triterpenoids and the taraxarane and ursanetriterpenoids lies in the position of the methyl groups on ring E. In the oleananes there aretwo methyl groups at C-20 whereas in the taraxaranes and the ursanes, a methyl groupmigrates from the oleanyl cation (22), resulting in the methyl groups being on adjacentcarbon atoms, C-19 and C-20.The ursane, α-amyrin (29) is the third most commontriterpenoid apart from lupeol (17) and β-amyrin (23), being found in ten of the sixteenVernonia species studied phytochemically. The three compounds, lupeol (17), β-amyrin(23) and α-amyrin (29) form a suite of compounds found in nine of the sixteen species ofVernonia and may be used as a chemotaxonomic marker for the genus.

P a g e | 18In addition, α-amyrin acetate (30) is found in V. patula and together with α-amyrin (29) inV. saligna. Taraxasterol (32) is found together with α-amyrin (29) in V. incana and V.cognata, while V. chalybaea contains α-amyrin (29), pseudotaraxasteryl acetate (31) andtaraxasteryl acetate (33) and V. cinerea contains α-amyrin (29), α-amyrin acetate (30), 24-hydroxytaraxa-14-ene (34), 3β-acetoxyurs-13(18)-ene (35) and 3β-acetoxyurs-19-ene (36).Between the taraxaranes and the ursanes, there is a difference in the position of the doublebond, which is brought about by the way in which the taraxasteryl cation (28) is quenchedby the loss of the hydrogen with concomitant hydrogen migrations. In α-amyrin (29) andthe rest of the ursanes, the H-12 proton is lost and the double bond is formed at ∆ 12 .Acetylation of 29 leads to formation of α-amyrin acetate (30) (Scheme 7).HOHoleanyl cation (22)HHHHH1,2-methyl shiftHHOtaraxasteryl cation (28)HH29 191,2-hydride shift20 2112181117 2225 26 131 9 14 1628210 8 153 4 7 27R 562324α−amyrin R = OH (29)α−amyrin acetate R= OAc (30)30Scheme 7: Formation of taraxasteryl cation and the ursane class triterpenoidsIn the taraxaranes, the proton at either C-21 or C-30 is lost, leading to a double bond ateither ∆ 20 (31) or at 20(30) leading to taraxasterol (32) with acetylation of 32 resulting in 33(Scheme 8).It is highly likely that the compound classified as a taraxarene (34) does not belong in thisclass and should be grouped with other compounds with a double bond at ∆ 14 resulting from

P a g e | 19the loss of a proton at C-15 in the oleanyl cation (22) with hydride migrations quenching thecation (Scheme 8).HHHHHOtaraxasteryl cation (28)H2019 21acetylationH19 20R213 45251191023 24R= OH, taraxasterol (32)R = OAc, taraxasteryl acetate (33)6812267291918131427H153020 2117162228acetylation302111259103 465CH2OH2324-hydroxytaraxer-14-ene (34)81226714191813H1520 2117222816H3COCO213 45112591023 2468122672927191813141520 2117222816pseudotaraxasteryl acetate (31)Scheme 8: Formation of taraxastane class triterpenoids from taraxasteryl cationHCompounds 35 and 36 have double bonds in positions different to the ursanes or thetaraxaranes, 35 has a double bond between C-13 and C-18 and 36 has a double bond at ∆ 19and is more likely to be associated with the taraxaranes as the loss of the proton occursadjacent to the C-20 cation, just as that in 31.

P a g e | 203030AcO22313 4525119102468122672927191813141520 2117162228AcO213 45112591023 243β -acetoxyurs-13(18)-ene (35) 3β-acetoxyurs-19-ene (36)68122672927191813141520 21171622281.1.4.6 Friedoursane triterpenoidsUnlike the cases where the double bond at ∆ 14and 13(18) were not given their ownclassification, those with a double bond at the ∆ 7 position are classified as friedoursanetriterpenoids as in bauerenyl acetate (37) isolated from V. patula. They come about by lossof a proton at C-7 with methyl and hydrogen shifts quenching the cation in the taraxasterylcation (28) (Scheme 9).HOHHHHH-HAcO213 45251191023 2468127292719181314153020 2117162228taraxasteryl cation (28)bauerenyl acetate (37)Scheme 9: Formation of bauerenyl acetate from taraxasteryl cation1.1.4.7 The Friedelane triterpernoidsThe Friedelane triterpenoids, 3β-friedelanol (38) and friedelin (39) are found in V. patulawhile friedelin (39) alone is found in V. chalybaea and V. saligna.The friedelanetriterpenoids are characterised by methyl groups at C-4, C-5, C-9 and C-13, in addition to

P a g e | 21those at C-14, C-17 and C-20, which differ to the oleanane triterpenoids which have twomethyl groups at C-4 and a methyl group at C-10 instead of C-9 and a proton at C-13instead of the methyl group. This difference arises from the origin of the methyl migrationbeing from the methyl group at C-4 resulting in a series of methyl and hydrogen migrationsto quench the oleanyl cation (22) (Scheme 10). Friedelin (39) is brought about by the actionof an oxidoreductase oxidising the 3β-hydroxyl group of 3β-friedelanol (38) to a ketone(Corsino et al., 2000) (Scheme 10).H30293029HHOHoleanyl cation (22)HrearrangementHO3 41927 20 211218111317 221142816210 81525 726624233β−friedelanol (38)oxidoreductaseO211110 83 25 46232412719271813142615friedelin (39)20 2117222816Scheme 10: Formation of friedelane triterpenoids from the oleanyl cation (Corsino etal., 2000)The species of Vernonia from which these compounds have been isolated, including theparts of the plant that contained them are given in Table 3. The table revealed that lupeol(17), α-amyrin (23) and β-amyrin (29) were common to several species. V. paltula had thehighest number of triterpenoids followed by V. saligna and V. chalybaea. V. fasciculata andV. potamophilia each had only one triterpenoid from the tetracyclic and lupane classrespectively.

P a g e | 22Table 3: Tritepenoids contained in the different Vernonia speciesSpecies Triterpenoids isolated ReferencesV. arkansana 17 r , 18 r Bohlmann et al., 1981V. brasiliana 17 l , 23 l Alves et al., 1997V. chamaedrys 17 fl,s , 23 fl,s , 29 fl,s Catalán et al., 1988V. chalybaea 17 a , 18 a , 23 a , 29 a , 31 a 33 a , 39 a da Costa et al., 2008V. chunii 17 ns , 27 ns Yuan et al., 2008V. cinerea 18 r , 23 r , 29 r , 30 r , 34 r , 35 r , 36 r Misra et al., 1984a, 1984b, 1993V. cognata 17 fr,l , 23 fr,l , 29 fr,l , 32 fr,l Bardon et al., 1988bV. fasciculata 14 l Narain, 1979V. incana 17 l, fl , 23 l, fl , 29 l ,fl , 32 l, fl Bardón et al., 1990V. mollissima 17 a , 23 a , 24 a , 29 a Catalán and Iglesias, 1986V. nitidula 17 fl,l , 23 fl,l , 29 fl,l Bardon et al., 1988aV. patula 17 ns , 18 ns , 19 a ,23 ns 24 ns ,26 ns , Liang and Min, 2003;30 a , 37 wp , 38 wp , 39 wp Liang et al., 2010V. potamophilia 17 l Babady-Bila et al., 2003Huang and Liu, 2004V. saligna 19 wp , 20 wp , 21 wp , 23 wp ,25 wp ,29 wp , 30 wp , 39 wpV. squamulosa 17 a , 23 a , 29 a Catalán and Iglesias, 1986V. tweediana 17 l , 23 l , 29 l Zanon et al., 2008Key: superscripts, a = aerial parts, fl = flowers, fr = fruits, l = leaves, ns = not specified,r = roots, s = stems, wp = whole plant1.2 Introduction to the genus Vepris1.2.1 PhylogenyThe genus Vepris is a member of the Rutaceae family which consists of one hundred andsixty genera. In the classification proposed by Engler in 1931 and adapted by Schotz in1964, this genus was put together with the genera Acronychia, Araliopsis, Casimiroa,Halfordia, Hortia, Oriciopsis, Sargentia, Skimmia and Toddalia, as individual sub-tribes ofthe tribe Toddalioliineae, subfamily Toddalioideae (Fernandes et al., 1988).A more

P a g e | 23accepted taxonomic grouping of the Rutaceae, which included the genera Acronychia,Araliopsis, Diphasia, Oricia, Oriciopsis, Vepris and Teclea within the tribe Aronychia wasproposed in 1983 by Waterman and Grundon (Fernandes et al., 1988). Vepris species arewidely distributed all over the world and their morphology is diverse. They contain a widerange of secondary metabolites, which include limonoids, flavonoids, coumarins, volatileoils and alkaloids (Groppo et al., 2008).This genus is comprised of eighty species of trees and shrubs, occurring primarily in tropicalAfrica, and the Mascarene Islands, and to a lesser extent in tropical Arabia and SouthwestIndia where one species is found (Chaturvedula et al., 2003). African Vepris species arefound in Cameroon, Democratic Republic of Congo, Ethiopia, Ghana, Kenya, Madagascar,Mauritius, Mozambique, Rwanda, South Africa, Swaziland, Tanzania, Zambia, Zanzibarand Zimbabwe (Waterman, 1986). About sixteen species are endemic to Kenya and thirtyare found in Madagascar (Louppe et al., 2008).Vepris species are plants without prickles and the bark is pale to dark grey and fairlysmooth. The leaves are unusually glossy green, large, alternate, fairly smooth and drooping;when crushed they give an aromatic scent. The flowers have short auxiliary heads and aregreenish; the fruits are drupaceous, smooth and usually contain 2-4 seeds (Cheek et al.,2009).1.2.2. Ethnobotanical use of Vepris speciesOf the eighty Vepris species, only a few have documented use in herbal medicine. Speciesof this genus are mainly used in the treatment of respiratory infections, dermatologicalinfections, as analgesics, antipyretics and for oral health. The two species used for oralhealth, maintains oral cleanliness by contributing to antimicrobial activity and inhibiting

P a g e | 24plaque and also by dislodging cariogenic microorganisms when the plant is chewed. Veprisspecies are also reported to have anti-inflammatory and antimalarial properties (Table 4).Table 4 Traditional uses of plants belonging to the genus VeprisPlant species Plant part Traditional use Reference (s)V. ampody leaves andbarkantimalarial, muscularaches and analgesicRandrianarivelojosia et al.,2003V. elliotti leaves aphrodisiac Poitou et al., 1995V. eugenifolia bark liver protection,respiratory diseases andFratkin, 1996;Hedberg et al., 1983kidney disordersV. glomerata roots eye infection andantimalarialChhabra et al., 1991V. heterophylla leaves diuretic and antipyretic Gomes et al., 1983V. lanceolata leaves andstemsleaves androotsgynaecological diseases,heart disease, astringent,burns, eye problem, colic,anti-diarrhoeic, antiviral,antibacterial, antipyretic,analgesic, fortifier,antirheumatic, gout,stimulant, musculardisorder anddermatological infectionroots heart diseases, antiviral,analgesic, respiratoryinfections andgynaecologicalcomplicationsV. louisii stem bark dermatological diseasesand antifungalArnold and Gulumian 1984;Gurib-Fakim et al., 1996;Louppe et al., 2008;Poullain et al., 2004;Vera et al., 1990antimalarial Gessler et al., 1994, 1995a,1995bArnold and Gulumian 1984;Steenkamp, 2003Ayafor et al., 1982aV. nobilis leaves Antipyretic Louppe et al ., 2008leaf androotrespiratory diseasesantirheumatic and itchingroot Anthelminticstem respiratory disodersbark and Analgesicleavesroot and dental caretwigsV. paniculata leaves respiratory infections Gurib-Fakim et al., 1996V. simplifolia bark chest complaint Louppe et al ., 2008rootmuscular problems,

P a g e | 25leaffruitstwigsantibacterial, venerealdiseases anddermatological infectionsrespiratory infection anddermatological infectionsdental cariesdental care1.2.3. Biological activity of extracts from Vepris speciesThe biological activities of the extracts from various Vepris species have not been widelyinvestigated and reported. The few reports are diverse (Table 5) with antimalarial activitybeing the most studied. Antimalarial activity is reported in the ethanol and chloroformextracts of six species and also in the essential oil of Vepris elliotii. Antibacterial activity isreported in two species, Vepris lanceolata and Vepris laendriana, antifungal activity in V.laendriana and Vepris heterophylla and antioxidant activity in V. heterophylla, and V.lanceolata. Cytotoxic activity was reported in Vepris punctata to be mild and mainlyagainst human colon and ovarian cancer (Chaturvedula et al., 2003).Of the Vepris species, V. lanceolata has been studied the most; its leaf and root bark extractswere found to possess antiplasmodial activity whilst the aqueous leaf and stem extracts werefound to have antibacterial and antifungal properties.

P a g e | 26Table 5 Biological activities of extracts from Vepris speciesNarod et al., 2004Plant species Biological ExtractReferenceactivityV. ampody antimalarial ethanol s /chloroform s Rasoanaivo et al., 1999V. elliotii antiplasmodial essential oil l Ratsimbason et al., 2009V. fitoravina antimalarial ethanol s /chloroform s Rasoanaivo et al., 1999V. glomerata antimalarial aqueous l and ethanolic l Innocent et al., 2009V. heterophylla antifungal andantioxidantmethanol l,t and essentialoil l Momeni et al., 2010;Aoudou et al., 2010V. lanceolata antibacterial hexane s ,and methanol lmethanol:chloroform(1:1) s ,methanol:chloroform (1:1) lantibacterial,Gessler et al., 1994, 1995antifungal and Nsantimalarialantioxidant dichloromethane s Poullain et al., 2004V. leandriana antibacterial andantifungalessential oil lRakotondraibe et al.,2001V. macrophylla antimalarial ethanol/chloroform s Rasoanaivo et al., 1999V. punctata cytotoxic n-hexane/chloroform w Chaturvedula et al., 2003Key: ns = not specified, l = leaves, s = stem, w = wood, t = twigs1.2.4. A phytochemical review of Vepris speciesSixteen species of Vepris have been studied for their phytochemical properties thus far(Scifinder, 2009). Although furoquinoline alkaloids are the most common isolates, acridonealkaloids, quinol-2-one alkaloids, indoloquinazoline alkaloids, flavonoids, cinnamic acidderivatives, limonoids and terpenoids have also been found.The species of Vepris from which these compounds were isolated, including the parts of theplant that contained them are given in Table 6, with their structures given in the followingsubchapters.

P a g e | 27Table 6: Compounds isolated from Vepris speciesSpecies Compounds isolated ReferencesKan-Fan et al., 1970;Govindachari and Sundararajan,V. ampody 45 l&br , 46 l&br , 47 l&br , 49 l&br , 54109 l&br , 110 l&brl&br 75 sb ,76 sb , 77 sb , 82 l&br , 87 l&br Rasoanaivo et al., 1999V. bilocularis 48 sb , 49 sb , 54 ns , 55 sb , 65 l ,63 l , 66 l ,89 ns 1966; Brader et al., 199667 l , 78 sb , 79 sb , 80 l , 81 l , 83 l , 84 sb , 1961, 1964; Ganguly et al.,V. dainellii 48 l , 49 l Dagne et al., 1988V. fitoravina 54 l , 79 l , 83 l , 85 l , 86 l Koffi et al., 1987V. glomerata 48 l , 59 l Dagne et al., 1988V. heterophylla 49 l , 60 l , 61 l , 93 l , 103 l -108 l Gomes et al., 1983, 1994V. lanceolata 58 l , 59 l , 61 l , 62 l , 95 sb,w , 97 sb,w ,99 sb,w , 100 sb,w , 101 sb,w , 102 sb,w Mbala, 2005Ayafor et al., 1980, 1981,V. louisii 17 sb , 43 sb , 44 tb , 49 tb , 52 tb , 64 sb ,114 tb al., 198271 ns , 72 ns , 73 ns , 74 ns , 88 tb , 113 tb , 1982a, 1982b, 1982c; Ngadjui etV. macrophylla 54 l , 79 l , 83 l , 85 l , 86 l Koffi et al., 1987V. pilosa 49 r , 79 r , 80 r , 88 r Haensel and Cybulski, 1978Chaturvedula et al., 2003, 2004Mbala, 2005V. punctata 17 w , 18 w , 29 w , 32 w , 48 w , 49 w , 53 w ,117 w54 w , 55 w , 56 w , 57 w , 115 w , 116 w ,V. reflexa 17 sb w , 49 sb&w , 55 sb&w , 94 sb,w ,96 sb,w , 98 sb,wV. stolzii. 17 sb , 48 sb , 50 sb , 64 sb ,68 sb ,69 sb ,70 sb Khalid and waterman, 1982V. uguenensis 51 r , 55 r , 90 r , 96 r , 111 r , 112 r Cheplogoi et al., 2008Key: superscripts, a = aerial parts, br = branches, fl = flowers, fr = fruits, l = leaves, r = roots,s = stems, sb = stem bark, tb = tree bark, w = wood, wp = whole plant,1.2.4.1. Quinoline alkaloidsThe quinoline alkaloids are found in several Vepris species (Table 6), with two species,Vepris dainellii and Vepris glomerata exclusively containing compounds of this class.Quinoline alkaloids are nitrogenous compounds based on the benzo[b]pyridine or 1-azanaphthalene (40) skeleton. They occur abundantly in the family Rutaceae and have beenused in the chemotaxanomic classification of many species in the family. These compoundsare biosynthesized from anthranilic acid (41).

P a g e | 28The quinoline ring is formed by the condensation of anthranilic acid (41) and malonyl-CoAfollowed by cyclisation via intramolecular amide formation, which results in a heterocyclicsystem with the more stable 4-hydroxy-2-quinolone (42) form. The other form, the di-enolis highly nucleophilic at position 3 and susceptible to alkylation via DMAPP (2) (Scheme11).OH6543CO 2 H654378N2NH 278N2O4041H42OOOHCOSCoAMalonyl-CoAAmide f ormationdi-enoltautomerNH2anthraniloyl-CoANH2SCoAONONOHH6758OH4N32OOPPalkylation at C-3OHNfavoured 4-hydroxyquinolone tautomerOH42aH4-hydroxy-2-quinolone (42)Scheme 11: Biosynthesis of 2-quinolone alkaloids (Dewick, 2006)The 2-Quinolone alkaloidsN-methylpreskimmianine (43) and veprisilone (44) have been found in the stem and trunkbark of Vepris louisii. Both these compounds have arisen from the intermediate 42 beingmethylated both at the secondary amine and the hydroxy group at C-4 as well as beingprenylated at C-3. Veprisilone (44) is further oxidised in the prenyl ring.

P a g e | 29OCH 3OCH 3OHH 3 CON OH 3 CONOCH 3 CH 3 OCH 3 CH 3OO43 44The 4-Quinolone alkaloidsThe 4-quinolones 45-47 have been isolated from Vepris ampody and differ from the 2-quinolones by the position of the carbonyl which is at C-4. These compounds arise fromcarbon nucleophiles attacking the C-2 carbonyl group in the quinolodione system, followedby dehydration at C-2 and C-3 resulting in a ∆ 2 double bond with alkyl substituents at C-2(Scheme 12).ONRH45 2-(Nona-3’,6’-diene)-4-quinolone (CH 2 ) 2 CH=CHCH 2 CH=CHCH 2 CH 346 2-(Nona-9’-ol)-4-quinolone (CH 2 ) 8 CH 2 OH47 2-(Undeca-10’-one)-4-quinolone (CH 2 ) 9 COCH 3R

P a g e | 30COSCoAMalonyl-CoAclaisen reactionNH2ONH2OSCoAONHOCH 2 RO5463278N RHQuinolinesScheme 12: Formation of simple 4-quinoline alkaloids (Dewick, 2006).ONHHCH 2 ROHFuranoquinoline alkaloidsThis class of alkaloids possesses three rings; a benzopyridine group consisting of two ringsto which a furan ring, a third ring, is attached. They are formed from 42 through prenylation,cyclisation and then side chain cleavage of the three carbon fragment (Scheme 13). Varioushydroxylations and methylations to dictamine (Scheme 13), the simplest knownfuroquinoline alkaloid, results in several substituted furoquinoline alkaloids.Aromatichydroxylation followed by alkylation leads to skimmianine (48) found in five Veprisspecies, V. bilocularis, V. dainellii, V. glomerata, V. punctata and V. stolzii (Table 6).Kokusaginine (49) is found in the Vepris species of ampody, bilocularis, dainellii,heterophylla, louisii, pilosa, punctata and reflexa, making it the most commonfuranoquinoline alkaloid, being found in eight species. Vepris stolzii contains γ-fagarine(50) and maculosidine (51) was isolated from Vepris uguenensis. V. louisii containsveprisinium hydrochloride (52) and 4,5,6,7,8-pentamethoxymaculine (53) is found in Veprispunctata.

P a g e | 31OCH 3OCH 3H 3 COH 3 COOCH 3NOOCH 348H 3 CON49OOCH 3N50OH 3 COOCH 3OCH 3H 3 COOCH 3OCH 3OCH 3NOH 3 CO+N OCl -OCH 3 CH3OHH 3 CONO5152OCH 353OHOMeNHOMethylationNHOcyclization on todimethylallylside-chainOMeNOOHoxidative cleavageof side-chainH 3 CO675 4OCH 32NO12OCH 3skimmianine (48)8 9 10 113OMeNdictamineOScheme 13: Biosynthesis of furanoquinoline alkaloids (Dewick, 2006)The methoxylated methylenedioxy furanoquinolines, maculine (54) and flindersiamine (55)are found together in V. bilocularis and V. punctata with only one of them, 55 being foundin V. reflexa and V. uguenensis. In addition, 5-methoxymaculine (56) and 5,8-dimethoxymaculine (57), methoxylated at the 5-position as well as the 4- and 8-positions arecontained along with 54 and 55 in V. punctata. The introduction of a methylenedioxy groupat C-6 and C-7 usually arises by cyclisation when a methoxy and hydroxy group are presenton adjacent carbon atoms.

P a g e | 32OCH 3OCH 3OCH 3O654a43a3OOO78OCH 328aON 9a ON O ON OOCH54 3R5556. R= H57. OCH 3OCH 3OCH 3OCH 3OOHOONOON O OHO N OOCH 358OCH 359OCH 3 60H 3 COOCH 3OCH 3OHOHO61NOONO OCH 3 62OThe oxidised C-7 O-prenylated anhydroevoxine (58) and evoxine (59) is found in V.lanceolata with 59 also being present in V. glomerata. These compounds are the epoxidisedand dihydroxylated forms of isoisohaplopine-3,3’-dimethylallyl ether (not isolatedpreviously from Vepris species), which probably arises from the O-prenylation at C-7 in γ-fagarine (50) (Scheme 14).V. heterophylla contains a C-5 prenylated furanoquinoline, tecleaverdoornine (60),containing a methylenedioxy group at C-6 and C-7. Evolantine (61), an isomer of evoxine(59), with a methoxy group at C-7 instead of C-5 has been isolated along with 60 in V.

P a g e | 33heterophylla. Anhydroevoxine (58), evoxine (59), evolantine (61) and evoxoidine (62), allisolated in V. lanceolata, suggest that all these compounds are related biosynthetically.OCH 3OCH 3OCH 3PPO[O]ON OO N OO N OOCH 3 OCH 3OCH 350 haplopine-3,3'-dimethylallyl ether 585'23'OCH 34'5 4a 4 3a 36OH 1'8aOCH 3HO 2' O 7 8 N 9a O59Scheme 14: Biosynthetic pathway to the prenylfuranoquinoline alkaloids (Grundon,1988)Pyranoquinoline alkaloidsThe pyranoquinoline alkaloids 63-74, all derivatives of flindersine (63a) are found in threeof the Vepris species, V. bilocularis, V. louisii and V. stolzii. They are formed by cyclisationof the prenyl side-chain of 4-hydroxy-2-quinolone (42a) (Scheme 15) to form an additionalangular pyran ring on the quinoline skeleton. The various pyranoquinoline alkaloidderivatives arise as a result of either methylations or prenylations at the nitrogen, C-7 and C-8. The dimers vepridimerine A-D 71-74 were found only in V. louisii.

P a g e | 34R 35'O65 471R 28NR 1R63a23'34'2'1'OR R 1 R 2 R 363 6-methoxyflindersine H H H OCH 364 veprisine CH 3 OCH 3 OCH 3 H65 7-methoxyflindersine H H OCH 3 H66 7-prenyloxyflindersine H H OCH 2 CH=C(CH 3 ) 2 H67 7-prenyloxy N-CH 3 CH 3 OCH 2 CH=C(CH 3 ) 2 Hmethylflindersine68 N-methyl-8-(3’,3’-CH 3 OCH 2 CHC(CH 3 ) 2 H Hdimethylallyoxy)flindersine69 N-methyl-7-methoxy-8-(3’,3’- CH 3 OCH 2 CHC(CH 3 ) 2 OCH 3 Hdimethylallyoxy)flindersine70 N-methyl-7-methoxy-8-(2’,3’-epoxy-3’,3’-dimethylallyoxy)flindersineCH 3 OCH 2 CH(O)C(CH 3 ) 2 OCH 3 HONOCH 3CH 3 OCH 3OCH 3H 3 COOCH 3ONCH 3ROHO71. R = α-H72. R = β-HH 3 COOOCH 3O RNCH 3HON OOCH 3 CH 373. R= α-H74. R= β-H

P a g e | 35OHONHON OHangular pyranoquinolineScheme 15: Formation of angular pyranoquinoline alkaloids (Grundon, 1988)1.2.4.2. Acridone alkaloidsThere are five species of Vepris that contain acridone alkaloids, ampody, bilocularis,fitoravina, macrophylla and pilosa (Table 6), with fitoravina and macrophylla containingexclusively the same suite of acridone alkaloids, 1,3-dimethoxy-N-methylacridone (79),arborinine (83), 1,3-dimethoxy-10-methylacridan-9-one (85) and 1-hydroxy-2,3,4-trimethoxyacridan-9-one (86) (Koffi et al., 1987). Together with the furanoquinoline andpyranquinoline alkaloids, they make the quinoline alkaloids one of the taxonomic markersof the genus Vepris.Acridone alkaloids contain a tricyclic ring having nitrogen at position 10 and a carbonylgroup at C-9. They are formed by the addition of three malonyl units to anthranilyl-CoA(N-methyl derivative of anthranilic acid) and sequential Claisen reactions, followed byheterocyclic ring formation by nucleophilic addition of the secondary amine on the carbonylgroup followed by dehydration and enolisation to form the heterocyclic and the secondaromatic ring (Scheme 16). They differ from the quinolone alkaloids in that two extramalonyl units are incorporated into the skeletal backbone, resulting in a further aromaticring. Once again the acetate-derived ring is susceptible to electrophilic attack leading toalkylation (with DMAPP (1)) or further aromatic hydroxylation (Scheme 16).

P a g e | 36O R 1R 2788a91a12R 610655a N 4a4R 5 R R 43R 3R R 1 R 2 R 3 R 4 R 5 R 675 melicopicine CH 3 OCH 3 OCH 3 OCH 3 OCH 3 H H76 tecleanthine CH 3 OCH 3 OCH 2 O H OCH 3 H77 6-methoxytecleanthine CH 3 OCH 3 OCH 2 O H OCH 3 OCH 378 evoxanthine CH 3 OCH 3 OCH 2 O H H H79 1,3-dimethoxy-NmethylacridoneCH 3 OCH 3 H OCH 3 H H H80 1,2,3- trimethoxy-NmethylacridoneCH 3 CH 3 OCH 3 OCH 3 H H H82 1-hydroxy-3-methoxy- CH 3 OH H H H H H10-methylacridone83 arborinine CH 3 OH OCH 3 OCH 3 H H H84 1-hydroxy-2,3,dimethoxyacridan-9-one85 1,3-dimethoxy-10-methylacridan-9-one86 1-hydroxy-2,3,4-trimethoxyacridan-9-one87 2,4-dimethoxy-10-methylacridan-9-oneH OH OCH 3 OCH 3 H H HCH 3 OCH 3 H H H H HH OH OCH 3 OCH 3 H H HCH 3 H OCH 3 OCH 3 H H HThe acridone alkaloids in Vepris are mainly hydroxylated and methoxylated at variouspositions on both the aromatic rings except for C-7 and C-8. Only two of the alkaloids,tecleanthine (76) and its 6-methoxy derivative (77) were functionalised on the anthranilyl

P a g e | 37derived ring. The methylenedioxy group between C-2 and C-3 is also a common occurrenceand can be seen in 76 and 77 as well as in evoxanthine (78).COSCoA3 X malonyl-CoAOOClaisen reactionOONHMeClaisen reactionNHMeCoASOOONHMeONucleophilicadditionOOHOOHDehydration and enolizationto f orm aromatic ringOONMeOprenylationNMeOH-H 2 ONMeOHOVebilocine (81)1,3-dihydroxy-N-methylacridoneScheme 16: Formation of acridone alkaloids1.2.4.3. LimonoidsThree limonoids of the limonin type skeleton, including limonin (88) itself have beenisolated in three separate Vepris species. Limonin (88) was isolated from V. louisii and V.pilosa, veprisone (89) from V. bilocularis and methyl uguenenoate (90) from V. uguenensis.This could be the third chemotaxonomic marker for the genus Vepris, the quinolone andacridone alkaloids being the first two. Thus, Vepris species such as bilocularis and lousiicontain all three classes of compounds. Phytochemists therefore need to keep a careful eyeon all three classes of compounds when studying the phytochemical constituents of Vepris.

P a g e | 38232220 OO182112 171 OO211 30 131914 163910 8O15OO45 76 O292888H 3 COO1 219OOOOHH 3 CO 1O23OOOOOO89 90OOOThe three Vepris limonoids possess the limonin skeleton, which from the 7α-hydroxyapoeupholprecursor have modified rings A and D as well as side chain modification to a furanring. The triterpene precursor, butyrospermol (91) (Scheme 17) is derived directly from thedammarenyl cation (11) (from Scheme 4) by the loss of a proton from C-7, catalysing aseries of methyl and hydrogen migrations. Since all three compounds isolated previouslyhave a ketone at C-7, the key step in their biosynthesis is the epoxidation of the ∆ 7 doublebond followed by ring opening with subsequent loss of the H-15 proton and concurrentWagner-Meerwein shift of the methyl group to C-8, retaining the methyl group in the β-position. Subsequent oxidation to the ketone at C-7 and to the epoxide at ∆ 14 results in thetwo functional groups present in 88-90.

P a g e | 39HHHHHOHHHHOHOxidationHOHOHDammarenyl cation (11)butyrospermol (91)HHHOHOHHOH8714OH15H7α-hydroxyapo-euphol/tirucallol (92)Scheme 17: Formation of 7α-hydroxy-euphol/tirucallol via apo rearrangementThe furan ring is biosynthesized through stepwise oxidation of the protolimonoid’s sidechainto produce a hydroxyl group at C-23, a 24,25-epoxy group, and an aldehyde at C-21,followed by nucleophilic cyclisations from the oxygen at C-23 to the carbonyl group at C-21to form the hemiacetal ring. <strong>Open</strong>ing of the C-24,25 epoxide ring followed by oxidationproduces a ketone at C-24, which then undergoes Baeyer-Villiger oxidative cleavage of theC-23,24 bond to give a dihydrofuran ring, which finally forms the furan ring, with the lossof four carbon atoms (Scheme 18).

P a g e | 40HH21HOH2324OHO25C-21, C-23 nucleophiliccyclisationHOHOO24 25euphol/tirucallolside-chainAcOHturraeanthinHOHOHOOOHBaeyer-VilligeroxidationHOHOO2423OHC-24 oxidationHOH24,25 epoxide ringopeningO24OHOHcleavage of4C side-chainHOHOOHOCO 2 H-H 2 OAcOHScheme 18: Furan ring formation via side-chain oxidation (Bevan et al., 1967)In the expansion of the D ring, the key step is the allylic oxidation at C-16 of the D ringcontaining the ∆ 14 -double bond to form an α, β unsaturated ketone. This is followed byBaeyer-Villiger ring expansion to the lactone, which can occur either before or after theepoxidation of the ∆ 14 double bond (Taylor, 1984) (Scheme 19).

P a g e | 41OO1614 15OOOabOOOOO16O15OOScheme 19: Ring D lactone formation (Taylor, 1984).Ring A is contracted to a five membered ring followed by lactonization which involves themethyl group (CH 3 -19) and the carbonyl group at C-3 leading to the formation of A and A’rings (A-seco) in a sequence of events starting from the deacetylation of nomilin (Scheme20). Methyl uguenenoate (90) is a derivative of limonin (88) in which the A’ lactone ringhas undergone cleavage resulting in the hydroxyl-acid, which is followed by methylation.

P a g e | 42OOOOOOAcnomilinOOOOOOOOobacunoneOOOHOOOOOOOOHOHOOOOOOOichangin-H 2 OHOOHOHOHOOOOOOOOHOHOOOOO-H 2 OOOOOOOOOlimonin (88)Scheme 20: Biosynthesis of limonin (Adesogan and Taylor 1969)1.2.4.5. Cinnamic acid derivatives and simple aromaticsThree cinnamic acid derivatives (93-95) and six simple aromatic compounds (96-102) wereisolated from two species, V. lanceolata and V. reflexa. Cinnamic acids are plant derivedfragrances and flavourings having a common cinnamoyl functionality, which is present in avariety of secondary metabolites of phenyl propanoid biosynthetic origin (De et al., 2011).Chemically, cinnamic acids are aromatic fatty acids composed of an aromatic ringsubstituted with an acrylic acid group commonly in the more favoured trans geometry. Theacrylic acid group is a αβ-unsaturated carbonyl group which can be considered as a Michael

P a g e | 43acceptor; an active moiety which is often used in the design of anticancer drugs (Ahn et al.,1996). The main precursor in their biosynthesis is L-Phenylalanine which undergoesdeamination to form trans-cinnamic acid. Further hydroxylation and methylation reactionsresult in the large variety of known cinnamic acids from plant sources. The cinnamic acidsalso undergo esterification leading to cinnamic acid esters such as 94 and 95.OR 2ORHOR 1R R 1 R 293 trans-sinapic acid methyl ester CH 3 CH 3 CH 394 alkyl trans-4-hydroxycinnamate OCH 2 (CH 2 ) n CH 3 H H95 alkyl trans-4-hydroxy-3-methoxycinnamate OCH 2 (CH 2 ) n CH 3 H OCH 3Apart from the cinnamic acids, several simple aromatic compounds, includingsyringaldehyde (96) and syringic acid (97), the precursors to lignins as well as the aromaticacid, para-hydroxybenzoic acid (99), a preservative, an ester, methyl 2,4-dihydroxy-3,6-dimethylbenzoate (100) and ketone, 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone (101) as well as the quinone, 2,6-dimethoxy-1,4-benzoquinone (98) wereisolated. The isolation of syringaldehyde (96) from V. reflexa and syringic acid (97) from V.lanceolata is consistent with the isolation of meso-syringaresinol (102) from V. lanceolata,since both the syringaldehyde and syringic acid are precursors to the lignins.

P a g e | 44ROOCOOHOHOH 3 COCH 3H 3 COOCH 3HOCH 3OHH 3 CO OCH 3OHO96. R = H97. R = OH9899100H 3 COOOHMeOHOOMeOHHOHOOHOMe101MeO1021.2.4.6. FlavonoidsSurprisingly, only V. heterophylla was reported to contain flavonoids, which were allglycosides with the sugar moiety on the A ring, 103-108 (Gomes et al., 1983). Flavonoidsare derived from three malonyl CoA units which condense with each other and thecinnamoyl CoA unit derived from cinnamic acid which is reduced and ring closed formingthe chalcone, the precursor to the flavonoids (Scheme 21).OHR 2 OR 3ORR 1OH OR R 1 R 2 R 3103 vitexin H H H C-Glu104 7-O-acylscoparin H H CH 3 CO C-Glu105 Chrysoeriol 7-glucoside CH 3 O H O-Glu H106 2’’-O-glucosylvitexin H H H C-Glu(1→2)]107 2’’-O-glucosylisovitexin H C-[Glu(1→2)] H H108 Chrysoeriol 7-rhamnoside CH 3 H C-Rha(1→2)Gal] H

P a g e | 45OOOOOOOHOSCoAOHCoASOCoASOCoASOCoASOOOO+ 3CO 2reductaseNADPHHOOOH OFlavanoneOH-HHOOH+H OH OHChalconeOHOring closureand aromatisationOHOSCoAOOHScheme 21: Condensation of three molecules of malonyl CoA with activated cinnamicacid (Hahlbrock and Grisebach, 1975).1.2.4.7. Miscellaneous compoundsApart from the classes mentioned above, N,N-dimethyltryptamine (109) andphenylacetamide (110)) have been isolated in V. ampody and an azole, unguenenazole(111), and amide, unguenenamide (112) were isolated from V. uguenensis. Theindoloquinazoline alkaloids 1-hydroxyrutaecarpine (113) and 7,8-dehydro-1-hydroxyrutaecarpine (114) were found in V. lousii. The terpenoids, glechomanolide (115),1β,10β:4α,5α-diepoxy-7(11)-enegermacr-8α,12-olide (116) and 28-acetyloxy-6α,7α:21β,28-diepoxytaraxer-3α-ol (117) were isolated in V. punctata.NCH 2 CONH 2N109H110

P a g e | 46H 3 COOH 3 COOONN111 112H876NO5876NO5NHNNHNHO113 114HOOOOOOHOOCOCH 3O115 116HOO1171.3 Aim of the studyThe main aim of the study was to investigate the species of Vernonia and Veprisphytochemically in order to determine what secondary metabolites were contained in thevarious extracts of the plant in order to validate the use of the plants in traditional medicinein Kenya and to provide suggestions for further use of the plant ethnomedicinally by testingthe compounds isolated in various identified assays, determined by the class of compoundisolated.

P a g e | 471.3.1 ObjectivesThe research objectives were;1. To extract and isolate the phytochemicals from Vernonia auriculifera, Vernoniaurticifolia, Vepris glomerata and Vepris uguenensis leaves, stems and roots.2. To identify and characterise the isolated compounds using spectroscopictechniques (NMR, IR, UV, CD, and MS).3. Based on the isolates contained in each plant, identify suitable bioassays to testthe compounds and thereby provide information on the further use of the plant orvalidate its existing use.

P a g e | 48ReferencesAbosi, A. O., Raseroka, B. H. 2003. In vivo antimalarial activity of Vernonia amygdalina. Brit.J. Biomed. Sci., 60, 89-91.Adedapo, A. A., Otesile, T., Soetan, K. O. 2007. Assessment of the anthelmintic efficacy of anaqueous crude extract of Vernonia amygdalina. Pharm. Biol., 45, 564-568.Adesanoye, O. A., Farombi, E. O. 2009. Hepatoprotective effects of Vernonia amygdalina(Asteraceae) in rats treated with carbon tetrachloride. Exp. Toxicol. Pathol., 62, 197-206Adesogan, E. K., Taylor, D. A. H. 1969. Methyl ivorensate, an A-seco limonoid from Khayaivorensis. Chem. Comm., 889.Ahn, B. Z., Sok, D. E. 1996. Michael acceptors as a tool for anticancer Drug Design. Curr.Pharm. Des., 2, 247-262.Akah, P. A., Ekekwe, R. K. 1995. Ethnopharmacology of some of the Asteraceae family usedin Nigerian traditional medicine. Fitoterapia, 66, 352-355.Akah, P. A., Okafor, C. L. 1992. Blood sugar lowering effect of Vernonia amygdalina (Del.) inan experimental rabbit model. Phytother. Res., 6, 171-173.Aliyu, A. B., Musa, A. M., Abdullahi, M. S., Ibrahim, H., Oyewale, O. A. 2011. Phytochemicalscreening and antibacterial activities of Vernonia ambigua, Vernonia blumeoides, Vernoniaoocephala (Asteraceae). Acta Pol. Pharm. Drug Res., 68, 67-73.Alves, T. M., Nagem, T. J., De Carvalho, L. H., Krettli, A. U., Zani, C. L. 1997.Antiplasmodial triterpene from Vernonia brasiliana. Planta Med., 63, 554-555.

P a g e | 49Aoudou, Y., Léopold, T. N., Michael, J. D. P., Xavier, F., Moses, C. M. 2010. Antifungalproperties of essential oil and some constituents to reduce food borne pathogens. J. YeastFungal Res., 1, 1-8.Arnold, H. J., Gulumian, M. 1984. Pharmacopoeia of traditional medicine in Venda. J.Ethnopharmacol., 12, 35-74.Ayafor, J. F., Sondengam, B. L., Ngadjui, B. T. 1980. Veprisine and N-methylpreskimmianine:novel 2-quinolones from Vepris louisii. Tetrahedron Lett., 21, 3293-3294.Ayafor, J. F., Sondengam, B. L., Ngadjui, B. T. 1981. The structure of veprisinium salt.Tetrahedron Lett., 22, 2685-2688.Ayafor, J. F., Sondengam, B. L., Ngadjui, B. T. 1982a. Veprisinium salt, a novel antibacterialquaternary alkaloid from Vepris louisii. Planta Med., 44, 139-142.Ayafor, J. F., Sondengam, B. L., Ngadjui, B. T. 1982b. Veprisilone, a prenylated 2-quinolone,and limonin from Vepris louisii. Phytochemistry, 21, 955-956.Ayafor, J. F., Sondengam, B. L., Ngadjui, B. T. 1982c. Quinoline and indolopyridoquinazolinefrom Vepris louisii. Phytochemistry, 21, 2733-2736.Babady-Bila, Kilonda, A., Mihigo, S., Lohohola, O., Tshibey, T. S. Compernolle, F., Toppet,S., Hoornaert, G. J. 2003. Potamopholide, a new sesquiterpene lactone from Vernoniapotamophila. Eur. J. Org. Chem., 123-127.Barbastefano, V., Cola, M., Luiz-Ferreira, A., Farias-Silva, E., Hiruma-Lima, C. A., Rinaldo,D., Vilegas, W., Souza-Brito, A. R. 2007. Vernonia polyanthes as a new source of antiulcerdrugs. Fitoterapia, 78, 545-551.

P a g e | 50Bardón, A., Catalán, C. A. N., Gutiérrez, A. B., Herz, W. 1988a. Glaucolides and otherconstituents from Vernonia nitidula. Phytochemistry, 27, 2691-2694.Bardón, A., Catalán, C. A. N., Gutiérrez, A. B., Herz, W. 1988b. Piptocarphol esters and otherconstituents from Vernonia cognata. Phytochemistry, 27, 2989-2990.Bardón, A., Catalán, C. A. N., Gutiérrez, A. B., Herz, W. 1990. Glaucolides and relatedsesquiterpene lactones from Vernonia incana. Phytochemistry, 29, 313-315.Beentje, H. 1994. Kenya Trees, Shrubs and Lianas. National Museums of Kenya, Nairobi, pp,564-570.Bevan, C. W. L., Ekong, D. E. U., Halsall, T. G., Toft, P. 1965. West African timbers. PartXIV the structure of turraeanthin, a triterpene monoacetate from Turraeanthus africanus.Chem. Comm., 636-638.Bevan, C. W. L., Ekong, D. E. U., Halsall, T. G. Toft, P. 1967. West African timbers. Part XX.The structure of turraeanthin, an oxygenated tetracyclic triterpene monoacetate. J. Chem.Soc., 820-828.Bohlmann, F. Sigh, P., Borthakur, N., Jakupovic, J. 1981. Three bourbonenolides and othersesquiterpene lactones from two Vernonia species. Phytochemistry, 20, 2379-2381.Brader, G., Bacher, M., Greger, H., Hofer, O. 1996. Pyranoquinolones and acridones fromVepris bilocularis. Phytochemistry, 42, 881-884.Builders, M. I., Wannang, N. N., Ajoku, G. A., Builders, P. F., Orisadipe, A., Aguiyi, J. C.2011. Evaluation of the antimalarial potential of Vernonia ambigua Kotschy and Peyr(Asteraceae). Int. J. Pharm., 7, 238-247.

P a g e | 51Buskuhl, H., de Oliveira, L. F., Blind, Z. L., de Freitas, R. A., Barison, A.,Campos, F. R.,Corilo, Y. E., Eberlin, M. N., Caramori, F. G., Biavatti, M. W. 2010.Sesquiterpenelactones from Vernonia scorpioides and their in vitro cytotoxicity. Phytochemistry, 71,1539-1544.Catalán, C. A. N., de Iglesias, D. I. A. 1986. Sesquiterpene lactones and other constituents ofVernonia mollissima and Vernonia squamulosa. J. Nat. Prod., 49, 351-353.Catalán, C. A. N., De Iglesias, D. I. A., Kavka, J., Sosa, V. E., Herz, W. 1988. Glaucolides andrelated sesquiterpene lactones from Vernonia chamaedrys. Phytochemistry, 27, 197-202.Chaturvedula, V. S. P., Schilling, J. K., Miller, J. S., Andransiferana, R., Rasamison, V. E.,Kingston, D. G. I. 2003. New cytotoxic alkaloids from the wood of Vepris punctata fromthe Madagascar rainforest. J. Nat. Prod., 66, 532-534.Chaturvedula, V. S. P., Schilling, J. K., Miller, J. S., Andransiferana, R., Rasamison, V. E.,Kingston, D. G. I. 2004. New cytotoxic terpenoids from the wood of Vepris punctata fromthe Madagascar rainforest. J. Nat. Prod., 67, 895-898.Carvalho, P. B., Ferreira, E. I. 2001. Leishmaniasis phytotherapy. Nature’s leadership againstan ancient disease. Fitoterapia, 72, 599-618.Cheek, M., Oben, B., Heller, T. 2009.The identity of the West-Central African Oricialecomteana Pierre, with a new combination in Vepris (Rutaceae). Kew Bulletin 64, 509-512.Cheplogoi, P. K., Mulholland D. A., Coombes, P. H., Randrianarivelojosia, M. 2008. An azole,an imide and a limonoid from Vepris uguenensis (Rutaceae). Phytochemistry, 69, 1384-1388.

P a g e | 52Chhabra, S. C., Mahunnah, R. L. A., Mshiu, E. N. 1989. Plants used in traditional medicine inEastern Tanzania. II angiosperms (Capparidaceae-Ebenaceae). J. Ethnopharmacol., 25, 339- 359.Chhabra, S. C., Mahunnah, R. L. A., Mshiu, E. N. 1991. Plants used in traditional medicine inEastern Tanzania Angiosperms (Passifloraceae to Sapindaceae). J. Ethnopharmacol., 33,143-157.Chiu, N. Y., Chang, K. H. 1987. The Illustrated Medicinal plants of Taiwan. 1, 230. Southernmaterials centre, inc., Taipei, Taiwan.Chukwujekwu, J. C., Lategan, C. A., Smith, P. J. 2009. Antiplasmodial and cytotoxic activityof isolated sesquiterpene lactones from the acetone extract of Vernonia colorata. S. Afr. J.Bot., 75, 176-179.Cioffi, G., Sanogo, R., Diallo, D., Romussi, G., De Tommasi, N. 2004. New compounds froman extract of Vernonia colorata leaves with anti-inflammatory activity. J. Nat. Prod., 67,389-394.Corsino, J., De Carvalho, P. R. F., Kato, M. J., Latorre, L. R., Oliveira, O. M. M. F., Araújo, R.A., Bolzani, V. S., França, C. S., Pereira, A. M. S., Furlan, M. 2000. Biosynthesis offriedelane and quinonemethide triterpenoids is compartmentalized in Maytenus aquifoliumand Salacia campestris. Phytochemistry, 55, 741-748Da Costa, J. F., Bandeira, N. P., Rose, M., Albuquerque, J. R. 2008. Chemical constituents ofVernonia chalybacea. Mart. Quim. Nova., 31, 1691-1695.Dagne, E., Yenesew, A., Waterman, P. G., Gray, A. I. 1988. The chemical systematics of theRutaceae subfamily Toddalioideae, in Africa. Biochem. System. Ecol., 16, 179-188.

P a g e | 53De, P., Baltas, M., Bedos-Belval, F. 2011. Cinnamic acid derivatives as anticancer agents. Areview. Curr. Med. Chem., 18, 1672-1703.Deborah, A., Lachman-white, Charles, D. A., Ulric, O. T. 1992. A guide to medicinal plants ofcoastal Guyana. Commonwealth secretariat publications, London, 294.Dewick, P. M. 2006. Medicinal Natural products. A biosynthetic approach, 2 nd edition, JohnWiley & Sons, London, 141-146, 168-219.Erasto, P., Grierson, D. S., Afolayan, A. J. 2006. Bioactive sesquiterpene lactones from theleaves of Vernonia amygdalina. J. Ethnopharmacol., 106, 117-120.Fatima, S. S., Rajasekhar, M. D., Kumar, K. V., Kumar, M. T., Babu, K. R., Rao, C. A. 2010.Antidiabetic and antihyperglylipidemic activity of ethyl acetate: isopropanol (1:1) fractionof Vernonia anthelmintica seeds in streptozotocin induced diabetic rats. Food Chem.Toxicol., 48, 495-501.Fernandes, da Silva, M. F., Gottlieb, O. R., Ehrendorfer, F. M. 1988. Chemosystematics ofRutaceae: Suggestions for a more natural taxonomy and evolutionary interpretation of thefamily. Plant Syst. Evol., 161, 97-134.Fratkin, E. 1996. Traditional medicine and concepts of healing Among Samburu pastoralists ofKenya. J. Ethnobiol., 16, 63-97Freire, M. F. I., Abreu, H. S., Cruz, L. C. H., Freire, R. B. 1996. Inhibition of fungal growth byextracts of Vernonia scorpioides (Lam.) Pers. Microbiology, 27, 1-6.Frutuoso, V. S., Gurjao, M. R., Cordeiro, R. S., Martins M. A. 1994. Analgesic and antiulcerogeniceffects of a polar extract from leaves of Vernonia condensata. Planta Med., 60,21-25.

P a g e | 54Funk, V. A., Bayer, R. J., Keeley, S., Chan, R., Watson, L., Gemeinholzer, B., Schilling, E.,Panero, J. L., Baldwin, B. G., Garcia-Jacas, N., Susanna, A., Jansen, R. K. 2005.Everywhere but Antarctica: using a supertree to understand the diversity and distribution ofthe Compositae. Biol. Skr., 55, 343-374.Ganguly, A. K., Govindachari, T. R., Manmade, A., Mohamed, P. A. 1966. Alkaloids of Veprisbilocularis. Indian J. Chem., 4, 334-335.Gani, A. 1998. Medicinal plants of Bangladesh: Chemical Constituents and Uses, AsiaticSociety of Bangladesh, Dhaka, Bangladesh.Geissler, P. W., Harris, S. A., Prince, R. J., Olsen, A., Odhiambo, R. A., Oketch-Rabah, H.,Madiega, P. A., Andersen, A., Molgaard, P. 2002. Medicinal plants used by Luo mothersand children in Bondo District, Kenya. J. Ethnopharmacol., 83, 39-54.Gessler, M. C., Nkunya, M. H. H., Mwasumbi, L. B., Schar, A., Heinrich, M., Tanner, M. 1994.Screening of Tanzanian medicinal plants for antimalarial activity. Acta Trop., 56, 65-77.Gessler, M. C., Tanner, M., Chollet, J., Nkunya, M. H. H., Heinrich, M. 1995a. Tanzanianmedicinal plants used traditionally for the treatment of malaria: In vivo antimalarial and invitro cytotoxic activities. Phytother. Res., 9, 504-508.Gessler, M. C., Msuya, D. E., Nkunya, M. H., Mwasumbi, L. B., Schar, A., Heinrich, M.,Tanner, M. 1995b. Traditional healers in Tanzania: the treatment of malaria with plantremedies. J. Ethnopharmacol., 48, 131-144.Germano, M. P., De Pascuale, R., Iauk, L., Galati, E. M., Keita, A., Sanogo, R., 1996.Antiulcer activity of Vernonia kotschyana Sch. Bip. Phytomedicine, 2, 229-233.

P a g e | 55Gomes, E., Dellamonica, G., Gleye, J., Moulis, C., Chopin, J. Stanislas, E. 1983. Phenoliccompounds from Vepris heterophylla. Phytochemistry, 22, 2628-2629.Gomes, E. T., Travert, S., Gleye, J., Moulis, C., Fouraste, I., Stanislas, E. 1994. Furoquinolinealkaloids from Vepris heterophylla. Planta Med., 60, 338-339Govindachari, T. R., Sundararajan, V. N. 1961. Alkaloids of Vepris bilocularis. J. Sci. Ind.Res., 20B, 298-299.Govindachari, T. R., Joshi, B. S., Sundararajan, V. N. 1964.Structure of veprisone, aconstituent of Vepris bilocularis. Tetrahedron, 20, 2985-2990.Groppo, M., Pirani, J. R., Salatino, M. L. F., Blanco, S. R., Kallunki J. A. 2008. Phylogeny ofRutaceae based on two noncoding regions from cpdna. Am. J. Bot., 95, 985-1005.Grundon, M. F. 1988.The Alkaloids: Quinoline Alkaloids Related to Anthranilic Acid,Academic Press, London, 32, pp. 341.Gundidza, M. 1986.Screening of extracts from Zimbabwean higher plants II: antifungalproperties. Fitoterapia, 57, 111-114.Gurib-Fakim, A., Sweraj, M. D., Gueho, J., Dullo, E. 1996. Medicinal plants of Rodrigues. Int.J. pharmacogn., 34, 2-14.Gupta, M., Mazumder, U. K., Manikandan, L., Haldar, P. K., Bhattacharya, S., Kandar, C. C.2003a. Antibacterial activity of Vernonia cinerea. Fitoterapia, 74, 148-150.Gupta, M., Mazumder, U. K., Manikandan, L., Bhattacharya, S., Haldar, P. K., Roy, S. 2003b.Evaluation of Antipyretic Potential of Vernonia cinerea extract in rats. Phytother. Res., 17,804-806.

P a g e | 56Haensel, R., Cybulski, E. M. 1978. Alkaloids from the root bark of Vepris pilosa. Arch. Pharm.,(Weinheim, Germany), 311, 135-138.Hahlbrock, K., Grisebach, H. 1975. Biosynthesis of flavonoids, in the flavonoids Ed. Harborne,J. B., Mabry, T. J., Mabry H., Chapman and Hall, London, pp 866-900.Hedberg, I., Hedberg, O., Madati, P. J., Msigen, K. E., Mshiu, E. N., Samuelsson, G. 1983.Inventory of plants used in traditional medicine in Tanzania. Part III, Plants of the familiesPapilionaceae-Vitaceae. J. Ethnopharmacol., 9, 237-260.Huang, Y. Ding, Z. H., Liu, J. K. 2003. A New Highly Oxygenated Flavone from Vernoniasaligna. Z. Naturforsch., 58c, 347-350.Huang, Y., Liu, J. K. 2004. Triterpenoids from Vernonia saligna. Chinese J. Appl. Environ.Biol.,10, 51-52.Huffman, M. A., Gotoh, S., Izutsu, D., Koshimizu, K., Kalunde, M. S. 1993.Furtherobservations on the use of the medicinal plant Vernonia amygdalina (Del.), by a wildchimpanzee, its possible effect on parasite load, and its Phytochemistry. Afr. Stud. Monogr.,14, 227-240.Innocent, E., Moshi, M., Masimba, P., Mbwambo, Z., Kapingu, M., Kamuhabwa, A. 2009.Screening of traditionally used plants for in vivo antimalarial activity in mice. Afr. J. Trad.Complem. Altern. Med., 6, 163-167.Iroanya, O., Okpuzor, J., Mbagwu, H. 2010. Anti-nociceptive and antiphlogistic actions of apolyherbal decoction. Int. J. Pharmacol., 6, 31- 36.

P a g e | 57Izevbigie, E. B., Bryant, J. L., Walker, A. 2003. Edible Vernonia amygdalina leaf extractinhibits extracellular signal-regulated kinases and human breast cancer cell growth. J. Nutr.,133, 3860.Jones, S. B. 1977. Vernonieae-Systematic Review. In: Heywood, V.H., Harborne,J.B., Turner, B.L. (Eds.). The Biology and Chemistry of the Compositae. AcademicPress, London, pp. 503-521.Kan-Fan, C., Das Bhupesh, C., Boiteau, P., Potier, P. 1970. Madagascan plants:Alkaloids of Vepris ampody. Phytochemistry, 9, 1283-1291.Keeley, S. C., Forsman, Z. H., Chan, R. 2007. A phylogeny of the “evil” (Vernoniaea:Compositae) reveals Old/New World long distance dispersal: Support from separate andcombined congruent data sets. Mol. Phylogenet. Evol., 44, 89-103.Khare, C. C. 2007. Indian medicinal plants, An illustrated Dictonary Springer Verlag Berlin,pp. 699-700.Khalid, S. A., Waterman, P. G. 1982. Chemosystematics in the Rutaceae. 15. Furoquinolineand pyrano-2-quinolone alkaloids of Vepris stolzii. J. Nat. Prod., 45, 343-346.Koffi, Y., Gleye, J., Moulis, C., Stanislas, E. 1987. Acridones from Vepris fitoravina and Veprismacrophylla. Planta Med., 53, 570-571.Kumar, P. P., Kuttan, G. 2009. Vernonia cinerea L., scavenges free radicals and regulates nitricoxide and proinflammatory cytokines profile in carrageen, an induced paw edema model.Immunopharmacol. Immunotoxicol., 31, 94-102.

P a g e | 58Kupchan, S. M., Hemingway, R. J., Werner, D., Karim, A. 1969. Tumor inhibitors. XLVI.Vernolepin, a sesquiterpene dilactone inhibitor from Vernonia hymenolepsis A. Rich. J.Org. Chem., 34, 3903-3908.Latha, Y. L., Darah, I., Sasidharan, S., Jain, K. 2009.Antimicrobial activity of Emiliasonchifolia D C., Tridax procumbens L. and Vernonia cinerea L. of the Asteraceae Family:potential as food preservatives. Mal. J. Nutr., 15, 223-231.Latha, R. M., Geetha, T., Varalakshmi, P. 1998. Effect of Vernonia cinerea Less flower extractin adjuvant-induced arthritis. Gen. Pharmacol., 31, 601-606.Liang, Q. L., Min, Z. D. 2003. Studies on the Constituents from the Herb of Vernonia patula.Zhongguo Zhongyao Zazhi, 28, 235-237Liang, Q. L., Jiang, J. H., Min, Z. D. 2010. A Germacrane Sesquiterpenoid from Vernoniapaltula. Chinese J. Nat. Med., 8, 104-106.Lin, R., Chen, Y. L., Shi, Z. 1985. In flora of China (Zhongguo Zhiwu zhi) Science press:Beijing, 74, 21.Louppe, D., Oteng-Amoako, A. A., Brink, M. 2008.Plant Resources of Tropical Africa(PROTA) Timbers 1, 574-578.Malafronte, N., Pesca, M., Sabina, B., Angela, E., Luis, M. T. N. 2009. New flavonoidglycosides from Vernonia ferruginea. Nat. Prod. Comm., 4, 1639-1642.Marita, I. G., Keith, M., Colin, M. 1999. Piggin upland Rice weeds of South and SoutheastAsia, International Rice Research Institute, Manila, 26-27.

P a g e | 59Mazumder, U. K., Gupta, M., Manikandan, L., Bhattacharya, S., Haldar, P. K., Roy, S. 2003.Evaluation of anti-inflammatory activity of Vernonia cinerea Less. Extract on rats.Phytomedicine, 10, 185-188.Mbala, P. B. M. 2005. A chemical investigation of Two South African Vepris species. MastersThesis, University of KwaZulu-Natal, pp 22-73.Misra, T. N., Sigh, R. S., Upadhyay, J., Srivastava, R. 1984a.Chemical constituents ofVernonia cinerea, Part 1. Isolation and spectral studies of triterpenes. J. Nat. Prod., 47, 368-372.Misra, T. N., Sigh, R. S., Upadhyay, J., Srivastava, R. 1984b.Chemical constituents ofVernonia cinerea, Isolation and structure elucidation of a new pentacyclic triterpenoid. J.Nat. Prod., 47, 865-867.Misra, T. N., Sigh, R. S., Srivastava, R., Pandey, H. S., Prasad, C., Singh, S. 1993. A newtriterpenoid from Vernonia cinerea, Planta Med., 59, 458-460.Mollik, Md. A. H., Hossan, Md. S., Paul, A. K., Taufiq-Ur-Rahman, M., Jahan, R.,Rahmatullah, M. 2010. A Comparative analysis of medicinal plants used by Folk Medicinalhealers in three Districts of Bangladesh and inquiry as to mode of selection of medicinalplants. Ethnobot. Res. Appl., 8, 195-218.Momeni, J., Ntchatchoua, D. W. P., Fadimatou, A. M. T., Ngassoum, M. B. 2010. Antioxidantactivities of some Cameroonian plants extracts used in the treatment of intestinal andinfectious diseases. Indian J. Pharm. Sci., 72, 140-144.

P a g e | 60Moundipa, P. F., Flore, K. G. M., Bilong, C. F. B., Bruchhaus, I. 2005. In vitro amoebicidalactivity of some medicinal plants of the Bamun region (Cameroon). Afr. J. Trad. Compl. Alt.Med., 2, 113-121.Mungarulire, J., Franz, C. H., Seitz, R. Y., Verlet, N. 1993. Some developments in the searchfor cytotoxic constituents from Rwandese medicinal plants. Acta hort., 333, 211-216.Morales-Escobar, L., Braca, A., Pizza, C., Tommasi, N. 2007. New phenolic derivatives fromVernonia mapirensis Gleason. ARKIVOC (Gainesville, FL, United States), 7, 349-358.Narain, N. K. 1979. Fasciculatol, new tetracyclic triterpenoid ether from Vernonia fasciculatamichx. Can. J. Pharm. Sci., 14, 33-35.Narod, F. B., Gurib-Fakim, A., Subratty, A. H. 2004. Biological investigations into Antidesmamadagascariense Lam. (Euphorbiaceae), Faujasiopsis flexuosa (Lam.) C. Jeffrey(Asteraceae), Toddalia asiatica (L.) Lam. and Vepris lanceolata (Lam.) G. Don (Rutaceae).J. Cell Mol. Biol., 3, 15-21.Nergard, C. S., Diallo, D., Michaelsen, T. E., Malterud, K. E., Kiyohara, H., Matsumoto, T.,Yamada, H., Paulsen, B. S. 2004. Isolation, partial characterisation and immunomodulatingactivities of polysaccharides from Vernonia kotschyana Sch. Bip. Ex Walp. J.Ethnopharmacol., 91, 141-152.Nduagu, C., Ekefan, E. J., Nwankiti, A. O. 2008. Effect of some crude plant extracts on growthof Colletotrichum capsici (Synd.) Butler and Bisby, causal agent of pepper anthracnose. J.Appl. Biosci., 6, 184-190.Ngadjui, T. B., Ayafor, J. F., Sondengam, B. L., Connolly, J. D., Rycroft, D. S., Khalid, S. A.,Waterman, P. G., Brown, N. M. D., Grundon, M. F., Ramachandran, V. N. 1982. The

P a g e | 61structures of vepridimerines A-D, four new dimeric prenylated quinolone alkaloids fromVepris louisii and Oricia renieri (Rutaceae). Tetrahedron Lett., 23, 2041-2044.Ogbulie, J. N., Ogueke, C. C., Nwanebu, F. C. 2007. Antibacterial properties of Uvariachamae, Congronema latifolium, Garcinia kola, Vernonia amygdalina and Aframomiummelegueta. Afr. J. Biotechnol., 6, 1549-1553.Ohigashi, H., Takagaki, T., Koshimizu, K., Watanabe, K., Kaji, M., Hoshino, J., Nishida, T.,Huffman, M. A., Takasaki, H., Jato, J., Muanza, D. N. 1991. Biological activities of plantextracts from tropical Africa. Afr. Stud. Monogr., 12, 201-210.Oketch-Rabah, H. A., Lemmich, E., Dossaji, S. F., Theander, T. G., Olsen, C. E., Cornett, C.,Arsalan, K., Christensen, B. S. 1997. Two new antiprotozoal 5-methylcoumarins fromVernonia brachycalyx. J. Nat. Prod., 60, 458-461.Oketch-Rabah, H. A., Brøgger, C. S., Frydenvang, K., Dossaji, S. F., Theander, T. G., Cornett,C., Watkins, W. M., Kharazmi, A., Lemmich, E. 1998. Antiprotozoal properties of 16,17-dihydrobrachycalyxolide from Vernonia brachycalyx. Planta Med., 64, 559-562.Olorode, O. 1984. Taxanomy of West Africa Flowering Plants, 1 st edn., pp. 98-100, Longman,London.Otari, K. V., Shete, R. V., Upasani, C. D., Adak, V. S., Bagade, M. Y., Harpalani, A. 2010.Evaluation of anti-inflammatory and anti-arthritic activities of the ethanolic extract ofVernonia anthelmintica seeds, J. Cell Tissue Res., 10, 2269-2280.Pagno, T., Blind, L. Z., da Saúde, C. C., Biavatti, W., Kreuger, M. R. O. 2006. Cytotoxicactivity of the dichloromethane fraction from Vernonia scorpioides (Lam.) Pers.(Asteraceae) against Ehrlich’s tumor cells in mice. Braz. J. Med. Biol. Res., 39, 1483-1491.

P a g e | 62Parekh, J., Chanda, S. V. 2008. Antibacterial activity of aqueous and alcoholic extracts of 34Indian Medicinal Plants against some Staphylococcus species. Turk J. Biol., 32, 63-71.Pereira, N. A., Pereira, B. M. R., Nascimento, M. C., Parente, J. P., Mors, W. B. 1994. Folkmedicine as snake venomantidotes: protection against jararaca venom by isolatedconstituents, Planta Med., 60, 99.Poitou, F., Masotti, V., Viano, J., Gaydou, E. M., Andriamahavo, N. R., Mamitiana, A.,Rabemanantsoa, A., Razanamahefa, B. V., Andriantsiferana, M. 1995.ChemicalComposition of Vepris elliotii essential oil. J. Essential Oil Res., 7, 447-449.Poullain, C. Girard-Valenciennes, Smadja, J. 2004. Plant from Reunion Island: evaluation oftheir free radical scavenging and antioxidant activities. J. Ethnopharmacol., 95, 19-26.Pratheeshkumar, P., Kuttan, G. 2010. Modulating of immune response by Vernonia cinerea L.inhibits the proinflammatory cytokines profile, iNOS, and COX-2 expression in LPSstimulatedmacrophages. Immunopharmacol. immunotoxicol., 6, 1-11.Rabe, T., Mullholland, D., van Staden, J. 2002. Isolation and identification of antibacterialcompounds from Vernonia colorata leaves. J. Ethnopharmacol., 80, 91-94.Rakotondraibe, L. H. R., Rakotovao, M., Ramanandraibe, V., Ravaonindrina, N., Frappier, F.,Brouard, J. P. 2001.Composition and antimicrobial activity of leaf oils of Veprisleandriana H. Perr. J. Essential Oil Res., 13, 467-469.Randrianarivelojosia, M., Rasidimananal, V. T., Rabarison, H., Cheplogoi, P. K., Ratsimbason,M., Mulholland, D. A., Mauclère, P. 2003. Plants traditionally prescribed to treat tazo(malaria) in the Eastern region of Madagascar. Malaria J., 2, 1-9.

P a g e | 63Rasoanaivo, P., Ratsimamanga-Urverg, S., Ramanitrahasimbola, D., Rafatro, H., Rakoto-Ratsimamanga, A. 1999.Criblage déxtraits de plantes de Madagascar pour recherchedáctivité antipaludique et déffet potentialisateur de la chloroquine. J. Ethnopharmacol., 64,117-126.Ratsimbason, M., Ranarivelo, L., Juliani, H., Simon, J. E. 2009. African Natural plant products.New discoveries and challenges in Chemistry and quality. Antiplasmodial Activity ofTwenty Essential Oils from Malagasy Aromatic Plants. Chapter 11, 209-215.Risso, W. E., Scarminio, I. S., Moreira, E. G. 2010.Antinociceptive and acute toxicityevaluation of Vernonia condensata Baker leaves extracted with different solvents and theirmixtures. Indian J. Exp. Biol., 48, 811-816.Rotimi, V. O., Mosadomi, H. A., 1987. The effect of crude extracts of nine African chewingsticks on oral anaerobes. J. Med. Microbiol., 23, 55-60.Sanogo, R., Germano, M. P., de Tommasi, N., Pizza, C., Aquino, R. 1998. Vernoniosides andan androstane glycoside from Vernonia kotschyana. Phytochemistry, 47, 73-78.Sanogo, R., de Pasquale, R., Germano, M. P. 1996.Vernonia kotschyana Sch. Bip.:Tolerability and Gastroprotective Activity. Phytother. Res., 10, 169-171.Sikkema, J., De Bont, J. A. M., Poolman, B. 1995. Mechanisms of membrane toxicity ofhydrocarbons. Microbiol. Rev., 59, 201-222.Steenkamp, V. 2003.Traditional herbal remedies used by South African women forgynaecological complaints. J. Ethnopharmacol., 86, 97-108.

P a g e | 64Sy, G. Y., Nongonierma, R. B., Sarrr, M., Cisse, A., Faye, B. 2004. Antidiabetic activity of theleaves of Vernonia colorata (Wilid) Drake (Composes) in alloxan-induced diabetic rats.Dakar Med., 49, 36-39.Taylor, D. A. H. 1984. Progress in the Chemistry of Organic Natural Products, Herz, W.,Grisebach, H. and Kirby, G. W. Eds., Springer, New York. 45, pp. 1-56.Taiwo, O., Xu, H. X., Lee, S. F. 1999. Antibacterial activities of extracts from Nigerianchewing sticks. Phytotherapy Res., 13, 675-679.Taiwo, I. A., Odeigah, P. G. C., Ogunkanmi, L. A. 2009. The glycaemic effects of Vernoniaamygdalina and Vernonia tenoreana with tolbutamide in rats and the implications for thetreatment of diabetes mellitus. J. Sci. Res. Dev., 11, 122-130.Tekobo, A. M., Onabanjo, A. O., Amole, O. O., Emeka, P. M., 2002. Analgesic and antipyreticeffects of the aqueous extract of Vernonia amygdalina. West Afri. J. Pharm., 16, 68-74.Tripathi, R. N., Tripathi, R. K. R., Pandey, D. K. 1981. Assay of antiviral activity in the crudeleaf sap of some plants. Envir. India, 4, 86-87.Tchinda, A. T., Tsopmo, A., Tane, P., Ayafor, J. F., Connolly, J. D., Sterner, O. 2002.Vernoguinosterol and Vernoguinoside, a trypanocidal stigmastane derivative from Vernoniaguineensis (Asteraceae), Phytochemistry, 59, 371-374.Vera, R., Smadja, J., Conan, J. Y. 1990. Preliminary assay of plants with alkaloids fromReunion Island. Plant. Med. Phytother., 24, 50-65.Vlietinck, A. J., Hoof, L. V., Totte, J., Lasure, A., Berghe, D. V. 1995. Screening of hundredRwandese medicinal plants for antimicrobial and antiviral properties. J. Ethnopharmacol.,46, 31-47.

P a g e | 65Waterman, P. G. 1986. The dimeric alkaloids of the Rutaceae derived by Diels-Alder addition,in alkaloids: chemical and biological perspectives, (S.W Pelletier, Ed). Wiley New York. 4,pp. 331-387.Williams, R. B., Norris, A., Slebodnick, C., Merola, J., Miller, J. S., Andriantsiferana, R.,Rasamison, V. E., Kingston, D. G. I. 2005. Cytotoxic sesquiterpene lacones from Vernoniapachyclada from the Madagascar rainforest. J. Nat. Prod., 68, 1371-1374.Yeap, S. K., Ho, W. Y., Beh, B. K., Liang, W. S., Ky, H., Yousr, A. H. N., Alitheen, N. B.2010. Vernonia amygdalina, an ethnoveterinary and ethnomedical used green vegetablewith multiple bioactivities. J. Med. Plant Res., 4, 2787-2812.Yuan, K., Jian, A., Cai, X. I. 2008. A new triterpene from Vernonia chunii. Chinese Tradit.Herb. Drugs, 39, 2, 168-169.Zanon, R. B., Pereira, F. D., Boschetti, K. T., dos Santos, M., Athayde, L. M. 2008.Fitoconstituintes isolados da fracão em dichlorometano das folhas de Vernonia tweedianaBaker. Revista Brasileira de Farmacognosia Brazilian J. pharmacog., 18, 226-229.Zhonghuabencao Editorial board, Zhonghuabencao, Shanghai Scientific and TechnologicalPress, Shanghai, 1999, Vol. 7, pp. 1001.Zulak, K. G., Bohlmann, J. 2010. Terpenoid Biosynthesis and Specialized Vascular Cells ofConifer Defense. J. Integr. Plant Biol., 52, 86-97.

P a g e | 66CHAPTER TWOTRITERPENOIDS FROM VERNONIA AURICULIFERAHIERN EXHIBIT ANTIMICROBIAL ACTIVITYJoyce Jepkorir Kiplimo 1* , Hafizah Chenia 2 , Neil Anthony Koorbanally 11 School of Chemistry, University of KwaZulu–Natal, Durban 4000, South Africa2 School of Biochemistry, Genetics and Microbiology, University of KwaZulu–Natal, SouthAfrica*Author to whom correspondence should be addressed; E-Mail: jjkiplimo@yahoo.com;Tel: +27-720-334-548ABSTRACTPhytochemical investigation ofVernonia auriculifera afforded farnesylamine, asesquiterpene amine that has not been found previously in plant species, together withlupenyl acetate, oleanolic acid, β-amyrin acetate, β-amyrin, friedelanone, friedelin acetate,α-amyrin and β-sitosterol. The compounds were characterized using NMR spectroscopyand by comparison with literature values. The isolated triterpenoids exhibited moderateantibacterial activity, α and β-amyrin had MIC values of 0.25 mg/mL against S. aureus, B.subtilis, E. faecium and S. saprophyticus while lupenyl acetate and oleanolic acid exhibitedMIC values of 0.25 mg/mL against S. maltophilia. Sub-MIC exposure of β-amyrin acetatewas effective in decreasing adhesion of S. aureus, K. pneumonia and E. faecium whileoleanolic acid decreased adhesion of K. pneumonia and P. aeruginosa significantly at sub-MIC concentrations. These compounds show potential for synergistic coupling withantimicrobial agents to improve therapeutic efficiency in the face of rising bacterialresistance.

P a g e | 67Keywords: Vernonia auriculifera; triterpenoids; farnesylamine; antibacterial activityINTRODUCTIONThe genus Vernonia (Asteraceae family) has more than 1000 species growing all over theworld with more than 30 species growing in Kenya (Beentje, 1994; Oketch-Rabah et al.,1997). Vernonia auriculifera is a small tree or woody herb that grows between 1-7.5 m highand is easily recognizable by its deep purple flowers. V. auriculifera has a wide variety ofapplications in traditional medicine. A drop of the juice squeezed from the crushed stembark, inserted into the nostrils, is known to relieve headache (Kusamba, 2001). The Kikuyupeople of central Kenya use the leaves of this plant as a wrap for pounded material used as apoultice (Kokwaro, 1976). Heated crushed leaves of Aspilia mossambicensis, are tied in theleaf of V. auriculifera, and then applied over the eyes to treat conjunctivitis (Muthaura et al.,2007). A cold water infusion of V. auriculifera is administered orally in Uganda and Kenyato treat fever associated with viral and bacterial infections (Muthaura et al., 2007;Freiburghaus et al., 1996). In Ethiopia, the roots are used to treat toothache (Mirutse et al.,2009) and snake poison (Mesfin et al., 2009).Hydroperoxides of unsaturated fatty acid methyl esters previously isolated from V.auriculifera were found to have lethal toxicity (Keriko et al., 1995a).Plant growthstimulators have also been identified from this plant (Keriko et al., 1995b). Other Vernoniaspecies that have received extensive phytochemical and pharmacological research include:V. galamensis (Miserez et al., 1996), V. brachycalyx (Oketch-Rabah et al., 1997), V.colorata (Rabe et al., 2002), V. amagdalina (Erasto et al., 2006), V. cinerea (Chen et al.,2006), V. mapirensis (Morales-Escobar et al., 2007), V. cumingiana (Mao et al., 2008), V.ferruginea (Malafronte et al., 2009) and V. scorpioides (Buskuhl et al., 2010). Members of

P a g e | 68the genus Vernonia are an excellent source of sesquiterpene lactones which includevernolide, vernolepin, vernodalin and hydroxyvernolide (Kupchan et al., 1969; Jisaka et al.,1993; Koshimizu et al., 1994). Other compounds have also been isolated from this genussuch as triterpenoid glycosides, flavonoids, coumarins and benzofuranones (Miserez et al.,1996; Oketch-Rabah et al., 1997; Mao et al., 2008).The current study was undertaken primarily to investigate the phytochemistry of V.auriculifera from which only fatty acids were previously isolated and to test the isolatedcompounds for antimicrobial activity since extracts of Vernonia species have been cited asantimicrobials in traditional medicine (Kokwaro, 1976).MATERIALS AND METHODSGeneral experimental procedureNMR spectra were recorded using a Bruker Avance III 400 MHz spectrometer. All thespectra were recorded at room temperature with all chemical shifts (δ) recorded against theinternal standard, tetramethylsilane (TMS). IR spectra were recorded on a Perkin ElmerSpectrum 100 FT-IR spectrometer with universal ATR sampling accessory. For GC-MSanalyses, the samples were analysed on an Agilent GC–MSD apparatus equipped with DB-5SIL MS (30 m x 0.25 mm i.d., 0.25 µm film thickness) fused-silica capillary column.Helium (at 2 ml/min) was used as a carrier gas. The MS was operated in the EI mode at 70eV. Optical rotation was recorded using a PerkinElmerTM, Model 341 Polarimeter.Melting points were recorded on an Ernst Leitz Wetzlar micro-hot stage melting pointapparatus.

P a g e | 69Plant MaterialThe leaves, stem bark and root bark of V. auriculifera were collected in August, 2009 fromEgerton University Botanical Garden, Rift Valley Province in Kenya.The plant wasidentified by taxonomist, Dr S. T. Kariuki, of the Botany Department, Egerton University,Kenya and a voucher specimen (Kiplimo, 02) was deposited in the Ward Herbarium,University of KwaZulu-Natal Westville, Durban, South Africa.Extraction and isolationThe air-dried and ground plant material of V. auriculifera (823 g leaves, 710 g roots, 600 gstems) was sequentially extracted with organic solvents in order of increasing polarity viz;hexane, dichloromethane, ethyl acetate and methanol using a Soxhlet apparatus for 24 h ineach case. The yields obtained for each solvent were, hexane 66.68 g (leaves), 9.10 g(roots), 16.73 g (stems); dichloromethane 16.77 g (leaves), 5.13 g (roots), 9.25 g (stems);ethyl acetate 8.28 g (leaves), 0.93 g (roots), 5.02 g (stems) and methanol, 27.01 g (leaves),20.28 g (roots), 15.09 g (stems).Isolation and purification of compounds 1, 3, 4, 5, 8 and 9The hexane extract from the leaves (30 g) was separated by column chromatography using astep gradient of hexane: dichloromethane: ethyl acetate gradient, starting with 100% hexanestepped to 10%, 20%, 30%, 50%, 80% and 100% dichloromethane, followed by 20% and30% ethyl acetate in dichloromethane. Twenty fractions of 100 mL each were collected ineach step. Fractions 5-12 were combined and purified using 100% hexane, to producefarnesylamine (9) (12 mg).Fractions 21-25 were recrystallised in methanol to yieldsitosterol (8) (78 mg). Fractions 41-67 were combined and separated with 20% and 30%

P a g e | 70dichloromethane in hexane. Lupenyl acetate (1) (52 mg) was obtained in fractions 8-12while fraction 18-35 was further purified using 20% ethyl acetate in hexane where fractions5-9 afforded β-amyrin acetate (4) (150 mg) and fractions 11-18 afforded a mixture of α-amyrin (5) and β-amyrin (3) (89 mg).Isolation and purification of compound 2The ethyl acetate extract (0.93 g) from the roots was dissolved in dichloromethane andseparated with a mobile phase consisting of a hexane: ethyl acetate step gradient 1:0(fractions 1-10), 9:1 (fractions 11-20), 7:3 (fractions 21-38), 6:4 (fractions 52-64) and 3:7(fractions 65-70). Fractions 22-27 were further purified with 20% ethyl acetate in hexane.Oleanolic acid (2) (25 mg) was obtained in fractions 9-13.Isolation and purification of compounds 6 & 7The hexane extract from the stems (16.73 g) was subjected to column chromatography. Themobile phase consisted of a hexane: dichloromethane step gradient; 1:0 (fractions 1-45), 9:1(fractions 46-66), 8:2 (fractions 67-80), 7:3 (fractions 81-98) and 1:1 (fractions 99-121).Friedelin acetate (7) (31 mg) was eluted in fraction 24-32 and the pure compound wasobtained by recrystallisation in methanol. Friedelanone (6) (120 mg) was obtained bypurification of fractions 55-80 using 10% dichloromethane in hexane as the mobile phasewhere the compound was eluted in fraction 7-15, followed by recrystallisation in methanol.Farnesylamine (9)White crystals, m/z (rel %): 221 [M] + , 206 (1), 189 (3), 179 (3), 161 (3); IR spectra (V maxcm -1 ): 3413, 2919, 1357, 1053; 1 H NMR spectral data (400 MHz, CDCl 3 ) δ H 5.11(H-2, 6,10), 2.07 (2H-1), 2.06 (2H-4), 2.05 (2H-9), 2.03 (2H-5), 1.69 (3H-12), 1.62 (3H-13, 3H- 14,

P a g e | 713H-15), 1.28 (2H-8); 13 C NMR spectral data (400 MHz) 134.82 (C-3), 134.16 (C-7), 130.97(C-11), 124.12 (C-6), 124.02 (C-10), 123.98 (C-2), 39.45 (C-1, 4), 29.42 (C-8), 27.99 (C-5),26.48 (C-9), 25.41 (C-12), 17.39 (C-13), 15.75 (C-14), 15.71 (C-15).BIOLOGICAL STUDIESMinimum inhibitory concentration (MIC)Four strains of Gram-negative bacteria (Escherichia coli ATCC 25922, Pseudomonasaeruginosa ATCC 35032, Klebsiella pneumonia ATCC 700603 and Stenotrophomonasmaltophilia ATCC 13637) and five Gram-positive bacteria (Staphylococcus aureus ATCC25923, Bacillus subtilis ATCC 6051, Enterococcus faecium ATCC 19434, Staphylococcusepidermidis ATCC 14990 and Staphylococcus saprophyticus ATCC 35552) were selectedfor the determination of antimicrobial activity.The antibacterial activities of the compounds were determined using the broth microdilutionmethod as described by Andrews (2001). Bacterial strains were cultured for 18 h at 37 o C inTryptone Soy Broth (TSB) and standardized to a final cell density of 1.5×10 8 cfu/mLequivalent to 0.5 McFarland Standard. The 96-well plates were prepared by dispensing intoeach well, 90 µL Muller-Hinton (MH) broth and 10 µL of the bacterial inoculum. Testcompounds were dissolved in dimethyl sulfoxide (DMSO) to a concentration of 10 mg/mLwhile tetracycline (a broad-spectrum antimicrobial agent) the positive control was dissolvedin ethanol. Serial two-fold dilutions were made in a concentration range of 0.002 to 2mg/mL.Wells containing MH broth only were used as a medium control and wellscontaining medium and cultures without the test compound were used as the growth control.Plates were covered to avoid contamination and evaporation and incubated for 24 h at 37 o C.The minimum inhibitory concentration (MIC) was described as the lowest concentration of

P a g e | 72the test compounds that completely inhibited the growth of microorganisms. The tests weredone in triplicate on two separate occasions and the results are as shown in table 2.Anti-biofilm activity evaluationTo determine the anti-biofilm activity of β-amyrin acetate and oleanolic acid, three strains ofGram-negative bacteria (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC35032 and Klebsiella pneumonia ATCC 700603) and four Gram-positive bacteria(Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 43300, Enterococcusfaecium ATCC 19434, and Staphylococcus saprophyticus ATCC 35552) were used.Bacterial isolates were cultured overnight in TSB to determine the effect of MIC, sub-MIC(0.5×MIC) and supra-MIC (2×MIC) exposures on biofilm formation. Cells were washedand resuspended in distilled water to a turbidity equivalent to a 0.5 McFarland standard.Wells of sterile, 96-well U-bottomed microtiter plates were each filled with 90 µL LuriaBertani broth (LB) and 10 µL of cell suspension, in triplicate. Based on individual MICs foreach isolate the effect of MIC, sub-MIC and supra-MIC of β-amyrin acetate and oleanolicacid on bacterial adhesion was investigated. Plates were incubated aerobically at 37 ˚C for24 h with shaking on an Orbit P4 microtitre plate shaker (Labnet).The contents of each well were aspirated and then washed three times with 250 µL of steriledistilled water. To remove all the non-adherent bacteria, the plates were vigorously shakenand the remaining attached cells were fixed with 200 µL of 99% methanol per well. After15 min, plates were left to dry and then stained for 5 min with 150 µL of 2% Hucker crystalviolet. Excess stain was washed with running tap water and plates were left to air dry(Basson et al., 2008). The bound stain was resolubilised with 150 µL of 33% (v/v) glacial

P a g e | 73acetic acid per well. The Optical Density (OD) of the contents of each well was obtained at595 nm using the Fluoroskan Ascent F1 (Thermolabsystems).Tests were done in triplicate on two separate occasions and the results were averaged(Stepanović et al., 2000). The negative control for both assays was un-inoculated LB, whilethe positive control was tetracycline, with respective cell suspensions without β-amyrinacetate or oleanolic acid. OD 595nm values of treated cells were compared with untreated cellsto investigate the increase/decrease of biofilm formation as a result of antimicrobial agentexposure. Treated and untreated samples were compared statistically using paired t-testsand Wilcoxon signed rank tests if normality failed using a SigmaStat V3.5, Systat Software.RESULTS AND DISCUSSIONThe phytochemical investigation of V. auriculifera led to the isolation of eight triterpenoids1-8 and a sesquiterpene amine (9) (Figure 1). Extracts from the leaves were found tocontain the sesquiterpene amine along with one lupane-type triterpenoid (lupenyl acetate 1),one ursane-type triterpenoid (α-amyrin 5), two oleanane-type triterpenoids (β-amyrin 3 andβ-amyrin acetate 4) and a common steroid (sitosterol 8) (Figure 1). The stem bark affordedfriedelanone (6) and friedelin acetate (7) belonging to the friedelane class. From the roots,oleanolic acid (2), the parent oleanane type triterpene, was isolated. Compounds 1-8 wereidentified using 2D NMR spectral data and by comparison with literature values, whichsupported the structures as lupenyl acetate (Jamal et al., 2008), oleanolic acid (Seebacher etal., 2003), β-amyrin, β-amyrin acetate, friedelin acetate and α-amyrin (Mahato and Kundu,1994), friedelanone (Igoli and Gray, 2008) and sitosterol (Kamboj and Saluja, 2011).Although farnesylamine (9) was previously reported (Jones et al., 2003), here we arereporting the complete data for the first time.

P a g e | 74Compound 9 was isolated as a colourless oily liquid; its molecular formula was assigned asC 15 H 27 N. The IR spectrum showed the presence of a primary amine (3413 cm -1 ) and (1375cm -1 ). The 13 C NMR spectrum showed the presence of six olefinic carbon resonances, 3-protonated carbon resonances at δ C 124.12 (C-6), 124.02 (C-10) and 123.98 (C-2) and 3-non-protonated carbons at δ C 130-135. The olefinic methine resonances could also be seenat δ H 5.11 in the 1 H NMR spectrum. The methylene carbon resonances were observedbetween δ C 26.48 and δ C 29.42 except for the methylene bonded to the amine group whichwas observed downfield at δ C 39.47. All the methylene proton resonances, including 2H-1were present at δ H 2.05 except for one methylene resonance which appeared upfield at δ H1.27. Three of the four methyl proton resonances overlap at δ H 1.62 (3H-13, 14, 15) and oneis in a different chemical environment at δ H 1.69 (3H-12). The methyl carbon resonancescan be seen in the 13 C NMR spectrum between δ C 25.41 and δ C 15.71. This compound hasbeen detected in an extract of the ant Monomorium fieldi Forel from Australia (Jones et al.,2003) and has only now been found in a plant species.

P a g e | 75301229201918212230202925 11 2612910 83H 3 COCO57632 31 423 2413 171416152728R 1R 21 lupenyl acetate 2 R 1 = OH; R 2 = COOH (oleanolic acid)3 R 1 = OH; R 2 = CH 3 (β−amyrin)4 R 1 = OAc; R 2 = CH 3 (β−amyrin acetate)302927HOR265 α-amyrin6 friedelanone7 friedelin acetateR = =OR = OCOCH 3NH 2HO8 sitosterol9 FarnesylamineFigure 1: Structures of compounds (1-9) isolated from Vernonia auriculifera.The triterpene family of compounds to which all the isolated compounds belong arereported to possess antibacterial activity (Collins and Charles, 1987). The sesquiterpene,farnesylamine, could not be screened for antibacterial activity due to sample decomposition.MIC values recorded for all tested compounds (Table 2) suggested moderate antibacterialactivity. The most active compounds were amyrins (mixture of α-and β-), with MICs of0.12 mg/mL against E. coli, 0.25 mg/mL against S. aureus, B. subtillis, E. feacalis, S.

P a g e | 76saprophyticus, and 0.5mg/mL against S. epidermis, K. pneumonia, and S. maltophilia. Theother compounds 3-7 had a MIC of 0.5 mg/mL against S. maltophilia. The least activecompounds were 6 and 7 with a MIC of 1.0 mg/mL against six microorganisms. All testedcompounds had MICs of 1.0 mg/mL for P. aeruginosae and 0.5 mg/mL for S. maltophilia.The oleanane triterpernoids (2-4) displayed better antibacterial activity than the friedelanetriterpenoids (6-7). It is reported that the 28-COOH and ester functionality at C-3contributes to pharmacological activities of pentacyclic triterpenes (Mallavadhi et al., 2004)like lupenyl which has greater antimutagenic activity than lupenyl acetate (Guevara et al.,1996). These effects are observed for friedelanone and friedelin acetate where the ketonehas higher activity against Bacillus subtilis than the ester.Table 1: Minimum inhibitory concentrations (MIC in mg mL -1 ) of compounds isolatedfrom V. auriculiferaMIC (mg mL -1 ) of the test organismsMicrooganism 1 2 3 & 5 4 6 7S. aureus 1.0 0.5 0.25 1.0 1.0 1.0B. subtilis 1.0 0.5 0.25 1.0 0.25 1.0E. faecium 0.5 1.0 0.25 0.5 1.0 1.0S. epidermidis 1.0 0.25 0.5 1.0 0.5 0.5S. saprophyticus 0.25 1.0 0.25 1.0 1.0 1.0E. coli 0.12 1.0 0.12 0.5 1.0 0.5K. pneumonia 1.0 0.5 0.5 1.0 1.0 1.0P. aeruginosa 1.0 1.0 1.0 1.0 1.0 1.0St. maltophilia 0.25 0.25 0.5 0.5 0.5 0.5Key: 1= lupenyl acetate, 2 = oleanolic acid, 3 = β-amyrin, 4 = β-amyrin acetate,5 = α−amyrin, 6 = friedlanone, 7 = friedlin acetateBiofilm is a microbially derived sessile community characterized by cells that areirreversibly attached to a substratum or interface or to each other and are embedded in amatrix of extracellular polymeric substances they have produced. When planktonic bacteriaadhere to surfaces, they initiate biofilm formation. The nature of biofilm structure and

P a g e | 77physiological attributes of biofilm organisms confer an inherent resistance to antimicrobialagents such as antibiotics, disinfectants or germicides (Donlan et al., 2002).β-amyrin acetate and oleanolic acid were tested for antibiofilm activity against seven strainsof bacteria. β-amyrin acetate decreased adhesion of S. aureus (ATCC 43300), K.pneumonia and E. faecium significantly at sub-MIC concentrations (figure 2). For K.pneumonia, this decreased adhesion was also seen at MIC concentrations and in S.saphrophyticus a marked decrease in adhesion was seen at MIC and supra MICconcentrations. Sub-MIC oleanolic acid exposure also decreased adhesion of K. pneumoniaand P. aeruginosa significantly (figure 3), but MIC and supra-MIC exposures of oleanolicacid increased adhesion of all tested bacterial strains. These results suggest that oleanolicacid and β-amyrin acetate that are relatively abundant, can be used at low concentrations todecrease adhesion of certain bacterial strains to abiotic surfaces. Since bacterial resistanceto antibiotics and their survival are associated with their ability to form biofilms (Donlan etal., 2002), compounds which decrease biofilm formation would be useful in being used inconjunction with other antibiotics to decrease bacterial resistance. Agents that decreaseadhesion of bacteria may also be useful in improving the efficacy of antibiotics and hygienein hospitals that have devices such as incubation tubes, catheters, artificial heart valves,water lines and cleaning instruments on which bacterial biofilm have been found (Donlan etal., 2002).

P a g e | 783.02.5UNTREATEDSUB-MICMICSUPRA-MICA dh eren ce (O D 595 n m )2.01.51.0E . coli A T C C 25922K . pneum oniae A T C C 700603P . aeruginosa A T C C 35032E . faecium A T C C 19434S . aureus A T C C 25923S . aureus A T C C 43300S . saprophyticus A T C C 355520.50.0IsolatesFigure 2: Antibiofilm results for β-amyrin acetate (4)

P a g e | 794.03.5UNTREATEDSUB-MICMICSUPRA-MIC3.0Adherence (OD 595 nm)2.52.01.51.0E. coli ATCC 25922K. pneumoniae ATCC 700603P. aeruginosa ATCC 35032E. faecium ATCC 19434S. aureus ATCC 25923S. aureus ATCC 43300S. saprophyticus ATCC 355520.50.0IsolatesFigure 3: Antibiofilm results for oleanolic acid (2)CONCLUSIONThis is the first report of a phytochemical investigation of V. auriculifera. The finding of asesquiterpene amine in V. auriculifera is unique as it has not been isolated from a plantspecies before. Although the genus Vernonia is known to be a rich source of sesquiterpenelactones, none were isolated from V. auriculifera. However, eight pentacyclic compoundswith moderate antibacterial activity were isolated. Oleanolic acid and β-amyrin acetateexhibited moderate anti-adhesion properties.These compounds show potential forsynergistic coupling with antimicrobial agents to improve therapeutic efficiency, in the faceof rising bacterial resistance, however this needs further investigation.

P a g e | 80ACKNOWLEDGEMENTSThe authors are thankful to the Organization for Women in Science for the DevelopingWorld (OWSDW) for the financial support and Chester Everia for organizing the collectionof the plant material.

P a g e | 81REFERENCESAndrews JM (2001).Determination of inhibitory concentrations. J. Antimicrob.Chemother., 48: 5-16.Beentje H (1994). Kenya Trees, Shrubs and Lianas. National Museums of Kenya, Nairobi,pp. 564-570.Buskuhl H, Oliveira LF, Blind ZL, Freitas RA, Barison A, Campos FR, Corilo YE, EberlinNM, Caramori GF, Biavatti MW (2010).Sesquiterpene lactones from Vernoniascorpioides and their in vitro cytotoxicity. Phytochemistry, 71:1539-1544.Chen X, Zhan ZJ, Yue JM (2006). Sesquiterpenoids from Vernonia cinerea. Nat. Prod. Res.Part B: Bioact. Nat. Prod., 23:1160.Collins MA, Charles HP (1987). Antimicrobial activity of carnosol and ursolic acid: twoanti-oxidant constituents of Rosmarinus officinalis L. Food Microbiol., 4: 311-315.Donlan RM, Costerton JW (2002). Biofilms: survival mechanisms of clinically RelevantMicro-organisms. Clin. Microbiol. Rev., 15: 167-193.Erasto P, Grierson DS, Afolayan AJ (2006). Bioactive sesquiterpene lactones from theleaves of Vernonia amygdalina. J. Ethnopharmacol., 106:117-120.Freiburghaus F, Ogwa NE, Nkunya MHH, Kaminsky R, Reto B (1996).In vitroantitrypanosomal activity of African plants used in traditional medicine in Uganda totreat sleeping sickness. Trop. Med. Intl. Health., 6: 765-777.Guevara AP, Amor E, Russell GR (1996). Antimutagens from Plumeria acunimata Ait.Mutation Research/Environmental mutagenesis and related subjects. 36: 67-72.

P a g e | 82Igoli OJ, Gray IA (2008). Friedelanone and other triterpenoids from Hymenocardia acida.Int. J. Phys. Sci., 3:156-158.Jamal AK, Yaacob WA, Din LBA (2008). Chemical Study on Phyllanthus reticulatus. J.Phys. Sci. 19:45-50.Jisaka M, Ohigashi H, Takegawa K, Huffman MA, Koshimizu K (1993). Antitumoral andantimicrobial activities of bitter sesquiterpene lactones of Vernonia amygdalina, apossible medicinal plant used by wild chimpanzees. Biochemistry, 57: 833-834.Jones TH, Clark DA, Heterick BE, Snelling RR (2003).Farnesylamine from the antMonomorium fieldi Forel. J. Nat. Prod., 66: 325-326.Kamboj A, Saluja AK (2011). Isolation of stigmasterol and β-sitosterol from petroleumether extract of aerial parts of Ageratum conyzoides Asteraceae). Int. J. Pharm. Pharm.Sci., 3:94-96.Keriko JM, Nakajima S, Baba N, Isozaki Y, Ikeda K, Iwasa J,Alam MK (1995a).Hydroperoxides of unsaturated fatty acid methyl esters from the Kenyan plant Vernoniaauriculifera (Asteraceae). Yukagaku, 44: 338-340.Keriko JM, Nakajima S, Baba N, Isozaki Y, Ikeda K, Iwasa J, Karanja PN (1995b). A plantgrowth regulator from Vernonia auriculifera (Asteraceae). Z. Naturforsch., 50: 455-458.Kokwaro JO (1976). Medicinal Plants of East Africa. East Africa Education Publishers, pp.56-70.Koshimizu K, Ohigashi H, Huffman MA (1994). Use of Vernonia amagdalina by wildchimpanzee: Possible roles of its bitter and related constituents. Physiol. Behav.,56:1209-1216.

P a g e | 83Kupchan SM, Hemingway RJ, Karim A, Werner D (1969). Tumor inhibitors. XLVII.vernodalin and vernomygdalin, two new cytotoxic sesquiterpene lactones from Vernoniaamygdalina Del. J. Org. Chem., 34: 3908-3911.Kusamba C (2001). Contribution to the inventory of medicinal plants from the Bushi area,South Kivu Province, Democratic Republic of Congo. Fitoterapia, 72: 351-368.Mahato SB, Kundu AP (1994).13 C NMR spectra of pentacyclic triterpenoids. ACompilation and some salient features. Phytochemistry, 37: 1517-1575.Malafronte N, Pesca M, Sabina B, Angela E, Luis MTN (2009). New flavonoid glycosidesfrom Vernonia ferruginea. Nat. Prod. Comm., 4: 1639-1642.Mallavadhi UV, Mahapatra A, Jamil K, Reddy PD (2004). Antimicrobial activity of somepentacyclic triterpenes and their synthesized 3-O-lipophilic chains. Biol. Pharm. Bull.,27:1576-1579.Mao RS, Jun SY, Ze Sheng Z (2008). Two new compounds from the stem of Vernoniacumingiana. Chin. Chem. Lett., 19:180-182.Mesfin F, Demissew S, Teklehaymanot T (2009). An Ethnobotanical study of medicinalplants in Wonago Woreda, SNNPR, Ethiopia. J. Ethnobiol. Ethnomed., 5: 28.Mirutse G, Zemede A, Zerihun W (2009). Medicinal plants of the Meinit ethnic group ofEthiopia: An ethnobotanical study. J. Ethnopharmacol., 124:513–521.Miserez F, Potterat O, Marston A, Mungai GM, Hostettmann K (1996).Flavonolglycosides from Vernonia galamensis ssp. Phytochemistry, 1996, 43: 283-286.

P a g e | 84Morales-Escobar L, Braca A, Pizza C, Tommasi N (2007). New phenolic derivatives fromVernonia mapirensis Gleason. ARKIVOC (Gainesville, FL, United States), 7: 349-358.Muthaura CN, Rukunga GM, Chhabra SC, Mungai GM, Njagi ENM (2007). Traditionalphytotheraphy of some remedies used in treatment of malaria in Meru District of Kenya.S. Afr. J. Bot., 73: 402-411.Oketch-Rabah HA, Lemmich E, Dossaji SF, Theander TG, Olsen CE, Cornett C, Arsalan K,Christensen BS (1997).Two new antiprotozoal 5-methylcoumarins from Vernoniabrachycalyx. J. Nat. Prod., 60: 458-461.Rabe T, Mullholland D, van Staden J (2002). Isolation and identification of antibacterialcompounds from Vernonia colorata leaves. J. Ethnopharmacol., 80:91-94.Seebacher W, Simic N, Weis R, Saf R, Kunert O (2003). Complete assignments of 1 H and13 C NMR resonances of oleanolic acid, 18α-oleanolic acid, ursolic acid and their 11-oxoderivatives. Magn. Reson. Chem., 41:636-638.Stepanović S, Vuković D, Davić I, Savić B, Švabić-Vlahović M (2000).A modifiedMicrotiter-plate test for quantification of staphylococcal biofilm formation. J. Microbiol.Meth., 40:175-179.

P a g e | 85CHAPTER THREEA NOVEL POLYENE FROM VERNONIA URTICIFOLIA(ASTERACEAE)J. J. Kiplimo 1* , C. A. Everia 2 and N. A. Koorbanally 11 School of Chemistry, University of KwaZulu –Natal, Durban, South Africa2 Department of Chemistry, Egerton University, P.O.BOX 536 Njoro, Kenya*Corresponding author; e-mail: jjkiplimo@yahoo.comABSTRACTA new polyene metabolite, urticifolene together with a known carotenoid, lutein andsitosterol were isolated from the leaves of Vernonia urticifolia and fully characterized.Lutein (2) inhibited the growth of Enterococcus faecium at a low concentration (MIC of 8µg/mL), while urticifolene (1) inhibited the growth of Enterococcus faecium andPseudomonas aeruginosa at low concentrations (MIC of 16 and 32 µg/mL respectively). Incontrast to the related polyenes, nystatin and amphotericin B, which exhibit no activityagainst bacteria, urticifolene exhibited inhibitory property against all the bacteriainvestigated.Keywords: Urticifolene, lutein, Vernonia urticifolia, Asteraceae, antibacterial.INTRODUCTIONEthnomedicinally, Vernonia species are employed in the treatment of a diverse range ofailments, including measles, skin rashes, backache, malaria (Anoka et al., 2008), asthma,bronchitis, dysentery and worms (guinea, round and thread) (Misra et al., 1984). The root ofV. cinerea is used as an anthelmintic and diuretic (Misra et al., 1984) and to treat, coughs

P a g e | 86intestinal colics and chronic skin diseases (Dastur, 1977). V. calvoana, a leafy vegetable isfound to be a rich source of provitamin A, particularly cis-β-carotene (Ejoh et al., 2010).Several Vernonia species have previously been investigated, of which V. amagdalina isextensively investigated for its pharmacological properties (Kupchan et al., 1963, Ohigashiet al., 1994). V. brasiliana and V. brachycalyx have shown potential antiprotozoal activity(Almeida Alves de et al., 1997; Oketch-Rabah et al., 1997). V. urticifolia, the subject of thepresent study is known as Motoiyokwo by the Kalenjin tribe of Kenya, who use it to treatsinuses, allergy and skin rashes (Kokwaro, 1976).There are no previous phytochemical or pharmacological studies carried out on this plant.The current study was undertaken to investigate the phytochemistry of V. urticifolia and totest the isolated compounds for antibacterial activity since extracts of Vernonia species havebeen reported to possess antimicrobial activities and are used in traditional medicine(Kokwaro, 1976). We present herein a detailed isolation, characterization and antibacterialactivity of the isolated compounds.MATERIALS AND METHODSPlant materialThe leaves of Vernonia urticifolia (762.87 g) were collected from Nakuru District, Kenya inJune 2010. The plant was identified by Mr Ezekiel Cheboi of the Department of NaturalResources, Egerton University, Kenya. A voucher specimen (Kiplimo 03) was retained atthe Ward Herbarium University of KwaZulu-Natal Westville, Durban.Extraction and Isolation of CompoundsThe air-dried and ground leaves were extracted sequentially using a soxhlet apparatus withsolvents of increasing polarity viz: hexane, dichloromethane, ethyl acetate and methanol.

P a g e | 87The dichloromethane extract was concentrated to yield a crude extract (6.94 g) which wasseparated using column chromatography over silica gel (Merck 9385) and eluted with ahexane/ethyl acetate step gradient, to yield three subfractions D 1 -D 3 . Compound 1 (32 mg)was obtained in fraction D 2 and compound 2 (43 mg) was obtained in fraction D 3 .GENERAL EXPERIMENTAL PROCEDUREMelting points were determined on an Ernst Leitz Wetziar micro-hot stage melting pointapparatus. NMR spectra were recorded at room temperature on a 400MHz varian UNITY–INOVA spectrometer. 1 H NMR spectra were referenced against the CHCl 3 signal at δ H 7.24and 13 C NMR spectra against the corresponding signal at δ C 77.0. Coupling constants aregiven in Hz. For GC-MS analyses, samples were analysed on an Agilent GC–MSDapparatus equipped with a DB-5SIL MS (30 m x 0.25 mm i.d., 0.25 µm film thickness)fused-silica capillary column. The MS was operated in the EI mode at 70 eV, in m/z range42–350. IR spectra were recorded on a Nicolet impact 400D Fourier Transform Infrared(FT-IR) spectrometer, using NaCl windows with CHCl 3 as solvent against an airbackground. UV spectra were obtained on a Varian DMS 300 UV-visiblespectrophotometer.BIOLOGICAL STUDIESTest organismsThree strains of Gram-negative bacteria (Escherichia coli ATCC 25922, Pseudomonasaeruginosa ATCC 35032, Klebsiella pneumonia ATCC 700603), and three Gram-positivebacteria (Staphylococcus aureus ATCC 25923, Enterococcus faecium ATCC 19434,Staphylococcus saprophyticus ATCC 35552) were selected to determine the antibacterialactivity of the isolated compounds.

P a g e | 88Determination of minimum inhibitory concentration (MIC)The antibacterial activities of the compounds were determined using the broth microdilutionmethod as described by Andrews, 2001; Bacterial strains were cultured for 18 h at 37 o C inTryptone Soy Broth (TSB) and standardized to a final cell density of 1.5×10 8 cfu/mlequivalent to 0.5 McFarland standard. The 96-well plates were prepared by dispensing intoeach well, 90 µl Muller-Hinton (MH) broth and 10 µl of the bacterial inoculum. Testcompounds were dissolved in dimethyl sulfoxide (DMSO) to a concentration of 10 mg/mlwhile tetracycline (a positive control) was dissolved in ethanol. Serial two-fold dilutionswere made in a concentration range of 0.002 to 1 mg/ml. Wells containing MH broth onlywere used as medium control and wells containing medium and cultures without the testcompound were used as the growth control. Plates were covered to avoid contamination andevaporation and incubated for 24 h at 37 o C. The minimum inhibitory concentration (MIC)was described as the lowest concentration of the test compounds completely inhibiting thegrowth of microorganisms. The tests were done in triplicate on two separate occasions(Table 2).RESULTS AND DISCUSSIONThe dichloromethane extract of the powdered leaves of V. urticifolia afforded a newpolyene, (urticifolene, 1), known carotenoid (lutein, 2) and the known triterpenoid,(sitosterol) that is ubiquitous in the Vernonia family. This is the first report of compound 1;a yellow oily solid which has been assigned the trivial name urticifolene. The molecularformula was established to be C 31 H 52 O 4 , as indicated by the M + ion at m/z 488 in the massspectrum, which implied six degrees of unsaturation.The IR spectrum revealed thepresence of hydroxyl (3396 cm -1 ) and carbonyl (1713 cm -1 ) functional groups. The UVspectrum of urticifolene, 1 showed maxima with three shoulders at 237, 245 and 322 nm

P a g e | 89consistent with the conjugated double bonds present in the compound. The 13 C NMRspectrum of urticifolene, 1 (Table 1) showed ten carbon resonances in the olefinic region(range 123-135 ppm) indicating five double bonds, a carbonyl carbon resonance at δ C179.23 (accounting for the six degrees of unsaturation) and two oxygenated methine carbonresonances at δ C 72.95 and 72.18. 1 H NMR data displayed one methyl group bound to analiphatic carbon at δ H 0.89 and overlapping olefinic resonances which appeared between δ H5.0 and 7.0, characteristic of polyenes. Also present in the 1 H NMR spectrum were twooverlapping signals at δ H 4.17 and δ H 4.22, their deshielded chemical shifts confirming thepresence of two hydroxyl groups at C-3 and C-29.The COSY spectrum revealed a correlation between H-3 and H-2 in the olefinic region andH-2 showed correlations with C-3 (δ C 72.95) in the HMBC spectrum. The geometry of thedouble bonds at ∆ 5 and ∆ 8 were determined to be trans based on the coupling constants ofJ 5,6 = 15.0 Hz and J 8,9 = 15.5 Hz. The geometry of the other three double bonds could not bedetermined as all the resonances overlapped and the coupling constants could not be clearlydetermined. These were however given the more stable trans configuration. Only theolefinic proton resonances of H-5, H-6, H-8 and H-9, the deshielded methine resonances ofH-3 and H-29, the methylene resonances at C-2 and C-30 (which overlap) and the methylresonance at δ H 0.89 can be distinguished along with their corresponding carbon resonancesby using the HSQC spectrum.All the other methylene proton resonances as well as theolefinic resonances of H-11-H-14 as well as that of H-26 and H-27 all overlapped in theirrespective regions of the NMR spectrum and could not be distinguished from each other.The double bonds at ∆ 5 and ∆ 8 were separated by a methylene resonance as these twodouble bonds were not conjugated as evidenced by separate resonances that coalesced forboth H-5 and H-6 and for H-8 and H-9. The olefinic resonances of H-11 to H-14 all

P a g e | 90overlapped in the olefinic region of the spectrum into more complex splitting patterns,which will only result if the two double bonds at ∆ 11 and ∆ 13 are conjugated. With regard tothe double bond at ∆ 26 , one of the olefinic resonances in overlapping region at δ 5.75 showsa HMBC correlation to the methine proton resonance of H-29. This indicates that thedouble bond is in close proximity to the oxygenated methine group and was therefore placedat ∆ 26 as a HMBC correlation to the carbony resonance was absent for it to be placed at ∆ 27 .There is however a lack of correlations in the spectra to assign a structure to this compoundunequivocally, however the proposed structure best fits the data.Urticifolene (1) displayed high structural similarity to laetiporic acid isolated fromLaetiporus sulphureus with the exception of the aliphatic methylene chain containing 13carbon atoms in urticifolene (1) that is absent in laetiporic acid (Davoli et al., 2005).Compound 2 was identified as Lutein due to characteristic peaks observed in thespectroscopic data. The UV spectrum showed absorption maxima at 454 nm, 480 nm and430 nm. This was consistent with the UV data of (9Z, 9’Z, 3R, 3’R, 6’R)-lutein isolatedfrom Brassica napus (Rape) (Kull and Pfander, 1997).

P a g e | 911OH2 3 456789101112131415161719OOH21HO31302826Urticifolene (1)2422OH5431217186 167 820911 131514'12 1019'1910121415'13'11' 9'Lutein (2)2016'8'17'1'2'7'6'3'5'4'18'OHFigure 1. Structures of compounds (1 & 2) isolated from Vernonia urticifolia

P a g e | 92Table 1. 1 H and 13 C NMR spectral data of compound 1 and 2 (400 or 100 MHz)urticifolene (1) lutein (2)position δ C δ H position δ C δ H1 14.19 0.89 1 37.13 -2 35.22 2.33 2 48.45 178, 1504,7,10, 20.74-33.93 1.20-2.30 3 65.10 4.0315-25,283 72.95 4.17 4 42.56 2.05, 2.425 125.82 6.52 5 126.17 -6 125.91 6.48 6 138.00 -8 127.84 6.02 7 128.73 6.129 127.78 5.98 8 130.81 6.6511-14, 132.80-135.70 5.25-5.75 9 135.07 -26-2729 72.18 4.22 10 130.04 6.0730 37.25 2.34 11 124.49 6.7331 179.23 12 137.57 6.2813 136.42 -14 132.58 6.2315 130.09 6.6316 30.26 1.0817 28.73 1.0918 21.62 1.7819 29.70 1.9720 12.81 1.971’ 34.04 -2’ 44.64 1.37, 1.853’ 65.93 4.254’ 128.81 5.505’ 137.77 -6’ 54.97 2.477’ 131.30 5.478’ 130.09 6.659’ 135.70 -10’ 125.60 6.0511’ 124.94 6.7412’ 138.50 6.2813’ 136.49 -14’ 137.73 6.2315’ 130.81 6.6316’ 29.50 0.8517’ 24.29 1.0318’ 22.86 1.6419’ 12.76 1.9020’ 13.11 1.95

P a g e | 93The antibacterial activity results (Table 2) shows that urticifolene, 1 and lutein, 2 had broadspectrum antibacterial activity. The minimum inhibitory concentration (MIC) determinationshowed that lutein at low concentration of 8 µg/mL completely inhibited the growth ofEnterococcus faecium (ATCC 19434) and at 32 µg/mL it inhibited the growth ofStaphylococcus aureus (ATCC 29212 ), Escherichia coli (ATCC 25922 ) and Klebsiellapneumonia (ATCC 700603). Urticifolene inhibited the growth of Pseudomonas aeruginosa(ATCC 35032) and Enterococcus faecium (ATCC 19434) at low concentrations of 32µg/mL and 16 µg/mL respectively, but the MIC for Escherichia coli (ATCC 25922),Klebsiella pneumonia (ATCC 700603) and Staphylococcus aureus (ATCC 29212,) wererecorded as 256 µg/mL. These findings indicated that urticifolene (1) and lutein (2)possessed inhibitory activity against bacteria.Other investigations showed that thecarotenoid methanol extract of the citrus the peel of Shatian pummel exhibited inhibitoryproperties within a range of 18.75-140.00 µg/mL against E. coli, S. aureus and B. subtilis(Tao et al., 2010).The constituents of citrus carotenoids cover a wide range of compounds such as β-cryptoxanthin, violaxanthin isomers, lycopene and β-carotene (Tao et al., 2010) thereforethe synergistic effect of these compounds could have possibly enhanced the activity of themethanol carotenoid extract.It has also been reported that some polyenes (of whichurticifolene, 1 belongs) such as amphotericin B and nystatin have no antibacterial activitywhilst others such as faeriefungin inhibit the growth of a variety of bacterial isolates. Themode of action of carotenoids is not clearly understood but Cucco et al., 2007) suggest thatβ-carotene could lead to the accumulation of lysozyme (a bacterial immune enzyme thatdigests bacterial cell walls).But the mode of action of polyenes against bacteria isunknown, though the fungicidal activity involves interaction of the lipophilic polyene

P a g e | 94structure with the cytoplasmic membrane sterol found in fungi and mammalian cellsresulting in alteration of the cell membrane, leakage of cell constituents and cell death(Mulks et al., 1990).Table 2 Minimum inhibitory concentration (MIC) of urticifolene and luteinConcentration (µg/ml)Test organism Urticifolene luteinE. faecium, (ATCC 19434) 16 8S. aureus, (ATCC 29212) 256 32S. saprophyticus, (ATCC 35552) 128 256E. coli, (ATCC 25922) 256 32K. pneumonia, (ATCC 700603) 256 32P. aeruginosa, (ATCC 35032) 32 256CONCLUSIONThe phytochemical study of this plant resulted in the isolation of two compounds, a polyeneand a carotenoid. These compounds exhibited moderate antibacterial activity. In contrast tothe related polyene antibiotics, nyastin and amphotericin B, which are inactive againstcertain bacteria, urticifolene exhibited inhibitory activity against all the bacteriainvestigated. These results provide scientific validity and credence to the ethnomedicinaluse of this plant in the treatment of ailments caused by some of the bacteria used in thisstudy and highlights the usefulness of V. urticifolia in the treatment of bacterial infections.ACKNOWLEDGEMENTSFinancial support from organization for Women in Science for the Developing World(OWSDW) is gratefully acknowledged. We thank Mr Ezekiel Cheboi of Department ofNatural Resources Egerton University for the identification of the plants.

P a g e | 95ReferencesAlmeida Alves de TM, Nagem TJ, Carvalho de LH, Krettli AU, Zani CL (1997).Antiplasmodial triterpenes fom Vernonia brassiliana. Planta. Med. 63, 554-555.Andrews JM (2001). Determination of inhibitory concentrations. J. Antimicrob. Chemother.48, 5-16.Anoka AN, Bulus A, Amon GA, Byarugaba D, Silvia Dl, Bangsbrg DR (2008). Theanalgesic and antiplasmodial activities and toxicology of Vernonia amygdalina. J. Med.Food. 11, 574-581.Cucco M, Guasco B, Malacarne G, Ottonelli R (2007). Effects of β-carotene on adultimmune condition and antibacterial activity of eggs of Grey Partridge, Perdix perdix.Comp. Biochem. Physiol., A, 147, 1038-1046.Dastur JF, (1977). Medicinal plants of India and Pakistan D. B. Taraporevala sons and co.Private Ltd India, pp. 174.Davoli P, Mucci A, Schenetti L, Weber RS, (2005). Laetiporic acids, a family of noncarotenoidpolyene pigments from fruit-bodies and liquid cultures of Laetiporussulphureus (Polyporales, Fungi). Phytochemistry, 66, 817-923.Ejoh RA, Dever JT, Mills JP, Tanumihardjo SA, (2010). Small quantities of carotenoid-richgreen leafy vegetables indegenous to Africa maintain Vitamin A status in Mongoliangerbils (Meriones unguiculatus). Br. J. Nutr. 103, 1594-1601.Kokwaro J O, (1976). Medicinal plants of East Africa. East African Literature Bureau,Nairobi.

P a g e | 96Kull DR, Pfander H, (1997). Isolation and structure elucidation of two (Z)-isomers ofLutein from petals of Rape (Brassica napus). J. Agric. Food Chem. 45, 4201-4203.Kupchan SM, Hemmingway RJ, Karim A, Wener D, (1963). Tumor inhibitors XLVII.Vernodalin and Vernomygdin, new cytotoxic sesquiterpene lactones from Vernoniaamygdalina. J. Org. Chem. 34, 3908-3911.Misra TN, Singh RS, Upadhyay J, Srivastava R, (1984). Chemical constituents of Vernoniacinerea part 1. Isolation and spectral studies of triterpenes. J. Nat. Prod. 47, 368-372.Mulks MH, Nair MG, Putnam AR, (1990). In vitro antibacterial activity of Faeriefungin, anew broad-spectrum polyene macrolide antibiotic. Antimicrob. Chemother. 34, 1762-1765.Ohigashi H, Huffman MA, Daisuku I, Koshimizu K, Kawanaka M, Sugiyama H, Kirby GC,Wahurst DC, Allen D, Eright CW, Phillipson DJ, David PT, Delmas F, Elias R,Balansard G, (1994).Toward the chemical ecology of medicinal plant use inchimpanzees. The case of Vernonia amygdalina a plant used by wild chimpanzeespossibly for parasite related diseases. J. Chem. Ecol. 20, 541-553.Oketch-Rabah HA, Lemmich E, Dossaji SF, Theander TG, Olsen EC, Cornett C, KharazmiA, Christensen BS, (1997). Two new antiprotozoal 5-methylcoumarins from Vernoniabrachycalyx. J. Nat. Prod. 60, 458-461.Tao N, Gao Y, Liu Y, and Ge F, (2010). Carotenoids from the peel of Shatian pummel(Citrus grandis Osbeck) and its Antimicrobial Activity. J. Agric. and Environ. Sci., 7,110-115.

P a g e | 97CHAPTER FOURA NOVEL FLAVONOID AND FUROQUINOLINEALKALOIDS FROM VEPRIS GLOMERATA AND THEIRANTIOXIDANT ACTIVITYJoyce J. Kiplimo a , Md. Shahidul Islam b and Neil A. Koorbanally a*aSchool of Chemistry, University of KwaZulu –Natal, Westville Campus , Private BagX54001, Durban 4000, South Africab School of Biochemistry, Genetics and Microbiology, University of KwaZulu–Natal,Westville Campus, Durban 4000, South AfricaAuthor to whom correspondence should be addressed; E-Mail: Koorbanally@ukzn.ac.zaAbstractThe dichloromethane extract of the aerial part of the plant Vepris glomerata (Rutaceae)yielded a new flavonoid, which was accorded the trivial name veprisinol (1), together withfour known furoquinoline alkaloids: isohaplopine-3,3’-dimethylallyl ether (2), tecleoxine(3), nkolbisine (4) and skimmianine (5). The structures of the compounds were establishedby 1D and 2D NMR spectroscopy, as well as HREIMS. Compounds 1 and 2 have strongantioxidant potential, similar to and in some instances better than ascorbic acid and can beused as beneficial additives to antioxidant supplements.Keywords: Vepris glomerata, veprisinol, furoquinoline alkaloids, antioxidant activity.IntroductionThe African Vepris species have proved to be a good source of furoquinoline and acridonealkaloids that typify the genus as a whole. V. bilocularis has been found to have both

P a g e | 98furoquinoline as well as acridone alkaloids [1, 2], while furoquinoline alkaloids alone havebeen found in V. ampody [3], V. heterophylla [4], V. punctata [5] and V. stolzii [6], andacridone alkaloids alone in V. fitoravina and V. macrophylla [7]. The alkaloids are reportedto possess broad spectrum antimicrobial [8], antiradical [9], antioxidant [10], antiplasmodial[11], anticancer [12] and antimutagenic [13] activities. V. glomerata is used in Africantraditional medicine, where its aqueous root extract is used to treat malaria, epilepsy,psychosis and stroke, when mixed with tea [14]. Earlier pharmacological studies on thisplant reported antiplasmodial activities of the ethanol extract [15].Since the species of Rutaceae are often cited as antimalarials or febrifuges in Africantraditional medicine [14] and the antioxidant activity of alkaloids [10] and flavonoids [16]has previously been demonstrated, all the five compounds isolated were assessed forantioxidant activity using three methods.Here we report on the isolation and structure elucidation of a new flavonoid, in addition tofour known furoquinoline alkaloids: isohaplopine-3,3’-dimethylallyl ether (2), tecleoxine(3), nkolbisine (4) and skimmianine (5) from the dichloromethane extract of V. glomerata,together with their antioxidant activities in vitro. The structures of the known compounds 2-5 were determined by comparison of their physical and spectroscopic data with thosereported in literature; 2 and 3 [17], 4 [18] and 5 [19]. Only skimmianine and evoxine(montrifoline) were previously reported from the leaves of V. glomerata endemic toEthiopia [20]. It is not apparent if the additional compounds found in this study are as aresult of either geographical or seasonal differences.

P a g e | 99Results and Discussion5''4''2''OH3''1''8O 765OH5'6' OCH 364'9 O1' 3' OH2 2'H 3 CO 71034O1 2OCH 354a 43a8a8 N 9aO1'4'2'3'3O25'3'4'4' 5'3'5'2'O1'OCH 3HO2'OH1'OCH 3O654a43a32O654a43a32H 3 CO788aN9aOH 3 CO788aNO34654a43a3H 3 CO78a9aOCH 3ON8OCH 3 52Compound 1 was obtained as a yellow solid. Its mass was established to be 388.1573 amu,based on HREIMS data, corresponding to a molecular formula of C 21 H 24 O 7 , which indicatesa double bond equivalence of 10, eight being due to the aromatic rings, one being due to thecarbonyl group and one to ring C of the flavanone skeleton. The IR spectrum showed acarbonyl stretching band at 1705 cm -1 and a hydroxyl absorption band at 3364 cm -1 . Thiscompound was identified as a flavanone based on its characteristic 1 H NMR spectral pattern.

P a g e | 100The characteristic ABX coupling system of H-2β, H-3α and H-3β appeared at δ H 5.29 (1H,dd, J = 12.84, 2.84 Hz, H-2β), δ H 3.04 (1H, dd, J = 17.12, 2.84 Hz, H-3α) and δ H 2.75 (1H,dd, J = 17.12, 12.84, Hz, H-3β). These signals also showed COSY and NOESY correlationswith each other.Another characteristic pattern was that of the trisubstituted aromatic B ring. The protonresonances of this ring occurred as a singlet at δ H 7.00 (s, H-2’) and doublets at δ H 6.89 and6.84 (1H each, d, J = 8.48 Hz, H-5’ and H-6’). The small coupling constant of about 2 Hzfor J H2’,H6’ could not be detected for the H-2’ resonance. The 1 H NMR spectrum alsoshowed the presence of a methoxy group at δ H 3.88 (s), its position at C-4’ being confirmedby both a 1D NOE and a NOESY correlation with the resonances at δ H 6.89 and 6.84 (H-5’and H-6’). Five aromatic C-O resonances were seen at δ C 164.0, 167.2, 162.8, 145.0 and147.0 attributed to oxygenation at C-5, 7, 9, 3’ and 4’.A pair of doublets at δ H 6.02 (1H, d, J = 1.76 Hz, H-6) and δ H 6.00 (1H, d, J = 1.76 Hz, H-8)were attributed to the meta coupled, H-6 and H-8 protons on ring A. These two protonresonances showed NOESY correlations to 2H-1’’ at δ H 4.02, confirming the position of theside chain at C-7. Its corresponding carbon resonance showed HMBC correlations to twomultiplets at δ H 1.87 (overlapping resonances of H-2’’a and H-3’’) and δ H 1.61 (H-2’’b).The H-2’’ resonances were diastereotopic and appeared as two separate resonances. COSYcorrelations were also observed between H-1’’ and H-2’’a and H-2’’b and between H-2’’band H-3’’. The H-3’’ methine proton was coupled to the methyl proton resonance at δ H 0.95(d, J = 6.52 Hz) attributed to 3H-5’’ and the methylene proton at δ H 3.50 (2H-4’’) in theCOSY spectrum. These correlations formed a side chain which was attached to ring A byan ether linkage at C-7. Compound 1 was thus identified as 4H-1-benzopyran-4-one-2,3-

P a g e | 101dihydro-5-hydroxy-2-(4’-methoxy-3’-hydroxybenzyl)-7-O-(2-methyl butanol) ether, andgiven the trivial name veprisinol.The results of the reducing potential (transformation of Fe 3+ -Fe 2+ ) of the standard (ascorbicacid) and compounds 1-5 are shown in figure 1.The activity of isohaplopine-3,3’-dimethylallyl ether, 2 and veprisiniol (1) was significantly higher than the activity of theother three alkaloids at all concentrations. However, the reducing power of compound 1was significantly lower than that of compound 2. The reducing power of the compoundsand standard followed the order: ascorbic acid > 2 > 1 > 3 > 4 > 5.Figure 1: Free radical reducing potential of compounds 1-5 and standard ascorbic acid asevaluated by the spectrophotomeric detection of the Fe 3+ -Fe 2+transformation (FRAPmethod).The DPPH radical scavenging assay results are shown in figure 2. The results revealed thatthe scavenging activity of the standard ascorbic acid was significantly higher than all othercompounds tested. At concentrations of 62.5 µg mL -1 and above, the activity decreased inthe order ascorbic acid > 1 > 2 > 4 > 3, whereas at the lower concentrations, 31.25 and

P a g e | 10215.625 µg mL -1 , nkolbisine (4) had the highest percentage antioxidant activity of 41%. Theactivity of compounds 1 and 2 was increased with their concentration and significantlyhigher than other compounds, particularly at higher concentrations (Figure 2).Figure 2: Antioxidant activity of compounds 1-4 and ascorbic acid standard, as measuredby the DPPH method.The hydroxyl radical scavenging activities in the deoxyribose assay are shown in figure 3.The results revealed that compound 1 possesed significantly higher activity than all the othercompounds tested, including the standard, ascorbic acid, at most concentrations.Compounds 1, 2 and 4 had hydroxyl radical scavenging activity comparable with and in thecase of 1 and 2, better than that of ascorbic acid. Skimmianine (5) was not tested in eitherthe DPPH or deoxyribose assays due to insufficent amount.The three assays revealed that compounds 1 and 2 are good antioxidant compounds, whilecompound 4 shows high activity at a lower concentration in the DPPH assay. Flavonoids

P a g e | 103are known to be potent antioxidants and their activity is dependent on their molecularstructure. The activity of 1 could be attributed to the hydroxyl (OH) groups in the molecule,which donate hydrogen to reduce the DPPH radical to DPPH-H. The alkaloids 2-5 have thesame basic skeleton, the only difference being in their side chain.Figure 3: Hydroxyl radical scavenging activity of compounds 1-4 and standard ascorbicacid as measured by the deoxyribose method.The reductive ability of 2 may be attributed to the double bond of the isoprenyl unit, rich indelocalized pi-electrons, which are easily donated during reduction of Fe 3+ to Fe 2+ . Sang etal. also reported that the double bond of the isoprenyl group was responsible for theantioxidant activity of garcinol [21]. The antioxidant activity of 2 in the DPPH assay, like 1,could also be attributed to the hydroxyl groups in the molecule.In conclusion, five compounds were isolated (a flavonoid and four alkaloids) from the aerialparts of V. glomerata. Verification of their antioxidant activities, as well as comparison

P a g e | 104with known antioxidants, will provide herbalists and traditional healers with scientificevidence for the use of the aerial parts of this plant as natural antioxidants.ExperimentalGeneral experimental procedures: The melting points were recorded on an Ernst LeitzWetzer micro-hot stage melting point apparatus and are uncorrected. UV spectra wereobtained on a Varian Cary UV-VIS Spectrophotometer in chloroform. IR spectra wererecorded on a Perkin-Elmer Universal ATR Spectrometer. The 1D and 2D NMR spectrawere recorded using a Bruker Avance III 400 MHz NMR spectrometer. All the spectra wererecorded at room temperature using deuterated chloroform (CDCl 3 ) as solvent.TheHREIMS was measured on a Bruker Micro TOF-QII instrument. Specific rotations weremeasured at room temperature in chloroform on a PerkinElmerTM, Model 341 Polarimeterwith a 10 mm flow tube. The separation, isolation and purification of compounds werecarried out by gravity CC and monitored by TLC. Merck silica gel 60 (0.040-0.063 mm)was used for CC. Merck 20 × 20 cm silica gel 60 F 254 aluminum sheets were used for TLC.TLC plates were analyzed under UV light (254 and 366 nm) before being sprayed withanisaldehyde: concentrated sulfuric acid: methanol [1:2:97] spray reagent and then heated.Plant material: Vepris glomerata was collected from the Rift Valley province of Kenya andidentified by Dr S. T. Kariuki from the Department of Botany, Egerton University, Kenya.A voucher specimen (Kiplimo 01) was deposited at the University of KwaZulu-Natal WardHerbarium, Westville Campus, Durban, South Africa.Extraction and isolation: The air-dried aerial parts (980 g) of V. glomerata weresequentially extracted with n-hexane, followed by dichloromethane in a Soxhlet apparatusfor 48 h, yielding crude extracts of 46 and 32 g, respectively. The oily residue of thedichloromethane extract obtained after evaporation under vacuum, was separated by CC on

P a g e | 105silica gel with n-hexane and then increasing the concentration of ethyl acetate from 10 to80% in n-hexane, to give 10 fractions (fr.); fr. 8-16 (1.27 g), fr. 17-19 ( 0.5 g), fr. 20-26(2.36 g), fr. 27-32 (2.35 g), fr. 33-39 (1 g), fr. 40-43 (2.1 g), fr. 44-49 (0.5 g), fr. 52-56 (3.9g), fr. 57-62 (1.75 g) and fr. 63-67 (5.1 g).Fraction 52-56 was separated by CC with n-hexane/EtOAc (7:3) as the solvent to affordsub-fractions A-C. Sub-fraction A was further purified using 100% dichloro-methane toafford compound 2, a green solid (51 mg). Sub-fraction B yielded compound 3, a brownishsolid (43 mg), which needed no further purification. Sub-fraction C was crystallized inmethanol to afford 4 (62 mg). Fraction 44-49 was purified using 100% dichloromethane toafford 5 (60 mg). Fraction 63-67 was separated with n-hexane/EtOAc (4:1) to yield 4 subfractionsA-D. Sub-fraction B was crystallized in methanol to afford yellow crystals ofcompound 1 (18 mg).Veprisinol (1); 4H-1-Benzopyran-4-one, 2, 3-dihydro-5-hydroxy-2-(4’-methoxy-3’-hydroxylbenzyl)-7-O-(2-methyl butanol) ether.Yellow solid.M.p: 78-80 o C[α] 20 D: +55.30 (c 0.056 CHCl 3 )IR: 3364 (O-H), 2928, 1705 (C=O), 1636, 1512, 1162 cm -1UV λ max (CHCl 3 ) nm (log ε): 337 (4.45), 285 (5.13), 239 (5.44)1 H NMR (400 MHz, CDCl 3 ): 11.97 (H, s, OH), 7.00, (H, s, H-2’), 6.89 (H, d, J = 8.28 Hz,H-5’), 6.84 (H, d, J = 8.28 Hz, H-6’), 6.02 (H, d, J = 1.76 Hz, H-6), 6.00 (H, d, J = 1.76 Hz,H-8), 5.29 (H, dd, J = 12.84, 2.84 Hz, H-2β), 4.02 (2H, dd, J = 12.88, 6.24 Hz, 2H-1’’), 3.88

P a g e | 106(3H, s, OCH 3 ), 3.50 (2H, d, J = 5.68 Hz, 2H-4’’), 3.04 (H, dd, J = 17.12, 12.84 Hz, H-3α),2.75 (H, dd, J = 17.12, 2.84, Hz, H-3β), 1.87 (2H, m, H-2’’a and H-3’’), 1.61 (H, m, H-2’’b), 0.95 (3H, d, J = 6.52 Hz, H-5’’).13 C NMR: 195.97 (C, C-4), 167.29 (C, C-7), 164.05 (C, C-5), 162.85 (C, C-9), 147.02 (C,C-4’), 145.93 (C, C-3’), 131.52 (C, C-1’), 118.15 (CH, C-6’), 112.71 (CH, C-2’), 110.71(CH, C-5’), 103.11 (C, C-10), 95.54 (CH, C-6), 94.60 (CH, C-8), 78.92 (CH, C-2), 67.86(CH 2 , C-4’’), 66.70 (CH 2 , C-1’’), 56.06 (OCH 3 ), 43.15 (CH 2 , C-3), 32.94 (CH, C-3’’), 32.37(CH 2 , C-2’’), 16.60 (CH 3 , C-5’’).HREIMS m/z 388.1573 [M] + (calcd. for C 21 H 24 O 7 , 388.1522)Antioxidant activity: The total reducing power was determined according to the methoddescribed previously [22]. The free radical scavenging activity (antioxidant capacity) of theplant phytochemicals on the stable radical 2, 2-diphenyl-β-picrylhydrazyl (DPPH) wasevaluated by the method established by Shirwaikar et al. [23], and the deoxyribose assay forhydroxyl radical scavenging activity was performed as described previously by Chung et al.[24].Statistical analysis: The data in figures 1-3 are presented as mean ± SD of triplicates. a-d Values with different superscript letters for a given concentration are significantly diferentfrom each of the other compounds. The data were statistically analyzed using a statisticalsoftware program SPSS (SPSS for Windows, version 18, SPSS Science, Chicago, IL, USA).One-way analysis of variance (ANOVA) followed by Tukey’s multiple range post-hoc testwas employed to find the differences. The data were considered significantly different at p< 0.05.

P a g e | 107Acknowledgments: The authors wish to acknowledge the financial support received fromthe Organization for Women in Science for the Developing World (OWSDW).References1. Govindachari TR, Sundararajan VN. (1961) Alkaloids of Vepris bilocularis. Journalof Scientific and Industrial Research, 20, 298-299.2. Ganguly AK, Govindachari TR, Manmade A, Mohamed PA. (1966) Alkaloids ofVepris bilocularis. Indian Journal of Chemistry, 4, 334-5.3. Kan-Fan B, Das C, Boiteau P, Potier P. (1970) Alcaloïdes de Vepris ampody(Rutacées). Phytochemistry, 9, 1283-1291.4. Gomes E, Dellamonica G, Gleye J, Moulis C, Chopin MJ, Stanislas E. (1983) Phenoliccompounds from Vepris heterophylla. Phytochemistry, 22, 2628-2629.5. Chaturvedula VSP, Schilling JK, Miller JS, Andransiferana R, Rasamison VE, KingstonDGI. (2003) New cytotoxic alkaloids from the wood of Vepris punctata from theMadagascar rainforest. Journal of Natural Products, 66, 532-534.6. Khalid SA, Waterman PG. (1982) Chemosystematics in the Rutaceae 15. Furoquinolineand pyrano-2-quinolone alkaloids of Vepris stolzii. Journal of Natural Products, 45,343-346.7. Koffi Y, Gleye J, Moulis C, Stanislas E. (1987) Acridones from Vepris fitoravina andVepris macrophylla. Planta Medica, 53, 570-571.

P a g e | 1088. Karou D, Savadogo A, Canini A, Yameogo S, Montesano C, Simpore J, Colizzi V,Traore AS. (2006) Antibacterial activity of alkaloids from Sida acuta. African Journalof Biotechnology, 5, 195-200.9. Račková L, Májeková M, Košt’álová D, Štefek M, (2004) Antiradical and antioxidantactivities of alkaloids isolated from Mahonia aquifolium. Structural aspects. Bioorganic& Medicinal Chemistry, 12, 4709-4715.10. Chung HS, Woo SO. (2001) A quinolone alkaloid with antioxidant activity from thealeurone layer of anthocyanin-pigmented rice. Journal of Natural Products, 64, 1579-1580.11. Waffo KFA, Coombes PH, Crouch NR, Mulholland DA, Amin SMM, Smith PJ. (2007)Acridone and furoquinoline alkaloids from Teclea gerrardii (Rutaceae: Toddalioideae)of Sourthern Africa. Phytochemistry, 68, 663-667.12. Iwasa K, Moriyasu M, Yamori T, Turuo T, Lee DU, Eigrebe W. (2001) In vitrocytotoxicity of the protoberberine-type alkaloids. Journal of Natural Products, 64, 896-898.13. Fukuda K, Hibiya Y, Mutoh M, Koshiji M, Akao S, Fujiwara H. (1999) Inhibition byberberine of cyclooxygenase-2 transcriptional activity in human colon cancer cells.Journal of Ethnopharmacology, 66, 227-233.14. Moshi JM, Mbwambo ZH, Nondo RSO, Masimba PJ, Kamuhabwa A, Kapingu MC,Thomas P, Richard M. (2006) Evaluation of ethnomedical claims and brine shrimptoxicity of some plants used in Tanzania as Traditional Medicines. African Journal ofTraditional Complementary and Alternative Medicine 3, 48-58.

P a g e | 10915. Innocent E, Moshi M, Masimba P, Mbwambo Z, Kapingu M, Kamuhabwa A. (2009).Screening of traditionally used plants for in vivo antimalarial activity in mice. AfricanJournal of Traditional Complementary and Alternative Medicine, 6, 163-167.16. Dimitrios B. (2006) Sources of natural phenolic antioxidants. Trends in Food ScienceTechnology, 17, 505-512.17. Al-Rehaily AJ, Ahamad MS, Muhammad IM, Al-Thukair AA, Perzanowski HP. (2003)Furoquinoline alkaloids from Teclea nobilis. Phytochemistry, 64, 1405-1411.18. Ayafor JF, Okogun JI. (1982) Nkolbisine, a new furoquinoline alkaloid, and 7-deacetylazadirone from Teclea verdoorniana. Journal of Natural Products, 45, 182-185.19. Latip J, Ali MNH, Yamin BM. (2005) 4,6,7-Trimethoxyfuro[2,3-b]quinoline-water(2/3). Acta Crystallographica. E61, 2230-2232.20. Dagne E, Yenesew A, Waterman PG, Gray AI. (1988) The chemical systematics of theRutaceae subfamily Toddalioideae, in Africa. Biochemical Systematics and Ecology, 16,179-188.21. Sang S, Liao CH, Pan MH, Rosen RT, Lin-Shiau SY, Lin JK, Ho CT. (2002) Chemicalstudies on antioxidant mechanism of garcinol: analysis of radical reaction products ofgarcinol with peroxyl radicals and their antitumor activities. Tetrahedron, 58, 10095-10102.22. Ferreira ICFR, Baptista M, Vilas-Baos, Barros L. (2007) Free radical scavengingcapacity and reducing capacity of wild edible mushrooms from northeast Portugal:individual cap and stipe activity. Food Chemistry, 100, 1511-1516.

P a g e | 11023. Shirwakar A, Shirwakar AR, Rajendran K, Punitha IRS. (2006) In vitro antioxidantstudies on the benzyltetraisoquinoline alkaloid berberine. Biological andPharmaceutical Bulletin, 29, 1906-1910.24. Chung SK, Osawa T, Kawakishi S. (1997) Hydroxyl radical-scavenging effects ofspecies andscavengers from brown mustard (Brassica nigra). Bioscience,Biotechnology and Biochemistry, 61, 118-123.

P a g e | 111CHAPTER FIVEANTIBACTERIAL ACTIVITY OF AN EPOXIDISEDPRENYLATED CINNAMALDEHDYE DERIVATIVE FROMAbstractVEPRIS GLOMERATAJoyce J. Kiplimo and Neil Koorbanally*School of Chemistry, University of KwaZulu –Natal, Private Bag X54001, Durban,4000, South Africa*corresponding author. Tel.: +27312603189, Fax: +27312603091. E-mail address:Koorbanally@ukzn.ac.zaA prenylated cinnamaldehyde derivative (glomeral), together with the known p-hydroxycinnamic acid, caffeic acid, methyl caffeate, hesperetin, scoparone, skimmianine,syringaresinol and two limonoids (limonin and limonyl acetate) were isolated from the rootsand stem bark of V. glomerata. The antibacterial assay of the isolated compounds indicatedan inhibition zone, ranging from 8 to 16 mm, against standard strains of Staphylococcusaureus (ATCC 29213, 25923) and Shigella dysentrieae. Glomeral inhibited the growth ofStaphylococcus aureus and Shigella dysentrieae at low concentrations (MIC of 2 µg mL -1and 0.4 µg mL -1 respectively). Of the other compounds tested, hesperetin displayed goodantibacterial activity, the limonoids, scoparone and skimmianine displayed moderateantibacterial activity and the cinnamic acid derivatives were inactive against the testpathogens. This study provides a rationale for the use of V. glomerata in its treatment ofbacterial infections.Keywords: Vepris glomerata; Rutaceae; glomeral; antibacterial activity.

P a g e | 112IntroductionThere are about eighty species of the genus Vepris, formerly known as Teclea distributed inTropical Africa, Madagascar, the Mascerenes, Tropical Arabia and Southwest India(Chaturvedula et al., 2003). Vepris glomerata is a small tree that grows to a height ofapproximately 7.5 m. In Africa it is distributed in the dry bushland of Sudan, Ethiopia,Somalia, Kenya and Tanzania (Beentje, 1994). A decoction of the roots is used traditionallyin the treatment of malaria, whilst the vapour is used to treat eye problems. A decoction ofthe bark is used in the treatment of cardiac pain while epilepsy, stroke and psychosis istreated using an aqueous root extract of the plant mixed with tea (Innocent et al., 2009;Moshi et al., 2006).Previous investigations of Vepris species indicated that it is rich in furoquinoline alkaloids(Govindachari and Sundararajan, 1961; Ganguly et al., 1966; Rudolf and Eva Maria, 1978;Dagne et al., 1988; Chaturvedula et al., 2003), acridone alkaloids (Rasoanaivo et al., 1999),pyranoquinoline alkaloids (Brader et al., 1996), triterpenoids and sesquiterpenoids (Khalidand Waterman, 1982; Chaturvedula et al., 2004), limonoids (Ngadjui et al., 1982; Cheplogoiet al., 2008), azoles and imides (Cheplogoi et al., 2008) and essential oils (Sidibe et al.,2001; Poitou et al., 1995; Denise and Favre-Bonvin, 1973).With regard to V. glomerata, only one phytochemical study has been undertaken where twofuroquinoline alkaloids (skimmianine and montrifoline) have been isolated from the aerialparts of V. glomerata indigenous to Ethiopia (Dagne et al., 1988).In terms ofpharmacological activity, the ethanolic crude extract of V. glomerata collected from Tabora(Tanzania) exhibited antimalarial activity against Plasmodium berghei (Innocent et al.,2009). The current study was undertaken primarily to re-investigate the phytochemistry ofV. glomerata since only two alkaloids were previously found and to test the isolated

P a g e | 113compounds for antimicrobial activity since extracts of Rutaceae species have been cited asantimicrobials in traditional medicine (Kokwaro, 1976). Furthermore, limonoids whichwere isolated in this work are reported to have antifungal activity (Govindachari et al.,1998) and natural phenyl propanoids to which compounds 1-4 are structurally related arewell known antibacterial agents (Jimenez and Riguera, 1994).This is the firstphytochemical investigation of the roots and stem bark of V. glomerata.Results and discussionOf the two furoquinoline alkaloids previously isolated, only skimmianine (7) (Latip et al.,2005) was isolated in this work. Montrifoline was not detected in the stem bark and rootsand may be confined to the aerial parts of the plant. Several other compounds, including anew prenylated cinnamaldehyde derivative (glomeral 1), known aromatic acids, (phydroxycinnamicacid 2 and caffeic acid 3 (Lu et al., 1999)), an aromatic ester (methylcaffeate 4 (Lu et al., 1999)), a flavonoid (hesperetin 5 (Wawer and Zielinska, 2001)), acoumarin (scoparone 6 (Lee et al., 2002)), a lignin (syringaresinol 8 (Ouyang et al., 2007)),and two limonoids (limonin 9 (Khalil et al., 2003) and limonyl acetate 10 (Ruberto et al.,2002)) (Figure 1) were isolated from the stem bark and roots of V. glomerata. Compounds2-10 were identified by their 1 H and 13 C NMR spectra and by comparison withspectroscopic data in literature.5'4'3'2'O1'HO879 34256OCH 31. GlomeralO1H2 7R 119 OR3386R 2 452. R 1= H, R 2= OH, R 3= H3. R 1 = OH, R 2 = OH, R 3 = H4. R 1 = OH, R 2 = OH, R 3 = CH 3O

P a g e | 114HOHOH 3 CO764385OH910OO4232'1'5. HesperetinOH6'3'5'OCH 3H 3 COH 3 CO67581094O6. Scoparone32OH 3 CO6754aOCH 38aN 9a O8OCH 343a7. Skimmianine2322OCH 320 O5O18612 1721H9 O1 OO211 30 1387' 6'1911' 5' OCH 14 1632 7 8'39O10 8 15O HOO4'9'42'OH5 73'R612928OCH 38. Syringaresinol 9. R 1 = O (Limonin)10. R 1 = OAc (Limonyl acetate)32Figure 1: Structures of compounds isolated from Vepris glomerataThe HREIMS data of compound (1) showed a molecular ion peak at m/z 263.1278 [M+H] + ,corresponding to the molecular formula of C 15 H 18 O 4 and indicating seven double bondequivalents. The IR spectrum of compound 1 revealed the presence of OH and carbonylstretching frequencies at 3348 cm -1 and 1713 cm -1 respectively. The 1 H NMR spectrum ofcompound 1 showed characteristic resonances for prenylated cinnamaldehydes withresonances at δ H 6.57 (1H, dd, J = 15.80, 7.74 Hz), δ H 7.37 (1H, d, J = 15.80 Hz) and δ H9.59 (1H, d, J = 7.74 Hz), with the large coupling constant of 15.80 Hz indicative of transolefinic protons and were attributed to H-2, H-3 and the aldehyde proton, H-1, respectively.This coupling was also evident in the COSY spectrum.An ABX coupled system could be seen in the second proton system, which consisted of atriplet at δ H 4.74 (1H, t, J = 9.15 Hz, H-2’), which coupled in the COSY spectrum to the two

P a g e | 115doublet resonances at δ H 3.18 and δ H 3.21, H-1’a and H-1’b, both with J = 9.15 Hz. The H-1’a and H-1’b resonances occurred separately, since these were diastereotopic protons. Thetwo methyl resonances at δ H 1.34 and δ H 1.24 showed HMBC correlations to the C-2’methine carbon resonance at δ C 91.09 and a quaternary carbon resonance at δ C 71.74, C-3’.These COSY and HMBC correlations indicated the presence of a prenyl group with anepoxide ring at C-2’ and C-3’, also confirmed by the molecular mass of the compound.Also present in the 1 H NMR spectrum were two aromatic resonances at δ H 6.91 and δ H 7.02and an aromatic methoxy proton resonance at δ H 3.93. Apart from the C-6 resonance, afurther deshielded aromatic C-O resonance was detected at δ C 151.23 indicating thepresence of a further aromatic hydroxyl group. The fifteen carbon resonances detected inthe 13 C NMR spectrum indicated that the substituents on the benzene ring were thecinnamaldehyde group, the prenyl group, the two aromatic hydrogen atoms, the methoxyand hydroxyl group. The prenyl group was placed at C-8 because 2H-1’ showed an HMBCcorrelation to C-9. The remaining hydroxyl group was then placed at C-7.The absolute configuration of glomeral (1) was established by the CD spectrum whichshowed a positive cotton effect at 328 nm (∆ε +3.0) due to the n→π* transition of thecarbonyl group, a negative cotton effect at 292 nm (∆ε -0.5) due to the π→π* transition of thedouble bond and a positive cotton effect at 218 nm (∆ε +6.5) due to the π→π* transition ofthe α, β unsaturated aldehydic carbonyl group. The configuration of the chiral carbon C-2’was established to be R and the compound identified as 3-[3-(3-methyl-(2R)-2,3-epoxybutyl)-4-hydroxy-5-methoxyphenyl]-propenalin comparison with the CD spectrum of methyl12,14-dihomojuvenate which was determined to be (E,E)-(10R,11S)-10,11-epoxy-7-ethyl-3,11-dimethyl-2,6-tridecadienoate (Meyer et al., 1971).

P a g e | 116This is the first report of compound 1 and has been given the trivial name glomeral.Glomeral (1) shared high structural resemblance to 3-(3-methyl-2-epoxy-butyl-)-P-coumaricacid methyl ester isolated from Psoralea plicata (leguminosae) except that glomeral (1) hasan additional methoxy group at C-6 as well as the cinnamaldehyde group at C-4 instead ofthe ester group (Hamed et al., 1997).The antibacterial results of the pure compounds (1, 5, 6, 7, 9 and 10) indicate a broadspectrum activity against the test organisms Staphylococcus aureus (ATCC 29213 and25923) and Shigella dysentrieae (Table 1). No results were recorded for Escherichia coliand the fungal strains investigated since all compounds tested were inactive against thesemicrobes. Compounds 2-4 and 8 were also inactive against all tested strains and thereforethe results are not included in the table. The inhibition zone determination shows that thenew compound, glomeral (1) exhibited good activity with inhibition zones of 16 mm againstS. dysentrieae, and 15 mm and 14 mm against S. aureus (ATCC 29213) and S. aureus(ATCC 25923) respectively. Hesperetin (5) and scoparone (6) also showed good activitywith inhibition zones of between 9 and 14 mm against S. dysentrieae and the two cultures ofS. aureus. The observed results for these compounds were comparable with that ofchloroamphenicol with an inhibition zone of 20 mm against S. aureus and 18 mm against S.dysentrieae. The remaining compounds (skimmianine (7), limonin (9), and limonyl acetate(10)) did not show good antibacterial activities.

P a g e | 117Table 1: Antibacterial activity of the isolated compoundsS.aATCC29213Inhibition zones(mm)S.aATCC25923S.dMinimum inhibitoryconcentration MIC (µg/mL)S.a S.a S.dATCC ATCC29213 25923CompoundGlomeral 1 15 ± 1.0 14 ± 1.2 16 ± 1.0 2 2 0.4Hesperetin 5 12 ± 1.3 11 ± 1.0 14 ± 1.1 0.2 0.2 4Scoparone 6 10 ± 1.1 09 ± 1.5 16 ± 1.6 16 16 0.2Skimmianine 7 09 ± 1.2 06 ± 1.2 10 ± 1.3 16 >32 16Limonin 9 08 ± 1.6 07 ± 1.3 08 ± 1.4 >32 >32 16Limonin acetate 10 08 ± 1.4 08 ± 1.2 10 ± 1.5 >32 >32 32Chloroamphenicol 20 ± 1.0 20 ± 1.3 18 ± 1.7 2 2 8Key: S.a –Staphylococcus aureus, S.d - Shigella dysentrieaeThe minimum inhibitory concentration (MIC) determination (Table 1) showed thathesperetin (5) completely inhibited the growth of S. aureus (ATCC 29213 and ATCC25923) and S. dysentrieae, at low concentrations of 0.2 µg/mL and 4.0 µg/mL respectively,even lower than that of chloroamphenicol. The MIC value of glomeral was the same as thatof chloroamphenicol for S. aureus at 2.0 µg/mL and twenty times lower (0.4 µg/mL) thanchloroamphenicol against S. dysentrieae.Scoparone (6) was the most active of all thecompounds tested against S. dysentrieae at 0.2 µg/mL being forty times lower than that ofchloroamphenicol in the same assay. The rest of the compounds (7, 9 and 10) had MICvalues of either 16 µg/mL or greater than 32 µg/mL.These results are consistent with previous findings that showed phenyl propanoidderivatives (which are structurally related to glomeral) isolated from Ballota nigra(Lamiaceae) to possess moderate activity against S. aureus (Didry et al., 1999).Experimental3.1 General experimental procedures. The 1 H, 13 C and all 2D NMR spectroscopy wererecorded using a Bruker Avance III 400 MHz spectrometer. The spectra were referencedaccording to the deuteriochloroform signal at δ H 7.24 (for 1 H NMR spectra) and δ C 77.0 (for

P a g e | 11813 C NMR spectra). The HREIMS was measured on Bruker Micro TOF-QII instrument. IRspectra were recorded using a Perkin Elmer Universal ATR spectrometer. Optical rotationswere measured at room temperature in a PerkinElmerTM, Model 341 Polarimeter with 10cm flow tube. The Circular Dichroism (CD) spectrum was recorded on chirascan plusspectropolarimeter by applied photophysics at wavelengths 190-400 nm. The melting pointswere determined on an Ernst Leitz Wetziar micro-hot stage melting point apparatus. Mercksilica gel 60 (0.040-0.063 mm) was used for column chromatography and Merck 20 × 20 cmsilica gel 60 F 254 aluminium sheets were used for thin-layer chromatography. The TLCplates were analysed under UV (254 and 366 nm) before being sprayed and developed witha [1:2:97] anisaldehyde: concentrated sulphuric acid: methanol spray reagent and thenheated.3.2 Plant material. The stem bark and roots of V. glomerata were collected in December,2009 in Rift Valley Province of Kenya. The plant was identified by Dr S. T. Kariuki of theDepartment of Botany, Egerton University. A voucher specimen (Kiplimo 01) has beenretained at the University of KwaZulu-Natal Ward Herbarium, Westville, Durban.3.3 Serial extraction and isolation. The air-dried and ground plant material of V. glomerata(823.3 g stem bark and 719.3 g roots) was sequentially extracted for 24 hours using asoxhlet apparatus with solvents of increasing polarity; hexane, dichloromethane, ethylacetate and methanol. The collected solution of each extract was individually evaporatedusing a rotavapor.Crude extracts were loaded onto a column (4.5 in diameter) packed with a silica gel slurry toa height of 30 cm. For each elution system 2 L volumes were used and 100 mL fractionscollected. The collected fractions were analysed using TLC to determine if separation hadoccurred. Similar fractions were combined and concentrated using a rotavapor.

P a g e | 119The dichloromethane extract from the roots was separated with a hexane: ethyl acetate stepgradient starting from 100% hexane to 80% ethyl acetate in hexane. The following fractionswere combined and purified using the given solvent systems; 19-28 (hexane: ethyl acetate,80:20) which yielded glomeral (1) (56 mg), 52-55 ( hexane: ethyl acetate, 70:30) whichyielded scoparone (6) (19.7 mg), 60-70 (dichloromethane: ethyl acetate, 80:20) yieldedskimmianine (7) (23 mg), 85-92 (dichloromethane: ethyl acetate, 70:30) yielded mesosyringaresinol(8) (5.3 mg).The ethyl acetate extract from the stems yielded compounds 2-5. The mobile phase in thecrude separation consisted of a hexane: ethyl acetate step gradient. Compound 5 (19.3 mg)was obtained from fraction 36-39 and further purified with (70:30) hexane: ethyl acetate.Trans-p-hydroxy cinnamic acid compound 2 (9.2 mg) was obtained from fraction 27-31 andpurified by 100% dichloromethane. Compound 3 (22.3 mg) was obtained from fraction 84-93 and purified with a 1:1 ratio of ethyl acetate and hexane. Fraction 40-48 was purified by30% ethyl acetate in dichloromethane to yield compound 4 (11.3 mg).The dichloromethane extract of the stems afforded two limonoids, after the crude extractwas separated using a hexane: ethyl acetate step gradient.Both were obtained fromfractions 32-49 using hexane: ethyl acetate (70:30). Fraction 32-45 was purified usinghexane: ethyl acetate, 80:20 to afford limonin, 9 (21 mg). Fraction 46-49 was purified with100% dichloromethane which yielded limonyl acetate 10 (33 mg).3.3.1 Glomeral (1) reddish brownish solid; mp 58-60 o C; [α] 20 D +27.8° (c 0.016, CHCl 3 );UV λ max (CHCl 3 ) nm (log ε) 253 (1.92); IR (cm -1 ): 3348 (-OH), 2927 (-CHO), 2854, 1713(>C=O), 1616, 1490; 1 H NMR spectral data ( 400 MHz, CDCl 3 ) δ H 9.59 (1H, d, J = 7.80Hz, H-1), 7.37 (1H, d, J = 15.80 Hz, H-3), 7.02 (1H, s*, H-9), 6.91 (1H, s*, H-5), 6.57(1H, dd, J = 15.80, 7.80 Hz, H-2), 4.74 (1H, t, J = 9.15, Hz, H-2’), 3.93 (3H, s, OCH 3 ),

P a g e | 1203.18 (1H, d, J = 9.15 Hz, H-1’a), 3.21 (1H, d, J = 9.15 Hz, H-1’b), 1.35 (3H, s, CH 3 -5’),1.21 (3H, s, CH 3 -4’); 13 C NMR spectral data (100 MHz, CDCl 3 ) δ C 193.74 (C-1), 153.38(C-3), 151.23 (C-7), 144.46 (C-6), 129.37 (C-8), 127.88 (C-4), 126.28 (C-2), 118.69 (C-9),111.57 (C-5), 91.09 (C-2’), 71.74 (C-3’), 56.05 (OCH 3 ), 30.64 (C-1’), 26.12 (C-5’), 24.18(C-4’); EIMS (70 eV) m/z (rel. int): 262 [M + ] (100), 229 (13), 203 (30), 191 (13), 173 (24),161 (15), 115 (17); HREIMS (70 eV) 263.1254 [M+1] + (Calc. for C 15 H 19 O 4 ).*These resonances were not resolved into doublets with J = 2-3 Hz as expected for metacoupling, however the resonances were slightly broadened and also coupled in the COSYspectrum, indicating meta coupling.3.4 Antimicrobial assay. Gram-negative bacteria, Escherichia coli and Shigelladysentrieae, and Gram-positive bacteria, Staphyloccocus aureus ATCC 29213 and ATCC25923 were used for the antibacterial assay and fungal strains, Candida albicans, Candidaparapsilosis, Cryptococcus neoformans and Trichophyton mentagrophytes were used for theantifungal assay. The bacterial and fungal strains were cultured for 18 hours at 37 o C onMuller Hinton agar (and saboraud dextrose agar for fungi) and standardized to a final celldensity of 1.5x10 8 cfu/ml using a barium sulphate standard equivalent to McFarland No. 0.5standard or its optical equivalent.Disc diffusion method: Blank discs were sterilized by air drying at 160 o C for 1 h. Testcompounds were dissolved in DMSO and 20 µl of the solution (compound and DMSO)impregnated onto the blank sterile discs and placed asceptically onto the inoculated petridish, which were then incubated at 37 o C for 24 hours for the bacteria while the fungi wereincubated for 72 hours at 25 o C. The activity of the compounds was determined by thepresence of measureable inhibition zones.

P a g e | 121Broth microdilution method (Andrews, 2001): The test compounds and the standardchloroamphenicol (fluconazole for the fungi) were dissolved in dimethyl sulfoxide (DMSO)to a concentration of 10 mg/ml. Serial two fold dilutions were made ranging from 0.002µg/ml to 32 µg/ml in sterile test tubes. The 96-well plates were prepared by dispensing intoeach well, 90 µl Muller Hinton and 10 µl of the bacterial inoculums. The first three wellscontaining Muller Hinton broth were used as a negative control and the next threecontaining 90 µl Muller Hinton broth and 10 µl of the bacterial inoculums without the testcompound were used as growth control. The standard antibiotic tetracycline was used as apositive control. The plates were covered to avoid contamination and evaporation andincubated for 24 hours at 37 o C.All procedures were done in triplicate. The lowestconcentration of each compound showing no visible growth was taken as its minimuminhibitory concentration (MIC).ConclusionThe phytochemical analysis of V. glomerata showed the existence of the antimicrobialcompounds, glomeral (a new cinnamaldehyde), hesperetin and scoparone. Skimmianine,limonin and limonyl acetate showed low to moderate antibacterial activity. These resultsprovide scientific validity and credence to the ethnomedicinal use of this plant in thetreatment of ailments caused by some of the pathogenic microbes used in this study andhighlights the efficacy of V. glomerata in the treatment of bacterial infections. Furtherstudies on structure-activity relationships and mode of action may be a guide to a betterunderstanding of the relationships between the structures and antimicrobial activities ofthese compounds.

P a g e | 122AcknowledgmentsWe thank Ms Tricia Naicker and Dr Jaco Brand (University of Stellenbosch) for runningHREIMS and CD spectra respectively.Dr Christine Bii of Kenya Medical ResearchInstitute (KEMRI)-Nairobi for the antimicrobial testing and the Organisation for Women inScience for the Developing World (OWSDW) for financial support.

P a g e | 123ReferencesAndrews, J. A., 2001. Determination of inhibitory concentrations. J. Antimicrob.Chemother. 48, 5-16.Beentje, H. J., 1994. Kenya Trees, Shrubs and Lianas, National Museum of Kenya, Nairobi,Kenya, pp. 17-22.Brader, G., Bacher, M., Greger, H., Hofer, O., 1996. Pyranoquinolones and acridones fromVepris bilocularis. Phytochemistry 42, 875-879.Chaturvedula, V. S. P., Schilling, J. K., Miller, J. S., Andransiferana, R., Rasamison, V. E.,Kingston, D. G. I., 2003. New cytotoxic alkaloids from the wood of Vepris punctata fromthe Madagascar rainforest. J. Nat. Prod. 66, 532-534.Chaturvedula, V. S. P., Schilling, J. K., Miller, J. S., Andransiferana, R., Rasamison, V. E.,Kingston, D. G. I., 2004. New cytotoxic terpenoids from the wood of Vepris punctatafrom the Madagascar rainforest. J. Nat. Prod. 67, 895-898.Cheplogoi, P. K., Mulholland, D. A., Coombes, P. H., Randrianarivelojosia, M., 2008. AnAzole, an amide and a limonoid from Vepris uguenensis (Rutaceae). Phytochemistry 69,1384-1388.Dagne, E., Yenesew, A., Waterman, P. G., Gray, A. I., 1988. The chemical systematics ofthe Rutaceae subfamily Toddalioideae, in Africa. Biochem. System. and Ecol. 16, 179-188.Denise, B., Farve-Bonvin, J., 1973. Essential oil constituents of Vepris madagascarica.Phytochemistry 12, 1194.

P a g e | 124Didry, N., Seidel, V., Dubreuil, L., Tillequin, F., Bailleul, F., 1999. Isolation andantibacterial activity of phenylpropanoid derivatives from Ballot nigra. J.Ethnopharmacol. 67, 197-202.Ganguly, A. K., Govindachari, T. R., Manmade, A., Mohamed, P. A., 1966. Alkaloids ofVepris bilocularis. Indian J. Chem. 4, 334-335.Govindachari, T. R., Sundararajan, V. N., 1961. Alkaloids of Vepris bilocularis. J. Sci.Industr. Res. 20, 298-299.Govindachari, T. R., Suresh, G., Gopalakrishnan, G., Banumathy, B., Masilamani, S., 1998.Identification of antifungal compounds from the seed oil of Azidirachta indica A Juss.Phytoparasit. 26, 109-116.Hamed, A. I., Springuel, I., El-Emary, N. A., Mitome, H., Yamada, Y., 1997. A phenoliccinnamate dimer from Psoralea plicata. Phytochemistry, 45, 1257-1261.Innocent, E., Moshi, M., Masimba, P., Mbwambo, Z., Kapingu, M., Kamuhabwa, A., 2009.Screening of traditionally used plants for in vivo antimalarial activity in mice. Afr. J.Trad. Complem. Altern. Med. 6, 163-167.Jimenez, C., Riguera, R., 1994. Phenylethanoid glycosides in plants: structure and biologicalactivity. Nat. Prod. Rep. 11, 591-606.Khalid, S. A., Waterman, P. G., 1982. Chemosystematics in the Rutaceae 15. Furoquinolineand pyrano-2-quinolone alkaloids of Vepris stolzii. J. Nat. Prod. 45, 343-346.Khalil, A. T., Maatooqa, G. T., El Sayeda, K. A., 2003. Limonoids from Citrus reticulate. Z.Naturforsch 58c, 165-170.

P a g e | 125Kokwaro, J. O., 1976. Medicinal plants of East Africa. East African Literature Bureau,Nairobi.Latip, J., Ali, M. N. H., Yamin, B. M., 2005. 4,6,7-Trimethoxyfuro[2,3-b]quinoline-water(2/3). Acta. Cryst. E61, 2230-2232.Lee, S., Kim, B., Cho, S. H., Shin, K. H., 2002. Phytochemical constituents from the fruitsof Acanthopanax sessiliflorus. Arc. Pharm. Res. 25, 280-284.Lu, Y., Foo, Y. L., Wong, H., 1999. Sage coumarin, a novel caffeic acid trimer from Salviaofficinalis. Phytochemistry 52, 1149-1152.Meyer, A. S., Hanzmann, E., Murphy, R. C., 1971. Absolute configuration of Cecropiajuvenile hormone. Proc. Nat. Acad. Sci. USA, 68, 2312-2315.Moshi, J. M., Mbwambo, Z. H., Nondo, S. O. R., Masimba, P. J., Kamuhabwa, A., Kapingu,M. C., Thomas, P., Richard, M., 2006. Evaluation of ethnomedical claims and brineshrimp toxicity of some plants used in Tanzania as traditional medicines. Afr. J. Trad.Complem. Altern. Med. 3, 48-58.Ngadjui, T. B., Ayafor, J. F., Sondengam, B. L., Connolly, J. D., Rycroft, D. S., Khalid, A.,Waterman, P. G., Brown, N. M. D., Grundon, M. F., Ramachandran, V. N., 1982. Thestructures of vepridimerines A-D, four new dimeric prenylated quinolone alkaloids fromVepris louisii and Oricia renieri (Rutaceae). Tetrahedron Lett. 23, 2041-2044.Ouyang, M., Wein, Y., Zhang, Z., Kuo, Y., 2007. Inhibitory activity against TobaccoMosaic Virus (TMV) replication of Pinoresinol and Syringaresinol Lignans and theirGlycosides from the root of Rhus javanica Var. Roxburghiana. J. Agric. Food Chem. 55,6460-6465.

P a g e | 126Poitou, F., Masotti, V., Viano, J., Gaydou, E. M., Adriamahavo, N. R., Mamitiana, A.,Rabemanantsoa, A., Razanamahefa, B. V., Andriantsiferana, M., 1995. Chemicalcomposition of Vepris elliotii essential oil. J. Essential Oil Res. 7, 447-449.Rasoanaivo, P., Federici, E., Palazzino, G., Galeffi, C., 1999. Acridones of Veprissclerophylla; their 13 C NMR data. Fitoterapia 70, 625-627.Ruberto, G., Renda, A., Tringali, C., Napoli, E. M., Simmonds M. S. J. 2002. CitrusLimonoids and their Semisynthetic derivatives as antifeedant agents against Spodopterafrugiperda Larvae. A Structure-Activity Relationship study. J. Agric. Food Chem. 50,6766-6774.Rudolf, H., Eva Maria, C., 1978. Alkaloids from the root bark of Vepris pilosa. Archiv.Pharm. (Weinheim, Germany) 311, 135-138.Sidibe, L., Charchat, J. C., Garry, R. P., Harama, M., 2001. Composition of essential oil ofVepris heterophylla (Engl.). J. Essential Oil Res. 13, 183-184.Wawer, I. and Zielinska, A., 2001. 13 C CP/MAS NMR studies of flavonoids. Magn. Reson.Chem., 39, 374–380.

P a g e | 127CHAPTER SIXRING A, D-SECO LIMONOIDS AND FLAVONOID FROMTHE KENYAN VEPRIS UGUENENSIS ENGL. AND THEIRANTIOXIDANT ACTIVITYJoyce J. Kiplimo † Md. Shahidul Islam ‡ and Neil A. Koorbanally †*† School of Chemistry, University of KwaZulu –Natal, Private Bag X54001, Durban 4000, South Africa‡ School of Biochemistry, Genetics and Microbiology, University of KwaZulu–Natal, Private Bag X54001,Durban 4000, South AfricaCorresponding Author: E-mail address: Koorbanally@ukzn.ac.zaTel.: +27312603189; Fax: +27312603091.ABSTRACTTwo new A, D-seco-limonoids, accorded the trivial names, uguenensene (4) anduguenensone (5) and a new C-7 prenylated flavonoid, uguenenprenol (8) were isolated fromVepris uguenensis (Rutaceae). In addition, eleven known compounds, niloticin (1),chisocheton A (2), kihadalactone A (3), limonyl acetate (6), methyl uguenenoate (7), 7-Omethylaromadenrin(9), flindersiamine (10), 8α,11-elemodiol (11), tricoccin S 13 acetate,skimmianine, and lupeol were isolated.The structures of the new compounds wereelucidated and characterized by spectroscopic analyses (NMR, GC-MS and IR).Antioxidant activity of the isolated compounds using the 2,2-diphenyl-β-picrylhydrazyl(DPPH), Deoxyribose and Ferric Reducing Antioxidant Power (FRAP) assays showed thatuguenenprenol (8) and 7-O-methylaromadenrin (9) are good antioxidant agents.Significantly high antioxidant activity was also exhibited by 8α,11-elemodiol (11), whichwas 72% at 250 µg mL -1 and 57% at 15.62 µg mL -1 when tested with the Deoxyribose

P a g e | 128method. The current contribution adds uguenensene (4) and uguenensone (5) to the class ofcitrus limonoids common to the Rutaceae which can be used not only for medicinalpurposes but also as an antioxidant supplement.O12HO29422 O20181221O171130 1319R9 14162810 8H5H67O4. R = 2H5. R = O15OCOCH 323OH4''3''5''2''1''O6789O4105OH O8236'OH5'2'OH4'3'INTRODUCTIONLimonoids are tetranortriterpenoids derived from the acetate-mevalonate pathway with thetriterpenoids euphane or tirucallane being the key intermediates 1,2 . Citrus limonoids havebeen shown to originate from nomilin which is biosynthesized in the phloem region of stemsand then migrate to other tissues such as leaves, fruits and seeds, where other limonoids arebiosynthesized 3 . Limonoids are the main constituents of the Rutaceae and are known tohave a wide range of biological activities. The biological activities of limonoids haveattracted widespread scientific interest; they are reported to exhibit antifungal 4 ,antibacterial 5 , antimalarial 6 , antifeedant 7 , antiprotozoan 8 , antiviral 9 and anti-inflammatory 10activities, and recently, the antioxidant capacity of citrus limonoids and limonoid-containingextracts have been evaluated using the racimat experiment, superoxide radical quenchingand the DPPH radical scavenging assays 11 . Limonoids have also been known to inhibit thedevelopment of cancer in laboratory animals and in human breast cancer cells 12 .The plant Vepris uguenensis is known as ‘Chemchir’ by the Pokot tribe of Kenya, who useit to treat malaria 13 . Previous phytochemical reports indicated that a limonoid (methyl

P a g e | 129uguenenoate), an azole (uguenenazole) and an imide (uguenenonamide) were isolated fromthe roots of V. uguenensis 13 . Methyl uguenenoate displayed mild antimalarial activity whilethe azole and the imide were found to be completely inactive 13 .No phytochemicalcompounds from the stem bark and leaves of V. uguenensis have been reported thus far.Oxidative stress is a situation where there is an imbalance between the production ofreactive oxygen species (ROS) that can damage cell structures and the body's ability todetoxify these molecules or repair the resulting damage. These reactive oxygen species(ROS) have been found to mediate neurological injury in cerebral malaria (CM) which is themost severe neurological complication of infection with Plasmodium falciparum 14 . Duringan attack of malaria, the parasite breaches the blood–brain barrier to cause cerebral malaria(CM), resulting in a life-threatening crisis 14 . With suitable treatment, patients do recoverbut often do so with lasting damage to their brain, resulting in a loss of mental function 14 .Studies have shown that the lives of many African children have been shattered in this way.It is reported that 21% of children with CM had cognitive deficits six months after dischargefrom hospital in Uganda 15 . This is because cerebral malaria leads to increased production ofmolecules indicative of oxidative stress in the brain 14 . Treatment with a combination ofchloroquine and two antioxidant agents, desferoxamine and N-acetylcysteine, at the firstsigns of cerebral malaria prevents both inflammatory and vascular changes in the tissues ofthe brain, as well as the development of persistent cognitive damage 14 . The addition ofantioxidants does not weaken the efficacy of chloroquine in eliminating Plasmodia from theblood.Combination therapy with antioxidants has been effective in treating cerebralmalaria and preventing subsequent cognitive impairment in mice 14 .Since V. uguenensis is used as an antimalarial in African traditional medicine, andlimonoids are reported to possess antioxidant activity 11 , all the isolated compounds were

P a g e | 130subjected to antioxidant assays using the ferric reducing antioxidant power (FRAP), the 2,2-diphenyl-β-picrylhydrazyl (DPPH) and the deoxyribose methods. We present herein thedetails of the isolation and structure elucidation of three new compounds, a C-7 prenylatedflavonoid (8) and two limonoids (4 and 5) as well as the in vitro antioxidant activities of theisolated compounds and a brief discussion on the biogenetic relationship of the limonoids(1-7).RESULTS AND DISCUSSIONRepeated column chromatography of the hexane and dichloromethane extract of the leaves,stem bark and roots yielded eleven known compounds; a triterpenoid (lupeol), asesquiterpene (8α,11-elemodiol (11)), two furoquinoline alkaloids (skimmianine andflindersiamine (10)), a flavonoid (7-O-methylaromadenrin (9)), two proto-limonoids(niloticin (1) and chisocheton A (2)) and four limonoids (tricoccin S 13 acetate, kihadalactoneA (3), limonyl acetate (6) and methyl uguenenoate (7)). A further two novel A, D-secolimonoids and a prenylated flavonoid, which were accorded the trivial names; uguenensene(4), uguenensone (5) and uguenenprenol (8) were isolated. Structures of known compoundswere determined using NMR and MS techniques and confirmed by comparison of physicaland spectroscopic properties against literature data 16-21 .The isolated compounds (1-7) displayed an interesting limonin biogenetic sequence withstructural features expected of intermediates in the limonin biosynthetic pathway (Figure 1).According to the generally accepted scheme, the ∆ 7 bond as in niloticin (1), is epoxidized tothe 7-epoxide and then opened, inducing a Wagner-Meerwein shift of the methyl group atC-14 to C-8 leading to the formation of a double bond at ∆ 14 in 2, together with modificationof the side chain 2 . This is followed by side chain cleavage of the four carbon epoxide groupand the expansion of the carbocyclic A-ring in 2 to a seven membered δ-unsaturated lactone

P a g e | 131ring as seen in tricoccin S 13 acetate. Reduction of the side chain lactone ring to form thefuran ring, as well as acetylation at C-1 results in 3. Contraction of ring A to a fivemembered ring and lactonization involving the methyl group (CH 3 -19) and the carbonylgroup at C-3 leading to the formation of A and A’ rings (A-seco), occurs with a concurrentepoxidation at ∆ 14 , producing 4. Ring contraction and lactone formation may occursimultaneously. Oxidation at C-16 results in 5, which is followed by a Baeyer-villageroxidation of the D ring to a lactone resulting in 6. Methanolysis of the A ring lactone resultsin 7. Figure 1 shows the postulated biogenetic pathway for the formation of the protolimonoidsand limonoids 1-7, with 4 and 5 taking its place in the biosynthetic scheme.Compound 4 was isolated as a reddish brown solid and had a molecular formula ofC 28 H 36 O 7 as determined by HREIMS, with a molecular ion M +at m/z 484.2461 (asexpected) indicating 11 degrees of unsaturation. The IR spectrum showed the presence ofcarbonyl (C=O) stretches at 1733 and 1739 cm -1 ; epoxide (C-O) stretches at 1231 cm -1 andβ-substituted furan ring C-H bending vibrations at 726 cm -1 . The 1 H NMR data (Table 1)indicated the presence of a characteristic epoxy group methine proton at δ H 3.39 (s, H-15)and oxygenated protons at δ H 4.01 (d, J = 3.96 Hz, H-3), δ H 4.71 (t, J = 2.2 Hz, H-7), δ H4.49 (d, J = 13.21 Hz, H-19 exo) and δ H 4.34 (d, J = 13.21 Hz, H-19 endo). The acetylmethyl proton resonance occurred at δ H 2.06. The three furan proton resonances occurred atδ H 7.07 (s, H-21), 6.11 (s, H-22) and 7.33 (s, H-23), with the corresponding carbonresonances of H-21 and H-22 showing HMBC correlations to a methine proton resonance atδ H 2.57 (dd, J = 11.25, 6.04 Hz), attributed to H-17. Beside the methyl acetate, there werefour other tertiary methyl resonances at δ H 0.88 (3H-30), 0.95 (3H-18), 1.20 (3H-29) and1.07 (3H-28). The position of the methyl groups were supported by HMBC data; 3H-28 and

P a g e | 1323H-29 with C-4 and C-5; 3H-18 with C-14, C-13 and C-12; 3H-30 with C-7, C-8 and C-9.The methine proton at δ H 3.39 (s, H-15) showed HMBC correlations with C-16 and C-17.Cyclisation between C-19, and C-3 which incorporated C-1 and C-2 was supported by 2DNMR correlations. The H-3 and both H-19 doublets (H-19 endo and exo) showed HMBCcorrelations to the carbonyl resonance of C-1 and the formation of the lactone ring wasfurther supported by HMBC correlations between H-3 and both C-19 and C-9. The H-19doublets also showed HMBC correlations to C-5, C-9 and C-10 as expected and to C-3, onlypossible with cyclisation between C-19 and C-3.The selected 1D gradient NOE (GOESY) experiments on compound 4 revealed a twist inthe rearranged A-ring structure; this twist is clear in a 3-D model. NOE correlations wereobserved between H-9 and H-5, H-3 and 3H-18, which were on the backface in the 3-Dmodel. The methyl group 3H-30 showed NOE correlations to H-7, H-6β, H-11β, and H-19exo; these were on the top face in the 3-D model. This results in H-19 endo beingsituated proximal to the 4β methyl (3H-29) and H-6β. The H-17β resonance exhibited NOEcorrelations with the furanyl protons H-21 and H-22 and the H-16β proton resonances.The absolute configuration was determined by circular dichroism (CD). The CD spectrumof 4 showed a positive cotton effect at 197 nm (∆ε +8.5) and a negative cotton effect 210nm (∆ε -22.0). The furan chromophore (λ max 206 nm) was probably masked by the cottoneffect of the ester and lactone groups. A positive cotton effect was also observed at 328 nm,(∆ε +1.2) due to the n→π* transition of the carbonyl groups of 4 and was in accordancewith the CD spectra observed for limonoids from Turraea pubescens 22 .Thestereochemistry of the epoxide was shown to be β, in accordance with the orientationpreviously described for limonoids with a ∆ 14 , epoxide 23,24 . The absolute configuration of

P a g e | 133the ten chiral centers in 4 was thus determined as 3R, 5R, 7R, 8R, 9R, 10S, 13R, 14R, 15Rand 17R.The spectroscopic data and functionalities established that compound 4 was an A, D-secolimonoid, which are common to the Rutaceae and rare in the Meliaceae 25 .Ring Dresembled evodulone isolated from Carapa procera 21 , whereas ring A resembled limonin(A, A’ ring system). The other two rings, B and C of compound 4 remained carbocyclic.Comparison of the 1 H NMR data of compound 5, a brown solid with that of compound 4,indicated the notable absence of the methylene protons, H-16α and H-16β, which occurredat δ H 2.13 and δ H 1.59 in 4. This occurred with an additional carbonyl resonance at δ H208.5, indicating that a ketone functionality was now present at position 16. This wassupported by the HREIMS, which showed a molecular ion peak at m/z 498.9120 forC 28 H 34 O 8 , two hydrogen atoms less and one oxygen atom more than 5. The presence of thecarbonyl functionality at C-16 also affected the proton and carbon chemical shifts atposition 17, shifting them downfield to δ H 3.81 from 2.57 and to δ C 50.85 from 39.63. Therelative stereochemistry of 5 was deduced to be the same as that of 4 based on their almostsuperimposable 1 H and 13 C NMR data and similar NOESY and NOE correlation patterns tothose of 4. The Absolute configuration of 4 and 5 were also identical as indicated by similarCD curves.

P a g e | 134O229119103428H3COCO21H22 O23 26H11 12 1820 242517 OH279 14 168 Wagner-Meerwein15 methyl shift and side5HH307chain modification OOHH 6H1 2OBaeyer villager oxidationof ring A and sidechain cleavageO8 14 15HHHOHHOOO3OOCOCH 310HH314OCOCH3HOreduction to furan ringand acetylation at C-13OHH148 15OCOCH 3tricoccin S 13 acetateEpoxidation, ring contraction andlactone formationHO2O22 O2018 211 12HO 113019 133 9 16104 529 28HH6874O15OCOCH 323oxidation at C-16HOOOHHOHOOOCOCH 35Baeyer-Villager oxidation of ring DOOOH 3COOHOOHHOHOmethanolysis of theA ring lactoneOHOOHOHOOH7OH6OCOCH 3Figure 1. Plausible Biogenetic Pathway for the Formation of Limonoids 1-7 fromVepris uguenensis.

P a g e | 135Table 1. NMR spectroscopic Data (400 MHz, CDCl 3 ) for uguenensene (4) and uguenensene (5)uguenensene (4) uguenensone (5)Position δ C, type δ H (J in Hz) HMBC a δc, type δ H (J in Hz)1 169.94, C - - 169.80, C -2α 35.95, CH 2 2.92, dd (16.48, 3.96) 1, 3 35.92, CH 2 2.93, dd (16.56, 3.96)2β - 2.60, d (16.48) 1, 3 - 2.62, d (16.56)3 80.26, CH 4.01, d ( 3.96) 1, 9, 19 80.07, CH 4.01, d (3.96)4 80.58, C - - 80.60, C -5 54.19, CH 2.22, m 3, 4, 10, 54.16, CH 2.21, dd (13.48, 3.08)19, 28, 296 α 24.41, CH 2 1.81, m 5 24.27, CH 2 1.84, m6β - 1.72, m - - 1.78, m7β 73.53, CH 4.71, t (2.20) 5, 9, 31 73.35, CH 4.66, t (2.36)8 42.79, C - - 43.10, C -9 44.03, CH 2.79, dd (11.8, 4.20) 3, 8, 11, 19 43.31, CH 2.84, dd (13.20, 4.72)10 45.62, C - - 45.58, C -11α 18.55, CH 2 1.96, m - 18.27, CH 2 1.95, m11β - 1.71, m - - 1.76, m12 α 29.64, CH 2 1.83, m - 28.30, CH 2 2.19, m12 β - 1.80, m - - 1.73, m13 41.82, C - - 42.50, C -14 72.40, C - - 71.62, C -15 57.58, CH 3.39, s 16, 17 57.29, CH 3.34, s16 α 31.95, CH 2 2.13, dd (13.35, 6.04) 13, 14,15, 17 208.05, C -16 β - 1.59, dd (13.35, 11.25) 17 - -17 39.63, CH 2.57, dd (11.25, 6.04) 12, 13, 16,18, 20, 21,2250.85, CH 3.81, s18 21.36, CH 3 0.95, s 12, 13, 24.21, CH 3 1.02, s14,1719 exo 66.04, CH 2 4.49, d (13.12) 1, 3, 5, 10 65.85, CH 2 4.50, d (13.08)19 endo - 4.34, d (13.12) 5, 9 - 4.36, d (13.08)20 123.46, C - - 116.27, C -21 139.57, CH 7.07, s 20, 22, 23 141.60, CH 7.50, s22 110.85, CH 6.11, s 20, 21, 23 110.84, CH 6.17, s23 143.01, CH 7.33, s 20, 21, 22 142.56, CH 7.35, s28 21.36, CH 3 1.07, s 4, 5, 29 21.25, CH 3 1.06, s29 30.17, CH 3 1.20, s 4, 5, 28 30.14, CH 3 1.19, s30 19.38, CH 3 0.88, s 7, 8, 9, 14 19.39, CH 3 0.96, s31 169.55, C - - 169.50, C -OCOCH 332 21.37, CH 3 2.06, s 31 21.32, CH 3 2.01, sOCOCH 3a HMBC- correlations are from proton(s) to the indicated carbon (H→C)Compound 8 was identified as a flavanone by its NMR, UV, IR and mass data.Itsmolecular formula was established to be C 20 H 20 O 7 based on HREIMS data of 372.1158 amuwith a double bond equivalence of 11, eight being due to the aromatic rings, one being due

P a g e | 136to the carbonyl group, one due to the double bond on the side chain and the remaining onebeing due to ring C of the flavanone skeleton. The IR data showed the presence of O-H(3330 cm -1 ), and C=O (1708 cm -1 ) stretching bands. The 1 H NMR data showed acharacteristic AB coupling system of H-2 and H-3 which appeared at δ H 4.58 (1H, d, J =11.65 Hz, H-2) and δ H 4.82 (1H, d, J = 11.65 Hz, H-3), which was supported by COSYcorrelations between the two resonances. Their coupling constants were characteristic oftrans protons 26 , and this trans stereochemistry was confirmed by NOESY correlationsbetween H-3 and H-2’/6’.The second characteristic pattern was that of an AA’BB’ system of a 1,4-disubstitutedaromatic ring (ring B). These proton resonances occurred as two doublets at δ H 7.36 (2H, d,J = 8.52 Hz, H-2’/6’), and δ H 6.85 (2H, d, J = 8.52 Hz, H-3’/5’). COSY coupling betweenH-2’/6’ and H-3’/5’ were also observed. Also present in the 1 H NMR data were twoaromatic protons at δ H 6.09 (1H, d, J = 2.2 Hz, H-6) and δ H 6.04 (1H, d, J = 2.2 Hz, H-8)attributed to ring A. The observed coupling constant of 2.2 Hz was indicative of metacoupling.The third pattern was that of the isoprenol group in ring A.The 2H-1’’ and H-2’’resonances were observed at δ H 4.66 (2H, d, J = 6.32 Hz, H-1’’) and δ H 5.70 (1H, t, J = 6.32Hz, H-2’’). A singlet was also observed at δ H 3.76 attributed to 2H-4’’, which showedHMBC correlations to the quaternary carbon resonance at δ C 142.04 (C-3’’), the methinecarbon resonance at δ C 119.65 (C-2’’) and the methyl carbon resonance at δ C 14.03 (C-5’’).These correlations indicated that one of the methyl protons in an isoprenyl unit had beenoxidized to a primary alcohol and that the isoprenyl group was attached to C-7 by an etherlinkage as the 2H-1’’ resonance was seen to be deshielded.

P a g e | 137The isoprenol group was placed at C-7 since H-6 and H-8 both showed NOESY correlationswith 2H-1’’. This would not be possible if the isoprenol group was located at C-5. Thehydroxyl groups were thus placed in the remaining C-4’, C-3 and C-5-positions, which arealso the most common positions to be hydroxylated. The carbon resonances of δ C 165.02(C-4’), 85.10 (C-3) and 164.36 (C-5) support these assignments.The absolute configuration of C-2 and C-3 were determined by CD analysis in comparisonwith literature values 26,27 . The CD spectrum showed a positive cotton effect at 205 nm, ∆ε+8.8, (π→π* for the double bond), 290 nm, ∆ε +4.0 (π→π*) and 335 nm, ∆ε +2.0 (n→π* ofthe carbonyl group). The absolute configuration of the chiral centers was assigned as 2R,3R. Compound 8 was thus identified as (2R,3R)-3,5-dihydroxy-7-((E)-4-hydroxy-3-methylbut-2-enyloxy)-2-(4-hydroxyphenyl)chroman-4-one.Eleven of the fourteen compounds isolated, were evaluated for their antioxidant potentialsusing three different antioxidant assays; FRAP, DPPH and deoxyribose methods. Two ofthe compounds, tricoccin S 13 acetate and skimmianine were not tested for their antioxidantactivity as they were isolated in very small quantities, 23 mg and 13 mg respectively. Onlycompounds in excess of 30 mg were of sufficient quantities to use in the three assays.Lupeol however did not show any activity.The results obtained (Table 2) shows that the reducing powers of all of the compoundsincreased with increasing concentration. The reducing power of the standard ascorbic acidwas significantly higher than that of all compounds tested in this assay except foruguenenprenol, (8) and 7-O-methylaromadenrin (9), whose values, although lower thanascorbic acid were found to be significantly higher than the rest of the compounds testedand exhibited good activity even at lower concentrations. The reducing power of thedifferent compounds tested decreased in the following order: uguenenprenol (8) > 7-O-

P a g e | 138methylaromadenrin (9) > 8α, 11-elemodiol (11) > chisocheton (2) > uguenensone (5) =flindersiamine (10) > niloticin (1) > limonyl acetate (6) > kihadalactone A (3) > methyluguenenoate (7) > uguenensene (4). The high antioxidant activity of the flavonoids incomparison to the other compounds was expected, since flavonoids are known antioxidants.Flavonoids are known to suppress reactive oxygen species formation either by inhibition ofenzymes or chelating trace elements involved in free radical production, scavenging ofreactive oxygen species and upregulating or protecting antioxidant defences 28 . The presenceof 3-hydroxyl groups as in compounds 8 and 9 in the heterocyclic ring also increases theradical scavenging activity (RSA) 28 .Table 2: Reducing power of compounds from V. uguenensis as evaluated by the FRAPmethod.AbsorbanceConcentration µg/mL 15.6 31.1 62.5 125 250Niloticin (1) 0.01±0.00 d 0.03±0.00 be 0.05±0.00 be 0.10±0.01 b 0.21±0.00 bgChisocheton (2) 0.02±0.01 bd 0.03±0.01 be 0.06±0.01 b 0.11±0.01 b 0.27±0.02 bKihadalactone A (3) 0.01±0.00 d 0.02±0.00 e 0.04±0.01 ge 0.07±0.00 be 0.13±0.00 egUguenensene (4) 0.01±0.01 d 0.02±0.00 e 0.03±0.00 g 0.05±0.00 e 0.11±0.00 eUguenensone (5) 0.03±0.00 b 0.05±0.00 bc 0.07±0.00 b 0.13±0.00 f 0.23±0.00 bgLimonyl acetate (6) 0.06±0.01 c 0.07±0.00 c 0.11±0.03 f 0.11±0.01 f 0.20±0.03 beMethyl uguenenoate (7) 0.01±0.00 d 0.02±0.00 e 0.03±0.01 g 0.05±0.00 e 0.11±0.00 eUguenenprenol (8) 0.06±0.01 c 0.12±0.00 d 0.35±0.02 d 0.87±0.00 d 1.27±0.10 d7-O-methylaromadenrin (9) 0.07±0.00 c 0.07±0.00 c 0.14±0.00 c 0.36±0.00 c 0.86±0.02 cFlindersiamine (10) 0.02±0.00 bd 0.04±0.00 be 0.05±0.01 be 0.08±0.00 be 0.23±0.02 bg8α,11-elemodiol (11) 0.03±0.00 b 0.04±0.01 be 0.05±0.00 be 0.10±0.01 b 0.28±0.01 bAscorbic Acid 0.15±0.02 a 0.33±0.06 a 0.60±0.01 a 1.05±0.01 a 1.97±0.15 aData are presented as means ±SD of triplicate. a-g Values with different superscript lettersfor a given concentration are significantly different from each of the other compounds (Oneway ANOVA followed by Tukey’s multiple range post-hoc test, p < 0.05).The RSA results shown in table 3 revealed that the scavenging ability of the novelflavonoid, uguenenprenol (8) and 7-O-methylaromadenrin (9) was 94% at 250 µg mL -1 .Their scavenging effects was marginally higher than that of ascorbic acid in almost allconcentrations except at the lower concentrations 15.62 µg mL -1 and 31.12 µg mL -1 where

P a g e | 139ascorbic acid was marginally higher. Again, the antioxidant activity of the flavonoids 8 and9 in this assay were significantly higher than the rest of the compounds. Of the limonoidstested, 2, 5 and 7 displayed significantly higher activities compared to the rest of thelimonoids. The RSA of all compounds in decreasing order was: 7-O-methylaromadenrin (9)> uguenenprenol (8) > uguenensone (5) > methyl uguenenoate (7) > chisocheton (2) >limonyl acetate (6) > uguenensene (4) > flindersiamine (10) > niloticin (1) > 8α,11-elemodiol (11) > kihadalactone A (3).Table 3: Results of the antioxidant activity of compounds isolated from Veprisuguenensis as measured by the DPPH methodPercentage scavenging activityConcentrations (µg/mL) 15.6 31.1 62.5 125 250Niloticin (1) 2.00±1.00 b 8.33±0.58 c 12.00±1.00 b 14.67±0.58 b 22.00±3.61 iChisocheton (2) 24.00±1.00 i 39.67±0.58 h 43.33±0.58 e 47.67±1.53 d 54.67±1.53 hKihadalactone A (3) 1.33±0.58 b 6.00±1.00 g 9.00±1.00 b 11.33±0.58 f 13.67±0.58 gUguenensene (4) 4.00±2.00 h 8.33±1.53 c 21.33±1.53 c 32.33±2.08 e 38.67±4.04 fUguenensone (5) 16.00±0.00 g 22.00±1.73 f 42.00±6.93 e 51.33±3.79 d 68.00±1.73 dLimonyl acetate (6) 13.00±1.00 f 22.33±0.58 f 26.00±1.73 f 29.00±1.00 e 49.67±0.58 eMethyl uguenenoate (7) 29.67±2.08 d 35.67±1.15 e 45.33±2.52 e 50.33±0.58 d 64.67±0.58 dUguenenprenol (8) 41.00±1.00 e 72.00±1.00 d 87.00±1.00 d 91.33±1.53 a 93.67±1.53 a7-O-methylaromadenrin (9) 30.67±0.58 d 75.00±2.65 d 91.00±1.00 d 92.67±1.15 a 94.33±0.58 aFlindersiamine (10) 7.00±0.00 c 9.00±1.00 c 19.00±1.00 c 20.67±0.58 c 28.67±2.31 c8α,11-elemodiol (11) 0.33±0.58 b 1.67±0.58 b 9.00±1.00 b 14.67±2.52 b 18.00±1.00 bAsorbic Acid 37.67±0.58 a 78.00±058 a 79.67±0.58 a 91.00±0.58 a 92.67±0.58 aData are presented as means ± SD of triplicate. a-i Values with different superscript lettersfor a given concentration are significantly different from each of the other compounds (Oneway ANOVA followed by Tukey’s multiple range post-hoc test, p < 0.05).The fact that 2, 5 and 7 have some antioxidant activity in this assay is worth noting aslimonoids are not common antioxidants. Due to the low redox potential of flavonoids theyare thermodynamically able to reduce highly oxidizing free radicals such as superoxide,peroxyl, alkoxyl and hydroxyl radicals by hydrogen atom donation 28 . Besides scavenging,flavonoids may stabilize free radicals involved in oxidative processes by complexing with

P a g e | 140them 29 . The flavanols containing a catechol group in ring B as in compound 8 and 9 arehighly active because of the presence of the 3-hydroxyl group. Additionally, the catecholgroup present in these compounds has strong electron donating properties and target freeradicals 29 .The results of the deoxyribose test are presented in table 4. The results revealed that thehydroxyl radical scavenging activities of the isolated compounds increased with increasingconcentrations. As in the other two assays, the highest activity was observed for theflavonoids, 8 (77% at 250 µg mL -1 ) and 9 (83% at 250 µg mL -1 ) and showed higher activitythan ascorbic acid at all concentrations. The sesquiterpenoid (11), and the alkaloid 10, bothhad comparable activities to that of ascorbic acid of 72% and 69% at 250 µg mL -1 . Ascorbicacid had an activity of 71% at 250 µg mL -1 . The new limonoid uguenensone (5) exhibitedan activity of 44% at 15.62 µg mL -1 , 50% at 31.25 µg mL -1 and 59% at 62.50 µg mL -1 , whichwas significantly higher than that of ascorbic acid at the corresponding concentrations,however it did not increase at the same rate with increasing concentration as ascorbic acid.Although flindersiamine (10) and 8α-11-elemodiol (11) did not show high radicalscavenging activity by the DPPH method, these two compounds showed potency whenassayed using the deoxyribose method. Although both of these methods measure hydroxylradical scavenging activity, a plausible explanation for this finding is that these compounds(alkaloid and sesquiterpenoid) efficiently target different radicals i.e the DPPH methodmeasures the ability to scavenge a phenolic radical whereas the deoxyribose methodmeasures the scavenging activity of aliphatic hydroxyl radicals 30 . Generally, the scavengingactivity of the compounds was found to decrease in the order: 7-O-methylaromadenrin (9) >uguenenprenol (8) > 8α,11-elemodiol (11) > ascorbic acid > flindersiamine (10) >

P a g e | 141uguenensone (5) > chisocheton (2) > methyl uguenenoate (7) > limonyl acetate (6) >kihadalactone A (3) > niloticin (1) > uguenensene (4). Higher hydroxyl radical scavengingactivity in flindersiamine could be attributed to electron rich benzene rings and the doublebond at ∆ 2 in the structure which is responsible for electron delocalization upon donation ofa proton 29 .Table 4: Results of the percentage hydroxyl radical scavenging activity of compoundsfrom V. uguenensis and ascorbic acid measured by the deoxyribose method.Percentage scavenging activityConcentrations (µg/mL) 15.6 31.1 62.5 125 250Niloticin (1) 13.33±1.53 f 19.67±1.53 g 27.00±1.00 f 29.00±2.00 f 36.67±1.53 fChisocheton (2) 34.33±2.52 d 40.67±1.53 a 43.67±1.53 g 47.67±1.53 h 55.33±1.53 dKihadalactone A (3) 21.33±1.53 e 22.67±1.53 f 27.00±1.00 f 30.00±1.00 f 43.33±3.06 eUguenensene (4) 5.33±3.06 a 11.00±1.00 de 19.33±1.53 e 21.00±1.00 e 34.00±3.61 fUguenensone (5) 43.67±2.08 c 50.33±1.53 c 59.33±1.53 c 56.67±1.53 g 59.67±2.52 dLimonyl acetate (6) 3.00±1.00 a 12.67±1.15 e 20.00±1.00 e 32.00±2.00 f 45.33±1.53 eMethyl uguenensoate (7) 5.33±2.08 a 10.00±1.00 d 14.00±1.00 d 22.67±0.58 e 53.33±3.06 dUguenenprenol (8) 37.33±1.53 d 58.00±2.00 b 63.33±1.53 b 73.00±1.00 d 76.67±1.15 c7-O-Methylaromadenrin (9) 46.00±1.00 c 50.33±0.58 c 54.67±2.52 a 76.33±3.21 c 83.00±1.73 bFlindersiamine (10) 43.00±2.00 c 49.67±1.53 c 58.67±3.21 c 62.00±1.73 b 69.00±1.00 a8α,11-elemodiol (11) 56.67±1.53 b 59.00±1.00 b 64.67±1.53 b 68.33±1.53 a 71.67±1.53 aAsorbic Acid 23.33±1.53 a 39.67±1.53 a 54.00±1.00 a 67.67±0.58 a 70.67±1.53 aData are presented as means ± SD of triplicate. a-h Values with different superscript lettersfor a given concentration are significantly different from each of the other compounds (Oneway ANOVA followed by Tukey’s multiple range post-hoc test, p < 0.05).In conclusion, the results of this study show that the classes of compounds andconcentrations have significant effects on their antioxidant activity. The flavonoids, asexpected, showed high activity in all of the antioxidant assays, and in some cases evenhigher than ascorbic acid. Unexpectedly, the limonoids 2, 5 and 7 showed some significantantioxidant activity in the DPPH assay and the alkaloid 10 and sesquiterpene 11 showedsignificantly higher activity in the deoxyribose assay compared to many other compounds.Whereas the flavonoids are non selective for any particular radical, the limonoids, alkaloids

P a g e | 142and sesquiterpene may be more selective to certain radicals, hence their activities were notconsistently observed in all three assays used in this study.These results provide scientific validity and credence to the ethnomedicinal use of this plantin the treatment of malaria as the antioxidants could reduce the ROS linked to cerebralmalaria in children in Africa. This study also highlights the efficacy of compounds from V.uguenensis as antioxidant additives.Further to this, the current contribution addsuguenensene (4) and uguenensone (5), both of the limonin-type limonoids to the list ofpreviously identified Rutaceae limonoids.EXPERIMENTAL SECTIONGeneral Experimental ProceduresThe melting points were recorded on an Ernst Leitz Wetzer micro-hot stage melting pointapparatus.Specific rotations were measured at room temperature in chloroform on aPerkinElmerTM, Model 341 Polarimeter with a 10 mm flow tube.UV spectra wereobtained on a Varian Cary UV-VIS Spectrophotometer. The Circular Dichroism (CD)spectra were recorded on chirascan plus spectropolarimeter by applied photophysics atwavelengths 190-400 nm. IR spectra were recorded on a Perkin-Elmer Universal ATRSpectrometer. The 1 H, 13 C and all 2D NMR spectra were recorded using a Bruker Avance III400 MHz spectrometer. All the spectra were recorded at room temperature using deuteratedchloroform (CDCl 3 ) as solvent. High-resolution mass data was obtained using a BrukermicroTOF-Q II ESI instrument operating at ambient temperatures, with a sampleconcentration of approximately 1 ppm.The separation, isolation and purification of compounds were carried out by gravity columnchromatography and monitored by thin layer chromatography (TLC). Merck silica gel 60

P a g e | 143(0.040-0.063 mm) was used for column chromatography and Merck 20 × 20 cm silica gel 60F 254 aluminum sheets were used for thin-layer chromatography.Plant materialThe leaves, stem bark and roots of V. uguenensis were collected from the Rift Valleyprovince of Kenya in December 2010 and were identified by Mr Ezekiel Cheboi of theDepartment of Natural Resources, Egerton University, Kenya. A voucher specimen (PKC02 NH) was deposited at the Natal Herbarium, Durban.Extraction and IsolationThe air dried powder of V. uguenensis leaves (400 g), stem bark (980 g) and roots (900 g)were each sequentially extracted for 24 hours using a soxhlet apparatus with solvents ofvarying polarity; hexane, dichloromethane, ethyl acetate and methanol. Each extract wasconcentrated using a rotavapor prior to being separated. The crude extracts were separatedby column chromatography (CC). The hexane extract from the leaves (13 g) was separatedwith a hexane: dichloromethane and dichloromethane: ethyl acetate step gradient. For thecrude extract, a 4.5 cm diameter column was used and fractions of 100 mL were collectedand monitored by thin layer chromatography (TLC). Fractions with similar TLC profileswere combined.From this column separation, eleven fractions (A-K) were obtained.Fraction C was recrystallised in methanol to yield lupeol (118 mg) while fraction G waspurified using hexane: dichloromethane (7:3) to afford three subfractions G 1 -G 3 of whichsubfraction G 3 yielded 8α,11-elemodiol (11) (59 mg).Fraction H was purified usinghexane: EtOAc (8:2) to yield subfractions, H 1 -H 4 of which subfraction H 4 was washedseveral times with hexane to yield niloticin (1) (583 mg). Fraction J was purified with100% dichloromethane and then EtOAc: dichloromethane (1:4) to afford five subfractions,J 1 -J 5. Subfraction J 3 contained chisocheton (2) (83 mg) while subfraction J 5 was purified

P a g e | 144further using dichloromethane: EtOAc (9:1) to give limonyl acetate (6) (134 mg). Thedichloromethane extract of the leaves (9 g) was also separated using a hexane: EtOAc andEtOAc: methanol step gradient from 100% hexane to 1:4 (methanol: EtOAc) to give 9fractions (A-I).Fraction E was purified with hexane: EtOAc (4:1) to afford 7-Omethylaromadenrin(9) (51 mg), while fraction G was purified using hexane:dichloromethane (1:1) to give four subfractions G 1 -G 4 of which fraction G 2 was furtherpurified with 100% dichloromethane to yield uguenensone (5) (92 mg). Fraction H waspurified with dichloromethane: EtOAc (4:1) to give uguenenprenol (8) (38 mg).The hexane extract (32 g) from the stems yielded the same compounds as the hexane extractof the leaves. The dichloromethane extract of the stems (15 g) was eluted with a hexane:EtOAc and EtOAc: methanol step gradient starting from 100% hexane and stepped up to100% EtOAc and then to 10% MeOH in EtOAc to give nine fractions A-I. Fraction H waspurified with hexane: EtOAc (6:4) to give five subfractions H 1 -H 5 . Subfraction H 2 wasfurther purified using dichloromethane: EtOAc (4:1) to yield uguenensene (4) (241 mg).Subfraction H 1 was purified with dichloromethane: EtOAc (4:1) into four other subfractionsH 1a -H 1d . Fraction H 1a yielded tricoccin S 13 acetate (23 mg) with 100% dichloromethane.Fraction H 1c was purified using dichloromethane: EtOAc (7:3) to afford three fractions aftercombination, H 1ca -H 1cc . Kihadalactone (3) (117 mg) was contained in fraction H 1ca whilemethyl uguenenoate (7) (120 mg) was obtained in fraction H 1cc .The dichloromethane extract from the roots was separated with hexane: EtOAc and thenEtOAc: MeOH. More methyl uguenenoate (7) was obtained after purification of fraction Dwith dichloromethane: EtOAc (4:1) and the two alkaloids skimmianine and flindersiamine(10) were contained in fraction H. Their separation was achieved after repeated column

P a g e | 145chromatography using dichloromethane: EtOAc (1:1), where fraction H 2c yieldedskimmianine (13 mg) and H 2d yielded flindersiamine (10) (45 mg).Compound 4: Reddish brown; mp 105-109 o C; [α] 20 D -62.50 (c 0.056 CHCl 3 ); UV (CHCl 3 )λ max (log ε) 240 (1.28) nm; CD (CH 3 CN) λ max (∆ε) 197 (+8.5), 210 (-22.0), 328 (+1.2) nm;IR ν max 2926, 1733, 1370, 1231, 1031, 729 cm -1 ; 1 H NMR and 13 C NMR data: see Table 1;EIMS m/z (rel. int.) 484 [M] + (33), 469 (10), 441 (10), 424 (100), 409 (88), 347 (50), 330(68); HREIMS m/z 484.2324 [M] + (calcd. for C 28 H 36 O 7, 484.2461).Compound 5: Dark brown solid; mp 132-134 o C; [α] 20 D -14.85 (c 0.404, CHCl 3 );UV(CHCl 3 ) λ max (log ε) 246 (0.72) nm; CD (CH 3 CN) λ max (∆ε) 197 (+8.5), 210 (-22.0), 328(+1.2) nm ; IR ν max 2960, 1732, 1369, 1230, 1028, 726 cm -1 ; 1 H NMR and 13 C NMR data:see Table 1; EIMS m/z (rel. int.) 498 [M] + (100), 482 (25), 467 (25), 438 (38), 423 (50),384 (38), HREIMS 498.9120 [M] + (calcd. for C 28 H 34 O 8 , 498.5648).Compound 8: Light yellow crystals; mp 75-78 o C; [α] 20 D +52.30 (c 0.056 CHCl 3 ); UV(CHCl 3 ) λ max (log ε) 337 (4.45), 285 (5.13), 239 (5.44) nm; CD (CH 3 CN) λ max (∆ε) 205(+8.8), 290 (+4.0), 335 (+2.0) nm; IR ν max 3330, 2924, 1708, 1634 cm -1 ; 1 H NMR (CD 3 OD,400 MHz) δ 7.36 (2H, d, J = 8.56 Hz, H-2’/6’), 6.85 (2H, d, J = 8.56 Hz, H-3’/5’), 6.09 (1H,d, J = 2.20 Hz, H-6), 6.04 (1H, d, J = 2.20 Hz, H-8), 5.70 (1H, t, J = 6.32 Hz, H-2’’), 4.82(1H, d, J = 11.65 Hz, H-2), 4.66 (2H, d, J = 6.32 Hz, 2H-1’’), 4.58 (1H, d, J = 11.65 Hz, H-3), 3.76 (2H, s, H-4’’), 1.54 (3H, s, H-5’’); 13 C NMR (CD 3 OD, 100 MHz) δ 198.98 (C, C-4), 168.97 (C, C-7), 165.01 (C, C-5), 164.36 (C, C-4’), 159.28 (C, C-9), 142.04 (C, C-3’’),130.40 (2CH, C-2’/6’), 129.16 (C, C-1’), 119.65 (CH, C-2’’), 116.18 (2CH, 3’/5’), 102.61(C, C-10), 96.71 (CH, C-6), 95.64 (CH, C-8), 85.10 (CH, C-2), 73.72 (CH, C-3), 67.63(CH 2 , C-4’’), 66.25 (CH 2 , C-1’’), 14.03 (CH 3 , C-5’’); HREIMS m/z 372.1158 [M] + (calcd.for C 20 H 20 O 7 , 372.1209).

P a g e | 146ANTIOXIDANT ACTIVITYDetermination of the reducing potential using the Ferric Reducing Antioxidant Power(FRAP) assayThe total reducing power of each compound from Vepris uguenensis was determinedaccording to the Ferric Reducing Antioxidant Power (FRAP) method as described 31 . A 2.5mL volume of different concentrations of the compounds (250, 125, 62.5, 31.25 and 15.625µg mL -1 ) were mixed with 2.5 mL phosphate buffer solution (0.2 M, pH = 6.6) and 2.5 mLof 1% potassium ferriccyanide [K 3 Fe(CN) 6 ] in test tubes. The mixture was placed in awater bath of 50 o C, for 20 minutes. A volume of 2.5 mL of 10% trichloroacetic acid (TCA)was added to the mixture and mixed thoroughly. A volume of 2.5 mL of this mixture wasthen mixed with 2.5 mL distilled water and 0.5 mL FeCl 3 of 0.1% solution and allowed tostand for 10 min. The absorbance of the mixture was measured at 700 nm using a UV-VISspectrophotometer (UV mini 1240, Shimadzu Corporation, Kyoto, Japan); the higher theabsorbance of the reaction mixture, the greater the reducing power. Ascorbic acid was usedas a positive control for this assay. All procedures were performed in triplicate.Measurement of free radical scavenging activity using the DPPH assayThe free radical scavenging activity (antioxidant capacity) of the plant phytochemicals onthe stable radical, 2,2-diphenyl-β-picrylhydrazyl (DPPH) was evaluated by the methodestablished by Shirwaikar et al. 32 . In this assay, a volume of 1.5 mL of methanolic solutionof the compound at different concentrations was mixed with 0.5 mL of the methanolicsolution of DPPH (0.1 mM). An equal amount of methanol and DPPH without sampleserved as a control. After 30 minutes of reaction at room temperature in the dark, theabsorbance was measured at 517 nm against methanol as a blank using a UV

P a g e | 147spectrophotometer as mentioned above. The percentage free radical scavenging activity wascalculated according to the following equation:% Scavenging activity = [(Ac-As) / Ac] × 100Where Ac = Absorbance of control and As = Absorbance of sampleDetermination of hydroxyl radical scavenging activity using the Deoxyribose assayThe deoxyribose assay for hydroxyl radical scavenging activity was performed as describedpreviously by Chung et al. 33 . A mixture of 2-deoxyribose (5.6 mM, 500 µL), Fe 3+ chloride(0.1 mM, 100 µL) and EDTA (0.1 mM, 100 µL), H 2 O 2 (1 mM, 100 µL), 100 µL of sample(concentrations ranging from 15.625 to 250 µg/mL), 500 µL of 50 mM potassium phosphatebuffer (pH 7.4) and 100 µL of vitamin C (1 mM) was made and incubated for 30 min at 50°C. The Fe 3+ chloride and EDTA was mixed prior to combining the other reagents. Asolution of thiobarbituric acid (TBA) (1 mL, 1% w/v) and trichloroacetic acid (TCA) inaqueous solution (1 mL, 2.8% w/v) was added, and the mixture was heated for 30 minutesin a water bath at 50 o C. The absorbance of the amount of chromogen produced wasmeasured at 532 nm using a UV spectrophotometer as mentioned above. The hydroxylradical scavenging activity (percentage inhibition rate) was calculated according to theequation as follows:% Scavenging activity = [(Ac-As) / Ac] × 100Where Ac = Absorbance of control and As = Absorbance of sampleAll experiments were performed in triplicate.Statistical AnalysisThe data are presented as mean ± SD of triplicates. The data were statistically analyzed byusing a statistical software program SPSS (SPSS for windows, version 18, SPSS Science,

P a g e | 148Chicago, IL, USA). The one-way analysis of variance (ANOVA) followed by the Tukey’smultiple range post-hoc test was employed to find the differences.The data wereconsidered significantly different at p < 0.05.ASSOCIATED CONTENTSupporting Information1D and 2D NMR spectra of compounds 5, 6 & 9 are available free of charge via the internetat http://pubs.acs.org.ACKNOWLEDGMENTFinancial support from the Organization for Women in Science for the Developing World(OWSDW) is gratefully acknowledged. We thank Mr E. Cheboi of the Department ofNatural Resources, Egerton University for the identification of the plants, Mr D. Jagjivan,Mr M. Sibonelo and Dr J. Brand for running of NMR, HREIMS and CD spectrarespectively.The cooperation of Mr A. Ibrahim in antioxidant assays is highlyacknowledged.

P a g e | 149REFERENCES1. Ekong, D. E. U.; Ibiyemi, S. A.; Olagbemi, E. O. J. Chem. Soc. Chem. Comm. 1971,1117-1118.2. Champagne, D. E.; Koul, O.; Isman, M. B.; Scudder, G. G.; Towers, N. G. H.Phytochemistry, 1992, 31, 377-394.3. Moriguchi, T.; Kita, M.; Hasegawa, S.; Omura, M. J. Food Agri. Environ. 2003, 1,22-25.4. Abdelgaleil, S. A. M.; Hashinaga, F.; Nakatani, M. Pest Manag. Sci. 2005, 61, 186-190.5. Rahman, A. K. M. S.; Chowdhury, A. K. A.; Ali, H. A.; Raihan, S. Z.; Ali, M. S.;Nahar, L.; Sarker, S. D. J. Nat. Med. 2009, 63, 41-45.6. Bickii, J.; Tchouya, G. R. F.; Tchouankeu, J. C.; Tsamo, E. Afr. J. Tradit.Complement. Altern. Med. 2007, 4, 135 – 139.7. Abdelgaleil, S. A. M.; Okamura, H.; Iwagawa, T.; Doe, M; Nakatani M. Heterocycles,2000, 53, 2233–2240.8. Khalid, S. A.; Friedrichsen, G. M.; Kharazmi, A.; Theander, T. G.; Olsen, C. E.;Christensen, S. B. Phytochemistry, 1998, 49, 1769-1772.9. Nakatani, M.; Abdelgaleil, S. A. M.; Kurawaki, J.; Okamura, H.; Iwagawa, T.; Doe,M. J. Nat. Prod. 2001, 64, 1261-1265.10. Akihisa, T.; Noto, T.; Takahashi, A.; Fujita, Y.; Banno, N.; Tokuda, H.; Koike, K.;Suzuki, T.; Yasukawa, K.; Kimura, Y. J. Oleo Sci. 2009, 58, 581-594.11. Breksa, A. P.; Manners, G. D. J. Agric. Food Chem. 2006, 54, 3827-383112. Zhang, H.; Wang, X.; Chen, F.; Androulakis, X. M.; Wargovich, M. J. Phytother.Res. 2007, 21, 731–734.

P a g e | 15013. Cheplogoi, P. K.; Mulholland, D. A.; Coombes, P. H.; Randrianarivelojosia, M.Phytochemistry, 2008, 69, 1384-1388.14. Reis, P.A.; Comim, C. M.; Hermani, F.; Silva, B.; Barichello, T.; Portella, A. C.;Gomes, F. C. A.; Sab, I. M.; Frutuoso, V. S.; Oliveira, M. F.; Bozza, P. T.; Bozza, F.A.; Pizzol, F.D.; Zimmerman, G. A.; Quevedo, J.; Castro-Faria-Neto, H. C.; PLoSPathog. 2010, 6, e100096315. Boivin, M. J.; Bangirana, P.; Byarugaba, J.; Opoka, R. O.; Idro, R.; Jurek, A. M.;John, C. C.; Pediatrics, 2007, 119, e360.16. Wansi, J. D.; Mesaik, A. M.; Chiozem, D. D.; Devkota, P. K.; Gaboriaud-Kolar, N.;Lallemand, M. C.; Wandji, J.; Choudhary, M. I.; Sewald, N. J. Nat. Prod. 2008, 71,1942-1945.17. Latip, J.; Ali, M. N. H.; Yamin, B. M. Acta. Cryst. 2005, E61, 02230-02232.18. Ruberto, G.; Renda, A.; Tringali, C.; Napoli, E. M.; Simmonds M. S. J. J. Agric.Food Chem. 2002, 50, 6766-6774.19. Barrero, A. F.; Moral, Q. F. J.; Herrador, M. M.; Akissira, M.; Bennamara, A.;Akkad, S.; Aitigri, M. Phytochemistry, 2004, 65, 2507-2515.20. Liu, H.; Heilmann, J.; Rali, T.; Sticher, O. J. Nat. Prod. 2001, 64, 159-163.21. Sondengam, B. L.; Kamga, C. S.; Connolly, J. D. Tetrahedron Lett. 1979, 15, 1357-1358.22. Wang, N. X.; Yin, S.; Fan, C.; Lin, L. P.; Ding, J.; Yue, J. M. Tetrahedron, 2007, 63,8234-8241.23. Tada, K.; Takido, M.; Kitanaka, S. Phytochemistry, 1999, 51, 787-791.24. Torto, B.; Hassanali, A.; Nyandat, E.; Bentley, D. M. Phytochemistry, 1996, 42, 1235-1237.

P a g e | 15125. Hasegawa, S.; Bennett, R. D.; Herman, Z.; Fong, C. H.; Ou, P. Phytochemistry, 1989,28, 1717-1720.26. Grade, M.; Piera, F.; Cuenca, A.; Torres, P.; Bellido, I. S. Planta Med. 1985, 51, 414-419.27. Slade, D.; Ferreira, D.; Marais, J. P. J. Phytochemistry, 2005, 66, 2177-2215.28. Shimada, V. L.; Fujikawa, K.; Yahara, K.; Nakamura, T. J. Agric. Food Chem. 1992,40, 945-948.29. Pietta, P. G. J. Nat. Prod. 2000, 63, 1035-1042.30. Ajisaka, K.; Agawa, S.; Nagumo, S.; Kurato, K.; Yokoyama, T.; Arai, K.; Miyazaki,T. J. Agric. Food Chem. 2009, 57, 3102-3107.31. Ferreira, I. C. F. R.; Baptista, M.; Vilas-Baos.; Barros, L. Food Chem. 2007, 100,1511-1516.32. Shirwakar, A.; Shirwakar, A. R.; Rajendran, K.; Punitha, I. R. S. Biol. Pharm. Bull. 2006,29, 1906-1910.33. Chung, S. K.; Osawa, T.; Kawakishi, S. Biosci. Biotech. Biochem. 1997, 61, 118-123.

P a g e | 152CHAPTER SEVENSUMMARY AND CONCLUSION7.1 SummaryThis study focused on the isolation and characterisation of secondary metabolites from fourplants belonging to the genera Vernonia and Vepris which are used in Kenyan traditionalmedicine as an analgesic, antibacterial, antimalarial, anti-allergy medication, and antivenom(snake bite) and for dermatological infection, dental caries, respiratory tract infections, eyeproblems, cardiovascular complications, neurological disorders and psychotic disorders.The study was undertaken in order to validate the use of these plants in what they are beingtraditionally used for as well as to provide the traditional healers with a scientific basis forusing the plants to treat additional medical symptoms and diseases.In order to do this, the plants were investigated for their phytochemical constituents andbased on what was present, a literature search on the medicinal properties of the compoundswere undertaken to provide a rationale for the use of the plants in different medicinal areas.In addition, bioassays were identified to test the activity of the isolated compounds, therebyproviding additional information on the use of the plant in traditional medicine.7.1.1 Recommendations for Vernonia auriculiferaVernonia auriculifera is used in Kenyan traditional medicine for the treatment of headache,conjunctivitis, toothache, snake bite and bacterial infection (wound infection). Previousphytochemical studies have found hydroperoxides of unsaturated fatty acid methyl estersand plant growth stimulators from V. auriculifera leaves. Our studies have revealed theleaves to contain a sesquiterpene amine (farnesylamine), four triterpenoids, (lupenyl acetate,α-amyrin, β-amyrin and β-amyrin acetate) and a steroid (β-sitosterol). The stem bark

P a g e | 153afforded two triterpenoids, friedelanone and friedelin acetate.From the roots, onetriterpenoid, oleanolic acid was isolated. The triterpenoids have been reported to be used asantibacterials, antifungals, anti-inflammatories, anti-leishmanials and antimalarials. Theplant could therefore be used in traditional medicine to treat the symptoms of inflammationand infections by bacteria, fungus and parasites (malaria and leishmania).In addition we have tested the compounds for antibacterial and antibiofilm activities usingthe broth microdilution method and found that β-amyrin, α-amyrin and β-amyrin acetatehad moderate antibacterial activity. At sub-MIC concentrations, oleanolic acid and β-amyrin acetate exhibited antibiofilm activity. Due to the inavailability of health facilitiesand expensive pharmaceutical supplies in most of rural Kenya, the plant V. auriculiferacould replace common commercial antibiotics.The isolation of pentacyclic triterpenoids in V. auriculifera also provides a chemotaxonomiclink to the genus Vernonia as pentacyclic triterpenoids could be an additionalchemotaxanomic marker to the sesquiterpene lactones.7.1.2 Recommendations for Vernonia urticifoliaVernonia urticifolia is used in Kenyan traditional medicine for the treatment of sinuses,allergy, respiratory tract infection and skin infections. There are no previous phytochemicalor pharmacological studies carried out on this plant. Our studies have indicated the leavesto contain, a polyene, urticifolene, a carotenoid and a streroid, β-sitosterol. Polyenes areknown to be used as anti-fungal agents and carotenoids are known to have antibacterial,anticancer and antioxidant properties. The plant could therefore be used in traditionalmedicine to treat the symptoms of bacterial infections, fungal infections and cancer. It can

P a g e | 154also be used as an antioxidant supplement due to its carotenoid component which arereported to be antioxidants.We assesed the compounds for antibacterial activity using the broth microdilution methodsand found that lutein and urticifolene possessed antibacterial activity. This plant couldsubstitute/supplement modern antibiotics in most of rural Kenya where modern healthfacitilities are inaccessible. The compounds isolated from V. urticifolia add to the existingcompounds from the genus Vernonia.7.1.3 Recommendations for Vepris glomerataVepris glomerata is used in Kenyan traditional medicine for the treatment of malaria, eyeproblem, cardiac pain, epilepsy, stroke and psychosis. Previous phytochemical studies havefound the leaves of the plant to contain two furoquinoline alkaloids, skimmianine and(montrifoline) evoxine.Our studies have indicated the leaves to contain a flavonoid (veprisinol) and fourfuroquinoline alkaloids, haplopine-3,3’-dimethylallyl ether, anhydroevoxine, evoxine, andskimmianine. The stem bark was found to contain two cinnamic acid derivatives, (phydroxycinnamicacid and caffeic acid), an aromatic ester (methyl caffeate), a flavonoid(hesperetin) and two limonoids (limonin and limonyl acetate).The roots contained acinnamaldehyde derivative (glomeral), a coumarin (scoparone) and a lignin (syringaresinol).Surpringly most of this compounds are derived from cinnamic acids, a key intermediate inthe shikimate and phenylpropanoid pathways where these classes of compounds arebiosynthesized. Cinnamic acids have been known to possess a wide range of biologicalactivities such as anti TB, antidiabetic, antioxidant, antimicrobial, hepatoprotective, CNSdepressant, anticholesterolemic, antifungal, antihyperglycemic, antimalarial, antiviral, cytotoxic,

P a g e | 155anti-inflammatory as well as to absorb UV rays. Flavonoids have been known to be used asantioxidants while alkaloids are reported to possess antimicrobial, antiradical, antioxidant,antiplasmodial, anticancer, and antimutagenic activities. Limonoids are reported to exhibitantifungal, antibacterial, antimalarial, antiprotozoan, antiviral, anti-inflammatory andantioxidant activities.The parts of this plant could therefore be used in traditional medicine to treat a wide rangeof diseases including; the symptoms of malaria, bacterial and fungal infection, cancer,diabetes, TB and inflammation and could also be taken as an antioxidant supplement due toits flavonoid component.In addition we have tested the flavonoids and alkaloids for antioxidant activity using theDPPH, FRAP and deoxyribose methods and found that two of the compounds were evenmore active than ascorbic acid as an antioxidant in the deoxyribose test. Compoundsisolated from the stem bark and roots were also assessed for antibacterial activity using thedisc diffusion and broth microdilution methods. Glomeral and hesperetin were found to beactive. Due to the inaccessibility of pharmaceutical supplies in most of rural Kenya, V.glomerata could replace common commercial antibiotics and antioxidants such as vitamin Cin these communities.Since veprisinol and haplopine-3, 3’-dimethylallyl ether shows high antioxidant activitywhereas glomeral exhibited good antibacterial activity, further work is necessary to firstinvestigate a possible synthetic scheme for the synthesis of these compounds and thendetermine whether veprisinol and haplopine-3, 3’-dimethylallyl ether can be developed intoa commercial antioxidant supplement.Glomeral can be developed into a commercialantibiotic in the face of rising bacterial resistance

P a g e | 156The isolation of limonin-type limonoids in V. glomerata also provides a chemotaxonomiclink to the family Rutaceae as this class of limonoids are exclusive to members of the familyRutaceae. The isolation of furoquinoline alkaloids provides a chemotaxonomic link to thegenus Vepris.7.1.4 Recommendations for Vepris uguenensisVepris uguenensis is used in Kenyan traditional medicine for the treatment of malariaPrevious phytochemical studies have found the root bark of this plant to contain methyluguenenoate, uguenenazole, uguenenonamide, flindersiamine, maculosidine andsyringaldehyde. Our studies have indicated the leaves to contain a triterpenoid (lupeol), twoprotolimonoids (niloticin and chisocheton), limonoids (uguenensone and limonyl acetate),flavonoids (uguenenprenol and 7-O-methylaromadenrin) and sesquiterpenoid, 8α,11-elemodiol.The stem bark contained the protolimonoid (chisocheton) limonoids(kihadalactone A, uguenensene and tricoccin S 13 acetate) and a furoquinoline alkaloid,(skimmianine) and the roots were found to contain a limonoid (methyl uguenenoate) andfuroquinoline alkaloid, flindersiamine. Alkaloids, flavonoids and limonoids have a widerange of biological activities as discussed above (section 7.3).V. uguenensis could therefore be used in traditional medicine to treat the symptoms ofmalaria, inflammation, bacterial, viral and fungal infections and could also be taken as anantioxidant supplement due to its flavonoid component.Since uguenenprenol and 7-O-methylaromadenrin shows high antioxidant activity, furtherwork is necessary to investigate the synthesis of these compounds and then develop theseflavonoids into commercial antioxidant supplements.

P a g e | 157The isolation of six limonoids of in V. uguenensis also provides a chemotaxonomic link tothe family Rutaceae as this class of limonoids are common to members of the familyRutaceae and rare in other limonoid producing families such as the Meliaceae family. Thelimonoids and the protolimonoids isolated, altogether formed an interesting limoninbiosynthetic pathway. The isolation of uguenensene and uguenensone provided the missingcompounds in this biosynthetic scheme.7.2 ConclusionThe two Vernonia and Vepris species studied in this work produced a range of secondarymetabolites, which have shown to have pharmaceutical effects, both in the literature and byour own bioassays. This knowledge is important for the ethnomedicinal healers in that theycould use this as a rationale for using the plants in African traditional medicine.Furthermore, these plants provide an inexpensive alternative to the pharmaceuticals alreadyon the market, which in many cases is inaccessible to the local communities in Kenya.Our work has shown, in particular that these plants could be used as antioxidantsupplements and antibacterial agents. It is worthwhile to take selected molecules which arehighly active from these results and test them in suitable animal modes. Further to this itwould also be important to test both the extracts and the active isolates for cytotoxicity todetermine whether or not it would be feasible to develop these further into drugs.

P a g e | 158SUPPORTING INFORMATIONSupporting information includes 1D NMR, 2D NMR, IR, UV, MS and CD experiments.Chapter 2: Lupenyl acetate P1-1, oleanolic acid P1-2, β-amyrin & α-amyrin P1-3 &5,β-amyrin acetate P1-4, friedelanone P1-6, friedelin acetate P1-7, β-sitosterolP1-8 and farnesylamine P1-9.Chapter 3: Urticifolene P2-1 and lutein P2-2.Chapter 4: Veprisinol P3-1, isohaplopine-3,3’-dimethylallyl ether P3-2, tecleoxine P3-3,nkolbisine P3-4 and skimmianine P3-5.Chapter5: Glomeral P4-1, p-hydroxycinnamic acid P4-2, caffeic acid P4-3, methylcaffeate P4-4, hesperetin P4-5, scoparone P4-6, syringaresinol P4-7, limoninP4-8 and limonyl acetate P4-9.Chapter 6: Niloticin P5-1, chisocheton P5-2, kihadalactone A P5-3, uguenensene P5-4,uguenensene P5-5, methyl uguenensoate P5-6, uguenenprenol P5-7, 7-OmethylaromadenrinP5-8, flindersiamine P5-9, 8α,11-elemodiol P5-10,tricoccin S 13 acetate P5-11 and lupeol P5-12.

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