ABSTRACT OF THE DISSERTATION ISOLATION AND CHARACTERIZATION OF NATURAL PRODUCTS FROM GINGER AND ALLIUM URSINUM BY HOU WU Dissertation Director: Dr. Chi-Tang Ho Phenolic compounds from natural sources are receiving increasing attention recent years since they were reported to have a remarkable spectrum of biological activities including antioxidant, anti-inflammatory and anti-carcinogenic activities. They may have many health benefits and can be considered possible chemo- preventive agents against cancer. In this research, we attempted to isolate and characterize phenolic compounds from two food sources: ginger and Allium ursinum. Solvent extraction and a series of column chromatography methods were used for isolation of compounds, while structures were elucidated by integration of data from MS, 1H-NMR, 13C-NMR, HMBC and HMQC. Antioxidant activities were evaluated by DPPH method and anti- inflammatory activities were assessed by nitric oxide production model. Ginger is one of most widely used spices. It has a long history of medicinal use dating back 2500 years. Although there have been many reports concerning ii chemical constituents and some biological activities of ginger, most works used ginger extracts or focused on gingerols to study the biological activities of ginger. We suggest that the bioactivities of shogaols are also very important since shogaols are more stable than gingerols and a considerable amount of gingerols will be converted to shogaols in ginger products. In present work, eight phenolic compounds were isolated and identified from ginger extract. They included 6-gingerol, 8-gingerol, 10- gingerol, 6-shogaol, 8—shogaols, 10-shogaol, 6-paradol and 1-dehydro-6-gingerdione. DPPH study showed that 6-shogaol had a comparable antioxidant activity compared with 6-gingerol, the 50% DPPH scavenge concentrations of both compounds were 21 µM. All of the eight isolated compounds had effects on inhibiting LPS-induced NO production, and 6-shogaol showed more inhibitory effect than 6-gingerol with reducing nitrite production by 85 % compared with 35 % by 6-gingerol at 5 µM. Flavonoids are a group of phenolic compounds that occur naturally in food of plant origin. Compelling data indicated that flavonoids had important effects on cancer chemoprevention and chemotherapy. Among 10 compounds we isolated from Allium ursinum, a widely used spice, eight of them were flavonoids. Three of them, kaempferol 3-O-α-L-rhamnopyranosyl (1→2)-[3-O-acetyl]-β-D-glucopyranoside, kaempferol 3-O-α-L-rhamnopyronosyl (1→2)-[6-O-acetyl]-β-D-glucopyranoside and 6’-O- acetyl kaempferol-3-O-L-rhamnopyranosyl(1→2)-β-D-glucopyranoside- 7-O- [2-O-(trans-p-coumaroyl)]-β-D-glucopyranoside were new natural products. iii ACKNOWLEDGEMENTS Words are not enough for expressing my gratitude to my advisor, Dr. Chi-Tang Ho, for his inspiring guidance and sincere encouragement throughout my study. He was always generous providing his valuable time for discussion. His encouragement, patience and trust always bring me through all the hardship I have encountered. My gratitude extends to my dissertation committee member, Dr. Henryk Daun, Dr. Thomas G. Hartman, and Dr. Slavik Dushenkovf, for their invaluable contributions to my research. Special thanks goes to Dr. Shenming Sang for his tremendous help with spectral analysis; Dr. Min-Hsiung Pan for his expertise in anti-inflammatory study. Finally, I would like to express my deepest appreciation to my beloved wife, Guanying Sun, for her understanding and sacrifice; and my dear mother and father, for their endless love and support in the world; also my adorable son, Evan Y. Wu, for the wonderful happiness he has been bringing to me. iv DEDICATION To my dear wife, Guanying Sun, son, Evan Wu, mother, Zhennv Zou and father Xingfu Wu v TABLE OF CONTENTS ABSTRACT OF THE DESSERTATION……………………………………ii ACKNOWLEDGEMENT………………………………………………………..iv DEDICATION………………………………………………………………….. v TABLE OF CONTENTS…………………………………………………………..vi LIST OF TABLES……………………………………………………..…………viii LIST OF FIGURES…………………………………………………………..……..ix LIST OF APPENDICES…………………………………………….……………xi 1. INTRODCUCTION……………………………………………….1 1.1. Food and health………………………………………………………………….1 1.2. Inflammation and some anti-inflammatory compounds in food……………..1 1.3. Antioxidants in food …………………………………………………..………….9 1.4. Flavonoids in food…………………………………………………..10 2. LITERATURE REVIEW ……………………………………………...12 2.1. Chemical components of ginger…………………………………..…………….12 2.1.1. Bioactive constituents of ginger………………………………………..……14 2.1.2. Stability of 6-gingerol…………………………………………….……15 2.2. Components in Allium ursinum………………………….………………………18 3. HYPOTHESIS AND OBJECTIVES………………………………….20 4. EXPERIMENTAL…………………………………………………..……………21 4.1. Plant materials……………………………………………………………….…..21 vi 4.2. Solvents, reagents and chromatographic supplies………………………………21 4.3. Instruments…………………………………………….……………………22 4.4. Extraction and Isolation procedures………………..……………………………23 4.4.1. Isolation of compounds from ginger………………..…………………………23 4.4.2. Isolation of compounds from Allium ursinum……………..……………….25 4.5. Nitrite assay……………………………………………………………….….27 4.6. Antioxidant assay: 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay……….….28 5. RESULTS AND DISCUSSSION…………………………………………..…30 5.1. Isolation of components from ginger and their biological activity study……….30 5.1.1. Method of separating 6-gingerol and 6-shogaol from ginger extract……….30 5.1.2. Identification of constituents from ginger extract…………………………….32 5.1.3. Quantification of 6-gingerol and 6-shogaol in different ginger products…….39 5.1.4. Antioxidant activity of 6-gingerol and 6-shogaol……………………..……….42 5.1.5. Anti-inflammatory activities of compounds from ginger……………..……….44 5.2. Isolation of flavonoids of Allium ursinum……………………………………47 6. CONCLUSION ………………………………………………………………….69 7. FUTURE PERSPECTIVES…………………………………………………..71 8. REFERENCE……………………………………………………….…………..73 9. APPENDIX……………………………………………………………………..81 CURRICULUM VITAE………………………………………………..…………125 vii LIST OF TABLES Table 1. The levels of 6-gingerol and 6-shogaol in ginger tea products……….41 Table 2. 1H NMR and 13C NMR assignments for Aub 30-3-2……………….50 Table 3. 1H NMR and 13C NMR assignments for Aub 30-3…………………..52 Table 4. 1H NMR and 13C NMR assignments for Aub 1……………………..54 Table 5. 1H NMR and 13C NMR assignments for Aub-30-1………………….56 Table 6. 1H NMR and 13C NMR assignments for Aub-1-1……………………59 Table 7. 1H NMR and 13C NMR assignments for Aub-3………………………62 Table 8. 1H NMR and 13C NMR assignments for Aub-7…………………………64 Table 9. 1H NMR and 13C NMR assignments for Aub-5…………………………67 viii LIST OF FIGURES Figure 1. Structure of curcumine………………...…………………………………….5 Figure 2. Structures of theaflavins.................................................................................6 Figure 3. Structure of garcinol……………………………………..…………….….7 Figure 4. Structure of carnosol…………………………………………………….….7 Figure 5. Structure of genistein……………………………………………….…….8 Figure 6. Basic structure of flavonoids………………………………………………11 Figure 7. Non-volatile pungent compounds of ginger……………………………….13 Figure 8. Schematic degradation process of gingerol in aqueous acidic environmen.15 Figure 9. Time dependent reversible degradation of [6]-gingerol…………….....16 Figure 10. Time dependent reversible degradation of [6]-shogaol .............................16 Figure 11. Volatile compounds of Allium ursinum………………………………19 Figure 12. Structures of known flavonoids in Allium ursinum………………….19 Figure 13. The procedure of isolation of compounds from ginger extract…………24 Figure 14. Procedure for isolation of compounds from Allium ursinum…………26 Figure 15. Reaction of DPPH with an antioxidant………………………………28 Figure 16. Isolation scheme of 6-gingerol and 6-shogaol from ginger extract……31 Figure 17. Structure of 6-gingerol………………………………………………..32 Figure 18. Structure of 8-gingerol…………………………………………………33 Figure 19. Structure of 10-gingerol…………………………………………………..34 Figure 20. Structure of 6-shogaol…………..………………………………………..34 Figure 21. Structure of 8-shogaol…………………………………………………..35 ix Figure 22. Structure of 10-shogaol…………………………………………………36 Figure 23. Structure of 6-paradol…………………………………………………….37 Figure 24. Structure of 1-dehydro-6-gingerdione………………………………..38 Figure 25. DPPH free radicals scavenge activity of 6-gingerol and 6-shogaol…….43 Figure 26. Effects of tested compounds on LPS-induced nitrite formation………46 Figure 27. Structure of p-coumaric acid………………………………………….47 Figure28. Structure of L-tryptophan……………………………………………..48 Figure 29. Structure of Aub 30-3-2……………………………………………….49 Figure 30. Structure of Aub 30-3………………………………………………..51 Figure 31. Structure of Aub-1……………………………………………………..53 Figure 32. Structure of Aub 30-1………………………………………………….55 Figure 33. Structure of Aub 1-1………………………………………………….58 Figure 34. Structure of Aub-3……………………………………………………..61 Figure 35. Structure of Aub-7…………………………………………………….63 Figure 36. Structure of Aub-5…………………………………………………….67 x LIST OF APPENDICES Appendix 1. 1H-NMR of 6-gingerol………………………………………………81 Appendix 2. 13C-NMR of 6-gingerol………………………………………………82 Appendix 3. 1H-NMR of 8-gingerol………………………………………………..83 Appendix 4. 13C-NMR of 8-gingerol……………………………………………….84 Appendix 5. 1H-NMR of 10-gingerol………………………………………………..85 Appendix 6. 13C-NMR of 10-gingerol………………………………………………86 Appendix 7. 1H-NMR of 6-shogaol………………………………………………….87 Appendix 8. 13C-NMR of 6-shogaol………………………………………………88 Appendix 9. 1H-NMR of 8-shogaol………………………………………………….89 Appendix 10. 13C-NMR of 8-shogaol………………………………………………90 Appendix 11. 1H-NMR of 10-shogaol……………………………………………….91 Appendix 12. 13C-NMR