phytochemistry and pharmacology of plants from the ginger family
TRANSCRIPT
Southern Cross UniversityePublications@SCU
Theses
2008
Phytochemistry and pharmacology of plants fromthe ginger family, ZingiberaceaeHans WohlmuthSouthern Cross University
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Publication detailsWohlmuth, H 2008, 'Phytochemistry and pharmacology of plants from the ginger family, Zingiberaceae', PhD thesis, Southern CrossUniversity, Lismore, NSW.Copyright H Wohlmuth 2008
PHYTOCHEMISTRY AND PHARMACOLOGY OF PLANTS FROM THE GINGER FAMILY, ZINGIBERACEAE
Hans Wohlmuth, BSc
Submitted for the Degree of Doctor of Philosophy
Department of Natural and Complementary Medicine Southern Cross University
Lismore, Australia
April 2008
i
Flowering Curcuma australasica, an endemic Australian Zingiberaceae with potential anti-inflammatory activity, cultivated at Southern Cross University, Lismore, Australia.
ii
Declaration
certify that the work presented in this thesis is, to the best of my knowledge and belief,
original, except as acknowledged in the text, and that the material has not been
submitted, either in whole or in part, for a degree at this or any other university.
I acknowledge that I have read and understood the University's rules, requirements,
procedures and policy relating to my higher degree research award and to my thesis. I certify
that I have complied with the rules, requirements, procedures and policy of the University (as
they may be from time to time).
Name: Hans Wohlmuth
Signature:…………………………………………………………………..
Date: ………………………………………………………………………..
I
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SYNOPSIS This thesis reports on a series of investigations into the phytochemistry and pharmacology of
plants belonging to the ginger family, Zingiberaceae (incl. Costaceae). The work falls into
two main parts. The first part examines the pungent compounds and essential oil in 17
clones of ginger (Zingiber officinale) with a view to identify one or more with unique
chemistry and consequent particular therapeutic (or flavouring) prospects. The second part
comprises the screening of 41 taxa for inhibition of PGE2 and other biological activities, with
the primary aim of identifying species with potential anti-inflammatory activity. This part
tested the hypothesis that the combination of ethnobotanical and taxonomic information is a
productive strategy to identify previously unrecognised plant species with therapeutic
potential.
Chapter 1 provides a general introduction to plants as medicines and the field of
ethnopharmacology. It also provides an overview of the process of inflammation, in
particular arachidonic acid metabolism.
The main literature review is presented in Chapter 2. It reviews the literature relating to the
chemistry and pharmacology of 15 genera in the Zingiberaceae, with the focus on species
included in the experimental work. The Zingiberaceae is rich in species used as traditional
medicines or spices, but extensive information about their chemistry and pharmacology is
available only for a few species, most notably ginger and turmeric (Curcuma longa). These
attributes make the family an ideal target for a screening project, since phylogenetically
related plant species usually display a significant degree of similarity in the kinds of
secondary metabolites they produce.
Chapter 3 describes the preliminary experimental work with ginger. This work aimed at
determining a suitable extraction solvent and method, guided by the activity of the extracts in
a cyclooxygenase-1 (COX-1) bioassay. It also established an HPLC method suitable for the
quantification of gingerols and shogaols in the extracts.
Seventeen ginger clones, including commercial cultivars and 12 experimental clones, were
analysed by HPLC for their content of pungent compounds. The result of this work is
reported in Chapter 4. Because ginger is a sterile cultigen, there is an increased likelihood
that chemically distinct and genetically stable clones may exist. This work identified one
cultivar that when compared with other clones contained a significantly higher level of
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pungent gingerols. The analysis included 12 tetraploid clones, but these did not display
elevated gingerol production compared with their diploid parent cultivar.
The essential oils obtained from the same 17 ginger clones by steam distillation were
analysed by GC-MS. These results are presented in Chapter 5. The oil from one particular
clone was distinctly different from the others; this was the same clone that had a very high
gingerol content. The essential oil of this clone differed from the others by having a lower
citral content and higher levels of sesquiterpene hydrocarbons. The unique chemistry of this
clone in terms of aroma, pungency and flavour should make it of interest to the flavour,
fragrance and pharmaceutical industries.
Chapter 6 presents the results of the screening of 41 taxa in an in vitro cell-based bioassay
for inhibition of PGE2 production. A number of the samples were also tested for antioxidant
activity in the oxygen radical absorbance capacity (ORAC) assay, for inhibition of nitric
oxide production and for modulation of natural killer cell activity. Known medicinal plants,
in particular ginger and turmeric, emerged as the most active in these assays. Included in the
work were seven native Australian species not previously investigated for pharmacological
activity. Two of these species showed good activity in the PGE2 assay and were selected for
further investigations.
Chapter 7 reports on the bioactivity-guided fractionation of these two native Australian
species, Curcuma australasica and Pleuranthodium racemigerum. Inhibition of PGE2 was
used as the primary bioassay in this process, but fractions with high activity in that assay
were also tested for cytotoxic properties. This work succeeded in isolating and structurally
characterising a novel curcuminoid compound with potent PGE2 inhibitory activity from P.
racemigerum as well as two known compounds from C. australasica.
The final chapter (Chapter 8) provides a short summary and concluding remarks, and
identifies areas for future research arising from the present work.
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PUBLICATIONS ARISING FROM THE WORK IN THIS THESIS
Wohlmuth, H, Leach, DN, Smith, MK, Myers, SP (2005). Gingerol content of diploid and
tetraploid clones of ginger (Zingiber officinale Roscoe). Journal of Agricultural and
Food Chemistry 53: 5772-5778.
Wohlmuth, H, Smith, MK, Brooks, LO, Myers, SP, Leach, DN (2006). Essential oil
composition of diploid and tetraploid clones of ginger (Zingiber officinale Roscoe)
grown in Australia. Journal of Agricultural and Food Chemistry 54: 1414-1419.
Wohlmuth, H, Deseo, MA, Brushett, DJ, MacFarlane, G, Waterman, PG, Stevenson, LM,
Leach, DN (2007). Biological activity and novel cytotoxic curcuminoid from
Pleuranthodium racemigerum - an Australian Zingiberaceae. Planta Medica 73: 947
[Poster abstract].
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ACKNOWLEDGMENTS Many individuals have supported the work presented in this thesis in a variety of ways, and
without their assistance, guidance, help and support the project would not have been
possible. I am grateful to them all.
First and foremost, my warm thanks and appreciation go to my principal supervisor,
Associate Professor David Leach, for his guidance, support, patience and friendship. I also
thank my associate supervisor, Dr Lesley Stevenson, whose valuable input was most
appreciated, even with she moved across the Tasman. My thanks also go to Professor
Stephen Myers, who inspired and encouraged me to do a PhD, whose idea it was to work on
ginger, and who was my supervisor in the early days.
Most of the work presented in this thesis was carried out in the laboratories of the Centre for
Phytochemistry and Pharmacology at Southern Cross University, and in addition to my
supervisors I wish to thank staff and fellow students of the Centre for their invaluable
assistance during the project. No one deserves more thanks and credit than Don Brushett,
who tirelessly and patiently introduced me to the intricacies of analytical chemistry, and I am
also deeply grateful to Dr Myrna Deseo (NMR), Paul Connellan (flow cytometry), Ashley
Dowell (analytical chemistry) and Dion Thompson (pharmacology) for their generous
assistance. I also thank Linda Banbury, Karen Beattie, Bill Eickhoff, Yen Lin Ho, Dr Denise
Hunter, Dr Gloria Karagianis, Kate MacFarlane, Aaron Pollack, Kelly Shepherd, Emeritus
Professor Peter Waterman and Kelly Winter for their support.
The plant materials I worked on in this project came from a variety of sources. I am
indebted to Mike Smith, who so generously made available ginger cultivars and
experimental clones arising from his work at the Maroochy Research Station (Queensland
Department of Primary Industries and Fisheries). I am equally grateful to Dr Greg Leach of
the Northern Territory’s Department of Natural Resources, Environment and the Arts, who
facilitated access to the living collection of Zingiberaceae in the George Brown Darwin
Botanic Gardens. Similarly, through the generosity of Curator David Warmington, I was
able to access the extensive living collection of Zingiberaceae in the Flecker Botanic
Gardens in Cairns. I wish to thank Rigel Jensen of Malanda, whose impressive botanical and
local knowledge of the North Queensland Wet Tropics made the task of collecting native
Australian species in the wild a surprisingly easy one.
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Many of the plants were grown for several years at Southern Cross University, and I owe the
Curator of the Medicinal Plant Garden, Geoff Callan, and his staff much thanks for the
construction of purpose build beds and for general support and friendship. I also gratefully
acknowledge the support of the university’s Ground Staff who let me have a large number of
plants growing in pots in their nursery for several years.
I acknowledge and appreciate the support of my employer, Southern Cross University, in
particular that of Professor Iain Graham, Head of the School of Health and Human Sciences.
I also thank my colleagues in the Department of Natural and Complementary Medicine, in
particular Sue Evans and Holly Muggleston, who shouldered most of the burden during the
times I was away working on my research and thesis.
I also thank Dr Myrna Deseo and Associate Professor John Stevens who provided valuable
comments on the final draft.
Finally, thanks to Catherine for her love and support, and for never resenting me being busy
with my gingers.
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ABBREVIATIONS
5-HETE 5-hydroxyeicosatetraenoic acid
12-HETE 12-hydroxyeicosatetraenoic acid
12-HTT 12-hydroxy-5,8,10-heptadecatrienoic acid
ADP Adenosine diphosphate
ATCC American Type Culture Collection
AP-1 Activator protein-1
BCE Before current era (= B.C.)
B.P. British Pharmacopoeia
CE Current era (= A.D.)
CONSORT Consolidated Standards of Reporting Trials
COX Cyclooxygenase
ED50 Median effective dose
ELAM-1 Endothelial leukocyte adhesion molecule-1
EtOH Ethanol
g Gram
GC Gas chromatography
GC-MS Gas chromatography – mass spectrometry
GM-CSF Granulocyte-macrophage colony-stimulating factor
HCl Hydrochloric acid
HDL High density lipoprotein
HETEs Hydroxyeicosatetraenoic acids
HPETEs Hydroperoxyeicosatetraenoic acids
HPLC High-performance liquid chromatography
IC50 Median inhibition concentration
ICAM-1 Intracellular adhesion molecule-1
IFN-β Interferon-beta
IFN-γ Interferon-gamma
IL-1 Interleukin-1
IL-1β Interleukin-1-beta
IL-2 Interleukin-2
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IL-6 Interleukin-6
IL-9 Interleukin-9
iNOS Inducible nitric oxide synthase
JNK c-Jun N-terminal kinase
LC-MS Liquid chromatography – mass spectrometry
LDL Low density lipoprotein
IP-10 Interferon-γ activated protein
L Litre
LD50 Median lethal dose
LOX Lipoxygenase
5-LOX 5-Lipoxygenase
LPS Lipopolysaccharide
LTC4 Leukotriene C4
LTD4 Leukotriene D4
LTE4 Leukotriene E4
M Molar
MAPK, MAP kinase Mitogen-activated protein kinase
MIC Minimum inhibitory concentration
MCP-1 Macrophage (monocyte) chemotactic protein-1
MCSF Macrophage colony-stimulating factor
mg Milligram
MIP-1α Monocyte inflammatory protein-1-alpha
mL Millilitre
MMPs Metalloproteinases
MS Mass spectrometry
NFκB Nuclear factor kappa B
NK cells Natural killer cells
NMR Nuclear magnetic resonance
NO Nitric oxide
NSAIDs Non-steroidal anti-inflammatory drugs
ORAC Oxygen radical absorption capacity
PAF Platelet-activating factor
PBS Phosphate-buffered saline
PG Prostaglandin
x
PGD2 Prostaglandin D2
PGE2 Prostaglandin E2
PGF2α Prostaglandin F2-alpha
PGH2 Prostaglandin H2
PGI2 Prostaglandin I2 (prostacyclin)
PLA2 Phospholipase A2
PMNs Polymorphonuclear neutrophils
q.i.d. quarter in die (four times a day)
RCF Relative centrifugal force
SD Standard deviation
TFA Trifluoroacetic acid
t.i.d. ter in die (three times a day)
TLC Thin layer chromatography
TNF Tumour necrosis factor
TNF-α Tumour necrosis factor-alpha
TxA2 Thromboxane A2
TxB2 Thromboxane B2
UV Ultraviolet
UV-VIS Ultraviolet-visible
UVB Ultraviolet B
VAS Visual analogue scale
VCAM-1 Vascular cell adhesion molecule-1
VLDL Very low density lipoprotein
VR1 Vanilloid receptor-1
WOMAC Western Ontario and McMaster Osteoarthritis Index
μg Microgram
μL Microlitre
μM Micromolar
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TABLE OF CONTENTS PHYTOCHEMISTRY AND PHARMACOLOGY OF PLANTS FROM THE GINGER
FAMILY, ZINGIBERACEAE I
SYNOPSIS III
SYNOPSIS III
PUBLICATIONS ARISING FROM THE WORK IN THIS THESIS V
ACKNOWLEDGMENTS VI
ABBREVIATIONS VIII
TABLE OF CONTENTS XI
LIST OF TABLES XIX
LIST OF FIGURES XXI
1. GENERAL INTRODUCTION 1
1.1 Plants as medicines 1
1.2 Ethnobotany and ethnopharmacology 3 1.2.1 Ethnobotany 3 1.2.2 Ethnopharmacology 3 1.2.3 Ethnopharmacology and bioprospecting 4 1.2.4 Ethnobotany and ethnopharmacology as strategies in bioprospecting 5 1.2.5 Ethnopharmacology and phytotherapy 5
1.3 Inflammation 6 1.3.1 Overview over the inflammatory process 6 1.3.2 Cellular products as inflammatory mediators 7 1.3.3 Arachidonic acid metabolism 8
1.3.3.1 Prostaglandins 11 1.3.3.2 Thromboxanes 12
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1.3.3.3 Leukotrienes 12
1.4 Plants as anti-inflammatory agents 13
2. LITERATURE REVIEW 16
2.1 Introduction 16
2.2 The ginger family, Zingiberaceae Martinov 16 2.2.1 Zingiberaceae in Australia 17
2.3 Genus Zingiber Boehmer 18 2.3.1 Ginger (Zingiber officinale Roscoe) 19
2.3.1.1 Ginger chemistry 20 2.3.1.1.1 Non-volatile compounds 20
2.3.1.1.1.1 Historical background 21 2.3.1.1.1.2 Gingerols 22 2.3.1.1.1.3 Gingerols in fresh ginger 23 2.3.1.1.1.4 Gingerol degradation products 23 2.3.1.1.1.5 Other non-volatile compounds 26
2.3.1.1.2 Volatile oil 27 2.3.1.2 Pharmacokinetics of ginger 30 2.3.1.3 Pharmacodynamics of ginger 32
2.3.1.3.1 Anti-oxidant activity 32 2.3.1.3.2 Effects on arachidonic acid metabolism and eicosanoids 34
2.3.1.3.2.1 Assays using isolated enzymes 34 2.3.1.3.2.2 Cell-based assays 35 2.3.1.3.2.3 Animal studies 37 2.3.1.3.2.4 Summary: Effects on arachidonic acid metabolism and eicosanoids 39
2.3.1.3.3 Effects on cytokines and chemokines 40 2.3.1.3.4 Anti-cancer activity 41 2.3.1.3.5 Anti-microbial activity 41 2.3.1.3.6 Immunomodulatory effects 42 2.3.1.3.7 Gastrointestinal effects 42 2.3.1.3.8 Metabolic effects 43 2.3.1.3.9 Anti-atherosclerotic effects 43 2.3.1.3.10 Other cardiovascular effects 44 2.3.1.3.11 Analgesic effects 44 2.3.1.3.12 Antipyretic activity 45
2.3.1.4 Safety data 45 2.3.1.4.1 Acute toxicity 45
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2.3.1.4.2 Teratogenicity and embryotoxicity 45 2.3.1.4.3 Mutagenicity 46
2.3.1.5 Clinical studies of ginger 46 2.3.1.5.1 Clinical studies of ginger in arthritis 46
2.3.2 Other Zingiber species 49 2.3.2.1 Thai ginger (Zingiber montanum) 49 2.3.2.2 Zingiber ottensii 51 2.3.2.3 Beehive ginger (Zingiber spectabile) 51
2.4 Genus Curcuma L. 52 2.4.1 Curcuma longa 52
2.4.1.1 Chemistry 52 2.4.1.2 Pharmacology 53
2.4.1.2.1 Pharmacokinetics of curcumin 54 2.4.1.2.2 Pharmacodynamics of curcumin 54
2.4.1.2.2.1 Anti-oxidant activity 54 2.4.1.2.2.2 Anti-inflammatory activity 57 2.4.1.2.2.3 Anti-carcinogenic and anti-tumour activity 59 2.4.1.2.2.4 Other pharmacological activities 61
2.4.2 Curcuma parviflora 62 2.4.3 Other Curcuma species 62
2.5 Genus Boesenbergia Kuntze 63 2.5.1 Chemistry 63 2.5.2 Pharmacology 64
2.6 Genus Hedychium Koenig 65 2.6.1 Chemistry 66 2.6.2 Pharmacology 66
2.7 Genus Kaempferia L. 67 2.7.1 Chemistry 67 2.7.2 Pharmacology 67
2.8 Genus Scaphochlamys Baker 68
2.9 Genus Alpinia Roxburgh 68 2.9.1 Chemistry 69 2.9.2 Pharmacology 70
2.10 Genus Pleuranthodium (K. Schum.) R. M. Smith 72
2.11 Genus Etlingera Giseke 72
xiv
2.12 Genus Elettaria Maton 73 2.12.1 Chemistry 73 2.12.2 Pharmacology 74
2.13 Genus Hornstedtia Retz. 75
2.14 Genus Renealmia L.f. 75
2.15 Genus Costus L. 75 2.15.1 Chemistry 76 2.15.2 Pharmacology 77
2.16 Genus Tapeinochilos Miquel 77
2.17 Summary 77
2.18 Aims and research questions 78 2.18.1 Part 1: Phytochemical investigations of 17 ginger clones 78
2.18.1.1 Research questions 78 2.18.1.2 Specific aims 79 2.18.1.3 Research methods 80
2.18.2 Part 2: Screening Zingiberaceae for pharmacological activity 80 2.18.2.1 Research questions 81 2.18.2.2 Specific aims 81 2.18.2.3 Research methods 82
3. INHIBITION OF CYCLOOXYGENASE-1 BY GINGER RHIZOME EXTRACTS 83
3.1 Introduction 83
3.2 Materials and Methods 83 3.2.1 Plant material 83 3.2.2 HPLC analysis 84 3.2.3 Cyclooxygenase-1 assay 84
3.2.3.1 Enzyme reaction 85 3.2.3.2 Extraction of arachidonic acid and metabolites 85 3.2.3.3 Separation of arachidonic acid and metabolites 85
3.2.4 Statistical analysis 86
3.3 Results and Discussion 87 3.3.1 Experiment 1 87 3.3.2 Experiment 2 89
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4. GINGEROL CONTENT OF SEVENTEEN GINGER (ZINGIBER OFFICINALE) CLONES 92
4.1 Introduction 92
4.2 Materials and Methods 93 4.2.1 Plant materials 93 4.2.2 Sample preparation 95 4.2.3 HPLC methods 95 4.2.4 Statistical analysis 96
4.3 Results 97 4.3.1 Measurement reproducibility 97 4.3.2 Quantification of gingerols in test samples 97 4.3.3 Correlation between gingerols 100 4.3.4 Gingerol ratios 102 4.3.5 Stability of gingerols 103 4.3.6 Gingerol content of tetraploid clones 103
4.4 Discussion 104 4.4.1 Gingerols 104 4.4.2 Shogaols 106 4.4.3 Ploidy 106
5. ESSENTIAL OIL COMPOSITION OF SEVENTEEN GINGER (ZINGIBER OFFICINALE) CLONES 108
5.1 Introduction 108
5.2 Materials and Methods 109 5.2.1 Plant materials and distillation 109 5.2.2 Chemical analysis 109 5.2.3 Statistical analysis 110
5.3 Results 110 5.3.1 Oil composition 110 5.3.2 Principal components analysis 113 5.3.3 Cluster analysis 115 5.3.4 Citral content 116 5.3.5 Neral to geranial ratio 117
5.4 Discussion 118
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5.4.1 Oil composition 118 5.4.2 Citral content 120 5.4.3 Neral to geranial ratio 121
6. PHARMACOLOGICAL ACTIVITY OF ZINGIBERACEAE SPECIES 123
6.1 Introduction 123
6.2 Materials and Methods 125 6.2.1 Plant materials 125 6.2.2 Extraction methods 128
6.2.2.1 Extraction method 1 (ethanolic) 128 6.2.2.2 Extraction method 2 (ethanolic) 129 6.2.2.3 Extraction method 3 (ethanolic) 129 6.2.2.4 Extraction method 4 (ethanolic) 129 6.2.2.5 Extraction method 5 (aqueous) 130 6.2.2.6 Extraction method 6 (aqueous) 130
6.2.3 Bioassay methods 130 6.2.3.1 Inhibition of PGE2 production 130 6.2.3.2 Oxygen Radical Absorbance Capacity (ORAC) 132 6.2.3.3 Inhibition of nitric oxide production 132 6.2.3.4 Modulation of natural killer cell activity 133
6.3 Results 136 6.3.1 Inhibition of PGE2 production 136 6.3.2 Oxygen Radical Absorbance Capacity (ORAC) 138 6.3.3 Inhibition of nitric oxide production 139 6.3.4 Modulation of natural killer cell activity 140
6.4 Discussion 142 6.4.1 Inhibition of PGE2 production 142 6.4.2 Oxygen Radical Absorbance Capacity (ORAC) 144 6.4.3 Inhibition of nitric oxide production 145 6.4.4 Modulation of natural killer cell activity 146 6.4.5 Conclusion 147
7. BIOACTIVITY-GUIDED FRACTIONATION OF TWO NATIVE AUSTRALIAN ZINGIBERACEAE 149
7.1 Introduction 149
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7.2 Materials and Methods 150 7.2.1 Plant material 150 7.2.2 Extraction methods 150
7.2.2.1 Curcuma australasica 150 7.2.2.2 Pleuranthodium racemigerum 150
7.2.3 Fractionation by preparative HPLC 151 7.2.3.1 Curcuma australasica 151 7.2.3.2 Pleuranthodium racemigerum 151
7.2.4 Nuclear magnetic resonance spectroscopy 152 7.2.4.1 One-dimensional NMR spectroscopy 152
7.2.4.1.1 1H NMR 152 7.2.4.1.2 J-modulated 13C NMR 152
7.2.4.2 Two-dimensional homonuclear correlation NMR spectroscopy 153 7.2.4.2.1 1H-1H Correlation spectroscopy (COSY) 153
7.2.4.3 Two-dimensional heteronuclear correlation spectroscopy 153 7.2.4.3.1 Heteronuclear single quantum correlation (HSQC) spectroscopy 153 7.2.4.3.2 Heteronuclear multiple-bond correlation (HMBC) spectroscopy 153
7.2.5 Determination of accurate mass 154 7.2.6 UV spectroscopy 154 7.2.7 Cytotoxicity assays 154 7.2.8 PGE2 assay 155
7.3 Results 155 7.3.1 Curcuma australasica 155
7.3.1.1 Activity-guided fractionation 155 7.3.1.2 Cytotoxic activity of Compound 1 160 7.3.1.3 Structural elucidation of Compound 1 nd 2 160
7.3.2 Pleuranthodium racemigerum 167 7.3.2.1 Activity-guided fractionation 167 7.3.2.2 Cytotoxic activity of Compound 3 170 7.3.2.3 Structural elucidation of Compound 3 172 7.3.2.4 Accurate Mass 176 7.3.2.5 UV spectrophotometric data and extinction coefficient 176
7.4 Discussion 178 7.4.1 Curcuma australasica 178 7.4.2 Pleuranthodium racemigerum 179
8. CONCLUDING REMARKS 180
8.1 Phytochemical investigations of ginger (Zingiber officinale) 180
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8.2 Screening Zingiberaceae for pharmacological activity 182
8.3 Direction of future research 183
APPENDIX A: CLINICAL EFFICACY TRIALS OF GINGER PREPARATIONS 185
APPENDIX B: QUALITY ASSESSMENT OF CLINICAL TRIALS OF GINGER IN OSTEOATHRITIS 190
APPENDIX C: INHIBITION OF PGE2 IN 3T3 CELLS 192
REFERENCES 196
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LIST OF TABLES
Table 1-1. Cytokines involved in inflammation. 8
Table 1-2. Examples of plant compounds with in vitro anti-inflammatory activity. 15
Table 2-1. Taxonomy of the family Zingiberaceae. 17
Table 2-2. Subfamilies, tribes and representative genera of the Zingiberaceae. 17
Table 2-3. Zingiberaceae species occurring in Australia. 18
Table 3-1. Effect of extraction method: cyclooxygenase-1 inhibitory activity and content of major pungent compounds of ginger extracts.
87
Table 3-2. Effect of extraction solvent: cyclooxygenase-1 inhibitory activity and content of major pungent compounds of ginger extracts.
89
Table 3-3. Gingerol ratios in ginger extracts compared with values from the literature. 91
Table 4-1. Ginger clones studied, their genotype and origin. 94
Table 4-2. Results of pairwise analyses of 17 ginger clones in terms of [6]-, [8]- and [10]-gingerol content.
100
Table 4-3. Pearson Product-Moment correlations between the concentration of gingerols in fresh rhizomes of seventeen ginger clones assayed by HPLC at zero and five months.
102
Table 4-4. Mean±SE (range) concentrations of gingerols in two commercial ‘Queensland’ clones and 12 tetraploid clones (µg/g).
103
Table 4-5. Literature data on gingerol content of fresh ginger rhizomes. 105
Table 5-1. Composition of essential oils of 17 clones of ginger analysed by GC-MS on a BPX-5 column.
112
Table 5-2. Content of 14 constituents in essential oils of 16 ‘typical’ clones of ginger and one ‘atypical’ clone, ‘Jamaican’ (Z46).
113
Table 5-3. Varimax rotated component matrix for two component solution for the ten most abundant ginger essential oil constituents.
114
Table 6-1. Zingiberaceae samples screened for biological activity. 126
Table 6-2. Inhibition of PGE2 production in 3T3 murine fibroblasts. 137
Table 6-3. Oxygen radical absorbance capacity (ORAC). 138
Table 6-4. Inhibition of nitric oxide production in LPS-stimulated RAW264 macrophages.
139
Table 6-5. Effect of ethanolic extracts on natural killer cell activity against K562 leukaemia cells.
140
Table 6-6. Effect of aqueous extracts on natural killer cell activity against K562 leukaemia cells.
141
Table 7-1. Inhibition of PGE2 production in 3T3 murine fibroblast cells by fractions of Curcuma australasica extract.
157
Table 7-2. Cytotoxicity of Compound 1 from Curcuma australasica in P388D1 murine lymphoma cells.
160
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Table 7-3. 1H and 13C NMR spectral data of Compound 1 (zederone) from Curcuma australasica.
162
Table 7-4. 1H and 13C NMR spectral data of Compound 2 (1(10)E,4E-furanodien-6-one) from Curcuma australasica.
164
Table 7-5. 1H NMR spectral data from the literature for the isomers isofuranodienone and 1(10)Z,4Z-furanodien-6-one.
166
Table 7-6. Inhibition of PGE2 production in 3T3 murine fibroblast cells by combined fractions of Pleuranthodium racemigera extract.
168
Table 7-7. LD50 (µM) values for cytotoxic activity of Compound 3 and curcumin against five cancer cell lines.
172
Table 7-8. 1H and 13C NMR data from one-dimensional (1H and J-modulated 13C NMR) and two-dimensional correlation (COSY, HSQC and HMBC) NMR spectroscopy experiments on Compound 3 from Pleuranthodium racemigerum.
173
Table 7-9. Accurate mass determination for Compound 3. 176
Table 7-10. Peak absorption (λmax) and extinction coefficients (ε) of Compound 3 at 203 nm, 225 nm and 278 nm.
177
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LIST OF FIGURES
Fig. 1-1. Schematic representation of the metabolism of arachidonic acid catalysed by cyclo-oxygenase (COX) and lipoxygenase (LOX) enzymes.
10
Fig. 2-1. Structure of [6]-gingerol, the most abundant gingerol in ginger rhizome. 21
Fig. 2-2. Structure of zingerone. 21
Fig. 2-3. Structure of [6]-shogaol. 21
Fig. 2-4. Structures of the major gingerols in ginger. 22
Fig. 2-5. Dehydration of gingerols to shogaols. 24
Fig. 2-6. Conversion of gingerols to zingerone and aliphatic aldehydes at high temperature.
25
Fig. 2-7. Paradols, gingerdiols and gingerdiones from Zingiber officinale. 27
Fig. 2-8. Volatile sesquiterpenes from Zingiber officinale. 28
Fig. 2-9. Volatile monoterpenoids from Zingiber officinale. 29
Fig. 2-10. Curcuminoids from Zingiber montanum. 50
Fig. 2-11. Structural diagrams of the major curcuminoids in turmeric rhizome: curcumin, demethoxycurcumin and bisdemethoxycurcumin.
53
Fig. 2-12. Parviflorene A and F from Curcuma parviflora. 62
Fig. 2-13. Boesenbergin A and B from Boesenbergia rotunda. 64
Fig. 2-14. 1’-acetoxychavicol acetate from Alpinia galanga. 70
Fig. 2-15. Terpinyl acetate and 1,8-cineole from Elettaria cardamomum. 74
Fig. 2-16. Diosgenin from Costus malortieanus. 76
Fig. 3-1. Gingerol content (mg/mL extract) of ginger extracts. 90
Fig. 4-1. Typical HPLC chromatogram of ethanolic extract of fresh ginger rhizome (Z44) obtained with HPLC Method 2.
98
Fig. 4-2. Concentrations of [6]-, [8]- and [10]-gingerol in fresh rhizomes of 17 ginger clones.
99
Fig. 4-3. Scatter plots illustrating the correlation between concentrations (µg/g) of [6]- and [8]-gingerol (A), [6]- and [10]-gingerol (B), and [8]- and [10]-gingerol (C) in fresh ginger rhizomes.
101
Fig. 5-1. Volatile constituents from Zingiber officinale essential oil. 111
Fig. 5-2. Component plot in rotated space showing Varimax rotated data on two components based on the ten most abundant constituents of ginger essential oil.
115
Fig. 5-3. Dendrogram of hierarchical cluster analysis of 17 essential oils of ginger. 116
Fig. 5-4. Scatter plot showing the relationship between the percentage content of the stereoisomers neral and geranial in essential oils from 17 clones of ginger.
117
Fig. 7-1. LC-MS chromatogram of Curcuma australasica extract redissolved in methanol.
156
xxii
Fig. 7-2. LC-MS chromatogram for Compound 1 (Fraction 17) from Curcuma australasica.
158
Fig. 7-3. Mass spectrum (M+1; left) and UV spectrum (right) for Compound 1 (Fraction 17) from Curcuma australasica.
158
Fig. 7-4. LC-MS chromatogram for Compound 2 (Fraction 20) from Curcuma australasica.
159
Fig. 7-5. Mass spectrum (M+1; left) and UV spectrum (right) for Compound 2 (Fraction 20) from Curcuma australasica.
159
Fig 7-6. Structure of zederone (Compound 1). 161
Fig. 7-7. Structure of 1(10)E,4E-furanodien-6-one (Compound 2) from Curcuma australasica.
165
Fig. 7-8. LC-MS chromatogram of Pleuranthodium racemigerum extract redissolved in 90% aqueous methanol.
167
Fig. 7-9. LC-MS chromatogram for Compound 3 from Pleuranthodium racemigerum. 169
Fig. 7-10. Mass spectrum (M+1; top) and UV spectrum (bottom) for Compound 3 from Pleuranthodium racemigerum.
169
Fig. 7-11. Cytotoxic effect of fraction Compound 3 from Pleuranthodium racemigerum on 3T3 murine fibroblasts.
170
Fig. 7-12. Dose-response curves for cytotoxic activity of Compound 3 from Pleuranthodium racemigerum against five cell lines.
171
Fig. 7-13. Heteronuclear correlations in Ring A. 174
Fig. 7-14. Heteronuclear correlations in Ring B. 175
Fig. 7-15. Chemical structure of Compound 3 from Pleuranthodium racemigerum, 1-(4”-methoxyphenyl)-7-(4’-hydroxyphenyl)-2E-heptene.
175
Fig. 7-16. UV spectrum of 1-(4”-methoxyphenyl)-7-(4’-hydroxyphenyl)-2E-heptene (Compound 3).
177
1
1. GENERAL INTRODUCTION This thesis reports on research into the chemistry and pharmacology of plants from the
Zingiberaceae family, in particular in terms of their potential use as anti-inflammatory
agents.
The experimental work presented in this thesis is divided into two main parts. The first part
focuses on the common ginger (Zingiber officinale), which in recent years has attracted
attention as a potential anti-inflammatory agent. Seventeen clones of ginger were analysed
in terms of their content of pungent compounds with a view to identify any with a profile
that might be of particular interest from a pharmacological point of view. Because ginger is
also an important flavouring commodity, profiling of the essential oil was also carried out.
The second part of the work comprises the screening of some 43 Zingiberaceae taxa,
representing 14 genera, for potential anti-inflammatory activity in a whole cell assay. Simple
chemical profiling of these extracts was carried out, and two were subjected to activity-
guided fractionation.
This chapter provides a brief introduction to the use of plants as medicines and the process of
inflammation. Chapter 2 provides a review of the plant species investigated during the
course of this work.
1.1 Plants as medicines
Plants have provided humans with medicines since time immemorial. The oldest known
document concerning medicinal plants and their uses is the Chinese Pen Ts’ao, which was
written 4800 years ago and describes no less than 360 plants, suggesting that herbal medicine
was already at an advanced stage in China at this time (Mann, 1992). In Mesopotamia (part
of present-day Iraq), 4600-year old clay tablets inscribed with cuneiform characters have
been found that contain references to familiar medicinal plants such as myrrh, licorice and
the opium poppy (Cragg & Newman, 2002). Another famous early document detailing the
use of plants as medicines is the Ebers papyrus from Egypt, which was written about 3500
years ago (Mann, 1992).
2
Much more ancient, albeit less conclusive, evidence suggests that humans might have
employed the pharmacological properties of plants much earlier. At the famous burial site in
the Shanidar Cave in the northern part of Iraq, a Neanderthal (Homo neanderthalensis) was
laid to rest with bunches of flowers about 60,000 years ago (Solecki, 1975). Of the eight
plants identified in the grave from preserved pollen, seven are considered medicinal plants
today. There is of course no way of knowing with certainty whether they were placed in the
grave because of their medicinal properties, to serve the dead man on his final journey, or
whether they simply were used for decorative purposes.
The more recent discovery of the ‘Iceman’ on the Italian-Austrian border in the Alps
provides intriguing evidence of early use of medicinal fungi in Europe. This hunter, who
had been lying well preserved in the ice for about 5300 years, was found to be in possession
of a fungus, the birch polypore (Piptoporus betulinus), which is known to have purgative and
antibiotic properties, and which he might well have been using to treat the whipworm
infestation of his intestines (Heinrich et al., 2004).
Plants play a key role in sophisticated ancient traditional medical systems such as traditional
Chinese medicine and Ayurveda of India, and have also been central in the Greco-Roman
medical tradition, which developed into modern biomedicine. Hippocrates (468-377 BCE),
used more than 400 plant species for therapeutic purposes, and it was a Roman army surgeon
by the name of Dioscorides (c. 40-80 CE) who wrote the most influential early European
manual of medicinal plants, De Materia Medica, in the first century CE (Griggs, 1997). This
comprehensive work included illustrations and descriptions of about 600 plant species, along
with text detailing their uses, doses and potential toxic effects. The writings of Galen (c.
129-199 CE), who classified herbs according to their humoral properties, had a profound and
almost unimaginable impact on medical thought in Europe for about 1500 years (Griggs,
1997). The English apothecary Nicholas Culpeper (1616-1654) wrote many herbal books,
the most famous being The English Physician (1653), in which he presented herbal medicine
in an astrological framework (Griggs, 1997).
Although modern biomedicine to a significant degree employs synthetic drugs as therapeutic
agents, plants still occupy a prominent place in contemporary pharmacy, either as sources of
pharmaceutical drugs in the form of isolated plant compounds, as sources of precursors to
drugs, or as sources of compounds that have served as models for synthetic or semisynthetic
drugs. It has been estimated that about one-half of all drugs in current use are natural
3
compounds or derivatives thereof (Iwu, 2002). It is however important to realise that despite
the many advances of biomedicine, the progress afforded residents of first world countries is
beyond the reach of the majority of the world’s population. For the majority of people, many
of whom live in miserable poverty, crude plants preparations are still the main form of
medicine. In acknowledgment of this situation, the World Health Organization (WHO) is
actively promoting the development of traditional medicine (Anonymous, 2002).
1.2 Ethnobotany and ethnopharmacology
1.2.1 Ethnobotany
The term ethnobotany lacks a singular, uniformly agreed definition. The term was coined by
J. W. Harshberger, who defined it as “…the use of plants by aboriginal peoples”
(Harshberger, 1896). Since then, ethnobotany has been redefined and reinterpreted by many
scholars in the area (for an overview, see Cotton, 1996). One of the broadest definitions of
ethnobotany is that provided by Martin, who described it as the subdiscipline of
ethnoecology that is concerned with local people’s interaction with plants (Martin, 1995).
Early ethnobotany was focused on plants of economic significance or potential, while
contemporary ethnobotany tends to have a far broader scope and include, for example,
traditional agricultural knowledge and traditional vegetation management (Cotton, 1996).
Throughout the history of formal ethnobotany, medicinal plants have been an area of keen
interest to many ethnobotanists.
1.2.2 Ethnopharmacology
Ethnopharmacology is a multidisciplinary field devoted to the study of pharmacologically
active agents traditionally used by humans. The term ethnopharmacology was coined as
recently as 1967 by Efron, who used the term in the context of hallucinogenic substances
(Heinrich & Gibbons, 2001).
4
More recently, ethnopharmacology has been defined as “… the interdisciplinary scientific
exploration of biologically active agents traditionally employed or observed by man”
(Bruhn & Holmstedt, 1981).
Ethnopharmacology applies conventional chemical and pharmacological analysis to
traditional medicines and in doing so differs from two related disciplines: medical
anthropology, which examines health and disease from a cultural perspective, and medical
ethnobotany, which is concerned with the use of plants within traditional medical systems
(Cotton, 1996).
Although ethnopharmacology is not exclusively concerned with plants or plant products, the
plant kingdom is the major focus of the discipline, because this kingdom has provided
humans with the greatest number of pharmacologically active substances throughout history.
The multidisciplinary nature of ethnopharmacology is evidenced by the important roles
played by fields such as botany, pharmacognosy, natural product chemistry, pharmacology,
toxicology, anthropology and others (Heinrich & Gibbons, 2001; Houghton, 2002).
1.2.3 Ethnopharmacology and bioprospecting
Heinrich and Gibbons (2001) have noted the differences between ethnopharmacology and
bioprospecting, while acknowledging that the two approaches are not mutually exclusive. In
brief, ethnopharmacology aims to develop (through increased knowledge and understanding)
the use of crude plant preparations in local communities, whereas the goal of bioprospecting
is the identification and development of compounds from nature as pharmaceutical drugs in
the international market place (Heinrich & Gibbons, 2001). Although the aims of these two
approaches to natural products research are vastly different from a socio-economic
viewpoint, many of the methodologies will often be the same, and work focussed on one
approach might yield results that are relevant to the other. For example, phytochemical and
pharmacological investigations of a traditional medicine might lead to the identification of a
compound that can be developed into a pharmaceutical drug. Well known examples of this
include ephedrine (from Ephedra spp.), atropine (from Atropa bella-donna and other
Solanaceae) and more recently the development of the anti-malarial drug artemether from the
5
lead compound artemisinin in the traditional Chinese herb Artemisia annua (Evans, 2002;
Wright, 2002).
1.2.4 Ethnobotany and ethnopharmacology as strategies in bioprospecting
Different approaches can be employed in the process of bioprospecting. Cotton (1996)
outlined three main approaches to the collection of plants for screening: the random method,
where every species in a given area is included; phylogenetic targeting, where a particular
taxon (such as a family) is targeted, because it is already known to be good source of
pharmacologically active metabolites; and the ethno-directed sampling, which is guided by
traditional plant use. The latter approach is based on the notion that initial screening and
selection has already been conducted effectively by the owners of the traditional knowledge.
The ethno-directed approach to identifying plants with biological activity has been shown in
a number of studies to be more efficient than the random method at identifying plants with
promising pharmacological activity. Such studies include one aimed at identifying plants
with anti-HIV activity from Central America (Balick, 1990) and another that showed that
plants used ethnomedically to treat viral infections were more than 100 times more likely to
yield compounds with anti-viral activity than randomly collected plants (Carlson, 2002).
However, it has been argued that the successful development of drugs from traditional
medicines is most likely for conditions such as inflammation, gastrointestinal or nervous
system disorders, because these pathologies are widely recognised and treated in indigenous
systems of medicine (Cox, 1994).
1.2.5 Ethnopharmacology and phytotherapy
The term phytotherapy is used to describe the use of plant-based, chemically complex
therapeutic agents in contemporary, mostly industrialised societies. Phytotherapy is usually
based on a history of traditional use, but it differs from traditional indigenous herbal
medicine by employing industrialised extraction and manufacturing methods and by being
cosmopolitan in scope. Hence phytomedicines made from plants from around the globe are
6
available in most industrialised countries. Ethnopharmacology has the potential to increase
our knowledge and understanding of traditional herbal medicines, how they work, how they
are best prepared, and how they can be applied in a safe and efficacious manner. Due to the
chemical complexity of both traditional herbal medicines and modern phytomedicines, the
task of elucidating their pharmacology is a complex one indeed. A full understanding of
how complex mixtures of plant compounds interact with the human body and with each
other is probably not achievable, and pharmacological investigations of plant extract almost
always focus on one or a few ‘active’ constituents, i.e. compounds with profound biological
activity. It should always be borne in mind that many other compounds present in a plant
extract could potentially play a role in the overall activity of that extract, for example by
modulating the pharmacokinetic and/or pharmacodynamic properties of the ‘actives’.
Despite this caveat, ethnopharmacological investigations clearly have much to offer modern
phytotherapy, and the long-term success of the ‘herbal renaissance’ currently experienced in
most of the industrialised world undoubtedly depends on the scientific underpinning of
traditional or anecdotal uses.
1.3 Inflammation
Inflammation is being implicated in the pathophysiology of an increasing number of
diseases. In addition to conditions traditionally considered to be inflammatory in nature,
inflammation is now considered to have a role in a wide range of pathologies, including
cardiovascular disease (Hansson, 2005; Kaperonis et al., 2006), cancer (Zhang & Rigas,
2006), diabetes (Deans & Sattar, 2006; Duncan & Schmidt, 2006), age-related macular
degeneration (Rodrigues, 2007), Parkinson’s disease (Hald et al., 2007), Alzheimer’s disease
(Eikelenboom et al., 2006), and possibly depression (Kulmatycki & Jamali, 2006).
1.3.1 Overview over the inflammatory process
Inflammation is a rapid and non-specific response to cellular injury in vascularised tissues.
The inflammatory response is produced and controlled by complex interactions between
7
cellular and plasma protein components. The cellular component involves intercellular
communication effected by a range of cytokines.
The inflammatory response commences with a brief constriction of arterioles followed by
vasodilation and exudation of protein-containing plasma and blood cells into the injured
tissue. This causes swelling and oedema. Meanwhile leukocytes adhere to vessel walls and
cause the endothelial cells to contract, creating enough space between these cells for the
leukocytes to enter the extravascular tissue. Increased vascular permeability is maintained
until the inflammatory state is resolved, and it is the interplay between blood cells and
plasma proteins in the affected tissue that controls the inflammatory response and interacts
with part of the immune response.
The principal cell types involved in inflammation are mast cells, endothelial cells,
phagocytic leukocytes (polymorphonuclear neutrophils, macrophages, and eosinophils), and
platelets.
Mast cells play a key role in the initiation of the inflammatory response. Degranulation leads
to the release of stored chemicals such as histamine, which causes increased vascular
permeability, and mast cells also synthesise pro-inflammatory mediators such as
prostaglandins, leukotrienes and platelet-activating factor (PAF).
Endothelial cells express adhesion molecules (selectins) for leukocytes and platelets, and
also produce nitric oxide (NO), which causes vasodilation but also may play a regulatory
role by suppressing mast cell and platelet function. Endothelial cells also produce two
prostaglandin derivatives with opposite action: the vasoconstrictor thromboxane A2 (TxA2)
and the vasodilator prostacyclin (PGI2), and it is the interplay between these two regulatory
compounds that allows for platelet aggregation to occur only at the site of injury (McCance
& Huether, 2002).
1.3.2 Cellular products as inflammatory mediators
The various cells involved in the inflammatory response produce a range of compounds that
act as inflammatory mediators, including cytokines and products of arachidonic acid
metabolism, i.e. prostaglandins and leukotrienes.
8
Cytokines are proteins produced by a range of different cell types. The major types of
cytokines are the interleukins and the interferons, but the class also includes tumour necrosis
factors, colony-stimulating factors, transforming growth factor, and others (McCance &
Huether, 2002).
Table 1-1 lists cytokines that play an important role in inflammation.
Table 1-1. Cytokines involved in inflammation.
(After McCance et al. 2002).
Cytokine Source Main actions relevant to inflammation
IL-1 Mainly macrophages Inflammatory mediator; increases prostaglandin production
IL-6 Monocytes, macrophages, T and helper T cells
Stimulates inflammatory response
IL-9 Helper T cells T cell and mast cell growth factor IFN-γ (IL-18) T and helper T cells, NK cells Activates macrophages TNF-α Macrophages, mast cells,
lymphocytes Increases other cytokines; increases inflammatory and immune responses
Macrophage colony-stimulating factor
Inflamed endothelial cells, monocytes, lymphocytes, fibroblasts
Macrophage growth factor
Transforming growth factor-β Macrophages, platelets, lymphocytes
Chemotactic for macrophages; increases IL-1 production
1.3.3 Arachidonic acid metabolism
Products of arachidonic acid metabolism such as prostaglandins and leukotrienes play key
roles in inflammation, and their syntheses are well established targets in the pharmacological
treatment of inflammation.
Arachidonic acid (5Z,8Z,11Z,14Z-eicosatetraenoic acid) is an unsaturated, 20-carbon,
omega-6 fatty acid found in cell membranes. It can be obtained from the diet or be derived
9
from linoleic acid. In the cell membrane, arachidonic acid is esterified to phospholipid, and
arachidonate must be liberated from phospholipid before it can act as a substrate for
enzymatic modification. These modifications, catalysed by various enzymes, are known as
arachidonic acid metabolism and can lead to the formation of inflammatory mediators
collectively known as eicosanoids, i.e. prostaglandins (PGs), thromboxanes (TXs),
hydroxyeicosatetraenoic acids (HETEs) and leukotrienes (LTs) (Calder, 2005; Eberhart &
Dubois, 1995).
Arachidonic acid is released from the cell membrane by phospholipase enzymes, in
particular phospholipase A2, and the free acid can be metabolised to eicosanoids by cyclo-
oxygenase (COX) and lipoxygenase (LOX) enzymes. Metabolism catalysed by COX
enzymes gives rise to prostaglandins of the 2-series as well as thromboxanes, while LOX
metabolism leads to the formation of leukotrienes (Fig. 1-1).
COX is also known as prostaglandin endoperoxide synthase. This protein possesses two
discrete activities: the cyclo-oxygenase activity first inserts two oxygen molecules into
arachidonic acid resulting in PGG2; this is followed by the reduction of PGG2 to PGH2, a
result of the protein’s peroxidase activity (Needleman et al., 1986). Since prostaglandins of
the 2-series, including PGE2, are involved in many inflammatory processes, the inhibition of
the COX pathway of arachidonic acid metabolism is a prime pharmacological target and one
that has been exploited long before the pathway was known. Non-steroidal anti-
inflammatory drugs (NSAIDs) inhibit the cyclo-oxygenase activity (but not the peroxidase
activity) of prostaglandin endoperoxide synthase (Marnett et al., 1999). Acetylsalicylic acid
(aspirin) does so in an irreversible fashion, by acetylating the enzyme (Foster, 1996;
Needleman et al., 1986).
Two isoforms of the COX enzyme are known; these are known as COX-1 and COX-2 and
their respective protein products show differential distribution (Marnett et al., 1999). COX-1
is constitutively expressed in most tissues, where it catalyses the biosynthesis of eicosanoids
(prostaglandins and thromboxanes) that regulate numerous cellular processes. In contrast,
COX-2 activity is generally undetectable in most tissues, but the expression of COX-2 can
be rapidly induced in inflammatory cells in response to stimulation by pro-inflammatory
cytokines or by growth factors (Calder, 2005). A new class of anti-inflammatory and
analgesic agents that are selective COX-2 inhibitors were developed in the 1990s (e.g.
celecoxib, refecoxib). The rationale for this drug development was that selective COX-2
10
inhibitors were not expected to interfere with homoeostatic physiological processes and
should therefore be less likely than non-selective COX inhibitors to cause the unwanted side-
effects typical of traditional NSAIDs (Lipsky, 1999; Sautebin, 2000; Simon et al., 1999).
Fig. 1-1. Schematic representation of the metabolism of arachidonic acid catalysed by cyclo-oxygenase (COX) and lipoxygenase (LOX) enzymes.
Already in the late 1990s there was growing evidence, however, that viewing COX-2 as
solely pro-inflammatory and COX-1 as essentially benign was too simplistic. COX-2 has
now been shown to be involved in normal physiology such as the regulation of vascular and
renal blood flow (Brater et al., 2001), and Sautebin reviewed work suggesting that COX-2
could have anti-inflammatory action, while COX-1 could contribute to inflammation under
certain circumstances (Sautebin, 2000). Nevertheless, COX-2 inhibitory drugs (coxibs) were
aggressively marketed and became very widely prescribed for inflammatory conditions in
industrialised countries in the early 2000s, only for some of them to be withdrawn from the
5-HPETE
PGH2 15-HETE 12-HETE LTA4
15-HPETE 12-HPETE
5-HETE
PGE2 PGD2 PGF2α PGI2 TX2
PGG2
Lipoxin A4 LTB4 LTC4
LTD4
LTE4
Phospholipase A2
15-LOX 12-LOX 5-LOX COX
PGJ4
Arachidonic acid in cell membrane
Free arachidonic acid
PEROXIDASE
11
market a few years later (2004) due to the increased cardiovascular risks associated with
their use (rofecoxib [Vioxx®] was withdrawn; celecoxib [Celebrex®] is still used, but in a far
more discriminate manner) (Fitzgerald, 2004; Yoon & Baek, 2005).
A so-called splice variant of COX-1, known as COX-3, COX-1b or COX-1v, is also known,
but after initial speculation that it might be a target for paracetamol, medical interest in it has
waned (Hersh et al., 2005).
The alternative metabolic pathway of arachidonic acid is controlled by the lipoxygenase
(LOX) enzymes (Fig. 1-1). They catalyse the insertion of molecular oxygen into
polyunsaturated fatty acids with a 1Z,4Z-pentadiene system. Depending of the location of
the oxygen insertion into arachidonic acid, mammalian lipoxygenase enzymes are classified
as 5-, 12- or 15-lipoxygenases. The primary products are 5-, 12- and 15-
hydroperoxyeicosatetraenoic acids (HPETEs), which are subsequently reduced to the
corresponding hydroxyeicosatetraenoic acids (HETEs) or, in case of the 5-LOX pathway,
converted to the eicosanoids known as leukotrienes (Calder, 2005; Needleman et al., 1986;
Schneider & Bucar, 2005).
1.3.3.1 Prostaglandins
The prostaglandins are a group of cyclic, 20-C unsaturated fatty acids, which all share a
double-bond at C13-C14. Based on structural differences, the prostaglandins (PGs) are
divided into 9 groups, named A-I. A subscript numeral indicates the number of unsaturated
carbon bonds in the compound, and the subscripts α- and β- denotes the orientation of a
hydroxyl-group on C9 below or above the molecular plane (e.g. PGF2α) (Foster, 1996).
Originally discovered in seminal fluid from the prostate (giving rise to their name),
prostaglandins are known to be synthesised and released in virtually all body tissues. No
tissue (except seminal fluid) appears to have the capacity to store prostaglandins, so rate of
release reflects the rate of biosynthesis. Prostaglandins are produced in response to a variety
of stimuli, including inflammation, allergic responses and trauma, and they usually exert
their action locally, close to their site of release. Several prostaglandins are known to be
metabolised rapidly in the liver, kidneys and lungs (Foster, 1996).
12
Five prostanoid receptors named DP, EP, FP, IP and TP have been identified that show some
degree of selectivity for PGD2, PGE2, PGF2α, PGI2 and thromboxane A2 (TxA2),
respectively. The existence of several subtypes of the EP receptor has been proposed and it
is believed that PGE2 exerts different actions at these (Foster, 1996).
The formation of different PGs from the unstable metabolite PGH2 is cell-specific. For
example, PGI2 (prostacyclin) is the predominant prostaglandin in the vascular endothelium,
where it inhibits platelet aggregation and causes vasodilation. In mast cells, PGD2 is the
major COX product, while PGE2 is the prevailing prostaglandin in the kidney (Needleman et
al., 1986). PGE2 has several pro-inflammatory activities, including vasodilation and
increasing vascular permeability, inducing fever, and enhancing pain and oedema caused by
other mediators such as bradykinin and histamine. Large amounts of PGE2 and PGF2 can be
produced by stimulated monocytes and macrophages, whereas stimulated neutrophils
produce moderate amounts of PGE2 (Calder, 2005).
1.3.3.2 Thromboxanes
The thromboxanes are eicosanoids arising from the COX metabolic pathway like the
prostaglandins. Thromboxane A2 (TxA2) is the major metabolite of PGH2 in platelets.
Unlike its metabolite thromboxane B2 (TxB2), which is inactive, TxA2 contracts vascular
smooth muscle, induces platelet aggregation and causes serotonin release (Needleman et al.,
1986).
1.3.3.3 Leukotrienes
Leukotrienes (LTs) are potent biologically active compounds produced by the 5-LOX
pathway via 5-HPETE. LTC4, LTD4, and LTE4 are produced mainly by mast cells,
basophils, eosinophils and endothelial cells from the precursor LTA4, while LTB4 is
produced in neutrophils, monocytes and macrophages (Calder, 2005; Jala & Haribabu,
2004).
LTB4 is a potent chemotactic agent for leucocytes, it increases vascular permeability, causes
the release of lysosomal enzymes, and promotes the generation of reactive oxygen species
13
and the production of inflammatory cytokines including tumour necrosis factor α (TNFα),
interleukin-1 (IL-1) and interleukin-6 (IL-6). LTC4, LTD4, and LTE4 increase vascular
permeability, but also cause bronchoconstriction and promote hypersensitivity reactions
(Calder, 2005).
Leukotrienes have been associated with a range of inflammatory conditions including
asthma, colitis, dermatitis, rheumatoid arthritis and septic peritonitis, and recent work has
suggested an important role for leukotrienes in the development and progression of
atherosclerosis (Jala & Haribabu, 2004). The modification of leukotrienes as a
pharmacological target has only been achieved relatively recently, but several drugs
(inhibitors of leukotrienes biosynthesis and receptor antagonists) are now approved for the
treatment of asthma and allergic rhinitis (Jala & Haribabu, 2004; Valli, 2003).
1.4 Plants as anti-inflammatory agents
When you examine a man with an irregular wound…and that wound is
inflamed…[there is] a concentration of heat; the lips of that wound are reddened
and that man is hot in consequence…Then you must make cooling substances for
him to draw the heat out…leaves of willow.
(Translated entry from the Ebers papyrus, as quoted in Mann 1992)
The bark and leaves of willow (Salix spp.) are among the most famous plants with anti-
inflammatory properties. A source of a range of salicylates, mostly in glycoside form,
willow has been used medicinally for millennia. The parent compound of these salicylates,
salicylic acid, was shown to be highly effective in the treatment of rheumatic fever in one of
the earliest clinical trials in history. Its corrosive effects in the gastrointestinal tract lead to
the development of a derivative, acetylsalicylic acid, which was marketed by the German
pharmaceutical company Bayer in 1899 under the name Aspirin (Mann, 1992). The most
successful pharmaceutical drug of all time, acetylsalicylic acid became the prototype for the
class of pharmaceuticals known as non-steroidal anti-inflammatory drugs (NSAIDs).
However, it was not until 1971 that the main mechanism of action for Aspirin was
elucidated. In groundbreaking work led by John Vane, who was later rewarded with a Nobel
14
Prize, the key to Aspirin’s anti-inflammatory action was shown to be its potent ability to
inhibit cyclooxygenase (Mann, 1992).
Over the past two decades, numerous plant extracts and plant compounds have been
investigated for their ability to modulate inflammation. Most of these investigations have
been conducted in vitro or in animal models, while only a relatively small number of human
trials have been conducted in this area. Plant compounds with anti-inflammatory activity
have been reviewed and arachidonic acid metabolism, nitric oxide and nuclear factor kappa
B (NFκB) identified as major targets (Bremner & Heinrich, 2002; Calixto et al., 2003). In
many cases such anti-inflammatory activity appears to be the result of the ability of a
compound to inhibit the action and/or biosynthesis of pro-inflammatory cytokines,
chemokines or adhesion molecules involved in the inflammatory process, for example by
activating transcription factors (incl. NFκB) and protein kinases (Calixto et al., 2004). Table
1-2 provides examples of anti-inflammatory plant compounds and their targets.
15
Table 1-2. Examples of plant compounds with in vitro anti-inflammatory activity.
Compound(s) Plant source Molecular target Reference
Isocoumarins Hydrangea dulcis Mast cells (Matsuda et al., 1999)
Resveratrol Vitis vinifera Mast cells (Baolin et al., 2004)
Terminoside A Terminalia arjuna Nitric oxide (Ali et al., 2003)
Orixalone A Orixa japonica Nitric oxide (Ito et al., 2004)
Eugenol Ocimum sanctum COX-1 (Kelm et al., 2000)
Ursolic acid Plantago major COX-2 (Ringbom et al., 1998)
Wogonin Scutellaria baicalensis
COX-2 expression (Chen et al., 2001)
Rhamnetin Guiera senegalensis 5-lipoxygenase (Bucar et al., 1998)
Maesanin Maesa lanceolata 5-lipoxygenase (Abourashed et al., 2001)
Ugandensidial Warburgia ugandensis
5-lipoxygenase 12-lipoxygenase
(Wube et al., 2006)
In terms of arachidonic acid metabolism, it is noteworthy that a number of plant compounds
and extracts have been shown to be dual inhibitors of cyclo-oxygenase and 5-lipoxygenase
enzymes in vitro (Liu et al., 1998; Resch et al., 1998).
16
2. LITERATURE REVIEW 2.1 Introduction
Many plants belonging to the ginger family, Zingiberaceae, have a history of medicinal use
in systems of traditional medicine. Best known are ginger (Zingiber officinale) and turmeric
(Curcuma longa), both of which has been the subject of substantial pharmacological and
clinical investigations over the last three decades, but many lesser known species are also
used, mostly in tropical Asia, where the majority are native. Several species in the family are
also important spices.
This chapter provides background information on the genera included in the present work,
with emphasis on chemistry and pharmacology, in particular as it relates to potential anti-
inflammatory activity. Ginger has been given special attention in this review. Zingiberaceae
genera not included in the present study have not been reviewed.
2.2 The ginger family, Zingiberaceae Martinov
The ginger family, Zingiberaceae, is a monocotyledenous family in the order Zingiberales.
The family comprises some 52 genera with a total about 1100 species. The family is
essentially tropical in distribution, with few species occurring in temperate climates, and is
particularly richly represented in the Indomalesian flora, i.e. from India to New Guinea.
Zingiberaceae species typically have thickened rhizomes with secretory cells producing
essential oil (Mabberley, 1997). The taxonomy of the Zingiberaceae according to K.
Kubitzki’s The Families and Genera of Vascular Plants as presented by Mabberley (1997) is
outlined in Table 2-1.
17
Table 2-1. Taxonomy of the family Zingiberaceae. (After Mabberley 1997).
Phylum Angiospermae Class Monocotyledonae (Liliopsida) Subclass Zingiberidae Order Zingiberales Family Zingiberaceae
The Zingiberaceae is divided into two subfamilies (Table 2-2). The subfamily
Zingiberoideae comprises four tribes. The subfamily Costoideae is commonly treated as a
separate family, Costaceae (Meissner) Nakai (Mabberley, 1997).
Table 2-2. Subfamilies, tribes and representative genera of the Zingiberaceae. (After Mabberley 1997.) Genera in bold were included in the present work.
Subfamily Tribe Representative genera
Zingiberoideae Hedychieae Boesenbergia, Curcuma, Hedychium, Kaempferia, Scaphochlamys
Zingibereae Zingiber Alpinieae Aframomum, Alpinia, Amomum, Elettaria,
Etlingera, Hornstedtia
Globbeae Globba Costoideae Costus, Tapeinochilus
2.2.1 Zingiberaceae in Australia
In Australia, the Zingiberaceae is represented by 14 native species from 8 genera. In
addition, 7 introduced species from 5 genera also occur (Smith, 1987) (Table 2-3).
18
Table 2-3. Zingiberaceae species occurring in Australia.
Native species in bold were included in the present study. (After Smith 1987).
Genus Native species Introduced and naturalised species
Alpinia A. arctiflora (F. Muell.) Benth. A. arundelliana (Bailey) Schumann A. caerulea (R. Br.) Benth. A. hylandii R. M. Smith A. modesta F. Muell. ex Schumann Amomum A. dallachyi F. Muell. A. queenslandicum R. M. Smith Costus C. potierae F. Muell. C. dubius (Afzel.) Schumann Curcuma C. australasica J. D. Hook C. longa L. Etlingera E. australasica (R. M. Smith) R. M. Smith Globba G. marantina L. Hedychium H. coronarium Koenig H. gardnerianum Sheppard ex
Ker Gawler Hornstedtia H. scottiana (F. Muell.) Schumann Kaempferia Kaempferia sp. Pleuranthodium P. racemigerum (F. Muell.) R. M. Smith Tapeinochilos T. ananassae (Hassk.) Schumann Zingiber Z. officinale Roscoe
Z. zerumbet (L.) Smith
2.3 Genus Zingiber Boehmer
The genus Zingiber comprises approximately 60 species ranging from India through tropical
Asia (Mabberley, 1997; Smith, 1987). There are no native Australian representatives of this
genus (despite the erroneous statement to the contrary by Mabberley, 1997), but two species,
Z. officinale Roscoe and Z. zerumbet (L.) Smith, have been reported as naturalised in tropical
north Queensland (Hnatiuk, 1990; Smith, 1987).
19
The generic name comes from the Greek Zingiberi, which in turn is derived from an Indian
word meaning ‘root’ (Smith, 1987).
Several species of Zingiber have a history of traditional medicinal, culinary or other
ethnobotanical uses. These include Japanese or mioga ginger (Z. mioga (Thunb.) Roscoe),
used against malaria and as a vermifuge in China (Mabberley, 1997); Z. montanum (J.
König) Theilade (syn. Z. cassumunar, Z. purpureum), used for a wide range of complaints in
India and South-East Asia (Johnson, 1999); and Z. zerumbet (L.) Smith, which has also been
employed for a range of conditions in Asia and the Pacific (Johnson, 1999).
The common ginger (Z. officinale Roscoe), which has been a major focus of the present
work, is treated in detail below. In addition, other Zingiber species included in this work are
briefly reviewed.
2.3.1 Ginger (Zingiber officinale Roscoe)
Ginger (Zingiber officinale Roscoe) is a sterile, reed-like plant with a pungent and aromatic
rhizome on which it relies for vegetative propagation. The plant is a cultigen, that is, it is
only known from cultivation. Its wild origins are not known with certainty but are believed
to be India or South-East Asia (Mabberley, 1997; Vaughan & Geissler, 1997). Ginger has a
very long history of use, both as a spice and as a medicinal plant, and is mentioned in ancient
Sanskrit texts and in classical Buddhist, Arabic, Greek and Roman literature (Govindarajan,
1982a). It was used widely in Europe by the tenth century (Vaughan & Geissler, 1997) and
was first exported from Jamaica, where it became a significant agricultural crop, in 1547
(Mabberley, 1997). It is now cultivated in many tropical and subtropical regions including
India, Africa, China, the West Indies and Australia, with the annual world production
estimated at 100,000 tons in 2000 (Bartley & Jacobs, 2000; Evans, 2002).
Ginger rhizome is valued as a spice for its combination of pungent and aromatic qualities,
which arise from its content of phenolic compounds and essential oil, respectively. Ginger is
used as flavouring in a vast array of foods, including savoury dishes such as curries, and
sweets such as cakes and biscuits, and also in beverages such as ginger ale, ginger beer and
ginger wine.
20
Ginger rhizome (known as Rhizoma Zingiberis in pharmacy) is used in several traditional
systems of medicine, including Traditional Chinese Medicine, Ayurveda and Western herbal
medicine (WHO, 1989; Williamson, 2002). Its traditional uses cover a great variety of
complaints including dyspepsia, flatulence and colic, nausea and vomiting, colds and flu,
migraine, as well as muscular and rheumatic disorders (WHO, 1999).
2.3.1.1 Ginger chemistry
The secondary metabolites found in the rhizome of ginger that are of primary interest can
broadly be divided into volatile compounds (extractable by steam distillation) and non-
volatile phenolic compounds, the major ones of which have pungent properties. It is
generally considered that the pharmacological activity of ginger rhizome resides with
compounds from these classes, in particular the non-volatile pungent phenolic compounds.
The term oleoresin, when applied to ginger, refers to the volatile oil, the pungent compounds
and other compounds extracted by means of solvents (ethanol or acetone) (Connell, 1969;
Govindarajan, 1982a).
2.3.1.1.1 Non-volatile compounds
Ginger owes its pungency to phenolic compounds. In the fresh rhizome the major type
comprises a series of homologous phenolic alkanones known as gingerols and derivatives
thereof such as gingerdiols. The principal of these compounds is [6]-gingerol with 8- and 10-
gingerol occurring in lower concentrations (Connell & Sutherland, 1969; Denniff et al.,
1981). When subjected to heat or alkali treatment, however, gingerols are converted to a
corresponding series of homologous shogaols by dehydration and/or to the compound
zingerone (Connell, 1969; Connell & Sutherland, 1969). The shogaols possess greater
pungency than the corresponding gingerols (Denniff et al., 1981).
21
2.3.1.1.1.1 Historical background
In 1879, Tresh isolated an oily pungent concentrate from ginger oleoresin and called it
gingerol (Tresh, 1879). In 1917, English and Japanese researchers independently isolated
two pungent ginger compounds, gingerol (Fig. 2-1) and zingerone (Fig. 2-2) (Lapworth et
al., 1917).
In 1927, the Japanese group published the structural characterisation of another pungent
ginger compound, shogaol (Fig. 2-3) (after shoga, the Japanese word for ginger) (Connell,
1969; Govindarajan, 1982a).
H3CO
HO
CH3
O
Fig. 2-2. Structure of zingerone.
H3CO
HO
(CH2)4
O OH
CH3
Fig. 2-1. Structure of [6]-gingerol, the most abundant gingerol in ginger rhizome.
Fig. 2-3. Structure of [6]-shogaol.
H3CO
HO
(CH2)4
O
CH3
22
Little progress on the chemistry of pungent ginger compounds was made until the 1960s,
when D. W. Connell of the Queensland Department of Primary Industries commenced his
work in the area.
2.3.1.1.1.2 Gingerols
Comparing the chemical composition of commercially prepared ginger oleoresin and an
oleoresin extracted with cold solvent, Connell's group found surprising differences. The
major pungent compound identified in the commercial oleoresin was shogaol and despite
repeated attempts, gingerol could not be isolated from this sample. The freshly prepared
oleoresin extracted with cold solvent, however, had a different major constituent, which was
isolated and identified as gingerol (Connell, 1969; Connell & Sutherland, 1969).
Extensive work on ginger oleoresin led Connell and Sutherland to suggest that although both
shogaol and zingerone had been isolated from oleoresins, they were in fact both artefacts, or
at the most very minor constituents of ginger rhizomes. The presence of shogaol and
zingerone in easily detectable quantities, according to Connell and Sutherland, were
indicative of the oleoresin having been exposed to excessive heat in the course of extraction
(Connell & Sutherland, 1969).
Although gingerol is normally an oily substance, Connell and Sutherland were able to obtain
a crystalline solid when storing the gingerol in hexane at –30°C. This solid was shown to
consist of a mixture of homologous phenolic ketones, identified as [6]-, [8]- and [10]-
gingerol (Fig. 2-4). [4]- and [12]-gingerols were not identified, but their presence in trace
amounts could not be excluded.
H3CO
HO
(CH2)n
O OH
CH3
[6]-gingerol: n = 4[8]-gingerol: n = 6[10]-gingerol: n = 8
Fig. 2-4. Structures of the major gingerols in ginger.
23
He and coworkers also identified [6]-, [8]- and [10]-gingerol in a methanol extract of fresh
ginger rhizome analysed by HPLC-electrospray mass spectrometry (He et al., 1998).
2.3.1.1.1.3 Gingerols in fresh ginger
Several studies have reported on the concentration of gingerols in fresh ginger rhizomes.
Zhang and colleagues analysed by reversed-phase high-performance liquid chromatography
(HPLC) freshly harvested rhizomes from Hawaii extracted with methanol, and found the
concentration of [6]-gingerol to be 2100 µg per gram fresh rhizome. The corresponding
concentrations of [8]- and [10]-gingerol were 288 and 533 µg/g, respectively (Zhang et al.,
1994). Fresh rhizomes purchased locally in the United States, extracted by methylene
chloride and analysed by HPLC, yielded 880, 93 and 120 µg per gram fresh rhizome of [6]-,
[8]- and [10]-gingerol, respectively (Hiserodt et al., 1998). Fresh rhizome from Taiwan was
reported to have a [6]-gingerol concentration of 806 µg/g by HPLC (Young et al., 2002). A
supercritical CO2 extract of ginger grown on the Australian east coast and analysed by
negative ion electrospray-MS contained 120, 19 and 24 µg per gram fresh rhizome of [6]-,
[8]- and [10]-gingerol, respectively (as well as shogaols that may have formed from
gingerols during processing) (Bartley, 1995). The considerable variation in gingerol
concentrations across these studies may reflect genetic or environmental differences, as well
as the variable methodological approaches, or a combination of these. Because ginger is a
sterile cultigen with a very long history of cultivation in different parts of the world, genetic
differences between clones are likely to be an important determinant of variation in
secondary metabolites.
2.3.1.1.1.4 Gingerol degradation products
Connell (1969) commented on the remarkable extent to which chemical changes occur in
ginger. It is interesting to note that these changes are reflected in the different therapeutic
applications of fresh and processed ginger in Oriental medicine (Hikino, 1985).
24
The main pungent compounds in fresh ginger, [6]-, [8]- and [10]-gingerol, are thermally
unstable and can undergo at least two reactions (Connell, 1969; Connell & Sutherland,
1969).
Firstly, [6]-, [8]- and [10]-gingerol can undergo dehydration and convert to [6]-, [8]- and
[10]-shogaol, respectively, when exposed to high temperature or subjected to prolonged
storage (Fig. 2-5) (He et al., 1998; Zhang et al., 1994).
Secondly, a retro-aldol reaction can give rise to zingerone and a series of aliphatic aldehydes,
which can cause undesirable flavours in the oleoresin (Fig. 2-6) (Connell, 1969).
Experiments showed that aldehyde formation occurred when oleoresin was heated to
temperatures above 200°C.
+ H2O H3CO
HO
(CH2)n
O
CH3
[6]-shogaol: n = 4[8]-shogaol: n = 6[10]-shogaol: n = 8
H3CO
HO
(CH2)n
O OH
CH3
Fig. 2-5. Dehydration of gingerols to shogaols.
Heat
25
In studies of the fate of pure gingerol at pH 3-5, which is the typical pH-range for oleoresin
samples, the conversion to shogaol was found to proceed at a very slow rate at room
temperature. At higher temperature, however, the reaction occurred more rapidly. Under
alkaline conditions, the dehydration reaction proceeded readily at room temperature,
following first order reaction kinetics. Studying this dehydration reaction in the oleoresin at a
constant temperature of 120°C, the gingerol content was found to decrease, with the rate of
loss increasing with decreasing pH. This reaction also followed first order kinetics. The
half-life of gingerol in ginger oleoresin at 120°C was found to range from approximately 3
hours at pH 2.4 to 15 hours at pH 7.2 (Connell, 1969; Connell & Sutherland, 1969).
Connell also extracted an oleoresin from freshly harvested ginger rhizome, using cold
acetone, which was subsequently removed from the extract at or below room temperature
(Connell, 1969). This extract showed a high gingerol content with little or no shogaol
present. From the same batch of ginger rhizome, two other oleoresins were prepared, one
from the whole dried rhizome and another from the sliced dried rhizome. Whole rhizomes
required longer heating time to dry than did sliced rhizomes. As expected, the oleoresin
from the whole dried rhizome had a higher shogaol content.
One of these oleoresins, with a natural pH of 3.5, was stored at ambient temperature for a
period of 18 months. During this time, a large proportion of the gingerol was converted to
H3CO
HO
(CH2)n
O OH
CH3
H3CO
HO
CH3
O
+ CH3(CH2) nCHO
200°C+
Fig. 2-6. Conversion of gingerols to zingerone and aliphatic aldehydes at high temperature.
zingerone
26
shogaol, and whereas gingerol was the major pungent compound in the original oleoresin,
shogaol was predominant in the stored product (Connell, 1969).
Whether shogaols occur in fresh ginger rhizome or form as artefacts of various extraction
and manufacturing processes has been a matter of some contention. Govindarajan, in his
comprehensive review of ginger from 1982, stated that several thin-layer chromatography
(TLC) studies had shown that shogaol is ‘definitely’ present in the fresh rhizome as a minor
component (Govindarajan, 1982a). This appeared to have been confirmed by liquid
chromatography – mass spectrometry (LC-MS) by He and co-workers who reported a small
amount of [6]-shogaol (He et al., 1998), but their sample was a methanolic extract prepared
by reflux, a process that would likely have provided the necessary heat for [6]-shogaol to
form from [6]-gingerol. [6]-Shogaol (and in one case also 8- and 10-shogaol) have also been
reported from supercritical carbon dioxide extracts of fresh ginger by GC-MS (Bartley,
1995; Bartley & Jacobs, 2000), but the high temperatures involved in this analytical method
(up to 290ºC) readily account for the formation of shogaols from gingerols. Chen and co-
workers found only a trace amount of [6]-shogaol in a supercritical CO2 extract prepared
from freeze-dried fresh rhizomes and analysed by HPLC, which does not involve high
temperatures (Chen et al., 1986b), and TLC of extracts of fresh Australian ginger did not
demonstrate the presence of shogaol (Connell & Sutherland, 1969).
More recent studies have indicated that in aqueous solution [6]-gingerol and [6]-shogaol
exist in a pH-dependent equilibrium. One study found that [6]-gingerol was most stable at
pH 4, while at pH 1 and 100ºC conversion of [6]-gingerol to [6]-shogaol was relatively fast
and reached equilibrium within two hours (Bhattarai et al., 2001). Another study found that
in simulated gastric fluid (pH 1 at 37ºC) [6]-gingerol and [6]-shogaol underwent fist order
reversible dehydration and hydration reactions to form [6]-shogaol and [6]-gingerol,
respectively (Bhattarai et al., 2007). The same study found that in simulated intestinal fluid
(pH 7.4 at 37ºC) insignificant interconversion of the two compounds took place.
2.3.1.1.1.5 Other non-volatile compounds
A range of other compounds related to gingerols as well as several diarylheptanoids have
been isolated from ginger rhizome. These include [4]-, [6]-, [8]- and [10]-gingerdiol, [6]-
methylgingerdiol, [6]-methylgingerdiacetate, [6]-paradol, gingerdiones, [6]-gingesulfonic
27
acid, [6]-hydroxyshogaol, [6]-, [8]- and [10]-dehydroshogaol and hexahydrocurcumin
(Kikuzaki, 2000 and references therein).
More recently, workers at the University of Arizona have published numerous compounds
from ginger, including many novel compounds (Jolad et al., 2004; Jolad et al., 2005). These
include paradols, dihydroparadols, acetyl derivatives of gingerols, 3-dihydroshogaols,
gingerdiols, mono- and diacetyl derivatives of gingerdiols, 1-dehydrogingerdiones,
diarylheptanoids, methyl [8]-paradol, methyl [6]-isogingerol, methyl [4]-shogaol, [6]-
isoshogaol, 6-hydroxy-[8]-shogaol, 6-hydroxy-[10]-shogaol, 6-dehydro-[6]-gingerol, three 5-
methoxy-[n]-gingerols (n = 4, 8 and 10), 3-acetoxy-[4]-gingerdiol, 5-acetoxy-[6]-gingerdiol,
diacetoxy-[8]-gingerdiol, methyl diacetoxy-[8]-gingerdiol, 5-acetoxy-3-deoxy-[6]-gingerol,
1-hydroxy-[6]-paradol and others. Some of these compounds could potentially be artifacts.
Some of the compounds reported from ginger are shown in Fig. 2-7 below.
(CH2)n
HO
On=6 [6]-paradoln=8 [8]-paradoln=10 [10]-paradol
HO
OH
(CH2)n
OHn=2 [4]-gingerdioln=4 [6]-gingerdioln=6 [8]-gingerdioln=8 [10]-gingerdiol
HO
O
(CH2)n
On=4 [6]-gingerdionen=6 [8]-gingerdionen=8 [10]-gingerdionen=10 [12]-gingerdione
CH3
CH3
CH3
MeO
MeO
MeO
Fig. 2-7. Paradols, gingerdiols and gingerdiones from Zingiber officinale.
2.3.1.1.2 Volatile oil
Commercial ginger oil is obtained by steam distillation of coarsely ground dried ginger
rhizome, and most published compositional analyses refer to oil prepared from dried raw
28
material. Ginger oil distilled from dry material is characterised by a high proportion of
sesquiterpene hydrocarbons and relatively small amounts of monoterpene hydrocarbons and
oxygenated compounds (Govindarajan, 1982a). The major sesquiterpene hydrocarbons are
zingiberene, ar-curcumene, β-bisabolene, (-)-β-sesquiphellandrene and (E,E)-α-farnesene
(Lawrence, 1995b), although published data indicate the relative abundance of these
compounds varies greatly (Fig. 2-8).
H3C
ar-curcumene
zingiberene
H
β-sesquiphellandrene
β-bisabolene
(E,E)-α-farnesene
α-copaene
Fig. 2-8. Volatile sesquiterpenes from Zingiber officinale.
Both zingiberene and (-)-β-sesquiphellandrene can be oxidised to ar-curcumene in oil stored
under unfavourable conditions (Connell & Jordan, 1971; Govindarajan, 1982a). High levels
of ar-curcumene may therefore be an indicator of degraded oil, but could also possibly
reflect distillation conditions (Govindarajan, 1982a).
29
Other constituents of ginger essential oil widely reported include α-pinene, camphene, 6-
methyl-5-hepten-2-one, myrcene, α- and β-phellandrene, limonene, 1,8-cineole, linalool,
borneol, α-terpineol, citronellol, neral, geraniol, geranial, bornyl acetate, 2-undecanone,
citronellyl acetate, α-copaene and geranyl acetate (Lawrence, 1995b; Lawrence, 1997;
Lawrence, 2000). Some of the monoterpenoids in the oil are shown in Fig. 2-9.
myrcene
α-pinene
α-phellandrene β-phellandrene
OO
bornyl acetate
O
O
citronellyl acetate
O
Ogeranyl acetate
Fig. 2-9. Volatile monoterpenoids from Zingiber officinale.
Australian ginger grown in Queensland yields a steam-distilled oil known for its citrus-like
aroma. Connell and Jordan compared 35 samples of commercially and laboratory prepared
Australian ginger oils with ten samples of commercial oils from other major ginger-
producing areas (Jamaica, Nigeria, Sierra Leone, China and India) (Connell & Jordan, 1971).
The compounds chiefly responsible for this citrus-like aroma were identified as the
monoterpene aldehydes geranial and neral (the mixture of which is known as citral). The 35
Australian oil samples analysed by Connell and Jordan contained 3-20% geranial and 1-10%
neral, whereas the oils from other parts of the world contained these compounds from trace
amounts to 3 and 1%, respectively (Connell & Jordan, 1971). The same study found that
30
apart from their high citral content, Australian ginger oils were similar to other oils in terms
of their major constituents, these being (-)-zingiberene (20-28%), (-)-β-sesquiphellandrene
(7-11%), ar-curcumene (6-10%) and β-bisabolene (5-9%). These sesquiterpenes, however,
are only weakly odorous and are believed to make only minor contributions to the aroma and
flavour of the oil (Connell & Jordan, 1971).
It is clear from more recent studies, however, that ginger oils from regions other than
Australia can also have a high citral content. The highest citral content of ginger oil reported
in the literature was 66% in one of five samples (ranging from 28% to 66%) prepared from
fresh rhizomes from Fiji (Smith & Robinson, 1981). Rhizomes (probably fresh) from the
Central African Republic steam-distilled for five hours yielded an oil containing 34.6% citral
(Menut et al., 1994), and a Mauritian ginger oil distilled from freshly harvested rhizomes
contained 26.6% citral (Gurib-Fakim et al., 2002). Ekundayo and colleagues compared oils
distilled for three hours from fresh and sun-dried Nigerian ginger (Ekundayo et al., 1988).
The oil made from fresh and dried plant material contained 23.9% and 14.3% citral,
respectively. Some Asian ginger oils have been reported to have low levels of citral
(Macleod & Pieris, 1984; Vernin & Parkanyi, 1994), but an Indian oil distilled (4 hours)
from fresh rhizomes contained 25% (Sharma et al., 2002), and an oil from Taiwan reportedly
had a citral content of 25% (Lawrence, 1995a).
Indian investigators have reported that the citrus-like aroma of ginger oil (which is due to
citral) is characteristic of oil distilled from fresh or quickly dried ginger, whereas commercial
oils and oils made from sun-dried ginger lack the lemony note (Govindarajan, 1982a). Thus
it is presently unclear whether the reported variability in citral content is due to genetic,
environmental or ontogenic factors, to factors relating to post-harvest handling or processing,
or to a combination of these.
2.3.1.2 Pharmacokinetics of ginger
No information is available regarding the pharmacokinetics of crude ginger preparations.
Several in vitro and animal studies and one in vitro study employing human hepatic and
intestinal microsomes have investigated the metabolism of the major pungent compound in
31
ginger, [6]-gingerol. One in vitro study has explored the hepatic metabolism of [6]-shogoal.
These studies are outlined below.
Experiments in rats with acute renal or hepatic failure showed that the elimination half-life
and plasma concentration of intravenously administered [6]-gingerol increased significantly
after hepatic failure was induced with carbon tetrachloride. The induction of renal failure,
however, did not affect clearance of [6]-gingerol from plasma. These results suggest that the
liver but not the kidney is involved in the elimination of [6]-gingerol (Naora et al., 1992).
The same study found more than 90% of [6]-gingerol present in plasma was bound to serum
protein. [6]-Gingerol incubated with phenobarbital-induced rat liver supernatant containing
the NADPH-generating system was converted to two diastereomers of [6]-gingerdiol (Surh
& Lee, 1994a).
Another study characterised a number of metabolites following the oral administration of
[6]-gingerol to rats (Nakazawa & Ohsawa, 2002). The major metabolite in the bile was (S)-
[6]-gingerol-4’-O-β-glucuronide, while the β-glucuronidase-treated urine contained vanillic
and ferulic acids, 9-hydroxy-[6]-gingerol, (S)-(+)-[6]-gingerol and two substituted organic
acids. These results demonstrated that orally administered [6]-gingerol undergoes
conjugation, and ω-1 oxidation and β-oxidation of a phenolic side chain. About half of the
oral dose of [6]-gingerol was excreted in the bile as the glucuronide. Incubation of [6]-
gingerol with rat liver produced only (S)-[6]-gingerol-4’-O-β-glucuronide and 9-hydroxy-
[6]-gingerol (glucuronidation and ω-1 oxidation), suggesting that metabolism must also
occur outside the liver. The contribution of gut microflora was demonstrated by the fact that
gut sterilisation decreased the amount of urinary metabolites.
In a recent study, [6]-gingerol incubated with human hepatic microsomes fortified with
uridine diphosphoglucuronic acid (UDPGA; a source of glucuronic acid) was glucuronidated
primarily at the phenolic hydroxyl group with a small amount of glucuronidation also
occurring at the aliphatic hydroxyl group, while human intestinal microsomes formed the
phenolic glucuronide only (Pfeiffer et al., 2006).
[6]-shogaol incubated with rat liver supernatant containing the NADPH-generating system
was converted to saturated ketone and reduced alcohol metabolites as well as an allyl
alcohol, 1-(4'-hydroxy-3'-methoxyphenyl)-deca-4-ene-3-ol (Surh & Lee, 1994b).
32
It is clear from the above that the current state of knowledge of the pharmacokinetics of
ginger compounds in humans is embryonic. Expanding this knowledge and including
information about oral bioavailability of compounds with known pharmacological activity
should be a priority and ought to precede further clinical trials of ginger for inflammatory
conditions.
2.3.1.3 Pharmacodynamics of ginger
The pharmacology and therapeutic use of ginger have been the subject of several recent
reviews (Afzal et al., 2001; Chrubasik et al., 2005; Grzanna et al., 2005).
Studies conducted in vitro have shown gingerols to act as agonists of the vanilloid receptor
(VR1) (Dedov et al., 2002). This receptor is also activated by capsaicin, the major pungent
principle in cayenne and chilli pepper, which shares structural features with the gingerols
(vanillin-derived compounds with phenolic 3-methoxy-, 4-hydroxy-groups). Earlier work
suggested that [6]-shogaol might share its site of action with capsaicin (Onogi et al., 1992);
if this were true, [6]-shogaol might also be a VR1 agonist.
Ginger extracts and various ginger constituents have demonstrated a wide range of
pharmacological activities in vitro and in vitro. Here the emphasis will be on the activities
relevant to inflammation, while other pharmacological activities will be briefly summarised.
2.3.1.3.1 Anti-oxidant activity
Ginger extracts have demonstrated anti-oxidant activity in a number of in vitro assay
systems, including 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity,
inhibitory effect on oxidation of methyl linoleate under aeration and heating by the Oil
Stability Index (OSI) method, inhibitory effect on oxidation of liposome induced by 2,2'-
azobis(2-amidinopropane)dihydrochloride (AAPH) (Masuda et al., 2004), oxygen radical
absorbance capacity (ORAC) (Ninfali et al., 2005), Fe2+-induced lipid peroxidation, 1,1-
diphenyl-2-picrylhydrazyl (DPPH) radical quencher assay (Kuo et al., 2005), β-carotene
bleaching method (Kaur & Kapoor, 2002) and ferric reducing ability of plasma (FRAP)
(Halvorsen et al., 2002).
33
[6]-Gingerol, [6]-shogaol, [6]-gingerdiol, two other gingerol-related compounds as well as 8
arylheptanoids delayed the oxidation of linoleic acid in the ferric thiocyanate assay
(Kikuzaki & Nakatani, 1993). More recently, Masuda and coworkers have identified more
than 50 compounds with anti-oxidant activity from ginger rhizome (Masuda et al., 2004).
They were either related to gingerols or diarylheptanoids. Structure-activity studies of the
gingerol-type compounds suggested that both the substituents on and the length of the alkyl
chain might contribute to the anti-oxidant activity. A glucoside of [6]-gingerdiol has also
been found to possess strong anti-oxidant activity in vitro (Sekiwa et al., 2000).
Several animal studies have demonstrated the anti-oxidant activity of ginger in vivo.
Lipid peroxidation was significantly lowered in rats fed ginger (1%); the activities of the
antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione
peroxidase were maintained, and the blood glutathione content was significantly increased.
Similar effects were achieved with ascorbic acid (100 mg/kg) treatment, suggesting that at
least in this assay ginger was comparable to ascorbic acid as an antioxidant (Ahmed et al.,
2000a).
Rats on a high fat diet supplemented with ginger (35 mg/kg and 70 mg/kg) had lowered
levels of tissue thiobarbituric acid reactive substances (TBARS) and hydroperoxides, raised
activities of SOD and CAT, and raised levels of reduced glutathione in the aorta, liver,
kidney and intestine, compared with unsupplemented controls (Jeyakumar et al., 1999).
In another study, rats fed dried ginger (1%) showed significantly attenuated oxidative stress
and lipid peroxidation in response to exposure to the organophosphorous pesticide malathion
for 4 weeks (20 ppm) (Ahmed et al., 2000b).
Atherosclerotic, apolipoprotein E-deficient mice fed ginger extract showed a significant
reduction in the LDL basal oxidative state, with reduced susceptibility to oxidation and
aggregation (Fuhrman et al., 2000).
Pre-treatment with a hydroethanolic extract of ginger reduced the severity of radiation
sickness and the mortality in mice exposed to gamma irradiation (Jagetia et al., 2003).
Elevated lipid peroxidation and depletion of glutathione following irradiation were both
reduced by the ginger treatment. Treatment with ginger after irradiation was not effective
(Jagetia et al., 2004).
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No studies have as yet directly assessed the anti-oxidant potential of ginger in humans, but
based on the substantial amount of evidence from both in vitro and in vivo studies, there are
sound reasons to predict that ginger might be a potent anti-oxidant in humans, although any
systemic effects could be limited if the compounds responsible have poor oral
bioavailability.
2.3.1.3.2 Effects on arachidonic acid metabolism and eicosanoids
Arachidonic acid metabolism is a major inflammatory pathway, and most of the work
relevant to the anti-inflammatory activity of ginger has focused on its effects on this
pathway. This substantial body of work is reviewed below under the headings (1) Assays
using isolated enzymes, (2) Cell-based assays, and (3) Animal studies.
2.3.1.3.2.1 Assays using isolated enzymes
Kiuchi and coworkers were the first group to publish details of the ability of ginger
compounds to modulate arachidonic acid metabolism (Kiuchi et al., 1982). They worked on
fresh ginger rhizomes obtained from a Japanese market and isolated [6]-gingerol, [6]-
gingerdione, [10]-gingerdione, [6]-dehydrogingerdione and [10]-dehydrogingerdione. All of
these compounds were shown to be potent inhibitors of prostaglandin-synthetase
(cyclooxygenase) in vitro (although details of the assay method were not provided), with
IC50 values comparable to that of indomethacin. Kiuchi's group subsequently reported the
potent inhibitory activity on prostaglandin-synthetase of [6]-gingerol, [10]-gingerol, [6]-
shogaol and numerous gingerol derivatives (Kiuchi et al., 1992). Very strong inhibitory
activity was found for [10]-gingerol, [6]-acetylgingerol, [6]-shogaol, [6]-
dehydrogingerdione, [10]-dehydrogingerdione, [6]-gingerdione and [10]-gingerdione, all of
which had IC50 values <2.5 µM.
[8]-Paradol has also been shown to be a potent inhibitor of ovine cyclooxygenase-1 (COX-1)
with an IC50 value of 4±1 μM (Nurtjahja-Tjendraputra et al., 2003).
Of particular interest was the finding by Kiuchi’s group that the homologous series of
synthetic gingerols (of which [6]-, [8]- and [10]-gingerol occur in ginger) were dual
35
inhibitors of arachidonic acid metabolism, i.e. inhibitors of both the cyclooxygenase and 5-
lipoxygenase pathways (Kiuchi et al., 1992). This finding appears to confirm an earlier
finding suggesting that several ginger compounds inhibit both pathways (Flynn et al., 1986),
while being at odds with the finding by Srivastava (see below), that fractionated ginger
preparations increased the formation of lipoxygenase products (Srivastava, 1984b;
Srivastava, 1986). It is possible, however, that these seemingly incongruous results are in
fact all accurate and reflect chemical differences between the test preparations.
Dual inhibitors of arachidonic acid metabolism are of significant interest as potential
therapeutic substances, because their inhibitory effect on the formation of eicosanoids is
likely to some extent to resemble the therapeutic effects of steroidal drugs, which inhibit
phospholipase A2 and therefore inhibit the release from phospholipid membranes of
arachidonic acid itself, resulting in potent anti-inflammatory activity.
2.3.1.3.2.2 Cell-based assays
Srivastava published two studies describing the effects of a ginger extract and its fractions on
arachidonic acid metabolism and platelet aggregation in washed human platelets (Srivastava,
1984a; Srivastava, 1984b). The preparation used in these studies was described as an
aqueous extract, but it is unclear whether it was prepared from fresh or dried plant material.
Regardless, thromboxane B2 (TxB2) formation was reduced by 73%, while formation of
prostaglandin F2α (PGF2α), prostaglandin E2 (PGE2) and prostaglandin D2 (PGD2) and the
prostaglandin endoperoxides, prostaglandin G2 (PGG2) and prostaglandin H2 (PGH2) was
also significantly reduced.
Additional data published by Srivastava and colleagues confirmed the previously observed
effects on the production of TxB2 and also found inhibition of lipoxygenase products by
ginger components at higher concentrations (Bordia et al., 1997; Srivastava, 1986).
An 80% ethanolic ginger extract (uncertain whether from fresh or dried material) was found
to significantly and dose-dependently inhibit prostaglandin release (expressed in terms of
PGE2) by stimulated rat peritoneal leukocytes (Mascolo et al., 1989).
Further evidence of ginger’s inhibitory activity on thromboxane formation was provided by
Bordia and coworkers who showed that an n-hexane extract of ginger powder dose-
36
dependently inhibited thromboxane formation in washed platelets challenged with (1-14C)-
labelled arachidonic acid (IC50 =16±8µg/mL, n=3). Using autoradiography, the
investigators found that 12-hydroxy-5,8,10-heptadecatrienoic acid (12-HHT) was reduced
with a concomitant increase in 12-hydroxyeicosatetraenoic acid (12-HETE) (Bordia et al.,
1997).
Inhibition of LPS-induced PGE2 production in human leukaemia HL-60 cells with IC50
values comparable to that of indomethacin was demonstrated for fractions of fresh ginger
(Jolad et al., 2004) and commercially processed dry ginger (Jolad et al., 2005; Lantz et al.,
2007).
Flynn and colleagues were the first to study the effects of pure synthetic ginger compounds
on arachidonic acid metabolism (Flynn et al., 1986). This study used intact human
leukocytes and found gingerdione to be a potent inhibitor of both cyclooxygenase and 5-
lipoxygenase. [6]-Shogaol and a related compound preferentially inhibited 5-
hydroxyeicosatetraenoic acid (5-HETE) formation (i.e. the 5-lipoxygenase pathway), while
[6]-gingerol and dehydroparadol appeared to be more potent inhibitors of PGE2 formation
(i.e. the cyclooxygenase pathway). All compounds studied possessed a 4-hydroxy-3-
methoxyphenyl moiety (which is also a major determinant of flavour). The same study also
found curcumin from turmeric (Curcuma longa) to be a dual inhibitor of arachidonic acid
metabolism.
Guh et al. investigated the anti-platelet mechanism of ‘gingerol’ isolated from ginger in
washed rabbit platelets (Guh et al., 1995). It is not clear from the report whether the plant
material was fresh or dried, and the method of isolation is not described. Neither is it clear
what is meant by ‘gingerol’. The report includes a diagram of [8]-gingerol that carries the
caption ‘Chemical structure of gingerol’. However, since [8]-gingerol is not the major
gingerol present in ginger, some uncertainty as to the exact identity of ‘gingerol’ remains.
‘Gingerol’ and indomethacin (a COX inhibitor) were found to significantly inhibit the
arachidonic acid-induced formation of TxB2 and PGD2 in a concentration-dependent manner.
In contrast, imidazole, which is a thromboxane synthase inhibitor, reduced TxB2 formation
while causing a highly significant rise in PGD2 production. These findings are important,
because they demonstrate that ‘gingerol’, like indomethacin, inhibits cyclooxygenase rather
than thromboxane synthase. It was also demonstrated that the anti-platelet activity of
37
‘gingerol’ was due to the subsequent reduced production of TxA2, a potent platelet
aggregating agent.
Venkateshwarlu was the first to study the effects of ginger compounds on eicosanoid
metabolism in whole blood (Venkateshwarlu, 1997). He showed that [6]- and [10]-gingerol
isolated from fresh ginger dose-dependently inhibited the formation of TxB2 in horse blood.
Curcumin and another compound isolated from dried rhizomes of turmeric (Curcuma longa)
as well as piperine isolated from dried pepper (Piper nigrum) fruits were also found to be
active.
[8]-gingerol and [8]-gingerdiol were potent inhibitors of COX activity in RBL-2H3 cells
with IC50 values of 1.54 and 3.3 μM, respectively, which was similar to that of indomethacin
(0.76 μM) (Koo et al., 2001). The inhibitory activity of the major pungent phenolic in fresh
ginger, [6]-gingerol, was an order of magnitude weaker (50 μM) in this study.
Tjendraputra and co-workers showed that a number of naturally occurring gingerol-type
compounds ([8]- and [10]-gingerol, [6]-shogaol, [8]-gingerdiol and [8]-paradol, as well as
several synthetic analogues) inhibited COX-2 activity in cultured human airways epithelial
A549 cells, with IC50 values in the low micromolar range (Tjendraputra et al., 2001).
Interestingly, [6]-gingerol did not inhibit COX-2 in this assay.
2.3.1.3.2.3 Animal studies
Sharma and coworkers examined the effects of a steam-distilled ginger oil on chronic
adjuvant arthritis in rats (Sharma et al., 1994). The oil, which was obtained from a company
in Germany, contained camphene, limonene, 1,8-cineole, linalool, citral and borneol. It is
noticeable that three of the major constituents of most ginger oils, zingiberene, ar-
curcumene, and β-sesquiphellandrene, were not listed as constituents of this oil. Rats in the
verum group (n=8) were given ginger oil (33 mg/kg) orally once daily for 26 days,
commencing the day prior to the induction of adjuvant arthritis. Ginger oil treatment
produced significant suppression of knee joint swelling on day 2 after induction of oedema
(p<0.05) and an even greater suppression of joint swelling on day 26 (p<0.001). In terms of
paw swelling, a significant inhibitory effect was evident at 18-26 days (p<0.001). It is
noteworthy that this study used a steam-distilled oil, which should lack gingerols and
38
shogaols. If the ginger preparation indeed was devoid of gingerols and shogaols, these
findings indicate that steam-distilled ginger oil contains other compounds with potential anti-
inflammatory activity.
In 1997, Sharma et al. re-published the findings of the study first published in 1994 (Sharma
et al., 1997). Included were new data on plasma and tissue kallikrein (also known as
kallidinogenase) levels in synovial tissue. The kallidinogenases are enzymes responsible for
the formation of kinins from their precursors, kininogens and kallidinogens. Kinins have
been implicated in many physiological functions, including the regulation of blood flow and
blood pressure, allergy and inflammation. Increases in kinin formation or kinin release have
been shown to occur in many inflammatory conditions. The presence of high levels of
plasma kallidinogenase-like enzyme has previously been demonstrated in blood-free paw
tissue from rats with adjuvant inflammation. The study found that raised levels of plasma
kallidinogenase fell significantly in rats treated with ginger oil for 26 days (p<0.01). Ginger
oil treatment produced no change in synovial tissue kallidinogenase levels. According to the
authors, these findings suggest that ginger oil might possess anti-rheumatic and anti-
inflammatory properties by inhibiting the kinin-forming kallidinogenase enzyme.
An aqueous preparation made from fresh ginger was given to rats in a study examining the
ex vivo effects on TxB2 and PGE2 synthesis (Thomson et al., 2002). Administered orally or
intraperitoneally at two doses (50 mg/kg and 500 mg/kg), the low dose significantly reduced
serum PGE2 levels only when given orally, whereas the higher dose was effective with either
route of administration. Serum TxB2 levels were reduced significantly only with oral
administration of the higher dose.
[6]-Gingerol has been tested for anti-inflammatory activity against carrageenan-induced paw
oedema in rats (Young et al., 2005). At doses of 50 mg/kg, 100 mg/kg and 250 mg/kg, [6]-
gingerol was effective in reducing oedema; an ED50 value of 85 mg/kg suggested a
considerable anti-inflammatory effect in this assay.
Penna and co-workers found that rat paw oedema induced by carrageenan, compound 48/80
(a synthetic mast cell-degranulating compound) and serotonin was significantly inhibited by
intraperitoneal administration of a 70% aqueous ethanolic extract made from fresh ginger
rhizomes (Penna et al., 2003). Local application of the extract also inhibited 48/80-induced
(but not Substance P- and bradykinin-induced) skin oedema, and intraperitoneal
39
administration one hour prior to serotonin injection significantly reduced serotonin-induced
skin oedema. It was concluded that the anti-oedematogenic activity of the ginger extract was
at least in part due to serotonin receptor antagonism.
A study investigating the anti-tumour promoting effects of [6]-gingerol found that when
applied topically to mouse skin stimulated with a tumour promoter, the compound inhibited
COX-2 expression in vivo (Kim et al., 2005b). The suggested mechanism of this effect was
blocking of the p38 mitogen-activated protein (MAP) kinase–nuclear factor kappa B (NFκB)
signalling pathway (NFκB regulates COX-2 induction).
2.3.1.3.2.4 Summary: Effects on arachidonic acid metabolism and eicosanoids
There is a substantial body of evidence from in vitro studies suggesting that ginger extracts
and a range of ginger constituents (including [6]-, [8]- and [10]-gingerol, and [6]-shogaol)
inhibit cyclooxygenase (COX). This causes a reduction in the formation of prostanoids of
the 2-series, including PGE2, which has potent pro-inflammatory properties in certain
circumstances, and TxA2, which causes platelet aggregation and serotonin release, and
contracts smooths vascular muscle. Some phenolic ginger constituents, including [8]- and
[10]-gingerol and [6]-shogaol (but not [6]-gingerol), were relatively potent inhibitors of
COX-2 in vitro.
Animal studies have confirmed the ability of ginger extracts to inhibit PGE2, thromboxane
and inflammation, and studies using essential oil suggest that compounds other than those of
gingerol type possess anti-inflammatory activity. Unlike the finding from the in vitro study
of COX-2 inhibition, [6]-gingerol was found to inhibit COX-2 when applied topically to
mouse skin. In this instance COX-2 inhibition appeared to be caused by the blocking of a
signalling pathway involving NFκB. Serotonin antagonism has also been suggested as a
possible mechanism of anti-inflammatory action.
Thus the emerging picture of ginger as an anti-inflammatory agent is consistent with that of a
typical phytomedicine: it contains multiple active compounds and as such it appears to have
multiple pharmacological targets.
40
2.3.1.3.3 Effects on cytokines and chemokines
Cytokines are immunoregulatory proteins (such as interleukins, tumour necrosis factor, and
interferons) secreted by cells, especially of the immune system. They provide
communication between macrophages and various subsets of lymphocytes; all of them act in
a non-specific manner to enhance the inflammatory response (McCance & Huether, 2002).
A hot extract of dried ginger in 50% aqueous ethanol was tested for its effects on cytokine
secretion by human peripheral blood mononuclear cells (Chang et al., 1995). The secretion
of IL-1β, IL-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) was
increased in the presence of the ginger extract at low concentrations (10-30 mg/mL), but
only after prolonged reaction time (18 or 24 hours); higher concentrations were less
effective. There was no significant effect on tumour necrosis factor-alpha (TNF-α).
Chemokines are chemotactic molecules, at least 50 of which are known to play a role in
inflammation. Joint chondrocytes and synoviocytes produce a range of chemokines, which
cause migration of leukocytes into the inflamed tissue and mediate cartilage degradation by
decreasing proteoglycan synthesis and activating matrix metalloproteinases (MMPs), which
catalyse the breakdown of cartilage (Phan et al., 2005).
An extract prepared from dried ginger rhizomes, described as having a high (sic) content of
[6]-, [8]- and [10]-gingerol, gingerdione and gingerdiol, was tested in vitro for its effects on
the expression of two chemokines, macrophage chemotactic factor (MCP-1) and interferon-γ
activated protein (IP-10), in human synoviocytes (Phan et al., 2005). These two chemokines
have been shown to be involved in the pathogenesis of both osteoarthritis and rheumatoid
arthritis by inducing the migration of leukocytes and monocytes from the blood to areas of
inflammation. MCP-1 also has been found to induce MMPs. IP-10 is a chemoattractant for
monocytes, natural killer cells and activated T-cell, in particular type 1 helper cells, causing
upregulation of interferon-γ. The experiment used two cultivated synoviocyte cell lines
obtained from patients with osteoarthritis during knee replacement surgery. Incubation with
ginger extract inhibited the secretion of both MCP-1 and IP-10 (by 40% and 54%,
respectively), but only in one of the two cell lines tested. Incidentally, the study
demonstrated a synergistic effect when extracts of Zingiber officinale and Alpinia galanga
(galangal) were combined.
41
These findings suggest that some aspects of ginger’s anti-inflammatory activity may be
mediated by effects on cyto- and chemokines.
2.3.1.3.4 Anti-cancer activity
An ethanolic ginger extract applied topically to mouse skin provided a highly significant
protective effect against the development of skin tumours, and this was associated with the
inhibition of 12-O- tetradecanoylphorbol-13-acetate (TPA)-caused induction of epidermal
ornithine decarboxylase, cyclooxygenase and lipoxygenase activities (Katiyar et al., 1996).
A subsequent study showed [6]-gingerol to have similar activity (Park et al., 1998).
A more recent study showed that topical application of [6]-gingerol inhibited COX-2
expression in mouse skin stimulated with the tumour promoter TPA (Kim et al., 2005b).
Results from this study suggested that the inhibition of COX-2 expression was the result of
the blocking of the p38 MAP kinase- NFκB signalling pathway.
A cytotoxic or cytostatic effect mediated by apoptosis was found for [6]-gingerol and [6]-
paradol in human promyelocytic leukaemia HL-60 cells (Lee & Surh, 1998), and also for
four diarylheptanoids and two shogaols (Wei et al., 2005).
2.3.1.3.5 Anti-microbial activity
Ginger extracts have demonstrated antimicrobial activity against a wide range of pathogenic
micro-organisms; these include both Gram-positive and Gram-negative bacteria and the
yeast Candida albicans, but the antimicrobial activity of ginger extracts appears to be
moderate in comparison with highly antimicrobial plant extracts.
Of particular interest is an in vitro study showing that a crude methanolic extract (MIC 6-50
µg/mL) and a gingerol-containing fraction (MIC 0.8-12.5 µg/mL) significantly inhibited the
growth of 19 strains of Helicobacter pylori, the micro-organism associated with peptic ulcer
disease as well as gastric and colon cancer (Mahady et al., 2003).
42
2.3.1.3.6 Immunomodulatory effects
Few studies have examined the potential immunomodulatory activity of ginger.
Non-specific immunity was increased in rainbow trout eating a diet containing 1% of a dried
aqueous ginger extract for three weeks (Dugenci et al., 2003). Mice fed a 50% ethanolic
ginger extract (25 mg/kg) for seven days had higher haemagglutinating antibody titre and
plaque-forming cell counts, consistent with improved humoral immunity (Puri et al., 2000).
One in vitro study found that ginger suppressed lymphocyte proliferation; this was mediated
by decreases in IL-2 and IL-10 production (Wilasrusmee et al., 2002). Another study found
an aqueous ginger extract significantly increased the production of IL-1β, IL-6 and TNF-α in
activated peritoneal mouse macrophages (Ryu & Kim, 2004). The same study found that
splenocyte proliferation and cytokine production were stimulated in a dose-dependant
manner when mice were given an aqueous ginger extract for 4 weeks (50-500 mg/kg).
2.3.1.3.7 Gastrointestinal effects
An orally administered acetone extract of ginger (75 mg/kg) as well as [6]-shogaol (2.5
mg/kg) and [6]-, [8]- and [10]-gingerol (5 mg/kg) enhanced gastric motility (charcoal meal)
in mice (Yamahara et al., 1990). Both acetone and 50% ethanolic extracts (100-500 mg/kg)
and ginger juice (2-4 mL/kg) reversed cisplatin-induced delay in gastric emptying in rats
when given orally (Sharma & Gupta, 1998). The effect on gastric motility may at least in
part explain the anti-emetic properties of ginger.
Both ginger extracts and several ginger compounds including 6-gingerol have demonstrated
ulcer-protective activity in rats (Agrawal et al., 2000; Sertie et al., 1992; Yamahara et al.,
1988; Yoshikawa et al., 1992). Ginger stimulated bile secretion in rats, with [6]- and [10]-
gingerol being chiefly responsible for this activity (Yamahara et al., 1985), and ginger has
also been shown to stimulate intestinal lipase, trypsin, chymotrypsin, amylase, sucrase and
maltase activity when fed to rats for an 8-week period (Platel & Srinivasan, 1996; Platel &
Srinivasan, 2000). These findings support the traditional use of ginger as a digestive
stimulant (Mills & Bone, 2000). Ginger may also increase the conversion of cholesterol to
43
bile acids by increasing the activity of hepatic cholesterol-7-α-hydroxylase, the rate-limiting
enzyme of bile acid biosynthesis (Srinivasan & Sambaiah, 1991).
2.3.1.3.8 Metabolic effects
Significant hypoglycaemic activity in rabbits was produced by an ethanolic Soxhlet extract
of ginger (Mascolo et al., 1989), and two animal studies have suggested that ginger may
improve insulin sensitivity (Goyal & Kadnur, 2006; Kadnur & Goyal, 2005).
2.3.1.3.9 Anti-atherosclerotic effects
A number of animal studies have demonstrated hypocholesterolemic action of ginger and
ginger extracts. These studies have shown decreased levels of total cholesterol, low-density
lipoprotein (LDL)-, very low-density lipoprotein (VLDL)-cholesterol and triglycerides, and
increases in high-density lipoprotein (HDL)-cholesterol (Ahmed & Sharma, 1997; Bhandari
et al., 1998; Fuhrman et al., 2000; Giri et al., 1984; Murugaiah et al., 1999).
In a more recent study, air-dried ginger powder (100 mg/kg orally daily) fed to rabbits with
experimentally induced atherosclerosis for 75 days inhibited atherosclerotic changes in the
aorta and coronary arteries by about 50% (Verma et al., 2004). In this study the ginger
treatment did not cause any significant lowering of serum lipids, but lipid peroxidation was
decreased and fibrinolytic activity increased.
The effects of an aqueous ginger extract, administered orally or intraperitoneally to rats for 4
weeks, on serum cholesterol and triglyceride levels and on PGE2 and TxB2 have been
reported (Thomson et al., 2002). At 500 mg/kg both modes of administration led to a
significant reduction in serum cholesterol levels, whereas only intraperitoneal administration
led to a reduction with a daily dose of 50 mg/kg. Neither dose caused significant changes to
serum triglyceride levels. The higher dose administered orally significantly lowered serum
PGE2 and TxB2 levels, and the lower dose also reduced PGE2 levels.
44
It is evident from this that ginger has demonstrated considerable potential as an anti-
atherosclerotic agent in animal studies, but as yet this promise has not been confirmed in
human trials.
2.3.1.3.10 Other cardiovascular effects
Ginger extracts as well as [6]- and [8]-gingerol have been shown to modulate eicosanoid
responses in smooth vascular muscles ex vivo (Hata et al., 1998; Kimura et al., 1989; Pancho
et al., 1989).
[6]- and [8]-gingerol and related analogues inhibited arachidonic acid-induced serotonin
release by human platelets in a dose range similar to the effective dose aspirin (IC50 values
between 45 and 83 μM), and the same compounds were also effective inhibitors of
arachidonic acid-induced platelet aggregation (Koo et al., 2001). COX inhibition was
demonstrated in RBL-2H3 cells, suggesting this might be the underlying mechanism.
An early study found a dose-dependent positive inotropic action of [6]-, [8]- and [10]-
gingerol on isolated guinea pig left atria (Shoji et al., 1982), and ‘gingerol’ stimulated the
Ca2+-pumping ATPase activity of fragmented sarcoplasmic reticulum prepared from
mammalian skeletal and cardiac muscle (Kobayashi et al., 1987).
[6]-Gingerol and [6]-shogaol lowered systemic blood pressure in anaesthetised rats at doses
of 10-100 μg/kg and caused bradycardia when administered intravenously (Suekawa et al.,
1984; Suekawa et al., 1986).
In a recent study a crude extract (70% aqueous methanol) of fresh ginger induced a dose-
dependent fall in arterial blood pressure of anaesthetised rats; this effect was shown to be
mediated through blockade of voltage-dependent calcium channels (Ghayur & Gilani, 2005).
2.3.1.3.11 Analgesic effects
[6]-gingerol had analgesic effects in mice in both the acid-induced writhing test and the
formalin test, suggesting the analgesic activity resulted from peripheral and possible anti-
inflammatory action (Young et al., 2005). [6]-shogaol has also been shown to inhibit acetic
45
acid-induced writhing in mice and to elevate the nociceptive threshold of the yeast-inflamed
paw (Suekawa et al., 1984). Experiments carried out by Onogi and co-workers suggested
that [6]-shogaol inhibits the release of Substance P by stimulation of the primary afferents
from their central terminal and hence shares this site of action with capsaicin (Onogi et al.,
1992).
2.3.1.3.12 Antipyretic activity
A Soxhlet extract of ginger in 80% ethanol reduced yeast-induced fever in rats by 38% when
administered orally (100 mg/kg) (Mascolo et al., 1989). This was comparable to the anti-
pyretic effect of acetylsalicylic acid at the same dose. The ginger extract did not affect the
temperature of normothermic rats. This anti-pyretic activity may be mediated by COX
inhibition.
2.3.1.4 Safety data
2.3.1.4.1 Acute toxicity
A concentrated Soxhlet extract of ginger in 80% ethanol was well tolerated orally in mice at
doses up to 2.5 g/kg, but doses of 3.0 and 3.5 g/kg caused 10-30% mortality. Death was
caused by involuntary contractions of skeletal muscle; other symptoms included
gastrointestinal spasm, hypothermia, diarrhoea and anorexia (Mascolo et al., 1989).
Another study found that a hydroethanolic ginger extract was nontoxic in mice when
administered by oral gavage up to a dose of 1.5 g/kg body weight (Jagetia et al., 2004).
2.3.1.4.2 Teratogenicity and embryotoxicity
Pregnant rats administered 20 g/L or 50 g/L ginger tea via their drinking water from
gestation day 6 to 15 showed no gross malformation on fetuses (Wilkinson, 2000).
However, embryonic loss in the ginger-treated group was twice that of controls. Surviving
46
fetuses in the ginger group were significantly heavier and showed more advanced skeletal
development compared with controls.
A patented ginger extract was tested for teratogenic potential in pregnant rats (Weidner &
Sigwart, 2001). The extract caused neither maternal nor developmental toxicity at daily
doses of up to 1 g/kg body weight.
2.3.1.4.3 Mutagenicity
[6]-gingerol and to a far lesser extent [6]-shogaol were shown to have mutagenic properties
in an assay using Escherichia coli Hs30 as an indicator strain of mutagenesis (Nakamura &
Yamamoto, 1983). Despite this finding, ginger is not considered a mutagenic substance,
presumably due to its long history of safe use.
2.3.1.5 Clinical studies of ginger
Data from clinical trials support the use of ginger and ginger preparations in motion sickness
and seasickness, morning sickness, hyperemesis gravidarum, postoperative nausea and
osteoarthritis, although not all clinical trials have produced positive outcomes. The clinical
efficacy trials on ginger published prior to 2008 are summarised in Appendix A.
2.3.1.5.1 Clinical studies of ginger in arthritis
Few clinical studies have examined the efficacy of ginger in osteoarthritis. Preceding proper
clinical trials, Srivatava and Mustafa published in 1989 a case series of 7 patients suffering
from rheumatoid arthritis who had reported improvements in their condition following the
consumption of ginger (Srivastava & Mustafa, 1989). The same investigators followed up
these observations with a questionnaire-based survey of people who were self-medicating
ginger for their condition (Srivastava & Mustafa, 1992). This survey provided data from a
total of 56 patients, including the 7 people whose cases had been published in the first report.
Of the 56 patients, 28 suffered from rheumatoid arthritis, 18 from osteoarthritis and 10 from
47
muscular discomfort. The majority of patients reported marked relief in symptoms of pain
and swelling as a result of ginger consumption.
The results obtained by Srivastava and Mustafa in their survey were promising. However,
the fact that the data came from an uncontrolled study with self-selected subjects meant that
these results offered little in terms of proper evidence for the efficacy of ginger in arthritic
and rheumatic conditions.
The first proper clinical trial to examine the usefulness of ginger in osteoarthritis was
conducted by a Danish group and published in 2000 (Bliddal et al., 2000a). This
randomised, placebo-controlled, cross-over study compared the effects of a standardised
ginger extract with those of ibuprofen in osteoarthritis. Fifty-six out-patients (41 women, 15
men) completed the study. Of these, 36 had osteoarthritis of the knee and 20 osteoarthritis of
the hip. Mean duration of osteoarthritis was 7.7 years (range 1-30 years). One week prior to
randomisation, treatment with analgesics and NSAIDs was discontinued, but acetaminophen
(paracetamol) was allowed as a rescue drug for pain relief throughout the study, up to a
maximum dose of 3 g daily. Subjects were randomised to three treatment periods of three
weeks each, during which they received (in different order) capsules containing either 170
mg of a standardised ginger extract t.i.d., ibuprofen 400 mg t.i.d., or placebo t.i.d. The ginger
extract had a standardised content of hydroxy-methoxy-phenyl compounds, but no further
details were provided, making it impossible to estimate the dose on a fresh or dried
equivalent basis. There were no wash-out periods between the three treatments. The primary
outcome variable was pain assessed using a visual analogue scale (VAS); secondary
outcomes were the Lequesne-index (a questionnaire-based disability score) for either hip or
knee, and range of motion.
The results produced a ranking in terms of efficacy on pain level and function with ibuprofen
being more effective than the ginger and placebo. The same ranking applied to the
consumption of the rescue medication, with ibuprofen treatment being associated with the
lowest consumption of the rescue medication. No difference between the ginger extract and
placebo was found in a test for multiple comparisons. However, explorative statistical testing
of the first period of treatment (before cross-over) found a significantly better effect of both
ginger and ibuprofen compared with placebo (p<0.05). This finding is particularly
interesting, because the design of this cross-over study was marred by the lack of wash-out
periods between treatments. The lack of wash-out periods and the short duration (three
48
weeks) of each treatment means that the trial carried out by Bliddal's group failed to
convincingly establish whether or not ginger might be useful in the treatment of
osteoarthritis.
In 2003, Wigler and co-workers published the results of small clinical trial of ginger in
osteoarthritis of the knee (Wigler et al., 2003). This was a randomised, double-blind,
placebo-controlled study of 6 months’ duration. Twenty-nine subjects were divided into
verum and placebo groups, which were crossed over after 12 weeks. The intervention was a
ginger extract produced by supercritical carbon dioxide extraction and formulated into an
enteric coated capsule containing 250 mg extract including 10 mg ‘gingerol’. The capsule
was designed to release 20% of its content under the acidic conditions of the stomach and the
remainder in the intestine. Subjects took one capsule (or identical placebo) four times daily
(equivalent to 40 mg ‘gingerol’ daily). Primary outcome measures were pain on movement
and handicap as assessed by subjects on a visual analogue scale based on the Western
Ontario and McMaster Osteoarthritis Index (WOMAC). Both groups showed a significant
decrease in both outcome measures after 12 weeks, but there was no significant difference
between the groups. After 24 weeks, however, the difference between the ginger and
placebo groups was highly significant for both outcomes in favour of the ginger intervention
(p>0.01).
Although the study conducted by Wigler and colleagues also did not include a wash-out
phase between treatments, the duration (12 weeks) of each treatment means that this was less
likely to invalidate the findings. In fact, the group that started on the active treatment
continued to improve for 2 weeks after being switched to placebo, suggesting the effects of
the ginger treatment continued after the treatment was withdrawn.
A third larger trial (n=247), rather misleadingly published in 2001 under the title ‘Effects of
a ginger extract on knee pain in patients with osteoarthritis’, is often cited as evidence for the
efficacy of ginger in osteoarthritis of the knee (Altman & Marcussen, 2001). This assertion
is not strictly valid, however, as the intervention employed in this trial was a patented extract
containing not only Zingiber officinale but also Alpinia galanga (galangal). This
randomised, double-blind, placebo-controlled, parallel-group, 6-week study found a
statistically significant effect of the extract in reducing symptoms of osteoarthritis of the
knee.
49
The reports of the only two randomised controlled trials of ginger extracts as the sole active
in the treatment of osteoarthritis (Bliddal et al., 2000b; Wigler et al., 2003) were scored
against the CONSORT (Consolidated Standards of Reporting Trials) (Moher et al., 2001)
and the elaborated CONSORT for herbal interventions (Gagnier et al., 2006) (see Appendix
B). Each item was given a score of 0, 0.5 or 1, depending on whether the information was
provided not at all or to an unsatisfactory extent (0), to some extent (0.5), or to a satisfactory
or mostly satisfactory extent (1) in the trial report. With total scores of 17.5 (Bliddal) and
17.0 (Wigler) out of a possible 27, the reporting of the two trials was of equal and reasonable
quality, although neither provided qualitative chemical information (such as an HPLC
chromatogram) about the intervention used, and the study by Bliddal and colleagues did not
use the binomial for ginger to unambiguously define the material taxonomically.
The results of the only two randomised controlled trials of ginger in osteoarthritis are
encouraging, if not conclusive. The results of the most recent trial suggest that treatment for
up to 24 weeks is required for the full benefits to manifest. A large, rigorous trial with a
chemically well-defined intervention is now warranted to more conclusively establish the
role (if any) of ginger in the treatment of osteoarthritis.
2.3.2 Other Zingiber species
Other Zingiber species included in this work are reviewed below.
2.3.2.1 Thai ginger (Zingiber montanum)
Zingiber montanum (J. König) Theilade is more widely cited in the literature under the
synonyms Z. cassumunar Roxb. and Z. purpureum Roscoe.
This species is used in the treatment of asthma in traditional Thai medicine and has been
shown to have an anti-histamine effect in asthmatic individuals (500 mg orally) (Piromrat et
al., 1986). A methanolic extract inhibited PGE2 production by in human promonocytic U937
cells (IC50 = 7.7 µg/mL) (Jiang et al., 2006b). The rhizome contains a number of
phenylbutanoid compounds (Han et al., 2004; Jitoe et al., 1993; Lu et al., 2005; Masuda &
Jitoe, 1995), some of which have been shown to have potent anti-inflammatory activity both
50
in vitro and in vivo through inhibition of COX and lipoxygenase (LOX) pathways
(Jeenapongsa et al., 2003; Panthong et al., 1997), including inhibition of COX-2 in
lipopolysaccharide (LPS)-stimulated RAW 264.7 cells (Han et al., 2005). One
phenylbutenoid dimer had anti-proliferative activity in several human cancer cell lines (Lee
et al., 2007). Also present are curcuminoids (cassumunin A and B, cassumunarin A, B and
C; Fig. 2-10) with potent anti-oxidant activity (Masuda & Jitoe, 1994; Nagano et al., 1997).
The cassumunarins were also shown to have anti-inflammatory activity in vivo (Masuda &
Jitoe, 1994). The sesquiterpene zerumbone has antifungal activity (Kishore & Dwivedi,
1992).
Extracts of Z. montanum have demonstrated in vitro antioxidant activity (Chirangini et al.,
2004; Habsah et al., 2000a; Jitoe et al., 1992), anti-allergic activity (Tewtrakul &
Subhadhirasakul, 2007) and anti-tumour promoter activity (Vimala et al., 1999). Extracts
also inhibited P-glycoprotein in human uterine sarcoma cells (Go et al., 2004) and
significantly inhibited CYP3A4 in human liver microsomes (Subehan et al., 2006). In
animals, extracts have shown anti-inflammatory and analgesic effects (Ozaki et al., 1991;
Pongprayoon et al., 1997).
H3CO
RH3CO HO
OCH3
O O
OCH3
OH
cassumunin A: R = Hcassumunin B: R = -OCH3
H3CO
OCH3
OCH3
OH
O O
OH
OCH3
cassumunarin A
Fig. 2-10. Curcuminoids from Zingiber montanum.
51
The essential oil, which primarily consists of monoterpenoids and phenylbutanoids (Taroeno
et al., 1991), was active against a wide range of Gram-positive and Gram-negative bacteria,
dermatophytes and yeasts (Pithayanukul et al., 2007).
2.3.2.2 Zingiber ottensii
Zingiber ottensii Valeton is another species native to South-east Asia. The rhizome is
reportedly used to treat lumbago and is an ingredient in a sedative lotion used in the
treatment of convulsions (Sirirugsa, 1999).
The rhizome contains a number of terpenoids and diarylheptanoids (Akiyama et al., 2006;
Sirat, 1994). Zerumbone is the major constituent of the essential oil (Sirat & Nordin, 1994),
which had moderate cytotoxicity in the brine shrimp assay (Thubthimthed et al., 2005).
Extracts of the rhizome have been found to possess antimicrobial (Azmi Muda et al., 2002;
Mohtar et al., 1998) and anti-oxidant (Habsah et al., 2000b) activities. No other information
about the pharmacological activity of this species was located.
2.3.2.3 Beehive ginger (Zingiber spectabile)
Zingiber spectabile Griff. is native to Thailand and the Malayan peninsula and is widely used
as an ornamental in tropical and subtropical areas. An infusion of the leaves has been
reported to have been used to treat infected eyelids (Sirirugsa, 1999).
The rhizome contains an essential oil reported to contain terpinen-4-ol (23.7%), labda-8
(17),12-diene-15,16-dial (24.3%), α-terpineol (13.1%), and β-pinene (10.3%) as major
constituents (Sirat & Leh, 2001). A methanolic extract inhibited PGE2 production in human
promonocytic U937 cells (IC50 = 1.2 µg/mL) (Jiang et al., 2006b), and a solvent extract
demonstrated antimicrobial activity (Ghosh et al., 2000). No other information pertaining to
the chemistry and pharmacology of this species was located.
52
2.4 Genus Curcuma L.
The genus Curcuma contains approximately 40 mostly tropical Asian species. Best known is
turmeric (C. longa L.), a cultigen of likely Indian origin, which is widely used as a spice and
as an orange and yellow dye (Mabberley, 1997). Several other species are also used for
culinary purposes including mango ginger (C. amada Roxb.), Bombay or Indian arrowroot
(C. angustifolia Roxb.) and zedoary (C. zedoaria (Christm.) Roscoe, syn. C. zerumbet)
(Mabberley, 1997).
In Australia, the genus is represented by a single native species, C. australasica J. D. Hook,
which is sometimes, rather misleadingly, called Cape York lily, and finds use as an
ornamental plant. Its natural habitat is shady rainforest margins in tropical parts of
Queensland and the Northern Territory, and it also occurs in New Guinea (Smith, 1987).
More than a dozen species of Curcuma have been used in traditional systems of medicine
(Johnson, 1999).
2.4.1 Curcuma longa
Turmeric (C. longa L., syn. C. domestica Val.) and its major active constituent curcumin
have been the subject of hundreds of scientific studies, the majority of which have been
published since 2000. The interest has focused on curcumin and its anti-oxidant, anti-
carcinogenic and anti-inflammatory actions.
2.4.1.1 Chemistry
The major constituents of interest in turmeric are the coloured diarylheptanoids known as
curcuminoids, which constitute around 5% of the dried rhizome. The major of these is the
unsaturated β-diketone curcumin (diferuloylmethane), which together with
desmethoxycurcumin and bisdesmethoxycurcumin make up 50-60% of the curcuminoids
present in the rhizome (Fig. 2-11). Dihydrocurcumin has also been reported (Evans, 2002;
WHO, 1999).
53
Turmeric also contains 5-6% volatile oil made up of mono- and sesquiterpenes including
zingiberene, curcumene, α- and β-turmerone (Evans, 2002; WHO, 1999).
O O
HO OH
H3CO OCH3
Curcumin
O O
HO OH
H3CO OCH3
H
bis-keto form(pH 3-7)
enolate form(pH >8)
O O
HO OH
OCH3
Demethoxycurcumin
O O
HO OHBisdemethoxycurcumin
Fig. 2-11. Structural diagrams of the major curcuminoids in turmeric rhizome: curcumin, demethoxycurcumin and bisdemethoxycurcumin. Curcumin exists in a pH-dependent equilibrium between its bis-keto and enolate forms. (After Higdon, 2007; Sharma et al., 2005).
2.4.1.2 Pharmacology
The biological and pharmacological activities of curcumin have been the subject of many
reviews. These have included general reviews (Bengmark, 2006; Maheshwari et al., 2006;
Sharma et al., 2005) and reviews focussing on anti-carcinogenic and chemopreventive
54
activities (Duvoix et al., 2005; Karunagaran et al., 2005; Leu & Maa, 2002; Lin, 2004;
Narayan, 2004; Thangapazham et al., 2006), mechanism of action (Joe et al., 2004), and the
potential role of curcumin in the treatment of Alzheimer’s disease (Ringman et al., 2005).
2.4.1.2.1 Pharmacokinetics of curcumin
In rodents, curcumin has low oral bioavailability and may undergo partial intestinal
metabolism. Following absorption, curcumin undergoes extensive first-pass metabolism and
excretion with the bile (Sharma et al., 2005). The major curcumin metabolites in
suspensions of both human and rat hepatocytes were hexahydrocurcumin and
hexahydrocurcuminol and these were less potent inhibitors of PGE2 production in human
colonic epithelial cells than curcumin itself (Ireson et al., 2001).
In humans, curcumin also has low oral bioavailability. In one study, a dose of up to 200 mg
failed to produce serum levels detectable at the level of detection (0.63 ng/mL) (Ruffin et al.,
2003). Administration of 2 g turmeric powder to fasting volunteers resulted in curcumin
plasma concentrations below 10 ng/mL, but this was increased 20-fold when piperine (the
pungent alkaloid from Piper species) was co-administered (Shoba et al., 1998). Curcumin
sulfate and curcumin glucuronide have been identified as metabolites in human urine
(Sharma et al., 2004) and in intestinal tissues of patients with colorectal cancer (Garcea et
al., 2005).
2.4.1.2.2 Pharmacodynamics of curcumin
2.4.1.2.2.1 Anti-oxidant activity
Numerous studies have shown curcumin to possess potent anti-oxidant activity both in vitro
and in vivo. Mechanistic studies have found that curcumin is a phenolic chain-breaking anti-
oxidant, which donates hydrogen atoms from its phenolic groups (Barclay et al., 2000;
Priyadarsini et al., 2003; Sun et al., 2002). The resulting phenoxyl radical can be repaired by
water-soluble anti-oxidants such as vitamin C (Jovanovic et al., 2001).
55
Curcumin can act directly as a scavenger of free radicals such as singlet oxygen (Das & Das,
2002), superoxide (Biswas et al., 2005; Mishra et al., 2004) and hydroxyl radical (Biswas et
al., 2005), but also exerts its anti-oxidant activity by enhancing endogenous defenses against
oxidative damage. Cultured alveolar epithelial cells (A549) showed increased glutathione
expression compared with controls (Biswas et al., 2005), and mice receiving dietary
curcumin (2%) had significantly increased activities of glutathione peroxidase, glutathione
reductase, glucose 6-phosphate dehydrogenase and catalase in the kidneys compared with
controls (Iqbal et al., 2003). This induction of detoxification enzymes may play a role in the
chemopreventive effects of curcumin.
The anti-oxidant activity of turmeric and curcumin has been demonstrated in a variety of in
vitro assay systems, including oxygen radical absorbance capacity (ORAC), radical
scavenging assays using 1,1-diphenyl-2-picryl hydrazyl (DPPH) and 2,2'-azobis-3-
ethylbenzthiazoline-6-sulfonic acid (ABTS), the ferric reducing antioxidant power (FRAP)
assay (Tilak et al., 2004), and the Trolox equivalent antioxidant capacity (TEAC) assay
(Betancor-Fernandez et al., 2003) .
Radical scavenging and anti-oxidant activities have been confirmed in vivo in a variety of
animal models. Some of these studies are briefly summarised here.
Curcumin was shown to protect rat brain homogenate against lipid peroxidation caused by
lead and cadmium (Daniel et al., 2004), and pre-treatment with curcumin significantly
reduced or abolished cadmium-induced hepatic oxidative damage in rodents (Eybl et al.,
2004; Eybl et al., 2006). Curcumin protected the rat forebrain against cerebral
ischaemia/reperfusion oxidative injury (Ghoneim et al., 2002).
A hydroethanolic turmeric extract fed to rabbits (1.66 mg/kg) receiving a high-fat,
atherogenic diet for 30 days significantly reduced the oxidative damage to erythrocyte and
liver microsome membranes (Mesa et al., 2003), and dietary curcumin (0.2%) reduced lipid
peroxidation and the depletion of intracelluluar anti-oxidants (thiols and glutathione) in
erythrocytes of rats fed a high-fat diet for 8 weeks (Kempaiah & Srinivasan, 2004). Mice fed
a turmeric extract (1% of diet) for just one week had reduced levels of erythrocyte
phospholipid hydroperoxides and liver triacylglycerol levels about half those of controls
(Asai et al., 1999).
56
Curcumin (200 mg/kg) significantly reduced lipid peroxidation in chronic streptozotocin-
diabetic rats, but had no significant effect on superoxide dismutase, catalase, or reduced
glutathione levels (Majithiya & Balaraman, 2005). The same curcumin dose administered
for 8 weeks to diabetic rats had a preventive effects on the cross-linking of collagen and the
formation of advanced glycation end products, a process linked to oxidative stress (Sajithlal
et al., 1998). Low dietary levels of curcumin (0.002% and 0.01%) delayed the progression
of diabetic cataract in the lens of diabetic rats (Kumar et al., 2005).
Rats subjected to oxidative stress caused by dietary ethanol or oxidised polyunsaturated fatty
acids had elevated liver marker enzymes and lipid peroxidative indices, but curcumin
abrogated this effect, suggesting it potentially could provide some protection against
alcoholic liver disease (Rukkumani et al., 2004). Lipid peroxidation was significantly
modulated and anti-oxidant status improved in rats suffering lung toxicity caused by
subcutaneous injection of nicotine (Kalpana & Menon, 2004).
Curcumin has also been shown to protect rodents against drug-induced oxidative organ
injury. Gentamycin-induced renal oxidative damage in rats (Farombi & Ekor, 2006),
nephro- and cardiotoxicity in rats caused by adriamycin (Venkatesan, 1998; Venkatesan et
al., 2000), and inflammatory and oxidative lung injury caused by bleomycin in rats
(Venkatesan et al., 1997) were all reduced by curcumin. Curcumin also reduced cisplatin-
induced clastogenesis in rats through it radical scavenging activity (Antunes et al., 2000), but
did not protect against cisplatin-induced lipid peroxidation and nephrotoxicity (Antunes et
al., 2001).
Several studies have found that curcumin can also promote the generation of reactive oxygen
species (ROS) under some circumstances (Ahsan et al., 1999; Atsumi et al., 2005a; Atsumi
et al., 2005b). One such study found that in human myeloid leukemia (HL-60) cells, low
curcumin concentration reduced ROS generation, while high concentrations promoted it
(Chen et al., 2005). A recent study using myelomonocytic U937 cells found that the anti-
oxidative action of curcumin was preceded by a time and dose dependent oxidative stimulus
and suggested that excessive curcumin concentrations may be harmful to cells (Strasser et
al., 2005).
57
In a placebo-controlled study involving 20 subjects with tropical pancreatitis, 500 mg of
curcumin (with 5 mg piperine to enhance the bioavailability of curcumin) reduced markers
of erythrocyte lipid peroxidation, but did not improve pain (Durgaprasad et al., 2005).
2.4.1.2.2.2 Anti-inflammatory activity
Numerous studies have indicated that the key to the anti-inflammatory activity of curcumin
is its ability to inhibit the activation of the transcription factor NFκB (Brennan & O'Neill,
1998; Funk et al., 2006; Jobin et al., 1999; Jung et al., 2006; Kang et al., 2004; Kumar et al.,
1998; Moon et al., 2005; Singh & Aggarwal, 1995; Strasser et al., 2005). NFκB plays a
critical role in the transcriptional regulation of pro-inflammatory gene expression in various
cells. Jobin and colleagues demonstrated that curcumin blocks a signal upstream of NFκB-
inducing kinase and IκB kinase, both of which are involved in the activation of NFκB itself
(Jobin et al., 1999).
Curcumin has also been found to inhibit the production of inflammatory cytokines involved
in NFκB activation. In human peripheral blood monocytes and alveolar macrophages
stimulated with phorbol ester or lipopolysaccharide (LPS), curcumin inhibited IL-8,
monocyte inflammatory protein-1 (MIP-1α), monocyte chemotactic protein-1 (MCP-1), IL-
1β, and TNF-α (Abe et al., 1999). At a concentration of 5 μM, curcumin inhibited LPS-
induced production of TNF and IL-1 by a human monocytic macrophage cell line (Chan,
1995), and in rats with adjuvant inflammation curcumin reduced levels of IL-1β and C-
reactive protein (Banerjee et al., 2003). Curcumin also inhibited IL-1β-mediated
intracellular adhesion molecule-1 (ICAM-1) and IL-8 gene expression in several epithelial
intestinal cell lines, resulting in the inhibition of NFκB activation (Jobin et al., 1999).
Crude organic turmeric extracts inhibited LPS-induced TNF-α and PGE2 production in HL-
60 cells with IC50 values of 15.2 μg/mL and 0.92 μg/mL, respectively (Lantz et al., 2005).
An important aspect of the inflammatory response is the migration of endothelial cells from
the vascular system into the tissues, following their recruitment of leukocytes. Treatment of
endothelial cells with TNF caused monocytes to adhere as a result of NFκB-dependent
expression of ICAM-1, vascular cell adhesion molecule-1 (VCAM-1), and endothelial
leukocyte adhesion molecule-1 (ELAM-1); pre-treatment with curcumin completely blocked
58
adhesion (Kumar et al., 1998). These findings suggest that inhibition of leukocyte
recruitment by endothelial cells (as a consequence of NFκB inhibition) may contribute to the
anti-inflammatory activity of curcumin.
The inhibition of NFκB activation also leads to subsequent reduction in the expression of
COX-2 and other inflammatory mediators whose genes are regulated by NFκB, and several
studies have shown curcumin and turmeric to reduce PGE2 levels (Funk et al., 2006; Hong et
al., 2004; Huang et al., 1991; Ireson et al., 2001; Lantz et al., 2005). Although one study
reported the direct inhibition of prostaglandin E synthase-1 and COX-2 (Moon et al., 2005),
other studies have suggested that PGE2 levels are reduced not through inhibition of COX
catalytic activity, but as a result of upstream inhibition of COX-2 expression (Funk et al.,
2006; Hong et al., 2004). Curcumin strongly inhibited COX-2 mRNA and protein
expression in UVB-irradiated keratinocytes; the mechanism appeared to involve the
suppression of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase
(JNK) activities (Cho et al., 2005).
In addition to inhibiting COX-2 expression, curcumin also affects arachidonic acid
metabolism by blocking the phosphorylation and thus the activation of PLA2, the enzyme
controlling the release of arachidonic acid from membranes, and by inhibiting the activity of
5-LOX (Hong et al., 2004).
Curcumin inhibits DNA binding of another transcription factor, activator protein-1 (AP-1),
which together with NFκB controls a key mediator of coagulation, endothelial tissue factor
(Bierhaus et al., 1997). Curcumin also inhibited DNA binding of AP-1 in microglial cells
(Kang et al., 2004), but did not reduce activation of AP-1 in mice suffering non-alcoholic
steatohepatitis, although NFκB activation was prevented (Leclercq et al., 2004).
Curcumin also inhibits the expression of nitric oxide (NO), which along with its derivatives
acts as an inflammatory mediator. The derivatives nitrite and peroxynitrite are capable of
damaging DNA and are therefore carcinogenic. The inhibition of nitric oxide production by
stimulated RAW 264.7 macrophages when cells were treated with curcumin (10 μM) was
shown to result from reduced expression of inducible nitric oxide synthase (iNOS) (Brouet &
Ohshima, 1995). Similar inhibition of nitric oxide production has been observed in mouse
peritoneal cells (Chan et al., 1995), while inhibition of both NO production and expression
of iNOS was demonstrated in rat primary microglial cells (Jung et al., 2006). In the latter
59
study, several signaling pathways (incl. NFκB, p38 MAPK and JNK) were found to be
involved in curcumin’s inhibition of NO, suggesting that the compound has multiple
molecular targets.
In an in vivo study with two oral treatments of curcumin (92 ng/g body weight), the
expression of hepatic iNOS RNA was reduced by 50-70% in mice injected with LPS (Chan
et al., 1998). Interestingly, inhibition was seen only when the curcumin was administered
after fasting, suggesting that concurrent food intake interfered with the bioavailability of
curcumin. This study is also important because is showed that curcumin can exert important
pharmacological activity when administered orally in very low doses, at least in mice.
In rodent brain microglial cells, curcumin reduced the inflammatory response (induction of
COX-2 and iNOS in response to stimuli) by inhibiting Janus kinase-STAT signalling (Kim et
al., 2003).
2.4.1.2.2.3 Anti-carcinogenic and anti-tumour activity
The chemopreventive and anti-tumour properties of curcumin (and turmeric) have been the
subject of numerous studies and, as previously mentioned, a number of reviews (Duvoix et
al., 2005; Karunagaran et al., 2005; Leu & Maa, 2002; Lin, 2004; Narayan, 2004;
Thangapazham et al., 2006).
Anti-tumour activity of curcumin has been demonstrated in many human cancer cells lines,
including colorectal (Goel et al., 2001; Lev-Ari et al., 2006), renal (Jung et al., 2005), lung
(Lee et al., 2005a; Shishodia et al., 2003), oral (Sharma et al., 2006), breast epithelial (Lee et
al., 2005b), pancreatic (Li et al., 2005) and bladder cells (Park et al., 2006). This anti-
tumour activity has been confirmed in several animal studies (Pal et al., 2005; Rao et al.,
1995; Volate et al., 2005).
Few human studies have been conducted in this area; those that have been carried out have
mostly been small and explorative in nature. Colorectal cancer patients who ingested 3.6 g
curcumin daily for 7 days had significantly fewer nucleotide adducts in malignant tissue
(p<0.05) but unchanged levels of COX-2 (Garcea et al., 2005). Both an ethanolic turmeric
extract and a curcumin-containing ointment was reported to have provided remarkable
symptomatic relief (smell, itch) in 62 patients with external cancers (Kuttan et al., 1987).
60
Numerous studies on curcumin suggest that it exerts its anti-cancer activities through a
variety of mechanisms at the initiation, promotion and progression stages of carcinogenesis
(Chauhan, 2002; Kawamori et al., 1999).
A major mechanism appears to be the induction of apoptosis (Anto et al., 2002; Atsumi et
al., 2005b; Divya & Pillai, 2006; Li et al., 2005). This occurs via the mitochondrial
pathway, where curcumin reduces mitochondrial membrane potential (Banjerdpongchai &
Wilairat, 2005; Karunagaran et al., 2005; Volate et al., 2005). Curcumin-induced apoptosis
in human renal cancer cells was found to be associated with upregulation of death receptor 5
expression (Jung et al., 2005), and in human colon cancer cell lines the induction of
apoptosis by curcumin was accompanied by down-regulation of COX-2 and reduced PGE2
synthesis (Lev-Ari et al., 2006).
Elevated levels of COX-2 are commonly seen in various types of malignant cells, and COX-
2 is therefore recognized as a molecular target in chemoprevention (Lee et al., 2005b).
COX-2 has also been implicated in tumour growth and invasiveness (Li et al., 2005). In
particular, COX-2 is selectively over-expressed in colon tumours and believed to play an
important role in colon carcinogenesis (Plummer et al., 1999). Many studies have shown
that curcumin down-regulates COX-2 (and subsequent PGE2 production) in cancer cells
(Atsumi et al., 2005b; Divya & Pillai, 2006; Goel et al., 2001; Lee et al., 2005a; Lee et al.,
2005b; Lev-Ari et al., 2006; Plummer et al., 1999; Sharma et al., 2006; Tunstall et al., 2006;
Yoysungnoen et al., 2006; Zhang et al., 1999). As outlined in the review of curcumin’s anti-
inflammatory activity above, it appears that the inhibition of the transcription factor NFκB is
the central mechanism by which curcumin inhibits the expression of COX-2 in cancer cells
(Divya & Pillai, 2006; Lee et al., 2005a; Lee et al., 2005b; Li et al., 2005; Plummer et al.,
1999; Sharma et al., 2006; Shishodia et al., 2003).
Curcumin has also demonstrated other anti-cancer mechanisms. These include protection of
DNA against genotoxic agents (Polasa et al., 2004), cytotoxicity (Chen et al., 1999; Divya &
Pillai, 2006; Su et al., 2006), causing cell cycle arrest (at G2/M) (Chauhan, 2002; Chen et
al., 1999; Park et al., 2006), inhibition of viral oncogenes (Divya & Pillai, 2006), inhibition
of tumour angiogenesis (Li et al., 2005; Yoysungnoen et al., 2006), production of reactive
oxygen species (Atsumi et al., 2005a; Banjerdpongchai & Wilairat, 2005), and the induction
of detoxification enzymes (glutathione S-transferases) (Piper et al., 1998; Singhal et al.,
1999).
61
2.4.1.2.2.4 Other pharmacological activities
Curcumin has demonstrated a range of other pharmacological activities. These include
immunological effects (Kang et al., 1999; Kobayashi et al., 1997; Ranjan et al., 1998; Youn
et al., 2006), anti-diabetic effects (Mahesh et al., 2005; Sharma et al., 2006), and
hepatoprotective action (Kang et al., 2002; Nanji et al., 2003; Park et al., 2000; Sreepriya &
Bali, 2006; Sugiyama et al., 2006; Xu et al., 2003).
Histamine release by leukemia cells was markedly decreased by both curcumin and
tetrahydrocurcumin (Suzuki et al., 2005). Curcumin is a potent inhibitor of Kv1.4 K+
channels (Liu et al., 2006) and inhibits arachidonic acid- and PAF-induced platelet
aggregation and platelet TxA2 synthesis (Shah et al., 1999). Curcumin reversed endothelial
dysfunction caused by homocysteine (Ramaswami et al., 2004) and had anti-atherogenic
effects in animals (Olszanecki et al., 2005; Ramirez-Tortosa et al., 1999). Curcumin had
anti-fibrotic action in rats with glomerular disease (Gaedeke et al., 2005) and inhibited or
reduced a range of inflammatory molecules and signals (incl. NFκB, AP-1, IL-6, TNF-α and
iNOS) in rat models of both ethanol and non-ethanol pancreatitis (Gukovsky et al., 2003).
Dietary curcumin (0.002%) protected rats against galactose-induced cataract formation
(Suryanarayana et al., 2003), and curcumin had potent anti-ulcer effects in a rat model by
preventing glutathione depletion, lipid peroxidation, and protein oxidation (Swarnakar et al.,
2005).
Curcumin improved wound healing following gamma-irradiation (Jagetia & Rajanikant,
2004) and also facilitated wound healing in diabetic rodents (Sidhu et al., 1999).
Curcumin has produced positive results in animal models of inflammatory bowel disease;
this effect is associated with the inhibition of NFκB (Jian et al., 2005; Jiang et al., 2006a;
Salh et al., 2003; Ukil et al., 2003; Zhang et al., 2006). A small pilot study with patients
with ulcerative proctitis or Crohn’s disease has yielded promising results (Holt et al., 2005).
Several in vitro and in vivo studies have identified curcumin as a promising agent for the
prevention and treatment of Alzheimer’s disease (Baum & Ng, 2004; Lim et al., 2001; Ono
et al., 2004; Yang et al., 2005). Curcumin has also been proposed as an agent worth
investigating for the treatment of cystic fibrosis (Davis & Drumm, 2004), but the interest in
pursuing this appears to have waned following further studies (Mall & Kunzelmann, 2005;
Song et al., 2004).
62
2.4.2 Curcuma parviflora
Curcuma parviflora Wallich is native to Thailand, where is has been used in the topical
treatment of cuts (Sirirugsa, 1999) and for the detoxification of scorpion bites (Takahashi et
al., 2003). It has been shown to contain a series of dimeric sesquiterpenes, parviflorenes A-
H with cytotoxic properties (Takahashi et al., 2003; Toume et al., 2004; Toume et al., 2005).
Parviflorene F (Fig. 2-12) induced apoptosis in HeLa cells (Ohtsuki et al., 2008).
OH
OH
OH
OH
parviflorene A
parviflorene F
OH
Fig. 2-12. Parviflorene A and F from Curcuma parviflora.
2.4.3 Other Curcuma species
No information regarding the chemistry or pharmacology was identified for the other two
Curcuma species included in this work, C. australasica J. D. Hook and C. cordata Wallich.
Several other species of Curcuma are used for medicinal purposes, particularly in Asia.
These include Javanese turmeric C. xanthorrhiza Roxb. (included in the British
Pharmacopoeia (Anonymous, 2007), C. kwangsiensis S. G. Lee & C. F. Liang, C.
63
phaeocaulis Valeton, C. wenyujin Y. H. Chen & C. Ling (all three are included in the
Pharmacopoeia of the People’s Republic of China (Anonymous, 2005), and C. zedoaria
Roscoe (included in the Japanese Pharmacopoeia (Anonymous, 2001)).
However, since these species were included in the present work, they are not reviewed here.
2.5 Genus Boesenbergia Kuntze
The genus Boesenbergia comprises about 30 species and is distributed from India to New
Guinea (Mabberley, 1997). There are no native or naturalised members of this genus in
Australia.
Fingerroot (Boesenbergia rotunda (L.) Mansf. (syn. Boesenbergia pandurata Schltr.,
Kaempferia pandurata Roxb., Gastrochilus panduratus (Roxb.) Ridl.) is used as a culinary
and medicinal herb in Southeast Asia. Fresh rhizomes are slightly pungent and have a
characteristic aroma. They are used for the treatment of colic, fungal infections, dry cough,
rheumatism and muscular pains, and for their reputed aphrodisiac properties (bin Jantan et
al., 2001; Murakami et al., 1993; Trakoontivakorn et al., 2001).
Boesenbergia rotunda was included in the present work.
2.5.1 Chemistry
A range of flavonoids have been identified in the rhizome of B. rotunda, notably chalcones
(including boesenbergin A and B [Fig. 2-13], panduratin A, cardamomin and
dihydromethoxychalcone) and flavanones (including pinostrobin, pinocembrin, alpinetin and
5-hydroxy-7-methoxyflavanone) (Murakami et al., 1993; Trakoontivakorn et al., 2001). A
chalcone derivative, 4-hydroxypanduratin A, has also been identified (Trakoontivakorn et
al., 2001).
Steam distilled rhizome oils from B. rotunda rhizomes from Malaysia, Indonesia and
Thailand showed considerable qualitative and quantitative differences in composition (bin
64
Jantan et al., 2001). The major volatile oil constituents identified were camphor (16.1-
32.1%), geraniol (16.2-26.0%), (E)-β-ocimene (19.0-23.7%) and 1,8-cineole (7.5-13.9%).
O
O
OMe
HO
boesenbergin A
O
OH
MeO
O
boesenbergin B
Fig. 2-13. Boesenbergin A and B from Boesenbergia rotunda.
2.5.2 Pharmacology
Fingerroot has attracted attention as a potential chemopreventive agent. Both chalcones and
flavanones from this species have demonstrated anti-mutagenic activity against mutagenic
heterocyclic amines in Salmonella typhimurium TA98 assays (Trakoontivakorn et al., 2001).
An ethanolic extract of rhizomes had potent inhibitory activity (similar to that of turmeric,
Curcuma longa) against human HT-29 colon cancer and MCF-7 breast cancer cell lines
(Kirana et al., 2003).
A methanolic extract of fresh B. rotunda rhizomes showed strong inhibition of tumour
promoter-induced Epstein-Barr virus activation (Murakami et al., 1993). This in vitro assay
is used to screen for agents with possible anti-tumour promoting properties. The chalcone
cardamonin was isolated and identified as having potent inhibitory activity in this assay (IC50
65
= 3.1 µM). Panduratin A has also been shown to induce apoptosis in human colon cancer
HT-29 cells (Yun et al., 2005).
These in vitro findings are at odds with a medium-term bioassay of hepatocarcinogenesis in
rats (Tiwawech et al., 2000). This study found that the administration of B. rotunda dried
rhizome, combined with a hepatocarcinogenic heterocyclic amine found in cooked meats,
resulted in an increase in the genesis of preneoplastic liver cell foci, suggestive of a possible
cancer promoting effect.
Panduratin A isolated from B. rotunda potently and dose-dependently inhibited nitric oxide
(IC50 = 0.175 µM) and PGE2 (IC50 = 0.0195 µM) production in RAW264.7 cells, suppressed
the expression of iNOS and COX-2 and reduced NFκB transcriptional activity without
significant cytotoxicity (Yun et al., 2003).
2.6 Genus Hedychium Koenig
The genus Hedychium comprises approximately 50 species distributed from the Himalayas
to New Guinea. The genus is also possibly native to Madagascar. Many species, including
H. coronarium Koenig, are cultivated as ornamental plants and often called ginger lilies
(Mabberley, 1997). There are no Hedychium species native to Australia, but two species, H.
coronarium and H. gardnerianum Sheppard ex Ker-Gawler, have become naturalised
(Smith, 1987).
Several species of Hedychium have been reported to have ethnobotanical uses including H.
coronarium, which has been employed for rhinitis, tonsillitis, halitosis, swelling and tumours
(Johnson, 1999). In Mauritius, the rhizomes of H. coronarium and H. flavescens are both
used in traditional medicine as topical applications for rheumatism (Gurib-Fakim et al.,
1997). Hedychium species are not used for culinary purposes (Omata et al., 1991).
The ground rhizome of H. spicatum Koenig is the major component of abir, a scented
powder used in Hindu ceremonies (Mabberley, 1997). This species has also been used
medicinally. The rhizome has been employed as a stomachic, carminative and stimulant,
whereas the root and stalk are reported to have analgesic and anti-inflammatory actions,
having been used in liver, urinary and respiratory disorders (Tandan et al., 1997).
66
Only Hedychium coronarium was included in the present work.
2.6.1 Chemistry
The steam-distilled volatile oil from the rhizome of H. gardnerianum was found to contain
67% monoterpenes and 31% sesquiterpenes, including 18% cadinane derivatives
(Weyerstahl et al., 1998). The major constituents of the oil were β-pinene (24.5%), α-pinene
(21.5%), p-cymene (6.2%), α-cadinol (4.9%), 10-epi-α-cadinol (2.4%), ar-curcumene
(2.3%) and linalool (2.1%).
Rhizome oil of H. coronarium from India was found to contain α- and β-pinene, limonene,
δ-3-carene, β-phellandrene, p-cymene, 1,8-cineole, β-caryophyllene, β-caryophyllene oxide,
linalool and elemol in varying quantities (Dixit et al., 1990). In addition to these
constituents, the following compounds also occur in the oil: myrcene, α-phellandrene,
borneol, methyl salicylate and eugenol (Omata et al., 1991).
Steam-distilled essential oil of fresh H. coronarium rhizome from Tahiti, to where the
species has been introduced from Indo-China, contained 1,8-cineole (40.2%), β-pinene
(24.8%), α-pinene (7.8%) and α-terpineol (5.4%) as major constituents (Lechat-Vahirua et
al., 1993).
In a study related to the present work steam-distilled oil from rhizomes of H. coronarium
grown in Mauritius was characterised by α-muurolol (17%), α-terpineol (16%) and 1,8-
cineole (11%) (Gurib-Fakim et al., 2002).
Seven labdane diterpenes have also been isolated from the rhizome of H. coronarium
(Nakatani et al., 1994).
2.6.2 Pharmacology
Anthelmintic properties in vitro (Dixit & Varma, 1975) and fungitoxicity against Aspergillus
flavus (Singh et al., 1984) have been reported for the essential oil of H. coronarium. The
ethanolic extract of the rhizome of this species does not appear to have been studied
67
previously, but in vivo analgesic and anti-inflammatory effects have been reported for an
ethanolic extract of H. spicatum (Tandan et al., 1997).
2.7 Genus Kaempferia L.
The genus Kaempferia contains approximately 50 species ranging from India to Southern
China and Malaysia. Among these, K. rotunda L. and in particular K. galanga L. are used
for medicinal and culinary purposes (Johnson, 1999; Mabberley, 1997).
The rhizome of lesser galangal (K. galanga) (also known as East Indian galangal, kacholam,
kentjur, chekur, and sometimes, misleadingly, simply ‘galangal’) is used medicinally and
cultivated in several countries, including India, China, Vietnam, Indonesia and the Sudan
(Luger et al., 1996; Vimala et al., 1999). It is considered to be warming, promoting vital
energy circulation, and is used in the treatment of complaints caused by cold, including
headache, dyspepsia, vomiting, diarrhoea, toothache, and abdominal and pectoral pain, while
a liniment applied topically through massage is used in rheumatism (Luger et al., 1996).
It should be noted that the binomial Kaempferia pandurata is a synonym for Boesenbergia
rotunda (refer to Section 2.4 above).
Kaempferia galanga and K. rotunda were included in the present work.
2.7.1 Chemistry
The rhizome of K. galanga contains a volatile oil with ethyl-p-methoxy-E-cinnamate as a
constituent (Luger et al., 1996). Other constituents reported from the rhizome of this species
are ethyl cinnamate, cinnamaldehyde, camphene, l-∆3-carene, borneol, p-methoxystyrene
and pentadecane (Noro et al., 1983).
2.7.2 Pharmacology
Several studies have investigated the biological activity of K. galanga. An ethanolic extract
exhibited moderate activity in a cell assay of anti-tumour promoter activity (inhibition of 12-
O-tetradecanoyl phorbol-13-acetate (TPA)-induced Epstein-Barr virus early antigen (EBV-
68
EA) in Raji cells) (Vimala et al., 1999). Another study showed moderate inhibition of
tumour promoter-induced Epstein-Barr virus activation (Murakami et al., 1993). This is a
similar in vitro assay, which like the first is used to screen for agents with possible anti-
tumour promoting properties.
A methanolic extract of K. galanga rhizomes showed in vitro cytotoxic activity against HeLa
cells, with an IC50 value of 80 µg/mL (Kosuge et al., 1985). Ethyl-p-methoxy-E-cinnamate
was isolated from this extract and identified as a cytotoxic compound, with an IC50 value of
35 µg/mL.
Ethyl-p-methoxy-trans-cinnamate isolated from K. galanga rhizomes has also been shown to
inhibit monoamine oxidase in vitro, with an IC50 value of 6.8 x 10−5 M (Noro et al., 1983).
2.8 Genus Scaphochlamys Baker
The genus Scaphochlamys includes 30 species, which occur in India, Southeast Asia and
New Guinea (Mabberley, 1997).
Scaphochlamys biloba and S. kunstleri were included in the present work.
No information about the ethnobotany, chemistry or pharmacology of this genus was located.
2.9 Genus Alpinia Roxburgh
The genus Alpinia comprises some 200 species distributed mainly in Asia and the Pacific
(Mabberley, 1997). Five species of Alpinia are indigenous to Australia: A. arctiflora (F.
Muell.) Benth., A. arundelliana (F. M. Bailey) Schumann, A. caerulea (R. Br.) Benth. (syn.
A. coerulea Benth.), A. hylandii R. M. Smith and A. modesta Schumann (Hnatiuk, 1990).
The species formerly known as A. racemigera F. Muell. is now named Pleuranthodium
racemigerum (F. Muell.) R. M. Smith (refer to Section 2.10). Both A. caerulea (commonly
known as ‘native ginger’ in eastern Australia) and A. arundelliana grow in rainforest and wet
sclerophyll forest in coastal New South Wales, north from the Central Coast, and in
69
Queensland, while the other three species are restricted to north-eastern Queensland
(Hnatiuk, 1990; Wilson & Hastings, 1993).
Many members of the genus are used as spices and/or as medicinal plants in indigenous
systems of traditional medicine around the world. This is particularly the case in China,
India and South-East Asia, but medicinal use of Alpinia species has also been reported from
Saudi Arabia and Brazil (Tewari et al., 1999). Galangal (greater galangal), A. galanga (L.)
Swartz is widely cultivated in Southeast Asia, where it is used as a spice and a traditional
medicine (Kubota et al., 1998; Someya et al., 2001).
The following Alpinia taxa were included in the present work: A. arctiflora (F. Muell.)
Benth., A. caerulea (R. Br.) Benth., A. calcarata Rosc., A. galanga (L.) Swartz, A.
luteocarpa Elmer, A. malaccensis Rosc., A. modesta Schumann, A. mutica Roxb., A.
purpurea ‘Eileen McDonald’, A. spectabile ‘Giant Orange’ and A. zerumbet (Pers.) B.L.
Burtt & R.M. Sm.
2.9.1 Chemistry
The chemistry of a range of Alpinia species has been reviewed by Tewari and co-workers
(Tewari et al., 1999), although it is evident that this review was not comprehensive.
Essential oil from Alpinia species, mostly extracted from rhizomes but also in some cases
from leaves, stems and seeds, contain terpenoids and phenylpropanoids typical of essential
oils. This includes monoterpenoids such as α- and β-pinene, geraniol, borneol, citronellol,
linalool, 1,8-cineole and camphor, sesquiterpenoids including eudesmol, β-
sesquiphellandrene and curcumene, and phenylpropanoids such as methyl eugenol (Tewari et
al., 1999). Four acetoxy-cineole isomers, viz. the (trans and cis)-2- and 3-acetoxy-1,8-
cineoles, have been identified as the major aroma constituents in the rhizome of A. galanga
(Kubota et al., 1998; Kubota et al., 1999). The major pungent compound in A. galanga is
1’-acetoxychavicol acetate (Fig. 2-14), which is at least ten times less pungent and produces
a more short-lived pungent sensation than capsaicin (Gautschi et al., 1999). Several pungent
diarylheptanoids have also been isolated from Alpinia species (Tewari et al., 1999).
70
O
O
O
O
Fig. 2-14. 1’-acetoxychavicol acetate from Alpinia galanga.
Two kava-pyrones, 5,6-dehydrokawain and dihydro-5,6-dehydrokawain, have been isolated
from the leaves of A. zerumbet (Pers) B. L. Burtt & R. M. Smith (syn. A. speciosa K.
Schum.), a species used as a diuretic and hypotensive agent in Brazil (Kuster et al., 1999).
Computerised searches of the databases Chemical Abstracts, MEDLINE, CAB Abstracts,
Current Contents Connect and Web of Science using the search terms ‘Alpinia caerulea’ and
‘Alpinia coerulea’ yielded no results relating the chemistry or pharmacology of this
Australian species.
2.9.2 Pharmacology
Several species of Alpinia are used in traditional medicine. Greater galangal (A. galanga) is
used for the treatment of stomach ache in China and Thailand (Yang & Eilerman, 1999), and
a number of species are used in traditional Ayurvedic medicine in India for inflammation,
cancer, urinary calculi, and as a digestive, liver and spleen tonic for the treatment of various
digestive complaints (Tewari et al., 1999). A. zerumbet is used medicinally in Brazil (Kuster
et al., 1999).
Alpinia galanga has demonstrated gastric anti-secretory and anti-ulcer effects in
experimental animals (Tewari et al., 1999) and is reported to possess anti-tumour (Itokawa et
al., 1987), anti-fungal (Haraguchi et al., 1996; Scheffer et al., 1981) and anti-oxidant activity
(Cheah & Abu, 2000). An extract of A. galanga in combination with a ginger (Zingiber
officinale) rhizome extract is marketed as an anti-inflammatory agent for use in arthritis
under the name Zinaxin (http://www.zinaxinrapid.com/). This product was the study
71
medication in one clinical trial in patients with osteoarthritis (Altman & Marcussen, 2001)
(refer to Section 2.2.1.5.1) (Altman & Marcussen, 2001).
A methanolic extract of A. galanga rhizome showed strong inhibitory effect in an in vitro
assay using inhibition of Epstein-Barr virus activation as a means of identifying potential
anti-tumour promoters (Murakami et al., 1994). This study identified 1’-acetoxychavicol
acetate, the major pungent principle in A. galanga, as the active principle with an IC50 value
of 1.5 µM.
Alpinia malaccensis and A. mutica had in vitro anti-bacterial and potent anti-oxidant activity
(Habsah et al., 2000b).
In Brazil, A. zerumbet is used as a diuretic and anti-hypertensive agent (Kuster et al., 1999).
The anti-hypertensive effect is likely due to the presence of several flavonoids with calcium
channel blocking activity. The kava pyrones present (see above) also have anti-platelet, anti-
convulsant, analgesic, sedative and anxiolytic actions. An aqueous extract of the leaves was
found to promote central nervous system excitation, followed by depression and
hypokinesia.
The major pungent principle of A. galanga, 1’-acetoxychavicol acetate, possesses
considerable biological activity, with anti-ulcer, anti-tumour and anti-microbial properties, as
well as inhibition of xanthine oxidase having been demonstrated (Kubota et al., 1998). Most
recently, the compound has been shown to inhibit the replication of human
immunodeficiency virus type 1 (Ye & Li, 2006).
Acute (24 h) and chronic (90 days) oral toxicity studies on the ethanolic extract of A.
galanga rhizome were carried out in mice (Qureshi et al., 1992). Acute extract dosages were
0.5, 1.0, and 3 g per kg body weight, while the chronic dosage was 100 mg/kg/day. No
significant mortality was observed compared with controls, but galangal treated mice
experienced a significant weight gain and an increased level of red blood cells. In male
mice, a highly significant increase in sperm count, sperm motility and weight of sexual
organs was recorded.
Diarylheptanoids possess potent anti-inflammatory properties. An early study found that
diarylheptanoids including yakuchinone A isolated from A. officinarum were dual inhibitors
of arachidonic acid metabolism, i.e. they inhibited both prostaglandin synthetase (COX) and
72
5-lipoxygenase (Kiuchi et al., 1992). The anti-inflammatory activity of diarylheptanoids
may in part result from their suppressive effect on the surface expression of inducible
adhesion molecules in endothelial cells and subsequent leukocyte adhesion (Yamazaki et al.,
2000), but other mechanisms may also be involved. Two diarylheptanoids isolated from A.
oxyphylla Miquel, yakuchinone A and B, were found to down-regulate the expression of
COX-2 and iNOS through suppression of NFκB in mouse skin treated with a tumour
promoter (Chun et al., 2002).
2.10 Genus Pleuranthodium (K. Schum.) R. M. Smith
The genus Pleuranthodium comprises some 23 species distributed in New Guinea, Australia
and the Pacific (Mabberley, 1997). In Australia, Pleuranthodium racemigerum (F. Muell.)
R. M. Smith (syn. Alpinia racemigera F. Muell., Psychanthus racemiger (F. Muell.) R. M.
Smith) occurs in the Wet Tropics region of Far North Queensland and was included in the
present work. No information about the chemistry or pharmacology of this species was
located in the literature.
2.11 Genus Etlingera Giseke
The genus Etlingera is made up of around 60 species distributed from India to New Guinea.
The genus is closely allied to, and sometimes included in, the genus Amomum (Mabberley,
1997).
One species is native to Australia, E. australasica R. M. Smith, which is confined to
rainforest in the north-eastern Cape York Peninsula in North Queensland (Smith, 1987).
Torch ginger (E. elatior (Jack) R. M. Smith) from Malesia is cultivated as an ornamental,
and its flowers are used as a condiment in cooking (Mabberley, 1997). The leaves of a
Tahitian species, E. cevuga Smith (syn. Amomum cevuga, Geanthus cevuga), are used as a
yellow dyeing material for dying vegetable fibres for traditional dresses (Lechat-Vahirua et
al., 1993).
73
The present work included Etlingera australasica R.M. Sm. and E. elatior ‘Burma Torch’.
No information about the chemistry and pharmacology of these taxa was identified, but the
essential oil of E. cevuga has been reported to contain methyl eugenol (47%) and (E)-methyl
isoeugenol (18%), as principal constituents (Lechat-Vahirua et al., 1993). No
pharmacological data pertaining to the genus Etlingera was located.
2.12 Genus Elettaria Maton
The genus Elettaria comprises seven species and is distributed from India to western
Malesia. Cardamom (E. cardamomum Maton), a native of India, produces aromatic seeds,
which are used in cooking and medicine (Mabberley, 1997). Today cardamom seeds are
obtained from the cultivated variety E. cardamomum var. minuscula, and the main producing
countries are Sri Lanka, India and Guatemala (Evans, 2002).
The medicinal use of cardamom seeds dates back several thousand years in the Indian system
of Ayurveda, and they were known to the Greeks in the 4th century BCE. In India,
cardamom is used for a variety of complaints including digestive problems, asthma,
bronchitis, kidney stones, anorexia and debility (Chevallier, 2001).
Cardamom fruit is included in the British Pharmacopoeia (Anonymous, 2007), and the seed
was approved by the German Commission E for the treatment of dyspepsia (Blumenthal,
1998).
2.12.1 Chemistry
Cardamom seeds yield 2.8-6.2% volatile oil with a high content of terpinyl acetate and 1,8-
cineole (Fig. 2-15) and smaller amounts of other monoterpenes including alcohols and esters
(Evans, 2002; Lawrence, 1998).
74
O
O
terpinyl acetate
O
1,8-cineole
Fig. 2-15. Terpinyl acetate and 1,8-cineole from Elettaria cardamomum.
No information on the chemical constituents of the underground parts of cardamom was
identified.
2.12.2 Pharmacology
Cardamom seed oil had anti-inflammatory activity against acute carrageenan-induced paw
oedema in the rat and analgesic activity in mice, producing 50% protection against writhing
induced by intraperitoneal administration of 0.02% β-benzoquinone (Al-Zuhair et al., 1996).
The same authors also reported anti-spasmodic activity in rabbit intestine in vitro resulting
from muscarinic receptor blockage. The seed oil administered intravenously (5-20 µL per
kg) caused dose-dependent decrease in arterial blood pressure in the rat, and large doses
relaxed spontaneously contracting rabbit jejunum in vitro (El Tahir et al., 1997).
The volatile oil of cardamom has been shown to enhance transdermal absorption of
indomethacin, as a result of its monoterpene content (Huang et al., 1993; YawBin et al.,
1999).
An aqueous cardamom seed extract (10% w/v) was shown to increase gastric acid secretion
almost three-fold in anaesthetised rats (Vasudevan et al., 2000).
Ethanolic extracts of combinations of cardamom leaves and root and also of roots and seeds
inhibited the growth of Staphylococcus aureus (Daswani & Bohra, 2000). No other
pharmacological information pertaining to root or rhizome preparations was located.
75
2.13 Genus Hornstedtia Retz.
The 24 species in the genus Hornstedtia occur in South-east Asia, Papua New Guinea and
Australia (Mabberley, 1997).
The species included in the present work, Hornstedtia scottiana (F. Muell.) Schum., is found
in New Guinea and Far North Queensland (Anonymous, 2008). It is reportedly edible (Dick,
1992), but no information about its chemical composition or biological activity was located.
A labdane diterpene, labda-8(17),12-diene-15,16-dial, has been reported from H. scyphifera
(Koenig) Steud. (Sirat et al., 1994), but no other information about the chemistry or
pharmacology of the genus was located.
2.14 Genus Renealmia L.f.
The genus Renealmia consists of some 60 species in tropical parts of the Americas and
Africa (Mabberley, 1997). Several species are reported to be used locally as medicinal
plants (Dos Santos & Sant'Ana, 2000; Lans et al., 2001; Otero et al., 2000; Zhou, 1997).
Diterpenoids (Boukouvalas et al., 2006; Sekiguchi, 2001; Yang, 1999), dihydrochalcones
(Gu, 2002), diarylheptanoids (Sekiguchi, 2002) and sesquiterpenes (Kaplan et al., 2000)
have been reported from the genus.
The Central American species Renealmia cernua (Sw. ex Roem. & Schult.) J.F. Macbr. was
included in the present work. No information about the chemistry or pharmacology of this
species was identified.
2.15 Genus Costus L.
The genus Costus is a tropical genus with 42 species and is placed in the family Costaceae
by some authorities (Mabberley, 1997). There is one endemic species in Australia, C.
potierae (Smith, 1987). Various species have been used in traditional medicine in Europe
and the Americas for a wide range of ailments (Johnson, 1999). C. speciosus (Koenig) Sm.
from Indomalesia is a commercial source of diosgenin, which occurs in the rhizome in a
76
concentration up to 2.6% on a dry weight basis (Gupta et al., 1981; Kaphai et al., 1977;
Mabberley, 1997).
It should be noted that the Asian drug ‘costus root’ is not derived from the genus Costus but
from Saussurea lappa (Asteraceae) (de Kraker et al., 2001; Moeslinger et al., 2000).
The following species of Costus were included in the present work: C. barbatus Suess., C.
leucanthus Maas, C. malortieanus H. Wendland, C. productus Gleason ex Maas, C.
pulverulentus C. Presl and C. tappenbeckianus J. Braun et K. Schum.
2.15.1 Chemistry
Diosgenin (Fig. 2-16) has been reported from C. malortieanus (Prasad & Ammal, 1983), and
other steroidal glycosides (including furostanol and spirostanol glycosides) have been
identified in the rhizomes of several other species (Agrawal et al., 1984; Inoue et al., 1995;
Pereira et al., 1999; Rui et al., 1997; Silva et al., 1998; Willuhn & Pretzsch, 1985).
O
HO
H
H
O
H
Fig. 2-16. Diosgenin from Costus malortieanus.
No further information was located pertaining to the chemistry of the species included in the
present work.
77
2.15.2 Pharmacology
No information about the pharmacology of any of the species included in the present work
was located, but C. spiralis, which is used in Brazilian folk medicine for urinary tract
complaints and calculi, reduced the growth of foreign body-induced calculi in the bladder of
rats (Viel et al., 1999), and C. lucanusianus, a traditional anti-abortion drug in the Ivory
Coast, produced complete inhibition of oxytocin-induced contractions in isolated rat uterus
(Foungbe et al., 1987).
2.16 Genus Tapeinochilos Miquel
The genus Tapeinochilos comprises approximately twelve species distributed in Indonesia,
New Guinea and Australia (Mabberley, 1997; Smith, 1987). The genus is closely related to
Costus, and is placed in the family Costaceae by some taxonomists (Mabberley, 1997). One
species, T. ananassae (Hassk.) K. Schum. (syn. T. queenslandiae K. Schum) occurring in
Queensland and New Guinea (Smith, 1987), was included in the present work.
No information about the chemistry or pharmacological activity of T. ananassae or any other
members of this genus was located.
2.17 Summary
The Zingiberaceae contains many species that have been used as medicinal plants or spices,
in particular in Southeast Asia. Extensive information about the chemistry and
pharmacology of these plants is available only for a few species, in particular ginger and
turmeric. For the majority of species, including many species traditionally used as
medicines, limited or no information is available, and this is also true for whole genera in
some cases.
Since the family clearly produces compounds with interesting pharmacological action, the
investigation of little known species should prove rewarding.
78
2.18 Aims and research questions
The overall aim of this work has been to contribute to the existing body of knowledge about
members of the Zingiberaceae as medicinal plants, in particular in terms of their potential
use as anti-inflammatory agents. The work comprised two distinct parts. The first part
focused on the phytochemistry of a number of ginger (Zingiber officinale) clones with the
aim of identifying one or more with unique chemistry and consequent particular therapeutic
prospects. The second part involved the screening of species from various genera in a
number of bioassays relevant to potential anti-inflammatory activity, with the aim of
increasing the understanding of established medicinal species and the potential identification
of species with novel therapeutic prospects.
2.18.1 Part 1: Phytochemical investigations of 17 ginger clones
That pharmacologically active constituents in medicinal plants can vary both qualitatively
and quantitatively is well established, and a range of genetic, environmental, ontogenic and
biologic factors can affect the production of secondary metabolites in plants (Evans, 2002;
Wijesekera, 1991).
Ginger is particularly interesting in this context, because the species is sterile and can only
propagate by vegetative means. This, combined with the fact that the plant has been widely
cultivated in tropical regions around the world for several hundreds of years, makes it
probable that genetically stable and potentially chemically distinct clones exist.
Access to 17 ginger clones (including 12 of tetraploid genotype) through the Queensland
Department of Primary Industries represented an excellent opportunity to investigate
phytochemical variability resulting from genotypic differences.
2.18.1.1 Research questions
A number of research questions arose from the literature review in relation to the
phytochemical variability of ginger clones:
79
1. Are there significant qualitative and/or quantitative phytochemical differences
between different clones of ginger that are genetically determined and independent of
environmental, ontogenic or biologic factors?
2. If such genetically determined phytochemical differences exist, would it be possible
to select one or more clones with characteristics that would make them particularly
suitable for pharmaceutical or other specific uses?
3. Does fresh ginger contain shogaols, or do they only form from gingerols post
harvest?
4. Does the essential oil composition vary significantly between clones and if so, how?
2.18.1.2 Specific aims
Based on the research questions above, a number of specific aims were formulated for this
part of the project. They were:
1. To assess ginger extracts, in a pilot study, for in vitro COX-1 inhibitory activity,
determine their content of gingerols and shogaols, and examine whether a correlation
between activity and levels of gingerols/shogaols exist.
2. To compare the 17 ginger clones in terms of their yield of pungent gingerols and
shogaols, both of which have demonstrated anti-inflammatory activity in vitro and
which are responsible for the pungency of ginger.
3. To identify one or more clones that yield particularly high amounts of these
compounds and therefore would be suitable for pharmaceutical use.
4. To determine whether fresh ginger contains shogaols.
5. To compare the 17 clones in terms of their essential oil composition.
6. To determine if all clones have high citral content (Australian ginger is said to
possess a particular ‘lemony’ aroma).
80
2.18.1.3 Research methods
The following methods were employed in order to meet the specific aims of this part of the
project:
1. Cultivation of ginger clones under uniform conditions.
2. Standardised extraction of ginger rhizomes.
3. Assay of COX-1 inhibition of in vitro.
4. Quantification of gingerols and shogaols by HPLC.
5. Extraction of essential oil from ginger rhizomes by steam distillation.
6. Analysis of essential oils by GC-MS.
2.18.2 Part 2: Screening Zingiberaceae for pharmacological activity
The second part of this work involved the screening of a number of Zingiberaceae species in
a range of bioassays. This work adds to the existing knowledge base regarding the
pharmacology of several traditional medicinal plants and provides novel information about
species that have not previously been investigated in this way.
Also, the work was designed to test the hypothesis that the combination of ethnobotanical
and taxonomic information is a productive strategy to identify previously unrecognised plant
species with therapeutic potential, either in the form of phytomedicines or by providing lead
compounds for subsequent drug development.
As evidenced by the literature review above, the Zingiberaceae contains many species that
have been used as traditional medicines. Among the most well known medicinal species
from the family are ginger (Zingiber officinale), turmeric (Curcuma longa), galangal
(Alpinia galanga) and fingerroot (Boesenbergia rotunda). Thus ethnobotanical information
has been used to identify a plant family and specific genera with significant pharmacological
and therapeutic activity. Subsequently, taxonomic information (based on phylogenetic
relationships) has been applied to identify species related to established medicinal species at
81
the generic or family level. This approach is based on the well established concept that
phylogenetically related plant species usually display a significant degree of similarity in the
kinds of secondary metabolites they produce (Evans, 2002).
2.18.2.1 Research questions
A number of research questions arose in relation to the second part of this work:
1. Can the mode of action of well known medicinal species from the Zingiberaceae be
further elucidated through bioassays?
2. Can Zingiberaceae species with previously unrecognised pharmacological activity be
identified through screening in bioassays?
3. Can species with previously unrecognised pharmacological activity be identified
from genera with recognised medicinal species (such as Zingiber, Curcuma and
Alpinia)?
4. If species with previously unrecognised pharmacological activity are identified, do
they contain active compounds that are similar to active compounds in related
medicinal species?
2.18.2.2 Specific aims
In accordance with the research questions, the specific aims of this part of the work were:
1. To screen a number of Zingiberaceae species in a range of bioassays (inhibition of
PGE2, antioxidant activity, inhibition of nitric oxide, stimulation of natural killer cell
activity, cytotoxicity).
2. Through bioassay work, to contribute to the understanding of the mode of action of
established medicinal plants.
3. To identify previously unrecognised species with pharmacological activity that
makes them candidates for novel phytomedicines or drug leads.
82
4. To isolate and determine the structure of major active compounds in previously
unrecognised medicinal species.
2.18.2.3 Research methods
To address the specific aims of this part of the project, the following methods were
employed:
1. Inhibition of PGE2 in 3T3 murine fibroblasts.
2. Oxygen radical absorption capacity (ORAC) assay.
3. Nitric oxide production by RAW 264 murine leukemic monocytes.
4. Cytotoxic activity of human natural killer (NK) cells against K562 human myeloid
leukemic cells by flow cytometry.
5. Cytotoxicity in murine and human cell lines.
6. Chemical profiling of extracts by liquid chromatography – mass spectrometry (LC-
MS).
7. Bioactivity-guided fractionation by semi-preparative high-performance liquid
chromatography (HPLC).
8. Structural elucidation by nuclear magnetic resonance (NMR) spectroscopy.
83
3. INHIBITION OF CYCLOOXYGENASE-1 BY GINGER RHIZOME EXTRACTS
3.1 Introduction
The aims of this study were: (i) to determine the most suitable method for extraction of
ginger rhizome; (ii) to determine the most suitable extraction medium for ginger rhizome;
(iii) to establish a protocol for the quantitative determination of major pungent ginger
compounds by high-performance liquid chromatography (HPLC); and (iv) to establish a
protocol for the determination of the biological activity of ginger rhizome extracts in a
cyclooxygenase-1 (COX-1) bioassay. The determination of the most suitable extraction
method and medium was guided by the biological activity observed in the bioassay.
3.2 Materials and Methods
3.2.1 Plant material
A single rhizome of Zingiber officinale 'Queensland', grown outdoors under sprinklers at
Southern Cross University over the previous 7 months, provided the raw material for all
ginger extracts used in this study. The plant was grown in a black 300 mm plastic pot in a
sand-peat 1:1 growth medium containing the following nutrients (g per m3): dolomite 3600,
ammonium sulfate 544, superphosphate 184, potassium sulfate 248, magnesium sulfate 472,
copper sulfate 7.2, zinc sulfate 9.6, iron sulfate 7.2 (Smith & Hamill, 1996).
The freshly harvested rhizome was peeled and chopped finely with a knife. For hot
extractions, 10 g of chopped rhizome was added to 150 ml of extraction medium (water or
ethanol), ground with a tissue tearer (Heidolph Diax 600) and extracted in a Soxhlet
apparatus for 5 hours. Extracts were reduced to near dryness under vacuum on a rotary
evaporator, then reconstituted in 100 ml fresh extraction medium. In Experiment 1, residues
from ethanolic extractions were dissolved in 75% ethanol and 25% water, whereas residues
from aqueous extractions were dissolved in 75% water and 25% ethanol. Laboratory grade
potable alcohol (96%) was used in the preparation of ginger extracts.
84
For extractions at ambient temperature (approximately 23°C), 1 g of chopped rhizome was
added to 10 ml of extraction medium, ground with a tissue tearer (Heidolph Diax 600), then
placed in a sonicator and macerated at ambient temperature for 20 minutes. The macerate
was then centrifuged (1800 RCF for 5 minutes), the supernatant removed, and the pellet
extracted again with 10 ml fresh extraction medium following the same procedure. The
supernatant fluids from both extractions were combined, reduced to near dryness under
vacuum on a rotary evaporator, then reconstituted in 10 ml fresh extraction medium. In
Experiment 1, residues from ethanolic extractions were dissolved in 75% ethanol and 25%
water, whereas residues from aqueous extractions were dissolved in 75% water and 25%
ethanol.
A preliminary experiment showed that two subsequent extractions with ethanol yielded 96%
of the substances extracted by 4 subsequent extractions (data not shown).
3.2.2 HPLC analysis
Reversed-phase HPLC analysis of the plant extracts was performed on a Hewlett Packard
1100 HPLC fitted with a HP LiChrospher 100 RP-18e (5µm) column. Mobile phase was
pumped at 1mL/min (90% H2O/10% acetonitrile to 10% H2O/90% acetonitrile over 20 min).
Injection volume was 10 µL. Data were collected using a UV/visible diode array detector
scanning from 195 to 330 nm.
Identification of [6]-gingerol, [8]-gingerol, [10]-gingerol, [6]-shogaol and [8]-shogaol was
based on comparisons of retention time and UV-spectra with pure synthesised standards
obtained from the Department of Pharmacy at the University of Sydney. Absolute ethanol
(99%) analytical grade was used in the preparation of standards. Quantification of the
compounds mentioned above was based on standard curves prepared with pure standards and
eugenol as an external standard. Duplicate injections were analysed of all samples.
3.2.3 Cyclooxygenase-1 assay
The COX-1 assay was carried out using 14C-labelled arachidonic acid as a substrate. Ginger
extracts were incubated with labeled arachidonic acid and cyclooxygenase-1 for 15 min at
85
37° C, after which the reaction was stopped. Arachidonic acid metabolites were extracted
and separated by thin layer chromatography and quantified using a liquid scintillation
counter. Samples were assayed in triplicates. Details of the assay are given below.
3.2.3.1 Enzyme reaction
40 µl Cox-buffer (0.1 M TRIS at pH 8.0 containing 0.5mM sodium EDTA and 0.5 mM
phenol), 10 µl co-factor-1 (aqueous solution of 10 µM hematin [Sigma] and 0.02 M NaOH)
and 10 µl co-factor-2 (0.1 mM TRIS buffer containing 10 mM reduced glutathione [Sigma]
and 10 mM (–)-epinephrine-(+)-bitartrate [Sigma] adjusted to pH 8.0) was added to
Eppendorf tubes and incubated for 5 minutes at 37°C. 10 µl dilute ovine cyclooxygenase-1
solution (Cayman Chemicals; 20 µl in 190 µl Cox-buffer) was added to each tube followed
by 10 µl sample or control solvent. 40 µl 14C-arachidonic acid (Amersham, Australia) was
diluted with 360 µl Cox-buffer and 20 µl of this solution added to each tube. Tubes were
vortexed gently after each addition of material. Tubes were incubated for 15 minutes at 37°C
with gentle shaking after which the reaction was stopped by the addition of 40 µl formic acid
solution to each tube.
3.2.3.2 Extraction of arachidonic acid and metabolites
After the enzymatic reaction was stopped, 200 µl chloroform (CHCl3) was added to each
tube, which was then vortexed at high speed for approximately 10 seconds. The chloroform
(bottom) layer was then transferred to a clean Eppendorf tube using a micropipette, and the
chloroform evaporated under a nitrogen gas stream.
3.2.3.3 Separation of arachidonic acid and metabolites
This separation was carried out by means of thin-layer chromatography (TLC). Two mobile
phases were prepared as follows: Mobile Phase-1 (hexane 70 ml, diethyl ether 30 ml, glacial
acetic acid 1 ml) and Mobile Phase-2 (ethyl acetate 40 ml, methanol 10 ml, distilled water 25
86
ml, combined in a separation funnel). Extracted substances were reconstituted in 20 µl
CHCl3 and applied to a 200 x 200 mm TLC plate (0.2mm Silicon F254; Merck, Germany).
Two plates were used at a time. Standards (arachidonic acid 2 µl and prostaglandin E2/D2 10
µl [Sigma]) were applied to one lane on each plate. After the application of samples and
standards, the plates were allowed to dry, then run in a TLC tank (CAMAG) containing
Mobile Phase-1 until the solvent front reached a groove cut in the gel at 10 cm. Plates were
removed from the tank, allowed to dry for 15 minutes, then inserted into another TLC tank
containing Mobile Phase-2 and run until the solvent front reached a pencil mark at 3 cm.
Plates were again removed from the tank, allowed to dry for 15 minutes and then placed in
an iodine chamber for approximately 45 minutes. After this development, the arachidonic
acid standard (Rf 65-70) and the prostaglandin standard (just below the 3 cm line) were
marked with a pencil on both plates. Two parallel lines, 10-13 mm apart and equidistant
from the marked prostaglandin standard, were drawn horizontally across the plate and the
number of each lane written between the lines. The plate was then cut up along the parallel
lines and the grooves separating the lanes, resulting in 9 pieces of silica gel containing the
prostaglandin metabolites of arachidonic acid from each plate. The silica gel from each
numbered piece was then scraped off the aluminium plate and transferred to a scintillation
tube to which 200 µl distilled water and 200 µl Soluene-350 (Packard, Australia) was added.
After thorough vortexing, tubes were left to stand for 30 minutes, after which 2 ml Insta-Gel
(Packard, Australia) and 2 ml Hionic-Fluor (Packard, Australia) was added to each tube.
Tubes were vortexed for 10 seconds, then placed in a liquid scintillation counter (Minaxiβ
Tri-carb® 4000; United Technologies Packard) running Program 4. Results were recorded as
counts per minute (CPMA/K).
3.2.4 Statistical analysis
Data were analysed using Excel 97-SR-1 (Microsoft Corporation, Redmond, WA). Statistics
calculated were Student's 2-tailed t-test and the correlation coefficient.
87
3.3 Results and Discussion
3.3.1 Experiment 1
This experiment aimed to determine the most efficient extraction method for gingerols. Hot
Soxhlet extraction was compared with maceration/sonication at room temperature (23°C)
using two different solvents, water and ethanol. The biological activity in the
cyclooxygenase-1 assay and the content of assayed pungent compounds of these extracts are
shown in Table 3-1.
Table 3-1. Effect of extraction method: cyclooxygenase-1 inhibitory activity and content of major pungent compounds of ginger extracts.
All extracts were prepared from fresh rhizome; the rhizome:solvent ratio was 1:4 (w/v). a mean±s.d of 6 injections; b mean±s.d. of 2 injections, c mean±s.d of 4 samples; d mean±s.d of 3 samples; e mean±s.d. of 2 samples; f 75% ethanol-25% water; g 75% water-25% ethanol. CPMA/K: counts per minute. * Two-tailed t-test for COX-inhibitory effect, sample v control.
Cold EtOH Hot EtOH EtOH Controlf
Cold Water Hot Water Water Controlg
6-gingerol (mg/mL)
0.2922 ±0.0005a
0.2841 ±0.0005b
0.1913 ±0.012b
0.2398 ±0.0001b
8-gingerol (mg/mL)
0.0842 ±0.0005a
0.0829 ±0.0006b
0.0491 ±0.0013b
0.0356 ±0.0001b
10-gingerol (mg/mL)
0.0868 ±0.0036a
0.0810 ±0.0003b
0.0402 ±0.0016b
0b
6-shogaol (mg/mL)
0a 0.0282 ±0.0000b
0b 0.0476 ±0.0001b
CPMA/K 12791 ±2199c
12225 ±942c
58496 ±9886c
51878 ±2595d
41476 ±1832d
78417 ±4502e
Inhibition of Cox-1 (%)
78.13±17.19 79.10±7.71 0±16.90 33.84±5.00 47.11±4.42 0±5.74
p-value* 0.002 0.002 0.038 0.036
All extracts demonstrated statistically significant inhibition of cyclooxygenase-1 in the
bioassay (two-tailed t-test; Table 3-1). The inhibitory effect ranged from approximately 34%
for the cold aqueous extract to almost 80% for both ethanolic extracts, compared with
controls. The data show that the choice of solvent was a stronger determinant of biological
88
activity than was the extraction method. The cold ethanolic extract showed significantly
more potent inhibitory activity than did the cold aqueous extract (p= 0.00003), while the hot
Soxhlet ethanolic extract was also significantly more potent than the hot Soxhlet aqueous
extract (p= 0.00022). Overall, the ethanolic extracts were significantly more potent in terms
of cyclooxygenase-1 inhibition than were the aqueous extracts (p= 0.00002).
It is interesting to note that while the extraction method appeared to have little effect on the
inhibitory activity when ethanol was used as the solvent, the aqueous hot Soxhlet extract was
significantly more active than the cold aqueous extract (47% vs 34% inhibition, p= 0.00653).
The [6]-gingerol content of the extracts showed a very high correlation to the inhibitory
activity in the cyclooxygenase-1 assay (r= 0.976). The [8]-gingerol content also showed
considerable correlation to the inhibitory activity (r= 0.891).
The amount of [10]-gingerol in the ethanolic extracts was approximately twice the amount
measured in the cold aqueous extract, while no [10]-gingerol was identified in the hot
Soxhlet extract. Being less polar than [6]- and [8]-gingerol, smaller amounts of [10]-gingerol
would be expected to be extracted in an aqueous solvent. It is possible that what small
amount of [10]-gingerol may have been extracted was degraded by the heat in the hot
Soxhlet extraction process.
It is noteworthy that [6]-shogaol was identified only in the hot Soxhlet extracts. This is in
agreement with the notion that [6]-shogaol is a degradation product of [6]-gingerol that
forms when [6]-gingerol is exposed to excessive heat (Connell & Sutherland, 1969; He et al.,
1998; Zhang et al., 1994). The present data also suggest that 6-shogaol is in fact absent from
fresh ginger rhizome.
Based on the findings of this experiment, it was decided to employ maceration with
sonication at ambient temperature in subsequent experiments, since hot Soxhlet extraction is
considerably more time consuming and was not found to provide additional extraction
efficiency.
89
3.3.2 Experiment 2
This experiment aimed to determine what extraction medium would yield the most active
extract in terms of inhibitory activity in the cyclooxygenase-1 bioassay. Water and ethanol
were identified as potential solvents because of their widespread use and acceptance as
solvents for herbal medicines. Five different extraction media were compared: ethanol
(96%), 75% ethanol-25% water, 50% ethanol-50% water, 25% ethanol-75% water, and
water (100%). All extractions were carried out as macerations with sonication at room
temperature (23°C) as described earlier.
The biological activity in the cyclooxygenase-1 assay and the content of assayed pungent
compounds of these extracts is shown in Table 3-2.
Table 3-2. Effect of extraction solvent: cyclooxygenase-1 inhibitory activity and content of major pungent compounds of ginger extracts.
All extracts were prepared from fresh rhizome; the rhizome:solvent ratio was 1:4 (w/v). a mean±s.d of 2 injections; b mean±s.d of 3 samples; c mean±s.d of 2 samples; d control values calculated on the basis of values for ethanol and water; CPMA/K: counts per minute. * Two-tailed t-test for COX-inhibitory effect, sample v control.
EtOH (96%) 75%EtOH+
25%Water 50%EtOH+ 50%Water
25%EtOH+ 75%Water
100% Water mm
6-gingerola (mg/mL)
0.3701±0.0005 0.3211±0.0028 0.3003±0.0008 0.2744±0.0016 0.1772±0.0007
8-gingerola (mg/mL)
0.0648±0.0008 0.0577±0.0006 0.0482±0.0008 0.0440±0.0010 0.0155±0.0002
10-gingerola (mg/mL)
0.1076±0.0009 0.0941±0.0003 0.0754±0.0005 0.0570±0.0001 0.0136±0.0002
CPMA/Kb 3396±390 6609±993 12507±2270 21353±5213 23030±5348
Control value 41197±6165b 40303d 39410d 38516d 37622±1951c
Inhibition (%) 91.76±11.49 83.60±15.02 68.26±18.16 44.56±24.41 38.78±23.22
p-value* 0.009 0.009 0.008 0.014 0.029
All extracts demonstrated statistically significant inhibition of COX-1 in the bioassay (two-
tailed t-test; Table 3-2). The ethanol content of the solvent was highly correlated to
inhibitory activity (r = 0.983) as well as [6]-, [8]- and [10]-gingerol content (r= 0.956, 0.939
90
and 0.971, respectively). This clearly shows that pure ethanol is a superior solvent to hydro-
ethanolic mixtures with respect to COX-1 inhibitory activity of the resulting extracts, as well
as in terms of gingerol content of these extracts.
Both [6]-, [8]- and [10]-gingerol content of the extracts showed high correlation to inhibitory
activity in the bioassay (r = 0.904, 0.889 and 0.941, respectively). It is not possible from this
experiment to determine to what extent each of these compounds contributes to the
inhibitory activity. Kiuchi and colleagues found the IC50 values of [6]-, [8]- and [10]-
gingerol to be 4.6, 5.0 and 2.5µM, respectively, against prostaglandin synthase
(cyclooxygenase), suggesting that [10]-gingerol may be the most potent of the three
gingerols (Kiuchi et al., 1992). If this is the case, [10]-gingerol may contribute significantly
to the inhibitory activity of the extracts, despite its relative low concentration compared with
the major gingerol, [6]-gingerol.
The inhibitory activity of more than 90% recorded for the ethanolic extract was very
promising and encouraging for future studies.
The gingerol content of the extracts is shown in Fig. 3-1. [6]-Shogaol was not detected in any
of the extracts, a further confirmation that this compound does not occur in fresh ginger (see
above).
Fig. 3-1. Gingerol content (mg/mL extract) of ginger extracts.
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In Table 3-3, the ratios of [6]-gingerol : [8]-gingerol : [10]-gingerol in the extracts are given
and compared to values from the literature.
Table 3-3. Gingerol ratios in ginger extracts compared with values from the literature.
a (Zhang et al., 1994); b (Chen et al., 1986b); c (Connell & Sutherland, 1969)
[6]-gingerol:[8]-gingerol:[10]-gingerol
Ethanol (96%) 5.7 : 1 : 1.7
75% Ethanol + 25% Water 5.6 : 1 : 1.6
50% Ethanol + 50% Water 6.2 : 1 : 1.6
25% Ethanol + 75% Water 6.2 : 1 : 1.3
100% Water 11.4 : 1 : 0.9
Fresh ginger, Hawaiia 7 : 1 : 2
Freeze-dried ginger, Taiwanb 7 : 1 : 1.4
Ginger oleoresinc 4.3 : 1 : 2.4
From Table 3-3 it can be seen that the ratio between the three gingerols is relatively stable
when the extraction medium contains at least 25% ethanol. In pure water, however, the ratio
is shifted significantly in favor of the most polar of the compounds, [6]-gingerol.
The results obtained in this experiment clearly show that of the extraction media tested,
ethanol (96%) was the most effective. The ethanol extract showed the greatest inhibitory
activity in the COX-1 assay and had the highest concentration of the putative active
compounds.
In conclusion, this study found that fresh ginger rhizome extracted by maceration with
sonication at room temperature, using ethanol (96%) as the extraction medium, resulted in
extracts with a high concentration of pungent gingerols and potent inhibitory activity in the
cyclooxygenase-1 bioassay.
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4. GINGEROL CONTENT OF SEVENTEEN GINGER (ZINGIBER OFFICINALE) CLONES
4.1 Introduction
As outlined in Chapter 2, the main pungent compounds in fresh ginger are a series of
homologous phenolic ketones known as gingerols. The major gingerol is [6]-gingerol, while
[8]- and [10]-gingerol occur in smaller quantities. The gingerols are thermally unstable and
are converted under high temperature to [6]-, [8]- and [10]-shogaol (after shoga, the
Japanese word for ginger) (He et al., 1998). Shogaols, which are more pungent than
gingerols, are the major pungent compounds in dried ginger rhizome. Both gingerols and
shogoals have pharmacological activity including inhibiting COX in vitro (refer to Chapter
2).
In Australia, the most widely grown ginger cultivar is ‘Queensland’, and it is estimated that
40% of the world’s confectionary ginger products are prepared from this cultivar (Smith et
al., 2004b). The origin of this cultivar remains uncertain. Various authors have suggested
that it arrived in Australia from the Cochin coast of India (Connell, 1986), from Fiji
(Leverington, 1975), or from China in the early 1900s (Miles, 1980).
This chapter reports on the analysis of 17 ginger clones grown in Eastern Australia,
including commercial cultivars and 12 experimental tetraploid clones derived from
‘Queensland’, and the quantification by HPLC of the major pungent phenolic compounds,
viz. gingerols and shogaols. The objectives of this study were to explore the variability of
Australian ginger clones in terms of their content of pungent phenolic compounds with a
view to identify one or more high-yielding clones as candidates for commercial cultivation
for flavour or pharmaceutical use.
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4.2 Materials and Methods
4.2.1 Plant materials
Seventeen clones of ginger (Table 4-1) were obtained from the Queensland Department of
Primary Industries & Fisheries, Maroochy Research Station at Nambour, Queensland. They
included two selections of the cultivar ‘Queensland’, which is grown commercially in
Queensland, the cultivars ‘Jamaican’, ‘Brazilian’ and ‘Canton’, which were introduced to
Queensland between 1970 and 1972 for cultivar evaluation studies, and twelve experimental
clones developed at the Maroochy Research Station at Nambour, including the newly
released cultivar ‘Buderim Gold’ (Smith & Hamill, 2002). The experimental clones were
obtained after in vitro colchicine treatment of shoots of diploid (2n=22) ‘Queensland’ parent
material provided by J. Roscoe. They were confirmed as solid tetraploids except for one
(Z30), which proved to be a periclinal chimera with both diploid and tetraploid tissues
(Smith et al., 2004b).
94
Table 4-1. Ginger clones studied, their genotype and origin.
Ploidy: *: confirmed as solid tetraploids by flow cytometry; $: chimera with both diploid and tetraploid tissue sectors; ^: presumed to be tetraploid from stomatal measurements; #: unknown but presumed to be diploid. BGL = Buderim Ginger Ltd.
ID Genotype Cultivar name Origin
Z22 Tetraploid^ (Unnamed) Derived from ‘Queensland’ (Selection 1) by colchicine treatment
Z23 Tetraploid^ (Unnamed)
Z24 Tetraploid* (Unnamed)
Z25 Tetraploid^ (Unnamed)
Z26 Tetraploid* ‘Buderim Gold’
Z27 Tetraploid* (Unnamed)
Z28 Tetraploid^ (Unnamed)
Z29 Tetraploid^ (Unnamed)
Z30 Tetraploid$ (Unnamed)
Z31 Tetraploid* (Unnamed)
Z32 Tetraploid^ (Unnamed)
Z33 Tetraploid* (Unnamed)
Z44 Diploid ‘Queensland’ (Selection 1)
Selected by J. Roscoe, BGL
Z45 Diploid ‘Queensland’ (Selection 2)
Selected by L. Palmer, BGL
Z46 Diploid ‘Jamaican’ Imported from Jamaica
Z47 Diploid# ‘Brazilian’ Imported from Brazil
Z58 Diploid ‘Canton’ Imported from China
The clones were grown from rhizome stock in raised, outdoor beds under uniform, irrigated
conditions at Southern Cross University, Lismore, New South Wales (28° 49’ S, 153° 18’ E)
for approximately eight months.
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4.2.2 Sample preparation
Fresh rhizomes were washed and a cylindrical sample was taken from the thickest part of the
rhizome using an apple corer. The epidermis was removed and the sample cut into cubes
approximately 1.5 × 1.5 × 1.5 mm. A five-gram sample was placed in a large centrifuge tube
to which twice the sample mass of 99% ethanol was added. The preparation was sonicated
for 20 minutes (Ultrasonic Cleaner 50 Hz, Unisonics Pty Ltd, Manly Vale, Australia) and
subsequently centrifuged for 5 minutes at 4000 rotations per minute (Hettich Universal 16A
Centrifuge, Tuttlingen, Germany). The supernatant was transferred to a brown glass vial
using a transfer pipette, stored at 4° C and filtered through a Zymark/Millipore Automation
Certified Filter (Glassfiber APFB 1.0 µm prefilter, hydrophilic PTFE membrane 0.45 µm)
before being injected onto the HPLC column.
In order to take account of variation between individual plants, three samples from different
rhizomes were prepared for each clone.
4.2.3 HPLC methods
The same extracts were analyzed by HPLC on two occasions, approximately 5 months apart,
during which period they were stored at 4° C. Measurement reproducibility was determined
by repeated analyses of a mixed standard solution (see below).
Method 1. The first (baseline) reversed-phase HPLC analysis of the extracts was performed
on an Agilent (Palo Alto, California) 1100 HPLC fitted with a HP LiChrospher 100 RP-18e
(5µm) column. Mobile phase A consisted of HPLC-grade water obtained from an in-house
Milli-Q system (Waters, Milford, MA), mobile phase B consisted of HPLC-grade
acetonitrile (EM Science, Gibbstown, NJ); both contained 0.05% trifluoroacetic acid (Sigma-
Aldrich, St. Louis, MO). The gradient eluting mobile phase was A:B (70:30, v/v) to A:B
(10:90, v/v) over 20 min followed by A:B (10:90, v/v) for 10 min. Mobile phase was
pumped at 1.00 mL/min, the column temperature was 40 ºC, and the injection volume was
10.0 µL. Data were collected using a UV/visible diode array detector collecting absorption
spectra from 200 to 400 nm with quantification performed at 228 nm.
96
Method 2. The second analysis (at 5 months) was carried out on an Agilent 1100 Series
LC/MSD system fitted with a Phenomenex (Torrance, California) Luna® 5µ (150 x 4.6mm)
C-18 column. Mobile phase A and mobile phase B were as described above. The gradient
eluting mobile phase was A:B (70:30, v/v) to A:B (10:90, v/v) over 20 min followed by A:B
(10:90, v/v) for 5 min. Mobile phase was pumped at 0.40 mL/min, the column temperature
was 40 ºC, and the injection volume was 10.0 µL. Data were collected using a UV/visible
diode array detector collecting absorption spectra from 200 to 400 nm with quantification
performed at 228 nm.
Standards. Synthetic standards of [6]-gingerol (99%), [8]-gingerol (99%), [10]-gingerol
(98%), [6]-shogaol (99%) and [8]-shogaol (99%) were obtained from the Department of
Pharmacy, University of Sydney (Sydney, NSW). A known quantity of each standard was
dissolved in 99% ethanol and made up to 10 mL in a volumetric flask. A mixed standard
solution was prepared by combining 100 µL of each of the individual standard solutions of
[6]-, [8]-, and [10]-gingerol, and [6]- and [8]-shogaol. A ten-fold dilution of this mixed
standard was employed in the analyses. It contained the standard compounds in
concentrations ranging from 15 to 25 µg/mL. Identification of gingerols and shogaols in
samples was based on comparisons of retention time and UV-spectra with the standards.
Quantification was based on standard curves prepared with pure standards.
4.2.4 Statistical analysis
Statistical analyses were performed using SPSS (Chicago, Illinois) for Windows Release
11.0.0. The mean, standard deviation and coefficient of variance were calculated for
repeated measurement of the standard solution. The mean and standard error were calculated
for the analyses of the samples. Two-factor repeated measure analysis of variance was used
to compare the content of the three gingerols (‘gingerol’ = the within factor) from the 17
clones (‘clone’ = the between factor). The analysis was based on three replicates for each
clone. The assumption of compound symmetry was not met, so tests of effects were adjusted
using the Greenhouse-Geisser method. Pairwise comparisons were also employed to
compare the gingerol content across the clones. The correlation between the different
gingerols was examined by way of scatter plots and Pearson Product-Moment correlations.
The stability over time of the gingerols was explored by repeated measure t-tests. The mean,
97
standard error and range were calculated for the gingerol content of diploid and tetraploid
clones, respectively.
4.3 Results
4.3.1 Measurement reproducibility
In order to determine the reproducibility of the HPLC measurements, repeated analyses of
the mixed standard solution were carried out by both methods. The analysis by Method 1
showed a high degree of reproducibility when the same sample was injected 9 times over a
5-day period. The coefficient of variance ranged from 2.2% to 6.4% for the five standard
compounds. The reproducibility of the measurements by Method 2 was assessed by
comparing eleven injections of the mixed standard over a 37-hour period. This analysis
showed a very high degree of reproducibility with the coefficient of variance being less than
2% for all compounds.
4.3.2 Quantification of gingerols in test samples
Rhizome extracts of seventeen clones of ginger were analyzed twice, approximately five
months apart. For each clone, three extracts were prepared from different rhizomes. The
mean concentrations of [6]-gingerol, [8]-gingerol and [10]-gingerol in three rhizomes of each
clone were calculated from the peak areas obtained at 228 nm. A typical HPLC trace is
shown in Fig. 4-1.
98
min16 18 20 22 24 26 28 30 32
mAU
0
50
100
150
200
250
300
[6]-g
inge
rol (
17.3
4)
[8]-g
inge
rol (
21.2
4)
[10]
-gin
gero
l (24
.80)
Fig. 4-1. Typical HPLC chromatogram of ethanolic extract of fresh ginger rhizome (Z44) obtained with HPLC Method 2.
The concentrations of gingerols in freshly extracted ginger are shown in Fig. 4-2. The most
abundant gingerol in all clones was [6]-gingerol, which occurred at concentrations ranging
from 132 to 339 µg per gram fresh rhizome (mean 215±54 µg/g). [8]-Gingerol and [10]-
gingerol occurred in lower concentrations; [8]-gingerol ranged from 40 to 177 µg/g (mean
75±31), while [10]-gingerol ranged from 37 to 148 µg/g (mean 73±26). The cultivar
‘Jamaican’ (Z46) had the highest total gingerol content of any clone (664 µg/g). Neither [6]-
nor [8]-shogaol was identified in any of the samples (freshly prepared or stored 5 months).
99
0
100
200
300
400
500
600
700
22 23 24 25 26 27 28 29 30 31 32 33 44 45 46 47 58
Clone ID (Z no.)
mic
rogr
am/g
ram
6-gingerol 8-gingerol 10-gingerol
Fig. 4-2. Concentrations of [6]-, [8]- and [10]-gingerol in fresh rhizomes of 17 ginger clones. Values are means of three separate rhizomes. Error bars indicate standard deviation (only positive half shown).
Two-factor repeated measure analysis of variance (Greenhouse-Geisser test) of the clone by
gingerol interaction effect showed that the mean values of [6]-, [8]- and [10]-gingerol varied
significantly across the 17 clones (F=2.335, p=0.01). When the mean total gingerol content
of each clone was compared with the mean value for all clones, only two clones showed a
statistically significant difference from the overall mean. These were the cultivar ‘Jamaican’
(Z46), which contained a significantly higher concentration of gingerols (p=0.002), and one
of the experimental tetraploid clones (Z25), which had a gingerol content that was
significantly lower than the overall mean (p=0.028).
The difference in gingerol content between the clones was also explored by way of pairwise
comparisons on each of the three gingerols. This analysis is summarised in Table 4-2, which
shows the number of significantly (p<0.05) different cases between each clone and the other
16 clones for each of the three gingerol compounds. The pairwise analysis confirms that the
cultivar ‘Jamaican’ (Z46) is the most outstanding clone in terms of gingerol content. This is
100
particularly true in terms of [10]-gingerol concentration, which is significantly higher in
‘Jamaican’ than in all but one other clone (Z31).
Table 4-2. Results of pairwise analyses of 17 ginger clones in terms of [6]-, [8]- and [10]-gingerol content.
Each clone is compared with all other clones and the number of significantly (p<0.05) different cases (max. 16) is shown for each gingerol. Note that Clone Z46 (‘Jamaican’) showed 32 significantly different comparisons, distinguishing it from all other clones.
Clone (Z no.) 22 23 24 25 26 27 28 29 30 31 32 33 44 45 46 47 58
[6]-gingerol 2 2 - 7 2 - 4 2 1 1 2 2 1 8 8 2 2
[8]-gingerol 1 5 1 3 2 - 1 - 1 6 - 2 - 2 9 2 2
[10]-gingerol 1 4 1 2 1 2 1 1 1 2 1 1 1 1 15 1 1
Total number 4 11 2 12 5 2 6 3 3 9 1 5 2 11 32 5 5
4.3.3 Correlation between gingerols
The correlations between the mean concentrations of [6]-, [8]- and [10]-gingerol in the
freshly prepared extracts of 17 clones are illustrated by way of scatter plots in Fig. 4-3. A
linear relationship between the concentrations of [6]-, [8]- and [10]-gingerol is apparent. The
cultivar ‘Jamaican’ (Z46) stands out from the others by containing higher concentrations of
[8]- and [10]-gingerol relative to [6]-gingerol (Fig. 4-3A-B).
101
6-gingerol
400300200100
8-gi
nger
ol
180
160
140
120
100
80
60
40
20
'Jamaican'
A
6-gingerol
400300200100
10-g
inge
rol
160
140
120
100
80
60
40
20
B 'Jamaican'
8-gingerol
18016014012010080604020
10-g
inge
rol
160
140
120
100
80
60
40
20
C 'Jamaican'
Fig. 4-3. Scatter plots illustrating the correlation between concentrations (µg/g) of [6]- and [8]-gingerol (A), [6]- and [10]-gingerol (B), and [8]- and [10]-gingerol (C) in fresh ginger rhizomes. Based on HPLC data from 17 different clones.
102
The Pearson Product-Moment correlations between the concentrations of [6]-, [8]- and [10]-
gingerol in the rhizomes at zero and five months were calculated (Table 4-3). Since the
scatter plots clearly show a positive correlation between the different gingerols a one-tailed
test for significance was chosen. The highly significant correlations confirm the strong
positive correlation between the three gingerols.
Table 4-3. Pearson Product-Moment correlations between the concentration of gingerols in fresh rhizomes of seventeen ginger clones assayed by HPLC at zero and five months.
*: p<0.0005 (one-tailed)
[6]-gingerol [8]-gingerol
0 months 5 months 0 months 5 months
[8]-gingerol 0.882* 0.850*
[10]-gingerol 0.880* 0.803* 0.959* 0.914*
4.3.4 Gingerol ratios
The ratio of [6]-gingerol:[8]-gingerol:[10]-gingerol was calculated for the 17 clones based on
the HPLC data. The mean ratio of [6]-gingerol:[8]-gingerol:[10]-gingerol was 3:1:1 across
the clones, but some clones deviated considerably from this ratio, for example Z29
(4.4:1:1.2) and Z46 (‘Jamaican’) (1.9:1:0.8). Since both [8]- and [10]-gingerol possess
considerable pungency (albeit less than [6]-gingerol) (Govindarajan, 1982b), the relatively
high levels of [8]- and [10]-gingerol present in ‘Jamaican’ in addition to the high
concentration of [6]-gingerol make this clone by far the most pungent of the 17 clones
assayed. This was confirmed organoleptically.
103
4.3.5 Stability of gingerols
Ethanolic extracts were assayed twice approximately 5 months apart. Between analyses the
extracts were refrigerated at 4º C. The concentrations of [6]-, [8]- and [10]-gingerol at zero
and five months were compared by way of repeated measure (paired samples) t-tests. The
mean concentrations of [6]- and [8]-gingerol did not change significantly in the 17 clones
over the 5-month period, and these compounds therefore appear to be stable in ethanolic
solution at 4º C for this period of time. The concentration of [10]-gingerol, however, showed
a small (5%) but statistically significant (p=0.01) decrease over the same period.
4.3.6 Gingerol content of tetraploid clones
The mean, standard error and ranges for gingerol concentrations in two commercial
selections of the diploid ‘Queensland’ cultivar and twelve experimental tetraploid clones are
shown in Table 4-4. ‘Queensland’ (Selection 1) is the parent clone from which the
tetraploids were generated by colchicine treatment. Both diploid and tetraploid clones
displayed considerable variation in gingerol concentrations. On average, the diploid
‘Queensland’ clones contained higher levels of all three gingerols but the differences were
not statistically significant.
Table 4-4. Mean±SE (range) concentrations of gingerols in two
commercial ‘Queensland’ clones and 12 tetraploid clones (µg/g).
[6]-gingerol [8]-gingerol [10]-gingerol
‘Queensland’ clones (n=2)
245.2±77.7 (139.3-339.4)
92.2±49.9 (50.9-176.6)
83.2±39.9 (45.2-147.9)
Tetraploid clones (n=12)
202.8±37.9 (131.6-252.5)
68.1±18.2 (40.2-92.0)
67.7±18.4 (36.6-98.5)
p-value (t-test) 0.058 0.279 0.471
104
4.4 Discussion
An extensive survey of fresh rhizomes of five diploid ginger cultivars, eleven experimental
tetraploid clones, and one recently released tetraploid clone grown in Australia was
conducted with respect to their content of pungent gingerols and shogaols. Ethanol was
chosen as the extraction solvent, as it is the solvent of choice for herbal medicine
preparations.
4.4.1 Gingerols
The three pungent compounds, [6]-, [8]- and [10]-gingerol, were identified and quantified in
all samples. [6]-Gingerol was the most abundant gingerol in all clones, which is in
accordance with the literature (Bartley, 1995; Chen et al., 1986a; Govindarajan, 1982a). The
mean ratio of [6]-gingerol:[8]-gingerol:[10]-gingerol was 3:1:1 across all 17 clones. A
strong, positive, linear correlation between levels of the three gingerols was found in all
clones, reflecting the close biosynthetic relationship between these compounds.
The mean content of gingerols obtained in the present study are considerably higher than
those found by Bartley in a supercritical CO2 extract of Australian ginger (Bartley, 1995),
but lower than levels reported from other parts of world (Table 4-5). This variability may
reflect genetic differences between clones in different regions or physiological responses to
environmental factors such as climate, soil characteristics or predation, but they may also be
due to differences in extraction and analytical methodologies.
105
Table 4-5. Literature data on gingerol content of fresh ginger rhizomes.
Values are µg per gram fresh rhizome. n.d. = not determined.
Country (reference)
Solvent/ extraction
Analytical method
[6]-gingerol [8]-gingerol [10]-gingerol
Australia (present study)
Ethanol HPLC 215 75 72
Hawaii (Zhang et al., 1994)
Methanol HPLC 2100 288 533
United States (Hiserodt et al., 1998)
Methylene chloride
HPLC 880 93 120
Taiwan (Young et al., 2002)
Ground; acetate buffer solution (pH 4.0) added
HPLC 806 n.d. n.d.
Australia (Bartley, 1995)
Supercritical CO2
NIES-MS
120 19 24
The cultivar ‘Jamaican’ contained the highest concentration of all three gingerols on a fresh
weight basis and was therefore the most pungent of the clones assayed. It also contained
higher levels of [8]- and [10]-gingerol relative to [6]-gingerol than any other clone.
‘Jamaican’ may thus be suitable for commercial production of highly pungent ginger
rhizomes with potential application in both the pharmaceutical and flavour industries, even
though, eventually, the viability of commercial production of this clone will depend on
biomass yield.
Repeated analyses of ethanolic extracts five months apart showed that [6]- and [8]-gingerol
did not degrade during this period when stored at 4º C. Concentrations of [10]-gingerol
showed a small but statistically significant decrease (5%). It is not known whether [10]-
gingerol is in fact less stable than [6]- and [8]-gingerol under these conditions or this finding
represents a type 1 error resulting from the small sample size.
106
4.4.2 Shogaols
Neither [6]- nor [8]-shogaol were identified in these samples, which were prepared at
ambient temperature from fresh rhizomes. In contrast, [6]-shogaol was identified in fresh
rhizome extracts prepared by hot Soxhlet extraction (data not shown, refer to Chapter 3).
These findings support the hypothesis that shogaols are not native constituents of fresh
ginger rhizomes but form from gingerols by dehydration as a result of heat treatment or
alkaline or acidic conditions (Connell & Sutherland, 1969; Vesper et al., 1995; Zhang et al.,
1994). Earlier reports of shogaols in fresh ginger extracts analyzed by GC-MS (Bartley,
1995; Bartley & Jacobs, 2000) can probably be explained by the high temperatures samples
are exposed to during this form of analysis, resulting in the formation of shogaols as artifacts
of analysis.
4.4.3 Ploidy
The mean concentrations of all three gingerols were lower for the tetraploid clones than for
the parent diploid ‘Queensland’ cultivar, although the differences were not statistically
significant. This observation is in marked contrast to the findings of Nakasone and
colleagues who reported that tetraploid clones of three Japanese ginger cultivars contained
higher concentrations of total gingerols ([6]-, [8]- and [10]-gingerol) and in particular of
[10]-gingerol than did their parent diploid genotypes (Nakasone et al., 1999).
Comparative quantitative studies of secondary metabolites in diploid versus polyploid
genotypes have been conducted on numerous medicinal plants. Although polyploidy often
appears to result in increased expression of secondary metabolites, this is not always the
case, and the effects of polyploidy are not predictable (Evans, 2002).
The findings of this study do not therefore preclude the possibility of identifying a tetraploid
clone with elevated gingerol biosynthesis. In this context it would be of particular interest to
monitor experimental tetraploid clones derived from the cultivar ‘Jamaican’, which are
currently under development at the Maroochy Research Station.
This study has described the variability of gingerol compounds in Australian commercial and
experimental ginger clones. When combined with agronomic data, the present information
107
should allow for selection of clones with specific levels of pungency, a characteristic which,
along with the aroma produced by the essential oil, determines the flavour characteristic of
ginger.
108
5. ESSENTIAL OIL COMPOSITION OF SEVENTEEN GINGER (ZINGIBER OFFICINALE) CLONES
5.1 Introduction
Ginger owes its unique flavour properties to the combination of pungency and aroma. The
pungency is provided by non-volatile phenolic compounds, whereas the essential oil gives
ginger its characteristic aroma. Ginger rhizome yields two primary extracts: oleoresin and
essential (or volatile) oil. The oleoresin is a solvent extract (usually in acetone or ethanol)
containing both essential oil and the phenolic compounds responsible for the pungency of
ginger, chiefly [6]-gingerol and to a lesser extent [8]- and [10]-gingerol. The corresponding
shogaols, which are dehydration products of gingerols formed in heat-treated ginger, are also
found in the oleoresin. Ginger oleoresin is used extensively as a flavouring agent in the food
and beverage industries.
Commercial ginger oil is normally extracted by steam distillation from dried rhizomes.
Typical ginger oil is characterized by a high content of sesquiterpene hydrocarbons, in
particular zingiberene, ar-curcumene, β-bisabolene, and β-sesquiphellandrene, while
important monoterpenoids normally include geranial, neral, and camphene (Lawrence, 1997;
Lawrence, 2000; Martins et al., 2001; Vernin & Parkanyi, 1994). Although these
compounds are characteristic of ‘typical’ ginger oils, the literature clearly shows that ginger
oil composition is highly variable (Asfaw & Abegaz, 1995; Connell & Jordan, 1971;
Ekundayo et al., 1988; Gurib-Fakim et al., 2002; Lawrence, 1995a; Lawrence, 1997;
Lawrence, 2000; Macleod & Pieris, 1984; Martins et al., 2001; Menut et al., 1994; Vernin &
Parkanyi, 1994). Factors such as geographical origin, whether the oil is distilled from fresh
or dried rhizome material, drying process and temperature, and analytical methodology may
all contribute to the disparity of published ginger oil analyses.
Ginger ‘oil’ obtained by supercritical fluid extraction using carbon dioxide is also
commercially available, but this product differs radically from steam-distilled oil due to the
presence of the pungent gingerols and shogaols (Bartley & Jacobs, 2000).
109
Ginger oil is used in the beverage and fragrance industries, and the world production of
ginger oil was estimated at 100-200 t in 2000 (Weiss, 2002). In the classic work, Perfume
and Flavor Materials of Natural Origin, Arctander described the odour of ginger oil as
“warm, but fresh-woody, spicy and with a peculiar resemblance to orange, lemon, lemon-
grass, coriander weed oil, etc. in the initial, fresh topnotes […the] sweet and heavy undertone
is tenacious, sweet and rich, almost balsamic-floral” (Arctander, 1994).
This chapter reports the essential oil composition (analysed by GC-MS) of the clones of
ginger previously analysed for their gingerol content (refer to Chapter 4). This is the first
comprehensive survey of steam-distilled Australian ginger oils since the early work by
Connell and Jordan (Connell & Jordan, 1971).
5.2 Materials and Methods
5.2.1 Plant materials and distillation
Details of the seventeen commercial and experimental clones of ginger included in this study
were provided in Section 4.2.1 of the previous chapter. Rhizomes were harvested in late July
and stored at ambient temperature for approximately seven weeks before being distilled.
Prior to distillation, unpeeled rhizomes were washed and had any diseased tissue removed
before being chopped into pieces approximately 1 3 7 mm. Equal parts of rhizome
from three different plants were pooled, and approximately 200 g of this material was
hydrodistilled in a Clevenger distillation apparatus for three hours.
5.2.2 Chemical analysis
Oils were analyzed on an Agilent Technologies (Palo Alto, California) 6890/5973 GC-MSD
system using helium as the carrier gas at a constant linear flow velocity of 29 cm/s. Oil
samples (150 µL) were diluted with pentane (1500 µL) and 1 µL of this solution was
injected. The column was a 50 m 0.22 mm capillary column, 1.00 µm film thickness
(BPX-5, SGE Ltd., Melbourne). The split ratio was 25:1. The column oven was
programmed from 70°C to 280°C at a ramp rate of 4°C/min (final hold time 4 minutes), and
110
the injector temperature was 250°C. Composition values were recorded as percentage area
based on the total ion current chromatogram. Retention indices were determined using a C-8
to C-22 n-alkane mixture. Compound identification was based on comparisons with mass
spectra and retention indices of authentic reference compounds where possible and by
reference to WILEY275, NBS75K and Adams terpene library (Adams, 1995) and published
data. Myrcene, 1,8-cineole, linalool, α-terpineol, β-citronellol, citral, geraniol, (–)-bornyl
acetate, citronellyl acetate, geranyl acetate and (E)/(Z)-nerolidol were obtained from Aldrich
Chemical Co. Inc. (Milwaukee, WI); (–)-borneol and (Z)-nerolidol were obtained from Fluka
Chemie (Buchs, Switzerland); 6-methyl-5-hepten-2-one was obtained from ants
(Formicidae) (Tomalski et al., 1987) and (E,E)-α-farnesene from the peel of ‘Granny Smith’
apples (Pechous & Whitaker, 2004). Germacrene-D and elemol were identified by
comparison with these compounds in authentic clary sage (Salvia sclarea L.) oil
(Anonymous, 2004) and elemi (Canarium luzonicum (Miq.) Asa Gray; Berjé Inc.,
Bloomfield NJ) oil (Villanueva et al., 1993), respectively.
5.2.3 Statistical analysis
Statistical analyses were performed using SPSS (Chicago, Illinois) for Windows Release
11.5. Mean values, standard deviations and ranges were calculated for major oil
constituents. The oil composition of the 17 clones was the subject of principal components
analysis and cluster analysis based on the ten most abundant constituents. The relationship
between neral and geranial was examined by way of a scatter plot and Pearson’s correlation
coefficient.
5.3 Results
5.3.1 Oil composition
The essential oil composition for the 17 clones is shown in Table 5-1. The mean, standard
deviation and range for the 14 most abundant constituents in the 16 homogenous or ‘typical’
clones are shown in Table 5-2, which also gives the percentage content for the atypical oil
from the cultivar ‘Jamaican’ (Z46). The ‘typical’ oils had a mean citral (neral + geranial)
111
content of 58%, whilst the five major sesquiterpene hydrocarbons typically found in ginger
oil (ar-curcumene, (E,E)-α-farnesene, zingiberene, β-bisabolene and β-sesquiphellandrene
(Lawrence, 1995b)) made up only 17%. In contrast, the oil from ‘Jamaican’ had a
comparatively low citral content (28%) and contained 35% of the main sesquiterpene
hydrocarbons. Some of the major oil constituents are shown in Fig. 5-1.
H3C
curcumene
zingiberene
H
β-sesquiphellandrene
β-bisabolene
(E,E)-α-farnesene
OH
borneol
CH2OH
geraniol geranial neral
CHO
OH
α-terpineol
OH
citronellol
CHO
Fig. 5-1. Volatile constituents from Zingiber officinale essential oil.
113
Table 5-2. Content of 14 constituents in essential oils of 16 ‘typical’ clones of ginger and one ‘atypical’ clone, ‘Jamaican’ (Z46).
Mean, standard deviation and range are shown for the 16 clones. All values are percentage content.
16 ‘typical’ clones mean±SD (range)
‘Jamaican’ (Z46)
1,8-cineole 1.52±0.52 (0.39-2.63) 0.79
linalool 1.21±0.20 (0.97-1.55) 1.02
borneol 2.41±0.39 (1.84-2.94) 3.91
α-terpineol 1.81±0.21 (1.49-2.18) 1.13
citronellol 1.93±0.28 (1.48-2.49) 1.09
neral 21.44±1.63 (19.39-26.49) 10.60
geraniol 4.91±1.28 (2.73-7.30) 1.54
geranial 36.50±3.26 (31.29-44.31) 17.51
geranyl acetate 2.02±0.92 (0.52-3.45) 0.26
ar-curcumene 3.24±0.73 (2.43-5.31) 5.72
(E,E)-α-farnesene 3.02±0.68 (2.10-4.30) 4.35
zingiberene 4.82±2.03 (1.86-9.00) 11.24
β-bisabolene 1.51±0.31 (0.97-2.16) 4.05
β-sesquiphellandrene 4.36±0.85 (2.93-5.62) 9.40
5.3.2 Principal components analysis
The percentage composition of the 17 oil samples was subjected to principal components
analysis based on the ten most abundant oil constituents (borneol, neral, geraniol, geranial,
geranyl acetate, ar-curcumene, (E,E)-α-farnesene, zingiberene, β-bisabolene and β-
sesquiphellandrene). This analysis revealed the presence of three components with
eigenvalues exceeding one. These components explained 61.6%, 18.8% and 10.7% of the
variability, respectively. Two components (together explaining 80% of the variability) were
retained for further investigation, and Varimax rotation was performed to assist in the
interpretation (Table 5-3).
114
Table 5-3. Varimax rotated component matrix for two component solution for the ten most abundant ginger essential oil constituents.
Component
1 2
neral -.96 -.25
geranial -.96 -.15
β-bisabolene .89 .39
borneol .87 -.21
β-sesquiphellandrene .78 .61
ar-curcumene .61 .30
zingiberene .55 .54
geraniol -.23 -.89
geranyl acetate .12 -.87
(E,E)-α-farnesene .45 .74
Percentage of variance explained 49.3% 31.2%
The rotated solution revealed a complex structure in which both components had several
strong loadings (coefficients relating the variables to the components), but some compounds
loaded substantially on both components. Neral, geranial, β-bisabolene, borneol and β-
sesquiphellandrene were strongly associated with component 1, indicating a high degree of
interrelationship (positive or negative) between the concentrations of these compounds.
Similarly, geraniol, geranyl acetate and (E,E)-α-farnesene were strongly associated with
component 2. More broadly, an inverse relationship between levels of citral (geranial +
neral) and the sesquiterpene hydrocarbons (zingiberene, ar-curcumene, β-
sesquiphellandrene, β-bisabolene and (E,E)-α-farnesene) was evident by inspection of the
component plot in Fig. 5-2.
115
Component 1
1.0.50.0-.5-1.0
Com
pone
nt 2
1.0
.5
0.0
-.5
-1.0
geranialneral
geraniol geranyl acetate
borneol
b-sesquiphellandrenezingiberene
b-bisabolenear-curcumene
(E,E)-a-farnesene
Fig. 5-2. Component plot in rotated space showing Varimax rotated data on two components based on the ten most abundant constituents of ginger essential oil.
5.3.3 Cluster analysis
In order to examine the degree of similarity displayed by the 17 clones in terms of oil
composition, a hierarchical, between-groups linkage, cluster analysis based on the ten most
abundant constituents was performed. This is a multivariate procedure that allows for the
classification of cases (or variables) into groups based on Euclidian distances between cases.
For each constituent, percentage values were rescaled to have a mean value of one, so that all
constituents were equally weighted for the purpose of the analysis, and Euclidian distances
were calculated between pairs of clones.
Fig. 5-3 shows a dendrogram of the 17 essential oils. This diagram confirms the unique
nature of the essential oil from the cultivar ‘Jamaican’ (Z46) when compared with oils from
the other 16 clones. The dendrogram also shows that the oil from the clone Z22 stands out
from the others. This oil had an extremely high citral content (71%). The remaining 15
116
clones fall into two clusters. The similarity between these two clusters was examined using a
multivariate general linear model, comparing the set of clones in Cluster 1 (Z25, Z32, Z33,
Z27, Z26, Z31, Z30, Z58, Z28, Z29, Z45) and Cluster 2 (Z24, Z44, Z47, Z23). Cluster 1
and Cluster 2 showed a near-significant difference (Wilks’ Lambda = 0.064; F = 5.838; df =
10 and 4; p = 0.052). In terms of individual constituents, six (borneol, geraniol, geranyl
acetate, (E,E)-α-farnesene, β-bisabolene, β-sesquiphellandrene) were significantly different
between clusters at p ≤ 0.05 and four (neral, geranial, ar-curcumene, zingiberene) were not.
Fig. 5-3. Dendrogram of hierarchical cluster analysis of 17 essential oils of ginger.
The diagram shows average linkage (between groups), and values shown along the horizontal axis are Euclidian distances rescaled to an arbitrary scale showing the levels of relative similarity where clusters join.
5.3.4 Citral content
The ginger oils analyzed in the present study had citral contents ranging from 28% in the
‘Jamaican’ cultivar (Z46) to 71% in the tetraploid clone Z22. The mean citral content of the
16 ‘typical’ ginger oils (‘Jamaican’ excluded) was 57.9±4.9% (range: 50.7-70.8%), which
was more than double the corresponding value for ‘Jamaican’ (28%). A similar trend was
117
evident for geraniol, which is a precursor to citral, with a mean concentration of 4.9%±1.3%
in the ‘typical’ oils compared with 1.5% in ‘Jamaican’.
5.3.5 Neral to geranial ratio
The 17 ginger oils contained neral and geranial in a ratio that was remarkably constant. The
neral to geranial ratio ranged from 0.55 to 0.64, with a mean value of 0.61±0.01. This fixed,
linear relationship between the two isomers, which persisted regardless of the percentage
content of citral, is illustrated in Fig. 5-4. The very strong correlation between the two
compounds is quantified by the Pearson’s correlation coefficient of 0.987 (p<0.001).
It is particularly interesting to note that the neral to geranial ratio also was 0.6 in the cultivar
‘Jamaican’ (Z46), which otherwise yielded an oil that was distinctly different from those of
the other 16 clones.
Neral (%)
302010
Ger
ania
l (%
)
50
40
30
20
10
'Jamaican'
Z22
Fig. 5-4. Scatter plot showing the relationship between the percentage content of the stereoisomers neral and geranial in essential oils from 17 clones of ginger.
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5.4 Discussion
Essential oils of 17 clones of Australian ginger were prepared by hydrodistillation of
rhizomes and analyzed by GC-MS. Compared with values in the literature (Asfaw &
Abegaz, 1995; Connell & Jordan, 1971; Ekundayo et al., 1988; Gurib-Fakim et al., 2002;
Lawrence, 1995b; Lawrence, 1997; Lawrence, 2000; Macleod & Pieris, 1984; Martins et al.,
2001; Menut et al., 1994; Vernin & Parkanyi, 1994), all the samples were characterized by a
very high citral content (28% or greater) and a relatively low content of sesquiterpene
hydrocarbons.
5.4.1 Oil composition
There was no distinct difference in the composition of the oils of the tetraploid clones
compared with the diploid parent cultivar ‘Queensland’ or the cultivars ‘Canton’ and
‘Brazilian’. These oils were characterized by very high levels of citral (geranial + neral) (51-
71%) and relatively low levels of the sesquiterpene hydrocarbons characteristic of ginger oil
(Lawrence, 1995b), namely zingiberene (4.8±2.0%), ar-curcumene (3.2±0.7%), β-
sesquiphellandrene (4.4±0.9%), β-bisabolene (1.5±0.3%) and (E,E)-α-farnesene (3.0±0.7%).
These values contrast with those reported in 1971 by Connell and Jordan, whose analysis of
35 Australian ginger oils found citral levels ranging from 4 to 30% and much higher
concentrations of sesquiterpene hydrocarbons (zingiberene 20-28%, ar-curcumene 6-10%, β-
sesquiphellandrene 7-11% and β-bisabolene 5-9% (but no (E,E)-α-farnesene) (Connell &
Jordan, 1971). The results obtained in the present study are better aligned with more recent
analyses of ginger oils from Mauritius (Gurib-Fakim et al., 2002), Sao Tomé e Príncipe
(Martins et al., 2001) and Nigeria (Ekundayo et al., 1988) by being high in citral, relatively
low in the major sesquiterpene hydrocarbons and by containing (E,E)-α-farnesene. In
general, the published values for these compounds vary greatly; whether this is due to true
natural variability or differences in raw material (fresh or dried, time of harvest), distillation
conditions or analytical methodologies is uncertain.
An inverse relationship between levels of citral on the one hand and β-bisabolene, β-
sesquiphellandrene and borneol on the other was demonstrated by principal components
analysis. Likewise, an inverse relationship was shown to exist between geraniol/geranyl
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acetate and (E,E)-α-farnesene. Terpenoids are derived from C5 isoprene units in the form of
the diphosphate (pyrophosphate) esters dimethylallyl diphosphate and isopentenyl
diphosphate and are products of the mevalonate pathway (Dewick, 1997). The common
biosynthetic origin of monoterpenoids (such as geranial, neral, geraniol and geranyl acetate)
and sesquiterpenoids (including β-bisabolene, β-sesquiphellandrene and (E,E)-α-farnesene)
provides a possible explanation for the inverse relationship seen between mono- and
sesquiterpenoids, as the pathways to mono- and sesquiterpenes would compete for common
precursors. Similarly, the fact that citral and borneol share the monoterpene precursor
geranyl pyrophosphate could explain the inverse relationship observed between these
compounds.
The oils analysed in this study were prepared from fresh rhizomes seven weeks post-harvest
by a short distillation process and analysed within a short period of time. They may differ
significantly from oils prepared from dried rhizome material by longer distillation processes
followed by prolonged storage under conditions that favour oxidation. It has been reported
that both zingiberene and β-sesquiphellandrene can undergo oxidation to ar-curcumene
(Connell & Jordan, 1971; Govindarajan, 1982a), and high levels of this compound may
therefore be indicative of a degraded oil.
The essential oil from the cultivar ‘Jamaican’ (Z46) differed distinctly in composition from
all the others, primarily by having lower citral content and higher sesquiterpene hydrocarbon
content. This oil contained 28% citral while the total amount of the five main sesquiterpene
hydrocarbons was 35%, which is more than double the mean concentration found in the 16
other clones. The distinctiveness of the oil of ‘Jamaican’ was confirmed by hierarchical
cluster analysis. As reported in Chapter 4, this cultivar was also the most pungent of the 17
clones tested due to its high content of gingerols. The dissimilar essential oil composition
coupled with its high pungency gives ‘Jamaican’ flavour and aroma qualities that are distinct
from the other clones examined.
Cluster analysis identified one other distinct clone (Z22), characterized by extremely high
(71%) citral content, while the remaining clones fell into two clusters. These two clusters
did not appear to reflect breeding history, as the different tetraploid clones produced from the
same parent cultivar (‘Queensland’) were split between the two clusters, as were the other
two cultivars, ‘Brazilian’ and ‘Canton’.
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5.4.2 Citral content
The ginger oils analysed in this study contained remarkably high levels of citral (above 50%
in all samples except ‘Jamaican’ and up to 71% in one sample). The highest citral content
described previously in ginger oil was 56% in a sample from Fiji (Smith & Robinson, 1981).
Although Australian ginger has a reputation for its ‘lemony’ aroma, a 1971 study of 35
Australian ginger oils by Connell and Jordan found considerably lower citral levels, ranging
from 4% to 30% (Connell & Jordan, 1971). The same study found only very low citral
levels in oils from other parts of the world, and many other studies have reported very low
citral levels in non-Australian ginger oils (Asfaw & Abegaz, 1995; Humphrey et al., 1993;
Lawrence, 1995b; Lawrence, 2000; Macleod & Pieris, 1984). Conversely, several more
recent investigations have found substantial citral levels in ginger oils from other parts of the
world, for example from the Central African Republic (35%) (Menut et al., 1994), Mauritius
(27%) (Gurib-Fakim et al., 2002), Sao Tomé e Príncipe (22-25%) (Martins et al., 2001),
Nigeria (24%) (Ekundayo et al., 1988) and Tahiti (14-25%) (Lawrence, 1997; Lechat-
Vahirua et al., 1996).
There are several possible, not mutually exclusive, explanations for these somewhat
incongruent findings regarding citral content. One relates to the changes in gas
chromatography technology (especially detection systems) since the early 1970s, which
might explain some of the differences between the results of the current study and those of
Connell and Jordan (Connell & Jordan, 1971). It is also likely that ginger oils distilled from
fresh rhizomes generally have a higher citral content than oils obtained from dried rhizomes.
The notion that sun drying and storage of ginger could cause a loss of citral was put forward
by Govindarajan more than two decades ago (Govindarajan, 1982a), and studies from Sri
Lanka (Macleod & Pieris, 1984) and Nigeria (Ekundayo et al., 1988) have documented citral
losses in ginger oils ranging from 40% to 74%, when rhizomes were dried before distillation.
A significant difference in citral content has also been observed in supercritical fluid extracts
of fresh and dried Australian ginger, which contained 19.9% and 6.2% citral, respectively
(Bartley & Jacobs, 2000). These data would seem to suggest that many ginger oils contain
considerable amounts of citral, provided they are distilled from fresh rhizomes or rhizomes
dried at low temperature. On the other hand, the existence of genuine low-citral genotypes
of ginger is supported by a recent study from India, where the essential oil distilled from
fresh rhizomes contained less than 4% citral (Singh et al., 2005).
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A number of other factors could also have contributed to the unexpectedly high citral values
obtained in this study. They include the particular growth period (8 months), the harvest time
(mid-winter), the 7-week delay between harvest and distillation, and the conditions of
cultivation (including climate) at Southern Cross University, which is located some 250 km
south of the commercial ginger-growing area in Australia.
5.4.3 Neral to geranial ratio
Neral (Z-citral) and geranial (E-citral) are stereoisomers occurring in many plants in mixtures
often referred to simply as citral. The neral to geranial ratios found in ginger oils in this
study were remarkably constant and close to 2:3 (mean 0.61±0.01, range 0.55-0.64). The
ratios reported in the literature for ginger oil vary greatly, from 0.3 in a Taiwanese oil
(Lawrence, 1995b) to 4.7 in a Chinese oil (Lawrence, 2000). However, a ratio of 0.6 was
also reported for a Mauritian oil made from fresh rhizomes (Gurib-Fakim et al., 2002), a
Nigerian oil also prepared from fresh rhizomes (Ekundayo et al., 1988) and an oil made from
dried rhizomes from Sao Tomé e Príncipe (Martins et al., 2001), while oils from Madagascar
(Möllenbeck et al., 1997) and the Central African Republic (Menut et al., 1994) were
reported to contain neral to geranial ratios of 0.4 and 0.9, respectively. The data obtained in
the current study support the hypothesis that neral and geranial occur in ginger in a relatively
fixed ratio of approximately 2:3 and calls into question the validity of analyses suggestive of
very different ratios.
Neral and geranial also occur in other plants in a similar ratio. Species of lemongrass
produce essential oil with a citral content typically exceeding 70%. Nine samples of West
Indian lemongrass (Cymbopogon citratus, family Poaceae) oil from Africa, China, New
Zealand and Cuba had neral to geranial ratios ranging from 0.61 (New Zealand) to 0.96
(China) with a mean of 0.76, while four Indian cultivars of East Indian lemongrass (C.
flexuosus) produced oils with a neral to geranial ratio ranging from 0.61 to 0.67 (Lawrence,
2002).
This study has shown that essential oil from the three ginger cultivars ‘Queensland’,
‘Canton’ and ‘Brazilian’ and twelve tetraploid clones derived from ‘Queensland’ had a high
degree of compositional similarity and were characterized by a very high citral content.
Tetraploid clones were shown to have retained the characteristics of the parent cultivar in
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terms of essential oil composition. In contrast, the cultivar ‘Jamaican’ produced an oil that
was distinctly different, characterized by a lower citral content and higher levels of
sesquiterpene hydrocarbons. The distinctive aroma profile of this cultivar, which also
contains significantly higher levels of pungent gingerols, should make it of commercial
interest to the flavour and fragrance industries.
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6. PHARMACOLOGICAL ACTIVITY OF ZINGIBERACEAE SPECIES
6.1 Introduction
Forty-one taxa were screened for inhibition of prostaglandin E2 (PGE2) production. Some of
the samples were also screened for antioxidant activity in the oxygen radical absorbance
(ORAC) assay, for inhibition of nitric oxide production, and for modulation of natural killer
cell activity. This work should be regarded as a preliminary survey, and it was constrained
by the cost of some of the assays.
Prostaglandin E2 is a largely pro-inflammatory eicosanoid produced from arachidonic acid
by the action of cyclooxygenase (COX). COX, in particular the isoform COX-2, is an
important pharmacological target for the mitigation of inflammation, which is an underlying
pathological mechanism in many chronic diseases including arthritic disorders,
cardiovascular disease, diabetes, some types of cancer, and osteoporosis (Libby, 2007).
Salicylates and other non-steroidal anti-inflammatory drugs target COX, and many plant
extracts and compounds have been shown to inhibit COX and the generation of PGE2
(Calixto et al., 2003; Calixto et al., 2004). Prostaglandins and COX were described in more
detail in Chapter 1.
Reactive oxygen species and oxidative stress have been linked to a large number of
pathologies including inflammation, cancer, cardiovascular disease, Parkinson’s disease and
Alzheimer’s disease, and the importance of antioxidant defenses (both endogenous and
exogenous) is well recognised (Gilbert, 1999). Higher plants are rich sources of antioxidant
compounds (well known examples are carotenoids and polyphenols), and plant extracts with
potent antioxidant activity are of interest as sources of potential therapeutic substances.
Nitric oxide (NO) is a free radical with numerous functions in both normal and pathological
processes. Nitric oxide is produced by the oxidation of L-arginine by inducible nitric oxide
synthase (iNOS) and is involved in pathological processes associated with an overproduction
of nitric oxide including inflammatory disorders of the joint, gut and lungs. Nitric oxide
functions as a messenger molecule in the immune system, and many immune cells produce
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and respond to nitric oxide (Morikawa et al., 2005; Sharma et al., 2007; Tripathi et al.,
2007). Nitric oxide and iNOS play key roles in the up-regulation of the inflammatory
response, and nitric oxide mediates many of the destructive effects of interleukin-1 (IL-1)
and tumour necrosis factor-alpha (TNF-α) on cartilage in osteoarthritis. The inhibition of
nitric oxide and iNOS therefore represent an attractive therapeutic target in inflammatory
conditions (Cuzzocrea, 2006; Sharma et al., 2007; Vuolteenaho et al., 2007).
Inhibition of nitric oxide production and/or iNOS expression has been demonstrated for a
number of medicinal plants and plant compounds in recent years, for example Andrographis
paniculata (Batkhuu et al., 2002), Salvia miltiorrhiza (Jang et al., 2003), Hypericum
perforatum (Di Paola et al., 2007), Citrus reticulata (Jung et al., 2007) as well as the reishi
mushroom (Ganoderma lucidum) (Woo et al., 2005). It is widely considered that this
property likely contributes to the anti-inflammatory activity of these plants.
Natural killer (NK) cells are specialised bone marrow-derived lymphocytes that form part of
the innate immune system. NK cells have the ability to lyse virally infected and tumour
cells. They comprise approximately 5-20% of the peripheral lymphocyte population in the
peripheral blood, liver and spleen and are present at lower frequencies in the lymph nodes,
bone marrow, and thymus. The anticancer activity of NK cells in the form of tumour
rejection has been well established in a variety of animal models of cancer. In addition to
their anti-tumour and anti-viral activity, NK cells are also involved in other
pathophysiological processes such as inflammation and autoimmunity, and stimulated NK
cells can produce cytokines including interferon-γ (IFN-γ), tumour necrosis factor-α (TNF-
α), and granulocyte-macrophage colony stimulating factor (GM-CSF) (Wallace & Smyth,
2005; Yokoyama et al., 2004).
Little has been published on the effects of medicinal plant extracts on NK cell activity.
Medicinal plants for which an ability to increase NK cell activity has been demonstrated
include garlic (Allium sativum) (Abuharfeil et al., 2001), Viscum album (Schink, 1997),
Echinacea spp. (Currier & Miller, 2000; Zhai et al., 2007) and Nigella sativa (Abuharfeil et
al., 2001).
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6.2 Materials and Methods
6.2.1 Plant materials
A total of 53 samples representing rhizome extracts of 43 Zingiberaceae taxa were included
in this study, although not all taxa were screened in all assays. Plant material was obtained
from a variety of sources. Nineteen samples were originally obtained from the live
collection in the George Brown Darwin Botanic Garden, Darwin, Northern Territory
(latitude S 12° 26'; longitude E 130° 50'), and a number of these were cultivated in pots at
Southern Cross University, Lismore, New South Wales (latitude S 28° 49'; longitude E 153°
18') before harvest of the rhizome material. Twelve samples were obtained from the living
collection in the Flecker Botanic Gardens, Cairns, Queensland (latitude S 16° 54'; longitude
E 145° 45'). Z129 Alpinia arctiflora, Z127 Alpinia modesta, Z126 Hornstedtia scottiana,
Z128 Pleuranthodium racemigerum, and Z125 Tapeinochilos ananassae were collected in
the wild in the Wet Tropics region of North Queensland under a permit issued by the
Queensland Environmental Protection Agency (voucher specimens were deposited in the
Queensland Herbarium; voucher number AQ736264, AQ736265, AQ736255, AQ736263,
AQ736256, respectively). Z107 Etlingera australasica was obtained from a tub specimen
grown in Brisbane, Queensland from wild stock collected at Bog Hole Creek, Silver Plains,
Queensland (latitude S 13° 42'; longitude E 143° 29') by Paul Forster of the Queensland
Herbarium (PIF30425 (BRI)). Z45 Zingiber officinale ‘Queensland’ rhizome stock was
obtained from the Queensland Department of Primary Industries & Fisheries Maroochy
Research Station at Nambour, Queensland (latitude S 26° 37’; longitude E 152° 57’) and
grown in raised beds at Southern Cross University. J-2 and J-4-4 Zingiber officinale
‘Jamaican’ were grown at the Maroochy Research Station. Z108/9 Zingiber officinale was
obtained from a produce store in Lismore, NSW. Twelve samples were obtained from the
living collections of the Medicinal Plant Garden and the Lismore campus grounds of
Southern Cross University.
Table 6-1 provides a list of the samples, their origin and methods of extraction.
126
Table 6-1. Zingiberaceae samples screened for biological activity.
ID Taxon Source Extraction method±
Z129$ Alpinia arctiflora (F. Muell.) Benth. Danbulla State Forest, c. 18 km WSW of Gordonvale, North Qld. (Qld Herbarium AQ736264)
3
Z13^ Alpinia caerulea (R. Br.) Benth. Darwin Botanic Garden (D94418) 1 Z102# Alpinia caerulea (R. Br.) Benth. Southern Cross University (NCM03-029) 2 Z49^ Alpinia calcarata Roscoe Southern Cross University 1 Z07^ Alpinia galanga (L.) Swartz Darwin Botanic Garden (D94418) 1 Z103# Alpinia galanga (L.) Swartz Southern Cross University (NCM04-002) 2, 5 Z111 Alpinia luteocarpa Elmer Flecker Botanic Gardens, Cairns 3 Z48^ Alpinia malaccensis Roscoe Southern Cross University 1 Z127$ Alpinia modesta Schumann Nyleta Creek, 16 km NNE of Tully, North
Qld. (Qld Herbarium AQ736265) 3
Z08* Alpinia mutica Roxb. Darwin Botanic Garden (D950346) 1 Z11* Alpinia purpurea ‘Eileen McDonald’ Darwin Botanic Garden (D93105) 1 Z53^ Alpinia spectabile Griff. ‘Giant Orange’ Southern Cross University 1 Z52^ Alpinia zerumbet (Pers.) B.L. Burtt & R.
M. Sm. Southern Cross University 1
Z104#+ Boesenbergia rotunda (L.) Mansf. Southern Cross University (NCM04-121) 2, 6 Z03^ Costus barbatus Suess. Darwin Botanic Garden (D971488) 1 Z112 Costus barbatus Suess. Flecker Botanic Gardens, Cairns 3 Z113 Costus leucanthus Maas Flecker Botanic Gardens, Cairns 3 Z114 Costus malortieanus H. Wendl. Flecker Botanic Gardens, Cairns 3 Z14^ Costus productus Gleason ex Maas Darwin Botanic Garden (D971499) 1 Z116 Costus productus Gleason ex Maas Flecker Botanic Gardens, Cairns 3 Z115 Costus pulverulentus C. Presl Flecker Botanic Gardens, Cairns 3 Z15* Costus tappenbeckianus J. Braun et K.
Schum. Darwin Botanical Garden (D971491) 1
Z59# Curcuma australasica J.D. Hook Southern Cross University (NCM03-036) 1 Z101# Curcuma australasica J.D. Hook Southern Cross University (NCM03-036) 2, 5 Z117 Curcuma cordata Wallich Flecker Botanic Gardens, Cairns 3 Z18^ Curcuma longa L. Darwin Botanic Garden (D970132) 1 Z106# Curcuma longa L. Southern Cross University (NCM04-013) 2, 5 Z118 Curcuma parviflora Wallich Flecker Botanic Gardens, Cairns
(NCM05-040) 3
Z12^ Elettaria cardamomum (L.) Maton Darwin Botanic Garden (D95162) 1 Z107 Etlingera australasica (R.M. Smith) R.M.
Smith Brisbane (PIF30425 (BRI)) 2, 5
127
Z04* Etlingera elatior (Jack) R.M. Smith ‘Burma Torch’
Darwin Botanic Garden (D95335) 1
Z10^ Hedychium coronarium Koenig Darwin Botanic Garden (D960188) 1 Z126$ Hornstedtia scottiana (F.Muell.)
Schumann Nyleta Creek, 16 km NNE of Tully, North Qld. (Qld Herbarium AQ736255)
3
Z05* Kaempferia galanga L. Darwin Botanic Garden (D970234) 1 Z120 Kaempferia rotunda L. Flecker Botanic Gardens, Cairns 3 Z128$ Pleuranthodium racemigerum (F. Muell.)
R.M. Smith Gillies Range, c. 13 km NE of Yungaburra, North Qld. (Qld Herbarium AQ736263)
3
Z20* Pleuranthodium racemigerum (F. Muell.) R.M. Smith
Darwin Botanic Garden (D971598) 1
Z121 Renealmia cernua (Sw. ex Roem. & Schult.) J.F. Macbr.
Flecker Botanic Gardens, Cairns 3
Z01* Scaphochlamys biloba (Ridley) Holttum Darwin Botanic Garden (D950355) 1 Z16* Scaphochlamys kunstleri (Baker)
Holttum Darwin Botanic Garden (D950403) 1
Z122 Scaphochlamys kunstleri (Baker) Holttum
Flecker Botanic Gardens, Cairns 3
Z09* Tapeinochilos ananassae (Hassk.) K. Schum.
Darwin Botanic Garden (D971993) 1
Z125$ Tapeinochilos ananassae (Hassk.) K. Schum.
Mt. Bellenden Ker, North Qld. (Qld Herbarium AQ736256)
3
Z06* Zingiber longipedunculatum Ridley Darwin Botanic Garden (D970235) 1 Z45^ Zingiber officinale Roscoe ‘Queensland’ Maroochy Research Station, Nambour, Qld. 1 Z108/9 Zingiber officinale Roscoe Purchased fresh in produce store, Lismore
(NCM04-207) 2, 5
J-2 Zingiber officinale Roscoe ‘Jamaican’, diploid
Maroochy Research Station, Nambour, Qld. 4
J-4-4 Zingiber officinale Roscoe ‘Jamaican’, tetraploid
Maroochy Research Station, Nambour, Qld. 4
Z124 Zingiber ottensii Valeton Flecker Botanic Gardens, Cairns 3 Z02* Zingiber montanum (J. König) Link ex.
A. Dietr. Darwin Botanic Garden (D970237) 1
Z105 Zingiber montanum (J. König) Link ex. A. Dietr.
Southern Cross University (NCM04-122) 2, 5
Z17 Zingiber spectabile Darwin Botanic Garden (D95357) 1 Z50 Zingiber spectabile Southern Cross University 1 Z55^ Zingiber ‘Dwarf Apricot’ Southern Cross University 1 Z19* Zingiber/Etlingera ‘Aniseed Ginger’ Darwin Botanic Garden (D95331) 1
± refer to Section 6.2.2 for details of extraction methods ; ^: cultivated in tubs at Southern Cross University; #: cultivated in the grounds of Southern Cross University; *: extract prepared from frozen rhizome material; +: ex George Brown Botanical Garden, Darwin; $: collected in the wild under permit.
128
6.2.2 Extraction methods
Although it would have been ideal for all extracts to have been prepared in the exact same
manner, this was not possible for practical reasons. Plant material was obtained over a
period of several years from various sources, in fresh, frozen or dried form. In some
situations it was also decided to compare extracts made with different solvents. This section
details the extraction methods employed.
6.2.2.1 Extraction method 1 (ethanolic)
All extracts were prepared from frozen rhizome material, except that of Curcuma
australasica, which was prepared from freshly harvested material.
Where possible, material that had been grown at Southern Cross University under uniform
conditions was used, but in cases where plants did not grow successfully, a frozen sample of
the material grown in Darwin was used (Table 1).
Rhizomes were cleaned of dirt and old leaf bases, where present, were removed. A healthy
tissue sample comprising a transection of the rhizome was chopped finely into pieces
approximately 1 mm3 in size. To chopped samples weighing between 0.9 g and 7.0 g was
added double the mass of 99% ethanol. Samples were then sonicated for 20 minutes
(Ultrasonic Cleaner 50 Hz, Unisonics Pty Ltd, Manly Vale, Australia) and subsequently
centrifuged for 10 minutes at 4000 rpm (Hettich Universal 16A Centrifuge, Tuttlingen,
Germany). The supernatant was retained and filtered through Zymark/Millipore Automation
Certified Filters (glassfiber APFB 1.0 µm prefilter, hydrophilic PTFE membrane 0.45 µm).
One mL aliquots of the extracts were taken to dryness on a rotational vacuum concentrator
(Christ Alpha 2-4, Osterode, Germany) 1 h at 35°C, 240 mbar; 1 h at 35°C, 150 mbar; 1 h at
35°C, 90 mbar; 4.5h at 45°C, 15 mbar). The resulting dry extracts were dissolved in DMSO
to a final concentration of 50 mg/mL.
129
6.2.2.2 Extraction method 2 (ethanolic)
Fresh rhizomes were sliced and dried at 40°C (except sample Z109 which was dried at 90°C
in order to examine the effect of drying temperature). The dried rhizomes were ground to a
coarse powder in a Retsch SM 2000 hammermill (Haan, Germany) fitted with a 4 mm mesh
and extracted with 10 parts (by mass) ethanol. The plant material was macerated for 24
hours in a stoppered conical flask while being agitated on a Bioline Orbital Shaker BL 4236.
The resulting extract was filtered through filter paper (Whatman No. 1) in a Buchner
apparatus and the solvent removed under vacuum on a Büchi rotary evaporator (Rotavapor
R114, Büchi Labortechnik, Flawil, Switzerland). The dry extract was weighed, redissolved
in ethanol to a known concentration and stored at –20°C until use. The dry extract yield as
percentage of dry herb mass was calculated.
6.2.2.3 Extraction method 3 (ethanolic)
Fresh rhizomes were sliced and dried at 40°C. The dried rhizomes were ground to a coarse
powder in a Waring blender and extracted with 4 parts (by mass) ethanol. The plant material
was macerated for 24 hours in a stoppered conical flask while being agitated on a Bioline
Orbital Shaker BL 4236. The resulting extract was filtered through filter paper (Whatman
No. 1) in Buchner apparatus and stored –20°C. Prior to testing in bioassays a small quantity
of extract was dried under a nitrogen stream and resolved to a known concentration.
6.2.2.4 Extraction method 4 (ethanolic)
Approximately 100 g fresh rhizome material (from 3-4 different rhizomes and incorporating
different rhizome parts) was chopped finely (approximately 1 mm3 cubes) and macerated
with 2 parts (by mass) ethanol in an agitated conical flask for 24 hours as described above.
The resulting extract was filtered through filter paper (Whatman No. 1) in Buchner apparatus
and stored –20°C. Prior to testing in bioassays a small quantity of extract was dried under a
nitrogen stream and resolved to a known concentration.
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6.2.2.5 Extraction method 5 (aqueous)
Dried milled rhizome material (5.0 g) was macerated with 25 mL Milli-Q water in a
stoppered conical flask placed on an orbital shaker for 6 hours. The resulting extracts were
stored at –20°C. Prior to use, the extracts were spun down in a centrifuge, and filtered
through a double-layered glass filter-paper and a Millipore Millex-HN Nylon 0.45 µm filter.
6.2.2.6 Extraction method 6 (aqueous)
Freshly harvested rhizome and root tuber (5:1) were crushed in a mortar with a little water
and left to macerate for 15 min. The resulting aqueous juice was stored and filtered as
described for Extraction method 5.
6.2.3 Bioassay methods
6.2.3.1 Inhibition of PGE2 production
The bioassay was carried out in 96-well plates using Swiss albino mouse embryo fibroblast
cells 3T3 (American Type Culture Collection (ATTC), Manassas, VA) and a PGE2 enzyme
immuno-assay kit (Prostaglandin E2 EIA Kit – Monoclonal, Cayman Chemical Company,
Ann Arbor, MI, Catalog No. 514010).
Cell culture. Murine 3T3 fibroblasts were grown in 96-well plates (Nunclon, Thermo
Fischer Scientific, Rochester, NY). The growth medium consisted of Dulbecco’s Modified
Eagle Medium (colourless) containing 2 mM L-glutamine, 100 mM pyruvate, 4.5 g/L
glucose, 10% (v/v) foetal bovine serum and 100 U/mL each of penicillin G and
streptomycin. The concentration of the cell suspension was determined using a cell counter
(Beckman Coulter ActDiff Haematology Analyser, Fullerton, CA) and the suspension
diluted with growth medium to give a cell concentration of 2 105 cells/mL. 90 µL of this
suspension was pipetted into each well and the plate incubated at 37°C and 5% CO2 (Sanyo
CO2 MCO-17 AIC Incubator). After incubation for approximately 20 hours, plant extracts
and controls were added to the plate. In experiment series A plant extracts were tested a two
concentrations (50 mg/mL and 0.5 mg/mL, giving final well concentrations of 5000 µg/mL
and 500 µg/mL. In experiment series B plant extract were tested at three concentrations (1
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mg/mL, 0.5 mg/mL and 0.1 mg/mL, giving final well concentrations of 100 µg/mL, 50
µg/mL and 10 µg/mL). Ethanolic plant extracts were diluted in the growth medium to an
ethanol concentration of 1% before being added to the wells.
Acetylsalicylic acid (Sigma A5376) was used as positive control. After addition of samples
and controls the plate was incubated for a further 3 hours. After 3 hours incubation, 1 µL
calcium ionophore A21387 (calcimycin; Sigma C7522) solution (5 mM in DMSO) was
added to each well to stimulate PGE2 synthesis, the plate shaken gently on an orbital shaker
for 20 seconds and incubated for a final 20 minutes. Immediately following incubation, the
plate was centrifuged (Hettich Universal 16A Centrifuge, Tuttlingen, Germany) for 3
minutes at 1000 RCF and the supernatant transferred to a clean 96-well plate, sealed and
frozen at –80°C until the enzyme immunoassay was performed.
Enzyme immunoassay. The PGE2 enzyme immunoassay was carried out using the thawed
cell culture supernatant, which was diluted 500-fold with the buffer provided with the kit.
50µL of diluted sample was added to each well. In experiment series A samples and controls
were run in duplicate, in experiment series B in triplicate. A PGE2 standard was run at 8
concentrations in duplicate on each plate in all experiments. PGE2 acetylcholine esterase
tracer and PGE2 monoclonal antibody were added (50 µL of each to each well) and the
sealed plate incubated at 4ºC for 18-20 hours. Following incubation the plate was washed 5
times with the wash buffer provided in the kit, dried and Ellman’s Reagent was added (200
µL per well). The plate was sealed, covered in alfoil and placed on an orbital shaker
(moderate speed) for 75-90 minutes, after which the absorbance at 405 nm of the developed
plate was read in a Wallac Victor 2 Plate Reader (Perkin Elmer, Waltham, MA). All
samples, standards and controls were assayed in triplicate.
Data analysis. Raw data were obtained as concentrations of PGE2 (pg/well). Standard
curves for PGE2 were plotted and R2 values calculated to confirm linearity. Mean, standard
deviation and coefficient of variance were calculated for replicates of samples, standards and
controls. Inhibition of PGE2 release by stimulated 3T3 cells was reported as percentage
inhibition compared with stimulated cells treated with solvent controls.
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6.2.3.2 Oxygen Radical Absorbance Capacity (ORAC)
This assay is based on the work of Cao and Prior (Cao & Prior, 1999).
Ethanolic plant extracts were taken to dryness under a nitrogen stream and resolved and
diluted in AWA solvent (acetone 70.0 : Milli-Q water 29.5 : acetic acid 0.5) to give
concentrations of 100 µg/mL, 50 µg/mL, 25 µg/mL and 12.5 µg/mL.
Trolox was used as a standard and BHT as a positive control; stock solutions of both in 75
mM phosphate buffer pH 7.4 were diluted in AWA solvent to give Trolox concentrations of
100 µM, 50 µM, 25 µM, and 12.5 µM and butylated hydroxytoluene (BHT) concentrations
of 500 µM, 250 µM, 125 µM, and 62.5 µM.
The assay was carried out in black 96-well fluorescence assay plates (Perkin Elmer
Optiplate). Into each well was added 10 µL fluorescein solution (6.0 x 10-7 M in 75 mM
phosphate buffer [NaH2PO4 17.25 g : Na2HPO4 86.25 g, adjusted to pH 7.4]); 20 µL sample,
Trolox, BHT or solvent control solution; and 170 µL AAPH (2,2’-azobis-2-methyl-
propanimidamide, dihydrochloride; Cayman) solution (20 mM in 75 mM phosphate buffer,
pH 7.4). Fluorescein control wells received 10 µL fluorescein solution and 190 µL
phosphate buffer. Immediately after the addition of the AAPH solution, the assay plates
were placed in Wallac Victor 2 Plate Reader (Perkin Elmer, Waltham, MA) and the
fluorescence recorded at 37°C every minute for 35 minutes with the integral of these
readings representing the total antioxidant capacity. All samples, standards and controls
were assayed in quadruplicate.
Data analysis. Mean and standard deviation were calculated for replicates. Standard curves
for Trolox standards were plotted and R2 values calculated to confirm linearity.
6.2.3.3 Inhibition of nitric oxide production
This assay measures the release of nitric oxide by murine monocytes stimulated by
lipopolysaccharide (LPS).
Cell culture. RAW264 murine leukemic monocyte-macrophages (ATCC, Manassas, VA)
were grown in clear 96-well plates (Nunclon). The growth medium was the same as that
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used in the PGE2 assay (see above). 100 µL of cell suspension (106 cells/mL) was added to
each well and the plate incubated at 37°C and 5% CO2 (Sanyo CO2 MCO-17 AIC Incubator).
After incubation (approx. 20 hours), 15 µL of plant extracts and controls were added to the
wells and the plate incubated for a further 1 hour. Ethanolic plant extracts were diluted in
the growth medium to an ethanol concentration of 1% before being added to the wells.
Extracts were screened at 10% and 20% of stock extract concentrations, which ranged from
35 – 50 mg/mL. One active extract was also assayed at 2% and 1% of stock extract
concentration. After incubation (1 hour), 5 µL of LPS solution (1 µg/mL in growth medium)
was added to each well and the plate returned to the incubator for a further 18 – 20 hours. At
the completion of the incubation period, the plate was centrifuged (3 min. at 1500 RCF) and
50 µL of the supernatants transferred to a clear assay plate (Spectra Plate, PerkinElmer).
Nitrite assay. Nitrite standards (7) were prepared by serial dilution of sodium nitrite in Milli-
Q water; standards were further diluted 10-fold in growth medium (final nitrite well
concentrations ranging from 1 to 500 µM). 50 µL of each standard concentration was
transferred to the designated wells. 50 µL of Griess Reagent (0.1% N-1-
napthylethylenediamine dihydrochloride, 1% sulphanilic acid in 5% phosphoric acid) was
added to all wells on the plate, the content mixed gently by placing the plate on an orbital
shaker (ensuring no bubbles were present), and the plate incubated at room temperature for
15 – 20 min protected from light. Following incubation the absorbance at 550 nm was read
in a Wallac Victor 2 Plate Reader. All samples, standards and controls were assayed in
triplicate.
Data analysis. Mean and standard deviation were calculated for replicates. Standard curves
for nitrite were plotted and R2 values calculated to confirm linearity. The nitric oxide
(nitrite) production in sample wells was calculated as a percentage of the production in
solvent control wells.
6.2.3.4 Modulation of natural killer cell activity
Target cells
Target cells were K562 cells (ATCC, Monassas, VA, USA), a leukemic cell line derived
from an individual with chronic myeloid leukaemia in terminal blast crisis. K562 cells lack
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major-histocompatibility-complex class I and II antigens, which makes them a sensitive
target for human NK cells. K562 cells were stored under liquid nitrogen and prior to use
rapidly thawed in 37°C water bath, then incubated in medium (RPMI-1640 (Invitrogen,
Auckland, New Zealand) 87%, FBS 10% (Invitrogen), L-glutamine (Invitrogen) 1%,
Penicillin/Streptomycin (Invitrogen) 2%) for 1 hour at 37°C and 5% CO2 (Sanyo CO2 MCO-
17 AIC Incubator). Following incubation, the cell suspension was centrifuged for 5 minutes
at 120 RCF at room temperature, the supernatant discarded, and the cell pellet resuspended
in 1 mL RPMI-1640. Cells were then stained with 5 µL of a fluorescent membrane dye
(DiO Vybrant Cell Labelling Solution, Molecular Probes, Eugene, OR, USA, cat. no. V-
22886), incubated for a further 5 minutes at 37°C and 5% CO2, then centrifuged for 5
minutes at 120 RCT and washed twice with medium to remove excess dye before being
resuspended in 1 mL medium. The cell concentration of the suspension was determined
using an electronic cell counter (Beckman Coulter ActDiff Haematology Analyser, Fullerton,
CA) and adjusted with medium to 0.1 109 cells/L.
Effector cells
Human peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood
collected by venesection into 4-mL Lithium-Heparin collection tubes. The whole blood was
diluted 1:1 with sterile phosphate-buffered saline (PBS), layered in 15-mL Falcon tubes on a
cushion of Isopaque-Ficoll (Amersham Biosciences, Uppsala, Sweden), and centrifuged for
20 minutes at 700 RCT (no brake, 0 deceleration). Following centrifugation, the upper
serum layer was removed, the PBMC layer collected and transferred to a clean 15-mL
Falcon tube, to which 12 mL of PBS was added. This cell suspension was centrifuged for 10
minutes at 250 RCF, the supernatant removed, and the PBMC pellet resuspended in 1 mL
medium. The cell concentration of this suspension was determined and adjusted with
medium to between 1 109 and 5 109 cells/L, dependent on the NK cell activity of the
donor blood used.
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Pre-incubation of effector cells with extracts
In the case of aqueous extracts, 196 µL of cell suspension was incubated with 4 µL extract.
In order to minimise the solvent effects of the ethanolic extracts, 398 µL of PBMC
suspension was incubated with 2 µL extract for 2 hours at 37°C and 5% CO2.
Incubation of effector cells with target cells
Following the pre-incubation of effector cells with test extracts, effector and target cell
suspensions were combined in 5-mL Falcon tubes and incubated for 2 hours at 37°C/5%
CO2. For the ethanolic extracts, 200 µL of the pre-incubated effector cell suspension was
combined with 200 µL target cell suspension, resulting in a final effector to target cell ratio
of approximately 25:1 (subsequently reduced to 12:1 due to high activity of NK cells) and a
solvent concentration of 0.25%. For the aqueous extracts, 100 µL effector cell suspension
was combined with 200 µL target cell suspension and 100 µL medium, resulting in an
effector : target cell ratio of approximately 12:1 and a solvent concentration of 0.5%. No-
treatment and solvent controls were included in each experiment. After incubation for 2
hours the tubes were placed on ice (protected from light) followed by the addition of 100 µL
DNA stain (propidium iodide, Molecular Probes, Eugene, OR, cat. no. P-3566; 20 µL
stock/mL in PBS, final propidium iodide concentration 4 µg/mL) to each tube, after which
the tubes were again placed on ice for 5 minutes, then assayed within 30 minutes.
Control tubes containing target cell suspension and test extract but no effector cells were run
for all samples and the no-treatment and solvent controls in order to monitor any mortality of
the target cells caused by the extracts and/or solvent, independent of effector cell activity.
The kill rate of the control was subtracted from the kill rate of each test sample (see below).
Flow cytometric assay
The assay was carried out on a FACSCalibur (Becton Dickinson, Franklin Lakes, NJ) flow
cytometer linked to a MacIntosh computer OS 9.1 running CellQuest Pro Software (Becton
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Dickinson). The percentage kill of target cells was based on a total count of 2500 target
cells. The specific cytotoxicity was calculated as follows:
Specific cytotoxicity (%) = Dead target cells(Sample) (%) − Dead target cells(Control)
6.3 Results
6.3.1 Inhibition of PGE2 production
A total of 42 samples representing 41 taxa and 39 species from a total of 14 genera were
screened for inhibition of PGE2 production. Samples were tested at two or three
concentrations. The data thus generated did not allow for accurate determination of IC50
values, but estimates were made where possible based on the available data (Table 6-2). For
the full data set, refer to Appendix B.
Only 9 of the 42 samples demonstrated 50% inhibition of PGE2 in the concentration range
tested. The species showing the most potent and dose-dependent inhibition were Alpinia
galanga, Boesenbergia rotunda, Curcuma australasica, C. longa, C. parviflora, Kaempferia
galanga, Pleuranthodium racemigerum, Zingiber officinale and Z. montanum. Most of these
are known medicinal plants, but nothing has been reported previously about the biological
activity of the two native Australian species, C. australasica and P. racemigerum.
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Table 6-2. Inhibition of PGE2 production in 3T3 murine fibroblasts.
Estimated IC50 values are shown. NA: the estimated maximum inhibition was less than 50%. Concentrations refer to final concentrations in assay well. n=3 expect: * (n=2), + (n=4) and ^ (n=6).
Taxon ID Estimated IC50 (µg/mL) Curcuma longa^ Z106 c. 10 Curcuma australasica^ Z101 c. 50 Curcuma parviflora Z118 c. 50 Alpinia galanga Z103 >92 Boesenbergia rotunda Z104 c. 94 Pleuranthodium racemigerum^ Z128 50-100 Zingiber officinale Z108 50-100 Zingiber montanum Z105 50-100 Zingiber montanum* Z02 c. 500 Kaempferia galanga* Z05 c. 500 Alpinia calcarata* Z49 >100 Alpinia mutica* Z08 >500 Alpinia purpurea 'Eileen McDonald'* Z11 >500 Alpinia zerumbet* Z52 >500 Elettaria cardamomum* Z12 >500 Hedychium coronarium* Z10 >500 Scaphochlamys biloba* Z01 >500 Zingiber longipedunculatum* Z06 >500 Zingiber spectabile Z17 >500 Zingiber/Etlingera (Aniseed ginger)* Z19 >500 Alpinia arctiflora Z129 NA Alpinia caerulea Z102 NA Alpinia luteocarpa Z111 NA Alpinia malaccensis* Z48 NA Alpinia modesta Z127 NA Alpinia spectabile 'Giant Orange'* Z53 NA Costus barbatus Z112 NA Costus leucanthus Z113 NA Costus malortieanus Z114 NA Costus productus Z116 NA Costus pulverulentus Z115 NA Costus tappenbeckianus* Z15 NA Curcuma cordata Z117 NA Etlingera australasica Z107 NA Etlingera elatior 'Burma torch'* Z04 NA Hornstedtia scottiana Z126 NA Kaempferia rotunda Z120 NA Renealmia cernua Z121 NA Scaphochlamys kunstleri* Z122 NA Tapeinochilos ananassae Z125 NA Zingiber ottensii Z124 NA Zingiber sp. 'Dwarf Apricot'+ Z55 NA
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6.3.2 Oxygen Radical Absorbance Capacity (ORAC)
Fifteen species were tested for antioxidant activity in the ORAC assay (Table 6-3).
Table 6-3. Oxygen radical absorbance capacity (ORAC).
Antioxidant activity is expressed in micromol Trolox equivalents (TE). Values shown are means±SEM for between 2 and 4 concentrations; each concentration was done in 4 replicates. ^: Drying temperature for plant material.
Taxon ID TE (µmol) per g extract
TE (µmol) per g dry herb equivalent
Curcuma longa Z106 3504±1141 528±172
Zingiber officinale (40°C)^ Z108 4166±63 262±4
Zingiber montanum Z105 2707±509 156±29
Boesenbergia rotunda Z104 2889±338 155±18
Zingiber officinale (90°C)^ Z109 3323±65 131±3
Alpinia galanga Z103 933±36 61±2
Curcuma australasica Z101 2357±479 54±11
Hornstedtia scottiana Z126 2284±275 47±6
Zingiber ottensii Z124 757±37 40±2
Tapeinochilos ananassae Z125 1257±125 37±4
Curcuma parviflora Z118 1452±191 37±5
Etlingera australasica Z107 778±75 36±4
Pleuranthodium racemigerum Z128 1418±221 34±5
Alpinia caerulea Z102 390±36 23±2
Alpinia luteocarpa Z111 1540±253 22±4
Scaphochlamys kunstleri Z122 944±60 18±1
The samples most active in this assay were Curcuma longa, Zingiber officinale, Z.
montanum and Boesenbergia rotunda. This was the case whether the samples were ranked
by Trolox Equivalence (TE, µmol) per gram extract or TE per gram dried herb equivalent.
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6.3.3 Inhibition of nitric oxide production
Eight species were tested for their ability to inhibit nitric oxide production in RAW264 cells
(Table 6-4).
Table 6-4. Inhibition of nitric oxide production in LPS-stimulated RAW264 macrophages.
Values are percent inhibition compared with solvent control ± SEM. Concentrations shown are final concentrations in assay well.
Taxon ID Concentration (µg/mL)
Percent inhibition of nitric oxide
Alpinia galanga Z103 4.6 57±4 9.2 78±2 23.0 100±2 46.0 92±3 92.1 97±2 Curcuma australasica Z101 34.7 86±7 69.4 95±1 Zingiber officinale Z109 50.2 85±5 100.3 95±1 Boesenbergia rotunda Z104 47.3 67±1 94.52 100±3 Alpinia caerulea Z102 47.6 57±9 95.1 63±1 Zingiber montanum Z105 50.2 35±2 100.3 72±1 Curcuma longa Z106 49.7 35±1 99.3 51±2 Etlingera australasica Z107 46.4 0±0 92.7 16±1
Several of the tested extracts showed potent inhibition on nitric oxide at the concentrations
tested. At a concentration of 95-100 µg mL-1 Boesenbergia rotunda, Curcuma australasica
and Zingiber officinale caused almost complete inhibition of nitric oxide production. The
most potent of the extracts was Alpinia galanga, which demonstrated dose-dependent
inhibition in the range 5-92 µg mL-1 with complete inhibition achieved at 23 µg mL-1.
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6.3.4 Modulation of natural killer cell activity
The results of the screening of 9 ethanolic extracts for modulation of NK cell activity are
shown in Table 6-5.
Table 6-5. Effect of ethanolic extracts on natural killer cell activity against K562 leukaemia cells.
^: Drying temperature for plant material.
Taxon ID Sample conc.
(µg/mL)
Conc. in pre-incubation tube with
effector cells (µg/mL)
Increase in specific cytotoxicity
compared with solvent control (%)
Untreated control 88.6 Solvent control (ethanol)
0.5% 0.0
Boesenbergia rotunda Z104 188.4 0.94 18.5 Curcuma longa Z106 198.0 0.99 10.8 Zingiber officinale (90°C)^
Z109 200.0 1.00 8.6
Etlingera australasica Z107 184.8 0.92 6.8 Zingiber officinale (40°C)^
Z108 173.6 0.87 0.0
Zingiber montanum Z105 200.0 1.00 -4.6 Curcuma australasica Z101 138.4 0.69 -4.9 Alpinia caerulea Z102 189.6 0.95 -4.9 Alpinia galanga Z103 91.8 0.46 -28.4
None of the ethanolic extracts tested produced a strong increase in NK cell activity. The
most effective extracts were Boesenbergia rotunda and Curcuma longa, which increased NK
cell activity by 18.5% and 10.8%, respectively. Alpinia galanga (tested at half the
concentration of the other extracts) caused an almost 30% decrease in activity.
Eight aqueous extract were also assayed for modulation of NK cell activity (Table 6-6).
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Table 6-6. Effect of aqueous extracts on natural killer cell activity against K562 leukaemia cells.
*: percentage of stock extract. ^: Drying temperature for plant material.
Taxon ID Sample conc.*
Increase in specific cytotoxicity compared
with solvent control (%)
Untreated control -2.0 Solvent control (ethanol) 0.0 Curcuma longa Z106 0.1% 11.3 0.2% 10.7 0.4% 12.8 1.0% 6.5 10.0% 8.0 Curcuma australasica Z101 0.1% 2.0 1.0% 0.3 10.0% -0.5 Etlingera australasica Z107 0.1% -0.9 1.0% -9.9 10.0% 1.2 Zingiber officinale (40°C)^ Z108 0.1% 1.0 1.0% -11.9 10.0% 7.0 Zingiber officinale (90°C)^ Z109 0.1% -1.9 1.0% 9.9 10.0% -0.7 Zingiber montanum Z105 0.1% -0.3 1.0% 2.9 10.0% -11.9 Boesenbergia rotunda Z104 0.1% -6.0 1.0% -3.9 10.0% -18.8 Alpinia galanga Z103 0.1% 1.5 0.2% 9.2 0.4% 4.4 1.0% 0.2 10.0% -77.0
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None of the aqueous extracts stimulated NK cell activity against K562 cells in a substantial
or dose-dependent manner. Alpinia galanga and to a lesser extent Boesenbergia rotunda
dramatically decreased the activity of natural killer cells at the highest concentration tested
(10% of stock extract), while these concentrations did not kill the target cells.
6.4 Discussion
6.4.1 Inhibition of PGE2 production
This study has for the first time demonstrated in vitro PGE2 inhibition by extracts of the two
native Australian Zingiberaceae, Curcuma australasica and Pleuranthodium racemigerum.
The estimated IC50 for the two extracts were c. 50 µg mL-1 and c. 75 µg mL-1, respectively,
which was similar to that of Zingiber officinale. These findings suggested the presence of
compounds with potent biological activity, and the extracts were therefore subsequently the
subject of bioactivity-guided fractionation (see Chapter 7). This is also the first report of
PGE2 inhibition by the South-east Asian species Curcuma parviflora.
Panduratin A isolated from Boesenbergia rotunda (syn. Kaempferia pandurata) has
previously been show to inhibit PGE2 and nitric oxide production as well as iNOS and COX-
2 expression in RAW264.7 cells (Yun et al., 2003), but the present study is the first to
demonstrate PGE2 inhibition by a crude extract of the plant.
PGE2 inhibition has also previously been shown for an extract of Zingiber montanum (syn. Z.
cassumunar) and several phenylbutanoid compounds isolated from this species (Jeenapongsa
et al., 2003; Jiang et al., 2006b; Panthong et al., 1997).
Extracts of Zingiber officinale, Curcuma longa and to a lesser degree Alpinia galanga were
included in this assay primarily as positive controls, as they are well established inhibitors of
PGE2 production in vitro and hence confirmed the validity of the assay.
The inhibitory effects of ginger (Z. officinale) and several ginger constituents on COX
activity and subsequent PGE2 production are well known and have been demonstrated in
isolated enzyme assays, cell-based models and animals. The early work by Kiuchi and
colleagues found that [6]-gingerol, [6]-gingerdione, [10]-gingerdione, [6]-
dehydrogingerdione and [10]-dehydrogingerdione isolated from fresh ginger were potent
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inhibitors of COX in vitro with IC50 values comparable to that of indomethacin (Kiuchi et
al., 1982). Subsequent work by the same group reported potent inhibition by [10]-gingerol,
[6]-shogaol, [10]-gingerol, [6]-acetylgingerol, [6]-shogaol, [6]-dehydrogingerdione, [10]-
dehydrogingerdione, [6]-gingerdione and [10]-gingerdione, many of which had IC50 values
<2.5 µM (Kiuchi et al., 1992), and [8]-paradol was shown to be a potent inhibitor of COX in
vitro with an IC50 of 4 µM (Nurtjahja-Tjendraputra et al., 2003).
Although gingerols (in particular [6]-gingerol) are commonly referred to as the main active
component in ginger extracts, there are clearly numerous other compounds with anti-
inflammatory activity present. This was demonstrated by a recent study from the University
of Arizona showing that no correlation existed between PGE2 inhibitory activity of ginger
extracts and their gingerol content (Jiang et al., 2006b). This study found IC50 values for
methanolic ginger extracts ranging from 0.058 to 0.629 µg mL-1, which is much lower than
the estimated IC50 obtained in the present study (50-100 µg mL-1). This difference may be
due to the different cell lines and assay methods used in the two studies (Jiang and
colleagues used the human promonocytic cell line U937 and an immunoassay kit from R&D
System, while the present study used murine 3T3 fibroblasts and the PGE2 immunoassay kit
from Cayman Chemical Co.). The magnitude of difference seems unlikely to be due to
phytochemical differences between the plant materials used in the two studies.
The same University of Arizona study also found methanolic extracts of Alpinia galanga,
Zingiber montanum and Z. spectabile to be potent inhibitors of PGE2 production (IC50 values
of 0.06, 7.68 and 1.17 µg mL-1, respectively (Jiang et al., 2006b). In the present study A.
galanga and Z. montanum were both found to have some inhibitory activity (albeit again at
much higher concentrations), while Z. spectabile was not active.
Curcuma longa was the most potent inhibitor of PGE2 activity in the present study (IC50 = c.
10 µg mL-1). Rhizome extracts have been shown to inhibit PGE2 production in HL-60 cells
(IC50 = 0.92 µg mL-1) (Lantz et al., 2005), and the principal constituent, curcumin, is a well
documented inhibitor of COX expression and PGE2 production (Funk et al., 2006; Hong et
al., 2004; Ireson et al., 2001).
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6.4.2 Oxygen Radical Absorbance Capacity (ORAC)
The most potent plant materials in terms of oxygen radical absorbance capacity (ORAC)
were turmeric (Curcuma longa) and ginger (Zingiber officinale) with values of 528 µmol TE
and 262 µmol TE per gram dry herb equivalent, respectively. The antioxidant properties of
both of these plants are well known (refer to Chapter 2, Sections 2.2.1.3.1 and 2.3.1.2.2.1).
Another study from the same laboratory reported a very similar ORAC value (557 µmol TE
per gram dry herb equivalent) for a turmeric extract prepared using a sequential three-solvent
extraction process (Wojcikowski et al., 2007). Another study reported an ethanolic extract
of fresh turmeric as having an ORAC value of 19.5 µmol TE per gram fresh weight (Tilak et
al., 2004). With fresh turmeric rhizome typically having a water content of about 75% (data
not shown), this is equivalent to approximately 80 µmol TE per gram dry herb. This value is
considerably lower than the one obtained in the present study (528 µmol TE), but differences
in extraction method and efficiency (stirring versus sonication) may at least partly explain
the discrepancy. Quantitative phytochemical differences between the two plant samples may
also have contributed.
One study has reported an ORAC value of 148 µmol TE per gram for fresh ginger (Zingiber
officinale) (Ninfali et al., 2005). Given that ginger rhizome typically has a water content
close to 90% (data not shown), this is equivalent to an ORAC value of approximately 1500
µmol TE per gram dry herb equivalent, which is an order of magnitude greater than the value
obtained in the present study (262 µmol TE per gram for ginger dried at 40° C). This
difference may reflect that the fresh rhizome has significantly greater antioxidant capacity
than the dried, but phytochemical differences in the plant material assayed and differences in
experimental methodology could also be factors.
It is interesting to note the difference in ORAC value for the two ginger samples, which were
prepared from the same raw material. Z108 was dried at 40° C, while Z109 was dried at 90°
C. Z108 had twice the ORAC value of Z109 (262 versus 131 µmol TE per gram dry herb
equivalent), clearly demonstrating that antioxidant activity is lost when plant material is
dried at high temperature. This is not unexpected, as oxidative processes catalysed by high
temperature are likely to deplete antioxidant capacity.
145
Curcuminoids isolated from Zingiber montanum (syn. Z. cassumunar) have been reported to
possess potent antioxidant activity (Masuda & Jitoe, 1994; Nagano et al., 1997). In the
present study the crude rhizome extract was found to be quite potent in the ORAC assay
(ORAC value of 156 µmol TE per gram dry herb equivalent).
Boesenbergia rotunda had an ORAC value similar to that of Z. montanum. B. rotunda (syn.
B. pandurata) has previously demonstrated potent antioxidant activity in a rat brain
homogenate assay, and several antioxidant compounds including panduratin A have been
isolated (Shindo et al., 2006).
Galangal (Alpinia galanga) has been shown to exert antioxidant activity in raw minced beef
(Cheah & Abu, 2000) but has not previously been tested in the ORAC assay. The observed
activity (61 µmol TE per gram dry herb equivalent) was quite modest.
None of the other species tested have been reported as having antioxidant activity. The two
native Australian species that displayed good activity in the PGE2 assay, Curcuma
australasica and Pleuranthodium racemigerum, had only modest antioxidant activity in the
ORAC assay (54 and 34 µmol TE per gram dry weight equivalent, respectively).
6.4.3 Inhibition of nitric oxide production
The ethanolic extract of Alpinia galanga was found to be a potent inhibitor of nitric oxide
production. At a concentration of 4.6 µg mL-1 it caused 57% inhibition, which corresponds
well with a reported IC50 = 7.3 µg mL-1 for an 80% aqueous acetone extract in LPS-activated
mouse peritoneal macrophages (Morikawa et al., 2005). 1’S-1’-acetoxychavicol acetate
isolated from A. galanga was found to be active in a peritoneal macrophage assay (Matsuda
et al., 2005).
Inhibition of nitric oxide production in vitro has been demonstrated for several other Alpinia
species and a considerable number of compounds isolated from them. These include
diarylheptanoids, a curcuminoid and flavonoids from A. blepharocalyx (Kadota et al., 1996;
Prasain et al., 1998), diarylheptanoids from A. officinarum (Lee et al., 2006; Matsuda et al.,
2006) and sesquiterpenes from A. oxyphylla (Muraoka et al., 2001). Inhibition of inducible
146
nitric oxide synthase (iNOS) has been reported for isolates from A. katsumadai (Hong et al.,
2002), A. officinarum (Yadav et al., 2003) and A. oxyphylla (Chun et al., 2002).
A methanolic extract of Curcuma longa (10 µg mL-1) was reported to cause 88% inhibition
of nitric oxide production in LPS-stimulated RAW264.7 cells (Hong et al., 2002). In
comparison, the present study found only 35% inhibition by C. longa at a concentration of
50 µg mL-1 (and 51% inhibition at 99 µg mL-1).
Inhibition of nitric oxide production and suppression of iNOS expression has previously
been demonstrated for methanolic extracts of C. longa, C. comosa and C. zedoaria
(Camacho-Barquero et al., 2007; Hong et al., 2002; Jang et al., 2004; Jantaratnotai et al.,
2006). Active compounds identified from these species include curcumin from C. longa and
two sesquiterpenoids from C. zedoaria. The sesquiterpenoid xanthorrhizol isolated from C.
xanthorrhiza has also been shown to inhibit iNOS activity and expression (Chung et al.,
2007; Lee et al., 2002).
In the present study ginger (Zingiber officinale) extract was a potent inhibitor of nitric oxide
at the concentrations tested (50 and 100 µg mL-1), while Z. montanum was less potent.
While inhibitory effects on nitric oxide production have not previously been reported for the
latter, this activity has been described for Z. officinale in osteoarthrotic sow chondrocytes
(Shen et al., 2005), and [6]-gingerol (the major pungent compound in ginger) has shown
dose-dependent inhibition of nitric oxide production and reduction in iNOS in LPS-
stimulated J774.1 mouse macrophages (Ippoushi et al., 2003). From other Zingiber species,
sesquiterpenoids from Z. zerumbet and the labdane diterpene aframodial from Z. mioga have
been shown to inhibit the expression of iNOS (Kim et al., 2005a; Murakami et al., 2002).
6.4.4 Modulation of natural killer cell activity
Only modest increases in NK cell activity were observed in this study for certain extracts,
while others were inactive. Best results were seen for the ethanolic extracts of Boesenbergia
rotunda (18.5% increase compared with solvent control) and Curcuma longa (10.8%
increase). The aqueous extract of C. longa also produced a modest increase (about 10%), but
this was not dose dependent in the concentration range tested. In contrast, both the ethanolic
extract of Alpinia galanga and the aqueous extract of fresh Boesenbergia rotunda caused
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marked inhibition of NK cell activity (by 28.4% and 18.8%, respectively). There are no
previous reports concerning the effects of A. galanga or B. rotunda on NK cell activity.
Curcumin (0.01 µg mL-1) has previously been shown to significantly increase the
cytotoxicity of human NK cells ex vivo, using K562 cells as a target (Yadav et al., 2005).
However, in that study effector cells were incubated with curcumin (solvent unknown) for 18
hours as opposed to 2 hours in the present study, and the effector cells were subsequently
incubated with the target cells for 4 hours compared with 2 hours in the present study.
Curcumin was also found to partially reverse breast tumour exosome-mediated inhibition of
NK cell tumour cytotoxicity in vitro (Zhang et al., 2007). However, NK cell activity did not
change significantly compared with controls in rats fed dietary curcumin (1, 20 or 40 mg per
kg bodyweight) for 5 weeks (South et al., 1997).
In the present study the presence of ethanol even at a very low concentration (0.25%) in the
target cell suspension caused a significant (47%) decrease in specific NK cell cytotoxicity
compared with the untreated control. The sizeable effect of the solvent may have affected
the results of this assay for the ethanolic extracts. Further work should explore the use of
alternative extraction solvents (less polar than water) that may interfere less with the assay
than does ethanol.
6.4.5 Conclusion
This study has confirmed the Zingiberaceae as a rich source of compounds with interesting
biological and pharmacological actions. Of some 40 species tested in at least one assay, the
established medicinal species turmeric (Curcuma longa) and ginger (Zingiber officinale)
emerged as the most active. Two other plants with traditional medicinal uses, galangal
(Alpinia galanga) and Boesenbergia rotunda also showed good activity in several assays.
Of the 41 taxa tested turmeric was the most potent inhibitor of PGE2, and it was also the best
antioxidant in the ORAC assay. It caused only moderate inhibition of nitric oxide
production, but other studies suggest that this may be an aberrant result. The well known
ability of ginger to inhibit PGE2 production in vitro was confirmed, and ginger also showed
good antioxidant capacity, although rhizome material dried at 90° C had only half the
activity of material dried at 40° C. Ginger was also a potent inhibitor of nitric oxide
148
production, but like turmeric did not affect NK cell activity significantly. Boesenbergia
rotunda inhibited PGE2 production and showed antioxidant activity. The ethanolic extract of
the dried material caused an increase in NK cell activity, while an aqueous extract of the
fresh material caused a marked decrease. The explanation for this is unknown and warrants
further investigation.
Of particular interest was the screening of seven native Australian species in the PGE2 assay,
as none of these had previously been investigated for pharmacological activity. Two of these
species, Curcuma australasica and Pleuranthodium racemigerum, showed good activity in
this assay and were selected for further investigations (see Chapter 7).
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7. BIOACTIVITY-GUIDED FRACTIONATION OF TWO NATIVE AUSTRALIAN ZINGIBERACEAE
7.1 Introduction
In the previous chapter, two native Australian species, Curcuma australasica and
Pleuranthodium racemigerum, were identified as having significant PGE2 inhibitory activity
in vitro. In this respect they demonstrated potency similar to well known medicinal species
such as Curcuma longa and Alpinia galanga.
Due to the paucity of information about the chemistry and pharmacological activity of both
Curcuma australasica and Pleuranthodium racemigerum, these species were selected for
further work with a view to extending the knowledge of the Zingiberaceae and about native
Australian plants.
The process of bioactivity-guided fractionation was applied and led to the isolation of
pharmacologically active compounds from both species. Inhibition of PGE2 was used as the
primary bioassay in this process, but fractions with high activity in this assay were also
tested for cytotoxic properties, primarily to ensure that the activity observed in the cell-based
PGE2 assay was not simply due to cytotoxic effects. Testing for cytotoxicity included the
murine cell line P388D1, which in the laboratory’s experience is highly sensitive to cytotoxic
agents. It should be noted that significant cytotoxic activity may potentially be of interest in
itself.
From C. australasica two known bioactive sesquiterpene compounds, zederone and
furanodien-6-one were isolated. In the case of P. racemigerum a novel curcuminoid
compound with significant pharmacological activity was isolated and structurally
characterised. The isolated compounds could potentially become candidates for further work
and preclinical testing.
150
7.2 Materials and Methods
7.2.1 Plant material
Fresh rhizome of Curcuma australasica was obtained from the George Brown Botanic
Gardens in Darwin (S 12° 27’; E 130° 50’) as a gift from Dr Greg Leach who also identified
the material.
Pleuranthodium racemigerum was collected in rainforest (828 m above sea level) in the Wet
Tropics Region of North Queensland in Gillies Range (S 17° 13'; E 145° 40') under a permit
issued by the Queensland Environmental Protection Agency. Voucher specimens have been
deposited in the Medicinal Plant Herbarium at Southern Cross University (NCM05-047) and
the Queensland Herbarium (AQ736263).
7.2.2 Extraction methods
7.2.2.1 Curcuma australasica
Fresh rhizomes were cleaned, sliced and extracted in double the mass of ethanol (99.7%) in a
sonicating bath for 30 min. The extract was filtered through filter paper (Whatman No. 3) in
a Buchner apparatus. The biomass was again covered with ethanol, left to steep for 22 hours
at 5° C, sonicated for 10 min, then filtered. The two filtrates were combined, and the extract
taken to dryness under vacuum on a Büchi Rotavapor R-114 (Switzerland) with the water
bath temperature at 40° C. The resulting semi-dry extract was dried on a rotational vacuum
concentrator for 48 hours at –88° C and 0.140 mbar (Christ Alpha 2-4, Osterode, Germany).
The dry extract was stored at –20° C until use.
7.2.2.2 Pleuranthodium racemigerum
Fresh rhizomes were cleaned, sliced and dried at 40° C. The dried rhizomes were ground to
a coarse powder in a Waring blender and extracted with 4 parts (by mass) ethanol. The plant
material was steeped for 24 hours in a stoppered conical flask while being agitated on a
Bioline Orbital Shaker BL 4236 (Edwards Instrument Company, Australia). The resulting
151
extract was filtered through a Quickfit glass filter and stored at –20° C. Prior to
fractionation, the extract was dried under vacuum on a Büchi Rotavapor R-114 (Switzerland)
with the water bath temperature at 45° C. The resulting extract, which was oily, was
redissolved in 91% aqueous methanol and partitioned with hexane in a separating funnel.
The methanolic phase was dried under vacuum. Prior to testing in bioassays a small quantity
of extract was dried under a nitrogen stream and redissolved to a known concentration.
7.2.3 Fractionation by preparative HPLC
7.2.3.1 Curcuma australasica
The dry extract was redissolved in 75% aqueous methanol (1 part extract to 10 parts solvent
by mass) and filtered through an Acrodisc CR PTFE 0.45 µm microfilter (Pall Gelman Lab.,
Ann Arbor, MI). Fractionation was carried out on a Gilson Preparative HPLC system fitted
with a Gilson FC204 Fraction Collector and an Altima C-18 5 µm column (150 mm long;
internal diameter 22 mm) (Alltech, Kentucky). Mobile phase A consisted of HPLC-grade
water obtained from an in-house Milli-Q system (Waters, Milford, MA), mobile phase B
consisted of HPLC-grade methanol (EM Science, Gibbstown, NJ); both contained 0.05%
trifluoroacetic acid (TFA). The mobile phase gradient was A:B (70:30, v/v) to A:B (5:95,
v/v) over 20 min followed by A:B (5:95, v/v) for 10 min, with a flow rate of 20 mL/min.
UV-VIS detection was at 210 nm and 360 nm. Twenty-nine 0.8-minute fractions were
collected, commencing at 2 min. Another fractionation of the same extract was carried out,
this time collecting 18 0.5-min fractions, starting at 12 min (3 1-mL injections of sample
were fractionated). Fractions were dried on a rotational vacuum concentrator for 48 hours at
–88° C and 0.140 mbar (Christ Alpha 2-4, Osterode, Germany).
7.2.3.2 Pleuranthodium racemigerum
The methanolic extract residue (700 mg) was redissolved in 3.5 mL acetonitrile with a few
drops of water and fractionated on a Gilson Preparative HPLC system as described in the
previous section. Mobile phase A consisted of HPLC-grade water, mobile phase B consisted
of HPLC-grade acetonitrile; both contained 0.05% TFA. The gradient eluting mobile phase
152
was A:B (70:30, v/v) to A:B (5:95, v/v) over 20 min followed by A:B (5:95, v/v) for 5 min,
with a flow rate of 20 mL/min. UV-VIS detection was at 210 nm and 360 nm. Twenty-nine
1-minute fractions were collected, commencing at 1 min. Two 1-mL injections of the
sample were fractionated. Another fractionation of the same extract was carried out, again
collecting 29 1-min fractions. Fractions were dried on a rotational vacuum concentrator as
described in the previous section.
7.2.4 Nuclear magnetic resonance spectroscopy
Nuclear magnetic resonance (NMR) spectra were obtained using a Bruker AVANCE
DRX500 (1H at 500.13 MHz; 13C at 125.77 MHz; 5mm QNI probe) spectrometer with
XWin-NMR software. The 1H and 13C NMR spectra were recorded using deuterated
chloroform (CDCl3) with the residual solvent peaks as reference (7.27 ppm for 1H and 77.2
ppm for 13C). In one case deuterated pyridine (pyridine-d5) was used instead (residual
solvent peaks 8.74 ppm for 1H and 150.35 ppm for 13C). The chemical shifts were expressed
in parts per million (ppm) as δ values and the coupling constants (J) in Hertz (Hz). All
experiments were carried out using the Bruker pulse programs.
7.2.4.1 One-dimensional NMR spectroscopy
7.2.4.1.1 1H NMR
The 1H NMR spectra were obtained for the isolated compounds. The chemical shifts,
coupling constants, peak intensities and splitting patters of each of the proton signals
provided information about the type of protons present in the molecule and their chemical
environments (Williams & Fleming, 1989).
7.2.4.1.2 J-modulated 13C NMR
The carbon resonances were distinguished according to their proton attachments (C, CH,
CH2, CH3) using the APT pulse sequence. The J-modulated 13C NMR spectra displayed the
153
CH3 and CH resonances arbitrarily pointing down and inverted with respect to the CH2 and
the quaternary carbons, which were arbitrarily pointing up (Sanders & Hunter, 1987).
7.2.4.2 Two-dimensional homonuclear correlation NMR spectroscopy
In the two-dimensional experiments, the 1H NMR spectra were displayed along both axes
and the proton correlations were indicated in the contour plot by cross-peaks along the
diagonal.
7.2.4.2.1 1H-1H Correlation spectroscopy (COSY)
This two-dimensional technique was used to correlate the chemical shifts of 1H nuclei that
were coupled to one another. A magnitude sequence with a final pulse angle of 45°
(COSY45) was used to obtain a COSY spectrum. The cross-peaks produced indicated which 1H nuclei were J-coupled.
7.2.4.3 Two-dimensional heteronuclear correlation spectroscopy
7.2.4.3.1 Heteronuclear single quantum correlation (HSQC) spectroscopy
This two-dimensional method was used to determine which 1H nuclei were bonded to which 13C nuclei (1JCH) in a molecule using an INEPT pulse sequence.
7.2.4.3.2 Heteronuclear multiple-bond correlation (HMBC) spectroscopy
This two-dimensional method was used for determining long-range 1H-13C connectivities
and was useful in determining the C assignments in a molecule through its correlation with a
proton.
154
7.2.5 Determination of accurate mass
A sample of the isolated Compound 3 from Pleuranthodium racemigerum was sent to the
School of Molecular and Microbial Sciences, University of Queensland, where determination
of accurate mass by high-resolution mass spectroscopy was carried out by Mr Graham
Macfarlane.
Electron impact ionization (EI) experiments were conducted on a Kratos MS25 RFA
instrument via a direct insertion probe at 70 eV and source temperature of 200° C.
Perfluorokerosene (PFK) was used as reference for magnet scan accurate mass in EI.
Electrospray ionization (ESI) experiments were conducted on a Finnigan MAT 900 XL-Trap
instrument with a Finnigan API III electrospray source, using MeOH as the solvent and
polypropylene glycol and polyethylene glycol as references for accurate mass data, acquired
by electric sector scan.
7.2.6 UV spectroscopy
UV absorbance data were obtained on a Hewlett-Packard Spectrophotometer 8453 (Palo
Alto, CA) controlled by UV-Visible ChemStation software (Rev. A.0803; Dayton, Ohio).
Absorbance data were acquired in the range 190-1100 nm at 1 nm intervals. The novel
Compound 3 from Pleuranthodium racemigerum was dissolved in methanol at a
concentration of 0.014 mg/mL (4.723 10–5 M). The determination of peak absorption
(λmax) was based on the mean value of five measurements. The extinction coefficient
(wavelength-dependent molar absorptivity coefficient), ε, was calculated from the Beer-
Lambert Law, A = ε c d, where A is absorbance, c the molar concentration and l the
distance (cuvette path) in cm.
7.2.7 Cytotoxicity assays
Cytotoxicity in 3T3 murine fibroblasts, P388D1 murine lymphoblasts, Caco-2 human
colonic adenocarcinoma, PC3 human prostate adenocarcinoma, HepG2 human hepatocyte
carcinoma, and MCF7 human mammary adenocarcinoma (all cell lines originating from the
155
American Type Culture Collection, ATCC) was assayed in 96-well plates using the ATPLite
kit (PerkinElmer, Waltham, MA) with chlorambucil and curcumin as reference compounds.
Cells were cultivated in the appropriate medium and test and reference compounds added at
five different concentrations and incubated for 24 hours (5% CO2). All samples were
assayed in triplicate.
The ATPLite assay was carried out according to the manufacturer’s instructions. Briefly, the
kit components were equilibrated to room temperature. The lyophilised substrate solution
was reconstituted with buffer and this reagent added to the plate containing the cells
incubated with test substances and standards. The plate was then shaken on an orbital
microplate shaker (700 rpm) for 2 minutes before luminescence was measured on a Wallac
Microbeta Scintillation and Luminescence Counter (PerkinElmer, Waltham, MA). ATP was
quantified using an ATP standard curve based on luminescence measurements of ATP
standards. Median lethal dose (LD50) values were calculated using Excel 2003 (Microsoft
Corporation, Redmond, WA) and GraphPad Prism 4 (San Diego, CA).
7.2.8 PGE2 assay
Inhibition of PGE2 production by 3T3 murine fibroblast cells (ATCC) was assayed using the
Prostaglandin E2 EIA Kit (Cayman Chemical, Ann Arbor, MI) as described in the previous
chapter.
7.3 Results
7.3.1 Curcuma australasica
7.3.1.1 Activity-guided fractionation
The extract was profiled by LC-MS prior to fractionation (Fig. 7-1).
156
min2 4 6 8 10 12 14 16 18 20
mAU
-100
102030405060
DAD1 A, Sig=210,8 Ref=off (D:\020821A\011-0101.D)
min2 4 6 8 10 12 14 16 18 20
mAU
-5
0
5
10
15
DAD1 C, Sig=280,8 Ref=off (D:\020821A\011-0101.D)
min2 4 6 8 10 12 14 16 18 20
0
20000
40000
60000
80000
MSD1 TIC, MS File (D:\020821A\011-0101.D) APCI, Pos, Scan, Frag: 150, "pos 150"
Fig. 7-1. LC-MS chromatogram of Curcuma australasica extract redissolved in methanol. Top: 210 nm; centre: 280 nm; bottom: total ion chromatogram.
Based on the chromatograms of the whole extract and the individual fractions, some
fractions were combined before being tested for their ability to inhibit PGE2 production in
murine fibroblasts. The results of this assay are shown in Table 7-1.
157
Table 7-1. Inhibition of PGE2 production in 3T3 murine fibroblast cells by fractions of Curcuma australasica extract. The final concentration of test substances in the assay well was 1mg/mL.
Fraction no. Percent inhibition
(mean±SD)
1 16±8
2 12±9
3-9 18±11
10 19±3
11-13 13±11
14 26±5
15 47±6
16 80±10
17 82±6
18 64±4
19 57±8
20 61±9
21-28 17±15
6-gingerol 94±4
8-gingerol 96±3
10-gingerol 92±2
8-shogaol 82±5
Two fractions, F16 and F17, showed strong inhibition of PGE2 production (80% and 82%
inhibition, respectively). F17 proved to contain a single major compound, Compound 1
(Figs. 7-2, 7-3), while F16 contained multiple compounds and included overlap with the
compound in F17. On this basis it was decided to proceed with structural elucidation of the
major compound in F17.
158
min16 16.5 17 17.5 18 18.5 19 19.5 20 20.5
mAU
0250500750
100012501500
DAD1 A, Sig=210,8 Ref=off (D:\020830\001-0101.D)
min16 16.5 17 17.5 18 18.5 19 19.5 20 20.5
mAU
050
100150200250300
DAD1 C, Sig=280,8 Ref=off (D:\020830\001-0101.D)
min16 16.5 17 17.5 18 18.5 19 19.5 20 20.50
2000000
4000000
6000000
8000000
10000000
MSD1 TIC, MS File (D:\020830\001-0101.D) APCI, Pos, Scan, Frag: 150, "pos 150"
Fig. 7-2. LC-MS chromatogram for Compound 1 (Fraction 17) from Curcuma australasica.
m/z100 200 300
0
20
40
60
80
100
*MSD1 SPC, time=19.660:19.785 of D:\020830\001-0101.D APCI, Pos, Scan, F
Max: 3.24134e+006
139
.2
201
.3
229
.2 2
30.1
245
.1 2
47.2
248
.1
nm200 225 250 275 300 325 350 375
mAU
0
200
400
600
800
1000
DAD1, 19.742 (1158 mAU,Dn1) of 001-0101.D
Fig. 7-3. Mass spectrum (M+1; left) and UV spectrum (right) for Compound 1 (Fraction 17) from Curcuma australasica.
Another active fraction, F20, was found to consist of another almost pure compound
(Compound 2), which was also the subject of structural elucidation, although it displayed
less potent activity in the PGE2 assay (61% inhibition). The LC-MS chromatogram for F20
is shown in Fig. 7-4 and the mass and UV spectra in Fig. 7-5.
159
min18 19 20 21 22 23
mAU
0
500
1000
1500
2000
2500
DAD1 A, Sig=210,8 Ref=off (D:\020829\008-0801.D)
min18 19 20 21 22 23
mAU
0
500
1000
1500
2000
DAD1 C, Sig=280,8 Ref=off (D:\020829\008-0801.D)
min18 19 20 21 22 230
10000002000000300000040000005000000600000070000008000000
MSD1 TIC, MS File (D:\020829\008-0801.D) APCI, Pos, Scan, Frag: 150, "pos 150"
Fig. 7-4. LC-MS chromatogram for Compound 2 (Fraction 20) from Curcuma australasica.
m/z100 200 300
0
20
40
60
80
100
*MSD1 SPC, time=22.680:22.804 of D:\020829\008-0801.D APCI, Pos, Scan, F
Max: 4.14733e+006
149
.1
213
.3
231
.3 2
32.2
nm200 225 250 275 300 325 350 375
mAU
0
200
400
600
800
1000
1200
1400
DAD1, 22.818 (1588 mAU,Dn1) of 008-0801.D
Fig. 7-5. Mass spectrum (M+1; left) and UV spectrum (right) for Compound 2 (Fraction 20) from Curcuma australasica.
160
7.3.1.2 Cytotoxic activity of Compound 1
In order to ensure that the observed activity in the PGE2 was not simply due to cytotoxic
effects, Compound 1 was also tested for cytotoxicity in the sensitive P388D1 murine
lymphoma cell line and compared to a number of other compounds and extracts (Table 7-2).
Table 7-2. Cytotoxicity of Compound 1 from Curcuma australasica in P388D1 murine lymphoma cells.
Values are mean±SD percentage survival compared to DMSO-treated (n=3).
Test substance Percent survival (mean±SD) 500 µg/mL 150 µg/mL 100 µg/mL 50 µg/mL 10 µg/mL
DMSO (control) 100±8 100±8 100±8 100±8 100±8 Alpinia galanga 1±0 - - 48±7 - Alpinia caerulea 68±2 - - 78±6 - Zingiber officinale 48±6 - - 68±5 - Curcuma longa 28±1 - - 62±1 - Curcuma australasica 34±3 - - 72±3 - Compound 1 - - 25±1 - 77±6 Curcumin - - 1±0 - 61±4 6-shogaol - - 1±0 - 50±5 Chlorambucil - 40±2 - - -
Compound 1 was found to be considerably less cytotoxic than curcumin and [6]-shogaol.
The cytotoxicity of the crude extract of Curcuma australasica was similar to that of Zingiber
officinale and less than that of C. longa.
7.3.1.3 Structural elucidation of Compound 1 and 2
Compounds 1 and 2 were the subjects of structural elucidation experiments by NMR
spectroscopy.
161
Compound 1 was identified as the sesquiterpene ketone zederone (Fig. 7-6) by comparison
of its spectral data with those reported in the literature (Hikino et al., 1966; Shibuya et al.,
1987). These data are presented in Table 7-3.
O
OO
43
21
10
15
98
7
6
12
13
115
14
Fig 7-6. Structure of zederone (Compound 1).
162
Table 7-3. 1H and 13C NMR spectral data of Compound 1 (zederone) from Curcuma australasica.
δ = chemical shift (ppm), int = integration, mult = multiplicity, J = coupling constant, s = singlet, d = doublet, dd = doublet of a doublet, ddd = doublet of a doublet of a doublet, dddd = doublet of a doublet of a doublet of a doublet, dt = doublet of a triplet, dq = doublet of a quartet, m = multiplet, br = broad
* Assignment could have been interchanged.
Compound 2 was identified as another sesquiterpene ketone, 1(10)E,4E-furanodien-6-one,
also by comparison with spectral data from the literature (Dekebo et al., 2000; Hikino et al.,
1975; Makabe et al., 2006). These data are presented in Table 7-4.
Differences greater than 1 ppm exist between the observed δC values and those reported by
Dekebo et al. (2000) for C-6 (1.2 ppm), C-7 (1.8 ppm), C-9 (1.3 ppm) and C-11 (1.2 ppm),
but these can be reasonably explained by the differences in instrumentation. It should be
Compound 1 CDCl3
Zederone (Shibuya et al. 1987) CDCl3
Position δC 126 MHz δH 500 MHz (int, mult, J in Hz)
δH, 500 MHz
1 131.5 5.49, 1H, dd (11.9, 4.0) 5.48, dd (11.5, 4.0) 2 24.9 2.25, 1H, m
2.53, 1H, m 2.23, br d 2.52, dddd (11.5, 12.0, 3.5, 13.5)
3 38.2 1.29, 1H, m 2.31, 1H, dt (13.0, 3.5)
1.29, ddd (13.5, 4.0, 13.0) 2.29, ddd (13.0, 3.5, 3.5)
4 64.2 5 66.8 3.82, 1H, s 3.81, s 6 192.5 7 123.5* 8 157.4 9 42.1 3.73, 2H, dd (16.5) 3.69, 3.75 (16.5)
10 131.3 11 122.5* 12 138.3 7.10, 1H, s 7.08, q (1.2) 13 10.5 2.13, 3H, d (1.2) 2.11, d (1.2) 14 15.4 1.35, 3H, s 1.34, br s 15 15.9 1.61, 3H, s 1.60, br s
163
noted that one of the coupling constants of the olefinic proton H-1 was found to be 11.5 Hz.
Dekebo et al. (2000) and Hikino et al. (1975) reported coupling constants of 6.6 and 4.5 Hz,
and 7.5 and 7.5 Hz, respectively. The large J value observed with this compound (2) has to
be a coupling of H-1 to one of the protons at the 2-position (H-2), probably the proton
resonating at 2.32 ppm (CDCl3) and for the J value to be that high, both H-1 and one of the
methylene protons must have assumed an axial-axial orientation. With the methyl group at
the 10-position trans to H-1, and the methyl group at the 4-position trans to H-5, the
structure could have formed a rigid configuration making H-1 almost axial to one of the
methylene protons of H-2.
165
The observed 1H and 13C chemical shift assignments for this compound (2) agree with the
literature values for 1(10)E,4E-furanodien-6-one as shown in Table 7-4. The structure of
1(10)E,4E-furanodien-6-one is shown in Fig. 7-7.
O
O
1
2
34 5 6
78
9
10
11
12
1314
15
Fig. 7-7. Structure of 1(10)E,4E-furanodien-6-one (Compound 2) from Curcuma australasica.
There are other reported isomers of this compound such as the 1(10)E,4Z isomer, which is
isofuranodienone (Hikino et al., 1975) and the 1(10)Z,4Z isomer (Brieskorn & Noble, 1983).
The NMR spectral data for these two isomers are summarised in Table 7-5. It is evident
from inspection of Table 7-4 and Table 7-5 that the NMR data of the isolated compound (2)
are consistent with those of the E,E isomer.
166
Table 7-5. 1H NMR spectral data from the literature for the isomers isofuranodienone and 1(10)Z,4Z-furanodien-6-one.
δ = chemical shift (ppm)
Isofuranodiene
Hikino et al. (1975) CDCl3
1(10)Z,4Z-furanodien-6one
Brieskorn & Noble (1983) CDCl3
Position δH 100 MHz δH, 90 MHz
1 5.20 5.29 5 6.10 5.88 9 3.13, 3.49 3.34
12 7.00 7.08 13 1.91 2.20 14 1.89 1.76 15 1.55 1.65
167
7.3.2 Pleuranthodium racemigerum
7.3.2.1 Activity-guided fractionation
An HPLC chromatogram of the extract redissolved in 90% aqueous methanol is shown in
Fig. 7-8.
min2 4 6 8 10 12 14 16 18 20
mAU
0200400600800
100012001400
DAD1 A, Sig=210,8 Ref=off (Z:\CHROMO~1\001-0101.D)
min2 4 6 8 10 12 14 16 18 20
mAU
0
200
400
600
800
1000
DAD1 C, Sig=280,8 Ref=off (Z:\CHROMO~1\001-0101.D)
min2 4 6 8 10 12 14 16 18 20
mAU
-15
-10
-5
0
5
DAD1 E, Sig=360,8 Ref=off (Z:\CHROMO~1\001-0101.D)
Fig. 7-8. LC-MS chromatogram of Pleuranthodium racemigerum extract redissolved in 90% aqueous methanol. Top: 210 nm; centre: 280 nm; bottom: 360 nm.
Based on the chromatograms of the whole extract and the individual fractions, fractions were
combined prior to testing for their ability to inhibit PGE2 production in murine fibroblasts.
The results of this assay are shown in Table 7-6.
168
Table 7-6. Inhibition of PGE2 production in 3T3 murine fibroblast cells by combined fractions of Pleuranthodium racemigera extract. Fractions and reference compounds were dissolved in DMSO at a concentration of 10mg/mL prior to being assayed. * Concentrations are final concentrations of test substance in assay well.
Combined fraction no. (fractions) Percent inhibition (mean±SD)
1µg/mL* 5µg/mL* 10µg/mL*
C1 (1-8) -35±3 -20±3 -33±3
C2 (9-13) 4±0 17±2 27±1
C3 (14-16) 10±1 24±6 28±3
C4 (17-18) 15±1 41±9 47±4
C5 (19-22) -1±0 19±2 -4±0
C6 (23-29) -5±0 13±1 -11±2
Aspirin (0.05mM*) 30±8
The most potent combined fraction, C4, was found to consist of a pure compound
(Compound 3). At a concentration of 10 µg/mL (subsequently calculated to be equivalent to
33.7 µM), this compound inhibited PGE2 production in 3T3 cells by close to 50%.
The LC-MS chromatogram for Compound 3 is shown in Fig. 7-9 and the mass and UV
spectra in Fig. 7-10.
169
min10 11 12 13 14 15 16 17
mAU
0
500
1000
1500
2000
DAD1 A, Sig=210,8 Ref=off (C:\DOCUME~1\ALLUSE~1\DOCUME~1\NEWCPP~1\DATA20~3\0509DATA\050929\021-0301.D)
min10 11 12 13 14 15 16 17
mAU
0
500
1000
1500
2000
DAD1 C, Sig=280,8 Ref=off (C:\DOCUME~1\ALLUSE~1\DOCUME~1\NEWCPP~1\DATA20~3\0509DATA\050929\021-0301.D)
min10 11 12 13 14 15 16 170
5000
10000
15000
20000
25000
30000
MSD1 TIC, MS File (C:\DOCUME~1\ALLUSE~1\DOCUME~1\NEWCPP~1\DATA20~3\0509DATA\050929\021-0301.D) APCI, Pos, Scan, Fra
Fig. 7-9. LC-MS chromatogram for Compound 3 from Pleuranthodium racemigerum.
m/z100 200 300 400
0
20
40
60
80
100
*MSD1 SPC, time=15.620:15.637 of C:\DOCUME~1\ALLUSE~1\DOCUME~1\NEWCPP~1\DA
Max: 6092 297
.2
107
.2 1
21.2
298
.4 2
87.2
147
.2
122
.4
189
.2
105
.2
213
.2
288
.4
351
.2
nm200 225 250 275 300 325 350 375
mAU
0
250
500
750
1000
1250
1500
1750
DAD1, 15.722 (1948 mAU,Dn1) of 021-0301.D
Fig. 7-10. Mass spectrum (M+1; top) and UV spectrum (bottom) for Compound 3 from Pleuranthodium racemigerum.
Compound 3 was subjected to further testing for cytotoxic activity and structural elucidation.
170
7.3.2.2 Cytotoxic activity of Compound 3
Compound 3 was screened for cytotoxic effect in 3T3 murine fibroblast cells. As
considerable cytotoxicity was detected (LD50 = 52.8 µM) after incubation for 3 hours (Fig 7-
11), further testing was undertaken.
Cytotoxicity of Compound 3 against 3T3 cells after 3 h
-20%
0%
20%
40%
60%
80%
100%
120%
0 20 40 60 80 100
Conc (ug/mL)
% In
hibi
tion
Fig. 7-11. Cytotoxic effect of fraction Compound 3 from Pleuranthodium racemigerum on 3T3 murine fibroblasts.
Compound 3 was tested for cytotoxic activity against one other murine cells line (P388D1
lymphoblast) and four human cell lines (Caco-2 colonic adenocarcinoma, PC3 prostate
adenocarcinoma, HepG2 hepatocyte carcinoma, and MCF7 mammary adenocarcinoma).
The cytotoxicity of Compound 3 was compared with that of the related compound curcumin
and the chemotherapeutic drug chlorambucil. The results are shown in Fig. 7-12.
171
Caco-2 (Human colonic adenocarcinoma)
-20%
0%
20%
40%
60%
80%
100%
0 1 10 100 1000
ug/mL
% in
hibi
tion
Compound 3CurcuminChlorambucil
PC3 (Human prostate adenocarcinoma)
-20%
0%
20%
40%
60%
80%
100%
0 1 10 100 1000
ug/mL
% in
hibi
tion
Compound 3CurcuminChlorambucil
HepG2 (Human hepatocyte carcinoma)
-20%
0%
20%
40%
60%
80%
100%
0 1 10 100 1000
ug/mL
% in
hibi
tion
Compound 3CurcuminChlorambucil
MCF7 (Human mammary adenocarcinoma)
-20%
0%
20%
40%
60%
80%
100%
1 10 100 1000
ug/mL
% in
hibi
tion
Compound 3CurcuminChlorambucil
P388 (Murine lymphoblast)
-20%
0%
20%
40%
60%
80%
100%
1 10 100 1000
ug/mL
% in
hibi
tion
Compound 3CurcuminChlorambucil
Fig. 7-12. Dose-response curves for cytotoxic activity of Compound 3 from Pleuranthodium racemigerum against five cell lines.
Curcumin was included for comparison and chlorambucil as a positive control.
172
LD50 values based on the dose-response curves were calculated for Compound 3 and curcumin (Table 7-7).
Table 7-7. LD50 (µM) values for cytotoxic activity of Compound 3 and curcumin against five cancer cell lines. 95% confidence intervals are shown in brackets
Cell line Compound 3 Curcumin
Caco-2 44.8 (32.6-61.8) 48.6 (38.9-60.4)
PC3 23.6 (20.6-26.9) 22.7 (17.7-28.9)
HepG2 40.6 (28.0-58.9) 43.8 (25.6-75.2)
MCF7 56.9 (45.3-71.1) 51.5 (34.8-76.0)
P388D1 117.0 (53.4-255.6) 75.7 (65.9-86.8)
The cytotoxic effects of Compound 3 closely reflected those of curcumin in the four human
cancer cell lines. In the case of the murine P388D1 cell line the LD50 for Compound 3 was
55% higher than for curcumin.
7.3.2.3 Structural elucidation of Compound 3
The NMR data obtained for Compound 3 from Z128 Pleuranthodium racemigerum are
summarised in Table 7-8.
173
Table 7-8. 1H and 13C NMR data from one-dimensional (1H and J-modulated 13C NMR) and two-dimensional correlation (COSY, HSQC and HMBC) NMR spectroscopy experiments on Compound 3 from Pleuranthodium racemigerum. δ = chemical shift (ppm), int = integration, mult = multiplicity, J = coupling constant, s = singlet, d = doublet, t = triplet, dt = doublet of a triplet, tt = triplet of a triplet
Position Chemical shift JHH (COSY) HMBC (ppm)
δH, 500 MHz (int, mult, J in Hz)
δC 126 MHz
2JCH 3JCH 4JCH
1 3.29 d (6.4) 38.3 H-2 133.4 129.6
129.6 131.6
32.5
2 5.55 dt (15.2, 6.4) 129.6 H-1, H-3 38.3 131.6
32.5 133.4
3 5.51 dt (15.2, 6.4) 131.6 H-2, H-4 32.5 129.6
29.2 38.3
4 2.07 dt (6.4, 7.3) 32.5 H-3, H-5 29.2 131.6
31.4 129.6
38.3
5 1.43 tt (7.3, 7.7) 29.2 H-4, H-6 32.5 35.0 131.6
6 1.61 dt (7.7, 7.8) 31.4 H-5, H-7 29.2 35.0
32.5 135.1
7 2.55 t (7.8) 35.0 H-6 31.4 135.1
29.2 129.6
1’ - 135.1 - 2’ 7.04 d (8.5) 129.6 H-3’ 115.3 35.0
129.6 153.8
3’ 6.76 d (8.5) 115.3 H-2’ 153.8 115.3 135.1
4’ - 153.8 - 5’ 6.76 d (8.5) 115.3 H-6’ 153.8 115.3
135.1
6’ 7.04 d (8.5) 129.6 H-5’ 115.3 35.0 129.6 153.8
1” - 133.4 - 2” 7.11 d (8.7) 129.6 H-3” 114.0 38.3
129.6 158.0
3” 6.86 d (8.7) 114.0 H-2” 158.0 114.0 133.4
4” 158.0 - 5” 6.86 d (8.7) 114.0 H-6” 158.0 114.0
133.4
6” 7.11 d (8.7) 129.6 H-5” 114.0 38.3 129.6 158.0
–OCH3 3.82 s 55.5 - 158.0
174
The 1H NMR spectrum showed signals in the region of 6.7-7.2 ppm, indicative of aromatic
protons. Signals were consistent with two aromatic rings.
Ring A. It was observed that the proton at 6.76 ppm (H-3’/H-5’) showed coupling to the
proton at 7.04 ppm (H-2’/H-6’) with a J value (coupling constant) of 8.5 Hz, suggesting
ortho-coupling. These signals each integrated to two protons, suggesting two sets of
identical protons in an aromatic ring.
The HSQC showed that the protons H-3’/H-5’ and H-2’/H-6’ were directly correlated to the
carbon signals at 115.3 and 129.6 ppm, respectively. HMBC showed long-range correlations
of the protons to the quaternary carbon signals at 153.8 ppm (C-4’) and 135.2 ppm (C-1’),
which made up the aromatic ring system with the carbon at position 4’ having a hydroxyl
substituent, as signified by its resonance in the downfield region (Fig. 7-13).
Fig. 7-13. Heteronuclear correlations in Ring A.
Ring B. Similar to what was observed for Ring A, the proton at 6.86 ppm (H-3”/H-5”)
showed coupling to the proton at 7.11 ppm (H-2”/H-6”) with a J value of 8.7 Hz, indicative
of ortho-coupling. Again, these signals integrated to two protons each, suggesting the
presence of a second aromatic ring.
Analogous to the findings for Ring A, the HSQC showed protons H-3”/H-5” and H-2”/H-6”
to be directly correlated to the carbon signals at 114.0 ppm and 129.6 ppm, respectively, and
HMBC showed long-range correlations to quaternary carbon signals at 158.0 ppm (C-4”)
and 133.4 ppm. In contrast to Ring A, a singlet that integrated to 3H was observed in the 1H
spectrum resonating at 3.82 ppm, consistent with the presence of a methoxy substituent.
HMBC showed correlation of the methoxy protons to the quaternary carbon at 158.0 ppm
(C-4”), placing the methoxy group at the C-4” position (Fig. 7-14).
1' 2' 3'4'
5'6'OH
HH
HH
175
Fig. 7-14. Heteronuclear correlations in Ring B.
Carbon bridge. The proton signals at 5.55 ppm (H-2) and 5.51 ppm (H-3) indicated olefinic
protons coupling to each other in an E position (J = 15.2 Hz). Five methylene proton signals
(H-1, H-4, H-5, H-6 and H-7) were also present in the region from 1.4 ppm to 3.3 ppm, and
the COSY experiment showed that H-1 and H-4 were vicinal to H-2 and H-3, respectively.
H-5 also showed JHH correlation to H-4 and H-6, and H-6 to H-7, as shown in Fig. 7-15.
HMBC showed correlation of H-1 to the quaternary carbon at 133.4 ppm (C-1”), which
connected C-1 to C-1”. Likewise, H-7 showed correlation to the quaternary carbon at 135.1
ppm (C-1’), linking C-7 to C-1’.
The structure of Compound 3 as shown in Fig. 7-15 was given the systematic name 1-(4”-
methoxyphenyl)-7-(4’-hydroxyphenyl)-2E-heptene. The APCI-MS data showed a (M+1)+
value of 297.2, which is consistent with a molecular formula of C20H24O2.
H3CO OH
1
2
3
4
5
6
71'
2'3'
4'
5'6'
1"
2"3"
4"
5"6"
Fig. 7-15. Chemical structure of Compound 3 from Pleuranthodium racemigerum, 1-(4”-methoxyphenyl)-7-(4’-hydroxyphenyl)-2E-heptene.
OH3C
1"2"
3"4"
5" 6"
H
HH
H
176
7.3.2.4 Accurate Mass
The accurate mass for the Compound 3 was experimentally determined to be 319.1683,
which is consistent with the formula C20H24O2Na and the theoretical mass of 319.1674 as
shown in Table 7-9. This confirmed the molecular formula of the novel Compound 3 as
C20H24O2.
Table 7-9. Accurate mass determination for Compound 3.
Mass determined by MS C20H24O2Na
319.1683
Theoretical mass* C20H24O2Na
319.1674
Difference (δ) 0.0009 Exact mass of C20H24O2* 296.18 Molecular weight of C20H24O2* 296.40
* Data from ChemDraw Pro v. 4.01 (CambridgeSoft Corporation, Cambridge, Massachusetts, 1997)
7.3.2.5 UV spectrophotometric data and extinction coefficient
The UV spectrum of the novel Compound 3, 1-(4”-methoxyphenyl)-7-(4’-hydroxyphenyl)-
2E-heptene, is shown in Fig. 7-16. The peak absorption (λmax) of the compound was at 203
nm, with other absorption maxima at 225 and 278 nm.
177
Wavelength (nm)200 225 250 275 300 325 350
Abs
orba
nce
(AU
)
0
0.2
0.4
0.6
0.8
1
1.2 203
225
278
334
245
214
Fig. 7-16. UV spectrum of 1-(4”-methoxyphenyl)-7-(4’-hydroxyphenyl)-2E-heptene (Compound 3).
The extinction coefficient (wavelength-dependent molar absorptivity coefficient), ε, was
calculated for the novel Compound 3 from Pleuranthodium racemigerum using ε = A/(c
l), where A is the absorbance, c the molar concentration (4.7233468 10–5 M), and l the
distance (cuvette path; 1 cm). The peak absorbance readings and extinction coefficients ε for
Compound 3, 1-(4”-methoxyphenyl)-7-(4’-hydroxyphenyl)-2E-heptene at 203 nm, 225 nm
and 278 nm are shown in Table 7-10.
Table 7-10. Peak absorption (λmax) and extinction coefficients (ε) of Compound 3 at 203 nm, 225 nm and 278 nm.
Abs203nm Abs225nm Abs278nm
1 1.19440 0.82368 0.16267 2 1.19610 0.82355 0.16243 3 1.18990 0.82464 0.16273 4 1.19790 0.82434 0.16282 5 1.19570 0.82616 0.16307 Mean 1.19480 0.82447 0.16274 SD 0.00301 0.00105 0.00023 ε 2.53 x 104 1.75 x 104 3.45 x 103
log(ε) 4.40 4.24 3.54
178
7.4 Discussion
Biologically active compounds were successfully isolated from two native Australian
Zingiberaceae species using bioactivity-guided fractionation. Bioactivity-guided
fractionation is a widely used method in natural products research. It is an effective means
of isolating the most active major compounds in a complex mixture, while it might be less
suitable for the identification of highly active compounds that occur in very low
concentrations. As the present work was of a preliminary nature, carried out on species that
had never been studied previously, bioactivity-guided fractionation was deemed an
appropriate methodology.
7.4.1 Curcuma australasica
From Curcuma australasica the sesquiterpene ketones zederone (Compound 1, Fig. 7-6) and
furanodien-6-one (Compound 2, Fig. 7-7) were isolated for the first time. Zederone has
previously been reported from the rhizomes of C. zedoaria (Christm.) Roscoe (Hikino et al.,
1966; Makabe et al., 2006) and C. phaeocaulis Valeton (Hou et al., 1997), and from
Chloranthus serratus (Thunb.) Roem. et Schult. (Chloranthaceae) (Kawabata et al., 1985),
while furanodien-6-one has been reported from C. zedoaria (Hikino et al., 1975; Makabe et
al., 2006), Commiphora molmol Engler (myrrh) (Brieskorn & Noble, 1983) and other
Commiphora species (Dekebo et al., 2000).
Zederone was isolated from the almost pure fraction of Curcuma australasica that most
potently inhibited the production of PGE2, suggesting this compound confers anti-
inflammatory activity to C. australasica. Zederone was found to be moderately cytotoxic
against P388D1 cells, but its toxicity was less than that of both curcumin and [6]-shogaol.
Compared with zederone, furanodien-6-one was a less potent inhibitor of PGE2. These
findings are in contrast to those of Makabe et al. (2006), who tested 11 sesquiterpenoids
isolated from C. zedoaria for in vivo anti-inflammatory activity in the 12-O-
tetradecanoylphorbol-13-acetate (TPA)-induced mouse ear oedema model and found
furanodien-6-one but not zederone to be a potent topical anti-inflammatory agent. As
oedema induced by TPA is reduced or delayed by COX inhibitors (Young et al., 1983), an
anti-inflammatory effect of zederone in this model would have been expected, given the
179
good in vitro activity in the PGE2 seen in the present study. Given the purity of Fraction 17
from which zederone was isolated, it seems unlikely that a compound other than zederone
contributed significantly to the inhibition of PGE2. It is possible that the seemingly
incongruent results obtained in the two studies reflect differences between an in vitro model
and an in vivo situation, or that stability issues played a role.
7.4.2 Pleuranthodium racemigerum
From Pleuranthodium racemigerum, the extract of which was a potent inhibitor of PGE2
production (see Chapter 6), a novel bioactive curcuminoid compound was isolated
(Compound 3, Fig. 7-15). This compound, 1-(4”-methoxyphenyl)-7-(4’-hydroxyphenyl)-2E-
heptene, also strongly inhibited PGE2 production (IC50 ≅ 34 µM). In addition, it displayed
considerable dose-dependent cytoxicity against four human and two murine cells lines. Its
potency as a cytotoxic agent was similar to that of the related curcuminoid compound,
curcumin. These findings suggest this novel compound could warrant further investigations,
both as a potential anti-inflammatory agent and as a cytotoxic agent. Whether its
cytotoxicity precludes it from being used as an anti-inflammatory agent would need to be
determined in the first instance by animal studies, but the fact that the cytotoxic potency
resembled that of curcumin suggests that it may exert anti-inflammatory activity in vivo at
sub-toxic concentrations.
This is the first report concerning the chemistry and pharmacology of Pleuranthodium
racemigerum. As this species grows in rainforest and has a limited distribution, it is doubtful
that wild stock would be able to sustain a potential development of this plant as a source of a
new medicinal substance, but the plant could potentially be cultivated in tropical areas,
should further work indicate that an efficacious and safe medicine can be derived from it.
180
8. CONCLUDING REMARKS This chapter discusses the findings in terms of the research questions and aims of the project
(refer to Chapter 2, Section 2.16). It also identifies areas of future research arising from the
present work.
8.1 Phytochemical investigations of ginger (Zingiber officinale)
This part of the work made a significant and original contribution to the knowledge of the
phytochemistry of ginger. It included the first extensive survey of Australian commercial
and experimental ginger clones in terms of the main pungent compounds, and also provided
the first survey of steam-distilled Australian ginger oils for more than three decades.
Seventeen clones of ginger were examined for gingerol and shogaol content and in terms of
essential oil composition (Chapters 4 and 5). The aim of identifying a clone yielding
particularly high levels of pharmacologically active gingerols was achieved. The cultivar
known as ‘Jamaican’ produced significantly higher concentrations of [6]-, [8]- and [10]-
gingerol than did any other clone, and it contained these compounds in a somewhat different
ratio. A linear relationship existed between the content of [6]-, [8]- and [10]-gingerol, which
occurred in the approximate ratio of 3:1:1 in all clones except ‘Jamaican’, which contained
relatively higher levels of [8]- and [10]-gingerol.
Given that gingerols appear to confer many of the pharmacological effects of ginger (refer to
Chapter 2, Section 2.2.1.3), ‘Jamaican’ may well prove to be of particular pharmaceutical
interest, although agronomic studies would be required to confirm its suitability as a viable
source of raw material for pharmaceutical use.
‘Jamaican’ also yielded an essential oil that was distinct from that of other clones. It was
characterised by a relatively high content of sesquiterpene hydrocarbons (incl. zingiberene,
β-sesquiphellandrene and ar-curcumene) and a relatively low content of monoterpenoids
such as geranial, neral and geranyl acetate, compared with the other clones. The
combination of high gingerol levels (pungency) with distinct essential oil composition
(aroma) should make ‘Jamaican’ a cultivar of interest also to the flavour and fragrance
industry.
181
The question of whether significant phytochemical differences that are genetically
determined exist between genotypes of ginger was answered in the affirmative with the
identification of the cultivar ‘Jamaican’ and its distinct phytochemical profile, which set it
apart from 16 other clones that were grown, harvested and extracted under identical
conditions.
Apart from the ‘Jamaican’, none of the clones stood out phytochemically. This included the
experimental tetraploid clones, which did not differ significantly from their parent cultivar in
terms of gingerol content or essential oil composition. This is noteworthy, because a
previous study in Japan found tetraploid gingers to contain increased levels of gingerols
(Nakasone et al., 1999), and it demonstrates that the effects of polyploidy on secondary
metabolites are unpredictable.
With the exception of ‘Jamaican’, the steam-distilled essential oils were characterised by
very high levels (>50%) of citral (neral + geranial) and correspondingly low levels of
sesquiterpenes, when compared with published ginger oil analyses generally and the
previous survey of Australian ginger essential oil (Connell & Jordan, 1971) in particular.
These findings confirm the reputation of Australian ginger as having a ‘lemony’ aroma due
to its high citral content. Neral and geranial occurred in a fixed ratio of 2:3 in all clones.
Both aqueous and ethanolic extracts of ginger were shown to inhibit COX-1 in vitro (Chapter
3), which is in accordance with the literature (refer to Chapter 2, Section 2.2.1.3.2). A strong
positive correlation existed between inhibitory activity and the content of both [6]- and [8]-
gingerol, confirming that these compounds inhibit COX-1. Ethanol extracted gingerols more
effectively than did water. Hot water was more effective than cold water, but when ethanol
was used as a solvent, temperature did not affect the extraction efficiency, suggesting that
ethanol at ambient temperature is a highly efficient solvent of gingerols.
Most of the phytochemical investigations of ginger in the present study were carried out on
extracts prepared from fresh rhizomes at ambient temperature. Shogaols were not detected
in these extracts; [6]-shogaol was identified only in hot Soxhlet extracts. This would appear
to confirm that shogaols are not native constituents of ginger rhizome, but form as
degradation products of gingerols.
182
8.2 Screening Zingiberaceae for pharmacological activity
In this part of the work 41 taxa were screened for in vitro inhibition of PGE2 production, and
a number of the samples were also tested for antioxidant activity, inhibition of nitric oxide
production, and for modulation of natural killer cell activity. The work succeeded in making
a substantial and original contribution to the knowledge of the Zingiberaceae as medicinal
plants and a source of pharmacologically active compounds.
The hypothesis that the combination of ethnobotanical and taxonomic information is a
productive strategy to identify previously unrecognised plants with therapeutic potential was
supported by the work, which identified two native Australian species that had not
previously been known to possess pharmacological activity.
The work also provided new insights into the pharmacological activity of several known
medicinal plants. Inhibition of PGE2 was demonstrated for the first time for extracts of the
South-east Asian medicinal plants Boesenbergia rotunda and Curcuma parviflora. This
supports the traditional use of B. rotunda in the treatment of rheumatism and muscular pains
(bin Jantan et al., 2001). The inhibition of nitric oxide by Zingiber montanum was shown for
the first time. This property supports the use of the species in traditional Thai medicine for
the treatment of asthma (Piromrat et al., 1986), given that nitric oxide acts as an
inflammatory mediator in the lung, and concentrations of nitric oxide correlate with airway
inflammation in this condition (Stewart & Katial, 2007). Few Zingiberaceae species have
previously been tested for modulation of natural killer cell activity, and the finding that both
Alpinia galanga and the aqueous extract of fresh B. rotunda caused inhibition of natural
killer cell activity is novel and warrants further investigation. It would also be desirable to
conduct further work to confirm whether ethanolic extracts of B. rotunda and C. longa do in
fact significantly increase the activity of NK cells, as suggested by the present work.
Two native Australian species, Curcuma australasica and Pleuranthodium racemigerum,
were identified in this work as being potent inhibitors of PGE2 (IC50 values being similar to
that of Zingiber officinale). Neither species has a recorded history of use as a medicinal
plant, nor has either of them been the subject of previous pharmacological or phytochemical
investigations. From C. australasica two known sesquiterpene ketones, zederone and
furanodienone, were isolated. Both inhibited PGE2 production, zedorone more potently so.
These compounds have previously been reported from other Curcuma species. From P.
183
racemigerum a novel curcuminoid compound, 1-(4”-methoxyphenyl)-7-(4’-hydroxyphenyl)-
2E-heptene, was isolated. This compound was a potent inhibitor of PGE2 production (IC50 ≅
34 µM), and its structure was successfully elucidated using NMR techniques. It also
displayed dose-dependent cytotoxicity against six different cell lines, its potency being
similar to that of the related compound, curcumin. Curcuminoids and other diarylheptanoids
are widespread in some Zingiberaceae genera, such as Curcuma, Zingiber and Alpinia.
Thus this work has contributed to the understanding of the pharmacology of the
Zingiberaceae. It has also successfully identified species with previously unrecognised
pharmacological activity from genera with recognised medicinal species (Pleuranthodium
racemigerum was previously placed in the genus Alpinia) and isolated active compounds that
were chemically related to other active compounds in related species.
8.3 Direction of future research
The Zingiberaceae, with about 1100 species in more than 50 genera, clearly represents a rich
source of secondary metabolites. For most of these species little or no information about
their phytochemistry or pharmacological properties exist, so there is vast scope for further
investigations of the family as a whole. In terms of future research arising more directly
from the present work, there is also significant scope.
The ginger cultivar ‘Jamaican’ was identified as being phytochemically unique among the
clones investigated, but whether it would prove a viable source of raw material for the
pharmaceutical and/or flavour and fragrance industries would depend on its agronomic
attributes. Agronomic trials with ‘Jamaican’ would be able to determine its suitability as a
commercial crop in terms of yield, resistance to disease, etc.
Although none of the tetraploid clones included in this work proved to have altered
phytochemical characteristics, it would be worthwhile to monitor new experimental
polyploid clones, in particular with respect to increased production of gingerols.
It would also be of interest to investigate the environmental effects, in particular climate and
latitude, as well as the effect of growth period and harvest time, on the phytochemistry of
184
ginger. Such work could clarify, for example, if any of these parameters impact on the citral
content of the essential oil.
With respect to the pharmacological activity of the species included in this work, there is
clearly much more to be done. The PGE2 assay employed was expensive and time
consuming to the point of being limiting. With the initial screening done, the species that
showed inhibition greater than 50% should be subjected to further work in order to establish
accurate IC50 values. Several other bioassays relevant to potential anti-inflammatory activity
could be applied. These include inhibition of phospholipase A2 (PLA2), 5-lipoxygenase (5-
LOX) and TNF-α, and effects on COX-1 and COX-2 expression and on the key transcription
factor nuclear factor kappa B (NFκB).
The most promising samples in these assays could be targeted for bioassay-guided
fractionation provided the active compounds were not already identified. As only a subset of
species were included in the assays for antioxidant activity, inhibition of nitric oxide and
modulation of natural killer cell activity, the remainder could be screened in these assays. It
would also be desirable to screen them for cytotoxic activity.
In terms of the two native Australian species that were found to possess good PGE2
inhibitory activity, further pharmacological investigations as outlined above would be highly
desirable. Both species should also undergo further phytochemical studies in an attempt to
isolate and characterise more novel bioactive compounds. Given the paucity of information
on any Australian Zingiberaceae, it would be highly desirable to carry out a comprehensive
survey using bioassay-guided fractionation of the remainder 12 native species.
oooOooo
190
APPENDIX B: QUALITY ASSESSMENT OF CLINICAL TRIALS OF GINGER IN OSTEOATHRITIS
Assessment of the quality of the reporting of two randomised trials of ginger in osteoarthritis (with ginger extract as the sole active intervention). Checklist based on the revised CONSORT Statement (Moher et al., 2001) except items marked with an asterisk (*), which were adapted from the CONSORT Statement elaborated for the reporting of herbal interventions (Gagnier et al., 2006). Each item was given a score of 0, 0.5 or 1, depending on whether the information was provided not at all or to an unsatisfactory extent (0), to some extent (0.5), or to a satisfactory or mostly satisfactory extent (1). PAPER SECTION And topic
Item Description
Bliddal et al. 2000
Wigler et al. 2003
TITLE & ABSTRACT
1 How participants were allocated to interventions (e.g., "random allocation", "randomized", or "randomly assigned").
1
1
INTRODUCTION Background
2 Scientific background and explanation of rationale.
0 0
METHODS Participants
3 Eligibility criteria for participants and the settings and locations where the data were collected.
0.5
0.5
Interventions 4 Precise details of the interventions intended for each group and how and when they were actually administered.
Elaborated in 4A-4F
Herbal medicinal product name
4A* E.g. Latin binomial, extract name, brand name, name of manufacturer
0.5 1
Characteristics of the herbal product
4B* Incl. plant parts, fresh or dried or extract, solvent details, method of authentication, lot number of raw material, details of voucher specimen or retention sample
0
0.5
Dosage regimen and qualitative description
4C* Dosage, duration of administration, how these were determined; content of quantified herbal constituents per dosage unit; details of additives; for standardised products, the quantity of each active/marker per dosage unit
0
1
Qualitative testing 4D* Chemical fingerprint and methods for obtaining this; description of any special/purity testing; standardisation: what to standardise and how
0
0
Placebo/control group
4E* The rationale for the type of control/placebo used
1 1
Practitioner 4F* A description of the practitioners (e.g., training and practice experience) that are a part of the intervention
0.5
0
Objectives 5 Specific objectives and hypotheses. 0.5 0.5 Outcomes 6 Clearly defined primary and secondary
outcome measures and, when applicable, any methods used to enhance the quality of measurements (e.g., multiple observations, training of assessors).
1
1
Sample size 7 How sample size was determined and, when applicable, explanation of any interim analyses and stopping rules.
1
0
191
Randomization -Sequence generation
8 Method used to generate the random allocation sequence, including details of any restrictions (e.g., blocking, stratification)
1
1
Randomization -Allocation concealment
9 Method used to implement the random allocation sequence (e.g., numbered containers or central telephone), clarifying whether the sequence was concealed until interventions were assigned.
0
0
Randomization - Implementation
10 Who generated the allocation sequence, who enrolled participants, and who assigned participants to their groups.
0
0
Blinding (masking) 11 Whether or not participants, those administering the interventions, and those assessing the outcomes were blinded to group assignment. When relevant, how the success of blinding was evaluated.
0.5
0.5
Statistical methods 12 Statistical methods used to compare groups for primary outcome(s); Methods for additional analyses, such as subgroup analyses and adjusted analyses.
1
1
RESULTS Participant flow
13 Flow of participants through each stage (a diagram is strongly recommended). Specifically, for each group report the numbers of participants randomly assigned, receiving intended treatment, completing the study protocol, and analyzed for the primary outcome. Describe protocol deviations from study as planned, together with reasons.
1
1
Recruitment 14 Dates defining the periods of recruitment and follow-up.
0 0
Baseline data 15 Baseline demographic and clinical characteristics of each group.
1 1
Numbers analyzed 16 Number of participants (denominator) in each group included in each analysis and whether the analysis was by "intention-to-treat". State the results in absolute numbers when feasible (e.g., 10/20, not 50%).
1
1
Outcomes and estimation
17 For each primary and secondary outcome, a summary of results for each group, and the estimated effect size and its precision (e.g., 95% confidence interval).
1
1
Ancillary analyses 18 Address multiplicity by reporting any other analyses performed, including subgroup analyses and adjusted analyses, indicating those pre-specified and those exploratory.
1
-
Adverse events 19 All important adverse events or side effects in each intervention group.
1 1
DISCUSSION Interpretation
20 Interpretation of the results, taking into account study hypotheses, sources of potential bias or imprecision and the dangers associated with multiplicity of analyses and outcomes.
1
1
Generalizability 21 Generalizability (external validity) of the trial findings.
1 1
Overall evidence 22 General interpretation of the results in the context of current evidence.
1 1
Total score 17.5 17.0
192
APPENDIX C: INHIBITION OF PGE2 IN 3T3 CELLS Concentrations shown are final concentrations in assay well. Mean value±SEM; n=3 expect * (n=2), + (n=4) and ^ (n=6).
Taxon ID Concentration (µg/mL)
Percent inhibition of PGE2
Alpinia arctiflora Z129 100.0 -18.51±1.81 50.0 12.08±1.39 10.0 1.41±0.26 Alpinia caerulea Z102 94.8 33.81±8.99 47.4 35.37±5.61 9.5 -15.16±2.85 Alpinia calcarata* Z49 500.0 54.96±4.66 5.0 8.52±0.25 Alpinia galanga Z103 91.8 47.02±0.53 45.9 42.88±4.34 9.2 22.80±4.23 Alpinia luteocarpa Z111 100.0 0.77±0.08 50.0 20.31±0.35 10.0 1.03±0.07 Alpinia malaccensis* Z48 500.0 28.98±1.62 5.0 -22.37±0.29 Alpinia modesta Z127 100.0 -3.60±0.73 50.0 23.65±1.49 10.0 -10.67±0.80 Alpinia mutica* Z08 500.0 48.04±0.17 5.0 -29.26±2.99 Alpinia purpurea 'Eileen McDonald'* Z11 500.0 47.36±0.99 5.0 15.09±0.13 Alpinia spectabile 'Giant Orange'* Z53 500.0 18.23±1.49 5.0 -18.08±0.76 Alpinia zerumbet* Z52 500.0 42.98±2.80 5.0 18.70±2.47 Boesenbergia rotunda Z104 94.2 50.52±1.69 47.1 45.60±2.53 9.4 21.77±0.84
193
Costus barbatus Z112 100.0 -45.67±2.76 50.0 -30.74±1.78 10.0 -59.31±2.29 Costus leucanthus Z113 100.0 -28.14±1.20 50.0 -7.79±0.29 10.0 -25.76±1.79 Costus malortieanus Z114 100.0 -33.55±2.37 50.0 -16.45±2.00 10.0 -17.10±1.25 Costus productus Z116 100.0 -0.22±0.05 50.0 6.28±1.00 10.0 -27.06±1.34 Costus pulverulentus Z115 100.0 -40.48±2.31 50.0 7.14±0.16 10.0 -20.56±0.76 Costus tappenbeckianus* Z15 500.0 -4.66±0.52 5.0 -24.54±1.38 Curcuma australasica^ Z101 100.0 56.21±7.41 50.0 51.11±11.81 10.0 19.90±3.53 Curcuma cordata Z117 100.0 7.63±0.51 50.0 14.61±0.10 10.0 9.33±0.51 Curcuma longa^ Z106 100.0 60.15±4.64 50.0 57.69±6.45 10.0 49.83±14.91 Curcuma parviflora Z118 100.0 66.72±4.75 50.0 51.38±1.59 10.0 30.11±1.79 Elettaria cardamomum* Z12 500.0 44.29±0.00 5.0 14.91±0.01 Etlingera australasica Z107 92.4 28.50±6.56 46.2 22.41±9.84 9.2 -14.12±3.28 Etlingera elatior 'Burma torch'* Z04 500.0 -0.37±0.01 5.0 -14.36±0.80 Hedychium coronarium* Z10 500.0 41.60±0.96 5.0 -9.02±0.15
194
Hornstedtia scottiana Z126 100.0 0.97±0.09 50.0 10.15±0.52 10.0 3.33±0.22 Kaempferia galanga* Z05 500.0 61.31±5.48 5.0 7.39±0.35 Kaempferia rotunda Z120 100.0 -39.61±2.58 50.0 -25.32±1.49 10.0 -52.60±3.67 Pleuranthodium racemigerum^ Z128 100.0 59.40±3.76 50.0 46.85±6.85 10.0 39.00±5.69 Renealmia cernua Z121 100.0 17.45±1.63 50.0 18.26±0.30 10.0 11.36±0.53 Scaphochlamys biloba* Z01 500.0 35.21±2.07 5.0 -33.13±4.92 Scaphochlamys kunstleri* Z122 100.0 26.13±3.43 50.0 31.09±0.22 10.0 24.67±1.17 Tapeinochilos ananassae Z125 100.0 4.30±0.31 50.0 16.31±0.68 10.0 14.28±0.65 Zingiber longipedunculatum* Z06 500.0 26.99±0.50 5.0 -4.23±0.11 Zingiber officinale Z108 100.0 50.44±1.69 50.0 47.62±5.69 10.0 11.02±2.53 Zingiber ottensii Z124 100.0 -28.17±2.77 50.0 -19.11±4.05 10.0 -3.62±0.18 Zingiber montanum* Z02 500.0 64.72±5.97 5.0 9.40±0.33 Zingiber montanum Z105 100.0 49.35±4.12 50.0 47.54±1.80 10.0 22.93±1.80 Zingiber spectabile Z17 500.0 33.99±1.91 5.0 -17.48±0.62
195
Zingiber sp. 'Dwarf Apricot'+ Z55 500.0 11.65±1.82 5.0 -7.49±0.48 Zingiber/Etlingera (Aniseed ginger)* Z19 500.0 38.83±0.90 5.0 10.92±0.50
196
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