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Antioxidant Activities of Sonchus Oleraceus L

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Antioxidant Activities of Sonchus Oleraceus L Antioxidant Activities of Sonchus oleraceus L. Sundara Mudiyanselage Maheshini Rangika Mawalagedera A thesis submitted to the Victoria University of Wellington in fulfilment of the requirements for the degree of Doctor of Philosophy in Ecology and Biodiversity Victoria University of Wellington 2014 ABSTRACT Supernumerary free radicals and other reactive species can cause oxidative damage in animal cells, potentially leading to non-infectious diseases. Diets rich in low molecular weight antioxidants (LMWAs) may prevent or arrest the pathogenesis of these diseases. Leaves of Sonchus oleraceus L. may be an excellent dietary LMWA source for humans given their apparent strong antioxidant activities in vitro. However, different S. oleraceus plants vary in their antioxidant capacity. Nothing is known of possible environmental effects on antioxidant potential. Equally, the effects of cooking and gastrointestinal digestion are unknown. The goals of this research were: (i) to study the effects of plant age, locality, and abiotic stressors on antioxidant potential; (ii) to study the effects of cooking and in vitro gastrointestinal digestion on antioxidant activity and uptake in human cells; and (iii) to study extractable antioxidant activities of S. oleraceus cell suspension cultures in relation to abiotic stressors. Antioxidant activities and levels of total phenolics, hydroxycinnamic acids and ascorbate increased as plants aged. An ecotype from Acacia Bay had a higher phenolic content and antioxidant activities than one from Oamaru; these differences were maintained across generations as well as in calli from in vitro cultures. This indicates heritability and genetic fidelity of antioxidant potential. Chilling and salinity had variable effects on concentrations of phenolics and antioxidant activities in plants, and the combination of the two stressors was not synergistic. This indicates that these two stressors share signalling and response pathways. Stressor- induced increases in antioxidant activities of leaf extracts correlated with improved cellular antioxidant activities (CAA) inside HepG2 cells. Antioxidants were released from leaves following in vitro gastrointestinal digestion, which were then subsequently uptaken by Caco2 and HepG2 cells wherein they displayed CAAs. Thus, elevated levels of antioxidants in stressor-imposed plants provide potentially more antioxidant protection to live human cells. Caftaric, chlorogenic and chicoric acids accounted for 92% of the phenolic compounds in S. oleraceus leaves. Of these, only chlorogenic acid was inducible by stressors, both in ii intact plants and in calli. In young stressor-applied plants, chlorogenic acid was enhanced to the levels achievable with plant ageing. Boiling leaves prior to digestion did not diminish the caftaric and chlorogenic acid levels released through digestion, but chicoric acid levels were. Out of the nine phenolic compounds in leaves, only chicoric, chlorogenic and caftaric acids were released into the medium during in vitro gastrointestinal digestion. Digestion of leaves resulted in effective release of caftaric and chlorogenic acids from leaves but the levels of released chicoric acid were diminished by digestion. This study offers insights into the factors that influence the antioxidant potential of S. oleraceus L. in vivo, in vitro, during cooking and in vitro gastrointestinal digestion. These results provide the foundation for: (1) encouraging the consumption of its fresh shoots as an antioxidant rich food; (2) further improving its antioxidant activities through manipulation of agronomy, ecotype and breeding; (3) developing its cell cultures as a commercial platform for phyto-antioxidant production aimed at formulating dietary supplements or food additives in biopharmaceutical industry. iii ACKNOWLEDGEMENTS It is with deep gratitude that I express my sincere thanks to my supervisor Prof. Kevin S. Gould for the tremendous advice, encouragement, guidance and supervision extended to me throughout the research and made this thesis possible. Despite his busy schedule, he was always there to listen, motivate me and give feedback. My secondary supervisor, Dr Arlene McDowell welcomed me into her lab at the School of Pharmacy, University of Otago which is greatly appreciated. I am truly thankful for her support and guidance provided for laboratory work and for her valuable feedback on the thesis. My gratitude also goes to Dr Lesley Milicich, Derek Heath, Jack Cameron, Alan Howard (School of Biological Sciences, Victoria University of Wellington, New Zealand), Zongquan Ou, Julie Harwood, Len Stevenson, Mike Broughton and Kevin Crump (School of Pharmacy, University of Otago, New Zealand) for their assistance and guidance with laboratory work. I wish to thank Kevin Gould lab group (Kate, Iggy, Luke, Karl and Gagan) for always providing fresh insights into problems and for just being there to answer my questions. I am grateful for the funding by a New Zealand International Doctoral Research Scholarship, a Faculty Strategic Research Grant and three Wellington Botanical Society student grants which aided major and complementary financial requirements during past three years. I am grateful for the moral support, friendly banter and concern shown by my adoptive family in New Zealand: Damayanthi, Prasad, Sandra, Trinh, Bruce, Kosala and Chathuri. I would like to thank my parents and Uday for giving me their care, love and unprecedented understanding throughout my life. I could not have done it without their moral and financial support over all these years. Finally, Waruna Thank you for making me your priority and putting up with me during past seven years. iv TABLE OF CONTENTS ABSTRACT ......................................................................................................................... ii ACKNOWLEDGEMENTS ................................................................................................ iv TABLE OF CONTENTS ..................................................................................................... v LIST OF FIGURES ........................................................................................................... xii LIST OF PLATES ............................................................................................................ xvi LIST OF TABLES ........................................................................................................... xvii CHAPTER 1: REVIEW OF LITERATURE ....................................................................... 1 1.1 REACTIVE SPECIES AND ANTIOXIDANT DEFENCES ............................... 1 1.2 LOW MOLECULAR WEIGHT ANTIOXIDANTS OF DIETARY ORIGIN .... 3 1.2.1 Ascorbic acid ................................................................................................. 3 1.2.2 Carotenoids .................................................................................................... 4 1.2.3 Vitamin E ....................................................................................................... 5 1.2.4 Phenolic compounds ...................................................................................... 6 1.2.4.1 Phenolic acids ......................................................................................... 6 1.2.4.2 Flavonoids .............................................................................................. 7 1.2.4.3 Stilbenes .................................................................................................. 9 1.2.4.4 Antioxidant activities of phenolic compounds ..................................... 10 1.3 EVIDENCE FOR INFLUENCE OF DIETARY LMWAS ON OXIDATIVE STRESS-INDUCED DISEASES .................................................................................. 12 1.4 THE BIOACCESSIBILITY, BIOAVAILABILITY, BIOACTIVITY AND STABILITY OF PHENOLIC COMPOUNDS .............................................................. 16 v 1.5 CHEMICAL ANTIOXIDANT ACTIVITY ASSAYS ....................................... 24 1.5.1 DPPH radical scavenging assay ................................................................... 24 1.5.2 ORAC assay ................................................................................................. 26 1.6 THE CELLULAR ANTIOXIDANT ACTIVITY (CAA) ASSAY .................... 27 1.6.1 Cytotoxicity .................................................................................................. 29 1.7 TRADITIONAL DIET OF MĀORI AND INCIDENCE OF COLORECTAL CANCER ....................................................................................................................... 30 1.8 Sonchus oleraceus L. ........................................................................................... 30 1.8.1 Biology of S. oleraceus ................................................................................ 31 1.8.2 Nutrients in leaves of S. oleraceus ............................................................... 32 1.8.3 LMWAs of S. oleraceus .............................................................................. 33 1.8.4 Variation in antioxidant potential of S. oleraceus ....................................... 34 1.9 EFFECT OF STRESS ON ANTIOXIDANT LEVELS IN PLANTS ................ 36 1.9.1 Drought stress on LMWA production in plants ........................................... 38 1.9.2 Chilling stress on LMWA production in plants ........................................... 38 1.9.3 Light stress on LMWA production in plants ..............................................
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