anti-aging and anti-oxidant phytochemical compounds ziyun wu national university of singapore ...

259 Pages·2013·4.5 MB·English

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

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