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ANTI-AGING AND ANTI-OXIDANT PHYTOCHEMICAL COMPOUNDS ZIYUN WU NATIONAL UNIVERSITY OF SINGAPORE 2013 ANTI-AGING AND ANTI-OXIDANT PHYTOCHEMICAL COMPOUNDS ZIYUN WU (M. Agr. & B.Sc., Hainan University, China) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY FACULTY OF SCIENCE NATIONAL UNIVERSITY OF SINGAPORE 2013 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety, under the supervision of associate professor Dejian Huang and the co-supervision of assistant professor Shao Quan Liu, (in the laboratory S13- 05-02 and S9-02-01), Chemistry Department, National University of Singapore, between August, 2009 and July, 2013. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. The content of the thesis has been partly published in: 1. Wu Z, Song L, Liu S and Huang D. Independent and additive effects of glutamic acid and methionine on yeast longevity. PLoS One. 2013, 8(11): e79319.doi:10.1371/journal.pone.0079319. 2. Wu Z, Liu S and Huang D. Dietary restriction depends on nutrient composition to extend chronological lifespan in budding yeast Saccharomyces cerevisiae. PLoS One. 2013, 8(5): e64448. doi:10.1371/journal.pone.0064448. 3. Wu Z, Song L, Feng S, Liu Y, He G, Yioe Y, Liu S and Huang D. Germination dramatically increases isoflavonoid content and diversity in chickpea (Cicer arietinum L.) seeds. Journal of Agricultural and Food Chemistry. 2012, 60, 8606−8615. 4. Wu Z, Song L, Liu S and Huang D. A high throughput screening assay for determination of chronological lifespan of yeast. Experimental Gerontology. 2011, 46, 915-922. 5. Wu Z, Song L and Huang D. Food grade fungal stress on germinating peanut seeds induced phytoalexins and enhanced polyphenolic antipxidants. Journal of Agricultural and Food Chemistry. 2011, 59, 5993−6003. Name Signature Date I ACKNOWLEDGEMENTS It is difficult to overstate my gratitude to my PhD supervisor, Professor Dejian Huang. With passion, he has helped me to develop scientific instincts. Through my years in his lab, he has provided sound advice, innovative ideas and rigorous training. My research would have been at a loss without his continuous guidance. I thanks for all his guidance, support and encouragement throughout my PhD research work. My special thanks go to my co-supervisor, Professor Shao Quan Liu for his guidance during my PhD study. I would like to show my greatest gratitude to my wife for helping me through the difficult times with her emotional support and love. I also wish to thank my father. I appreciate his unconditional love and support, without which my life would not go on. To them I dedicate this thesis. I would like to express my deepest appreciation to Dr Wei Yao for help in doing the NMR, and for his knowledge and expertise in the isolation of glyceollins. Next, I would like to express my heartfelt appreciation to Miss Seu Lan Lee, Mr Yitohiya Yoichi, Miss Yi Ling Quek, Miss Wei Chen, Miss Pei Chun Yip, Mr Caili Fu, Miss Hongyu Wang, Miss TingTing Liu, Miss Yan Yan, Miss Ik Chian Wong, Mr Restituto T. Tocmo and Mr Zhongwei Liu for their help, technical support, advice and assistance rendered throughout the project. I am grateful for the learning opportunity at National University of Singapore. I thank Ms Chooi Lan Lee, Ms Huey Lee Lew, Miss Xiaohui Jiang and FST staff for their excellent technical support, advice and help throughout the project. Special thanks also go to Professor Weibiao Zhou, Dr. Hyun-Gyun Yuk, Dr. Lai Peng Leong, and NUS staff for their teaching and support. I wish to thank my NUS friends Liangliang Wang, Jie Han, Lihua Ding, Dan Zhang and FST friends for their help, support and camaraderie. Last but not least, the financial support from National University of Singapore is greatly appreciated. II TABLE OF CONTENTS DECLARATION .................................................................................................................................... I ACKNOWLEDGEMENTS .................................................................................................................. II TABLE OF CONTENTS .................................................................................................................... III SUMMARY .......................................................................................................................................... IX LIST OF TABLES ............................................................................................................................... XI LIST OF FIGURES ......................................................................................................................... XIII LIST OF ABBREVIATIONS ......................................................................................................... XVII LIST OF PUBLICATIONS .............................................................................................................. XIX LIST OF PRESENTATIONS ............................................................................................................ XX Part I SCREENING OF ANTI-AGING FACTORS USING A HIGH THROUGHPUT ASSAY . 1 Chapter 1 INTRODUCTION ............................................................................................................... 2 1.1 Background ................................................................................................................................ 2 1.2 Objectives .................................................................................................................................. 5 Chapter 2 LITERATURE REVIEW ................................................................................................... 7 2.1 Aging models in budding yeast ................................................................................................. 7 2.2 Methods for measuring yeast CLS ............................................................................................. 8 2.2.1 Traditional colony count plating method ....................................................................... 8 2.2.2 Flow cytometry method ............................................................................................... 10 2.2.3 High throughput method based on outgrowth of aging cells ....................................... 11 2.3 Nutrients and longevity ........................................................................................................... 12 2.3.1 Carbohydrates .............................................................................................................. 12 2.3.2 Amino acids ................................................................................................................. 13 2.3.3 Other nutrients ............................................................................................................. 15 2.4 Natural products for lifespan extension ................................................................................... 15 2.4.1 Resveratrol ................................................................................................................... 19 2.4.2 Rapamycin ................................................................................................................... 22 2.5 The evolutionarily conserved signaling pathways ................................................................... 25 2.5.1 TOR/Sch9 pathway ...................................................................................................... 25 2.5.2 Ras/AC/PKA pathway ................................................................................................. 27 III 2.5.3 Sirtuins ......................................................................................................................... 29 Chapter 3 DEVELOPMENT OF HTS ASSAY FOR DETERMINATION OF CHRONOLOGICAL LIFESPAN OF YEAST .................................................................................. 31 3.1 Introduction ............................................................................................................................. 31 3.2 Materials and methods ............................................................................................................. 32 3.2.1. Materials ..................................................................................................................... 32 3.2.2 Modification of a shaker incubator for aging culture ................................................... 34 3.2.3 High-throughput assay procedure ................................................................................ 34 3.2.4 Data analysis ................................................................................................................ 35 3.3 Results ..................................................................................................................................... 38 3.3.1 Evaluation of reproducibility, precision and accuracy of HTS assay .......................... 38 3.3.2 Initial population size modulates CLS independent of CR .......................................... 39 3.3.3 Effect of cellular state in YPD medium on Yeast CLS ................................................ 41 3.3.4 Calorie level optimizes yeast CLS but not biomass production ................................... 42 3.3.5 Identification of calorie restriction mimetic gene ........................................................ 42 3.4 Discussion ................................................................................................................................ 45 Chapter 4 DIETARY RESTRICTION DEPENDS ON NUTRIENT COMPOSITION TO EXTEND CHRONOLOGICAL LIFESPAN IN BUDDING YEAST .............................................. 50 4.1 Introduction ............................................................................................................................. 50 4.2 Materials and methods ............................................................................................................. 52 4.2.1 Materials ...................................................................................................................... 52 4.2.2 Experimental design and statistical analysis ................................................................ 52 4.2.3 Lifespan, biomass and yeast cell growth assay ............................................................ 53 4.2.4 Data analysis ................................................................................................................ 53 4.3 Results ..................................................................................................................................... 58 4.3.1 DR regime is dependent on nutrients in media ............................................................ 58 4.3.2 Development of statistical design of experiments for evaluation of nutrition, biomass and lifespan ........................................................................................................................... 61 4.3.3 Nutrient composition is a key factor for longevity of yeast ......................................... 62 4.3.4 Biomass production of the four strains has similar changes in response to nutrient composition ........................................................................................................................... 67 4.3.5 sch9Δ is more sensitive to nutrients than the other three strains .................................. 69 4.3.6 Aging media pH is dependent on glucose concentration and has no correlation with lifespan .................................................................................................................................. 71 4.3.7 The optimal SD medium for yeast ............................................................................... 72 IV 4.4 Discussion ................................................................................................................................ 74 4.5 Conclusion ............................................................................................................................... 80 Chapter 5 INDEPENDENT AND ADDITIVE EFFECTS OF GLUTAMIC ACID AND METHIONINE ON YEAST LONGEVITY ....................................................................................... 81 5.1 Introduction ............................................................................................................................. 81 5.2 Materials and methods ............................................................................................................. 83 5.2.1 Materials ...................................................................................................................... 83 5.2.2 Lifespan, biomass and yeast cell growth assay ............................................................ 83 5.2.3 Acetic acid analysis ...................................................................................................... 83 5.2.4 Data analysis ................................................................................................................ 84 5.3 Results ..................................................................................................................................... 84 5.3.1 Amino acids regulate lifespan and biomass changes in yeast ...................................... 84 5.3.2 Methionine and glutamic acid cause lifespan and biomass alterations in yeast ........... 89 5.3.3 Independent and additive effects of glutamic acid, methionine and glucose on lifespan extension ............................................................................................................................... 94 5.3.4 Conserved protein kinase Gcn2 mediates amino acids induced lifespan extension ..... 99 5.4 Discussion .............................................................................................................................. 105 5.5 Conclusion ............................................................................................................................. 110 Chapter 6 CRYPTOTANSHINONE EXTENDS CHRONOLOGICAL LIFESPAN IN THE BUDDING YEAST ............................................................................................................................. 111 6.1 Introduction ........................................................................................................................... 111 6.2 Experimental Procedures ....................................................................................................... 113 6.2.1 Materials .................................................................................................................... 113 6.2.2 Lifespan and yeast cell growth assay ......................................................................... 113 6.2.3 HPLC chromatogram analysis of compounds from Danshen .................................... 113 6.2.4 Intracellular ROS quantification and fluorescence images of yeast cells .................. 114 6.2.5 Data analysis .............................................................................................................. 115 6.3 Results ................................................................................................................................... 115 6.3.1 A high throughput assay identifies cryptotanshinone as the key compound from Danshen to extend yeast lifespan in a concentration and the time of addition dependent manner ................................................................................................................................ 115 6.3.2 Cryptotanshinone induced CLS extension is prevented by amino acid restriction .... 120 6.3.3 Essential amino acid sufficiency is required for cryptotanshinone induced longevity ............................................................................................................................................ 122 6.3.4 Cryptotanshinone requires Tor1 and Sch9 for CLS extension ................................... 125 V 6.3.5 Tanshinones extend yeast lifespan via similar mechanisms ...................................... 129 6.3.6 Gcn2 regulates essential amino acids and cryptotanshinone induced CLS extension 130 6.3.7 Cryptotanshinone extend lifespan without reduce in ROS level in sod2Δ ................. 132 6.4 Discussion .............................................................................................................................. 133 6.5 Conclusion ............................................................................................................................. 139 Chapter 7 HORMESIS OF GLYCEOLLIN I, AN INDUCED PHYTOALEXIN FROM SOYBEAN, ON BUDDING YEAST CHRONOLOGICAL LIFESPAN EXTENSION .............. 140 7.1 Introduction ........................................................................................................................... 140 7.2 Experimental Procedures ....................................................................................................... 141 7.2.1 Materials .................................................................................................................... 141 7.2.2 Isolation of glyceollins ............................................................................................... 141 7.2.3 Lifespan and yeast cell growth assay ......................................................................... 142 7.2.4 Data analysis .............................................................................................................. 142 7.3 Results and Discussion .......................................................................................................... 142 7.3.1 Antiproliferation activity of glyceollins ..................................................................... 142 7.3.2 Glyceollin I extends yeast CLS by CR-dependent regime ......................................... 145 7.3.3 Hormetic effect of glyceollin I on yeast lifespan ....................................................... 147 7.4 Conclusion ............................................................................................................................. 148 Chapter 8 CONCLUSIONS AND FUTURE OUTLOOK ............................................................. 150 Part II ANTIOXIDANTS FROM GERMINATED LEGUME SEEDS ....................................... 154 Chapter 9 INTRODUCTION ............................................................................................................ 155 9.1 Introduction ........................................................................................................................... 155 9.1.1 Grain legume is important for human diet ................................................................. 155 9.1.2 Phytochemical profiling is significant for quality and safety control ........................ 156 9.1.3 Antioxidant capacity is a popular criterion of functional food................................... 156 9.2 Objectives .............................................................................................................................. 158 Chapter 10 POLYPHENOLIC ANTIOXIDANTS AND PHYTOALEXINS CHANGES IN GERMINATING LEGUME SEEDS WITH FOOD GRADE FUNGAL RHIZOPUS OLIGOPORUS STRESS .................................................................................................................... 160 10.1 Introduction ......................................................................................................................... 160 10.2 Materials and Methods ........................................................................................................ 161 10.2.1 Materials .................................................................................................................. 161 10.2.2 Germination and fungal inoculations ....................................................................... 162 VI 10.2.3 Quantification of antioxidant capacity and total phenolics ...................................... 162 10.2.4 Phytochemicals and phytoalexins identification ...................................................... 163 10.2.5 Data analysis ............................................................................................................ 163 10.3 Results and Discussion ........................................................................................................ 164 10.3.1 Comprehensive evaluation of antioxidant contents of fungal-stressed sprouts ........ 164 10.3.2 Phytochemical and phytoalexin changes in germinating legume seeds ................... 167 10.4 Conclusion ........................................................................................................................... 173 Chapter 11 GERMINATION DRAMATICALLY INCREASES ISOFLAVONOID CONTENT AND DIVERSITY IN CHICKPEA (CICER ARIETINUM L.) SEEDS ......................................... 174 11.1 Introduction ......................................................................................................................... 174 11.2 Materials and Methods ........................................................................................................ 176 11.2.1 Materials .................................................................................................................. 176 11.2.2 Instruments ............................................................................................................... 176 11.2.3 Seed germination ..................................................................................................... 176 11.2.4 Sample preparation procedures ................................................................................ 177 11.2.5 Quantification of antioxidant capacity and TPC ...................................................... 177 11.2.6 Detection of isoflavonoids by PDA and MS ............................................................ 178 11.2.7 Isolation and identification of biochanin A and formononetin ................................ 179 11.2.8 Identification and quantification of isoflavonoids from chickpea and soybean ....... 179 11.2.9 Statistical analysis .................................................................................................... 180 11.3 Results and Discussion ........................................................................................................ 180 11.3.1 Antioxidant capacity and TPC ................................................................................. 180 11.3.2 Profiles of isoflavonoids in germinated chickpea seeds ........................................... 182 11.3.3 Quantification of isoflavonoids in chickpea and soybean ........................................ 188 11.4 Conclusion ........................................................................................................................... 193 Chapter 12 FOOD GRADE FUNGAL STRESS ON GERMINATING PEANUT SEEDS INDUCED PHYTOALEXINS AND ENHANCED POLYPHENOLIC ANTIOXIDANTS ........ 195 12.1 Introduction ......................................................................................................................... 195 12.2 Materials and Methods ........................................................................................................ 196 12.2.1 Reagents ................................................................................................................... 196 12.2.2 Instruments ............................................................................................................... 196 12.2.3 Peanut germination and fungal inoculations ............................................................ 196 12.2.4 Sample preparation procedures ................................................................................ 197 VII 12.2.5 Quantification of antioxidant capacity and total phenolics ...................................... 197 12.2.6 Detection of phytochemical by PDA and MS .......................................................... 198 12.2.7 Phytoalexin extraction and detection by HPLC and LC-MS ................................... 199 12.3 Results and Discussion ........................................................................................................ 199 12.3.1 Profiles of phenolic acids in peanut seeds ................................................................ 200 12.3.2 Polyphenolic profiles in germinated peanuts ........................................................... 207 12.3.3 Phytoalexins in germinated peanuts ......................................................................... 211 12.4 Conclusion ........................................................................................................................... 214 Chapter 13 CONCLUSIONS AND FUTURE OUTLOOK ........................................................... 216 BIBLIOGRAPHY .............................................................................................................................. 218 VIII

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appreciate his unconditional love and support, without which my life would not go on. Thus, looking for the elixir of youth is an emergency task for (Longo et al. 1996a, Longo 1999). In this protocol, the inoculation time point is day 0. Every 2-4 days, aliquots from aging culture are diluted and
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