Isolation and Characterization of Natural

Home , Garcinia indica, Gingerol, Paradol, Shogaol, Zingerone, Zingibain

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 of 10-shogaol……………………………………………92

Appendix 13. 1H-NMR of 6-paradol………………………………………………93

Appendix 14. 13C-NMR of 6-paradol………………………………………………94

Appendix 15. 1H-NMR of 1-dehydro-6-gingerdione………………………………95

Appendix 16. 13C-NMR of 1-dehydro-6-gingerdione………………………………96

Appendix 17. Standard curve of 6-gingerol……………………………………..….97

Appendix 18. Standard curve of 6-shogaol……………………………….………….97

Appendix 19. HPLC profile of 6-gingerol………………………………………….98

Appendix 20. HPLC profile of 6-shogaol………………………………………….98

Appendix 21. 1H-NMR of Aub 30-2-3…………………………………………….99

xi Appendix 22. 1H-NMR of Aub 30-3-1……………………………………………100

Appendix 23. 13C-NMR of Aub 30-3-1……………………………………………101

Appendix 24. 1H-NMR of Aub 30-3-2…………………………………………….102

Appendix 25. 13C-NMR of Aub 30-3-2…………………………………………….103

Appendix 26. 1H-NMR of Aub 30-3………………………………………………104

Appendix 27. 13C-NMR of Aub 30-3……………………………………………….105

Appendix 28. 1H-NMR of Aub-1…………………………………………………..106

Appendix 29. 13C-NMR of Aub-1…………………………………………………107

Appendix 30. 1H-NMR of Aub 30-1……………………………………………….108

Appendix 31. 13C-NMR of Aub 30-1……………………………………………….109

Appendix 32. 1H-NMR of Aub-1-1……………………………………………….110

Appendix 33. 13C-NMR of Aub-1-1……………………………………………….111

Appendix 34. HMQC of Aub-1-1………………………………………………….112

Appendix 35. HMBC of Aub-1-1…………………………………………………..113

Appendix 36. 1H-NMR of Aub-3…………………………………………………114

Appendix 37. 13C-NMR of Aub-3…………………………………………………115

Appendix 38. HMQC of Aub-3……………………………………………………116

Appendix 39. HMBC of Aub-3…………………………………………………….117

Appendix 40. 1H-NMR of Aub-7…………………………………………………118

Appendix 41. HMQC of Aub-7……………………………………………………119

Appendix 42. HMBC of Aub-7……………………………………………………120

Appendix 43. 1H-NMR of Aub-5………………………………………………….121

xii Appendix 44. 13C-NMR of Aub-5………………………………………………….122

Appendix 45. HMQC of Aub-5…………………………………………………….123

Appendix 46. HMBC of Aub-5…………………………………………………….124

xiii 1

1. INTRODCUCTION

1.1. Food and health

From l990s, consumers began to view food not only as a means to satisfy hunger, prevent diet-deficiency diseases or to provide essential nutrition (e.g., water, protein, carbohydrate, fat, vitamins and minerals), but also as an important vehicle to keep us healthy (Hasler, 2000). We have begun to believe that a good diet could potentially help us reduce the risk for a variety of chronic disease such as cancer, heart diseases, osteoporosis, arthritis and age-related macular degeneration (Hasler, 2000; Hasler, 2002).

Many components have been evaluated as potential chemopreventive agents in diets. For examples, more than 4000 flavonoids in foods or plant origin were found to have a variety of biological effect, including serving as antioxidants, influencing drug detoxification mechanisms, and altering cell proliferation (Milner, 2000).

And more and more foods are under chemical and biological investigation for their potential role in disease prevention and health promotion. In our study, we have focus on investigation of isolation and biological activity of components in ginger and Allium ursinum.

1.2. Inflammation and some anti-inflammation agents in food

1.2.1. Inflammation and chronic diseases

Inflammation is a complex process initiated by bacterial infection, injury, trauma,

or ultraviolet light irradiation (Huang et al., 2004). This process involves activation and directed migration of leukocytes (neutrophils, monocytes and eosinophils); the killing of microbes and host cells they effect; liquefaction of surrounding tissue to prevent microbial metastasis; and the healing of tissues damaged by trauma or by the host’s response (Coussens and Werb; 2002; Nathan, 2002).

Acute inflammatory response is believed to be a defense mechanism to aid in the

killing of bacteria, virus and/or parasite while facilitating wound repair. On the other

hand, persistent and prolonged chronic inflammation increase the risk associated with

developing certain degenerative diseases such as arthritis, atherosclerosis, heat disease,

asthma, diabetes and cancer (Huang et al., 2004; O’ Byrne and Dalgleish, 2001).

Recent data have expanded the concept that inflammation is strongly related with

tumor progression (Coussens and Werb, 2002; O’Byrne and Dalgleish, 2000).

Inflammation process provides the prerequisite environment for the development of

cancer. This environment includes suppressing cell mediated immune response and

promoting angiogenesis, inhibition of apoptosis and induction of cell proliferation, production of reactive oxygen species and metabolites that may in turn induce DNA damage and mutations and as a result, be carcinogenic (O’Byrne, and Dalgleish,

2001).

Since the inflammation is essential for the progression of cancer, chemical agents that have anti-inflammatory activities may have role in reducing the incidence of cancer.

1.2.2. Pro-inflammatory targets and their inhibition

During the inflammation process, the level of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (1L-1β) and interleukin-6 (1L-6) are higher elevated. These cytokines further trigger the up-regulation of other pro- inflammatory cytokines and chemokines, as well as increase the expression of many cellular adhesion molecules, selectins, integrins and immunoglobulins (Huang et al.,

2004).

On the other hand, there is an increase in the expression of phospholipase A2, cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX), nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase, xanthine oxidase and myeloperoxidase during the inflammation process (Huang et al., 2004; Hong et al., 2004).

Furthermore, the activation of the nuclear factor kappa B (NFkB) was observed in inflammatory process. NFkB play a pivotal role in the regulation of inducible enzymes, inflammatory cytokines, Cell adhesion molecules (CAMs), and other substances that are initiators or enhancers of the inflammatory process (Huang et al., 2004; Bremner and

Heinrich, 2002).

Huang et al. proposed that anti-inflammatory agents reduced the inflammatory process via the following mechanism ((Huang et al., 2004):

1. Inhibition of activation of NFkB

2. Blocking the over-expression of pro-inflammatory cytokines such as TNF-α,

1L-1, and 1L-6

3. Down-regulation of over-expression of CAMs and enzymes

4. Inhibit enzyme activity such as phospholipase A2, COX-2, 5-LOX, iNOS and

myeloperoxidase

5. Inhibit ROS generating enzyme activity

6. Increase ability to scavenge ROS (Huang et al., 2004)

Compounds isolated from ginger will be evaluated as potential anti-inflammatory agents in this study.

1.2.3. Nitric Oxide and nitric oxide synthase in inflammation process

Nitric oxide is a short-lived free radical. In low concentration, it is involved in

various physiological and pathophysiology processes, such as vasodilatin, inhibition

of platelet function, macrophage cytotoxicities, synaptic neurotransmission as well as

host defense (Hong et al., 2006; Lin et al., 1999; Ho et al., 2002). However, in

inflammation, macrophages simultaneously produce high concentration of NO, which

has been shown to cause deamination of deoxynucleotides and bases with intact DNA

in vitro and are cytotoxic and mutagenic (Lin et al., 1999; Ho et al., 2002; Jun et al.,

2005).

Nitric oxide is synthesized in vivo from L-arginine by NO synthase with NADPH

and oxygen (Lin et al., 1999). The formation of NO from arginine is catalyzed by

three different types of NO synthase ((NOS): endothelial NOS, neuronal NOS and

iNOS. iNOS is the enzyme stimulated by inflammatory cytokines for NO production

in macrophages and many other cell types (Hong et al., 2006). Endothelial NOS and

neuronal NOS are constitutive and Ca2+/calmodulin-dependent. This constitutive

enzyme synthesizes small amounts of NO triggered by various agonists (Lin et al.,

1999; Pan et al., 2000).

On the other hand, inducible NOS is calcium-independent and expressed in response to certain stimuli such as endotoxin or Escherichia coli lipopolysaccharide

(LPS) and cytokines (Jun et al., 2005). Increased NOS expression and/or activity were

reported in human gynecological, breast, and central nervous system tumors (Pan et

al., 2000).

In the present study, we examined effects of compounds isolated from ginger on

NO production in a murine macrophages cell line, RAW 264.7, and on the iNOS gene expression.

1.2.4. Anti-inflammation agents

A great number of anti-inflammation agents were found in the food, such as curcumin, theaflavins, garcinol, α-tocopherol, ascorbic acid, (-)-epigallocatechin gallate (EGCG), genistein, resveratrol, and omega-3 polyunsaturated fatty acid.

Curcumin (Figure1) is a food coloring and flavoring agent. It is a major constituent found in the spice turmeric. Studies showed that curcumin suppress the over expression of pro-inflammatory cytokine, down regulate over expression of phospholipids A2, COX-2, LOX, iNOS and myeloperoxidase (Hong et al., 2004;

Huang et al., 2004)

Figure 1. Structure of curcumin

Theaflavins (Figure 2) inhibited NO generation in lipopolysaccharide-activated macrophage, inhibited arachidonic acid metabolism via lipoxygenase and COX pathway in vivo and inhibited leukocyte infiltration and expression of ICAM-1, COX-

2, and iNOS in injured brain (Lin et al., 1999; Cai et al., 2006; Huang et al., 2006).

Figure 2. Structures of theaflavins. (A) theaflavin, (B) theaflavin-3-O-gallate, (C) theaflavin-3’-O-gallate, and (D) theaflavin-3,3’-O-digallate

Garcinol (Figure 3) is a polyisoprenylated benzophenoe derivative which is

purified from Garcinia indica fruit rind. Studies found that garcinol inhibited the

expression of inducible nitric oxide synthase and cyclooxygenases-2 in

lipopolysaccharide-activated macrophage. It also inhibited the NF-κB activation,

7 reduced the LPS-induced increase of intracellular reactive oxygen species (ROS)

(Liao et al., 2005; Hong et al., 2006; Liao et al., 2004).

Figure 3. Structure of garcinol

Carnosol (Figure 4) is a natural occurring phytopolyphenol found in rosemary.

Studies showed that it reduced lipopolysaccharid (LPS)-stimulated NO production in macrophage RAW 264.7, inhibited phorbol ester tetradecanoyl phorbol acetate (TPA) induced ear inflammation (Huang et al., 1994; Chan et al., 1995). It also decreased

LPS-induced iNOS mRNA and protein expression, inhibited iNOS and NF-κB promoter activity (Lo et al., 2002).

Figure 4. Structure of Carnosol

Genistein (Figure 5) is an isoflavone found in great abundance in soybeans. It

8 is found that genistein inhibited LPS-induced production of pro-inflammatory cytokines, TNF-α, IL -1β, and IL-6 in micoglia or astrocyte-enriched cultures; It also inhibited the expression of COX-2 (Huang et al., 2004).

Figure 5. Structure of Genistein

There are also a myriad of other anti-inflammation agents in food besides the above five. In our work, we intend to isolate potential anti-inflammation compounds from ginger.

1.3 Antioxidants in food

Free radical formed during normal metabolic processes can initiate the

peroxidation of membrane lipids, leading to the accumulation of lipid peroxide, which

is related to aging and other diseases. Antioxidants are compounds that inhibit or

delay the oxidation of other molecules by inhibiting the initiation or propagation of

oxidizing chain reaction (Catherin and Rice, 2003; Bergman et al., 2001).

The main role of antioxidants is to help the body protect itself against damage

caused by reactive oxygen species and degenerative diseases (Bergman et al., 2001).

Free radicals and reactive species attack lipids, proteins and DNA and induce their

oxidation, which may result in oxidative damage (Sang et al., 2002). And oxidative damage is associated with an elevated risk of chronic diseases, such as cardiovascular morbidity and cancer (Ninfali et al., 2005). Antioxidants can protect human being from oxidative damage by adsorbing and neutralizing free radical, quenching singlet and triplet oxygen, or decomposing peroxide by their redox properties (Sang et al.,

2002a ).

Food plants, such as fruits, vegetables, nuts and spices are the primary sources of naturally occurring antioxidants for human (Sang et al., 2002a). The most important groups are polyphenolics, such as flavonoids (comprising flavonols, isoflavones, and anthocyans). Also some pigments such as chlorophyllins, phytosterines and allysulfides were found to have antioxidant activities. Many of these compounds are currently marketed as food supplements and are used for the production of functional foods (Hoelzl et al., 2005).

A great number of antioxidants have been isolated from food sources, e.g. EC,

EGC, ECG, EGCG from green tea; theaflavins from black tea; garcinol from Garcinia indica; curcumin from turmeric; carnosol, carnosic acid and rosmarinic acid from rosemary (Jhoo et al., 2005; Sang et al., 2002b; Hong et al., 2004; Sang et al., 2003;

Lo et al., 2002). In our study, we attempted to isolate potential antioxidant from ginger and Allium ursinum.

1. 4. Flavonoids in food

Polyphenols are the most abundant antioxidants in the diet. The main dietary

sources of polyphenols are fruits, vegetables, cereals, chocolate, dry legumes as well

as plant-derived beverages such as fruit juices, tea, coffee, and red wine (Scalbert et

al., 2005). The dietary intake of polyphenols could be as high as 1 g/d (Scalbert et al.,

2005). They are very important antioxidants for our health. Flavonoids are a group of

polyphenolic compounds isolated from a wide range of plants (Pietta et al., 2000;

Catherin and Rice 2003). Their antioxidant properties and effects in diseases

prevention were not truly been studied until 1995 (Scalbert et al., 2005).

Flavonoids have their basic structure: the flavan nucleus (Figure 6.), which

consists of 15 carbon atoms arranged in three rings (C6-C3-C6), which are labeled A, B, and C. Flavonoids may be further divided into several subclasses, i.e., flavones (and isoflavones), flavanones, flavonols, flavanols (also called catechins), and anthocyanidins (Lotito 2006).

Studies found that consumption of flavonoid-rich foods, in particular fruits and

11 vegetables, is associated with a lower incidence of heart disease, stroke, cancer, and other chronic diseases (Lotito 2006). It has been suggested that the health benefits of dietary flavonoids are due to their antioxidant properties (Catherin and Rice 2003).

Many foods in human diets such as vegetables, fruits, tea and wine, contribute to the total daily intake of flavonoids in human. In this study, we attempted to find some new flavonoids in Allium ursinum.

Figure 6. Basic structure of flavonoids

2. LITERATURE REVIEW

In this chapter, two topics will be discussed. In the first topic, previous work regarding ginger, especially the chemistry and bioactivities of important compounds and stability of 6-gingerol will be reviewed. While the bioactive compounds especially flavonoids will be reviewed in the second topic.

2.1. Chemical components of ginger

Ginger (Zingiber officinale) is cultivated in some temperate zone or tropical zone countries, such as China and India (Nakatani et al., 2001). It is one of the most widely used spices in the world. Pungency is an important sensory character of ginger and is attributed to the presence of gingerol compounds and their decomposition product, zingerone, and the dehydration products, shogaols. It can also be used in the pharmaceutical industry because they contain active compounds that have effects on certain physiological process (Onyenekwe, 2000; Shukla and Singh, 2006; Park and

Pizzuto, 2002, Nakatani et al., 2001).

The components in ginger include: extractable oleoresins, many fats, carbohydrates, vitamins, minerals and a potent proteolytic enzyme called zingibain

(Shukla and Singh, 2006).

Oleoresins contribute to the sensory perception of ginger (Shukla and Singh,

2006). There are 5-8% of oleoresins in crude ginger, which consist of two distinct groups of chemicals: volatile oils and non-volatile pungent compounds (Chrubasik et al., 2005; Shukla and Singh, 2006).

More than 66 compounds have been identified in volatile oil. They include

zingeberene, curcumene and farnesene, β-sesquiphelandrene, bisabolene, 1, 8-cineole,

linalool, borneol, nerol, geraniol, camphen, limonen, myrcen, β-phellandren, α-pinen, citronellol, geranial, neral and others (Chrubasik et al., 2005; Shukla and Singh, 2006).

Many of these constituents contribute to the distinct aroma and taste of ginger (Shukla and Singh, 2006).

Non-volatile pungent compounds of ginger consist mainly of gingerols,

shogaols, paradols and zingerone (Figure 7). These pungent principles produce a

“hot” sensation in the mouth.

H CO 3 CH3 (CH2)4 6-Shogaol

O OH

H CO 3 CH3 6-Gingerol (CH2)4

H3CO CH3 Zingerone

HO O

H3CO CH3 (CH2)6 6-Paradol

Figure 7. Non-volatile pungent compounds of ginger

2.1.1. Bioactive constituents of ginger

Ginger has been used as a medicine in Asian countries since ancient times

(Altman and Marcussen, 2001; Nakatani and Kikuzaki, 2001). It has been used to cure

cold, inflammation, gastrointestinal discomfort, rheumatic disorder, neuralgia and

motion sickness (Park et al., 2006; Kim et al., 2005; Wei et al., 2005).

Many bioactive compounds have been found in ginger, such as gingerols,

shogaols and paradols. Gingerols, especially 6-gingerol have been of intense interest

because of their biological properties. It has been shown that 6-gingerol has

antioxidant activity and anti-inflammation activity (Kuo et al., 1999; Ippoushi, 2003;

Reddy and Lokesh, 1992; Krishnakantha and Lokesh, 1993; Young et al., 2005). 6-

gingerol was also found to have anti-tumor activities which are related to cytotoxic

and antiproliferative effects and inhibit angiogenesis invitro and in vivo (Lee and Surh,

1998; Keum et al., 2002; Kim et al., 2005). 6-paradol is also known to have anti-

tumor effects, antioxidative activity and anti-inflammation activities (Lee and Surh,

1998; Keum et al., 2002, Chung et al., 2001; Surh et al., 1999).

While 6-shogaol has shown to exhibit antioxidative activity and cytotoxic activity

(Zaeoung et al., 2005), its biological activities are far from completely studied. In our

study, we will study the bioactivities of shogoals and gingerols we isolated.

2.1.2. Stability of gingerols

Gingerols are main pungent compounds in fresh ginger and are regarded as major

active components in ginger (Bhattarai et al., 2001). They contribute to many

pharmacological and physiological effects of ginger and their derived products

(Bhattarai et al., 2001). However, gingerols are thermally unstable due to their β-

hydroxy keto group in the structure and are easily converted to shogaols under high

temperature (Wohlmuth et al., 2005; He et al., 1998). Bhattarai et al. found that 6-

gingerol and 6-shogaol exhibit reversible kinetics in aqueous solution (Figures 8, 9,

10).

Figure 8. Schematic degradation process of gingerol in aqueous acidic environment.

Kf and Kr are first order rate constant for forward and reverse reactions respectively (Bhattarai et al., 2001).

Figure 9. Time dependent reversible degradation of [6]-gingerol at 80°C in 0.1 M HCl ( [6]-gingerol; [6]-shogaol). Data are presented as the mean of four independent determinations (Bhattarai et al., 2001).

Figure 10. Time dependent reversible degradation of [6]-shogaol at 80°C in 0.1 M HCl ( [6]-gingerol; [6]-shogaol). Data are presented as the mean of three independent determinations (Bhattarai et al., 2001).

In our study, we tested a ginger extract and several ginger products and found out that the concentration of 6-shogaols is comparable to 6-gingerol in these samples

(table 1).

Since the high levels of 6-shogaol in ginger products, we proposed to compare the anti-inflammation activities and antioxidant activities of 6-shogaol with 6-gingerol.

2.2. Components in Allium ursinum

Allium ursinum L. is a wild-growing Allium species in the forest. It is also called ramson bear’s garlic, alliaceae and subgenus Amerallium Traub (Schmitt et al., 2005).

It is widely used as spice as well as traditional medicine. The herbaceous plants grow up to a height of 50 cm and feature with white flowers. The bulbs don’t exceed 6 cm in size (Schmitt et al., 2005).

Allium ursinum is found throughout Europe, Asia, the Caucasus and Siberia. Its vegetation cycle starts in the spring and ends at the beginning of summer (Djurdjevic et al., 2004).

Many volatile compounds such as sulfides and disulfides have been identified in

Allium ursinum (Figure11) (Schmitt et al., 2005). Cysteine sulfoxides and lectin all exist in Allium ursinum (Smeet et al., 1997a; Smeet et al., 1997b; Schmitt et al.,

2005).

Moreover, there are phenolic compounds in Allium ursinum. The bulbs and the leaves were found to contain 2.3 mg/g and 3.24 mg/g (dry weight) of total free phenolics, respectively, and the same amount of bound phenol forms (1.0 mg/g)

(Djurdjevic et al., 2004). These phenolic compounds could be important because the phenolic compounds have antioxidant effects that are effective in prevention and treatment of different diseases (Stajner et al., 2003). The phenolic compounds in

Allium ursinum may be flavonoids. There are five flavonoids separated from Allium ursinum (Figure 12) (Carotenuto et al., 1996).

In present study, more flavonoids were isolated from Allium ursinum.

Figure 11. Volatile compounds of Allium ursinum: 1, methyl 2-propenyl sulfide

(AlMeS); 2, propylthiol (PrSH); 3: dimethylthiophene (Me2Thio); 4, 2- methylpentanal (MePent); 5, hex-3-en-1-ol; 6, 2-hexenal; 7, dimethyl disulfide

(Me2S2); 8, (E)-methyl 1-propenyl disulfide [(E)-PeMS2]; 9, (Z)-methyl 1-propenyl disulfide [(Z)-PeMS2]; 10, methyl propyl disulfide (MePrS2); 11, methyl 2-propenyl disulfide (MeAlS2); 12, dipropyl disulfide (Pr2S2); 13, 2-propenyl propyl disulfide (AlPrS2); 14, (Z)-1-propenyl propyl disulfide [(Z)-PePrS2]; 15, (E)-1-propenyl propyl disulfide [(E)-PePrS2]; 16, (E)-1-propenyl 2-propenyl disulfide [(E)-PeAlS2]; 17, di-2- propenyl disulfide (Al2S2); 18, dimethyl trisulfide (Me2S3); 19, methyl propyl trisulfide (MePrS3); 20, dipropyl trisulfide (Pr2S3); 21, di-2-propenyl sulfide (Al2S).

Figure 12. Structures of known flavonoids in Allium ursinum

3. HYPOTHESES AND OBJECTIVES

3.1. Hypotheses

3.1.1. Isolation components of ginger and their biological activities

There have been many reports concerning biological activities of 6-gingerol.

However, 6-gingerol is unstable and will be converted to 6-shogaol. We hypothesize that there are considerable amount of 6-shogaols in ginger products and there are some other phenolic compounds besides 6-gingerol that have biological activities.

3.1.2. Isolation flavonoids of Allium ursinum

There are 3.24 mg phenolic components in 1 gram of leaves of allium ursinum

(Djurdjevic et al., 2004). The phenolic compounds in Allium ursinum may be flavonoids. There are five flavonoids isolated from Allium ursinum by Carotenuto

(Carotenuto et al., 1996).We hypothesize that a systematic investigation of chemical constituents of Allium Ursinum would result in the discovery of more flavonoids that may have utility for the treatment and prevention of adverse health conditions.

3.2. Objectives

3.2.1. Isolation components of ginger and their biological activities

1). Isolation and identification phenolic compounds in ginger

2) Quantification of 6-gingerol and 6-shogaol in some ginger products

3). Comparing the antioxidant activity of 6-shogaol with 6-gingerol

4). Confirmation of promising compounds for their anti-inflammatory activity

3.2.2. Isolation flavonoids of Allium ursinum

Isolation and identification of flavonoids of Allium ursinum

4. EXPERIMENTAL

4.1. Plant materials

Ginger extract was provided by Sabinsa Corp (Piscataway, NJ).

Allium ursinum was provided by WellGen, Inc.

4.2. Solvents, reagents and chromatographic supplies, cell cultures

Solvents including ethanol, methanol, acetone, ethyl acetate, hexane, n-butanol,

acetonitrile were purchased from Fisher Scientific (Springfield, NJ) and were of

HPLC grade.

DPPH and H2O2 were purchased from Sigma Chemical Co. (St. Louis MO).

CD3OD, CDCl3, DMSO-d6 were purchased from Aldrich Chemical Co.

Silica gel (60 Å, 32-63 µm) for normal phase chromatography was purchased from Sorbent Technology Inc. (Atlanta, GA)

Thin Layer chromatography (TLC) plates were purchased from Fisher Scientific

(Springfield, NJ).

C18 silica gel (60 Å), Sephadex LH-20 and Diaion HP-20 for column chromatography were purchased from Sigma (St. Louis, MO)

Lipopolysaccharide (LPS) (Escherichia coli 0127: E8), sufanilamide, naphthylethylenediamine dihydrochloride, and dithiothreitol (DTT) were purchased from Sigma Chemical Co. (St. Louis, MO). Acrylamide was purchased from E. Merck

Co. (Darmstadt, Germany).

RAW 264.7 cells, which were derived from murine macrophages, were obtained from the American Type Culture Collection (Rockville, MD).

4.3. Instruments

1H NMR, 13C NMR and 2 D NMR were recorded on a U-400 instrument (Varian

Inc., California). Chemical shifts were expressed in parts per million (δ) using TMS as internal standard.

LC-MS data were obtained on a HP-59980 B particle beam LC/MS interface with

HP 1100 HPLC instrument.

HPLC system was composed of a Hitachi LC 6200A intelligent pump, Waters 490

E programmable multiple wavelength detector, Water 717 auto-sample injector and a

Supelcosil LC-18 HPLC column ( 15 cm × 4.6 mm, 5 µm)

4.4. Isolation procedures

4.4.1. Isolation of compounds from Ginger (Figure 13)

a) Ginger extract was added to a Sephadex LH-20 column for fractionation. 95% ethanol was used as eluting solvent. The eluate was collected and concentrated to a residue. TLC was used to check the procedure.

b) Residue from step “a” was subjected to Diaion HP-20 column for further isolation. The column was eluted with water first, followed by 50% ethanol, 70% ethanol and 95% ethanol and the eluents were collected and concentrated. Water fraction, 50% ethanol fraction (Compound 1), 70% fraction and 95% fraction were obtained respectively.

c) 70% ethanol fraction from step “b” was subjected to a normal phase silica gel column for further isolation. The column was eluted with hexane/ethyl acetate (9:1). Two pure compounds, compounds 2 and 3 were obtained.

d) 95% ethanol fraction from step “b” was subjected to a reverse phase column for further isolation. The column was eluted with methanol/water (3:2). Five pure compounds, compounds 4, 5, 6, 7 and 8 were obtained.

Ginger extract

Sephadex LH-20

Residue

Diaion HP-20

50% ethanol fraction Water fraction 70% ethanol fraction 95% ethanol fraction (Compound 1)

Silica gel RP-18

Compounds 2-3 Compounds 4-8

Figure 13. The procedure of isolation of compounds from ginger extract

4.4.2. Isolation of compounds from Allium ursinum (Figure 14)

a) Allium ursinum (387 g) were extracted with 95% ethanol (4 L) at room

temperature for 1 week. This procedure was repeated three times. The ethanol

fraction were combined and concentrated under reduced pressure. Ethanol

fraction residue was obtained.

b) Ethanol fraction residue from procedure “a” was added 200 mL water. The

water solution was extracted with hexane and then with ethyl acetate followed

by butanol, three times each.

c) The butanol fraction from step b was subjected to MCI HP-20 column for

further separation. The column was eluted with water first, followed by 30 %

ethanol, 70% ethanol and 95% ethanol.

d) The 30% ethanol fraction from step “c” was subjected to Sephadex LH-20

column followed by RP-C18 column for further isolation. Compounds 1-4 were

obtained.

e) The 70% ethanol fraction from step “c” was subjected to Silica gel column

followed by RP-C18 column for further isolation. Compounds 5-10 were

obtained.

Allium ursinum

Extracted with 95% ethanol for three times

Ethanol fraction

Extracted with hexane, ethyl acetate and n- butanol respectively

Hexane fraction Ethyl acetate fraction Butanol fraction Water fraction

MCI HP-20 gel (Water and ethanol solvent system)

Water fraction 30% ethanol fraction 70% ethanol fraction 95% ethanol fraction

LH-20 Silica gel RP-C18 RP-C18 Compounds 1-4 Compounds 5-10

Figure 14. Procedure for isolation of compounds from Allium ursinum

4.5. Nitrite assay

RAW 264.7 cells were cultured in RPMI-1640 (without phenol red)

supplemented with 10% endotoxin-free, heat-inactivated fetal calf serum (GIBCO,

Grand Island, NY), 100 units/ml penicillin, and 100µg/mL streptomycin. When the cells reached a density of 2-3×106 cells/ml, they were activated by incubation in medium containing E. coli LPS (100 ng/mL). Various concentrations of test compounds dissolved in dimethylsulfoxide were added together with LPS.

The nitrite concentration in the culture medium was measured as an indicator of

NO production, according to the Griess reaction. One hundred microliters of each supernatant was mixed with the same volume of Griess reagent (1﹪sulfanilamide in

5% phosphoric acid and 0.1% naphthylethylenediamine dihydrochloride in water).

Absorbance of the mixture at 550 nm was determined with an enzyme-linked immunosorbent assay plate reader (Dynatech MR-7000; Dynatech Labs, Chantilly,

VA) .

4.6. Antioxidant assay: 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay

DPPH assay is one of methods used to examine the efficiency of the antioxidants.

DPPH is a stable free radical with violet color. When DPPH is mixed with a phenolic compound that can donate a hydrogen atom, it will transfer into a reduced form with loss of color. The reaction is shown in Figure 15 (Molyneux, 2004; Son et al., 2002).

OH O N N N NH + + O2NNO2 O2NNO2

NO2 NO2

DPPH Phenol (DPPH)H 517 nm (purple) Antioxidant yellow Figure 15. Reaction of DPPH with an antioxidant

The radical form of DPPH is purple and has maximum absorbance at 517 nm.

When it reacts with an antioxidant, a reduced DPPH is formed, and the absorbance intensity decreases. The stronger the antioxidant is, the more the intensity decrease will occur. Based on the decrease of the absorbance intensity, we can judge the potential of the antioxidant.

The percent inhibition of DPPH radical by sample was calculated according to

formula (Yen and Duh, 1994).

% DPPH free radicals scavenge activity = [(A0-At)/A0] × 100

Where A0 is the absorbance of the DPPH radical without antioxidant at t=0 min and At is the absorbance of the antioxidant at t = 30 minutes.

The method is that 2 mL sample alcohol solution is added to 2 mL DPPH alcohol solution and reacts for 30 min in a dark condition. The DPPH concentration is

100 µM. IC50 was obtained by extrapolation from linear regression analysis.

5. RESULTS AND DISCUSSSION

5.1. Isolation of components from ginger and their biological activity

study

Following a serious of column chromatography separation of ginger extract

(Figure 13), 8 compounds were isolated.

5.1.1. Method of separating 6-gingerol and 6-shogaol from ginger extract

A simple and effective method was developed to isolate compounds, especially major compounds, gingerols and shogaols from ginger extract.

Ginger extract was subjected to a Sephadex LH-20 column first, followed by a

Diaion HP-20 column. After the Diaion HP-20, 6-gingerol can be separated. Then the sample was subjected to normal phase silica gel columns and a reverse column. 6- shogaol was obtained (Figure 16).

Ginger Extract

Sephadex LH-20 Column

Fractions

6-Gingerol Diaion HP-20 Column

Fractions

Reverse Phase Column

6-Shogaol

Figure 16. Isolation scheme of 6-gingerol and 6-shogaol from ginger extract.

5.1.2. Identification of components isolated from ginger extract.

5.1.2.1. Identification of 6-gingerol.

Compound 1 from ginger extract is a pale yellow liquid. Its structure (Figure 17.)

was assigned as 6-gingerol on the basis of its 1H NMR and 13C NMR (100MHz) data and comparison with the literature data (Agarwal et al., 2001; Shoji et al., 1982).

1H NMR (400 MHz, Appendix 1) δ: 0.88 (3H, t, J=6.8 Hz, H-10), 1.23-1.68 (8H, m, H-6~H-9), 2.52 (1H, d, J=8.4 Hz, H-4a), 2.54 (1H, d, J=3.6 Hz, H-4b), 2.73 (2H, brd, J=6.8 Hz, H-2), 2.81 (2H, brd, J=6.8 Hz, H-1), 3.84 (3H, s, OCH3), 4.03 (1H, m,

H-5), 6.64 (1H, dd, J=8, 2 Hz, H-6’), 6.67 (1H, s, H-2’), 6.81 (1H, d, J=8 Hz, H-5’)

13C NMR (100 MHz, Appendix 2) δ: 13.8 (C-10, q), 22.3 (C-9, t), 24. 9 (C-7, t),

29.0 (C-8, t), 31.1 (C-1, t),, 36.2 (C-6, t), 45.2 (C-2, t), 49.1 (C-4, t), 55.6 (OCH3, q),

67.5 (C-5, d), 110.9 (C-2’, d), 114.3 (C-5’,d ), 120.4 (C-6’, d), 132.4 (C-1’, s), 143.7

(C-4’,s ), 146.3 (C-3’, s), 211.3 (C-3, s)

O OH 2' 1 3 10 H CO 5 3 CH3 3' (CH2)4 1' 2 4 6' HO 4' 5'

Figure 17. Structure of 6-gingerol

5.1.2.2. Identification of 8-gingerol

Compound 2 from ginger extract is a pale yellow liquid. It was elucidated as 8-

gingerol (Figure 18) by 1H NMR and 13C NMR, and confirmed by comparing with

literature data (Shoji et al., 1982).

O OH 2' 1 3 5 12 H3CO CH3 (CH ) 3' 1' 2 4 2 6 6' HO 4' 5'

Figure 18. Structure of 8-gingerol

1H NMR (400 MHz, Appendix 3) δ: 0.90 (3H, t, J=6.4 Hz, H-12), 1.27-1.46 (10H,

m, H-7~ H-11), 1.49 (2H, m, H-6), 2.48 (1H, d, J= 8.8 Hz, H-4b), 2.55 (1H, m, H-

4ba), 274 (2H, dd, J= 7.6 Hz and 6.8 Hz, H-2), 2.85 (2H, dd, J=7.2, 6.8 Hz, H-1), 3.89

(3H, s, OCH3), 4.05 (1H, m, H-5), 6.68 (1H, brd, J=8Hz, H-6’), 6.7 (1H, s, H-2’), 6.84

(1H,d, 7.6, H-5’).

13C NMR (100 Hz, Appendix 4) δ: 13.8 (C-12, q), 22.4 (C-11, t), 25.2 (C-7, t),

29.0 (C-9. t; C-8, t), 29.3 (C-10, t), 31.6 (C-1, t), 36.2 (C-6, t), 45.2 (C-2, t), 49.1 (C-4, t), 55.6 (OCH3, q), 67.4 (C-5, d), 110.7 (C-2’, d), 114.1 (C-5’, d), 132.4 (C-1’, s),

143.7 (C-4’, s), 146.2 (C-3’), 211.3 (C-3, s).

5.1.2.3. Identification of 10-gingerol

Compound 3 from ginger extract is a pale yellow liquid. It was elucidated as 10- gingerol (Figure 19) by 1H NMR and 13C NMR, and confirmed by comparing with

literature data (Shoji et al., 1982).

O OH 2' 1 3 14 H3CO 5 (CH ) CH3 3' 1' 2 4 2 8 6' HO 4'

Figure 19. Structure of 10-gingerol

1H NMR (400 MHz, Appendix 5) δ: 0.87 (3H, t, J=6.4 Hz, H-14), 1.31-1.46 (14H,

m, H-7 ~H-14), 1.49 (2H, m, H-6), 2.42-2.65 (2H, m, H-4), 2.75 (2H, dd, J=10 Hz

and 7.8 Hz, H-2), 2.85 (2H, dd, J=20, 7.8 Hz, H-1), 3.88 (3H, s, OCH3), 4.07 (1H, m,

H-5), 6.67 (1H, dd, J=9.6, 2 Hz, H-6’), 6.71 (1H, s, H-2’), 6.84 (1H, d, 8, H-5’)

13C NMR (100 MHz, Appendix 6) δ: 13.9 (C-14, q), 22.5 (C-13, t), 25.2 (C-7, t),

29.0, 29.1 (C-11, t; C-10, t), 29.3 (C-9, t), 29.3 (C-8, t), 31.7 (C-1, t), 36.2 (C-6, t),

45.2 (c-2, t), 49.1 (C-4, t), 55.6 (OCH3, q), 67.4 (C-5, d), 110.7 (C-2’, d), 114.1 (C-5’, d), 132.4 (C-1’, s), 143.7 (C-4’, s), 146.2 (C-3’), 211.3 (C-3, s)

5.1.2.4. Identification of 6-shogaol

Compound 4 from ginger extract is a pale yellow liquid. It was elucidated as 6- shogaol (Figure 20) by 1H NMR and 13C NMR, and confirmed by comparing with

literature data (Connell and Sutherland, 1969).

O 2' 1 510 H CO 3 3 CH3 (CH ) 3' 1' 2 4 2 4 6' HO 4' 5' Figure 20. Structure of 6-shogaol

1H NMR (400 MHz, Appendix 7.) δ: 0.88 (3H, t, J=6.8 Hz, H-10), 1.26-1.31 (4H,

m, H-8 and H-9), 1.42 (2H, m, H-7), 2.17 (2H, m, H-6), 2.84 (4H, m, H1 and H-2),

3.82 (3H, s, OCH3), 6.09 (1H, dt, J=16, 1.6 Hz, H-4), 6.66 (1H, dd, J=8, 2Hz, H-6’),

6.70 (1H, d, J= 1.6 Hz, H-2’), 6.81 (1H, d, J= 7.6 Hz, H-5’) 6.83 (1H, d, J=16.0 Hz,

H-5).

13C NMR (100 MHz, Appendix 8) δ: 13.7 (C-10, q), 22.2 (C-9, t), 27.5 (C-7, t),

29.6 (C-8, t), 31.1 (C-1, t), 32.2 (C-6, t), 41.6 (C-2, t), 55.5 (OCH3, q), 111.0 (C-2’, d),

114.2 (C-5’,d ), 120.5 (C-6’, d), 130.8 (C-4, d) 132.8 (C-1’, s), 143.7 (C-4’,s ), 146.3

(C-3’, s), 147.4 (C-5, d), 199.9 (C-3, s)

5.1.2.5. Identification of 8-shogaol

Compound 5 from ginger extract is a pale yellow liquid. It was elucidated as 8- shogaol (Figure 21) by 1H NMR and 13C NMR, and confirmed by comparing with

literature data (Connell and Sutherland, 1969).

O 2' 5 12 H CO 1 3 3 3' 1' CH3 (CH2)6 2 4 4' 6' HO 5'

Figure 21. Structure of 8-shogaol

1H NMR (400 MHz, Appendix 9) δ: 0.90 (3H, t, J= 6.0 Hz, H-12), 1.29-1.44 (8H, m, H-8 ~ H-11), 1.45 (2H, m, H-7), 2.21 (2H, m, H-6), 2.87 (4H, m, H-1 and H-2),

3.90 (3H, s, OCH3), 6.11 (1H, dt, J= 16, 1.6 Hz, H-4), 6.70 (1H, dd, J=8, 2 Hz, H-6’),

6.73 (1H, d, J= 1.6 Hz, H-2’), 6.83 (1H, brd, J= 7.6 Hz, H-5’) 6.86 (1H, H-5).

13C NMR (100 MHz, Appendix 10) δ: 13.9 (C-12, q), 22.4 (C-11, t), 27.8 (C-9, t),

28.1 (C-7, t), 28.9 (C-8, t), 29.6(C-10, t), 31.5 (C-1, t) 32.2 (C-6, t), 41.7 (C-2, t), 55.6

(OCH3, q), 110.8 (C-2’, d), 114.0 (C-5’, d), 120.5 (C-6’, d), 130.0 (C-4, d) 133.0 (C-1’,

s), 143.6 (C-4’, s ), 146.1 (C-3’, s), 147.4 (C-5, d), 199.9 (C-3, s)

5.1.2.6. Identification of 10-shogaol

Compound 6 from ginger extract is a pale yellow liquid. It was elucidated as 10-

shogaol (Figure 22) by 1H NMR and 13C NMR, and confirmed by comparing with

literature data (Connell and Sutherland, 1969).

O 5 2' 1 3 14 H3CO 3' 1' (CH ) CH3 2 4 2 8 4' 6' HO 5'

Figure 22. Structure of 10-shogaol

1HNMR (400 MHz, Appendix 11) δ: 0.89 (3H, t, 8.4, H-14), 1.27-1.31 (12H,

m, H-8~H-13), 1.45 (2H, m, H-7), 2.21 (2H, m, H-1), 2.86 (4H, m, H-1 and H-2),

3.85 (3H, s, OCH3), 6.11(1H, brd, J=16 Hz, H-4), 6.68 (1H, dd, J= 8, 2 Hz, H-6’),

6.72 (1H, d, J= 1.6 Hz, H-2’), 6.83 (1H, d, J= 8 Hz, H-5’), 6.86 (1H, d, J= 7.2 Hz, H-

5).

13C NMR (100 MHz, Appendix 12) δ: 13.9 (C-14, q), 22.4 (C-13, t), 27.8 (C-11, t), 28.9, 29.1, 29.2 29.6 (C-7, t; C-8, t; C-9, t; C-10, t), 31.6 (C-1, t) 32.3 (C-6, t), 41.7

(C-2, t), 55.6 (OCH3, q), 110.9 (C-2’, d), 114.2 (C-5’,d ), 120.5 (C-6’, d), 130.0 (C-4, d) 132.9 (C-1’, s), 143.7 (C-4’,s ), 146.2 (C-3’, s), 147.8 (C-5, d), 199.7 (C-3, s)

5.1.2.7. Identification of 6-paradol

Compound 7 from ginger extract is a pale yellow liquid. It was elucidated as 6- paradol (Figure 23) by 1H NMR and 13C NMR, and confirmed by comparing with literature data (Tachie et al., 1975)

O 2' H CO 1 3 10 3 CH (CH ) 3 3' 1' 2 2 6 6' HO 4' 5'

Figure 23. Structure of 6-paradol

1H NMR (400 MHz, Appendix 13) δ: 0.89 (3H, t, J = 6.8 Hz, H-10), 1.27-1.32

(8H, m, H-6, H-7, H-8, H-9), 1.56 (2H, brt, J = 6.4Hz, H-5), 2.39 (2H, t, J = 7.6 Hz,

H-4), 2.71 (2H, t, J = 7.6 Hz, H-2), 2.84 (2H, t, J = 7.6, H-1), 3.88 (3H, s, OCH3),

6.67 (1H, brd, J = 8 Hz, H-6’), 6.71 ( 1H, brs, H-2’), 6.83 (1H, d, 8, H-5’)

13C NMR (100 MHz, Appendix 14) δ: 13.8 (C-10, q), 22.4 (C-9, t), 23.6 (C-5, t),

28.8 (C-7, t), 28.9 (C-6, t), 29.3 (C-8, t), 31.4 (C-1, t) 42.9 (C-2, t), 44.4 (C-4, t), 55.6

(OCH3, q), 110.8 (C-2’, d), 114.1 (C-5’,d ), 120.5 (C-6’, d), 132.9 (C-1’, s), 143.6 (C-

4’,s ), 146.1(C-3’, s), 211.3 (C-3, s)

5.1.2.8. Identification of 1-dehydro-6-gingerdione

Compound 8 from ginger extract is yellow crystal. It was elucidated as 1- dehydro-6-gingerdione (Figure 24) by 1H NMR and 13C NMR, and confirmed by

comparing with literature data (Charles et al., 2000)

O O 2' 1 10 H CO 3 5 3 CH3 (CH ) 3' 1' 2 4 2 4 6' HO 4' 5'

Figure 24. Structure of 1-dehydro-6-gingerdione

1H NMR (400 MHz, Appendix 15) δ: 0.92 (3H, t, J= 6.4 Hz, H-10), 1.34 (4H, m,

H-8, H-9), 1.66 (2H, m, H-7), 2.40 (2H, m, H-6), 3.93 (3H, s, OCH3), 5.64 (2H, s, H-

4), 6.36 (1H, dd, J= 15.6, 6 Hz, H-2), 6.94 (1H, dd, J= 8, 4.8 Hz, H-5’), 7.03 (1H, d, J

=1.6 Hz, H-2’), 7.09 (1H, dd, J= 8, 2 Hz, H-6’), 7.55 (1H, dd, J= 15.2, 4.8 Hz, H-1)

13C NMR (100 MHz, Appendix 16) δ: 13.7 (C-10, q), 22.2 (C-9, t), 25.1 (C-8, t),

31.2 (C-7, t), 39.9 (C-6, t), 55.7 (OCH3, q), 99.9 (C-4, t), 109.2 (C-2’, d ), 116.6 (C-5’,

d), 120.2 (C-2, d) 122.4 ( C-6’, d) 127.4 (C-1’, s), 139.7 (C-1, d), 146.6 (C-4’,s ),

147.5 (C-3’, s), 177.5 (C-3, s), 200.0 (C-5, s)

5.1.3. Quantification of 6-gingerol and 6-shogaol in different ginger

products

6-Gingerol and 6-shogaol levels in ginger extract (Sabinsa Corp. Piscataway, NJ) and several other ginger products were quantified by isocratic HPLC method. 6-

Gingerol and 6-shogaol standards were isolated in our laboratory. Their standard

curves were shown in appendix 17 and 18. Their purities were confirmed by HPLC

(Appendixes 19 and 20).

HPLC condition:

Column: Supelcosil LC-18

Injection volume: 50 µL

Length: 15 cm × 4.6 mm, 5 µm

Detector set at: 228 nm and 280 nm

Flow rate: 1 mL/min

Mobile phase:

A: 95 % water, 5 % acetonitrile, 0.2% acetic acid

B: 95 % acetonitrile, 5 % water, 0.2 % acetic acid

Profile of gradient elution:

0 minute: A: 100 %; B: 0 %

36 minutes: A: 0 %; B: 100%

44 minutes: A: 0%; B: 100%

45 minutes: A: 100%; B: 0%

It was reported that gingerols were main pungent components while only a very small amount of shogaols existed in fresh ginger (Bhattarai et al., 2001; Shukkla and

Singh 2006). However, gingerols were unstable and were easily converted to shogaols

(Wohlmuth et al., 2005; He et al., 1998).

Ginger extract we analyzed was from Sabinsa Corp (Piscataway, NJ). This ginger extract was claimed to have about 20% gingerols. However, our results indicated that it contained 5.37% 6-gingerol and 1.84% 10-gingerol while it also contained 6.73% 6- shogaol and 1.05% 10-shogaol. Apparently, about half of gingerols had already converted to 6-shogaol.

The other ginger products were randomly selected from supermarket. The results shown in table 1 indicated that ginger green tea (Beauti Leaf), sugar free ginger tea

(Kinsway Trading Inc.), ginger drink (Gold Kiki), instant ginger tea (Rhee Bros., Inc.) contained higher levels of 6-shogaol than 6-gingerol. Although tenren ginger tea (Ten

Ren Tea Co. Ltd), herbal ginger (Midori Trading Inc.), ginger honey crystal (Prince of

Peace) contained lower levels of 6-shogaol than 6-gingerol, the differences were not big.

In summary, all of these products had considerable high levels of 6-shogaol compared with 6-gingerol. Since fresh ginger only contained low level of 6-shogaol compared with 6-gingerol. The high levels of 6-shogaol in these products were mostly due to the conversion of 6-gingerol.

The results of this study confirmed the report that gingerols were easily converted

to shogaols (Bhattarai et al., 2001). While most of studies have focus on the

bioactivities of gingerols, 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.

Table 1. The levels of 6-gingerol and 6-shogaol in ginger products

Ginger tea 6-Gingerol 6-Shogaol percentage percentage

Tenren ginger tea (Ten Ren Tea Co. 0.127 ± 0.006 0.084 ± 0.007 Ltd)

Herbal ginger (Midori Trading Inc.) 0.054 ± 0.007 0.030 ± 0.004

Ginger green tea (Beauti Leaf) 0.058 ± 0.029 0.124 ± 0.020

Sugar free ginger tea (Kinsway 0.112 ± 0.010 0.130 ± 0.020 Trading Inc.)

Ginger honey crystal (Prince of 0.020 ± 0.006 0.018 ± 0.004 Peace)

Ginger drink (Gold Kiki) 0.016 ± 0.006 0.028 ± 0.002

Instant ginger tea (Rhee Bros., Inc.). 0.016 ± 0.004 0.020 ± 0.001

Note. The tests were performed in triplicates. Data are reported as means ± standard deviation.

5.1.4. Antioxidant activity of 6-gingerol and 6-shogaol

The antioxidant activity of 6-gingrol and 6-shogaol isolated from ginger extract

was assayed by DPPH free radicals scavenge. Various concentrations of 6-gingerol, 6-

shogaol and Vitamins E were mixed with 100 µM (final) DPPH dissolved in 95 %

alcohol. Absorption at 517 was measured after 30 minutes.

6-gingerol and 6-shogaol showed concentration-dependent free radical

scavenging ability (shown in Figure 25). The antioxidant activity of 6-shogaol is

comparable to 6-gingerol. At the concentration under 12 µM, they are more potent

than Vitamin E. While at concentration higher than 13 µM, their antioxidant activities

are not as good as Vitamin E. The 50% DPPH scavenge concentrations are: 6-

gingerol: 21 µM, 6-shogaol: 21 µM, Vitamin E: 18 µM

Phenolic substances are the most effective antioxidants from natural sources. The

phenolic antioxidants include alkoxyl phenols that containing one free and one

alkylated hydroxyl group, usual methoxy, or polyphenols with ortho- or para- dihydroxylic groups, or phenols containing condensed ring (Rice-Evans, C.A., et al.,

1996). The antioxidative activity of phenolic compounds is related to the hydroxyl group and the presence of a second hydroxyl group in the ortho or para postition (Ho. et al., 2000; Chen and Ho 1997). Furthermore, the antioxidant efficiency of phenolics is increased substantially by methoxy substitution in position ortho to the hydroxyl group because the electron-donating methoxy group allows increased stabilization of the resulting aryloxyl radical through electron delocalization after hydrogen donation by hydroxyl group (Rice-Evans et al, 1996). Both of the 6-gingerol and 6-shogaol

43 contain a same aromatic moiety with a free hydroxyl group and a methoxyl group in the position ortho to hydroxyl group. Therefore, it is no surprise that they have strong antioxidative activity.

The structure difference between 6-gingerol and 6-shogaol is that 6-gingerol has a hydroxyl group on decane while 6-shogaol is dehydrated form of 6-gingerol, resulting from the elimination of OH group at C-5 with the formation of a double bond between

C-4 and C-5. According to Sekiwa, side chain of phenol does not influence the antioxidant activity (Sekiwa et al., 2000). This supports our results that 6-gingerol and

6-shogaol almost have the same antioxidative activities.

100

40 6- Gi nger ol

activity % 6- Shogaol 20 Vi t amin E

0 DPPH free radicals scavenge 0 20406080100 Conc ent r at i on ( µ M)

Figure 25. DPPH free radicals scavenge activity of 6-gingerol, 6-shogaol and Vitamins E. Data represent one of three similar results.

5.1.5. Anti-inflammatory activities of compounds from ginger

Ginger has been reported to have anti-inflammatory effects and has been used to treat many inflammatory conditions (Lantz et al., 2007; Tjendraputra et al., 2001).

Aqueous and CH2Cl2 ginger extracts have shown to reduce LPS-stimulated PGE2 production (Jolad et al., 2005; Tjendraputra et al., 2001). Ethanol extract of ginger was purported to inhibited fresh egg albumin-induced acute inflammation (Ojewole

2006). Moreover, 6-gingerol was reported to inhibit nitric oxide synthesis in activated

J774.1 mouse macrphage and reduce phorbol ester induced inflammation in mice

(Aktan et al., 2006; Tjendraputra et al., 2001).

While most works used ginger extracts to study the anti-inflammatory effects of ginger or focused mostly on the anti-inflammatory effects of gingerols. No systematic work has been performed to study the anti-inflammatory activities of a series of individual compounds isolated from ginger.

In present study, the effects of a series compounds from ginger on NO production were evaluated. Nitric oxide is a free radical generated from conversion of L-arginine to L-citrulline by No synthases (Jun et al., 2005). Overproduction of NO contributes to numerous diseases such as inflammation and cancer. The suppression of NO production by phytochemicals might be a promising strategy to treat inflammation and cancer.

We used nitrite production as an indicator of NO release in LPS-activated macrophages since the half-life of NO is very short. The concentration-response relationships (Figure 26) were determined 24 hours after treatment of LPS (100

ng/mL) only or along with the tested compounds. 100 µL of the culture medium was

collected for nitrite assay.

6-gingerol and 6-shogaol inhibited NO generation in a concentration-dependent

manner. 6-shogaol showed more inhibitory effect than 6-gingerol with reducing nitrite

production by 85 % at 5 µM while 6-gingerol reducing nitrite production by 35 % at

5µM.

8-shogaol, 10-shogaol, 8-gingerol, 10-gingerol, 6-paradol and 1-dehydro-6-

gingerdione also have effect on inhibiting LPS-induced NO production. The

inhibitory potency was as follows: 8-shogaol > 8-gingerol ≈ 6-shogaol ≈ 10-shogaol >

8-gingerol > 6-paradol > 1-dehydro-gingerdione > 6-gingerol, at 5 µM.

These findings provided a significant basis on the anti-inflammatory activities of ginger:

1. There are many compounds contributing to anti-inflammatory activities of

ginger and most of them are very efficient in inhibiting NO generation.

This explains why ginger has strong anti-inflammatory activities.

2. 6-shogaol showed more inhibitory effect on nitrite production than 6-

gingerol. This explains why when dry ginger decreased more than 50 % of

gingerols, it is still have comparable anti-inflammatory activity to fresh

ginger (Jolad, et al., 2005). Because when ginger was dried, gingerols

converted to shogaols and shogaols still had strong inflammatory activity.

Figure 26. Effects of tested compounds on LPS-induced nitrite formation in RAW 264.7 cells. 1. 6-paradol; 2. 1-dehydro-6-gingerodione; 3. 6-gingerol; 4. 8-gingerol; 5. 10- gingerol; 6. 6-shogaol; 7. 8-shogaol; 8. 10-shogoal.

5.2. Isolation of flavonoids of Allium ursinum

Following a series of extraction and column chromatographic separation of the

ethanol fraction of Allium ursinum (Figure 14), a total of 10 compounds were

identified. Eight of them belong to flavonoids.

5.2.1. Identification of compound 1 (Aub 30-2-3)

Compound 1 from Allium ursinum was elucidated as p-coumaric acid (Figure

27) by 1H NMR, and confirmed by comparing with literature data (Christophoridou et al., 2005). p-Coumaric acid is first time identified in this plant.

1H NMR (600 MHz, Appendix 21) δ: 6.27 (1H, d, J= 10.4 Hz, H-8), 6.75 (2H, d,

J= 5.6 Hz, H-3, H-5), 7.41 (2H, d, J= 5.6 Hz, H-2, H-6), 7.60 (1H, d, J= 10.8 Hz, H-7)

H O 2 7 8 9 3 1 H OH 6 HO 4 5

Figure 27. Structure of p-coumaric acid

5.2.2. Identification of compound 2 (Aub 30-3-1)

Compound 2 from Allium ursinum was identified as L-tryptophan (Figure 28) by

1H NMR and 13C NMR, and confirmed by comparing with literature data (Morris et al., 1998). L-tryptophan is first time identified in this plant.

4 COOH β 5 9 3 CH2 CH α

8 NH 6 2 2 N 1 7 H

Figure28. Structure of L-tryptophan

1H NMR (600 MHz, Appendix 22 ) δ: 2.96 (1H, dd, J = 10, 6 Hz, Hβ-1), 3.30 (1H, dd, J = 10, 2.4 Hz, Hβ-2), 3.44 (1H, dd, J = 5.6 Hz, 2.4, H-α), 6.96 (1H, t, J = 4.8 Hz,

H-5), 7.05 (1H, t, J = 4.8 Hz, H-6), 7.34 (1H, dd, J = 5.6, 0.4 Hz, H-2), 7.56 (1H, d, J

= 5.2 Hz, H-7)

13C NMR (150 MHz, Appendix 23) δ: 27.1 (C-α, d), 54.8 (C-β, t), 109.6 (C-3, s),

111.4 (C-7, d), 118.3 and 118.4 (C-4 or C-6, d, d), 120.9 and 124.1 (C-2 or C-5, d,d),

127.3 (C-9, s), 136.4 (C-8, s), 170.1 (CO, s)

5.2.3. Identification of compound 3 (Aub 30-3-2)

Compound 3 isolated from Allium ursinum was identified as kaempferol- 3,7-di-

O-β-D-glucopyranoside (Figure 29) by 1H NMR and 13C NMR (Table 2, Appendix 24,

25) and confirmed by comparing with literature data (Gall et al., 2003). It is first time to identify this compound in Allium ursinum.

6 OH

6' 5' 5 O 1 O 8 O 1' 4' 4 OH 9 2 1 7 OH

OH 3 2 3 6 10 2' 3' 4 OH 5 O

OH 6 O OH

5 O H2O 4 OH 1

OH 2 3 OH

Figure 29. Structure of Aub 30-3-2

Table 2. 1H NMR and 13C NMR assignments for Aub 30-3-2. a,b,cSignals with the same superscript are interchangeable Compound 3: Aub 30-3-2 Pos. δH δC 2 155.5 (s) 3 130.5 (s) 4 177.5 (s) 5 160.9 (s) 6 6.75 (1H, d, J = 2.4 Hz) 100.3 (d) 7 162.6 (s) 8 6.97 (1H, d, J = 2.4Hz) 94.7 (d) 9 156.3 (s) 10 105.5 (s) 1’ 120.4 (s) 2’-6’ 8.37 (2H, d, J = 9.0 Hz) 129.5 (d) 3’-5’ 7.14 (2H, d, J = 9.0 Hz) 114.8 (d) 4’ 160.5 (s) 3-O-Glc 1 6.36 (1H, d, J = 7.2 Hz) 99.0 (s) 2 4.26 (1H, m) 74.7 (d) 3 4.25 (1H, m) 77.2 (d)b 4 4.19 (1H, m) 70.1 (d) 5 4.04 (1H, m) 77.9 (d)a 6 4.56 (1H,m) 61.2 (t)c 4.40 (1H, m) 7-O-Glc 1 5.80 (1H, d, J = 7.8 Hz) 102.1 (s) 2 4.33 (1H, m) 73.4 (d) 3 4.35 (1H, m) 77.1 (d)b 4 4.19 (1H, m) 69.7 (d) 5 4.42 (1H, m) 77.9 (d)a 6 5.07 (1H, m) 61.0 (t)c 4.57 (1H, m)

5.2.4. Identification of compound 4 (Aub 30-3)

Compound 4 isolated from Allium ursinum was identified as 7-O-β-D- glucopyranosyl kaempferol 3-O-α-L-rhamnopyranosyl- (1→ 2)-β-D-glucopyranoside

(Figure 30) by 1H NMR and 13C NMR (Table 3, Appendix 26, 27), and confirmed by comparing with literature data (Carotenuto et al., 1996).

OH 6

6' 5' O 5 O 8 O OH 7 9 2 4' 4 1 1' OH

OH 3 2 10 3 6 2' 3' 5 4 OH O OH O 6 OH

5 O

OH 4 1

OH 2 3 5 HO O O

4 6 1 3 2

OH OH Figure 30. Structure of Aub 30-3

Table 3. 1H NMR and 13C NMR assignments for Aub 30-3

compound 4: Aub-30-3 Pos. δH δC 2 159.5 (s) 3 134.3 (s) 4 179.2 (s) 5 164.3 (s) 6 6.44 (1H, d, J = 1.8 Hz) 101.5 (d) 7 164.3 (s) 8 6.72 (1H, brs) 95.7 (d) 9 157.8 (s) 10 107.6 (s) 1’ 121.2 (s) 2’-6’ 8.05 (2H, d, J = 9Hz) 132.2 (d) 3’-5’ 6.78 (2H, d, J = 9 Hz) 117.1 (d) 4’ 162.8 (s) 3-O-Glc 1 5.68 (1H, d, J = 7.8 Hz) 100.6 (d) 2 3.60 (1H, dd, J = 9.6, 7.2 Hz) 79.9 (d) 3 3.55 (1H, t, J = 9.6 Hz) 78.8 (d) 4 3.34 (1H, t, J = 9.6 Hz) 71.8 (d) 5 3.29 (1H, m) 78.2 (d) 6 3.84 (1H, dd, J = 10.2, 3 Hz) 62.6 (t) 3.37 (1H, brd, J =10.2 Hz) Rha 1 5.24 (1H, brs) 102.5 (d) 2 4.05 (1H, m) 72.3 (d) 3 3.72 (1H, m) 72.2 (d) 4 3.38 (1H, m) 74.0 (d) 5 4.07 (1H, m) 69.9 (d) 6 1.0 (1H, d, J=6 Hz) 17.6 (q) 7-O-Glc 1 5.05 (1H, d, J = 7.2 Hz) 100.3 (d) 2 4.06 (1H, dd, J = 8.4, 7.2 Hz) 74.6 (d) 3 3.68 (1H, t, J = 8.4 Hz0 77.7 (d) 4 3.44 (1H, t, J = 8.4 Hz) 71.2 (d) 5 3.62 (1H, ddd, 12, 8.4, 2.4 Hz) 78.2 (d) 6 3.92 (1H, brd, J = 12 Hz) 62.4 (t) 3.72 (1H, m)

5.2.5. Identification of compound 5 (Aub 1)

Compound 5 isolated from Allium ursinum was identified as kaempferol 3-O-β-

D-glucopyranoside (Figure 31) by 1H NMR and 13C NMR (Table 4, Appendix 28, 29) and confirmed by comparing with literature data (Nakano et al., 1981). It is first time to identify this compound in Allium ursinum.

6' 5' 8 HO O 7 9 2 4' OH 1' 3 6 2' 3' 10 5 4 O

OH O 6 OH

5 O

OH 4 1

2 OH 3

OH Figure 31. Structure of Aub-1

Table 4. 1H NMR and 13C NMR assignments for Aub 1

Compound 5: Aub 1 Pos. δH δC 2 159.1 (s) 3 135.1 (s) 4 179.4 (s) 5 162.9 (s) 6 6.19 (1H, d, J = 1.8 Hz) 104.0 (d) 7 165.9 (s) 8 6.39 (1H, d, J = 1.8 Hz) 95.2 (d) 9 158.5 (s) 10 106.1 (s) 1’ 123.4 (s) 2’-6’ 8.04 (2H, d, J = 9 Hz) 133.2 (d) 3’-5’ 6.87 (2H, d, J = 9 Hz) 116.2 (d) 4’ 161.5 (s) 3-O-Glc 1 5.23 (1H, d, J = 7.8 Hz) 100.4 (d) 2 3.52 (1H, dd, J = 9, 7.8 Hz) 78.4 (d) 3 3.41 (1H, t, J = 9 Hz) 75.7 (d) 4 3.20 (1H, t, J = 9 Hz) 71.3 (d) 5 3.30 (1H, brdd, J = 9, 5.4 Hz) 78.0 (d) 6 3.69 (1H, dd, J = 12, 1.8 Hz) 62.5 (t) 3.41 (1H, dd, J = 12, 5.4 Hz)

5.2.6. Identification of compound 6 (Aub 30-1)

Compound 6 isolated from Allium ursinum was identified as kaempferol 3-O-α-

L-rhamnopyranosyl- (1→ 2)-β-D-glucopyranoside (Figure 32) by 1H NMR and 13C

NMR (Table 5, Appendix 30, 31) and confirmed by comparing with literature data

(Nakano et al., 1981)

6' 5' HO 8 1 O 1' 9 2 4' 7 OH

3 6 2' 3' 10 5 4 O

OH O 6 OH

5 O

4 OH 1

2 OH 3 5 HO O O

4 61

3 2

OH OH

Figure 32. Structure of Aub 30-1

Table 5. 1H NMR and 13C NMR assignments for Aub-30-1

Compound 6: Aub-30-1 Pos. δH δC 2 158.2 (s) 3 134.4 (s) 4 179.2 (s) 5 163.0 (s) 6 5.97 (1H, brs) 99.8 (d) 7 165.6 (s) 8 6.09 (1H, brs) 94.7 (d) 9 158.5 (s) 10 105.8 (s) 1’ 123.1 (s) 2’-6’ 7.98 (2H, d, J = 8.4 Hz) 132.1 (d) 3’-5’ 6.80 (2H, d, J = 8.4 Hz) 116.1 (d) 4’ 161.1 (s) 3-O-Glc 1 5.62 (1H, d, J = 7.8 Hz) 100.3 (d) 2 3.53 (1H, dd, J = 9.0, 7.8 79.8 (d) Hz) 3 3.64 (1H, t, J = 9.0 Hz) 78.8 (d) 4 3.53 (1H, t, J = 9.0) 71.7 (d) 5 3.19 (1H, brdd, J = 9, 6 Hz) 78.0 (d) 6 3.70 (1H, brd, J = 12 Hz) 62.6 (t) 3.52 (1H, brd, J =12 Hz) Rha 1 5.24 (1H, brs) 102.5 (d) 2 4.0 (1H, m) 72.3 (d) 3 3.82 (1H, m) 72.2 (d) 4 3.32 (1H, m) 74.0 (d) 5 4.09 (1H, m) 69.9 (d) 6 1.02 (3H, d, J = 6.6 Hz) 17.6 (q)

5.2.7. Identification of compound 7 (Aub 1-1)

Compound 7, a yellow powder, had a molecular formula of C29H32O16 determined by negative ion ESI-MS (at m/z 635 [M-H]-) as well as 13C NMR data. Its 1H and 13C

NMR spectra (Table 6) showed the signals for kaempferol. The structure of 7 was determined by its 1H-NMR, 13C-NMR, HMQC, and HMBC spectra (Appendix 32-35).

1 The H-NMR spectrum of 7 exhibited signals for A ring (H-6, 8 at δH 6.15 1H, d,

J = 2.4 Hz and 6.34 1H, d, J = 2.4 Hz, respectively) and B ring (H-2', 6' and H-3', 5' at

13 δH 8.02 2H, d, J = 9.0 Hz and 6.89 2H, d, J = 9.0 Hz, respectively). The C NMR spectrum of 7 showed signals for A ring (δC 163.0 C-5, 99.7 C-6, 165.6 C-7, 94.6 C-8,

158.3 C-9, and 105.9 C-10), B ring (δC 123.0 C-1', 132.1 C-2', 6', 116.1 C-3', 5', and

161.2 C-4'), and C ring (δC 158.5 C-2, 134.3 C-3, and 179.1 C-4). Therefore, the aglycone of 7 was identified as kaempferol.

The identity of the sugars and the sequence of the oligosaccharide chain were determined by the analysis of a combination of its HMQC and HMBC NMR spectra.

The β-anomeric configurations for the glucose unit were determined from its large

3 JH1, H2 coupling constant (7.2 Hz). The α-anomeric configuration for the rhamnose was determined by its chemical shifts at C-5 (δC 69.6). The HMBC spectrum showed cross peaks between C-3 (δ 134.3) and H-G1 (δ 5.82), C-G2 (δ 79.0) and H-R1 (δ 4.87).

Therefore, the glucose unit located at the C-3 position of kamepferol, and the rhamnose unit connected at the C-2 position of the glucose.

In comparison to the 1H and 13C NMR spectra of the known compound Aub-30-1,

compound 7 showed the signals for one acetyl group (δH 2.16, 3H, s and δC 21.8 q and

172.3 s). In addition, the molecular weight of 7 was 42 mass units higher than that of

Aub 30-1. All these spectral features supported compound 7 was the mono-acetylated

derivative of Aub 30-1. The HMBC spectrum showed the cross peak between δC

172.3 and H-G3 (δH 5.13 t 9.0) indicating the acetyl group was located at the position

C-3 of the glucose unit. Thus, ursinoside A (7) was determined as kaempferol 3-O-α-

L-rhamnopyranosyl (1→2)-[3-O-acetyl]-β-D-glucopyranoside (Figure 33). It is a new natural product.

6' 5' 1 HO 8 O 7 2 1' 4' 9 OH

3 2' 6 10 3' 5 4 O

OH O 6 OH

5 O 7 8 OCOCH 4 3 1

OH 3 2

5 O HO O

4 6 1

3 2

OH OH

Figure 33. Structure of Aub 1-1

Table 6. 1H NMR and 13C NMR assignments for Aub-1-1

Compound 7: Aub-1-1 Pos. δC 2 158.5 (s) 3 134.3 (s) 4 179.1 (s) 5 163.0 (s) 6 6.15 (1H, d, J =2.4 Hz) 99.7 (d) 7 165.6 (s) 8 6.34 (1H,d, J = 2.4 Hz) 94.6 (d) 9 158.3 (s) 10 105.9 (s) 1’ 123.0 (s 2’-6’ 8.02 (2H,d, J = 9 Hz) 132.1 (d) 3’-5’ 6.89 (2H,d, J = 9 Hz) 116.1 (d) 4’ 161.2 (s) 3-O-Glc 1 5.82 (1H, d, J = 7.2 Hz) 100.1 (d) 2 3.72 (1H, dd, J = 9, 7.2 Hz) 79.0 (d) 3 5.13 (1H, t, J = 9 Hz) 79.0 (d) 4 3.54 (1H, t, J = 9 Hz) 70.2 (d) 5 3.38 (1H, ddd, J = 9, 5.4, 2.4 78.0 (d) Hz) 6 3.74 (1H, dd, J = 11.4, 2.4 Hz)) 62.2 (t) 3.54(1H, dd, J = 11.4, 5.4) Acetyl group 7 172.3 (s) 8 2.16 (3H, s) 21.8 (q) Rha 1 4.87 (1H, Brs) 102.8 (d) 2 3.79 (1H, brs) 72.5 (d) 3 3.72 (1H, m) 72.1 (d) 4 3.30 (1H, m) 73.8 (d) 5 4.00 (1H, m) 69.6 (d) 6 0.97 (3H, d, J = 6.6 Hz) 17.5 (q)

5.2.8. Identification of compound 8 (Aub- 3)

The negative-ion ESI-MS of Aub-3 (8) displayed a molecular ion peak at m/z [M-

- H] 635, supporting a molecular formula of C29H32O16, as noted above for Aub-1-1.

The structure of 7 was determined by its 1H-NMR, 13C-NMR, HMQC, and HMBC spectra (Appendix 36-39).

The NMR spectra of 8 displayed signal patterns similar to those of 7 (Tables 7).

1 The H NMR spectrum of 8 showed signals for A ring (H-6, 8 at δH 6.15 1H, brs and

6.35 1H, brs, respectively) and B ring (H-2', 6' and H-3', 5' at δH 7.99 2H, d, J=9.0 Hz and 6.87 2H, d, J=9.0 Hz, respectively). The 13C NMR spectrum of 8 showed signals

for A ring (δC 163.0 C-5, 99.9 C-6, 166.5 C-7, 94.8 C-8, 158.4 C-9, and 105.5 C-10),

B ring (δC 123.1 C-1', 132.0 C-2', 6', 115.9 C-3', 5', and 161.2 C-4'), and C ring (δC

158.8 C-2, 134.1 C-3, and 179.1 C-4). Therefore, the aglycone of 8 was also identified as kaempferol. It also had one acetyl group, a glucose unit, and one rhamnose unite

(Tables 1 and 2). The HMBC spectrum of 8 showed cross peaks between C-3 (δ

134.1) and H-G1 (δ 5.59), C-G2 (δ 79.8) and H-R1 (δ 5.23). Therefore, the sequence of the oligosaccharide chain in compound 8 was the same as that in compound 7. The major differences between 7 and 8 were the location of the acetyl group. In compound

8, the acetyl group located at C-6 position of glucose unit, rather than the C-3 position

in 7. Compound 8’s HMBC spectrum showed correlations between δC 172.3 and H-G6

(δH 4.20 brd 11.4 and 4.06 m). This confirmed that the acetyl group was located at C-6 position of glucose unit in 8. Therefore, compound 8 was identified as kaempferol 3-

O-α-L-rhamnopyranosyl (1→2)-[6-O-acetyl]-β-D-glucopyranoside (Figure 34). It is a

61 new natural product.

6' 5' 8 1 HO O 7 9 2 4' OH 1' 10 6 3 2' 3' 5 4 O

OH 7 O 6 OCOCH3 8 5 O

OH 4 1

OH 2 3 5 O HO O

4 6 1

3 2

OH OH

Figure 34. Structure of Aub-3

Table 7. 1H NMR and 13C NMR assignments for Aub-3

Compound 8: Aub-3 Pos. δH δC 2 158.8 (s) 3 134.1 (s) 4 179.1 (s) 5 163.0 (s) 6 6.15 (1H, brs) 99.9 (d) 7 166.5 (s) 8 6.35 (1H, brs) 94.8 (d) 9 158.4 (s) 10 105.5 (s) 1’ 123.1 (s) 2’-6’ 7.99 (2H, d, J= 9 Hz) 132.0 (d) 3’-5’ 6.87 (2H, d, J = 9Hz) 115.9 (d) 4’ 161.2 (s) 3-O-Glc 1 5.59 (1H, d, J = 7.2 Hz) 100.2 (d) 2 3.6 (1H, dd, J = 9, 7.2 Hz) 79.8 (d) 3 3.55 (1H, t, J = 9 Hz) 78.7 (d) 4 3.31 (1H, t, J = 9 Hz) 71.5 (d) 5 3.37 (1H, brdd, J=9, 5.4 Hz) 75.3 (d) 6 4.20 (1H, brd, J = 11.4 Hz) 63.8 (t) 4.06 (1H, brd, J = 11.4, 5.4 Hz)) Acetyl 7 172.3 (s) 8 1.75 (3H, s) 20.3 (q) Rha 1 5.23 (1H, s) 102.5 (d) 2 4.0 (1H, s) 72.3 (d) 3 3.79 (1H, dd, J = 8.4, 2 Hz) 72.2 (d) 4 3.34 (1H, m) 74.0 (d) 5 4.07 (1H, m) 69.8 (d) 6 1.00 (3H, d, J = 6 Hz) 17.5 (q)

5.2.9. Identification of compound 9 (Aub-7)

Compound 9 isolated from Allium ursinum displayed a molecular formula of

- 13 C42H46O22 determined by negative ion ESI-MS (at m/z 901 [M-H] ) as well as C-

NMR data. It was identified as kaempferol 3-O-α-L-rhamnopyranosyl (1→2)-β-D- glucopyranoside -7-O-[2-O-(trans-p-coumaroyl)] -β-D-glucopyranoside) (Figure 35) by 1H NMR , 13C NMR, HMQC and HMBC (Table 8, appendix 40-42) and confirmed by comparing with literature data (Carotenuto et al., 1996).

6' 5' O O 8 1 O 7 9 2 4' OH OH 1'

OH 6 10 3 2' 3' 5 4 O O H 9 OH O 6 OH 8 O 5 O 7 OH 1 H 4 1 2 6 OH 3 2

5 O 3 5 HO O 4 4 6 1 OH 3 2 OH OH

Figure 35. Structure of Aub-7

Table 8. 1H NMR and 13C NMR assignments for Aub-7

Compound 9 : Aub-7 Pos. δH δC 2 160.6 (s) 3 134.6 (s) 4 179.0 (s) 5 163.0 (s) 6 6.35 (1H, d, J=2.4 Hz) 100.0 (d) 7 163.9 (s) 8 6.65 (1H, d, J=2.4 Hz) 95.8 (d) 9 157.5 (s) 10 107.9 (s) 1’ 122.7 (s) 2’-6’ 8.04 (2H, d, J = 9 Hz) 132.3 (d) 3’-5’ 6.87 (2H, d, J = 9 Hz) 116.2 (d) 4’ 161.5 (s) 3-O-Glc 1 5.72 (1H, d, J = 7.2 Hz) 100.4 (d) 2 3.61 (1H, dd, J = 9.0, 7.2 Hz) 79.8 (d) 3 3.54 (1H, t, J = 9 Hz) 78.5 (d) 4 3.27 (1H, t, J = 9.0 Hz) 71.5(d) 5 3.23 (1H, ddd, J = 9.0, 4.8, 2.4 Hz) 77.8 (d) 6 3.71 (1H, dd, J = 12.6, 2.4 Hz) 62.2 (t) 3.48 (1H, dd, J = 12.6, 4.8, Hz) Rha 1 5.23 (1H, brs. ) 102.8 (d) 2 4.00 (1H) 71.9 (d) 3 3.82 (1H) 71.8 (d) 4 3.35 (1H) 73.4 (d) 5 4.02 (1H) 69.5 (d) 6 0.95 (3H, d, J = 6 Hz) 17.5 (q) 7-O-Glc 1 5.32 (1H, d, J = 7.8 Hz) 99.6 (d) 2 5.11 (1H, dd, J = 9, 7.8 Hz) 74.4 (d) 3 3.77 (1H, t, J = 9 Hz) 75.5 (d) 4 3.53(1H, t, J = 9 Hz) 71.0 (d) 5 3.62 (1H, brdd, J = 11.4, 9 Hz) 78.0 (d) 6 3.95 (1H, dd, J = 11.4, 1.8 Hz) 61.9 (t) 3.82 (1H) TPC 1 127.0 (s) 2-6 7.42 (2H, d, J = 8.4 Hz) 131.2 (d) 3-5 6.76 (2H, d, J = 8.4 Hz) 116.8 (d) 4 161.0 (s) 7 7.67 (1H, d, J = 15.6 Hz) 147.3 (d) 8 6.37 (1H, d., J = 15.6 Hz) 114.8 (d) 9 168.0 (s)

5.2.10. Identification of compound 10 (Aub-5).

Compound 10 had a molecular formula of C44H48O23 determined by negative ion

ES-MS (at m/z 943 [M-H]- as well as 13C NMR data. The structure of 10 was determined by its 1H-NMR, 13C-NMR, HMQC and HMBC spectra (Table 9, appendix

43-46).

1 The H-NMR spectrum of 10 exhibited signals for A ring (H-6, 8 at δH 6.34 1H, d,

J=1.6 Hz and 6.61 1H, d, J=1.6 Hz, respectively) and B ring (H-2', 6' and H-3', 5' at δH

8.03 2H, d, J=9.0 Hz and 6.89 2H, d, J=9.0 Hz, respectively). The 13C NMR spectrum

of compound 10 showed signals for A ring (δC 162.8 C-5, 100.0 C-6, 163.8 C-7, 95.8

C-8, 157.7 C-9, and 107.8 C-10), B ring (δC 122.7 C-1', 132.3 C-2', 6', 116.2 C-3', 5', and 161.5 C-4'), and C ring (δC 159.2 C-2, 134.6 C-3, and 179.2 C-4). Therefore, the aglycone of 10 was identified as kaempferol.

Analysis of 1H NMR and 13 C NMR spectrum of 10 also revealed the presence of two glucose units, one rhamnose unit and a p-coumaric acid (table 9 ). In the p- coumaric acid moiety, the 7 and 8 protons had a large coupling constant (J= 10.8).

Therefore, the olefinic part of the p-coumaric acid moiety was deduced to have the trans-configuration.

The β-anomeric configurations for the two glucose units were determined

3 3 from its large JH1, H2 coupling constant (7.2 and 8.4 Hz for 3-O- JH1, H2 and 7-O-

3 JH1, H2 respectively). The α-anomeric configuration for the rhamnose was

determined by its chemical shifts at C-5 (δC 69.5).

The HMBC spectrum showed cross peaks between C-3 (δ100.4) and H-G1

(δ5.81) of glucose A, C-7 (δ99.6) and H-G1 (δ5.29) of glucose B. Therefore, the glucose units located at C-3 and C-7 position.

The HMBC spectrum also showed the cross peaks between C-R1 (δ102.8) and H-G2 (δ3.6) of glucose A, carbonyl carbon C-9 (δ168.2) of trans-p- coumaric acid and H-G2 (δ5.13) of glucose B. Therefore, the rhamnose unit connected at the C-2 position of glucose A and the trans-p-coumaric acid connected at C-2 position of glucose B.

In comparison to the 1H and 13C NMR spectra of the known compound 9, compound 10 showed the signals for one acetlyl group (δH 2.16 3H s and δC

21.1 q and 172.3 s). In addition, the molecular weight of 10 was 42 mass units higher than that of 9. These entire spectral features supported compound 10 was the mono-acetylated derivative of 9. The HMBC spectrum showed for C-9’ (δ

172.3), the only cross peak with H is H-8’ (δ2.16). The down field shift of H-3’ and 5’ (δ6.89 ) indicated that the acetyl group was connected to C-6’. Thus, compound 10 was determined as 6’-O-acetyl kaempferol 3-O-α-L- rhamnopyranosyl(1→2)-β-D-glucopyranoside-7-O-[2-O-(trans-p-coumaroyl)]-

β-D-glucopyranoside (Figure 36). It is a new natural product.

6' 5' O O O 8 1 O 7 9 2 4' OH O C CH 1' 3 7' 8' OH 6 10 3 2' 3' 5 4 O O H 9 OH O 6 OH 8 O 5 O 7 OH 1 H 4 1 2 6 OH 3 2

5 O 3 5 HO O 4 4 6 1 OH 3 2

OH OH

Figure 36. Structure of Aub-5

Table 9. 1H NMR and 13C NMR assignments for Aub-5

Compound 10: Aub-5 Pos. δH δC 2 159.2 (s) 3 134.6 (s) 4 179.2 (s) 5 162.8 (s) 6 6.34 (1H, d, J = 2.4 Hz) 100.0 (d) 7 163.8 (s) 8 6.61 (1H, d, J = 2.4 Hz) 95.8 (s) 9 157.7 (s) 10 107.8 (s) 1’ 122.7 (s) 2’-6’ 8.03 (2H, d, J = 9 Hz) 132.3 (d) 3’-5’ 6.89 (2H, d, J = 9 Hz) 116.2 (d) 4’ 161.5 (s) Acetyl group 7’ 172.3 (s) 8’ 2.16 (3H, s) 21.1 (q) 3-O-Glc 1 5.81 (1H, d, J = 7.2 Hz) 100.4 (d) 2 3.60 (1H, dd, J = 9, 7.2 Hz) 80.0 (CH) 3 3.52 (1H, t, J = 9 Hz) 79.0 (CH) 4 3.33 (1H, t, J = 9 Hz) 71.2(CH) 5 3.30 (1H, ddd, J = 9, 5.4, 1.8 Hz) 77.9 (CH) 6 3.72 (1H, dd, J = 12.0, 1.8 Hz). 62.3 (CH2) 3.51 (1H, dd, J = 12.0, 5.4 Hz) Rha 1 4.87 (1H, brs) 102.8 (d) 2 3.96 (1H) 72.5 (d) 3 3.81 (1H) 72.1 (d) 4 3.37 (1H) 73.7 (d) 5 3.99 (1H) 69.5 (d) 6 0.95 (3H, d, J = 6 Hz) 17.5 (q) 7-O-Glc 1 5.29 (1H, d, J = 8.4 Hz) 99.6 (d) 2 5.13 (1H, dd, J = 9,8.4 Hz) 74.7 (d) 3 3.78 (1H, t, J = 9 Hz) 75.9 (d) 4 3.50 (1H, t, J = 9 Hz) 70.2 (d) 5 3.61 (1H, brdd, J = 11.4, 9 Hz) 78.4 (d) 6 3.95 (1H, brd, J = 11.4 Hz) 62.1 (t) 3.81 (1H) TPC 1 127.0 (s) 2-6 7.40 (2H, d, J = 9 Hz) 131.2 (d) 3-5 6.74 (2H, d, J = 9 Hz) 116.8 (d) 4 161.2 (s) 7 7.67 (1H, d, J = 16.2 Hz) 147.3 (s) 8 6.37 (1H, J = 16.2 Hz) 114.8 (s) 9 168.2 (s)

6. CONCLUSION

6.1. Study of Ginger

Gingerols are major bioactive principles in fresh ginger. However, they are easily converted to shogaols. Our studies showed that the levels of shogaols in ginger products are comparable to gingerols. While most of studies of ginger were concerned with the bioactive of gingerols, we focused the bioactivities of shogaols in this study.

A column chromatography method was developed to isolate gingerols and shogaols from ginger extract. By using this method, 8 compounds, 6-gingerol, 8- gingerol, 10-gingerol, 6-shogaol, 8-shogaol, 10-shogaol, 6-paradol, and 1-dehydro-6- gingerdione were isolated from ginger. These structures were elucidated by 1H-NMR,

13C-NMR and MS analysis.

The antioxidant activity of 6-gingrol and 6-shogaol isolated from ginger extract was assayed by DPPH free radicals scavenge. It was found that the antioxidant activity of 6-shogaol was comparable to that of 6-gingerol.

The anti-inflammatory activities of compounds isolated from ginger were evaluated by nitrite assay. The results showed that there were many compounds contributing to anti-inflammatory activities of ginger and most of them were very efficient in inhibiting NO generation. 6-gingerol and 6-shogaol inhibited NO generation in a concentration manner. 6-shogaol showed more inhibitory effect than

6-gingerol with reducing nitrite production by 85 % while 6-shogaol reducing nitrite production by 35 % at 5 µM. 8-shogaol, 10-shogaol, 8-gingerol, 10-gingerol, 6-

70 paradol and 1-dehydro-6-gingerdione also have effect on inhibiting LPS-induced NO production. The inhibitory potency was: 8-shogaol > 8-gingerol ≈ 6-shogaol ≈ 10- shogaol > 8-gingerol > 6-paradol > 1-dehydro-gingerdione > 6-gingerol, at 5 µM

6.2. Study of Allium ursinum

Ten compounds were isolated from Allium ursinum and elucidated with 1H-

NMR, 13C-NMR, HMQC and HMBC spectra. They were p-coumaric acid, L-

Tryptophan, kaempferol-3, 7-di-O-β-D-glucopyranoside, 7-O-β-D-glucopyranosyl kaempferol 3-O-α-L-rhamnopyranosyl- (1→ 2)-β-D-glucopyranoside, kaempferol 3-

O-β-D-glucopyranoside, kaempferol 3-O-α-L-rhamnopyranosyl- (1→ 2)-β-D- glucopyranoside, 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, kaempferol 3-O-α-L-rhamnopyranosyl (1→2)-β-D- glucopyranoside-7-O-[2-O-(trans-p-coumaroyl)]-β-D-glucopyranoside, 6’-O- acetyl kaempferol 3-O-α-L-rhamnopyranosyl (1→2)-β-D-glucopyranoside-7-O-[2-O-(trans- p-coumaroyl)]-β-D-glucopyranoside.

Eight of them are 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, 6’-O- acetyl kaempferol-3-O-α-L-rhamnopyranosyl(1→2)-β-D-glucopyranoside-7-O-[2-O-(trans- p-coumaroyl)]-β-D-glucopyranoside, are new compounds.

7. FUTURE PERSPECTIVES

a. Ginger has been reported to have many biological activities including anti- inflammatory property (Ojewole 2006; Shuka, Y, and Singh, M. 2006; Jolad et al.,

2005; Tjendraputra et al., 2001). However, most of works focused on the anti- inflammatory activities of gingerols from ginger (Ojewole 2006; Aktan et al., 2006;

Jolad et al., 2005). Ojewole speculated that the anti-inflammatory effects of ginger ethanol extract were largely attributed to 6-gingerol constituent of the extract. Our study, however, showed that there are other constituents in ginger besides gingerols also having strong anti-inflammatory activities toward inhibition of NO production.

Moreover, shogaols have stronger anti-inflammatory activities than gingerols toward inhibition of No production. But the mechanism for the inhibition of NO formation by shogaols is not clear. Shogaols may inhibit either iNOS gene expression or enzyme activity. Further studies are needed to determine the mechanism of action of shogaols.

b. Studies showed that some compounds in ginger exerted cancer preventive effects (Lee and Surh 1998; Lee et al., 1998; Keum et al., 2002). 6-gingerol and 6- paradol suppressed proliferation of human cancer cells through the induction of apoptosis and inhibited the viability of human HL-60 cells (Lee and Surh 1998; Lee et al., 1998). 10-paradol, 6-dehydroparadol, and 6-paradol induced apoptosis in an oral squamous carcinoma cell line (Keum et al., 2002). Our study showed that shogaols had stronger anti-inflammatory activities than gingerols and 6-gingerol on inhibition of NO production. Therefore, shogaols may be potential anti-cancer compounds since inflammation is closely related to cancer (Huang et al., 2004; O’Byrne and Dalgleish

2001). However, only a paper reported that 6-shogaol induced apoptosis of human hepatoma p53 mutant hahlavu subline. In order to fully understand the anti-cancer activities of shogaols, much more work should be done. For example, the cytotoxic effect of shogaols (6-shogal, 8-shogaol, 10-shogaol and others) on different cell lines and the effects of shogaols on inducing apoptosis of different cell lines.

c. Many studies suggested that flavonoids exhibit biological activities, including antioxidative, anti-inflammatory and anti-carcinogenic activities (Pietta

2000; Ren et al., 2003). Therefore, they may have health benefits and can be potential chemopreventive or therapeutic agents against cancer. Eight flavonoids were identified in Allium ursinum in this study. All the eight compounds have a kampherol group. The hydroxyl group at position 5 of A ring in all the eight compounds, the hydroxyl group at position 7 of A ring in Aub –1, Aub 30-1, Aub-1-1, Aub-3 and the hydroxyl group at position 4’ of B ring in all compounds except Aub-5 should have electron-donating properties. The 2, 3-double bond conjugated with the 4-oxo group in C ring in all flavonoids we isolated may contribute to electron delocalization. Base on these structure analyses, the flavonoids we isolated from Allium ursinum are very possible to have anti-oxidative activities. Additional work should be performed to confirm the anti-oxidative activities of these compounds. Furthermore, it was found that many antioxidants possessed anti-inflammatory effects (Lo et al., 2002; Liang et al., 1999). It will be interesting and promising to do some works on the anti- inflammatory activities of the flavonoids we isolated.

8. References

Agarwal, M., Walia, S., Dhingra, S. and Khambay, B. P. S. 2001. Insect growth inhibition, antifeedant and antifungal activity of compounds isolated/derived from Zingiber officinale Roscoe (ginger) rhizomes. Pest Management Science 57: 289-300.

Aktan, F., Henness, S., Tran, V. H., Duke, C. C., Roufogalis, B. D. and ammit, A. 2006. Gingerol metabolite and a synthetic analogue capsarol inhibit macrophage NF- κB-mediated iNOS gen expression and enzyme activity. Planta Med. 72: 727-734.

Bergman, M., Varshavsky, L., Gottlieb, H. E. and Grossman, S. 2001. The antioxidant activity of aqueous spinach extract: chemical identification of active fractions. Phytochemistry 58: 143-152

Bhattarai, S., Tran, Van H. and Duke, C. C. 2001. The stability of gingerol and shogaol in aqueous solution. J. Pharm. Sci. 90: 1658-164

Brenmer, P. and Heinrich, M. 2002. Natural products as targeted modulators of the nuclear factor-kappa B pathway. Journal of Pharmacy and Pharmacology 54: 453-472

Cai F., Li, C. R., Wu, J. L., Chen, J. G., Liu, C., Min, Q., Yu, W., Quyang, C. H. and Chen, J. H. 2006. Theaflavin ameliorate cerebral ischemia-reperfusion injury in rats through its anti-inflammatory effect and modulation of STAT-1. Mediators Inflammations 2006 (5): 1-9

Carotenuto, A.; Feo, V. D.; Fattorusso, E.; Lanzotti, V.; Magano, S. and Circala, C. 1996. The flavonoids of Allium ursinum. Phytochemistry 41(2): 531-536

Catherin A. and Rice-Evan. 2003. Flavonoids in Health and Disease. Marcel Dekker, Inc, New York.

Chan, M.M.Y., Ho, C. T. and Huang H. I. 1995. Effects of three dietary phytochemicals from tea, rosemary and turmeric on inflammation-induced nitrite production. Cancer Letters 96 (1): 23-29

Chen, J.H. and Ho, C.T. 1997. Antioxidant activities of caffeic acid and its related hydroxycinnamic acid compounds. J. Agric. Food Chem. 45: 2374-2378.

Christophoridou, S., Dais, P., Tseng, L. H. and Spraul, M. 2005. Separation and identification of phenolic compounds in olive oil by coupling high-performance liquid chromatography with postcolumn solid-phase extraction to nuclear magnetic resonance spectroscopy (LC-SPE-NMR). J. Agric. Food Chem. 53: 4667-4679

Chrubasik, S., Pittler, M. H. and Roufogalis, B. D. 2005. Zingiberis rhizoma: a comprehensive review on the ginger effect and efficacy profiles. Phytomedicine 12: 684-701

Chung, W. Y., Jung, Y. J., Surh, Y. J., Lee, S. S., Park, K. K. 2001. Antioxidative and antitumor promoting effects of 6-paradol and iths homogs. Mutat. Res. 496: 268-270.

Coussen, L. M. and Werb, Z. 2002. Inflammation and cancer. Nature 420: 860-867

Djurdjevic, L.; Dinic, A.; Pavlovic, P.; Mitrovic, M.; Karadzic, B. and Tesevic, V. 2004. Allelopathic potential of Allium ursinum L. Biochemical Systematics and Ecology 32(6): 533-544

Gall,G. L., Dupont, M. S., Mellon, F. A., Davis, L. A., Collins, G. J., Verhoeyen, M. V. and Colquhoun, I. J. 2003. Characterization and content of flavonoid glycosides in genetically modified tomato (Lycopersicon esculentum) fruit. J. Agri. Food Chem. 51: 2438-2446

Hasler, C. M. 2000. The changing face of functional foods. J. American College of Nutrition19: 499-506.

Hasler, C. M.2002. Functional foods: Benefits, concerns and challenges--a position paper from the American Council on Science and Health. J. Nutr. 132: 3772-3781

He, X. G., Bernart, W., Lian, L. Z. and Lin L. Z. 1998. High-performance liquid chromatography-electrospray mass spectrometric analysis of pungent constituents of ginger. Journal of Chromatography A 796: 327-334

Ho, C. T. 1997. Antioxidant properties of plant flavonoids. In Ohigashi, H., Ssawa, T., Terao, S., Watanabe, S. and Yoshikswa, T. (eds) Food Factors for cancer prevention. Springer-Verlag, Tokyo: 593-597

Ho, C. T., Wang, M., Wei, G. J., Huang, T. C. and Huang, M. T. 2000. Chemistry and antioxidative factors in rosemary and sage. Biofactors 13: 161-166

Ho, Y. S., Liou, H. B., Lin, L. K., Jeng, J. H., Pan, M. H., Lin, Y. P., Guo, H. R., Ho, S. Y., Lee, C. C., and Wang , Y. J. 2000. Lipid peroxidation and cell death mechanisms in pulmonary epithelial cells induced by peroxynitritrite and nitric oxide. Arch Toxicol 76: 484-493

Hong J., and Sang, S., Park, H. J., Kwon, S. J., Suh, N., Huang, M. T., Ho, C. T. and Yang, C. S. 2006. Modulation of arachidonic acid metabolism and nitric oxide synthesis by garcinol and its derivatives 27 (2): 278-282

Hong J., Bose, M., Ju, J., Ryu, J. H., Chen, X., Sang, S. Lee, M. J. and Yang, C. S. 2004. Modulation of arachidonic acid metabolism by curcumin and related β-diketone derivatives: effects on cytosolic phospholipase A2, cyclooxygenases and 5- lipoxygenase. Carcinogenesis 25 (9): 1671-1679

Huang, M. T., Liu, Y., Ramji, D., Lo, C. Y., Ghai, G., Dushenkov, S. and Ho, C. T. 2006. Inhibitory effects of black tea theaflavin derivatives on 12-O- tetradecanoylphorbol-13-acetate-induced inflammation and arachidonic acid metabolism in mouse ears. Mol. Nutr. Food Res. 50: 115-122

Huang, M. T., Ghai, G. and Ho, C. T. 2004. Inflammatory process and molecular targets for anti-inflammatory nutraceuticals. Comprehensive Reviews in Food Science and Food Safety 3: 127-139

Huang, M. T., Ho, C. T., Wang Z. Y., Ferraro, T., Lou, Y. R., Stauber, K., Ma, W., Georgiadis, C., laskin, J. D. and Conney, A. H. 1994. Inhibition of skin tumorigenesis by rosemary and its constituents carnosol and ursolic acid. Cancer Research 54: 701- 708

Ippoushi, K; Azuma, Ito H.; Horie H. and Higashio H. 2003. 6-Gingerol inhibits nitric oxide synthesis in activated J774.1 mouse macrophages and prevents peroxynitrite- induced oxidation and nitration reactions. Life Sci. 73: 3427–3437

Jhoo, J. W., Lo, C. Y., Li, S. M., Sang, S., Ang, C. Y. W., Heinze, T. M. and Ho, C. T. 2005. Stability of black tea polyphenol, theaflavin, and identification of theanaphthoquinone as its major radical reaction product. J. Agric. Food Chem. 53: 6146-6150

Jolad, S. D.; Lantz, R. C.; Chen, G. J.; Bates, R. B. and Timmermann, B. N.2005. Commercially processed dry ginger (Zingiber officinale): Composition and effects on LPS-stimulated PGE2 production. Phytochemistry 66(13): 1614-1635.

Jun, M., Hong, J., Jeong, W. S. and Ho, C. T. 2005. Suppression of arachidonic acid metabolism and nitric oxide formation by kudzu isoflavone in murine macrophages. Mol. Nutr. Food Res. 49: 1154-1159

Keum, Y. S.; Kim J; Le K. H.; Park, K.K.; Surh Y. J.; Lee, J. M.; Lee, S. S.; Yoon, J. H.; Joo, S. Y.; Cha. I. H. and. Yook, J. I. 2002. Induction of apoptosis and caspase-3 activation by chemopreventive [6]-paradol and structurally related compounds in KB cells. Cancer Lett. 177: 41–47

Kim, E. C., Min, J. M., Lee, S. J., Yang, H. O., Han, S., Kim, Y. M. and Kwon, Y. G. 2005. 6-gingerol, a pungent ingredient of ginger, inhibits angiogenesis in vitro and in vivo. Biochemical and Biophysical Research Communications 335: 300-308

Kuo, J. M.; Yeh, D .B. and Pan, B. S. 1999. Rapid photometric assay evaluating antioxidative activity in edible plant material. J. Agric. Food Chem. 47: 3206–3209.

Krishnakantha, T. P and Lokesh, B. R. 1993. Scavenging of superoxide anions by spice principles. Indian J. Biochem. Biophys. 30: 133–134

Lantz, R. C., Chen, G. J., Sarihan, M., Solyom, A. M., Jolad, S. D. and timmermann, B. N. 2007. The effect of extracts from gigner rhizome on inflammatory mediator production. Phytomedicine 14: 123-129.

Leal, P. F., Braga, M. E. M., Sato, D. N., Carvalho, J. E., Marques, M. O. M. and Meireles, M. A. A. 2003. Functional properties of spice extracts obtained via supercritical fluid extraction, Journal of Agricultural and Food Chemistry 51: 2520– 2525

Lee, E. and Surh, Y. J. 1998. Induction of apoptosis in HL-60 cells by pungent vanilloids, [6]-gingerol and [6]-paradol. Cancer Lett. 134: 163–168

Liang, Y. C., huang, Y. T., Tsai, S.H., Lin-Shiau, S. Y., Chen, C. F. and Lin, J. K. 1999. Suppression of inducible cyclooxygenase and inducible nitric oxide synthase by apigenin and related flavonoids in mouse macrophages. Carcinogenesis 20: 1945- 1952

Liao, C. H., Ho, C. T. and Lin, J. K. 2005. Effects of garcinol on free radical generation and NO production in embryonic rat cortical neurons and astrocytes. Biochemical and Biophysical Research Communication 329: 1306-1314

Liao, C. H., Sang, S., Liang Y. C., Ho, C. T. and Lin, J. K. 2004. Suppression of inducible nitric oxide synthase and cyclooxygenase-2 in downregulating nuclear factor-Kappa B pathway by garcinol. Molecular Carcinogenesis 41: 140-149

Lin, Y. L., Tsai, S. H., Lin-Shiau, S. Y., Ho, C. T. and Lin, J. K. 1999. Theaflavin-3,3’ –digallate from black tea blocks the nitric oxide synthase by down-regulating the activation of NF-κB in macrophages. European Journal of Pharmacology 367: 379-88

Lo, A. H., Liang, Y. C., Lin-Shiau, S. Y., Ho, C. T. and Lin, J. K. Carnosol, an antioxidant in rosemary, suppresses inducible nitric oxide synthase through downregulating nuclear factor-κB in mouse macrophages. Carcinogenesis 23(6): 983-991

Milner, J. A. 2000. Functional foods: the US perspective. Am J Clin Nutr. 71(suppl): 1654-1659

Lo, A. H., Liang, Y. C., Lin-Shiau, S. Y., Ho, C. T. and Lin, J. K. 2002. Carnosol, an antioxidant in rosemary, suppress inducible nitric oxide synthase through downregulating nuclear factor-κB in mouse macrophage. Carcinogenesis 23 (6): 983-991

Lotito, S .B. and Frei, B. 2006. Consumption of flavonoid-rich foods and increased plasma antioxidant capacity in humans: cause, consequence, or epiphenomenon? Free Radical Biology & Medicine 41: 1727-1746

Morris, C. F., Mueller, D. E., faubion, J. M. and Paulsen, G. M. 1988. Identification of L-tryptophan as an endogenous inhibitor of embryo germination in white wheat. Plant Physiol. 88: 435-440.

Molyneux, P. 2-004. The us aubion, J. M. and Paulsen, G. M. 1988. Identification of L-tryptophan as an endogenous inhibitor of embryo germination in white wheat. Plant Physiol. 88: 435-440e of the stable radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity. Songklanakarin J. Sci. Technol. 26 (2): 211-219

Nakano, N., Murakami, K., Nohara, T., Tomimatsu, T. and Kawasaki, T. 1981. The constituents of Paris verticillata M. v. Bibe. Chem. Pharm. Bull 29 (5): 1445-1451

Nakatani, N. and Kikuzaki, H. 2001. Antioxidant in Ginger family; Eds. Ho, C. T. and Zheng, Q. Y. ACS Smposium Series 803: 230-239

Ninfali, P., Mea, G., Giorgini, S., Rocchi, M. and Bacchiocca, M. 2005. Antioxidant

78 capacity of vegetable, spices and dressings relevant to nutrition. British Journal of Nutrition 93: 257-266

Nathan, C. 2002. Points of control in inflammation. Nature 420: 846-852

O’Byrne K. J and Dalgleish A G. 2001. Chronic immune activation and inflammation as the cause of malignancy. Br J Cancer 85: 473-483

Ojewole, J. A. O. 2006. Analgesic, antiinflammatory and hypoglycaemic effects of ethanol extract of Zingiber officinale (Roscoe) rhizomes (Zingiberaceae) in mice and rat. Phytother. Res. 20: 764-772

Onyenekwe, P. C. 2000. Assessment of oleoresin and gingerol contents in gamma irradiated ginger rhizomes. Nahrung 44 (2): 130-132.

Pan, M. H., Lin-Shiau, S. Y. and Lin, J. K. 2000. Comparative studies on the suppression of nitric oxide synthase by curcumin and its hydrogenated metabolites through down-regulation of IκB kinase and NFκB activation in macrophages. Biochemical Pharmacology 60: 1665-1676

Park, E. J. and Pezzuto, J. M. 2002. Botanicals in cancer chemoprevention. Cancer and Metastasis Reviews 21: 231-255

Park, Y. J., Wen, J., Bang, S., Park, S. W. and Song, S. Y. 2006. 6-gingerol induces cell cycle arrest and cell death of mutant p-53-expressing pancreatic cancer cells. Yonsei Medical Journal 47(5): 688-697

Pietta, P. G. 2000. Flavonoids as antioxidants. J. Nat. Prod. 63: 1035-1042

Reddy, A. C. and Lokesh, B. R. 1992. Studies on spice principles as antioxidants in the inhibition of lipid peroxidation of rat liver microsomes. Mol. Cell. Biochem. 111: 117–124.

Ren, W., Qiao, Z., Wang, H., Zhu, L. and Zhang, L. 2003. Flavonoids: Promising anticancer agents. Medicinal Research Review 23: 519-534

Rice-Evans, C.A., Miller N.J. and Paganga., G. 1996. Structure-antioxidant activity relationship of flavonoids and phenolic acids. Free Radical Biol. Med 20 970; 933- 956

Sang, S., Tian, S., Wang, H., Stark, S. E., Rosen, R. T., Yang, C. S. and Ho, C. T. 2003. Chemical studies of the antioxidant mechanism of tea catechins: radical reaction of epicatechin with peroxyl radical. Bioorganic & Medicinal Chemistry 11: 3371-3378

Sang, S., Lapsley, K., Jeong, W. S., Lachance, P. A., Ho, C. T. and Rosen, R. 2002a. Antioxidative phenolic compounds isolated from almond skin (prunus amygdalus batsch). Journal of Agricultural and Food Chemistry 50: 2459-2463

Sang, S., Liao, C. H., Pan, M. H., Rosen, R. T., Lin-Shiau, S. Y., Lin, J. K. and Ho, C. T. 2002b. Chemical studies on antioxidant mechanism of garcinol: analysis of radical reaction products of garcinol with peroxyl radicals and their antitumor activites. Tetrahedron 58: 10095-10102

Scalbert, A., Johnson, I. T. and Saltmarsh, M. 2005. Polyphenols: antioxidants and beyond. Am J Nutr 81 (suppl): 215-217

Schmitt, B.; Schulz, H.; Storsberg, J. and Keusgen, M. 2005. Chemical characterization of Allium ursinum L. depending on harvesting time. J. Agric. Food Chem. 53: 7288-7294

Sekiwa, Y., Kubota, K. and Kobayash, A. 2000. Isolation of novel glucosides related to gingerdiol from ginger and their antioxidative activities. J. Agric. Food Chem. 48: 373-377

Shoji, N., Iwasa, A., Takemoto, T. and Ishida, Y. 1982. Cardiotonic principles of ginger (Zingiber officinale Roscoe). J. Pharm. Sci. 71 (10): 1174-1175.

Shukla, Y. and Singh, M. 2006. Cancer preventive properties of ginger: a brief review. Food and chemical toxicology. (in press)

Smeett, K., Damme, E. J. M. V., Leuven, F. V. and Peumans, W. J. 1997. Isolation and characterization of lectins and lectin-alliinase complexes from bulbs of garlic (Allium sativum) and ramsons (Allium ursinum). Glycoconjugate Journal 14: 331-343

Smeets, K., Damme, E. J. M. V., Leuven, F. V. and Peumans, W. J. 1997. Isolation, characterization and molecular cloning of a leaf-specific lectin from ramsons (Allium ursinum L.). Plant Molecular Biology 35: 531-535

Son, S. and Lewis, B. A. 2002. Free radical scavenging and antioxidant activity of

80 caffeic acid amide and ester analogues: structure-activity relationship. J. Agric. Food Chem. 50: 468-472

Stajner, D. and Varga, I. S. 2003. An evalution of the antioxidant abilities of Allium species. Acta Biologica Szegediensis 47 (1-4): 103-106

Surh, Y. J., Park, K. K., Chun, K, S., Lee, L. J., Lee, E. and Lee, S. S. 1999. antitumor-promoting activities of selected pungent phenolic substances present in ginger. J. Environ. Pathol. Toxicol. Oncol. 18(2): 131-139

Surh Y.J., Lee, E. and Lee, J.M. 1998 Chemoprotective properties of some pungent ingredients present in red pepper and ginger, Mutation Research 402 : 259–267.

Tachie, A. N., Dwuma-Badu, D., Ayim, J. S. K., Dabra, T. T. 1975. Hydroxyphenylalkanones from Amomun melegueta. Phytochemistry 14: 853-854

Tjendraputra, E., Tran, V. H., Liu-Brennan, D., Roufogalis, B. D. and Duke,C. C. 2001. Effect of ginger constituents and synthetic analogues on cyclooxygenase-2 enzyme in intact cells, Bioorganic Chemistry 29: 156–163

Wei, Q. Y., Ma, J. P., Cai, Y. J., Yang, L. and Liu Z. L. 2005. Cytotoxic and apoptotic activities of diarylheptanoids and ginger related compounds from the rhizome of Chinese ginger. Journal of Ethnopharmacology 102: 177-184

Wohlmuth, H., Leach, D. N., Smith, M. K. and Myers, S. P. 2005. Gingerol content of diploid and tetraploid clones of ginger (Zingiber officinale Roscoe). J. Agric. Food Chem 53: 5772-5778

Young, H. Y.; Luo, Y. L.; Cheng; H. Y.; Hsieh, W. C.; Liao, J. C. and Peng, W. H. 2005. Analgesic and anti-inflammatory activities of [6]-gingerol. J Ethnopharmacol. 96(1-2): 207-210.

Zaeoung, S. plubrukarn, A. and Keawpradub, N. 2005. Cytotoxic and free radical scavenging activities of Zingiberaceous rhizomes. J. Sci. Technol. 27(4): 799-812.

9. APPENDIX

Appendix 1. 1H-NMR of 6-gingerol

Appendix 2. 13C-NMR of 6-gingerol

Appendix 3. 1H-NMR of 8-gingerol

Appendix 4. 13C-NMR of 8-gingerol

Appendix 5. 1H-NMR of 10-gingerol

Appendix 6. 13C-NMR of 10-gingerol

Appendix 7. 1H-NMR of 6-shogaol

Appendix 8. 13C-NMR of 6-shogaol

Appendix 9. 1H-NMR of 8-shogaol

Appendix 10. 13C-NMR of 8-shogaol

Appendix 11. 1H-NMR of 10-shogaol

Appendix 12. 13C-NMR of 10-shogaol

Appendix 13. 1H-NMR of 6-paradol

Appendix 14. 13C-NMR of 6-paradol

Appendix 15. 1H-NMR of 1-dehydro-6-gingerdione

Appendix 16. 13C-NMR of 1-dehydro-6-gingerdione

0. 6 y = 4E- 08x - 0. 031 0. 5 R2 = 0. 9976 0. 4

0. 3 6- ginger ol 0. 2 0. 1

concentration (mg/mL) 0 0 5000000 10000000 15000000 20000000 Area (uV * sec)

Appendix 17. Standard curve of 6-gingerol

0. 3 L)

m y = 5E- 09x - 0. 0301 0. 25

g/ 2

m R = 0. 9972

( 0. 2 n o i

t 0. 15 a

r t 0. 1

n 6- shogaol e c

n 0. 05

Co 0 0 10000000 20000000 30000000 40000000 50000000 60000000 Ar ea(uV * sec)

Appendix 18. Standard curve of 6-shogaol

Appendix 19. HPLC profile of 6-gingerol

Appendix 20. HPLC profile of 6-shogaol

Appendix 21. 1H-NMR of Aub 30-2-3

100

Appendix 22. 1H-NMR of Aub 30-3-1

101

Appendix 23. 13C-NMR of Aub 30-3-1

102

Appendix 24. 1H-NMR of Aub 30-3-2

103

Appendix 25. 13C-NMR of Aub 30-3-2

104

Appendix 26. 1H-NMR of Aub 30-3

105

Appendix 27. 13C-NMR of Aub 30-3

106

Appendix 28. 1H-NMR of Aub-1

107

Appendix 29. 13C-NMR of Aub-1

108

Appendix 30. 1H-NMR of Aub 30-1

109

Appendix 31. 13C-NMR of Aub 30-1

110

Appendix 32. 1H-NMR of Aub-1-1

111

Appendix 33. 13C-NMR of Aub-1-1

112

Appendix 34. HMQC of Aub-1-1

113

Appendix 35. HMBC of Aub-1-1

114

Appendix 36. 1H-NMR of Aub-3

115

Appendix 37. 13C-NMR of Aub-3

116

Appendix 38. HMQC of Aub-3

117

Appendix 39. HMBC of Aub-3

118

Appendix 40. 1H-NMR of Aub-7

119

Appendix 41. HMQC of Aub-7

120

Appendix 42. HMBC of Aub-7

121

Appendix 43. 1H-NMR of Aub-5

122

Appendix 44. 13C-NMR of Aub-5

123

Appendix 45. HMQC of Aub-5

124

Appendix 46. HMBC of Aub-5

125

CURRICULUM VITAE

HOU WU

EDUCATION

PH.D in Food Science May 2007 Rutgers University, New Brunswick, New Jersey

M. S. in Food Science & Technology, March 2000 Zhengzhou Grain College, Zhengzhou. China

B.S. in Food Science & Technology, July 1995 Zhengzhou Grain College, Zhengzhou. China

WORKING EXPERIENCE

September 2003-present Ph.D student, Department of Food Science, Rutgers University, New Brunswick, NJ

May 2006-November 2006 Research Intern, International Flavor and Fragrances, Union Beach, NJ

September 2005-April 2006 Research Assistant, Department of Food Science, Rutgers University, New Brunswick, NJ

September 2004-August 2005 Teaching Assistant, Department of Food Science, Rutgers University, New Brunswick, NJ

April 2004-August 2003 Faculty, Department of Life Sciences, Shanghai University, Shanghai, China

August 1995-August 1997 Engineer, Changzhou Jili edible oil company, Changzhou, China

126

Scientific Publication

1,1-Di (2’, 5’-dihydroxy-4’-tert-butylphenyl) Ethane: a novel antioxidant. 2006. Li, J. Y., Wang, T., Wu, H., Ho, C. T. and Weng, X. C. Journal of Food Lipids 13: 331-340

Coco butter equivalent from enzymatic interestification of tea seed oil and fatty acid methyl esters. Wang, H.X.; Wu, H.; Ho, C,T. and Weng, X.C. Food Chemistry, 2006, 97 (4), 661-665

Antioxidant from a Chinese medicine-Psoralea Corylifolia, L. Guo, J. N.; Weng, X.C.; Wu, H.; Li, Q.H. and Bi, K.S. Food Chemistry. 2005, 97: 287-292

Two novel synthetic antioxidants for deep frying oils. Zhang C. X.; Wu, H. and Weng X. C. Food Chemistry, 2004, 84 (2): 219-222

Antioxidative effect of Psoralea corylifolia L. on Lard. Guo J. N.; Weng X. C. and Wu. H. China Oil and Fat, 2004, 29 (3)

Studies on extraction and isolation of active constituents from Psoralen corylifolia L. and the antitumor effect of the constituents in vitro. Guo J. N.; Wu. H. ; Weng X. C.; Yan, J.H. and Bi, K. S. Zhong Yao Cai, 2003, 26(3), 185-187

Antioxidant effects of Officinal Magnolia, Chinese Traditional and Herbal Drugs. Wu, H., Weng, X. C. and Guo, J. N., , 2002, suppl.: 65 – 67

Effect of chinese ganoderma lucid Essence on telomerase activity and apoptosis of the hepatic cancer cell BEL-7402. Zou, C.Z., Wu, H., Yu, Z. G. and Weng, X. C. Shangxi Oncology Medecine. 2002, 10(2): 134- 155

Oxidation stablility of cojugated linoleic acid. Li, K., Weng X. C. and Wu, H. Journal of Shanghai University, 2001, 7(6): 547-549

Extraction of tocopherols from soybean oil deodorizer distillation. Wu, H. and Weng, X. C. Journal of Shanghai University. 2001, 7(4): 331-333

Antioxidant activities of natural tocopherols. Wu, H. and Weng, X. C., Journal of Shanghai University. 2001, 7(2): 142-146

Determination methods of antioxidant activity and their evaluation. Weng, X. C. and Wu, H., China Oil and Fat, 2000, 25(6): 119-122

Science and technology of fats and oils when the new century is coming. Weng, X. C, and Wu, H. China Oil and Fat, 2000, 25(3): 3-4