The chemical and biological characterization of South African helichrysum species

Olugbenga Popoola

The genus Helichrysum Mill comprises hundreds of species that are mostly flowering perennial shrubs. Some of these plants that belong to the Helichrysum species are used in traditional medicine to treat cough, back pain, diabetes, asthma, digestive problems, menstrual pain, chest pain, kidney disorders, skin disorders, wounds, open sores, among other conditions, but, only a few scientific studies are reported in the literature with sufficient information that validates the acclaimed folkloric benefits of these plants. This review, therefore, provides a comprehensive update of the available information on the cytotoxicity, genotoxicity, anti-proliferative, anti-bacterial, anti-fungal, anti-viral, anti-HIV, anti-malarial, anti-ulcerogenic, anti-tyrosinase, anti-inflammatory, and anti-oxidant activities of selected Helichrysum species of interest: H. petiolare, H. cymocum, H. foetidum, and H. pandurifolium Schrank, using scientific databases as well as electronic and print sources. The...

Helichrysum decorum DC, Helichrysum lepidissimum S. Moore, and Helichrysum umbraculigerum are three species traditionally used in the South African medicine. The present work deals with the investigation of the spontaneous emission and the essential oils obtained from these plants cultivated in open field under uniform conditions. Fractions of the volatile organic compounds of the three species were rich in monoterpene hydrocarbons, representing more than 70% of the total composition. Pinene isomers were the most representative compounds: β-pinene in H. decorum (53.0%), and α-pinene in H. lepidissimum (67.9%) and H. umbraculigerum (54.8%). These latter two species evidenced an important amount of sesquiterpene hydrocarbons (SH) especially represented by γ-curcumene (H. lepidissimum) and α- and β-selinene (H. umbraculigerum). On the contrary, in the EOs, sesquiterpenes compounds prevailed, representing more than 64% of the identified fraction to reach more than 82 and 87% in H. umbra...

Native South Africans make use of Helichrysum aureonitens Sch. Bip. extracts for the treatment of a variety of infections and they are important in traditional medicinal preparations. This study investigated the effect of seasonal variation and geographical location on the antibacterial and antifungal activities of H. aureonitens. Material was collected in two different seasons: early spring, with high rainfall and high temperatures (October), and late autumn, with low rainfall and lower temperatures (May). Further analysis was carried out using 1H-NMR based metabolomics to analyse and compare the chemical profiles of the plants in both seasons and locations. Plant materials were collected from two sites for each season, at Wakefield farm (KwaZulu-Natal), representing a colder, wetter environment, and Telperion (Mpumalanga), representing a drier and warmer environment. Leaves of H. aureonitens were tested against bacteria (Proteus vulgaris (P. vulgaris) and Bacillus subtilis (B. sub...

CHEMICAL AND BIOLOGICAL CHARACTERIZATION OF SOUTH AFRICAN HELICHRYSUM SPECIES By Olugbenga Kayode Popoola B.Sc (Chemistry, Ado-Ekiti); M.Sc (Organic Chemistry, Ibadan) A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy Department of Chemistry Faculty of Natural Sciences University of the Western Cape Supervisor: Dr. Ahmed Mohammed Co-Supervisor: Prof. Jeanine L. Marnewick September 2015 ABSTRACT South Africa has immensely rich natural flora diversity with more than 20 000 species of higher plants. Asteraceae is one of the biggest families of flowering plants with about 246 genera and 2,300 species in southern Africa. South Africa being home to more than 35 % of the world's Helichrysum species (c.a. 244) of which many are used in traditional medicine, and can be considered as a potential resource for new bioactive chemical entities. Chemical studies on the total extract of the South African Helichrysum species viz: H. teretifolium, H. niveum and H. rutilans resulted in the isolation of twenty eight [14 flavonoids (C1-C10; C22C25), 10 phloroglucinols (C11-C20) and 4 terpenoids (C21, C26-C28)] pure compounds. The chemical structures of the newly isolated compounds were elucidated on the basis of their 1D and 2D-NMR, HRMS, IR and UV spectroscopic data as heliteretifolin (C1), 1-benzoyl-3-(3-methyl2-butenylacetate)-phloroglucinol (helinivene A, C11), 1-benzoyl-3-(2-hydroxyl-3-methyl-3butene-1-yl)-phloroglucinol (helinivene trihydroxyl-2,2-dimethoxychromone C12) B, (helinivene C, and 8-(2-methyl-1-propanone)-3,5,7- C13), while occurrence of 7- methoxyisoglabranin (C6), 4-methoxyquercetin (C8), 4`-methoxykaempferol (C9), mosloflavone (C10), 3β-24-dihydroxyterexer-14-ene (C21), 5,7,8-trihydroxy-3,6-dimethoxyflavone-8-O-2methyl-2-butanoate (C22) and 15--hydroxy-(-)-kaur-16-en-19-oic acid (C28), from Helichrysum genus were reported for the first time. In vitro inhibition of oxidative stress by the isolated compounds were measured as total antioxidant capacity using the FRAP, TEAC, ORAC (hydroxyl and peroxyl radicals) as well as Fe 2+-induced microsomal lipid peroxidation assays. Inhibitory activities against skin-diseases related enzymes i were evaluated in a tyrosinase and elastase non-biological system, while In vitro prooxidant behavior of the compounds was also investigated in the presence of copper (II). Compounds C7, C8, C11 and C12 in comparison with the commercial antioxidant EGCG demonstrated TEAC (4529.01 ± 2.44; 4170.66 ± 6.72; 19545.00 ± 10.25; 43615.73 ± 6.66; vs 11545.40 ± 17.28) μM TE/g respectively, and ORAChydroxyl radical (7.265 ± 0.71; 6.779 ± 3.40; 64.85 ± 10.95; 94.97 ± 5.80; vs 3.91 ± 4.65) X106 μM TE/g capacities, respectively. Inhibition of Fe2+induced microsomal lipid peroxidation demonstrated by C7, C8, C11 and C12 expressed as IC50 values included: 2.931 ± 0.64; 6.449 ± 3.16; 5.115 ± 0.90; 3.553 ± 1.92 µg/mL respectively. Additionally, the total antioxidant capacities measured as FRAP (4816.31 ± 7.42; 3584.17 ± 0.54) µMAAE/g, and ORACperoxyl radical (17.836 ± 2.90; 12.545 ± 5.07) X 103 µMTE/g were also observed for compounds C7 & C8, respectively. Compound C7 demonstrated potent anti-tyrosinase activity with IC50 8.092 ± 7.14, while mild anti-tyrosinase activities were demonstrated by compounds C8, C11, C12, C22 and C23 and expressed as IC50 values (IC50 = 27.573 ± 3.11; 35.625 ± 4.67; 26.719 ± 5.05; 25.735 ± 9.62; 24.062 ± 0.61) µg/mL respectively. Anti-elastase activity with IC50 values of 25.313 ± 7.85 µg/mL was observed for C13. This is the first scientific report to be carried out on the chemical and biological profiles of H. teretifolium.H. niveum and H. rutilans. The results suggest that these isolated compounds might become natural agents to inhibit oxidative stress and skin disease-related enzymes, with the prospect of being utilized in cosmetic products formulation upon further biological and clinical investigations. KEYWORDS: H. teretifolium; H. niveum; H. rutilans; Flavonoids; Phloroglucinols; Terpenoids; Oxidative stress; Antioxidants; Skin anti-aging; Cosmetics. ii DECLARATION I, Olugbenga Kayode POPOOLA hereby declare that “The Chemical and Biological Characterization of South African Helichrysum species” is my original work and to the best my knowledge, that it has not been submitted before for any degree or assessment in any other University, and that all the sources I have used or quoted have been indicated and acknowledged by means of complete references. Date …………………… signed …………………………….. iii ACKNOWLEDGMENTS First and foremost, I want to thank my amiable Supervisor Dr. Ahmed Mohammed for his wonderful encouragement, suggestion, and undiluted motivation during the course of this work. I am indeed delighted to him for introducing me into the field of cosmetics chemistry and skin health as an opportunity to rejuvenate my research career. It is a remarkable honour to be his first PhD student at University of the Western Cape. Your persistence criticism can never be forgotten. I am also greatly indebted to my wonderful Co-Supervisor Professor Jeanine L. Marnewick, Director of Oxidative Stress Research Centre, for motherly role she played during the course of this work. She improvises solution to the biological application of this work, through her encouragement and provision of materials. Again, I am very thankful for her painstakingly speed up detailed editing of this thesis and allowing me to experience the different field of in vitro evaluation of cellular oxidative stress and skin anti-aging. I would like to thank the HOD Chemistry Prof Farouk Ameer for his academic support and Prof Emmanuel Iwuoha for his financial support. To other staff of the Department of Chemistry, most especially, Prof M. Onani, Dr. Fanelwa Ajayi, Dr. Edith Antunes, Mr. T. Lesch and the Departmental Secretary Mrs Wilman Jackson. I extend thanks to Dr. Stefan Abel, (IBMB, CPUT, South Africa), for the provision of S-9 Rat liver fraction and facilities for the isolation of microsomes, using gel filtration chromatography. Dr. Christopher Cupido, SANBI (Kirstenbosch, South Africa), for his assistance in identification of the plant species used for this research, Zandile Mthembu (senior Technician, Chemistry Dept., CPUT) for her assistance in some of the spectroscopic (UV & IR) analyses of my samples. iv I would like to thank the management of Ekiti State University, Ado-Ekiti, Ekiti State, Nigeria for my Study Leave I enjoyed while away for this work. I wish to express my gratitude to Prof. Emmanuel Adeyeye and Prof. Olorunfemi Olaofe for their financial assistance, encouragement and moral support. I really say thank you for your leadership role they are indeed playing in the Department of Chemistry, Ekiti State University. I also extend my thanks to the Vice Chancellor of Ekiti State University Prof Oladipo Aina for an uncommon favour I enjoyed through my study leave. So also the Dean of Science Prof Joshua Kayode, Ass. Prof S.O. Adefemi, Dr. F. Tinuola and Dr R.O. Akinyeye for their encouragement and support. To my mentors Dr. (Mrs.) Folakemi Olomojobi, Permanent Secretary, Ekiti State Ministry of Health for her prayers, encouragement and financial assistance and Dr. ‘Jide Faleye, Chemistry Department, Ekiti State University for introducing me into the field of Natural Products at my early age. Special thanks to, Abdulrahman Elbagory for being a considerate roommate. To all my colleagues in Organic Chemistry unit and wonderful people I met in the Chemistry UWC and Oxidative Stress Research Centre, CPUT, most especially, Fanie Rautenbach, Olivia Parbhunath, Dr. Olawale Ajuwon, Dr. M. Opuwari, Dr. Abiodun Badmus, I acknowledged them for their individual training and assistance. I will like to acknowledge with special thanks to Pastor A. Adeleye and Pastor (Dr) & Pastor (Mrs) O. Fatoba for their prayers. My colleague names are numerous to mention, but special thanks to Dr. Bunmi Omoyeni (who initiated my admission at UWC), Dr. K.K. Agbele, Seyi Abegunde, Emmanuel Ameh, Dr. Femi Alamu, ‘Niyi Pereao, Vodah Sunday, Albert Amosu, Ayoola Jegede, ‘Toyin Ayodele, v Slyvester Omoruyi, Mathew Omosulewa, Victor Onwu, Ademola and Kehinde Aseperi, Mr. Gbenga Apara, Vivian John, Ninon, Jimoh Oladejo and Dr. Bulelwa Ntsendwana. My deepest gratitude to my beloved wife (a lady more than men), Bolanle Tosin Popoola, for her undiluted love, prayers, financial support and encouragement, and to my wonderful gifts Dolapo and Damilola, I will forever indebted to say thank you for your understanding and settled mind despite your daddy is far away from home, and to my mother Chief (Mrs.) Alice Ibisola Popoola I thank you for your prayers and your fulfilled dream that one day your last child will meet you alive with his PhD. Above all, I give all the glory to GOD ALMIGHTY (the Beginning and the Ending) for the provision of wisdom and strength towards successful completion of this work. vi DEDICATION To my family ‘Bolanle, ‘Dolapo & ‘Damilola (BDD) POPOOLAs For their intense prayers, undiluted love and care vii TABLE OF CONTENTS ABSTRACT ……………………………………………………………………….... i DECLARATION …………………………………………………………………… iii ACKNOWLEDGEMENTS ……………………………………………………….... iv DEDICATION …………………………………………………………………….... vii TABLE OF CONTENTS …………………………………………………………… viii LIST OF ABBREVIATION ………………………………………………………... xiv LIST OF FIGURES …………………………………………………………………. xvii LIST OF TABLES ………………………………………………………………….. xxi LIST OF SCHEMES ………………………………………………………………... xxiv LIST OF ISOLATED COMPOUNDS ……………………………………………... xxv LIST OF PUBLICATIONS …………………………………………………………. xxviii CHAPTER ONE: INTRODUCTION ……………………………………………… 1 1.1 Natural products as drugs ……………………………………………………….. 1 1.2 Skin aging ………………………………………………………………………. 2 1.3 Oxidative stress …………………………………………………………………. 4 1.4 Skin enzymes …………………………………………………………………… 5 1.4.1Tyrosinase ……………………………………………………………............... 5 1.4.2 Elastase ……………………………………………………………………….. 6 1.4.3 Hyaluronidase ………………………………………………………………… 7 1.5 General overview of the use of plants as source of medicines …………………. 8 1.6 Why investigate the genus Helichrysum? ………………………………………. 9 1.7 Natural products as inhibitors against oxidative stress and skin aging …………. 10 1.8 Rationale for the study ………………………………………………………….. 11 1.9 Aims of this study ………………………………………………………………. 12 1.10 Objectives of this study ………………………………………………………… 12 References …………………………………………………………………………... 14 viii CHAPTER TWO: CHEMISTRY OF HELICHRYSUM LITERATURE REVIEW 18 2.1 Aim of this chapter …………………………………………………………….... 18 2.2 Asteraceae family ……………………………………………………………….. 18 2.3 Helichrysum genus …………………………………………………………….... 19 2.4 Traditional uses of Helichrysum genera ……………………………………….... 19 2.5: Chemistry of Helichrysum ……………………………………………………… 22 2.6 Antioxidant activities of Helichrysum genus …………………………………..... 23 2.7 Skin anti-aging properties of Helichrysum genus ……………………………….. 24 References …………………………………………………………………………... 69 CHAPTER THRE: CHEMICAL AND BIOLOGICAL CHARACTERIZATION 83 OF HELICHRYSUM TERETIFOLIUM CONSTITUENTS ………………………... 3.1 Abstract …………………………………………………………………………. 83 3.2 Background information on Helichrysum teretifolium …………………………. 84 CHEMICAL CHARACTERIZATION OF HELICHRYSUM TERETIFOLIUM 86 CONSTITUENTS …………………………………………………………………... 3.3. General experimental procedure ………………………………………………... 86 3.3.1 Reagents and solvents ………………………………………………………. 86 3.3.2 Chromatography ……………………………………………………………. 86 3.3.2.1 Thin layer chromatography (TLC) …………………………………….... 86 3.3.2.2 Column chromatography ………………………………………………... 87 3.3.2.3 High Pressure Liquid Chromatography (HPLC) ……………………….. 87 3.3.3 Spectroscopy ……………………………………………………………….. 88 3.3.3.1 Nuclear magnetic resonance (NMR) spectroscopy …………………….. 88 3.3.3.2 Mass spectroscopy (MS) ……………………………………………….. 88 3.3.3.3 Infrared (IR) spectroscopy ……………………………………………... 88 3.3.3.4 Ultra violet (UV) spectroscopy ……………………………………….... 88 3.4 Collection and identification of plant material …………………………………. 89 3.5 Extraction and fractionation of total extract ……………………………………. 89 3.6 Isolation of pure compounds ……………………………………………………. 92 3.6.1 Isolation of compound C9 - Column chromatography of main fraction XX 92 3.6.2 Isolation of compounds C1 & C4 - Column chromatography of main fraction III ………………………………………………………………… 94 ix 3.6.3 Isolation of compounds C5 & C6 - Column chromatography of main fraction X …………………………………………………………………... 94 3.6.4 Isolation of compounds C2 & C10 - Column chromatography of main fraction XII ……………………………………………………………….... 95 3.6.5 Isolation of compounds C7 & C8 - Column chromatography of main fraction XXIII ……………………………………………………………... 97 3.6.6 Isolation of compound C3 - Column chromatography of main fraction XVIII ………………………………………………………………………. 98 BIOLOGICAL CHARACTERIZATION OF HELICHRYSUM TERETIFOLIUM CONSTITUENTS …………………………………………………………………... 101 3.7 General experimental procedure for biological assays …………………………. 101 3.7.1 Reagents ………………………………………………………………….... 101 3.7.2 Antioxidant assays …………………………………………………………. 101 3.7.2.1 Ferric-ion reducing antioxidant power (FRAP) assay ……………….... 101 3.7.2.2 Automated oxygen radicals absorbance capacity (ORAC) assay …….. 102 3.7.2.3 Trolox equivalent absorbance capacity (TEAC) assay ………………… 103 3.7.2.4 Inhibition of Fe (II)-induced microsomal lipid peroxidation assay …… 104 3.7.3 Skin enzymes inhibitory assays ……………………………………………. 105 3.7.3.1 Tyrosinase enzyme assay ……………………………………………..... 105 3.7.3.2 Elastase inhibition assay ……………………………………………….. 106 3.8 Statistical analysis ……………………………………………………………….. 107 3.9 Chemical evaluations: Results and discussion …………………………………... 107 3.9.1 Summary of the isolated compounds ……………………………………….. 107 3.9.2 Spectroscopic data of compound C1 ………………………………………… 108 3.9.3 Analysis of prenylated chalcones ………………………………………….... 108 3.9.3.1 Analysis of compound C1 (O-prenylated chalcone) …………………….. 108 3.9.3.2 Analysis of compounds C2 & C3 (prenylated chalcones) ……………..... 109 3.9.4 Analysis of compounds C4, C5 and C6 (prenylated flavanones) …………… 110 3.9.5 Analysis of compounds C7, C8, C9 and C10 (flavones) ………………….... 111 3.10 Biological evaluations: Results and discussion ………………………………… 114 3.10.1 Evaluating the ORAC activities of the isolated compounds ………………. 114 x 3.10.2 Evaluating the FRAP and TEAC activity of the isolated compounds …...... 116 3.10.3 Evaluating the anti-lipid peroxidation activity of the isolated compounds 118 3.10.4 Evaluating the anti-tyrosinase activity of the isolated compounds ……..... 120 3.10.5 Evaluating the anti-elastase activity of the isolated compounds ……….... 122 3.11 Conclusion ……………………………………………………………………... 123 References …………………………………………………………………………... 124 CHAPTER FOUR: CHEMICAL AND BIOLOGICAL CHARACTERIZATION OF HELICHRYSUM NIVEUM CONSTITUENTS ………….... 128 4.1 Abstract …………………………………………………………………………. 128 4.2 Background information on Helichrysum nivuem …………………………….... 129 4.3 Chemical characterization of Helichrysum niveum: General experimental procedure ……………………………………………………………………….. 131 4.3.1 Reagents and solvents ………………………………………………………. 131 4.3.2 Thin layer chromatography (TLC) ………………………………………….. 131 4.3.3 Spectroscopy ………………………………………………………………... 132 4.3.4 Optical rotation measurements ……………………………………………... 132 4.4 Collection and identification of plant material …………………………………. 132 4.5 Extraction and fractionation of total extract ……………………………………. 132 4.6 Isolation of pure compounds ……………………………………………………. 135 4.6.1 Isolation of compounds C11, C15 & C16 - Column chromatography of main fraction XI ……………………………………………………………. 135 4.6.2 Isolation of compound C13 - Column chromatography of sub fraction XI7 138 4.6.3 Isolation of compound C14 - Column chromatography of main fraction IX 139 4.6.4 Isolation of compounds C17 & C19 - Column chromatography of main fraction VII …………………………………………………………………. 140 4.6.5 Isolation of compound C18 - Column chromatography of main fraction VI 142 4.6.6 Isolation of compounds C12 & C20 - Column chromatography of main fraction III ………………………………………………………………….. 142 4.6.7 Isolation of compound C21 - Column chromatography of main fraction IV 143 4.7 Biological characterization of Helichrysum niveum: General experimental procedure ………………………………………………………………………... 145 4.7.1 Reagents …………………………………………………………………….. 145 xi 4.7.2 Antioxidant assays ………………………………………………………….. 145 4.7.3 Skin enzyme inhibitory assays …………………………………………….... 145 4.7.4 Acetylcholinesterase inhibition assay ………………………………………. 145 4.8 Statistical analysis ………………………………………………………………. 146 4.9 Chemical evaluations: Results and discussion ………………………………….. 147 4.9.1 Summary of the isolated compounds ……………………………………….. 147 4.9.2 Spectroscopic data of the isolated compounds C11-C13 ………………….... 147 4.9.3 Analysis of compound C11 ……………………………………………….... 151 4.9.4 Analysis of compound C12 ……………………………………………….... 152 4.9.5 Analysis of compound C13 ……………………………………………….... 152 4.9.6 Analysis of compounds C14 and C15 ……………………………………… 154 4.9.7 Analysis of compounds C16 – C20 ………………………………………… 155 4.9.8 Analysis of compound C21 …………………………………………………. 156 4.9 Biological evaluations: Results and discussion …………………………………. 157 4.10.1 Evaluating the ORAC activities of the isolated compounds …………….... 157 4.10.2 Evaluating the FRAP and TEAC activities of the isolated compounds …… 159 4.10.3 Evaluating the anti-lipid peroxidation activities of the isolated cpds …….. 161 4.10.4 Evaluating the anti-tyrosinase activities of the isolated compounds ……… 162 4.10.5 Evaluating the anti-elastase activities of the isolated compounds ………… 163 4.11 Conclusion …………………………………………………………………….. 164 References………………………………………………………………………….... 165 CHAPTER FIVE: CHEMICAL AND BIOLOGICAL CHARACTERIZATION OF HELICHRYSUM RUTILANS CONSTITUENTS ……….... 5.1 Abstract …………………………………………………………………………. 167 167 5.2 Background information on Helichrysum rutilans ……………………………... 168 5.3 Chemical characterization of Helichrysum rutilans constituents ………………. 169 5.3.1 Reagents and solvents ………………………………………………………. 169 5.3.2 Chromatography ……………………………………………………………. 169 5.3.3 Spectroscopy ………………………………………………………………... 170 5.4 Collection and identification of plant material …………………………………. 170 5.5 Extraction and fractionation of total extract ……………………………………. 171 xii 5.6 Isolation of pure compounds ……………………………………………………. 173 5.6.1 Isolation of compounds C23 and C28 - Column chromatography of main fraction X ………………………………………………………………….. 173 5.6.2 Isolation of compound C22 - Column chromatography of main fraction VIII ………………………………………………………………………….. 176 5.6.3 Isolation of compound C24 and C25 - Column chromatography of main fraction VI …………………………………………………………………... 177 5.6.4 Isolation of compounds C26 and C27 ……………………………………… 178 5.7 Biological characterization of Helichrysum rutilans ………………………….... 180 5.7.1 Reagents and solvents ………………………………………………………. 180 5.7.2 Antioxidant assays ………………………………………………………….. 180 5.7.3 Skin enzyme inhibitory assays …………………………………………….... 180 5.8 Statistical analysis ………………………………………………………………. 180 5.9 Chemical evaluations: Results and discussion ………………………………….. 180 5.9.1 Summary of the isolated compounds ……………………………………….. 180 5.9.2 Spectroscopic data of C22 ………………………………………………….. 181 5.9.3 Analysis of compound C22 ……………………………………………….... 182 5.9.4 Analysis of compounds C23, C24 and C25 ……………………………….. 183 5.9.5 Analysis of compounds C26, C27 and C28 ……………………………….. 184 5.10 Biological evaluations: Results and discussion ……………………………….. 186 5.10.1 Evaluating the total antioxidant capacities of the isolated compounds …... 186 5.10.2 Evaluating the anti-lipid peroxidation activity of the isolated compounds 188 5.10.3 Evaluating the anti-tyrosinase activity of the isolated compounds ……….. 189 5.10.4 Evaluating the anti-elastase activity of the isolated compounds ………….. 190 5.11 Conclusion ……………………………………………………………………... References …………………………………………………………………………... CHAPTER SIX: CONCLUSION AND RECOMMENDATIONS Annexures …………………………………………………………………………... xiii 191 192 194 199 LIST OF ABBREVIATIONS 13 Carbon-13 nuclear magnetic resonance 1D-NMR One-dimensional nuclear magnetic resonance 1 Proton nuclear magnetic resonance C-NMR H-NMR 2D-NMR Two-dimensional nuclear magnetic resonance AAE/g Ascorbic acid per gram AAPH 2, 2’-Azobis (2-methylpropionamidine) dihydrochloride, perchloric acid ABTS 2, 2’-Azino-bis (3-ethylbenzo thiazoline-6-sulfonic acid) diammonium salt AChE Acetylcholineesterase BHT Butylated hydroxytoluene Br Broad CDCl3 Deuterated chloroform d Doublet DCM Dicholoromethane DIW De-ionized water DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid ECM Extra cellular matrix EDTA Ethylenediaminetetraacetic acid EGCG Epigallocatechingallate ESI Electrospray ionization EtOAc Ethyl acetate Fig Figure FRAP Ferric-ion reducing antioxidant power FT-IR Fourier transform infrared g Gram HMBC Heteronuclear multiple bond coherence xiv H2SO4 Sulphuric acid HD Helichrysum rutilans HF Helichrysum niveum HIV Human immune virus HPLC High pressure liquid chromatography HRMS High resolution mass spectroscopy HT Helichrysum teretifolium IC50 Half maximal inhibitory concentration IR Infrared J Coupling constant in Hz KCl Potassium chloride L Litre MeOH Methanol mg Milligram mL Millilitre min Minute MS Mass spectroscopy NMR Nuclear magnetic resonance ORAC Oxygen radicals absorbance capacity q Quartet ROS Reactive oxygen specie Rt Retention time s Singlet SANA N-succ-(Ala) 3-nitroanilide SANBI South African National Biodiversity Institute SD Standard deviation spp. Species TBARs Thiobarbituric acid reagents xv TCA Trichloroacetic acid td Triplet of doublets TE/g Trolox equivalent per gram TEAC Trolox equivalent absorbance capacity TLC Thin layer chromatography TPTZ (2,4,6-tri[2-pyridyl]-s-triazine, Iron (III) chloride hexahydrate Trolox 6-Hydroxyl-2, 5,7,8-tetramethylchroman-2-carboxylic acid USA United States of America UV Ultra violet xvi LIST OF FIGURES Figure 1.1: Effects of UV radiation on skin ……………………………………….. 4 Figure 1.2: Chemistry of metal chelation between tyrosinase and polyphenols …... 6 Figure 1.3: Damage caused by UV to the structural elements of the skin …………. 8 Figure 3.1: H. teretifolium description (A), and distribution along South African Coastal area (B) ………………………………………………………... 85 Figure 3.2: TLC profile of the main fractions under UV (254 nm; A&B), and after Spraying with H2SO4/Vanillin and then heated (C&D) ……………… Figure 3.3: TLC chromatogram of XXD3 (Fig. 3.3A) and HPLC spectrum of C9 92 93 Figure 3.4: Chromatogram of sub fraction XII …………………………………….. 96 Figure 3.5: HPLC spectrum of compounds C7 and C8 …………………………… 97 Figure 3.6: HPLC spectrum of compound C3 ……………………………………... 99 Figure 3.7: Chemical structure of heliteretifolin …………………………………... 109 Figure 3.8: Chemical structure of 2`,6`-dihydro-4`-methoxy-3-prenylchalcone ….. 110 Figure 3.9: Chemical structure of 2`,4`,6` -trihydroxy-3`-prenylchalcone …………. 110 Figure 3.10: Chemical structure of isoglabranin ……………………………………. 110 Figure 3.11: Chemical structure of glabranin ……………………………………….. 111 Figure 3.12: Chemical structure of 7-methoxyisoglabranin ………………………… 111 Figure 3.13: Chemical structure of quercetin ……………………………………….. 112 Figure 3.14: Chemical structure of 4-methoxyquercetin ……………………………. 112 Figure 3.15: Chemical structure of 4`-methoxykaempferol ………………………… 112 Figure 3.16: Chemical structure of mosloflavone …………………………………... 112 Figure 3.17: Effects of H. teretifolium constituents on inhibition of Fe2+-induced microsomal lipid peroxidation ………………………………………… 119 Figure 3.18: Effects of H. teretifolium constituents on inhibition of tyrosinase activity ………………………………………………………………… 121 Figure 3.19: Effects of H. teretifolium constituents on inhibition of elastase activity ………………………………………………………………… 122 xvii Figure 4.1: H. niveum description (A), and distribution along South African Coastal area (B) ………………………………………………………... 130 Figure 4.2: TLC profile of the collected fractions (1-44) under UV (254 nm; A-C), and after spraying with H2SO4/Vanillin and then heated (D-F) ……….. 134 Figure 4.3: TLC profile of the collected sub fractions XI (15-51) after spraying with H2SO4/Vanillin and then heated ……………………………………….. 136 Figure 4.4: TLC profile of the collected sub fractions XI-6 (1-24) after spraying with H2SO4/vanillin and then heated …………………………………… 137 Figure 4.5: HPLC spectrum of compounds C11, C15 & C16 ……………………... 137 Figure 4.6: TLC profile of the collected sub fractions XI-7 (16-30) after spraying with H2SO4/vanillin and then heated ……………………………………. 138 Figure 4.7: HPLC spectrum of compound C13 …………………………………….. 138 Figure 4.8: TLC profile of the collected fractions IX (1-24) after spraying with H2SO4/vanillin and then heated ……………………………………….... 139 Figure 4.9: HPLC spectrum of compound C14 …………………………………….. 139 Figure 4.10: TLC profile of the collected fractions IX (1-24) after spraying with H2SO4/vanillin and then heated ……………………………………….. 141 Figure 4.11: HPLC spectrum of compounds C17 & C19 …………………………... 141 Figure 4.12: HPLC spectrum of compounds C12 & C20 …………………………... 142 Figure 4.13: TLC profile of the collected fractions IV (1-32) after spraying with H2SO4/vanillin and then heated ………………………………………... 143 Figure 4.14: Chemical structure of 1-benzoyl-3 (3-methyl-2-butenylacetate)Phloroglucinol …………………………………………………………. 151 Figure 4.15: Structure of 1-benzoyl-3 (2-hydroxyl-3-methyl-3-butene-1-yl)phloroglucinol …………………………………………………………. 152 Figure 4.16: Possible absolute configurations of C13 at C-3 ……………………….. 153 Figure 4.17: Chemical structure of 8-(2-methyl-1-propanone)-3,5,7-trihydroxyl-2,2 dimethoxychromone …………………………………………………... 154 Figure 4.18: Chemical structure of 1-(2-methylbutanone)-4-O-prenylphloroglucinol …………………………………………………………. 154 Figure 4.19: Chemical structure of 1-(2-methylpropanone)-4-O-prenylxviii phloroglucinol …………………………………………………………. 154 Figure 4.20: Chemical structure of 1-(butanone)-3-prenyl-phloroglucinol …………. 155 Figure 4.21: Chemical structure of 1-(2-methylbutanone)-3-prenyl-phloroglucinol 155 Figure 4.22: Chemical structure of 1-butanone-3-(3-methyl-2-butenyl-acetate)phloroglucinol …………………………………………………………. 156 Figure 4.23: Chemical structure of 1-(2-methylpropanone)-3-prenylphloroglucinol 156 Figure 4.24: Chemical structure of caespitate ………………………………………. 156 Figure 4.25: Chemical structure of 3β-24-dihydroxyterexer-14-ene ………………... 157 Figure 4.26: Effects of H. niveum constituents on inhibition of Fe2+ - induced Microsomal lipid peroxidation ………………………………………... 161 Figure 4.27: Effects of H. niveum constituents on inhibition of tyrosinase activity ………………………………………………………………… 162 Figure 4.28: Effects of H. niveum constituents on inhibition of elastase activity ………………………………………………………………… 163 Figure 5.1: Helichrysum rutilans description (A), and distribution along South African mountain and coastal areas (B) ………………………………… 168 Figure 5.2: TLC profile of the collected fractions (1-58) under UV (254 nm; A -E), and after spraying with H2SO4/vanillin and then heated (F-J) …………. 172 Figure 5.3: TLC profile of the main fractions (I-XVI) under UV (254 nm) and after spraying with H2SO4/vanillin and gentle heating …………….. 173 Figure 5.4: TLC profile of sub fractions of X after spraying with H2SO4/vanillin and then heated …………………………………………………………. 174 Figure 5.5: TLC profile of sub fractions of XC after spraying with H2SO4/vanillin and then heated ………………………………………………………… 175 Figure 5.6: HPLC spectrum of compound C23 …………………………………….. 175 Figure 5.7: TLC profile of sub fractions of VIII after spraying with H2SO4/vanillin and then heated …………………………………………………………. 176 Figure 5.8: HPLC spectrum of compound C22 ……………………………………... 177 Figure 5.9: TLC profile of sub fractions of VI after spraying with H2SO4/vanillin and then heated …………………………………………………………. xix 177 Figure 5.10: HPLC spectrum of compounds C24 and C25 …………………………. 178 Figure 5.11: Structure of 5,7,8-trihydroxy-3,6-dimethoxyflavone-8-O-2-methyl-2Butanoate ……………………………………………………………… 182 Figure 5.12: Structure of 5,7-dihydroxy-3,6,8-trimethoxyflavone ………………….. 182 Figure 5.13: Structure of 5-hydroxy-3,6,7,8-tetramethoxyflavone …………………. 184 Figure 5.14: Structure of 5-hydroxy-3,6,7-trimethoxyflavone ……………………… 184 Figure 5.15: Chemical structure of ent-kaurenoic acid ……………………………... 185 Figure 5.16: Chemical structure of ent-kauran-18-al ……………………………….. 185 Figure 5.17: Chemical structure of 15-β-hydroxy-(-)-kaur-16-en-19-oic acid ……… 186 Figure 5.18: Effects of H. rutilans constituents on inhibition of Fe2+-induced microsomal lipid peroxidation ………………………………………… 189 Figure 5.19: Effects of H. rutilans constituents on inhibition of tyrosinase activity ………………………………………………………………… 190 Figure 5.20: Effects of H. rutilans constituents on inhibition of elastase activity ………………………………………………………………… 191 xx LIST OF TABLES Table 2.1: Traditional uses of some Helichrysum species ………………………… 20 Table 2.2.1: Monoterpenes …………………………………………………………. 25 Table 2.2.2: Sesquiterpenes ………………………………………………………... 25 Table 2.2.3: Diterpenes …………………………………………………………….. 28 Table 2.2.4: Triterpenes ……………………………………………………………. 33 Table 2.3.1: Flavones ………………………………………………………………. 35 Table 2.3.2: Flavonols ……………………………………………………………... 36 Table 2.3.3: Flavanones ……………………………………………………………. 39 Table 2.3.4: Chalcones …………………………………………………………….. 44 Table 2.3.5: Dihydrochalcones …………………………………………………….. 48 Table 2.4.1: Monomeric phloroglucinols ………………………………………….. 50 Table 2.5: Benzopyrone ……………………………………………………………. 55 Table 2.6: Coumarins ……………………………………………………………… 56 Table 2.7: Benzofuran ……………………………………………………………... 57 Table 2.8: Chromones ……………………………………………………………... 59 Table 2.9: Acetylene derivatives …………………………………………………... 60 Table 2.10: Quinones ……………………………………………………………… 61 Table 2.11: Miscellaneous …………………………………………………………. 62 Table 3.1: TLC solvent system …………………………………………………….. 87 Table 3.2: Fractionation of the methanol extract of H. teretifolium ……………….. 89 Table 3.3: Main fractions obtained upon fractionation of the total extract of H. teretifolium …………………………………………………………….. Table 3.4: Fractions grouped from the column ……………………………………. 91 93 Table 3.5: Sub fractions from III …………………………………………………… 94 Table 3.6: Sub fractions from X …………………………………………………… 95 Table 3.7: Sub fractions from XII …………………………………………………. 96 Table 3.8: Sub fractions from XXIII ………………………………………………. 97 Table 3.9: Sub fractions from XVIII ………………………………………………. 98 xxi Table 3.10: 1H (400 MHz: m, J Hz) and 13C (100 MHz) NMR spectral data of isolated compounds C1 - C6 in CDCl3 ………………………………. 113 Table 3.11: Oxygen radicals’ antioxidant capacity of H. teretifolium constituents …………………………………………………...... 116 Table 3.12: Ferric ion reducing and trolox equivalent antioxidant capacities of H. teretifolium constituents ………………………………………………. 118 Table 4.1: TLC solvent system …………………………………………………….. 131 Table 4.2: Fractionation of the methanol extract of H. niveum ……………………. 133 Table 4.3: Main fractions obtained upon fractionation of the total extract of H. niveum …………………………………………………………………. 134 Table 4.4: Sub fractions from XI …………………………………………………... 135 Table 4.5: Sub fractions obtained upon fractionation of the main fraction XI ……. 136 Table 4.6: Chromatographic fractionation of main fraction VII ………………….. 140 Table 4.7: 1H (400 MHz: m, J Hz) and 13C (100 MHz) NMR spectral data of compound C13 in CD3COCD3 ………………………………………... 148 Table 4.8: 1H (400 MHz: m, J Hz) and 13C (100 MHz) NMR spectral data of compounds C11, C12, C16, C18 & C19 in CD3COCD3 ……………… 149 Table 4.9: 1H (400 MHz: m, J Hz) and 13C (100 MHz) NMR spectral data of isolated compounds C14, C15, C17 & C20 in CDCl3 ………………… 150 Table 4.10: Oxygen radical absorbance capacity of H. niveum constituents ……… 159 Table 4.11: Ferric ion reducing and trolox equivalent absorbance capacities, and anti-acetylcholineesterase (AChE) inhibitory capacity of 160 H. niveum constituents ………………………………………………... Table 5.1: TLC solvent system …………………………………………………….. 170 Table 5.2: Fractionation of the methanol extract of H. rutilans …………………… 171 Table 5.3: Main fractions obtained upon fractionation of the total extract of H. Rutilans …………………………………………………………………. 173 Table 5.4: 1H (400 MHz: m, J Hz) and 13C (100 MHz) NMR spectral data of isolated compounds C22-C25 in CDCl3 ……………………………… 181 Table 5.5: Total antioxidant capacity assay for isolated compounds ……………… 186 xxii Table 5.6: Oxygen radical absorbance capacity and anti-acetylcholineesterase Activity ………………………………………………………………… 187 Table 6.1: Isolated compounds from the three selected Helichrysum species …….. xxiii 195 LIST OF SCHEMES Scheme 3.1: A flow diagram of experimental procedure for the isolation of constituents from H. teretifolium ………………………………… 100 Scheme 4.1: A flow diagram of experimental procedure for the isolation of constituents from H. niveum …………………………………… 144 Scheme 5.1: A flow diagram of experimental procedure for the isolation of constituents from H. rutilans …………………………………….. xxiv 179 PLANT 1: Helichrysum teretifolium (HT) xxv PLANT 2: Helichrysum niveum (HF) xxvi PLANT 3: Helichrysum rutilans (HD) xxvii LIST OF PUBLICATIONS 1. Olugbenga K. Popoola, Abdulrahman M. Elbagory, Farouk Ameer and Ahmed A. Hussein. (2013). Marrubiin. Molecules, 18, pp. 9049-9060. 2. Olugbenga K. Popoola, Jeanine L. Marnewick, Fanie Rautenbach, Farouk Ameer, Emmanuel I. Iwuoha and Ahmed A. Hussein. (2015). Inhibition of Oxidative Stress and Skin Aging-Related Enzymes by Prenylated Chalcones and Other Flavonoids from Helichrysum teretifolium. Molecules, 20, pp. 7143-7155. 3. Olugbenga K. Popoola, Jeanine L. Marnewick, Fanie Rautenbach, Emmanuel I. Iwuoha and Ahmed A. Hussein. Acylphloroglucinol Derivatives from the South African Helichrysum niveum and their Biological Activities. Molecules, submitted LIST OF PROPOSED PUBLICATIONS 1. Olugbenga K. Popoola, Jeanine L. Marnewick, Fanie Rautenbach, Farouk Ameer, Emmanuel I. Iwuoha and Ahmed A. Hussein. In vitro investigation of natural antioxidants and other skin disease-related inhibitory activities of constituents from South African Helichrysum spp. Manuscript in preparation 2. Olugbenga K. Popoola, Jeanine L. Marnewick, Fanie Rautenbach, Farouk Ameer, Emmanuel I. Iwuoha and Ahmed A. Hussein. The review of Chemistry of Helichrysum. Manuscript in preparation. CONFERENCE PRESENTATION 1. 2014: RSC/PACN congress on biodiversity and global challenges: A chemical science Approach (30-2 December, 2014, Addis Ababa, Ethiopia). Title of presentation: Inhibition of oxidative stress and tyrosinase in vitro of South African Helichrysum teretifolium constituents. Award: 2nd best presenter. xxviii CHAPTER ONE: INTRODUCTION CHAPTER ONE INTRODUCTION Why Natural Product Research in Cosmetics? “In general, people want to appear younger because a youthful appearance can result in better mental and physical health” – Sparavigna et al 1.1 Natural products as drugs The ethno-medicinal approach to drug discovery represents one of the most important sources of new and safe therapeutic agents to the challenges confronting modern medicine and daily life. Many of the traditionally important medicinal plants contain active compounds or ones that serve as precursors to biosynthesized secondary metabolites to which the biological activity could be attributed (Karthikeyan & Balasubramanian, 2014). Natural products derived from plant sources have assumed greater importance in recent days, due to the tremendous potential they offer in formulating new drugs which may protect humankind against many diseases (Balunas & Kinghorn, 2005; Khalid, et al., 2013). Further evidence of the importance of natural products is provided by the fact that close to half of the best-selling pharmaceuticals in 1991 were either natural products or their derivative analogues (Ashour, et al., 2013). Herbal products today is majorly important to the world population, mainly those in the developing countries for primary health care, due to a better cultural acceptability, better compatibility with the human body, and P a g e 1 | 238 CHAPTER ONE: INTRODUCTION less side effects when compared to the synthetics that are regarded as unsafe to humans and the environment (Vinha, et al., 2012; Nag, et al., 2013; Popoola, et al., 2013). 1.2 Skin aging The skin is the largest organ of the human body, both in terms of surface area (covering about 1.8 m2 in an average adult) and weight. It serves as an important environmental interface providing a protective envelope that is crucial for homeostasis (Chompo, et al., 2012). The structure of the skin is made primarily of two layers. The outer layer is the epidermis, consisting mainly of keratinocytes responsible for protecting the body against environmental damage, while the inner part, the dermis constitutes connective tissue and structural components such as collagen (responsible for the skin firmness), elastic fibers (responsible for skin elasticity), and extracellular matrix (ECM) also known as structural components (Sparavigna, et al., 2013). The process of aging in humans is complex with underlying multiple influences including the probable involvement of inheritable and various environmental factors (Zhang, et al., 2014). Aging is attributed to, but not limited to, the excessive accumulation of free radicals and other forms of reactive oxygen species (ROS) including hydroxyl radicals, peroxyl radicals, and hydrogen peroxide, which are primarily generated in the body as a result of physiological and biochemical processes (Aiyegoro & Okoh, 2010). Other notable ways of accumulating ROS in the body, is through continuous body contact with a series of environmental cues (such as UV radiation and pollution), and lifestyle choices including (but limited to) the diet, smoking, status of concurrent diseases (e.g. diabetes), exercise and alcohol consumption (Fearon & Faux, 2009; Chompo, et al., 2012). The free radical accumulation, when above threshold level in the body, can cause oxidative damage (Fig. 1.1) to important macromolecules such as proteins, lipids and DNA, eventually leading to many chronic diseases such as cancer, diabetes, aging, atherosclerosis, P a g e 2 | 238 CHAPTER ONE: INTRODUCTION neurodegenerative disorders (Niki, et al., 2005; Tepe, et al., 2005; Evgenia & Zouboulis, 2007; Carocho & Ferreira, 2013), and other degenerative diseases in humans (Aiyegoro & Okoh, 2010). Other significant pathological implications of ROS in the body is the activation of skin enzyme’s degenerative actions, resulting in early or premature skin aging processes such as pigmentation (Corstjens, et al., 2007; Ndlovu, et al., 2013), sagging and wrinkle formation (Porcheron, et al., 2014). Melanin which plays an important role in protecting human skin from the harmful effects of UV radiation from the sun, also causes the formation of pigmented patches (hyperpigmentation) on the skin surface as a result of the accumulation of an abnormal amount of melanin production (Chang, 2009). UV- exposed human skin may be an accessible model system in which to characterize the role of oxidative damage in both internal and external tissue, given the compelling evidence for the role of ROS as mediators of photoaging (Watson, et al., 2014). P a g e 3 | 238 CHAPTER ONE: INTRODUCTION Figure 1.1: Effects of UV radiation on skin. *source: www.healthyfellow.com 1.3 Oxidative stress The formation of free radicals is a continuous process and adaptation which occurred through evolution. These molecules play vital roles in cell signaling, controlling vascular tone, defense against microorganisms, cell generation and degeneration, and basal regulation of homeostasis (Basu, 2010). However, when in excess, ROS are also involved in the pathogenesis of diseases by damaging to DNA (deoxyribonucleic acid) and proteins causing gene modifications resulting in altered protein structures and functions, while glycoxidative damage and oxidative degradation of lipids in cell membranes can also be a result (Carocho & Ferreira, 2013). The body’s defense mechanism generally declines with age, and can be compromised by various forms of oxidative P a g e 4 | 238 CHAPTER ONE: INTRODUCTION stress resulting from environmental factors to cancer, diabetes, atherosclerosis, and neurodegenerative disorders. All these conditions, as well as the aging process, are associated with oxidative stress due to elevation of ROS or insufficient ROS detoxification (Limon-Pacheco & Gonsebatt, 2009; Igwe & Echeme, 2014). Oxidative stress therefore occurs when the formation of bioactive oxidative products such as oxidizing agents, free radicals and reactive oxygen species, greatly overwhelms the capacity of the endogenous cellular antioxidant defense system, thus leading to potential damage of the cells and organs, and to the progression of degenerative diseases in humans (Schrader & Fahimi, 2006; Basu, 2010). 1.4 Skin enzymes 1.4.1 Tyrosinase Tyrosinase is a copper-containing enzyme which catalyzes the first two stages during the process of melanogenesis (Chang, 2009). Melanin plays a vital role as photo protective agent against the harmful effects of UV radiation, and also determines our phenotypic outlook. However, over accumulation of melanin in specific parts of the skin result in undesirable and abnormal secretion of dark macromolecular pigment called skin hyperpigmentation (Wangthong, et al., 2007; Chompo, et al., 2012). Since the accumulation of excessive epidermal pigmentation leads to various dermatological disorders, tyrosinase inhibitors (such as phenolic compounds, Fig. 1.2) have become increasingly important in medication and in cosmetics to prevent hyperpigmentation through the inhibition of enzymatic oxidation, via its direct chelation to the central metal (copper ion) in tyrosinase (Moon, et al., 2010). P a g e 5 | 238 CHAPTER ONE: INTRODUCTION Phenolic compound Tyrosinase enzyme Figure 1.2: Chemistry of metal chelation between tyrosinase and polyphenols *source: www.pub.rsc.org 1.4.2 Elastase Elastase is a proteolytic enzyme involved in the degradation of elastin, leading to skin aging. (Ndlovu, et al., 2013). In normal condition, skins produce enzymes such as elastase at indistinguishable rate as aging process occurs and age increases. However, with overexposure to UV radiation and the presence of excessive ROS, the enzymes are produced at a rapid rate, resulting in early degradation of elastin (Chompo, et al., 2012; Kammeyer & Luiten, 2015). Degradation of elastin is a major part of what causes visible signs of aging (wrinkles, sagging) and P a g e 6 | 238 CHAPTER ONE: INTRODUCTION tissue injury in the skin (Castelletto, et al., 2014). Several studies have demonstrated that both skin-aging and anti-wrinkle effects are significantly correlated with decreased elastase activity (Moon, et al., 2010). 1.4.3 Hyaluronidase Hyaluronic acid plays a role in retaining the moisture of the skin, as well as its structure and elasticity. It facilitates the exchange of nutrients and waste products and is involved in rapid tissue proliferation, regeneration and repair. This compound is also involved in organization and structural maintenance of the ECM (Ndlovu, et al., 2013). Hyaluronidase plays a crucial role in degradation of ECM leading to bacterial invasion, envenomation of various toxins including honeybee toxins, snake toxins, promote tumor growth and angiogenesis. The potent hyaluronidase inhibitors are useful as contraceptives, antitumor agents and have antibacterial and anti-venom properties (Satardekar & Deodhar, 2010). Other enzymes that are pathologically implicated as a result of excessive accumulation of ROS in the body include collagenase, 5α reductase (responsible for hair loss) and acetylcholinesterase among others. P a g e 7 | 238 CHAPTER ONE: INTRODUCTION UV radiation from the sun Increase in free radicals Over production of skin enzymes Epidermis Dermis Figure 1.3: Damage caused by UV to the structural elements of the skin *source: www.leadinginstanteyelift.com 1.5 General overview of the use of plants as source of medicines The use of plants as a source of medicine has been inherited from the onset of human civilization and is an important component of the healthcare system (Fernnell, et al., 2004). Medicinal foods and plants have been widely used as foods, dietary supplements, or medicines to visiting western health care in South Africa due to its large cultural and floral biodiversity health benefits with a long history. Over 20,000 species of higher plants are present, out of which 10 % of these species have been found to be used in traditional medicine across the country to treat disease ailments (Thring & Weitz, 2006). In South Africa, most of the population in urban as well as smaller rural communities is dependent on plant remedies for their medical requirements. Herbal remedies, apart from their traditional and P a g e 8 | 238 CHAPTER ONE: INTRODUCTION cultural significance, are generally more accessible and affordable. Therefore the trend to integrate traditional Western medicine, especially in the primary health care setting, is becoming increasingly important (Meissner, 2004). Medicinal plants have curative properties due to the presence of various important bioactive constituents (such as phenolics, flavonoids, alkaloids, tannins, and terpenoids) known as secondary metabolites that have a definite physiological action in the human body (Shaukat, et al., 2013). Since time immemorial and in the present day traditional medicine as practiced in many countries, medicinal plants are taken in as their crude forms and prepared in the form of decoctions, infusions or powders. The isolation of plant metabolites started in the nineteenth century. At the beginning, structural elucidation of isolated compounds were limited by the technology available at that time in which case, it was done by classical and exhaustive degradative methods and the structures emanated from such were confirmed through synthesis and in most cases followed by biological activity determinations. Currently, with advent of technology, the structural elucidations of chemical compounds is done with only a few milligrams of pure material using sophisticated instruments such as HPLC, NMR (1D and 2D), HRMS with many new compounds generated each year. Some of these compounds are antimalarial, anti-aging, anticancer enzyme inhibitors and some are known to bind to specific receptors of pharmacological interest. However, there are some compounds isolated through chemically guided isolation that have proved to be biologically active. 1.6 Why investigate the genus Helichrysum? Helichrysum (Mill.) is classified in the tribe Gnaphalieae and as current circumscribed contains 500-600 species which are mainly found in Africa and Madagascar, but also in Europe, Asia and P a g e 9 | 238 CHAPTER ONE: INTRODUCTION Australia (Hilliard, 1983). The approximately 250 southern African species are widespread and display enormous morphological diversity. Among such medicinal value, is in the treatment of wounds. Also in Angola, the aromatic fruiting head is used for cosmetic purposes. The leaf of Helichrysum specie is regarded as a “Zulu love philter” resulting in the desired lady finding the man irresistible, by applying an ointment from the dried leaf to the body (www.herbafrica.co.za/herbahelichrysum). Other notable economic importance of Helichrysum species are highlighted in the comprehensive literature review presented in the next Chapter. 1.7 Natural products as inhibitors against oxidative stress and skin aging Plants have been used in the cosmetic industry as amongst others, skin lighteners and sun-screen agents. In vitro scientific evidence has shown that plants possess antioxidant capacity by converting free radicals to stable products, and inhibiting the degenerative activity of certain skin enzymes (Ndlovu, et al., 2013). The skin antioxidant system has an extensive area exposed to the environment to protect and, as a consequence, highly exposed to exogenous radical attacks, resulting in the defense system to be constantly challenged. Incorporation of plant secondary metabolites with effective natural antioxidant and skin anti-aging activities into cosmetic products therefore has significant role in the prevention of skin aging (Tavares da Mota, et al., 2014). Polyphenolic substances possess many biochemical properties, but the best described property of almost every group of phenolics is their capacity to act as an antioxidant. The antioxidant activity of phenols depends upon the arrangement of functional groups around the nuclear structure. The configuration, substitution, and total number of hydroxyl groups substantially influence the different mechanism of antioxidant activity such as radical scavenging and metal chelating ability (Kumar & Pandey, 2013). P a g e 10 | 238 CHAPTER ONE: INTRODUCTION 1.8 Rationale for the study The process of skin aging due to free radical attacks and exposure to unavoidable and irreversible ultra-violet radiation is the cause of major concern for human health. In particular, damages related to the skin have great relevance. To maintain the youthful appearance of the skin, there are two methods viz: 1. To promote the synthesis of matrix proteins in the skin and / or 2. To inhibit matrix protein degrading enzymes like elastase, collagenase, tyrosinase and hyaluronidase. Therefore, there is a need to overcome the problem associated with skin degenerative processes, caused by involvement of exogenous radical attacks and other oxidative species, through the study of isolated secondary metabolites from natural origin with effective antioxidant and skin anti-aging activities. People living on the African continent are exposed to the harsh sun and rarely use skin protective agents as they are not affordable. It is for this reason that South African Helichrysum species, widely distributed and readily available for human exploration for their potential application as antioxidant and skin anti-aging sources were selected for this study. Helichrysum species are wide spread with well documented chemistry, but scant biological and pharmacological information to support their varying ethno-medicinal uses. It is an established fact that this genus contains more phenolic compounds which are found to possess antioxidant characteristics. Figure 1.3 illustrates the activation of the degenerative effects of skin enzymes as a result of over accumulation of free radicals in the body. Propositions were therefore made to investigate South African Helichrysum species for possible antioxidant activity, in order to complement the body’s natural antioxidant defense mechanisms against deleterious effects posed P a g e 11 | 238 CHAPTER ONE: INTRODUCTION by free radicals. Further hypothesis was made to investigate the degree of chelation of the phenolic compounds present in Helichrysum with copper (the central metal of tyrosinase enzyme) and determine their possible applications as skin depigmentation products. This theorem therefore underpins the need to formulate cosmetic products with skin anti-aging properties using bioactive secondary metabolites isolated from South African Helichrysum species selected for this study. 1.9 Aims of this study The main aim of this project is directed towards the chemical studies on three Helichrysum species (H. teretifolium, H. nevium and H. rutilans) to isolate the main chemical constituents, as well as the biological profiling of the total extracts and the isolated compounds for their potential antioxidative stress and their inhibitory activity against some skin-aging enzyme. 1.10 Objectives of this study The main objectives of the study are: Collection of samples of H. teretifolium, H. niveum and H. rutilans, from their natural habitats, documentation and identification. Preparation of methanolic extracts from each species and isolation of secondary constituents in pure form. Elucidation of chemical structures of bioactive constituents isolated from the selected species of Helichrysum using different spectroscopic techniques (e.g. 1D, 2D-NMR, HRMS, UV, and IR) as well as Polarimeter. Investigation of total antioxidant capacities of the isolated chemical constituents. P a g e 12 | 238 CHAPTER ONE: INTRODUCTION Exploration of the inhibitory action of each of the identified constituents against skindiseases related enzymes in in vitro system. Investigation of the structure-activity relationship of the isolated compounds with their respective biological specificity. Conduct comparative phytochemical and biological analyses on the isolated chemical constituents present in the selected Helichrysum species to the existing chemical compounds isolated from the genus. Recommendations of all bioactive constituents so far isolated and identified from the selected Helichrysum species, for further biological characterizations and clinical studies. 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Limon-Pacheco, J. and Gonsebatt, M.E. (2009). The role of antioxidants and antioxidant-related enzymes in protective responses to environmentally induced oxidative stress. Mutation Research, 674, pp. 137-147. P a g e 15 | 238 CHAPTER ONE: REFERENCES Meissner, O. (2004). Editorial: The traditional healer as part of the primary health care team? South African Medical Journal, 94, pp. 901-902. Moon, J., Yim, E., Song, G., Lee, N.N. and Hyun, C. (2010). Screening of elastase and tyrosinase inhibitory activity from Jeju Island plants. EurAsian Journal of Biosciences, 4, pp. 41-53. Nag, S., Paul, A. and Dutta, R. (2013). Phytochemical analysis of methanol extracts of leaves of some medicinal Plants. International Journal of Science Research Publication, 3(1), pp. 1-5. Ndlovu, G., Fouche, G., Tselanyane, M., Cordier, W. and Steenkamp, V. (2013). In vitro determination of the anti-aging potential of four southern African medicinal plants. 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Shaukat, K., Ghazala, H.R., Hina, Y., Rehana, P., Hina, A., Huma, S., Kaneez, F. and Maryam, A. (2013). Medicinal importance of Holoptela integrifolia (Roxb.) Planch - its biological and pharmacological activities. Natural Products Chemistry & Research, 2(1), pp. 1-4. P a g e 16 | 238 CHAPTER ONE: REFERENCES Sparavigna, A., Tenconi, B., Deponti, I., Scarci, F. and Caserini, M. (2013). An innovative concept gel to prevent skin aging. Journal of Cosmetics, Dermatological Sciences and Applications, 3, pp. 271-280. Tavares da Mota, G.S., Arantes, A.B., Sacchetti, G., Spagnoletti, A., Ziosi, P., Scalambra, E., Vertuani, S. and Manfredini, S. (2014). Antioxidant activity of cosmetic formulations based on novel extracts from seeds of Brazilian Araucaria angustifolia (Bertoll) Kuntze. Journal of Cosmetics, Dermatological Sciences and Applications, 4, pp. 190-202. Tepe, B., Sokmen, M., Akpulat, H.A. and Sokmen, A. (2005). In vitro antioxidant activities of the methanol extracts of four Helichrysum species from Turkey. Food Chemistry, 90, pp. 685-689. Thring, T. and Weitz, F. (2006). Medicinal plant use in the Bredasdorp/Elim region of the Southern Overberg in the Western Cape Province of South Africa. Journal of Ethnopharmacology, 103, pp. 261-275. Vinha, A.F., Soares, M.O., Castro, A., Santos, A., Oliveira, M.B. and Machado, M. (2012). Phytochemical characterization and radical scavenging activity of aqueous extracts of medicinal plants from Portugal. European Journal Medicinal Plants, 2(4), pp. 335-347. Wangthong, S., Tonsiripakdee, l., Monhaphol, T., Nonthabenjawan, R. and Wanichwecharungruang, P.S. (2007). TLC developing technique for tyrosinase inhibitor detection. Biomedical Chromatography, 21, pp. 94-100. Watson, R.E.B., Gibbs, N.K., Griffiths, C.E.M. and Sheraft, M.J. (2014). Damage to skin extracellular matrix induced by UV exposure. Antioxidant & redox signaling, 21(7), pp. 10631077. Zhang, S., Dong, Z., Peng, Z. and Lu, F. (2014). Anti-aging effect of adipose-derived stem cells in a mouse model of skin aging induced by D-Galactose. PLoS One, 9(5), pp. e97573/1 - e97573/7. P a g e 17 | 238 CHAPTER TWO: LITERATURE REVIEW CHAPTER TWO CHEMISTRY OF HELICHRYSUM - LITERATURE REVIEW 2.1 Aim of this chapter This chapter summarizes existing data from SciFinder and the dictionary of natural products on phytochemistry of notable constituents of Helichrysum species. Ethno medicinal uses as applied to some geographic locations are briefly described. The biological importance of some of these notable chemical constituents as applicable to this study is also described. 2.2 Asteraceae family Asteraceae is the largest family of flowering in the world, comprising 23 600 species assigned to 1620 genera and have a cosmopolitan distribution, but is especially diverse in the tropical and subtropical regions of southern Africa, the Mediterranean region, central Asia, south-western China, and Australia (Funk, et al., 2009). Although vegetatively varied, it is recognisable by capitulate and involucrate inflorescences in which numerous small flowers open first on the outside and only sometimes subtended by bracts. The anthers are fused and form a tube through which the style extends before the two stigmatic lobes separate and become recurved, while the single-seeded fruit usually have a plumose pappus (Galbany-casals, et al., 2014). The family is divided into 13 subfamilies (Panero, et al., 2014) of which the Asteroideae contains more than 70 % of the species currently recognised. Recent molecular studies revealed three main lineages within the Asteroideae that have been recognised at the supertribe level (Robinson, 2004). P a g e 18 | 238 CHAPTER TWO: LITERATURE REVIEW 2.3 Helichrysum genus Helichrysum (Mill.) is classified in the tribe Gnaphalieae and as currently circumscribed contains 500-600 species which are mainly found in Africa and Madagascar, but also in Europe, Asia and Australia (Hilliard, 1983; Manning & Goldbatt, 2012). Approximately 250 southern African species are widespread and display enormous morphological diversity. Helichrysum species are generally regarded as aromatic perennial herbs with dense hair or woolly leaves and persistent flower heads of different varieties (Wyk, et al., 2009). Among such, the medicinal value of South African Helichrysum species is in the treatment of conditions associated with infections of the skin, such as circumcised wounds and wound dressings (Lourens, et al., 2008). Helichrysum species are generally enriched with constituents naturally present in the plant as secondary metabolites, like phenolics (phloroglucinol derivatives, flavonoids and chalcones), α-pyrone derivatives, and acetophenones derivatives (Lourens, et al., 2008), which give them a veritable icon for global acceptability and large application in the cosmetic and pharmaceutical fields as anti-inflammatory (Viegas, et al., 2014), antibacterial and antioxidant agents (Kolayli, et al., 2010; Mari, et al., 2014; Rigano, et al., 2014). 2.4 Traditional uses of Helichrysum genera Helichrysum species have been used in traditional medicine globally and were already known in the Greek-Roman period (Ruberto, et al., 2002). Plants have been used since time immemorial in folk medicine in Europe, Egypt, North America, China, and Australia (Jakupovic, et al., 1989a; Lwande, et al., 1993). They are well-known in South African traditional medicine, and their usage is often linked to their geographical location. Detailed information about the traditional uses of South African Helichrysum species has been documented (Lourens, et al., 2008). Effort was therefore directed towards the global ethnomedicinal uses and chemistry of Helichrysum species P a g e 19 | 238 CHAPTER TWO: LITERATURE REVIEW from 3 major locations: South African, Australian and European species as presented in Tables 1.1 and 1.2 In the Mediterranean region for instance, Helichrysum is known as aromatic plant widely used in traditional medicine as skin repair, due to its effectiveness in the treatment of scars by prevention of bleeding and the formation of scar tissue and all kinds of cuts (Kladar, et al., 2015). The genus is described to possess anti-inflammatory, antimicrobial, and antioxidant properties and find large application as food supplement as well as in cosmetic and pharmaceutical field (Cavar, et al., 2015). Commercially, the drug derived from this genus is widely used as a liver stimulant and diuretic. The anti-inflammatory and tissue-regenerating properties as well as the health benefits in the treatment of some cardiovascular conditions are confirmed (Kladar, et al., 2015). H. microphyllum growing in Sardinia, Corsica and Balearic Islands is used traditionally to nurse cough, burns and as antirheumatic and analgesic. It is found to contain antispasmodic, antioxidant, antibacterial and antiallergic (Ornano, et al., 2015). Table 2.1: Traditional uses of some Helichrysum species Plant source Location Traditional uses References H. pedunculatum South Africa Antioxidant, antibacterial, wound healing Aiyegoro & Okoh, 2009; Aiyegoro, et al., 2010 H. aureonitens South Africa Antiviral, antifungal and antibacterial Ziaratnia, et al., 2009 H. petiolare South Africa Coughs, colds, catarrh, headache, fever, menstrual Lourens, et al., 2008 disorders, urinary tract infections, Antiseptic wound dressing H. longifolium South Africa Treatment of circumcised wound; antioxidant Aiyegoro & Okoh, 2010 H. splendinum Southern Fuel source and for the treatment of various conditions Mashigo, et al., 2015 African associated with rheumatism, combat colds, flu and pneumonia, and as a general antiseptic as a perfume amongst rural communities H. italicum Mediterranean Skin disease, antieczematic, bronchial mucus Melito, et al., 2013; expectoration, antimicrobial, Taglialatela-Scafati, et P a g e 20 | 238 CHAPTER TWO: LITERATURE REVIEW al., 2013; Mari, et al., 2014 ; Kladar, et al., 2015 H. microphyllum Mediterranean cough, antirheumatic Cavar, et al., 2015 H. devium Portugal respiratory diseases, such as bronchitis and pharyngitis Gouveia & Castilho, 2012 H. obconicum Portugal Stomach and intestinal pain relieve Gouveia-Figueira, et al., 2014 Herbal tea & salads, bronchitis & pharyngitis, Gouveia-Figueira, et al., melaleucum; H. cardiotonic and cough relief, Respiratory diseases, 2014 obconicum; H. cough relieve H. devium; H. Portugal monizii H. stoechas Portugal Cold, bronchitis and fever, antioxidant potential of Barroso, et al., 2014 extracts H. zivojinii Yugoslav Cough relief and Antibacterial Aljancic, et al., 2014 H. arenarium Poland, China, prevention of age-related diseases, heart, cardiovascular Morikawa, et al., 2009a, Japan ailments, gastro-intestinal disorders, rheumatism and 2009b; Wang, et al., respiratory infection, as food additives, antibacterial and 2009; Yong, et al., anti-oxidant, choleretic, diuretic, and detoxify function, 2011; Jarzycka, et al., antitumor, choleretic, hepatoprotective, and detoxication 2013 H. graveolens Turkey Jaundice, for wound-healing and as a diuretic Suntar, et al., 2013 H. plicatum Turkey Diabetes disease, hepatits, kidney stones Polat, et al., 2013 H. chasmolycicum Turkey Antimicrobial Suezgec-Selcuk & Birteksoez, 2011 Antioxidant and antimicrobial; gastric and hepatic Demir, et al., 2009 ; armenium; H. disorders Albayrak, et al., 2010 plicatum Antimicrobial H. arenarium; H. Turkey H. tuberosus Turkey Antihypertensive, diabetes disease Polat, et al., 2013 H. cameroonense Cameroon Antibacterial Antoine, et al., 2010 ; 2011 H. foetidum Cameroon influenza, infected wounds, herpes, eye problems, Zanetsie Kakam, et al., menstrual pain and to induce trance 2011 H. italicum Italy Antioxidant Rosa, et al., 2011 H. oligocephalum Iran Antibacterial Ebrahim Sajjadi, et al., 2009 P a g e 21 | 238 CHAPTER TWO: LITERATURE REVIEW 2.5: CHEMISTRY OF HELICHRYSUM The chemistry of Helichrysum genus is complex with occurrence of variety of organic compounds identified by previous work as flavonoids, phloroglucinol derivatives, terpenoids among others by two major researchers (Bohlmann and Jakupovic). Some of the major compounds, categorized in accordance to their respective carbon skeletal backbones are summarized in Tables 2.2.1 – 2.2.11. Detailed phytochemical investigation of South African Helichrysum species have been documented (Lourens, et al., 2008). This chapter therefore focus on chemistry of selected Helichrysum species widely used in traditional medicine from three different geographical locations viz: South Africa, Mediterranean region and Australia. Widespread occurrence of terpenoids of various chemical classes in Helichrysum genus is an indication that characterized the plant from this genus with aromatic fragrance smell (Asekun, et al., 2007; Lourens, et al., 2008). Sesquiterpenes (Table 2.2.2) of close skeletal type chemical constituents exist in both Australian and South African Helichrysum species with occurrence of variety of bicyclic derivatives of alcohol and mono acid (Bohlmann, et al., 1978a; Bohlmann & Abraham, 1979a; Jakupovic, et al., 1986; Jakupovic, et al., 1987c; 1989a; 1989b). Kaurane diterpenes (ent- and nor- derivatives) including its acids derivatives (Table 2.2.3) are widely found in different species belonging to Asteraceae family (Lloyd & Fales, 1967; Bohlmann & Zdero, 1980a; Jakupovic, et al., 1989a). Such occurrence is also observed in Helichrysum genus. Kaurenoic acid were found to demonstrate antibacterial and moluscicide activities due to their existence as intermediate compounds in the biosynthesis of diverse kaurane diterpene including gibberellins, which make them to acts as plant growth inhibitors. P a g e 22 | 238 CHAPTER TWO: LITERATURE REVIEW The presence of hydroxylated/methoxylated flavonoids (Tables 2.3.1-2.3.5) are generally present in all species (Bohlmann & Abraham, 1979a; 1979c; Forkmann, 1983; Jakupovic, et al., 1987b; Jia & Zhao, 2005; Lall, 2006), while ring-A methoxylated flavonols occurrence is well pronounced in both European and South African Helichrysum species (Bohlmann, et al., 1978b; Bohlmann & Abraham, 1979c; 1980a; Tomas-Barberan, et al., 1988). Prenylated and O-prenylated flavonoids were also common to the genus inrespective of locations (Bohlmann, et al., 1979a; Bohlmann & Misra, 1984a; Bohlmann & Ates, 1984a). The presence of different class of flavonoids in the genus gave them significant global acceptability and large application in cosmetic and pharmaceutical field as anti-inflammatory (Viegas, et al., 2014), antibacterial, and antioxidant agent (Kolayli, et al., 2010; Mari, et al., 2014; Rigano, et al., 2014). H. italicum is found to be rich in phenolic compounds such as flavonoids, coumarins and chemical composition of volatiles is well documented in the literature (Kladar, et al., 2015). Occurrence of Arzanol (205), a novel antiviral lead compound according to literature was found to inhibit HIV1 replication in T cells made another biological significance for this genus (Appendino, et al., 2007; Rosa, et al., 2011). Other notable heterodimeric acylphloroglucinols of this genus were also documented (Bohlmann & Zdero, 1980a; Randriaminahy, et al., 1992; Appendino, et al., 2007). Occurrence of unusual compounds is also documented to include thiophenes (285, 286), epoxide (275) and furan (271) substituted acetylenics with different moieties. 2.6 Antioxidant activities of Helichrysum genus Previous data documented on the antioxidant activities of this genus were directed towards fractions obtained directly from the crude plant materials, nevertheless, the compounds responsible for the antioxidant activities have been documented only in a few cases (Albayrak, et al., 2010). P a g e 23 | 238 CHAPTER TWO: LITERATURE REVIEW Some famous medicinal plant from the Mediterranean region was found to demonstrate good antioxidant activity using the DPPH assay with an IC50 value 100-fold higher than that of trolox. It also has moderate anti-aging properties due to its skin tissue regeneration ability and alleviation of inflammation through its antioxidant properties, protecting the skin from the damaging effects of free radicals (Ornano, et al., 2015). Other Helichrysum spp. investigated for its antioxidant capacity using the DPPH assay is H. italicum (Kladar, et al., 2015). 2.7 Skin anti-aging properties of Helichrysum species Few chemistry of metal chelating ability of flavonoids with copper (II) in tyrosinase have been investigated with mild inhibitory activities demonstrated by compounds 114, 147 and 148. Other previous anti-tyrosinase activity data documented, were carried out using extracts obtained from plant material and therefore, details of the mechanism of chelation between copper (II) and the chemical constituents of Helichrysum species, remain untraceable in the SciFinder database. P a g e 24 | 238 CHAPTER TWO: LITERATURE REVIEW Table 2.2: TERPENOIDS Table 2.2.1: MONOTERPENES s/n Compound Plant source H. heterolasium OR 1 2 Biological activity Reference Bohlmann & Abraham, 1979b R =H; R = Ac Table 2.2.2: SESQUITERPENES s/n 3 Compound 4 Plant source H. heterolasium, H. nuifolium, H. mimetes H OH O 5 Reference Jakupovic, et al., 1989a H. krausii Jakupovic, et al., 1989b H. ambiguum Jakupovic, et al., 1989a H. petiolare 6 Biological activity Antiseptics Jakupovic, et al., 1989b O H. dasyanthum 7 HO OH Jakupovic, et al., 1989b H H. dasyanthum 8 HO OH Jakupovic, et al., 1989b OH H P a g e 25 | 238 CHAPTER TWO: LITERATURE REVIEW Jakupovic, et al., 1986 H. mimetes 9 H HO H. nudifolium H Antiseptics Bohlmann, et al., 1978a R1 R2 10 11 12 13 R1 = R2 =H R1=H; R2 = OH R1 = H; R2 = OAc R1 =OAc; R2 = H 14 OH H. bilobum Jakupovic, et al., 1989a H. chionosphaerum Jakupovic, et al., 1989b H. italicum Jakupovic, et al., 1986 H. nudifolium Jakupovic, et al., 1986 18 H. nudifolium Jakupovic, et al., 1986 19 H. italicum Leimner, et al., 1984 15 COOH 16 O 17 O P a g e 26 | 238 CHAPTER TWO: LITERATURE REVIEW 20 OH 21 H. davyi Jakupovic, et al., 1987c H. albirosulatum H. chionosphaerum Bohlmann, et al., 1978a; Bohlmann & Zdero, 1980a O Bohlmann & Suwita, 1979a; Bohlmann & Zdero, 1980c R1 H. splendidum, H. tenuifolium, H. heterolasium H. glomeratum, H. drakenbergense H. chionosphaerum R2 22 23 24 R1= R2 = CH3 R1= H; R2= CH3 R1= CH3; R2= CH2OH 25 H. petiolare, H. chionospaerum, H. kraussii Jakupovic, et al., 1989b 26 H. chionosphaerum Bohlmann, et al., 1980b H. dasyanthum Jakupovic, et al., 1989b H. dasyanthum Jakupovic, et al., 1989b H. dasyanthum Jakupovic, et al., 1989b H. splendidum Bohlmann & Suwita, 1979a COOH 27 H O OH 28 O OH H O O OH OH 29 H O O OH OH 30 O O P a g e 27 | 238 CHAPTER TWO: LITERATURE REVIEW O 31 H. splendidum Bohlmann & Suwita, 1979a H. dasyanthum Jakupovic, et al., 1989b H. splendidum Jakupovic, et al., 1989b H. splendidum Jakupovic, et al., 1989b O O HO O O OH 33 O O O 34 O OH Table 2.2.3: DITERPENES s/n Compound Plant source 35 Bohlmann & Zdero, 1980c; Jakupovic, et al., 1986 H. oreophilum H. krebsanum Bohlmann & Zdero, 1980c; Jakupovic, et al., 1986 H. krebsianum, H. odoratissimum H. cephalsideum, H. nudifolium Bohlmann & Abraham, 1979d; Bohlmann, et al., 1978a; 1980b OH 36 OH 37 Reference H. oreophilum O HO Biological activity P a g e 28 | 238 CHAPTER TWO: LITERATURE REVIEW 38 H. chionosphaerum Bohlmann, et al., 1980a H. chionosphaerum H. candolleanum Bohlmann, et al., 1980 H. refluxum Bohlmann, et al., 1985 H. heterolasium Bohlmann & Abraham, 1979b H 39 H COOH 40 COOH 41 O R 42 43 Jakupovic, et al., 1986 H. nudifolium; H. odoratissimum H. nudifolium R= H; R= OH 44 H. nudifolium Bohlmann, et al., 1978a H. formosissinum, I Jakupovic, et al., 1990 OH H R 45 R=H 46 R = OH P a g e 29 | 238 CHAPTER TWO: LITERATURE REVIEW 47 H. confertum OH Bohlmann, et al., 1978a OH H. albirosulatum Bohlmann, et al., 1978a; Bohlmann & Zdero, 1980c H. tenax Drewes, et al., 2006 H. chionosphaerum Jakupovic, et al., 1989a OH R1 48 49 50 R1= O R1= H R1= -OH 51 O HO 52 H H H COOH R5 R7 R6 R8 53 54 55 56 57 R9 R3 R1R2 R1= R2= R4= R5= R6= R7= R8= H; R3= CH2; R9= COOH R1= R2= R4= R5= R6= R8= H; R3= CH2; R7= OAc; R9= COOH R1= R4= R5= R6= R7= R8= H; R2= OH; R3= CH2; R1= COOH R1= R2= R4= R5= R7= R8= H; R3= CH2OH; R4= OH; R9= CH3 R1= R2= R6= OH; R4= R5= R7= R8= H; R3= CH2; R9= COOH R3 R1 58 59 Bohlmann, et al., 1980a R4 H. chionosphaerum; H. aureum, H. pallidum, H. kraussii, H. fulvum H. chionosphaerum H. aureum H. dasyanthum H. dendroideum Lloyd & Fales, 1967 R2 R1= R2 =H; R3= CH2OH R1= OH; R2= CH2OH; R3=CH3 P a g e 30 | 238 CHAPTER TWO: LITERATURE REVIEW 60 61 OAc COOH AcO H. chionosphaerum Jakupovic, et al., 1989b H. aureum and H. cooperi Bohlmann, et al., 1978a H. dendroideum Bohlmann, et al., 1978a H. heterolasium Bohlmann & Abraham, 1979b H. dasyanthum Jakupovic, et al., 1989b H. bracteatum Bohlmann & Zedro, 1973 H. foetidum Barrero, et al., 1998 COOH 62 OH 63 AcO HOOC 64 OH HOOC OH OH 65 OH 66 OH COOH H. athrixia, 67 Trypanosomici dal and antibacterial Bohlmann, et al., 1978a COOH P a g e 31 | 238 CHAPTER TWO: LITERATURE REVIEW 68 Jakupovic, et al., 1989a H. davenportii OH COOH H. tenax 69 HO Antimicrobial Drewes, et al., 2006 H. diosmifolium Lassak & Pinhey, 1968; Jakupovic, et al., 1987b H. setosum Jakupovic, et al., 1986 H. subfalcatum Bohlmann & Zdero, 1980c 75 H. kraussii, Bougatsos, et al., 2003 76 H. argyrophyllum Jakupovic, et al., 1989b 70 OH OH COOH R1 R2 71 R2 = R1 = CH3 O O O 72 R1 = O 73 OH O O R1= CH2OH R2 = CH3 OH R2 = CH3 74 OH P a g e 32 | 238 CHAPTER TWO: LITERATURE REVIEW 77 O O Drewes, et al., 2006 H. tenax Drewes, et al., 2006 H2 C CC O O O O 78 H. tenax H2 C CC O O O Table 2.2.4: TRITERPENES s/n Compound Plant source 79 Biological activity Reference H. callicomum H. heterolasium Bohlmann & Abraham, 1979b H. chrysargyrum Bohlmann & Abraham, 1979a HO 80 COOH AcO P a g e 33 | 238 CHAPTER TWO: LITERATURE REVIEW 81 H. mundtii Bohlmann, et al., 1980b H. tenuiculum Bohlmann & Abraham, 1979a H. panduratum, H. mundtii Bohlmann, et al., 1980b H. chrysargyrum Bohlmann & Hoffmann, 1979b COOH HO 82 COOH HO 83 HO 84 COOH AcO H. italicum Exhibits spasmolytic activity Mezzitti, et al., 1970 O O RO 85 R=H 86 R = Ac P a g e 34 | 238 CHAPTER TWO: LITERATURE REVIEW H. arenarium 87 Glu O GROUP 2: FLAVONOIDS Table 2.3.1: FLAVONES s/n 88 Compound Plant source H. viscosum Biological activity Antimutagenic Reference Geissman, et al., 1967 H. nitens Pesticide, antiviral activity against HSV-1. Inhibitor of PGE2 production. Tomas-Barberan, et al., 1988 OH HO O O O OH O 89 O O O O O 90 H. mundii Bohlmann, et al., 1978b H. herbaceum Bohlmann, et al., 1979c H. herbaceum, H. mimetes Bohlmann, et al., 1979c O O O O 91 O O O HO O O O 92 O O HO O O OH O P a g e 35 | 238 CHAPTER TWO: LITERATURE REVIEW 93 O O H. nitens Bohlmann, et al., 1979c H. herbaceum Bohlmann, et al., 1979c H. herbaceum, H. nitens Tomas-Barberan, et al., 1988 H. mundii Bohlmann, et al., 1978b O O OH O 94 O HO O O O O 95 O O O O O 96 O O O O O O Table 2.3.2: FLAVONOLS s/n 97 Compound OH HO O Plant source H. melanacme, H. bracteatum OH Biological activity Anticarcinogenic, antitumour, Inhibitor of various enzymes, algaecide, antioxidant, antiHIV activity; enhances apoptosis in colon cancer cells Reference Lall, 2006; Liu, et al., 2007 OH OH O OH 98 HO O H. odoratissimum Van Puyvelde, et al., 1989 H. picardii De La Puerta, et al., 1990 OH O OH O 99 HO O O OH O P a g e 36 | 238 CHAPTER TWO: LITERATURE REVIEW 100 H. herbaceum, H. chrysargyrum Bohlmann, et al., 1979c 101 H. nitens Bohlmann, et al., 1979c H. italicum, H. picardii De La Puerta, et al., 1990 H. decumbens TomasLorente, et al., 1989 H. nitens Bohlmann, et al., 1979c H. italicum Wollenweber, 2005 H. chrysargyrum, TomasLorente, et al., 1989 O O O O O 102 O HO O O OH O 103 O O O OH OH O 104 O O O O O O 105 O O O OH OH O 106 O O HO O OH O P a g e 37 | 238 CHAPTER TWO: LITERATURE REVIEW 107 O Van Puyvelde, et al., 1989 H. odoratissimum O O O OH OH O H. nitens 108 O O O O antifungal TomasBarberan, et al., 1988 O O O 109 O HO H. argyrophyllum Jakupovic, et al., 1989b H. cephaloideum Bohlmann, et al., 1980a H. krausii Candy, et al., 1975 O O OH O O 110 O O O O O OH O 111 OH OH HO O O OH O HO HO O OH O O OH P a g e 38 | 238 CHAPTER TWO: LITERATURE REVIEW Table 2.3.3: FLAVANONES s/n Compound Plant source Biological activity Jakupovic, et al., H. stirlingii 112 Reference 1987b O OH O 113 O HO OH H. Iryanthera, H. Strong antimicrobial and Bohlmann & acutatum, H. antibacterial activity, Abraham, 1979c; Jia tenuifolium, H. ketosteroid reductase & Zhao, 2005 cymosum inhibitor H. Larix Ketosteroid reductase Bohlmann & inhibitor, Abraham, 1979a O 114 OH immunostimulant. O Inhibits murine B16 HO melanoma and O 115 mushroom tyrosinase OH H. bracteatum Forkmann, 1983 H. herbaceum. Bohlmann, et al., OH HO O OH OH O 116 HO 1979c O O O H. polycladum 117 O O Inhibits aromatase, Bohlmann, et al., Cytotoxic to human 1980b breast cancer, SF-268, NCI-H460 cells OH O P a g e 39 | 238 CHAPTER TWO: LITERATURE REVIEW 118 HO Bohlmann, et al., H. glaciale O 1980b O OH O 119 OH O H. mimetes Jakupovic, et al., 1986 H. nitens, H. Bohlmann, et al., herbaceum 1979c H. thapsus Bohlmann & Zdero, O O OH O 120 O O O O 121 HO 1983 O OH OH O H. forskahlii 122 Antibacterial activity Al-Rehaily, et al., against Staphylococcus 2008 aureus and Bacillus HO O subtilis OH O H. arthrixiifolium 123 OH HO O Phytoestrogen, cytotoxic, Bohlmann & Ates, antifungal, cancer 1984a; Stevens & chemopreventive, Page, 2004 antiosteoporosis, aromatase inhibitor. OH O 124 H. hypocephalum HO Bohlmann & Abraham, 1979d HO O OH O P a g e 40 | 238 CHAPTER TWO: LITERATURE REVIEW 125 HO O H. hypocephalum, Bohlmann & H. polycladum, H. Abraham, 1979d; rugulosum, H. Bohlmann, et al., thapsus, 1980b; Bohlmann & OH O Zdero, 1983; Bohlmann & Misra, 1984a H. hypocephalum 126 Strong inhibitory effects Bohlmann & on aminopeptidase N Abraham, 1979d activity HO O OH O H. thapsus 127 HO Bohlmann & Zdero, 1983 O OH O 128 HO Bohlmann, 1984b H. rugulosum Bohlmann & Misra, O OH O 129 O 1984a O O O OH 130 O H. rugulosum, O H. athrixiifolium Bohlmann & Ates, 1984a OH O P a g e 41 | 238 CHAPTER TWO: LITERATURE REVIEW 131 O O H. athrixiifolium, Bohlmann & Ates, H. rugulosum 1984a; Bohlmann & Misra, 1984a OH O H. rugulosum 132 Bohlmann & Misra, 1984a O O OH O H. retrorsum 133 Randriaminahy, et al., 1992 O O OH O H. hypocephalum 134 O Bohlmann & Abraham, 1979d O HO OH O 135 O O OH OH OH Jakupovic, et al., 1986 H. lepidissimum Jakupovic, et al., 1986 O OH O 136 O H. lepidissimum HO O O OH O P a g e 42 | 238 CHAPTER TWO: LITERATURE REVIEW Al-Rehaily, et al., H. forskahlii 137 2008 O O HO O O 138 Bohlmann, et al., H. cymosum O O 1979a O OH O Bohlmann & Zdero, H. thapsus 139 HO 1983 O OAc OH 140 O OH OH O Yong, et al., 2011 H. arenarium O O OH O H. gymnocomum 141 O 142 O OH HO Glu Drewes & van Vuuren, 2008 O O Antimicrobial activity H. arenarium Yong, et al., 2011 O O O P a g e 43 | 238 CHAPTER TWO: LITERATURE REVIEW 143 OH HO Glu Yong, et al., 2011 H. arenarium Morikawa, et al., O O O 144 Glu O HO Glu H. arenarium 2009a O O O 145 OH HO Morikawa, et al., H. arenarium 2009a O Lactose O O 146 OH Morikawa, et al., H. arenarium 2009a O HO O O Rhamose Table 2.3.4: CHALCONES s/n 147 Compound HO OH Plant source H. acucatum, H. kraussii, H. oreophilum, H. cymosum Biological activity antimutagenic, inhibits recombinant PTP1B, mushroom tyrosinase Reference Bohlmann & Abraham, 1979c H. bracteatum Protein tyrosine phosphatase, antioxidant, antidepressant, potent 5lipoxygenase inhibitor Liu, et al., 2007 OH O 148 OH HO OH OH OH O P a g e 44 | 238 CHAPTER TWO: LITERATURE REVIEW 149 OH O Antileishmaniai, antiproliferative Van Puyvelde, et al., 1989 OH OH O 150 H. odoratissimum, H. heterolasium O Bohlmann, et al., 1978b H. sutherlandii, OH O O O H. rugulosum, 151 O Exhibits weak antineoplastic activity against sarcoma 180 Bohlmann & Misra, 1984a OH O Bohlmann, et al., 1979a H. cymosum, H. tenuiculum 152 O OH OH 153 HO H. athrixiifolium OH Antiviral and antituberculosis Bohlmann & Ates, 1984a; Bohlmann & Misra, 1984a OH 154 HO H. polycladum Bohlmann, et al., 1980b H. umbraculigerum, H. retrorsum Bohlmann & Hoffmann, 1979b; Randriaminahy, et al., 1992 OH O OH O 155 HO OH OH O P a g e 45 | 238 CHAPTER TWO: LITERATURE REVIEW 156 O H. rugulosum Bohlmann, 1984b H. athrixiifolium, H. rugulosum Bohlmann & Ates, 1984a H. rugulosum Bohlmann & Misra, 1984a H. krausii, H. melanacme Jakupovic, et al., 1989b H. forskahlii Al-Rehaily, et al., 2008 H. aphelexioides Randriaminahy, et al., 1992 H. cymosum Bohlmann, et al., 1979a OH O 157 O OH OH O 158 O O OH O 159 O OH HO OH O 160 HO OH O O 161 HO OH O HO O 162 O HO OH O O P a g e 46 | 238 CHAPTER TWO: LITERATURE REVIEW 163 HO H. aphelexioides Randriaminahy, et al., 1992 H. teretifolium Popoola, et al., 2015 H. glomeratum Bohlmann & Suwita, 1979b H. mundii; H. polycladum Bohlmann, et al., 1978b H. sutherlandii Bohlmann, et al., 1978b; 1980b H. gymnocomum Drewes & van Vuuren, 2008 H. bracteatum Rimpler & Haensel, 1965 O O O HO 164 O O OH O 165 OH O OH O O 166 O O O O O 167 O O O O O O 168 O OH O O OH 169 OH HO OGlu OH OH O P a g e 47 | 238 CHAPTER TWO: LITERATURE REVIEW OH 170 HO OH O OH H. cooperi Wright, 1976 OH H. arenarium Morikawa, et al., 2009a OH OGlu O 172 Rimpler & Haensel, 1965 OH OGlu O 171 H. bracteatum HO OH O Lactose O Table 2.3.5: DIHYDROCHALCONES s/n Compound Plant source H. tenuifolium 173 HO Reference Bohlmann & Abraham, 1979a OH OH O 174 Biological activity HO H. forskahlii Al-Rehaily, et al., 2008 H. argyrolepis Bohlmann, et al., 1984a OH O 175 OH O HO OH OH O P a g e 48 | 238 CHAPTER TWO: LITERATURE REVIEW 176 HO H. aphelexioides Randriaminahy, et al., 1992 H. monticola Jakupovic, et al., 1989b H. monticola Bohlmann & Zdero, 1980c H. polycladum Bohlmann, et al., 1980b H. forskahlii Jakupovic, et al., 1990 OH MeO O 177 OH OH HO OH O O 178 OH HO OH HO OH O 179 HO OH OH O 180 HO OH HO O O P a g e 49 | 238 CHAPTER TWO: LITERATURE REVIEW 181 H. polycladum, H. cymosum (Bohlmann, et al., 1979a; 1980b H. polycladum, H. tenuiculum Bohlmann, et al., 1979a; 1984a H. polycladum Bohlmann, et al., 1980b OH HO MeO O O 182 HO OH O 183 O O OH OH O H. forskahlii 184 O Antibacterial activity Al-Rehaily, et al., 2008 OH HO O O H. sutherlandii 185 Bohlmann, et al., 1978b O O O O O P a g e 50 | 238 CHAPTER TWO: LITERATURE REVIEW Table 2.4.1: PHLOROGLUCINOLS s/n Compound Plant source 186 Biological activity H. callicomum Bohlmann & Abraham, 1979b H. candolleanum, H. oreophilum Jakupovic, et al., 1989b O HO Reference OH OH 187 O OH HO OH H. caespititium. 188 Antimicrobial Dekker, et al., 1983 O HO OH OH 189 HO OH H. gymnoconum, H. bellum OH O H. paronychioides 190 HO O OH O 191 O HO Bohlmann & Mahanta, 1979; Bohlmann & Zdero, 1979; Bohlmann & Suwita, 1979b; Jakupovic, et al., 1986 Mutanyatta-Comar, et al., 2006 H. gymnocomum Bohlmann & Mahanta, 1979; Drewes & van Vuuren, 2008 OH OH OH P a g e 51 | 238 CHAPTER TWO: LITERATURE REVIEW 192 O HO H. mimetes Jakupovic, et al., 1986 H. natalitium, H. ballum, H. stoechas Bohlmann & Zdero, 1979; Rios, et al., 1991 OH OH 193 HO OH OH O H. natalitium, H. ballum, H. stoechas 194 HO OH Antiinflammatory Bohlmann & Zdero, 1979; Bohlmann, et al., 1980b; Rios, et al., 1991 OH O 195 O HO H. caespititium Jakupovic, et al., 1986 H. gymnocomum Bohlmann & Mahanta, 1979 H. infaustum Bohlmann & Suwita, 1979b H. gymnocomum Drewes & van Vuuren, 2008 OH OH OAC 196 HO O O OH 197 O HO OH OH 198 O OH OH O Glu O 199 HO OH Helichrysum sp. O P a g e 52 | 238 CHAPTER TWO: LITERATURE REVIEW Randriaminahy, et al., 1992 H. triplinerve 200 O OH O OH H. spathulatum 201 HO Cytotoxic O O Randriaminahy, et al., 1992 OH 202 HO H. triplinerve Randriaminahy, et al., 1992 H. monticola Bohlmann & Suwita, 1979b; Bohlmann & Zdero, 1980c H. arenarium, H. italicum Appendino, et al., 2007 O O O 203 HO OH O OH 204 OH HO O OH OH O O 205 OH HO O OH OH H. arenarium, H. italicum var. microphyllum Anti-HIV-1, antibacterial antiinflammatory, antioxidant, Taglialatela-Scafati, et al., 2013 O O OH 206 HO O OMe OH H. auriceps H. odoratissimum Bohlmann & Zdero, 1980a; Haensel, et al., 1980 O O P a g e 53 | 238 CHAPTER TWO: LITERATURE REVIEW OH 207 HO OH H. decumbens O OH Antifungal activity and fairly weak activity against Grampositive bacteria Tomas-Lorente, et al., 1989; Tomas-Lorente, et al., 1990 Antifungal Tomas-Lorente, et al., 1989; Tomas-Lorente, et al., 1990 H. cameroonense Antialgae Antoine, et al., 2010 H. decumbens Antifungal Tomas-Lorente, et al., 1989; 1990 O O OH 208 OH H. decumbens HO O OH O O 209 O O O OGlu O OH OH OH OH 210 HO O OH O O Bohlmann & Zdero, 1980c H. monticola 211 O OH OH O 212 OH O OH H. plicatum H. stoechas HO OHO O Shows antibacterial activity Haensel, et al., 1980; Rios, et al., 1991 O 213 H. diosmifolium Jakupovic, et al., 1987b H. cephaloideum Jakupovic, et al., 1986 OH OH O 214 O O O HO O OH OH O O O P a g e 54 | 238 CHAPTER TWO: LITERATURE REVIEW 215 Bohlmann & Zdero, 1980a H. auriceps O O O O O AcO 216 HO OH O OAc O H. italicum Haensel, et al., 1980 OH O O HO O OH O Table 2.5: BENZOPYRONE s/n Compound Plant source 217 Biological activity Reference H. stoechas Garcia de Quesda, et al., 1972 H. platypterum Jakupovic, et al., 1986 H. platypterum Jakupovic, et al., 1986 H. platypterum Jakupovic, et al., 1986 O OH O O 218 OH HO O O 219 OH O O OH 220 HO O O P a g e 55 | 238 CHAPTER TWO: LITERATURE REVIEW O 221 OH H. platypterum Jakupovic, et al., 1986 H. platypterum Jakupovic, et al., 1989b H. cerastioides Bohlmann, et al., 1984a H. mixtum Jakupovic, et al., 1986 H. mixtum Jakupovic, et al., 1986 H. auriceps Bohlmann & Zdero, 1980a OH O O 222 O HO O O 223 OH O HO O OH O O 224 OH O HO O OH O O 225 OH O OH O HO O O 226 OH O OH O O OH O P a g e 56 | 238 CHAPTER TWO: LITERATURE REVIEW Table 2.6: COUMARINS s/n 227 Compound Plant source H. zeyheri O Biological activity Reference Jakupovic, et al., 1986 O OH 228 Appendino, et al., 2007 H. italicum var. microphyllum O O O H. arenarium; H. stoechas 229 OH O O O OH O O OH O H. arenarium; H. stoechas Vrkoc, et al., 1975 H. arenarium Rios, et al., 1970 H. diosmifolium Bohlmann & Zder, 1980b H. diosmifolium Bohlmann & Zder, 1980b O O O O O OH O O 233 O Haensel, et al., 1980 OH COOH 232 H. italicum; H.stoechas OH 231 O Vrkoc, et al., 1975 O 230 O Antibacterial OH O O O O OH 234 O O O O O O P a g e 57 | 238 CHAPTER TWO: LITERATURE REVIEW Bohlmann & Zder, 1980b ` H. serpyllifolium 235 O O O O OH Table 2.7: BENZOFURAN s/n Compound Plant source H. arenarium 236 OH O O Biological activity Antibacterial and antiinflammatory agent Reference Vrkoc, et al., 1973 OH 237 H. arenarium, H. polyphyllum Taglialatela-Scafati, et al., 2013 H. italicum Taglialatela-Scafati, et al., 2013 O O O 238 OH O O O O H. stoechas 239 O Insecticidal Rios, et al., 1991; ElDahmy, 1993 O O 240 H. argrophyllum Bohlmann, et al., 1984a H. italicum Haensel, et al., 1980 OH O O OH 241 O OH O O O P a g e 58 | 238 CHAPTER TWO: LITERATURE REVIEW O 242 H. acuminatum Jakupovic, et al., 1987b H. italicum and H. stoechas Sala, et al., 2001 H. platypterum Jakupovic, et al., 1987a O O O O O 243 O O 244 Benzofurane part, move O O O OH 245 O O H. italicum Antioxidant Rigano, et al., 2014 H. italicum Ant inflammation Sala, et al., 2001; Daniel, et al., 2014 O OH O O 246 O Glu O O 247 Glu O HO H. arenarium Lv, et al., 2009; Zhang, et al., 2009 H. arenarium Eshbakova & Aisa, 2009 H. arenarium Eshbakova & Aisa, 2009 O O O 248 Glu O O OH O O 249 O Glu O O O P a g e 59 | 238 CHAPTER TWO: LITERATURE REVIEW Eshbakova & Aisa, 2009 H. arenarium 250 Lactose O O O O Table 2.8: CHROMONES s/n Compound Plant source 251 O O Reference H. paronychioides Mutanyatta-Comar, et al., 2006 H. spp. Bohlmann, et al., 1984a Helichrysum spp. Bohlmann, et al., 1984a OH OH 252 O O OH OH 253 HO Biological activity O O 254 OH OH HO O O H. italicum, H. stoechas Antibacterial Haensel, et al., 1980; Rios, et al., 1991 O OH OH O P a g e 60 | 238 CHAPTER TWO: LITERATURE REVIEW Table 2.9: ACETYLENE DERIVATIVES s/n Compound Plant source 255 256 Biological activity H. vestitum Bohlmann & Zder, 1980b H. aureonitens Ziaratnia, et al., 2009 H. coriaceum Bohlmann, et al., 1984 OH 257 Cl O 258 Cl O Cl Bohlmann & Zedro, 1973 H. bracteatum 259 H. vestitum Bohlmann & Zder, 1980b H. adenocarpum Bohlmann, et al., 1980b H. panduratum Bohlmann & Abraham, 1979a O HO Reference O 260 O O OMe 261 HO Cl S O Table 2.10: QUINONES s/n Compound Plant source H. paronychioides 262 HO O Biological activity Reference Mutanyatta-Comar, et al., 2006 OH O P a g e 61 | 238 CHAPTER TWO: LITERATURE REVIEW 263 H. nudifolium Jakupovic, et al., 1986 H. nudifolium Jakupovic, et al., 1986 H. nudifolium Jakupovic, et al., 1986 O O HO OH O 264 O O O O 265 OH O O O O Table 2.11: MISCELLANEOUS s/n 266 Compound Plant source H. italicum, O HO 267 O Biological activity Antifungal, antiseptic, dental analgesic. Reference H. arenarium Zhang, et al., 2009 H. mundii Bohlmann, et al., 1978b OH O O OH 268 O HO O O OH O P a g e 62 | 238 CHAPTER TWO: LITERATURE REVIEW H. umbraculigerum 269 HO Bohlmann & Hoffmann, 1979b OH 270 O HO H. umbraculigerum Bohlmann & Hoffmann, 1979b OH OH 271 H. aureonitens. Bohlmann & Ziesche, 1979 H. acuminatum Jakupovic, et al., 1987b H. bracteatum Bohlmann & Zedro, 1973 H. bracteatum Powell, et al., 1965 H. bracteatum Conacher & Gunstone, 1970 H. umbraculigerum Bohlmann & Hoffmann, 1979b HO O 272 O O 273 O O O 274 HOOC OH 275 O 276 COOH OH COOH OH P a g e 63 | 238 CHAPTER TWO: LITERATURE REVIEW O 277 H. umbraculigerum Bohlmann & Hoffmann, 1979b H. umbraculigerum Bohlmann & Hoffmann, 1979b H. umbraculigerum Bohlmann & Hoffmann, 1979b H. umbraculigerum (Bohlmann & Hoffmann, 1979b) O HO COOH 278 O O HO COOH 279 O O HO COOH 280 COOH HO OH 281 COOH HO OH OH P a g e 64 | 238 CHAPTER TWO: LITERATURE REVIEW O O O O O HO C19H40CH3 O HO NH OH (CH2)6 (CH2)8CH3 OH O C6H12C=CC8H17 O HO S Cl HO O S Cl 287 H. acuminatum Jakupovic, et al., 1987c H. arenarium Morikawa, et al., 2009b OH OH O O O O OH 288 O Glu H. arenarium 289 O Glu O O P a g e 65 | 238 CHAPTER TWO: LITERATURE REVIEW 290 O H. italicum Sala, et al., 2001 H. platypterum Jakupovic, et al., 1987a OH Glu O 291 O O O OH Hansel, et al., 1960 292 HO O HO OH O OH O Glu 293 O Glu Wang, et al., 2009 H. arenarium Morikawa, et al., 2009b H. arenarium Morikawa, et al., 2009b H. arenarium Morikawa, et al., 2009b H. arenarium Morikawa, et al., 2009b OH HO OH 294 O Lactose H. arenarium O 295 COOH O OH O OGlu HO 296 OH Lactose 297 O HO O Lactose P a g e 66 | 238 CHAPTER TWO: LITERATURE REVIEW 298 H. arenarium Wang, et al., 2009 H. arenarium Morikawa, et al., 2009b OH O HO HO O O O O Glu 299 OH O 300 OH OGlc O HO H. zivojinii Anticancer Aljancic, et al., 2014 H. zivojinii Anticancer Aljancic, et al., 2014 H. italicum Anti-biofilm D'Abrosca, et al., 2013 H. italicum Anti-biofilm D'Abrosca, et al., 2013 OGlc OH OH O HO HO OGlc O 301 HO OH OH OH OH CH2 HO OGlc O O O O OR OH O OH O R = 3-hydroxy-3-methyl)glutaryl 302 303 304 R = malonyl MeO manonyl O O O HO OH O O OH P a g e 67 | 238 CHAPTER TWO: LITERATURE REVIEW Me 305 Glu O H. italicum Anti-biofilm D'Abrosca, et al., 2013 OH 306 H. italicum TaglialatelaScafati, et al., 2013 H. italicum TaglialatelaScafati, et al., 2013 H. italicum TaglialatelaScafati, et al., 2013 H. italicum TaglialatelaScafati, et al., 2013 OH O C10H23 O O O 307 OH O O O O 308 O OC11H23 O O O 309 O O O C11H23 O O O *H: Helichrysum; Glu/Glc: glucose; Me: methyl; Ac: acetate P a g e 68 | 238 CHAPTER TWO: REFERENCES REFERENCES Aiyegoro, O.A., Afolayan, A.J. and Okoh, A.I. (2010). Interactions of antibiotics and extracts of Helichrysum pedunculatum against bacteria implicated in wound infections. Folia Microbiologica, 55(2), pp. 176-180. Aiyegoro, O.A. and Okoh, A.I. (2009). Phytochemical screening and polyphenolic antioxidant activity of aqueous crude leaf extract of Helichrysum pedunculatum. International Journal of Molecular Sciences, 10(11), pp. 4990-5001. Aiyegoro, O.A. and Okoh, A.I. (2010). Preliminary phytochemical screening and in vitro antioxidant activities of the aqueous extract of Helichrysum longifolium DC. BMC Complementary and Alternative Medicine, 10(21), pp 1-8. Albayrak, S., Aksoy, A., Sagdic, O. and Budak, U. (2010). Phenolic compounds and antioxidant and antimicrobial properties of Helichrysum species collected from eastern Anatolia, Turkey. Turkish Journal of Biology, 34(4), pp. 463-473. Aljancic, I.S., Vuckovic, I., Jadranin, M., Pesic, M., Djordjevic, I., Podolski-Renic, A., Stojkovic, S., Menkovic, N., Vajs, V.E. and Milosavljevic, S.M. (2014). Two structurally distinct chalcone dimers from Helichrysum zivojinii and their activities in cancer cell lines. Phytochemistry, 98, pp. 190-196. Al-Rehaily, A.J., Albishi, O.A., El-Olemy, M.M. and Mossa, J.S. (2008). Flavonoids and terpenoids from Helichrysum forskahlii. Phytochemistry, 69(9), pp. 1910-1914. Antoine, K.Z., Hussain, H., Dongo, E., Kouam, S.F., Schulz, B. and Krohn, K. (2010). Cameroonemide A: a new ceramide from Helichrysum cameroonense. Journal of Asian Natural Products Research, 12(7), pp. 629-633. Antoine, K.Z., Hussain, H., Dongo, E., Krohn, K. and Schulz, B. (2011). Cameroonenoside A: a new antialgal phenolic glycoside from Helichrysum cameroonense. Records of Natural Products, 5(4), pp. 305-308. P a g e 69 | 238 CHAPTER TWO: REFERENCES Appendino, G., Ottino, M., Marquez, N., Bianchi, F., Giana, A., Ballero, M., Sterner, O., Fiebich, B.L. and Munoz, E. (2007). Arzanol, an Anti-inflammatory and Anti-HIV-1 Phloroglucinol α-Pyrone from Helichrysum italicum ssp. microphyllum. Journal of Natural Products, 70(4), pp. 608-612. Asekun, O.T., Grierson, D.S. and Afolayan, A.J. (2007). Characterization of essential oils from Helichrysum odoratissimum using different drying methods. Journal of Applied Sciences, 7(7), pp. 1005-1008. Barrero, A.F., Arteaga, P. and Herrador, M.M. (1998). Ent-Kaurene diterpenoids from Helichrysum foetidum. Fitoterapia, 69(1), pp. 83-84. Barroso, M.R., Barros, L., Duenas, M., Carvalho, A.M., Santos-Buelga, C., Fernandes, I.P., Barreiro, M.F. and Ferreira, I. (2014). Exploring the antioxidant potential of Helichrysum stoechas (L.) 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P a g e 81 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM CHAPTER THREE CHEMICAL AND BIOLOGICAL CHARACTERIZATION OF HELICHRYSUM TERETIFOLIUM CONSTITUENTS 3.1 Abstract Ten (10) flavonoid-related structures viz heliteretifolin (C1), isoxanthohumol (C2), 2`,4`,6`trihydroxy-3`-prenylchalcone (C3), isoglabranin (C4), glabranin (C5), 7-methoxy-isoglabranin (C6), quercetin (C7), 4`-methoxyquercetin (C8), 4`-methoxykaempferol (C9) and mosloflavone (C10) were isolated and identified from H. teretifolium methanol extract. One of them (C1) was reported for the first time from a natural source, while compounds C6, C8-C10 were isolated for the first time from Helichrysum genus. Compound C3 possess moderate biological activity compared to C2 when tested for total antioxidant capacity, displaying some of the highest TEAC values (4529.01 ± 2.44; 4170.66 ± 6.72) µM/TEg, respectively. Compounds C7 & C8 demonstrated the highest inhibitory activities against Fe2+-induced lipid peroxidation (IC50= 2.931; 6.449 g/mL); tyrosinase (8.092; 27.573 g/mL) and elastase (43.342; 86.548 g/mL). Additionally, the total antioxidant capacities measured as FRAP (4816.31 ± 7.42; 3584.17 ± 0.54) µMAAE/g, and ORAC for hydroxyl radical (7.265 ± 0.71; 6.779 ± 3.40) X 106 and peroxyl radical (17.836 ± 2.90; 12.545 ± 5.07) X 103 µMTE/g were also observed for compounds C7 & C8, respectively. The H. teretifolium total extract presents a rich source of bioactive constituents with potent antioxidant and moderate anti-tyrosinase and anti-elastase activities that can help to avert accumulation of free radicals in the body, and could therefore be good candidates for the P a g e 82 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM prevention and/or treatment of skin-related conditions, such as aging. This is the first scientific report on the chemical and biological profile of H. teretifolium. Keywords: Helichrysum teretifolium; Flavonoid; Antioxidant; Anti-tyrosinase; Skin aging. 3.2 Background information on Helichrysum teretifolium Plants have long been used in the cosmetic industry, as amongst others as tyrosinase inhibitors which have become increasingly important to prevent hyperpigmentation through the inhibition of enzymatic oxidation (Chang, 2009; Moon, et al., 2010). Since neutralization of free radicals usually comes from phenolic compounds such as flavonoids, plants rich in phenolic like Helichrysum genus can also contribute. Helichrysum teretifolium (L.) D. Don (Asteraceae) is a straggling subshrub up to 300 mm tall with cream colored bracts, occasionally tinged pink flowers, widely distributed along the coast of South Africa, often on sandy flats and mountain slopes from western Cape to the eastern Cape, thence to Kwazulu-Natal (Goldblatt & Manning, 2000). The plants are called “Everlastings” because the flowers dry well and retain their shape and color for many years. Traditionally, many people believe that this shrub has magical properties and can be used to protect a house from lighting strikes. Apart from the “magical power” of this plant, there is no scientific work to ascertain its medicinal value unlike other plants of the same family. Our initial motivation for the medicinal possibilities of this plant was attributed to the biological and pharmacological importance of other South African Helichrysum species of the same family. Among such is the extensive usage of Helichrysum species in the treatment of respiratory diseases and wound dressing, antiinflammatory, anti-bacterial, antidiuretic, antidiabetic, and other skin conditions. Hence, the genus P a g e 83 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM generally finds large application in the cosmetic and pharmaceutical field (Lourens, et al., 2008; Kolayli, et al., 2010; Tirillini, et al., 2013; Viegas, et al., 2014; Mari, et al., 2014). A B Western Cape region Figure 3.1: H. teretifolium description (A), and distribution along South African Coastal area (B) http://keys.lucidcentral.org This chapter describes the:  Isolation of bioactive constituents present in a methanol extract of H. teretifolium, using chromatographic methods.  Identification of isolated constituents using different spectroscopic techniques.  Investigation of total antioxidant capacities of the isolated constituents.  Determination of inhibitory activities of the H. teretifolium constituents against skin diseases related enzymes.  Structure-activity relationship (mechanism of action) of the H. teretifolium bioactive constituents responsible for specific biological activity. P a g e 84 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM CHEMICAL CHARACTERIZATION TERETIFOLIUM CONSTITUENTS OF HELICHRYSUM 3.3 General experimental procedures 3.3.1 Reagents and solvents Organic solvents of methanol, acetonitrile (HPLC graded), ethanol, ethyl acetate, dichloromethane, n-hexane (redistilled), vanillin, deuterated chloroform and acetone were supplied by Merck (Darmstadt, Germany). Sulphuric acid and acetic acid were secured from Kimix (Cape Town, South Africa). 3.3.2 Chromatography 3.3.2.1 Thin layer chromatography (TLC) Pre-coated plates of silica gel 60 F254 (Merck, Germany) was used for TLC analysis. Visualization of TLC plates was done by observing the bands “spots” after development under UV at λ254 nm and λ366 nm using UV lamp (CAMAG, Switzerland), followed by spraying with the vanillin/sulphuric acid reagent (Wagner, et al., 1984). Chemical profiles of the fractions were identified based on the colour produced after viewing under UV and then spraying with the spray detecting reagent (vanillin/sulphuric). Unless otherwise stated, the solvent systems generally used for the TLC development of H. teretifolium fractions are indicated in Table 3.1 P a g e 85 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM Table 3.1: TLC solvent system Solvent system Ratio Assigned code Hexane - ethylacetate 9:1 A Hexane – ethylacetate 7:3 B DCM - methanol 95:5 C DCM - methanol 90:10 D 3.3.2.2 Column chromatography Column chromatography were performed using silica gel 60 H (0.040 − 0.063 mm particle size, Merck, South Africa) and sephadex LH-20 (Sigma-Aldrich, South Africa) as stationary phases, supported with glass column of different diameters. 3.3.2.3 High Pressure Liquid Chromatography (HPLC) Sample purification was carried out using Agilent Technologies 1200 series, equipped with UV detector, manual injector, quaternary pump (G1311A), vacuum degasser (G1322A), column compartment (G1316A) (all experiments were done at room temperature) and reversed phase C18 column SUPELCO (25 x 2.1 cm, 5 μm). The flow rate was set at 1.5 mL/min. 3.3.3 Spectroscopy 3.3.3.1 Nuclear magnetic resonance (NMR) spectroscopy NMR spectra were recorded at 25 oC, using deuterated chloroform and acetone as solvents, on a Bruker Avance 400 MHz NMR spectrometer (Germany). Chemical shifts of 1H (δH) and 13C (δC) in ppm were determined relative to tetramethylsilane as internal reference. P a g e 86 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM 3.3.3.2 Mass spectroscopy (MS) High resolution mass spectroscopy (HRMS) analysis was conducted on a Dionex Ultimate (Sunnyvale, CA) 3000 LC coupled to Bruker QTOF with electrospray ionization (ESI) interface working in the positive mode. 3.3.3.3 Infrared (IR) spectroscopy Attenuated total internal reflectance FTIR measurements were carried out using Spectrum 100 (Perkin Elmer Corp.). Spectra recording were accomplished using the interface “Spectrum”. Dicholoromethane was used to dissolve the samples. 3.3.3.4 Ultra violet (UV) spectroscopy UV Nicolet Evolution (EV-100 ver. 4.60) spectrophotometer (Therma Electron Corporation, Madison, USA) was used for measurement of absorbance maxima between the wavelenghts 200450 nm. 3.4 Collection and identification of plant material The plant material (Helichrysum teretifolium) was collected in October 2012, from Jonkershoek (about 9 km SE Stellenbosch) nature reserve, Western Cape, South Africa. The voucher specie was identified by Dr. Christopher Cupido (SANBI, Kirstenbosch), and a copy has been deposited at the Compton Herbarium, South African National Biodiversity Institute, Kirstenbosch, South Africa, with herbarium number NBG145880. P a g e 87 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM 3.5 Extraction and fractionation of total extract The plant material (stem, leave and flower; 450 g) was air dried at room temperature, blended and extracted with methanol (2.5 L) at room temperature (25 oC) for 48 hours. Methanol extract was then evaporated till dryness using a rotary evaporator at 40 oC to yield 16 g (3.556 %). A portion of the total extract of H. teretifolium (15 g) was applied to a silica gel column (30 x 18 cm) and eluted using a gradient of hexane and ethyl acetate in the following order of increasing polarity as indicated in Table 3.2. Seventy four (74) fractions (250 mL each) were collected during the process and numbered 1 -74. P a g e 88 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM Table 3.2: Fractionation of the methanol extract of H. teretifolium Solvent system Hexane Solvent volume Fraction collected 1L 1-4 Hexane - ethylacetate 95:5 1L 5-8 Hexane – ethylacetate 90:10 1L 9-12 Hexane – ethylacetate 85:15 1L 13-16 Hexane – ethylacetate 80:20 1L 17-20 Hexane – ethylacetate 75:25 1L 21-24 Hexane – ethylacetate 70:30 1L 25-28 Hexane – ethylacetate 65:35 1L 29-32 Hexane – ethylacetate 60:40 1L 33-36 Hexane – ethylacetate 55-45 1L 37-40 Hexane – ethylacetate 50:50 1L 41-44 Hexane – ethylacetate 40:60 1L 45-48 Hexane – ethylacetate 20:80 1L 49-52 Hexane – ethylacetate 10:90 1L 53-56 Ethylacetate 1L 57-60 Ethylacetate - methanol 95:5 1L 61-64 Ethylacetate – methanol 90:10 1 L 65-68 Ethylacetate – methanol 50:50 1 L 69-72 Methanol 500 mL 73,74 P a g e 89 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM The collected fractions (1-74) were concentrated in vacuo and combined according to their TLC (using solvent systems A - D) profiles to yield 28 main fractions (Fig. 3.2). The main fractions obtained were coded by roman numbers (I – XXVIII) and the results ae summarized in Table 3.3. The general scheme of the fractionation of the total extract is given in Scheme 3.1. Table 3.3: Main fractions obtained upon fractionation of the total extract of H. teretifolium Combined fraction 3-6 Designated number I Combined fraction 22 Designated number XI Combined fraction 42, 43 Designated number XXI 7, 8 II 23-26 XII 44 XXII 9, 10 III 27-29 XIII 45, 46 XXIII 11 IV 30, 31 XIV 47-51 XXIV 12 V 32 XV 52-57 XXV 13 VI 33 XVI 58-62 XXVI 14 VII 34 XVII 63 XXVII 15 VIII 35, 36 XVIII 64-74 XXVIII 16-18 IX 37 XIX - - 19-21 X 38-41 XX - - P a g e 90 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM A C B D 3.6 Isolation of constituents (pure compounds) 3.6.1 Isolation of compound C9 - Column chromatography of main fraction XX Main fraction XX (1.4 g) was rechromatographed on silica gel using gradient elution of hex/EtOAc (1 L, 9:1; 1 L, 7:3; & 1 L, 100%). 100 mL each of the fraction were collected, and evaporated using rotary evaporator. Fractions obtained were developed on TLC (using solvent system C) and fraction displayed same profiles were combined as indicated in Table 3.4. P a g e 91 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM Table 3.4: Fractions grouped from the column Fraction Weight Assigned number 3-6 211 mg XXA 7-16 309 mg XXB 17-24 34 mg XXC 25-30 97 mg XXD The chromatography of sub fraction XXD (85 mg) on sephadex column (5 % aqeuous ethanol, TLC solvent system C) afforded a pure compound labelled XXD3 (C9, 32 mg, 0.0071 %, Fig. 3.3A). The purity of C9 was further confirmed by direct injection to the HPLC, and eluted using gradient solvent system of ACN and de-ionized water (90:10 to 100% ACN in 30 mins), as indicated in Figure 3.3B. A XXD3 B XXD3 XXA XXB XXC XXD FR Figure 3.3: TLC chromatogram of XXD3 (Fig. 3.3A) and HPLC spectrum of C9 (Fig. 3.3B) *Conditions Solvent Column Flow rate Detection ACN:DIW 90:10 to 100 % in 30 min SUPELCO, RP-18 (25 X 2.1 cm) 1.5 mL/min UV at 254 nm P a g e 92 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM 3.6.2 Isolation of compounds C1 & C4 - Column chromatography of main fraction III Main fraction III (466 mg) was chromatographed on silica gel using gradient elution of hex/EtOAc (2 L, 95:5; 500 mL, 90:10; 500 mL, 1:1; & 500 mL, 100% ethylacetate). 100 mL each of the fraction were collected, and evaporated using rotary evaporator. Fractions obtained were developed on TLC (using solvent system A) and fraction displayed same profiles were combined as indicated in Table 3.5. Table 3.5: Sub fractions from III Fraction Weight Designated number 6-8 68 mg IIIA 19-21 53 mg IIIB 22-30 64 mg IIIC Injection of sub fraction IIIA (60 mg) to the HPLC and eluted using gradient solvent system of ACN and de-ionized water (90:10 to 100% ACN in 30 mins) afforded two prominent peaks (Fig. not shown), collected and labelled as IIIA-HPLC-2 (C4, Rt 15 min, 19 mg, 0.0042 %), and IIIAHPLC-5 (C1, Rt 23 min, 15 mg, 0.0033 %). 3.6.3 Isolation of compounds C5 & C6 - Column chromatography of main fraction X Main fraction X (450 mg) was chromatographed on sephadex column (using isocratic elution of 5 % aqeuous ethanol). Fractions of 100 mL each was collected from the column. The TLC (using solvent system B) profile of the fractions resulted into four sub fractions (X1-X4) and the result is indicated in Table 3.6 below. P a g e 93 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM Table 3.6: Sub fractions from X Fraction Weight Designated number 4-9 19 mg X1 11-15 26 mg X2 17-21 52 mg X3 22-29 33.2 mg X4 Sub fraction X3 (35 mg) was injected to the HPLC and eluted using gradient solvent system of ACN and de-ionized water (70:30 to 90 % ACN in 20 min, then 100 % for 20 min). One prominent peak was collected and labelled as X3-HPLC-3 (C5, Rt 27 min, 15 mg, 0.0033 %). Sub fraction X4 (30 mg) was injected to the HPLC and eluted using gradient solvent system of ACN and deionized water (70:30 to 90 % ACN in 20 min, then 100 % for 20 min). The prominent peak collected was labelled as X4-HPLC-2 (C6 , Rt 32 min, 17 mg, 0.0038 %). 3.6.4 Isolation of compounds C2 & C10 - Column chromatography of main fraction XII Main fraction XII (505 mg) was chromatographed on sephadex column (5 % aqeuous ethanol). Sub fractions (50 mL) collected were combined as indicated in Table 3.7, and developed on TLC using solvent system C as indicated in Figure 3.4 P a g e 94 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM Table 3.7: Sub fractions from XII Fraction Weight Designated number 10-16 34 mg XIIA 17-20 115 mg XIIB 21-26 87 mg XIIC 27-29 36 mg XIID 30 24 mg XIIE XIIC XIIA XIIB XIIC XIID XIIE Figure 3.4: Chromatogram of sub fraction XII Sub fraction XIIC (73 mg) was injected to the HPLC and eluted using gradient solvent system of ACN and de-ionized water (80:20 to 95% ACN in 30 min, then 100 % for 20 min). Two promient peaks were collected, labelled as XIIC-HPLC-1 (C10, Rt 16, 13 mg, 0.0028 %), and XIIC-HPLC2 (C2, Rt 30.5, 14.8 mg, 0.0033 %). P a g e 95 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM 3.6.5 Isolation of compounds C7 & C8 - Column chromatography of main fraction XXIII Fraction XXIII (170 mg) was chromatographed on sephadex using isocratic 10 % aqueous ethanol, the collected fractions (50 mL each) were combined according to their TLC (using solvent system C & D) characteristics to yield sub fractions XXIIIA – XXIIIC (Table 3.8). Table 3.8: Sub fractions from XXIII Fraction Weight Designated number 9-20 83 mg XXIIIA 21-48 25 mg XXIIIB 49-58 34 mg XXIIIC The precipitate (73 mg) of sub fraction XXIIIA was dissolved in methanol, injected to the HPLC and eluted using gradient solvent system of ACN and de-ionized water (50:50 to 60% ACN in 5 min, then 60 % for 20 min, followed by 100 % for 3 min). Two prominent peaks (Fig. 3.5) were collected and labelled as XXIIIAppt-HPLC-1 (C7, Rt 22, 24 mg, 0.0053 %), and XXIIIAppt-HPLC2 (C8, Rt 24.5, 19 mg, 0.0042 %). P a g e 96 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM XXIIIpptHPLC-1 XXIIIpptHPLC-2 Figure 3.5: HPLC spectrum of compounds C7 and C8 *conditions Solvent Column Flow rate Detection ACN:DIW 50:50 to 60 % in 25 min, then to 100 % in 3 min SUPELCO, RP-18 (25 X 2.1 cm) 1.5 mL/min UV at 254 nm 3.6.6 Isolation of compound C3 - Column chromatography of main fraction XVIII Fraction XVIII (270 mg) was chromatographed on sephadex using isocratic 5 % aqueous ethanol, the collected fractions (50 mL each) were combined according to their TLC (using solvent system C) profiles to yield 5 sub fractions (Table 3.9). Table 3.9: Sub fractions from XVIII Fraction Weight Designated number 19-25 14.8 mg XVIIIA 26-30 27 mg XVIIIB 31-36 44 mg XVIIIC 37-44 74 mg XVIIID 45-48 20.2 mg XVIIIE P a g e 97 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM Sub fraction XVIIID (63 mg) was injected to the HPLC and eluted using gradient solvent system of ACN and de-ionized water (90:10 to 100% ACN in 30 min)]. One prominent peak (Fig. 3.6) was collected and labelled as XVIIID-HPLC-2 (C3, Rt 15, 21 mg, 0.0047 %). XVIIID-HPLC-2 Figure 3.6: HPLC spectrum of compound C3 *conditions Solvent Column Flow rate Detection ACN:DIW 90:10 to 100 % in 30 min SUPELCO, RP-18 (25 X 2.1 cm) 1.5 mL/min UV at 254 nm P a g e 98 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM HELICHRYSUM TERETIFOLIUM METHANOL EXTRACT (HT) OPEN COLUMN (SILICA GEL) III X XII SILICA XX XVIII SEPHADEX IIIA X3 XXIII SILICA GEL XIIC XVIIID SEPHADEX XXD XXIIIpptA HPLC FRACTIONATION (ACN/DIW) 4''' 3''' MeO OH HO OH OH 4` 1''' O 3'' 1'' O 5'' 4  HO O 8 9 1 2 OH 1` 3`` 1`` 10  OH OH O OH 4 5 2 3`` O 1`` O OH C2 C5 O H 9 C7 MeO 2 9 2 5 4 HO C6 OH O C10 OH OMe O OH 4 OH O OMe O 1` 10 OH O C4 HO O 8 1` 10 5 O 4` H 4` O 8 OH OH C3 C1 HO O HO HO H O OH OH OH O OH C9 O C8 Scheme 3.1: A flow diagram of experimental procedure for the isolation of constituents from H. teretifolium *All silica gel column fractionations were done using gradient solvent system of hex/EtOAc/MeOH. * All sephadex column fractionations were done using isocratic elution of aq. EtOH. * Compounds derived from same sub-fractions are indicated with same color box. P a g e 99 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM BIOLOGICAL CHARACTERIZATION TERETIFOLIUM CONSTITUENTS OF HELICHRYSUM 3.7 General experimental procedure for biological assays 3.7.1 Reagents Standards (purity > 99.0%) for antioxidant, inhibition of Fe2+ induced lipid peroxidation, antielastase and anti-tyrosinase assays such as kojic acid, oleanolic acid, EGCG, trolox (6-Hydroxyl2, 5,7,8-tetramethylchroman-2-carboxylic acid), and other reagents including ABTS (2,2’-Azinobis (3-ethylbenzo thiazoline-6-sulfonic acid) diammonium salt), potassium peroxodisulphate, fluorescein sodium salt, AAPH (2, 2’-Azobis (2-methylpropionamidine) dihydrochloride, perchloric acid, TPTZ (2,4,6-tri[2-pyridyl]-s-triazine, Iron (III) chloride hexahydrate, sepharose (wet bead diameter, 60-200 µm), copper sulphate, hydrogen peroxide, N-succ-(Ala)3-nitroanilide (SANA) were secured from Sigma-Aldrich, Inc. (St. Louis, MO, USA). All antioxidant assays including FRAP, TEAC, lipid peroxidation, and enzyme inhibition (tyrosinase and elastase) were measured by Multiskan spectrum plate reader, while automated ORAC assays were determined by Floroskan spectrum plate reader. 3.7.2 Antioxidant assays 3.7.2.1 Ferric-ion reducing antioxidant power (FRAP) assay Working FRAP reagent was prepared in accordance to the method described previously (Benzie & Strain, 1996). In a 96-well clear microplate (visible range), 10 μL of the stock solution (1mg/mL, w/v) of the isolated compounds (C1-C10) and a methanol extract (HT) were mixed with 300 μL FRAP reagent. The FRAP reagent was a mixture (10:1:1, v/v/v) of acetate buffer (300 mM, pH 3.6), tripyridyl triazine (TPTZ) (10 mM in 40 mM HCl) and FeCl3.6H2O (20 mM). After P a g e 100 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM incubation at room temperature for 30 min, the plate was read at a wavelength of 593 nm in a Multiskan Spectrum plate reader (Thermo Fisher Scientific). L-Ascorbic acid (Sigma Aldrich, South Africa) was used as a standard with concentrations varying between 0 and 1000 μM. Further dilutions were done to the samples that were highly concentrated and such dilution factors were recorded and used for calculations of the affected samples. The results were expressed as μM ascorbic acid equivalents per milligram dry weight (μM AAE/g) of the test samples. 3.7.2.2 Automated oxygen radicals absorbance capacity (ORAC) assay ORAC was done according to the previous method (Prior, et al., 2003) with some modifications (Cao et al, 1997; 1998). The method measures the antioxidant scavenging capacity of thermal decomposition generated by (a) peroxyl radical of 2,2’-azobis (2-amino-propane) dihydrochloride (AAPH; ORACROO. assay), (b) hydroxyl radical (ORACOH. assay), generated by H2O2-Cu2+ (H2O2, 0.3%; Cu2+ [as CuSO4], 18 µM, or (c) Cu2+ [as CuSO4], 18 µM as a transition metal oxidant at 37 o C. Fluorescein was used as the fluorescent probe. The loss of fluorescence of fluorescein was an indication of the extent of its oxidation through reaction with the peroxyl or the hydroxyl radical. The protective effect of an antioxidant was measured by assessing the fluorescence area under the curve (AUC) plot relative to that of a blank in which no antioxidant was present. The analyzer was programmed to record the fluorescence of fluorescein every 2 minutes after AAPH, H2O2Cu2+, or Cu2+ was added. The fluorescein solution and sample were added in the wells of an illuminated 96 well plate, 12 µL of each of our sample (in stock solution of 1 mg/mL) was combined with 138 µL of a fluorescein working solution followed by addition of 50 µL of 150 mg of AAPH prepared in-situ in 6 mL Phosphate buffer. Absorbance was measured with Fluoroskan spectrum plate reader with the excitation wavelength set at 485 nm and the emission wavelength P a g e 101 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM at 530 nm. A calibration curve was used, using a Trolox stock solution of concentrations in the range of 83 - 417 µM (R2 = 0.9514). The ORAC values were calculated using a regression equation (Y = a + bX + cX2) between Trolox concentration (Y in µM) and the net area under the fluorescence decay curve (X). ORAC values were expressed as micromoles of Trolox equivalents (TE) per milligram of test sample, except when Cu2+ (without H2O2) was used as an oxidant in the assay. In the presence of Cu2+ without H2O2, test samples acted as a prooxidant rather than antioxidants in the ORAC assay. The copper-initiated prooxidant activity was calculated using [(AreaBlank – AreaSample)/AreaBlank] X 100 and expressed as prooxidant units where one unit equals the prooxidant activity that reduces the area under the fluorescein decay curve by 1% in the ORAC assay. Samples without perfect curve were further diluted and the dilution factors were used for the calculation of such samples. 3.7.2.3 Trolox equivalent absorbance capacity (TEAC) assay The total antioxidant activity of the test sample was measured using previously described methods (Pellegrini, et al., 1999; Re, et al., 1999). The stock solutions which contains 7 mM ABTS and 140 mM potassium-peroxodisulfate (K2S2O8) (Merck, South Africa) was prepared and kept at -2 oC. The working solution was then prepared by adding 88 μl K2S2O8 solution to 5 mL ABTS solution. The two solutions were mixed well and allowed to react for 24 hours at room temperature in the dark. Trolox (6-hydrox-2,5,7,8-tetramethylchroman-2-carboxylic acid) was used as the standard with concentrations ranging between 0 and 500 μM. After 24 hours, the ABTS mix solution was diluted with ethanol to read a start-up absorbance (control) of approximately 2.0 (± 0.1). The stock solution (1 mg/mL) of a methanol extract (HT) and purified compounds (25 μL) were allowed to react with 300 μL ABTS in the dark at room temperature for 30 min. The absorbance was read at P a g e 102 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM 734 nm at 25 °C in the plate reader. The results were expressed as μM Trolox equivalents per milligram dry weight (μM TE/g) of the test samples. 3.7.2.4 Inhibition of Fe (II)-Induced Microsomal Lipid Peroxidation Assay Rat liver microsomes were first isolated from an S9 liver fraction using a sepharose column with 0.01M potassium phosphate buffer; pH 7.4, supplemented with 1.15 % KCl. The isolation of microsomes was done at -5 0C. A glass column (24/29) was parked with sepharose (30 cm) and eluted with phosphate buffer as described above. Microsomes obtained were homogenized and distributed into 2 mL eppendorf tube and kept at -80 0C prior experiment. The protein content of the homogenized microsome was determined in situ. A method described by (Snijman, et al., 2009) with a few modifications was adopted. The reaction mixture contained microsomes (1 mg of protein/mL in 0.01M potassium phosphate buffer; pH 7.4, supplemented with 1.15 % KCl). The positive control included microsomes, buffer and ferrous sulphate, in the absence of the samples to be tested. The sample stock solutions (HT and C1-C10) were prepared in methanol (1 mg/mL, w/v). The working sample solutions were prepared in 0.01M potassium phosphate buffer; PH 7.4, supplemented with 1.15 % KCl diluted to 26.750, 13.375, 6.688, 3.344, 1.672, and 0.836 µg/mL concentrations. 100 µL of each sample (working solutions) were dissolved in potassium phosphate buffer and pre-incubated with 500 µL microsomes at 37 0C for 30 minutes in a shaking water bath. 200 µL of KCl-buffer were added to the mixture, followed by 200 µL of a 2.5 mM ferrous sulphate solution and incubated at 37 0C for 1 hour in a shaking water bath. The reaction was terminated with 10% trichloroacetic acid (TCA) solution (1 mL) containing 125 µL butylated hydroxytoluene (BHT, 0.01 %) and 1mM ethylenediaminetetraacetic acid (EDTA). Samples were centrifuged at 2000 rpm for 15 minutes, 1 mL of supernatant was mixed with 1 mL of 0.67 % thiobarbituric acid P a g e 103 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM (TBA) solution. The reaction mixture was then incubated in a water bath at 90 0C for 20 minutes and the absorbance were measured at 532 nm using plate reader. The percentage inhibition of TBARS formation relative to the positive control was calculated by: [(Acontrol – A sample) / Acontrol] X 100 (1) 3.7.3 Skin enzymes inhibitory assays 3.7.3.1 Tyrosinase enzyme assay This assay was performed using the method described by (Chompo, et al., 2012; Vardhan, et al., 2014) with slight modifications. Samples were dissolved in DMSO (dimethyl sulphoxide) to a stock solution of 1 mg/mL (w/v). Further dilutions were done with 50 mM sodium phosphate buffer (pH 6.5) for all working solutions to the concentrations of 100.00, 50.00, 25.00, 12.50, 6.25, 3.12, and 1.56 µg/mL. Kojic acid was used as a control drug. In the wells of a 96-well plate, 70 µL of each sample working solution was combined with 30 µL of tyrosinase (from mushroom, 500 Units/mL in sodium phosphate buffer) in triplicate. After incubation at room temperature for 5 minutes, 110 µL of substrate (2 mM L-tyrosine) was added to each well. Final concentrations of the crude extract, isolated compounds, and positive control ranged from 1.0 - 100 µg/mL. The sample control was made up of each sample with sodium phosphate buffer. The reacting mixture was then incubated for 30 minutes at room temperature. The enzyme activity was determined by measuring the absorbance at 490 nm using plate reader. The percentage of tyrosinase inhibition was calculated as follows: [(A – B) – (C – D)] / (A – B) X 100 (2) P a g e 104 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM Where A is the absorbance of the control with the enzyme, B is the absorbance of the control without the enzyme, C is the absorbance of the test sample with the enzyme and D is the absorbance of the test sample without the enzyme. 3.7.3.2 Elastase inhibition assay Inhibition of elastase by the test samples was assayed using N-succ-(Ala)3-nitroanilide (SANA) as the substrate, monitoring the release of p-nitroanilide by the method described (Chompo, et al., 2012) with little adjustment. The inhibitory activity determined the intensity of color released during cleavage of SANA by the action of elastase. The working reagent for anti-elastase assay was prepared in line with literature. The preparation involved 1 mM SANA in 0.1 M tris-HCl buffer pH 8.0. The sample stock solutions were prepared as 1 mg in 1 mL methanol. 200 µL of already prepared SANA was added to the 20 µL of sample solution (diluted to 100.00, 50.00, 25.00, 12.50, 6.25, and 3.12 µg/mL concentrations in tris-HCl buffer as working solutions) in a 96-Well plate. The mixtures were vortexed and preincubated for 10 minutes at 25 0C and then 40 µL of elastase from porcine pancrease (0.03 Units/mL) was added. The mixtures were further incubated for 10 minutes and the absorbance was measured at 410 nm. Tris-HCl buffer was used as control, while oleanolic acid used as a positive control. The sample control involved the smple and SANA (uninhibited sample). The percentage of elastase inhibition was calculated as follows: (1 – B/A) X 100 (3) Elastase inhibition (%), where A is the enzyme activity without sample and B is the activity in the presence of the sample. P a g e 105 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM 3.8 Statistical analysis All skin enzyme inhibitory assays and Fe2+-induced lipid peroxidation assay calculations, expressed as percentage inhibitions ± SD, were performed using MS Excel 2013, while the final values expressed as IC50, were determined using GraphPad prism 5.0. The data presented are means ± SD obtained from 96 well plate readers for all in vitro experiments in triplicate. Differences between the means were considered to be significant if p < 0.05 according to Prism’s one way ANOVA. ORAC, FRAP, and TEAC values were determined using their respective templates. 3.9 Chemical evaluations: Results and discussion 3.9.1 Summary of the isolated compounds Chromatographic purification of a methanol extract of H. teretifolium using different techniques including semi-prep HPLC yielded ten pure compounds categorized into the following class of flavonoids: prenylated chalcones (C1 – C3), prenylated flavanones (C4 – C6), and flavones (C7 – C10). Compound C1 was isolated as a new compound and its occurrence is hereby reported for the first time from nature. 3.9.2 Spectroscopic data of isolated compound C1 Compound C1 was isolated as yellow amorphous powder; UV λmax (MeOH) nm: 340 nm; IR (KBr) cm-1: 3450, 1800, 1600, 1410, and 1150. HRMS m/z 391.1889 [M + 1]+, (calcd: 390.1831). 1Hand 13C-NMR data, see Table 3.10. Other spectra data obtained for compound C1 were attached as Annexure I. P a g e 106 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM 3.9.3 Analysis of prenylated chalcones 3.9.3.1 Analysis of compound C1 (O-prenylated chalcone) Compound C1 was isolated as an amorphous yellow powder, the HRMS of C1 showed a [M + 1]+ peak at m/z 391.1889, corresponding to the molecular formula C25H26O4. Absorption in the IR (KBr) spectrum were attributed to the hydroxyl functions (3450 cm-1), C=O of ketone (1800 cm1 ), conjugated C=C (1600 cm-1), aromatic ring (1410 cm-1), as well as C-O ether (1150 cm-1). UV λmax (MeOH) nm: 340 nm. The NMR spectra (Annexure I, Fig. 1-4) demonstrated chalcone skeleton features with an un-substituted ring B. 1H NMR showed singlet at 6.03 (H3`), aromatic multiplet of mono-substituted phenyl group 7.38 - 7.35 (m, H2-H6); trans coupled protons at 8.11, 7.72 (d each, J = 15.6 Hz), in addition to signals of two prenyl groups, one of them (at C-5`) forming a pyrane ring with 6`-OH and contains signals of two cis olefinic protons at 6.58, 5.45 (d, J = 9.6 Hz) and two methyls at 1.52. The other prenyl group forming ether bond with 4`-OH and showed signals of a methylene group at 4.53 (2H, d, 6.4 Hz), a proton at 5.56 (t, J = 6.4 Hz) and two methyls at 1.77 s, 1.72 s. The 13C NMR with DEPT-135 confirmed the above data and showed 25 carbons, 15 of them belong to the main chalcone skeleton and the other 10 carbons belong to the two prenyl groups (Table 3.10). 2D NMR spectra established structure of C1 in particular HMBC correlations spectrum (Annexure I, Fig. 4) which showed cross peaks between H1``/C5`, C6`, C4`; H1```/C4`; 5-OH/C1`, C2`, C3`; H3`/C2`, C4`, C1`, C5` among others and confirmed the structure given in Figure 3.7 as 2'-hydroxy-5',6'-(2,2-dimethylpyrano)-4`-(O-prenyl)-chalcone and given the name heliteretifoline (C1). P a g e 107 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM 4''' 3''' 3'' 1''' 1'' O O 5'' 4  1  2 OH O Heliteretifoline (C1) Figure 3.7: Chemical structure of heliteretifoline. To the best of our knowledge and Scifinder database, compound C1 is only described once as synthetic products but not isolated before from natural source (Xia, et al., 2010). 3.9.3.2 Analysis of compounds C2 & C3 (prenylated chalcones) Isoxanthohumol (C2) and 2`,4`,6`-trihydroxy-3`-prenylchalcone (C3) were isolated previously from H. polycladum, identification of C2 and C3 were done by correlating our spectral data to the existing data (Bohlmann, et al., 1978). Isoxanthohumol (C2) was also isolated and identified (Fig. 3.8) alongside with 2`,4`,6`-trihydroxy-3`-prenylchalcone (C3, Fig. 3.9), and glabranin (C5) from H. cymosum (Bohlmann, et al., 1979; Jakupovic, et al., 1989). It is of interest to note that the NMR of compound C2 showed duplication of some signals (Table 3.10) because of the free rotation of the single bonds around the carbonyl. This duplication is not observed for the other prenylated chalcones (C1 and C3). MeO 3`` OH 1`` OH O 2`,6`-dihydroxy-4`-methoxy-3` prenylchalcone (C2) Figure 3.8: Chemical structure of 2`,6`-dihydro-4`-methoxy-3-prenylchalcone P a g e 108 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM HO 3`` OH 1`` OH O 2`,4`,6`-trihydroxy-3`-prenylchalcone (C3) Figure 3.9: Chemical structure of 2`,4`,6` -trihydroxy-3`-prenylchalcone 3.9.4 Analysis of compounds C4, C5 and C6 (prenylated flavanones) 7-methoxyisoglabranin (C6) demonstrated very similar NMR spectra to that of Isoglabranin (C4) and glabranin (C5), however, the distinction between structure C4 (Fig. 3.10) and C6 were confirmed by HMBC through H1`` and 5-OH cross peaks with C5 and C6, while C5 (Fig. 3.11) showed shift of some signal (C6 and C2``) from C4 (Table 3.10). The structure of C6 (Fig. 3.12) was finally confirmed from the previous data (Braz, et al., 1975). 4` HO O 8 9 2 1` 10 4 5 OH O isoglabranin (C4) Figure 3.10: Chemical structure of isoglabranin 4` HO O 8 9 2 1` 10 5 4 OH O glabranin (C5) Figure 3.11: Chemical structure of glabranin P a g e 109 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM 4` H MeO O 8 9 2 1` 10 5 4 OH O 7-methoxyisoglabranin (C6) Figure 3.12: Chemical structure of 7-methoxyisoglabranin 3.9.5 Analysis of compounds C7, C8, C9 and C10 (flavones) The spectroscopy properties of isolated flavones such as Quercetin (C7, Fig. 3.13) isolated form H. arenarium was correlated with previous data (Prokopenko, et al., 1972), while that of 4`methoxyquercetin (C8, Fig. 3.14) was confirmed from (Servettaz, et al., 1984). Other flavones isolated and identified based on previous work include 4`-methoxykaempferol (C9, Fig. 3.15), and mosloflavone (C10, Fig. 3.16) identified from (Gowsala & Uvais, 1975) and (Donald, et al., 1988) respectively. It is worthy to indicate the isolation of compounds C6, C8-C10 from Helichrysum species for the very first time. OH OH HO O H OH OH O quercetin (C7) Figure 3.13: Chemical structure of quercetin P a g e 110 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM OH OMe HO O OH OH O 4-methoxyquercetin (C8) Figure 3.14: Chemical structure of 4-methoxyquercetin OMe HO O H OH OH O 4'-methoxykaempferol (C9) Figure 3.15: Chemical structure of 4`-methoxykaempferol HO O OH OH O mosloflavone (C10) Figure 3.16: Chemical structure of mosloflavone P a g e 111 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM Table 3.10: 1H (400 MHz: m, J Hz) and 13C (100 MHz) NMR spectral data of isolated compounds C1 - C6 in CDCl3 No. C1 13 1 2 C 135.6 128.2 3 128.9 4 5 6 130.0 128.9 128.2 1 H 7.58 dd, 7.6, 1.6 C2 13 C 136.6 129.23 β CO 1`` 2`` 3`` 4`` 5`` 1``` 2``` 3``` 4``` 5``` 5-OH 7-OMe H 7.55 d, 6.2 129.88 7.37 m 7 8 9 10 1’ 2’ 3’ 4’ 5’ 6’ α C3 1 7.58 dd, 7.6, 1.6 1 128.6 C 135.6 128.3 C4 13 1 7.53 dd, 1.6, 7.2 7.31 m 79.07 43.44 7.31 m 7.31 m 7.53 dd, 1.6, 7.2 195.9 161.2 107.0 H C 7.29 m 131.0 129.9 129.2 106.2 167.3 95.5 160.6 103.3 155.7 127.5 6.03 s 8.11 d 15.6 106.2 164.7/164.6 109.2/109.1 160.9 91.6 164.3/163.9 128.7/128.8 142.1 192.8 117.0 124.4 77.9 27.9 27.9 65.5 118.8 138.6 25.8 18.3 - 7.72 d 15.6 6.58 d, 9.6 Hz 5.45 d, 9.6 Hz 142.8/142.7 193.8 22.0 124.0 136.6 26.0 17.9 1.52 s 1.52 s 4.53 d, 6.4 4.56 t, 6.4 1.77 s 1.72 s 14.19 s 13 56.6 7.55 d, 6.2 6.05 s 8.10/8.12 d, 15.7 7.65 d, 15.7 129.7 128.6 128.3 105.2 163.4 106.7 162.0 94.4 160.0 127.9 141.7 192.9 163.8 95.5 161.0 102.9 138.5 126.1 C6 1 13 1 5.37 dd, 3.2, 13.2 79.1 5.30 dd, 2.5, 13.1 79.3 5.38 dd 3.0, 13.2 3.05 dd, 13.2, 17.2 2.79 dd, 3.2, 17.2 - 43.4 3.05 dd, 3.1, 17.2 2.71 dd, 13.3, 17.2 43.5 3.05 dd 13.2, 17.1 2.78 dd 3.0, 17.1 5.99 s C H 195.5 161.1 108.6 164.2 94.8 160.6 102.3 138.5 126.1 7.30 m 5.80 s 128.7 126.1 126.1 H 195.8 161.3 110.1 5.93 s 7.42 m 128.8 C 165.5 91.0 160.3 102.9 138.5 126.1 128.9 6.07 s - 7.37 m 126.1 8.07 d 15.6 7.68 d, 15.6 3.22 d, 6.9 5.09 t, 6.9 21.1 121.4 135.7 25.8 17.9 1.48 s 1.60 s 13.61 3.70 s C5 13 H 13.30 3.33 d, 7.2 5.28 t/, 7.2 1.74 s 1.79 s 20.9 122.1 132.4 25.6 17.7 3.21 d, 6.9 5.17 t, 6.9 1.63 s 1.72 s 21.0 122.2 131.7 25.8 17.7 3.24 d, 6.8 5.16 tt, 6.8, 1.4 1.65 s 1.75 s 55.8 12.03 3.81 s 12.36 *C1-C6: isolated compounds 1-6 P a g e 112 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM 3.10 Biological evaluations: Results and discussion In this study, an in vitro investigation was carried out to determine the antioxidant capacity of a methanol extract of H. teretifolium and its ten isolated constituents (C1-C10). The ORAC (peroxyl and hydroxyl), FRAP, TEAC, and Fe2+-induced lipid peroxidation assays were used to determine the antioxidant and oxidative damage modulatory capacities of the extract, while trolox, ascorbic acid and EGCG were used as references. 3.10.1 Evaluating the ORAC activities of the isolated compounds Accelerated skin aging is a consequence of direct continuous contact with the environment due to accumulation of reactive oxygen species (ROS). Since aging is becoming a major concern, it is important to focus on the causes and its cure. Although a wide range of factors contribute to skin aging, environmental factors are majorly involved in inducing the stress and enhancing the effect of internal factors in causing aging. Natural antioxidants being cost effective and safer are proposed as good alternatives towards modulating the stress induced by the gerontogens. Plant extracts or compounds thus offer new effective treatment options to minimize the effects of UV stress and harmful compounds. Bio-prospecting of natural resources for antioxidants has hence intensified, as a great deal of research is being carried out to identify plants with potent antioxidant activity to act against skin aging. Table 3.11 indicate the ORAC for both hydroxyl and peroxyl radicals demonstrated by the isolated compounds and total extract (HT). Compounds C7 & C8 were isolated as active constituents with significant peroxyl (17.836 ± 2.90; 12.545 ± 5.07) X 103 and hydroxyl (7.26 ± 0.71; 6.779 ± 3.40) X 106 µM TE/g radical absorbance capacities. In general, a hydroxyl on the B-ring are the most important active group, for donation of hydrogen to hydroxyl and peroxyl radicals, thereby P a g e 113 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM stabilizing them and giving rise to a relatively stable flavonoid radical (Wolfe & Liu, 2008). Our result is therefore an indication that C7 and C8 have the capacities of donating hydrogen atom(s) from B-hydroxyl groups to stablilize free radicals. The result we obtained in Table 3.11 indicated that the copper-initiated prooxidant activity, expressed as arbitrary units, were very low when compared with the peroxyl and hydroxyl radical absorbance capacities expressed in 103 and 106, respectively. This result suggests none of the compounds possessed prooxidant activity, possibly due to the non-existence of pyrogallol groups in their respective ring-B substitution (Heim, et al., 2002). This finding seemed exciting to us because many of the antioxidants have been reported to have adverse effect through their antioxidant-prooxidant behavior during absorption, metabolism and excretion (Nijvedt, et al., 2001; Carocho & Ferreira, 2013). P a g e 114 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM Table 3.11: Oxygen radicals’ antioxidant capacity of H. teretifolium constituents Sample - Automated oxygen radical absorbance capacity (ORAC μMTE/g ) . OH (μM/g X 106) ROO. (μM/g X 103) Prooxidant HT 1.313 ± 7.54 3.016 ± 5.90 4.163 ± 0.83 C1 2.833 ± 3.88 2.998 ± 1.67 2.036 ± 2.98 C2 3.113 ± 17.59 2.910 ± 6.00 3.601 ± 2.23 C3 5.025 ± 6.16 3.771 ± 3.02 4.704 ± 0.27 C4 1.063 ± 33.50 2.918 ± 4.13 3.947 ± 0.29 C5 0.856 ± 17.35 2.997 ± 0.36 2.971 ± 1.10 C6 3.854 ± 5.14 2.955 ± 3.41 3.799 ± 0.60 C7 17.836 ± 2.90 7.265 ± 0.71 4.361 ± 0.78 C8 12.545 ± 5.07 6.779 ± 3.40 8.963 ± 2.79 C9 10.491 ± 0.97 3.675 ± 1.40 3.790 ± 1.15 C10 2.403 ± 2.50 2.909 ± 8.41 6.482 ± 1.55 EGCG 14.970 ± 5.53 3.911 ± 4.65 7.281 ± 1.42 EGCG: Epigallocatechingallate; Isolated compounds C1-C10; HT: methanol crude extract 3.10.2 Evaluating the FRAP and TEAC activity of the isolated compounds The FRAP assay measures the sample’s ability to reduce the intense blue ferric TPTZ complex to its ferrous form in acidic medium, thereby changing its absorbance (Benzie & Strain, 1996). The FRAP activity demonstrated by C7 and C8 (4816 ± 7.42; 3584.17 ± 0.54) μΜ AAE/g, respectively, with almost a similar activity when compared to the commercial antioxidant derived from green P a g e 115 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM tea, EGCG (Table 3.12). Compound C7 demonstrated highest FRAP value due to the presence of 3`, 4`-dihydroxy group in the B-ring. Further explanation to validate our result was documented that greater radical stability was due to an increased electron delocalization and intramolecular hydrogen bonding between the 3`- and 4`-hydroxyls (Wolfe & Liu, 2008). Replacement of one of the hydroxyl groups by a methoxyl group (C8) also contributed to the significant value recorded, possibly due to the presence of a lone pair of electron on methoxyl (OMe) group which can keep intramolecular hydrogen bond with C3`-OH. The reducing capacity of compounds C7 and C8 could be used as an important indicator of possible antioxidant activity in acidic conditions. On the other hand, C2 and C3 demonstrated the highest TEAC value (Table 3.12). The significance of the hydroxyl configurations in ring-A for such TEAC activity observed for C2 & C3 is less clear to us, except a supporting evidence that 5,7-m-di/tri-hydroxyl arrangement in ring-A increases TEAC, but such mechanism was not fully explained (Heim, et al., 2002). P a g e 116 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM Table 3.12: Ferric ion reducing and trolox equivalent antioxidant capacities of H. teretifolium constituents. Sample FRAP TEAC - μM AAE/g μM TE/g HT 511.89 ± 4.61 1179.60 ± 8.20 C1 nd nd C2 619.91 ± 1.97 4170.66 ± 6.72 C3 817.94 ± 4.26 4529.01 ± 2.44 C4 7.052 ± 3.76 43.17 ± 6.26 C5 67.79 ± 14.27 204.15 ± 2.04 C6 104.09 ± 4.64 519.25 ± 3.66 C7 4816.31 ± 7.42 1361.70 ± 1.98 C8 3584.17 ± 0.54 1009.01 ± 1.98 C9 191.47 ± 1.39 261.30 ± 4.02 C10 544.60 ± 6.98 699.66 ± 2.28 EGCG 3326.45 ± 5.76 11545.4 ± 17.28 * nd: not detected; EGCG: Epigallocatechingallate; HT: methanol crude extract; C1-C10: Isolated compounds 1-10 3.10.3 Evaluating the Fe2+ induced anti-lipid peroxidation activity of the isolated compounds Oxidative degradation of lipids is a common consequence of oxidative stress, a process whereby polyunsaturated lipids of the membranes are susceptible to oxidative damage via the reaction of free radicals, which can lead to lipid peroxidation. Products of lipid peroxidation such as malondialdehyde (MDA), 4-hydroxyl 2-nonenal, and some other alkanals react with cell P a g e 117 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM macromolecules to form adducts with significant irreversible effects on cellular functions, and could also promote the aging process. We observed Compounds C7 - C10 (IC50=2.931; 6.449; 10.520; 10.720 μg/ml, respectively, Fig. 3.17) to be good inhibitors of lipid peroxidation. Their significant (p<0.05) values recorded were due to the presence of , β-unsaturated double bond in conjunction with 4-keto function in their respective structures. An existing literature established the significance of these features through delocalization of the electron on the keto group which resulted in resonance stabilization energy (ring current) in both rings A & B, stabilizing them and giving rise to relatively stable flavonoid radicals formed after the transfer of hydrogen and/or electron (Kumar & Pandey, 2013). Figure 3.17: Effects of H. teretifolium constituents on inhibition of Fe2+ induced microsomal lipid peroxidation. (p<0.05) *Data are expressed as IC50 with isolated compounds and a methanol extract (HT) screened at 26.750 µg/mL P a g e 118 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM 3.10.4 Evaluating the anti-tyrosinase activity of the isolated compounds The skin diseases-related enzyme assays revealed that compound C7 has strong activity in among of isolated compounds tested. Recently, tyrosinase inhibitors have received special attention, due to its alleviating property to deliver skin lightening and anti-aging benefits, caused by undesirable skin hyperpigmentation (Narayanaswamy, et al., 2011). Our findings demonstrated anti-tyrosinase activity in order of C7>C8>C9>C10 for our samples. From the previous skin enzymes assays investigated on flavonoids, the tyrosinase inhibitory activities of flavones were ascribed to their ability to chelate copper in the enzyme (Chang, 2009). Since the partial structure (3, 5-dihydroxy-4-keto moiety) which is responsible for the ability to form chelation can be found in our isolated compounds C7 - C10, it appears very likely that the copper chelation is the main inhibition mechanism of flavones as long as their 3, 5-dihydroxyl groups are free (Kumar & Pandey, 2013). P a g e 119 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM Figure 3.18: Effects of H. teretifolium constituents on inhibition of anti-tyrosinase activity (p<0.05) *Data are expressed as IC50 with isolated compounds screened at 50 µg/mL while the methanol extract (HT) screened at 100.00 µg/mL In addition to the above features exhibited by flavones, the results (Fig. 3.18) further showed C7 bearing catechol group at the B-ring was the most effective inhibitor of tyrosinase and lipid peroxidation. Other compounds (C8 - C10), not bearing a catechol group, were not significantly active under the condition assessed. C7 exhibited potent (p<0.05) anti-tyrosinase inhibitory activity as shown in Figure 3.18 in accordance with reported data (Heim, et al., 2002; Masuda, et al., 2009; Zheng, et al., 2013). P a g e 120 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM 3.10.5 Evaluating the anti-elastase activity of the isolated compounds No significant IC50 value was observed by the tested samples when compared to oleanolic acid except Compound C7 that demonstrated anti-elastase (though weak but significant at IC50 = 43.342 µg/mL, Fig. 3.19) activity. No previous study has been reported the activity of flavones to exhibit anti-elastase and our findings cannot be used to establish that compound C7 bearing catechol group at the B-ring was responsible for such anti-elastase activity. Figure 3.19: Effects of H. teretifolium constituents on inhibition of anti-elastase activity (p<0.05) *Data are expressed as IC50 with isolated compounds screened at 50 µg/mL while the methanol extract (HT) screened at 100.00 µg/mL P a g e 121 | 238 CHAPTER THREE: HELICHRYSUM TERETIFOLIUM 3.11 Conclusion Ten flavonoid related structures were isolated as constituents from a methanol extract of H. teretifolium (HT). The determinations of the structures were carried out by 1D and 2D-NMR as well HRMS. There are many assumptions with regards to structural activity relationship of flavonoids. The numbering, positioning and substituting patterns displayed by phenyl rings A & B in the isolated compounds resulted in different biological activities. It may be collectively stated that most of the H. teretifolium constituents exert efficient hydroxyl and peroxyl radical absorbance capacities, inhibiting lipid peroxidation as well serve as a good source with anti-tyrosinase activity in the in vitro systems. This could be attributed to its good quality flavonoids it contained which may also contribute to its biomedical applications. The present work is the first scientific report on H. teretifolium and the results suggesting that the extract of this plant or its constituents might become natural agents to inhibit oxidative stress and tyrosinase activity, both playing an important role in skin aging. P a g e 122 | 238 CHAPTER THREE: REFERENCES REFERENCES Benzie, I.F.F. and Strain, J.J. (1996). The ferric reducing ability of plasma (FRAP) as a measure of "antioxidant power": The FRAP Assay. Analytical Biochemistry, 238, pp. 70-76. Bohlmann, F., Zdero, C., Hoffmann, E., Mahanta, P.K. and Dorner, W. (1978). 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Antioxidant activity applying an improved ABTS radical cation assay. Free radical Biology and Medicine, 26, pp. 1231-1237. Servettaz, O., Colombo, M.L., De Bernardi, M., Uberti, E., Vidari, G. and Vita-Finzi, P. (1984). Flavonol glycosides from Dryas octopetala. Journal of Natural Products, 47(5), pp. 809814. Snijman, P.W., Joubert E., Ferreira D., Li, X., Ding, Y., Green, I.R. and Gelderblom, W.C.A. (2009). Antioxidant activity of the dihydrochalcones aspalathin and nothofagin and their corresponding flavones in relation to other rooibos (Aspalathus linearis) flavonoids, epigallocatechin gallate, and trolox. Journal of Agricultural and Chemistry, 57, pp. 6678-6684. P a g e 125 | 238 CHAPTER THREE: REFERENCES Tirillini, B., Menghini, L., Leporini, L., Scanu, N., Marino, S. and Pintore, G. (2013). Antioxidant activity of methanol extract of Helichrysum foetidum Moench. Natural Product Research, 27(16), pp. 1484-1487. Vardhan, A., Khan, S. and Pandey, B. (2014). Screening of plant parts for anti-tyrosinase activity by tyrosinase assay using mushroom tyrosinase. Indian Journal of Science Research, 4(1), pp. 134-139. Viegas, D.A., Palmeira-de-Oliveira, A. and Salgueiro, L. (2014). Helichrysum italicum: From traditional use to scientific data. Journal of Ethnopharmacology, 151, pp. 54-65. Wagner, H., Bladt, S. and Zgainski, E. (1984). Plant drug analysis: A thin layer chromatography. 1st ed. Berlin: Springer-Verlag: Atlas. Wolfe, K.L. and Liu, H.R. (2008). Structure-activity relationship of flavonoids in the cellular antioxidant activity assay. Journal of Agricultural and Food Chemistry, 56, pp. 8404-8411. Xia, L., Narasimhule, M., Li, X., Shim, J. and Lee, Y. (2010). New synthetic routes to biological interesting geranylated acetophenones from Melicope semecarpifolia and their unatural prenylated and fernesylated derivatives. Bulletin-Korean Chemical Society, 31(3), pp. 664-669. Zheng, Z., Tan, H., Chen, J. and Wang, M. (2013). Characterization of tyrosinase inhibitors in the twigs of Cudranis tricuspidata and their structure-activity relationship study. Fitoterapia, 84, pp. 242-247. P a g e 126 | 238 CHAPTER FOUR: HELICHYSUM NIVEUM CHAPTER FOUR CHEMICAL AND BIOLOGICAL CHARACTERIZATION OF HELICHRYSUM NIVEUM CONSTITUENTS 4.1 Abstract Phytochemical investigation of aerial parts of Helichrysum niveum (HF) using different chromatographic methods including semi-preparative HPLC afforded three new (C11-C13) and six known (C14-C20) acylphloroglucinols alongside a known dialcohol triterpene (C21). The structures of the isolated compounds were characterized accordingly as 1-benzoyl-3 (3-methylbut2-enylacetate)-phloroglucinol (helinivene A, C11), 1-benzoyl-3 (2S-hydroxyl-3-methylbut-3enyl)-phloroglucinol (helinivene B, C12), 8-(2-methylpropanone)-3S,5,7-trihydroxyl-2,2- dimethoxychromane (helinivene C, C13), 1-(2-methylbutanone)-4-O-prenyl-phloroglucinol (C14), 1-(2-methylpropanone)-4-O-prennyl-phloroglucinol (C15), 1-(butanone)-3-prenyl- phloroglucinol (C16), 1-(2-methylbutanone)-3-prenyl-phloroglucinol (C17), 1-butanone-3-(3methylbut-2-enylacetate)-phloroglucinol (C18), 1-(2-methylpropanone)-3-prenylphloroglucinol (C19), caespitate (C20) and 3β-24-dihydroxyterexer-14-ene (C21). Excellent total antioxidant capacities were demonstrated by helinivenes A and B (C11 & C12) when measured as ORAC, FRAP, TEAC and including the inhibition of Fe2+-induced lipid peroxidation (IC50 = 5.115 ± 0.90; 3.553 ± 1.92) µg/mL, while anti-tyrosinase activity at IC50 = 35.625 ± 4.67 & 26.719 ± 5.05 µg/mL were also observed for C11 & C12 respectively. This is the first chemical and in vitro biological study on H. niveum. These findings underpin new perspectives for the exploitation of these natural P a g e 127 | 238 CHAPTER FOUR: HELICHYSUM NIVEUM phenolic compounds in applications such as in the natural cosmeceutical and pharmaceutical sectors. Keywords: Helichrysum niveum; Phloroglucinols; Triterpene; Antioxidant; Anti-tyrosinase; Skin aging; Cosmetics. 4.2 Background information on Helichrysum niveum Helichrysum niveum (L.) Less [synonyms; Gnaphalium niveum L., Helichrysum ericifolium Less. var. metalasioides (DC.) Harv., Helichrysum metalasioides DC.] is an indigenous plant widely distributed along the Western Cape coast of South Africa (Figure 4.1: A & B), often on dunes and sandy soil. It has distinguishable acute white bracts where its name niveum, or “snow white” plant (www.keys.lucidcentral.org) originated from. From SciFinder and the dictionary of natural products database, no reports on scientific or ethno-medicinal value have been recorded. Our proposition was based on the chemotaxonomic relationship which may possibly exist between H. niveum and other Helichrysum species. Previous findings expanded the knowledge about the phenolic profile of Helichrysum to be richly blessed with a large proportion of phloroglucinol derivatives (Bohlmann & Abraham, 1979a; Haensel, et al., 1980; Dekker, et al., 1983; Jakupovic, et al., 1989; Tomas-Lorente, et al., 1989; Drewes & van Vuuren, 2008) and terpenoids (Bohlmann & Abraham, 1979b) among others. Dimeric phloroglucinols including arzanol with notable antioxidant, anti-inflammatory and antibacterial activities (Dekker, et al., 1983; Rios, et al., 1991; Taglialatela-Scafati, et al., 2013), has been documented in several Helichrysum species. The objective of this chapter was also directed to investigate the constituents of a methanol extract of H. niveum with a particular focus on the phenolic compounds and investigate them for possible P a g e 128 | 238 CHAPTER FOUR: HELICHYSUM NIVEUM total antioxidant capacity, as well as the tyrosinase, elastase and cholinesterase inhibitory activities. . B A Western Cape region This chapter describes for the:  Isolation of bioactive constituents present in methanol extract of H. niveum, using different chromatographic methods.  Identification of isolated constituents using different spectroscopic techniques.  Determination of total antioxidant capacities of the constituents. P a g e 129 | 238 CHAPTER FOUR: HELICHYSUM NIVEUM  Determination of inhibitory activities of the H. niveum constituents against skin diseases related enzymes.  Structure-activity relationship (mechanism of actions) of the H. niveum bioactive constituents responsible for specific biological activity. CHEMICAL CHARACTERIZATION OF HELICHRYSUM NIVEUM CONSTITUENTS 4.3 General experimental procedure 4.3.1 Reagents and solvents All organic solvents described in section 3.3.1 were used. 4.3.2 Thin layer chromatography (TLC) Unless otherwise stated, the solvent systems generally used for the TLC development of H. niveum fractions are indicated in Table 4.1 Table 4.1: TLC solvent system Solvent system Ratio Assigned code Hexane - ethylacetate 9:1 A Hexane – ethylacetate 7:3 B Hexane – ethylacetate 3:2 C DCM-methanol 99:1 D DCM-methanol 95:5 E P a g e 130 | 238 CHAPTER FOUR: HELICHYSUM NIVEUM All other chromatographic of TLC, column, and HPLC techniques and procedures described in section 3.3.2 were followed. 4.3.3 Spectroscopy All analytical procedure of spectroscopic (NMR, MS, UV, and IR) techniques described in section 3.3.3 were followed. 4.3.4 Optical rotation measurements Optical activity measurements were conducted using Autupol III Polarimeter (Rudolph research analytical, Hackettstown, MA, USA). 4.4 Collection and identification of plant material The plant material was collected in October 2012, from Jonkershoek (about 9 km SE Stellenbosch) nature reserve, Western Cape, South Africa. Voucher specie was identified by Dr. Christopher Cupido (SANBI, Kirstenbosch), and a copy has been deposited at the Compton Herbarium, South African National Biodiversity Institute, Kirstenbosch, South Africa, with herbarium number NBG1458801 4.5 Extraction and fractionation of total extract The aerial (stem, leave and flower; 400 g) part of the plant was air dried at room temperature, blended and extracted with methanol (2.5 L X 2) at room temperature (25 oC) for 48 hours. Methanol extract was evaporated till dryness with rotary evaporator at 40 oC to yield 15.7 g (3.925 %). P a g e 131 | 238 CHAPTER FOUR: HELICHYSUM NIVEUM Portion of total extract (15 g) was applied to silica gel column (30 x 18 cm) and eluted using gradient of hexane and ethyl acetate in the order of increasing polarity as indicated in Table 4.2. Forty eight (48) fractions (500 mL each) were collected and numbered 1 - 48. Table 4.2: Fractionation of the methanol extract of H. niveum Solvent system Solvent volume Fraction collected Hexane 1L 1, 2 Hexane - ethylacetate 90:10 2L 3-6 Hexane – ethylacetate 80:20 2L 7-10 Hexane – ethylacetate 70:30 3L 11-16 Hexane – ethylacetate 60:40 3L 17-22 Hexane – ethylacetate 40:60 2L 23-26 Hexane – ethylacetate 20:80 2L 27-30 Ethylacetate 6L 31-42 Ethylacetate - methanol 90:10 1L 43, 44 Ethylacetate - methanol 70:30 1L 45, 46 Methanol 1L 47, 48 The collected fractions (1 – 48) were concentrated on the rota-vapor and combined according to their TLC profiles (using solvent systems A – D, Fig. 4.2). Seventeen (17) main fractions were and labelled with designated roman numbers I – XVII (Table 4.3). P a g e 132 | 238 CHAPTER FOUR: HELICHYSUM NIVEUM Table 4.3: Main fractions obtained upon fractionation of the total extract of H. niveum Combined fraction 1-5 Designated number I Combined fraction 19-22 Designated number VII Combined fraction 32, 33 Designated number XIII 6-8 II 23 VIII 34, 35 XIV 9-11 III 24, 25 IX 36, 37 XV 12-14 IV 26, 27 X 38-43 XVI 15, 16 V 28, 29 XI 44 XVII 17, 18 VI 30, 31 XII - - A 1 2 3 4 5 6 7 8 9 10 11 12 13 14 FR D 1 2 3 4 5 6 7 8 9 10 11 12 13 14 FR C B 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 FR 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 FR F E 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 FR 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 FR Figure 4.2: TLC profile of the collected fractions (1-44) under UV (254 nm; A -C), and after spraying with H2SO4/vanillin and then heated (D-F). *TLC plate (A & D) of fractions 1-15 (reference to total extract FR) developed using solvent system A *TLC plate (B & E) of fractions 15-29 (reference to total extract FR) developed using solvent system B * TLC plate (C & F) of fractions 29-44 (reference to total extract FR) developed using solvent system D *FR refers to the total extract of H. niveum. P a g e 133 | 238 CHAPTER FOUR: HELICHYSUM NIVEUM 4.6 Isolation of constituents (pure compounds) 4.6.1 Isolation of compounds C11, C15 & C16 - Column chromatography of main fraction XI Main fraction XI (1.6 g) was applied to silica gel column (24 x 1.4 cm), eluted using gradient of hexane-ethyl acetate in the following order of increasing polarity as indicated in Table 4.4, with fifty (50) fractions (100 mL each) collected and numbered 1 - 50. Table 4.4: Sub fractions from XI Solvent system Solvent volume Fraction collected Hexane 500 mL 1-5 Hexane - ethylacetate 95:5 500 mL 6-10 Hexane – ethylacetate 90:10 500 mL 11-15 Hexane – ethylacetate 85:15 500 mL 16-20 Hexane – ethylacetate 80:20 500 mL 21-25 Hexane – ethylacetate 75:25 500 mL 26-30 Hexane – ethylacetate 70:30 1L 31-40 Hexane – ethylacetate 60:40 500 mL 41-45 Hexane – ethylacetate 50:50 500 mL 46-50 The collected sub fractions (1 – 50) were concentrated in vacuo and combined according to their TLC (using solvent systems A - D) profiles. (Fig. 4.3) to yield Eight (8) sub fractions were obtained, labelled as XI1-XI8 (Fig. 4.3 and Table 4.4). P a g e 134 | 238 CHAPTER FOUR: HELICHYSUM NIVEUM Table 4.5: Sub fractions obtained upon fractionation of the main fraction XI Combined fraction Designated number Weight 26-31 XI-1 16 mg 32-35 XI-2 19 mg 36, 37 XI-3 34 mg 38 XI-4 47 mg 39-41 XI-5 119 mg 42 XI-6 257 mg 43-48 XI-7 209 mg 49-51 XI-8 49 mg Sub fraction XI-6 (250 mg) was chromatographed on sephadex column (5 % aqeuous ethanol). Fractions (1-24) collected, were developed on TLC (solvent system B), and combined as indicated in Figure 4.4. P a g e 135 | 238 CHAPTER FOUR: HELICHYSUM NIVEUM XI-6A, 189mg 12 3 4 5 6 7 8 9 10 11 12 13 13 14 15 16 17 18 19 20 21 22 23 24 25 Figure 4.4: TLC profile of the collected sub fractions XI-6 (1-24) after spraying with H2SO4/vanillin and then heated. *TLC plate comprised of fractions 1-24, using solvent system B Sub fraction XI-6A (105 mg) was injected to the HPLC and eluted using gradient solvent system of ACN and de-ionized water (60:40 to 80 % ACN in 30 min, then 100 % for 10 min). Three (3) promient peaks were collected, labelled as XI-6A-HPLC-1 (C11, Rt 25, 29 mg, 0.0073 %), XI6A-HPLC-3 (C15, Rt 34, 31 mg, 0.0075 %), and XI-6A-HPLC-4 (C16, Rt 37, 29 mg, 0.0073 %) as shown in Figure 4.5 XI-6A-HPLC-3 XI-6A-HPLC-1 XI-6A-HPLC-4 Figure 4.5: HPLC spectrum of compounds C11, C15 & C16 Solvent Column Flow rate Detection ACN:DIW 60:40 to 80 % in 30 min, then to 100 % in 10 min SUPELCO, RP-18 (25 X 2.1 cm) 1.5 mL/min UV at 254 nm P a g e 136 | 238 CHAPTER FOUR: HELICHYSUM NIVEUM 4.6.2 Isolation of compound C13 - Column chromatography of sub fraction XI-7 Sub fraction XI-7 (180 mg) was chromatographed on sephadex column (5 % aqeuous ethanol). Fractions (1-30) collected, were developed on TLC (using solvent system B). fracions of same TLC profile were combined as indicated in Figure 4.6 below. XI-7B, 62 mg 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Figure 4.6: TLC profile of the collected sub fractions XI-7 (16-30) after spraying with H2SO4/vanillin and then heated. *TLC plate comprised of fractions 16-30, using solvent system B Sub fraction XI-7B (52 mg) was injected to the HPLC and eluted using gradient solvent system of ACN and de-ionized water (75:25 to 90 % ACN in 30 min, then 100 % for 10 min). A peak was collected (Fig. 4.7), labelled as XI-7B-HPLC-1 (C13, Rt 17, 44 mg, 0.011 %). XI-7B-HPLC-1 Figure 4.7: HPLC spectrum of compound C13 *Conditions Solvent Column Flow rate Detection ACN:DIW 75:25 to 90 % in 30 min, then to 100 % in 10 min SUPELCO, RP-18 (25 X 2.1 cm) 1.5 mL/min UV at 254 nm P a g e 137 | 238 CHAPTER FOUR: HELICHYSUM NIVEUM 4.6.3 Isolation of compound C14 - Column chromatography of main fraction IX Sub fraction IX (305 mg) was chromatographed on sephadex column (5 % aqeuous ethanol). Fractions (1-24) collected, were developed on TLC (using solvent system B, Fig. 4.8) IX-B, 59 mg 1 2 3 4 5 6 7 8 9 10 11 12 13 14 14 15 16 17 18 19 20 21 22 23 24 Figure 4.8: TLC profile of the collected fractions IX (1-24) after spraying with H2SO4/vanillin and then heated. *TLC plate comprised of fractions 1-24, using solvent system B Sub fraction IX-B (50 mg) was injected to the HPLC and eluted using gradient solvent system of ACN and de-ionized water (60:40 to 80 % ACN in 30 min, then 100 % for 10 min). A peak was collected (Fig. 4.9), labelled as IX-B-HPLC-2 (C14, Rt 35, 23 mg, 0.0058 %). IX-B-HPLC-2 Figure 4.9: HPLC spectrum of compound C14 *Conditions Solvent Column Flow rate Detection ACN:DIW 60:40 to 80 % in 30 min, then to 100 % in 10 min SUPELCO, RP-18 (25 X 2.1 cm) 1.5 mL/min UV at 254 nm P a g e 138 | 238 CHAPTER FOUR: HELICHYSUM NIVEUM 4.6.4 Isolation of compounds C17 & C19 - Column chromatography of main fraction VII Main fraction VII (2.6 g) was chromatographed on silica gel column (Table 4.6). The collected sub fractions (1 – 55, 100 mL each) were concentrated in vacuo. Sub fractions obtained were developed and combined according to their TLC (using solvent systems D, Fig. 4.10) profiles to yield 8 sub fractions indicated in Figire 4.6. Table 4.6: Chromatographic fractionation of main fraction VII Solvent system Solvent volume Fraction collected Hexane 500 mL 1-5 Hexane - ethylacetate 95:5 500 mL 6-10 Hexane – ethylacetate 90:10 1L 11-20 Hexane – ethylacetate 85:15 500 mL 21-25 Hexane – ethylacetate 80:20 1L 26-35 Hexane – ethylacetate 75:25 500 mL 36-40 Hexane – ethylacetate 70:30 1.5 L 41-55 P a g e 139 | 238 CHAPTER FOUR: HELICHYSUM NIVEUM VII-5, 73 mg 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 FR 36 37 38 39 40 41 42 43 44 45 46 47 48 49 FR Figure 4.10: TLC profile of the collected fractions IX (1-24) after spraying with H2SO4/vanillin and then heated. *TLC plate comprised of fractions 1-24, using solvent system B Sub fraction VII-5 (72 mg) was injected to the HPLC and eluted using gradient solvent system of ACN and de-ionized water (70:30 to 90 % ACN in 20 min, then 100 % for 15 min). Two prominent peaks were collected (Fig. 4.11), labelled as VII-5-HPLC-1 (C17, Rt 22, 17 mg, 0.0043%), and VII-5-HPLC-3 (C19, Rt 25, 24 mg, 0.006 %). VII-5-HPLC-3 VII-5-HPLC-1 Figure 4.11: HPLC spectrum of compounds C17 & C19 *Conditions Solvent Column Flow rate Detection ACN:DIW 70:30 to 90 % in 20 min, then to 100 % in 15 min SUPELCO, RP-18 (25 X 2.1 cm) 1.5 mL/min UV at 254 nm P a g e 140 | 238 CHAPTER FOUR: HELICHYSUM NIVEUM 4.6.5 Isolation of compound C18 - Column chromatography of main fraction VI Main fraction VI (200 mg) was chromatographed on sephadex column (5 % aqeuous ethanol). Fractions collected were developed on TLC (using solvent system B), and combined to form VI (1–4). Sub fraction VI-3 (45 mg) was injected to the HPLC and eluted using gradient solvent system of ACN and de-ionized water (80:20 to 90 % ACN in 20 min, then 100 % for 15 min). A peak was collected, labelled as VI-3C2-HPLC-3 (C18, Rt 23, 21 mg, 0.0053 %). 4.6.6 Isolation of compounds C12 & C20 - Column chromatography of main fraction III Main fraction III (130 mg) was chromatographed on sephadex column (5 % aqeuous ethanol). Fractions collected were developed on TLC (using solvent system A), and combined to form III (A–C). Sub fraction III-A (51 mg) was injected to the HPLC and eluted using gradient solvent system of ACN and de-ionized water (75:25 to 90 % ACN in 30 min, then 100 % for 10 min). Two peaks (Fig. 4.12) were collected, labelled as IIIA-HPLC-1 (C20, Rt 22, 13 mg, 0.0033 %), and IIIA-HPLC-3 (C12, Rt 28, 15 mg, 0.0038 %). IIIA-HPLC-1 IIIA-HPLC-3 Figure 4.12: HPLC spectrum of compounds C12 & C20 *Conditions Solvent Column Flow rate Detection ACN:DIW 75:25 to 90 % in 30 min, then to 100 % in 10 min SUPELCO, RP-18 (25 X 2.1 cm) 1.5 mL/min UV at 254 nm P a g e 141 | 238 CHAPTER FOUR: HELICHYSUM NIVEUM 4.6.7 Isolation of compound C21 - Column chromatography of main fraction IV Main fraction IV (155 mg) was chromatographed on sephadex column (5 % aqeuous ethanol). Fractions collected when developed on TLC (using solvent system A, Fig. 4.13) afforded IVC (C21, 53 mg, 0.0133 %). IVC 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Figure 4.13: TLC profile of the collected fractions IV (1-32) after spraying with H2SO4/vanillin and then heated. *TLC plate comprised of fractions 1-32, using solvent system A P a g e 142 | 238 CHAPTER FOUR: HELICHYSUM NIVEUM HELICHRYSUM NIVEUM METHANOL EXTRACT (HF) OPEN COLUMN (SILICA GEL) VI IV III SEPHADEX IIIA 5 1` OH 1 3 6` 1` O 1 3` O 3 7 OH C12 HO 9 C18 11 7 4` XI-6 3` 7 1 9 11 O 6 1` OH SEPHADEX 1` 1 O 3 O 9 7 OH OH 11 OH 5 HO OH OH XI-7 HPLC FRACTIONATION (ACN/DIW) C14 C17 5 HO OH 1` 5 HO 9 1` C20 O 1 3 OH HO OH C21 C19 7 9 1` 1 O 3 9 7 OH HO OAc 1 O 3 OH 7 9 2 O 7 11 1` C11 11 4 8 3` OH OH 5 OH 6` OAc O OH 5 HO SILICA GEL COLUMN IXB HO OH OAc XI SEPHADEX VI-3 VII-5 HPLC FRACTIONATION (ACN/DIW) HO HO IX SILICA GEL COLUMN IVC 5 VII 10 11 O 3` C13 HO O 6 11 4` 1` 3` 7 C16 1 O OH C15 Scheme 4.1: A flow diagram of experimental procedure for the isolation of constituents from H. niveum *All silica gel column fractionations were done using gradient solvent system of Hex/EtOAc * All sephadex column fractionations were done using isocratic elution of aq. EtOH * Compounds isolated from same sub-fraction are indicated with same color box P a g e 143 | 238 CHAPTER FOUR: HELICHYSUM NIVEUM BIOLOGICAL CHARACTERIZATION OF HELICHRYSUM NIVEUM 4.7 General experimental procedure for biological assays 4.7.1 Reagents Standards (purity > 99.0%) for acetylcholinesterase assay as galanthamine, EGCG, acetylthiocholineiodide (ACTI), 5,5’-Dithiobis-(2-nitrobenzoic acid) and tris-HCl. All other reagents described in section 3.7.1 were secured from Sigma-Aldrich, Inc. (St. Louis, MO, USA). 4.7.2 Antioxidant assays All experimental procedure for the various antioxidant capacity assays (FRAP, ORAC, and TEAC), and inhibition of Fe2+-induced lipid peroxidation as described in section 3.7.2, were followed. 4.7.3 Skin enzyme inhibitory assays Skin enzymes inhibitory assays described in section 3.7.3 were carried out. 4.7.4 Acetylcholinesterase inhibition assay Acetylcholinesterase (AChE) inhibitory activity was measured using Ellman’s method as previously described (Zengin, et al., 2014) with little modification. AChE catalysis the hydrolysis of acetythiocholine iodide (ACTI as substrate) to thiocholine which can then react with Ellman’s reagent (DTNB) to produce 5-thio-2-nitrobenzoate (yellow colour). In the presence of samples (inhibitors), the release of 5-thio-2-nitrobenzoate (yellow colour) is reduced and it is monitored by measuring the absorbance at 450 nm. The samples (isolated compounds C11-C21, and total extract HF) were weighed and dissolved in DMSO (1 mg/mL, w/v) as stock solution. Then the samples were diluted to 500, 250, 100, 50, 10 P a g e 144 | 238 CHAPTER FOUR: HELICHYSUM NIVEUM and 1 µg/mL concentrations in tris-HCl buffer as working solution. Micro molar (µM) concentrations for the tested samples (C11-C21 and HF) could not be calculated, since the molecular weights of the compounds were not yet known at the time of carrying out this assay. In a 96 Well plate, the reaction mixture contains 50 µL of samples pre-incubated with 125 µL of 3 mM 5,5’-Dithiobis-(2-nitrobenzoic acid), and 25 µL AChE (2.0 U/mL), in 50 mM tris-HCl buffer (pH 8.0), with 0.1 % bovine serum albumin (BSA) for 15 minutes at 25 oC. Subsequently, 25 µL (15 mM) ACTI was added to the incubation mixture and further incubated for 10 minutes at 25 o C. Sample control was prepared by adding sample solution to all reagents without AChE, blank contained tris-HCl in the presence of AChE, while galanthamine was used as reference. Absorbance was recorded at 450 nm. The percent AChE inhibitory activity is given by: [A0 – (B – C)] / A0 X 100 (4) Where A0 = full enzymatic reaction; B = Activity in the presence of sample; C =Activity without sample. 4.8 Statistical analysis Statistical analyses described in section 3.8 was followed P a g e 145 | 238 CHAPTER FOUR: HELICHYSUM NIVEUM 4.9 Chemical evaluations: Results and discussion 4.9.1 Summary of the isolated compounds The presence of terpenes and high amount of phenolics was first recognised by preliminary thin layer chromatographic screening of a methanol extract (Fig. not shown). Ten phloroglucinols derivatives (C11-C20) and a triterpene (C21) were isolated from H. niveum. Their chemical structures were elucidated by extensive analyses of spectroscopic data (Tables 4.7-4.9) as well as correlations with published data on literature. Three of the isolated compounds, helinivenes A-C (C11-C13) were reported for the first time. The total antioxidant capacities measured as FRAP, TEAC, ORAC and the inhibition of Fe (II)-induced microsomal lipid peroxidation ability of the total extract and its constituents are presented in Tables 4.10 & 4.11, and Figure 4.26, while the inhibition of aging-related enzymes measured using mushroom tyrosinase, elastase from pancreas porcine and acetylcholinesterase are presented in Figures 4.27 & 4.28, and Table 4.11. 4.9.2 Spectroscopic data of the isolated compounds C11-C13 Compound C11, obtained as yellow amorphous powder; UVλmax (MeOH) nm: 300; IR (KBr) cm1 3300, 1800, 1680, 1410, 990. HRMS m/z 357.1330 [M+1]+ (calcd: 356.1260). 1H- and 13C-NMR data, see Table 4.8. Full NMR spectra (1H- and 13C-, HSQC and HMB) are attached as Annexure II, Figures 1-4. Compound C12 was obtained as a yellow amorphous powder; UV λmax (MeOH) nm: 220, 280; IR (KBr) cm-1: 3300, 3100, 1800, 1410, 990. HRMS m/z 299.1220 [M+1]+ (calcd. 298.1205). 1H- and 13 C-NMR data, see Table 4.8. Full NMR spectra (1H- and 13C-, HSQC and HMB) are attached as Annexure III, Figures 1-4. P a g e 146 | 238 CHAPTER FOUR: HELICHYSUM NIVEUM Compound C13, obtained as a pale amorphous powder; [α]25 D = +0.039 (MeOH); UV λmax (MeOH) nm: 314; IR (KBr) cm-1: 3300, 1800, 1735, 1330, 990+750. HRMS m/z 279.1596 [M+1]+ (calcd. 298.1205). 1H- and 13 C-NMR data, see Table 4.7. Full NMR spectra (1H- and 13 C-, HSQC and HMB) are attached as Annexure IV, Figures 1-4. Table 4.7: 1H (400 MHz: m, J Hz) and 13C (100 MHz) NMR spectral data of compound C13 in CD3COCD3 13C 1H 1 - - 2 27.09 - 3 79.98 3.81br t 6.3 4 69.20 2.87 dd (5.5, 16.6); 2.51 dd (7.4, 16.6) 5 157.24 - 6 163.43 5.99 s 7 167.01 - 8 105.64 - 9 96.55 - 10 100.61 - 11 20.30 1.37 s 12 20.20 1.45 s 1’ 211.13 2’ 40.26 3’ 26.12 4’ 21.13 3.92 sep 6.7 1.15 d 6.7 P a g e 147 | 238 CHAPTER FOUR: HELICHYSUM NIVEUM Table 4.8: 1H (400 MHz: m, J Hz) and 13C (100 MHz) NMR spectral data of compounds C11, C12, C16, C18 & C19 in CD3COCD3 C11 C12 C16 C18 C19 13C 1H 13C 1H 13C 1H 13C 1H 13C 1H 1 104.5 s - 104.9 s - 105.1 s - 105.4 s - 104.3 s - 2 163.4 s - 163.2 s - 165.2 s - 165.4 s - 165.2 s - 3 106.4 s - 106.5 s - 107.9 s - 107.0 s - 108.0 s - 4 163.2 s - 161.2 s - 160.5 s - 162.8 s - 162.5 s - 5 96.0 d 6.07 s 96.6 d 6.03 s 95.0 d 6.05 s 95.3 d - 95.1 d 6.06 s 6 160.0 s - 160.1 s - 162.6 s - 161.1 s - 160.1 s - 7 21.91 t 3.38 d, 7.5 28.9 t 3.02 dd, 15.8, 2.0 22.0 t 3.22 d, 6.5 22.1 t 3.45 d, 7.6 22.1 t 3.23 d, 6.8 8 129.2 d 5.54 t, 7.5 77.7 d 4.34 br d, 7.8 124.3 d 5.20 t, 6.5 129.6 d 5.51 t, 7.6 124.2 d 5.21 t, 6.8 9 130.4 s - 148.8 s - 130.8 s - 130.4 s - 130.8 s - 10 63.7 t 4.82 s 110.8 t 4.97 s, 4.79 s 26.0 q 1.61 s 64.0 t 4.82 s 17.9 q 1.73 s 11 21.6 q 1.71 s 19.0 q 1.81 s 17.9 q 1.72 s 21.8 q 1.71* 25.9 q 1.62 s 1` 200.3 s - 200.1 s - 206.5 s - 206.9 s - 210.9 s - 2` 143.0 s - 143.4 s - 46.5 t 3.04 t, 7.4 46.8 t 3.08 t, 7.2 39.7 d 3.98 sept, 6.8 3` 128.4 d 7.59 m 128.9 d 7.49 br. d, 7.2 19.0 t 1.67 sext, 7.4 19.2 t 1.71* 19.8 q 1.12 s 4` 129.1 d 7.38 m 129.6 d 7.40 br. t, 7.6 14.4 q 0.94 t, 7.4 14.6 q 0.99 t, 7.2 19.8 q 1.12 s 5` 131.6 d 7.47 m 132.4 d 7.45 br t, 7.6 - - - - - - 6` 129.1 d 7.38 m 129.6 d 7.40 br. t, 7.6 - - - - - - 7` 128.4 d 7.59 m 128.9 d 7.49 br. d, 7.2 - - - - - - CO 171.3 s - - - - - 171.5 s - -- - CH3 20.9 q 2.02 s - - - - 21.1 q 2.07 s - - 2-OH - 12.27 - - - 14.07 s - 14.05 - 14.13 s 4-OH - 9.28 S - - - 9.31 br s - - - 9.50 s 6-OH - 8.99 - - - - - - - 5.61 2.77 dd, 15.8, 7.8 *overlapped signals. P a g e 148 | 238 CHAPTER FOUR: HELICHYSUM NIVEUM Table 4.9: 1H (400 MHz: m, J Hz) and 13C (100 MHz) NMR spectral data of isolated compounds C14, C15, C17 & C20 in CDCl3 C14 C15 C17 C20 13C 1H 13C 1H 13C 1H 13C 1H 1 104.5 s - 104.0 s - 105.7 s - 104.0 s - 2 164.6 s - 164.7 s - 162.6 s - 163.5 s - 3 95.1 d 5.91 s 95.1 d 5.91 s 104.7 s - 105.8 s - 4 164.6 s - 164.7 s - 159.9 s - 160.7 s 5 95.1 d 5.91 s 95.1 d 5.91 s 95.4 d 5.83 s 95.3 d 5.88 s 6 164.6 s - 164.7 s - 160.7 s - 159.4 s - 7 210.1 s - 210.2 s - 21.6 t 3.32 d, 7.0 21.0 t 3.42 d, 7.0 8 45.9 d 3.70 sext , 6.4 39.3 d 3.83 sept, 6.4 121.6 d 5.21 t, 7.0 128.9 d 5.38 t, 7.0 9 26.9 t 1.38 m 19.2 q 1.59 d, 6.4 135.9 s - 129.9 s - 10 11.9 q 1.14 d, 6.4 19.2 q 1.59 d, 6.4 25.8 q 1.73 s 64.1 t 4.73 s 11 16.6 q 0.89 t, 7.6 - - 17.9 q 1.78 s 21.1 q 1.70 s 1` 65.1 t 4.47 d, 6.7 65.1 t 4.47 d, 6.7 210.8 s - 210.8 s - 2` 118.7 d 5.42 t, 6.7 118.7 d 5.42 t, 6.7 45.9 d 3.74 sext, 6.8 39.2 d 3.89 sept, 6.8 3` 139.2 s - 139.2 s - 26.9 t 1.37 m 19.3 q 1.43 s 4` 18.2 q 1.77 s 18.2 q 1.77 s 11.9 q 0.88 t, 7.2 19.3 q 1.43 s 5` 25.8 q 1.71 s 25.8 q 1.71 s 16.9 q 1.13 d, 6.8 - - 6` - - - - - - - - 7` - - - - - - - - CO - - - - - - 172.6 s - CH3 - - - - - - 21.2 q 2.05 s 2-OH - - - 9.92 s - - - - 4-OH - - - - - - - - 6-OH - - - 9.92 s - - - - P a g e 149 | 238 CHAPTER FOUR: HELICHRYSUM NIVEUM 4.9.3 Analysis of compound C11 Compound C11 was obtained as yellow amorphous powder. It showed UV (MeOH) spectrum characteristics of phloroglucinol derivative (λmax 300 nm), IR (KBr) spectrum showed absorption bands due to hydroxyl (3300 cm-1), carbonyl (1800 cm-1), isolated C=C (1680 cm1 ), aromatic (1410 cm-1), and C-H of m-trisubstituted (990 cm-1) benzene ring. HRMS indicated molecular formular of C20H20O5 and [M+1]+ 357.1330. NMR showed signals corresponding to phloroglucinol skeleton (Table 4.8), benzoyl group and a prenyl group. The 1H NMR is very similar with 1-benzoyl-3-prenyl phloroglucinol (Bohlmann & Suwita, 1978). The only difference we observed between NMR data of C11 to that of previous data is the presence of a signal at 4.82 (2H, s) and acetyl group which indicate the hydroxylation of a methyl group from the prenyl side chain of C11. The low field shift of the CH2 at 4.82 indicated acetylation. This fact is supported by the absence of the second olefinic methyl terminal of the prenyl group. HMBC correlation showed correlations H-8/C-10, C-11, C-7, C-3 and H-11/C-9, C-10, and C8. Other 2D NMR spectra attached as Annxure II, Figures 1-4, supported the structure given in Figure 4.14 as 1-benzoyl-3 (3-methyl-2-butenylacetate)-phloroglucinol denoted by C11. 5 HO OH 6` 1` 3` O 1 3 OH 7 9 OAc 11 C11 Figure 4.14: Chemical structure of 1-benzoyl-3 (3-methyl-2-butenylacetate)-phloroglucinol (heinivene A) P a g e 150 | 238 CHAPTER FOUR: HELICHRYSUM NIVEUM 4.9.4 Analysis of compound C12 Compound C12 was obtained as a yellow amorphous powder, with UV spectrum characteristics of phloroglucinol derivative (λmax 220, 280 nm). Its IR (KBr) spectrum showed bands of a hydroxyl group (3300 cm-1), terminal C=C (3100 cm-1), carbonyl (1800 cm-1), aromatic system (1410 cm-1), and C-H m-trisubstituted (990 cm-1) benzene ring. HRMS indicated the molecular formula of C18H18O4 with [M+1]+ 299.1220. The NMR of C12 is very similar to compound C11 except the presence of signals of terminal olefinic methylene group at H 4.97, 4.79 each singlet (C 109.6, 147.6) and the absence of signals of H-8 olefinic proton and H-10 acetylated methylene group (Table 4.8). The HMBC correlations (Annexure III, Figure 4) of C12 supported the structure as shown in Figure 4.15 and showed correlations between H-11 / C-9, C-10, C-8 andH-7/C-3,C-4, C-5 (among others). Other 2D spectra (Annexure III, Figures 1-4) supported the proposed structure given in Figure 4.15 as 1-benzoyl3 (2-hydroxyl-3-methyl-3-butene-1-yl)-phloroglucinol, denoted by C12. 5 HO OH OH 6` 1` 1 3 3` O OH 7 9 11 C12 Figure 4.15: Structure of 1-benzoyl-3 (2-hydroxyl-3-methyl-3-butene-1-yl)-phloroglucinol (helinivene B) 4.9.5 Analysis of compound C13 Compound C13 was obtained as a pale amorphous powder. It showed similar spectroscopic data of phloroglucinol derivatives like compounds C11 and C12. Compound C13 showed UV spectra characteristics of phloroglucinol derivative (λmax 314 nm). The IR (KBr) spectra showed bands of a hydroxyl group (3300 cm-1), carbonyl (1800 cm-1), C-O lactone (1735 cm1 ), aromatic system (1330 cm-1), and m-disubstituted (990,750 cm-1) benzene ring. The P a g e 151 | 238 CHAPTER FOUR: HELICHRYSUM NIVEUM molecular formula of C13 was determined to be C15H20O5 by HRMS analysis with m/z 279.1596. The NMR data showed signals of 1-methyl-1-propanone side chain (Table 4.7), an aromatic proton at 5.99 s, and two oxygenated carbons at 80.0, 69.2, one of them showed a proton at δH 3.81, cyclic methylene group at δH 2.87, 2.51. The above data and the absence of olefinic double bond at C-8 (of C11) indicated the formation of a pyrane ring between C-3 (of the prenyl group) and hydroxyl at C-3 of the phloroglucinol nucleus. Furthermore, the compound showed similar 1H-NMR spectra to that of 8-benzoyl-3,5,7-trihydroxy-2,2dimethoxychroman isolated from Leontonyx spathulatus (Bohlmann & Suwita, 1978). The only difference is the nature of the side chain at C-8. The HMBC showed correlations between H-4/C-9, C-5, C-8, C-2, C-3; H-6/C-5, C-7, C-9 C-1 and H-2’/C-1. Other 2D spectra, thereby supported the structure of C13 as 8-(2-methyl-1-propanone)-3,5,7-trihydroxyl-2,2- dimethoxychromone (Fig. 4.17). All NMR spectral used for structural identification of compound C13 were attached as Annexure IV, Figures 1-4. The absolute configuration at C-3 could not be ascertained directly from the data obtained however, the coupling constant between hydrogens of C-3 and C-4 indicated e-OH position accordingly, one of the following tautomer (in Fig. 4.16) can be proposed O H C-3a O or H H OH C-3b OH H Figure 4.16: Possible absolute configurations of C13 at C-3 Comparing the [α]25 D (+0.039) value of compound C13 with similar structure “containing identical stereo centre” and the coupling constant values of H-3 and H-4 indicated 3S isomer of C13 (Lim, et al., 2001). P a g e 152 | 238 CHAPTER FOUR: HELICHRYSUM NIVEUM OH OH 5 9 4 2 8 HO 10 O 7 1` 11 O 3` C13 Figure 4.17: Chemical structure of 8-(2-methyl-1-propanone)-3,5,7-trihydroxyl-2,2 Dimethoxychromone (helinivene C) 4.9.6: Analysis of compounds C14 and C15 Compounds C14 and C15 showed very similar spectra. 1H NMR showed signals of two aromatic protons at δH 5.91 s, and a typical signals of O-prenyl group. The only difference between C14 and C15 is the side chain at C-1, in case of compound C14, IH NMR showed a typical 2-methylbutanone, while compound C15 showed 2-methylpropanone (Table 4.9). Compound C14 was identified as 1-(2-methylbutanone)-4-O-prenyl-phloroglucinol (Fig. 4.18) and compound C15 as 1-(2-methylpropanone)-4-O-prennyl-phloroglucinol (Fig. 4.19). Both compounds C14 and C15 were previously isolated from H. crispum (Bohlmann & Suwita, 1979b). HO O 6 4` 1` 3` 7 1 O OH C14 Figure 4.18: Chemical structure of 1-(2-methylbutanone)-4-O-prenyl-phloroglucinol HO O 6 4` 1` 3` 7 1 O OH C15 Figure 4.19: Chemical structure of 1-(2-methylpropanone)-4-O-prenyl-phloroglucinol P a g e 153 | 238 CHAPTER FOUR: HELICHRYSUM NIVEUM 4.9.7: Analysis of compounds C16 – C20 Compound C16 showed a typical NMR signals like that of 1-(butanone)-3-prenylphloroglucinol (Jakupovic, et al., 1989b), while compound C17 identified as 1-(2methylbutanone)-3-prenyl-phloroglucinol (Fig. 4.21). Both compounds (C16 and C17) were isolated previously from H. gymnoconum (Bohlmann & Mahanta, 1979). Compound C18 identified as 1-butanone-3-(3-methyl-2-butenyl-acetate)-phloroglucinol (Jakupovic, et al., 1989b). Compound C19 identified as 1-(2-methylpropanone)-3-prenylphloroglucinol and previously isolated from H. mimetes (Jakupovic, et al., 1986) and compound C20 identified as caespitate, was first isolated from H. caespititium (Jakupovic, et al., 1986). 1H and 3C NMR spectra data of C16 - C20 are shown in Tables 4.8, 4.9. 5 HO 1` 1 O OH 3 7 OH 9 11 C16 Figure 4.20: Chemical structure of 1-(butanone)-3-prenyl-phloroglucinol 5 HO 1` O 1 OH 3 OH 7 9 11 C17 Figure 4.21: Chemical structure of 1-(2-methylbutanone)-3-prenyl-phloroglucinol P a g e 154 | 238 CHAPTER FOUR: HELICHRYSUM NIVEUM 5 HO 1` OH OAc 1 3 O 9 7 OH 11 C18 Figure 4.22: Chemical structure of 1-butanone-3-(3-methyl-2-butenyl-acetate)phloroglucinol 5 HO 1` OH 1 3 O 9 7 OH 11 C19 Figure 4.23: Chemical structure of 1-(2-methylpropanone)-3-prenylphloroglucinol 5 HO 1` O OH OAc 1 3 OH 7 9 11 C20 Figure 4.24: Chemical structure of caespitate 4.9.8: Analysis of compound C21 Compound C21 was obtained as white solid and is the only alcohol triterpene detected in this study. Previous study indicated the occurrence of C21 in Erythroxylum passerium, identified as 3β-24-dihydroxyterexer-14-ene (Barreirosa, et al., 2002). According to SciFinder and the dictionary of natural products, occurrence of compound C21 is reported in our findings for the first time. P a g e 155 | 238 CHAPTER FOUR: HELICHRYSUM NIVEUM HO OH C21 Figure 4.25: Chemical structure of 3β-24-dihydroxyterexer-14-ene 4.10 Biological evaluations: Results and discussion 4.10.1 Evaluating the ORAC activities of the isolated compounds Chapter 2 established the occurrence of Helichrysum species that are generally rich in phenolic compounds, especially with mono- and bicyclic aromatic rings (88-216), bearing one or more hydroxyl groups per molecule. These phenolic compounds were found to exhibit good antioxidant activities acting as hydrogen donors and or electron donating agents, and metal ionchelators. Our findings also noticed that the greater the number of hydroxyl groups in these phenolic compounds, the higher is the antioxidant activity. Hydroxyl radicals are an extremely reactive oxygen species, capable of modifying almost every molecule in the living cell. Moreover, hydroxyl radicals are capable of quick initiation of the lipid peroxidation process by abstracting hydrogen atoms from unsaturated fatty acids (Kang, et al., 2011). Compounds C11 & C12 in comparison with (EGCG), displayed excellent hydroxyl radical absorbance capacity (64.85 ± 10.95; 94.97 ± 5.88; 3.862 ± 4.65) X 106 µM TE/g, respectively. The same trend of high activity for compounds C11 & C12 than EGCG in peroxyl radical absorbance capacity assay was observed (22671.78 ± 26.72; 45095.82 ± 31.99; 14693 ± 5.53) µM TE/g respectively, while compounds C16 – C20 possess mild peroxyl absorbance capacity P a g e 156 | 238 CHAPTER FOUR: HELICHRYSUM NIVEUM (range 13544 - 16735 µM TE/g), and hydroxyl absorbance capacity (range 16.26 - 24.24) X 106µM TE/g respectively in Table 4.10. The results of Table 4.10 are in support of previous reports on structure-activity relationship of antioxidant activities of monomeric phenolic compounds, polyphenols, prenylated coumarins and phloroglucinol derivatives, where their respective antioxidants were found to depend on the degree of hydroxylation and extent of conjugation (Yang, et al., 2005; Jangu, 2012; Queguineur, et al., 2012). The potential antioxidant activities of compounds C11 & C12, as well as moderate antioxidant activities of C16 - C20 may have been attributed to the presence of hydroxyl groups which can act as good hydrogen donors, thereby forming phenolate ions as intermediates. These intermediates are stabilized by resonance, when unpaired electrons at the m- positions of the aromatic ring are delocalized (Queguineur, et al., 2012). P a g e 157 | 238 CHAPTER FOUR: HELICHRYSUM NIVEUM Table 4.10: Oxygen radical absorbance capacity of H. niveum constituents Sample Peroxyl Hydroxyl X 106 Prooxidant µMTE/g HF 4553.13 ± 17.77 53.77 ± 8.42 17.298 ± 0.99 C11 22671.78 ± 26.72 64.85 ± 10.95 8.921 ± 1.15 C12 45095.82 ± 31.99 94.97 ± 5.88 756.9 ± 1.98 C13 3937.78 ± 25.85 5.99 ± 4.52 41.732 ± 8.20 C14 5406.65 ± 1.39 3.92 ± 14.47 7.023 ± 4.02 C15 6053.87 ± 18.67 5.60 ± 0.79 12.330 ± 6.62 C16 13544.93 ± 12.13 18.07± 3.47 64.376 ± 0.22 C17 14139.74 ± 5.96 23.12 ± 18.94 73.871 ± 1.10 C18 14218.17 ± 12.36 16.70 ± 1.25 76.591 ± 0.27 C19 13639.27 ± 17.62 24.24 ± 1.71 69.543 ± 0.29 C20 16735.55 ± 21.72 16.26 ± 15.39 92.311 ± 1.59 C21 1193.10 ± 1.68 0.895 ± 7.34 31.38 ± 7.00 EGCG 4970.09 ± 5.53 0.39 ± 4.65 6.483 ± 1.19 EGCG: Epigallocatechingallate; HF: methanol extract of H. niveum; C11-C21: Isolated compounds from H. niveum 4.10.2 Evaluating the FRAP and TEAC activities of the isolated compounds To further elucidate the mechanism of ation of the isolated compounds, we investigate whether our compounds can have the ability of transferring electron to free radicals by measuring their antioxidant capacities on FRAP and TEAC. The results in Table 4.11 shows as expected, that phloroglucinols containing aromatic acyl groups (C11 & C12) possessed higher antioxidant (ORAC, FRAP, TEAC) activities relative to their monocyclic (C14-C20), and bicyclic (C13) P a g e 158 | 238 CHAPTER FOUR: HELICHRYSUM NIVEUM counterparts with aliphatic acyl substitutions. The observed tendencies demonstrated by these compounds are in accordance with data reported in the literature (Sun, et al., 2014; Roleira, et al., 2010). It is evident therefore that the isolated compounds C11 and C12 possessed interesting antioxidant activities, expressed by their trolox (TEAC, ORAC) and ascorbic acid (FRAP) equivalents. Table 4.11: Ferric ion reducing and trolox equivalent absorbance capacities and antiacetylcholineesterase (AChE) inhibitory capacity of H. niveum constituents Sample HF FRAP µMAAE/g 437.64 ± 6.86 TEAC µMTE/g 1449.54 ± 3.09 Anti-AChE activity IC50 (µg/mL) 257.98 ± 4.01 C11 2530.54 ± 0.92 19545.00 ± 10.25 267.96 ± 3.88 C12 4950.08 ± 0.65 43615.73 ± 6.66 267.96 ± 1.15 C13 155.65 ± 12.02 6001.40 ± 5.63 272.95 ± 4.61 C14 194.27 ± 1.78 1629.10 ± 2.03 272.95 ± 1.67 C15 446.64 ± 8.07 2673.62 ± 1.68 252.99 ± 2.41 C16 1183.52 ± 6.20 4316.61 ± 1.06 252.99 ± 2.13 C17 1029.03 ± 0.66 9998.71 ± 2.66 260.48 ± 5.04 C18 1203.02 ± 2.07 6757.40 ± 4.69 248.00 ± 5.41 C19 1096.01 ± 1.12 4423.32 ± 3.11 250 50 ± 2.49 C20 1019.28 ± 1.79 8705.14 ± 1.83 250.50 ± 2.21 C21 197.99 ± 3.90 1323.61 ± 1.76 - EGCG 3326.45 ± 5.76 11545.44 ± 17.28 - - 10.981 ± 1.03 Galanthamine - AChE: acetylcholinesterase; HF: methanol crude extract; C11-C21: Isolated compounds from H. niveum P a g e 159 | 238 CHAPTER FOUR: HELICHRYSUM NIVEUM 4.10.3 Evaluating the anti-lipid peroxidation activities of the isolated compounds Over production of ROS results in an attack of not only DNA, but also other cellular components including the PUFAs, which are highly sensitive to oxidation (Lee, et al., 2013). Therefore, unsaturated fatty acids in cell membranes are susceptible to free-mediated oxidation. Compounds C11 & C12 demonstrated potent inhibitory activity against the Fe2+-induced lipid peroxidation (IC50 = 5.115 ± 0.90; 3.553 ± 1.92) µg/mL respectively (Fig. 4.26) in a competitive manner to that of EGCG (IC50 1.044 µg/mL). This result is in agreement with previous work, where phloroglucinol were found to possess similar Fe2+ induced inhibition against lipid peroxidation to that of BHA and α-tocopherol (Lee, et al., 2003). Our results further explain how the poly-hydroxylated ring A of C11 & C12 can be an active anti-lipid peroxidation skeleton. However, it is possible that the second aromatic ring in C11 & C12 can enhance the contact of these antioxidants with lipid, and consequently resulting in an efficient termination of the chain reaction. Figure 4.26: Effects of H. niveum constituents on inhibition of Fe2+ induced microsomal lipid peroxidation. (p<0.05) *Data are expressed as IC50 with isolated compounds and a methanol extract (HF) screened at 50.00 µg/mL P a g e 160 | 238 CHAPTER FOUR: HELICHRYSUM NIVEUM 4.10.4 Evaluating the anti-tyrosinase activities of the isolated compounds Moderate inhibition of tyrosinase activity demonstrated by these compounds (most especially, C11 & C12, Fig. 4.27), might depend on the substitution pattern of the hydroxyl groups. We expect such anti-tyrosinase activity, due to the non-existence of the catechol group, which may form hydrogen bonds to a chelating site of the enzyme (Prasad, et al., 2009; Chang, 2009). We therefore made further attempts were made to correlate the chemistry of phenol-metal chelation between Fe2+ (in lipid peroxidation assay) to that of Cu2+ (in tyrosinase), but the anti-tyrosinase result indicated that kojic acid is eight times more active than C11 and C12. We therefore proposed that the mechanism of Fe2+ chelation in the lipid peroxidation assay may be totally different from that of Cu2+ chelation in the tyrosinase assay, despite both metal ions (Fe2+ and Cu2+) being bivalent in nature. Figure 4.27: Effects of H. niveum constituents on inhibition of anti-tyrosinase activity (p<0.05) *Data are expressed as IC50 with isolated compounds and methanol extract (HT) screened at 100.00 µg/mL P a g e 161 | 238 CHAPTER FOUR: HELICHRYSUM NIVEUM 4.10.5 Evaluating the anti-elastase activities of the isolated compounds Our results in Figure 4.28 demonstrated a less potent inhibition against elastase (with IC50 ranged 35.469 – 69.611 µg/mL), in comparison to oleanolic acid (IC50 10.080 µg/mL). No scientific report has pointed out the reason for such activity, except previous data indicating a less potent activity of non-prenylated acylphloroglucinol (isobutyropenone) against inhibition of the release of leukocyte elastase (Feisst, et al., 2005). Figure 4.28: Effects of H. niveum constituents on inhibition of anti-elastase activity (p<0.05) *Data are expressed as IC50 with isolated compounds and methanol extract (HF) screened at 100.00 µg/mL None of the compounds demonstrated activity against acetylcholinesterase (Table 4.11) except at a high concentration of 250.0 µg/mL, possibly due to the absence of nitrogen atomcontaining compounds in our isolated products (Colovic, et al., 2013). P a g e 162 | 238 CHAPTER FOUR: HELICHRYSUM NIVEUM 4.11 Conclusion Fractionation of a methanol extract of HF was arried out using standard chromatographic methods. A total of eleven pure compounds (C11-C21) were isolated fro which only three (3) were new. Their chemical structures were established by spectroscopic methods. Several reports have convincingly shown a close relationship between antioxidant activity and the structural activity of phenolic compounds. On the basis of our findings, the prerequisite for a strong antioxidant activity assessed in these assays was the presence of an aromatic acylsubstituent, which may be valuable as a potential antioxidant and lipid peroxidation preventer, in the absence of prooxidant behaviour. However, we had fully established the mode of actions through which the isolated compounds behave as good source of antioxidant and moderate anti-tyrosinase and anti-elastase activities demonstrated by the Helichrysum niveum constituents. There is no synergistic effects between the methanol extract (HF) and the isolated compounds. No significant observation made for anti-acetylcholineesterase activity for all samples. All results indicated in Figures 4.26-4.28 are very good dose-response. This study is the first report on antioxidant and skin enzyme inhibitory activity of a methanol extract of H. niveum and its constituents. The anti-elastase activity demonstrated thereof can be considered significant and informative as a guide for further studies. P a g e 163 | 238 CHAPTER FOUR: REFERENCES REFERENCES Barreirosa, M.L., Davida, J.M., de Pereiraa, R.A., Guedesb, M.L.S. and David, J.P. (2002). Fatty acid esters of triterpenes from Erythroxylum passerinum. Journal of Brazilian Chemical Society, 13(5), pp. 669-673. Bohlmann, F., Abraham, W.R. (1979a). Polyacetylenic compounds. 250. New chlorosubstituted acetylenic thiophenes with unusual structures from Helichrysum species. Phytochemistry, 18, 839-842. Bohlmann, F., Abraham, W.F. (1979b). Neue diterpene ud weitere inhaltsstoffe aus Helichrysum calliconum und Helichrysum heterolasium. Phytochemistry, 18, 889-891. Bohlmann, F. and Mahanta, P.K. (1979). Further phloroglucinol derivatives from Helichrysum gymnoconum. Phytochemistry, 18(2), pp. 348-350. Bohlmann, F. and Suwita, A. (1978). 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Phloroglucinol derivatives and other constituents from South African Helichrysum species. Phytochemistry, 25, 1133-1142. Jakupovic, J., Zdero, C., Grenz, M., Tsichritzis, F., Lehmann, L., Hashemi-Nejad, S.M., Bohlmann, F. (1989). Twenty-one acylphloroglucinol derivatives and further constituents from South African Helichrysum species. Phytochemistry, 28, 1119-1131. Kang, S-M., Lee, S-H., Heo, S-J., Kim, K-N. and Jeon, Y-J. (2011). Evaluation of antioxidant properties of a new compound, pyrogallol-phloroglucinol-6,6'-bieckol isolated from brown algae, Ecklonia cava. Nutrition Research Practice, 5(6), pp. 495-502. Lee, D.-S., Cho, Y.-S. and Je, J.-Y. (2013). Antioxidant and antibacterial activities of chitosan-phloroglucinol conjugate. Fisheries and Aquatic Sciences, 16(4), pp. 229-235. Lee, S-M., Na, M-K., An, R-B., Min, B-S. and Lee, H-K. (2003). Antioxidant activity of two phloroglucinol derivatives from Dryopteris crassirhizoma. Biological & Pharmaceutical Bulletin, 26(9), pp. 1354-1356. Lim, J., Kim, I-H., Kim, H.H., Ahn, K-S. and Han, H. (2001). Enantioselective syntheses of decursinol angelate and decursin. Tetrahedron Letters, 42, pp. 4001-4003 Prasad, K.N., Yang, B., Yang, S., Chen, Y., Zhao, M., Ashraf, M. and Jiang, Y. (2009). Identification of phenolic compounds and appraisal of antioxidant and antityrosinase activities from litchi (Litchi sinensis sonn.) seeds. Food Chemistry, 116, pp. 1-7. P a g e 165 | 238 CHAPTER FOUR: REFERENCES Queguineur, B., Goya, L., Ramos, S., Martin, A.M., Mateos, R. and Bravo, L. (2012). Phloroglucinol: Antioxidant properties and effects on cellular oxidative markers in human HepG2 cell line. Food and Chemical Toxicology, 50, pp. 2886-2893. Rios, J.L., Recio, M.C., Villar, A. (1991). Isolation and identification of the antibacterial compounds from Helichrysum stoechas. Journal of ethnopharmacology, 33, 51-55. Roleira, M.F.F., Siquet, C., Orru, E., Garrido, M., Garrido, J., Milhazes, N., Podda, G., Paiva-Martins, F., Reis, S., Carvalho, A.R., Tavares da Silva, J.E. and Borges, F. (2010). Lipophilic phenolic antioxidants: correlation between antioxidant, partition coefficients and redox properties. Bioorganic & Medicinal Chemistry, 18, pp. 5816-5825. Sun, Q., Schmidt, S., Tremmel, M., Heilmann, J. and Konig, B. (2014). Synthesis of natural-like acylphloroglucinols with anti-proliferative, anti-oxidative and tube-formation inhibitory activity. European Journal of Medicinal Chemistry, 85, pp. 621-628. Taglialatela-Scafati, O., Pollastro, F., Chianese, G., Minassi, A., Gibbons, S., Arunotayanun, W., Mabebie, B., Ballero, M., Appendino, G. (2013). Antimicrobial phenolics and unusual glycerides from Helichrysum italicum subsp. microphyllum. Journal of Natural Products, 76, 346-353. Tomas-Lorente, F., Iniesta-Sanmartin, E., Tomas-Barberan, F.A. (1989). TrowitzschKienast, W.; Wray, V. Antifungal phloroglucinol derivatives and lipophilic flavonoids from Helichrysum decumbens. Phytochemistry, 28, 1613-1615. Yang, H., Protiva, P., Gil, R.R., Jiang, B., Baggett, S., Basile, J.M., Reynertson, A.K., Weinstein, B. and Kennelly, J.E. (2005). Antioxidant and cytotoxic isoprenylated coumarins from Mammea americana. Planta Medica, 71(9), pp. 852-860. Zengin, G., Sarikurkcu, C., Aktumsek, A., Ceylan, R. and Ceylan, O. (2014). A comprehensive study on phytochemical characterization of Haplophyllum myrifolium Bioss. endemic to Turkey and its inhibitory potential against key enzymes involved in Alzheimer, skin diseases and type II diabetes. Industrial Crops and Products, 53, pp. 244-251. P a g e 166 | 238 CHAPTER FIVE: HELICHRYSUM RUTILANS CHAPTER FIVE CHEMICAL AND BIOLOGICAL CHARACTERIZATION OF HELICHRYSUM RUTILANS CONSTITUENTS 5.1 Abstract Chromatographic fractionation of a methanol extract of H. rutilans followed by semi-prep HPLC afforded isolation of four known flavonol derivatives (C22-C25) and 3 known kaurane diterpene derivatives (C26-C28). C22 is a rare compound and reported once previously, the structure of C22 was established by extensive analyses of its 1D and 2D-NMR, HRMS, IR, UV spectral data as 5,7,8-trihydroxy-3,6-dimethoxyflavone-8-O-2-methyl-2-butanoate, while the structures of other known compounds were identified by comparing their respective spectroscopic data with those reported in the literature as 5,7-dihydroxy-3,6,8trimethoxyflavone (C23), 5-hydroxy-3,6,7,8-tetramethoxyflavone (C24), 5-hydroxy-3,6,7trimethoxyflavone (C25), ent-kaurenoic acid (C26), ent-kauran-18-al (C27), and 15-hydroxy-(-)-kaur-16-en-19-oic acid (C28). C22-C25 demonstrated high antioxidant capacities on ORAChydroxyl radical (2.114 ± 4.01; 2.413 ± 6.20; 1.924 ± 16.40; 1.917 ± 3.91) X 106; ORACperoxyl radical (3.523 ± 3.22; 2.935 ± 0.13; 2.431 ± 8.63; 2.814 ± 5.20) X 103 µMTE/g and FRAP (1251.45 ± 4.18; 1402.62 ± 5.77) µMAAE/g respectively. Moderate inhibitory activities against Fe2+-induced lipid peroxidation were observed for C22-C25 as IC50 values of 13.123 ± 0.34; 16.421 ± 0.92; 11.64 ± 1.72; 14.90 ± 0.06 µg/mL, respectively, while their respective anti-tyrosinase activities as IC50 values of 25.735 ± 9.62; 24.062 ± 0.61; 39.03 ± 13.12; 37.67 ± 0.98 µg/mL were also observed. All compounds demonstrated close TEAC values within the range 1105 - 1424 µMTE/g. P a g e 167 | 238 CHAPTER FIVE: HELICHRYSUM RUTILANS The result is an indication that a methanol extract of H. rutilans might possibly be a good source of natural antioxidants against aging related ailments caused by cellular oxidative stress and as inhibitors against skin depigmentation. Keywords: Helichrysum rutilans; Methoxylated flavonoids; Kaurane diterpenes; Antioxidant; Anti-tyrosinase; Skin aging. 5.2 Background information on Helichrysum rutilans Helichrysum rutilans is a dense twiggy shrublet (≤ 600 mm tall) with characteristic lemonyellow bracts (Fig. 5.1A). It occurs on sandy or stony soils from western and Eastern Cape and southern part of Free State (Fig. 5.1B) (www.keys.lucidcentral.org). No traditional or scientific information about this plant is documented in the literature according to SciFinder and the dictionary of natural products database. A B Figure 5.1: Helichrysum rutilans description (A), and distribution along South African mountain and coastal areas (B). P a g e 168 | 238 CHAPTER FIVE: HELICHRYSUM RUTILANS This chapter provide details on the:  Isolation of bioactive constituents present in methanol extract of H. rutilans, using chromatographic methods.  Identification of isolated constituents using different spectroscopic techniques.  Determination of total antioxidant capacities of the constituents.  Determination of inhibitory activities of the H. rutilans constituents against skin diseases related enzymes.  Structure-activity relationship (mechanism of action) of the H. rutilans bioactive constituents responsible for specific biological activity. CHEMICAL CHARACTERIZATION OF HELICHRYSUM RUTILANS CONSTITUENTS 5.3 General experimental procedure for biological assays 5.3.1 Reagents and solvents Organic solvents used were described in section 3.3.1 5.3.2 Chromatography Unless otherwise stated, the solvent systems generally used for the TLC development of H. rutilans fractions are indicated in Table 5.1 P a g e 169 | 238 CHAPTER FIVE: HELICHRYSUM RUTILANS Table 5.1: TLC solvent system Solvent system Ratio Assigned code Hexane - ethylacetate 9:1 A Hexane – ethylacetate 7:3 B Hexane – ethylacetate 3:2 C Hexane – ethylacetate 1:1 D DCM - methanol 99:1 E DCM - methanol 98:2 F DCM - methanol 97:3 G All other chromatographic methods of TLC, column, and HPLC techniques and procedures described in section 3.3.2 were followed. 5.3.3 Spectroscopy All analytical procedure of spectroscopic (NMR, MS, UV, and IR) techniques as well as optical rotation ([α]D) measurement described in sections 3.3.3 and 4.3.4 were followed. 5.4 Collection and identification of plant material The plant material was collected in October 2012 from Jonkershoek (approx. 9 km SE Stellenbosch) nature reserve, Western Cape, South Africa. Voucher specie was identified by Dr. Christopher Cupido (SANBI, Kirstenbosch), and a copy has been deposited at the Compton Herbarium, South African National Biodiversity Institute, Kirstenbosch, South Africa, with herbarium number NBG145882. P a g e 170 | 238 CHAPTER FIVE: HELICHRYSUM RUTILANS 5.5 Extraction and fractionation of total extract The whole plant material (400 g) was air dried at room temperature, blended and extracted with methanol at room temperature (25 oC) for 48 hours. Methanol extract was evaporated till dryness with rotary evaporator at 40 oC to yield 19 g (4.75 %). Table 5.2: Fractionation of the methanol extract of H. rutilans Solvent system Hexane Solvent volume 2L Fraction collected 1-8 Hexane – ethylacetate 90:10 2L 9-16 Hexane – ethylacetate 85:15 2L 17-24 Hexane – ethylacetate 80:20 2L 25-32 Hexane – ethylacetate 70:30 3L 33-44 Hexane – ethylacetate 60:40 2L 45-52 Hexane – ethylacetate 50:50 3L 53-63 Total extract was applied to silica gel column (30 x 18 cm) and eluted using gradient of hexane and ethyl acetate in the following order of increasing polarity as indicated in Table 5.2, with sixty three (63) fractions (250 mL each) collected and numbered 1 -63. P a g e 171 | 238 CHAPTER FIVE: HELICHRYSUM RUTILANS A C B D E 1 2 3 4 5 6 7 8 9 1011 12 13 14 15 FR 1516 17 1819 2021 22 23 24 25 26 27 28FR 2829303132333435363738ppt39FR3940414243444546474849505152FR525354555657585960616263 F G H I FR J 1 2 3 4 5 6 7 8 9 1011 12 13 14 15 FR 1516 17 1819 2021 22 23 24 25 26 27 28FR 2829303132333435363738ppt39FR3940414243444546474849505152FR525354555657585960616263 FR Figure 5.2: TLC profile of the collected fractions (1-58) under UV (254 nm; A -E), and after spraying with H2SO4/vanillin and then heated (F-J). *TLC plate (A & F) comprised of fractions 1-15 (ref. to total extract FR) developed using solvent system A *TLC plate (B & G) comprised of fractions 15-28 (ref. to total extract FR) developed using solvent system A * TLC plate (C & H) comprised of fractions 28-39 (ref. to total extract FR) developed using solvent system B *TLC plate (D & I) comprised of fractions 39-52 (ref. to total extract FR) developed using solvent system C *TLC plate (E & J) comprised of fractions 52 & 61 (ref. to total extract FR) developed using solvent system D *FR refers to methanol total extract of H. rutilans The collected fractions (1 – 63, Fig. 5.2) were concentrated on the rota-vapor and combined according to their TLC (using solvent systems A–D) profiles. The combined main fractions (IXVI, Table 5.3) were developed on TLC (using solvent system E) as indicated in Figure 5.3 Table 5.3: Main fractions obtained upon fractionation of the total extract of H. rutilans P a g e 172 | 238 CHAPTER FIVE: HELICHRYSUM RUTILANS Combined fraction Designated number Combined fraction Designated number 10-14 I 37 IX 15,16 II 38,39 X 17-20 III 40-43 XI 21-23 IV 44-48 XII 24-26 V 49-53 XIII 27,28 VI 54-56 XIV 29-32 VII 57-60 XV 33-36 VIII 61 XVI B A I II III IV V VI VII VIII IX X XI XII XIII XIV XV XVI I II III IV V VI VII VIII IX X XI XII XIII XIV XV XVI Figure 5.3: TLC profile of the main fractions (I-XVI) under UV (254 nm; Fig. 5.3A), and after spraying with H2SO4/vanillin and gentle heating (Fig. 5.3B). 5.6 Isolation of constituents (pure compounds) 5.6.1 Isolation of compounds C23 and C28 - Column chromatography of main fraction P a g e 173 | 238 CHAPTER FIVE: HELICHRYSUM RUTILANS X The crystal of main fraction X (411 mg) when washed with DCM, followed by methanol, afforded XAcrystal (C28, 90 mg, 0.0225 %), and other two supernantant as sub fractions (DCM supernantant-XB, and MeOH supernantant-XC). The sub frations (XB and XC) along with compound XA were developed on TLC (using solvent system E). Result obtained is indicated Figure 5.4. XA ppt XA XC XB Figure 5.4: TLC profile of sub fractions of X after spraying with H2SO4/vanillin and then heated. Sub fraction XC (70 mg) was rechromatographed on sephadex with isocratic 20 % aqueous ethanol and developed on TLC (using solvent system F) to give sub fractions XC1 (29 mg) as indicated in Figure 5.5. P a g e 174 | 238 CHAPTER FIVE: HELICHRYSUM RUTILANS XCI 1 3 4 5 6 7 8 9 10 11 12 13 14 Figure 5.5: TLC profile of sub fractions of XC after spraying with H2SO4/vanillin and heated Sub fraction XC1 (25 mg) was injected to the HPLC, and eluted using gradient solvent system of ACN and de-ionized water (55:45 to 65% ACN in 30 mins, then 100 % in 15 minutes). One prominent peak (Fig. 5.6) was collected and labelled as XC1-HPLC-2 (C23, Rt 26 min, 18 mg, 0.0045 %). XC1-HPLC-2 Figure 5.6: HPLC spectrum of compound C23 *Conditions Solvent Column Flow rate Detection ACN:DIW 55:45 to 65 % in 30 min, then to 100 % in 15 min SUPELCO, RP-18 (25 X 2.1 cm) 1.5 mL/min UV at 254 nm P a g e 175 | 238 CHAPTER FIVE: HELICHRYSUM RUTILANS 5.6.2 Isolation of compound C22 - Column chromatography of main fraction VIII Fraction VIII (291 mg) was chromatographed on sephadex using isocratic 10 % aqeuous ethanol, the collected fractions (50 mL each) were combined in accordance to their TLC (using solvent system F) profiles to yield sub fraction VIIIA as indicated in Figure 5.7. VIIIA Figure 5.7: TLC profile of sub fractions of VIII after spraying with H2SO4/vanillin and then heated. Sub fraction VIIIA (90 mg) was injected to the HPLC, and eluted using gradient solvent system of ACN and de-ionized water (50:50 to 70 % ACN in 30 mins, then 100 % in 15 minutes). One prominent peak (Fig. 5.8) was collected and labelled as VIIIA-HPLC-5 (C22, Rt 37 min, 22 mg, 0.0055 %). P a g e 176 | 238 CHAPTER FIVE: HELICHRYSUM RUTILANS VIIIA-HPLC-5 Figure 5.8: HPLC spectrum of compound C22 *Conditions Solvent Column Flow rate Detection ACN:DIW 50:50 to 70 % in 30 min, then to 100 % in 15 min SUPELCO, RP-18 (25 X 2.1 cm) 1.5 mL/min UV at 254 nm 5.6.3 Isolation of compound C24 and C25 - Column chromatography of main fraction VI Fraction VI (192 mg) was chromatographed on silica using isocratic 1 % methanol in DCM and 20 % hexane, the collected fractions (50 mL each) were combined according to their TLC (using solvent system E) profiles to yield sub fraction VIB (Fig. 5.9). VIB 1 2 3 4 5 6 7 8 9 10 11 12 13 Figure 5.9: TLC profile of sub fractions of VI after spraying with H2SO4/vanillin and then heated Sub fraction VIB (70 mg) was injected to the HPLC, and eluted using gradient solvent system of ACN and de-ionized water (70:30 to 80 % ACN in 30 mins, then 100 % in 15 minutes). Two P a g e 177 | 238 CHAPTER FIVE: HELICHRYSUM RUTILANS prominent peaks (Fig. 5.10) were collected and labelled as VIB-HPLC-1 (C25, Rt 18 min, 12 mg, 0.003 %), and VIB-HPLC-3 (C24, Rt 21 min, 22 mg, 0.0055 %). VIB-HPLC-3 VIB-HPLC-1 Figure 5.10: HPLC spectrum of compounds C24 and C25 *Conditions Solvent Column Flow rate Detection ACN:DIW 70:30 to 80 % in 30 min, then to 100 % in 15 min SUPELCO, RP-18 (25 X 2.1 cm) 1.5 mL/min UV at 254 nm 5.6.4 Isolation of compounds C26 and C27 Crystals formed from main fractions II and III were separately washed with hexane. White needle-like crystals obtained from main fractions II and III were dried and labelled as IIcrystal (C26, 62 mg, 0.0155 %) and IIIcrystal (C27, 65 mg, 0.01625 %) respectively. P a g e 178 | 238 CHAPTER FIVE: HELICHRYSUM RUTILANS HELICHRYSUM RUTILANS METHANOL EXTRACT (HD) OPEN COLUMN (SILICA GEL) II VIII VI III CRYSTALLIZATION II crystal III crystal SILICA COLUMN X CRYSTALLIZATION SEPHADEX HPLC FRACTIONATION VIB VIIIA METHANOL SUPERNANTANT OH H H COOH C26 COOH CHO C27 O O C28 3`` O O O SEPHADEX/HPLC 1`` O O OH O C24 O O OH O O HO 2` O 8 O 6` C25 O 3 5 O OH O C22 O HO O O O OH Scheme 5.1: A flow diagram of experimental procedure for the isolation of constituents from H. rutilans O C23 *Compounds isolated from same sub-fraction are indicated with same color box P a g e 179 | 238 CHAPETR FIVE: HELICHRYSUM RUTILANS BIOLOGICAL CHARACTERIZATION OF HELICHRYSUM RUTILANS 5.7 General experimental procedure 5.7.1 Reagents and solvents All reagents and solvents described in section 3.7.1 were used. 5.7.2 Antioxidant assays All experimental procedure for the various antioxidant capacity assays (FRAP, ORAC, and TEAC), and inhibition of Fe2+-induced lipid peroxidation as described in section 3.7.2, were followed. 5.7.3 Skin enzyme inhibitory assays Skin enzymes inhibitory assays measured on tyrosinase, elastase as described in sections 3.7.3 and AChE enzymes described in 4.7.4 were carried out. 5.8 Statistical analysis Statistical analysis described in section 3.8 was followed. 5.9 Chemical evaluation: Results and discussion 5.9.1 Summary of the isolated compounds Chromatographic purification of a methanol extract of H. ruilans using different chromatographic techniques including semi-prep HPLC resulted in the isolation of seven (7) pure compounds categorized into methoxylated flavonols (C22 – C25), and kaurane diterpene derivatives (C26 – C28). P a g e 180 | 238 CHAPETR FIVE: HELICHRYSUM RUTILANS 5.9.2 Spectroscopic data of C22 Compound C22 isolated as yellow solid; UV λmax (MeOH) nm: 280, 360; IR (KBr) cm-1: 3300, 1800, 1600, and 1150. HRMS m/z 413.1201 [M+1]+ (calcd. 413.1192). 1H- and 13C-NMR data, see Table 5.4. Other spectra of C22 are attached as Annexure V, Figures 1-4. Table 5.4: 1H (400 MHz: m, J Hz) and 13C (100 MHz) NMR spectral data of isolated compounds C22-C25 in CDCl3 C22 No. 13 C C23 1 H 13 C C24 1 H 13 C C25 1 H 13 C 1 H 2 155.8 s 155.8 s 156.1 s 3 139.2 s 139.3 s 139.5 s 4 179.1 s 179.5 s 179.5 s 5 149.9 s 148.9 s 153.1 s 6 131.2 s 130.5 s 130.6 s 7 149.6 s 148.1 s 149.2 s 8 118.2 s 127.2 s 136.2 s 9 144.3 s 145.1 s 145.1 s 10 104.3 s 105.2 s 107.6 s 1` 130.1 s 128.5 s 132.9 s 2` 128.1 d 7.29 br s 128.3 d 8.15 br s 128.4 d 8.17 br s 8.19 d, 7.2 3` 128.4 d 8.02 br s 128.8 d 7.65 br s 128.8 d 7.56 br s 7.45 m 4` 130.9 d 7.29 br s 131.1 d 7.65 br s 131.2 d 7.56 br s 7.45 m 5` 128.4 d 8.02 br s 128.8 d 7.65 br s 128.8 d 7.56 br s 7.45 m 6` 128.1 d 7.29 br s 128.3 d 8.15 br s 128.4 d 8.17 br s 8.19 d, 7.2 1`` 165.5 s 2`` 126.4 s 3`` 140.9 d 6.36 q, 6.8 4`` 15.8 q 2.13 d, 6.8 5`` 20.3 q 2.16 s OMe-3 60.2 q 3.88 s 60.4 q 3.90 s 60.4 q 3.91 s 3.89 s OMe-6 60.6 q 4.11 s 61.1 q 4.09 s 61.2 q 3.98 s 3.92 s 62.2 q 4.14 q 4.06 s 61.7 q 3.98 s OMe-7 OMe-8 61.8 q 4.02 s 5-OH 12.65 s 12.55 s 7-OH 6.58 br s 6.80 br s 6.61 s 12.37 11.37 P a g e 181 | 238 CHAPETR FIVE: HELICHRYSUM RUTILANS 5.9.3 Analysis of compound C22 HRMS established the molecular formula of C22 as C22H20O8 (m/z 413.1201). UV spectrum showed absorption at λmax 280 nm and 360 nm (MeOH), in addition to dark purple spot color under short UV (254 nm) as an indication of flavonoids characteristics. IR (KBr) spectrum showed bands of a hydroxyl group (3300 cm-1), carbonyl (1800 cm-1), conjugated C=C 1600 cm-1), and C-O side chain substituent (1150 cm-1). NMR spectra (Table 5.4, and Annexure V, Figures 1-4) showed 22 carbon signals, 15 of them belong to flavonoid skeleton with unsubstituted ring B [δH 8.02, br s, 2H; 7.29, br s, 2H; and 7.29, br s, 1H; δC: 128.1 (2XC), 128.4 (X 2C), and 130.9 (1C)]; Signals of two methoxyls at δH 3.88; and 4.11 and 5-OH signal at 12.65. Additional signals of a tigloyl side chain esterified with a hydroxyl group of ring A, [two olefinic methyls at δH 2.13 (d, 6.8 Hz; δC 15.8), 2.16 (s; δC 20.3), and an olefinic proton at 6.39 (q, 6.8 Hz; δC 140.9). The above data indicated the presence of a flavonol skeleton with two methoxyl and a tigloyl ester groups. The positions of the two methoxyls were evidenced from HMBC correlations which showed a cross peaks between O-methyl (δH 3.88) with C-3 (139.1) and O-methyl (δH 4.11) with C-6 (131.2). The fact that both C-5 and C-7 containing free hydroxyl group is evidenced from the OH signal at 12.65 (5-OH) and the C-7 chemical shift at 149.6. The extra side chain of the tigloyl group was positioned at C-8 from high field shift of C-8 to 118.2 ppm. The E geometry of the tigloyl group was supported by the coupling of the H-3`` with methyl 5`` (J = 6.8 Hz). The above data established the structure of C22 as 5,7,8-trihydroxy-3,6-dimethoxyflavone-8-O-2-methyl-2-butanoate (Fig. 5.11), and confirmed by comparison of the NMR data with the reported values (Urzua, et al., 1999). Isolation of compound 22 was reported once from Pseudognaphalium cheiranthyfolium collected from Chile. P a g e 182 | 238 CHAPETR FIVE: HELICHRYSUM RUTILANS 3`` 1`` O HO 2` O 8 O 6` O 3 5 O OH O C22 Figure 5.11: Structure of 5,7,8-trihydroxy-3,6-dimethoxyflavone-8-O-2-methyl-2-butanoate 5.9.4 Analysis of compounds C23, C24 and C25 Compounds C23 - C25 were identified as methoxylated derivatives of flavonols. Compound C23 showed NMR similar to C22, with unsubstituted ring B. The only difference is the absence of the tigloyl group and the presence of extra methoxyl group. The HMBC correlations showed cross peaks between the O-methyls (δH 3.90; 4.09; 4.02) with C-3 (139.3); C-6 (130.5) and C8 (127.2) respectively. Compound C23 showed typical 1H NMR data similar to compounds reported in literature (Urzua, et al., 1999; Tomas-Lorente, et al., 1989; Jakupovic, et al., 1986), and we established the chemical structure as 5,7-dihydroxy-3,6,8-trimethoxyflavone (as represented in Figure 5.12) from full analysis of 2D NMR. To the best of our knowledge and according to SciFinder, there is no any report documented for its 13 C NMR. The compound was identified previously from H. decumbens (Tomas-Lorente, et al., 1989). O HO O O O OH O C23 Figure 5.12: Structure of 5,7-dihydroxy-3,6,8-trimethoxyflavone P a g e 183 | 238 CHAPETR FIVE: HELICHRYSUM RUTILANS Compound C24 showed the same NMR pattern of compound C23, with extra methoxyl group at C-7. The structure was identified based on the analysis of 2D NMR spectra, and comparison with similar compounds reported in literature (Jakupovic, et al., 1986; Tomas-Lorente, et al., 1989; Urzua, et al., 1999), as 5-hydroxy-3,6,7,8-tetramethoxyflavone (Fig. 5.13). The compound was reported from H. cepaloideum (Jakupovic, et al., 1986). O O O O O OH O C24 Figure 5.13: Structure of 5-hydroxy-3,6,7,8-tetramethoxyflavone Compound C25 was identified as 5-hydroxy-3,6,7-trimethoxyflavone (Fig. 5.14) from careful analysis of the 1H NMR and comparison with the previously isolated compounds, C25 was reported from H. decumbens (Tomas-Lorente, et al., 1989). O O O O OH O C25 Figure 5.14: Structure of 5-hydroxy-3,6,7-trimethoxyflavone 5.9.5 Analysis of compounds C26, C27 and C28 Compounds C26-C28 showed typical diterpene NMR feature with 20 carbons. Compound C26 showed signals of two angular methyls at (1.14 s, δC 29.0, C-18), (0.92 s, 15.7, C-20); exomethylene double bond (δH 4.63 s, 4.69 s), (δC 103.0 t, 155.9 s) and a carboxylic group (184.6). C26 was identified as ent-kaurenoic acid (Fig. 5.15) after comparing the [α]D value P a g e 184 | 238 CHAPETR FIVE: HELICHRYSUM RUTILANS (- 0.078) and the 13 C NMR data with literature (Hutchison, et al., 1984). C26 is a common diterpene and was reported from many Helichrysum species e.g. H. fulvum (Bohlmann, et al., 1979) COOH C26 Figure 5.15: Chemical structure of ent-kaurenoic acid Compound C27 showed a typical NMR spectra to that of compound C26 except the presence of an aldehydic signal at δH 9.71 s, (δC 205.9 ), and the absence of the carboxyl group. C27 was identified as ent-kauren-18-al (as indicated in Fig. 5.16), with occurrence reported previously from H. pilosellum (Jakupovic, et al., 1986). CHO C27 Figure 5.16: Chemical structure of ent-kauran-18-al Compound C28 was identified as kaurenoic acid derivative. The compound showed typical NMR spectra similar to C26, with additional oxygenated methine signal at δH (3.50, overlapped signal with H2O), δC (81.03 d). The NMR data was identical with the same compound isolated from Mikania vitifolia (Lobitz, et al., 1998), and identified as 15-β-hydroxy-(-)-kaur-16-en-19oic acid (Fig. 5.17). P a g e 185 | 238 CHAPETR FIVE: HELICHRYSUM RUTILANS OH COOH C28 Figure 5.17: Chemical structure of 15-β-hydroxy-(-)-kaur-16-en-19-oic acid. 5.10 Biological evaluations: Results and discussion 5.10.1 Evaluating the total antioxidant capacities of the isolated compounds During the last decade, natural antioxidants particularly phenolic compounds, has been under very close scrutiny as potential therapeutic agents against a wide range of ailments including cardiovascular dysfunctions and aging (Soobrattee, et al., 2005). The type of flavonoids, the degree of methoxylation and the number of hydroxyl groups are some of the parameters that determine their antioxidant potentials. In general, the differences in antioxidant activity between polyhydroxylated and polymethoxylated flavonoids are most likely due to differences in both hydrophobicity and molecular planarity (Heim, et al., 2002). Flavonoid radical stability is thought to be increased by the creation of a completely conjugated electron system. This can be accomplished through structural planarity of the flavonoid due to the presence of a hydroxyl group at the C-3 position on the C-ring, resulting in a flavonol backbone structure. Replacement of 3-OH by a methoxy substituent at this position perturbs this planarity, due to steric hindrance imparted by the methyl group, thus render the flavonoids less active as antioxidants than their corresponding OH at C-3 (Dugas, et al., 2000). In agreement with literature data, the present study corroborate with the view that structural features of flavonols (3-OH) are important moiety for antioxidant efficacy (Burda & Oleszek, 2001; Abdul Karim, et al., 2014; Popoola, et al., 2015). P a g e 186 | 238 CHAPETR FIVE: HELICHRYSUM RUTILANS Table 5.5: Total antioxidant capacity assay for isolated compounds Sample HD FRAP µMAAE/g 906.71 ± 5.18 TEAC µMTE/g 765.23 ± 2.43 C22 1251.45 ± 4.18 1131.80 ± 6.41 C23 1402.62 ± 5.77 1276.11 ± 1.32 C24 1314.42 ± 2.42 1378.10 ± 9.06 C25 1119.44 ± 11.89 1207.11 ± 7.21 C26 19.66 ± 8.12 1105.00 ± 3.09 C27 29.39 ± 5.84 1361.90 ± 0.35 C28 60.90 ± 7.90 1424.51 ± 0.70 EGCG 3326.45 ± 5.76 11545.44 ± 17.28 HD: methanol extract from H. rutilans; EGCG: epigallocatechingallate; C22-C29: isolated compounds The total antioxidant activities (Tables 5.5 and 5.6) nominated C22-C25 as natural antioxidant but less active in comparison to their hydroxylated analogues in reported in chapter 3. Our result obtained on total antioxidant assays when compare with their corresponding hydroxylated derivatives therefore described methoxylation of flavonoids obviously weakened the antioxidant activity as observed by previous data (Xiao, et al., 2013). Our results are in agreement with the existing data which demonstrated that the replacement of the C-3 hydroxyl by methoxy group resulted in a reduction of antioxidant activity. P a g e 187 | 238 CHAPETR FIVE: HELICHRYSUM RUTILANS Table 5.6: Oxygen radical absorbance capacity and anti-acetylcholineesterase activity Hydroxyl X 106 AChE inhibitory assay Sample Peroxyl HD µMTE/g 2935.16 ± 3.92 1.817 ± 1.72 IC50 ± STDEV (µg/mL) 300.40 ± 3.49 C22 3523.51 ± 3.22 2.114 ± 4.01 310.38 ± 6.44 C23 2935.47 ± 0.13 2.413 ± 6.20 380.24 ± 0.93 C24 2431.30 ± 8.63 1.924 ± 16.40 375.25 ± 3.12 C25 2814.51 ± 5.20 1.917 ± 3.91 325.35 ± 14.31 C26 364.44 ± 6.71 0.429 ± 12.00 380.24 ± 5.23 C27 914.29 ± 2.74 0.531 ± 10.24 310.38 ± 11.65 C28 93.10 ± 13.68 0.845 ± 13.34 310.38 ± 7.00 EGCG 14693.09 ± 5.53 3.862 ± 4.65 Galanthamine - - 10.981 HD: methanol extract of H. rutilans; EGCG: epigallocatechingallate; C22-C29: Isolated compounds 5.10.2 Evaluating the anti-lipid peroxidation activity of the isolated compounds The ability of phenolic compounds to inhibit oxidative damage in lipids was assessed using thiobarbituric acid as a model system. Peroxidation was initiated by addition of FeSO4-EDTA mixture. It is well known that transition metal like iron may generate highly reactive hydroxyl or alkoxyl radicals (Fenton reaction). The result indicated compounds C22-C25 with mild inhibitory activities against the Fe2+-induced lipid peroxidation as expressed as IC50 values of 13.123 ± 0.34; 16.421 ± 0.92; 11.64 ± 1.72; 14.90 ± 0.06 µg/mL, respectively . This suggests that C22-C25 possess a feasible mechanistic pathway for iron-chelating and iron-stabilizing capacity due to the presence of 3-OMe and 5- OH in conjugation with 4-keto group. It is also an indication that the lone pair of electron on OMe contributes to the antioxidant activities of compounds investigated. Previous work illustrated the significant role of these features to have P a g e 188 | 238 CHAPETR FIVE: HELICHRYSUM RUTILANS more potent antioxidants due to the formation of hydrogen bonding between 5-OH and 4-keto group, which help to further stabilize flavone radicals formed (Catala, 2006; Wolfe & Liu, 2008; Georgiev, et al., 2013; Kumar & Pandey, 2013; Popoola, et al., 2015). Compounds C22C25 may therefore serve as good source of potent antioxidant due to their ability to stabilize Fe2+ thereby reduce the production of reactive hydroxyl radical (OH.) during Fenton reaction (Fahran, 2013). Figure 5.18: Effects of H. rutilans constituents on inhibition of Fe2+ induced microsomal lipid peroxidation. (p<0.05) *Data are expressed as IC50 with isolated compounds and a methanol extract (HD) screened at 50.00 µg/mL 5.10.3 Evaluating the anti-tyrosinase activity of the isolated compounds Due to everyday growing market of cosmetics, tyrosinase inhibitors have received a special attention because of alleviating of hyperpigmentation and undesirable browning of food products. Effort are geared in sourcing for a better option of getting compounds from natural origin due to excellent pharmacological properties with commendably high safety margins demonstrated by tyrosinase inhibitors isolated from nature. When tyrosinase enzyme activity is inhibited, melanin production is reduced, resulting in a fairer skin (Abdul Karim, et al., 2014). Amongst the H. rutilans constituents investigated for P a g e 189 | 238 CHAPETR FIVE: HELICHRYSUM RUTILANS anti-tyrosinase activity in Figure 5.19, where compounds C22-C25 demonstrated moderate inhibition against tyrosinase (with respective IC50 = 25.735 ± 9.62; 24.062 ± 0.61; 39.03 ± 13.12; 37.67 ± 0.98 µg/mL) enzyme, but remained less potent than kojic acid (IC50 = 3.511± 1.44 µg/mL). The presence of conjugation in C22-C25 rings further give rise to a resonance effect in both ring thereby provides stability to the respective flavone radicals formed (Georgiev, et al., 2013). Figure 5.19: Effects of H. rutilans constituents on inhibition of anti-tyrosinase activity (p<0.05) *Data are expressed as IC50 with isolated compounds and methanol extract (HT) screened at 100.00 µg/mL 5.10.4 Evaluating the anti-elastase activity of the isolated compounds No significant inhibitory activity was recorded by the samples tested against the inhibition of elastase. P a g e 190 | 238 CHAPETR FIVE: HELICHRYSUM RUTILANS Figure 5.20: Effects of H. rutilans constituents on inhibition of anti-elastase activity (p<0.05) *Data are expressed as IC50 with isolated compounds and methanol extract (HD) screened at 100.00 µg/mL 5.11 Conclusion Seven known compounds were isolated and characterized by our previous analytical tools. Flavonols have been shown to possess good antioxidant activity and have been implicated as inhibitors of lipid peroxidation. Indeed, the evidence presented herein suggests that a dietary intake of flavonoids-containing foods may be of benefit in lowering the risk of certain pathophysiologies associated with free radical mediated events. This study revealed that methanol extract of H. rutilans had indicated strong antioxidant and skin depigmentation activities. Possible cosmetic product formulation against photo-oxidation of skin can be made from the constituents of H. rutilans, upon the recommendation from clinical trials to ascertain their probable side effect on the skin. This present the first report to be documented on H. rutilans, a plant widely spread in coastal part of western and mountainous part of eastern province of South Africa. P a g e 191 | 238 CHAPTER FIVE: REFERENCES REFERENCES Abdul Karim, A., Azlan, A., Ismail, A., Hashim, P., Gani, S.S.A., Zainudin, B.H. and Abdullah, NA. (2014). Phenolic composition, antioxidant, anti-wrinkles and tyrosinase inhibitory activities of cocoa pod extract. BMC Complementary and Alternative Medicine, 14, pp. 1-28. Bohlmann, F., Zdero, C., Zeisberg, R. and Sheldrick, W.S. (1979). Naturally occurring terpene derivatives. Part 213. Hydroxyhelifulvanoic acid, a new diterpene with an anomalous carbon skeleton from Helichrysum fulvum. Phytochemistry, 18(8), pp. 1359-1362. Burda, S. and Oleszek, W. (2001). Antioxidant and antiradical activities of flavonoids. Journal of Agriculture and Food Chemistry, 49, pp. 2774-2779. Catala, A. (2006). An overview of lipid peroxidation with emphasis in outer segments of photoreceptors and chemiluminescence assay. International Journal of Biotechnology & Cell Biology, 38, pp. 1482-1495. Dugas, A.J., Castañeda-Acosta, J., Bonin, G.C., Price, K.L., Fischer, N.H. and Winston, G.W. (2000). Evaluation of the total peroxyl radical-scavenging capacity of flavonoids: structure-activity relationships. Journal of Natural Products, 63(3), pp. 327-331. Fahran, S. A. (2013). Study on the interaction of Copper (II) complex of morin and its antimicrobial effect. International Journal of Chemical Science, 11(3), pp. 1247-1255. Georgiev, L., Chochkova, M., Totseva, I., Seizova, K., Emma, M., Ivanova, G., Ninova, M., Najdenski, H. and Milkova, T. (2013). Anti-tyrosinase, antioxidant and antimicrobial activities of hydroxycinnamoylamdes. Medicinal Chemistry Research, 22, pp. 4173-4182. Heim, K.E., Tagliaferro A.R. and Bobilya D.J. (2002). Flavonoid antioxidants: chemistry, metabolism and structure-activity relationship. The Journal of Nutritional Biochemistry, 13, pp. 572-284. Hutchison, M., Lewer, P. and MacMillan, J. (1984). Carbon-13 nuclear magnetic resonance spectra of eighteen derivatives of ent-kaur-16-ene-19-oic acid. Journal of Chemical Society, 1, pp. 2363-2366. P a g e 192 | 238 CHAPTER FIVE: REFERENCES Jakupovic, J., Kuhnke, J., Schuster, A., Metwally, M.A. and Bohlmann, F. (1986). Phloroglucinol derivatives and other constituents from South African Helichrysum species. Phytochemistry, 25(5), pp. 1133-1142. Kumar, S. and Pandey, A.K. (2013). Chemistry and biological activities of flavonoids: An overview. The Scientific World Journal, 2013, pp. 1-16. Lobitz, G.O., Tamayo-Castillo, G., Poveda, L. and Merfort, I. (1998). Phytochemical and biological studies on Costa Rica Asteracea II. New kaurene derivatives from Milkania vitifolia. Phytochemistry, 49, pp. 805-809. Popoola, O.K., Marnewick, J.L., Rautenbach, F., Ameer, F., Iwuoha, E.I. and Hussein, A.A. (2015). Inhibition of oxidative stress and skin aging-related enzymes by prenylated chalcones and other flavonoids from Helichrysum teretifolium. Molecules, 20, pp. 7143-7155. Soobrattee, M.A., Neergheen, V.S., Luximon-Ramma, A., Aruoma, O.I. and Bahorun, T. (2005). Phenolics as potential antioxidant therapeutic agents: Mechanism and actions. Mutation Research, Fundamental and Molecular Mechanisms of Mutagenesis, 579(1-2), pp. 200-213. Tomas-Lorente, F., Iniesta-Sanmartin, E., Tomas-Barberan, F.A., Trowitzsch-Kienast, W. and Wray, V. (1989). Antifungal phloroglucinol derivatives and lipophilic flavonoids from Helichrysum decumbens. Phytochemistry, 28(6), pp. 1613-1615. Urzua, A., Mendoza, L., Tojo, E. and Real, M.E. (1999). Acetylated flavonoids from Pseudognaphalium species. Journal of Natural Products, 62, pp. 381-382. Wolfe, K.L. and Liu, R.H. (2008). Structure-activity relationship of flavonoids in the cellular antioxidant activity assay. Journal of Agriculture and food chemistry, 56, pp. 8404-8411. Xiao, J., Ni, X., Kai, G. and Chen, X. (2013). A review on structure-activity relationship of dietary polyphenols inhibiting α-amylase. Critical Reviews in Food Science and Nutrition, 53(5), pp. 497-506. P a g e 193 | 238 CHAPTER SIX: CONCLUSION CHAPTER SIX GENERAL DISCUSSION, CONCLUSION AND RECOMMENDATIONS Flavonoids, a multifunctional compounds are suggested to be potent modulators of oxidative stress and enzymatic oxidations due to their reducing, metal chelating, free radical quenching ability among others (Kelly, et al., 2008). Therefore, the implication of reactive oxygen species (ROS) in chronic degenerative and aging related diseases including skin aging continue to underpin the search for natural antioxidants with little or no side effects. Although, an extensive amount of phytochemical research has been done on the genus Helichrysum spp., only a few have been investigated for their biological and pharmacological importance, with an integral part of this genus left untapped. Among such are the three (3) unexplored Helichrysum species selected for this research viz: H. teretifolium (HT), H. niveum (HF) and H. rutilans (HD). Their selections were based on the non-availability of both scientific and ethnomedicinal information to support their respective traditional uses. This investigation therefore is the first scientific report on the phytochemical constituents and their possible biological application as antioxidants in cosmetics product formulations. A preliminary investigation by thin layer chromatography (TLC) revealed that extracts of HT, HF and HD (result not shown) exhibited yellow/red/purple staining of certain chemical compounds with vanillin/H2SO4 spray. The TLC results of these plant extracts indicated that they may exhibit an interesting chemical profile similar to those previously investigated. Repeated chromatographic purification procedures (CC, TLC, HPLC) on the methanol extracts and extensive structural determination of the isolated compounds were carried out using spectroscopic (1D and 2D-NMR, HRMS, IR, UV) data. Optical rotations of the new P a g e 194 | 238 CHAPTER SIX: CONCLUSION compounds with stereogenic centres were also done to support the proposed structures. The results emanated from the extensive chemical analyses of the isolated compounds are summarized as follows: Table 6.1: Isolated constituents from the three selected Helichrysum species Code Name Source Remark C1 Heliteretifolin H. teretifolium new C2 2`,6`-dihydroxy-4`-methoxy-3-prenychalcone H. teretifolium known C3 2`,4`,6`-trihydroxy-3`-prenyhalcone H. teretifolium known C4 isoglabranin H. teretifolium known C5 glabranin H. teretifolium known C6 7-methoxyisoglabranin H. teretifolium known* C7 quercetin H. teretifolium known C8 4-methoxyquercetin H. teretifolium known* C9 4`-methoxykaempferol H. teretifolium known* C10 mosloflavone H. teretifolium known* C11 1-benzoyl-3-(3-methyl-2-butenylacetate)- H. niveum new H. niveum new H. niveum new phloroglucinol (helinivene A) C12 1-benzoyl-3-(2-hydroxyl-3-methyl-3-butene-1-yl)phloroglucinol (helinivene B) C13 8-(2-methyl-1-propanone)-3,5,7-trihydroxyl-2,2 dimethoxychromone (helinivene C) C14 1-(2-methylbutanone)-4-O-prenyl-phloroglucinol H. niveum known C15 1-(2-methylpropanone)-4-O-prenyl-phloroglucinol H. niveum known C16 1-(butanone)-3-prenyl-phloroglucinol H. niveum known C17 1-(2-methylbutanone)-3-prenyl-phloroglucinol H. niveum known C18 1-butanone-3-(3-methyl-2-butenylacetate H. niveum known C19 1-(2-methylpropanone)3-prenylphloroglucinol H. niveum known C20 caespitate H. niveum known C21 3β-24-dihydroxyterexer-14-ene H. niveum Known* C22 5,7,8-trihydroxy-3,6-dimethoxyflavone-8-O-2-methyl- H. rutilans Known* 2-butanoate P a g e 195 | 238 CHAPTER SIX: CONCLUSION C23 5,7-dihydroxy-3,6,8-trimethoxyflavone H. rutilans Known C24 5-hydroxy-3,6,7,8-tetramethoxyflavone H. rutilans known C25 5-hydroxy-3,6,7-trimethoxyflavone H. rutilans known C26 ent-kauranoic acid H. rutilans known C27 ent-kauran-18-al H. rutilans known C28 15--hydroxy-(-)-kaur-16-en-19-oic acid H. rutilans Known* *compounds isolated for the first time from Helichrysum genus In vitro total antioxidant capacity was determined using FRAP, an automated ORAC (peroxyl and hydroxyl), TEAC and inhibition of Fe2+-induced lipid peroxidation assays. The possibilities of the isolated compounds from HT and HF to possess prooxidant behaviour in the presence of metal ions were also investigated. Inhibitions of skin disease-related enzymes (tyrosinase and elastase) were carried out while the acetylcholinesterase (AChE) inhibitory activity was also included (chapters 4 and 5). Micro molar concentrations for the isolated compounds could not be calculated, since the molecular weights of the compounds were not yet known at the time of performing the biological assays. The total antioxidant capacity assays measure ability of antioxidant to either transfer electron (s) and or hydrogen ion to free radicals thereby, converting them to stable and usable products as discussed in sections 3.10.1, 3.10.2; 4.10.1, 4.10.2 and 5.10.1. The result of these sections explained the stability of the compounds to act as antioxidants in different medium such as pH. Literature pointed out that both FRAP and TEAC assays have the same mechanism of antioxidants to act as reducing agent (electron transfer) (Pellegrini, et al., 1999; Re, et al., 1999; Prior, et al., 2003). The distinction between the two assays is that the former is acidic, while the latter is neutral. Compounds C11 and C12 in comparison with EGCG were found to be stable in acidic medium with observed FRAP values (2530.54 ± 0.92; 4950.08 ± 0.65 Vs 3326.45 ± 5.76) respectively, while high TEAC values were observed for C2 and C3. P a g e 196 | 238 CHAPTER SIX: CONCLUSION Oxidative degradation of lipids is a common consequence of oxidative stress, a process whereby PUFAs of the membranes are susceptible to oxidative damage via reaction of free radicals, which can lead to lipid peroxidation (Kumar & Pandey, 2013). In this study, the hydroxylated flavonoid with catechol moiety (C7) demonstrated highest activity against the inhibition of the Fe2+-induced microsomal lipid peroxidation with IC50 = 2.931 µg/mL, while acylphloroglucinol derivatives with phenyl side chain (C11 & C12) gave activity in the same manner. The systemic process of aging in humans results in an imbalance between synthesis and degradation of the extracellular matrix. Overproduction of degradative enzymes and oxygen free radicals during chronological and photo-induced aging leads to degradation of the network and elastic skin collagen and hyper-synthesis of melanin (Ndlovu, et al., 2013). It is a complex process underlying with multiple influences including the probable involvement of inheritable and various environmental factors (Zhang, et al., 2014) that represents a major burden to the health care system. Moreover, enzymatic browning in fruit, vegetable, and fungus, which decreases the commercial value of the product during processing or storage, is undesirable (Chang, 2009). Compounds C7, C11 & C12 demonstrated significant biological activity as anti-tyrosinase (IC50 = 8.092; 35.625; 26.719) µg/mL respectively, while compound C8 (a methoxylated analogue of C7) demonstrated a lower anti-tyrosinase activity (IC50 = 27.573 µg/mL). Other compounds with an absence of structural features highlighted in various sections of evaluations for anti-tyrosinase property demonstrated very weak activity. There are many assumptions with regards to structural activity relationship of flavonoids. The numbering, positioning and substituting patterns displayed by side chains in the isolated compounds resulted in different biological activities. One of the notable finding proposed Fe2+ (in case of anti-lipid peroxidation) and Cu2+ (in case of tyrosinase) undergo different P a g e 197 | 238 CHAPTER SIX: CONCLUSION mechanistic pathways through which phenolic-metal ion chelation occurs, despite that they are bivalent metals in nature. The presence of structural analogues between the existing data and the isolated compounds from these findings confirm the assumed chemotaxonomic relationship to other Helichrysum species. These findings expand the knowledge about the phenolic profile of Helichrysum and enhance the chemotaxonomic understanding. Interesting observations were made regarding correlations observed between the morphology and traditional uses as well as the phytochemistry of the South African species: there are indications that morphological relationships can serve as a guide towards expected phytochemical composition of related species. The expected hypotheses of the class of natural flavonoids and acylphloroglucinols of having potent antioxidant and metal ion chelation properties have been fully established. Different experimental data which are consonant with previous literature were demonstrated by the constituents of these selected Helichrysum species. Different propositions were therefore made for the mechanisms of action of the structure-activity relationship between the constituents and their respective biological properties demonstrated. From the above, it is recommended that 1- Biological studies of the isolated compounds against other biological target (especially HIV), to explore their potential as bioactive compounds. 2- Applications of these chemical constituents in cosmetics and pharmaceutical industry will require proof of its antioxidant and skin-enzyme inhibitory effects in in vivo models. Further analyses such as cytotoxicity determination in melanoma cell lines as well as clinical trials are hereby recommended in order to translate the current findings into final cosmetic- and/or pharmaceutical product formulations. P a g e 198 | 238 CHAPTER SIX: CONCLUSION REFERENCES Chang, Te-Sheng. (2009). An update review of tyrosinase inhibitors. International Journal of Molecular Sciences, 10, pp. 2440-2475. Kumar, S. and Pandey, A. (2013). Chemistry and biological activities of flavonoids: An overview. The Scientific World Journal, 2013, pp. 1-16. Ndlovu, G., Fouche, G., Tselanyane, M., Cordier, W. and Steenkamp, V. (2013). In vitro determination of the anti-aging potential of four southern African medicinal plants. BMC Complementary and Alternative Medicine, 13(304). 1-7. Pellegrini N., Re. R., Yang, M. and Rice-Evans C.A. (1999). Screening of dietary carotenoidrich fruit extracts for antioxidant activities applying ABTS radical cation decolorisation assay. 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P a g e 199 | 238 ANNEXURE ANNEXURE I: Spectra of compound C1 Figure 1: 1H NMR spectrum of compound C1 in CDCl3 Figure 2: 13C NMR spectrum of compound C1 in CDCl3 P a g e 200 | 238 ANNEXURE Figure 3: HSQC NMR spectrum of compound C1 in CDCl3 Figure 4: HMBC NMR spectrum of compound C1 in CDCl3 P a g e 201 | 238 ANNEXURE ANNEXURE II: Spectra of compound C11 Figure 1: 1H NMR spectrum of compound C11 in CD3COCD3 Figure 2: 13C NMR spectrum of compound C11 in CD3COCD3 P a g e 202 | 238 ANNEXURE Figure 3: HSQC NMR spectrum of compound C11 in CD3COCD3 Figure 4: HMBC NMR spectrum of compound C11 in CD3COCD3 P a g e 203 | 238 ANNEXURE ANNEXURE III: Spectra of compound C12 Figure 1: 1H NMR spectrum of compound C12 in CD3COCD3 Figure 2: 13C NMR spectrum of compound C12 in CD3COCD3 P a g e 204 | 238 ANNEXURE Figure 3: HSQC NMR spectrum of compound C12 in CD3COCD3 Figure 4: HMBC NMR spectrum of compound C12 in CD3COCD3 P a g e 205 | 238 ANNEXURE ANNEXURE IV: Spectra of compound C13 Figure 1: 1H NMR spectrum of compound C13 in CD3COCD3 Figure 2: 13C NMR spectrum of compound C13 in CD3COCD3 P a g e 206 | 238 ANNEXURE Figure 3: HSQC NMR spectrum of compound C13 in CD3COCD3 Figure 4: HMBC NMR spectrum of compound C13 in CD3COCD3 P a g e 207 | 238 ANNEXURE ANNEXURE V: Spectra of compound C22 Figure 1: 1H NMR spectrum of compound C22 in CDCl3 Figure 2: 13C NMR spectrum of compound C22 in CDCl3 P a g e 208 | 238 ANNEXURE Figure 3: HSQC NMR spectrum of compound C22 in CDCl3 Figure 4: HMBC NMR spectrum of compound C22 in CDCl3 P a g e 209 | 238

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