Potential for Ginkgo Biloba As a Functional Food

POTENTIAL FOR GINKGO BILOBA

AS A FUNCTIONAL FOOD

LENA MEI LING GOH

(B.SC. (HONS.), LEEDS, UK)

A THESIS SUBMITTED

FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

FOOD SCIENCE AND TECHNOLOGY PROGRAMME C/O DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE

2004

ACKNOWLEDGEMENTS

This study was carried out at the National University of Singapore, Department of Chemistry. This academic dissertation was produced under the Food Science and

Technology Programme of the Chemistry Graduate School of Science.

I wish to express my deepest gratitude to my supervisor, Dr Philip J. Barlow, for his encouragement to start this work and for the opportunity to be a member of the inspiring research group. His endless support and constructive criticism has been precious during these years. I thank him also for his inventive comments and suggestions, especially during the writing phase. His never failing support and patience during these years were vital to the success of the study. The knowledge you shared with me and your scientific criticism are gratefully acknowledged.

I owe my thanks to Professor Hian Kee Lee, Head of the Department of

Chemistry, for providing the facilities for my work in his department and for his support.

I wish to thank him also, for his support and advice during my first steps as a Ph.D. student. I would also like to thank Dr. Leong Lai Peng, A/P Weibiao Zhou and A/P

Perera Conrad O., from the Food Science and Technology Programme for providing various research equipment for my investigations in their laboratories. Their valuable advice and knowledge have sparked off many research ideas for my work.

I am grateful to Mdm. Lee Chooi Lan, Ms Lew Huey Lee, Mrs Lim Guek Choo,

Frances, Mdm Toh Soh Lian and Ms Tang Chui Ngoh for their excellent technical assistance. My colleagues and friends in the Food Science and Technology Programme deserve warm thanks, for making my work easier during these years, for giving a hand in solving problems, and for providing a pleasant working atmosphere. My special thanks go to Anne Bruneau, M.Sc., and Chuan Hong Tu, Ph.D. for pleasant and inspiring working atmosphere in the lab. It was indeed a pleasure to work with people that have

Page 2of 201 such a good sense of humour! My warm thanks go to my colleagues Sok Li Tay, M.Sc.,

Yean Yean Soong, M.Sc., Cui Min, and Tze Han Lim for inspiring discussions and sharing good moments when writing this thesis. I am also indeed grateful to have good friends like David J. Young, Ph.D., Suna Goh, Chia Lee Koh, Jing Xuan Xie, Yi Qin Pan

& Hui Ying Lee for being around me and being ever so patient and understanding for the times that I am so attached to my work.

Special thanks to Dr. Yong Eu Leong and his students from the Department of

Obstetrics and Gynaecology for the collaborative work in investigation for the oestrogenic activity in the various Ginkgo biloba plant parts’ extracts. Their efforts and time put in is greatly appreciated.

Special thanks also go to Martin Fletcher from California Gardens for making the effort in helping and supplying me with the constant source of Ginkgo leaves for my experiments.

My warmest thanks belong to my parents, William and Ann Goh and my godma,

Lai Eng Heng for their confidence in me and for being always so supportive and interested in my work and well being. I would like to thank them, my sister Laura Goh, and my close friends for providing unfailing support to finish this work. My brother

Lawrence Goh, M.Computing. deserves special thanks for his friendship, advice, prayers and continuous encouragement during my academic career.

Finally, my dearest thanks are addressed to my fiancée, Meng Yew Leong, M.BA

(Finance) for his love and tireless support.

Page 3 of 201 TABLE OF CONTENTS

ACKNOWLEDGEMENTS...... 2 LIST OF ABBREVIATIONS...... 11 SUMMARY...... 12 1 INTRODUCTION ...... 14 2 LITERATURE REVIEW AND BACKGROUND TO THE STUDY...... 16

2.1 GINKGO BILOBA ...... 16 2.1.1 Phytobiology ...... 17 2.1.2 Ginkgo biloba leaves ...... 19 2.1.2.1 Traditional Chinese medicine approach ...... 19 2.1.2.2 Controversies ...... 21 2.1.2.3 Folklore vs. Scientific evidence...... 22 2.1.3 Ginkgo leaf extract - EGb 761 ...... 23 2.1.3.1 Description of each component ...... 24 2.1.3.2 Biological action of Ginkgo biloba leaf constituents ...... 25 2.1.3.3 Polyvalent mechanism of action of EGb 761 ...... 32 2.1.3.4 Adverse effects...... 32 2.1.4 Ginkgo biloba nuts...... 34 2.1.4.1 Nutritional composition...... 35 2.1.4.2 Popularity of consumption ...... 35 2.1.4.3 Health related functions ...... 36 2.1.4.4 Adverse effects...... 36 2.1.5 Leaf extract vs. nuts ...... 38 2.1.5.1 Similarities, differences and future potential...... 39

2.2 FLAVONOIDS AND PHENOLIC ACIDS...... 41 2.2.1 Chemistry of flavonoids...... 42 2.2.2 Flavonoids as antioxidants...... 51 2.2.2.1 Mechanisms of action of antioxidants ...... 52 2.2.2.2 Determination and measurement of antioxidation activity...... 56 2.2.3 Structure-Antioxidant activity relationship ...... 66

2.3 TERPENOIDS FRACTION...... 67 2.3.1 Chemistry of Terpenoids ...... 67 2.3.2 Bioactivity ...... 69

2.4 POTENTIAL TOXIC COMPOUNDS ...... 70 2.4.1 Alkaloids ...... 70 2.4.2 Alkylphenols...... 72 3 AIMS & OBJECTIVES...... 79

3.1 MAIN FOCI OF THE RESEARCH ...... 79

3.2 HYPOTHESES PROPOSED ...... 81 4 MATERIALS & EQUIPMENT ...... 84

4.1 MATERIALS...... 84 4.1.1 Test specimens ...... 84 4.1.2 Other test specimens for comparison studies ...... 85 4.1.3 Solvents...... 85 4.1.4 Chemicals ...... 85

Page 4 of 201 4.2 EQUIPMENT USED...... 88 5 PROBLEM BASED APPROACH – ANALYTICAL CHOICES & METHODOLOGIES ...... 90

5.1 PROBLEM BASED APPROACH...... 91 5.1.1 Problem 1 - Compositional analysis of Ginkgo biloba specimens...... 91 5.1.2 Problem 2 - Vitamin analysis - The natural antioxidant present ...... 91 5.1.3 Problem 3 - Effect of heat over time on AOC of Ginkgo biloba Nuts...... 91 5.1.4 Problem 4 - Effect of preparation procedure on AOC of Ginkgo biloba leaf infusions 92 5.1.5 Problem 5 – In vitro, Simulated Digestion studies ...... 92 5.1.6 Problem 6 –Toxicity consideration ...... 93 5.1.6.1 Colchicine determination ...... 93 5.1.6.2 GA – An allergen...... 93 5.1.7 Problem 7 – Hormonal studies on the Ginkgo biloba specimens ...... 94

5.2 METHODOLOGIES ADOPTED...... 95 5.2.1 Sample Treatment & Preparation ...... 95 5.2.2 Proximate analysis...... 96 5.2.2.1 Lipids determination...... 96 5.2.2.2 Protein...... 96 5.2.2.3 Moisture ...... 96 5.2.2.4 Ash...... 96 5.2.3 Vitamins analysis ...... 97 5.2.3.1 Vitamin C...... 97 5.2.3.2 Vitamin E ...... 97 5.2.4 Fermentation techniques...... 98 5.2.5 Sensory evaluation ...... 99 5.2.6 Extraction techniques...... 100 5.2.6.1 Antioxidants - Flavonoids...... 100 5.2.6.2 Alkaloids ...... 100 5.2.6.3 Alkylphenols...... 101 5.2.7 AOC determination ...... 104 5.2.7.1 ORAC – Oxygen radical absorbance capacity ...... 105 5.2.7.2 FRAP – Ferric reducing antioxidant power ...... 106 5.2.7.3 ABTS cation decolorisation assay...... 107 5.2.8 Statistical analysis...... 109 5.2.9 Qualification and Quantification techniques...... 110 5.2.9.1 High Pressure Liquid Chromatography technique ...... 110 5.2.9.2 Capillary Electrophoresis determination...... 116 5.2.9.3 GCMS ...... 117 5.2.10 Digestive system simulation studies...... 119 5.2.10.1 Gastric fluids ...... 119 5.2.10.2 Intestinal fluids...... 119 6 RESULTS AND DISCUSSIONS...... 121

6.1 COMPOSITIONAL ANALYSIS OF GINKGO BILOBA SPECIMENS...... 121

6.2 EFFECT OF HEAT APPLICATION ON GINKGO NUTS WITH REGARDS TO AOC...... 124

6.3 EFFECT OF PREPARATION PROCEDURE ON AOC FOR LEAF INFUSION ...... 131 6.3.1 Comparison of AOC results with ABTS and FRAP ...... 133 6.3.2 Comparison studies among Ginkgo Leaves, Green & Black Tea Leaves...... 140 6.3.3 Caffeine determination...... 141 6.3.4 Sensory Evaluation ...... 142

Page 5 of 201 6.4 IN VITRO, SIMULATED DIGESTION STUDIES ...... 145 6.4.1 HPLC analysis for determination of stability of Glycosides and Aglycones ...... 151 6.4.1.1 Qualification ...... 151 6.4.1.2 Quantification...... 152

6.5 TOXICOLOGICAL DETERMINATION ...... 157 6.5.1 Colchicine determination ...... 157 6.5.2 Ginkgolic acids – as allergen...... 158 6.5.2.1 SAMPLE PREPARATION ...... 158 6.5.2.2 HPLC ANALYSIS...... 161 6.5.2.3 GC-MS ANALYSIS...... 168 7 CONCLUSIONS...... 174

7.1 POTENTIAL FOR THIS RESEARCH ...... 179 8 SUGGESTIONS FOR FURTHER STUDIES...... 180

8.1 GINKGO NUTS...... 180

8.2 GINKGO LEAF INFUSION...... 180

8.3 IN VIVO VS IN-VITRO ANTIOXIDANT DETERMINATION ASSAY ...... 180

8.4 SIMULATED DIGESTION PROCESS...... 180

8.5 HORMONAL STUDIES...... 180 REFERENCES ...... 181 APPENDICES...... 197

Page 6 of 201 LIST OF FIGURES

Fig 1. The extraction procedure for EGb761 from Ginkgo biloba leaves...... 23 Fig 2. Flavonol aglycons that may be present in trace amounts (<0.1%) in EGb761...... 24 Fig 3. Chemical structures of terpene constituents of EGb 761 – Ginkgolide...... 24 Fig 4. Chemical structures of terpene constituents of EGb 761 - Terpenoid...... 25 Fig 5. Shikimic acid pathway...... 43 Fig 6. Phenylpropanoid metabolism leading to flavonoids, stilbenes, styrylpyrones and coumarins as well as the precursors for lignin and lignan synthesis...... 44 Fig 7. Biosynthesis pathways of flavonoids...... 45 Fig 8. Interconnections of flavonoids subgroups...... 66 Fig 9. Chemical Structure and proposed fragmentation pattern of Colchicine...... 71 Fig 10. Structures of alkylphenols isolated from G. biloba leaves ...... 72 Fig 11. Synthesis of GAs via the polyacetate pathway ...... 73 Fig 12. Generic schematic of the fluorescence system...... 104 Fig 13. Trolox concentration effect on FL fluorescence decay curve...... 106 Fig 14. Calculation of ORAC values...... 106 Fig 15. Typical UV spectrum of Ginkgolic acids...... 113 Fig 16. Structure of external standard (syn. 6-heptadecenylsalicylic acid)...... 113 Fig 17. Major TMS derivatives of GAs, bilobols and cardanols...... 117 Fig 18. Nuts and Leachate extract and summation of the both to express the total AOC in mg AEAC per 100 g homogenate vs cooking time ...... 125 Fig 19. Composition of heat stable and heat labile antioxidant in Ginkgo dessert during heating...... 126 Fig 20. AOC contribution by vitamin C in the nuts and in the leachate...... 127 Fig 21. Absorbance drop vs ferrous sulphate standard concentration ...... 133 Fig 22. Absorbance drop vs ascorbic acid standard concentration ...... 133 Fig 23. FRAP Equivalent (mg/100g) of Ginkgo leaves infusion with respect to temperatures (°C)...... 135 Fig 24. Electropherogram of the capillary electrophoresis determination of the Ginkgo leaves profile ...136 Fig 25. FRAP Equivalent (mg/100g) with respect to Time of Infusion (min)...... 137 Fig 26. FRAP Equivalent (mg/100g) with respect to Time of Infusion (min)...... 138 Fig 27. Ranking of preferences amongst the three samples ...... 144

Fig 28. Chromatogram of a standardised extract of Ginkgo biloba leaves (control sample: blank alcohol). 151 Fig 28a. HPLC chromatogram of Ginkgo R.P.C. leaf extract with acetonitrile-0.01% formic acid eluent...... 162 Fig 28b. HPLC chromatogram of Ginkgo R.P.C. leaf extract with acetonitrile-0.1% formic acid eluent.163 Fig 28c. HPLC chromatogram of Ginkgo R.P.C. leaf extract with acetonitrile-0.5% acetic acid eluent. .163 Fig 28d. HPLC chromatogram of Ginkgo R.P.C. leaf extract with acetonitrile-5% acetic acid eluent.....163 Fig 29(a-c). Comparative gradient elution profiles of Ginkgo nut extract using 0.01% formic acid in acetonitrile solvent based gradients...... 165 Fig 30 a. UV spectrum of ginkgolic acids in HPLC chromatogram of Ginkgo U.S.A. leaf extract, Rt = 33.353 min...... 165 Fig 30 b. UV spectrum of ginkgolic acids in HPLC chromatogram of Ginkgo R.P.C leaf extract, at Rt = 33.748 min...... 166

Page 7 of 201 Fig 30 c. UV spectrum of ginkgolic acids in HPLC chromatogram of Ginkgo nut extract, Rt = 30.782 min ...... 166 Fig 30 d. UV spectrum of ginkgolic acids in HPLC chromatogram of Ginkgo capsule extract, Rt = 32.662 min ...... 166 Fig 30 e. UV spectrum of ginkgolic acids in HPLC chromatogram of Ginkgo EGb 761 extract, Rt = 30.656 min ...... 166 Fig 31 a. Gas Chromatogram of U.S.A leaf extract, Rt = 27.068 min (Peak 4), 28.753 (Peak 5), 28.894 (Peak 6) and Rt = 31.278 (Peak 7)...... 168 Fig 31 b. Gas Chromatogram of R.P.C. leaf extract, Rt = 27.019 (Peak 4), 28.709 (Peak 5), 28.853 (Peak 6) and Rt = 31.218 (Peak 7)...... 169 Fig 31 c. Gas Chromatogram of Ginkgo capsule extract, Rt = 27.013 (Peak 5) and 28.827 (Peak 7)...... 169 Fig 31 d. Gas Chromatogram of Ginkgo EGb 761 extraction, Rt = 27.025 (Peak 5), 28.723 (Peak 6) and Rt = 28.871 (Peak 7)...... 170 Fig 32 a. Mass spectra of external standard at Rt = 27.017 min...... 172 Fig 32 b. Mass spectra of external standard at Rt = 28.708 min, strong ions at m/z 208 and 221...... 172

Page 8 of 201 LIST OF TABLES

Table 1. List of Flavonoids in the leaves of Ginkgo biloba...... 48 Table 2. Selected protocols used to evaluate free radical scavenging properties of antioxidants...... 60 Table 3. Some applications of some plant terpenoids...... 68 Table 4. Scientific interest in terpenoids ...... 68 Table 5. Some economical aspects of terpenoids ...... 68 Table 6. Distribution of GAs in Ginkgo biloba...... 75 Table 7. Experimental samples obtained from various Ginkgo biloba extracts ...... 120 Table 8. Comparison of Nutritional values from Ginkgo biloba samples ...... 121 Table 9. Ascorbic acid Antioxidant Equivalent Capacity for leachate and nut extract sample and summation of the two...... 124 Table 10. Antioxidant capacity from various Ginkgo biloba samples using ABTS cation decolorisation assay ...... 128 Table 11. Antioxidant capacity from various Ginkgo biloba samples using ORAC method ...... 129 Table 12. Various leaf infusion samples used .for AOC determination ...... 132 Table 13. Particle size distribution of the leaves passing through various sieve sizes...... 132 Table 14. FRAP Equivalent (mg/100g) of fermented and unfermented Ginkgo leaves infusion...... 134 Table 15. AOC of the different test subjects ...... 140 Table 16. Determined caffeine content in selected Beverages ...... 141 Table 17. Summary of total sores based on the criteria tested. Statistical analysis ...... 143 Table 18. Tabulation of the ranking amongst the three samples...... 143 Table 19 Comparison of the amount of the various constituents (mg/g) present in the various Ginkgo biloba samples studied both in the aqueous & alcohol extracts...... 147 Table 20. Percentage recovery of the various compounds from raw Ginkgo biloba leaves following specific treatment ...... 148 Table 21. Percentage recovery of the various compounds from the Standardised Ginkgo Extract following various treatments...... 149 Table 22. Percentage recovery of the various compounds from commercial Ginkgo capsules following specified treatments...... 150 Table 23. Sample preparation of Ginkgo extracts for HPLC analysis...... 158 Table 24. Sample preparation of Ginkgo extracts for GC-MS analysis ...... 159

Page 9 of 201 TABLE OF PLATES

Plate 1. Ginkgo trees growing along the streets of Japan during the winter and summer seasons ...... 18 Plate 2. Ginkgo fruit, seeds and Ginkgo leaf extract powder ...... 74

Page 10 of 201 LIST OF ABBREVIATIONS

No Description Acronyms 1 β-phycoerythrin β-PE 2 2,2'-azobis(2-amidinopropane) dihydrochloride AAPH 3 2,2'-azobis(2-amidinopropane) hydrochloride ABAP 4 2,2’-azinobis-(3-ethylbenzothiazoneline-6-sulfonic acid) assay ABTS 5 Ascorbic acid equivalent antioxidant capacity AEAC 6 Antioxidant AH 7 Antioxidant capacity AOC 8 Capillary Electrophoresis CE 9 Clastogenic factors CFs 10 a terpene-free extract of Ginkgo biloba CP205 11 cytochromes P450 CYPs 12 1,1-Diphenyl-2-Picryl-Hydrazyl DPPH 13 Ginkgo biloba leaf extract EGb 761 14 (3’,6’dihydroxyspiro [isobenzofuran-1[3H],9’[9H]-xanthen]-3-one) FL 15 Ferric Reducing Ability of Plasma FRAP 16 Ginkgolic acids GAs 17 Gas Chromatography-Mass Spectrometer GC-MS 18 limits of detection LOD 19 microwave-assisted extraction MAE 20 Oxygen radical absorbance capacity ORAC 21 platelet activating factor PAF 22 Photodiode array PDA 23 reversed phase high pressure liquid chromatography RP-HPLC 24 Standard Deviation SD 25 supercritical fluid extraction SFE 26 traditional Chinese medicine and pharmacology TCMP 27 Trolox equivalent antioxidant capacity TEAC 28 trimethylsilyl TMS 29 Total radical-trapping parameter assay TRAP

Page 11 of 201 SUMMARY

This work set out to examine the potential of Ginkgo biloba as a functional food

and to draw distinctions between Ginkgo nuts and the leaves. Whilst traditionally

Chinese medicine has claimed and regarded Ginkgo biloba nuts as having numerous

therapeutic functions, there is also growing speculation as to the potential benefits of the

leaves and their therapeutic properties, however scientific evidence is needed to back up

all these claims and this study attempts to address this need. Suggested toxicity of the

plant has also been highlighted and this issue is also addressed in the current work.

A problem-based approach was undertaken to tackle the theme of the research.

The work progresses from the basic study of nutritional values of Ginkgo biloba, through its measurement of antioxidant capacity (AOC), stability of its compounds subjected to a simulated digestion process, determination of toxic compounds to suggested new frontiers of hormonal function based on components in Ginkgo biloba that may lead to new evidence of its functionality.

One of the finding of this research is that the glycosides within the Ginkgo leaf are actually available for absorption to fulfil their ascribed antioxidant function. This result is significant as it indicates that by ingesting the raw materials, (in this case, the leaves of the Ginkgo biloba plant) as compared to its commercial products, there is better

provision for a more efficient uptake by the body of the potentially beneficial flavonoids.

The potential for Ginkgo biloba leaf infusion was also investigated and shown to

provide an antioxidant-rich beverage that had the added advantage of being caffeine free.

The work also established a new HPLC method capable of identifying and quantifying in one run, 8 standard reference flavonoids (5 glycosides and 3 aglycones).

With this method established, the conversion of glycosides to aglycones in many substrates is more easily monitored.

Page 12 of 201 Lastly, from the toxicity studies, it was established that colchicine is absent in

Ginkgo leaf, nuts, standardised extract and commercial capsules. The worry about presence of colchicine in Ginkgo biloba may just be an unfounded consideration.

Page 13 of 201 1 INTRODUCTION

This research work was carried out between Jan 2001 and Jan 2004. Background

studies were undertaken to understand and put in context the subject of the research. An

extensive literature review of reported health effects of Ginkgo biloba, in particular

related to Chinese folklore, was carried out. In the literature review, the underlying

science of the claims made and the active ingredients reported to have beneficial effect

were also examined along with possible analytical procedures.

Ginkgo biloba has, over recent years, been popularized as a health supplement in

the Western world. However, it is only over the last few years, that leaf extracts have

been important in the production of dietary supplements. Tracing back to the Yuan

dynasty (1280-1368 A.D.), it was the nuts that were mooted as having health benefits,

and these products are still used extensively in the preparation of Traditional Chinese

Dishes. This study aims to consider, in a scientific manner, the potential for Ginkgo

biloba to be recognised as a functional food. Detailed comparisons have been carried out

between the nuts and leaves to give a better picture of what this whole plant may have to

offer.

Work carried out as part of this study has shown that the Antioxidant Capacity

(AOC) determined by ABTS cation decolorisation assay is relatively high in both leaf

and nut extracts. When these results were related to the Vitamin C content of nuts, they

showed that the responsible constituents contributing to AOC are additional to the

Vitamin C content. This led to further research into the additional antioxidants that are apparently active in spite of prolonged heating of the nuts and which are most likely phenolic compounds that contribute to therapeutic function. Leaf infusion of Ginkgo biloba has also been studied to establish the possibility of development into a functional

beverage.

Page 14 of 201 Considering that absorption from the diet is normally a prerequisite for the

potential in vivo beneficial role of flavonoids, research to determine the flavonoids

stability and recovery from Ginkgo biloba when subjected to simulated gastrointestinal

fluids was carried out. The results indicate a trend of conversion from the glycosides to

the aglycones for some samples and subsequent degradation of the aglycones. This may indicate a need to further investigate the reported benefits of Ginkgo flavonoids as in-

vivo antioxidants and/or to consider the antioxidant activity of the resulting digestion

derived compounds.

The potential toxicity of Ginkgo biloba was also investigated and determination

of colchicine and ginkgolic acids was carried out and a comparison made amongst the

Ginkgo biloba samples, i.e. the Ginkgo nuts, leaves from China and USA, standardised

Ginkgo leaf extract and lastly the commercial Ginkgo capsules available as dietary

supplements.

Another hypothesis explored was that an extract of Ginkgo biloba, which

contains several antioxidative polyphenolics, might have wide potential utility as pro- or

anti-fertility agents or for hormone replacement therapy. The aim is to use receptor

studies by collaborating with other departments within NUS for detecting testosterone-

like compounds in Ginkgo biloba.

During the research a range of analytical procedures have been modified for

particular use and these include Gas Chromatography Mass Spectrophotometry,

Capillary Electrophoresis, High performance Liquid Chromatography, Fluorescence

Spectrometer, UV-VIS spectrophotometer, Atomic Absorption, ICP-MS and Nuclear

Magnetic Resonance.

Page 15 of 201 2 LITERATURE REVIEW AND BACKGROUND TO THE STUDY

2.1 GINKGO BILOBA

From the beginning of ancient civilization, Ginkgo biloba has been used

extensively. Visionary healers have tapped the resources of this amazing herb since the

beginning of recorded history. This plant may be traced back more than 200 million

years and the tree has been described as a rare and precious “fruit” in a region of eastern

China (Li, 1956).

There are several claims made about its beneficial effects for humans. Some of these claims are supported by scientific evidence while others currently remain only a

“legend”. Now, with the aid of advanced diagnostic equipment and devices, there is the possibility to more scientifically examine some of these legends. Different parts of the plant are reported to have various health benefits namely the Ginkgo leaf standardised extract termed as EGb 761, and the Ginkgo semen also know as the Ginkgo nut. Ginkgo history suggests a subject worthy of further studies on the nutritional and therapeutic

actions that Ginkgo biloba might offer.

Page 16 of 201 2.1.1 Phytobiology

The Ginkgo tree (Ginkgo biloba), also known as the maidenhair or kew tree, is

the only living member of the Ginkgoaceae family. It is a dioecious plant i.e. having

male reproductive organs in one individual and female in another. Fossils of the trees

have been dated from as far back as 250 million years. The Ginkgo tree may be the

longest living tree, and was referred to by Darwin as “a living fossil” (Major, 1967). It

appears to have curiously strong resistance to a wide range of insect pests and fungi, a

fact that likely contributes to its longevity.

The Ginkgo tree is indigenous to Korea, China, and Japan. It is popular for lining

the streets and parks, although its seeds are reported to give off a foul odour when ripe.

The tree may grow to 40m in height and can live for more than 1000 years (See Plate 1).

It flowers for the first time when it is about 25 years old. The seeds, often incorrectly

referred to as the fruit, become fleshy and plum like, with a light green or yellow colour.

These seeds are usually about 3 cm in diameter and contain a two-edged edible nut.

However the sizes of the seeds differ depending on country of origin. In California, where many Ginkgo trees are harvested, they have smaller seeds about 1 cm in diameter.

A wide variety of compounds are extracted from the seeds, leaves and bark. The leaves are fan shaped, with bifurcated ribs, and glabrous. The leaves turn from bright green in the spring to a beautiful yellowish green in the fall.

Page 17 of 201

Plate 1. Ginkgo trees growing along the streets of Japan during the winter and summer seasons

Page 18of 201 2.1.2 Ginkgo biloba leaves

Ginkgo biloba standardised extract prepared from the leaves has received much attention in recent decades (DeFeudis, 1998; Packer, 1999). Many researchers have reported the leaf extracts of Ginkgo biloba to be useful as regards their medicinal properties and the active ingredients of the leaves are widely reported. (DeFeudis, 1998;

Packer, 1999). In the past three decades, Ginkgo leaves have attracted attention as agents for improving circulation, particularly cerebral circulation, which may lead to improved mental function (Wadsworth & Koop, 2001; Kleijnen & Knipschild, 1992a;

Oken et. al., 1998; Bradly et. al., 2000). A standardised extract of the leaves, EGb761,

has been shown to have numerous therapeutic effects for human health and many papers

have cited studies utilising this standardised extract.

2.1.2.1 Traditional Chinese medicine approach

Early humans gained knowledge of herbs through trial and error, and by

comparing and translating herbs’ visual appearance, odor, and smell. Ginkgo was no exception: early records show that its thorns could pierce and drain an abscess and were

boiled in drinks to help reduce swellings. They also learned that the Ginkgo fruit caused

skin rashes but that the seeds inside were medicinally valuable, along with the leaves.

The history of Ginkgo’s use in China is well recorded, with information dating back

5000 years in many texts including the I Ching: The Book of Changes. The tree has a phenomenal record of resistance to insects, pollution and parasitic invasion. The coating of the nut was found to be microbially resistant, which demonstrates the survival mechanism attributable to the whole plant. Ginkgo’s resilience is also evidenced by reports that Ginkgo nuts were found alive after the atomic bomb explosion in Hiroshima,

Japan, where even damaged trees soon began to sprout and grow (DeFeudis, 1998).

Page 19 of 201 The traditional Chinese medicine approach is that every therapeutic effect takes place with the balance of the yin and yang. Traditional Chinese Medicine has relied on

the theory of yin-yang balance in the diagnosis and treatment of disease for over 2,000

years and foods may be classified as yin or yang and used to create balance in a person.

Ou et. al., 2003 have attempted to relate the Yin and Yang aspect to the oxidative state

of a food or system. They propose that yin-yang balance is antioxidation-oxidation

balance with yin representing antioxidation and yang as oxidation. This they

investigated by determining the antioxidant activity (using the oxygen radical absorbance

capacity, ORAC) of a range of representative herbs classed as yin or yang as described in

the Zhong Hua Ben Cao, an authoritative book on Chinese herbs, and found on average that those classified as yin had about six times more antioxidant activity and polyphenol

content than the yang herbs. According to Zheng & Wang, 2001, Ginkgo biloba is

reported to have 1.57±0.05 milligrams of gallic acid (GAE) equivalents (GAE) per gram

of fresh weight of total phenolic and 13.18±0.24 micromoles of Trolox equivalents per

gram of fresh weight of antioxidant activity (ORAC). With the study by Zheng &

Wang, 2001, Ginkgo biloba would then be ranked as a yang-tonic herb. However this

deduction may not be conclusive as the later study may not be appropriate for

comparison and correlation to the earlier study by Ou et. al., 2003. Ou et. al., 2003 used

fluorescein (FL), whilst, Zheng & Wang, 2001 use β-phycoerythrin to measure the

inhibition of damage to the respective fluorescence probe. Ou et. al., 2001 reported to

have results demonstrating that FL is superior to B-phycoerythrin. They also concluded

that, unlike other popular methods, the improved ORACFL assay provides a direct

measure of hydrophilic chain-breaking antioxidant capacity against peroxyl radicals.

Traditional Chinese folklore regards food as warm/hot and cool/cold and neutral.

However in modern context, these will be termed as yin, being cool/cold and yang, being

warm/hot. The neutral would then be balance of yin and yang. According to the Zhong

Page 20 of 201 Hua Ben Cao, an authoritative book on Chinese herbs, Ginkgo biloba leaf has not been classified but Ginkgo biloba nuts also known as “bai guo”, have the property of neutral.

Many Chinese use bai guo with lotus seeds, bean curd skin (fu zhu) to make cooling soup. The cooling properties of this dish could mainly be contributed by the bean curd skin and the lotus seeds and not from bai guo. From the usage of bai guo in treating chronic asthma of old people and other chronic problems, it seems that bai guo is actually on the warm side (i.e possessing the yang i.e. the warm aspect, property instead of neutral). To date, however there is virtually no study on the chemical nature of yin and yang. It is definitely of interest to know how the phytochemicals in yang-tonic herbs work to boost the energy metabolism efficiency.

On the whole, there is much focus on yin and yang vs antioxidant and oxidant,

and there is scope to try and understand what such an approach means and to see if it has

any bearing on more accepted nutrition/dietary approaches to prevention or amelioration

of disease.

2.1.2.2 Controversies

Often the promotional literature for Ginkgo purports or implies Ginkgo leaf extract as having been used for thousand of years in China. However, until the last 20-

30 years, traditional Chinese herbal medicines rarely used Ginkgo leaves for treating

occlusive circulatory disorders, medical conditions and for other therapeutic purposes

(Robert, 1994). It is the nuts that have been traditionally consumed and reports of such were first mentioned in herbals in the Yuan dynasty [1280-1368 A.D], published in 1350

A.D. (Del Tredici, 1991; Foster & Chongxi, 1992). Hence, it seems that the therapeutic effects of nut and leaves may be different and they are used differently for treatment/prevention of diseases.

Page 21 of 201 Ginkgo biloba is often used as a broad term, which is then misunderstood when

applied. In the ASEAN region, the majority of people would interpret it to refer to the

nuts, as this is the common commodity consumed. It is only since the 1980s when the

discovery of the leaf’s fraction potent action on the human cardiovascular system,

particularly on cerebral vascular activity, made it popular that its use has grown dramatically in the Western medicinal field. It is necessary to correct the misunderstanding of Ginkgo nuts being able to improve memory and children being made to eat lots of them! In fact, over-consumption of the nuts may result in convulsions or even death (see Section 2.1.4.4). It is the leaves that have been demonstrated to have positive effects on memory.

2.1.2.3 Folklore vs. Scientific evidence

Ginkgo nuts were used in the treatment of bronchial disorders and to aid circulation and the digestion process as early as a thousand year ago, however all this postulation has never been proven by scientific research and evidence. Only in recent times has the scientific community paid more attention to the therapeutic functions of

Ginkgo biloba, but to date, little conclusive evidence has been found to support its historical usage and mechanistic actions. In many instances the effectiveness and mechanism of herbal compounds has never been understood, yet used extensively for treatments of specific diseases such as quinine for malaria or ginseng for fatigue.

Page 22 of 201 2.1.3 Ginkgo leaf extract - EGb 761

Dr. Willmar Schwabe developed an extract from the leaves of Ginkgo biloba for

use in the treatment of cerebral insufficiency in the 1960s (Maurer et. al., 1997). He

represented a German company and later this German company formed a partnership

with Laboratories Beaufour in France who defined the extract EGb 761. The preparation of this involves the following procedures: The dried leaves received from the plantations

are blended prior to extraction. They are then extracted with acetone/water (60:40)

(Drieu, 1986) to produce EGb761. EGb761 is obtained via an 18-step extraction

procedure, briefly outlined in Figure 1 (DeFeudis, 1998).

Ginkgo biloba leaves

Extraction with acetone/water

Total Extract

Removal of strongly lipophilic components

Water soluble components and polar compounds

Enrichment of active components

Active components/condensed polyphenolic comp ounds

Removal of condensed polyphenolic EGb761

Fig 1. The extraction procedure for EGb761 from Ginkgo biloba leaves. (Modified from DeFeudis 1998)

Page 23 of 201 2.1.3.1 Description of each component

The main active ingredients of medicinal Ginkgo leaf extracts (i.e. the EGb 761) are standardised and reported as 22-27% flavonoid glycosides [See Fig 2], determined by high-performance liquid chromatography (Hasler et. al, 1992; Pietta & Mauri, 1988;

Pietta et. al. 1991) as quercetin and kaempferol including isorhamnetic and calculated as acyl flavonol glycosides with a molar mass of Mr = 756.7 (quercetin glycosides) and Mr

= 740.7 (Kaempferol glycosides); 5-7% terpene lactones [See Fig 3], which consists of about 2.8-3.4% Ginkgolides A, B and C and [Figure 4] 2.6-3.2% bilobalide; and less

than 5µg/g Ginkgolic acids (DeFeudis, 1998).

R1 R1 R2 OH Kaempferol H H B HO O Quercetin OH H R2 A C Isorhamnetin OCH3 H OH Myricetin OH OH OH O

Fig 2. Flavonol aglycons that may be present in trace amounts (<0.1%) in EGb761. (Modified from DeFeudis 1998)

H O O

O H R1 R2 R3 R4 O R4 H R2 Ginkgolide A (GA) H OH H OH H Ginkgolide B (GB) H OH OH OH H3C O C(CH3)3 Ginkgolide C (GC) OH OH OH OH R Ginkgolide J (GJ) OH OH H OH H 1 O O H R3 Ginkgolide M (GM) OH H OH OH H H Fig 3. Chemical structures of terpene constituents of EGb 761 – Ginkgolide. Ginkgolides A, B, C and J have been isolated from the leaves of Ginkgo biloba; ginkgolides A, B, C, taken together, account for about 3.1% of EGb761; ginkgolides J accounts for ≤0.5%. Ginkgolide M is present only in roots of the Ginkgo biloba tree (Modified from DeFeudis 1998)

Page 24 of 201 O O

O H O Bilobalide

O H H

H O H O (H 3 C) 3 C O

Fig 4. Chemical structures of terpene constituents of EGb 761 - Terpenoid Bilobalides, which accounts for about 2.9% of EGb761, is also shown. Chemical structures modified from DeFeudis 1998.

2.1.3.2 Biological action of Ginkgo biloba leaf constituents

Many isolated active phytochemical components are found ubiquitously in the leaves of higher plants, however to date, no other source other than Ginkgo biloba is known to contain certain specific flavonoids and the unique terpene trilactones.

As described earlier, the Ginkgo biloba leaf extract is a complex mixture, however some of the mechanisms that underlie the pharmacological and therapeutic actions of the modern extract (EGb761) may be related to specific molecular constituents of the extract that have been shown to possess biological activity. However, prudence must be exercised even when such biological activities have been identified as properties of individual constituents of EGb761, since they may not necessarily contribute to the pharmacological or therapeutic effects that are observed following in vivo administrations of the total EGb761 preparation. Furthermore, the demonstration that a given constituents of EGb761 is biologically active in vitro does not provide sufficient evidence for therapeutic activity, since the bioavailability of the substance after oral or parenteral administration must also be established.

Page 25 of 201 Detailed examination of all the chemical constituents of Ginkgo biloba plant parts

presents an extremely difficult task due to the complexity of the extract. Hence,

quantification of the major compounds and possibly those with the most clinical

relevance, is presumed to be sufficient and adequate to explain and established the

significant pharmacological mechanisms of action in vivo and clinical effects of the total

EGb761 preparation. However it seems necessary to emphasize that the biological

activities of the various constituents of EGb761 cannot be determined by simply

examining their individual biological or pharmacological activities, as they might exist in

the total EGb761 extract. The actions of any one of these constituents observed in basic

pharmacological experiments could be greatly modified (intensified or reduced) by the presence of other constituents, or even by the drug vehicle that is employed in pharmacological testing. Detailed below are the major identified roles for Ginkgo’s efficacy.

No Identified roles for Ginkgo’s Efficacy Reference 1 Ginkgo helps to improve circulation to the brain, which enhances memory, mental 137 agility, alertness, and other brain functions, and eases some types of depression. 2 As a general circulatory herb, Ginkgo increase the supply of oxygen and heals damage 63 to the heart veins, and capillary system, as well as assisting general health by improving energy levels and by slowing down the aging process. 3 Helpful for the senses, especially hearing and eyesight, and treats a wide range of 77 connected disorders. Ginkgo also helps in the treatment of any balance or vertigo problems, such as Ménière’s disease. 4 As a radical scavenger, Ginkgo protects cell membranes in the brain and other brain 108, 153, tissues from damage, which helps to keep the body and mind young, alert and healthy. 234 5 Aids the work of neurotransmitters in the brain, which in turn helps memory agility 137 and enhancement. This is useful against dyslexia, memory loss, depression and senility. 6 Helps fight disorders of the central and peripheral nervous system, partly by protecting 63 verve coverings against some types of damage or wear and tear (often associated with stress). The coverings on the nerve ending are called “myelin sheaths”. The function of the nervous system is further helped by well-circulated blood. 7 Balances and regulates brain metabolism partly because of the increase flow of blood 153 and the utilisation of oxygen and glucose. As mentioned earlier, the mechanism is not yet fully understood, however the following are some suggested ways of how Gingko affects the body:

Increases oxygen and glucose levels throughout the body, together with additional blood flow, which optimises physical and mental activities in the body.

Page 26 of 201 Increase blood flow, which leads to positive, results in the vascular system. For instance, the consistency of the blood is altered favourably, making the blood thinner if it is too thick and sticky. Inappropriately sticky blood, clumping together unnecessarily, causes poor blood flow (platelet aggregation). This can results in painful muscular cramps (premenstrual and otherwise), clots, varicose veins, embolism, and thrombosis.

Helps to protect the actual makeup of the red blood cell membranes and capillaries, improving their efficiency.

Ginkgo has antibacterial, antifungal and antiviral qualities that are able to deal with a range of disorders, diseases and allergies, including asthma, hay fever, and Candida infections.

Enables the white blood cells to fight a whole range of minor and major infections. Helps reduce the immune system’s overreaction of which inflammation is a by- product, for example in arthritis, rheumatism, asthma, and Hepatitis B infection.

The leaf extract has been postulated to possess numerous therapeutic effects. The

most prominent ones, discussed in more detail later, are antioxidant properties (see

Section 2.1.3.2.1) and its ability to increase flow of blood through aging blood vessels,

particularly in the brain (see Section 2.1.3.2.2).

2.1.3.2.1 Antioxidants properties

Antioxidants have become an important consideration in health and disease.

Halliwell 1990 has suggested that in order to characterise a substance as an “antioxidant” it is necessary to assess its capacity to interact with a variety of substances capable of causing oxidative damage. This term, antioxidant, will be used throughout this thesis to refer to agents that act by inhibiting the generation of free radicals. It is widely accepted that highly reactive free radicals such as the superoxide anion radical (O2-·), hydroxyl radical (OH·) and hydrogen peroxide (H2O2) can have deleterious effect on various

tissues, which appear to be involved in acute tissue injury and in chronic disease states.

Page 27 of 201 A similar term to antioxidants is free radical scavengers. Free radical scavengers

are agents, which reduce free radical mediated deleterious effects by scavenging free

radicals that are already formed. Recent studies have also shown that EGb 761 meets all

of the criteria that are required for characterizing it as an antioxidant as well as a free

radical scavenger.

Ginkgo biloba demonstrates antioxidant effects against several species of free

radicals in vitro as reported below. Studies of such activity on the various flavonoids or terpenes provide clues to the mechanisms of antioxidant properties of Ginkgo biloba.

A. Superoxide-Scavenging Activity

Superoxide anion (O2-·) can be generated in several ways such as irradiation with gamma rays or with phenazine methosulfate-NADH in a food system. Ginkgo biloba is

known to be able to scavenge such a radical.

Ginkgo biloba extract scavenges O2-·in a dose–dependent manner (Gardes-

Albert et. al., 1993; Marcocci et. al, 1994a; Pincemail et. al, 1985). Flavonoids in

Ginkgo biloba, such as quercetin and rutin can act as scavengers of superoxide anion.

Therefore, the flavonoids in Ginkgo biloba may be responsible, in part, for its superoxide

anion scavenging activity. However, these studies suggest that extracts of Ginkgo biloba

leaves might scavenge the anion by a combination of mechanisms.

B. Hydroxyl Radical-Scavenging Activity

Hydroxyl radicals (OH·) are highly reactive and toxic active oxygen species. OH·

can be produced following irradiation with gamma rays or the Fenton reaction in a food

system but are also capable of scavenging by Ginkgo biloba

Page 28 of 201 EGb 761 scavenges OH· in a dose-dependent manner (Gardes-Albert et. al.,

1993, Marcocci et. al., 1994b). Flavonoids have been reported to scavenge hydroxyl

radicals (Husain et. al., 1987; Bors et. al., 1990). The scavenging activity of these flavonoids is correlated with the number of hydroxyl groups substituted in the aromatic

B-ring, indicating that myricetin and quercetin have strong scavenging properties.

Therefore, flavonoids may be responsible for the hydroxyl radical-scavenging activity of

EGb 761.

C. Inhibition of Lipid Peroxidation and Quenching of other Radicals

Lipid peroxide radicals can be produced by chemiluminescence from luminol in the presence of DOPC liposomes during reaction with an azo initiator 2,2’-azobis (2,4- dimethylvaleronitrile). It is reported that EGb 761 is capable of quenching such lipid peroxide radicals in a dose-dependent manner (Marcocci et. al, 1994b; Maitra et. al.,

1995).

EGb 761 contains flavonoids, which have been shown to inhibit lipid peroxidation (Bors et. al., 1990; Yuting et. al., 1990; Takahama, 1985; Das & Ray,

1988). Flavonoids can also chelate metal ions, e.g. iron, which prevents them from acting as a catalyst in the initiation of lipid peroxidation (Afans, et. al., 1989). It is these properties of flavonoids, alone or in combination that seem to contribute to the inhibition of lipid peroxidation by Ginkgo biloba extract.

EGb 761 also scavenges nitric oxide (Marcocci, et. al., 1994a). Nitric oxide induces the oxidation of oxyhemoglobin and the production of nitrate, which is an oxidized product of nitric oxide induced by sodium nitroprusside. Such activity is suppressed by incubation with EGb 761 in a concentration dependent manner.

Page 29 of 201 The compounds responsible for such an action have usually been associated with

flavonoids as they possesses a hydroxyl group in carbon position three, a double bond

between carbon positions two and three, a carbonyl group in carbon position four, and

polyhydroxylation of the A and B aromatic rings.

Recently in vivo and in vitro studies have demonstrated that the terpene constituents of EGb 761, especially ginkgolides, have potent antioxidant properties as more fully described in Section 3.1.3. (Pietri et. al., 1997; Yao, et. al., 1998; Yao et. al.,

1999). Terpenoids have also been shown to possess antioxidative properties in different situations (Baratta et. al., 1998; Grassmann, et. al., 2001; Grassmann et. al., 2000;

Choi et. al., 2000; Teissedre & Waterhouse, 2000), particularly against lipid peroxidation as a result of their high lipophilicity.

2.1.3.2.2 Effects on the brain

Many studies report that extracts of Ginkgo leaves enhance brain blood

circulation (Larsen, et. al., 1978; Rapin & Le Poncin-Lafitte, 1979; Le Poncin-Lafitte

et. al., 1980), have peripheral vasodilative and hypotensive effects (Auguet et. al.,

1982), increase the brain’s tolerance to hypoxia (Le Poncin-Lafitte et. al., 1982;

Schaffler & Rech, 1985), reduce venule thrombi formation (Borzeix et. al., 1980),

increase venular and capillary blood flow and reactivate capillaries that were formerly shut off from circulation (Piovella, 1973), protect retinal injury from free radicals

(Clairambault et. al., 1986), render the erythrocyte membrane more resistant to hemolysis (Etienne et. al., 1982) enable patients with peripheral arterial insufficiency to walk longer distances and improve their blood flow through the lower limbs (Bauer,

1984), and improve cerebral hemodynamics and ameliorate mental deficit (Gessner et. al., 1985).

Page 30 of 201 Analysis of the available scientific information, suggests four major “concepts of action” for the constituents of EGb 761, not all of which are as antioxidants (DeFeudis,

1998). These are:

Vasoregulatory action which protects blood vessels and various tissues and which is useful in explaining its uses in managing cerebral insufficient states, neurosensory disturbances and peripheral occlusive arterial disease;

Cognitive-enhancing action, which relates to its use in treating Alzheimer’s disease and various dementias;

“Stress-alleviating” action which is useful in explaining its “anxiolytic- like”/”antidepressant-like” effects;

Gene-regulating action which provides a basis for explaining why most of the clinically beneficial effects of the extracts require repeated administration.

There are reported recommended doses and ways of application for Ginkgo

(Davies, 1999; Pedersen, 2001), these authors suggest taking 40mg of Ginkgo extract three times a day before meals which will result in the expected remedy. Another suggestion is to make it into a tincture, which is useful in cases of dyslexia, by combining 2 parts of Ginkgo, leaves, 1 part of Gotu Kola root, and 1 part of prickly ash bark. The dosage recommendation is adults: 1 tsp (5ml) three times daily and for children: over 12 years, adult dose; 3-7 years, a quarter of the adult dose; younger than 3,

3 drops three to four times daily.

Ginkgo generally promotes good circulation but it also has a special property of aiding circulation in the brain by providing those particular blood vessels, arteries, veins, and capillaries with oxygen. This can help to ease short-term memory problems.

Ginkgo also increases the receptivity of neurotransmitters in the brain and enables them to transmit messages and impulses more efficiently (Davies, 1999). Gotu Kola root can assist Ginkgo by promoting mental calm and clarity. In India, Gotu Kola is revered as a prime tonic for the nervous system. In the West, this herb is often combined with

Page 31 of 201 Ginkgo because the Gotu Kola calms, clears, and organises the person who becomes

flustered when not remembering things (Davies, 1999). Prickly Ash Bark promotes capillary circulation over the entire body and its effects are felt almost instantly, not least in the brain where it can improve clarity and remove clouded thoughts and uncertainty.

With the combination of herbs, taken as a tincture, the tincture is postulated to be useful in cases of dyslexia (Davies, 1999).

2.1.3.3 Polyvalent mechanism of action of EGb 761

EGb 761 is a complex product since it contains several active chemical constituents and, as discussed earlier in Section 1.1.3.1, it is a combined action that brings about the therapeutic function. Additive, antagonistic and synergistic effects probably occur in pharmacological experiments as a result of interaction of the various active constituents with diverse sites in different organs and tissues. In most cases for plant extracts, their action depends upon a combination of constituents, a criterion that cannot possibly be satisfied by a single pure compound to achieve the same therapeutic

benefits. This is also most likely the case for EGb 761 since several of its constituents

are pharmacologically active and it is their combined activity, or polyvalent action that is

reported to be responsible for the therapeutic benefits that are achieved in treating

clinical disorders and illness (DeFeudis, 1998).

2.1.3.4 Adverse effects

Ginkgo biloba leaf extracts are reported to be relatively safe and remarkably free

of side effects when taken as directed at recommended doses (Kleijnen & Knipschild,

1992b; O’Reilly 1993; Newall et. al, 1996). Studies carried out to test its toxicology

Page 32 of 201 report rare occurrences of adverse effects in patients treated with Ginkgo biloba.

Adverse effects when reported are stomach complaints, nausea, headache, dizziness, heart palpitations, gastrointestinal problems and dyspepsia (Pittler & Ernst, 2000;

DeFeudis, 1991). Woerdenbag & De Smet, 2000 have presented a detailed report on the adverse effects of Ginkgo biloba.

Page 33 of 201 2.1.4 Ginkgo biloba nuts

The seeds of Ginkgo biloba are a commodity that is freely available and

affordable. They are usually categorised as “nuts” but their characteristics differ from

true nuts. Most nuts can be eaten directly without cooking but not Ginkgo “nuts”. The

point of purchase is also usually different as Ginkgo nuts are frequently seen in medicine

halls or medical product shops. Whilst many people believe Ginkgo nuts do bring about a health benefit, the mechanism of this benefit is not yet fully understood. In addition, the determination of the active constituents present in Ginkgo nuts is still in its infancy and few reliable assays exist to qualify or quantify them (Chu, 1999). Although such reports have been published over the Internet, it cannot be treated as an authoritative information source. The facts stated in such publications are not substantiated and may be misleading and totally fictitious.

The female trees of Ginkgo biloba are able to bear fruits that can be picked (while wearing latex gloves – in view of their hyper allergenic properties) and the nuts removed from the pungent smelling meat of the fruit. The nuts will not be viable seeds unless there is a male tree nearby. Ginkgo trees take about 20 –25 years of growth before they start to produce nuts. The flowers of the trees are rather inconspicuous and appear in

Spring. Subsequently the male trees produce cones and females the fruit.

Ginkgo biloba nuts are in season from middle to end of winter. They are usually harvested during the colder times of the year. They should not be refrigerated for more than a few days, or they will dry out and become hard whether shelled or not. However with freezing at –4 °C or below they can be stored and the nuts remain in a sound condition. The fruit is soft and contains one nut in the centre; however the fruit is known to cause a contact allergic response in susceptible individuals (Brill, 1994; Irie et. al.,

1996) – hence the need to wear gloves during harvesting.

Page 34 of 201 2.1.4.1 Nutritional composition

Ginkgo nuts are well recognised for their nutritional properties (Del Tredici,

1991; Foster & Chongxi, 1992). Ginkgo nuts make a good snack; they have a relatively high carbohydrate content and are relatively low in fat (USDA Nutrient Database for

Standard Reference, 1999) (See Table 8).

2.1.4.2 Popularity of consumption

Ginkgo biloba seed is a food commonly consumed in China and many parts of

the SEA region and usually in small quantities. The cooked nuts are softened by the

cooking process and lack the texture normally associated with crunchy nuts, due to their

high moisture content. The nuts are edible only after they are cooked. The shells are

thin enough to open with gentle pressure applied. Undercooked nuts are relatively hard

and slightly unpleasant in taste. Ginkgo nuts are frequently consumed and are used in a

variety of dishes.

They are more like a soft, dense savoury vegetable. They can be eaten in a soup,

as a stuffing or vegetable dish. Most Asians eat them in the form of a sweet dessert e.g. with yam paste or as “Cheng Teng” (i.e. boiled Ginkgo nuts with other Chinese ingredients in sugar water) or Ginkgo nuts with beancurd strands.

Antioxidants are increasingly being recognised as important health promoters in such conditions as cardiovascular problems, many forms of cancer and even aging. The antioxidants are able to reduce the effect of free radicals formed in the body either due to exposure to environment pollutants or because the bodies own defence mechanisms are reduced in dealing with the natural production of these compounds. Whilst an antioxidant property is suspected for the Ginkgo nuts, there is limited information on the

Page 35 of 201 base constituents. Apparently, no studies have been conducted on the effectiveness as

regards antioxidant capacity, of Ginkgo biloba nuts when consumed as a food product.

Therefore, in view of the popularity of the consumption of Ginkgo nuts in the

South East Asia region, it was considered of value to study their AOC. Apart from studying the AOC, this study also examines the nature and chemical characteristics of

the component(s) responsible for such an action.

2.1.4.3 Health related functions

Gingko nuts are claimed to have therapeutic functions to treat coughing with

sputum, cloudy urine and incontinence (Dai & Luo, 1996; Anon, 1997). In Chinese

folklore, the nuts are valued as promoting digestion and diminishing the effects of

excessive alcohol. They are also applied to treat polyuria, tuberculosis, leukorrhoea,

miction problems, pollakiuria and spermatorrhoea (Wada et. al., 1985; Wada et. al.,

1988; Tang & Eisenbrand, 1992; Bauer & Zschocke, 1996; Newall et. al., 1996).

Nowadays, Ginkgo nuts are consumed with the intention that they possesses

health benefits as they contain a range of phytochemicals, which are reported to possess

anti-cancer activity and treat neurological dysfunctions (Youdim & Joseph, 2001) and postulated to aid in other health promotion activities and disease prevention.

Ginkgo seeds are also used as a medicine for treating various conditions, including certain types of asthma, leukorrhoea, and polyuria (Anon., 1997).

2.1.4.4 Adverse effects

The central kernel of the Ginkgo seed is edible when cooked but may give rise to toxicity. The symptoms of poisoning by Ginkgo seeds are primarily convulsion and loss

Page 36 of 201 of consciousness or even death (Wada et. al., 1985; Wada et. al., 1988; Wada 2000;

Wada & Haga, 1997). Of most cases in Japan, infants, and in particular children under

six years of age, made up approximately 74% of the patients. Ginkgo seeds contain

certain toxic substances; collectively know as ginkgotoxins, which deter excessive

consumption (Arenz et. al., 1996; Scott et. al., 2000). Food poisoning from Ginkgo

seeds is reported to result from ingestion of 20 to 50 seeds (Wada & Haga, 1997). The

minimum ingestion of Ginkgo seeds to have caused food poisoning was six seeds

(Takano et. al., 1953). Normally consumption below six Ginkgo seeds would have

insufficient toxin to cause any detrimental effect. (Arenz et al, 1996; Scott et. al., 2000;

Wada & Haga, 1997, Wada, 2000).

Food poisoning due to over- consumption of Ginkgo nuts, especially in children,

is caused by the compound, 4-O-methylpyridoxine found in Ginkgo seeds. In the past,

some Japanese people have died from over-consumption of Ginkgo seeds. This

compound is an antagonist of vitamin B6. It occurs in Ginkgo leaves as well, but it is in

such low concentrations that it poses no toxicological threat (Arenz et. al., 1996).

It is proposed that this toxic principle causes food poisoning not only by antagonizing vitamin B6 in the body but also by inhibiting the formation of 4- aminobutyric acid from glutamate in the brain (Wada et. al., 1988).

Page 37 of 201 2.1.5 Leaf extract vs. nuts

In Europe and, to a lesser degree, in the United States, Ginkgo leaf extracts are

promoted in the health food market and/or drug stores as popular dietary supplements

(Eisenberg et. al, 1998; Kaye et. al, 2000). Ginkgo leaf extracts have been reported useful for their medicinal properties and as an aid in therapeutic functions. The active ingredients of the leaves are widely reported but there is limited information on the nutritional value of these plant parts i.e. the leaves and the nuts (DeFeudis, 1998;

Packer, 1999; Bradly et. al., 2000). It seems that the therapeutic effects of the nuts and leaves are different and they are used differently. The nuts are presented in the form of food and the leaves in the form of capsules, tablets or liquid herbal concentrates.

Consumers’ preference is usually for eating of the product rather than taking them in the form of a tablet, a method of ingestion usually associated with feeling unwell (Anon.,

2001).

There seem to be a controversy over the nutritional and therapeutic functions of

Ginkgo biloba leaves extract versus nuts. In addition Ginkgo nuts and Ginkgo leaf

extracts show great differences in their proven effectiveness and benefits through

medical diagnostics. Hence they cannot belong to the same category when it comes to

application in medical treatment.

Demand for nutraceuticals and functional foods are growing dramatically

(Hasler, 1996; Malaspina, 1996; Xu, 2001). Products which may be able to enhance

health, but which are not officially drugs, are now more appealing to consumers than

prescription drugs. However, consumers are not just interested in food as medication,

the organoleptic properties of a food are still important. Consumers would rather enjoy

the taste of food and a balance of health benefits and organoleptic properties must be

provided.

Page 38 of 201 Health promoting and “healing” foods have a long history in Eastern cultures, particularly in ancient China, where food and medicine are considered to be equally important in supporting health and preventing diseases. The saying that medicine and food come from the same origin and perform the same functions has been passed down to the present from an ancient legend, described by the herbalist, Shennong who was said

to have tested a hundred types of herbs (Zhang, 1990).

In addition, according to Dai & Lou, 1996 in traditional Chinese medicine and

pharmacology (TCMP), foods play as an important role as medicine in the treatment of

diseases; in many cases food is considered even more important than medicine. Food

offers great potential to prevent and moderate a host of chronic and genetically regulated

genetotrophic diseases, from heart diseases and cancer to Alzheimer’s and arthritis

(Camire et. al., 2001). Today the concept of foods having both preventive and

therapeutic effects is being increasingly recognised throughout the world. Hence the

impetus for this study is to further examine the possibility of introducing Ginkgo nuts as

a potential functional food.

2.1.5.1 Similarities, differences and future potential

EGb 761 has a strong potential as a therapeutic agent. A good deal of further

research and analysis needs to be carried out to elucidate the efficacy of this extract

before hoping to evolve a new Nutraceutical/Functional food to the consumer market. In

addition the main aim will be to focus on the area of functional food, hoping to

contribute to the consumer’s market in the form of food products rather than in the form

of a dietary supplement i.e. tablets or capsules.

A functional food is a product that can offer some function other than its basic

nutrition and it should improve health and well-being or reduce the risk of a disease

Page 39 of 201 condition. Some natural foods do exhibit functionality e.g. the presence of lycopene in tomatoes, which is an effective anti-cancer compound, at least for some forms of cancer.

These days functional foods may also be engineered and this can be by a number of ways

– its composition may be influenced by growing conditions or by genetic modification

(Golden Rice), it may have specific components added to it (addition of probiotics to promote gut health), it may have a component reduced (reduction of saturated fatty acids), it may have had one or more components chemically modified to improve a health benefit (hydrolysis of certain proteins to decrease allergenicity) or have the bio- availability of a particular component enhanced (bioactive peptides in milk to increase calcium bio-availability). The potential uses of Ginkgo in food products are wide and it might find usage in fortified food such as bread, flour, pasta, wheat, cereals, etc.

With more sophisticated methods of assessing nutrient deficiency and of analysing the components present in a food, it is increasingly possible to identify the missing or active ingredient affecting health. This means that the over emphasis on harmony and balance (perhaps a method of saying there is something good there but we are not sure what) has perhaps become less important, but this is not to say that balance is unimportant. Synergistic effects do occur between components. An opportunity to bring the rich knowledge of traditional Chinese folk medicine and the sophisticated methods of modern science and technology together to produce foods/medicines that can help many people stay healthy for longer is there waiting to be realised and underlies the work presented in this thesis.

Page 40 of 201 2.2 FLAVONOIDS AND PHENOLIC ACIDS

The flavonoids are polyphenolic compounds possessing 15 carbon atoms; two

benzene rings joined by a linear three-carbon chain.

The skeleton above can be represented as the C6 - C3 - C6 system.

Flavonoids constitute one of the most characteristic classes of compounds in higher plants. Many flavonoids are easily recognised as flower pigments in most angiosperm families (flowering plants). However, their occurrence is not restricted to flowers but includes all parts of the plant. Flavonoids are ubiquitous in nature and are categorized, according to chemical structure, into flavonols, flavones, flavanones, isoflavones, catechins, anthocyanidins and chalcones.

Page 41 of 201 2.2.1 Chemistry of flavonoids

The chemical structure of flavonoids are based on a C15 skeleton with a

CHROMANE ring bearing a second aromatic ring B in position 2, 3 or 4.

In a few cases, the six-membered heterocyclic ring C occurs in an isomeric open

form or is replaced by a five - membered ring. The oxygen bridge involving the central

carbon atom (C2) of the 3C - chain occurs in a rather limited number of cases, where the

resulting heterocyclic is of the FURAN type.

AURONE (2-benzyl-coumarone) Flavonoids are produced via the metabolic pathway illustrated in Fig 5. The

metabolic pathway in plants and microorganisms is via the shikimic acid pathway, by

which the aromatic amino acids (phenylalanine, tyrosine and trytophan) are formed from

phosphoenolpyruvate and erythrose 4 phosphate via shikimic acid. The aromatic amino

acids in turn serve as precursors for the formation of lignin and other phenolic

compounds in plants (Fig 6). Phenylalanine and tyrosine are converted to cinnamic acid

and p-coumaric acid respectively, by the enzyme PAL (phenylalanine (tyrosine)

ammonia-lyase). Aromatic hydroxylations, O-methylations, CoA ligations and NADPH-

dependent reductions are the four types of enzymatic reactions that occur throughout this

pathway. Phenylpropanoid metabolism may lead to several families of phenols, such as

Page 42 of 201 flavonoids, stilbenes, styrylpyrones, coumarins and the precursors for lignin and lignan

synthesis illustrated in Fig 6. A schematic diagram of the biosynthesis of flavonoids is

illustrated as shown in Fig 7.

Phospoenolpyruvate + Erythrose 4-phosphate

Shikimate

Chorismate

Alkaloids Anthranilate Arogenate

Phenylalanine and Tyrosine Alkaloids Trytophan

Alkaloids Flavonoids

Fig 5. Shikimic acid pathway

Page 43 of 201

Fig 6. Phenylpropanoid metabolism leading to flavonoids, stilbenes, styrylpyrones and coumarins as well as the precursors for lignin and lignan synthesis (Figure adapted from Haslam 1993)

Page 44 of 201 erythrose-4- Glucose phosphate

phosphoenolpyruvate shikimic acid

pyruvate aromatic amino acids

Phenylpropanoids acetyl-CoA (p-coumaroyl-SCoA)

malonyl-CoA Cinnamate

p-Coumarate

4-Coumaroyl-CoA

Naringenin chalcone

Naringenin

Favonoids (Flavones, Flavonols, Anthocyanins, Isoflavonoids, Flavan-3ols, Proanthocyanidins)

Fig 7. Biosynthesis pathways of flavonoids. (Figure adapted from Haslam 1993)

Page 45 of 201 Various subgroups of flavonoids are classified according to the substitution

patterns of ring C. Both the oxidation state of the heterocyclic ring and the position of

ring B are important in the classification. Examples of the 6 major subgroups are as

shown below:

1. Chalcones

2. Flavone (generally in herbaceous families, e.g. Labiatae, Umbelliferae, Compositae). E.g. i. Apigenin (Apium graveolens, Petroselinum crispum), ii. Luteolin (Equisetum arvense)

3. Flavonol (generally in woody angiosperms). E.g Quercitol (Ruta graveolens, Fagopyrum esculentum, Sambucus nigra), ii. Quercetin (Ginkgo biloba), iii. Kaempferol (Ginkgo biloba Sambucus nigra, Cassia senna, Equisetum arvense, Lamium album, Polygonum bistorta), iv. Myricetin.

Page 46 of 201 4. Flavanone

5. Anthocyanins

6. Isoflavonoids

Most of these (flavanones, flavones, flavonols, and anthocyanins) have ring B attached at position 2 of the heterocyclic ring except for isoflavonoids, where ring B occupies at position 3.

To date, no other source other than Ginkgo biloba is known to contain certain specific flavonoids. The biochemical activities of flavonoids and their metabolites depend on their chemical structure and the relative orientation of various moieties on the molecule (Cody 1988). There are over 40 flavonoids that have been identified in the leaves of Ginkgo biloba and these are show in Table 1 (Yoshitama, 1997). Of all the major groups of flavonoids, flavonols are the most prominent flavonoids found in

Ginkgo biloba (Hasler et. al., 1992; Vanhaelen &Vanlaelen-Fastre, 1988; Vanhaelen

&Vanlaelen-Fastre, 1989; Victoire et. al., 1988).

Page 47 of 201 Table 1. List of Flavonoids in the leaves of Ginkgo biloba (Table modified from Yoshitama, 1997)

No. Flavonoids Flavonoids Identified Ref. Class 1 Biflavones Amentoflavone Baker, W. et. al. 1961 2 Bilodetin Baker, W. et. al. 1961 3 Isoginkgetin Baker, W. et. al. 1961 4 Ginkgetin Baker, W. et. al. 1961 5 Sciadopitysin Miura, H. et. al. 1969 6 Flavonol Glycosides Kaempferol Hasler, A. et. al. 1992a 7 Kaempferol 3-O-glucoside Vanhaelen, M. et. al. 1988 8 Kaempferol 3-O-rhamnoside Hasler, A. et. al. 1992a 9 Kaempferol 3-O-rutinoside Victoire, C. et. al. 1988 10 Kaempferol 3-[2''-glucosyl]rhamnoside Hasler, A. et. al.1992b 11 Kaempferol 3-O-a-(6' ''-p-coumaroylglucosyl-b-1,4- Nasr, C. et. al. 1986 rhamnoside) 12 Kaempferol 3-[2''-6'''{p-(7'''-glucosyl)coumaroyl}- Hasler, A. et. al. 1992b glucosyl]rhamnoside 13 Kaempferol 3-O-[a-rhamnosyl-(1--2)-a-rhamnosyl(1-- Vanhaelen, M. et. al. 1989 6)]-b-glucoside 14 Kaempferol 3-O-a-(6''-p-coumaroylglucosyl)-(1--2)- Hasler, A. et. al. 1992a rhamnoside 15 Kaempferol 7-O--glucoside Victoire, C. et. al. 1988 16 Quercetin Hasler, A. et. al. 1992a 17 Quercetin 3-O-glucoside Victoire, C. et. al. 1988 18 Quercetin 3-O-rhamnoside Victoire, C. et. al. 1988 19 Quercetin 3-O-rutinoside Pietta et. al. 1991 20 Quercetin 3-[2''-glucosyl]rhamnoside Hasler, A. et. al. 1992b 21 Quercetin 3-O-[a-rhamnosyl-(1--2)-a-rhamnosyl(1-- Vanhaelen, M. et. al. 1989 6)]-b-glucoside 22 Quercetin 3-O-a-(6' ''-p-coumaroylglucosyl-b-1,4- Nasr, C. . et. al. 1987 rhamnoside) 23 Quercetin 3-[2''-6'''{p-(7'''-glucosyl)coumaroyl}- Hasler, A. et. al. 1992b glucosyl]rhamnoside 24 Quercetin 3-O-a-(6''-p-coumaroylglucosyl)-(1--2)- Hasler, A. et. al. 1992b rhamnoside-7-glucoside 25 Quercetin 3-O-a-(6''-p-coumaroylglucosyl)-(1--2)- Hasler, A. et. al. 1992a rhamnoside 26 Isorhamnetin Hasler, A. et. al. 1992a 27 Isorhamnetin 3-O--glucoside Hasler, A. et. al. 1992a 28 Isorhamnetin 3-O-rhamnoside Hasler, A. et. al. 1992a 29 Isorhamnetin 3-O-rutinoside Pietta et. al. 1991 30 Isorhamnetin 3-rhamnosyl-(1--2)-rhamnosyl(1--6)]- Hasler, A. et. al. 1992a glucoside 31 Myricetin Geiger, H. 1979 32 Meyricetin 3-O-rutinoside Hasler, A. et. al. 1992a 33 3'-O-Methymyricetin 3-O--rutinoside Geiger, H. 1979 34 Flavone Apigenin Hasler, A. et. al. 1992a 35 Apigenin 7-O-glucoside Hasler, A. et. al. 1992a 36 Luteolin Hasler, A. et. al. 1992a 37 Luteolin 3'-O-glucoside Hasler, A. et. al. 1992a 38 Flavan-3-ol & Catechin Schrall, R. et. al. 1977 Proanthocyanidin 39 Epicatechin Stafford, H.A. et. al. 1986 40 Gallocatechin Stafford, H.A. et. al. 1986 41 Epicgallocatechin Stafford, H.A. et. al. 1986 42 Procyanidin Schrall, R. et. al. 1977 43 Prodelphinidin Stafford, H.A. et. al. 1986 44 Epigallocatechin-catechin Stafford, H.A. et. al. 1986 45 Flavanonol Dihydromyricetin Stafford, H.A. et. al. 1986

Page 48 of 201 Flavonoid glycosides and biflavones are the major constituents of the flavonoids

found in Ginkgo biloba leaf extract. Flavonols are an extended conjugation made up of

the aglycone of the flavonol glycoside. Other major sources of flavonols in the human

diet are from fruits, vegetables, and beverages such as tea and red wine. Food sources,

dietary intakes, and bioavailability of flavonols are strongly influenced by variations in

plant type and growth, season, light, degree of ripeness, food preparation, and

processing. In the past few years, a number of studies on the absorption and metabolism

of flavonols in humans have been published and the findings from these studies are

reviewed by Hollman, et. al., 1995.

Flavones, flavan-3-ols, and proanthocyanidins are also found as minor

components. As the flavonoids content varies with season and storage conditions; the various biological functions of the extracts such as UV-protection, radical scavenging, antimicrobial activities, and insecticidal properties will also vary. (Ellnain-Wojtaszek &

Zgorka, 1999; Ellnain-Wojtaszek, et. al., 2002)).

Flavonoids represent a group of phytochemicals exhibiting a wide range of biological activities arising mainly from their antioxidant properties and ability to modulate several enzymes or cell receptors. Flavonoids have been recognized to exert anti-bacterial and anti-viral activity (Xu, et. al., 2000; Spedding, et. al., 1989), anti- inflammatory, anti-allergic effects, hepatoprotective, cytostatic, apoptotic, estrogenic and anti-estrogenic properties.

However, not all flavonoids and their actions are necessarily beneficial. Some flavonoids have mutagenic and/or prooxidant effects and can also interfere with essential biochemical pathways. Among the proteins that interact with flavonoids, cytochromes

P450 (CYPs), monooxygenases metabolizing xenobiotics (e.g. drugs, carcinogens) and endogenous substrates (e.g. steroids), play a prominent role (Walle, et. al., 2001; Van der Weide & Steijns, 1999). Flavonoid compounds influence these enzymes in several

Page 49 of 201 ways: flavonoids induce the expression of several CYPs and modulate (inhibit or stimulate) their metabolic activity. In addition, some CYPs participate in metabolism of flavonoids.

Flavonoids enhance activation of carcinogens and/or influence the metabolism of drugs via induction of specific CYPs. On the other hand, inhibition of CYPs involved in carcinogen activation and scavenging reactive species formed from carcinogens by CYP- mediated reactions can be beneficial properties of certain flavonoids.

In addition, flavonoids show an estrogenic or anti-estrogenic activity (Chen,

1998; Lee et. al., 1996) owing to the structural similarity with the estrogen skeleton.

Mimicking natural estrogens, they bind to estrogen receptors and modulate its activity.

They also block CYP19, a crucial enzyme involved in estrogen biosynthesis. Flavonoids in the human diet may reduce the risk of various cancers, especially hormone-dependent breast and prostate cancers, as well as preventing menopausal symptoms. For these reasons, it would be useful to study the structure–function relationship of flavonoids to provide the basis for a rational drug and/or chemopreventive agent designed for future pharmaceutical use.

Page 50 of 201 2.2.2 Flavonoids as antioxidants

Over 4,000 flavonoids have been identified, many of which occur in fruits,

vegetables and beverages (tea, coffee, beer, wine and fruit juices). The flavonoids have

aroused considerable interest recently because of their potential beneficial effects on

human health, they have been reported to have antiviral, anti-allergic, antiplatelet

aggregation, anti-inflammatory, antitumor and antioxidant activities.

Antioxidants are compounds that protect cells against the damaging effects of

reactive oxygen species, such as singlet oxygen, superoxide, peroxyl radicals, hydroxyl

radicals and peroxynitrite. An imbalance between antioxidants and reactive oxygen

species results in oxidative stress, leading to cellular damage (Hertog & Katan, 1998).

Oxidative stress has been linked to cancer, aging, atherosclerosis, ischemic injury, inflammation and neurodegenerative diseases (Parkinson's and Alzheimer's). Flavonoids may help provide protection against these diseases by contributing, along with antioxidant vitamins and enzymes, to the total antioxidant defence system of the human body. Epidemiological studies have shown that flavonoid intake is inversely related to mortality from coronary heart disease and to the incidence of heart attacks (Hertog &

Katan, 1998).

The traditionally recognized dietary antioxidants are vitamin C, vitamin E, selenium, and the carotenoids. However, recent studies have demonstrated that flavonoids found in fruits and vegetables are also potent antioxidants. Van Acker et. al.

2000 report that flavonoids can replace vitamin E as chain-breaking antioxidants in liver microsomal membranes. The contribution of flavonoids to the antioxidant defence system may be substantial considering that the total daily intake of flavonoids can range from 50 to 800 mg. This intake is high compared with the average daily intake of other dietary antioxidants like vitamin C (70 mg), vitamin E (7-10 mg) or carotenoids (2-3

Page 51 of 201 mg). Flavonoid intake depends upon the consumption of fruits, vegetables, and certain

beverages, such as red wine, tea, and beer. The high consumption of tea and wine may be most influential on total flavonoid intake in certain groups of people.

Antioxidant flavonoids - listed in order of decreasing potency

Quercetin (a flavonol in vegetables, herbs e.g. Ginkgo biloba, fruit skins, onions, etc.)

Xanthohumol (a prenylated chalcone in hops and beer)

Isoxanthohumol (a prenylated flavanone in hops and beer)

Genistein (an isoflavone in soy)

(Grassmann et. al., 2002)

Pro-oxidant flavonoids

Chalconaringenin (a non-prenylated chalcone in citrus fruits)

Naringenin (a non-prenylated flavanone in citrus fruits)

The capacity of flavonoids to act as antioxidant depends upon their molecular

structure. The position of hydroxyl groups and other features in the chemical structure of

flavonoids are important for their antioxidant and free radical scavenging activities.

Quercetin, the most abundant dietary flavonol, is a potent antioxidant because it has all the right structural features for free radical scavenging activity.

2.2.2.1 Mechanisms of action of antioxidants

It has been proposed that antioxidant mechanisms underlie some of the therapeutic effects of EGb 761, as a growing body of evidence points toward free radical

Page 52 of 201 and lipid peroxidation reactions as participants in peripheral and central vascular diseases

and neuronal damage (Maitra et. al., 1995; Oyama et. al., 1994). Oyama et. al., 1994 report that the flavonoid constituents of EGb 761 quercetin, myricetin, kaempferol, and rutin scavenge radical oxygen species. The authors suggest that these flavonoids, particularly quercetin and myricetin, are the beneficial constituents of EGb 761 in preventing free radical-induced neuronal damage. Potent peroxyl radical scavenging activity was noted in a 1995 in vitro study, along with reduced LDL oxidative modification (Maitra et. al., 1995).

Diabetics characteristically exhibit signs of oxidative stress in the retina, resulting in thickened basement membranes and altered retinal vessel permeability. In animal models, EGb 761 have been shown to improve retinal functioning by decreasing oxidative retinal stress (Doly et. al., 1992; Droy-Lefaix et. al., 1991)

Recent theories regarding the etiology of macular degeneration, the most common cause of blindness in the elderly population, and for which there are no adequate allopathic medical treatments, center on free radical damage to the retina (van der Hagen et. al., 1993; Snodderly, 1995). Studies of antioxidant status and macular degeneration note that elderly subjects with a high antioxidant index (ascorbic acid, alpha-tocopherol, and beta-carotene status) have a reduced risk for development of macular degeneration (West et. al., 1994; Seddon et. al., 1994). A preliminary double- blind study on the use of Ginkgo extract in macular degeneration showed a statistically significant improvement in long distance visual acuity with EGb 761 vs. placebo. EGb

761 antioxidant activity was reported to be responsible for this change (Lebuisson et. al.,

1986). Cataracts, another common cause of visual impairment, have been associated

with free radical damage in numerous studies. Flavonoids do not appear to have been

studied as a preventative or treatment for cataracts; however, oxygen radicals do seem to

play a role in the formation of cataracts, and subjects with lower antioxidant status have

Page 53 of 201 been shown to have a greater incidence of cataract formation (Gerster, 1989; Robertson

et. al., 1991; Mittag 1984).

Clastogenic factors (CFs) long-term, persistent superoxide ion-induced DNA

damage is found commonly in the plasma of people irradiated by accident or

therapeutically, and are thought to be responsible for chromosomal abnormalities many

years after exposure. CFs were found in a high percentage of Chernobyl salvage

workers. In a recent study, ten of these workers who exhibited a high amount of CFs

were given EGb 761 for two months (40 mg TID), and showed no signs of clastogenic

activity following the treatment period (Emerit, et. al. 1995).

Ginkgo extract has also been found to have a protective effect against lipid peroxidation in hepatic microsomes secondary to cyclosporin treatment. Cyclosporin A is an immunosuppressive drug with a small therapeutic window; i.e., the difference between therapeutic blood levels and toxic levels is quite small. It is lipid-soluble, and thus concentrates in fatty tissue, causing lipid peroxidation. Ginkgo was shown to inhibit this lipid peroxidation in a dose-dependent manner in an in vitro study (Barth et. al.,

1991).

Two principle mechanisms of action have been proposed for antioxidants (Ingold

1968). The first is a chain-breaking mechanism, by which the primary antioxidant donates an electron to the free radical present in the system (e.g., lipid radical). The second mechanism involves removal of Reactive Oxygen Species/Reactive Nitrogen

Species initiators (secondary antioxidants) by quenching chain-initiating catalysts.

Antioxidants possess the ability to donate electrons and thereby act as reducing agents, to chelate metal ions and thereby remove potential radical initiators and to facilitate antioxidant activity by other compounds (co-antioxidants). Antioxidants can also affect directly or indirectly the expression of genes in tissues. A number of genes

Page 54 of 201 are regulated by changes in the cellular redox status (Allen, & Tresini, 2000). Cellular

redox status is determined, among other things, by the type and concentration of

ROS/RNS present in the tissue as well as by the type and levels of antioxidants. ROS

such as H2O2 (Nose et. al., 1991; Crawford et. al., 1996), UV radiation (Devary et. al.,

1991), HOCl (Ramana et. al., 1998), singlet oxygen (Scharffetter-Kochanek et. al.,

1993) and also oxidized LDL (Suc et. al., 1998) were all studied for their ability to stimulate the expression of genes and protein activity, in order to identify redox-sensitive genes. Oxidative stress was shown to increase Type III and Type IV collagen mRNA, the transcription of collagenase and the expression of ferritin. Antioxidants thus, may modulate signal transduction pathways and gene expression through their reducing properties, rather than through their ability (under certain conditions) to generate ROS, or through their metabolic products. It was shown that only polyphenols with relatively high antioxidant potential were able to induce c-foc mRNA, while those lacking potent antioxidant properties were inactive (Choi, & Moore, 1993).

Ginkgo biloba extract, EGb 761, may be affected through modulation of

transcriptional processes (Gohil & Packer, 2002). Various techniques for large-scale

mRNA expression analysis, including differential display of mRNAs, cDNA arrays and

high-density oligonucleotide arrays have been used, to evaluate the actions of EGb 761

on the activities of the genomes in vitro and in vivo. The results show broad but cell

specific actions of Ginkgo biloba extract that may enhance antioxidant defenses in vitro

in cancer cells and modulate neuronal functions in the cortex and hippocampus in brain

in vivo. The large scale analysis of mRNAs in response to Ginkgo biloba extract in vitro

and in vivo show that the standardized extract affects the activities of the mammalian

genome. The data provide some support for the concept that the actions of EGb 761 are

mediated through it effects on the process of gene transcription which plays a causative

role in chronic diseases.

Page 55 of 201 Antioxidants, like other signaling molecules, induce specific activity, which

depends not only on the structure of the stimulus, but also on the target place; the

specific tissue or the cell type. When tissue interacts with an antioxidant, the signal

formed must then translate into a biological response, and as tissues differ in their composition, the biological effect or response also vary. Antioxidants can thus mediate activities of biological systems, directly or indirectly, through involvement in one of the multiple stages associated with signal transduction pathways.

2.2.2.2 Determination and measurement of antioxidation activity

The interpretation of results obtained from in vitro measurements of antioxidant activity of a compound or of a crude plant extract, must be dealt with caution as the antioxidative effect of a tested compound may vary considerably with the method and conditions used. Thus, selection of the appropriate assay to be used should be based on the intended application of the antioxidant.

Factors that influence the efficiency of an antioxidant in vivo are complex and require consideration of its bioavailability, the site of action, the type of ROS that the antioxidant must react with, its pharmacokinetic character, its stability, its toxicity and its possible synergistic behavior with other compounds. One must take all these limitations into account when extrapolating the inhibitory effect of an antioxidant in vitro to an in

vivo application.

Methods to examine antioxidant activity of a sample can be divided in principle

into two major categories: 1) Measuring its ability to donate an electron (or hydrogen

atom) to a specific ROS or to any electron acceptor. 2) Testing its ability to remove any

source of oxidative initiation, e.g., inhibition of enzymes, chelation of transition metal

ions and absorption of UV radiation. Free radicals could also be reduced by other

mechanism. Example: 1). Reducing the rate at the initiation of new radicals (Burton &

Page 56 of 201 Ingold, 1981). 2) Metal chelation by complexing with transition metal ions, thereby

hindering metal-catalysed initiation reactions and decomposition of lipid hydroperoxides

(Frankel, 1998). 3). Other antioxidant mechanisms include singlet oxygen quenching, oxygen scavenging, and blocking the prooxidant effects by binding certain proteins containing catalytic metal sites.

Antioxidant function in biological systems is much more complicated than a simple free radical scavenging process. An antioxidant may affect biological system by suppressing the formation of ROS and RNS, by affecting enzyme activities (Tasinato et. al., 1995), by inducing biosynthesis of other defense enzymes, and thereby affecting other endogenous antioxidants (Hensley et. al., 2000), by preserving NO activity

(Stewart-Lee et. al., 1994), or by sequestering transition metal ions. It is apparent that some antioxidants have more than one mechanism for their effect on biological systems.

In many methods of detecting antioxidant activity, the ability of an antioxidant to retard the oxidation of PUFA such as linoleic acid (C-18:2), exposed to oxidative stress, is determined by exposing the linoleic acid to heat, oxygen, light, or to free radical generators.

There are a variety of methods that can be used for the determination of lipid peroxidation and these include:

Page 57 of 201 A. Conjugated Diene assay (Esterbauer et. al., 1989)

This method allows dynamic quantification of conjugated dienes (CD) formed as

a result of initial PUFA oxidation by measuring UV absorbance at λ = 234 nm. The

principle of this assay is that, during linoleic acid oxidation, the double bonds are

converted into conjugated double bonds, which are characterized by a strong UV

absorption at 234 nm.

B. Lipid Peroxide Assay

When linoleic acid is oxidized, oxidation starts at its allylic position in a non-

specific reaction, to form an unstable mixture of lipid peroxides. The total amount of lipid peroxides can be detected iodometrically by using the method originally developed by El-Saadani et al 1989. The amount of lipid peroxides accumulated first reaches a

maximum, thereafter declining (decomposition phase) while forming aldehydes.

C. Linoleyl Hydroperoxide (L-OOH) and Linoleyl Hydroxide (L-OH) Assay

(Aviram, et. al. 2000)

During the initial stage of lipid peroxidation, hydroperoxides and hydroxides of esterified fatty acids are mainly formed, with minor amounts of free oxidized fatty acids.

The position of the hydroperoxide and hydroxide along the fatty acid chain depends on

the type of fatty acid and on the nature of the oxidative stress. Various approaches have

been suggested for the quantitative determination of L-OOH and of L-OH but the most

direct one is by HPLC. This method is specific and does not require any derivatization,

as does the more sensitive GC-MS methods which involves esterification, silylation and,

in some instances, also a reduction of the double bonds.

Page 58 of 201 D. The β-Carotene-Linoleic Acid System (Dapkevicius et. al., 1998)

A modification of the above assays is based on the determination of the coupled

oxidation of β-carotene and linoleic acid. This assay is simple, reproducible and time- efficient for a rapid evaluation of antioxidant properties.

E. Cyclic Voltametry (CV)

This technique, developed by Kohen et. al., 2000, measure the antioxidant activity of tissue or plasma (Chevion et. al., 1997) on the bases of the reducing properties of the tested sample, by measuring their oxidation potential (E1/2, between 0-

1 V).

F. Radical Stability Calculation by Quantum Mechanical Parameters

The 1,1-Diphenyl-2-Picryl-Hydrazyl (DPPH) (or the galvinoxyl) method and the

CV method analyze the net ability of an antioxidant to donate an electron without interfering with other possible properties of the antioxidant (which might contribute to the total antioxidant activity), such as its ability to chelate metal ions. Calculation of heat

formation differences (Hf) between radicals and their parent compound is performed

using the PM3 semi-empirical Hamiltonian for energy optimized species (Stewart,

1989). The Hf value represents the relative stability of a radical with respect to its parent

compound and it enables a comparison between the stability of radical achieved after

hydrogen abstraction from parent molecule (toward radical formation) rather than; from

alternative positions within an individual molecule, as well as between molecules (van

Acker et. al., 1996a; Vaya et. al., 2000).

Other protocols classified as free radical-trapping methods to measure the ability

of antioxidants to intercept free radicals are shown in Table 2.

Page 59 of 201

Table 2. Selected protocols used to evaluate free radical scavenging properties of antioxidants. Table adopted from Frankel and Meyer, 2000

26,32, 221-223 269

22,52, 224

174-1745 213 40,41

13

23

The methods examined and used in this work for the in vitro investigation on the

antioxidant activity of Ginkgo biloba specimens are Trolox equivalent antioxidant capacity (TEAC) assay, Oxygen radical absorbance capacity (ORAC) assay and Ferric- reducing antioxidant power (FRAP) assay. Comparisons and conclusions were drawn from these three methods. The individual assays are further discussed in detail below and justifications are also given as to why they were chosen for the study.

A. TEAC assay

In this assay the radical cation 2,2'-azinobis(3-ethylbenzothiazoline- 6- sulphonate) (ABTS•+) is produced continuously by reaction between the ferrylmyoglobin

radical generated from metmyoglobin, H2O2 and peroxidase, and the activity of

Page 60 of 201 antioxidants to scavenge the ABTS•+ radical cation is measured by the decrease in its

absorbance at 734nm (Miller et. al., 1993; Miller et. al., 1995; Rice-Evans, & Miller,

1994).

The Trolox equivalent antioxidant activity (TEAC) reflects the amount of Trolox

(mM) required producing the same activity as 1mM of the test compound. The reaction of ferrylmyoglobin in this assay may be confounded by the reduction of the ferrylmyoglobin radical by the antioxidants to be tested. Since ABTS•+ is a relatively

long-lived radical, the TEAC assay was modified by generating the ABTS•+ radical by direct oxidation of ABTS with potassium persulphate prior to reaction with test antioxidants (Re, et. al. 1999). Thus the high TEAC values obtained for quercetin and cyanidin in the original myoglobin/ABTS assay were significantly lower with the modified persulphate-activated ABTS•+ assay. This result is consistent with some

interaction occurring between these flavonoid antioxidants and ferrylmyoglobin in the

original assay (Re, et. al. 1999).

Despite recent improvements and increased use, the TEAC assay has several

limitations. The ability of an antioxidant to scavenge the artificial ABTS•+ radical may

not reflect the antioxidant activity due to other mechanisms effective in complex food

lipids or physiologically relevant substrates, including metal chelation, and effects of

antioxidant partitioning among phases of different polarities. Despite its limitation,

TEAC assay was chosen for this work because of the reproducibility of the results

obtained as well as to improve and modify the assay in such a way that the assay is able to reflect the antioxidant activity in a lipid food matrix. This is achieved by first freeze drying the food matrix, then extracting the lipids and working in a solvent where even the non-polar compounds or lipid compounds are soluble.

Page 61 of 201 B. ORAC assay

The oxygen radical absorbance capacity (ORAC) method of Cao et. al., 1993 is a modification of the procedure measuring the fluorescence during 2,2'-azobis(2- amidinopropane) dihydrochloride (AAPH)-induced oxidation of porphyridium cruentum

β-phycoerythrin (β-PE), a phycobiliprotein containing a red photoreceptor pigment

(Delange, & Glazer, 1989; Glazer, 1988; Glazer, 1990). In the ORAC assay the

`antioxidant capacity' is quantified by calculating the net protection area under the time recorded fluorescence decay curve of β-PE in the presence of an antioxidant or serum.

The decrease in fluorescence is not linear with time, and the shapes of fluorescence

decay curves differ in the presence of different antioxidants and with different

concentrations of the same antioxidant (Cao et. al., 1993; Prior, & Cao, 1999). The

calculation of the area under the curve apparently circumvents erroneous assumptions of

linearity and response similarities.

However, in a recent application of this method to the evaluation of antioxidants

in oat extracts, the inhibition of AAPH-induced oxidation of β-PE was evaluated from

the midpoint (30min) of the assay (Handelman, et. al. 1999). A principal drawback of

the ORAC method is that it is assumed that the oxidative deterioration and, in turn, the

antioxidative mechanism and protection of the fluorescent protein β-PE can mimic

critical biological substrates (Cao et. al., 1993; Delange, & Glazer, 1989; Glazer,

1990). In the ORAC assay the effect of oxidation of the photoreceptor portion of

phycoerythrin (34 covalently attached linear tetrapyrrole prosthetic groups) on

fluorescence measurements does not necessarily reflect the extent of antioxidant

protection afforded against oxidative damage of the protein itself. However, the major

limitation of the ORACβ-PE assay is the use of β-PE as the probe. First, β-PE produces

inconsistency from lot to lot, which results in variable reactivity to peroxyl radical (Cao

& Prior, 1999). Second, β-PE is not photostable, and after exposure to excitation light

Page 62 of 201 or certain time, it can be photobleached. Thirdly it was observed that β-PE interacts with

polyphenols due to the nonspecific protein binding.

Hence, an improved method of oxygen radical absorbance capacity (ORAC)

assay has been developed and validated using fluorescein (3’,6’dihydroxyspiro

[isobenzofuran-1[3H],9’[9H]-xanthen]-3-one) as the fluorescent probe (Ou et. al., 2001).

The later authors reported to have results demonstrating that FL is superior to β-

phycoerythrin.

ORAC assay was chosen because it forms a standard measurement unit for the

United States of America’s market for antioxidant products, where most of the products

are measured against this unit. However due to lack of time and resource constraint, the

method mentioned by Ou et. al., 2001 were not carried out.

C. FRAP assay

The ferric-reducing antioxidant power (FRAP) assay measures directly the ability

of antioxidants to reduce a ferric tripyridyltriazine complex (Fe3+-TPTZ) to the ferrous

complex (Fe2+-TPTZ) at low pH (i.e at pH 3.6) (Benzie, & Strain, 1996; Benzie, &

Strain, 1999). The resulting blue colour measured spectrophotometrically at 593nm is taken as linearly related to the total reducing capacity of electron-donating antioxidants.

The main disadvantage of this approach is that the measured reducing capacity does not necessarily reflect antioxidant activity. Since the method does not include an oxidisable substrate, no information is provided on the protective properties of antioxidants.

However FRAP assay was chosen for this research even though final confirmation of its application still requires further investigation. This method is also easy to carry out and is an increasingly used technique reported in the literature.

Page 63 of 201 Some of the other methods listed in Table 2 are further discussed below with

respect to their limitations of the individual assay and reason why it was not chosen for

this study.

D. Reaction with 1,1-Diphenyl-2-Picryl-Hydrazyl (DPPH) (Blois, 1958).

The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical was one of the earliest

synthetic radicals used to study structural effects on the activity of phenolic antioxidants

(Blois, 1958). This commercially available radical serves as the oxidising radical to be

reduced by the antioxidant (AH) and as the indicator for the reaction DPPH• + AH

→DPPH-H + A•. The disappearance of the DPPH radical absorption at 515nm by the

action of antioxidants is measured spectrophotometrically in a methanolic solution until the absorbance remains constant. Assay time may vary from 10 mins up to ~ 6h (Brand-

Williams et. al, 1995). The assay was employed to determine the `antiradical efficiency'

of polyphenolic compounds, (Brand-Williams et. al, 1995; Sanchez-Moreno et. al,

1998) of caffeic acid and related hydroxycinnamates (Moon &Terao, 1998; Chen &

Ho, 1997) and of different wines, grape juices and grape pomace extracts (Sanchez-

Moreno et. al, 1999a; Sanchez-Moreno et. al, 1999b; Larrauri et. al, 1998; Larrauri

et. al, 1999). The decay slope and the absorbance level reached by the remaining DPPH

radicals vary significantly with different types and concentrations of antioxidants. An

antioxidant index parameter was proposed to determine `antiradical efficiency' based on

the amount of antioxidant required for a 50% decrease in initial DPPH radical

concentration and the time required to reach the steady state DPPH radical concentration

(Sanchez-Moreno et. al, 1998).

This assay is limited and was not chosen because DPPH radicals interact with

other radicals (alkyl), and the time response curve to reach the steady state is not linear

Page 64 of 201 with different ratios of antioxidant/DPPH (Brand-Williams et. al, 1995; Sanchez-

Moreno et. al, 1998).

E. Total radical-trapping parameter assay (TRAP)

The total radical-trapping parameter (TRAP) assay was developed to measure

`total antioxidant capacity' of plasma or serum (Wayner et. al, 1985). This assay uses peroxyl radicals generated by 2,2'-azobis(2-amidinopropane) hydrochloride (ABAP) to oxidise plasma antioxidants, and the oxidation is monitored by oxygen absorption. The induction period is compared to that of Trolox used as a reference water-soluble antioxidant. This method was later modified by adding linoleic acid as an oxidisable lipid substrate, before oxidation with ABAP (Wayner et. al, 1986). The decomposition of ABAP showed the following decreasing order of consumption of plasma antioxidants: ascorbate>thiols>bilirubin>urate>vitamin E. The use of an artificial water-soluble azo compound as a radical generator such as ABAP does not provide a useful estimate of the important protective activities of metal chelators such as urate and of lipophilic antioxidants such as vitamin E. To overcome the problem of an unsteady electrode endpoint, several modifications were developed by using chemiluminescence methods.

These methods were reviewed by Alho & Leinonen, 1999 who also described protocols for plasma and LDL. These authors concluded that the measurement of plasma

TRAP might not be valid, since free radical production would have to be sufficiently extensive to disturb the steady state level of antioxidants and disrupt the compartmentalisation protection afforded by cell membranes, hence because of this reason, it was not chosen for this work.

Page 65 of 201 2.2.3 Structure-Antioxidant activity relationship

The interconnections between the individual flavonoids subgroups are shown in

Fig 8. Individual differences with each group result from the variation in number and arrangement of the hydroxyl groups as well as from the nature and extent of alkylation and/or glycosylation of these groups. The antioxidant activity of the flavonoids depends greatly on their chemical structure and the relative orientation of various moieties on the molecule, particularly the number and position of hydroxyl groups within the molecule

(Cody, 1988). The presence of hydroxyl group in position three (3-OH) of the C ring,

(for example, the flavonoid aglycones such as fisetin, (+)-catechin, quercetin, myricetin and morin) are potent inhibitors of lipoxygenase compared with those that lack a 3-OH substitution such as diosmetin, apigenin (flavones), hesperetin, and naringenin

(flavanones) (Ratty & Das, 1988; Mora et. al, 1990).

Fig 8. Interconnections of flavonoids subgroups. Diagram adopted from Rice-Evans et. al, 1996.

Page 66 of 201 2.3 TERPENOIDS FRACTION

To date, no other source other than Ginkgo biloba is known to contain the unique

terpene trilactones. As a measure of potency, researchers have established a common

standard and thus the terpenoids fraction comprises approximately 5-7% of the total

Ginkgo biloba leaf extracts.

2.3.1 Chemistry of Terpenoids

Terpenoids are a class of natural products and related compounds formally

derived from isoprene units. They contain oxygen in various functional groups. This

class is subdivided according to the number of carbon atoms in the same manner, as are

those seen in terpenes. The skeleton of terpenoids may differ from strict additivity of

isoprene units by the loss or shift of a fragment, generally a methyl group.

Terpenes are another group of hydrocarbons of biological origin having carbon skeletons formally derived from isoprene [CH2=C(CH3)CH=CH2] as well. This class is subdivided into the C5 hemiterpenes, C10 monoterpenes, C15 sesquiterpenes, C20 diterpenes, C25 sesterterpenes, C30 triterpenes, C40 tetraterpenes i.e. carotenoids, and

C5n polyterpenes.

Terpenoids are found in all living organisms. They are biosynthesised from building blocks of five-carbon atoms, the isoprene units. Terpenoids are involved in vital physiological processes. Plant terpenoids find many applications in daily life

(Table 3). Terpenoids have also attracted much interest from the scientific community, where several branches of industry and research institutes are involved with terpenoids investigations (Table 4). Other than these, terpenoids have also been useful in a range of industrial application (Table 5).

Page 67 of 201 Table 3. Some applications of some plant terpenoids.

Application Plant terpenoid Industrial raw material Rubber Drug Contraceptives Anti-cancer (taxol, vinblastine) Vaccins, adjuvent Flavour & Fragrance Essential Oils Hop bitter acids Pigment Alizarin Carotenoids Food additives Vitamin A, K, E Crop protection Feromones Azadirachtine

Table 4. Scientific interest in terpenoids

Discipline Interest Biology Plant & Animal Physiology Ecology, Crop Protection and Improvement Medicine Cardiovascular (cholesterol) Contraceptives, Endocrinology, Cancer Research Pharmacy Drug Development, Vaccins Biological activity Chemistry Fine Chemicals Biochemistry Biosynthesis, enzymology, genetic engineering

Table 5. Some economical aspects of terpenoids

Branch of Industry Aspect Agriculture Product (Vegetable, fruit, flowers) Taste, smell, colour, life-time crop protection Alternative or novel crops/products Chemistry Bulk and fine chemicals Alternative raw materials Protection and Improvement Pharmacy Fine Chemicals, drugs Food Product quality Flavours, fragrances, pigments

Page 68 of 201 2.3.2 Bioactivity

It is suggested that the terpene fraction of Ginkgo biloba extract is mainly

responsible for the purported neuroprotective properties, since it is the terpene fraction that contains the potent PAF (platelet activating factor) antagonist (Smith et. al., 1996).

Ginkgolide B, which is a terpene, may provide some of the neuroprotective and

antithrombotic properties attributed to Ginkgo (Beek et. al., 1998; Reuter, 1996; Koltai,

1991). Several studies have also reported that ginkgolide B has the ability to protect

against neuronal damage following trauma (Kornecki & Ehrlich, 1988). For example,

it decreases the build-up of free polyunsaturated fatty acids during ischemia in the mouse

(Birkle et. al., 1988) and it reduces free fatty acid levels and improves blood flow to the

gerbil brain following ischemia-induced injury (Panetta et. al., 1987).

To define and differentiate the role of components, a study was carried out using

CP205 (a terpene-free extract of Ginkgo biloba) (Gardès-Albert et. al., 1993). CP205 did not react with superoxide radical, regardless of the concentration tested (25-200 mg/ml), hence suggesting that the terpene constituents of EGb 761 are apparently responsible for the action in superoxide radical scavenging (DeFeudis, 1998), in contrast

to the normally accepted role of flavonoids. However the true situation is most likely a synergistic effect between the two classes of compounds.

Recently in vivo and in vitro studies have demonstrated that the terpene constituents of EGb 761, especially ginkgolides, have potent antioxidant properties as more fully described in section 3.1.3. (Pietri et. al., 1997; Yao et. al., 1998; Yao et. al.,

1999). Terpenoids have been shown to possess antioxidative properties in different situations (Baratta et. al., 1998; Grassmann et. al., 2001; Grassmann et. al., 2000;

Choi et. al., 2000; Teissedre & Waterhouse, 2000), particularly against lipid

peroxidation as a result of their high lipophilicity.

Page 69 of 201 2.4 POTENTIAL TOXIC COMPOUNDS

There are two classes of potentially toxic compounds associated with EGb 761.

Recent reports have shown the presence of Colchicine, an alkaloid that will cause adverse effects, especially to pregnant women, as it know to cause prevention of mitosis.

Another class of compound that has gained much attention are the alkylphenols, i.e. the

ginkgolic acids (GAs). This group of compounds have mostly been associated with

allergenic effects.

2.4.1 Alkaloids

Colchicine, (-) -N-5,6,7,9-tetrahydro-1,2,3,10-tetramethoxy-9-oxobenzo [a]

heptalen -7-yl (S) -acetamide (Fig 9), is an alkaloid occurring in plants of the Liliaceae

including Colchicum autumnale (Autumn crocus) and Gloriosa superba (glory lily).

Clinically, colchicine has been used for the treatment of several diseases including acute

gouty arthritis, familial Mediterranean fever, and inflammatory diseases such as

scleroderma (Ellenhorn & Barceloux, 1988; Naidus et. al., 1977). Recently, colchicine

has been demonstrated to be a potent inhibitor of cellular mitosis by interfering with

microtubule polymerization (Kirshner, 1978). Colchicine poisoning is rare and begins

with symptoms such as nausea, vomiting, and diarrhoea, followed by multisystem organ

failure. Doses of colchicine exceeding 0.8 mg/kg are usually fatal (Marray et. al., 1987;

Hobson & Rankin, 1986; Allender, 1982; Luciani, 1989; Clevenger et. al., 1991;

Sauder et. al., 1983; McIntyre et. al., 1994; Stapczynski & Rothstein, 1981; Folpini

& Furfoni, 1995)

Page 70 of 201

Fig 9. Chemical Structure and proposed fragmentation pattern of Colchicine Preparations of the leaves of Ginkgo biloba have been used widely for the treatment of cerebrovascular insufficiency and peripheral circulatory problems and for slowing down the progression of cognitive deficits in Alzheimer’s disease. Ginkgo leaf extract is among the top 10 most popular dietary supplements in the United States (Mar

& Bent, 1999). Recently, Petty et. al., 2001 reported detecting colchicine in commercial dietary supplements of Ginkgo (26 ± 3 µg/tablet) and echinacea (2.0 ± 0.5

µg/tablet) and in the placental blood of pregnant women taking Ginkgo supplements (49-

763 µg/L). However, this report has been criticized for not disclosing the commercial products that were tested and for not confirming the results of the HPLC-UV assay by using an independent method (Borman, 2001). Since Ginkgo leaf extracts are not known to biosynthesize colchicine or any of its congeners (Farnsworth, 2002), the paper by Petty raises the concern that colchicine contamination might be a general problem in commercial dietary supplements containing Ginkgo and Echinacea. To address this concern, Li et. al., 2002 developed a highly sensitive and selective analytical method based on high-performance liquid chromatography. On the basis of the results obtained by Li et. al., 2002, they found Ginkgo supplements to contain no colchicine, which is consistent with the botanical literature and hence there no cause for concern regarding colchicine contamination of Gingko or Echinacea dietary supplements.

Page 71 of 201 2.4.2 Alkylphenols

The best-known sources of phenolic lipids in plants are the members of families

of Anacardiaceae (Cashew), Ginkgoaceae (Ginkgo biloba) and Gramineae (cereals).

Historically, most of the analytical work on alkylphenols was carried out on

Anacardium occidentale, due to its commercial value (Verotta et. al., 2000). Thus, the term anacardic acid is often used. Ginkgolic acids (GAs) are restricted only to those originating from the Maidenhair tree (Jaggy & Koch, 1997).

The three different classes of alkylphenols are: (1) phenolic acids (anacardic acids) (2) dihydroxyphenols (bilobols, also known as cardols: 5-alky resorcinols) and (3) mono-hydroxyphenols (cardanols, also known as ginkgols) (Fig 10) (Verotta et. al.,

2000). The phenolic lipids are biogenetically derived from the polyacetate pathway, i.e. they are non-isoprenoic compounds (Fig 11). The biosynthesis of long chain phenols takes into account the condensation of an activated unsaturated or saturated fatty acid.

The similarity of alkyl chains in anacardic acids and cardanols, allows one to prostulate cardanols as decarboxylated derivatives of the precursor anacardic acids (Verotta et. al.,

2000).

Ginkgolic acids Ginkgols Cardols

R= C13H27 (C13:0) R= C13H27 (C13:0) R= C15H31 (C15:0)

R= C15H31 (C15:0) R= C15H31 (C15:0)

R= C15H29 (C15:1) R= C15H29 (C15:1)

R= C17H33 (C17:1) R= C17H33 (C17:1)

R= C17H31 (C17:2)

Fig 10. Structures of alkylphenols isolated from G. biloba leaves (Figure adopted from van Beek 2002)

Page 72 of 201

Fig 11. Synthesis of GAs via the polyacetate pathway (figure adopted from Tyman & Morris 1967).

GAs are 6-alkyl salicylic acids with saturated or unsaturated long chain alkyl groups. The alkyl side chain varies from 13 to 17 carbons in length with zero or two double bonds. The double bonds possess the Z configuration and the position is frequently 8 if one double bond is present. Hence, GAs are phenolic and hydrophobic molecules. As mentioned earlier, GAs are suspected to possess undesirable side effects like cytotoxic (Siegers, 1999), mutagenic (Westendorf & Regan, 2000) and slight neurotoxic properties (Ahlemeyer et. al., 2001).

On the other hand, antitumour activities have been reported for these compounds

(Itokawa et. al., 1987). Furthermore, there is no solid proof of a strong allergic reaction to these alkylphenols when taken orally. Nevertheless, these compounds are suspect and the larger manufacturers limit the total alkylphenol concentration in the final standardised extract to between 5 to 10 ppm.

Page 73 of 201 A. Distribution of GAs

GAs were widely distributed in the Ginkgo fruit, seeds and Ginkgo leaf extract powder (see Plate 2). The seed-buds of Ginkgo are shed in autumn, they have an integument whose exterior layer, the sarcotesta, has become a fleshy resin-like substance which when ripe exudes an unpleasant smell of butyric acid. Adherent to the stony layer is an interior thin layer of thin walled cells, endotesta. In the seed-buds it is soft and juicy, but it dries up later and can be identified as a paper-like brown skin in the ripe seed

(Melzheimer & Lichius, 2000). GAs found in the Ginkgo nuts fraction was first described by Peschier 1818. The reason for isolation and structure determination of

GAs was the occurrence of pronounced skin inflammation as a result of contact with the sarcotesta or the fruit pulp of Ginkgo nuts (Kawamura, 1928).

Endotesta

Plate 2. Ginkgo fruit, seeds and Ginkgo leaf extract powder

Studies by Gellermann & Schlenk, 1968 concluded that Ginkgo leaves consist mainly of GAs with unsaturated C15-side chain at position 8 and GAs with unsaturated

Page 74 of 201 C15-side chain at position 10, {6-[8'(Z)- and 6-[10'(Z)pentadecenyl salicylic acid]}

(52%), GA with a C17-side chain and a double bonding at position 12 {6-[12'(Z)- heptadecenyl] salicyclic acid} (40%) and trace amounts of saturated C13-side chain ( 6- tridecanyl salicyclic acid), (8%).

Similar results were obtained in a later study by Verotta & Peterlongo, 1993 using trimethylsilyl derivatives of GAs and GC-MS coupling. In addition, GAs with a saturated C15-side chain and a double unsaturated C17-side chain were found as secondary components. In recent studies by (Irie et. al., 1996) using NMR, MS and oxidative decomposition, simple unsaturated GA with a C15-side chain in position 8 was identified as the main component in Ginkgo leaves. Unsaturated bond at position 8 of the C17-side chain was identified as the second most frequent component in Ginkgo leaves. C13:0-side chain and C17:2-side chains were identified to be minor components

(see Table 6).

Table 6. Distribution of GAs in Ginkgo biloba Adopted from Irie et. al., 1996

Compounds Leaf Sarcotesta Nut C13:0 0.40 ± 0.08 1.90 ± 0.20 n.d.' ∆8 15:1 12.0 ± 0.09 31.0 ± 2.5 n.d.' ∆9 ∆12 17:2 0.50 ± 0.05 0.30 ± 0.05 0.02 ± 0.01 ∆8 17:1 4.40 ± 0.08 2.20 ± 0.60 n.d.' n.d.': not detected

B. Cytotoxic, allergenic and neurotoxic properties of GAs

To assess the cytotoxic potential of GAs, their effect on the human keratinocyte

cell line, HaCaT and the rhesus monkey kidney tubular epithelial cell line LLC-MK2 has

been investigated (Ghadially, 1988a;). Morphological examination by electron

microscropy revealed that GAs induce structural changes in both cell types. The most

prominent morphological alteration was the formation of myelinosomes in the cytoplasm

of HaCaT cells. It is thought that myelinosomes are formed when amphiphilic

Page 75 of 201 compounds bind to lipids in cellular membranes and other cellular compartments. This alters the physiochemical properties of lipids and hence inhibits lipid digestion by lysosomal enzymes. Another hypothesis suggests that amphiphilic agents may selectively inhibit lysosomal enzymes. In any case, the accumulation of lipids in lysosomes may finally cause cell death (Ghadially, 1988a;). In contrast, if renal tubal epithel cells, LLC-MK2 were incubated together with GA, transformation of

mitochondria to a longitudinal oriented type was observed which can be interpreted as

the result of uncoupled oxidative phosphorylation (Ghadially, 1988b).

The fruit or sarcotesta of Ginkgo biloba has been known from ancient times to

cause skin irritation. Saito, 1929 reported the poisonous properties of the fruit

describing erythema, oedema, papules and vesicles were accompanied by intense itching

but disappearing seven to ten days after contact. Bolis, 1939 described the development

of a stinging, burning rash two days after contact with the pulp. Sowers et. al., 1965

described an epidemic of contact dermatitis among girls in a preparatory school due to

contact with sarcotesta of Ginkgo biloba. They demonstrated cross reactivity between

the Ginkgo sarcotesta and poison ivy. GAs are structurally similar to allergens from the

poison ivy family.

(Ahlemeyer et. al., 2001) reported that GAs resulted in the deaths of cultured

chick embryonic neurons in a concentration dependent way; in the presence and in the

absence of serum. GAs-induced death showed features of apoptosis as chromatin

condensation, shrinkage of the nucleus and reduction of the damage by the protein

synthesis inhibitor cycloheximide were observed. These demonstrated an active type of

cell death.

However, DNA fragmentation detected by the terminal-transferase-mediated

ddUTP-digoxigenin nick-end labeling (TUNEL) assay and caspase-3 activation, were

not seen after treatment with 150 microM GAs in serum-free medium; a dose which

Page 76 of 201 increased the percentage of neurons with chromatin condensation and shrunken nuclei to

88% compared with 25% in serum-deprived, vehicle- treated controls. This suggests that

GA-induced death shows signs of apoptosis as well as of necrosis.

C. Antibacterial and antifeedant activities of GAs

Although, GAs do not possess significant clinical properties, it is still worthwhile to study this alkylphenolic class because recent studies indicated useful GA biochemical and pharmacological properties. These may lead to possible future exploitation and uses.

Such properties include molluscicidal (Kubo et. al., 1986), antifeedant

(Matsumoto & Sei, 1987; Fu-shun et. al., 1990), antitumor (Kubo et. al., 1993), antibiotic and antiacne (Kubo et. al., 1994) properties of anacardic acids, cardols and cardanols. The antibacterial activity of anacardic acids against Gram-positive bacteria has been studied by Kubo et. al., (1995). Their activities were also found to be dependent on the alkyl side chain.

Adawadkar & El Sohly, 1981 extracted and isolated constituents from the fruits of Ginkgo biloba, in order to determine the constituents responsible for the antibacterial activity. A mixture of GAs, bilobols and small amounts of cardanols were obtained. The

GAs represented the major components of the extracts (4.1% w/w of the fresh deseeded fruits) while the bilobols fraction constituted 0.53%, only trace amounts of cardanols were isolated. Chromatographic analysis showed GA contained C13H27, C15H29 and

C17H33 side chains, while the bilobols turned out to contain mainly C13H27 and C15H29 side chains.

Matsumoto & Sei, 1987 examined Ginkgo biloba, which is resistant to insect attack, in search for natural pest/insect control agents.

Page 77 of 201 The crude methanol extract obtained from G. biloba leaves was found to possess

antifeedant activity against the third-instar larvae of the cabbage butterfly (Pieris rapae

crucivora) by using a leaf disk bioassay.

In context to the adverse effects of GAs, some studies have shown their ability to

inhibit glycerol-3-phosphate dehydrogenase (GPDH), a key enzyme in the synthesis of

triacylglycerol and cholesterol (Irie et. al., 1996). The regulation of the amount of these lipids in biological tissues is important in the control of obesity, a negative factor in various diseases such as hyperglycemia and high blood pressure.

Page 78 of 201 3 AIMS & OBJECTIVES

3.1 MAIN FOCI OF THE RESEARCH

Proximate analysis values of Ginkgo biloba specimens and comparison with the USDA Nutrient Database for Standard Reference 1999 for the Ginkgo nuts and comparison between the various specimens (i.e. USA Ginkgo leaves, USA Ginkgo nuts, China Ginkgo nuts, commercial Ginkgo capsules).

Determination of the ascorbic acid and α-Tocopherol content in Ginkgo biloba specimens in order to establish their contribution to the antioxidant activity of the product.

Determination of the selected minerals present in the various Ginkgo specimens.

Establishment of the most suitable and effective binary solvent system for extracting the Ginkgo biloba specimens in terms of producing AOC.

Optimisation of the existing ABTS method (Arnao, 2000; Cano et. al., 2000; Re et. al., 1998) for AOC determination followed by its application to measure AOC for Ginkgo biloba specimens.

Assessment of the antioxidant capacity AOC using ABTS·* decolorisation assay of Ginkgo biloba nuts over cooking time.

Determination of the nature and most likely reason for the dramatic fall in AOC during the initial stages of the heating process.

Correlation and comparison of results from the studies on AOC between the raw nuts, cooked for 1 hr nuts, leaves extract, standardised leaf extracts and the commercial Ginkgo biloba capsules.

To prepare a range of infusion of Ginkgo leaves and to determine their AOC.

Make comparison from the Ginkgo biloba leaves with various other leaves used for infusion eg. : Green tea, Black tea, etc as regards their AOC level.

Establishment of a new or modified method to separate, isolate and identify the component(s)/constituent(s) responsible for the beneficial health effect from Ginkgo biloba.

Page 79 of 201 Investigation of the in vitro stability and recovery of flavonoids from Ginkgo biloba when subjected to a simulated digestion process.

Examination of toxic compounds in Ginkgo biloba that may pose adverse effects following consumption.

To utilize receptor studies for detecting testosterone-like compounds in Ginkgo biloba.

Page 80 of 201 3.2 HYPOTHESES PROPOSED

A. Ginkgo nuts are a potential functional food with desired organoleptic properties that can

contribute to the nutraceutical (Functional food) industry.

Functional foods have been increasing in popularity (Hasler, 1996; Malaspina, 1996; Xu, 2001) but to be successful consumers still require to enjoy the taste of food with a balance of health benefits. A balance of therapeutic function and tastiness of the oral intake has to be achieved and organoleptic properties therefore must be provided to achieve this. Hence dietary supplements may not seem to be a good alternative to obtain substantial and essential amounts of desired therapeutic benefits in hand with organoleptic properties. Since Ginkgo nuts are described as healthy nuts that are sold in medical halls, more attention should be paid to these products similar to that already directed to Ginkgo biloba leaves in order to consider then as a potential functional food.

B. Ginkgo nut are an effective and suitable functional food as regards their AOC.

Ginkgo biloba has been around for many years and leaves have received much attention over the last 3 decades and are widely available in over the counter dietary supplements. Whilst Ginkgo nuts have also been popular and frequently consumed as a food product, little research has been carried out to study the Ginkgo nuts. Hence in this research, Ginkgo nuts will be the test subject of interest and focus, in particular as to whether the nuts have similar beneficial effects to that of Ginkgo leaves. Possible roles for Ginkgo leaf extract in the treatment of disease that could involve free radicals and oxidative damage have been widely studied (Bridi et. al., 2001; Pitchumoni & Doraiswamy, 1998).

C. Ginkgo nuts have a range of free radical scavenging compounds present, which have a

therapeutic function.

The active ingredients / components responsible for such a therapeutic function i.e. scavenging of free radicals have been extensively studied in the leaves however not in the case of the nuts, therefore in this research Ginkgo nuts will be examined in order to determine their contributing components for the aforementioned function.

D. Ginkgo infusions are a potential functional food in view of their antioxidant activity.

Tea is a common beverage and drinking tea provides the additional function of scavenging free radicals. Ginkgo leaves could well be developing into a functional beverage similar to tea as regards their antioxidant activity.

Page 81 of 201 E. Absorption from the diet is a prerequisite for the potential beneficial role of flavonoids

The experimental work carried out was designed to mimic the human gut condition. HPLC analysis was then conducted to determine the resulting breakdown compounds and intact flavonoids, thus indicating those compounds likely to be available for absorption.

F. Absorption of glycosides from Ginkgo biloba is superior to their aglycones

It has been reported that flavonoids present in foods cannot be absorbed from the intestine because they are bound to sugars as glycosides (Kuhnau 1976). Only free flavonoids without a sugar molecule, the so-called aglycones, are considered to be able to pass through the gut wall, and no enzymes that can split these predominantly β- glycosidic bonds are secreted into the gut or present in the intestinal wall. However, in contrast, Hollman & Katan, 1999 suggest that, certainly for quercetin glycosides from onions, the absorption of the intact glycosides is in fact far better then the pure aglycones. These workers suggest that absorption kinetics and bioavailability of the flavonoids are probably governed by the form of glycoside present. Hence there is a need to better understand which form of flavonoids is likely to be more available for absorption.

G. HPLC is a suitable method for analysing the Ginkgo biloba compounds as well as an

appropriate method to separate glycosides and aglycones

Flavonoids are a large family of compounds existing as glycosides or aglycones. With this method established, the conversion of glycosides to aglycones could then be monitored in a single chromatogram.

H. Colchicine, an anti-inflammatory agent that is fatal at high dosages, might be present in

commercially available EGb 761, Ginkgo leaves extract, and probably also the nuts

Recently, colchicine has been demonstrated to be a potent inhibitor of cellular mitosis by interfering with microtubule polymerization (Kirshner, 1978). Since Ginkgo leaf extract are not known to biosynthesize colchicine or any of its congeners (Farnsworth, 2002), the paper by Petty et. al. 2001 raises the concern that colchicine contamination might be a general problem in commercial dietary supplements containing Ginkgo and Echinacea. In this research the aim is to detect presence of colchicine in the nuts fraction.

Page 82 of 201 I. Ginkgolic acids (GAs) the alkylphenolic compounds having side chains (C13:0), (C15:0),

(C15:1), (C17:1) or (C17:2), which exhibit strong allergy reactions, but also, antimicrobial and

antitumour activities might be present in the leaves and nuts of Ginkgo biloba.

A rapid, simple and accurate routine analysis method capable of detection and identification of GAs from Ginkgo leaves, nuts and commercial extracts needs to be established.

J. Testosterone-like compounds might be present in Ginkgo biloba, and possess wide potential

utility as pro- or anti-fertility agents, for hormone replacement therapy.

An extract of Ginkgo biloba that contains several antioxidative polyphenolics may have wide potential utility as pro- or anti-fertility agents, for hormone replacement therapy, etc. Hormonal receptor studies could be a method for detecting testosterone- like compounds in Ginkgo biloba and eventually lead to new evidence of its functionalities.

Page 83 of 201 4 MATERIALS & EQUIPMENT

The various methods used to carry out the analytical work for the research were

either used directly or modified from previously established methods. In this Chapter,

discussion will focus on two aspects: Firstly, the material used with respect to the test

specimens as well as the solvents and chemicals that were used for the various

experiments. Secondly, it will describe the equipment that were used to meet the

investigative requirements of the research.

4.1 MATERIALS

4.1.1 Test specimens

A B C

D E F

A. Ginkgo nuts from China - China Forestry I&E Co., Ltd. were the main raw materials B. USA leaves used for were imported from Ventura County, California, United States of America. C. USA NUTS used for were imported from Ventura County, California, United States of America. D. China leaves used for comparison studies are from Voigt Global Distribution, Kansas City, MO, United States of America. E. The commercial Ginkgo leaf standardised capsules studied were purchased from a local General Nutrition Center, Greenville, SC, United States of America. F. Standardised Ginkgo biloba leaves (EGb 761 compliance) were purchased from Voigt Global Distribution, Kansas City, MO, United States of America.

Page 84 of 201 4.1.2 Other test specimens for comparison studies

G. Soya bean oil was purchased from a local supermarket (Brand: NTUC) H. Commercial Green Tea was purchased from a local supermarket (Brand: Teo Lain Yew Enterprise). I. Commercial Black Ceylon Tea was purchased from a local supermarket (Brand: Lipton).

4.1.3 Solvents

Ethanol (GR grade, 99.9%), nitric acid (analytical grade), sulphuric acid

(analytical grade), hydrochloric acid (analytical grade), formic acid (analytical grade)

and acetic acid (analytical grade) were purchased from Merck (Darmstadt, Germany);

diethyl ether used for chromatography analysis was of HPLC grade, petroleum ether and acetone used for extraction (analytical grade), n-hexane and sodium borate used for capillary zone electrophoresis (CZE) as a buffer were purchased from J.T. Baker (NJ,

USA); 2-Propanol, tetrahydrofuran (THF), acetonitrile and methanol used for chromatography analysis were of HPLC grade and were purchased from Merck

(Darmstadt, Germany).

4.1.4 Chemicals

Used for Proximate analysis

The standard AOAC methods (Nielsen, 1998) were used to determine ash,

carbohydrate, fat, moisture content and protein and comparison studies were made to compare these nutritional values between the leaves, nuts and the commercial capsules.

See Nielsen 1998 for the detailed chemicals used in the analysis.

Page 85 of 201 Used for Antioxidant testing

ABTS, [2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)]; potassium persulphate were purchased from Sigma (Jefferson City, MO, USA); iron (II) sulfate heptahydrate, TPTZ and gallic acid (analytical grade) were purchased from Argos

Organics (NJ, USA); Iron (III) choloride hexahydrate, were purchased from Merck

(Darmstadt, Germany); from J.T. Baker (NJ, USA); Porphyridium cruentum β- phycoerythrin (β-PE) was obtained from Boehringer Mannhein (Germany). 2,2'- azobis(2-amidinopropane) dihydrochloride (AAPH) was obtained from Polyscience

(Warrington, PA). 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) was obtained from Aldrich Chemical Co. (Milwaukee, WI, USA).

Used for Vitamins analysis

α-tocopherol standard was purchased from Argos Organics (Trenton, NJ, USA); sodium hydroxide was purchased from Merck (Darmstadt, Germany); L-ascorbic acid were purchased from Sigma Chemical Co. (Jefferson City, MO, USA).

Used for simulated digestion process

Authentic standard samples of quercetin-3-O-rutinoside (rutin), quercetin-3- galactoside, kaempferol-3-rutinoside, quercetin-3-rhamnoside (quercetrin), kaempferol-

3-O-rutinoside, kaempferol-3-(p-Coumaryl) glycoside], were obtained from Apin chemical, (Abingdon, UK). Quercetin, kaempferol, and isorhamnetin standards were purchased from Adrich Chemical Co, (Jefferson City, MO, USA). Reference solutions were prepared in methanol (varying concentration from 100-500ppm). Pepsin (analytical grade), monobasic potassium phosphate (analytical grade), pancreatin mix (analytical grade), sodium hydroxide (analytical grade), sodium chloride (analytical grade) and

Page 86 of 201 hydrochloric acid (analytical grade) used for the simulation of the gastrointestinal fluids were purchased from Sigma Aldrich, (Singapore).

Used for qualitative and quantitative analysis of colchicine

Authentic standard of colchicine was purchase from Fluka Chemical Corp,

Milwaukee, (WI, USA). Potassium phosphate (monobasic) buffer (LC grade) and phosphoric acid (analytical grade) was purchase from Sigma Chemical Co. (Jefferson

City, MO, USA),

Used for qualitative analysis of GAs

Reagents used in GC-MS analysis include: Sylon BTZ (N,O- bis(trimethylsily)acetamide (analytical grade): methylchlorosilane (analytical grade): N- trimethylsilylimidazole (analytical grade), 3: 2: 3 v/v) were purchased from Sigma

Chemical Co. (Jefferson City, MO, USA), pyridine (analytical grade) and white powdered (U8, C15:1) ginkgolic acid (external standard) were purchased from Apin

Chemicals, (Oxon, UK).

Page 87 of 201 4.2 EQUIPMENT USED

General

Rotary Evaporator (BUCHI R-200 Evaporators, Switzerland), Oven with

Temperature control (Memmert Universal Ovens Model U, Wisconsin Oven

Corporation, USA) Homogeniser (Braun, Multiquick MR 400, Germany) and Silverson

Batch mixers, USA) UV-VIS spectrophotometer (Shimadzu UV 1601, Japan), LS-5 fluorescence spectrophotometer (Perkin-Elmer, Norwalk, CT), Incubator with temperature control (CERTOMAT® IS, Germany), thermometer, Vortex, Centrifuge machine (Eppendorf Centrifuge 5804 Refrigerated, Hamburg, Germany), Tyler Standard

Screen Scale Sieves; Magnetic Stirrer, and Hot plate with Temperature Control.

Proximate analysis

Kjeldahl apparatus for protein analysis, above mentioned oven for moisture analysis, Soxhlet Apparatus set up for lipid analysis, and Muffle furnace for ash analysis.

(Pearson, 1970; Neilson, 1998).

Elemental Analysis

Inductively Coupled Plasma Optical Emission Spectrometer (Thermo Jarrell Ash, model: Duo Axial plasma, Franklin, MA, USA), CHNS Auto-analyser (Perkin Elmer

Series II CHNS/O Analyzer 2400, USA),

High performance liquid chromatography Analysis

HPLC system (Shimadzu HPLC, Shimadzu, Kyoto, Japan) equipped with a photo-diode array detector, Column: Shim-Pack VP-ODS column (250x4.6 mm i.d.)

(Shimadzu, Kyoto, Japan)

Page 88 of 201 Waters 2996 HPLC system with automatic sample injection, photodiode-array detector set at 210nm and Empower software, Column: Waters (Milford, MA, U.S.A)

Symmetry® Shield RP18, 5.0 µm, 4.6 mm x 250 mm i.d. column

Capillary Zone Electrophoresis Analysis

Hewlett-Packard 3D CE system supplied by HP (Waldbronn, Germany) equipped with a 48.5 cm fused silica capillary column (with effective length of 40 cm for separation), i.d. 50 µm, from Polymicro Technologies (Phoenix, AZ, USA). The data were collected and analysed using software for instrument control and a HP Chemstation

(Edition A. 06.04) data processor

GC-MS Analysis

Shimadzu GCMS-QP5000 Gas Chromatography- Mass Spectrometer (Kyoto,

Japan) equipped with a SuperIncos Data System was used with a fused silica capillary column (30 m x 0.25 mm i.d., 0.25 um film thickness) connected directly to the ion source; Helium was used as the carrier gas.

Page 89 of 201 5 PROBLEM BASED APPROACH – ANALYTICAL CHOICES &

METHODOLOGIES

In view of the perceived functionality of Ginkgo biloba, this section will justify

its efficacies vs its practical application as a functional food. This work was carried out using a problem-based approach with the aims of investigating the potential for Ginkgo

biloba as a functional food.

The work progresses from the basic study of nutritional values of Ginkgo biloba,

through its measurement of antioxidant capacity (AOC), stability of its compounds subjected to a simulated digestion process, determination of toxic compounds to suggested new frontiers of hormonal function based on components in Ginkgo biloba

that may lead to new evidence of its functionality.

Page 90 of 201 5.1 PROBLEM BASED APPROACH

5.1.1 Problem 1 - Compositional analysis of Ginkgo biloba specimens

The first problem or task for this research was to carry out a proximate analysis of the test specimens, to determine protein, ash, moisture, fat, and carbohydrate by

difference. In addition to that, selected minerals were also analysed.

From the chemical composition of a food a better understanding can be exercised

in areas including nutrition and health, toxicology and safety, and stability to

microbiological, chemical or physical changes.

5.1.2 Problem 2 - Vitamin analysis - The natural antioxidant present

The second problem approached was the role of Vitamin C and E’s role as in vivo

antioxidants. By determining the ascorbic acid and α-Tocopherol content in Ginkgo

biloba specimens it is possible to ascertain antioxidant functions as regards to these

compounds.

5.1.3 Problem 3 - Effect of heat over time on AOC of Ginkgo biloba Nuts

The third problem investigated was the effect that heat has on Ginkgo nuts particularly as regards prolonged heating as that is the common method of treating the nuts prior to consumption in the SE Asian region. It was soon established that there was a dramatic fall in AOC during the initial stages of the heating and the reason for this was further investigated.

It was suspected that this initial loss was the result of the heat instability of ascorbic acid hence determination of ascorbic acid over the time frame was determined.

Page 91 of 201 5.1.4 Problem 4 - Effect of preparation procedure on AOC of Ginkgo biloba

leaf infusions

As discussed in Section 2.1.5, consumers’ preference is usually for eating or

drinking a product rather than taking it in the form of a tablet, hence the need for

developing Ginkgo leaf into a functional beverage in the form of a leaf infusion. The

AOC of various Ginkgo biloba leaf infusion was compared with various other infusions i.e. Green tea and Black tea. A sensory evaluation using preference test with the various

Ginkgo infusions was carried out to determine the acceptance of the Ginkgo leaf infusion

by consumers. Fermentation is a common practice for the tea leaf infusion; hence a

separation technique was needed to determine the difference in the leaf profile with

regards to fermentation vs. non-fermentation.

5.1.5 Problem 5 – In vitro, Simulated Digestion studies

The fifth problem addressed was that absorption from the diet is normally a

prerequisite for the potential in vivo beneficial role of flavonoids. In order to ascertain

the role of dietary flavonoids as antioxidants in vivo it is necessary to understand the chemical nature of the absorbed forms in the circulation in vivo and how the multiplicity of research findings in vitro reflect the bioactivity of flavonoids in vivo. Only when adequate information on the circulating forms is available is it possible to understand the targeting to the tissues, whether flavonoids cross the blood-brain barrier, for example, and in what form. Flavonoids are powerful antioxidants in vitro, but their overall

function in vivo has yet to be clarified, whether antioxidant, anti-inflammatory, enzyme

inhibitor, enzyme inducer, inhibitor of cell division, or some other role. In addition there

is also debate about the stability and absorption of polyphenols under gastrointestinal

conditions.

Page 92 of 201 5.1.6 Problem 6 –Toxicity consideration

The sixth problem was to investigate whether Ginkgo biloba specimens possess

any toxic compounds. Two compounds worthy of consideration are as follows:

5.1.6.1 Colchicine determination

Colchicine, which is an alkaloid, as previously described in section 2.4.1 has been suspected to be presence in commercial dietary supplements of Ginkgo (26 ± 3

µg/tablet) Petty et. al., 2001 and in the placental blood of pregnant women taking

Ginkgo supplements (49-763 µg/L). To address this concern, Li et. al. 2002 developed a highly sensitive and selective analytical method based on high-performance. On the basis of the results obtained by Li et. al, 2002, they found Ginkgo supplements to contain no colchicine, which is consistent with the botanical literature and hence there no cause for concern regarding colchicine contamination of Gingko. To confirm this fact as well as to add on to Li’s group work, an investigation is needed to confirm the presence of colchicine in other Ginkgo specimens i.e. Ginkgo nuts.

5.1.6.2 GA – An allergen

Ginkgolic acids (GAs) the alkylphenolic compounds having side chains (C13:0),

(C15:0), (C15:1), (C17:1) or (C17:2), from the leaves and fruits of Ginkgo biloba have been shown to possess toxic effects, exhibit strong allergy reactions, but also, antimicrobial and antitumour activities. In view of its allergic reactions, it is therefore important to specifically identify their presence so that eventually the percentage can be regulated especially in commercial phytopharmaceutical extracts of Ginkgo biloba leaves. Hence there is a need to set up a rapid, simple and accurate routine analysis method capable of detection and identification of GAs in Ginkgo leaves, nuts and commercial extracts.

Page 93 of 201 5.1.7 Problem 7 – Hormonal studies on the Ginkgo biloba specimens

Estrogenic activities have been shown in many herbal products. The eighth problem-based approach, which was deemed worthy of investigation, was the examination of potential estrogenic activity present in Ginkgo biloba. To date no mechanism has been confirmed to explain how Ginkgo biloba acts as a therapeutic agent in many diseases, e.g. Alzheimer’s disease. The establishment of estrogenic activity by

Ginkgo biloba may help to explain some of its therapeutic functions. Unfortunately it was not possible to study this aspect within the time frame of this research programme.

Page 94 of 201 5.2 METHODOLOGIES ADOPTED

5.2.1 Sample Treatment & Preparation

Nut sample preparation

The edible nut samples were shelled and skinned by flash blanching (placing in boiling water for 1 min and removing the skin by hand). Subsequently the nuts were ground in a Braun grinder to a paste, and freeze-dried. The samples were then vacuum packaged and stored at –20°C prior to further extraction steps or analysis.

Ginkgo leaves preparation

After the leaves were harvested, they were dried in an oven where the air temperature was specifically controlled at 60°C for 24 hours. The dried leaves were then sorted, to separate twigs, branches, stalks and any other foreign matter that is unnecessary and which might interfere with the final extract. They were pressed into bales, kept under a constant temperature at 25°C and humidity at 20% to avoid moisture uptake and fermentation and then freeze-dried to wait for further extraction steps or analysis.

Commercial Ginkgo capsules & Standardised extract preparation

Upon receipt of samples, they were vacuum packaged and stored in the freezer at

–20 °C to await further extraction steps or analysis.

Page 95 of 201 5.2.2 Proximate analysis

5.2.2.1 Lipids determination

Lipid was extracted using Soxhlet apparatus using petroleum spirit as solvent.

An extraction time of 4 hours was used in a continuous extraction apparatus of the

Soxhlet

5.2.2.2 Protein

The Kjeldahl method was used and a conversion factor of 6.25 was used to

convert N2 to protein. The formula for the calculation of protein is as below

3 -3 Let, T cm be the titration value of the H2SO4 concentration, M mol dm be the

H2SO4 concentration, m g be the mass of the foodstuff.

Protein in food = T x M x (atomic mass of nitrogen = 14)

m x 1000

5.2.2.3 Moisture

Moisture determination was carried out by the measurement of weight loss

following heating to 100°C for 24 hrs or until a constant weight was achieved.

5.2.2.4 Ash

5g of the sample was weighed into a silica dish, which has previously been

ignited and cooled before weighing. Then the dish and contents were ignited, first gently

and then at 500 °C (loss of chlorides due to volatilisation tends to occur above 500 °C).

Care was taken when transferring dishes containing fluffy ashes (eg, Ginkgo nuts), which tended to blow away easily. Such ashes are covered with a Petri dish after placing

in the desiccator prior to weighting.

Page 96 of 201 5.2.3 Vitamins analysis

5.2.3.1 Vitamin C

Determination of vitamin C was carried out on a simple isocratic HPLC system with UV detection. Interfering components were removed by means of efficient protein precipitation, and stabilizing vitamin C simultaneously. For accurate quantification a stable internal standard is used. Sample were homogenised and extracted with 2% (v/v)

acetic acid for 30 min. Prior to injection into the HPLC for vitamin C analysis, (20 uL),

the samples were filtered through a 0.45 µm membrane filter. An HPLC system

(Shimadzu HPLC, Shimadzu, Kyoto, Japan) equipped with a photo-diode array detector

was employed. The separation was performed on a Shim-Pack VP-ODS column

(250x4.6 mm i.d.) (Shimadzu, Kyoto, Japan) using 2% (v/v) acetic acid/acetonitrile at a

flow-rate of 0.8 ml/min at 40°C

5.2.3.2 Vitamin E

Vitamin E was determined by the method of Furter-Meyer (Association of

Vitamin Chemists, 1966). This method makes use of the oxidation of the tocopherols

by nitric acid to the red tocoquinone. The tocopherols are oxidised readily in the

presence of alkali but are stable in acid so the initial saponification is carried out with

1M alcoholic sulphuric acid. Absorbance is measured at 470 nm using UV/Visible – spectrophotometer (1601 Shimadzu Corporation, Japan) against a blank containing 5 ml absolute alcohol and 1 ml conc. nitric acid treated in a similar manner. The unsaponifiable matter was extracted with diethyl ether and samples were protected from light at all times.

Page 97 of 201 5.2.4 Fermentation techniques

Unfermented leaves

Approximately 50 g of Californian Ginkgo biloba dried leaves were ground using a Braun grinder for 5 s. The ground leaves were transferred into a clean beaker and were kept in an oven at a temperature of 25°C. The above steps were repeated to prepare

Ginkgo biloba leaves ground for 20 s. The unfermented Ginkgo biloba leaves were kept in the oven at 25°C and were put in a desiccator for least 24 hours prior to analysis.

Fermented leaves

Approximately 10 g of unfermented Ginkgo biloba dried leaves (ground for 5 s) were weighed into a beaker. Distilled water (1 g of leaves: 2 ml of water) was added into the beaker and they were mixed and bruised thoroughly. The bruised Ginkgo leaves were put in an oven at 25°C and were allowed to ferment for two hours. After two hours, the leaves were taken out and they were dried in an oven for 24 hours at a temperature of 80°C.

The above steps were repeated to prepare Ginkgo biloba leaves (ground for 5 s) fermented for 6, 10 and 24 hours. The same method was used to prepare all the fermented (2, 6, 10, 24 hours) Ginkgo biloba leaves ground for 20 s. All fermented

Ginkgo biloba leaves were kept in a suitable desiccator after drying them in the oven.

Page 98 of 201 5.2.5 Sensory evaluation

Satisfaction of consumer needs is important when it comes to product development. Proper understanding of these needs is dependent upon a clear communication with consumers through a common language (Moskowitz, 1988).

In this sensory evaluation, three infusion products were compared. They were:

a) Commercial green tea (Uji No Tsuyu Sen-Cha Green Tea)

b) Commercial black tea (Bon Tea, Pure Ceylon Tea-Gold Blend)

c) Ginkgo biloba leaves

No additional flavours were applied to the leaves and the individual was asked to perform the sensory analysis using the Sensory Evaluation Form shown at Appendix 4.

During the sensory evaluation, each of the individuals was asked to evaluate every sample based on five criteria:

a) Flavour

b) Aroma

c) Colour

d) Overall liking

e) Likelihood of buying the product if it was available in the market

Lastly, the participants were also asked to rank the three samples based on their preferences.

The samples were prepared in a similar condition so as to eliminate any errors due to irregular infusion time and amount of leaves used.

Page 99 of 201 5.2.6 Extraction techniques

5.2.6.1 Antioxidants - Flavonoids

Flavonoids were extracted using organic solvents. Different commercial companies have perfected their own extraction techniques, and some of these processes are closely guarded secrets. Nevertheless the essentials steps to extract out flavonoids from Ginkgo leaves are to pulverize and mix with organic solvents, which liberate the chemical components of the leaves. The process is repeated a number of times to ensure purity. The crude extract is then further refined to a point where the flavonoids make up a precise 24% concentration of the mixture. As previously described in Fig 1 in section

2.3.1, the extraction procedures were followed in this research.

For the various specimens, Ginkgo biloba leaves, Ginkgo biloba standardised leaf extract, Ginkgo nuts and commercial Ginkgo biloba capsules, an optimised extraction process was developed and this was established as 60:40 acetone/water under reflux for

30 min. Prior to injection into the HPLC for the compounds analysis, (20 µL), the samples were filtered through a 0.45 µm membrane filter.

5.2.6.2 Alkaloids

A stock solution of colchicine (0.333mg/mL) was prepared in methanol and diluted to obtain working standard solutions of 0.1-100 ng/mL. All working solutions were stored at -20 °C and brought to room temperature immediately prior to use.

Note that the colchicine standard solutions and the Ginkgo dietary supplement extracts, standardised extract, leaves and nuts were prepared in separate laboratories to minimize the risk of contamination of the dietary supplements. As another precaution, only new glassware was used for the preparation and storage of each sample.

Page 100 of 201 One or more capsules, tablets, or caplets representing a single dose of a ginkgo

dietary supplement (as indicated on the product label) was weighed and placed into a 20- mL PTFE capped sample vial. Each sample was extracted twice with 15 mL of 80% methanol using a sonicator for 60 min at room temperature followed by filtration through

Whatman no. 40 filter paper into a 250-mL round-bottom flask. While on the filter paper, the solid material was washed three times with 15 mL portions of 80% methanol.

The methanol extracts were combined and evaporated to dryness using a rotary evaporator at 45-50 °C. The residue was redissolved in methanol (4 X 2 mL), transferred to a 10- mL volumetric flask, and made to volume with methanol. Prior to injection into the HPLC for the colchicine analysis, (20 µL), the samples were filtered through a 0.45

µm membrane filter.

Standardised Ginkgo extract, leaves and nuts were carried out in a similar method as described above.

5.2.6.3 Alkylphenols

The reasons for carrying out an extraction procedure in this experiment were to separate Ginkgolic acids (GAs) from interfering matrix compounds like starch, and to concentrate the desired analyte to an appropriate concentration for detection.

In general, many small extractions are much more effective than a few large extractions. Phenolic compounds can be dissociated to phenolate ions or protonated to phenoxonium ions depending on acidity of solution and their chemical structure. If the pH is too high, phenolic analytes will exist in dissociated phenolate form, which may interact strongly with solid matrix resulting in strong absorption. Their hydrophilic characteristics will prevent their extraction to the organic layer. Strong matrix absorption also results if pH is too low. If pH is too low, phenolic analytes will exist as

Page 101 of 201 positive phenoxonium ions that are also strongly hydrophilic. Hence, the recoveries of phenolic analytes are dependent on the polarity of the extracting solvent (Li et. al.,

2003). Better recoveries were obtained in polar, neutral 60% acetone because the

“phenol” forms of the phenolic analytes are slightly polar.

In this research, sequential extractions involving solid – liquid, liquid – liquid and continuous liquid – liquid (i.e. Soxhlet extraction) extractions were performed. Solid – liquid extraction involved using a liquid (i.e. neutral 60% acetone) to dissolve the analytes that were part of the solid material but not covalently bound within it.

Solid – acetone extraction was followed by liquid – liquid extraction. Liquid – liquid extraction is often used to extract organic analytes from aqueous solutions. Two immiscible liquids, i.e. aqueous acetone and hexane were intimately mixed by vigorous shaking in a separating funnel. One layer (i.e. the denser aqueous fraction) was drained off until the interface was reached. The collected hexane fraction was smaller in volume thus the analytes (i.e. alkylphenols) were isolated and concentrated. Prior to injection into the HPLC for the GA analysis, (20 µL), the samples were filtered through a 0.45 µm membrane filter.

The solvent boiled from the bottom, the vapor rose through the outer bypass tube and condensed into a reservoir that held a paper thimble. When the solvent neared the top, it was siphoned through the inner, U-shaped tube back to the bottom. By distillation and condensation of the solvent, the Soxhlet apparatus allowed multiple extractions to be done repeatedly using the same volume of hexane solvent.

The net result of these sequential extractions was the removal of as many non- lipid interfering species as possible. If aqueous acetone – hexane solvent extraction was not carried out, most of the extraneous non-lipid substances like the polar pigments and organic acids would be left behind with the hydrophobic compounds after the polar

Page 102 of 201 solvents were removed by evaporation. Soxhlet extraction is one of the most popular techniques used because of its simplicity and relative inexpensiveness.

Both polar and nonpolar solvents could be (Shang et. al., 1999) and were used in this experiment. However, this required a large amount of extracting solvents and was time consuming. Furthermore, elevated extraction temperature in Soxhlet extraction might cause the transformation or degradation of the analytes, resulting in lowered recoveries and reproducibility.

With recent developments in extraction methods like pressurized liquid extraction, supercritical fluid extraction (SFE) and microwave-assisted extraction (MAE)

(Avila et. al., 1995) techniques, it is possible to reduce the extraction time and the amount of extracting solvent, but expensive special equipment is still required. Hence, the extraction process is a limiting step in the analysis of phenolic analytes.

Page 103 of 201 5.2.7 AOC determination

In this research the AOC determination was carried out using two techniques

1). UV-Visible Spectroscopy (Wilson, 1994)

Absorption spectroscopy is based on the interaction between a source of energy in the correct frequency region and a sample. A spectrometer analyzes the transmitted energy and compares it with the incident energy. The ultraviolet UV/ visible region of the spectrum normally includes the region from approximately 200 to 800 nm.

2). Fluorescence Spectroscopy.

Fluorescence is a standard analytical technique that can be used to measure the concentration of various analytes in many different matrices. When light is passed through a sample, the sample emits light (fluoresces) proportional to the concentration of the fluorescent molecule (in this case, the antioxidant compounds) in the sample. This technique is based on the measurement of fluorescence observed following 540 nm excitation of the organic solvent extracts of Ginkgo biloba specimens. Fig 12 shows a generic schematic of the fluorescence system.

Fig 12. Generic schematic of the fluorescence system.

Page 104 of 201 5.2.7.1 ORAC – Oxygen radical absorbance capacity

The final reaction mixture for the assay contained 1.67 X 10-8 M β-PE & 3 x 10-3

M AAPH in 7.5 X 10-2 M phosphate buffer, pH 7.0 (Li et. al., 2002). A final volume of

2 ml was used in 10 mm wide cuvettes. Into each sample tube, 20 µL of the extracted

Ginkgo specimens were added. The plant samples (2.0g) were extracted with 15ml

phosphate buffer (75 mM, pH 7.0) from 30 mins and were then centrifuged at 20000g

from 30 mins. They were first dissolved in acetone and then added to reaction mixture.

For the blank in this situation, 20 µL acetone instead of phosphate buffer was used.

AAPH was used as the peroxyl radical generator to start the reaction. Once AAPH was added, the reaction mixture was incubated at 37 °C. Fluorescence was measured every 5 min at the emission of 565 nm and excitation of 540 nm using a fluorescence spectrophotometer until zero fluorescence occurred. For a standard, 20 µL of a 100 µM

(1 µM in final concentration) Trolox stock solution was assayed during each run.

Quantification

The ORAC value refers to the net protection area under the quenching curve of

β-PE in the presence of an antioxidant. One ORAC unit has been assigned the net protection area (S) provided by 1 µM Trolox in final concentration. The ORAC value

(units) of a sample is calculated on the basis of a Trolox standard curve, Fig 13 illustrates the FL fluorescence decay curves in the presence of Trolox and AAPH. Because of the linear correlation found between ORAC value and Trolox concentration, the results can also be calculated in the following way (Fig. 14):

ORAC Value (U/ml) = 50k(Ssample - - Sblank)/(STrolox - Sblank)

S refers to the area under the quenching curve of β-PE. This area is integrated by

a computer connected directly to the output of the fluorescence spectrophotometer.

Page 105 of 201

Fig 13. Trolox concentration effect on FL fluorescence decay curve.

Fig 14. Calculation of ORAC values

5.2.7.2 FRAP – Ferric reducing antioxidant power

The Ferric Reducing Ability of Plasma Assay (FRAP) is a simple automated test used for determining the antioxidant activity of a sample. At low pH (~3.6), ferric ion is reduced to ferrous ion forming a compound known as ferrous-tripyridyltriazine complex

(Benzie & Strain, 1996). Absorbance changes at 593 nm are monitored for 90 minutes and the FRAP values are obtained by comparing absorbance change in the test reaction

Page 106 of 201 mixtures with those containing ferrous ions in known concentrations. The FRAP assay is inexpensive, the procedure is straightforward and the results are highly reproducible.

The antioxidant activity of all the infusions was determined using a modification of FRAP assay of (Langley-Evans 2000). FRAP reagent was prepared from 300

mmol/L pH 3.6 acetate buffer, 20 mmol/L ferric chloride and 10 mmol/L 2, 4, 6-

tripyridyl-s-triazine made up in 40 mmol/L hydrochloric acid. All three solutions were mixed together in the ratio of 25: 2.5: 2.5 (v: v: v). The FRAP assay was performed using reagents preheated to 37°C. 3 ml of reagents were transferred to the glass cuvettes with one of them containing 3 ml acetate buffer as blank. The initial absorbance of the reagents in the glass cuvettes at 593 nm was noted. 40 µL of the infusions were

transferred into the cuvettes containing the reagent and the mixtures were shaken thoroughly. The mixtures in the cuvettes were examined over 90 minutes using a UV-

VIS spectrophotometer (Shimadzu UV 1601, Japan) and the absorbance of the mixture after 90 minutes were recorded. Each sample of infusions was repeated three times and the FRAP equivalent of each infusion or sample was calculated and compared.

FRAP = CFS x V x (100/W)

Where, CFS is the concentration of Ferrous Sulphate standard solution (mg/ml) corresponds to the absorbance change, V is the volume of infusion (ml) and W is the weight of leaves used for infusion preparation (g).

5.2.7.3 ABTS cation decolorisation assay

The 2,2’-azinobis-(3-ethylbenzothiazoneline-6-sulfonic acid) assay (ABTS) is

based on the relative ability of antioxidant substances to scavenge the 2,2’-azinobis-(3-

ethylbenzothiazoneline-6-sulfonate) free radical cation (Re et. al., 1998). The preformed

radical monocation is generated by oxidation of ABTS with potassium persulphate and is

Page 107 of 201 reduced in the presence of such hydrogen donating antioxidants. The concentration of antioxidant and the duration of reaction on the inhibition of the radical cation determine the antioxidant activity of the test reaction mixtures.

As mentioned earlier, ABTS [2,2’-azinobis-(3-ethylbenzothiazoneline-6-sulfonic acid)] was used as the free radical provider and was generated by reacting this compound

(7.4 mmol/L) with potassium persulphate (2.45 mmol/L) overnight. The solution was diluted to obtain an absorbance of between 1.5-2.2 at 414 nm [molar extinction coefficient ε = 3.6 x 104 mol-1 cm-1, (Forni et. al., 1986)] with 98% ethanol before use.

3 ml of reagents were transferred to the glass cuvettes with one of them containing 3 ml ethanol as blank. The initial absorbance of the reagents in the glass cuvettes at 414 nm were recorded. 40 µL of the infusions were transferred into the cuvettes containing the reagent and the mixtures were shaken thoroughly. The mixtures in the cuvettes were run over 90 minutes using a UV-Vis spectrophotometer (Shimadzu UV 1601, Japan) and the absorbance of the mixture after 90 minutes were recorded. Each sample of infusions were repeated three times and the Ascorbic acid equivalent antioxidant capacity (AEAC) of each infusion or sample was calculated and compared. The AEAC expressed as mg

Ascorbic Acid (AA) per 100 g of homogenate was calculated using the following equation:

AEAC = CAA x V x (100/W)

Where, CAA is the concentration of Ascorbic acid standard solution (mg/mL) corresponds to the absorbance change, V is the volume of infusion (mL) and W is the weight of leaves used for infusion preparation (g).

Page 108 of 201 5.2.8 Statistical analysis

Two-tailed analysis of variance was performed on the data. Student’s t-LSD

(least significant difference) (P=0.05) was calculated to compare means for the different teas and also on the fermented and unfermented Ginkgo leaves infusion.

Page 109 of 201 5.2.9 Qualification and Quantification techniques

5.2.9.1 High Pressure Liquid Chromatography technique

5.2.9.1.1 Caffeine determination

An HPLC system (Shimadzu HPLC, Shimadzu, Kyoto, Japan) equipped with a

photo-diode array detector was used and detection of caffeine was carried out at 280 nm.

The separation was performed on a Shim-Pack VP-ODS column (250x4.6 mm i.d.)

(Shimadzu, Kyoto, Japan) using Mobile Phase, 40:60 (v/v) methanol: water at a flow-

rate of 1.0 mL/min at 40°C. The solvent was filtered through a 0.45 µm membrane filter

and degassed using an ultrasonic bath or by flushing with helium before use.

Sample were homogenised and extracted with 60% (v/v) methanol for 30 min.

Prior to injection into the HPLC for caffeine analysis, (20 µL), the samples were filtered

through a 0.45 µm membrane filter. Caffeine standard solutions were prepared in a

range from 20 µL of a 100 µM and a standard calibration curve is established for quantification of caffeine in Ginkgo biloba leaves. The limits of detection (LOD) were determined by fitting interday back-calculated standard deviations of each calibration standard. The LOD is defined as the lowest determinable quantity that indicates the presence of an analyte at a given statistical level of confidence (3 SD). The LOD was calculated statistically and was found to be 1.4 x 10 –4 mgmL-1.

5.2.9.1.2 Stability of Glycosides and Aglycones in simulated digestive system

An HPLC system (Shimadzu HPLC, Shimadzu, Kyoto, Japan) equipped with a

photo-diode array detector was used and detection of the flavonoids was carried out at

360nm. The separation was performed on a Shim-Pack VP-ODS column (250 x 4.6 mm

i.d.) (Shimadzu, Kyoto, Japan) using Mobile Phase A: water: 2-propanol (95:5), eluent

Page 110 of 201 B: 2-propanol: THF: water (40:10:50), binary gradient from 20% - 55.2% B in 44 min at a flow-rate of 1.0 mL/min at 40°C. The use 2-propanol-tetrahydrofuran-water or 2- propanol-water systems were confirmed to be of great value for the sharp resolution of flavonoids from medicinal plants. The solvent was filtered through a 0.45 µm membrane filter and degassed using an ultrasonic bath or by flushing with helium before use. The mobile phase conditions were modified from a method previously reported by Pietta, et. al., 1991. This method is capable of analyzing in one run, 8 flavonoids (5 glycosides and

3 aglycones) in the various samples during one HPLC analysis; thereby having the ability to monitor the conversion of glycosides to aglycones. Samples were injected in amounts of 20µl and the various compounds identified by their retention times in comparison with standards. The determination was carried out by means of the external standard method. Authentic samples as previously described in section 4.1.4. were prepared in methanol (varying concentration from 100-500ppm).

Identification and determination of the glycosides and aglycones

The identification of the HPLC chromatographic peaks was established with external standards. Each sample was injected three times for HPLC profiling. The linearity of the determination of the 5 flavonol glycosides and 3 flavonol aglycones was verified by linear regression. The correlation coefficients were 0.9446 for rutin, 0.999 for quercetin-3galactosides, 0.998 for kaempferol-3-rutinoside, 0.997 for quercetin-3-O- rhamnoside, 0.997 for quercetin, 0.998 for isorhamnetin and 0.998 for kaempferol.

Standards such as quercetin, isorhamnetin and kaempferol used for calibration are reported to have a very high stability with a loss of < 4 % within 12 months after storage at 8°C in a refrigerator (Hasler & Sticher, 1992).

Purity assay of the chromatographic peaks

The UV spectra of each peak, after subtraction of the corresponding UV base spectrum of the standards, was normalized by the computer and their plots

Page 111 of 201 superimposed. Peaks were considered to be similar when there was exact matching among the corresponding standards spectra and also by comparison of retention times in both standards and samples.

5.2.9.1.3 Colchicine determination

An HPLC system (Shimadzu HPLC, Shimadzu, Kyoto, Japan) equipped with a photo-diode array detector was used and detection of caffeine was carried out at 254 nm.

The separation was performed on a Shim-Pack VP-ODS column (250x4.6 mm i.d.)

(Shimadzu, Kyoto, Japan) using Mobile Phase, as followed:

Methanol – 0.05M KH2PO4 (55+45), adjust to pH 5.5 (±0.05) with 4-10 drops of

0.5M H3PO4 at a flow-rate of 0.8 mL/min at 35 °C. The solvent was filtered through a

0.45 µm membrane filter and degassed using an ultrasonic bath or by flushing with helium before use.

Colchicine standard solutions were prepared in a range from 20 µL of a 100 µM and a standard calibration curve is established for quantification of colchicine in Ginkgo biloba specimens (i.e. leaves, nuts, standardised extract and commercial capsules).

5.2.9.1.4 GA determination

Qualitative analysis was done by employing sensitive and selective analytical methods, based on reversed phase high pressure liquid chromatography (RP-HPLC-UV) for GA determination.

In this research, separation was carried out using a polar stationary phase, C18 column, packed with microparticulate octadecyl silca and increasing non-polar acetonitrile eluent strength. RP chromatographic technique was used in this

Page 112 of 201 investigation; it is reversed in the sense that the elution order was the opposite of normal

phase (adsorption) chromatography.

Photodiode array (PDA) detector used in analysis allowed access to the entire UV

spectrum. A variety of tasks such as peak purity (comparing UV spectra at various

points along the peak) and compound confirmation (adding spectral information to the

retention time) could then be performed to identify the presence of GA.

However, UV spectrum of GAs (Fig. 15.) had limited specificity in identification

of the compounds. External standard (ginkgolic acid, C15:1-side chain or syn. 6-

heptadecenylsalicylic acid, shown in Fig. 16.) and mass spectra were required to confirm the identification. PDA detector used in analysis allowed access to the entire UV spectrum. A typical UV spectrum of GA is shown in fig 15. A variety of tasks such as peak purity (comparing UV spectra at various points along the peak) and compound confirmation (adding spectral information to the retention time) could then be performed to identify the presence of GA.

Fig 15. Typical UV spectrum of Ginkgolic acids (He et. al., 2000).

CO 2H

HO (CH 2)7 Z (CH 2)5 Me

Fig 16. Structure of external standard (syn. 6-heptadecenylsalicylic acid).

Page 113 of 201 Approximately 15 g of G. biloba ground U.S.A. leaves were weighed and

extracted with 100 mL of 60% acetone (C3H6O) at 50 °C for 30 min. Two rounds of

filtration were then carried out. The residue was collected and taken up with another

100ml of 60% acetone for another round of extraction at 50 °C for 30 min. A third round

of extraction was carried out by repeating the above steps.

The above steps were repeated to prepare China leaf extract, dried nut extract,

capsule extract and EGb 761 extract. However, approximately 30 g of nuts, 10 g of

capsule powder and 10 g of EGb powder were used instead of 20 g.

The pooled filtrates were mixed with 50ml of n-hexane in a separating funnel.

Vigorous shaking was carried out and the mixture was left to stand until two distinct

layers were formed. The lower polar layer was drained off and another 50ml of n- hexane was added to the organic layer, followed by shaking and standing until two distinct layers were formed.

The pooled organic layers were then collected and evaporated to dryness under vacuum using Rotary Evaporator (vacuum drying) at 55 °C. The dried extracts were weighed and exhaustively purified with n-hexane in a liquid/ liquid continuous extractor

(Soxhlet extraction) for approximately 12 hours at 100 °C.

The pooled hexane extracts from Soxhlet extraction were evaporated to dryness under vacuum using Rotary Evaporator (vacuum drying) at 55 °C. The oily residues were then stored in the freezer. The dried hexane fractions each reconstituted with 10.0 mL of 0.01% formic acid in acetonitrile (B) for HPLC analysis.

Chromatographic analysis was carried out using Waters 2996 HPLC system with automatic sample injection, photodiode-array detector set at 210 nm and Empower software. Samples (20 µL) were injected in triplicates for each extract onto a Waters

(Milford, MA, U.S.A) Symmetry® Shield RP18, 5.0 µm, 4.6 mm x 250 mm i.d. column.

Page 114 of 201 A step gradient of 0.1% formic acid in acetonitrile solvent (A) and 0.1% formic acid in

water (B); according to the following profile: 0 – 30 min, 35 – 90% A, 65 – 10% B; 30 –

40 min, isocratic 90% A, 10% B; 41 – 60 min, isocratic 35% A, 65% B was used. The flow rate was 1.0 ml/min. Column temperature was set at 35°C. UV spectra were set in the range of 200-500 nm.

Page 115 of 201 5.2.9.2 Capillary Electrophoresis determination

5.2.9.2.1 Fermented leaves vs Unfermented leaves

All separations were performed using a Hewlett-Packard 3D CE system as described in Section 4.2. The analysis buffer was 50 mM sodium borate (pH9.3) and

10% acetonitrile in water. The temperature was kept constant at 20 °C; separation voltage was 25 kV and detection was at 230 nm - the wavelength giving the best resolution. All buffers were filtered using a Sigma (St. Louis, MO, USA) 0.2 µm filter.

To maintain the capillary conditions, fresh buffer was introduced into the capillary between each run. The data were collected and analysed using software for instrument control and a HP Chemstation (Edition A. 06.04) data processor.

Page 116 of 201 5.2.9.3 GCMS

5.2.9.3.1 GA determination

GC allows for highly efficient gas-phase separation of components in complex mixtures but is itself not capable of providing qualitative identifications for unknown samples. Mass spectrometry is a technique in which gaseous molecules are ionised, accelerated by an electric field and then separated according to their mass. Mass spectrum is the appearance of the fragmentation pattern at specified energies and times.

The various fragmentation pathways together compose a fragmentation pattern characteristic of the compound under investigation.

Although the GC-MS is a combination of two powerful analytical tools, it is limited to components with sufficient volatility and thermal stability due to it being a gas-phase separation technique. Hence, silylation and volatilization of Gingko samples

was performed prior to their separation. Although mass spectra of trimethylsilyl (TMS)

derivatives obtained by Verotta & Peterlongo 1993 showed poor fragmentations and

low intensity molecular ions, alkylphenols could still be identified as GAs, bilobols or

cardanols due to presence of strong ions at m/z 219, 268 and 180 respectively (Fig. 17.).

Fig 17. Major TMS derivatives of GAs, bilobols and cardanols (adapted from Verotta et. al., 2000).

Page 117 of 201 The above steps mentioned in section 5.2.9.1.3 were repeated to prepare dried hexane fractions for GC-MS analysis. Each fraction was derivatized and analysed by

GC-MS.

Each purified hexane extract (0.034 – 0.217 g) was treated with 4.0 ml of silylating reagent (BTZ: pyridine, 7:3 v/v) and heated for 30 min at 60 °C. An aliquot (2

µL) was taken for each analysis. Each extract was injected three times. A Shimadzu

GCMS-QP5000 Gas Chromatography- Mass Spectrometer (Kyoto, Japan) equipped with a SuperIncos Data System was used with a fused silica capillary column (30 m x 0.25 mm i.d., 0.25 um film thickness) connected directly to the ion source. Helium was employed as the carrier gas at a pressure of 50 kPa; column programme was 80 °C for 5 min, then 10 °C/min to 300 °C followed by 5 min at 300 °C. Injections (2 µL) were carried out at 300°C in the splitter mode (split ratio 1:60). The electron impact (EI) mode was used as 70 eV. The source temperature was 100 °C and the interface temperature was 300 °C; the electron multiplier operated at 1800 V. Mass spectra were recorded by scanning from 40 to 700 mass units with a total cycle time of 1.0 s.

Page 118 of 201 5.2.10 Digestive system simulation studies

5.2.10.1 Gastric fluids

Simulated gastric fluid was prepared according to the procedure of the USP,

National Formulary: 2.0 g NaCl, 3.2 g pepsin and 3.0 mL concentrated HCL were

diluted to 1 L and adjusted to pH between 1.2-1.8. (U. S. Pharmacopoeia, 1995)

5.2.10.2 Intestinal fluids

Simulated intestinal fluid was prepared according to the procedure of the USP,

National Formulary: 6.8 g monobasic potassium phosphate, 650 mL water, 190 mL of

0.2 mol/L NaOH and pancreatin mix (10 g). (U. S. Pharmacopoeia, 1995)

In the experiment the simulated gastric juice, was made up as above and

incubation conditions with temperature control and vortex action were applied to mimic

the stomach conditions. The temperature was set at 37 °C and the churning action of the stomach was mimicked by vortexing the chyme at 100 r.p.m. In vivo, after residing in

the stomach for 1-2 hours, the chyme then proceeds on to the small intestine and for this

experiment intestinal fluids were made up to mimic the pancreatic fluids and allowed to

react for 2-4 hours in a vortexing bath at 37 °C corresponding to the average and

maximal transit time in the small intestine respectively.

1 g of Ginkgo biloba leaves (carried out in triplicate) was incubated with 10ml of

simulated gastric juice at 37 °C for 1 h (designated step 1). In step 2, the earlier amounts

from the simulated gastric juice (chyme) were then incubated with 10ml intestinal fluid

at 37 °C for 2 h and 4 h respectively. These steps were carried out so as to mimic the

digestion process as discussed earlier. The incubated samples from both steps 1 and 2

were then filtered and made up to 25ml with water. A duplicate of the supernatant from

both steps was shaken with methanol to extract the alcoholic soluble compounds. From

Page 119 of 201 this, two samples were obtained i.e an aqueous acidic extract and an alcoholic acid extract. Both solvent systems were used in order to track possible changes in the flavonoids as aglycones are more likely to be found in the alcohol medium whilst the glycosides are more likely to be associated with the aqueous medium.

Similar steps as detailed above were carried out on the commercials capsules but using 2 capsules in 25 mL and for EGb 761, using 0.25 g in 25 mL. This was deemed a realistic ratio of what might occur in vivo. This allowed comparison with the undigested samples. This experiment yields the range of samples shown in Table 7.

Table 7. Experimental samples obtained from various Ginkgo biloba extracts Type of Samples and treatment* Description Sample Step 1 Step 2 Code

Samples in aqueous extract - Control Blank aqueous left overnight Samples in alcohol extract - Control Blank alcohol left overnight Samples incubated with - To mimic food residing in the A aqueous gastric juice 1 h in aqueous stomach condition for 1 h extract Samples incubated with - To mimic food residing in the B alcohol gastric juice 1 h in alcohol stomach condition for 1 h extract Samples incubated with Followed by To mimic food residing in the C aqueous gastric juice 1 h incubation in stomach for 1 h then passing intestinal fluid for 2 through the intestinal fluids for h in aqueous extract 2 h Samples incubated with Followed by To mimic food residing in the D alcohol gastric juice 1 h incubation in stomach for 1 h then passing intestinal fluid for 2 through the intestinal fluids for h in alcohol extract 2 h Samples incubated with Followed by To mimic food residing in the E aqueous gastric juice 1 h incubation in stomach for 1 h then passing intestinal fluid for 4 through the intestinal fluids for h in aqueous extract 4 h Samples incubated with Followed by To mimic food residing in the F alcohol gastric juice 1 h incubation in stomach for 1 h then passing intestinal fluid for 4 through the intestinal fluids for h in alcohol extract 4 h

* Samples were prepared and analysed in triplicate

Page 120 of 201 6 RESULTS AND DISCUSSIONS

6.1 COMPOSITIONAL ANALYSIS OF GINKGO BILOBA SPECIMENS

Table 8 shows the nutritional analysis of Ginkgo biloba samples. The samples as previously described in section 4.1.1 underwent the various analyses techniques to obtain the results in Table 8. The analyses were carried out with the intention of providing a

better understanding on the base nutrient constituents in Ginkgo samples.

Table 8. Comparison of Nutritional values from Ginkgo biloba samples NUTRITIONAL ANALYSIS OF GINGKO BILOBA SAMPLES

a LOCAL NUTS FROM COMMERCIAL PROXIMATES (g/100g) USA LEAVES USA NUTS CHINA SOURCE CAPSULES Protein 1.11±0.79 0.72±0.03 0.74±0.37 1.01±0.91 Ash 19.07±0.02 3.72±0.12 4.4±0.14 1.17±0.08 Moisture 12.3±0.06 49.1±0.01 55.6±0.03 4.5±0.02 Fat 11.44±0.05 3.43±0.18 2.91±0.32 3.39±0.35 Carbohydrate (by difference) 56.08 43.03 36.35 89.93 b LOCAL NUTS FROM COMMERCIAL VITAMINS (mg/g) USA LEAVES USA NUTS CHINA SOURCE CAPSULES Vitamin C 1.36±0.06 0.38±0.03 0.90±0.02 0.50±0.04 Vitamin E 22.19±0.06 1.21±0.04 0.19±0.12 1±0.6

c LOCAL NUTS FROM COMMERCIAL MINERALS (mg/100g) USA LEAVES USA NUTS CHINA SOURCE CAPSULES Zinc 0.67 2.19 1.20 0.52 Iron 4.29 1.37 1.20 0.76 Sodium 89.57 58.55 64.50 179.57 Magnesium 598.40 91.07 78.12 44.30 Potassium 1513.54 989.26 1015.56 37.39 Calcium 2630.75 39.92 41.76 62.44

C LOCAL NUTS FROM COMMERCIAL NON-MINERALS (mg/100g) USA LEAVES USA NUTS CHINA SOURCE CAPSULES Carbon 222.66 232.56 313.48 259.97 Nitrogen 10.74 5.21 45.17 9.29 (From Goh & Barlow, 2002)

a Mean of 6 determinations ± SD (standard deviation) dry weight basis. b Mean of 6 determinations ± SD (standard deviation) dry weight basis. c Calculated per 100 grams of edible portion dry weight basis. c Calculated per 100 grams of edible portion dry weight basis.

Page 121 of 201 From Table 8, it can be observed that the moisture content of both sources of nuts

are relatively high as compared with other types of nuts eg. walnut, peanut, almonds,

having 4 g/100 g, 6.5 g/100 g and 5.3 g/100 g of water content respectively. The latter

mentioned group of nuts have a higher content of oil and carbohydrate as compared to

moisture. Ginkgo nuts on the other hand are juicy and fleshy due to their high moisture

content.

The leaves seem to be better balanced nutritionally, being high in vitamin C and

E, suggesting a high contribution to antioxidant activity. Wheat germ oil and Canola oil,

which commonly claim to contained high amounts of vitamin E having 1.2 mg/g

(National Institutes of Health, 2001) and 0.2 mg/g (Paas & Pierce, 2002) respectively,

would not be as good a source of these vitamins compared with Ginkgo leaves. From

this finding, it is suggested that Ginkgo leaves might prove to be a good source of

vitamin E for future commercial extraction. In addition, the vitamin E content of Ginkgo

nuts from USA having 1.21 mg/g is perhaps as good as the wheat germ oil. Both Ginkgo

nuts and leaves from USA have considerable amounts of vitamin E, which deserves

more attention, and further work needs to be done to investigate possible extraction and

utilisation.

The composition of the nuts from USA and China was similar for the various

nutrients examined except for vitamins C and E (significantly different at P=0.05). This

could be related to geographical difference, seasonal variations or variety difference.

The USA nuts were less mature and this may be the reason for the differences. Maturity of the nuts could yield different nutrient content. In this case, the less mature nuts from

USA yielded a higher level of vitamin E, while the more mature nuts from China gave a

higher level of vitamin C.

Boths vitamins will contribute to the natural antioxidant capacity present, as they are normally referred to as in vivo antioxidants. However care must be taken as such

Page 122 of 201 vitamins are heat labile and easily oxidised. The content of both vitamins in

commercials capsules were shown to be considerably low as compared with the raw

material, i.e. the Ginkgo leaves. This may be due to the extensive and intense extraction

procedures to obtain these dietary supplement capsules, which causes the destruction of

these vitamins.

Ginkgo leaves also contain a high level of calcium and potassium. The calcium

content in Ginkgo leaves is circa 42 times more than that found in commercial capsules

and circa 65 times more than that found in the nuts. However such mineral levels would

have insignificant therapeutic function.

As regards to nitrogen content of the nuts, it was noted that the China source nuts

had a much higher nitrogen content. The most likely explanation for this is a high level of available nitrogen in the growing medium.

Page 123 of 201 6.2 EFFECT OF HEAT APPLICATION ON GINKGO NUTS WITH REGARDS TO AOC

The antioxidant capacity, AOC of Ginkgo biloba nuts with various cooking time are shown in Table 9.

Table 9. Ascorbic acid Antioxidant Equivalent Capacity for leachate and nut extract sample and summation of the two.

Wet cooking

time (min) @ 90°C

Nut extract Leachate Summation 0 515.5±3.2 ---- 515.5 5 416.9±1.2 43.4±0.4 481.3 10 208.5±0.8 105.2±1.0 340.7 15 200.2±0.5 113.5±1.0 347.6 20 151.6±0.5 113.7±0.5 290.3 25 148.3±1.0 138.5±0.6 308.8 30 139.7±0.18 139.1±0.9 297.8 60 115.9±1.1 137.0±1.0 287.9 90 97.7±0.3 175.1±0.4 303.8 120 76.0±0.7 248.8±0.1 352.8 150 43.6±0.6 274.7±1.0 344.3 180 34.6±0.7 287.2±1.1 349.8 210 34.6±0.1 303.3±0.2 362.9 240 34.6±0.1 319.4±0.8 377.0

The raw nuts contained 515.5 mg AEAC per 100 g of homogenate. After dry heat treatment (25 min at 140 °C), the AOC level decreased to 275 mg AEAC. During wet cooking of the nuts, a rapid fall in AOC of the nuts + leachate was observed after 5 min, levelling off to circa 350 mg AEAC per 100g of homogenate after 10 min. The results indicate migration of the antioxidant capacity from the nut extract to the leached aliquot, with the total AEAC level remaining similar during cooking. This accounts for a loss of 59% AOC in the nuts from the raw samples. Fig 18 shows the AOC over the test

a Mean of 6 determinations ± SD expressed as mg AEAC per 100g of sample

Page 124 of 201 period of time. The results were calculated based on the slope y = 3.7732χ (R2 = 0.9982)

from the calibration curve of ascorbic acid (See Fig 22).

Fig 18. Nuts and Leachate extract and summation of the both to express the total AOC in mg AEAC per 100 g homogenate vs cooking time

It is known that Ginkgo nuts do contain a substantial level of Vitamin C (circa 15

mg/100g is reported for the raw nuts by (USDA Nutrient database, 1999) and up to 39 mg/100g found in this study). Thus it was suspected that the initial loss could be attributed to the heat instability of this vitamin.

In order to confirm this, a further investigation is needed to understand if the antioxidant activity is contributed by vitamin C. A calibration graph of vitamin C was then prepared. The level of vitamin C was determined using the same slope of the calibration graph with y = 3.7732χ (R2 = 0.9982) in the raw nuts and in the nuts after 10

min of heating. The relationship of the absorbance drop to the vitamin C content can

thus be calculated. The vitamin C level in the raw nuts determined by HPLC was 39

mg/100g. This amount of vitamin C in 40 µl of reaction solution was calculated to be

Page 125 of 201 1.5x10-2 mg. The change in absorbance after 10 min cooking was 1.17, corresponding to a loss of 1.24x10-2 mg of vitamin C. The measured vitamin C in Ginkgo nuts after heating for 10 mins was 14.26 mg/100g, and calculated as 0.57x10-2 mg of vitamin C/40

µl aliquot. Therefore the calculated loss of vitamin C was 0.95±0.2 x10-2 mg. The calculated and the actual experimental loss in vitamin C were therefore relatively similar.

Further studies were undertaken to determine the change in AOC contributed by the heat sensitive component(s), assumed to be vitamin C and heat-stable compounds.

Vitamin C contents in the nuts and in the leachate were found to decrease progressively

(Fig 19). After 2 hours of cooking, vitamin C level was almost completely absent, and the AOC activity will be independent of vitamin C content.

600

500

400

300 AEAC (mg/100g) 200

100

0 0 5 10 15 20 25 30 60 90 120 150 180 210 240 Cooking Time (min)

Fig 19. Composition of heat stable and heat labile antioxidant in Ginkgo dessert during heating. g AEAC - non vitamin C; g AEAC – vitamin C in nuts and leachate

Page 126 of 201

The AOC level starts to increase gradually again after 60 min at a rate of 0.5406 mg AEAC per 100 g homogenate per min. This strongly suggests that additional antioxidant components are formed and/or activated in spite of the prolonged heating. In addition, it was observed that after 5 min of cooking, vitamin C content decreased in the nut extract but remained unchanged in the leachate. After 20 min the vitamin C level in the leachate also decreased but then remained constant at around 1.5 mg/100g (Fig 20).

40

35

30

25

20 AEAC (mg/100g) AEAC

15

10

5

0 0 5 10 15 20 25 30 60 90 120 150 180 210 240 Cooking Time (min)

Fig 20. AOC contribution by vitamin C in the nuts and in the leachate.

g Vitamin C in leachate; Vitamin C in nut extract

The total AOC levels in the raw nuts, nuts cooked for 1 h (which is the normal

practice), leaf extract, standardised leaf extracts and the commercial Ginkgo biloba

capsules were determined and compared (Table 10). The results show the order of AOC

Page 127 of 201 activity as: cooked nuts < raw nuts < commercial capsules < leaves < standardised leaf

extract. The origin of the leaves does not greatly affect the AOC level. However, it is

interesting to note that the commercial capsules had a much lower AOC level than the

fresh samples.

Table 10. Antioxidant capacity from various Ginkgo biloba samples using ABTS cation decolorisation assay

Ginkgo biloba samples AEACa

China Ginkgo biloba nuts – Raw 515.5 (RSD%=2.13)

China Ginkgo biloba nuts – Cooked for 1 h 287.9 (RSD%=1.15)

USA Ginkgo biloba leaves 16288 (RSD%=2.44)

China Ginkgo biloba leaves 15509 (RSD%=2.75)

Standardised Ginkgo biloba leaf extract 41777 (RSD%=1.98)

Ginkgo biloba leaves commercial capsules 4440 (RSD%=2.56)

Another set of results using ORAC method is shown in Table 11.

a mg of ascorbic acid equivalents per 100g of homogenate

Page 128 of 201 Table 11. Antioxidant capacity from various Ginkgo biloba samples using ORAC method

Ginkgo biloba samples ORAC unitsb

China Ginkgo biloba nuts – Raw 1.29 (RSD%=7.23)

USA Ginkgo biloba leaves 6.60 (RSD%=5.12)

China Ginkgo biloba leaves 5.44 (RSD%=3.56)

Standardised Ginkgo biloba leaf extract 5.20 (RSD%=2.98)

Ginkgo biloba leaves commercial capsules 10.30 (RSD%=2.76)

This set of results seems to suggest the order of AOC activity as: Raw nuts < standardised leaf extract < leaves < commercial capsules. The origin of the leaves does not greatly affect the AOC level. However in contrast to the ABTS cation decolorisation assay, it is interesting to note that the commercial capsules had a much higher AOC level than the fresh samples. There are many reasons for such differences. As previously discussed in 2.2.2.2 the ORAC assay, possesses numerous drawbacks. On the other hand, ABTS cation decolorisation assay follows a single electron-transfer mechanism.

In comparison of the methods, the ORAC assay directly estimates the chain-breaking antioxidant activity, while the ABTS cation decolorisation assay actually measure the specific oxidant reducing power. Based on the mechanism of these two methods, it can be seen from the results in Table 10 and 11, Ginkgo raw leaves are a complex matrix, which has not been fully evaluated, it could contain numerous hydrogen donating compounds. This explains the vast difference in AOC between the leaves and commercial capsules that has been extracted and extensively cleaned up to contain the standardised content. ORACβ-PE may be an unreliable method due to the instability of the protein as it could have degenerated itself or by other factors and loses its

b µmol of Trolox/g of fresh weight)

Page 129 of 201 fluorescence. Hence further work is needed to examine the ORAC method before more conclusive results can be drawn.

In addition, another improved ORAC assay using fluorescein (FL) as the fluorescent probe has been developed (Ou, et. al., 2001) as compared to β-PE as the

fluorescent probe, FL does not interact with antioxidant samples. However, this method was not carried out due to resource and equipment constraints.

Page 130 of 201 6.3 EFFECT OF PREPARATION PROCEDURE ON AOC FOR LEAF INFUSION

In this investigation, antioxidant capacity, AOC, of Ginkgo biloba leaf infusions

was determined and compared using Ferric Reducing Antioxidant Power assay (FRAP)

and ABTS cation decolorisation assay. Capillary electrophoresis was used for the

determination of flavonoids in Ginkgo biloba leaves to confirm and elucidate the fingerprint compounds that contribute to the AOC. Various parameters in the

preparation of the infusion, such as degree of fermentation, surface area of leaves, temperature and time of leaves infusion has been considered to determine their effect on the AOC. In all the experiments, Ginkgo leaf infusions were prepared according to the following standard protocol. About 0.4 g of leaves were weighed into a beaker and 40 mL of deionised water was added (100 ml of water/g of leaves). Water temperatures and infusion times were varied between 60 oC and 100 oC and 1 min to 20 min respectively

to consider the effect of infusion temperature as well as the infusion time on the

antioxidant activity. The effect of the degree of fermentation and surface area of Ginkgo

leaves as well as comparison among different tea types on the antioxidant activity was

also determined using similar procedures. Table 12 illustrates the various samples used

for AOC determination. Further discussion of the results obtained follows:

For the purpose of investigating whether surface area of the ground leaves will

affect AOC, the particle size was investigated with the use of sieves to separate

particulate materials into fractions of different sizes. Sieving is one of the simplest

methods of determining particle size distributions and it is probably used in industrial

laboratories more than any other method. To increase the accuracy of the particle size

determination, sieving can be used along with optical or electron microscopy as a

method which classifies particles according to geometric similarity, regardless of density

or optical properties. The sieves used for the determination of particle size of the leaves

were the Tyler Standard Screen Scale and the U.S Sieve Series. The particle size

Page 131 of 201 distribution of the leaves was determined as follows, in decreasing size order: Ginkgo leaves (ground 5 s) > Ginkgo leaves (ground 20 s) > Commercial black tea leaves >

Commercial green tea leaves. See Table 13.

Table 12. Various leaf infusion samples used .for AOC determination

Samples description Fermented (F)/ unfermented (U) Grinding time (s)

Sample A (U) 5

Sample B (U) 20

Sample C (F) for 2 h 5

Sample D (F) for 2 h 20

Sample E (F) for 24 h 5

Sample F (F) for 24 h 20

Table 13. Particle size distribution of the leaves passing through various sieve sizes.

Sieve openinga

2000 (µ) 850 (µ) 354 (µ) 177 (µ) 150 (µ) <150 (µ)

Ginkgo leaves (ground for 5 s) 88 26 6 2 0 0

Ginkgo leaves (ground for 20 s) 100 76 12 2 0 0

Commercial Black Tea Leaves 100 90 10 0.8 0.7 0

Commercial Green Tea Leaves 100 93 53.2 15.2 12 0

a Percentage passing (%)

Page 132 of 201 6.3.1 Comparison of AOC results with ABTS and FRAP

The following are the standard calibration curves of the two standards i.e. ferrous sulphate standard use for FRAP determination and ascorbic acid standard curves used for

ABTS cation decolorisation assay respectively as shown in Fig 21 and 22.

0.35 y = 1.0951x 2 0.3 R = 0.9903

0.25

0.2

0.15 Abs. Drop Ferrous sulphate standards Linear (Ferrous Sulphate Standards) 0.1

0.05

0 0 0.05 0.1 0.15 0.2 0.25 0.3 Ferrous Sulphate Standards Conc. (mg/ml)

Fig 21. Absorbance drop vs ferrous sulphate standard concentration

1.2

1 y = 3.7732x R2 = 0.9982 0.8

0.6 Ascorbic acid Standards Abs. drop drop Abs. 0.4 Linear (Ascorbic acid Standards)

0.2

0 0 0.05 0.1 0.15 0.2 0.25 0.3 Concentration of Vit C mg/ml

Fig 22. Absorbance drop vs ascorbic acid standard concentration

Page 133 of 201 FRAP Determination.

AOC was demonstrated in the Ginkgo leaves infusates at all temperatures studied. Generally the FRAP values in Ginkgo leaves infusates increased in a linear manner across the range of temperatures studied. There is no significant difference in

FRAP (p=0.05) between unfermented and fermented leaves for 24 h and 2 h (with infusion conditions of 100°C and 10 min) as seen in Table 14.

Table 14. FRAP Equivalent (mg/100g) of fermented and unfermented Ginkgo leaves infusion

Type of Ginkgo Ground Temperature Time of FRAP Equivalent leaves Time of infusion Infusion mg/ 100g leaves (s) (oC) (min) (%RSD)

Fermented 2 h 20 100 10 6329 (5.48%)

Fermented 24 h 20 100 10 6409 (1.59%)

Unfermented 20 100 10 6825 (4.54%)

The above AOC results are generated from three determinations

The linear increase of FRAP, illustrated in Fig.23., is similar for leaves ground for 5 or 20 s, however the FRAP value of those ground for 20 seconds is consistently higher. In addtion, it also further demonstrated that fermentation does not affect the

FRAP results. This result also correlated well with the chromatograms obtained from capillary electrophoresis as shown in Fig. 24. These clearly show that the compounds profile is very similar for fermented and unfermented samples and there is no significant difference in the pattern of peaks obtained. It is generally suggested that unfermented tea leaves, e.g., green tea, should have a higher AOC (Von Gadow et. al., 1997) due to the flavonols present that are known potent antioxidants (Lunder, 1992; Xie et. al., 1993).

However, in this study it was shown otherwise. Further studies on Ginkgo leaves fermented for 6 h and 10 h were not continued.

Page 134 of 201

Ground for 20 s

Ground for 5 s

Fig 23. FRAP Equivalent (mg/100g) of Ginkgo leaves infusion with respect to temperatures (°C).

* (Sample A) Unfermented ground for 5 s; ■ (Sample B) Unfermented ground for 20 s. X (Sample C) Fermented 2 h ground for 5 s; ♦ (Sample D) Fermented 2 h ground for 20 s;

● (Sample E) Fermented 24 h ground for 5 s; ▲ (Sample F) Fermented 24 h ground for 20 s;

From Fig 23, It can be deduced that leaves ground for 20s having a larger surface area exposed, therefore producing a higher AOC.

Page 135 of 201 DAD1 C, Sig=230,30 Ref=off (FLAV\031101-2.D) mAU 10 A

8

6

4

2

0 4 6 8 min DAD1 C, Sig=230,30 Ref=off (FLAV\031103-2.D) mAU B 10

8

6

4

2

4 6 8 min Fig 24. Electropherogram of the capillary electrophoresis determination of the Ginkgo leaves profile A: Unfermented Ginkgo leaves, (See method for details) infusion 10 min, 0.4g/40 ml; B: Fermented 24 h Ginkgo leaves (See method for details) infusion 10 min 0.4g/40 ml; Capillary electrophoresis condition: buffer, 50 mM borate and 10 %ACN, injection: 50 mbar*4s, +25 kv

Page 136 of 201

Fig 25. FRAP Equivalent (mg/100g) with respect to Time of Infusion (min)

▲Unfermented Ginkgo leaves ground for 20 s (Sample B); ■ Unfermented Ginkgo leaves ground for 5 s (Sample A)

There was an observable increase in the FRAP value from Ginkgo leaves ground for 5 s and 20 s (Fig. 25) with respect to infusion times. Taking unfermented Ginkgo leaves infused for 10 min as an example, leaves that were ground for 20 s (at 100oC) gave a FRAP value of 6830 mg/100 g leaves, an increase of 2219 mg/100 g leaves from the FRAP value obtained from leaves that were ground for 5 s (at 100oC), (an increase of approximately 33%). As expected, Ginkgo leaves ground for 20 s had a higher FRAP value and that could be attributed to the larger surface area of the leaves that were ground for a longer time and its abitliy to release the antioxidant components more efficiently over the same period of time.

For the unfermented samples i.e. sample A & B, the FRAP values increased within the range of infusion time (1 min to 20 min) under study (Fig 25). For the fermented samples i.e. samples C & D, the FRAP values increased with infusion time for

Page 137 of 201 from 1 min to a maximum FRAP value at 10 min (Fig 26). The trend for unfermented leaves as seen in Fig. 25, on the other hand, was uncertain for the range of infusion time under study. For example, sample B exhibited an increase in the FRAP value obtained at t = 3 min (6478 mg/100 g leaves) but the value decreased at t = 5 min (6121 mg/100 g leaves) before having a global maxima increasing again at t = 10 min (6830 mg/100 g leaves). For sample A, the FRAP value was increasing within the range of infusion time

(1 min to 20 min) under study. However, most of the data (including 2-hour fermented

leaves) shows that the release of antioxidants from Ginkgo leaves were maximum at t =

10 min to t = 15 min as shown in Fig 26.

Fig 26. FRAP Equivalent (mg/100g) with respect to Time of Infusion (min)

▲Fermented 2 h Ginkgo leaves ground for 20 s (Sample D); ■ Fermented 2 h Ginkgo leaves ground for 5 s (Sample C).

ABTS•+ Determination.

This experiment was conducted on samples A and B. As fermentation showed no

significant difference in the FRAP value, only unfermented Ginkgo leaves were then

Page 138 of 201 used in determining the AOC using the ABTS method. The AEAC value of unfermented

Ginkgo leaves ground for 20 s was observed to be higher than leaves ground for 5 s, similar to the results obtained using FRAP method. However, the optimum infusion time in releasing the maximum antioxidants from the leaves was different for different leaf types. The results seem contradictory because the maximum AEAC value was observed at t = 10 min for leaves ground 5 s whereas for leaves ground 20 s, the maximum AEAC value was observed at t = 15 min. However, it was predicted that leaves of smaller particle size are capable of releasing the maximum antioxidants after a shorter time. The results of ABTS determination do need to be viewed with care. This is because the specificity of assay involving ABTS method in measuring the capacity of a sample to directly quench free radicals is not always guaranteed or reproducible (Cao & Prior,

1998). However the method has been successfully used for AOC determination in alcoholic extracts of Ginkgo biloba nuts and leaves. (Goh & Barlow, 2002).

Page 139 of 201 6.3.2 Comparison studies among Ginkgo Leaves, Green & Black Tea Leaves.

The AOC of the different beverages as determined by the FRAP assay and ABTS

decolorisation assay decreased in the order: commercial green tea leaves > commercial

fermented black tea > Ginkgo leaves infusion ground for 20 s as shown in Table 15. The

black and green tea leaves had an approximate FRAP value of 7 to 9-fold higher than

Ginkgo leaves. However, the difference of the comparison of these values between the

green and black tea leaves and Ginkgo leaves might be misleading because from the

particle size analysis of the leaves, the surface area of the green and black tea leaves was

smaller than the surface area of Ginkgo leaves ground for 20 s. From the results presented earlier, it is seen that leaves with a larger surface area have a significantly higher FRAP value (higher AOC). The difference of the AOC values amongst the test samples i.e Ginkgo leaves, black tea and green tea could be due to the presence of caffeine, which is present in considerable amounts in the commercial hot beverages whilst being absent in the Ginkgo leaves infusion.

Table 15. AOC of the different test subjects

Types of test samples FRAPa (%RSD) ABTSb (%RSD)

Green Tea 56810 (2.05%) 36461 (1.67%)

English Breakfast Tea 44745 (1.27%) 31883 (1.75%)

Ginkgo leaves infusion 6211 (1.40%) 3185 (1.88%)

The above AOC results are generated from three determinations.

a mg/100 g leaves b mg/100 g leaves

Page 140 of 201 6.3.3 Caffeine determination

To confirm the presence or otherwise of caffeine, which is present in considerable amounts in the commercial hot beverages whilst being absent in the Ginkgo leaves infusion, HPLC analysis was carried out on the Ginkgo leaves infusions and also to quantify the caffeine present in the commercial hot beverages. The results showed negative result for caffeine in Ginkgo leaves infusion and relatively high caffeine contents in green and black tea as shown in Table 16. The reported values i.e. 25 – 110 mg of caffeine/cup (Food Facts Asia Third Quarter, 2000) coincides with the experimental results.

Table 16. Determined caffeine content in selected Beverages

Types of test samples Caffeine contenta English Breakfast Tea 49.4 ± 1.98 Green Tea 52.0 ± 1.41 Ginkgo leaves infusion 0 The caffeine quantification are generated from three determinations

The ability of caffeine to scavenge the hydroxyl radical OH (Shi, et. al. 1991) displayed a reaction rate constant of approximately 5.9 x 109 M-1 sec-1 that is comparable with those of other efficient hydroxyl radical scavengers. Hence, caffeine does make a contribution to total AOC. However the degree of contribution of caffeine to total AOC in this study was not determined. In addition, there are also negative effects of caffeine including rapid/irregular heartbeat, elevated blood sugar and cholesterol, nervousness, agitation and insomnia (Iwaoka & Brewer, 2000), negative effects during pregnancy (Harland, 2000) and increased calcium loss (Lloyd, et. al.

1997; Harris & Dawson-Hughes, 1994; Barrett-Connor et. al. 1994). Ginkgo leaves infusion is caffeine-free yet capable of providing significant AOC.

a mg/cup (normal serving size of 250ml)

Page 141 of 201 6.3.4 Sensory Evaluation

The sensory evaluation was conducted on 50 individuals. A general description of the participants is:

Description Sex 19 males and 31 females Age Majority (>85%) age from 20 to 25-year-old Race Majority (>85%) of Chinese ethnic origin

For flavour, aroma, colour and overall liking, they were evaluated based on the scale below:

1 9

Least preferred Most preferred

Therefore, the higher the score (given by each participant) for each criteria above, the more preferred the particular sample is to them.

For likelihood of buying the product if it is available in the market, it was evaluated based on the scale below:

1 9

Most Likely Most Unlikely

The lower the score (given by each participant), the more likely that they will buy the product if it is available in the market.

The summary of total scores given by the 50 participants for each of the sample according to the criteria above is shown in Table 17:

Page 142 of 201 Table 17. Summary of total sores based on the criteria tested. Statistical analysis

Criteria Black Tea Green Tea Ginkgo Leaves

Flavour 267 232 187

Aroma 341 273 217 Colour 325 301 238

Overall liking 297 247 199

Likelihood 261 298 331

From the tabulated scores in Table 17, it is possible to rank the various criteria.

Black tea was the most preferred beverage having the highest scores in flavour, aroma, colour and overall liking. The second most preferred beverage is green tea having the second highest scores in all the criteria followed by the Ginkgo leaves as the least preferred. From the sensory evaluation, the participants also stated that the probability of them buying black tea (likelihood) will be the highest followed by green tea and lastly

Ginkgo leaves.

Apart from that, the participants were also asked to rank the samples from 1 to 3

(Most preferred to least preferred) at the end of the sensory evaluation.

Table 18. Tabulation of the ranking amongst the three samples.

Black Tea Green Tea Ginkgo leaves Ranking 1 28 13 9 Ranking 2 19 21 10 Ranking 3 3 16 31 Total Participants 50 50 50

Page 143 of 201

Fig 27. Ranking of preferences amongst the three samples

From the tabulated graph shown in Fig 27 and Table 18, more than 50% of the participants ranked black tea the first (most preferred), followed by green tea (26%) and

Ginkgo leaves (18%). Unfortunately, for the sensory evaluation, Ginkgo leaves were the ranked as least preferred. This means that additional flavours might be necessary to improve the acceptability of Ginkgo leaves as an infusion

Page 144 of 201 6.4 IN VITRO, SIMULATED DIGESTION STUDIES

The fate of phytochemicals in Ginkgo biloba leaves and its standardised extract in the human body after ingestion is of interest in order to investigate the potential role of flavonoids as regards their radical scavenging ability. Over recent years, Ginkgo biloba has achieved unprecedented popularity, as a health supplement. Concurrently, a major thrust of research has been the development of new food products, which have functional properties, which are truly expressed in the body.

An experiment (in vitro) to simulate the workings of the human gut during digestion was designed to study the recovery and stability of the flavonoids. The products of interest were raw Ginkgo biloba leaves, commercial Ginkgo biloba leaf extract capsules and standardised Ginkgo biloba extract. These products were subjected to conditions that followed closely those encountered in the gastrointestinal tract.

The aim of this study was to achieve a better understanding of what happens to the flavonoid compounds when they are placed in the mouth, carried by peristalsis to the stomach and the fate of these compounds when they come into contact with the upper gut secretions.

It has been reported that flavonoids present in foods cannot be absorbed from the intestine because they are bound to sugars as glycosides (Kuhnau 1976). Only free flavonoids without a sugar molecule, the so-called aglycones, are considered to be able to pass through the gut wall, and no enzymes that can split these predominantly β- glycosidic bonds are secreted into the gut or present in the intestinal wall. However, in contrast, Hollman & Katan, 1999 suggest that, certainly for quercetin glycosides from onions, the absorption of the intact glycosides is in fact far better then the pure aglycones. These workers suggest that absorption kinetics and bioavailability of the flavonoids are probably governed by the actual form of glycoside present. However, this

Page 145 of 201 current work seems to support the earlier report by Paganga & Rice-Evans, 1997 who showed the presence of glycosylated flavonoids present in human plasma, in that the digested raw leaf residues was high in glycosides content.

This study aims to investigate the stability of flavonoid compounds before considering whether they are absorbed. If the aglycones or their glycosides were totally degraded in the gastrointestinal fluids then their absorption would be impossible. This simulated digestion/absorption study was undertaken with two objectives; a) to determine the degree of degradation, if any, in the upper GI tract and b) in view of the difficulty of measuring flavonoids in the blood, and being able to relate such levels to specific foods in the diet to assess the availability of specific flavonoids for absorption.

An in vitro study allows a more controlled approach to test hypotheses, which can later be studied, in vivo. Other studies have been carried out to determine flavonols in body fluids and generally seem to indicate minimal degradation, at least for quercetin or its glycosides with gastrointestinal fluids (Hollman, et. al., 1995).

This study aims to provide further information on the stability of flavonoids in humans during the digestion process and to contribute to the development of Ginkgo biloba leaf products, which can later be directed to consumers with an increased knowledge on the content and biological activity of these natural antioxidants.

Section 5.2.10, and Table 7 show the various samples that were obtained. Table

18 shows the amount of constituents identified from the various control samples i.e. before simulated digestion. From these results, which were designated 100%, an increase or decrease in the percentage after the incubation procedures was obtained.

Tables 19, 20 and 21 show results for the different samples.

Page 146 of 201

Table 19 Comparison of the amount of the various constituents (mg/g) present in the various Ginkgo biloba samples studied both in the aqueous & alcohol extracts.

* Type of Samples Recovered amount (mg/g) of the constituents studied

Quercetin-3-O- Quercetin-3- Kaempferol-3- Quercetin-3- Kaempferol-3- Quercetin Isorhamnetin Kaempferol rutinoside galactosides rutinoside O-rhamnoside (p-coumaryl) (Rutin) (Quercetrin) glucoside Raw Ginkgo leaves

Blank aqueous 0.190(1.38%) 0.033(2.96%) 0.109(0.34%) 0.015(0.39%) N.D. N.D. N.D. N.D.

Blank alcohol 1.880(2.0%) 0.579(5.24%) 1.311(1.28%) 0.354(0.9%) 0.015(0.62%) 0.0243(3.16%) 0.002(7.35%) 0.015(7.4%)

EGb 761

Blank aqueous 4.468(0.67%) 6.257(2.21%) 3.673(0.26%) 4.575(0.08%) 1.632(1.19%) 0.212(5.77%) 0.027(1.97%) N.D.

Blank alcohol 9.278(3.22%) 4.599(3.73%) 1.668(7.1%) 0.738(5.81%) 1.604(0.98%) 0.046(13.22%) 1.243(3.77%) 0.06(8.8%) Commercial capsules

Blank aqueous 0.753(1.68%) 0.073(5.91%) 0.220(4.2%) 0.816(1.13%) 1.093(0.76%) 0.131(2.85%) N.D. N.D.

Blank alcohol 25.329(1.53%) 1.083(5.45%) 0.532(6.35%) 1.509(1.47%) 2.318(1.3%) 10.966(2.61%) 1.306(6.94%) 3.636(3.4%)

N.D. = Not detected.

* Mean of 3 determinations (%RSD)

Page 147 of 201

Table 20. Percentage recovery of the various compounds from raw Ginkgo biloba leaves following specific treatment

Sample code Treatment Quercetin- Quercetin-3- Kaempferol- Quercetin- Kaempferol- Quercetin Isorhamnetin Kaempferol 3-O- galactosides 3-rutinoside 3-O- 3-(p- rutinoside rhamnoside coumaryl) (Rutin) (Quercetrin) glucoside

A aqueous Gastric 1 h 73.205 6347.424 230.733 55.490 NIL. NIL. NIL. NIL.

B alcohol Gastric 1 h 7.752 375.943 22.784 3.651 NIL. NIL. NIL. NIL. C aqueous Gastric 1 h + 116.248 121.502 1018.613 40.384 NIL. NIL. NIL. NIL. Intestinal 2 h D alcohol Gastric 1 h + 9.218 6.295 74.442 5.777 NIL. NIL. NIL. NIL. Intestinal 2 h E aqueous Gastric 1 h + 125.413 10901.225 441.236 153.732 NIL. NIL. NIL. NIL. Intestinal 4 h F alcohol Gastric 1 h + 9.790 501.405 29.344 4.940 NIL. NIL. NIL. NIL. Intestinal 4 h

Page 148 of 201

Table 21. Percentage recovery of the various compounds from the Standardised Ginkgo Extract following various treatments.

Sample code Treatment Quercetin- Quercetin-3- Kaempferol- Quercetin- Kaempferol- Quercetin Isorhamnetin Kaempferol 3-O- galactosides 3-rutinoside 3-O- 3-(p- rutinoside rhamnoside coumaryl) (Rutin) (Quercetrin) glucoside A aqueous Gastric 1 h 85.124 13.580 119.438 4.545 9.700 78.388 NIL NIL

B alcohol Gastric 1 h 40.978 18.772 359.699 43.756 18.147 390.621 NIL 54.895

C aqueous Gastric 1 h + 88.615 17.269 175.641 6.380 17.690 90.344 NIL NIL Intestinal 2 h D alcohol Gastric 1 h + 46.248 25.183 421.600 43.127 18.672 415.965 NIL 25.916 Intestinal 2 h E aqueous Gastric 1 h + 98.538 18.518 190.411 7.739 20.337 105.746 NIL NIL Intestinal 4 h F alcohol Gastric 1 h + 40.056 22.320 381.791 30.615 2.894 79.530 NIL NIL Intestinal 4 h

Page 149 of 201

Table 22. Percentage recovery of the various compounds from commercial Ginkgo capsules following specified treatments.

Sample Treatment Quercetin- Quercetin-3- Kaempferol- Quercetin- Kaempferol- Quercetin Isorhamnetin Kaempferol code 3-O- galactosides 3-rutinoside 3-O- 3-(p- rutinoside rhamnoside coumaryl) (Rutin) (Quercetrin) glucoside A aqueous 7.422 3418.678 21.995 1.999 1.601 9.027 NIL NIL Gastric 1 h B 0.135 124.098 4.719 0.864 0.294 0.055 NIL 0.055 alcohol Gastric 1 h C aqueous Gastric 1 h + 7.542 39.664 0.813 1.208 0.389 0.000 NIL NIL Intestinal 2 h D alcohol Gastric 1 h + 0.207 3.174 39.365 0.000 0.909 0.110 NIL NIL Intestinal 2 h E aqueous Gastric 1 h + 6.283 43.336 96.664 1.197 0.222 0.000 NIL NIL Intestinal 4 h F alcohol Gastric 1 h + 0.220 3.452 36.543 0.810 0.621 0.118 NIL NIL Intestinal 4 h

Page 150of 201 6.4.1 HPLC analysis for determination of stability of Glycosides and

Aglycones

6.4.1.1 Qualification

The constituents of various Ginkgo biloba samples (i.e the raw Ginkgo leaves, standardised Ginkgo extract and the commercial Ginkgo capsules) were examined and 5 flavonoid glycosides and 3 aglycones were positively identified as shown in Figure 28.

These peaks were identified by comparison with authenticated standards. Together with the retention time data, these peaks were also identified by comparing their UV spectra with those of the corresponding standards.

Fig 28. Chromatogram of a standardised extract of Ginkgo biloba leaves (control sample: blank alcohol). The constituents determined are quercetin-3-O-rutinoside (rutin), quercetin-3-galactoside, kaempferol-3- rutinoside, quercetin-3-rhamnoside (quercetrin), kaempferol-3-O-rutinoside, kaempferol-3-(p-Coumaryl) glycoside, quercetin, kaempferol, and isorhamnetin. Shim-Pack VP-ODS column (250 x 4.6 mm i.d.); Mobile Phase A: water: 2-propanol (95:5), eluent B: 2-propanol: THF: water (40:10:50), binary gradient from 20% to 55.2% B in 44 min at a flow-rate of 1.0 mL/min at 40°C.

Page 151of 201 6.4.1.2 Quantification

Raw Ginkgo leaves

Reference to Table 19 shows that the aqueous and alcohol extracts of raw Ginkgo

leaves gave different values in the determined amounts of the various constituents. The

difference between the two extracts provides an understanding on the extraction power

of different solvents and also verified the nature of the compounds of interest. In the

aqueous extraction of raw leaves, flavonol aglycones could not be detected and there was

a lower extraction of the flavonoid glycosides as compared with the alcohol extract.

However, following incubation of the raw leaves with gastric fluids for 1 h and

subsequent intestinal fluids for 2 h and 4 h respectively, the aqueous extract seems to

display an increase in the amount of compounds identified (Table 20). Flavonoids are

ubiquitous and are found in the flowers, fruit and leaves of the Ginkgo biloba plant.

They are biosynthesed from the starting material glucose via the pathway reported by

Haslam 1993. Plant cells walls are mainly composed of linear chains of covalently

linked glucose residues. It is very stable chemically and extremely insoluble.

However, when subjected to prolonged incubation under digestion conditions,

even though the cellulose is not digested, there is likely to be cellular disruption and an

increased release of flavonoid glycosides available for extraction thus, explaining the

apparent increases (recovery of up to >1.25 times for rutin, >63 times for quercetin3- galactosides, >2.3 times for kaempferol-3-rutinosides and >1.53 times for quercetin-3-O- rhamnosides) in the results obtained. The findings also seem to suggest possibly low yields of flavonoids from undigested leaves because the compounds are chemically bound to the cell walls or within the intact cells and not available for extraction.

Page 152 of 201 EGb 761

For EGb 761, reference to Table 19 indicates a similar trend in the distribution of

glycosides and aglycones in the aqueous and alcoholic medium. Following the simulated

digestion procedure, the quantification of the various flavonoids in the aqueous extract of

EGb761, differs (Table 21). The following percentages compared with the undigested

samples of the various flavonoids identified were observed, rutin circa 80-90%, circa 10-

20% for quercetin-3-O-galactosides, circa 100-200% for kaempferol-O-rutinosides, circa

4-8% for quercetin-3-O-rhamnosides, circa 9-20% for kaempferol-3-(p-coumaryl)

glucoside and circa 70-105% for quercetin. Isorhamnetin and kaempferol were not

detected.

If the simulated digestion system does break down the glycosides this should be

reflected in an increase in aglycone recovery in the alcoholic medium after digestion.

The breakdown of the glycosides is indicated by a less than a 100% recovery of rutin,

quercetin-3-galactosides and quercetin-3-O-rhamnosides in the aqueous acidic extract.

Differences in the glycoside/aglycone ratio, most likely reflects differences in stability of

the glycosides.

Interestingly kaempferol-3-rutinoside, which appears to be a more stable

glycoside, does show an increased extraction following more severe digestion and hence

likely to be more available for potential absorption into the body. In contrast,

kaempferol-3-(p-coumaryl) glucoside, shows a poor recovery in both the alcoholic and

aqueous extract, indicating a likely total breakdown. However following the 4th hr of incubation, kaempferol-3-(p-coumaryl) glucoside seems to be degraded indicating some degree of instability of the aglycone previously formed.

In sample D (alcohol extract) (Table 21), it may be noted that the sum of the recoveries for kaempferol-3-rutinoside and quercetin adds up to 837.56%. As the starting material in this experiment was an extract, even if all eight compounds tested

Page 153 of 201 had been recovered in their entirety, the maximum total figure is in theory only 800%.

This apparent de novo production of flavonoids is most likely explained by the low yield

of recovery of flavonoids from the undigested samples.

Commercial capsules

Similar to the results seen in the pervious samples, the undigested aglycones were

better extracted into the alcoholic medium compared with the aqueous medium (Table

19). Isorhamnetin and kaempferol were not detected at all in the aqueous medium.

Following the simulated digestion (Table 22), the following recoveries were obtained, circa 6% and 0.2% respectively in the aqueous and alcoholic extract for rutin.

However for quercetin-3-galactosides an increase of 34 times and 1.24 times respectively for aqueous and alcoholic extract occurred after the 1st hr. After the full-simulated digestion procedure, there was a reduction in the amount to 30-40% and 3-3.5% recovery in the aqueous and alcoholic medium respectively. Recovery of quercetin-3-O- rhamnosides also gave a low percentage recovery circa 0-2% for the aqueous and alcoholic medium. Such low recovery seen in the quercetin glycosides would be expected to be accompanied by a higher recovery in the corresponding aglycones.

However, this was not observed. The recovery of quercetin was also low giving circa

9% recovery in the aqueous medium after the 1st hr of digestion and subsequently showing no recovery when subjected to the intestinal conditions. This would seem to indicate that there was little or no conversion of glycosides to aglycones and, in addition, any aglycones so formed appear to have been degraded in the simulated digestion process.

Similar to the above it would appear that the kaempferol-3-rutinoside is relatively stable. However the percentage recovery of kaempferol was almost insignificant

Page 154 of 201 following the simulated digestion. Thereby indicating similar phenomenon i.e. there was

little or no conversion of glycosides to aglycones and in addition it would seem that the

latter has been degraded in the simulated digestion process.

On the other hand, kaempferol-3-(p-coumaryl) glucoside, yielded very low

recovery circa 0-2% for the aqueous and alcoholic medium. This indicated that

kaempferol-3-(p-coumaryl) glucoside was fairly unstable and could have been degraded to yield non-active compounds.

Summary

Degradation of the flavonoid compounds in gastrointestinal fluids has been

reported to be minimal at circa 5 % in ileostomy subjects (n=6) (Hollman, et. al., 1995).

A similar finding was also observed in this work. In the digested Ginkgo leaves, a

higher concentration of flavonoid glycosides compared with the undigested leaves was seen. However, in contrast, this study seems to indicate that the recovery of selective

flavonoid compounds in EGb 761 and commercial capsules after incubation in vitro in

simulated gastrointestinal fluids is minimal. For quercetin-3-galactosides quercetin-3-O-

rhamnosides and kaempferol-3-(p-coumaryl) glucoside in EGb 761 and for all the eight

constituent of the Ginkgo capsules a very low recovery was observed of only circa 20%.

This observation would seem to suggest caution when expressing the beneficial

effect that these constituents may provide. From various reports based on

epidemiological research, a hypothesis has been proposed of an inverse relationship

between the dietary intake of some flavonoids and the incidence of several chronic

diseases (Knekt et. al., 2002; Hertog et. al., 1992). However, whether the hypothesis is

true or not has still to be confirmed in view of the controversial subject of flavonoid absorption (Hollman et. al., 1997; Hollman & Katan, 1997; Hollman et. al., 1997).

Page 155 of 201 This research has not addressed the absorption issue but merely the digestion aspect of the flavonoids.

Based on the above results, it would appear that some of the active flavonoid

compounds in the commercially prepared samples are produced in lower amounts after

the simulated digestion procedure compared with the digestion of the natural leaves.

This may be due to previous losses of these compounds during the preparation of the

commercial samples. By taking in the raw leaves as opposed to the commercial

preparations examined, there is likely to be a greater benefit in view of the higher level

of flavonoids detected.

From Table 18 it may be seen that the undigested commercial extracts do in fact

contain a higher proportion of aglycones than the raw Ginkgo leaves and thus, only when

the controversy of glycosides vs aglycones absorption has been fully resolved, will it

indicate which products are most likely to provide the greatest in vivo benefit.

Page 156 of 201 6.5 TOXICOLOGICAL DETERMINATION

6.5.1 Colchicine determination

Ginkgo nuts, an essential ingredient added to a traditional sweet dessert, Cheng

Teng, is very popular in the South East Asia region. Due to its popularity of consumption, attention is needed to ensure its safety for consumers. Hence colchicine determination was carried out to verify the presence of colchicine in Ginkgo nuts. Since colchicine is speculated to be present in Ginkgo leaves and its commercial capsules, the hypothesis is that Ginkgo nuts could also be a potential source of colchicine.

However, no colchicine was detected in any of the Ginkgo nut samples in this research study. A control (i.e, a known standard) was used to validate the results. The limits of detection (LOD) were determined by fitting interday back-calculated standard deviations of each calibration standard. The limits of detection LOD is defined as the lowest determinable quantity that indicates the presence of an analyte at a given statistical level of confidence (3SD). The LOD was calculated statistically and was found to be 9x 10 –5 mgml-1.

Page 157 of 201 6.5.2 Ginkgolic acids – as allergen

6.5.2.1 SAMPLE PREPARATION

In this part of the study, sample preparation is discussed. It is the process of

transforming a sample into a form that is suitable for analysis. This process might

involve extracting analyte from a complex matrix (i.e. solid – 60% acetone extraction),

pre-concentrating very dilute analytes to get a concentration high enough to measure (i.e.

60% acetone-hexane extraction), and removing interfering species (i.e. Soxhlet extraction). Micro filtering was carried out prior to reconstitution with 10 ml of acetonitrile, to remove interfering species or impurities. Derivatisation was carried out prior to GC-MS analysis, to transform analyte to trimethylsilyl derivative, which is a more easily detected form.

Obtaining and preparing samples are most important at the beginning of any analytical method and unless care and attention to details are taken into account, may provide major opportunities for inaccuracies and errors to be introduced.

The objectives were to obtain a representative sample and to reduce the number of sample handling steps as much as possible. Tables 23 and 24 show the weight of extracted material used.

Table 23. Sample preparation of Ginkgo extracts for HPLC analysis

Sample Raw weight (g) Dried weight after Soxhlet extraction (g) U.S. Ginkgo 15.763 0.148 biloba leaves P.R.C. Ginkgo 15.672 0.097 biloba leaves Freeze dried, raw 30.056 0.062 nuts Ginkgo biloba 10.106 0.033 capsules EGb 761 10.062 0.262

Page 158 of 201 Table 24. Sample preparation of Ginkgo extracts for GC-MS analysis

Sample Raw weight (g) Dried weight after Soxhlet extraction (g) U.S. Ginkgo 15.600 0.161 biloba leaves P.R.C. Ginkgo 15.425 0.109 biloba leaves Freeze dried, 30.223 0.053 raw nuts Ginkgo biloba 10.186 0.034 capsules EGb 761 10.358 0.217

In the sample protocol followed, small samples obtained were representative of

the larger population of material. For instance, leaves that weighed approximately 15 g

were randomly selected from a total weight of 2 kg of U.S.A leaves prior to grinding.

Grinding ensured sample homogeneity.

Samples were handled as little as possible. Initially, approximately 15 g of dried raw nuts and approximately 5 g of capsule powder were used. However, very small weights were extracted and these caused weighing uncertainties and inconsistencies. A

greater amount of nuts (approximately 30 g) were used compared with approximately 15

g of leaves because nuts generally contained much higher starch mass than lipid mass.

Cross contamination and exposure to the environment were reduced or avoided as much

as possible.

Due to the restriction of ginkgolic acids in products for human consumption to 5

µg/g by the Commission E of the former Federal German Health Authority founded in

1978, the amount of alkylphenols found in commercial extracts were significantly reduced. From the results obtained, EGb 761 yielded the largest alkylphenol containing extract with a dry mass of 0.217 - 0.262 g (i.e. this equalled 2.09 -2.63% of initial raw powder weight). This was followed by U.S. leaves, which yielded an alkylphenol enriched extract with a dry mass of 0.148 - 0.161 g (i.e. 0.94 – 1.03 % of raw weight).

Page 159 of 201 Thirdly, by P.R.C. leaves that gave 0.097 – 0.109 g of dried alkylphenol enriched extract

(i.e. 0.62 – 0.71 % of raw weight). Ginkgo capsules contained small amounts of alkylphenols, 0.033 – 0.034 g of dried extract (i.e. this equalled around 0.33% of initial powder weight). But it gave the highest recovery at Soxhlet extraction step as 0.033 -

0.034 g of dried extract was yielded from 0.039 – 0.040 g of hexane extract. This equalled to around 87% recovery. Nuts contained trace amounts of alkylphenols; a dried extract of 0.18- 0.21% of initial raw weight was obtained.

The main problem encountered was weighing inconsistencies. The weighing procedure followed involved tare weighing the empty container, adding the appropriate weight of samples, recording the total weight and calculating the weight of sample by difference. However, especially in the weighing of dried extracted samples, there was uncertainty in weighing small amounts. Moreover, the dried samples might absorb water from the atmosphere adding to experimental errors.

Page 160 of 201 6.5.2.2 HPLC ANALYSIS

6.5.2.2.1 SOLVENT OPTIMIZATION

An eluotropic series ranks solvents by their relative abilities to displace solutes

from a given adsorbent. The eluent strength is a measure of the solvent adsorption

energy. The less polar solvent has higher eluent strength in RP chromatography. RP

chromatography eliminates peak tailing because the stationary phase has few sites that

can strongly adsorb a solute to cause peak tailing. The more nonpolar the solvent the

greater is its eluent strength for RP chromatography. The greater the eluent strength is,

the more rapid the solutes will be eluted from the column.

From the literature searched (Fuzzati et. al., 2003; He et. al., 2000; Irie et. al.,

1996), acetonitrile solvent, which is relatively non-polar, was extensively used in the RP

chromatographic separation of GAs from G. biloba leaves or extracts. Its high eluent

strength enabled all compounds to be eluted rapidly via isocratic (Irie et. al., 1996) or

via gradient elution (Fuzzati et. al., 2003) with polar water solvent.

He et. al., 2000 used acetonitrile-water-acetic acid as the HPLC eluent in their

qualitative analysis of GAs in Ginkgo biloba extracts by HPLC-EI-MA technique. The mass detection sensitivity was higher than UV detection but relied heavily on the concentration of acetic acid in HPLC eluent. From their studies, increasing acetic acid concentration in the eluent increased HPLC peak sharpness in UV detection but decreased the MS sensitivity. Ginkgolic acids with unsaturated C15-side chain at position 8 {6-[8'(Z)- and 6-[10'(Z) pentadecenyl salicylic acid]} (i.e. which were used as

external standard in this protocol) separation was more affected by acetic concentration

in the eluent than, ginkgolic acid with a C17-side chain and a double bonding at position

12 {6-[12'(Z)-heptadecenyl] salicyclic acid}. Due to practical considerations, 1% acetic acid concentration in the eluent was used. Irie et. al., 1996 identified ∆8 15:1 ginkgolic

Page 161 of 201 acids as the main GA in G. biloba leaves, in their HPLC analysis with acetonitrile- 5% acetic acid as eluent. Fuzzati et. al., 2003 carried out HPLC analysis with 0.1% formic acid in acetonitrile and 0.1% formic acid in water solvents.

Hence, in this experimental protocol, acetonitrile and water were used as the binary mobile phase. Small amounts of polar solvents, formic acid or acetic acid were added to the binary mobile phase (See Fig 28a-d).

The addition of these polar modifiers was expected to decrease the mobile phase surface tension as well as to influence the characteristics of the RP adsorbent surface.

This also increased the accessibility of the hydrophobic regions on the solute surface.

From the results (Fig 28a-d), the polar modifiers may affect the surface tension, dielectric constant, and viscosity of eluent, UV absorbance and elutropic value. The results showed that lower concentration of formic acid significantly improved chromatographic separation (i.e. increased resolution) and HPLC peak sharpness.

Hence, 0.01% formic acid in acetonitrile solvent and 0.01% formic acid in water were used as the eluents in HPLC analysis.

1.00 31.446 AU

0.50 33.745 34.796 39.727 33.187

0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 Minutes

Fig 28a. HPLC chromatogram of Ginkgo R.P.C. leaf extract with acetonitrile-0.01% formic acid eluent.

1.00 AU 0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 Minutes Page 162 of 201

Fig 28b. HPLC chromatogram of Ginkgo R.P.C. leaf extract with acetonitrile-0.1% formic acid eluent.

0.50

AU 0.00

-0.50

0.00 10.00 20.00 30.00 40.00 50.00 60.00 Minut es

Fig 28c. HPLC chromatogram of Ginkgo R.P.C. leaf extract with acetonitrile-0.5% acetic acid eluent.

0.40

AU -0.20 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 Minutes

Fig 28d. HPLC chromatogram of Ginkgo R.P.C. leaf extract with acetonitrile-5% acetic acid eluent.

Page 163 of 201 6.5.2.2.2 GRADIENT OPTIMIZATION

Variation of the gradient steepness parameter significantly changes the selectivity

and bandspacing in a chromatographic separation. Isocratic elution is performed with a

single solvent or constant solvent mixture. If one solvent does not provide sufficiently

rapid elution of all components the gradient elution can be used. Increasing amount of

solvent A are added to solvent B to form a continuous gradient.

Based on the results of the three gradient experiments, resolution optimization

was best achieved by gradient elution profile described in Method C (see to Figs 29a-c).

This was because the peaks were well separated over a 30 – 40 min time interval with

isocratic 90% A (i.e. high eluent strength due to high percentage of non-polar acetonitrile

solvent as eluent in the binary mobile phase) and 10% B as compared with gradient elution profiles followed in Methods A and B.

0.40

0.20

AU 0.00

-0.20

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40 Minutes

(a)

0.20

AU 0.10

0.00 0.00 10.00 20.00 30.00 40.00 50.00 6 Minutes

(b)

Page 164 of 201 0.20

AU 0.10

0.00

0.00 10.00 20.00 30.00 40.00 50.00 6 Minutes

(c)

Fig 29(a-c). Comparative gradient elution profiles of Ginkgo nut extract using 0.01% formic acid in acetonitrile solvent based gradients.

Separations were carried out at 35°C, a flow rate of 1 ml/min on a Symmetry® Shield RP18, [5.0 µm, 4.6 mm x 250 mm (i.d.)] column and eluted with (a) Method A: 0.01% formic acid in acetonitrile solvent (A) and 0.01% formic acid in water (B); according to the following profile: 0 – 30 min, 75 – 90% A, 25 – 10% B; 30 – 40 min, isocratic 90% A, 10% B; 41 – 60 min, isocratic 75% A, 25% B (b) Method B: 0.01% formic acid in acetonitrile solvent (A) and 0.01% formic acid in water (B); according to the following profile: 0 – 30 min, 55 – 70% A, 45 – 30% B; 30 – 40 min, isocratic 90% A, 10% B; 41 – 60 min, isocratic 55% A, 45% B (c) Method C: 0.1% formic acid in acetonitrile solvent (A) and 0.1% formic acid in water (B); according to the following profile: according to the following profile: 0 – 30 min, 35 – 90% A, 65 – 10% B; 30 – 40 min, isocratic 90% A, 10% B; 41 – 60 min, isocratic 35% A, 65% B.

Moreover, the UV spectrum of peak in HPLC chromatogram of Ginkgo U.S.A. leaf, Ginkgo R.P.C. leaf, nut, capsule and EGb 761 extracts at Rt = 33.353 min, Rt =

33.748 min, Rt = 30.782 min, Rt = 32.662 min and Rt = 30.656 min respectively (See

Fig 30a-e), showed absorption maxima at 210, 245 and 310 nm, which is characteristic of ginkgolic acids (Fig 15).

1.00 210.5

AU 0.50 244.5 311.8 0.00 200.00 250.00 300.00 350.00 400.00 450.00 nm

Fig 30 a. UV spectrum of ginkgolic acids in HPLC chromatogram of Ginkgo U.S.A. leaf extract, Rt = 33.353 min

Page 165 of 201 0.20 191.8 209.3 AU 244.5 311.8 362.1 393.9 423.9 0.00 200.00 250.00 300.00 350.00 400.00 450.00 nm

Fig 30 b. UV spectrum of ginkgolic acids in HPLC chromatogram of Ginkgo R.P.C leaf extract, at Rt = 33.748 min

0.08 190.6 0.06 0.04 AU 0.02 244.5 310.6 448.0461.3 377.1 407.1 0.00 200.00 250.00 300.00 350.00 400.00 450.00 nm

Fig 30 c. UV spectrum of ginkgolic acids in HPLC chromatogram of Ginkgo nut extract, Rt = 30.782 min

0.20 191.8 209.3 AU 244.5 311.8 362.1 393.9 423.9 0.00 200.00 250.00 300.00 350.00 400.00 450.00 nm

Fig 30 d. UV spectrum of ginkgolic acids in HPLC chromatogram of Ginkgo capsule extract, Rt = 32.662 min

0.10 209.3

AU 0.05 244.5 311.8 471.0 378.3403.5 437.2 487.9 0.00 200.00 250.00 300.00 350.00 400.00 450.00 nm

Fig 30 e. UV spectrum of ginkgolic acids in HPLC chromatogram of Ginkgo EGb 761 extract, Rt = 30.656 min

Page 166 of 201 UV detection at 210 nm was chosen in order to obtain maximum sensitivity for

ginkgolic acids. Due to lower concentrations of extracts used in analysis (i.e. 0.148g of

hexane extract/ 15.763 g of U.S. leaves, 0.097g of hexane extract/ 15.672 g of R.P.C. leaves, 0.062g of hexane extract/ 30.056 g of freeze dried Ginkgo nuts, 0.033g of hexane extract/ 10.106 g of Ginkgo capsule and 0.262g of hexane extract/ 10.062 g of Ginkgo

EGb 761 extract, only small peaks were shown in UV detection. Hence, GAs could not be detected in sufficient amounts to deserve quantification.

Page 167 of 201 6.5.2.3 GC-MS ANALYSIS

6.5.2.3.1 RESOLUTION

The objective of chromatography is to separate the components of a mixture.

Hence, a measure of the extent of that separation is required.

The common measure used is peak resolution, R and its definition is:

where R = the distance of separation of two peaks, ∆Z = the difference of the 2 peaks’ retention time; tR = Retention time; W = Peak width. A resolution of 1.5 and any value greater than 1.5 designates complete peak separation. From the results obtained as shown in resolution values were mostly much greater than 1.5. Resolution values are tabulated as in Figs 31a-d:

150e6

125e6

100e6

75.0e6 22.405/407132420 27.068/156641574 50.0e6 28.894/114362240 28.753/75840620

25.0e6 31.278/62569318 21.567/95295621 22.617/22469664

10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0

Peak Retention time Resolution 1 21.567 - 2 22.405 5.421 3 22.617 1.720 4 27.068 53.910 5 28.753 18.960 6 28.894 1.440 7 31.278 21.840 Fig 31 a. Gas Chromatogram of U.S.A leaf extract, Rt = 27.068 min (Peak 4), 28.753 (Peak 5), 28.894 (Peak 6) and Rt = 31.278 (Peak 7).

Page 168 of 201 30e6 27.019/51253095 28.709/54288401

20e6 28.853/108263942 31.218/38181678 21.068/27693098 10e6 23.438/15015015 21.684/13198875

10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0

Peak Retention time Resolution 1 21.068 - 2 21.684 6.722 3 23.438 22.551 4 27.019 46.821 5 28.709 19.504 6 28.853 1.424 7 31.218 21.189

Fig 31 b. Gas Chromatogram of R.P.C. leaf extract, Rt = 27.019 (Peak 4), 28.709 (Peak 5), 28.853 (Peak 6) and Rt = 31.218 (Peak 7).

15.0e6

12.5e6

10.0e6 21.678/38395686

7500e3

5000e3 28.827/10173342 28.698/6890224 22.416/5784654 27.013/2395695 2500e3 21.079/3430499 21.592/2438899

10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0

Peak Retention time Resolution 1 21.079 - 2 21.592 - 3 21.678 - 4 22.416 9.265 5 27.013 54.656 6 28.698 18.633 7 28.827 1.303

Fig 31 c. Gas Chromatogram of Ginkgo capsule extract, Rt = 27.013 (Peak 5) and 28.827 (Peak 7)

Page 169 of 201

150e6

125e6

100e6

75.0e6 22.310/857334200 23.436/105672077 50.0e6 21.690/101686184 28.871/158691121 28.723/81348200 25.0e6 27.025/49386235 21.468/215277342

10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0

Peak Retention time Resolution 1 21.468 - 2 21.690 0.378 3 22.310 1.063 4 23.436 1.924 5 27.025 48.639 6 28.723 19.819 7 28.871 1.480

Fig 31 d. Gas Chromatogram of Ginkgo EGb 761 extraction, Rt = 27.025 (Peak 5), 28.723 (Peak 6) and Rt = 28.871 (Peak 7).

(i) R = undetectable to 53.910 in gas chromatogram of Ginkgo U.S. leaf extract.

(ii) R = undetectable to 46.821 in gas chromatogram of Ginkgo R.P.C. leaf extract.

(iii) R = undetectable to 34.002 in gas chromatogram of Ginkgo nut extract.

(iv) R = undetectable to 54.656 in gas chromatogram of Ginkgo capsule extract

(v) R = undetectable to 48.639 in gas chromatogram of Ginkgo EGb 761 extract.

Page 170 of 201 6.5.2.3.2 OPTIMIZATION AND IDENTIFICATION OF GINKGOLIC ACIDS

Prior to GC-MS analysis, samples were derivatized with the use of silylating

reagents. This resulted in the formation of derivatives (refer to Fig 17 for fragment ions

formed) or compounds that were more polar and more volatile than the original GA

compound.

This combination of properties allowed lower operational temperatures in GC

analysis while keeping adsorption of the compound onto the solid phase minimal,

resulting in increased sensitivity and better chromatography than the parent compound

under the same conditions.

Excess silylating reagents were used in this protocol compared with the study

carried out by Verotta et. al., 2000 due to lower column temperature used, i.e. 300 °C as compared to 340 °C used by them. This also ensured that GA analyte was the limiting reagent in the derivatisation procedure.

In this protocol, the column used had a thin film of stationary phase (low percentage of stationary phase on packed columns) and small inert diameters. Helium being a light gas gave the best results when used as a carrier gas in the least amount of time. Smaller injections of the concentrated extract (pre-concentration of the hexane fraction) gave better resolution in GC analysis.

Individual peaks were identified by comparing their retention time and mass spectrum to those of authentic samples. Their mass spectra were also compared with those stored in the database (Wiley library). Identification is only tentative when carried

out with one column, but is more conclusive when carried out on several columns with

different kinds of stationary phases for comparison. However this was not done in this

study due to lack of resources.

Page 171 of 201 Ginkgolic acid with unsaturated C15:1 at position 8 side chain (Irie et. al., 1996) was used as the external standard because of its highest abundance in Ginkgo biloba compared with other isomeric phenolic acids. Preliminary GC-MS analysis showed the presence of ginkgolic acid with unsaturated C15: 1 at position 8 side chain in all samples except the Ginkgo nut extract. This matched with results obtained by Irie et. al., 1996, who reported undetectable ∆8 15:1 side chain ginkgolic acid in Ginkgo nut extract.

Although the mass spectra of trimethylsilyl (TMS) derivatives obtained showed poor fragmentations and low intensity molecular ions, alkylphenols could still be identified as

GAs, due to the presence of strong ions at m/z 208 and 221. Fig. 32a-b showed the mass spectra of an authentic external standard used in GC-MS analysis. Presence of strong ions at m/z 208 & 221 was shown in Fig. 32a-b.

Fig 32 a. Mass spectra of external standard at Rt = 27.017 min

Fig 32 b. Mass spectra of external standard at Rt = 28.708 min, strong ions at m/z 208 and 221.

Page 172 of 201 Based on similar mass spectra or fragmentation pattern, GAs were found to be present at Rt = 27.068 min (Peak 4), 28.753 (Peak 5), 28.894 (Peak 6) and Rt = 31.278

(Peak 7) in gas chromatogram of U.S.A leaf extract (refer to Fig. 31a.). GAs were found to be present at Rt = 27.019 (Peak 4), 28.709 (Peak 5), 28.853 (Peak 6) and Rt = 31.218

(Peak 7) in gas chromatogram of R.P.C. leaf extract (refer to Fig. 31b.). GAs were found to be present at Rt = 27.013 (Peak 5) and 28.827 (Peak 7) in gas chromatogram of

Ginkgo capsule extract (refer to Fig. 31c.). GAs were found to be present at Rt = 27.025

(Peak 5), 28.723 (Peak 6) and Rt = 28.871 (Peak 7) in gas chromatogram of Ginkgo EGb

761 extract (refer to Fig. 31d.). Presence of strong ions at m/z 208 and 221 were close values to m/z 219 as obtained by Verotta & Peterlongo, 1993. The difference might be due to modifications carried out in this study. From this work, it is shown that GA content in Ginkgo products comply with the to 5 µg/g regulated by Commission E of the former Federal German Health Authority.

Both chromatographic techniques provide validated evidence for the presence of ginkgolic acids in Ginkgo biloba samples. Resolution is better achieved in GC as to

HPLC and mass detection sensitivity is higher than ultraviolet detection. Nevertheless, because GC-MS technique requires prior derivatisation and is not widely available in pharmaceutical quality control labs, a more convenient and less expensive approach is the HPLC-UV analytical technique. HPLC-UV is hence a more practical choice for industrial application and suitable for routine analysis because of its simplicity, sensitivity, accuracy and reproducibility.

Page 173 of 201 7 CONCLUSIONS

The major conclusions that may be drawn from this study are the following:

A. Ginkgo biloba has much potential as a functional ingredient in foods. The main

reasons for coming to this conclusion are that both the leaves and the nuts have

been shown to demonstrate antioxidant capacity (see Table 10), and high vitamins

content (see Table 8). Whilst a number of techniques for AOC measurement have

been examined, it was established that the ABTS cation decolorisation method was

the most appropriate in that it determines the scavenging ability of the free radicals

and the resulting products that are formed based on the decrease in the absorbance

unit at 414 nm (Section 6.1 and 6.2).

B. Heating of the Ginkgo nuts to above 90 °C causes a substantial loss of vitamin C

and produces a loss in AOC of around 40%. Vitamin C being a heat unstable

compound is lost during the early stages of heating but over 60% of the AOC of the

nuts remains and is therefore related to the heat-stable water-soluble compound(s),

most likely polyphenols. The data indicate that Ginkgo biloba nuts can be regarded

as nutritionally beneficial even when they have been cooked using the traditional

method of producing Cheng Teng, a traditional sweet dessert of Southeast Asia

(Section 6.2). Thus hypotheses A, B and C proposed in section 3.2 are true and

proven.

C. Ginkgo leaves infusion studies have concluded that the fermentation process had

no significant effect on the AOC of the Ginkgo biloba leaves. With the increased

surface area of leaves (leaves ground for 20 s), infusion temperature of 100 oC and

infusion time around 10 to 15 min it is able to yield the highest overall AOC

(section 6.3). This study has also investigated the presence of caffeine. Whilst,

Page 174 of 201 Ginkgo leaves infusion is caffeine free, it is capable of providing significant AOC.

Hypothesis D is thus true and Ginkgo leaves could well be developing into a

functional beverage similar to tea as regards their antioxidant activity.

D. Flavonoid recovery and stability from Ginkgo biloba when subjected to a

simulated digestion process is different depending on the types of product

examined. The results indicate a trend of conversion from the glycosides to the

aglycones and subsequent degradation for the commercial preparations. However,

for the raw leaves no conversion of glycosides to aglycones was observed. In

addition, the digestion process appears to increase the availability of the glycosides

compared with the undigested sample. For the EGb 761, the Quercetin glycosides

seem to have been converted to their aglycone, showing 4 times the amount

recovered from the control and lastly for the commercial capsules most of the

constituents were not recovered, indicating degradation of the flavonoids

constituents taking place. With the increase in popularity of dietary supplements as

a source of flavonoids with health benefits, this result is significant as it indicates

that ingesting the raw materials (in this case, the leaf of the Ginkgo biloba plant) is

a more efficient medium for the body to obtain the potentially beneficial flavonoids

(Section 6.4). However, whether hypothesis E or F is true or not has still to be

confirmed in view of the controversial subject of flavonoid absorption. This

research has not addressed the absorption issue but merely the digestion aspect of

the flavonoids. Hypothesis G is tested and proven to be true that HPLC is a

suitable method for analysing the Ginkgo biloba compounds as well as an

appropriate method to separate glycosides and aglycones.

E. The toxicological study in determining colchicine seems to suggest that Ginkgo is

relatively non-toxic when taken in moderation. It concludes absence of colchicine

Page 175 of 201 in Ginkgo leaf, nuts, standardised extract and commercial capsules. From this

research, no correlation is found to suggest taking of Ginkgo biloba leading to ill

effects that colchicine possess (Refer to section 2.4.1). The worry about presence

of colchicine in Ginkgo biloba may just be an unfounded consideration. This result

is consistent with Li et. al, 2002 as well the botanical literature which reports

Ginkgo supplements to contain no colchicine. This result provides further

assurance that the nuts fraction is free of colchicine and the myth that nuts

complicate pregnancy can be dispelled (Section 6.5). Hence hypothesis H tested, is

false and no colchicine, can be detected in the range of commercially available

Ginkgo capsules, Ginkgo leaves extract, and nuts examined

F. The analysis for GAs, using GC-MS, showed the presence of GA in all samples

except the Ginkgo nut extract. In contrast, HPLC analysis at 210nm detected GAs

in all Ginkgo samples. Therefore other forms of GAs may exist in Ginkgo nuts.

On the other hand, resolution is better achieved by GC compared with HPLC and

mass detection sensitivity is higher than ultraviolet detection. Nevertheless,

because GC-MS technique requires prior derivatisation, a more convenient and

cheaper approach is the HPLC-UV analytical technique. A better HPLC method

has been developed for the identification of GAs in all Ginkgo samples. Further

work is required for quantification and thus confirmation of regulation compliance

(Section 6.5). Hence hypothesis I is tested true to be presence of GA in a fast,

reproducible and accurate manner.

G. Whilst there are many reports on the benefits of Ginkgo biloba, its therapeutic

functions should now be considered in light of the data from the novelty of this

research on the simulated digestion process, as to which of the compounds within

the plant are actually available and absorbed to fulfil their ascribed properties.

Page 176 of 201 However care should be exercised in the interpretation of this study as no

consideration was given to the possible interaction that may exist with other

components present in the diet e.g. proteins, lipids, minerals etc. Also this study

did not investigate possible effects on flavonoids exerted by enzymes from the

Brush border membrane nor the microorganisms in the colon. This research has

also established a new HPLC method capable of identifying and quantifying in one

run, the 8 standard reference flavonoids (5 glycosides and 3 aglycones) used in the

study. With this method established, the conversion of glycosides to aglycones is

more easily monitored.

H. In view of time constraints and lack of availability of appropriates facilities it was

not possible to further develop the hormonal studies. (See Section 5.1.7.).

However this would seem to be an area worthy of more study and may provide

possible explanations on the beneficial mechanism of Ginkgo biloba in conditions

such as Alzheimer’s disease.

Through this work the author has been able to advance the knowledge base on

Ginkgo biloba. Following the extensive and critical literature review, areas of research were identified and addressed. Whilst it was possible to draw the conclusion identified above, the achieving of these has provided an excellent learning exercise in how to carry out research and the challenges that are faced during such a study. The execution of this work has provided insights into skills much beyond the technical and analytical skills, adaptability, disciplined, independence, flexibility and the ability to work with and learn from a variety of colleagues to name but a few. The importance of time management, being resourceful and working to tight deadlines has also been realised. Finally the

Page 177 of 201 ability to produce a concise and accurate report on experimental work and the ability to discuss results and draw conclusion has been developed.

Page 178 of 201 7.1 POTENTIAL FOR THIS RESEARCH

Information developed in this project has lead to an increased understanding on the antioxidant activity of Ginkgo nuts and other Ginkgo biloba parts that could bring about a therapeutic function and will help to ensure the sustained growth of this new segment of the market for functional foods. Information obtained in this research would also allow the understanding of the responsible component(s) in the Ginkgo nut fraction and make comparison to that of the standardised leaf extract where composition is available. Differences and similarities between leaf and nut composition have been examined. Increased comprehension on the responsible component(s) for the beneficial action Ginkgo biloba allows further understanding of the mechanisms that bring about the therapeutic function. Detailed studies on the antioxidant activity of Ginkgo nuts as a food product and Ginkgo leaves in the form of an infusion have increased awareness of its functional food potential. Examining the stability and recovery of flavonoids in Ginkgo biloba when subjected to a simulated digestive process have allowed an indication of whether those compounds are likely to be available for absorption. Research results may be useful to functional food companies and research & development personnel to directly enhance their knowledge and develop new products. Extensive understanding on the nuts antioxidant activity could well improve therapeutic benefits to consumers. This project may provide benefits to the following: 1. Functional food industry 2. Food development entrepreneurs 3. Consumers of functional foods 4. Research & Development scientists in food research 5. Food technologists 6. General public

Page 179 of 201 8 SUGGESTIONS FOR FURTHER STUDIES

8.1 GINKGO NUTS

To study the AOC of Ginkgo nuts cooked with sugar and water, which is the normal cooking practice for making dessert. This is to determine if sugar will bring about any implication or complication to the AOC level. Comparison could be made with the earlier studies to determine the difference in AOC level. This could also aid in probably changing the cooking methods or styles to optimise the AOC level.

To further establish the chemical profile of Ginkgo nuts so as to understand better the contributing compounds to its therapeutic function.

8.2 GINKGO LEAF INFUSION

To further establish and understand the chemical profile of Ginkgo leaf infusion, which later can be developed into a commercial functional beverage.

8.3 IN VIVO VS IN-VITRO ANTIOXIDANT DETERMINATION ASSAY

To elucidate a full profile of antioxidant activity against various reactive oxygen species (ROS)/reactive nitrogen species (RNS), such as O2 -•, HO•, and NO•, the development of different methods specific for each ROS/RNS is needed and to further investigate the reported benefits of Ginkgo flavonoids as in vivo antioxidants

8.4 SIMULATED DIGESTION PROCESS

It is important to note that the commercial products seem to have a higher level of aglycones. Thus, an important area of further research is to establish the validity of the absorption of glycosides vs aglycones in the human body and advice on the most effective form of Ginkgo biloba for functional food use

To further investigate the microbial changes to flavonoids in the lower gut

8.5 HORMONAL STUDIES

To understand and determine the estrogenic activities in Ginkgo biloba. The establishment of estrogenic activity by Ginkgo biloba may help to explain some of its therapeutic functions.

Page 180 of 201 REFERENCES

1) Adawadkar, P.D. and El Sohly, M. (1981). Isolation, purification and antimicrobial activity of ancardic acids from Ginkgo biloba fruits. Fitoterapia, 52, 129-135.

2) Afans, I.B., Dorozhko, A.L., Brodskii, A.V., Kostyuk, V.A., Potapovitch, A.I. (1989). Chelating and free radical scavenging mechanisms of inhibitory action of rutin and quercetin in lipid peroxidation. Biochem Pharmacol, 38, 1763-1769.

3) Ahlemeyer, B., Selke, D., Schaper, C., Klumpp, S., Krieglstein, J. (2001). Ginkgolic acids induce neuronal death and activate protein phosphatase type-2C. Eur. J. Pharmacol. 430, 1.

4) Alho, H., and Leinonen, J. (1999). Total antioxidant activity by chemiluminescence methods. Methods Enzymol 299:3-15.

5) Allen, R. G., Tresini, M. (2000). Pathways of Induction of Peroxiredoxin I Expression in Osteoblasts. Free Radic Biol Med., 28, 463-99.

6) Allender, W. J. (1982). Colchicine poisning as a mode of suicide. J. Forensic Sci. 27, 944-947.

7) Anonymous (1997). Great Pharmacopoeia of Traditional Chinese Medicine [in Chinese]. Shanghai, China: Shanghai Publishing House of Science and Technology.

8) Anonymous (2001). Combining nutrients for health benefits. Food Technology, 55, 2, 42-47.

9) Arenz, A. et. al., (1996). Occurrence of Neurotoxic 4’-O-Methylpyridoxine in Ginkgo biloba Leaves, Ginkgo Medications and Japanese Ginkgo Food. Planta Medica, 62, 548-551.

10) Arnao, M.B. (2000). Some methodological problems in the determination of antioxidant activity using chromogens radicals: a practical case. Trends in Food Science & Technology, 1-3.

11) Aruoma, O.I., Murcia, A., Butler, J. and Halliwell, B., (1993). Evaluation of the antioxidant and prooxidant actions of gallic acid and its derivatives. J Agric Food Chem 41:1880-1885.

12) Association of Vitamin Chemists (1966). Methods of Vitamin Assay. 3rd edition. USA: Interscience.

13) Auguet, M., De Feudis, V., & Clostre, F. (1982). Effects of Ginkgo biloba on arterial smooth muscles responses to vaso-active stimuli. Ge. Pharmacol. 13, 169-172.

14) Avila, V.L., Young, R., Benedicto, J., Ho, P., Kim, R. (1995). Extraction of Organic Pollutants from Solid Samples Using Microwave Energy. Anal. Chem., 67, p, 2096.

15) Aviram, M., Hardak, E., Vaya, J., Mahmood, S., Milo, S., Hoffman, A., Billicke, S., Draganov, D., Rosenblat, M. (2000). Human Serum Paraoxonases (PON1) Q and R Selectively Decrease Lipid Peroxides in Human Coronary and Carotid Atherosclerotic Lesions : PON1 Esterase and Peroxidase-Like Activities. Circulation May., 101, 2510-7.

16) Baker, W., & Ollis, W.D. (1961). Biflavonyls. In Ollis, W.D. (Ed.) Recent developments in the Chemistry of natural phenolic compounds. Pergamon, New York, 152-184.

17) Baratta, M.T., Dorman, H.J. Deans, S.G., Figueiredo, A.C., Barroso, J.G., & Ruberto, G., (1998). Antimicrobial and antioxidant properties of some commercial essential oils. Flavour Fragr. J., 13 235–244.

18) Barrett-Connor, E., Chang, J.C., & Edelstein, S.L. (1994). Coffee-associated osteoporosis offset by daily milk consumption. JAMA, 271, 280.

Page 181 of 201 19) Barth, S.A., Inselmann, G., Engemann, R., Heidemann, H.T. (1991). Influences of Ginkgo biloba on cyclosporin A induced lipid peroxidation in human liver microsomes in comparison to vitamin E, glutathione, and N-acetylcysteine. Biochem Pharmac, 41, 1521-1526.

20) Basaga, H., Tekkaya, C. and Acikel, F., (1997). Antioxidative and free radical scavenging properties of rosemary extract. Lebensm Wiss Technol 30:105-108.

21) Bauer, R., & Zschocke, S., (1996). Medisinische Anwendung von Ginkgo biloba L. Z.. Phytotherapy., 17, 275-283.

22) Bauer, U. (1984). 6-monthdouble-blind randomised clinical trial of Ginkgo biloba extract versus placebo in two parallel groups in patients suffering from peripheral arterial insufficiency. Arzneim. Forsch, 34, 716.

23) Benzie, I.F.F., and Strain, J.J. (1996). The ferric reducing ability of plasma (FRAP) as a measure of `antioxidant power': the FRAP assay. Anal Biochem 239:70-76.

24) Benzie, I.F.F., and Strain, J.J. (1999). Ferric reducing/antioxidant power assay: direct measure of total antioxidant activity in biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. Methods Enzymol 299:15-27.

25) Birkle, D.L., Kurian, P., Braquet, P., & Bazan, N.G., (1988). Platelet-activating factor antagonist BN52021 decreases accumulation of free polyunsaturated fatty acid in mouse brain during ischemia and electroconvulsive shock. Journal of Neurochemistry, 51(6), 1900-1905.

26) Blois, M.S. (1958). Antioxidant determination by the use of a stable radical. Nature 4617:1199- 1200.

27) Bolis, M. (1939). Dermatitis venenata due to ginkgo berries. Arch Derm Syph, 39, p, 530.

28) Borman, S. (2001). Toxin reported in supplements. Chem. Eng. News 79, 33-34.

29) Bors, W., Heller, W., Michel, C., & Saran, M. (1990). Flavonoids as antioxidants: determination of radical-scavenging efficiencies. Methods Enzymol., 189, 343-355.

30) Borzeix, M.G.; Labos, M., & Hartl, C. (1980). Recherches sur l’action antiagrepant de l’extrait de Ginkgo biloba, Activite au niveau des arteres et des veines de la piemaere chez le lapin. Sem. Hop. Paris, 56, 393.

31) Bradly, P., Jacobs, M.D., Warren, S., & Browner (2000). Ginkgo biloba: A Living Fossil. Am. J Med., 108, 341-342.

32) Brand-Williams, W., Cuvelier, M.E., and Berset, C. (1995). Use of a free radical method to evaluate antioxidant activity. Lebensm Wiss Technol 28:25-30.

33) Bridi, R., Croseetti, F.P., Steffen, V.M., & Henriques, A.T. (2001). The antioxidant activity of standardised extract of Ginkgo biloba (EGb761) in Rats. Phytotherapy Research, 15, 449-451.

34) Brill, S., & Dean, E. (1994). Identifying and harvesting edible and medicinal plants in wild (and not so wild) places. New York: Hearst Books, 1st Edition.

35) Burton, G.W., & Ingold, K.U. (1981). Autoxidation of biological molecules. I. The antioxidant activity of vitamin E and related chain-breaking phenolic antioxidants in vitro. J Am Chem Soc, 103:6472-6477.

36) Camire, et. al., (2001). Diet and health research needs. Food Technology, 55, 5, 189-191.

37) Cano, A., Acosta, M. & Arnao, M.B. (2000). A method to measure antioxidant activity in organic media: application to lipophilic vitamins. Redox report, 5, 6, 365-370.

38) Cao, G., & Prior, R. L. (1999). The measurement of oxygen radical absorbance capacity in biological samples. Methods Enzymol,. 299, 50-62.

Page 182 of 201 39) Cao, G., & Prior, R.L. (1998). “Comparison of different analytical methods for assessing total antioxidant capacity of human serum”, Clinical Chemistry, 44(6), 1309-1315.

40) Cao, G., Alessio, H.M., and Cutler, R.G. (1993). Oxygen-radical absorbance capacity assay for antioxidants. Free Radicals Biol Med 14:303-311.

41) Cao, Y., Lou, C., Fang, Y. & Ye, J. (2001). Determination of active ingredients of Rhododendron dauricum L. by capillary electrophoresis with electrochemical detection. Journal of Chromatography A, In Press, Uncorrected Proof, Available online 9 November 2001.

42) 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.

43) Chen, S. (1998). Aromatase and breast cancer, Front. Biosci. 3, 922–933.

44) Chevion, S., Berry, E. M., Kitrossky, N., Kohen, R. (1997). Evaluation of plasma low molecular weight antioxidant capacity by cyclic voltammetry. Free Radic Biol Med., 22, 411-21.

45) Choi, H. S., & Moore, D. D. (1993). Induction of c-fos and c-jun gene expression by phenolic antioxidants. Mol Endocrinol., 7, 1596-602.

46) Choi, H.S., Song, H.S., Ukeda, H., & Sawamura, M. (2000). Radical-scavenging activities of citrus essential oils and their components: detection using 1,1-diphenyl-2-picrylhydrazyl. J. Agric. Food Chem. 48, 4156–4161.

47) Chu (1999). Ginkgo biloba, Bai Guo. http://alternativehealing.org/ginkgo.htm

48) Clairambault, P. et. al., (1986). Effet de l’extrait de Ginkgo biloba sur les lesion induites par une photocoagulation au laser a l’argon sur la retine de lapin. Sem. Hop. Paris, 62, 57.

49) Clevenger, C. V., August, T. F., and Shaw, L. M. (1991). Colchicine poisoning: report of fatal case with body fluid analysis by GCMS and histopathologic examination of postmortem tissue. J. Anal. Toxicol. 15, 151-154.

50) Cody, V. (1988). Crystal and molecular structure of flavonoids. In Plant Flavonoids in Biology and Medicine II: Biochemical, Cellular, and Medicinal Properties, p. 29-44, Alan R. Liss. New York, NY.

51) Corradini, C. & Cavazza, A. (1998). Application of capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) in food analysis. Italian Journal of Food Science, 10, 4, 299-316.

52) Costantino, L., Albasino, A., Rastelli, G. and Benvenuti, S. (1992). Activity of polyphenolic crude extracts as scavengers of superoxide radicals and inhibitors of xanthine oxidase. Planta Med 58:342-344.

53) Crawford, D. R., Schools, G. P., Salmon, S. L., Davies, K. J. (1996). Hydrogen peroxide induces the expression of adapt15, a novel RNA associated with polysomes in hamster HA-1 cells. Arch Biochem Biophys., 325, 256-64.

54) Dadakova, E., Prochazkova, E., & Krizek, M. (2001). Application of micellar electrokinetic capillary chromatography for quantitative analysis of quercetin in plant materials. Electrophoresis, 22, 8, 2001, 1573-1578.

55) Dai, Y. & Luo, X.Y. (1996). Functional food in China. Nutrition reviews, 54, S21-S23.

56) Dapkevicius, A., van Beek, T.A., Linssen, J.P.H. & Venskutonis, R. (1998). Rapid spectroscopic screening for antioxidant activity in sage, thyme and oregano isolates with the b-carotene-linoleic acid model system, In: P. Schreier, M. Herderich, H.-U. Humpf and W. Schwab (eds.) Natural Product Analysis. Chromatography – spectroscopy – biological testing, Würzburg, 235-237.

Page 183 of 201 57) Das, M., Ray, P.K. (1988). Lipid antioxidant properties of quercetin in vitro. Biochem Int. 17, 203- 209.

58) Davies, J.R., (1999). In a nutshell Ginkgo – Ginkgo biloba, in Miranda Spicer (Ed.), Element Books Limited, Boston, USA.

59) DeFeudis, F.V. (1991). Ginkgo biloba Extract EGb761, Pharmacological Activities and Clinical Applications Elsevier, Paris.

60) DeFeudis, F.V. (1998). Ginkgo biloba extract (EGb 761) From Chemistry to the Clinic. Ullstein Medical, USA.

61) Del Tredici, P. (1991). Ginkgos and People – A thousand years of interaction. Arnoldia, 51, 2-15.

62) Delange, R.J. and Glazer, A.N. (1989). Phycoerythrin fluorescence-based assay for peroxyl radicals: a screen for biologically relevant protective agents. Anal Biochem 177:300-306.

63) Devary, Y., Gottlieb, R. A., Lau, L. F., Karin, M. (1991). Rapid and preferential activation of the c-jun gene during the mammalian UV response. Mol Cell Biol., 11, 2804-11.

64) Doly, M., Droy-Lefaix, M.T., Braquet, P. (1992). Oxidative stress in diabetic retina. EXS, 62, 299- 307.

65) Drieu, K. (1986). Preparation at definition de l’extrait de Ginkgo biloba. La Presse Medicale, 31, 1455-1457.

66) Droy-Lefaix, M.T., Bonhomme, B., Doly, M. (1991). Protective effect of Ginkgo biloba extract (EGB 761) on free radical-induced changes in the electroretinogram of isolated rat retina. Drugs Exp Clin Res, 17, 571-574.

67) Eisenberg, D.M. et. al., (1998). Trends in alternative medicine use in the United States. JAMA, 280, 18, 1569-1575.

68) Ellenhorn, M. J., and Barceloux, D.G. (1988). Medical Toxicology-Diagnosis and Treatment of Human Poisoning, Elsevier, Amsterdam.

69) Ellnain-Wojtaszek, M., Kruczyński, Z., & Kasprzak, J. (2002). Variations in the free radical scavenging activity of Ginkgo biloba L. leaves in the period of complete development of green leaves to fall of yellow ones. Food Chemistry, 79, 1, 79-84.

70) Ellnain-Wojtaszek, M.; Zgorka, G. (1999). High-performance liquid chromatography and thin-layer chromatography of phenolic acids from Ginkgo biloba L. leaves collected within vegetative period. Journal of Liquid Chromatography & Related Technologies, 22(10), 1457-1471.

71) el-Saadani, M., Esterbauer, H., el-Sayed, M., Goher, M., Nassar, A. Y., Jurgens, G. J. (1989). A spectrophotometric assay for lipid peroxides in serum lipoproteins using a commercially available reagent. Lipid Res., 30, 627-30.

72) Emerit, I., Arutyunyan, R., Oganesian, N., et. al., (1995). Radiation-induced clastogenic factors: anticlastogenic effect of Ginkgo biloba extract. Free Rad Biol Med,18, 985-991.

73) Ernst, E. and Stevinson, C. (1999). Ginkgo biloba for tinnitus: a review. Clinical Otolaryngology; 24, 164-7.

74) Esterbauer, H., Striegl, G., Puhl, H., Rotheneder, M. (1989). Continuous monitoring of in vitro oxidation of human low density lipoprotein. Free Radic Res Commun., 6, 67-75.

75) Etienne, et. al., (1982). Comparison des effetsd’un extrait de Ginkgo biloba et de la chloropromazine sur la fragilite osmotique, in vitro, d’erythrocytes de rat. J. Pharmacol Paris 13, 291.

Page 184 of 201 76) Farnsworth, N. R. (2002). NAPRALERT database. University of Illinois at Chicago, IL (an on-line database available directly through the University of Illinois at Chicago or through the Scientific and Technical Network (STN) of Chemical Abstracts Services) (last accessed May 1, 2002).

77) Folpini, A., and Furfoni, P. (1995). Colchicine toxicology-clinical features and treatment, massive overdose case report. Clin. Toxicol. 33, 71-77.

78) Fonseca, F.N., Kato, M.J., Oliveira Junior, L., Pinto Neto, N., & Tavares, M.F.M. (2001). Critical assessment of electrolyte systems for the capillary electrophoresis analysis of phenolic compounds in herbal extracts, Journal of Microcolumn Separations, 13, 6, 227-235.

79) Food Facts Asia 3rd Quarter (2000). Caffeine content of Food & Beverages. Issue 9, Available: http://www.afic.org/article.asp?SearchMethod=Search&ArticleId=187&CollectionType=1

80) Forni, L. G., Morla-Arellano, V. O., Packer, J. E, Willison, R. L (1986), “Nitrogen dioxide and related free radicals: electron transfer reactions with organic compounds in solutions containing nitrite and nitrate”, J. Chem. Soc., Vol 2, 1-6

81) Fosters, S., & Chongxi, Y. (1992). Herbal emissaries. Healing Arts Press, Rochester, Vermont.

82) Frankel, E.N. & Meyer, A.S. (2000). The problems of using one-dimensional methods to evaluate multifunctional food and biological antioxidants. J. Agric. Food Chem., 80, 1925-1941.

83) Frankel, E.N. (1998). Lipid Oxidation. The Oily Press, Dundee.

84) Fu-shun, Y., Evans, K.A., Stevens, L.H., van Beek, T.A. and Schoonhoven, L.M. (1990). Deterrents extracted from the leaves of Ginkgo biloba: effects on feeding and contact chemoreceptors. Entomol. exp. appl., 54, 57-64.

85) Fuzzati, N., Pace, R. and Villa, F. (2003). A simple HPLC-UV method for the assay of ginkgolic acids in Ginkgo biloba extracts. Fitoterapia. 1-10.

86) Gardes-Albert, M., Ferradini, C., Sekaki, A., & Droy-Lefaix, M.T. (1993). Oxygen-centered free radicals and their interactions with EGb 761 or CP 202. In: Ferradini C, Droy-Lefaix MT, Christen Y, (Eds). Advances in Ginkgo biloba extract research, Paris: Elsevier, 1-11.

87) Geiger, H. & De Groot- Pfleiferer, W. (1979). Die flavon- und flavonolglykoside von Taxodium distichum. Phytochemistry, 18, 1709-1710.

88) Gellerman, J.L. and Schlenk, H. (1968). Methods for isolation and determination of anacardic acids. Anal.Chem., 40, 739-743.

89) Gerster, H. (1989). Antioxidant vitamins in cataract prevention. Z Ernahrungswiss, 28, 56-75.

90) Gessner, B.; Voelp, P.l & Klesser, M. (1985). Study of the long-term action of a Ginkgo biloba extract on vigilance and mental performance as determined by means of quantitastive pharmaco- EEG abd psychometri measurements. Arzneim Forsch. 35, 1459.

91) Ghadially, F.N. (1988a). Myelinoid membranes, myelin figures and myelinosomes. In: Ghadially, F.N. (E.d.), Ultrastructural Pathology of Cell and Matrix, Third ed. Butterworks, London, p, 191- 328.

92) Ghadially, F.N. (1988b). Mitochondria. In: Ghadially, F.N. (E.d.), Ultrastructural Pathology of Cell and Matrix, Third ed. Butterworks, London, p, 191-328.

93) Glazer, A.N. (1988). Fluorescence-based assay for reactive oxygen species: a protective role for creatinine. FASEB J 2:2487- 2491.

94) Glazer, A.N. (1990). Phycoerythrin fluorescence-based assay for reactive oxygen species. Methods Enzymol 186:161-168.

Page 185 of 201 95) Goh, M. L., & Barlow P.J. (2002). Antioxidant capacity in Ginkgo biloba. Food Research International,35, 815-820.

96) Goh, M. L., & Barlow P.J. (2004). Flavonoid recovery and stability from Ginkgo biloba subjected to a simulated digestion process. Food Chemistry,86, 195-202.

97) Goh, M.L., Barlow, P.J. & Yong, C.S. (2003). Examination of Antioxidant Activity of Ginkgo biloba Leaf Infusions. Food Chemistry, 82, 275-282.

98) Gohil, K., & Packer, L. (2002). Global gene expression analysis identifies cell and tissue specific actions of Ginkgo biloba extract, EGb 761. Cell Mol Biol (Noisy-le-grand). Sep;48(6): 625-31.

99) Grassmann, J., Hippeli, S., Dornisch, K., Rohnert, U., Beuscher, N., & Elstner, E.F. (2000). Antioxidant properties of essential oils: possible explanation for their anti-inflammatory effects. Drug Res., 50, 135–139.

100) Grassmann, J., Hippeli, S., Elstre, E.F., (2002). Plant’s defence mechanism and its benefits for animals and medicine: role of phenolics and terpenoids in avoiding oxygen stress. Plant Physiology and Biochemistry, 40, 471–478.

101) Grassmann, J., Schneider, D., Weiser, D., & Elstner, E.F. (2001). Antioxidative effects of lemon oil and its components on copper induced oxidation of low density lipoprotein. Drug Res., 51, 799–805.

102) Halliwell, B. (1990). How to characterise a biological antioxidant. Free Rad. Res. Commun, 9, 1- 32.

103) Handelman, G.J., et.al. (1999). Antioxidant capacity of oat (Avena sativa L.) extracts. 1. Inhibition of low-density lipoprotein oxidation and oxygen radical absorbance capacity. J Agric Food Chem 47:4888-4893.

104) Haramaki, N. Aggarwal, S. Kawabata, T. Droy- Lefaix, M.T., Packer, L. (1994). Effects of natural antioxidant Ginkgo biloba extract (EGb 761) on myocardial ischemia-reperfusion injury. Free Radic Biol Med., 16, 789-794.

105) Harland B.F. (2000). Caffeine and Nutrition. Nutrition, 16, no. 7/8, 522-526.

106) Harman, D. (1996a). A hypothesis on the pathogenesis of Alzheimer’s disease. Ann. N. Y. Acad. Sci., 786, 152-168.

107) Harman, D. (1996b). Aging and disease: Extending the functional life span. Ann. N. Y. Acad. Sci., 786, 321-336.

108) Harris, S., & Dawson-Hughes, B. (1994). Caffeine and bone loss in healthy post-menopausual women. Am. J. Clin. Nutr., 60, 573.

109) Haslam E. Ed. (1993). Shikimic acid metabolism and metabolites. Wiley & Sons, Chichester:, UK.

110) Hasler, A., Gross, G., Meier, B., & Sticher, O. (1992). Complex flavonol glycosides from the leaves of Ginkgo biloba. Phytochemistry, 31, 1391-1394.

111) Hasler, A., Sticher, O., Meier, B., (1992). Identification and determination of the flavonoids from Ginkgo biloba by high-performance liquid chromatography, Journal of Chromatography, 605, 41- 48.

112) Hasler, C.M. (1996). Functional Foods: The Western Perspective. Nutrition Reviews, 54, 11, S6- S10.

113) He, X.G., Bernart, M.W., Nolan, G.S., Lin, L.Z., and Lindenmaier, M.P. (2000). High Performance Liquid Chromatography-Electrospray Ionisation-Mass Spectrometry Study of Ginkgolic Acid in the leaves and Fruits of the Ginkgo Tree (Ginkgo biloba). J. Chromatographic Science. 38:169-173.

Page 186 of 201 114) Hensley, K., Robinson, K. A., Gabbita, S. P., Salsman, S., Floyd, R. A. (2000). Reactive oxygen species, cell signaling and cell injury. Free Radic Biol Med., 29, 1456-62.

115) Hertog, M.G.L., & Katan, M.B. (1998), in Rice-Evans, C.A., & Lester, P. (Ed.) Flavonoids in Health and Disease, Marcel Dekker, New York, 447-467

116) Hertog, M.G.L., Hollman, P.C.H., & Katan, M.B. (1992). Content of potentially anticarcinogenic flavonoids of 28 vegetables and 9 fruits commonly consumed in the Netherlands. J Agric Food Chem; 40: 2379-83.

117) Hobson, C. H., and Rankin, A. P. (1986). A fatal colchicine overdose. Anaesth. Intensive Care 14, 453-455.

118) Hollman P.C.H., & Katan, M.B. (1997). Adsorption, metabolism and health effects of dietary flavonoids in man. Biomed & Pharmacother, 51, 305-310.

119) Hollman, P.C.H., & Katan, M.B. (1999). Dietary flavonoids: intake, health effects and bioavailability. Food Chem Toxicol, 37: 937-42

120) Hollman, P.C.H., de Varies, J.H.M., van Leeuwen, S.D., Mengelers, M.J.B., & Katan, M.B. (1995). Absorption of dietary quercetin glycosides and quercetin in healthy ileostomy volunteers. Am J Clin Nutr, 62: 1276-82.

121) Hollman, P.C.H., de Varies, J.H.M., van Leeuwen, S.D., Mengelers, M.J.B., & Katan, M.B. (1995). Absorption of dietary quercetin glycosides and quercetin in healthy ileostomy volunteers. Am J Clin Nutr, 62: 1276-82.

122) Hollman, P.C.H., et. al. (1997). Relative bioavailability of the antioxidant flavonoids quercetin from various foods in man. FEBS Letters, 418, 152-156.

123) Hollman, P.C.H., van Trijp, J.M.P. Mengelers, M.J.B. de Vries, J.H.M., Katan, M.B. (1997). Bioavailability of the dietary antioxidant flavonol quercetin in man. Cancer Letters, 114, 139-140.

124) Husain, S.R., Cillard, J., & Cillard, P. (1987). Hydroxyl Radical scavenging activity of flavonoids. Phytochemistry, 26, 2489-2491.

125) Ingold, K.U. (1968). Inhibition of antoxidation. Adv. Chem. Ser, 75, 296-305.

126) Irie, J., Murata, M., & Homma, S. (1996). Glycerol-3-phosphate dehydrogenase inhibitors, anacardic acids, from Ginkgo biloba. Bioscience, Biotechnology, and Biochemistry, 60, 240-243.

127) Itokawa, H., et. al. (1987). Antitumor principles from Ginkgo biloba L. Chem. Pharm. Bull., 35, 3016-3020.

128) Iwaoka, W.T., & Brewer, M.S. (2000). Toxicants, Chapter 15, pp. 241-260, In Food Chemistry: Principles and application, eds. Christen G.L. & Smith J.S. Science Technology System, West Sacramento, California.

129) Jaggy, H. and Koch, E. (1997). Chemistry and biology of alkylphenols from Ginkgo biloba L. Pharmazie, 52, 735-738.

130) Kawamura, J. (1928). Chemical constituents of the fruit of Ginkgo biloba. Jap. J. Chem. 3, 89-108.

131) Kaye, A.D. et. al., (2000). Herbal Medicines: Current trends in Anesthesiology Practice – A Hospital Survey. Journal of Clinical Anesthesia, 12, 468-471.

132) Kirshner, M. W. (1978). Microtubule assembly and nucleation. Int. Rev. Cytol. 54, 1-65.

133) Kleijnen, J., & Knipschild, P. (1992). Ginkgo biloba. Lancet, 340, 1136-1139.

134) Kleijnen, J., & Knipschild, P. (1992a). Ginkgo biloba for cereal insufficiency. Br J Clinical Pharmaco., 34, 352-358.

Page 187 of 201 135) Kleijnen, J., & Knipschild, P. (1992a). Ginkgo biloba for cereal insufficiency. Br J Clinical Pharmaco., 34, 352-358.

136) Kleijnen, J., & Knipschild, P. (1992b). Ginkgo biloba. Lancet, 340, 1136-1139.

137) Knekt, et. al. (2002). Flavonoids intake and risk of chronic diseases. Am J Clin Nutr; 76: 560-8.

138) Knekt, et. al. (2002). Flavonoids intake and risk of chronic diseases. Am J Clin Nutr; 76: 560-8.

139) Kohen, R., Vellaichamy, E., Hrbac, J., Gati, I., & Tirosh, O. (2000). Quantification of the overall reactive oxygen species scavenging capacity of biological fluids and tissues. Free Radic Biol Med., 28, 871-9.

140) Koltai, M. (1991). Platelet activating factor. A review of its effects, antagonists and possible future clinical implications (Part II). Drugs, 42, 174-204.

141) Kornecki, E., Ehrlich, Y.H. (1988). Neuroregulatory and neuropathological actions of the ether- phospholipid platelet-activating factor. Science, 240(4860), 1792-1794.

142) Kubo, I., Kinst-Hori, I. and Yokokawa, Y (1994). Tyrosinase inhibitors from Anacardium occidentale. J. Nat. Prod., 57, 545-551.

143) Kubo, I., Komatsu, S. and Ochi, M. (1986). Molluscicides from the cashew Anacardium occidentale and their large scale isolation. J. Agric. Food Chem, 34, 970-973.

144) Kubo, I., Muroi H and Kubo A (1995). Structural functions of antimicrobial long-chain alcohols and phenols. Bioorg. Med. Chem. 3, 873-880.

145) Kubo, I., Ochi, M., Viera P.C. and Komatsu, S. (1993). Antitumor agents from the cashew Anacardium occidentale apple juice. J. Agric. Food Chem. 41, 1012-1015.

146) Kuhnau, J. (1976). The flavonoids. A class of semi-essential food components: their role in human nutrition. World Rev. Nutr Diet; 24: 117-191.

147) Kulomaa, A., Sirén, H. & Riekkola, M-L. (1997). Identification of antioxidant compounds in plant beverages by capillary electrophoresis with the marker index technique. Journal of Chromatography A, 781, 523-532.

148) Langley-Evans, S.C. (2000), “Antioxidant potential of green and black tea determined using the ferric reducing power (FRAP) assay”, International Journal of Food Sciences and Nutrition, Vol 51, 181-188.

149) Larrauri, J.A., Sanchez-Moreno, C., and Saura-Calixto, F. (1998). Effect of temperature on the free radical scavenging capacity of extracts from red and white grape pomace peels. J Agric Food Chem 46:2694-2697.

150) Larrauri, J.A., Sanchez-Moreno, C., Ruperez, P., and Saura-Calixto, F. (1999). Free radical scavenging capacity in the aging of selected red Spanish wines. J Agric Food Chem 47:1603-1606.

151) Larsen, R.G., Dupeyron, J.P., and Boulu, R.G. (1978). Modele d’ischemie cerebrale experimentale par microsphere chez le rat. Etude de l’effect de deux extrait de Ginkgo biloba et du naftidrofuril. Therapie 33, 651.

152) Le Poncin-Lafitte, M., Martin, P., Lespinasse, P., & Rapin, J.R. (1982). Ischemie cerebral après ligature non-simultaneous des arteres carotids chez le rat: Effet de l’extrait de Ginkgo biloba. Sem Hop, Paris 58, 403.

153) Le Poncin-Lafitte, M., Rapin, J., & Rapin, J.R. (1980). Effects of Ginkgo biloba on changes induced by quantitative cerebral microembolization in rats. Arch.Int. Pharmacodyn. 243-236.

Page 188 of 201 154) Lebuisson, D.A., Leroy, L., Rigal, G. (1986). Treatment of senile macular degeneration with Ginkgo biloba extract. A preliminary double-blind drug vs. placebo study. Presse Med, 15, 1556- 1558.

155) Lee, Bee-Lan & Ong, Choon-Nam (2000). Comparative analysis of tea catechins and theaflavins by high-performance liquid chromatography and capillary electrophoresis. Journal of Chromatography A, 881, 1-2, 439-447.

156) Lee, Y.S., Lorenzo, B.J. , Koufis, T. , Reidenberg, M.M. (1996). Grapefruit juice and its flavonoids inhibit 11 beta-hydroxysteroid dehydrogenase, Clin. Pharmacol. Ther. 59, 62–71.

157) Li, D., Oh, J.R., Park, J. (2003). Direct extraction of alkylphenols, chlorophenols and bisphenol A from acid-digested sediment suspension for simultaneous gas- chromatographic-mass spectrometric analysis. J. Chromatogr. A. 1012, 207-214.

158) Li, H.L. (1956). A horticultural and botanical history of Ginkgo. Bull Morris Arb. 7, 3-12.

159) Li, Kwei-Lan & Sheu, Shuenn-Jyi (1995). Determination of flavonoids and alkaloids in the scute- coptis herb couple by capillary electrophoresis. Analytica Chimica Acta, 313, 1-2, 113-120.

160) Li, W., Sun, Y., Fitzloff, J.F., and van Breemen, R.B. (2002). Evaluation of Commercial Ginkgo and Echinacea Dietary Supplements for Colchicine Using Liquid Chromatography-Tandem Mass Spectrometry. Chem. Res. Toxicol., 15, 1174-1178.

161) Liebich, H.M. et. al., (1998). Analysis of traditional Chinese anticancer drugs by capillary electrophoresis. Journal of Chromatography A, 795, 388-393.

162) Lloyd, T., Rollings, N., Eggli, D.F., Kieselhorst, K., & Chinchilli, V.M. (1997). Dietary caffeine intake and bone status of postmenopausal women. Am. J. Clin. Nutr., 65, 1826.

163) Luciani, I. (1989). Case review: Fatal IV colchicine injection in a 60-year-old woman. J. Emerg. Nursing 15, 80-82.

164) Lunder, T.L. (1992). Catechins of green tea: antioxidant activity. In Phenolic compounds in Food and their Effects on Health. II. Antioxidants and Cancer Prevention, eds M.T. Huang, C.T. Ho and C.Y. Lee. American Chemical Society, Washington, pp. 114-120.

165) Maitra, I., Marcocci, L., Droy-Lefaix, M.T., Packer, L. (1995). Peroxyl radical scavenging activity of Ginkgo biloba extract EGb 761. Biochem Pharmac, 49, 1649-1655.

166) Maitra, I., Marcocci, L., Packer, L., & Droy-Lefaix, M.T. (1995). The nitric oxide-scavenging activity of Ginkgo biloba extracts EGb 761. Biochem Pharmacol., 45, 1649-1665.

167) Major, R.T. (1967). The Ginkgo, the most ancient living tree: The resistance of Ginkgo biloba L. to pests accounts in part for the longevity of this species. Science, 157, 1270-1273.

168) Malaspina, A. (1996). Functional Foods: Overview and Introduction. Nutrition Reviews, 54, 11, S4- S5.

169) Mar, C., and Bent, S. (1999). An evidence-based review of the 10 most commonly used herbs. West J. Med. 171, 168-171.

170) Marcocci, L., Maguire, J.J., Packer, L., & Droy-Lefaix. M.T. (1994a). The nitric oxide-scavenging properties of Ginkgo biloba extract EGb761. Biochem Biophys Res Commun, 201, 748-755.

171) Marcocci, L., Packer, L., Droy-Lefaix, M.T., Sekaki, A.H., & Gardes-Albert, M. (1994b). Antioxidant actions of Ginkgo biloba extract EGb761. In: Packer, L, (Ed). Methods in Enzymology, San Diego: Academic Press, 462-475.

172) Matsumoto, T. and Sei, T. (1987). Antifeedant activities of Ginkgo biloba L. Components against the Larva of Pieris rapae crucivora. Agric. Biol. Chem. 51, 249-250.

Page 189 of 201 173) Maurer, K., Ihl, R., Dierks, T. & Frolich L. (1997). Clinical efficacy of Ginkgo biloba special extract EGb761 in dementia of the Alzheimer type. J. Psychiat, 31, 6, 645-655.

174) McIntyre, I. M., Ruszkiewicz, A. R., Crump, K., and Drummer, O. H. (1994). Death following colchicine poisoning. J. Forensic Sci. 39, 280-286.

175) Melzheimer V. and Lichius J.J. (2000). Ginkgo biloba L.: Aspects of the Systematical and Applied Botany. Ginkgo biloba, in T.A van Beek (Ed.), Medicinal and Aromatic Plants – Industrial Profiles, Vol, 12, Harwood, Amsterdam, p, 25- 48.

176) Miller, N.J., Diplock, A.T., and Rice-Evans, C.A. (1995). Evaluation of the total antioxidant activity as a marker of the deterioration of apple juice on storage. J Agric Food Chem 43:1794- 1801.

177) Miller, N.J., Rice-Evans, C.A., Davies, M.J., Gopinathan, V., and Milner, A. (1993). A novel method for measuring antioxidant capacity and its application to monitoring the antioxidant status in premature neonates. Clin Sci 84:407-412.

178) Mittag, T. (1984). Role of oxygen radicals in ocular inflammation and cellular damage. Exp Eye Res, 39, 759-769.

179) Miura, H., Kihara, T., & Kawano, N. (1969). Studies on biflavones in the leaves of podocarpus macrophylla and P. nagi. Chem Pharm Bull, 17, 150-154.

180) Moon, J-H, and Terao J. (1998). Antioxidant activity of caffeic acid and dihydrocaffeic acid in lard and human low-density lipoprotein. J Agric Food Chem 46:5062-5065.

181) Morin, P.H., & Dreux, M. (1993). Factors influencing the separation of ionic and non-ionic chemical natural compounds in plant extracts by capillary electrophoresis. Journal of Liquid Chromatography,16, 17, 3735-3755.

182) Moro, A., Paya, M., Rios, J.L., & Alcarza, M.J. (1990). Structure-activity relationship pf polymethoxyflavones and other flavonoids as inhibitors of non-enzymic lipid peroxidation. Biochem. Pharmacol, 40, 793-797.

183) Moskowitz, Howard (1988), Applied Sensory Analysis of Foods Volume I, CRC Press.

184) Naidus, R. M., Rodvien, R., and Mielke, C. H. (1977) Colchicine toxicology. Arch. Inern. Med. 137, 394-396.

185) Nasr, C., Haag-Berrurier, M., Lobstein-Guth, A., & Anton, R. (1986). Kaempferol coumaroyl glucorhamnoside from Ginkgo biloba. Phytochemistry, 25, 770-771.

186) Nasr, C., Lobstein-Guth, A., Haag-Berrurier, M., & Anton, R. (1987). Quercetin coumaroyl glucorhamnoside from Ginkgo biloba. Phytochemistry, 26, 2869-2870.

187) National Institutes of Health. (2001). http://www.healthlink.mcw.edu/article/983211401.html

188) Neilson, S.S. (1998). Food Analysis. (2nd edition), Gaithersburg, MD : Aspen Publishers.

189) Newall, C.A., Anderson, L.A., Phillipson, J.D., (1996). Herbal medicines, The Pharmaceutical Press, London, pp. 138-140.

190) Nose, K., Shibanuma, M., Kikuchi, K., Kageyama, H., Sakiyama, S., Kuroki, T. (1991). Transcriptional activation of early-response genes by hydrogen peroxide in a mouse osteoblastic cell line. Eur J Biochem., 201, 99-106.

191) O’Reilly, J. (1993). Ginkgo biloba- cultivation, extraction and therapeutic use of the extract., in van Beek, T.A., and Breteler, H., (eds.), Phytochemistry and Agriculture, 34, Clarendon Press, Oxford, pp, 253-270.

Page 190 of 201 192) Oken, B.S., Storzbach, D.M., & Kaye, J.A. (1998). The efficacy of Ginkgo biloba on cognitive function in Alzheimer disease. Arch Neurol, 55, 1409-1415.

193) Ou, B., Hampsch-Woodill, M. & Prior, R.L. (2001). Development and Validation of an Improved Oxygen RadicalAbsorbance Capacity Assay Using Fluorescein as the Fluorescent Probe. J. Agric. Food Chem., 49, 4619-4626.

194) Ou, B., Huang, D., Hampsch-Woodill, M., & Flanagan, J.A. (2003). When east meets west: the relationship between yin-yang and antioxidation-oxidation. The FASEB Journal, 17, 127-129.

195) Oyama, Y., Fuchs, P.A., Katayama, N., Noda, K. (1994). Myricetin and quercetin, the flavonoid constituents of Ginkgo biloba extract, greatly reduce oxidative metabolism in both resting and Ca2+- loaded brain neurons. Brain Res; 635, 125-129.

196) Paas, E. & Pierce, G. (2002). Canola oil. http://www.sbrc.ca/ncarm/canolaoil.htm

197) Packer, L. (1999). The Antioxidant Miracle – Your complete plan for total health and healing, Wiley, USA.

198) Paganga, G., & Rice-Evans, C.A. (1997). The identification of flavonoids as glycosides in human plasma. FEBS Letters, 401: 78-82.

199) Panetta, T., Marcheselli, V.L., Braquet, P., Spinnewyn, B., & Bazan, N. G. (1987). Effects of a platelet activating factor antagonist (BN 52021) on free fatty acids, diacylglycerols, polyphosphoinositides and blood flow in the gerbil brain: inhibition of ischemia-reperfusion induced cerebral injury. Biophys. Res. Commun. 149, 580-587.

200) Pearson, D. (1970). The chemical analysis of foods, (Sixth Edition). J.A. Churchill, Gloucester, London, UK.

201) Pedersen, S. (2001). Ginkgo – Boost brain power and improve circulation in LaVonne Carlson (Ed.), Dorling Kindersley Limited, Great Britain.

202) Peschier. (1818). Recherches analytiques sur le fruit du Ginkgo. Bill. Uni. Genève, Sc. Arts., 7, 3me annèe, 29-34.

203) Petty, H. R., et. al. (2001). Identification of colchicine in placental blood from patients using herbal medicine. Chem. Res. Toxicol. 14, 1254-1258.

204) Pietri, S., Maurelli, E., Drieu, K., & Culcasi, M. (1997). Cardioprotective and Antioxidant effects of the terpenoids constituents of Ginkgo biloba Extract (EGb761). J Mol. Cell. Cardtol, 29,733-742.

205) Pietta, P. et. al.. (1991). Identification of flavonoids from Ginkgo biloba L., Anthemis nobilis L. and Equisetum arvense L. by high performance liquid chromatography with diode-array UV detection. Journal of Chromatography, 553, 223-231.

206) Pietta, P., & Mauri, P. (1988). Reversed-phase high-performance liquid chromatographic method for the analysis of biflavones in Ginkgo biloba L. extracts. Journal of Chromatography, 437, 453- 456.

207) Pincemail, J., Dupuis, M., Nasr, C., Hans, P., Haag-Berrurier, M., Anton, R., & Derby, C. (1985). Superoxide dismutase from a eucaryote, Ginkgo biloba. Arch. Biochem. Biophys., 243, 305-314.

208) Piovella, C. (1973). Effetti della Ginkgo biloba sui microvasi della congiuntiva bulbare. Min Med. 64, 4179.

209) Pitchumoni, S.S., & Doraiswamy, P.M. (1998). Current status of antioxidant therapy for Alzheimer’s disease. J. Am. Geriatric Soc., 46, 1566-1572.

210) Pittler, M.H., & Ernst, E. (2000). Ginkgo biloba Extract for the treatment of intermittent claudication: A meta-analysis of Randomised Trails. The American Journal of Medicine, 108, 276- 281.

Page 191 of 201 211) Prior, R. and Cao, G. (1999). In vivo total antioxidant capacity: comparison of different analytical methods. Free Radicals Biol Med 27:1173-1181.

212) Ramana, C. V., Boldogh, I., Izumi, T., Mitra, S. (1998). Adaptive response of mammalian cells to reactive oxygen species associated with activation of AP-endonuclease. Proc Natl Acad Sci U S A., 95, 5061-6.

213) Rapin, J.R., & Le Poncin-Lafitte, M. (1979). Modele experimental d’ischemic cerebrale. Action preventive de l’extrait de Ginkgo. Sem Hop Paris, 55, 2047.

214) Ratty, A.K., & Das, N.P. (1988). Effects of flavonoids on non-enzymic lipid peroxidation: Structure activity relationship. Biochem. Med. Metabol. Biol., 39, 69-79.

215) Re R, et.al. (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radicals Biol Med 26:1231- 1237.

216) Reuter, H.D. (1996). Ginkgo biloba-botany, constituents, pharmacology, and clinical trials. Br. J Phytotherapy, 4, 3-20.

217) Rice-Evans, C., Nicholas M.J., George, P (1996). Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radical Biology & Medicine, 20(7), 933-956.

218) Rice-Evans, C.A., and Miller, N.J. (1994). Total antioxidant status in plasma and body fluids. Methods Enzymol 234:279-293.

219) Robak, J. and Gryglewski, J.R., (1998). Flavonoids are scavengers of superoxide anions. Biochem Pharmacol 37:837-841.

220) Robert I-San Lin, (1994). Phytochemicals and Antioxidants. In: Israel Goldberg, ed. Functional foods, Great Britain: Chapman & Hall, 393-449.

221) Robertson, J.M., Donner, A.P., Trevithick, J.R. (1991). A possible role for vitamins C and E in cataract prevention. Am J Clin Nutr, 53, 346S-351S.

222) Saito, J. (1929). Research about skin burning from ginkgo meat. Jap. J. Dermatol. Urol., 129, p, 105-129.

223) Sanchez-Moreno, C. , Larrauri, J.A., and Saura-Calixto, F. (1998). A procedure to measure the antiradical efficiency of polyphenols. J Sci Food Agric 76:270-276.

224) Sanchez-Moreno, C., Larrauri, J.A., and Saura-Calixto, F. (1999a). Free radical scavenging capacity of selected red, rose and white wines. J Sci Food Agric 79:1301-1304.

225) Sanchez-Moreno, C., Larrauri, J.A., and Saura-Calixto, F. (1999b). Free radical scavenging capacity and inhibition of lipid oxidation of wines, grape juices and related polyphenolic constituents. Food Res Int 32:407-412.

226) Sauder, P., Kopferschmitt, J., Jaeger, A., and Mantz, J. M. (1983). Haemodynamic studies in eight cases of acute colchicine poisoning. Hum. Toxicol. 2, 169-173.

227) Schaffler, K., & Rech, P.W. (1985). Doppellblind studie zur hypoxieprotektiven wirkung eines standardieserten Ginkgo biloba Praeparates nach Mehrfachverabreitchung au gesunden Probanden. Arzneim. Forsch., 35, 1283.

228) Scharffetter-Kochanek, K., Wlaschek, M., Briviba, K., Sies, H. (1993). Singlet oxygen induces collagenase expression in human skin fibroblasts. FEBS Lett., 331, 304-6.

229) Schrall, R., Becker, H., (1977). Produktion von Catechinen und oligomeren Proanthocyanidinen in Callus- und Suspensionkulturen von Crataegus monogyna, C. oxyacantha und Ginkgo biloba. Planta Medica, 32, 297-307.

Page 192 of 201 230) Scott, P.M., Lau, B.P.-Y., Lawrence, G.A., & Lewis, D.A. (2000). Analysis of Ginkgo biloba for the Presence of Ginkgotoxin and Ginkgotoxin 5’-Glucoside. Journal of AOAC International, 83, 6, 1313-1320.

231) Seddon, J.M., Ajani, U.A., Sperduto, R.D., et. al., (1994). Dietary carotenoids, vitamins A, C, and E, and advanced age-related macular degeneration. Eye disease case-control study group. JAMA, 272, 1413-1420.

232) Shang, D .Y., Ikonomou, M.G., Macdonald, R.W. (1999). Quantitative determination of nonylphenol polyethoxylate surfacants in marine sediment using normal-phase liquid chromatography-electrospray mass spectrometry. J. Chromatogr.A, 849, P, 467.

233) Shen, J.G, Zhou, D.Y. (1995). Efficiency of Ginkgo biloba extract (EGb 761) in antioxidant protection against myocardial ischemia and reperfusion injury. Biochem Mol Biol Int, 35, 125-134.

234) Shi, X., Dalal, N. S., Jain, A. C. (1991). Antioxidant behavior of caffeine: efficient scavenging of hydroxyl radicals, Food Chem. Toxicology., 29(1), 1-6.

235) Siegers, C.P. (1999). Cytotoxicity of alkylphenols from Ginkgo biloba. Phytomedicine., 6, 281- 283.

236) Smith, P. F., Maclennan, K., Darlington, C.L. (1996). The neuroprotective properties of the Ginkgo biloba leaf: a review of the possible relationship to platelet-activating factor (PAF). Journal of Ethnopharmacology, 50(3), 131-139.

237) Snodderly, D.M. (1995). Evidence for protection against age-related macular degeneration by carotenoids and antioxidant vitamins. Am J Clin Nurtr, 62, 1448S-1461S.

238) Sowers, E.F., et. al., (1965). Ginkgo tree dermatitis. Arch Dermatol, 91, p, 452-456.

239) Spedding, G., Ratty, A., Middleton Jr., E. (1989). Inhibition of reverse transcriptases by flavonoids, Antiviral Res. 12, 99–110.

240) Stafford, H. A., Kreitlow, K.S., Lester, H.H. Comparison of proanthocyanidins and related compounds in leaves and leaf-derived cell cultures of Ginkgo biloba L., Pseudotsuga menziesii Franco, and Ribes sanguineum Pursh. Plant Physiology (1986), 82(4), 1132-8.

241) Stapczynski, J. S., and Rothstein, R. J. (1981). Colchicine overdose: report of tow cases and review of the literature. Ann. Emerg. Med. 10, 364-369.

242) Stewart, J. P. (1989). Optimization of Parameters for Semi-Empirical Methods I-Method. J Comp Chem., 10, 210-220.

243) Stewart-Lee, A. L., Forster, L. A., Nourooz-Zadeh, J., Ferns, G. A., Anggard, E. E. (1994). Vitamin E protects against impairment of endothelium-mediated relaxations in cholesterol-fed rabbits. Arterioscler Thromb., 14, 494-9.

244) Suc, I., Meilhac, O., Lajoie-Mazenc, I., Vandaele, J., Jurgens, G., Salvayre, R., Negre-Salvayre, A. (1998). Activation of EGF receptor by oxidized LDL. Faseb J., 12, 665-71.

245) Takahama, U. (1985). Inhibition of lipoxygenase-dependent lipid peroxidation by quercetin: mechanism of antioxidative function. Phytochemistry, 24, 1443-1446.

246) Takano, T., Kobayashi, M., Wada, I. (1953). Study on Gin-nan food poisoning in experimental animals (in Japanese). Jui Chikusan Sihinpo, 109, 353-357.

247) Tang, W., & Eisenbrand, W. (1992). Ginkgo biloba L. Chinese Drugs of Plant Origin, Springer- Verlag, Berlin, pp1583-1587.

248) Tasinato, A., Boscoboinik, D., Bartoli, G. M., Maroni, P., Azzi, A. (1995). d-α-Tocopherol inhibition of vascular smooth muscle cell proliferation occurs at physiological concentrations,

Page 193 of 201 correlates with protein kinase C inhibition, and is independent of its antioxidant properties. Proc Natl Acad Sci U S A., 92, 12190-4.

249) Teissedre, P.L., & Waterhouse, A.L. (2000). Inhibition of oxidation of human low-density lipoproteins by phenolic substances in different essential oils varieties. J. Agric. Food Chem., 48, 3801–3805.

250) U. S. Pharmacopoeia (1995) U. S. Pharmacopoeia & the National Formulary: USP 23-NF18, U. S. Pharmacopoeia.Adawadkar, P.D. and El Sohly, M. (1981). Isolation, purification and antimicrobial activity of ancardic acids from Ginkgo biloba fruits. Fitoterapia, 52, 129-135.

251) USDA Nutrient Database for Standard Reference, Release 13 (November 1999). http://www.nal.usda.gov/fnic/foodcomp/Data/SR15/reports/sr15fg12.pdf

252) van Acker, F.A., Schouten, O., Haenen, G.R., van der Vijgh, W.J., & Bast, A. (2000a). Flavonoids can replace alpha-tocopherol as an antioxidant. FEBS Lett, 473: 145–148.

253) van Acker, S. A., van den Berg, D. J., Tromp, M. N., Griffioen, D. H., van Bennekom, W. P., van der Vijgh, W.J., Bast, A. (1996a). Structural aspects of antioxidant activity of flavonoids. Free Radic Biol Med., 20, 331-42.

254) van Beek T.A.(2002). Chemical analysis of Ginkgo biloba leaves and extracts. J. Chromatogr. A., 967, 21-55.

255) van der Hagen, A.M., Yolton, D.P., Kaminski, M.S., Yolton, R.L. (1993). Free radicals and antioxidant supplementation: a review of their roles in age-related macular degeneration. J Am Optom Assoc; 64, 871-878.

256) Van der Weide, J., Steijns, L.S.W. (1999). Cytochrome P450 enzyme system: genetic polymorphisms and impact on clinical pharmacology, Ann. Clin. Biochem. 36, 722–729.

257) Vanhaelen, M., Vanlaelen-Fastre, R. (1988). Countercurrent chromatography for isolation of flavonol glycosides from Ginkgo biloba leaves. Journal of Liquid Chromatography, 11, 2969-2975.

258) Vanhaelen, M., Vanlaelen-Fastre, R. (1989). Flavonol triglycerides from Ginkgo biloba. Plant Medica, 55, 202.

259) Vaya, J., Mahmood, S., Goldblum, A., Shaalan, A., Tamir, S. (2000). Abstract 046; oxidative stress and atherosclerosis, Oslo, Norway.

260) Verotta L and Peterlongo F. (1993). Selective extraction of phenolic components from Ginkgo biloba extracts using supercritical carbon dioxide and off-line capillary gas chromatography/ mass spectrometry. Phytochem. Anal. 4:178-182.

261) Verotta L, Morazzoni P and Peterlongo F. (2000). Occurrence and Analysis of Alkyl Phenols in Ginkgo biloba. Ginkgo biloba. In: T.A van Beek (Ed.). Medicinal and Aromatic Plants – Industrial Profiles. Vol 12. Harwood, Amsterdam. p 203- 214.

262) Verotta, L and Peterlongo, F. (1993). Selective extraction of phenolic components from Ginkgo biloba extracts using supercritical carbon dioxide and off-line capillary gas chromatography/ mass spectrometry. Phytochem. Anal., 4, 178-182.

263) Victoire, C., Haag-Berrurier, M., Lobstein-Guth, A., Balz, J.P., & Anton, R. (1988). Isolation of flavonol glycosides from Ginkgo biloba leaves. Planta Medica, 54, 245-247.

264) Von Gadow, A., Joubert E., & Hansmann C. F. (1997) Comparison of the antioxidant activity of rooibos tea (Aspalathus linearis) with green, oolong and black tea, Food Chemistry, 60(1), 73-77.

265) Wada, K. (2000). Food poisoning by Ginkgo seeds: The role of 4-O-Methylpyridoxine. Ginkgo biloba, in T.A van Beek (Ed.), Medicinal and Aromatic Plants – Industrial Profiles, Vol, 12, Harwood, Amsterdam, 453.

Page 194 of 201 266) Wada, K. et. al., (1988). Studies on the constitution of edible and medicinal plants. I. Isolation and identification of 4’-O-methoxypyridoxine, toxic principle from seed of Ginkgo biloba L. Chem. Pharm. Bull., 36, 1779-1782.

267) Wada, K., & Haga, M. (1997). Food Poisoning by Ginkgo biloba Seeds, in Hori, T., Ridge, R.W., Tulecke, W., Del Tredici, P, Tremouillaux-Guiller, J., Tobe, H. (Ed.), Ginkgo biloba – A Global Treasure from Biology to Medicine, Springer – Verlag, Tokyu. 309-321.

268) Wada, K., Ishigaka, S., Ueda, K., Sakata, M., Haga, M. (1985). An antivitamin B6, 4’-O- methoxypyridoxine, from the seed of Ginkgo biloba L. Chem. Pharm. Bull., 33, 3555-3557.

269) Wadsworth, T.L., & Koop, D.R. (2001). Effects of Ginkgo biloba extract (EGb 761) and quercetin on lipopolysaccharide-induced release of nitric oxide. Chemico-Biological Interactions, 137, 1, 43- 58.

270) Walle, T. , Otake, Y. , Brubaker, J.A. , Walle, U.K. , Halushka, P.V. (2001). Disposition and metabolism of the flavonoid chrysin in normal volunteers, Br. J. Clin. Pharmacol. 5, 143–146.

271) Wayner, D.D.M., Burton, G.W., and Ingold, K.U. (1986). The antioxidant efficiency of vitamin C is concentration-dependent. Biochem Biophys Acta 884:119-123.

272) Wayner, D.D.M., Burton, G.W., Ingold, K.U., and Locke, S. (1985). Quantitative measurement of the total, peroxyl radical trapping antioxidant capacity of human blood plasma by controlled peroxidation. FEBS Lett 187:33-37.

273) West, S., Vitale, S., Hallfrisch, J., et. al., (1994). Are antioxidants or supplements protective for age-related macular degeneration? Arch Ophthalmol, 112, 222-227.

274) Westendorf, J., Regan, J. (2000). Genotoxic and tumor promoting activity of ginkgolic acids in primary rat hepatocytes. Pharmazie., 55, 864.

275) Wilson, R.H., (1994). Spectroscopic techniques for food analyses, Ed. VCH Publishers, Inc., pp 221-240.

276) Woerdenbag, H.J., & De Smet P.A.G.M. (2000) Adverse effects and toxicity of Ginkgo extracts. Ginkgo biloba, in T.A van Beek (Ed.), Medicinal and Aromatic Plants – Industrial Profiles, Vol, 12, Harwood, Amsterdam, p, 443

277) Xie, B., Shi, H., Chen, Q. and Ho, C.T. (1993). Antioxidant properties of the fractions and polyphenol constituents from green, oolong and back teas. Proc. Natl. Sci. Counc. Repub. China [B], 17, 77-84.

278) Xu, H.-X., Wan, M., Dong, H., But, P.P.-H., Foo, L.Y. (2000). Inhibitory activity of flavonoids and tannins against HIV-1 protease, Biol. Pharm. Bull. 23, 1072–1076.

279) Xu, Y. (2001). Perspectives on the 21st century development of functional foods: bridging Chinese medicated diet and functional foods. International Journal of Food Science and Technology, 36, 229-242.

280) Yao, Z., Boujrad, N., Drieu, K., & Papadopoulos, V. (1998). Protection of the cytochrome P450scc enzyme and steroidogenic ability of Leydig cells by Ginkgolide B. Adv. Ginkgo biloba Res., 7, 129–138.

281) Yao, Z., Drieu, K., Szweda, L.I., & Papadopoulos, V. (1999). Free radicals and lipid peroxidation do not mediate b-amyloid-induced neuronal cell death. Brain Res., 847, 203–210.

282) Yoshitama, K. (1997). Flavonoids of Ginkgo biloba, in Hori, T., Ridge, R.W., Tulecke, W., Del Tredici, P, Tremouillaux-Guiller, J., Tobe, H. (Ed.), Ginkgo biloba – A Global Treasure from Biology to Medicine, Springer – Verlag, Tokyu, 287 – 299.

Page 195 of 201 283) Youdim, K.A., & Joseph, J.A. (2001). A possible emerging role of phytochemicals in improving age-related neurological dysfunction: A multiplicity of effects. Free Radical Biology and Medicine, 30, 6, 583-594.

284) Yuting, C., Ronglinang, Z., Zhongjian, J., & Yong, J. (1990). Flavonoids as superoxide scavengers and antioxidants. Free Rad. Biol Med; 9, 19-21.

285) Zhang, E.Q. (1990). Chinese medicated diet Pp. 1-15. Shanghai: Publishing house of Shanghai College of traditional Chinese medicine.

Page 196 of 201 APPENDICES

Appendix 1: See article - Antioxidant capacity in Ginkgo biloba.

Appendix 2: See article - Examination of antioxidant activity of Ginkgo biloba leaf infusions.

Appendix 3: See article - Flavonoid recovery and stability from Ginkgo biloba subjected to a simulated digestion process.

Appendix 4: See Sensory evaluation form

Page 197 of 201

Appendix 1: Antioxidant capacity in Ginkgo biloba.

Page 198 of 201 Food Research International 35 (2002) 815–820 www.elsevier.com/locate/foodres

Antioxidant capacity in Ginkgo biloba

Lena M. Goh*, Philip J. Barlow Food Science and Technology Programme, Department of Chemistry, National University of Singapore, Singapore 117543

Received 18 February 2002; received in revised form 19 February 2002; accepted 19 February 2002

Abstract The antioxidant capacity, AOC, of Ginkgo biloba nuts was determined after various periods of cooking. The AOC was based on the ability of the sample to scavenge 2,20-azino-bis-(3-ethlybenzthiazoline-6-sulfonic acid) free radical ABTS+ . The heat-stable antioxidant components present in the nuts accounted for 60% AOC. Thus, around 40% of the AOC was lost when the nuts were cooked for 4 h. After an initial fall, related to a loss in vitamin C, the AOC remained constant. During heating, the non-ascorbic acid AOC compounds were transferred from the nuts to the leachate. A gradual formation of non-ascorbic acid antioxidants was observed in the nuts. The effect of cooking on AOC in the Ginkgo nuts was compared with Ginkgo biloba standardised leaf extracts, dried leaves extracts and Ginkgo commercial capsules. # 2002 Elsevier Science Ltd. All rights reserved.

Keywords: ABTS +; Antioxidant capacity; Functional foods; Ginkgo biloba nuts; Ginkgo biloba leaves; EGb 761

1. Introduction (Youdim & Joseph, 2001), and aid in disease preven- tion. There are considerable debates on the therapeutic and An extract from the leaves of Ginkgo biloba, used in nutritional effects of Ginkgo biloba nuts versus the stan- the treatment of cerebral insufficiency, was developed in dardised leaf extract. The promotional literature for the the 1960s (Maurer, Ihl, Dierks, & Frolich, 1997) and latter purports or implies that Ginkgo leaf extract has designated EGb 761. This preparation is an acetone/ been used for thousand of years in China. However, water (60:40) extract from the dried leaves (Drieu, until the last 20–30 years, Ginkgo leaves were rarely 1986). The main active ingredients of medicinal Ginkgo used in traditional Chinese herbal medicine for ther- leaf extracts include 22–27% flavonoid glycosides, 5– apeutic purposes. The nuts have been traditionally con- 7% terpene lactones, and less than 5 mg/g Ginkgolic sumed and their use was first mentioned in herbals in acids (DeFeudis, 1998). The flavonoid glycosides were the Yuan dynasty [1280–1368 AD], published in 1350 determined by high-performance liquid chromato- AD (Del Tredici, 1991; Foster & Chongxi 1992). Recent graphy as quercetin and kaempferol including iso- research suggests that Ginkgo biloba nuts do in fact have rhamnetic, and calculated as acyl flavonol glycosides a health giving property (Anonymous, 1997). with molar masses of 756.7 (quercetin glycosides) and Ginkgo biloba nuts are in season from the middle to 740.7 (kaempferol glycosides). The terpene lactones the end of winter. Ginkgo nuts are consumed with the include 2.8–3.4% Ginkgolides A, B and C and 2.6– intention that they possess health benefits. The nuts 3.2% bilobalide. contain a range of phytochemicals which are reported to Ginkgo biloba standardised leaf extract, Egb 761, possess anti-cancer activities, can treat neurological supplied as a dry powder, has received much attention dysfunction when tested both in vivo and in vitro in recent decades (DeFeudis, 1998; Packer, 1999). Many researchers suggested that the leaf extracts possess medicinal properties, and the active ingredients of the Abbreviations: EGb 761, Ginkgo biloba extract; AOC, antioxidant leaves are widely reported (DeFeudis, 1998; Packer, 0 capacity; TCM, traditional Chinese medicine; ABTS+ , 2,2 -azinobis- 1999). Ginkgo leaves have attracted much attention as (3-ethylbenzothiazoline-6-sulfonic acid); AEAC, ascorbic acid equiva- lent antioxidant capacity. agents for improving circulation, particularly cerebral * Corresponding author. Tel.: +65-8741655; fax: +65-7757895. circulation, which may lead to improved mental func- E-mail address: [email protected] (L.M. Goh). tion (Kleijnen & Knipschild, 1992a; Oken, Storzbach, &

0963-9969/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0963-9969(02)00084-4 816 L.M. Goh, P.J. Barlow / Food Research International 35 (2002) 815–820

Kaye, 1998; Wadsworth & Koop, 2001). Many resear- fornia, USA. Commercial capsules of Ginkgo biloba ches have been conducted on the role of the extract in leaves (claimed to be dried leaves) were purchased from the treatment of diseases involving free radicals and a local General Nutrition Center. Standardised Ginkgo oxidative damage (Bridi, Croseetti, Steffen, & Henri- biloba leaf extract was purchased from Voigt Global ques, 2001; Pitchumoni & Doraiswamy, 1998). Distribution, Kansas City, Kansas, USA. Although antioxidant action has been attributed to this extract, the mechanism of the multiple principles 2.2. Chemicals involved in this pharmacological activity has not been completely established. The pharmacokinetics of the 2,20-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic Ginkgo extracts has been studied in experimental ani- acid) (ABTS), l-ascorbic acid and potassium persul- mals and in humans (Hofferberth, 1994; Kanowski, phate were purchased from Sigma Chemical Co. (Jef- Hermann, & Stephan, 1996; Kleijnen & Knipschild, ferson City, MO, USA). a-Tocopherol standard was 1992b; Le Bars, Katz, & Berman, 1997). from Argos Organics (Trenton, NJ, USA) and other In Europe and, to a lesser degree, in the United chemicals used were of reagent grade. States, Ginkgo leaf extracts are promoted in the health food market and/or drug stores as popular dietary sup- 2.3. Nut sample preparation plements in the form of capsules, tablets or liquid herbal concentrates. In contrast, the nuts are only presented in The edible nut samples were shelled and skinned by the form of food. It seems that the therapeutic effects of flash blanching (placing in boiling water for 1 min and the nuts and the leaves are different. Consumers’ pref- removing the skin by hand). Samples were then heated erence is usually to consume products in the form of food under dry and wet conditions for periods between 5 min rather than tablet/capsule, a method of ingestion usually and 4 h according to the traditional method of prepar- associated with feeling unwell (Anonymous, 2001). ing Cheng Teng or other sweet desserts. Wet heating Antioxidants are increasingly being recognised as was carried out using distilled water at a ratio of 1:10 important health promoters in conditions such as car- (w/v) and at 80–90 C. diovascular problems, many forms of cancer and even aging (Packer, 1999). The antioxidants are able to 2.4. Proximate analysis reduce the effect of free radicals formed in the body either due to exposure to environmental pollutants or Standard methods of analysis (AOAC, 2000) were because the bodies’ own defense mechanisms are used to determine ash, carbohydrate, fat, moisture and reduced in dealing with the natural production of these protein content. compounds. Although Ginkgo nuts are suspected to possess antioxidant property, there is limited informa- 2.5. Elemental analysis tion on the constituents responsible for the property. No studies have been conducted on the antioxidant An inductively coupled plasma optical emission spec- capacity (AOC) of Ginkgo biloba nuts when consumed trometer (Thermo Jarrell Ash, model: Duo Axial as a food product. In view of the popularity of Ginkgo plasma, Franklin, MA, USA) was used to determine nuts in Southeast Asia, a study of their AOC is con- Zn, Fe, Na, Mg, K and Ca. A CHNS auto-analyser sidered important. (Perkin Elmer Series II CHNS/O Analyser 2400, USA) In this investigation, the total AOC of Ginkgo biloba was used to determine elemental C and N. nuts both in their raw state and following their pre- paration as a traditional sweet dessert was determined. 2.6. Determination of vitamins Ginkgo biloba nuts were subjected to a variety of condi- tions to simulate food product preparation. As Ginkgo The vitamin C content in nuts was determined nuts consumed in this region are excessively boiled to according to the method of Lambert and De Leenheer obtain the required softness, investigations were carried (1992). The nuts were homogenised and extracted with out to determine the AOC over a range of cooking time. 2% (v/v) acetic acid for 30 min. An HPLC system (Shi- madzuHPLC, Shimadzu,Kyoto, Japan) equipped with a photo-diode array detector was used. The separation was 2. Materials and methods performed on a Shim-Pack VP-ODS column (250Â4.6 mm i.d.; Shimadzu, Kyoto, Japan) using 2% (v/v) acetic 2.1. Materials acid/acetonitrile at a flow-rate of 0.8 ml/min at 40 C. Vitamin E was determined according to the Associ- Nuts imported from China were purchased from local ation of Vitamin Chemists method (1966) which deter- supermarkets. Leaves and nuts used for nutritional mines the oxidation of tocopherols by nitric acid to analysis were imported from Ventura County, Cali- tocoquinone. Absorbance was measured at 470 nm L.M. Goh, P.J. Barlow / Food Research International 35 (2002) 815–820 817

Table 1 Proximate and nutritional composition of Ginkgo biloba samples

Nuts from Nuts from Leaves from Commercial USA China USA capsules

Proximate (g/100 g)a Protein 0.7Æ0.03 0.7Æ0.37 1.1Æ0.07 1.0Æ0.91 Ash 3.7Æ0.12 4.4Æ0.14 19.1Æ0.02 1.2Æ0.08 Moisture 49.1Æ0.01 55.6Æ0.03 12.3Æ0.06 4.5Æ0.02 Fat 3.4Æ0.18 2.9Æ0.32 11.4Æ0.05 3.4Æ0.35 Carbohydrate (by difference) 43.0 36.4 56.1 89.9

Vitamins (mg/g)a Vitamin C 0.4Æ0.02 0.9Æ0.02 1.4Æ0.06 0.5Æ0.04 Vitamin E 1.2Æ0.03 0.2Æ0.03 22.2Æ0.06 1Æ0.6

Minerals (mg/100 g)a Zinc 2.2Æ0.14 1.2Æ0.02 0.7Æ0.16 0.5Æ0.18 Iron 1.4Æ0.02 1.2Æ0.01 4.3Æ0.02 0.8Æ0.31 Sodium 58.6Æ0.04 64.5Æ0.22 89.6Æ0.03 180Æ0.27 Magnesium 91.1Æ0.06 78.1Æ0.18 598Æ0.04 44.3Æ0.19 Potassium 989Æ0.44 1016Æ0.34 1514Æ0.41 37.4Æ0.12 Calcium 39.9Æ0.01 41.8Æ0.28 2631Æ0.16 62.4Æ0.03 Carbon 233Æ0.31 313Æ0.42 223Æ0.21 260Æ0.29 Nitrogen 5.2Æ0.01 45.2Æ0.02 10.7Æ0.16 9.3Æ0.16

a Mean of six determinationsÆSD (dry weight basis). against a blank containing 5 ml absolute alcohol and 1 biloba leaf capsules, were extracted with 60:40 acetone/ ml conc. nitric acid. The unsaponifiable matter was water under reflux for 30 min. Similar procedures as for extracted with diethyl ether and samples were protected AOC determination in the nuts were followed. from light at all times.

2.7. Determination of AOC in nuts 3. Results and discussion

The nuts were cooked for the appropriate time, and 3.1. Proximate and nutritional analysis both the cooking water and the drained nuts were col- lected. The nuts were homogenised and extracted with Table 1 shows the proximate and nutritional analysis 60:40 acetone/water under reflux. The antioxidant of Ginkgo biloba samples. The nutrient content of the capacity was determined using the standard ABTS+ decolorisation assay (Re et al., 1998). ABTS was used as Table 2 the free radical provider and was generated by reacting Antioxidant capacity of Ginkgo nut extract and leachate this compound (7.4 mM) with potassium persulphate (2.45 mM) overnight. The solution was diluted with Cooking time AEACa 98% ethanol to obtain an absorbance of 1.5 at 414 nm. (min) Nut extract Leachate Total An aliquot (3 ml) of the solution was added to 40 mlof sample and the standard curve was prepared using 0 515.5Æ3.2 – 515.5 similar volume of l-ascorbic acid. All readings were 5 416.9Æ1.2 43.4Æ0.4 481.3 10 208.5Æ0.8 105.2Æ1.0 340.7 taken after 90 min of reaction time when the absorbance 15 200.2Æ0.5 113.5Æ1.0 347.6 appeared to reach a plateau. The results were expressed 20 151.6Æ0.5 113.7Æ0.5 290.3 as mg ascorbic acid equivalent antioxidant capacity 25 148.3Æ1.0 138.5Æ0.6 308.8 (AEAC) per 100 g of homogenate. The AOC results 30 139.7Æ0.18 139.1Æ0.9 297.8 were based on 6 determinations and the relative stan- 60 115.9Æ1.1 137.0Æ1.0 287.9 90 97.7Æ0.3 175.1Æ0.4 303.8 dard deviation was determined as RSD%=(Standard 120 76.0Æ0.7 248.8Æ0.1 352.8 deviation/average of 6 determinations)Â100. 150 43.6Æ0.6 274.7Æ1.0 344.3 180 34.6Æ0.7 287.2Æ1.1 349.8 2.8. Determination of AOC in leaves 210 34.6Æ0.1 303.3Æ0.2 362.9 240 34.6Æ0.1 319.4Æ0.8 377.0

The various leaf samples, Ginkgo biloba leaves, Ginkgo a Mean of six determinationsÆSD expressed as mg ascorbic acid biloba standardised leaf extract and commercial Ginkgo equivalent antioxidant capacity (AEAC) per 100 g of sample. 818 L.M. Goh, P.J. Barlow / Food Research International 35 (2002) 815–820 nuts from USA and China was similar except for vita- 515.5 mg AEAC per 100 g of homogenate. After dry min C and E (significantly different at P=0.05). This heat treatment (25 min at 140 C), the AOC level could be related to geographical difference, seasonal decreased to 275 mg AEAC. During wet cooking, a variations or variety difference. The American nuts were rapid fall in AOC was observed after 10 min, leveling off less mature and this may attribute to the differences. to 350 mg AEAC per 100 g of homogenate. The results The leaves seems to be better balanced nutritionally, indicate migration of the antioxidant capacity from the being high in vitamin C and E, suggesting a high con- nut extract to the leached aliquot, with the total AEAC tribution to antioxidant activity. level remaining constant during cooking. Ginkgo biloba nuts contain a substantial level of vitamin C (USDA 3.2. AOC of Ginkgo biloba nuts Nutrient Database, 1999), and the initial loss could be attributed to the heat instability of this vitamin. The AOC of Ginkgo biloba nuts after various cooking A calibration graph of vitamin C was prepared. The time are shown in Table 2. The raw nuts contained level of vitamin C was determined using the slope of the

Fig. 1. Changes in heat-stable and heat-labile antioxidants in Ginkgo nuts (nut extract+leachate) during cooking. &, AEAC—non-vitamin C; &, AEAC—vitamin C.

Fig. 2. Antioxidant capacity contribution by vitamin C in the Ginkgo nut extract and leachate during cooking. 3, vitamin C in nut extract; &, vitamin C in leachate. L.M. Goh, P.J. Barlow / Food Research International 35 (2002) 815–820 819

Table 3 4. Conclusion Antioxidant capacity of various Ginkgo biloba samples

Ginkgo biloba samples AEACa The dramatic loss of AOC in Ginkgo nuts over the first 10 min of heating was most likely due to the loss of Ginkgo biloba nuts from China—raw 515.5 (RSD%=2.13) vitamin C, a heat-unstable vitamin. However, a sub- Ginkgo biloba nuts from China—cooked 287.9 (RSD%=1.15) for 1 h stantial amount of AOC still remained in the nuts, Ginkgo biloba leaves from USA 16 288 (RSD%=2.44) which must be due to heat-stable water-soluble com- Ginkgo biloba leaves from China 15 509 (RSD%=2.75) pound(s), most likely polyphenols. The nature and che- Standardised Ginkgo biloba leaf extract 41 777 (RSD%=1.98) mical characteristics of these component(s) required Commercial Ginkgo biloba leaf capsules 4440 (RSD%=2.56) further investigation. The present data indicate that a mg Of ascorbic acid equivalents per 100 g of homogenate. Ginkgo biloba nuts can be regarded as nutritionally beneficial even when they have been cooked using the traditional method of producing Cheng Teng, a tradi- tional sweet dessert of southeast Asia. calibration graph with y=3.7732x (R2=0.9982). The vitamin C level in the raw nuts was 39 mg/100 g, and the amount of vitamin C in 40 ml of reaction solution was References calculated to be 0.015 mg. The change in absorbance Anonymous. (1997). Great pharmacopoeia of traditional Chinese medi- after 10 min cooking was 1.17, corresponding to a cine (in chinese). Shanghai, China: Shanghai Publishing House of loss of 0.0124 mg of vitamin C. The vitamin C in Science and Technology (in Chinese). Ginkgo nuts after cooking was 14.26 mg/100 g, or Anonymous. (2001). Combining nutrients for health benefits. Food 0.057 mg/40 ml aliquot. The calculated loss of vitamin Technology, 55(2), 42–47. C was 0.095Æ0.002 mg. The calculated and the AOAC. (2000). Official methods of analysis of AOAC International (OMA)—(17th ed.). Horwitz, W. (Ed.). Arlington, VA: AOAC actual experimental loss in vitamin C were therefore International. similar. Association of Vitamin Chemists. (1966). Methods of vitamin assay Further studies were undertaken to determine the (3rd ed.). USA: Interscience. change in AOC contributed by the heat sensitive com- Bridi, R., Croseetti, F. P., Steffen, V. M., & Henriques, A. T. ponent(s), assumed to be vitamin C and heat-stable (2001). The antioxidant activity of standardised extract of Ginkgo biloba (EGb761) in Rats. Phytotherapy Research, 15, compounds. Vitamin C contents in the nuts and in the 449–451. leachate were found to decrease progressively (Fig. 1). DeFeudis, F. V. (1998). Ginkgo biloba extract (EGb 761) from chem- After 2 h of cooking, vitamin C level was almost com- istry to the clinic. Germany: Ullstein Medical. pletely absent, and the AOC activity will be independent Del Tredici, P. (1991). Ginkgos and people—a thousand years of of vitamin C content. interaction. Arnoldia, 51, 2–15. Drieu, K. (1986). Preparation et definition de l’extrait de Ginkgo The AOC level started to increase gradually again biloba. La Presse Medicale, 31, 1455–1457. after 60 min at a rate of 0.5406 mg AEAC per 100 g Fosters, S., & Chongxi, Y. (1992). Herbal emissaries. Rochester, Ver- homogenate per min. This strongly suggests that addi- mont: Healing Arts Press. tional antioxidant components were formed and/or Hofferberth, B. (1994). The efficacy of EGB 761 in patients with senile activated in spite of prolonged heating. In addition, it dementia of the Alzheimer type: double blind, placebo-controlled study on different levels of investigation. Human Psychopharmaco, was observed that after 5 min of cooking, vitamin C 9, 215–222. content decreased in the nut extract but remained Kanowski, S., Hermann, W. M., & Stephan, K. (1996). Proof of effi- unchanged in the leachate. After 20 min, the vitamin C cacy of the Ginkgo biloba special extract EGB 761 in outpatients level in the leachate also decreased but remained con- suffering from mild to moderate primary degenerative dementia of stant at around 1.5 mg/100 g (Fig. 2). the Alzheimer type or multi-infarct dementia. Pharmacopsychiatry, 29, 47–56. Kleijnen, J., & Knipschild, P. (1992a). Ginkgo biloba. Lancet, 340, 3.3. AOC in various Ginkgo samples 1136–1139. Kleijnen, J., & Knipschild, P. (1992b). Ginkgo biloba for cerebral The total AOC levels in the raw nuts, nuts cooked for insufficiency. British Journal of Clinical Pharmacology, 34, 352– 1 h (which is the normal practice), leaf extract, standar- 358. Lambert, W. E., & De Leenheer, A. (1992). Quantitative determina- dised leaf extracts and the commercial Ginkgo biloba cap- tion of water-soluble vitamins using HPLC. In Food analysis using sules were determined and compared (Table 3). The results HPLC. New York: Marcel Dekker. show the order of AOC activity as: cooked nuts

Ginkgo biloba on cognitive function in Alzheimer disease. Archives USDA Nutrient Database for Standard Reference, Release 13 (1999, of Neurology, 55, 1409–1415. November). Available: http://www.nal.usda.gov/fnic/cgi-bin/nut_- Packer, L. (1999). The antioxidant miracle—your complete plan for search.pl?ginkgo+nut. total health and healing. New York: Wiley. Wadsworth, T. L., & Koop, D. R. (2001). Effects of Ginkgo biloba Pitchumoni, S. S., & Doraiswamy, P. M. (1998). Current status of extract (EGb 761) and quercetin on lipopolysaccharide-induced antioxidant therapy for Alzheimer’s disease. Journal of the American release of nitric oxide. Chemico-Biological Interactions, 137(1), 43– Geriatrics Society, 46, 1566–1572. 58. Re, X., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice- Youdim, K. A., & Joseph, J. A. (2001). A possible emerging role of Evan, C. (1998). Antioxidant activity applying an improved ABTS phytochemicals in improving age-related neurological dysfunction: radical cation decolorisation assay. Free Radical Biology & Medi- a multiplicity of effects. Free Radical Biology & Medicine, 30(6), cine, 26(9/10), 1231–1237. 583–594.

Appendix 2: Examination of antioxidant activity of Ginkgo biloba leaf infusions.

Page 199 of 201 Food Chemistry 82 (2003) 275–282 www.elsevier.com/locate/foodchem

Examination of antioxidant activity of Ginkgo biloba leaf infusions

Lena M. Goh*, Philip J. Barlow, Chee S. Yong Department of Chemistry, Food Science and Technology Programme, National University of Singapore, 117543 Singapore

Received 11 July 2002; received in revised form 11 July 2002; accepted 11 July 2002

Abstract The antioxidant capacity, AOC, of Ginkgo biloba leaf infusions was determined and compared using FRAP and ABTSassays. Capillary electrophoresis was used for the determination of flavonoids in G. biloba leaves to confirm and elucidate the fingerprint compounds that contribute to the AOC. Various parameters in the preparation of the infusion, such as degree of fermentation, surface area of leaves, temperature and time of leaves infusion have been considered, in order to determine their effects on the AOC. It was found that fermentation had no significant effect on the AOC of the G. biloba leaves. However, increased surface area of leaves (leaves ground for 20 s), an infusion temperature of 100 C and infusion time around 10–15 min gave the highest overall AOC. # 2003 Elsevier Science Ltd. All rights reserved.

Keywords: ABTS +; Antioxidant capacity; Capillary electrophoresis; Ginkgo biloba leaves infusion

1. Introduction leaves infusion (1, 3, 5, 10, 15 and 20 min), have been examined. Of particular interest is the fermentation of In recent years, there has been an increasing aware- the leaves as this is the normal preparation practice for ness of the benefit of functional foods. A major thrust of many leaf infusions, including tea. current research has been the aim of developing new Pharmacologically, there is one significant group of food products, which have functional properties. With compounds found in Ginkgo leaves, the flavonoids, Ginkgo biloba achieving unprecedented popularity over which give Ginkgo its antioxidant activity and possible the past decade, and the recognition of the important protection against the damage caused by free radicals. therapeutic effects shown by this plant, there is a grow- These compounds scavenge and destroy free radicals ing market for phytomedicines based on its extracts. and reactive forms of oxygen, such as superoxide radical One current example of such a phytomedicine is a ( O2)(Gardes-Albert, Ferradini, Sekaki, & Droy- yellow-green tea infusion based on G. biloba leaves Lefaix, 1993; Marcocci, Packer, Droy-Lefaix, Sekaki, & which has a smooth, light bamboo taste. Available Gardes-Albert, 1994; Pincemail et al., 1985), hydroxyl commercially, it is postulated to be useful in the treat- radicals (OH) (Bors, Heller, Michel, & Saran, 1990; ment of arteriosclerosis, varicose veins and haemor- Husain, Cillard, & Cillard, 1987), lipid peroxide radicals rhoids (Tenney, 1996; Zuess, 1998). However, there is (Maitra, Marcocci, Packer, & Droy-Lefaix, 1995; Mar- limited information about how the methods of pre- cocci, Packer et al., 1994) and nitric oxide (Marcocci, paration of the infusion may alter the postulated ther- Maguire, Packer, & Droy-Lefaix, 1994). ABTSassays apeutic effects. Using FRAP and ABTSmethods, for have shown that Ginkgo leaves extract is an effective the determination of antioxidant activity (AOC), the free radical scavenger (Goh & Barlow, 2002). However, effects of various parameters in the preparation of the there are also considerations with regard to the seasonal infusion, such as degree of fermentation (unfermented, 2 variations and storage conditions of the leaves (Ellnain- and 24 h fermentation), surface area of leaves (ground 5 Wojtaszek & Zgorka, 1999; Ellnain-Wojtaszek, Kruc- and 20 s), temperature (60, 80 and 100 C) and time of zyn´ ski, & Kasprzak, 2002). In addition, the antioxidant activity of the flavonoids also depends greatly on their * Corresponding author. Tel.: +65-68741655; fax: +65-67757895. chemical structure and the relative orientation of var- E-mail address: [email protected] (L.M. Goh). ious moieties on the molecule, particularly the number

0308-8146/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0308-8146(02)00548-4 276 L.M. Goh et al. / Food Chemistry 82 (2003) 275–282

Approximately 50 g of leaves were ground using a Nomenclature Braun grinder for 5 s. The above steps were repeated to prepare G. biloba leaves ground for 20 s. The unfer- AOC antioxidant capacity mented G. biloba leaves were kept in the oven at 25 C ABTS+ 2,20-azinobis-(3-ethylbenzothiazoline-6- and put in a desiccator for at least 24 h prior to analysis. sulfonic acid) AEAC ascorbic acid equivalent antioxidant 2.1.2. Fermented leaves capacity Approximately 10 g of unfermented G. biloba leaves FRAP ferric reducing antioxidant power (ground for 5 s) were weighed into a beaker. Distilled HPLC high performance liquid chromatography water (1 g of leaves: 2 ml of water) was added and they were mixed and bruised thoroughly. The bruised Ginkgo leaves were put into an oven at 25 C and were allowed and position of hydroxyl groups within the molecule to ferment for 2 h, after which, the leaves were removed (Cody, 1988). The presence of a hydroxyl group in and dried in an oven for 24 h at a temperature of 80 C. position three (3-OH) of the C ring. For example, the The earlier steps were repeated to prepare fermented flavonoid aglycones, such as fisetin, (+)-catechin, quer- G. biloba leaves (ground for 5 s) fermented for 6, 10 and cetin, myricetin and morin, are more potent inhibitors 24 h. The same method was used to prepare all the fer- of LPO than those that lack a 3-OH substitution such as mented (2, 6, 10, 24 h) G. biloba leaves ground for 20 s. diosmetin, apigenin (flavones), hesperetin, and nar- All fermented G. biloba leaves were kept in a suitable ingenin (flavanones) (Mora, Paya, Rios, & Alcarza, desiccator after drying in the oven. 1990; Ratty & Das, 1988). Antioxidants are increasingly being recognised as 2.2. Sample preparation protocol important health promoters in conditions such as car- diovascular problems, treatments of many forms of In all the experiments, Ginkgo leaf infusions were cancer and even aging (Packer, 1999). Antioxidants are prepared according to the following standard protocol. able to reduce the effects of free radicals formed in the About 0.4 g of leaves were weighed into a beaker and 40 body, either by exposure to environmental pollutants or ml of deionised water was added (100 ml of water/g of because somatic defence mechanisms are reduced in leaves). Water temperatures and infusion times were dealing with the natural production of these com- varied between 60 and 100 C and 1–20 min, respec- pounds. Thus, it was decided that AOC would be the tively, to consider the effect of infusion temperature, as benchmark by which the therapeutic effects of the leaf well as the infusion time, on the antioxidant activity. infusion could be measured. The effect of the degree of fermentation and surface The AOC was based on the ability of the sample to area of Ginkgo leaves, as well as a comparison among scavenge 2,20-azino-bis-(3-ethlybenzthiazoline-6-sul- different tea types, on the antioxidant activity, was also fonic acid) (free radical ABTS+). The FRAP assay, is determined, using similar procedures. Table 1 illustrates based on low pH (3.6), where ferric ion is reduced to the various samples used for AOC determination. ferrous ion, forming a compound known as a ferrous– tripyridyltriazine complex (Benzie & Strain, 1996, 1999). 2.3. Particle size determination The main advantages and disadvantages of these two methods have recently been discussed (Frankel & Particle size determination was performed on Ginkgo Meyer, 2000). In this paper both methods are used and leaves (ground for 5 s, and 20 s), commercial green tea, compared. and black tea, using Tyler Standard Screen Scale Sieves This study will provide more information for epide- (Cadle, 1955). miological studies and aid in the product development Particle size and particle size distributions are gen- of G. biloba leaves which can later be directed to con- erally measured, because of the relationships they bear sumers with an optimal knowledge of the content and activity of these natural antioxidants. Table 1 Test samples 2. Materials and methods Samples Fermented (F)/ unfermented (U) Grinding time (s) A (U) 5 2.1. Ginkgo biloba leaves preparation B (U) 20 C (F) for 2 h 5 2.1.1. Unfermented leaves D (F) for 2 h 20 Gingko biloba leaves used for AOC determination E (F) for 24 h 5 F (F) for 24 h 20 were imported from Ventura County, California, USA. L.M. Goh et al. / Food Chemistry 82 (2003) 275–282 277 to other properties of such particles (Cadle, 1955). In were measured at 593 nm. The infusions (40 ml) were this case, the particle size difference between Ginkgo transferred into the cuvettes containing the reagent leaves ground for 5 and 20 s, commercial green and and the mixtures were shaken thoroughly. The mix- black tea might have an effect on the release of anti- tures in the cuvettes were examined after 90 min using oxidant compounds during infusion. a UV–vis spectrophotometer and the absorbance of the mixture, after 90 min was recorded. Each 2.4. Experiment 1: effect of infusion temperature measurement of sample infusions was analysed in tri- plicate and the FRAP equivalent, expressed as mg The effect of infusion temperatures was investigated FRAP per 100 g of leaves, was calculated using the using six types of Ginkgo leaves: samples A, B, C, D, E following equation: and F. The infusion of each was prepared by adding FRAP ¼ CFS Â V Â ðÞ100=W ð1Þ water at 60, 80 and 100 C. The mixtures of each type of leaves were stirred and allowed to stand for 10 min where CFS is the concentration of ferrous sulphate before filtering using filter paper (Whatman No. 1). standard solution corresponding to the absorbance The different infusions were collected in clean test change (mg/ml), V is the volume of infusion (ml) and W tubes. is the weight of leaves used for infusion preparation (g). Experiments 1, 2 and 3, mentioned earlier, were per- 2.5. Experiment 2: effect of infusion time formed using the FRAP method.

The effect of infusion time was investigated using four 2.8. ABTS+ determination types of Ginkgo leaves: samples A, B, C, and D. The infusion of each was prepared by adding water at ABTS[2,2 0-azinobis-(3-ethylbenzothiazoneline-6-sul- 100 C. They were stirred and allowed to stand for 1 fonic acid)] was used as the free radical provider and min before being filtered. All the infusions were col- was generated by reacting this compound (7.4 mM) with lected in clean test tubes. The steps were repeated for potassium persulphate (2.45 mM) overnight (Re et al., infusion times of 3, 5, 10, 15 and 20 min for all the 1999). The solution was diluted, to obtain an absor- leaves. bance of between 1.5 and 2.2 at 414 nm [molar extinc- tion coefficient, "=3.6 Â 104 mol1 cm1 (Forni et al., 2.6. Experiment 3: comparison studies among Ginkgo 1986) with 98% ethanol, before use. Reagents (3 ml) leaves, commercial green and black tea leaves were transferred to the glass cuvettes with one of them containing 3 ml ethanol as blank. The initial absorbance In order to benchmark the AOC of Ginkgo with of the reagents in the glass cuvettes at 414 nm was similar hot beverages, green tea and Ceylon tea were recorded. The infusions (40 ml) were transferred into the used for comparison. The antioxidant activities of three cuvettes containing the reagent and the mixtures were different types of leaves were investigated: unfermented shaken thoroughly. The mixtures in the cuvettes were Ginkgo leaves ground for 20 s (sample B), commercial examined after 90 min using a UV–vis spectro- green tea and commercial black tea. The infusion of photometer, and the absorbance of the mixture was each tea was prepared by adding water at 100 C. They recorded. were stirred and allowed to stand for 3 min before being Each infusion was analysed in triplicate and the filtered using filter paper as before. All the infusions ascorbic acid equivalent antioxidant capacity (AEAC) were collected in clean test tubes. Commercial green tea of each infusion or sample was calculated and com- infusion (1 ml) was diluted with 10 ml of distilled water pared. The AEAC, expressed as mg ascorbic acid (AA) and black tea with 20 ml of distilled water. per 100 g of leaves, was calculated using the following equation: 2.7. FRAP determination AEAC ¼ CAA Â V Â ðÞ100=W ð2Þ

The AOC of all the infusions were determined using a where CAA is the concentration of the AA standard modification of the FRAP assay previously described by solution corresponding to the absorbance change (mg/ Langley-Evans (2000). The FRAP reagent was prepared ml), V is the volume of infusion (ml) and W is the from 300 mM, pH 3.6, acetate buffer, 20 mM ferric weight of leaves used for infusion preparation (g). chloride and 10 mM 2, 4, 6-tripyridyl-s-triazine made up Experiment 2 (using only samples A and B) and 3 were in 40 mM hydrochloric acid. All three solutions were repeated using the ABTSmethod. Ascorbic acid was mixed together in the ratio of 25: 2.5: 2.5 (v:v:v). The used as a standard in this assay in view of the fact that FRAP assay was performed using reagents preheated to earlier work (Goh & Barlow, 2002) had shown a corre- 37 C. Prior to analysis, the initial absorbances of 3 ml lation between ascorbic acid content and total anti- of the reagents, and a 3 ml acetate buffer used as blank, oxidant capacity. 278 L.M. Goh et al. / Food Chemistry 82 (2003) 275–282

2.9. Fingerprint of Ginkgo biloba leaves infusion black tea leaves > commercial green tea leaves. (See compounds (unfermented vs. fermented) using capillary Table 2). zone electrophoresis (CZE) separation technique 3.2. FRAP determination 2.9.1. Materials AOC was demonstrated in the Ginkgo leaves infusates Sample A: unfermented Ginkgo leaves ground for 20 s. at all temperatures studied. Generally, the FRAP values Sample B: fermented 24 h Ginkgo leaves ground for in Ginkgo leaves infusates increased in a linear manner 20 s. across the range of temperatures studied. There was no significant difference in FRAP (P=0.05) between 2.9.2. Apparatus (CZE) unfermented leaves and leaves fermented for 24 and 2 h All separations were performed using a Hewlett- (with infusion conditions of 100 C and 10 min) as seen Packard 3D CE system supplied by HP (Waldbronn, in Table 3. The linear increase of FRAP, illustrated in Germany) equipped with a 48.5 cm fused silica capillary Fig. 1, was similar for leaves ground for 5 or 20 s; column (with effective length of 40 cm for separation), however, the FRAP value of those ground for 20 s was i.d. 50 mm, from Polymicro Technologies (Phoenix, AZ, consistently higher. In addition, fermentation did not USA). The analysis buffer was 50 mM sodium borate affect the FRAP results. This finding also correlated (pH 9.3) and 10% acetonitrile in water. The tempera- well with the chromatograms obtained from capillary ture was kept constant at 20 C; separation voltage was electrophoresis, as shown in Fig. 2. These clearly show 25 kV and detection was at 230 nm—the wavelength that the compound profiles are the same for samples A giving the best resolution. All buffers were filtered using and B and there is no significant difference between the a Sigma (St. Louis, MO, USA) 0.2-mm filter. To main- patterns of peaks obtained. It is generally accepted that tain the capillary conditions, fresh buffer was intro- unfermented tea leaves, for example, green tea, should duced into the capillary between runs. The data were have a higher AOC (Von Gadow, Joubert, & Hans- collected and analysed using software for instrument mann, 1997) due to the flavonols present that are known control and an HP Chemstation (Edition A. 06.04) data potent antioxidants (Lunder, 1992; Xie, Shi, Chen, & processor. Ho, 1993). However, this study showed otherwise. Fur- ther studies on Ginkgo leaves fermented for 6 and 10 h 2.10. Caffeine determination using HPLC were not continued. There was an observable increase in the FRAP value An HPLC system (Shimadzu HPLC, Shimadzu, of Ginkgo leaves ground for 5 s and 20 s (Fig. 3)with Kyoto, Japan) equipped with a photo-diode array respect to infusion times. Taking unfermented Ginkgo detector was used. The separation was performed on a leaves infused for 10 min as an example, leaves that Shim-Pack VP-ODS column (250Â4.6 mm i.d.) (Shi- madzu, Kyoto, Japan) using, as mobile phase, 40:60 Table 2 a (v.v) methanol:water at a flow-rate of 1.0 ml/min at Particle size distribution of the leaves passing through various sieve sizes 40 C. The solvent was filtered through a 0.45-mm membrane filter and degassed using an ultrasonic bath Sieve opening or by flushing with helium before use. 2000 850 354 177 150 <150

2.11. Statistical analysis Ginkgo leaves (ground for 5 s) 88 26 6 2 0 0 Ginkgo leaves (ground for 20 s) 100 76 12 2 0 0 Commercial black tea leaves 100 90 10 0.8 0.7 0 Two tailed analysis of variance was performed on the Commercial green tea leaves 100 93.2 53.2 15.2 12 0 data. Student’s t-LSD (least significant difference) (P=0.05) was calculated to compare means for the dif- a Percentage passing (%). ferent teas and also for the fermented and unfermented Ginkgo leaves infusion. Table 3 FRAP equivalent (mg/100 g) of fermented and unfermented Ginkgo leaves infusion

3. Results and discussion Type of Ground Temperature Time of FRAP equivalent Ginkgo leaves time (s) of infusion infusion (mg/100 g leaves) (C) (min) (% RSD) 3.1. Particle size distribution Fermented 2 h 20 100 10 6329 (5.48%) The particle size distribution of the leaves was deter- Fermented 24 h 20 100 10 6409 (1.59%) Unfermented 20 100 10 6825 (4.54%) mined as follows, in decreasing size order: Ginkgo leaves (ground 5 s) > Ginkgo leaves (ground 20 s) > commercial The AOC results are generated from three determinations. L.M. Goh et al. / Food Chemistry 82 (2003) 275–282 279

Fig. 1. FRAP equivalent (mg/100 g) of Ginkgo leaves infusion with respect to temperatures (C). * Sample A: unfermented, ground for 5 s; * sample B: unfermented, ground for 20 s. X sample C: fermented 2 h, ground for 5 s; ^ sample D: fermented 2 h, ground for 20 s; sample E: fermented 24 h, ground for 5 s; ~ sample F: fermented 24 h, ground for 20 s. were ground for 20 s (at 100 C) gave a FRAP value of FRAP value, only unfermented Ginkgo leaves were then 6830 mg/100 g leaves, an increase of 2219 mg/100 g leaves used in determining the AOC using the ABTSmethod. from the FRAP value obtained from leaves that were The AEAC value of unfermented Ginkgo leaves ground ground for 5 s, at 100 C (an increase of approximately for 20 s was observed to be higher than that for leaves 33%). As expected, Ginkgo leaves ground for 20 s had a ground for 5 s, similar to the results obtained using the higher FRAP value and that could be attributed to the FRAP method. However, the optimum infusion time, larger surface area of the leaves that were ground for a for releasing the maximum antioxidants from the leaves, longer time and their ability to release the antioxidant was different for different leaf types. The results seem components more efficiently over the same period of time. contradictory because the maximum AEAC value was The FRAP values increased with infusion time for observed at t=10 min for leaves ground for 5 s whereas, fermented leaves (samples C and D) from 1 min to a for leaves ground 20 s, the maximum AEAC value was maximum FRAP value at 10 min. Similarly, for sample observed at t=15 min. However, it was anticipated that A, the FRAP values increased within the range of infu- leaves of smaller particle size should be capable of sion time (1–20 min) under study. The trend for unfer- releasing the maximum antioxidants after a shorter mented leaves, as seen in Fig. 3, on the other hand, was time. The results of ABTSdetermination do need to be uncertain for the range of infusion time under study. viewed with care. This is because the specificity of assay For example, sample B exhibited an increase in the involving the ABTSmethod for measuring the capacity FRAP value obtained at t=3 min (6478 mg/100 g of a sample to directly quench free radicals, is not leaves) but the value decreased at t=5 min (6121 mg/ always guaranteed or reproducible (Cao & Prior, 1998). 100 g leaves) before reaching a global maximum, However the method has been successfully used for increasing again at t=10 min (6830 mg/100 g leaves). AOC determination in alcoholic extracts of Ginkgo For sample A, the FRAP value was increasing within biloba nuts and leaves (Goh & Barlow, 2002). the range of infusion time (1–20 min) under study. However, most of the data (including 2-h fermented 3.4. Comparison studies among Ginkgo leaves, commercial leaves) show that the release of antioxidants from green and black tea leaves Ginkgo leaves was maximum at t=10 min to t=15 min, as seen in Fig. 4. The AOC of the different beverages, as determined by the FRAP assay and ABTSdecolorisation assay, 3.3. ABTS+ determination decreased in the order: commercial green tea leaves > commercial fermented black tea > Ginkgo leaves infu- This experiment was conducted on samples A and B. sion ground for 20 s as shown in Table 4. The black and As fermentation showed no significant difference in the green tea leaves had an approximate FRAP value 7 to 280 L.M. Goh et al. / Food Chemistry 82 (2003) 275–282

Fig. 2. A: unfermented Ginkgo leaves (see Section 2 for details) infusion 10 min, 0.4 g/40 ml; B: fermented 24 h Ginkgo leaves (see Section 2 for details) infusion 10 min 0.4 g/40 ml; capillary electrophoresis conditions: buffer, 50 mM borate and 10% ACN, injection: 50 mbar*4 s, +25 kV.

Fig. 3. FRAP equivalent (mg/100 g) with respect to time of infusion (min): ~ unfermented Ginkgo leaves ground for 20 s (sample A); & unfer- mented Ginkgo leaves ground for 5 s (sample B). L.M. Goh et al. / Food Chemistry 82 (2003) 275–282 281

Fig. 4. FRAP equivalent (mg/100 g) with respect to time of infusion (min): ~ fermented 2 h Ginkgo leaves ground for 20 s (sample D); * fermented 2hGinkgo leaves ground for 5 s (sample C).

Table 4 Table 5 AOC of the different test samples Caffeine content in selected beverages

Types of test samples FRAPa (% RSD) ABTSb (% RSD) Types of test samples Caffeine contenta

Green tea 56 810 (2.05%) 36 461 (1.67%) English breakfast tea 49.41.98 English breakfast tea 44 745 (1.27%) 31 883 (1.75%) Green tea 52.01.41 Ginkgo leaves infusion 6211 (1.40%) 3185 (1.88%) Ginkgo leaves infusion 0

The AOC results are generated from three determinations. The caffeine quantifications are generated from three determinations. a mg/100 g leaves. a mg/cup (normal serving size of 250 ml). b mg/100 g leaves.

9-fold higher than Ginkgo leaves. However, the differ- constant of approximately 5.9 Â 109 mol1 s1 that is ence of these values, between the green and black tea comparable with those of other efficient hydroxyl radi- leaves and Ginkgo leaves, might be misleading because, cal scavengers. Hence, caffeine does make a contribu- from the particle size analysis of the leaves, the surface tion to total AOC. However the degree of contribution area of the green and black tea leaves was smaller than of caffeine to total AOC in this study was not deter- the surface area of Ginkgo leaves ground for 20 s. From mined. In addition, there are also negative effects of the results presented earlier, it is seen that leaves with a caffeine, including rapid/irregular heartbeat, elevated larger surface area have a significantly higher FRAP blood sugar and cholesterol, nervousness, agitation and value (higher AOC). insomnia (Iwaoka & Brewer, 2000), a negative effect The differences of the AOC values amongst the test during pregnancy (Harland, 2000) and increased cal- samples could be due to the presence of caffeine, which cium loss (Barrett-Connor, Chang, & Edelstein, 1994; is present in considerable amounts in the commercial Harris & Dawson-Hughes, 1994; Lloyd, Rollings, Eggli, hot beverages whilst being absent in the Ginkgo leaves Kieselhorst, & Chinchilli, 1997). Ginkgo leaves infusion infusion. To confirm this, HPLC analysis was carried is caffeine-free yet capable of providing significant out on the Ginkgo leaves infusions (and also to quantify AOC. the caffeine present in the commercial hot beverages). This showed a negative result for caffeine in Ginkgo leaves infusion and relatively high caffeine contents in 4. Conclusion green and black tea, as shown in Table 5. Reported values, i.e. 25–110 mg of caffeine/cup (Food Facts Asia Ginkgo biloba leaves ground for 5 s had a larger Third Quarter, 2000) coincide with the experimental average particle size distribution than leaves ground for results. 20 s. Fermentation had no significant effect on the AOC The ability of caffeine to scavenge the hydroxyl radi- of the G. biloba leaves. However, larger surface area of cal (Shi, Dalal, & Jain, 1991) displayed a reaction rate leaves (leaves ground for 20 s), infusion temperature of 282 L.M. Goh et al. / Food Chemistry 82 (2003) 275–282

100 C and an infusion time around 10–15 min gave the Harland, B. F. (2000). Caffeine and nutrition. Nutrition, 16(7/8), 522– highest AOC of Ginkgo leaves. 526. From the study, it is clearly shown that G. biloba leaf Harris, S., & Dawson-Hughes, B. (1994). Caffeine and bone loss in healthy post-menopausual women. American Journal of Clinical infusion has a lower AOC than the studied commercial Nutrition, 60, 573. hot tea beverages. However, it has an advantage over Husain, S. R., Cillard, J., & Cillard, P. (1987). Hydroxyl radical these in that caffeine is absent and this would definitely scavenging activity of flavonoids. Phytochemistry, 26, 2489–2491. be of benefit to people who show adverse effects to Iwaoka, W. T., & Brewer, M. S. (2000). Toxicants. In G. L. Christen, caffeine. & J. S. Smith (Eds.), Food chemistry: principles and application Chapter 15 (pp. 241–260). West Sacramento, California: Science Technology System. Langley-Evans, S. C. (2000). Antioxidant potential of green and black References tea determined using the ferric reducing power (FRAP) assay. International Journal of Food Sciences and Nutrition, 51, 181–188. Barrett-Connor, E., Chang, J. C., & Edelstein, S. L. (1994). Coffee- Lloyd, T., Rollings, N., Eggli, D. F., Kieselhorst, K., & Chinchilli, associated osteoporosis offset by daily milk consumption. JAMA, V. M. (1997). Dietary caffeine intake and bone status of post- 271, 280. menopausal women. American Journal of Clinical Nutrition, 65, Benzie, I. F. F., & Strain, J. J. (1996). The ferric reducing ability of 1826. plasma (FRAP) as a measure of ‘‘antioxidant power’’: the FRAP Lunder, T. L. (1992). Catechins of green tea: antioxidant activity. In assay. Analytical Biochemistry, 239, 70–76. M. T. Huang, C. T. Ho, & C. Y. Lee (Eds.), Phenolic compounds in Benzie, I. F. F., & Strain, J. J. (1999). Ferric reducing/antioxidant food and their effects on health. II. Antioxidants and cancer preven- power assay: direct measure of total antioxidant activity of biologi- tion (pp. 114–120). Washington: American Chemical Society. cal fluids and modified version for simultaneous measurement of Maitra, I., Marcocci, L., Packer, L., & Droy-Lefaix, M. T. (1995). The total antioxidant power and ascorbic acid concentration. Methods in nitric oxide-scavenging activity of ginkgo biloba extract EGb 761. Enzymology, 299, 15–27. Biochemical Pharmacology, 45, 1649–1665. Bors, W., Heller, W., Michel, C., & Saran, M. (1990). Flavonoids as Marcocci, L., Packer, L., Droy-Lefaix, M. T., Sekaki, A. H., & antioxidants: determination of radical-scavenging efficiencies. Gardes-Albert, M. (1994). Antioxidant actions of Ginkgo biloba Methods Enzymology, 189, 343–355. extract EGb761. In L. Packer (Ed.), Methods in enzymology Cadle, R. D. (1955). Particle size determination. New York: Inter- (pp. 462–475). San Diego: Academic Press. science Publishers. Marcocci, L., Maguire, J. J., Packer, L., & Droy-Lefaix, M. T. (1994). Cao, G., & Prior, R. L. (1998). Comparison of different analytical The nitric oxide-scavenging properties of Ginkgo biloba extract methods for assessing total antioxidant capacity of human serum. EGb761. Biochemical Biophysical Research Communications, 201, Clinical Chemistry, 44(6), 1309–1315. 748–755. Cody, V. (1988). Crystal and molecular structure of flavonoids. Moro, A., Paya, M., Rios, J. L., & Alcarza, M. J. (1990). Structure- InPlant flavonoids in biology and medicine II: biochemical, cellular, activity relationship pf polymethoxyflavones and other flavonoids as and medicinal properties (pp. 29–44). New York, NY: Alan R. Liss. inhibitors of non-enzymic lipid peroxidation. Biochemical Pharma- Ellnain-Wojtaszek, M., & Zgorka, G. (1999). High-performance liquid cology, 40, 793–797. chromatography and thin-layer chromatography of phenolic acids Packer, L. (1999). The antioxidant miracle—your complete plan for from Ginkgo biloba L. leaves collected within vegetative period. Journal total health and healing. New York: Wiley. of Liquid Chromatography & Related Technologies, 22(10), 1457–1471. Pincemail, J., Dupuis, M., Nasr, C., Hans, P., Haag-Berrurier, M., Ellnain-Wojtaszek, M., Kruczyn˜ ski, Z., & Kasprzak, J. (2002). Varia- Anton, R., & Derby, C. (1985). Superoxide dismutase from a tions in the free radical scavenging activity of Ginkgo biloba L. eucaryote, Ginkgo Biloba. Arch. Biochem. Biophys., 243, 305–314. leaves in the period of complete development of green leaves to fall Ratty, A. K., & Das, N. P. (1988). Effects of flavonoids on non-enzy- of yellow ones. Food Chemistry, 79(1), 79–84. mic lipid peroxidation: structure activity relationship. Biochem. Food facts Asia third quarter. (2000). Caffeine content of Food & Med. Metabol. Biol., 39, 69–79. Beverages. Issue 9, Available: http://www.afic.org/article.asp?Search Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yong, M., & Method=Search&ArticleId=187&CollectionType=1 Rice-Evans, C. (1999). Antioxidant activity applying an improved Forni, L. G., Morla-Arellano, V. O., Packer, J. E., & Willison, R. L. ABTSradical cation decolorisation assay. Free Radical Biology & (1986). Nitrogen dioxide and related free radical; electron transfer Medicine, 26(9/10), 1231–1237. reaction with organic compounds in solutions containing nitrite or Shi, X., Dalal, N. S., & Jain, A. C. (1991). Antioxidant behavior of nitrate. Journal of Chem. Soc., 2, 1–6. Perkin Trans. caffeine: efficient scavenging of hydroxyl radicals. Food Chemistry Frankel, E. N., & Meyer, A. S. (2000). The problems of using one- Toxicology, 29(1), 1–6. dimensional methods to evaluate multifunctional food and biologi- Tenney, L. (1996). Ginkgo biloba. Pleasant Grove, UT: Woodland cal antioxidants. Journal of the Science of Food and Agriculture, 80, Publishing. 1925–1941. (Online: 2000). Von Gadow, A., Joubert, E., & Hansmann, C. F. (1997). Comparison Gardes-Albert, M., Ferradini, C., Sekaki, A., & Droy-Lefaix, M. T. of the antioxidant activity of rooibos tea (Aspalathus linearis) with (1993). Oxygen-centered free radicals and their interactions with green, oolong and black tea. Food Chemistry, 60(1), 73–77. EGb 761 or CP 202. In C. Ferradini, M. T. Droy-Lefaix, & Xie, B., Shi, H., Chen, Q., & Ho, C. T. (1993). Antioxidant properties Y. Christen (Eds.), Advances in Ginkgo biloba extract research of the fractions and polyphenol constituents from green, oolong and (pp. 1–11). Paris: Elsevier. back teas. Proc. Natl. Sci. Counc. Repub. China [B], 17, 77–84. Goh, M. L., & Barlow, P. J. (2002). Antioxidant capacity in Ginkgo Zuess, J. (1998). Ginkgo the smart herb. New York: Three River Press. biloba. Food Research International, 35, 815–820.

Appendix 3: Flavonoid recovery and stability from Ginkgo biloba subjected to a simulated digestion process.

Page 200 of 201 Food Chemistry Food Chemistry 86 (2004) 195–202 www.elsevier.com/locate/foodchem

Flavonoid recovery and stability from Ginkgo biloba subjected to a simulated digestion process

Lena M.L. Goh *, Philip J. Barlow

Food Science and Technology Programme, Department of Chemistry, National University of Singapore, Science Drive 4, Science Block S3-06, Singapore 117543, Singapore

Received 21 January 2003; received in revised form 14 August 2003; accepted 14 August 2003

Abstract

Absorption from the diet is normally a prerequisite for the potential in vivo beneficial role of flavonoids. Antioxidant activity of flavonoids in vitro has been the subject of several studies, and important structure–activity relationships of the antioxidant activity have been established. However, there is still debate about the stability and absorption of polyphenols under gastrointestinal conditions. Ginkgo biloba, a product well known for its flavonoid content, was chosen for this study. Ginkgo biloba leaves, stan- dardised leaf extract EGb 761 and commercial tablets (containing EGb761) were incubated in simulated gastrointestinal fluids to determine the stability of their flavonoid profiles. The experiment was designed to mimic the human gut condition. HPLC analysis was then conducted to determine the resulting breakdown compounds and intact flavonoids after the incubation, thus indicating those compounds likely to be available for absorption. The different samples seem to react differently to the simulated digestion process. The results indicate a trend of conversion from the glycosides to the aglycones for some samples and subsequent degra- dation of the aglycones. This may indicate a need to further investigate the reported benefits of Ginkgo flavonoids as in vivo antioxidants and/or to consider the antioxidant activity of the resulting digestion-derived compounds. Ó 2003 Elsevier Ltd. All rights reserved.

Keywords: Ginkgo biloba leaves; Ginkgo biloba commercial capsules; Simulated gastrointestinal fluids; EGb 761; Flavonoids; HPLC

1. Introduction standardised Ginkgo biloba extract. These products were subjected to conditions that closely followed those en- The fate of phytochemicals in Ginkgo biloba leaves countered in the gastrointestinal tract. and their extract (EGB761) (DeFeudis, 1998) in the The aim of this study was to achieve a better under- human body after ingestion is of interest in order to standing of what happens to the flavonoid compounds investigate the potential role of flavonoids as regards when they are placed in the mouth, carried by peristalsis their radical-scavenging ability. Over recent years, to the stomach and the fate of these compounds when Ginkgo biloba has achieved unprecedented popularity, as they come into contact with the upper gut secretions. a health supplement. Concurrently, a major thrust of It has been reported that flavonoids present in foods research has been the development of new food prod- cannot be absorbed from the intestine because they are ucts, which have functional properties, which are truly bound to sugars as glycosides (Kuhnau, 1976). Only free expressed in the body. flavonoids without a sugar molecule, the so-called An experiment (in vitro) to simulate the workings of aglycones, are considered to be able to pass through the the human gut during digestion was designed to study gut wall, and no enzymes that can split these predomi- the recovery and stability of the flavonoids. The prod- nantly b-glycosidic bonds are secreted into the gut or ucts of interest were raw Ginkgo biloba leaves, com- present in the intestinal wall. However, in contrast, mercial Ginkgo biloba leaf extract capsules and Hollman and his team (1999) suggest that, certainly for quercetin glycosides from onions, the absorption of the * Corresponding author. Tel.: +65-68741655; fax: +65-67757895. intact glycosides is in fact far better then the pure E-mail address: [email protected] (L.M.L. Goh). aglycones. These workers suggest that absorption

0308-8146/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2003.08.035 196 L.M.L. Goh, P.J. Barlow / Food Chemistry 86 (2004) 195–202 kinetics and bioavailability of the flavonoids are prob- were prepared in methanol (varying concentration from ably governed by the form of glycoside present. How- 100 to 500 ppm). Pepsin, monobasic potassium phos- ever, this current work would seem to support the earlier phate, pancreatin mix, NaOH, NaCl and HCl, used for report by Paganga and Rice-Evans (1997) who showed the simulation of the gastrointestinal fluids, were pur- the presence of glycosylated flavonoids present in hu- chased from Sigma Aldrich, Singapore. 2-Propanol and man plasma, in that the digested raw leaf residues were tetrahydrofuran (THF), used for chromatography, were high in glycoside content. of HPLC grade. This study aims to investigate the stability of flavo- noid compounds before considering whether they are 2.2. Ginkgo biloba leaves preparation absorbed. If the aglycones or their glycosides were to- tally degraded in the gastrointestinal fluids then their The leaves were first ground for 20 s, a particle size absorption would be impossible. This simulated diges- distribution of 98% being retained in the 177 lm sieve. tion/absorption study was undertaken with two objec- The size of the leaf particles is 98% larger than 177 lm tives: (a) to determine the degree of degradation, if any, but 88% smaller than 354 lm. Sample extractions (w/v in the upper GI tract and (b) in view of the difficulty of 1:10) were carried out with two different solvents, i.e. measuring flavonoids in the blood, and being able to water and ethanol/water (80:20), via stirring overnight. relate such levels to specific foods in the diet, to assess These samples were used as controls for the profile the availability of specific flavonoids for absorption. An analysis. Prior to injection into the HPLC for the in vitro study allows a more controlled approach to test compounds analysis (20 ll), the samples were filtered hypotheses, which can later be studied, in vivo. Other through a 0.45 lm membrane filter. studies have been carried out to determine flavonols in body fluids and these generally seem to indicate minimal 2.3. Particle size determination degradation, at least for quercetin or its glycosides with gastrointestinal fluids (Hollman, de Varies, van Leeu- Particle size determination was performed on Ginkgo wen, Mengelers, & Katan, 1995). leaves using Tyler Standard Screen Scale Sieves (Cadle, This study aims to provide further information on the 1955). Particle size and particle size distributions are stability of flavonoids in the human organism during the generally measured because of the relationships they digestion process and to contribute to the development bear to other properties of such particles (Cadle, 1955). of Ginkgo biloba leaf products, which can later be di- In this case, particle size determination is used to set the rected to consumers with an increased knowledge of the baseline on the effect of release of antioxidant com- content and biological activity of these natural antioxi- pounds during extraction. dants. 2.4. Preparation of simulated gastric fluids and intestinal fluid 2. Materials and methods Simulated gastric fluid was prepared according to the 2.1. Materials procedure of the USP, National Formulary: 2.0 g NaCl, 3.2 g pepsin and 3.0 ml concentrated HCl. diluted to 1 l The dried Gingko biloba leaves used were imported and verified that the pH is 1.2–1.8. from Ventura County, California, USA. Ginkgo biloba Simulated intestinal fluid was prepared according to leaf commercial capsules (claimed to be dried leaves) the procedure of the USP, National Formulary: 6.8 g were purchased from a local General Nutrition Center. monobasic potassium phosphate, 650 ml water, 190 ml Each capsule is claimed to contain 50 mg of Ginkgo of 0.2 mol/l NaOH and pancreatin mix (10 g). biloba leaf extract, which has been ground to a fine In this experiment the simulated gastric juice, was powder. Standardised Ginkgo biloba leaf extract made up as above and incubation conditions with tem- (EGb761) used was purchased from Voigt Global Dis- perature control and vortex action were applied to mi- tribution Kansas City, USA. These three samples were mic the stomach conditions. The temperature was set at used to determine the flavonoid profiles based on their 37 °C and the churning action of the stomach was stability in the gastrointestinal fluids. mimicked by vortexing the chyme at 100 rpm. In vivo, Authentic samples of quercetin-3-O-rutinoside (ru- after residing in the stomach for 1–2 h, the chyme then tin), quercetin-3-galactoside, kaempferol-3-rutinoside, proceeds on to the small intestine and for this experi- quercetin-3-rhamnoside (quercetrin), kaempferol-3-O- ment intestinal fluids were made up to mimic the pan- rutinoside, kaempferol-3-(p-coumaryl) glycoside], were creatic fluids and allowed to react for 2–4 h in a obtained from Apin chemical, Abingdon, UK. Querce- vortexing bath, at 37 °C, corresponding to the aver- tin, kaempferol, and isorhamnetin standards were pur- age and maximal transit time in the small intestine, chased from Aldrich Chemical Co. Reference solutions respectively. L.M.L. Goh, P.J. Barlow / Food Chemistry 86 (2004) 195–202 197

2.5. Incubation of Ginkgo biloba leaves in simulated 2.7. Chromatographic conditions gastrointestinal fluids An HPLC system (Shimadzu HPLC, Shimadzu, Carried out in triplicate, 1 g of Ginkgo biloba leaves Kyoto, Japan), equipped with a photo-diode array de- was incubated with the simulated gastric juice at tector, was used and detection of the flavonoids was 37 °C for 1 h (designated step 1). In step 2, the earlier carried out at 360 nm. The separation was performed on amounts from the simulated gastric juice (chyme) were a Shim-Pack VP-ODS column (250 4.6 mm i.d.) then incubated with the intestinal fluid at 37 °C, for 2 (Shimadzu, Kyoto, Japan) using mobile phase A: water: and 4 h, respectively. These steps were carried out so 2-propanol (95:5), eluent B: 2-propanol: THF: water as to mimic the digestion process as discussed earlier. (40:10:50), binary gradient from 20% to 55.2% B in 44 The incubated samples from both steps, 1 and 2, were min at a flow-rate of 1.0 ml/min at 40 °C. The solvent then filtered and made up to 25 ml. A duplicate of the was filtered through a 0.45 lm membrane filter and supernatant from both steps was shaken with metha- degassed using an ultrasonic bath or by flushing with nol to extract the alcohol soluble compounds. From helium before use. The mobile phase conditions were this, two samples were obtained, namely an aqueous modified from a method previously reported by Pietta et acidic extract and an alcoholic acid extract. Both al. (1991). This method is capable of analyzing 8 flavo- solvent systems were used in order to track possible noids in the various samples during one HPLC analysis. changes in the flavonoids as aglycones are more likely to be found in the alcohol medium whilst the glyco- 2.8. Identification and determination of the glycosides and sides are more likely to be associated with the aque- aglycones ous medium. This allowed comparison with the undigested samples. This experiment yields the range The identification of the HPLC chromatographic of samples shown in Table 1. peaks was established with external standards. Each sample was injected three times for HPLC profiling. The 2.6. Incubation of Ginkgo biloba leaf commercial capsules linearity of the determination of the 5 flavonol glyco- and standardised Ginkgo biloba leaf extract (EGb761) in sides and 3 flavonol aglycones was verified by linear simulated gastrointestinal fluids regression. The correlation coefficients were 0.9446 for rutin, 0.999 for quercetin-3-galactosides, 0.998 for ka- Similar steps, as detailed in 2.4, were carried out on empferol-3-rutinoside, 0.997 for quercetin-3-O-rham- the commercial capsules but using 2 capsules in 25 ml noside, 0.997 for quercetin, 0.998 for isorhamnetin and and for EGb 761, using 0.25 g in 25 ml. This was deemed 0.998 for kaempferol. Standards, such as quercetin, a realistic ratio of what might occur in vivo. isorhamnetin and kaempferol, used for calibration, are

Table 1 Experimental samples obtained from standardised Ginkgo biloba extract, Ginkgo biloba leaves extract and Ginkgo biloba capsules Type of samples and treatmenta Description Sample code Step 1 Step 2

Samples in aqueous extract left overnight – Control Blankaqueous Samples in alcohol extract left overnight – Control Blankalcohol Samples incubated with gastric juice 1 h in – To mimic food residing in the Aaqueous aqueous extract stomach condition for 1 h

Samples incubated with gastric juice 1 h in – To mimic food residing in the Balcohol alcohol extract stomach condition for 1 h

Samples incubated with gastric juice 1 h Followed by incubation To mimic food residing in the Caqueous in intestinal fluid for 2 h stomach for 1 h then passing through in aqueous extract the intestinal fluids for 2 h

Samples incubated with gastric juice 1 h Followed by incubation To mimic food residing in the Dalcohol in intestinal fluid for 2 h stomach for 1 h then passing through in alcohol extract the intestinal fluids for 2 h

Samples incubated with gastric juice 1 h Followed by incubation To mimic food residing in the Eaqueous in intestinal fluid for 4 h stomach for 1 h then passing through in aqueous extract the intestinal fluids for 4 h

Samples incubated with gastric juice 1 h Followed by incubation To mimic food residing in the stomach Falcohol in intestinal fluid for 4 h for 1 h then passing through the in alcohol extract intestinal fluids for 4 h a Samples were prepared and analysed in triplicate. 198 L.M.L. Goh, P.J. Barlow / Food Chemistry 86 (2004) 195–202 reported to have a very high stability with a loss of <4% From these results, which were designated 100%, an within 12 months after storage at 8 °C in a refrigerator increase or decrease in the percentage after the incuba- (Hasler & Sticher, 1992). tion procedures was obtained. Tables 3–5 show results for the different samples. 2.9. Purity assay of the chromatographic peaks 3.2. Raw Ginkgo leaves The UV spectra of each peak, after subtraction of the corresponding UV base spectrum of the standards, was The blankaqueous and blankalcohol extract of raw normalised by the computer and their plots superim- Ginkgo leaves gave different values in the determined posed. Peaks were considered to be similar when there amounts of the various constituents. The difference was exact matching among the corresponding standard between the two extracts provides an understanding on spectra and also by comparison of retention times in the extraction power of different solvents and also both standards and samples. verifies the nature of the compounds of interest. In the aqueous extraction of raw leaves, flavonol aglycones could not be detected and there was a lower extraction 3. Results and discussion of the flavonoid glycosides than with the alcohol ex- tract (Table 2). 3.1. General However, following incubation of the raw leaves with gastric fluids for 1 h and subsequent intestinal fluids for The constituents of various Ginkgo biloba samples 2 and 4 h, respectively, the aqueous extract seems to were examined and 5 flavonoid glycosides and 3 agly- display an increase in the amounts of compounds cones were positively identified as shown in Fig. 1. These identified (Table 3). Flavonoids are ubiquitous and are peaks were identified by comparison with the authentic found in the flowers, fruits and leaves of the Ginkgo standards. Together with the retention time data, these biloba plant. They are biosynthesised from the starting peaks were also identified by comparing their UV material glucose via the pathway reported by Haslam spectra with those of the corresponding standards. (1993). Plant cells walls are mainly composed of linear Table 2 shows the amount of constituents identified chains of covalently linked glucose residues. They are from the various control samples, i.e. before incubation. very stable chemically and extremely insoluble.

Fig. 1. Chromatogram of a standardised extract of Ginkgo biloba leaves (control sample: blankalcohol). The constituents determined are quercetin-3-O- rutinoside (rutin), quercetin-3-galactoside, kaempferol-3-rutinoside, quercetin-3-rhamnoside (quercetrin), kaempferol-3-O-rutinoside, kaempferol-3- (p-coumaryl) glycoside, quercetin, kaempferol, and isorhamnetin. Shim-Pack VP-ODS column (250 4.6 mm i.d.); Mobile Phase A: water: 2-propanol (95:5), eluent B: 2-propanol: THF: water (40:10:50), binary gradient from 20% to 55.2% B in 44 min at a flow-rate of 1.0 ml/min at 40 °C. Table 2 Comparison of the amount of the various constituents (mg/g) present in the various Ginkgo biloba samples studied, both in the aqueous and alcohol extracts Type of samples Recovered amount (mg/g) of the constituents studieda Quercetin- Quercetin-3- Kaempferol-3- Quercetin-3-O- Kaempferol-3- Quercetin Isorhamnetin Kaempferol 3-O-rutinoside galactosides rutinoside rhamnoside (p-coumaryl) (Rutin) (Quercetrin) glucoside Raw Ginkgo leaves

Blankaqueous 0.190(1.38%) 0.033(2.96%) 0.109(0.34%) 0.015(0.39%) N.D. N.D. N.D. N.D. Blankalcohol 1.88(2.0%) 0.579(5.24%) 1.31(1.28%) 0.354(0.9%) 0.015(0.62%) 0. 0243(3.16%) 0.002(7.35%) 0.015(7.4%) EGb 761 ...Gh ..Bro odCeity8 20)195–202 (2004) 86 Chemistry Food / Barlow P.J. Goh, L.M.L. Blankaqueous 4.47(0.67%) 6.26(2.21%) 3.67(0.26%) 4.58(0.08%) 1.63(1.19%) 0. 212(5.77%) 0.027(1.97%) N.D. Blankalcohol 9.28(3.22%) 4.60(3.73%) 1.67(7.1%) 0.738(5.81%) 1.60(0.98%) 0. 046(13.22%) 1.24(3.77%) 0.06(8.8%) Commercial capsules

Blankaqueous 0.753(1.68%) 0.073(5.91%) 0.220(4.2%) 0.816(1.13%) 1.09(0.76%) 0. 131(2.85%) N.D. N.D. Blankalcohol 25.3(1.53%) 1.08(5.45%) 0.532(6.35%) 1.51(1.47%) 2.32(1. 3%) 11.0(2.61%) 1.31(6.94%) 3.64(3.4%) N.D. ¼ Not detected. a Mean of 3 determinations (% RSD).

Table 3 Percentage recovery of the various compounds from raw Ginkgo biloba leaves following specific treatment Sample Treatment Quercetin-3-O- Quercetin-3- Kaempferol-3- Quercetin-3-O- Kaempferol-3- Quercetin Isorhamnetin Kaempferol code rutinoside galactosides rutinoside rhamnoside (p-coumaryl) (Rutin) (Quercetrin) glucoside

Aaqueous Gastric 1 h 73.2 6347 231 55.5 NIL NIL NIL NIL Balcohol Gastric 1 h 7.75 376 22.8 3.65 NIL NIL NIL NIL Caqueous Gastric 1 h + Intestinal 2 h 116 122 1019 40.4 NIL NIL NIL NIL Dalcohol Gastric 1 h + Intestinal 2 h 9.22 6.30 74.4 5.78 NIL NIL NIL NIL Eaqueous Gastric 1 h + Intestinal 4 h 125 10901 441 154 NIL NIL NIL NIL Falcohol Gastric 1 h + Intestinal 4 h 9.79 501 29.3 4.94 NIL NIL NIL NIL 199 200

Table 4 Percentage recovery of the various compounds from the EGb 761 following various treatments Sample code Treatment Quercetin-3-O- Quercetin- Kaempferol- Quercetin-3-O- Kaempferol-3- Quercetin Isorhamnetin Kaempferol rutinoside 3-galactosides 3-rutinoside rhamnoside (p-coumaryl) (Rutin) (Quercetrin) glucoside

Aaqueous Gastric 1 h 85.1 13.6 119 4.55 9.70 78.4 NIL NIL Balcohol Gastric 1 h 41.0 18.8 860 43.8 18.2 391 NIL 54.9

Caqueous Gastric 1 h + Intestinal 2 h 88.6 17.3 176 6.38 17.7 90.3 NIL NIL 195–202 (2004) 86 Chemistry Food / Barlow P.J. Goh, L.M.L. Dalcohol Gastric 1 h + Intestinal 2 h 46.3 25.2 422 43.1 18.7 416 NIL 25.9 Eaqueous Gastric 1 h + Intestinal 4 h 98.5 18.5 190 7.74 20.3 106 NIL NIL Falcohol Gastric 1 h + Intestinal 4 h 40.1 22.3 382 30.6 2.89 79.5 NIL NIL

Table 5 Percentage recovery of the various compounds from commercial Ginkgo capsules following specified treatments Sample code Treatment Quercetin-3-O-rutino- Quercetin-3- Kaempferol-3- Quercetin-3-O- Kaempferol-3- Quercetin Isorhamnetin Kaempferol side galactosides rutinoside rhamnoside (p-coumaryl) (Rutin) (Quercetrin) glucoside

Aaqueous Gastric 1 h 7.42 341 22.0 2.00 1.60 9.03 NIL NIL Balcohol Gastric 1 h 0.135 124 4.72 0.864 0.294 0.055 NIL 0.055 Caqueous Gastric 1 h + 7.54 39.7 0.813 1.21 0.389 0.000 NIL NIL Intestinal 2 h

Dalcohol Gastric 1 h 0.207 3.17 39.4 0.000 0.909 0.110 NIL NIL + Intestinal 2 h

Eaqueous Gastric 1 h 6.28 43.3 96.7 1.20 0.222 0.000 NIL NIL + Intestinal 4 h

Falcohol Gastric 1 h 0.220 3.45 36.5 0.810 0.621 0.118 NIL NIL + Intestinal 4 h L.M.L. Goh, P.J. Barlow / Food Chemistry 86 (2004) 195–202 201

However, when subjected to prolonged incubation 3.4. Commercial capsules under digestion conditions, even though the cellulose is not digested, there is likely to be cellular disruption and Similar to the results seen in the pervious samples, the an increased release of flavonoid glycosides available for undigested aglycones were better extracted in the alco- extraction, thus explaining the apparent increases (re- holic medium compared with the aqueous medium covery of up to >1.25 times for rutin, >63 times for (Table 2). Isorhamnetin and kaempferol were not de- quercetin-3-galactosides, >2.3 times for kaempferol-3- tected in the aqueous medium. rutinosides and >1.53 times for quercetin-3-O-rhamno- Following the simulated digestion (Table 5), the rutin sides) in the results obtained. The findings also seem to recoveries were ca. 6% and 0.2%, respectively, in the suggest low yields of flavonoids from undigested leaves aqueous and alcoholic extracts. However for quercetin- because the compounds are chemically bound to the cell 3-galactosides an increase of 34 times and 1.24 times, walls or within the intact cells and not available for respectively, for aqueous and alcoholic extract occurred extraction. after the 1st h. After the full simulated digestion pro- cedure, there was a reduction in the amounts to 30–40% 3.3. EGb 761 and 3–3.5% recovery in the aqueous and alcoholic me- dia, respectively. Recovery of quercetin-3-O-rhamno- For EGb 761, reference to Table 2 indicates a similar sides also gave a low percentage recovery (ca. 0–2% for trend in the distribution of glycosides and aglycones in the aqueous and alcoholic media). Such low recovery, the aqueous and alcoholic medium. Following the sim- seen in the quercetin glycosides, would be expected to be ulated digestion procedure, the quantification of the accompanied by a higher recovery in the corresponding various flavonoids, in the aqueous extract of EGb761, aglycones. However, this was not observed. The recov- differs (Table 4). The following percentages, compared ery of quercetin was also low giving ca. 9% recovery in with the undigested samples of the various flavonoids the aqueous medium after the 1st h of digestion and identified, were observed, ca. 80–90% for rutin, ca. 10– subsequently showing no recovery when subjected to the 20% for quercetin-3-O-galactosides, ca. 100–200% for intestinal conditions. This seems to indicate that there kaempferol-O-rutinosides, ca. 4–8% for quercetin-3-O- was little or no conversion of glycosides to aglycones rhamnosides, ca. 9–20% for kaempferol-3-(p-coumaryl) and, in addition, any aglycones so formed appear to glucoside and ca. 70–105% for quercetin. Isorhamnetin have been degraded in the simulated digestion process. and kaempferol were not detected. As above, it appears that the kaempferol-3-rutinoside If the simulated digestion system does break down the is relatively stable. However, the percentage recovery of glycosides, this should be reflected in an increase in kaempferol was almost insignificant following the sim- aglycone recovery in the alcoholic medium after diges- ulated digestion, thereby indicating a similar phenome- tion. The breakdown of the glycosides is indicated by less non; i.e., there was little or no conversion of glycosides than 100% recovery of rutin, quercetin-3-galactosides to aglycones and, in addition, it seems that the latter has and quercetin-3-O-rhamnosides in the aqueous acidic been degraded in the simulated digestion process. extract. Differences in the glycoside/aglycone ratio, most On the other hand, kaempferol-3-(p-coumaryl) glu- likely reflect differences in stability of the glycosides. coside, showed very low recovery (ca. 0–2% for the Interestingly, kaempferol-3-rutinoside, which appears aqueous and alcoholic media). This indicated that ka- to be a more stable glycoside, does show an increased empferol-3-(p-coumaryl) glucoside was fairly unstable extraction following more severe digestion and hence is and could have been degraded to yield non-active likely to be more available for potential absorption into compounds. the body. In contrast, kaempferol-3-(p-coumaryl) glu- coside, shows a poor recovery in both the alcoholic and 3.5. Summary of effects aqueous extract, indicating a likely total breakdown. However, following the 4th h of incubation, kaempferol- Degradation of the flavonoid compounds in gastro- 3-(p-coumaryl) glucoside seems to be degraded, indi- intestinal fluids has been reported to be minimal, at ca. cating instability of the aglycone previously formed. 5%, in ileostomy subjects (n ¼ 6) (Hollman et al., 1995). In sample D (alcohol) (Table 4), it may be noted that A similar finding is also observed in this work. In the the sum of the recoveries for kaempferol-3-rutinoside digested Ginkgo leaves, a higher concentration of flavo- and quercetin adds up to 838%. As the starting material noid glycosides than with the undigested leaves was seen. in this experiment was an extract, even if all eight However, in contrast, this study seems to indicate that compounds tested had been recovered in their entirety, the recovery of selective flavonoid compounds in EGb the maximum total figure is in theory only 800%. This 761 and commercial capsules, after incubation in vitro in apparent de novo production of flavonoids is most likely simulated gastrointestinal fluids, is minimal. For quer- explained by the low yield of recovery of flavonoids cetin-3-galactosides, quercetin-3-O-rhamnosides and from the undigested samples. kaempferol-3-(p-coumaryl) glucoside in EGb 761, and 202 L.M.L. Goh, P.J. Barlow / Food Chemistry 86 (2004) 195–202 for all the eight constituents of the Ginkgo capsules, a However, it is important to note that the commercial very low recovery was observed of only ca. 20%. products seem to have a higher level of aglycones. Thus, This observation seem to suggest caution when ex- an important area of further research is to establish the pressing the beneficial effect that these constituents may validity of the absorption of glycosides vs aglycones in provide. From various reports, based on epidemiologi- the human body. While there is no doubt about the cal research, a hypothesis has been proposed of an in- benefits of Ginkgo biloba, its therapeutic functions verse relationship between the dietary intake of some should now be considered in light of the data from this flavonoids and the incidence of several chronic diseases study, and, in particularly, which of the compounds (Hertog, Hollman, & Katan, 1992; Knekt et al., 2002). within the plant are actually available and absorbed to However, whether the hypothesis is true or not has still bestow their physiological benefits. to be confirmed in view of the controversial subject of However, care should be exercised in the interpreta- flavonoid absorption (Hollman & Katan, 1997; Holl- tion of this study as no consideration was given to the man et al., 1997a; Hollman, van Trijp, Mengelers, de possible interaction that may exist with other compo- Vries, & Katan, 1997b). This paper has not addressed nents present in the diet, e.g., proteins, lipids and min- the absorption issue but merely the digestion aspect of erals, etc. Also, this study did not investigate possible the flavonoids. effects on flavonoids exerted by enzymes from the brush Based on the above results, it appears that some of border membrane nor the microorganisms in the colon. the active flavonoid compounds in the commercially prepared samples are produced in lower amounts after the simulated digestion procedure than after with the digestion of the natural leaves. This may be due to References previous losses of these compounds during the prepa- ration of the commercial samples. By taking in the raw Cadle, R. D. (1955). Particle size determination. New York: Inter- science Publishers. leaves as opposed to the commercial preparations ex- Haslam, E. (Ed.). (1993). Shikimic acid metabolism and metabolites. amined, there is likely to be a greater benefit in view of Chichester: Wiley. the higher level of flavonoids detected. Hasler, A., & Sticher, O. (1992). Identification and determination of From Table 2 it may be seen that the undigested com- flavonoids from Ginkgo biloba by high-performance liquid chro- mercial extracts do in fact contain a higher proportion of matography. Journal of Chromatography, 605, 41–48. Hertog, M. G. L., Hollman, P. C. H., & Katan, M. B. (1992). Content aglycones than the raw Ginkgo leaves and thus, only when of potentially anticarcinogenic flavonoids of 28 vegetables and 9 the controversy of glycoside vs aglycone absorption has fruits commonly consumed in the Netherlands. Journal of Agricul- been fully resolved, will it became clear which products are tural and Food Chemistry, 40, 2379–2383. most likely to provide the greatest in vivo benefits. Hollman, P. C. H., & Katan, M. B. (1997). Adsorption, metabolism and health effects of dietary flavonoids in man. Biomedicine & Pharmacotherapy, 51, 305–310. Hollman, P. C. H., & Katan, M. B. (1999). Dietary flavonoids: intake, 4. Conclusion health effects and bioavailability. Food and Chemical Toxicology, 37, 937–942. The different Ginkgo biloba samples seem to have Hollman, P. C. H., de Varies, J. H. M., van Leeuwen, S. D., reacted differently to the simulated gastrointestinal fluid. Mengelers, M. J. B., & Katan, M. B. (1995). Absorption of dietary quercetin glycosides and quercetin in healthy ileostomy volunteers. The results indicate a trend of conversion from the American Journal of Clinical Nutrition, 62, 1276–1282. glycosides to the aglycones and subsequent degradation Hollman, P. C. H. et al. (1997a). Relative bioavailability of the for the commercial preparations. However, for the raw antioxidant flavonoids quercetin from various foods in man. FEBS leaves, no conversion of glycosides to aglycones was Letters, 418, 152–156. observed. In addition, the digestion process appears to Hollman, P. C. H., van Trijp, J. M. P., Mengelers, M. J. B., de Vries, J. H. M., & Katan, M. B. (1997b). Bioavailability of the dietary increase the availability of the glycosides compared with antioxidant flavonol quercetin in man. Cancer Letters, 114, 139– the undigested sample. For the EGb 761, the quercetin 140. glycosides seem to have been converted to their agly- Knekt et al. (2002). Flavonoids intake and risk of chronic diseases. cone, showing four times the amount recovered from the American Journal of Clinical Nutrition, 76, 560–568. control while, for the commercial capsules, most of the Kuhnau, J. (1976). The flavonoids. A class of semi-essential food components: their role in human nutrition. World Review of constituents were not recovered, indicating degradation Nutrition and Dietetics, 24, 117–191. of the flavonoids constituents. Paganga, G., & Rice-Evans, C. A. (1997). The identification of With the increase in popularity of dietary supple- flavonoids as glycosides in human plasma. FEBS Letters, 401, 78– ments as a source of flavonoids with health benefits, this 82. result is significant as it indicates that the raw materials Pietta, P., Mauri, P., Bruno, A., Rava, A., Manera, E., & Ceva, P. (1991). Identification of flavonoids from Ginkgo biloba L., Anth- (in this case, the leaf of the Ginkgo biloba plant) is a emis nobilis L. and Equisetum arvense L. by high-performance more efficient medium for absorption of potentially liquid chromatography with diode array UV detection. Journal of beneficial flavonoids. Chromatography, 553, 223–231.

Appendix 4: Sensory evaluation form

Page 201 of 201

Paper No.:

Age: Race: Gender: F / M

SENSORY EVALUATION

PRODUCT: BEVERAGES

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4. Answer the questions accordingly and tick (√) where appropriate.

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Rank the three samples.

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