Plant substances as alternatives for animal products in traditional medicines

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Plant substances as alternatives
for animal products
in traditional medicines
Report submitted to the Department
for Environment Food and Rural Affairs
September 2006
Dr Celia M Bell,
Academic Group Chair, Human and Healthcare Sciences,
School of Health and Social Sciences,
Middlesex University
, UK
and
Professor Monique SJ Simmonds,
Head of Biological Interactions,
Jodrell Laboratory,
Royal Botanic Gar
dens (RBG), Kew, UK
Researchers:
Dr Sandra S Appiah, Middlesex University, UK
Dr Melanie-Jayne R Howes, Jodr
ell Laboratories, RBG, Kew, UK

Plant substances as alternatives
for animal products
in traditional medicines
Report submitted to the Department
for Environment Food and Rural Affairs
September 2006
Dr Celia M Bell,
Academic Group Chair, Human and Healthcare Sciences,
School of Health and Social Sciences,
Middlesex University, UK
and
Professor Monique SJ Simmonds,
Royal Botanic Gardens, Kew
, UK
Researchers:
Dr Sandra S Appiah, Middlesex University, UK
Dr Melanie-Jayne R Howes, Royal Botanic Gar
dens, Kew, UK

Department for Environment, Food and Rural Af
fairs
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London SW1P 3JR
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ebsite: www.defra.gov.uk
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Product code PB 11737

We thank DEFRA and IFAW Charitable Trust for their financial support for this project. We
also thank the CITES TEAM (Special Operations District) at Heathrow airport for the kind
donation of the rhino horn sample, and the National Museums of Scotland for providing
a sample of tiger bone.
Other contributors
Middlesex University, UK
Dr Henry Lee
Dr John Langley
Dr Huw Jones
Professor Mike Revitt
Peter Lister (Technician)
Alan LaGrue (Technician)
John Schmitt (Technician)
Jodrell Laboratory, Biological Interactions, RBG, Kew, UK
Dr Tetsuo Kokubun
Christine Leon
Dr Elaine Porter
Cheryl Waring
Dr Renée Grayer
Dr Geoffrey Kite
Dr Nigel C Veitch
Centre for Pharmacognosy and Phytotherapy, The School of Pharmacy,
University of London, UK
Prof. Michael Heinrich
Dr Paul Bremner
AstraZeneca, Research and Development, Charnwood, UK
David Wilkinson
Dr Craig Lambert
Herbal Suppliers
Jo Liu (Mayway Ltd, UK)
Paul Skipworth (Kingham herbs and Tinctures, UK)

Traditional Chinese Medicine Practitioners
Dr Bing Chung Chan (Practitioner, main advisor)
Professor Dang Yi (Middlesex University, UK)
Dr Lin Zhixiu (Middlesex University, UK)
Dr Kaicun Zhao (Middlesex University, UK)
Dr Charlie Buck (Practitioner)
Dr Ke Wang (Practitioner)
Dr Su (Practitioner)
Mrs Hong Wen

Contents i
Figures v
Tables vi
Executive summary vii
Section 1. Introduction and overview 1
1.1. Introduction 1
1.1.1. The popularity of traditional medicine 1
1.1.2. The threat to endangered species from their use in traditional medicine 1
1.1.3. The need for research into the use of substitutes
for specimens of endangered species 2
1.1.4. Middlesex University and traditional medicine 2
1.2. The current research project 3
1.2.1. Aims of the project 3
1.3. Structure of the report 4
Section 2. Rationale for selection of herbs as potential
alternatives to bear bile 5
2.1. Introduction 5
2.2. Sources of bear bile 5
2.3. Bear bile and its constituents in TCM and W
estern medicine 6
2.4. Bear bile in TCM 6
2.5.
Selection of herbs and herbal prescriptions for investigation 6
2.5.1. Criteria used for selection of single herbs 6
2.5.2. Criteria used for selection of herbal prescriptions for study 8
2.6. Single herbs chosen as potential alternatives to bear bile in TCM:
summary of reputed and pharmacological effects 9
2.6.1. Huang Qin (Radix Scutellariae) 9
2.6.2.
Huang Lian (Rhizoma Coptidis)
9
2.6.3. Huang Bai (Cortex Phellodendri) 9
2.6.4. Zhi Zi (Fructus Gardeniae) 10
2.6.5. Chuan Xin Lian (Herba Andrographis) 10
2.6.6. Zhi Mu (Rhizoma Anemarrhena) 11
2.6.7. Da Huang (Radix et Rhizoma Rhei) 11
Contents
i

2
.7. Prescriptions chosen for investigation as potential alternatives to bear bile in TCM 11
2
.7.1. Orengedokuto 11
2
.7.2. Dia-Orengedokuto 12
2
.8. Alternatives to bear bile: summary 13
Section 3. Rationale for selection of herbs as potential
alternatives to rhino horn 14
3.1. Introduction 14
3.2. Sources of rhino horn 14
3.3. Rhino horn and its constituents 14
3.4. Rhino horn in TCM 15
3.5. Selection of herbs and prescriptions for investigation 15
3.5.1. Criteria used for selection of single herbs 15
3.5.2. Criteria used for selection of herbal prescriptions 16
3.6. Single herbs as potential alternatives to rhino horn: reputed and pharmacological effects 18
3.6.1. Ban Lan Gen (Radix Isatidis) 18
3.6.2. Chi Shao (Radix Paeoniae Rubra) 18
3.6.3. Mu Dan Pi (Cortex Moutan) 19
3.6.4. Dan Shen (Radix Salvia Miltiorrhizae) 19
3.6.5. Jin Yin Hua (Flos Lonicerae) 20
3.6.6. Lian Qiao (Fructus Forsythia) 20
3.6.7. Sheng Di Huang (Radix Rehmanniae) 20
3.6.8. Xuan Shen (Radix Scrophulariae) 21
3.6.9. Zi Cao (Radix Lithospermi Arnebaie) 21
3.7. Prescriptions traditionally containing rhino horn: reputed and pharmacological effects 21
3.7.1. Qing Ying Tang 21
3.7.2. Qingwen Baidu Yin 21
3.7.3. Xi Jiao Dihuang Tang 22
3.7.4. Sheng Xi Dan 22
3.7.5. Qing Gong Tang 22
3.7.6.
Zhi Zi Jin Hua
22
3.8. Alternatives to rhino hor
n: summary 22
ii
Contents

Section 4. Rationale for selection of herbs as potential
alternatives to tiger bone 23
4
.1. Introduction 23
4
.2. Sources of tiger bone 23
4
.3. Tiger bone and its constituents 23
4.4. Tiger bone in TCM 24
4.5. Selection of herbs 24
4.5.1. Criteria used for selection of single herbs 24
4.5.2. Criteria used for the selection of prescriptions 25
4.6. Single herbs and other TCM remedies as potential alternatives to tiger bone:
reputed and pharmacological effects 31
4.6.1. Bai Shao (Radix Paeoniae Alba) 31
4.6.2. Bai Zhu (Rhizoma Atractylodis Macrocephalae) 31
4.6.3. Cang Zhu (Rhizoma Atractylodis) 31
4.6.4. Chuan Xiong (Rhizoma Chuanxiong) 31
4.6.5. Dang Gui (Radix Angelicae Sinensis) 32
4.6.6. Di Huang (Radix Rehmanniae) 32
4.6.7. Du Huo (Radix Angelicae Pubescentis) 32
4.6.8. Du Zhong (Cortex Eucommiae) 33
4.6.9. Fang Feng (Radix Saposhnikoviae) 33
4.6.10. Fu Ling (Poria) 33
4.6.11. Gui Zhi (Ramulus Cinnamomi) 33
4.6.12. Ji Xue Teng (Caulis Spatholobi) 34
4.6.13. Lu Lu Tong (Fructus Liquidambaris) 34
4.6.14.
Mu Gua (Fructus Chaenomelis) 34
4.6.15. Mu Xiang (Radix Aucklandiae) 34
4.6.16.
Niu Xi (Radix Achyranthis Bidentatae) 35
4.6.17.
Qin Jiao (Radix Gentiianae Macrophyllae) 35
4.6.18. Ren Shen (Radix Ginseng) 35
4.6.19. Rou Gui (Cortex Cinnamomi) 35
4.6.20. San Qi (Radix Notoginseng) 36
4.6.21. Sang Zhi (Ramulus Mori) 36
4.6.22.
W
ei Ling Xian (Radix Clematidis) 36
4.6.23. Xu Duan (Radix Dipsaci) 36
4.6.24. Yin Yang Huo (Herba Epimedii) 37
4.7. Alternatives to tiger bone: summary 37
Contents
iii

Section 5. Biological and chemical methods used to study
plant and fungal material 38
5
.1. Introduction 38
5
.2. Materials 39
5
.3. Authentication techniques for trade TCM material 39
5.3.1. HPLC (UV-DAD) method 40
5.3.2. LC-MS method 40
5.3.3. TD-GC-MS method 40
5.4. Methods for fractionation and isolation of compounds 41
5.4.1. Fractionations of
Scutellaria baicalensis and isolation of compounds 41
5.4.2. Fractionation of
Salvia miltiorrhiza and Qing Ying Tang 41
5.5. Method for anti-bacterial tests 42
5.6. NF
−κΒ Studies using the IL-6 promoter assay method 43
5.7. Method for cytochrome P450 3A4 inhibition studies 43
5.7.1. CYP3A4 LC-MS method 44
5.8. Statistical analysis 44
Section 6. Results and discussion 45
6.1. Bear bile: bioassay results and discussion 45
6.1.1. Anti-bacterial tests 45
6.1.2. Anti-inflammatory (NF−κΒ) tests 46
6.1.3. Cytochrome P450 3A4 tests 49
6.1.4. Conclusions 51
6.2. Rhino hor
n: bioassay r
esults and discussion
51
6.2.1. Anti-bacterial tests 51
6.2.2. Anti-inflammatory (NF
−κΒ) tests 53
6.2.3. Cytochrome P450 3A4 tests 55
6.2.4. Conclusions 57
6.3. T
iger bone: bioassay r
esults and discussion
57
6.3.1. Anti-inflammatory (NF
−κΒ) tests 58
6.3.2. Cytochrome P450 3A4 tests 60
6.3.3. Conclusions 60
Contents
iv

Section 7. Conclusions 61
7.1. General conclusions 61
7.2. Herbs selected as potential alternatives to animal products 61
7.2.1. Herbs proposed as potential alternatives to bear bile used in TCM 61
7.2.2. Prescriptions proposed as potential alternatives to bear bile used in TCM 62
7.2.3. Herbs proposed as potential alternatives to rhino horn used in TCM 62
7.2.4. Herbs proposed as potential alternatives to tiger bone used in TCM 63
7.3. Future work 64
References 65
Appendices:
Appendix 1. Fifty-four herbal alternatives to bear bile (IFAW report, 1994) 80
Appendix 2. Reference standards used in multi-residue pesticide screening of TCM herbs 82
Appendix 3. Results for metal analyses of extracts of rhino horn and TCM herbs 86
Figures
Fig. 6.1. The effects of six herbal extracts on NF−κΒ activity 46
Fig. 6.2. The effects of fractions obtained from methanol extract of scutellaria baicalensis
on NF
−κΒ activity 47
Fig. 6.3. The effects of flavonoids from
Scutellaria baicalensis, and salicyclic acid on
NF−κΒ activity 48
Fig. 6.4. The effects of six TCM herbs and ketoconazole on CYP3A4 activity 49
Fig. 6.5. The effects of fractions and flavonoid compounds of Scutellaria baicalensis (sb)
and ketoconazole on CYP3A4 activity
50
Fig. 6.6.
The ef
fects of rhino horn extract, TCM prescriptions (with and without rhino horn)
on NF
−κΒ activity 54
Fig. 6.7. The effects of TCM remedies on NF
−κΒ activity 55
Fig. 6.8. The effects of TCM remedies and ketoconazole on microsomal CYP3A4 activity 56
Fig. 6.9. The effects of TCM prescriptions, tiger bone and remedies traditionally found
in tiger bone prescriptions, on NF−κΒ activity 59
Fig. 6.10. The effects of water extracts of tiger bone and 13 TCM herbs on CYP3A4 activity 60
Contents
v

Tables
Table 2.1. Properties / functions of bear bile and udca used as criteria for herb selection 7
Table 2.2. TCM plants selected after consultation with TCM practitioners and from
evaluation of TCM literature and pharmacological and clinical data 7
Table 2.3. Herbal composition of prescription X 8
Table 2.4. Composition of Orengedokuto 12
Table 3.1. Properties / functions of rhino horn used as criteria for herb selection 15
Table 3.2. TCM plants selected after consultation with TCM practitioners and from
evaluation of TCM literature and pharmacological and clinical data 16
Table 3.3. The distribution of 24 herbs in six TCM prescriptions 17
Table 4.1. Properties / functions of tiger bone used as criteria for species selection 25
Table 4.2. TCM species identified from evaluation of TCM literature using criteria
based on the TCM functions and properties of tiger bone 26
Table 4.3. Composition of TCM prescriptions, traditionally containing tiger
bone, selected for biological activity tests 30
Table 4.4. Other species selected for biological activity tests (not listed in table 4.3.) 30
Table 6.1. Plant species investigated in biological activity tests 45
Table 6.2. TCM herbs investigated in biological assays 52
Table 6.3. Anti-bacterial activity of rhino horn and TCM prescriptions 53
Table 6.4. TCM samples studied in biological assays 58
vi
Contents

Executive summary
vii
An investigation of plant species as alternatives to the use of products obtained from
endangered animal species (bear bile, rhino horn and tiger bone) was undertaken with
financial support from the Department for Environment Food and Rural Affairs (DEFRA)
and the International Fund for Animal Welfare (IFAW) Charitable Trust. The research was
carried out by Middlesex University (UK) in collaboration with the Jodrell Laboratory, Royal
Botanic Gardens, Kew (UK).
Products from several endangered species are used in Traditional Chinese Medicine for a
variety of purposes. Bear bile (Xiong Dan) and rhino horn (Xi Jiao) are primarily classified as
anti-inflammatory and fever-reducing remedies and tiger bone (Hu Gu) has been used as
an anti-rheumatic/anti-arthritic remedy; the pathology of arthritis also involves inflammatory
mechanisms. With the popularity of Traditional Medicine increasing, a continued demand
for these products poses an ongoing and major threat to the survival of these species, all
of which are listed under Appendix 1 of CITES. This study was undertaken in response to
a recognised need for more research into possible herbal substitutes.
Based on both traditional use and scientific evidence for pharmacological actions, single
herbs and TCM ‘prescriptions’ (combinations of herbs) were selected for investigation as
alternatives to the use of bear bile, rhino horn and tiger bone in Traditional Chinese Medicine.
A selection of 7 single herbs and 2 prescriptions were chosen for investigation as potential
alternatives to bear bile; 9 single herbs and 6 prescriptions as potential alternatives to rhino
horn; and 19 single herbs and two prescriptions as potential alternatives to tiger bone.
As all three animal products are traditionally used to treat conditions associated with
inflammatory processes, this area was chosen for investigation. The inflammatory response
is a complex cascade of events, often trigger
ed by infection (commonly by bacteria) and is
one of the body’s defence mechanisms in fighting disease. The inflammatory response
forms one of the underlying pathologies of arthritis, fever, liver diseases, cancer and
cardiovascular diseases. Therefore, preliminary studies were conducted to assess the
effects of crude extracts, fractions and isolated compounds on bacterial growth and an
anti-inflammatory mediator, nuclear factor-kappaB (NF
−κΒ) in vitro. Cytochrome P450 3A4
inhibition tests were conducted to determine the effect of herbal extracts on this drug
metabolising enzyme
in vitro.
When recommending potential herbal alternatives it is essential to ensure that the correct
plant species is being proposed. Verification of the plant material was carried out by
comparing the chemical profiles of the samples obtained for r
esearch from commercial
sources with chemical profiles of authentic and reference material from the Chinese
Medicinal plant Authentication Centre, Royal Botanical Gardens, Kew. In addition, pesticide
residues and metal concentrations were determined to confirm the quality of the product.

Several of the herbs chosen for investigation as possible alternatives to bear bile were found
to possess anti-bacterial activity (
Anemarhena asphodeloides Bge, Gardenia jasminoides
Ellis, Scutellaria baicalensis Georgi, Phellodendon amurense Rupr., Coptis chinensis Franch.
and
Rheum palmatum L). Extracts of three herbs were also shown to have anti-inflammatory
properties through the inhibition NF
−κΒ activity (Scutellaria baicalensis, Rheum palmatum
and Coptis chinensis). Preliminary results from the cytochrome P450 3A4 inhibition studies
suggest that possible herb-herb interactions may occur in preparations containing both
Coptis chinensis and Scutellaria baicalensis (such as Dia-Orengedokuto and Orengedokuto).
Also, drug-herb interactions may occur when herbal preparations containing
Coptis chinensis
and/or Scutellaria baicalensis are co-administered with some pharmaceutical drugs metabolised
by this enzyme. Further work is required to investigate the extent of these effects.
Water extracts of rhino horn did not demonstrate anti-bacterial nor anti-inflammatory
properties, nor did they have any effect on the drug metabolising enzyme, cytochrome
P450 3A4. However, certain Traditional Chinese Medicine prescriptions, both with and
without rhino horn, did show anti-bacterial and anti-inflammatory properties in the assays
used in this study. Further work using other bioassays is required to ascertain the contribution
of the hor
n extracts to any activity shown by the prescriptions. The majority of herbs chosen
as possible alternatives to rhino horn showed some anti-bacterial activity (17 out of 20).
Extracts of several single herbs were also shown to have anti-inflammatory properties
through the inhibition NF
−κΒ activity (Paeonia suffruticosa Andr., Trichosanthes kirilowii
Maxim., Lophatherum gracile Brongn., Acorus calamus, Paeonia veitchii Lynch, Isatis
indigotica
Fort., Glycine max L. and Rehmannia glutinosa Steud) as well as extracts of two
prescriptions (Xi Jiao Dihuang Tang and Qing Ying Tang). To date, no scientific literature
has been found suggesting that
Lophatherum gracile has an anti-inflammatory effect and
further studies are required to confirm these findings.
Salvia miltiorrhiza, Rehmannia glutinosa,
as well as Scutellaria baicalensis and Coptis chinensis showed inhibition of cytochrome
P450 3A4. Since they are commonly used TCM herbs, further work may be required to
determine potential adverse interactions with other remedies or orthodox medicines.
Preliminary results suggest that tiger bone may possess some anti-inflammatory properties
through the inhibition of NF
−κΒ activity. Three herbs included in existing Traditional Chinese
Medicine prescriptions containing tiger bone also showed anti-inflammatory activity in the
same assay
Angelica dahurica Maxim., Taxillus chinensis (DC.) Danser and Angelica sinensis
(Oliv.). None of the herbs investigated as alternatives to tiger bone were found to affect
cytochrome P450 3A4 activity.
Supported by evidence of efficacy as anti-inflammatory and anti-bacterial agents as measured
in this study, by information obtained from the available scientific literature, and by
Traditional Chinese Medicine theory, a number of prescriptions and single herbs have been
selected as suitable alternatives to the use of bear bile, rhino horn or tiger bone in Traditional
Chinese Medicine. Most of the suggested herbal ‘alternatives’ to the animal products were
found to already form part of one or more traditional prescriptions containing the animal
products. This finding confirmed the practice in Traditional Chinese Medicine of combining
remedies with similar functions for their additive and synergistic effects.
Executive summary
viiiviii

The inflammatory response is a complex cascade of events and nuclear factor-kappaΒ
is only one anti-inflammatory mediator amongst many. Further studies are warranted
to assess other pharmacological mechanisms through which the plants might mediate
anti-inflammatory effects. Further work should also be carried out to investigate further
the effect of herbal extracts on drug metabolising enzymes such as cytochrome P450 3A4.
If the findings of this study are to impact upon the use of products from endangered
animal species in Traditional Chinese Medicine, the suggestions made for herbal
alternatives will need to be acceptable to practitioners in terms of philosophy as well
as potential efficacy. It will be necessary to discuss the findings of this study with TCM
practitioners to determine whether the selected plant species would be considered suitable
for use in Traditional Chinese Medicine as substitutes to bear bile, rhino horn and tiger
bone. The suggested herbs and the evidence to support these suggestions will then need
to be disseminated to practitioners and the public, both via scientific publications and
through the more popular Traditional Chinese Medicine literature.
Executive summary
ix

x

Summary
1
Introduction and overview
1.1. Introduction
1.1.1. The popularity of traditional medicine
The use of traditional medicine (TM) containing ingredients obtained from animals and
plants has maintained its popularity in all regions of the developing world and is gaining
in popularity in the industrialised countries. Countries in Africa, Asia and Latin America
use TM to help meet some of their primary health care needs while in industrialised
countries, traditional medicine use is seen as “Complementary“ or “Alternative” (CAM)
to orthodox or allopathic medicine. Thus, in Africa, up to 80% of the population uses
traditional medicine for primary health care and in China traditional herbal preparations
account for 30%–50% of the total medicinal consumption. In Europe, North America
and other industrialised regions, over 50% of the population have used complementary
or alternative medicine at least once. The popularity of TM has created a global market
for herbal medicines that currently stands at over US $ 60 billion annually and is growing
steadily (WHO, 2004).
1.1.2. The threat to endangered species from their use
in traditional medicine
The growing market in TM poses a major threat to the survival of many endangered
species, notably tigers, bears and rhinoceroses. Most TM “consumer” countries, including
China, Japan, the UK and the USA, are Parties to the Convention on International Trade
in Endangered Species (CITES), which bans international trade in these species between
CITES member states. However, demand for medicines containing them continues and
with it illegal trade in their parts and derivatives for the TM market. Tigers, rhinoceroses,
and three species of bear, are listed under CITES Appendix 1 (2004); species that are the
most endangered among CITES-listed animals and plants and are threatened with extinction.
Despite this protection, the number of tigers, bears and rhinoceroses in the wild continues
to fall. Tiger numbers have dropped from more than 100,000 to between 4,800 and
7,300 individuals over the last century, three tiger species have become extinct, and, as
tigers become increasingly difficult to find, other big cat species have begun to be hunted
as an alternative. Whilst several parts of the tiger are used in TM, tiger bone is the most
commonly used. It is believed to have an anti-inflammatory effect, particularly in cases
of arthritis.
Section 1

Section 1
2
Bear populations are also declining around the world to the extent that the immediate
survival of bears in key regions is threatened. It is recognised that bears are poached
and illegally traded for use in TM, but the extent of illegal poaching is difficult to assess.
Various bear parts are used in Asian TM, including the meat, gall bladder, brain, blood,
bone and paw. Bear bile, extracted from the gall bladder, is most commonly used in TM,
being prescribed for febrile diseases with high temperature and convulsions, inflammation
of the liver, laryngitis, conjunctivitis and to reduce swelling and pain (for trauma, sprains,
fractures and haemorrhoids).
Rhinoceros populations have also suffered severely in recent years. Three of the five species
of rhinoceros are now critically endangered and threatened with extinction, with more that
half of the world’s remaining rhinoceroses lost during the 1970s (WWF, 2002). The use
of rhinoceros horn in TM has largely been blamed for the decline in the population in
Asia. Rhinoceros horn is prescribed in Asian TM as a detoxifying, anti-inflammatory and
anti-convulsant agent and is used in the treatment of advanced stages of fever.
1.1.3. The need for research into the use of substitutes for specimens
of endangered species
Parties to CITES have expressed concern over the continued and uncontrolled use of several
endangered species in traditional medicine in view of the potential threat to the long-term
survival of these species and the need to ensure the continued use and development of
traditional medicines on a sustainable basis (CITES, 1997; Conf. 10.19). It has been recognised
that pr
oblems of overexploitation must be addressed within the context of an improved
understanding about the significance of traditional medicines in the world’s health care
systems. A resolution was therefore agreed at the 10th meeting of the Conference of
Parties 1997 (CITES, 1997) calling for more research into the use of substitutes for specimens
of endangered species in TM. The UK, represented by the Global Wildlife Division of the
Department for Environment, Food and Rural Affairs (DEFRA), the UK CITES Management
Authority, was instrumental in CITES in gaining agreement to this Resolution.
IFAW first began to address the issue of endangered species used in traditional medicine
products in 1983 and have also emphasised the importance of research into herbal alternatives.
1.1.4. Middlesex University and traditional medicine
Against this background, the increasing popularity of TM in the industrialised countries,
including the UK, led to the introduction of degree level education in several TM disciplines.
Middlesex University was at the forefront of these developments, validating a degr
ee
in Herbal Medicine in 1994 and in Traditional Chinese Medicine (TCM) in 1996. Both
programmes aim to provide an education and training to produce graduates who will be
competent, reliable and caring practitioners. The TCM programme was developed and is
delivered in collaboration with Beijing University of Chinese Medicine. The need to address
issues surrounding the use of endangered species in TCM practice and to bring endangered
species education into the curriculum is recognised by the academic group at Middlesex,
as is the need for appropriate research in this area.

1.2. The current research project
In 2001 DEFRA and IFAW jointly commissioned Middlesex University to undertake research
to investigate “Plant Substances as Alternatives for Animal Products Used in Traditional
Chinese Medicine”. The research was carried out at Middlesex and at the Jodrell
Laboratories, Royal Botanic Gardens, Kew. The Royal Botanic Gardens at Kew are the UK
appointed CITES Scientific Authority for Plants and carry out CITES projects and research
into the trade in certain CITES-listed plants. In addition, Dr Bing Chan acted as external
advisor on matters of TCM philosophy and practice.
The project was designed to be carried out in two parts. Project 1 was to investigate
anti-inflammatory and anti-pyretic herbs as alternatives for bear bile and rhino horn.
Project 2 was to investigate anti-inflammatory and anti-rheumatic herbs as alternatives for
tiger bone. As the project progressed, further collaborations were established in order to
achieve the aims of the project. These collaborations are listed in the acknowledgements.
All collaborators have been vital to the success of the project and their input greatly valued.
1.2.1. Aims of the project
The overall objective of the project is to provide original scientific data to support the
promotion of alternatives to the use of animal products in TM in order to help prevent
the further depletion of threatened and endangered wildlife.
The original aims for phase 1 and 2 of this research proposal were as follows:
i) To identify the active chemical components of products derived from bear (bile),
tiger (bone) and rhino (horn).
ii) Once identified, to find plant substitutes.
The following report summarises the work that has been carried out over the past three
years. From the report it can be seen that the initial aims of the project have been met.
Having conducted an extensive literature review and consultations with Chinese medicine
practitioners, the decision was taken to place the emphasis on the choice of herbs to
investigate as possible alternatives to animal products based on TCM theory and philosophy
rather than on similarities in chemical structure or in activity in Western pharmacological
terms. The latter was taken into account and investigated once the choice of herbs had
been made. This decision was taken as the team consider that the effective promotion
of the use of alternatives to practitioners and patients of TCM will depend on their
acknowledgment that the herbs possess the same or similar properties to the animal
products as described by TCM philosophy. Also taken into consideration was the common
usage in TCM of prescriptions consisting of a combination of herbs with a ‘principal’
ingredient. Several prescriptions, which include animal products as an ingredient, were
therefore also investigated.
Introduction and overview
3

Section 1
4
All three animal products under investigation (bear bile, rhino horn and tiger bone)
are used in TM medicines for a variety of reasons, but all share a common use as an
anti-inflammatory. Both bear bile (Xiong Dan) and rhino horn (Xi Jiao) are also classified
in TCM as anti-pyretic remedies and tiger bone (Hu Gu) as an anti-rheumatic/anti-arthritic
remedy. Herbs chosen for investigation based on TM theory were therefore investigated
in vitro for their efficacy as anti-inflammatory agents. Since fever is often initiated by
infection (commonly bacteria) anti-bacterial tests were also conducted as part of the bear
bile and rhino horn projects. There has been concern expressed over possible herb-herb
and drug-herb interactions and over the contamination of herbal medicines with metals
and pesticide residues. This was investigated using cytochrome P450 3A4 assays, and
quantitatively evaluating the chemical contaminants (metals and pesticide residues) in some
selected herbs. In addition, the importance of authentication of herb samples is recognised
and the plant species of the herbs in this study was investigated using chemical analysis.
An additional suggestion, though not included in the initial proposal for funding, was the
possibility of extending the research to include animal trials, followed by human trials using
volunteers to assess the effectiveness of herbal alternatives (Phase 4). It has not been felt that
ther
e is justification at this point to extend the project in this way and that this would, in any
case, conflict with the mission and vision of both the University and the funding bodies.
1.3. Structure of the report
The report is divided into 7 sections. Following this overview, Sections 2, 3, and 4 present
the rationale for the selection of herbs to investigate as potential alternatives to bear bile,
rhinoceros horn and tiger bone respectively, identifying both herbs and combinations
of herbs (prescriptions) chosen and briefly describing their properties as identified in the
literature. Section 5 describes the methods used to prepare, authenticate and assess the
biological activity of the chosen herbs. The assays allowed investigation of anti-bacterial
activity, anti-inflammatory activity (NF
−κΒ inhibition) and cytochrome P450 3A4 activity.
Section 6 presents the main findings of the investigation into biological activity. The results
of assays to measure contaminant levels (pesticide residues and metals) are shown in
Appendices 2, 3A and 3B. A summary of the main findings and suggested future work
is outlined in Section 7.

Summary
5
Rationale for selection of herbs as
potential alternatives to bear bile
2.1. Introduction
In order to identify which plants might be suitable to replace bear bile use in traditional
medicine (TM), in particular, traditional Chinese medicine (TCM), it is important to understand
the beliefs and evidence supporting its continued use. In this part of the report, sources
of bear bile, its uses in TM and the identification of active constituents used in ‘Western’
medicine are discussed. Based on the findings of this research, the criteria for choosing
herbs to investigate as potential alternatives were established, based on both the traditional
uses and knowledge about medicinal properties of the different species of plants. Whilst
single herbs have been selected for study, it is recognised that a mixture of different plant
species is commonly prescribed in TCM, with the prescription relying on purported
synergism of a combination of herbs, sometimes with animal parts and minerals.
Therefore, prescriptions or combinations of herbs were also investigated as potential
alternatives to the use of bear bile in TCM.
A summary of some of the known constituents and available research on the herbs and
prescriptions chosen for study as potential alternatives to the use of bear bile in TCM is
given at the end of this section.
2.2. Sources of bear bile
In TCM, bear bile was originally obtained from two members of the Ursidae family, namely,
Selenarctos thibetanus (Asiatic black bear) and Ursus arctos (brown bear) (Bensky and
Gamble, 1993; Chang and But, 1987). There is evidence to suggest that other species of
bears such as
Ursus americanus (American black bear) and Helarctos malayanus (sun bear)
have also been exploited (Lin
et al., 1997). Selenarctos thibetanus (Ursus thibetanus),
Helarctos malayanus and Melursus ursinus (sloth bear) are among bear species listed under
CITES Appendix 1 (2004), banning international trade in their parts in CITES member states.
However, due to demand for bear products in TCM, regulated bear farming is in operation
in China and the Republic of Korea, where bile is artificially drained from bear gall bladders
(Li, 2004). As a substitute to bear bile, bile derived from pig, water buffalo, goat, cattle
and chicken have also been used in TCM and several of these have been sold as ‘bear bile’
(Lin
et al., 1997).
Section 2

Section 2
6
2.3. Bear bile and its constituents in TCM and Western medicine
Bear bile is composed of deconjugated tauroursodeoxycholic acid (TUDC),
taurochenodeoxycholic acid (TCDC) and taurocholic acid (TC), of which the primary bile
acids are known as ursodeoxycholic acid (UDCA), chenodeoxycholic acid (CDCA) and cholic
acid, respectively (Espinoza
et al., 1993). In the UK, pharmaceutical products containing
UDCA (e.g. Destolit
, Urdox, Ursofalk, Ursogal), which may be obtained from other
animal sources (e.g. ox), are indicated for the dissolution of gallstones (British Pharmacopoeia,
2000). UDCA has also been used in the treatment of some chronic inflammatory disorders
such as liver fibrosis and chronic active hepatitis (Kowdley, 2000; Rolo
et al., 2000; Van
Den Bogaert, 2003).
2.4. Bear bile in TCM
Bear bile (Xiong Dan; Fel Ursi) is described in TCM as having a ‘bitter’ taste and being
‘cold’ in nature. There are several ailments associated with the use of bear bile (Bensky
and Gamble, 1993; Chang and But, 1987). These include gallstones, cholestatic hepatic
diseases, febrile diseases with high fever and convulsions, pharyngolaryngitis, conjunctivitis,
traumatic injuries, swelling, pain, sprains and fractures, haemorrhoids and cardiovascular
diseases. The Association of Chinese Medicine and Philosophy and Earthcare Society (Hong
Kong) in their report on ‘Herbal alternatives to bear bile in Chinese medicine’ included
syphilis and several cancers as being treated by bear bile (IFAW report, 1994). Bile obtained
from other species of animals has also been investigated; one study showed bear bile
and pig bile to demonstrate comparable anti-inflammatory, analgesic and anti-convulsant
properties and therefore, pig bile has been advocated as a suitable animal alternative to
bear bile (Li
et al., 1995).
2.5. Selection of herbs and herbal prescriptions for investigation
2.5.1. Criteria used for selection of single herbs
There is some scientific evidence to support the traditional use of bear bile in the treatment
of inflammatory conditions (Li
et al., 1995), and its chief active constituent, UDCA, in
the treatment of cholesterol gallstones and chronic liver inflammation (Van Den Bogaert,
2003), some cardiovascular diseases (Lee, 1999) and cancer (Im and Martinez, 2004). At
the beginning of this study an extensive literature survey was conducted to select herbs to
be investigated as possible alternatives to bear bile. One hundred and three plant species
used as ‘heat-clearing’ herbs in TCM were identified. In order to select suitable herbs,
criteria were then used which included herbs with constituents similar in structure and
function to UDCA, a tetracyclic compound. For example, pentacyclic triterpenoids such
as ursolic and oleanolic acid are attributed with anti-inflammatory, hepatoprotective and
anti-neoplastic activities (Saraswat
et al., 2000; Syrovets et al., 2000). However, these
compounds are found in several plant species. In consultation with TCM practitioners,
the criteria wer
e further refined (Table 2.1.) in order to select herbs with both TCM and
pharmacological properties similar to bear bile.

Rationale for selection of herbs as potential alternatives to bear bile
7
Criteria A – D: TCM properties of bear bile (Bensky and Gamble, 1993; Chang and But, 1987).
Criteria E – H: Pharmacological / clinical effects of bear bile and UDCA reported in the literature
(Li
et al., 1995; Lee, 1999; Van Den Bogaert, 2003; Im and Martinez, 2004).
Criteria A – D (r
efer to Table 2.1.): based on TCM literature (Chang and But, 1987
1
; Bensky and Gamble, 1993
2
;
Chang and But, 2001
3
; Chinese Pharmacopoeia, 2005
4
).
Criteria E – H (refer to Table 2.1.): based on pharmacological and clinical data (Section 2.4).
Table 2.2. TCM plants selected after consultation with TCM practitioners and from evaluation of TCM
literature and pharmacological and clinical data
Plant species with some similar properties to bear bile A B C D E F G H TCM references
1. Gardenia jasminoides Ellis (syn.: G. augusta Merr.)
(Rubiaceae) fruit = Zhi Zi
* * * * * * * 1, 2, 4
2. Anemarrhena asphodeloides Bge. (Anthericaceae) rhizome
= Zhi Mu
* * * * * 1, 2, 4
3. Scutellaria baicalensis Georgi (Lamiaceae) root =
Huang Qin
* * * * * * * 1, 2, 4
4. Coptis chinensis Franch. (Ranunculaceae) rhizome =
Huang Lian
* * * * * * * 1, 2, 4
5. Phellodendron amurense Rupr (Rutaceae) bark =
Huang Bai
* * * * * * 4
6. Andrographis paniculata Nees (Acanthaceae) aerial parts =
Chuan Xin Lian
* * * * * * 1, 2, 4
7. Rheum palmatum L. (Polygonaceae) root and rhizome =
Da Huang
* * * * * 1, 3
Table 2.1. Properties / functions of bear bile and UDCA used as criteria for herb selection
C
riteria
P
roperties and functions of bear bile and UDCA
A ‘Cold’ nature
B ‘Bitter’ taste
C ‘Heat clearing’
D ‘
Fire-purging’
E Anti-inflammatory properties
F Hepatoprotective properties
G Anti-neoplastic properties
H Cardiovascular protective properties

Out of the eight criteria listed (Table 2.1.) priority was given to plant species that complied
with the properties of bear bile as described in TCM [Table 2.1; criteria A – D]. Therefore,
all the herbs selected for study are traditionally used in the practice of Chinese medicine.
Additional criteria for choice were based on evidence from published scientific studies
[Table 2.1; E – H]. When assessing the suitability of the selected herbs as alternatives to bear
bile, the known relevant biological activities and their constituents were also considered.
The potential for using these species as alternatives to bear bile was then discussed with
TCM practitioners. These discussions resulted in a reduction in the number of plant species
from 103 species to 7 species (Table 2.2.).
2.5.2. Criteria used for selection of herbal prescriptions for study
In addition to the 7 selected herbs chosen for study, the use of prescriptions in TCM
was also considered. In TCM, a mixture of different plant species is commonly prescribed,
relying on the synergy between herbs, sometimes with animal parts and/or minerals. A
literature survey was therefore conducted on TCM prescriptions containing bear bile with
the aim of selecting prescriptions for investigation, to determine whether they demonstrate
biological activity without bear bile. A major criterion for choosing TCM prescriptions for
this study was that they contained not more than one animal product (i.e. bear bile). It
was also important to choose prescriptions that did not contain endangered plant species
(those restricted by CITES). A survey identified a TCM prescription (prescription X), which
complied with the criteria. Prescription X is used in the tr
eatment of laryngitis and contains
bear bile and six herbs (Zhu, 1989). The herbal composition of prescription X is described
in Table 2.3. In addition, two Chinese-Japanese (Kampo) patent medicines, Orengedokuto
and Diao-Orengedokuto are also proposed as possible replacements for prescriptions
containing bear bile. These two prescriptions were selected on the basis of being composed
of herbs proposed fr
om this investigation and also possessing some similar biological and
TCM functions as bear bile.
Section 2
8
Table 2.3. Herbal composition of prescription X
Herbs listed as being in prescription
Zhi Zi, fruit of Gardenia jasminoides (Rubiaceae)
Huang Lian, rhizome of Coptis chinensis (Ranunculaceae)
Ban Lan Gen, r
oot of
Isatis indigotica (Brassicaceae)
Jin Yin Hua, flower bud of Lonicera japonica (Caprifoliaceae)
Lian Qiao, fruit of Forsythia suspensa (Oleaceae)
Hu Po, fossil r
esin of
Pinus succinifer (Pinaceae)

Rationale for selection of herbs as potential alternatives to bear bile
9
2.6. Single herbs chosen as potential alternatives to bear bile
in TCM: summary of reputed and pharmacological effects
2.6.1. Huang Qin (Radix Scutellariae)
Huang Qin (skullcap) is prepared from the root of Scutellaria baicalensis Georgi (Lamiaceae).
It functions to ‘remove damp’, ‘quench fire’ and counteract toxins. It is used as an anti-
inflammatory agent and in the treatment of fever, hepatitis and acute conjunctivitis
(Bensky and Gamble, 1993; Chang and But, 1987). The anti-pyretic, analgesic (Huang
et al., 1990), anti-hypercholesterolemic (Yotsumoto et al., 1997), cardiovascular protective
(Wang
et al., 2004) and anti-neoplastic (Fukutake et al., 1998; Wozniak et al., 2004)
effects of
S. baicalensis have been reported. In addition, the anti-inflammatory effect of
S. baicalensis has been demonstrated in vitro and in vivo (van Loon, 1997; Cuellar et al.,
2001). Flavonoid constituents (baicalein, baicalin, wogonin and oroxylin A) of
S. baicalensis
have been researched for the mechanism of action of their anti-inflammatory effects (Chen
et al., 2000; Chen et al., 2001; Kang et al., 2003a; Huang et al., 2004).
2.6.2. Huang Lian (Rhizoma Coptidis)
Huang Lian (goldthread rhizome) is prepared from the dried rhizome of Coptis chinensis
Franch, C. deltoidea C.Y.Cheng.&Hsiao, or C. teeta Wallich (Ranunculaceae). It is used
traditionally in the treatment of some inflammatory diseases, fever, conjunctivitis and some
tumours (Hsu
et al., 1986; Chang and But, 1987). Some studies have associated Rhizoma
Coptidis with potential anti-inflammatory, anti-oxidant, anti-hypercholesterolemic, and
anti-neoplastic effects.
C. chinensis has shown anti-inflammatory activity in vivo (Cuellar
et al., 2001), which may be mediated through the inhibition of interleukin-8 (IL-8)
induction (Lee
et al., 1995). Berberine, a constituent of C. chinensis, is reported to inhibit
cyclooxygenase-2 (COX-2) activity (Fukuda
et al., 1999). C. chinensis is reported to be
anti-oxidant
in vitro and in vivo (Liu and Ng, 2000; Schinella et al., 2002) and has shown
cholesterol lowering effects (Yotsumoto
et al., 1997; Yokozawa et al., 2003). Rhizoma
Coptidis has also shown potential anti-neoplastic effects
in vitro (Fukutake et al., 1998;
Iizuka
et al., 2000). Anti-bacterial activity of Rhizoma Coptidis is cited in Chang and But
(1987) and Tang and Eisenbrand (1992). Adverse effects associated with Rhizoma Coptidis
include vomiting, dyspnoea and convulsions (Huang, 1999).
2.6.3. Huang Bai (Cortex Phellodendri)
Huang Bai (Amur corktree) is prepared from the dried stem bark of Phellodendron amurense
Rupr. (Rutaceae). It functions as a ‘heat-clearing’, anti-inflammatory and anti-bacterial
agent in TCM (Chang and But, 1987; Tang and Eisenbrand, 1992). Some studies indicate
that
P. amurense has anti-inflammatory (Cuellar et al., 2001), hepatoprotective (Yotsumoto
et al., 1997), anti-oxidant (Kong et al., 2001) and anti-bacterial (Chang and But, 1987)
activities. In one study, the hepatoprotective effects of Cortex Phellodendri were reported
to be less effective than Rhizoma Coptidis and Radix Scutellariae (Yotsumoto
et al., 1997).

2.6.4. Zhi Zi (Fructus Gardeniae)
Zhi Zi (cape jasmine fruit) is prepared from the fruit of Gardenia jasminoides Ellis (syn.:
G. augusta Merr.) (Rubiaceae). It is reputed to ‘reduce heat’, ‘remove heat from the blood’,
counteract toxicity and ease the mind (Pharmacopoeia of PRC, 2000). It is a ‘fire-purging’
febrifuge and is indicated in febrile diseases with restlessness. It is also used traditionally
in the treatment of conjunctivitis, some tumours and externally for sprains and bruises
(Chang and But, 1987; Bensky and Gamble, 1993). The anti-inflammatory, anti-neoplastic
(Fukutake
et al., 2000) and hepatoprotective (Chiu et al., 1989) activities of Fructus
Gardeniae have been reported. Genipin and geniposide, constituents of the herb, have
been r
eported to be analgesic (Harada
et al., 1974) and geniposide is r
eported to be anti-
inflammatory
in vivo (Yao et al., 1991). Genipin, as well as crocetin have been reported to
possess anti-neoplastic properties (Chang
et al., 1996; Kuo et al., 2004). Fructus Gardeniae
and its constituents, genipin and crocin, are reported to be choleretic (Harada
et al., 1974;
Tang and Eisenbrand, 1992). Crocin, a carotenoid constituent of
Gardenia jasminoides
possess anti-oxidant properties (Pham et al., 2000). However, reversible acute hepatic
damage has been observed with crocin (Tang and Eisenbrand, 1992) and diarrhoea was
observed as a side effect of geniposide in mice (Bensky and Gamble, 1993). Gardenic acid
and gardenodic acid A (constituents of
Gardenia jasminoides) can cause abortion in early
pregnancy (Pei-Gen and Nai-Gong, 1991).
2.6.5. Chuan Xin Lian (Herba Andrographis)
Chuan Xin Lian (green chiretta) is prepared from the dried aerial parts of Andrographis
paniculata
Nees (Acanthaceae). It is used in TCM to ‘remove heat’, ‘counteract toxicity’
and reduce swelling (Pharmacopoeia of PRC, 2000). It is used traditionally in the treatment
of inflammatory diseases, fever, hepatitis and pharyngolaryngitis (Chang and But, 1987;
Bensky and Gamble, 1993; Hocking 1997). There is some scientific evidence to support
the traditional use of
A. paniculata as an anti-pyretic, anti-inflammatory, hepatoprotective
and cardiovascular protective agent. Clinical trials indicate that
A. paniculata has some
efficacy in treating fever, sore throat and cold symptoms, but was not as effective as
paracetamol (Thamlikitkul
et al., 1991; Caceres et al., 1999). Diterpene lactones
(andrographolide, neoandrographolide, 14-deoxy-11, 12-didehydroandrographolide
and 14-deoxyandrographolide) from
A. paniculata have been reported to exert anti-
inflammatory effects through the inhibition of nitric oxide (Zhang and Tan, 1999; Chiou
et al., 2000; Batkhuu et al., 2002), and are also reputedly anti-pyretic (Chang and But,
1987). The anti-inflammatory action of andrographolide has been extensively researched
(Habtemariam, 1999; Amroyan
et al., 1999; Chiou et al., 2000; Shen et al., 2002) and
it is also reported to possess anti-neoplastic properties (Rajagopal
et al., 2003). The
hepatoprotective activities of
A. paniculata have been reported (Ram 2001; Trivedi et al.,
2001), and several hepatoprotective compounds have been isolated from
A. paniculata
(Jain et al., 2000). Extracts of A. paniculata and 14-deoxy-11,12-didehydroandrographolide
have demonstrated cardiovascular activity
in vivo (Guo et al., 1996; Zhang et al., 1998).
Overdose of the herb may cause dizziness, palpitations, gastric discomfort and loss of
appetite (Chang and But, 1987; Huang, 1999). The herb extract has shown contraceptive
effects
in vivo (Zoha et al., 1989).
Section 2
10

2.6.6. Zhi Mu (Rhizoma Anemarrhena)
Zhi Mu is prepared from the dried rhizome of Anemarrhena asphodeloides Bge
(Anthericaceae). It is classified as a ‘fire-purging’ anti-pyretic (Pharmacopoeia of PRC,
2000).
A. asphodeloides and its constituent, sarsapogenin, are reputed to reduce fever
in vivo (Chang and But, 1987; Huang, 1999). A xanthone-C-glucoside isolated from
A. asphodeloides, mangiferin, has shown anti-oxidant effects (Sanchez et al., 2000; Ma
et al., 2001). Mangiferin and other constituents of A. asphodeloides, cis-hinokiresinol,
tigogenin and hecogenin have demonstrated anti-neoplastic properties (Yoshimi
et al.,
2001; Corbiere
et al., 2003; Jeong et al., 2003). A. asphodeloides is reputed to have an
inhibitory ef
fect on several bacteria (Chang and But, 1987).
2.6.7. Da Huang (Radix et Rhizoma Rhei)
Da Huang (rhubarb) is prepared from the dried root and rhizome of Rheum palmatum L.,
R. tanguticum Maxim. and R. officinale Baill. (Polygonaceae). It is used traditionally mainly
as a laxative and also in the treatment of haemorrhoids, conjunctivitis and as a choleretic
agent (Hsu
et al., 1986; Tang and Eisenbrand, 1992; Huang, 1999). Radix et Rhizoma Rhei
has been shown to have cholesterol reducing ef
fects through the inhibition of 3-hydroxy-
3-methlyglutary-coenzyme A (HMG-CoA) reductase activity (Kim
et al., 2002). The rhizome
of
R. undulatum and constituent stillbenes (rhapontigenin, piceatannol, resveratrol,
chrysophanol 8-
O-β-D-(6’-galloyl)-glucopyranoside and aloe-emodin 1-O-β-D-
(glucopyranoside) have been shown to exert anti-inflammatory effects through the
inhibition of nitric oxide production
in vitro (Matsuda et al., 2000; Kageura et al., 2001).
Species of Rheum used in Da Huang listed above have shown anti-oxidant effects
in vitro
(Matsuda et al., 2001b).
2.7. Prescriptions chosen for investigation as potential
alternatives to bear bile in TCM
2.7.1. Orengedokuto
Orengedokuto (Huanglian-Jie-Du-Tang, TJ-15) is a traditional kampo patent medicine
which is approved as an ethical medicine by the Ministry of Health and Welfare of Japan,
and is listed in the Pharmacopoeia of Japan for the treatment of cerebrovascular disease,
hypertension, gastritis and liver diseases (Ohta
et al., 1998; Maclean and Taylor, 2000).
Orengedokuto is composed of four herbs (Table 2.4.).
In this current study, the four herbs were individually studied and proposed as herbal
alternatives to bear bile. There has been extensive scientific research, by other workers, into
the pharmacological properties of Orengedokuto. Similar to bear bile, it has been associated
with anti-inflammatory (Dai
et al., 2000; Fukutake et al., 2000), hepatoprotective (Ohta
et al., 1998; Seikiya et al., 2002) and anti-neoplastic (Fukutake et al., 2000) effects.
Rationale for selection of herbs as potential alternatives to bear bile
11

Water extracts of Rhizoma Coptidis, Radix Scutellariae and Fructus Gardeniae (three of the
components of orengedokuto) also showed some potential anti-neoplastic activity, but
with lower efficacy than the formula (Fukutake
et al., 1998; Fukutake et al., 2000). Fructus
Gardeniae has also been associated with potential anti-inflammatory activity (which may
be mediated via inhibition of COX-2 activity) (Fukutake
et al., 2000). Orengedokuto has
also been reported to inhibit hepatic cholesterol ester formation by inhibiting the activity
of acyl-coenzymeA:cholesterol acyltransferase (ACAT)
in vitro (Yotsumoto et al., 1997). In
addition, extracts of Radix Scutellariae, Rhizoma Coptidis and Cortex Phellodendri decreased
ACAT activity, whereas Fructus Gardeniae had no significant effect (Yotsumoto
et al., 1997).
However, oral administration of Orengedokuto was not able to significantly reduce fever
caused by a bacterial pyrogen
in vivo (Itami et al., 1992).
2.7.2. Dia-Orengedokuto
Dia-Orengedokuto contains the same herbs as Orengedokuto with an additional herb,
Radix et Rhizoma Rhei and is used in the treatment of atherosclerosis (Kim
et al., 2002).
Water and ethanol extracts of Dia-Orengedokuto inhibited the activity of HMG-CoA
reductase more potently than extracts of Orengedokuto (Kim
et al., 2002). Of the herbal
constituents of Dia-Orengedokuto, Rhizoma Coptidis was more potent at reducing HMG-
CoA reductase activity, followed by Radix et Rhizoma Rhei (Kim
et al., 2002). Constituents
of bear bile (CDCA and cholic acid) are also inhibitors of HMG-CoA reductase, which leads
to reduction in hepatic cholesterol levels (Björkhem
et al., 1993).
Section 2
12
Table 2.4. Composition of herbs in Orengedokuto (Seikiya et al., 2002)
H
erbs
R
atio
Huang Qin (Radix Scutellariae) 3.0
H
uang Lian (Rhizoma Coptidis)
2
.0
Huang Bai (Cortex Phellodendri) 1.5
Zhi Zi (Fructus Gardeniae) 2.0

2.8. Alternatives to bear bile: summary
Based on both traditional uses and knowledge of the medicinal properties of the different
species of plants, a selection of 7 single herbs and 2 prescriptions consisting of a combination
of herbs, were chosen as alternatives to bear bile in TCM. In addition, a TCM prescription
containing bear bile was considered for study (prescription X). From Table 2.2., it is apparent
that none of the 7 herbs listed as having similar properties to bear bile fulfil all the functions
reputedly associated with bear bile. In addition, some TCM prescriptions containing bear
bile also contain some of the suggested herbal replacements. It may be that, in some
circumstances combinations of herbs, based on TCM principles, may be more suitable
as replacements for bear bile in prescriptions containing the animal product.
The Association of Chinese Medicine and Philosophy and Earthcare Society (Hong Kong)
has published a report on ‘The Herbal Alternatives to Bear Bile in Chinese Medicine’ based
on TCM philosophy (IFAW report, 1994). They suggest 54 herbs as alternatives to bear bile
(Appendix 1). Five of the seven herbs listed in Table 2.2 were also proposed in the IFAW
report as alternatives to bear bile, Huang Qin (Radix Scutellariae), Huang Bai (Cortex
Phellodendri), Zhi Zi (Fructus Gardeniae), Chuan Xin Lian (Herba Andrographitis) and
Da Huang (Radix et Rhizoma Rhei). The underlying pathology of several of the ailments
treated with bear bile is inflammation. In humans, inflammation is often initiated by
infection, frequently caused by different species of bacteria (Moltz, 1993). Therefore, in
this study, the herbs and prescription selected for investigation as possible alternatives to
bear bile were tested in anti-bacterial and anti-inflammatory assays. The methods used and
the results obtained in the present study are presented in Sections 5 and 6.
Rationale for selection of herbs as potential alternatives to bear bile
13

Rationale for selection of herbs as
potential alternatives to rhino horn
3.1. Introduction
This section addresses the selection of potential herbal alternatives to rhino horn in TCM.
As with the choice of herbs to investigate as potential alternatives for the use of bear bile,
selection has been based on investigation of both traditional use and the medicinal properties
of the different species of plants. In this part of the report, sources of rhino horn, its uses
in TCM and its reported pharmacological activities are discussed. In addition, TCM
prescriptions traditionally containing rhino horn were investigated to determine their
pharmacological potential in the presence and absence of the animal product. A summary
of some of the known constituents and available research on the herbs and prescriptions
selected for further investigation is given.
3.2. Sources of rhino horn
The black rhino (Deceros bicornis, Rhinocerotidae) population decreased by 95% between
1970 and 1993 (WWF, 2002). Due to a decline in their population, all five species (
Deceros
bicornis, Cerathotherium sinum, Dicerorhinus sumatrensis, Rhinoceros sondaicus,
Rhinoceroos unicornis
) of Rhinocerotidae are listed under CITES Appendix 1 (2004), therefore
banning international commercial trade in their parts. Water buffalo horn has been used
as an animal substitute for rhino horn, but generally at higher doses (Bensky and Barolet,
1990). Other alternatives to rhino horn include horns from cattle and the Saiga antelope
(But
et al., 1990).
3.3. Rhino horn and its constituents
The primary constituent of rhino horn is keratin; constituents also include other proteins,
amino acids, peptides, sterols, amines and calcium (Ingaki and Oida, 1970; Lee and Kim,
1974; Chang and But, 1987). Aqueous extracts of horns from rhino, Saiga antelope
(Saiga
tatarica
), water buffalo (Bubalus bubalis) and cattle (Bos taurus domesticus) are reported
to be anti-pyretic (But
et al., 1990). But and Tam (1991) investigated the anti-pyretic
properties of herbal prescriptions containing either rhino horn or buffalo horn; separate
rhino and buffalo horn extracts were found to be anti-pyretic and the combined horn-herb
extracts were also anti-pyretic. In another study, rhino horn did not show anti-pyretic activity
in vivo (Laburn and Mitchel, 1997). But et al. (1990) cited other studies conducted in Asia
on the anti-pyretic properties of rhino horn with contradictory conclusions, but mainly with
negative results. Scientific research into the anti-pyretic properties of rhino horn has shown
that it is effective at reducing temperature in febrile animals only at high concentrations.
Summary
14
Section 3

3.4. Rhino horn in TCM
In TCM, rhino horn is considered as having a strong action of ‘clearing heat’, ‘removing
heat from the blood’, as well as arresting convulsions (Xu, 1994). The low efficacy of the
rhino horn extracts to reduce temperature in febrile animals could in part be explained by
the differences in concepts of the pathology of fever between Western medicine and TCM.
The major difference is that in TCM febrile diseases can manifest without an increase in
body temperature (Hsu
et al., 1986; Xu, 1994). In contrast, febrile diseases are associated
with an increase in body temperature in Western medicine (Moltz, 1993).
Rhino horn (Xi Jiao; Cornu Rhinocerotis) is used as a detoxifying, anti-convulsant and
anti-inflammatory agent. A major use of rhino horn is in the treatment of advanced stages
of fever in the ying and blood conformation, complicated by delirium or coma. It is often
used in combination with other TCM remedies and the horn is reputed to be a potent
anti-convulsant in these remedies (Chang and But, 1987). Haemorrhagic conditions (e.g.
erythema, haematemesis and epistaxis) sometimes manifest symptoms associated with
conditions treated by rhino horn. Rhino horn has also been associated with the treatment
of cardiovascular diseases (Chang and But, 1987).
3.5. Selection of herbs and prescriptions for investigation
3.5.1. Criteria used for selection of single herbs
After consulting TCM practitioners a set of criteria was developed (Table 3.1.) and used to
conduct a survey of TCM literature, to identify herbs to be investigated as possible alternatives
to rhino horn (Table 3.2.). This was used in consultation with TCM practitioners to select
nine plant species (Table 3.2.) for further investigation.
Rationale for selection of herbs as potential alternatives to rhino horn
15
Table 3.1. Properties / functions of rhino horn used as criteria for herb selection
Criteria Properties and functions of rhino horn
A ‘Cold’ nature
B ‘Bitter’ taste
C ‘Salty’ taste
D ‘Blood cooling’
E ‘Heat clearing’
F Anti-convulsant
G Anti-inflammatory properties
H Anti-pyr
etic pr
operties
I Reduce haemorrhage

Section 3
16
Table 3.2. TCM herbs selected after consultation with TCM practitioners and from evaluation
of TCM literature and pharmacological and clinical data
Herbs with some similar properties to rhino horn A B C D E F G H I TCM references
1. Scrophularia ningpoensis Hemsl. (Scrophulariaceae) root
= Xuan Shen
* * * * * * * 1, 2
2. Rehmannia glutinosa Steud (Scrophulariaceae) root
= Sheng Di Huang
* * * * * 1, 2
3. Paeonia suffruticosa Andr. (Paeoniaceae) root = Mu Dan Pi * * * * * * 1, 2
4. Paeonia veitchii Lynch or P. lactiflora Pall. (Paeoniaceae) root
= Chi Shao
* * * * * * * 2
5. Arnebia euchroma I.M.Johnst. (Boraginaceae) root = Zi Cao * * * * * 1
6. Isatis indigotica (Brassicaceae) root = Ban Lan Gen * * * * * * 1, 2
7. Lonicera japonica Thunb. (Caprifoliaceae) flower bud
= Jin Yin Hua
* * * * * 1, 2
8. Forsythia suspensa Vahl (Oleaceae) fruit = Lian Qiao * * * * * 1, 2
9. Salvia miltiorrhiza Bge (Lamiaceae) root = Dan Shen * * * * 1, 2
Criteria A – I (refer to Table 3.1.): based on TCM literature (Bensky and Gamble, 1993
1
; Chinese Pharmacopoeia, 2005
2
).
3.5.2. Criteria used for selection of herbal prescriptions
When combined with other TCM remedies to form prescriptions, rhino horn is reputed
to play an important role. Therefore, another literature survey was conducted to ascertain
TCM prescriptions containing rhino horn, which could be studied in biological assays with
and without rhino horn. Five TCM prescriptions were selected for study on the basis of
containing rhino horn as the only animal component. The prescriptions selected were Qing
Ying T
ang, Qingwen Baidu Yin, Xi Jiao Dihuang Tang, Sheng Xi Dan and Qing Gong Tang.
Also selected was a TCM prescription composed only of herbs, Zhi Zi Jin Hua, which was
used as a TCM ‘control’. All six prescriptions are used to treat epidemic febrile diseases and
their compositions are described in Table 3.3.

Rationale for selection of herbs as potential alternatives to rhino horn
17
Table 3.3. The distribution of 23 herbs and one mineral in six TCM prescriptions.
TCM control TCM prescriptions traditionally containing rhino horn
Zhi Zi Jin Hua Qingwen Baidu Yin Xi Jiao Dihuang Tang Qing Ying Tang Sheng Xi Dan Qing Gong Tang
1. Xuan Shen, root of Scrophularia ningpoensis (Scrophulariaceae) * * * *
2. Sheng Di Huang, root of Rehmannia glutinosa (Scrophulariaceae) * * * *
3. Mu Dan Pi, root of Paeonia suffruticosa Andr. (Paeoniaceae) * *
4. Chi Shao, root of Paeonia lactiflora, P. veitchii (Paeoniaceae) * *
5. Zi Cao, root of Arnebia euchroma (Boraginaceae) *
6. Ban Lan Gen, root of Isatis indigotica (Brassicaceae) *
7. Jin Yin Hua, flower bud of Lonicera japonica (Caprifoliaceae) * * *
8. Lian Qiao, fruit of Forsythia suspensa (Oleaceae) * * * *
9. Dan Shen, root of Salvia miltiorrhiza (Lamiaceae) *
10. Zhi Mu, rhizome of Anemarrhena asphodeloides (Anthericaceae) * *
11. Zhi Zi, fruit of Gardenia jasminoides (Rubiaceae) * *
12. Huang Qin, root of Scutellaria baicalensis (Lamiaceae) * * *
13. Huang Lian, rhizome of Coptis chinensis (Ranunculaceae) * * *
14. Huang Bai, cortex of Phellodendron amurense (Rutaceae) *
15. Da Huang, root and rhizome of
Rheum palmatum (Polygonaceae)
*
16. Tian Hua Fen, root of
Trichosanthes kirilowii (Cucurbitaceae)
* *
17. Lian Zi Xin, seed of Nelumbo nucifera (Nelumbonaceae) *
18. Mai Men Dong, root of Ophiopogon japonicus (Convallariaceae) * *
19. Dan Zhu Ye, aerial part of Lophatherum gracile (Poaceae) * * *
20. Jie Geng, root of Platycodon grandiflorum (Campanulaceae) *
21. Gan Cao, root of Glycyrrhiza uralensis or G. glabra (Leguminosae) *
22. Chang Pu, rhizome of Acorus calamus, A tatarinowii (Acoraceae) *
23. Dan Dou Chi, seed of Glycine max (Leguminosae) *
24. Shi Gao, calcium sulphate *
Zhi Zi Jin Hua was used as a TCM control prescription in biological tests. Herbs numbered 1 to 7 are also listed in Table 2.6 as possible alternatives to rhino horn.

The six prescriptions were made up of a total of 23 herbs and one mineral (Table 3.3.). It
became apparent from Table 3.3., that all nine possible herbal ‘alternatives’ to rhino horn
already existed in one or more prescriptions used in this study. This finding confirmed the
practice in TCM of combining remedies with similar functions for their additive and synergistic
effects. However, it challenged the logic of replacing one plant species with a specific range
of functions with another species that has different properties, and that might already exist in
a prescription. Therefore, the principles governing the composition of TCM prescriptions were
considered in determining the suitability of the selected herbs as alternatives to rhino horn.
3.6. Single herbs as potential alternatives to rhino horn:
reputed and pharmacological effects
3.6.1. Ban Lan Gen (Radix Isatidis)
Ban Lan Gen (Dyer’s woad) is prepared from the dried root of Isatis indigotica Fort.
(Brassicaceae). Similar to rhino horn, it is a ‘blood cooling’ febrifuge with the TCM properties
‘cold’ and ‘bitter’ (Hsu
et al., 1986). Ban Lan Gen is used traditionally as an anti-inflammatory,
anti-bacterial and anti-viral remedy, and is often used in the treatment of seasonal febrile
diseases (Hsu
et al., 1986; Ho and Chang, 2002). Ho and Chang (2002) reported that the
methanolic extract of the dried roots of
I. indigotica was anti-pyretic, anti-inflammatory
and analgesic. Ethanol extracts of the roots of
I. indigotica and organic acids isolated
from
I. indigotica (2-aminobenzoic acid, benzoic acid, salicylic acid, syringic acid and
3-(2’-carboxyphenyl)-4(3H)-quinazoline) showed anti-endotoxin activity (Wu
et al., 1997).
The alkaloid tryptanthrin, isolated from
I. indigotica, inhibited nitric oxide, PGE
2
(Ishihara
et al., 2000) as well as 5-lipoxygenase (5-LOX) and COX-2 activities (Danz et al., 2002),
indicating anti-inflammatory properties. Isaindigotone, also an alkaloid from
I. indigotica,
inhibited 5-LOX activity, prostaglandin E
2
(PGE
2
) generation and nitric oxide (Molina
et al., 2001).
3.6.2. Chi Shao (Radix Paeoniae Rubra)
Chi Shao is prepared from the dried roots of Paeonia lactiflora Pall. (white peony) or P. veitchii
Lynch (red peony) (Paeoniaceae). Similar to rhino horn, it is classified as a ‘blood cooling’
febrifuge in TCM (Hsu
et al., 1986; Wiseman and Ye, 1998). It has been reputed to possess
analgesic and anti-convulsive effects (Hsu
et al., 1986; Ding et al., 2000). P. lactiflora and
its constituent paeonol, are reported to be anti-oxidant (Goto
et al., 1999; Ohsugi et al.,
1999).
P. lactiflora demonstrated anti-inflammatory action in vitro (Huang et al., 1990) and
reduced liver damage
in vivo (Qi, 1991). Radix Paeoniae Rubra showed anti-neoplastic
properties
in vitro (Lee et al., 2002). Paeoniflorin isolated from P. lactiflora has been shown
to be an anti-hyperlipidemic agent
in vivo (Yang et al., 2004), reduce haemorrhage due
to bacterial infection and possess anti-inflammatory properties
in vitro (Ding et al., 2000).
Another compound, 1,2,3,4,6-penta-
O-galloyl-β-D-glucose isolated from the root of Paeonia
lactiflora
has been shown to possess anti-oxidant, anti-neoplastic and anti-inflammatory
effects (Lee
et al., 2003). Resveratrol, also isolated from the root of Paeonia lactiflora, has
been reported to have anti-oxidant and anti-neoplastic activities,
in vitro (Kang et al., 2003b).
Section 3
18

3.6.3. Mu Dan Pi (Cortex Moutan)
Mu Dan Pi is prepared from the dried root bark of Paeonia suffruticosa Andr. (Paeoniaceae).
Similar to rhino horn, it is used as a ‘blood-cooling’ febrifuge in TCM (Hsu
et al., 1986;
Wiseman and Ye, 1998). The TCM herbs Mu Dan Pi and Chi Shao have been shown to
have some similar pharmacological and phytochemical properties (Lin
et al., 1999;
Ding
et al., 2000). Both herbs contain the compounds paeoniflorin, resveratrol and
1,2,3,4,6-penta-
O-galloyl-β-D-glucose (pharmacological properties are listed under Chi
Shao). Recently, 1,2,3,4,6-penta-
O-galloyl-β-D-glucose has been demonstrated to exert
anti-inflammatory activity through the inhibition of IL-8 via NF
−κΒ binding inhibition
(Oh
et al., 2004) and inhibition of iNOS and COX-2 (Lee et al., 2003). The major lipophilic
compound from
P. suffruticosa, paeonol and minor constituents, 2,5-dihydroxy-4-
methoxyacetophenone and 2,5-dihydroxy-4-methylacetophenone have also demonstrated
anti-inflammatory effects (Lin
et al., 1999). Methanolic extracts of P. suffruticosa inhibited
IL-8 production (Oh
et al., 2003); water extracts showed anti-oxidant effects (Liu and Ng,
2000),
in vitro. Some compounds from P. suffruticosa namely suffruticosides A, B, C, and
D, galloyl-oxypaeoniflorin, and galloyl-paeoniflorin has been reported to exhibit more
potent anti-oxidant effects than
α-tocopherol (Matsuda et al., 2001a).
3.6.4. Dan Shen (Radix Salvia Miltiorrhizae)
Dan Shen (red sage root) is prepared from the root of Salvia miltiorrhiza Bge (Lamiaceae).
Tanshinones from
S. miltiorrhiza are reported to be anti-inflammatory in rats with infective
arthritis (Duke and Ayensu, 1985) and in mice with croton oil induced inflammation of the
ear (Tang and Eisenbrand, 1992), however the mechanism of action was not established
in these studies. A diterpene, tanshinone IIA, isolated from the root of
S. miltiorrhiza
demonstrated anti-inflammatory effects through the inhibition of iNOS expression and
production of TNF-
α, IL-1β and IL-6 (Jang et al., 2003). Tanshinones from S. miltiorrhiza
root have also demonstrated anti-inflammatory activity in mice and were active against
5-LOX in porcine leukocytes, but were not as active as the crude extracts (Chang and But,
1986; Paulus and Bauer, 2000).
Rationale for selection of herbs as potential alternatives to rhino horn
19

3.6.5. Jin Yin Hua (Flos Lonicerae)
Jin Yin Hua (honeysuckle) is prepared from the dried flower buds of Lonicera japonica
Thunb. (Caprifoliaceae). It is used traditionally as an anti-bacterial, anti-inflammatory and
anti-pyretic remedy (Chang and But, 1987; Tang and Eisenbrand, 1992). A water extract
of
L. japonica demonstrated anti-inflammatory properties by inhibiting NF–κΒ activity,
inducible nitric oxide and TNF-
α in vitro (Lee et al., 2001); anti-inflammatory effects of
aqueous extracts of
L. japonica have also been shown in vivo (Tae et al., 2003). Oral
administration of a butanol extract of
L. japonica had mild anti-inflammatory activity
against acute granulomatic and chronic inflammatory models
in vivo (Lee et al., 1998).
Other studies pr
ovide some information regarding the compounds that might be
responsible for the anti-inflammatory effects of the crude extracts. Lonicerosides A and C,
saponins from the aerial parts of L. japonica, caused inhibition of ear oedema in vivo
(Kwak et al., 2003). Some compounds (e.g. methyl caffeate, 3,4-di-O-caffeoylquinnic acid,
methyl 3,4-di-
O-caffeoylquinate) isolated from L. japonica, inhibited platelet aggregation
(Chang and Hsu, 1992); methyl caffeate and methyl 3,4-di-
O-caffeoylquinate also potently
inhibited thromboxane formation from endogenous arachidonic acid (Chang and Hsu,
1992) and ochnaflavone, also isolated from
L. japonica, strongly inhibited rat platelet
phospholipase A
2
(Chang et al., 1994).
3.6.6. Lian Qiao (Fructus Forsythia)
Lian Qiao (rengyo) is prepared from the dried fruit of Forsythia suspensa Vahl. (Oleaceae).
It is used traditionally as an anti-pyretic and an anti-inflammatory agent in the treatment
of bacterial infections (Chang and But, 1986; Tang and Eisenbrand, 1992). Water extracts
of Fructus Forsythia have been reputed to reduce inflammation and fever
in vivo (Chang
and But, 1987). Methanol and
n-hexane fractions of aqueous extracts of F. suspensa have
been shown to have anti-inflammatory effects
in vivo (Ozaki et al., 1997). One of the
anti-inflammatory constituents of the
n-hexane extract was found to be 3β-aceto-20,25-
epoxydammarane-24-ol (Ozaki
et al., 2000).
3.6.7. Sheng Di Huang (Radix Rehmanniae)
Sheng Di Huang (Chinese foxglove root) is prepared from the root of Rehmannia glutinosa
Steud (Scrophulariaceae). Similar to rhino horn, it is classified in TCM as an anti-inflammatory
and a ‘blood-cooling’ febrifuge (Hsu
et al., 1986). Due to its similarity in TCM functions as
rhino horn, when combined with rhino horn in TCM prescriptions, the two remedies are
often considered as the most important ingredients (Xu, 1994). However,
R. glutinosa is
not attributed with the anti-convulsant pr
operties of rhino horn (Xu, 1994).
Rehmannia
glutinosa
has shown potential anti-inflammatory effects through the inhibition of COX
(Prieto
et al., 2003), TNF-α and IL-1 (Kim et al., 1999) secretion, in vitro.
Section 3
20

3.6.8. Xuan Shen (Radix Scrophulariae)
Xuan Shen (figwort root), prepared from the dried roots of Scrophularia ningpoensis Hemsl
possesses anti-inflammatory properties and is a ‘blood-cooling’ febrifuge (Hsu
et al., 1986;
Wiseman and Ye, 1998). Phenylpropanoid glycosides (angoroside C and acteoside) isolated
from the root of
S. ningpoensis have demonstrated anti-oxidant effects in vitro (Li et al.,
2000). Constituent iridoids glycosides (aucubin, verbenalin, and loganin) are reported to
show anti-inflammatory effects
in vivo (Recio et al., 1994); aucubin has been shown to
exert anti-inflammatory activity through the inhibition of the leukotriene, LTC
4
, in vitro
(Bermejo et al., 2000).
3.6.9. Zi Cao (Radix Arnebiae)
Zi Cao (purple gromwell root) is prepared from the root of Arnebia euchroma I.M.Johns+
(Boraginaceae). Like rhino horn, it is classified as a ‘blood cooling’ febrifuge in TCM (Hsu
et al., 1986). Arnebia euchroma demonstrated anti-inflammatory activity, in vivo (Kaith
et al., 1996) and in vitro through the inhibition of COX-2 activity (Subbaramaiah et al.,
2001). Shikonin, a compound isolated from the root of Arnebia euchroma has also been
reported to demonstrate anti-inflammatory activity,
in vivo and in vitro (Wang et al., 1994;
Ko
et al., 1995).
3.7. Prescriptions traditionally containing rhino horn:
reputed and pharmacological effects
3.7.1. Qing Ying Tang
Qing Ying Tang is a decoction for ‘clearing heat’ in the ‘ying’ system and contains eight
herbs in addition to rhino horn (Zhu, 1989; Zou, 1989; Xu, 1994). Rhino horn (Rhinoceros
Cornu) and Sheng Di Huang (Radix Rehmanniae) are the two principal (important) remedies
and they function by ‘clearing heat’ from the ‘ying’ and blood systems. Other prescription
components are described in Table 3.3. The combined horn-herb extracts, and prescription
absent from animal product, were anti-pyretic
in vivo (But and Tam, 1991).
3.7.2. Qingwen Baidu Yin
The prescription Qingwen Baidu Yin is an anti-pyretic and anti-toxic decoction used to ‘clear
away’ heat from ‘qi’ and blood systems (Xu, 1994). The prescription contains 12 herbs, one
mineral and rhino horn. Five of the herbs (Sheng Di Huang, Xuan Shen, Lian Qiao, Huang
Lian and Dan Zhu Ye) are included in the Qing Ying Tang prescription. The two important
components are regarded as rhino horn and Sheng Di Huang. Other prescription components
are described in Table 3.3. A modified version of Qingwen Baidu Yin, with buffalo horn
substituted for rhino horn, was found to be effective in reducing fever
in vivo, but the
prescription without added animal product was not tested (Xie, 1993).
Rationale for selection of herbs as potential alternatives to rhino horn
21

3.7.3. Xi Jiao Dihuang Tang
Xi Jiao Dihuang Tang is a prescription used to ‘clear away’ heat from ‘qi’ and blood systems
(Zhu, 1989; Bensky and Barolet, 1990; Xu, 1994) It contains rhino horn (the principal
component) and three herbs, as described in Table 3.3. All four components of Xi Jiao
Dihuang Tang are also included in Qingwen Baidu Yin.
3.7.4. Sheng Xi Dan
Sheng Xi Dan is also known as ‘magical rhinoceros special pill’ (Bensky and Barolet, 1990).
It is composed of rhino horn and 10 herbs (Table 3.3.).
3.7.5. Qing Gong Tang
Qing Gong Tang is used in the treatment of epidemic febrile diseases. It is composed
of rhino horn and five herbs (Table 3.3.).
3.7.6. Zhi Zi Jin Hua
Zhi Zi Jin Hua is listed in the Pharmacopoeia of the People’s Republic of China (1997)
as an anti-pyretic agent and it is composed of eight herbs (Table 3.3.).
3.8. Alternatives to rhino horn: summary
Based on both traditional use and knowledge of the medicinal properties of the different
species of plants, a selection of 9 single herbs were chosen for investigation as alternatives
to rhino horn in TCM. Six TCM prescriptions were also investigated; five contained rhino
horn as the only animal component and one TCM prescription was composed only of
herbs. All six prescriptions are used in TCM to treat epidemic febrile diseases. The nine
single herbs chosen as possible herbal ‘alternatives’ to rhino horn were found to be
included in one or more prescriptions used in this study. This finding confirmed the practice
in TCM of combining remedies with similar functions for their additive and synergistic
effects. As with the bear bile research, it may be that combinations of the herbs, based
on TCM principles, may be more suitable as replacements for animal product use.
Rhino horn is used in TCM as a detoxifying, anti-convulsant and anti-inflammatory agent.
A major use of rhino horn is in the treatment of advanced stages of fever in the ‘ying’ and
blood conformation, complicated by delirium or coma. The herbs and prescriptions selected
for investigation were tested in anti-bacterial and anti-inflammatory assays. The methods
used and results obtained are reported in Sections 5 and 6 of this report.
Section 3
22

Rationale for selection of herbs as
potential alternatives to tiger bone
4.1. Introduction
This section addresses the selection of potential alternatives to tiger bone in TCM. In terms
of finding herbal alternatives to animal products in traditional practices of medicine, the
need is perhaps most urgent with that of tiger bone, where hunting has driven the species
to the brink of extinction. As with the herbs selected to investigate as potential alternatives
for bear bile and rhino horn, selection has been based on investigation of both traditional
use and the medicinal properties of the different species of plants and fungi. Sources of
tiger bone and the devastating effect of hunting on the tiger population, its uses in TCM
and its reported pharmacological activities are discussed. As with the research seeking
alternatives to bear bile and rhino horn, both single herbs and prescriptions were selected
for investigation. A summary of some of the known constituents and available pharmacological
and clinical research on the selected herbs / fungi, and the prescriptions, is given.
4.2. Sources of tiger bone
One of the world’s most endangered animal species is the tiger, Panthera tigris (Felidae).
There are only five out of eight remaining subspecies (
P. t. amoyensis, P. t. sumatrae, P. t.
altaica, P. t. corbetti and P. t. tigris
) surviving today, with an estimated population of 5,000
– 7,500 (WWF, 2000). Consequently,
P. tigris is included in Appendix 1 of CITES (2003).
In view of the concerns relating to the declining numbers of tigers, investigations were
conducted to identify plants or fungi that may be suitable alternatives to tiger bone in
TCM. Both TCM principles and pharmacological activities were considered to assist with
the identification of suitable species in this study.
4.3. Tiger bone and its constituents
Tiger bone is reported to contain collagen, fats, calcium phosphate, calcium carbonate and
magnesium phosphate; the gelatin is reputed to be composed of 17 amino acids (Chang
and But, 1987). Generally, bone is primarily composed of inorganic calcium salts (65–70%);
smaller amounts of choidroitin sulphate, keratin sulphate and phospholipids are also
reported to be present (Brody, 1994). In the UK, choidroitin sulphate is included in some
over-the-counter remedies that are used to relieve symptoms of arthritis.
Summary
23
Section 4

There is a general lack of published literature on tiger bone and there is limited evidence
to support the pharmacotherapeutic potential of tiger bone for alleviating symptoms of
arthritis (Chang and But, 1987). Suspensions of both tiger bone and dog bone are reported
to be anti-inflammatory in vivo (Chang and But, 1987); however, the doses used in this
study appeared to be much higher than would usually be administered therapeutically.
Tiger bone powder is reported to reduce total neutrophil concentration and to inhibit
leukocyte and lymphocyte proliferation
in vivo (Chang and But, 1987). Both tiger and
dog gelatin are reported to be analgesic
in vivo (Chang and But, 1987), and tiger and
dog bones are reported to be sedative
in vivo (Chang and But, 1987).
Chang and But (1987) also cite research conducted on a TCM prescription, ‘compound
union pill’, containing tiger bone which promoted healing of fractures in rabbits. Analysis
of each component of this prescription showed that tiger bone was one of the most effective
ingredients to replenish bone density and promote healing (Chang and But, 1987).
4.4. Tiger bone in TCM
Tiger bone (Hu Gu) is described in TCM as having a ‘pungent’ taste and ‘warm’ nature
(Chang and But, 1987; Bensky and Gamble, 1993). It is believed to ‘dispel wind-dampness’,
‘disperse wind cold’ and ‘strengthen the sinews and bones’ (Bensky and Gamble, 1993).
Tiger bone is used in TCM to treat symptoms such as bone and muscle pain, limb spasms,
lower back pain and chills. It is used to treat pathologic states classified under ‘painful
obstruction disorders’ in TCM. This group of disorders best fits the Western term ‘arthritic
disorders’ which includes various rheumatic diseases and osteoarthritis (Guilaume and
Chieu, 1996).
4.5. Selection of herbs
4.5.1. Criteria used for selection of single herbs
There is some limited scientific evidence to support the use of tiger bone as an anti-
inflammatory and an analgesic agent in traditional medicine. In order to identify species,
which possessed the appropriate TCM properties and functions, an extensive literature
survey was initially conducted using the criteria described in Table 4.1. Forty-six species
(Table 4.2.) were identified based on the criteria. Although, all the species listed in Table
4.2 may be used in combination with other TCM remedies in the treatment of arthritis and
rheumatism, several of them are not categorised as anti-rheumatics in TCM Materia
Medicas, and may have other functions (e.g. as a tonic).
Section 4
24

4.5.2. Criteria used for the selection of prescriptions
In addition to the literature survey conducted to identify appropriate species, another survey
was conducted to identify TCM prescriptions that included tiger bone as a component.
Those prescriptions identified were generally indicated for arthritis or related disorders. In
consultation with TCM practitioners, two TCM prescriptions that contained tiger bone as
the only animal component and non-endangered plant / fungal species (those not restricted
by CITES) were selected for further investigation (prescription compositions are described in
Table 4.3.). The two TCM prescriptions were composed of a combined total of 19 different
TCM herbs and one fungus; eight of these species were identified in the initial literature
survey of the herbs (Table 4.2.).
Since TCM often uses remedies with similar functions to form prescriptions, preliminary
pharmacological investigations were conducted to assess the potential anti-inflammatory
effects of the two TCM prescriptions; 19 species were also subjected to analysis in the
bioassays (Table 4.3. and 4.4.). This approach was designed to identify species used in the
prescriptions, which may have anti-inflammatory properties via the biological pathway tested.
In addition to the preliminary pharmacological investigations, which were conducted in this
study to identify any scientific basis for the reputed activities of the 19 selected remedies,
a literature search was also conducted. This aim of this investigation was to identify any
pharmacological or clinical studies relating to the potential anti-inflammatory / anti-rheumatic /
analgesic ef
fects of the 19 selected remedies (Tables 4.3. and 4.4.). This exercise was to
provide information previously reported that might also assist in the identification of possible
alternatives to tiger bone. The results of this study are summarised in section 4.6.
Rationale for selection of herbs as potential alternatives to tiger bone
25
Table 4.1. Properties / functions of tiger bone used as criteria for species selection
Criteria Properties and functions of tiger bone
A For arthritic and rheumatic conditions
B A
nalgesic
C ‘
Warm’ nature
D ‘
Pungent’ or ‘acrid’
E ‘
Sweet’ taste
F Heals wounds and fractures
G Alleviates pain in lower back and knees
H Expels wind dampness or cold

Section 4
26
Table 4.2. TCM species identified from evaluation of TCM literature using criteria based on the TCM functions and properties of tiger bone
TCM herbs A B C D E F G H
Arthritis
Rheumatic
Analgesic Warm Pungent
(acrid)
Sweet Wounds &
fractures
Lower back
& knee pain
Expels wind
dampness /
cold
1. Fang Feng, root of Saposhnikovia divaricata
(Turcz.) Schischk. (Apiaceae)
* * * * * *
2. Wei Ling Xian, root and rhizome of Clematis
chinensis Osb. (Ranunculaceae)
* * * * * *
3. Nao Yang Hua, aerial part of Rhododendron
molle Siebold & Zucc. (Ericaceae)
* * * * * *
4. Du Huo, root of Angelica pubescens Maxim.
(Apiaceae)
* * * * *
5. Fu Zi, prepared daughter root tuber of
Aconitum carmichaelii Debx. (Ranunculaceae)
* * * *
6. Cang Er Zi, fruit of Xanthium sibiricum Patrin
ex Widd. (Asteraceae)
* * * * *
7. Xi Xin, whole plant of Asarum
heterotropoides forma manshuricum
(Maxim.) Kitag. (Aristolochiaceae)*
* * * *
8. Zu Shi Ma, bark or root bark of Daphne giraldi
Nitsche (Thymelaeaceae)
* * * *
9. Yang Jin Hua, corolla of
Datura metel L.
(Solanaceae)
* * * *
10. Chuan Shan Long, rhizome of Dioscorea
nipponica Makino (Dioscoreaceae)
* * * * * *
11.
Ba Jiao Feng, leaves, stems and fibr
ous r
oots
of Alangium chinense (Lour.) Harms
(Alangiaceae)
* * * *
12.
Qi Ye Lian, roots, stems and leaves of
Schefflera arboricola Hayata (Araliaceae)
* * * *
Continued

Rationale for selection of herbs as potential alternatives to tiger bone
27
Table 4.2. TCM species identified from evaluation of TCM literature using criteria based on the TCM functions and properties of tiger bone
TCM herbs A B C D E F G H
Arthritis
Rheumatic
Analgesic Warm Pungent
(acrid)
Sweet Wounds &
fractures
Lower back
& knee pain
Expels wind
dampness /
cold
13. Chuan Xiong, rhizome of Ligusticum
chuanxiong Hort. (Apiaceae)
* * * *
14. Qing Feng Teng, stem of Sinomenium acutum
Rehd. (Menispermaceae)
* * * *
15. Xue Shang Yi Zhi Hao, root of Aconitum
brachypodum Diels (Ranunculaceae)
* * *
16. Qin Jiao, root of Gentiana macrophylla Pall.
(Gentianaceae)
* * * *
17. Fang Ji, root of Stephania tetrandra S.Moore
(Menispermaceae)
* * *
18. Lei Gong Teng, root of Tripterygium wilfordii
Hook.f. (Celastraceae)
* * *
19. Chou Wu Tong, twigs and leaves of
Clerodendron trichotonum Thunb.
(Verbenaceae)
* * *
20. Dang Gui, root of Angelica sinensis (Oliv.)
Diels (Apiaceae)
* * * *
21. Ji Xue Teng, root and stem of
Spatholobus
suberectus Dunn. (Fabaceae)
* * * * *
22. Yin Yang Huo, aerial parts of Epimedium
sagittatum Maxim. (Berberidaceae)
* * * *
23. Cang Zhu, rhizome of Atractylodes lancea
(Thunb.) DC. or A. chinensis (D.C.) Koidz.
(Asteraceae)
* * * *
24. Ding Gong Teng, root and stem of Erycibe
obtusifolia Benth. (Convolvulaceae)
* * * *
Continued

Section 4
28
Table 4.2. TCM species identified from evaluation of TCM literature using criteria based on the TCM functions and properties of tiger bone
TCM herbs A B C D E F G H
Arthritis
Rheumatic
Analgesic Warm Pungent
(acrid)
Sweet Wounds &
fractures
Lower back
& knee pain
Expels wind
dampness /
cold
25. Ci Wu Jia, root and rhizome of
Eleutherococcus senticosus (Rupr. & Maxim.)
Rupr. (Araliaceae)
* * *
26. Yun Xiang Cao, whole plant of Cymbopogon
distans (Nees ex Steudel) Will. (Poaceae)
* * *
27. Rou Gui, bark of Cinnamomum cassia D.Don
(Lauraceae)
* * *
28. Tou Gu Cao, whole plant of
Impatiens
balsamina L. (Balsaminaceae)
* * * *
29. Xu Chang Qing, root and rhizome of
Cynanchum paniculatum Bunge
(Asclepiadaceae)
* *
30. Xue Lian, root of
Saussurea laniceps
Hand.-Mazz (Asteraceae)
* * *
31. Xiang Jia Pi, root bark of Periploca sepium
Bunge (Asclepiadaceae)
* * *
32. Lu Xian Cao, whole plant, Pyrola calliantha
Andres and P. decorata Andres (Pyrolaceae)
* * *
33. Yi Ye Qiu, shoot and root of Securinega
suffruticosa (Pall.) Rehd. (Phyllanthaceae)
* * *
34. Huang Jing Zi, fruit of Vitex negundo L.
(Lamiaceae)
* *
35. Shi Diao Lan, whole plant of Lysionotus
pauciflorus Maxim. (Gesneriaceae)
* *
36. Kun Ming Shaun Hai Tang, root of
T
ripter
ygium hypoglaucum
Hutchinson
(Celastraceae)
* *
37. Mu Gua, fruit of Chaenomeles speciosa
(Sweet) Nakai (Rosaceae)
* *

Rationale for selection of herbs as potential alternatives to tiger bone
29
Table 4.2. TCM species identified from evaluation of TCM literature using criteria based on the TCM functions and properties of tiger bone
TCM herbs A B C D E F G H
Arthritis
Rheumatic
Analgesic Warm Pungent
(acrid)
Sweet Wounds &
fractures
Lower back
& knee pain
Expels wind
dampness /
cold
38. Sang Zhi, young branches of Morus alba L.
(Moraceae)
* * *
39. Fu Zi, prepared daughter root tuber of
Aconitum carmichaelii Debx. (Ranunculaceae)
* *
40. Nu Zhen Zi, fruit of Ligustrum lucidum Ait.
(Oleaceae)
* * *
41. Sang Ji Sheng is the stem and branch of
Taxillus chinensis (DC.) Danser (Loranthaceae)
* * *
42. Jin Gi Er, seed of Caragana microphylla Lam.
(Leguminosae)
* *
43. Man Shan Xiang, Lysimachia capillipes Hemsl.
(Primulaceae)
* *
44. Qian Cao, root and stem of Rubia cordifolia L.
(Rubiaceae)
* *
45. Jiu Jie Feng, root, aerial part or whole plant
of Sarcandra glabra (Thunberg) Nakai
(Chloranthaceae)
* *
46. Ma Qian Zi, seed of Strychnos nux-vomica L.
(F of China) (Loganiaceae)
* *
References: Chinese Pharmacopoeia, 2005; Chang and But, 1987; Bensky and Gamble, 1993 and 2004; Chang and But, 2001.

φ Species also tested individually in biological activity tests
+
Other plant species may be used for the Chinese name specified (not listed in T
able 4.4.)
Section 4
30
Table 4.4. Other species selected for biological activity tests (not listed in Table 4.3.)
Herbs
Cang Zhu, rhizome of Atractylodes lancea+ (Asteraceae)
Mu Gua, fruit of Chaenomeles speciosa (Rosaceae)
W
ei Lin Xian, root and rhizome of
Clematis chinensis (Ranunculaceae)
Yin Yang Huo, aerial parts of Epimedium sagittatum (Berberidaceae)
San Qi, root of Panax pseudoginseng (Araliaceae)
Table 4.3. Composition of herbs as described TCM prescriptions, traditionally containing tiger bone
Herbs Yang Xue Gu Feng Tang Du Huo Ji Sheng Tang
B
ai Shao, root of
P
aeonia lactiflora
(
Ranunculaceae)
φ
* *
Dang Gui, root of Angelica sinensis (Apiaceae) φ
* *
Du Huo, root of Angelica pubescens (Apiaceae) φ
* *
F
u Ling, sclerotium of
P
oria cocos
(
Polyporaceae) fungus
* *
Niu Xi, root of Achyranthes bidentata (Amaranthaceae) φ
* *
Qin Jiao, root of Gentiana macrophylla (Gentianaceae) φ
* *
Di Huang, root of Rehmannia glutinosa (Scrophulariaceae) * *
Bai Zhu, rhizome of Atractylodes macrocephala (Asteraceae) φ
*
Gui Zhi, twigs of Cinnamomum cassia (Lauraceae) φ
*
Mu Xiang, root of Saussurea costus (Falc.) Lipsch. (Asteraceae) φ
*
Sang Chi (Sang zhi), twig of Morus alba (Moraceae) *
Xu Duan, rhizome of Dipsacus asper (Dipsacaceae) *
Chuan Xiong, rhizome of Ligusticum chuanxiong (Apiaceae) φ
*
Du Zhong, bark of Eucommia ulmoides (Eucommiaceae) φ
*
Fang Feng, root of Saposhnikovia divaricata (Apiaceae) φ
*
Gan Cao, rhizome of Glycyrrhiza uralensis (Leguminosae) φ
*
Ren Shen, r
oot of
Panax ginseng (Araliaceae) φ
*
Rou Gui, bark of Cinnamomum cassia (Lauraceae) *
Sang Ji Sheng, stem and branch of T
axillus chinensis
(Loranthaceae) φ
*

4.6. Single herbs and other TCM remedies
as potential alternatives to tiger bone:
reputed and pharmacological effects
4.6.1. Bai Shao (Radix Paeoniae Alba)
Bai Shao is the dried root of Paeonia lactiflora Pall (Ranunculaceae) and is used to treat a
variety of disorders in TCM including spasmodic pain of the limbs;
Paeonia root may also be
used for analgesic effects (Pharmacopoeia of PRC, 2000; Tang and Eisenbrand, 1992). Few
studies have been conducted to investigate the anti-inflammatory potential of
P. lactiflora,
however, it has been associated with activity against COX (Prieto
et al., 2003). Paeoniflorin,
isolated from the root, is reported to be anti-inflammatory (Tang and Eisenbrand, 1992).
4.6.2. Bai Zhu (Rhizoma Atractylodis Macrocephalae)
Bai Zhu is the dried rhizome of Atractylodes macrocephala Koidz. (Asteraceae) and it is
indicated in TCM for a number of disorders, which include oedema (Tang and Eisenbrand,
1992; Pharmacopoeia of PRC, 2000). Limited research has been conducted to investigate
the anti-inflammatory potential of
A. macrocephala, but it has been associated with
activity against COX (Prieto
et al., 2003).
4.6.3. Cang Zhu (Rhizoma Atractylodis)
Cang Zhu is the dried rhizome of Atractylodes lancea DC. or A. chinensis Koidz (Asteraceae)
and it has been used in TCM for the treatment of rheumatic arthralgia (Pharmacopoeia of
PRC, 2000). Some compounds (including phenols, polyacetylenes, atractylon (sesquiterpene)
and osthole (coumarin)) from
A. lancea rhizomes and lipophilic extracts have been associated
with inhibition of COX-1 and 5-LOX (Resch
et al., 1998; Resch et al., 2001). A Japanese
prescription used traditionally for arthritis treatment and composed of seven crude drugs
including
A. lancea was anti-inflammatory in vivo (Kimura et al., 1991). Pharmacological
studies regarding
A. chinensis are lacking.
4.6.4. Chuan Xiong (Rhizoma Chuanxiong)
Chuan Xiong is the dried rhizome of Ligusticum chuanxiong Hort. (Apiaceae), which is
used in TCM for various conditions such as rheumatic arthralgia (Tang and Eisenbrand,
1992; Pharmacopoeia of PRC, 2000). There is a relative lack of research regarding the
pharmacological basis of the reputed anti-inflammatory / anti-rheumatic activity of
L. chuanxiong. An alcohol extract is reported as anti-inflammatory and analgesic (Chang
and But, 2001). Tetramethylpyrazine, an alkaloid from
L. chuanxiong, has been associated
with anti-inflammatory activity in both the early and late stages of inflammation (Ozaki, 1992).
Rationale for selection of herbs as potential alternatives to tiger bone
31

4.6.5. Dang Gui (Radix Angelicae Sinensis)
Dang Gui is prepared from the dried root of Angelica sinensis (Oliv.) Diels (Apiaceae) and it
is indicated in TCM for a number of disorders including rheumatic arthralgia and traumatic
injuries (Tang and Eisenbrand, 1992; Pharmacopoeia of PRC, 2000). There is a relative lack
of research regarding the pharmacological basis of the reputed anti-inflammatory / anti-
rheumatic activity of
A. sinensis. Ferulic acid, reported to occur in A. sinensis, has been
associated with anti-inflammatory activity in both the early and late stages of inflammation
(Ozaki, 1992).
4.6.6. Di Huang (Radix Rehmanniae)
Di Huang is the fresh or dried root tuber of Rehmannia glutinosa Steud. (Scrophulariaceae)
and is used in TCM to treat various disorders and may be used as a tonic (processed roots)
or haemostatic (fresh and dried roots) (Tang and Eisenbrand, 1992; Pharmacopoeia of PRC,
2000). An extract (100% methanol) of
R. glutinosa root showed inhibition of COX-2 and
iNOS activity (Hong
et al., 2002). An aqueous extract of R. glutinosa root has been suggested
to inhibit TNF-
α secretion by inhibiting IL-1 secretion and R. glutinosa root extract may have
anti-inflammatory activity in the CNS (Kim
et al., 1999). However, in vivo, an ethanolic
extract of
R. glutinosa root was ineffective on the development of oedema in arthritic rats
and on chronic and acute inflammation (Kubo
et al., 1994). It is also reported that it is only
the decoction which has an anti-inflammatory effect and not an alcohol extract (Chang
and But, 2001).
4.6.7. Du Huo (Radix Angelicae Pubescentis)
Du Huo is the dried root of Angelica pubescens Maxim. (Umbelliferae), which has been used
in TCM as an analgesic and anti-rheumatic agent (Tang and Eisenbrand, 1992; Pharmacopoeia
of PRC, 2000). Anti-inflammatory and analgesic activities of
A. pubescens root are well
documented (Chen
et al., 1995; Kosuge et al., 1985; Liu et al., 1998a; Liu et al., 1998b;
Prieto
et al., 2003). Methanol, chloroform and ethyl acetate extracts are reported to reduce
pain and oedema
in vivo; columbianadin, columbianetin acetate, bergapten, umbelliferone
and caffeic acid were anti-inflammatory and analgesic
in vivo; osthole and xanthotoxin
were anti-inflammatory
in vivo (Chen et al., 1995). A. pubescens, A. pubescens f. biserrata,
linoleic acid, osthole, osthenol and some polyacetylenes (e.g. falcarindiol) have also
been associated with inhibition of COX and 5-LOX (Liu
et al., 1998a; Liu et al., 1998b).
A. pubescens is also reported to be effective in attenuating persistent hindpaw inflammation
and hyperalgesia in rats (Wei
et al., 1999).
Section 4
32

4.6.8. Du Zhong (Cortex Eucommiae)
Du Zhong is the dried stem bark of Eucommia ulmoides Oliver (Eucommiaceae) and is reputed
in TCM to strengthen the tendons and bones (Tang and Eisenbrand, 1992; Pharmacopoeia
of PRC, 2000). The chemical composition of
E. ulmoides has been subjected to some
investigation and pharmacological activities have been associated with some constituents.
However, there is a comparative lack of research regarding pharmacological studies associated
with anti-inflammatory or anti-rheumatic activities. A decoction is reported to be analgesic
and anti-inflammatory (which may be related to enhancement of adrenocortical function)
in vivo (Chang and But, 2001).
4.6.9. Fang Feng (Radix Saposhnikoviae)
Fang Feng is the dried root of Saposhnikovia divaricata (Turcz.) Schischk. (Apiaceae) and it
is used in TCM to treat rheumatic arthralgia (Pharmacopoeia of PRC, 2000). The ethanol
extract of Fang Feng is reported to be analgesic and anti-inflammatory (Chang and But,
2001). Analgesic components of
S. divaricata are reported to be chromones, coumarins,
polyacetylenes and 1-acylglycerols; the most potent analgesia was associated with chromones
such as divaricatol, ledebouriellol and hamaudol (Okuyama
et al., 2001). Imperatorin and
deltoin, isolated from
S. divaricata root, inhibited the expression of the iNOS protein (Wang
et al., 1999).
4.6.10. Fu Ling (Poria)
Fu Ling is the dried sclerotium of the fungus Poria cocos (Polyporaceae), which is indicated
in TCM for a number of disorders and is reputed to cause diuresis and to calm the mind
(Pharmacopoeia of PRC, 2000).
P. cocos is reported to inhibit 5-LOX and phospholipase
A
2
activities and dehydrotumulosic and pachymic acids, which have been isolated from
P. cocos, are reported to inhibit leukotriene B
4
(LTB4) release and to inhibit phospholipase
A
2
activity (Cuellar et al., 1996; Giner et al., 2000; Giner-Larza et al., 2000; Prieto et al.,
2003). A triterpene derivative (3
β-p-hydroxybenzoyldehydrotumulosic acid) isolated from
P. cocos showed anti-inflammatory activity in vivo (Yasukawa et al., 1998).
4.6.11. Gui Zhi (Ramulus Cinnamomi)
Gui Zhi is the dried young branches of Cinnamomum cassia (Lauraceae), which has been
used in TCM for the treatment of arthralgia and oedema (Tang and Eisenbrand, 1992;
Pharmacopoeia of PRC, 2000). An extract (100% methanol) of
C. cassia twigs showed
inhibition of COX-2 and iNOS activity (Hong
et al., 2002), which may explain some of the
reputed effects. The active constituents responsible for activities observed in pharmacological
studies require further investigation.
Rationale for selection of herbs as potential alternatives to tiger bone
33

Section 4
34
4.6.12. Ji Xue Teng (Caulis Spatholobi)
Ji Xue Teng is the root and stem of Spatholobus suberectus Dunn (Fabaceae) and it has
been used in TCM for various conditions, which include knee pain or generalised joint
soreness (Bensky and Gamble, 1993).
Ji Xue Teng is reported to promote beneficial effects on artificially-induced arthritis
in vivo
(Bensky and Gamble, 1993). In addition, the alternative plant species used to prepare
Ji Xue Teng,
S. suberectus stem, was active against COX-1, phospholipase A
2
, 5-LOX and
12-LOX activities, but did not inhibit COX-2 activity (Li
et al., 2003).
4.6.13. Lu Lu Tong (Fructus Liquidambaris)
Lu Lu Tong is the dried ripe fruit of Liquidambar formosana Hance (Hamamelidaceae) and
it is indicated for arthralgia with numbness and muscular contracture (Pharmacopoeia of
PRC, 2000). Some studies have investigated the chemistry of
Liquidambar species, however,
pharmacological studies are limited; anti-inflammatory / anti-rheumatic effects have not
been substantially investigated.
4.6.14. Mu Gua (Fructus Chaenomelis)
Mu Gua is the dried nearly ripe fruit of Chaenomeles speciosa Nakai (Rosaceae) and it is
indicated in TCM for arthritis with ankylosis (Pharmacopoeia of PRC, 2000). Glucosides
from
C. speciosa were anti-inflammatory (effects included inhibition of TNF-α and PGE
2
)
in vivo (Chen and Wei, 2003; Dai et al., 2003).
4.6.15. Mu Xiang (Radix Aucklandiae)
Mu Xiang is the dried root of Saussurea costus (Falc.) Lipsch (Syn.: Saussurea lappa)
(Asteraceae) and it is used in TCM for treating some types of pain (Pharmacopoeia of PRC,
2000). An ethanolic extract of
S. lappa is reported to show anti-inflammatory and anti-
arthritic activity (Gokhale
et al., 2002). Sesquiterpene lactones (e.g. costunolide, cynaropicrin)
from
Saussurea lappa have been associated with anti-inflammatory activity (Cho et al.,
2000; Gokhale
et al., 2003; Matsuda et al., 2003; Cho et al., 2004). The anti-inflammatory
activity of the sesquiterpene lactone fraction of
S. lappa has been suggested to be due to
stabilisation of lysosomal membranes and an anti-proliferative effect (Gokhale
et al., 2003).
Cynaropicrin inhibited TNF-
α release, attenuated nitric oxide accumulation and dose-
dependently suppressed the proliferation of lymphocytes (Cho
et al., 2000); costunolide
inhibited IL-1
β gene expression (Kang et al., 2004). Some amino acid-sesquiterpene
conjugates (saussureamines A and B) from a methanolic extract of
S. lappa roots inhibited
activation of NF-
κΒ (Matsuda et al., 2003).

4.6.16. Niu Xi (Radix Achyranthis Bidentatae)
Niu Xi is prepared from the dried root of Achyranthes bidentata Blume (Amaranthaceae)
and is used in TCM as a tonic and for soreness of the lumbar and knee joints with weakness
in the legs (Tang and Eisenbrand, 1992; Pharmacopoeia of PRC, 2000). There is a general
lack of research regarding the study of potential anti-inflammatory / anti-rheumatic effects
of
A. bidentata. An oligosaccharide (AbPS) isolated from A. bidentata significantly
enhanced the humoral immune response and antagonised the immunosuppressive effects
of cyclosporin A; AbPS increased the production of TNF and the activity of natural killer
cells; in tumour patients treated by chemotherapy or radiotherapy, AbPS maintained their
peripheral white blood cell count and impr
oved the quality of life (Li, 2000). Root
polysaccharides induced IL-1 and TNF-
α synthesis and secretion from mouse peritoneal
macrophages
in vitro, indicating immunopotentiating activity (Xiang and Li, 1993).
Leflunomide (disease modifying anti-rheumatic drug in clinical use) and its active metabolite
are associated with inhibition of IL-1
β, TNF-α and NF–κΒ (Breedveld and Dayer, 2000;
Elkayam
et al., 2003), thus A. bidentata saccharides may be of no therapeutic benefit in
rheumatoid arthritis (RA) via these mechanisms (in view of the reported association with
immunopotentiating effects).
4.6.17. Qin Jiao (Radix Gentianae Macrophyllae)
Qin Jiao is the dried root of Gentiana macrophylla Pall. It has been used in TCM mainly
for the treatment of rheumatic or rheumatoid arthritis with muscular contracture and
severe joint pain (Tang and Eisenbrand, 1992; Pharmacopoeia of PRC, 2000). Compounds
identified in
Gentiana macrophylla include various secoiridoids. The secoiridoid glucoside,
gentiopicroside, is reported to show anti-inflammatory activity
in vivo (Tang and
Eisenbrand, 1992).
4.6.18. Ren Shen (Radix Ginseng)
Ren Shen is the dried root of Panax ginseng C.A.Mey (Araliaceae) and it is indicated for
various conditions, including general weakness with irritability and insomnia in chronic
diseases, and may be included in prescriptions as a tonic (Tang and Eisenbrand, 1992;
Pharmacopoeia of PRC, 2000). P. ginseng has been extensively researched regarding its
chemistry and its pharmacological and clinical effects. Ginsenoside Rg
3
inhibited COX-2
expression and NF–
κΒ activation (Keum et al., 2003).
4.6.19. Rou Gui (Cortex Cinnamomi)
Rou Gui is the dried stem bark of Cinnamomum cassia D.Don (Lauraceae), which was
used traditionally for cold and pain in the knees and for some inflammatory disorders
(Tang and Eisenbrand, 1992; Pharmacopoeia of PRC, 2000). An extract (70% methanol)
of
C. cassia cortex showed an inhibitory effect on acute inflammation in vivo (Kubo et al.,
1996). Further investigation is required to identify the active constituents r
esponsible for
these effects.
Rationale for selection of herbs as potential alternatives to tiger bone
35

Section 4
36
4.6.20. San Qi (Radix Notoginseng)
San Qi is the dried root of Panax notoginseng (Burkill) Chen (Araliaceae), which is used in
TCM to alleviate traumatic swelling and pain, amongst other conditions (Pharmacopoeia of
PRC, 2000; Tang and Eisenbrand, 1992). Saponins from
Panax notoginseng are reported to
be anti-inflammatory, which may be associated with inhibition of phospholipase A
2
activity
(Hao and Yang, 1986; Tang and Eisenbrand, 1992; Li and Chu, 1999). Another study
showed that
P. notoginseng did not produce any significant effect on inflammation and
hyperalgesia
in vivo (Wei et al., 1999).
4.6.21. Sang Zhi (Ramulus Mori)
Sang Zhi is the dried young branches of Morus alba L. (Moraceae) and is used for the
treatment of arthritis and rheumatism (Tang and Eisenbrand, 1992; Pharmacopoeia of PRC,
2000). An extract (100% methanol) of
M. alba twigs showed inhibition of iNOS activity
(Hong
et al., 2002). Mulberroside A and oxyresveratrol, obtained from M. alba cortex, have
been investigated for their anti-inflammatory activity and were shown to significantly reduce
paw oedema (Chung
et al., 2003). The anti-inflammatory properties of oxyresveratrol were
associated with inhibition of NOS expression through down-r
egulation of NF–
κΒ binding
and inhibition of COX-2 activity (Chung
et al., 2003).
4.6.22. Wei Ling Xian (Radix Clematidis)
Wei Ling Xian is the dried root and rhizome of Clematis chinensis Osb. (Ranunculaceae),
and it is indicated in TCM for rheumatic or rheumatoid arthralgia with numbness of the
limbs (Tang and Eisenbrand, 1992; Pharmacopoeia of PRC, 2000). Stems of
C. chinensis
have also been used in TCM to treat rheumatic arthritis and other inflammatory conditions
(Xu
et al., 1996). In one study, an ethanol extract of C. chinensis stems only inhibited COX-
1 activity
in vitro at high concentrations (Li et al., 2003). Few pharmacological studies have
been conducted to investigate any scientific basis for the reputed anti-inflammatory / anti-
rheumatic effects of
C. chinensis.
4.6.23. Xu Duan (Dipsaci Radix)
Xu Duan is the dried root of Dipsacus asperoides C.Y.Cheng & T.M.Ai (Dipsacaceae), which
is indicated in TCM for aching and weakness of the loins and knees, rheumatic arthralgia
and traumatic injuries (Pharmacopoeia of PRC, 2000).
D. asperoides has not been subjected
to substantial investigation regarding its chemistry and pharmacological activities.

4.6.24. Yin Yang Huo (Herba Epimedii)
Yin Yang Huo is prepared from the dried aerial parts of Epimedium sagittatum (S. et Z)
Maxim (Berberidaceae); it is indicated in TCM for weakness of the limbs and rheumatic or
rheumatoid arthralgia with numbness or muscle contracture, and has also been included
in prescriptions as a tonic (Tang and Eisenbrand, 1992; Pharmacopoeia of PRC, 2000).
A flavonoid extract from
E. sagittatum was effective in preventing osteoporosis in vivo,
which may be mediated by enhancement of osteoblast development (Chen
et al., 2004).
Other studies, relating to the potential anti-inflammatory / anti-rheumatic effects of the
species used to prepare Yin Yang Huo are limited, and further investigations are necessary
to establish the basis for their clinical use.
4.7. Alternatives to tiger bone: summary
Based on both traditional uses and evidence from scientific research, a selection of 19 single
herbs and 2 prescriptions (Tables 4.3. and 4.4.) were selected. Five other species present
in some TCM prescriptions also traditionally containing tiger bone, were chosen for further
study (T
able 4.4.). In TCM, tiger bone is traditionally used to treat conditions involving
inflammation and this property is supported by research suggesting that suspensions of
both tiger bone and dog bone are anti-inflammatory
in vivo. Preliminary pharmacological
investigations were therefore conducted to assess the potential anti-inflammatory effects
of the selected herbs and prescriptions. The methods used are reported in Section 5 of this
report and the findings are reported in Section 6. This approach was designed to identify
species used in the prescriptions, which may have anti-inflammatory properties via the
biological pathway tested.
Rationale for selection of herbs as potential alternatives to tiger bone
37

Summary
38
Biological and chemical methods used
to study plant and fungal material
5.1. Introduction
Both rhino horn and bear bile are primarily classified in TCM as anti-inflammatory and
fever-reducing remedies and tiger bone has been used as an anti-arthritic / anti-rheumatic
remedy (Hsu
et al., 1986); the pathology of arthritis also involves inflammatory mechanisms.
Thus, assays were selected to evaluate the anti-inflammatory potential of the TCM material.
The inflammatory response is a complex cascade of events, often triggered by infection
(commonly by bacteria) and is one of the body’s defence mechanisms in fighting disease.
The inflammatory response forms one of the underlying pathologies of arthritis (McEvoy,
2004); fever (Ivanov and Romanovsky 2004); liver diseases (Tanasescu, 2004), cancer (Ross
et al., 2004) and cardiovascular diseases (Br
own and Jones, 2004). Therefore, preliminary
studies were conducted to assess the effects of crude extracts, fractions and isolated
compounds on bacterial growth and an anti-inflammatory mediator, nuclear factor-kappa
Β
(NF−κΒ), in vitro. NF−κΒ is a transcriptional factor which regulates the genes of several
pro-inflammatory chemicals, such as cytokines (IL-1
β, IL-6, IL-8, TNF-α), enzymes (COX-2,
iNOS), adhesion molecules and in a self-regulatory way its inhibitory protein, I-
κΒ (Bremner
and Heinrich, 2002; Ross
et al., 2004). CYP3A4 inhibition tests, using testosterone
6
β-hydroxylation as a probe for enzyme activity, were conducted in human liver microsomes,
to determine the effect of herbal extracts on drug metabolising enzyme
in vitro.
In this study, TCM material was extracted using solvents of varying polarity and the resulting
extracts were tested in a range of bioassays. Preliminary studies were conducted using
aqueous extracts since this extraction procedure reflects the preparation of decoctions in
TCM. Prior to testing in the bioassays, the chemical profiles of the plant material, obtained
from commercial companies, were compared with the chemical profiles of authentic and
other reference material, obtained from the Chinese Medicinal Plant Authentication Centre
(CMPAC), Royal Botanic Gardens, Kew.
Section 5

Verification of the TCM material is essential to ensure that, when interpreting data
regarding the chemistry and pharmacological activities of each species, the results refer to
the correct species. This is of particular concern, as TCM material may be adulterated or
deliberately substituted with other plant species. A TCM name for a herb may refer to
more than one plant species, for example Chi Shao is prepared from the dried root of
Paeonia lactiflora or Paeonia veitchii. Thus, when recommending potential alternatives to
the animal material (bear bile, rhino horn, tiger bone), it is essential to ensure the correct
species is being proposed as an alternative. This process involved comparison of the
chemical profiles of the commercial material with verified TCM material (available from the
collections held at CMPAC, Royal Botanic Gardens, Kew) using various chromatographic
techniques. Some other concerns associated with the use of traditional herbal medicines
(in particular TCM) are the safety of the remedies (Koh and Woh, 2000). Therefore, in
addition, it was important to determine if the herbs contained high levels of pesticide
residues (Appendix 2) and heavy metals (Appendix 3).
5.2. Materials
Many of the samples of the TCM material were kindly donated by Jo Liu of Mayway (UK)
Ltd. (Hanwell, UK) and Paul Skipworth of Kingham Herbs and Tinctures (UK). Rhino horn
was kindly donated by the CITES team (Special Operations District, Heathrow airport) and
tiger bone (obtained from the rib of a male hybrid Amur tiger), was donated by the
National Museums of Scotland. Ursodeoxycholic acid (UDCA) was purchased from Sigma
chemicals (UK). Authentic and other reference (market) samples of TCM material, used to
assist with authentication of the trade TCM material, were obtained from CMPAC, Royal
Botanic Gardens, Kew.
5.3. Authentication techniques for trade TCM material
TCM material (trade samples and reference material) was ground using a pestle and
mortar or grinding equipment, to provide sufficient material for authentication procedures
and bioassays. Ground material was then extracted using aqueous 80% methanol, and as
different types of compounds were targeted to assist with the authentication process, the
ground material of some species was also extracted using other solvents (water, ethanol,
dichloromethane or hexane). Following extraction, extracts were filtered, evaporated to
dryness (aqueous extracts were freeze-dried) and reconstituted in the appropriate solvent,
prior to HPLC and LC-MS analysis.
Biological and chemical methods used to study plant and fungal material
39

5.3.1. HPLC (UV-DAD) method
Analytical HPLC was carried out using a Waters LC600 pump and a 996 photodiode array
detector. A Merck LiChrospher 100RP-18 (250 x 4.0 mm i.d. 5 µm particle size) column
(maintained at 30°C) was used for analysis with a flow rate of 1 ml/min. The mobile phase
consisted of 2% aqueous acetic acid (A) and methanol : acetic acid : water (18:1:1). Initial
conditions were 75% A and 25% B; the proportion of B increased with a linear gradient,
reaching 100% at t = 20 min. This was followed by an isocratic elution of 100% B until
t = 25 min. Injections (30 µl) were made by an autosampler.
5.3.2. LC-MS method
LC-MS analysis was conducted at the Royal Botanic Gardens, Kew, by Dr G. Kite. Aqueous
80% methanol extracts were analysed using a Thermo-Finnigan LC/MS/MS system consisting
of a ‘Surveyor’ autosampling LC system, interfaced to a ‘LCQ Classic’ quadrupole ion trap
mass spectrometer.
Chromatographic separation of compounds was performed on a 250 mm x 4.6 mm i.d.,
5 µm Supelco Discovery-C18 column using a 1 ml/min mobile phase gradient programmed
from water (A), methanol (B) and methanol containing 5% acetic acid (C). The gradient
programme (A:B:C) was 80:0:20 (t = 0 min), 0:80:20 (t = 20 min), 0:80:20 (t = 25 min),
80:0:20 (t = 27 min), 80:0:20 (t = 37 min). Data analysis was performed using Xcalibur
1.2 software (Thermo-Finnigan).
5.3.3. TD-GC-MS method
The TD-GC-MS system used consisted of a Perkin-Elmer ATD400 thermal desorption unit,
a Perkin-Elmer AutoSystem XL GC and a Perkin-Elmer TurboMass MS (quadrupole).
Chromatography was performed on a 30 m x 0.25 mm i.d. x 0.25 µm DB-5MS column
(J. & W. Scientific, USA) using an oven program of 60–300°C at 6°C/min. The carrier gas
was helium at a flow rate of 1 ml/min. The TCM material was analysed using desorption;
the inlet split flow was 0 ml/min and the outlet split flow was 18.75 ml/min, the desorption
flow was 60 ml/min, the desorption temperature was 150°C, the trap temperature was
4°C and the pressure was 14.6 psi. Detection was by mass-spectrometry; the MS was fitted
with an EI source operated at 70eV with a source temperature of 180°C, and mass spectra
were recorded in the range m/z 38–300. The software was Turbomass, version 4.1.1.
Approximately 2 mg of dried TCM material was desorbed for each analysis. Compounds
were identified by comparing mass spectra with published data (Ausloss
et al., 1992;
Adams, 2001).
Section 5
40

5.4. Methods for fractionation and isolation of compounds
5.4.1. Fractionations of Scutellaria baicalensis and isolation of compounds
Dried powdered root of S. baicalensis (35 g) was extracted in MeOH (800 ml) using a
Soxhlet apparatus for 19 h (64°C). The extract was concentrated to about 300 ml and
partitioned using hexane (100 ml). Column chromatography using normal phase silica gel
(60A S-230/70 mesh, SL06SA4, YMC Co. Ltd) was conducted on the dried methanol layer
(12 g). The mobile phase used was a step gradient elution of 10 combinations (each 500
ml) of CHCl
3
, Me
2
CO, MeOH and H
2
O in incr
easing polarity, starting with 100% CHCl
3
and ending with 95% MeOH in H
2
O. A total of 20 fractions were collected and concentrated
separately to dryness. HPLC-(UV-DAD) analysis, anti-bacterial and anti-inflammatory (NF
−κΒ)
tests were conducted on the crude extracts of
S. baicalensis and each of the 20 fractions.
Fraction 4 (SB4) was selected for further fractionation based on the results obtained from
NF
−κΒ tests. Preparative TLC was adopted as a method to fractionate SB4 using a mobile
phase of hexane: CHCl
3
: EtOAc (1:1:1). Nine fractions were obtained and tested in
anti-bacterial and anti-inflammatory (NF
−κΒ) assays. The fifth fraction of SB4 (SB4v)
demonstrated potent NF
−κΒ inhibition. Therefore SB4v was fractionated using preparative
HPLC-UV-DAD and NMR spectroscopy was used to identify the compounds isolated. NMR
data were acquired and interpreted at the Royal Botanic Gardens, Kew, by Dr N. Veitch.
5.4.2. Fractionation of Salvia miltiorrhiza and Qing Ying Tang
Dried powdered root of S. miltiorrhiza (72 g) was extracted in MeOH (800 ml) by using
a Soxhlet apparatus for 17 h (64°C). The extract was concentrated to dryness and 11 g
was subjected to silica column chromatography. A gradient elution of 8 combinations of
CHCl
3
, MeOH and H
2
O in increasing polarity
, starting with 100% CHCl
3
and ending with
85% MeOH in H
2
O, was employed. A total of 17 fractions were collected.
Lypophilised crude hot water extract of the TCM prescription, Qing Ying Tang (2.4 g) was
fractionated using flash chromatography (reversed-phase). A stepwise gradient elution system
was used starting with 5% MeOH in water and ending with 100% MeOH in volumes of
600 ml each. Twenty-four fractions were collected. Anti-bacterial tests were performed on
the fractions obtained from which
S. miltiorrhiza and Qing Ying Tang. The fractions which
showed some anti-bacterial activity were tested in an anti-inflammatory (NF
−κΒ) assay.
Biological and chemical methods used to study plant and fungal material
41

Section 5
42
5.5. Method for anti-bacterial tests
Assays were conducted at the Royal Botanic Gardens, Kew with Dr T. Kokubun. The
method for assessing anti-bacterial activity was a modification of that described by
Rehalison
et al. (1991). For this study, 20 µl aliquots of each herb extract (5 mg/ml) or
fraction (1 mg/ml) were applied to three replicate TLC plates (20 x 10 cm
2
, pre-coated
aluminium-backed silica gel, 60F
254
sheets, Merck, Germany). The TLC plates were developed
in a tank containing one of the following solvent systems: chloroform : acetone (4:1) or
(17:3), or chloroform : acetone : water (7:3:1). Developed plates were observed under
UV light (254 nm and 355 nm). One TLC plate was subjected to chemical analysis and two
were used to conduct anti-bacterial tests. The TLC plate for chemical analysis was sprayed
evenly with
p-anisaldehyde (0.5 ml in 50 ml HOAc and 1 ml conc. H
2
SO
4
); the chemical
profile of the sprayed plate was examined under UV light (355 nm). After heating, plates
were re-examined under UV light (355 nm).
Two of the developed TLC plates were fixed onto separate culture dishes and chloramphenical
(3 µg/ml; positive control) was applied to a solvent-free area on each TLC plate. A small
colony of previously cultured bacteria (
Pseudomonas syringae, ID No. IMI347448, CABI
Bioscience, UK or
Bacillus subtilis, ID No. IMI347329, CABI Bioscience, UK) was suspended
in water and added to 50 ml nutrient agar solution to form a seeded medium. This medium
was used as an overlay on the TLC plate to form bioautograms with a layer of approx
1 mm thickness of medium. The bioautographs were sealed and incubated overnight
(37°C, 100 % relative humidity).
After incubation the bioautographs were stained with
p-iodonitrotetradium violet
(2-[4-indophenyl]-3-[4-nitrophenyl]-5-phenyltetrazolium chloride, Sigma, USA) diluted in
ethanol (0.5 mg/ml, diluted 10-fold immediately prior to use). The dishes were incubated
(at 37°C) for a further two hours and then visually examined. Areas on the plate with
inhibited or reduced bacterial growth appeared as white spots against a pink background.
Areas of inhibition were compared to corresponding areas in the chemically treated plates
to ascertain groups of compounds responsible for the inhibition of bacterial growth.

5.6. NF–κΒ studies using the IL-6 promoter assay method
Dr P. Bremner conducted the assays as part of the collaboration with Professor M. Heinrich,
School of Pharmacy, London. The assay used was as described by Bork
et al. (1999). HeLa
S3 cells were stably transfected with IL-6 promoter fused with a Luciferase reporter gene
for 24 hours. Extracts/fractions (final incubation concentration of 100 µg/ml in DMSO)
and compounds (prepared in acetone) were placed in 96-well plates and incubated with
the transfected cells (at 37°C; 95% humidity) for 1 hour. The cells were then stimulated
with 50 ng/ml (final concentration) of either phorbol myristate acetate (PMA, Sigma, UK)
or TNF-
α (Sigma, UK) and incubated for a minimum of 7 hours (maximum 14 hours)
before harvesting. To each well, 100 µl of lysis buffer (25 mM Tris-phosphate pH 7.8, 8
mM MgCl
2
, 1 mM DTT, 1% Triton X-100 and 7% glycerol) was added and left for 15
minutes. After harvesting the cells, beetle Luciferin (50 µl, Promega, USA) was added to
the lysed cells (15 µl) in a 96-well plate by an automated Luminoter/photometer (Anthos
Lucy 1, Rosys Anthos, Switzerland) and the light emission was measured following a reaction
time of 10 seconds. The light emission of the lysis buffer was obtained as a background
reading and subtracted from each experimental value. Positive controls consisted of cells
stimulated with either PMA or TNF-
α only and negative controls involved cells subjected
to no stimulation.
5.7. Method for cytochrome P450 3A4 inhibition studies
A Tecan Genesis 150 RSP (Tecan UK Ltd, Reading, UK) was used to incubate the test solutions
in 96-well plates using an automated set-up and timed procedures. The following
sequence was performed, in duplicate, by the RSP. Ketoconazole (0.1, 0.3, 1, 3 and 10 µM),
herbal extracts and fractions (1000 µg/ml in water), compounds (200 µg/ml in 2.5%
acetone) and negative contr
ols (water or 2.5% acetone) were placed in 96-well plates.
Phosphate buffer (0.1 M; 245 µl) was added to human liver microsomes containing
testosterone (100 nM; 50 µl). A test solution (50 µl) was then added. The plates were
pre-incubated for 2 minutes. NADPH regenerating mixture (155 µl) was added to each
tube (to give a final volume of 0.5 ml) and incubated at 37
+ 2°C for 20 minutes.
Phosphoric acid (0.15 M; 0.1 ml) was added to terminate the reactions.
Instrumental quality control samples were performed (for each 96-well plate) by preparing
tubes containing the human liver microsomes (50 µl) (with no added testosterone) and
phosphate buffer (0.1 M; 245 µl). After the 2 minutes pre-incubation, NADPH mixture
(155 µl) was added to each tube (to a final volume of 0.45 ml) and incubated as
described above. On the termination of the reactions, by the addition of phosphoric acid,
6
β-hydroxytestosterone (50 µl; 10 and 100 µM) was used to generate duplicate quality
samples at two concentrations (1 µM and 10 µM). All the incubation tubes were centrifuged
at 3000 rpm for 10 minutes at ambient temperature. Aliquots of each sample were placed
in HPLC vials using the RSP.
Biological and chemical methods used to study plant and fungal material
43
–

The incubation procedure described above resulted in a 10 times dilution of the test solutions.
Therefore, the herbal extracts and compounds were tested at final concentrations of 100 µg/ml
and 20 µg/ml, respectively. Baicalin (45 µM), baicalein (74 µM), scutellarein (69 µM) and
chrysin (79 µM) were tested. The final concentrations for ketoconazole were 0.01, 0.03,
0.1, 0.3 and 1 µM.
5.7.1. CYP3A4 LC-MS method
Liquid chromatographic-mass spectrometric (LC-MS) analysis was conducted using a 1100
HPLC system (Agilent, Berks., UK) interfaced with mass spectr
ometer. The chromatographic
determination of 6
β-hydroxytestosterone was performed on a 150 mm x 4.6 mm i.d., 5 µm
Luna Phenyl Hexyl column (Phenomenex, Cheshire, UK) with a sentry 20 mm x 2.9 mm
Symmetry C
8
guard cartridge (Waters, Mass., USA) at 25°C. A flow rate of 1.5 ml/min was
used with mobile phase gradient programmed between ammonium acetate buffer (A) and
acetonitrile (B). The gradient programme (A:B) was 81:19 (t = 0); 73:27 (t = 3 minutes);
10:90 (t = 10 min) and then back to the original mobile phase ratio of 81:19 at t = 11
minutes till t = 11.5 minutes. The injection volume was 50 µl. The MS detection was made
in the positive ion mode, with TurboIonspray, 500°C, flow split 1:5. The nebuliser gas was
set at 8 (arbitrary scale), auxiliary gas at 6 L/min, and the curtain gas was set at 10 L/min.
The ionspray voltage was 4800 mV, orifice voltage (declustering potential) was 31 mV,
ring voltage (focussing potential) was 180 mV and the Q0 voltage (entrance potential)
was -5 mV. The ion was monitor
ed at m/z = 305.6 with a scan time of 500 ms. Data
were acquired for 10 minutes per sample. The approximate retention time for
6
β-hydroxytestosterone was 8.5 minutes.
5.8. Statistical analysis
The values obtained for the NF−κΒ and CYP450 assays were expressed as mean values +
SD. The Student’s 1-sample
t-test was used to determine statistical differences between test
and control groups. The difference was considered statistically significant when
p < 0.05.
Section 5
44
–

Results and discussion
6.1. Bear bile: bioassay results and discussion
Results of the authentication study indicate that at least five of the seven TCM herbs
(Table 6.1.) showed similar chemical profiles to the reference and authentic material,
thus indicating they were the correct plant species, as described in TCM.
6.1.1. Anti-bacterial tests
The seven herbs described in Table 6.1., as well as prescription X were tested for their
effect on Gram-positive and Gram-negative bacteria. Some components from ethyl acetate
extracts (100 µg) of the following six herbs, separated on TLC plates, showed inhibitory
action against the growth of
Bacillus subtilis (Gram-positive bacteria): Anemarrhena
asphodeloides, Gardenia jasminoides, Scutellaria baicalensis, Phellodendron amurense,
Coptis chinensis
and Rheum palmatum. In addition, some fractions from the ethyl acetate
extract (100 µg) of
Rheum palmatum and fractions from the methanol extract (20 µg) of
Scutellaria baicalensis also inhibited the growth of Pseudomonas syringae (Gram-positive
bacteria). Out of the seven herbs tested only
Andrographis paniculata showed no inhibitory
effect against either
Bacillus subtilis or Pseudomonas syringae at 100 µg. However, some
TCM literature cites studies conducted in China, which have shown that
Andrographis
paniculata
inhibits several Gram-negative and Gram-positive bacteria in vitro, but the
concentrations tested were not stated (Hsu
et al., 1986; Chang and But, 1987; Huang,
1999). Prescription X (100 µg) did not inhibit either
Bacillus subtilis or Pseudomonas
syringae
at the concentration tested.
Summary
45
T
able 6.1. TCM herbs for which the plant species were identified
Chuan Xin Lian, aerial part of Andrographis paniculata (CXL)
Zhi Mu, rhizome of Anemarrhena asphodeloides (ZM)
Zhi Zi, fruit of Gardenia jasminoides (ZZ)
Huang Qin, root of Scutellaria baicalensis (SB)
Huang Bai, cortex of Phellodendron amurense (HB)
TCM herbs for which the plant species were not verified
Huang Lian, Rhizoma Coptidis (HL)*
Da Huang, Radix et Rhizoma Rhei (DH)*
Section 6

6.1.2. Anti-inflammatory (NF−κΒ) tests
Herbal extracts (100 µg/ml) were tested in either TNF-α or PMA-stimulated HeLa cells using
an IL-6 promoter method to determine their effect on NF–
κΒ activity; results are shown in
Fig. 6.1. Water extracts of
Rheum palmatum (DH) showed significant (p<0.05) inhibition of
NF–
κΒ activity. Water extracts of Coptis chinensis (HL), Phellodendron amurense (HB) and
Anemarrhena asphodeloides (ZM) reduced NF–κΒ activity; however, these reductions were
not statistically significant. A water extract of
Andrographis panicalata (CXL) did not affect
NF–
κΒ production. Also, prescription X reduced Luciferase values by 16%, but this was not
statistically significant (data not shown).
Although potent inhibition of NF–
κΒ activity was measured for the ethyl acetate extract of
Scutellaria baicalensis (69%, p<0.001), stimulation was obtained for the corresponding water,
methanol (SBM) and hexane (SBH) extracts (Fig. 6.1.). The effects of fractions (SB1 – SB20;
obtained from methanol extract) of S.
baicalensis on NF–κΒ activity are shown in Fig 6.2.
Cells for water extracts (100 µg/ml) were stimulated with phorbol myristate acetate
(PMA; 50 ng/ml) and the ethyl acetate extract (100 µg/ml) with TNF-
α (50 µg/ml). Induced
IL-6 promoter activity was measured as light emission (Luciferase values) expressed as a
percentage relative to cells stimulated by PMA or TNF-
α only. The data represent mean
(n = 3) ± SD. *
p<0.05 and **p<0.001 indicate statistically significant differences from
cells treated with PMA or TNF-
α only.
Codes for herbs presented in Fig. 6.1: HB: Huang bai, cortex of
Phellodendron amurense;
HL: Huang lian, Rhizoma Coptidis ; DH: Da Huang, Radix et Rhizoma Rhei; ZM: Zhi Mu,
rhizome of
Anemarrhena asphodeloides; CXL: Chuan Xin Lian, aerial part of Andrographis
paniculata
; SB: Huang Qin, root of Scutellaria baicalensis.
Section 6
46
Fig. 6.1. The effects of six herbal extracts on NF−κΒ activity
92
183
65
73
108
82
148
173
*
*
31
0
50
100
150
200
250
HB HL DH ZM CXL SB
water
methanol hexane ethyl acetate
Mean % of (stimulatory) control

Extracts (100 µg/ml) were tested in PMA-stimulated cells. IL-6 gene promoter activity was
measured as outlined in the legend for Fig. 6.1. Negative controls: resting cells (RC;
unstimulated); positive controls: PMA stimulated cells. The data represent mean (n = 9
for controls and n = 3 for test samples) ± SD. *
p= 0.001, **p<0.001 indicate statistically
significant differences from PMA-stimulated cells. Colour code: yellow = positive control
(stimulatory); red = resting cells (inhibitory); purple = other fractions obtained from
fractionating the crude extract; orange = fraction further fractionated. Fraction 4v
(obtained from fractionating fraction 4) = 3%,
p<0.001.
Fractions SB4-SB13 demonstrated significant inhibition of NF
−κΒ activity (p< 0.001), in
contrast to the stimulatory effects of some fractions (SB14-SB17) and the crude methanolic
extract (SBM). Three compounds, chrysin, wogonin and oroxylin A, were isolated and
identified from SB4 using TLC, HPLC (UV-DAD) and NMR. These three flavonoids were
also identified from their characteristic UV profiles, as present in active fractions SB4
to SB12 from HPLC-(UV-DAD) analysis. When the constituents of
S. baicalensis (chrysin,
wogonin, oroxylin A, baicalein, baicalin and scutellarein) were tested, the NF
−κΒ inhibitory
and stimulatory activities of some of these compounds were found to be dependent on
their concentrations (Fig. 6.3). Salicylic acid, an anti-inflammatory compound, which
inhibits COX activity, was used as a further control in this assay. Chrysin inhibited NF
−κΒ
activity in a dose-dependant manner (at 50–393 µM, p<0.001), showing more inhibitory
activity than salicylic acid (145 µM,
p<0.01; Fig 6.3). Baicalein and wogonin demonstrated
a significant reduction in NF
−κΒ activity, only at 370 and 100 µM, respectively. However,
chrysin and baicalein had some associated cytotoxity at 393 µM and 370 µM, respectively.
Results and discussion
47
Fig. 6.2. The effects of fractions obtained from methanol extract of Scutellaria baicalensis on NF−κΒ activity
100
106
105
101
100
133
164
101
116
147
145
*
*
4.5
*
*
6
*
*
7
*
*
14
*
*
10
*
*
14
*
*
16
*
*
21
*
26
*
33
*
47
0
20
40
60
80
100
120
140
160
180
200
PMA
RC
1 2 3 4 5 6 7 8 9
10 11 12 13 14 15 16 17 18
19 20
Mean % of (stimulatory) control
Fractions showing anti-inflammatory effects
positive control
negative control
hexane
ethyl acetate

At lower concentrations chrysin (25 µM) and wogonin (25 and 50 µM) demonstrated
significant stimulation of NF
−κΒ activity (Fig. 6.3.). Oroxylin A also stimulated NF−κΒ
activity at 25–100 µM (p<0.05) but showed no significant effect at 352 µM. However,
oroxylin A (70 µM) has been reported to inhibit LPS-induced NF
−κΒ activity in RAW264.7
macrophages via the inhibition of NF
−κΒ complex (Chen et al., 2000). The anti-inflammatory
pathway tested in the current study was via the inhibition of NF
−κΒ activity (as assessed
using IL-6 promoter assay). Recently, baicalein (24, 48 and 96 µM) has been reported to
potently inhibit IL-12 production in LPS-activated macrophages via the inhibition of NF
−κΒ
binding activity (Kang et al., 2003a).
Compounds were tested in PMA-stimulated cells; IL-6 promoter activity was measured as outlined in the legend for
Fig.6.1. The data repr
esent mean (n = 8 for PMA, n = 9 for r
esting cells and n = 3 for test compounds)
±
SD.
*
p<0.01, **p<0.001 indicate statistically significant differences from PMA-stimulated cells. Scutellarein (5,6,7,4’-
tetrahydroxyflavone); baicalin (5,6-dihydroxy-7-glucuronide); chrysin (5,7-dihydroxyflavone); baicalein (5,6,7-
trihydroxyflavone); wogonin (5,7-dihydroxy-8-methoxyflavone); oroxylin A (5,7-dihydroxy-6-methoxyflavone).
Section 6
48
Fig. 6.3. The effects of flavonoids from Scutellaria baicalensis, and salicyclic acid on NF−κΒ activity.
344
*
41
*
17
*
65
*
*
10
*
*
2
86
*
*
9
118
263
*
*
23
*
*
37
119
153
99
135
172
267
0
100
200
300
400
PMA, 50 ng/ml
Unstimulated cells
PMA + Salicylic acid (145
µ
M)
PMA + Scutellarein (347
µ
M)
PMA + chrysin
PMA + Baicalein
PMA + Wogonin
PMA + Oroxylin A
controls 25 µM 50 µM 100 µM >300 µM
Mean % of PMA (stimulatory control)

6.1.3. Cytochrome P450 3A4 tests
Results for the CYP3A4 inhibition assay, using testosterone 6β-hydroxylation as a probe for
enzyme activity in human liver microsomes, are presented in Figs 6.4 and 6.5. Crude hot
water extracts of
Coptis chinensis (100 µg/ml) significantly reduced CYP3A4 activity by
37% (
p <0.01) compared to that of the negative (uninhibited) control (Fig. 6.4). Water
extracts of
Scutellaria baicalensis, Anemarrhena asphodeloides, Andrographis paniculata,
Phellodendron amurense
and Gardenia jasminoides showed no significant effect CYP3A4
activity at 100 µg/ml (Fig. 6.4).
The assay was conducted using testosterone 6β-hydroxylation as a probe for enzyme activity in human liver microsomes.
Results were calculated as mean % of uninhibited controls (water); ketoconazole (KC) was used as positive control. Data
represent mean (n = 2) ± SD. *
p<0.01 indicate statistically significant differences from groups only treated with water.
Codes for herbs presented in Fig. 6.4: HL: Huang lian, Coptis chinensis; Huang qin, root of
Scutellaria baicalensis; ZM:
Zhi Mu, rhizome of
Anemarrhena asphodeloides; CXL: Chuan Xin Lian, aerial part of Andrographis paniculata; SB: HB:
Huang Bai, cortex of
Phellodendron amurense; ZZ: Zhi Zi, fruit of Gardenia jasminoides.
Results and discussion
49
Fig. 6.4. The effects of six TCM herbs and ketoconazole on CYP3A4 activity.
100
97
90
74
93
95
102
104
106
*
63
*
16
92
0
20
40
60
80
100
120
Mean % of (uninhibited) control
Control
KC (0.01µM)
KC (0.03 µM)
KC (0.1µM)
KC (0.3 µM)
KC (1µM)
HL
SB
ZM
CXL
HB
ZZ

Section 6
50
The assay was conducted using testosterone 6β-hydroxylation as a probe for enzyme activity in human liver microsomes.
Results were calculated as mean % of uninhibited controls (water); ketoconazole (KC) was used as positive control. Data
represent mean (n = 2) + SD. *p<0.05, **p<0.01, ***p<0.001 indicate statistically significant differences from groups
only treated with water. Colour codes: red = unstimulated controls (water or 2.5% acetone); yellow = ketoconazole; blue
= known compounds of
S. baicalensis; plum = fractions obtained from fractionating S. baicalensis; green = crude extracts
of
S. baicalensis.
However, a methanolic extract of Scutellaria baicalensis (SBM), a fraction (SB4v) and
two constituent flavonoids, baicalein (74 µM) and scutellarein (69 µM), from
Scutellaria
baicalensis
all inhibited CYP3A4 activity by 30–40 % (p<0.05) compared to the negative
(uninhibited) control (Fig. 6.5). Chrysin (79 µM), showed greater inhibition by lowering
CYP3A4 activity to 74% (
p<0.001) compared to the negative (uninhibited) control (Fig.
6.5). The IC
50
value obtained for the positive control, ketoconazole in the CYP3A4 studies
was 0.6 µM, which was high, compared to reported values of 0.1 µM (McKillop
et al.,
1999) and 0.04 mM (Sai
et al., 2000). The high positive control value observed may
indicate that moderate inhibition might not have been detected with some of the
test extracts at the concentration tested.
Fig. 6.5. The effects of fractions and flavonoid compounds of Scutellaria baicalensis (SB) and ketoconazole
o
n CYP3A4 activity.
112
9
5
96
9
3
95
104
100
93
*
*
*
26
*
*
16
*
*
60
*
59
*
60
*
69
0
20
40
60
80
100
120
140
Control
KC (0.01µM)
KC (0.03 µM)
KC (0.1 µM)
KC (0.3 µM)
KC (1 µM)
Baicalein (74 µM)
Scutellarein (69 µM)
Baicalin (45 µM)
SB4v
SB14
SBM
SBW
Mean % of (uninhibited) control
Chrysin (79 µM)

6.1.4. Conclusions
Results from this study have shown that Anemarrhena asphodeloides, Gardenia jasminoides,
Scutellaria baicalensis, Phellodendron amurense,
Rhizoma Coptidis and Radix et Rhizoma
Rhei all possess some anti-bacterial activity. Also, results from this study have further
confirmed the anti-inflammatory properties of Radix et Rhizoma Rhei and
Scutellaria
baicalensis
through the inhibition NF−κΒ activity. The results from both the anti-bacterial
and anti-inflammatory tests have highlighted the herbs which may be investigated further
through bioactivity guided fractionations.
Preliminary results from the CYP3A4 studies suggest that possible herb-herb interactions
may occur in preparations containing both Rhizoma Coptidis and
Scutellaria baicalensis
(such as Dia-Orengedokuto and Orengedokuto). Also, drug-herb interactions may occur
when herbal preparations containing Rhizoma Coptidis and/or
Scutellaria baicalensis
are co-administered with some pharmaceutical drugs, which are metabolised by CYP3A4.
However, further work is required to investigate the extent of these effects.
6.2. Rhino horn: bioassay results and discussion
Twenty-two herbs that could be potential substitutes for rhino horn were assayed for
activity (Table 6.2). The extracts of the 22 herbs were chemically profiled along with
authenticated TCM material. At least 13 of these herbs showed similar chemical profiles
to the authenticated TCM samples, thus indicating they were the correct plant species,
as described in TCM. Further work is necessary on 9 of the plant species to confirm
their identification.
6.2.1. Anti-bacterial tests
Results for anti-bacterial activity of extracts (100 µg) of rhino horn and TCM prescriptions
ar
e summarised in Table 6.3. In addition, 24 fractions were obtained from flash
chromatography of water extracts of Qing Ying Tang; fractions QYT9, QYT15, QYT16
and QYT17 showed some inhibitory activity against
Bacillus subtilis but did not inhibit
Pseudomonas syringae. The anti-bacterial tests were qualitative, so although some
prescriptions with and without rhino horn demonstrated anti-bacterial activity, the contribution
of the horn extracts in the prescriptions could not be evaluated. However, rhino horn
alone did not inhibit the growth of either
Bacillus subtilis or Pseudomonas syringae.
Crude ethyl acetate extracts (100 µg) of Radix et Rhizoma Rhei and fractions from
methanolic extracts (20
µg) of
Salvia miltiorrhiza, Scutellaria baicalensis and Lonicera japonica
showed anti-bacterial activity against both Bacillus subtilis and Pseudomonas syringae.
Ethyl acetate extracts (100 µg) of 17 herbs showed some inhibitory activity against
Bacillus subtilis and are listed in Table 6.2 (samples 4–20).
Results and discussion
51

Section 6
52
(B) Results have also been presented in the bear bile project (section 6.1).
TCM pharmaceutical names are used in the text for herbs that require further investigation to assist with their authentication.
Table 6.2. TCM herbs investigated in biological assays
Abbreviation TCM herbs
MDP Mu Dan Pi, root of Paeonia suffruticosa
J
G
J
ie Geng, root of
P
latycodon grandiflorum
GC Gan Cao, root of Glycyrrhiza uralensis
XS Xuan Shen, root of Scrophularia ningpoensis
SDH Sheng Di Huang, root of Rehmannia glutinosa
JYH Jin Yin Hua, flower bud of Lonicera japonica
LQ Lian Qiao, fruit of Forsythia suspensa
DS Dan Shen, root of Salvia miltiorrhiza
DZY Dan Zhu Ye, aerial part of Lophatherum gracile
ZM (B) Zhi Mu, rhizome of Anemarrhena asphodeloides
ZZ (B) Zhi Zi, fruit of Gardenia jasminoides
HQ (B) Huang Qin, root of Scutellaria baicalensis
HB (B) Huang Bai, cortex of Phellodendron amurense
Abbreviation TCM herbs
HL (B) Huang Lian, Rhizoma Coptidis
DH (B) Da Huang, Radix et Rhizoma Rhei
ZC Zi Cao, Radix Arnebiae
BLG Ban Lan Gen, Radix Isatidis
MMD Mai Men Dong, Ophiopogonis Radix
DDC Dan Dou Chi, Semen Sojae Praeparatum
CP Chang Pu, Rhizoma Acori Graminei
CSY Chi Shao, Radix Paeoniae Rubra
THF Tian Hua Fen, Radix Trichosanthis

Results and discussion
53
nd: no inhibition detected
6.2.2. Anti-inflammatory (NF−κΒ) tests
Water extracts (100 µg/ml) of five TCM prescriptions containing rhino horn and five
without rhino horn, rhino horn alone, as well as Zhi Zi Jin Hua (composed only of herbs)
were tested in PMA-stimulated HeLa cells using IL-6 promoter assay (Fig 6.6). Only the
prescriptions Xi Jiao Dihuang Tang without rhino horn (XJDHT) and Xi Jiao Dihuang tang
with rhino horn (XJDHT+RH) showed significant inhibitory effect on NF
−κΒ activity (Fig
6.6.). XJDHT+RH and XJDHT reduced NF
−κΒ activity by 45 % (p<0.01) and 34 % (p<0.05),
respectively, compared to fully stimulated cells by PMA, indicating that rhino horn might
contribute to the inhibitory effect. However, since rhino horn extract alone did not show
any apparent effect on the NF
−κΒ activity and results from the other prescriptions were
not conclusive, further work is required to clarify the contribution of the horn extract
and whether there is a synergistic effect.
The TCM prescription Qing Ying Tang (QYT) demonstrated stimulatory effect on NF
−κΒ
activity (Fig. 6.6.). However, when fractions obtained from QYT (QYT9, QYT15, QYT16
and QYT17), that showed anti-bacterial activity were tested in the NF
−κΒ assay they
demonstrated significant inhibition of NF
−κΒ activity (Fig. 6.7(A)).
Table 6.3. Anti-bacterial activity of rhino horn and TCM prescriptions
Rhino horn and TCM prescriptions Anti-bacterial tests
B. subtilis P. syringae
R
hino horn
n
d
n
d
Qing Ying Tang plus rhino horn (QYT + RH) Inhibition nd
Qing Ying Tang without rhino horn (QYT) Inhibition nd
Sheng Xi Dan plus rhino horn (SXD+RH) Inhibition Inhibition
Sheng Xi Dan without rhino horn (SXD) Inhibition Inhibition
Qing Gong Tang plus rhino horn (QGT+RH) Inhibition nd
Qing Gong Tang without rhino horn (QGT) Inhibition Inhibition
Qingwen Baidu Yin plus rhino horn (QWBY + RH) nd nd
Qingwen Baidu Yin without rhino horn (QWBY) nd nd
Xi Jiao Dihuang Tang plus rhino horn (XJDHT + RH) nd nd
Xi Jiao Dihuang Tang without rhino horn (XJDHT) nd nd
Zhi Zi Jin Hua (ZZJH) nd nd

Section 6
54
Water extracts (100 µg/ml) were tested in PMA-stimulated cells as outlined in Fig. 6.1.
Resting cells were used as negative controls; cells stimulated with PMA alone were used
as positive controls.
The data represent mean (n = 5 for controls and n = 3 for test samples) ± SD. *
p<0.05,
**
p<0.01, ***p<0.001 indicate statistically significant differences from groups only treated
with PMA. The abbreviations for the prescriptions are described in Table 6.3.
Salvia miltiorrhiza is one of eight herbs making up the QYT prescription. Fractions SM6
and SM7 from the methanolic extract of
Salvia miltiorrhiza potently inhibited NF−κΒ activity
to below levels obtained for resting cells, and SM8 reduced NF
−κΒ activity to 52% of that
of PMA (fully stimulated cells) (Fig. 6.7.(A)).
Crude hot water extracts of some of the 16 herbs (found in some of the TCM prescriptions
investigated in this study) were also tested in PMA-stimulated HeLa cells in NF−κΒ tests
and the results are presented in Fig. 6.7(B).
Paeonia suffruticosa and Radix Trichosanthis
significantly reduced NF
−κΒ activity by about 50% of that obtained by cells fully stimulated
with PMA. Other herbs showing statistically significant r
eduction in NF
−κΒ activity wer
e
Lophatherum gracile, Radix Isatidis, Rhizoma Coptidis, Semen Sojae Praeparatum and
Rehmannia glutinosa (Fig 6.7.(B)).
Fig. 6.6. The effects of rhino horn extract, TCM prescriptions (with and without rhino horn) on NF−κΒ activity.
*
*
*
10
100
115
1
07
*
*
55
1
40
9
3
95
135
110
1
09
*
66
113
9
0
0
20
40
60
80
100
120
140
160
PMA Resting
cells
Rhino horn XJDHT SXD QGT QWBY QYT ZZJH
Mean % of (stimulatory) control
C
ontrols
P
lus rhino horn
W
ithout rhino horn

Results and discussion
55
(A) The effects of fractions (SM6, SM7 and SM8) of a methanol extract of Salvia miltiorrhiza (SM) and water extract of
Qing Y
ing T
ang (QYT).
(B) The effects of 12 TCM herbs (full names are described in Table 6.2).
Extracts (100
µg/ml) were tested in PMA-stimulated cells as outlined in Fig. 6.1. Resting cells (RC) were used as negative
controls; cells stimulated with PMA alone were used as positive controls. The induced IL-6 promoter activity was
measured as light emission (Luciferase values) and expressed as a percentage relative to cells stimulated with PMA
alone. The data represent mean (n = 5 for controls and n = 3 for test samples) ± SD. *
p<0.05, **p<0.01,
***
p<0.001 indicate statistically significant differ
ences fr
om groups only treated with PMA.
6.2.3. Cytochrome P450 3A4 tests
Water extracts (100 µg/ml) of rhino horn and TCM prescriptions Sheng Xi Dan and Qing
Ying Tang (with and without rhino horn) showed no apparent significant effect on
6
β-testosterone hydroxylation due to inhibition of CYP3A4 activity (Fig. 6.8. (A)).
The IC
50
value obtained for the positive control, ketoconazole, in these studies was 0.5 µM.
Water extracts (100 µg/ml) of 14 herbs were also tested and only Rhizoma Coptidis and
Rehmannia glutinosa showed any significant effect on CYP3A4 activity compared to the
control (containing no inhibitor) (Fig. 6.8.(B)). Fraction SM7 obtained from a methanolic
extract of
Salvia miltiorrhiza also demonstrated inhibitory activity against CYP3A4 activity
compared to the control (Fig. 6.8. (C)). The inhibitory effects of some
Scutellaria constituents
are discussed under the bear bile project (section 6.1.).
Fig. 6.7. The effects of TCM remedies on NF−κΒ activity.
100
*
4
8
*
*
2.0
*
*
1.1
*
*
28
*
*
5
4
*
47
*
69
*
*
17
-20
0
20
40
60
80
100
1
20
140
PMA Unstimulated QYT9 QY15 QYT16 QYT17 SM6
SM8
SM8
98
118
97
94
100
66
*
86
*
82
*
67
*
*
50
*
*
*
44
*
*
*
11
62
*
54
0
20
40
60
80
100
120
140
PMA RC MDP THF DZY CP CSY BLG DDC SDH XS JG JYH GC
Mean % of (stimulatory) control
Mean % of (stimulatory) control
A
B

Section 6
56
(A) Rhino horn (RH) and Shen Xi Dan (SXD) and Qing Ying Tang (GYT) with and without RH.
(B) Nine TCM herbs (full names ar
e described in Table 6.2.).
(C) Fractions (SM7 & SM8) of a methanol extract of
Salvia miltiorrhiza (SM).
The CYP3A4 inhibition assay was conducted using testosterone 6
β-hydroxylation as a probe for enzyme activity in human
liver microsomes. Results were calculated as mean percent of uninhibited control (water); ketoconazole (KC) was used
as positive contr
ol. The data r
epresent mean (n = 2) ± SD. *
p<0.05, **p<0.01 indicate statistically significant dif
fer
ences
from groups only treated with vehicle solution.
Fig. 6.8. The effects of TCM remedies and ketoconazole on microsomal CYP3A4 activity.
95
109
9
9
96
1
01
100
*
16
1
01
0
2
0
4
0
60
80
100
1
20
Control KC
(0.3 µM)
KC (1 µM) RH SXD SXD+RH QYT QYT+RH
100
74
90
91
92
108
*
88
*
16
111
113
102
89
0
20
40
60
80
100
120
140
Control KC
(0.3µM)
KC
(1µM)
SDH GC JG CP MDP XS JYH CSY BLG
100
95
*
*
16
*
64
76
102
0
20
40
60
80
100
120
Control KC (0.3 µM) KC (1 µM) SM7 SM8 SM
Mean % of (uninhibited) control
Mean % of (uninhibited) control
Mean % of (uninhibited) control
A
B
C

6.2.4. Conclusions
Water extracts of rhino horn did not demonstrate anti-bacterial or anti-inflammatory
properties, nor did they have any effect on the drug metabolising enzyme, CYP3A4.
However, some TCM prescriptions with and without rhino horn showed some anti-bacterial
and anti-inflammatory properties in the assays used in this study. However, further
work using other bioassays is required to ascertain the contribution of the horn extracts
to any activities of the TCM prescriptions.
Most of the herbs (17 out of 22) showed some anti-bacterial activity against
Bacillus
subtilis
, indicating potential pharmacological effects. Also, results from this study indicate
potential anti-inflammatory properties of four out of the nine herbs selected to study as
possible alternatives to rhino horn:
Paeonia suffruticosa, Radix Isatidis, Rehmannia glutinosa
and Salvia miltiorrhiza. In addition Radix Trichosanthis, Semen Sojae Praeparatum, Rhizoma
Coptidis and
Lophatherum graciIe, found in different prescriptions also showed anti-
inflammatory properties. To date no scientific literature in the English language has been
obtained for the anti-inflammatory effects of
Lophatherum gracile and therefore further
studies are required to verify the data obtained from this study. The TCM prescriptions Xi
Jiao Dihuang Tang and Qing Ying Tang also showed a significant anti-inflammatory effect
through the inhibition of NF
−κΒ activity.
Salvia miltiorrhiza, Rehmannia glutinosa, as well as Scutellaria baicalensis and Rhizoma
Coptidis
, showed inhibitory effects on CYP3A4 during preliminary studies. Since they are
commonly used TCM herbs, further work may be required to determine potential adverse
interactions with other remedies.
6.3. Tiger bone: bioassay results and discussion
The extracts of the twenty-three herbs and one fungus were chemically profiled along with
authenticated TCM material, using various analytical techniques (HPLC (UV-DAD), LC-MS
and GC-MS). At least 10 of the species showed similar chemical profiles to the authenticated
TCM samples, thus indicating they were the correct species, as described in TCM. Further
analysis of the remaining TCM material is necessary to confirm their identification.
Eighteen of the herbs were investigated in biological assays for the tiger bone project
(Table 6.4.).
Results and discussion
57

Section 6
58
(a) Herbs tested in NF–κB assay only; (b) Herbs tested CYP3A4 assay only
6.3.1. Anti-inflammatory (NF−κΒ) tests
Crude hot water extracts (100 µg/ml) of tiger bone and the prescription Yang Xue Gu
Feng Tang (without tiger bone) reduced NF
−κΒ activity (via the inhibition of IL-6 promoter
activity) by 31% and 34%, respectively (p<0.05), in PMA-stimulated HeLa cells (Fig. 6.9(A)).
However, the reduction of NF
−κΒ activity (26%) by the prescription, Du Huo Ji Sheng Tang
(without tiger bone) was not statistically significant (Fig. 6.9(A)). Results for crude hot water
extracts (100 µg/ml) of 17 herbs are shown in Fig 6.9(B). Four of these herbs produced a
statistically significant reduction in NF
−κΒ activity: Atractylodes macrocephala (Bai Zhu),
Saussurea costus
(Mu Xiang), Taxillus chinensis (Sang Ji Sheng) and Angelica sinensis
(Dang Qui).
Table 6.4. TCM samples studied in biological assays
TCM samples studied in biological assays
Tiger bone Panthera tigris
D
H(b)
D
u Huo, root of
A
ngelica pubescens
W
LX (a)
W
ei Lin Xian, root of
C
lematis chinensis
BS (a) Bai Shao, root of Paeonia lactiflora
YYH (a) Ying Yang Huo, aerial part of Epimedium sagittatum
FF (a) Fang Feng, root of Saposhnikovia divaricata
DZ (a) Du Zhong, bark of Eucommia ulmoides
MX Mu Xiang, root of Saussurea costus
CZ Cang Zhu, root of Atractylodes lancea
RS Ren Shen, root of Panax ginseng
SQ San Qi, root of Panax pseudoginseng
BZ Bai Zhu, roots of Atractylodes macrocephala
SJS Sang Ji Sheng, stem and branch of Taxillus chinensis
CX Chuan Xiong, rhizome of Ligusticum chuanxiong
QJ Qin Jiao, leaf of Gentiana macrophylla
DQ Dang Gui, root of Angelica sinensis
GZ Gui Zhi, twigs of Cinnamomum cassia
NX Niu Xi, root of Achyranthes bidentata
MG Mu Gua, fruit of Chaenomeles speciosa

Results and discussion
59
(A) The effects of tiger bone and two TCM prescriptions on NF−κΒ activity.
(B) The effects of 17 herbs on NF
−κΒ activity (full names are described in Table 6.4.).
Extracts (100 µg/ml) were tested in PMA-stimulated cells as outlined in Fig. 6.1. The full names of the herbs are as described
in Table 6.4. Resting cells (RC; unstimulated) were used as negative controls; cells stimulated with PMA were used as positive
controls. The induced IL-6 promoter activity was measured as light emission (Luciferase values) and expressed as a percentage
r
elative to cells stimulated by PMA only. The data represent mean (n = 7 for controls and n = 3 for test samples) + SD.
*
p<0.05 and **p<0.01 indicate statistically significant differ
ences from gr
oups only tr
eated with PMA.
Although, the inhibitory effects of the herbs were not potent at the concentrations
tested, the results have highlighted the herbs that could be investigated further, using
other assays to assess potential pharmacological mechanisms that could be related
to anti-inflammatory activity.
*
69
*
6
6
*
*
10
100
74
0
20
4
0
6
0
80
1
00
120
1
40
PMA Resting cells Tiger bone Yang Xue Gu
Feng Tang
Du Huo Ji
Sheng Tang
98
103
102
102
97 9792
90
89
84
84
81
78
*
88
83
*
75
*
73
*
*
10
100
0
20
40
60
80
100
120
140
PMA RC BZ MX SJS CZ CX WLX QJ DQ RS GZ NX SQ BS MG YYH FF DZ
Mean % of (stimulatory) control
Mean % of (stimulatory) control
A
B
Fig. 6.9. The effects of TCM prescriptions, tiger bone and remedies traditionally found in tiger bone
p
rescriptions, on NF
−
κΒ
a
ctivity.

Section 6
60
6.3.2. Cytochrome P450 3A4 tests
Crude hot water extracts (100 µg/ml) of tiger bone and thirteen individual herbs were