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BIOLOGICAL PROPERTIES of SELECTED FLAVONOIDS of ROOIBOS (Aspalathus Linearis)

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BIOLOGICAL PROPERTIES of SELECTED FLAVONOIDS of ROOIBOS (Aspalathus Linearis) BIOLOGICAL PROPERTIES OF SELECTED FLAVONOIDS OF ROOIBOS (Aspalathus linearis) Petra W Snijman Thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Chemistry at the University of the Western Cape Study Leader: Prof IR Green Co-study Leaders: Prof E Joubert Prof WCA Gelderblom June 2007 ii DECLARATION I, the undersigned, hereby declare that the work contained in this thesis is my own original work and that I have not previously in its entirety or in part submitted it at any university for a degree. _______________________________ ____________ Petra Wilhelmina Snijman Date Copyright © 2007 University of the Western Cape All rights reserved iii ABSTRACT Bioactivity-guided fractionation was used to identify the most potent antioxidant and antimutagenic fractions contained in the methanol extract of unfermented rooibos (Aspalathus linearis), as well as the bioactive principles for the most potent antioxidant fractions. The different extracts and fractions were screened using Salmonella typhimurium tester strain TA98 and metabolically activated 2- acetoaminofluorene (2-AAF) to evaluate antimutagenic potential, while the antioxidant potency was assessed by two different in vitro assays, i.e. the inhibition of Fe(II) induced microsomal lipid peroxidation and the scavenging of the 2,2'- azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation. The most polar XAD fraction displayed the most protection against 2-AAF induced mutagenesis in TA98. Successive fractionation of the two XAD fractions most active in the ABTS•+ assay led to both aspalathin and nothofagin being isolated for the first time to a purity of >95%. Thirteen flavonoids of rooibos were also compared in the two antioxidant assays in addition to a metal chelating assay in order to derive a possible comparative structure-activity profile between the dihydrochalcones aspalathin and nothofagin, their flavone analogues orientin and isoorientin (from the precursor aspalathin) and vitexin and isovitexin from nothofagin, the flavone aglycones luteolin and chrysoeriol as well as four flavonols from rooibos, i.e. quercetin, isoquercitrin (quercetin-3-O-glucoside), hyperoside (quercetin-3-O-galactoside) and rutin (quercetin-3-O-rutinoside). The flavanol (+)-catechin was also included while iv epigallocatechin gallate (EGCG), the major active principle from Camellia sinensis was used as benchmark. Aspalathin, the dihydrochalcone unique to rooibos, was a very efficient inhibitor of lipid peroxidation and scavenger of ABTS•+, while nothofagin was equipotent to aspalathin in the ABTS•+ assay, but had the lowest inhibitory effect of all the flavonoids tested in the lipid peroxidation assay. Aspalathin was shown to be the major contributor to the antioxidant activity of unfermented rooibos in quantity and potency. The same thirteen flavonoids of rooibos were also investigated in the Salmonella typhimurium mutagenicity assay, again using EGCG as benchmark. Strains TA98 and TA100 utilizing 2-AAF and aflatoxin B1 (AFB1) respectively, were used. Neither aspalathin nor nothofagin displayed potent antimutagenic properties against both the mutagens evaluated while luteolin was the most effective antimutagen. The antimutagenic behaviour of rooibos could not be solely attributed to any of these monomeric flavonoid constituents. No clear and direct link between the antioxidant and antimutagenic properties of the major rooibos flavonoids could be conclusively be established in the present studies. v AIMS AND OBJECTIVES 1. To investigate whether the same flavonoids are responsible for both the potent antioxidant and antimutagenic properties of unfermented rooibos. 2. To compare the activity of thirteen of the major flavonoids of unfermented rooibos in two antioxidant assays. 3. To compare the antimutagenic activity of the same thirteen flavonoids of unfermented rooibos in two different Salmonella typhimurium strains using two different metabolically activated mutagens. vi ACKNOWLEDGEMENTS This thesis is the result of many years of work and it realized thanks to the support of, amongst others, the following people and institutions: I would first-off like to express my deep and sincere gratitude towards Prof Ivan Green, my study leader, for being a true mentor to me. Thank you for your encouragement, understanding and personal guidance, always given in a positive, gentle and friendly manner. I have learnt so much from you. I would like to thank Dr Wentzel Gelderblom of the Medical Research Council, my former project leader, for defining this project and allocating me to this fascinating work. Thank you for the technical skills you taught me. Thank you for your patience with me and teaching me to have patience with columns and other laboratory routines. I owe a sincere thanks to Dr Lizette Joubert, not only for the weeks of training I spent at ARC Infruitec-Nietvoorbij and sharing her insights and expertise with me throughout this project, but also for always being ready to enthusiastically think up a new angle to an argument or another possible solution to a problem. I benefited much from her about rooibos, its flavonoids and analytical chemistry. I am also endebted to the following people: Prof Kayoko Shimoi of the University of Shizuoka, Japan, for her valuable contribution in optimising the lipid peroxidation assay; Ms Sonja Swanevelder of the Medical Research Council and Dr Martin Kidd of the Department of Statistics at the University of Stellenbosch for statistical analyses; Dalene de Beer of ARC Infruitec-Nietvoorbij for training and assistance in HPLC analysis and the ABTS•+ assay; Shamiel Joseph and Dr Jeanine Marnewick for their assistance in the Salmonella typhimurium assay; vii Dr BN Ames and colleagues of the University of California in Berkeley for kindly supplying the Salmonella typhimurium tester strains; Dr Ann Louw and her students Ciko Mfenyana and Steven Robertson of the Biochemistry Department at the University of Stellenbosch for valuable assistance with the Prism computer programme; Dr Thinus van der Merwe (then of the University of Stellenbosch) for LC-MS spectra of aspalathin and nothofagin; Dr Robert Vleggaar of the University of Pretoria for NMR-spectra of aspalathin; The Medical Research Council, Rooibos Tea Forum and THRIP for financial support; Mercia Mottie, Faculty Officer at the University of the Western Cape for always being friendly and helpful with registration and any administrative enquiry or problem; My former colleagues at the Medical Research Council where the experimental work was done, for their support, advice and inputs, and John for always having clean glassware. To my present colleagues at the University of Stellenbosch I have such appreciation and gratefulness: Prof Jan Dillen and Dr Catharine Esterhuysen: thank you for your support and flexibility in granting me leave from work; Japie, Glen and Jessie: thank you for your interest shown and for being prepared to fill in for me; Moebarick and Johannes for your assistance and support; Maryna, thank you for your encouragement and perspectives, and for reminding me about the Bigger Picture! And last but by no means least: I want to thank my mother, sisters and friends for their interest and support throughout this time. Yes, it is finally done! viii CONTENTS Chapter Page Abstract iii Aims and Objectives v Acknowledgements vi Key to abbreviations used in thesis ix List of Tables x List of Figures xii List of Addenda iv 1. Introduction 1 2. Literature Review 6 3. Bioguided fractionation and isolation of the two major 94 dihydrochalcones from unfermented rooibos (Aspalathus linearis) 4. Antioxidant activity of the major flavonoids of rooibos 134 (Aspalathus linearis) 5. Antimutagenic activity of the major flavonoids of 165 rooibos (Aspalathus linearis) 6. General discussion and conclusions 193 Addenda 198 Every chapter of this thesis has been written as an individual entity that could form the basis of a journal manuscript. Repetition between chapters has therefore been unavoidable. ix Key to abbreviations used in thesis • OH : hydroxyl radical 2-AAF : 2-acetylaminofluorene ABTS• : 2,2΄-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical AFB1 : aflatoxin B1 BHP : tert-butylhydroperoxide BHT : butylated hydroxytoluene CHP : cumene hydrogen peroxide COMT : catechol-O-methyl transferase DMSO : dimethyl sulfoxide DPPH• : 2,2’-diphenyl-1-picrylhydrazyl radical EDTA : ethylenediaminetetraacetic acid EGCG : epigallocatechin gallate, a green tea flavonoid used as reference in this study Fl-OH : flavonoid Fl-O• : aroxyl radical HPLC : High Performance Liquid Chromatography IQ : 2-amino-3-methylimidazo[4,5-ƒ]quinoline L• : alkyl radical LH : polyunsaturated fatty acid LO• : lipid alkoxyl radical LOO• : lipid peroxyl radical LOOH : lipid hydroperoxide PhIP : 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine • RO2 : peroxyl radical S9-mix : Aroclor-induced rat liver homogenate fraction used to metabolic activate mutagens in Salmonella mutagenicity assay •- O2 : superoxide radical TBA : thiobarbituric acid TBARS : thiobarbituric acid reactive substances TCA : trichloroacetic acid TLC : Thin Layer Chromatography x LIST OF TABLES Number Page 2.1 Some of the most popular Salmonella typhimurium strains 68 with their reversion event, tar gets, number of spontaneous revertants and a possible diagnostic mutagen for antimutagenicity purposes 2.2 Summary of general structural requirements for in vitro 78 biological activity of flavonoids 3.1 Composition of mobile
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