5.1 Introduction 111
5.2 Methods 114
5.2.1 Guinea pigs used in the study 114
5.2.2 CHT used in the study 114
5.2.3 Contact dermatitis in guinea pigs 115
5.2.4 Measurement o f the skin thickness 115
5.2.5 Control animals used in the study 116
5.2.6 Treatment o f guinea pigs with CHT 116
5.2.6 Statistical method 117
5.3 Results 118
5.3.1 Consumption o f water/CHT-containing water by guinea pigs 118 5.3.2 Skin reactions to different concentrations o f DNCB 120 5.3.3 Effect o f CHT prior to sensitisation on guinea pigs 122
5.3.4 CHT post sensitisation effects on guinea pigs 124
5.3.5 Effects o f CHT when given in the late phase 126
5.3.6 CHT post sensitisation effects on guinea pigs (No 2nd week
challenge) 128
5.3.7 Effects o f CHT on the recall skin reaction 130
5.3.8 The effects o f CHT following progressive treatment 132
5.3.9 Dose response (1) 135
5.3.10 Dose response (2) 138
5.3.11 Comparison o f the effects o f CHT using different administration
methods 141
5.3.12 Placebo controlled study o f the effects o f CHT 144 5.3.13 Comparison the effects o f CHT introduced at different times 147 5.3.14 Comparison the effects o f CHT(PSE222) and a fraction
ofPSE222 150
5.1
Introduction
The previous chapters address parameters of immune dysfunction in atopic
dermatitis and investigate at what level CHT is effective. In short the relationship
between CHT induced changes in lesional skin and clinical efficacy has been
explored. Immunohistochemistry and in-situ hybridisation have been used to
identify possible modulation of immune mechanisms by Chinese herbal therapy at
the cellular and molecular level. All such investigations have been conducted on
skin biopsies taken from patients. The use of human subjects made it difficult to
establish direct cause and effect relationships between CHT induced clinical effects
and immune changes, as placebo controls were not applied. Furthermore, all
patients had existing AD thus any possible effects on the induction of immune
reactivity could not be tested nor could dose response studies be performed. Other
considerations such as any possible side effects of CHT after prolonged treatment
are also not possible to examine in man. It was thus decided to use an animal model
of dermatitis to investigate these questions and if successful, to then initiate studies
of the effects of isolated purified fractions of the CHT in an attempt to identify the
‘active’ components of the decoction.
The dog is the only animal in which naturally occurring atopic disease with
hay fever-like symptoms has been identified (Patterson, 1969; Halliwell, 1971). In
this model, immediate skin test reactivity has been passively transferred to recipient
dogs, but other symptoms such as rhinitis, sneezing, asthma and conjunctivitis are
it is difficult or impossible to conduct systematic controlled experiments on large
numbers of dogs for both logistic and financial reasons.
However, research into the mechanisms of atopic dermatitis in man have
revealed that AD patients exhibit patch test reactivity to allergens that are
immunopathologically similar to clinical AD skin lesions (Buckley et al. 1993). The
local patch test to allergens produces an infiltrate of lymphocytes and macrophages
in the dermis and epidermis, which peaks at 24 - 48 hours. Thus although allergen
generated, the patch test, (as distinct from the prick test) represents an immunologic
recall reaction with the characteristics of type IV delayed type hypersensitivity.
For many years investigation of delayed-type hypersensitivity reactions has
been undertaken using animal models such as oxazolone sensitivity in mice
(Griswold and Walz, 1982; Griswold and Walz, 1977) and sensitivity to dinitro-
chlorobenzene (DNCB) in guinea pigs (Basketter et al. 1997; Rothman, 1966).
Topical application of DNCB on the guinea pig ear and subsequent challenge with
DNCB on the flank is a well established model of contact dermatitis (Maibach and
Maguire, 1963; Polak, 1976). In the absence of a specific allergic reaction in the
skin being available, this model of contact sensitivity was used. As the degree of
reactivity to the contact sensitivity is measured using an index of induration and
erythema this represented a comparable parameter to that used to determine CHT
efficacy in man. By using this model, it was possible to investigate the effects of
CHT on a controlled immune response. By altering the relationship between time of
in terms of induction and recall of the immune response. Further, this model could
be used to establish an animal system that allow selected components of the herbs in
the decoction to be assessed independently. Such investigation would not be
5.2
Methods
5.2.1 Guinea pigs used in the study
Male Dunkin-Hartley guinea pigs purchased from BK Universal, Hull, UK,
were used in the study. All animals weighed 200 - 250 grams when purchased.
Guinea pigs were housed in cages, 5 guinea pigs each cage, for 1 week to allow
health checks. All guinea pigs had free access to food and water or water containing
appropriate concentrations of CHT, (see results). A sterile electronic tag (Avid
identity tags, Labtrac Limited, East Sussex, UK) was implanted subcutaneously into
each guinea pig during the second week post arrival so that each animal could be
identified by using Avid tag readers.
5.2.2 CHT used in the study
CHT used (PSE222) in these studies was supplied by Phytopharm Co. Ltd
UK, as freeze-dried granules prepared from the decoction of the original 10 herbs.
This product is called ZEMAPHYTE™. Img PSE222 (batch no.9451) was quoted
as equivalent to approximately 6 g of the original herbs. For some studies a purified
fraction (Fraction F) was used. The fraction F used in the study was purified by
HPLC from PSE222. Img of fraction F is equivalent to 55.6 mg of PSF222. The
placebo used in the study was composed of Homulus lupulus, Hordeum distichon,
hordeum distichon ustum, baker’s bran, sucrose, Salivia spp. Thymus vulgaris,
Rosmarinus officianalis, Mentha piperita and clove oil. This formulation of placebo
5.2.3 Contact dermatitis in guinea pigs
At appropriate times, not less than 2 weeks or more than 4 weeks after
purchase, guinea pigs were sensitised by epicutaneous application of DNCB (1-
chloro-2,4-dinitrobenzene) (5 mg, 0.01ml of 50% DNCB in acetone) to the ear.
After various times (see specified experiments) the animals were challenged by
application of 0.025ml of a 0.15% DNCB solution in acetone to the shaved skin of
the flank. In all cases animals were challenged with the same volume of acetone
applied to the other flank as a control. Challenge sites were identified by ringing
with felt tip pen.
5.2.4 Measurement of the skin thickness
Before challenge, the thickness of the skin at the test site was measured
using a calibrated caliper gauge. The reactions to DNCB of guinea pigs were then
read after 24 and 48 hours of challenge and the thickness of the skin was recorded.
The thickness in the control side before and after applying acetone was also
recorded. The thickness of the skin fold was recorded in ‘units’ equivalent to
1/1000 inch. “Single” skin thickness was obtained by dividing the reading from the
caliper of the folded skin by 2. The results were presented as increase in skin
thickness. Increase of skin thickness = (Increase of skin thickness at challenge site
- Increase of skin thickness at the control site). Because the thickness of the skin
on both sides of the guinea pigs were the same before challenge or application of
acetone (demonstrated in previous studies), the result can also be presented as:
5.2.5 Control animals used in the study
Control animals were used in parallel with every group of CHT treated
animals. Unless specified in the placebo controlled experiments, all controls used in
the experiments are guinea pigs given normal drinking water only, instead of CHT.
All control animals received the same sensitisation and challenge as received in the
CHT treated guinea pigs.
5.2.6 Treatment of guinea pigs with CHT
Chinese herbs and/or placebo were given at various times before or after
sensitisation for defined periods (see specific experiments). Initially CHT was given
to the guinea pigs dissolved in the drinking water. In a pilot study performed, the
amount of water guinea pigs consumed per day was measured (see results section in
this chapter). The standard dosage of CHT in drink water was adjusted to the
equivalent dosage used in patients (428mg/kg, 150mg/per guinea pig/per day, based
on 350 gram/guinea pig). Dosages suggested by Phytopharm for guinea pigs are
between 150-1000 mg/kg.
Placebo was given in the same dosage. Control groups were given normal
drinking water only. In some experiments groups of guinea pigs were gavaged with
CHT using the same daily dosage as that added in drinking water. In this case the
herbs (150mg) were dissolved in 2 ml of drinking water and gavaged to guinea pigs
everyday for the experimental period. Guinea pigs were restrained by the assistant
during gavage, no anaesthetic agent was applied. Gavaged guinea pigs had free
In some experiments Fraction F was used. This was purified from CHT
PSE222 by HPLC (high pressure liquid chromatography) and was given as a gavage
of 1.5 mls/per guinea pig/per day. This was equivalent to 2.7 mg/per guinea pig/per
day. This amount of ‘F ’ was equivalent to the ‘F ’ contained in 150 mg PSE222, the
dose of full CHT normally given per day.
5.2.6 Statistical method
Mean and standard deviation of scores from the skin thickness of groups of
guinea pigs were presented. Comparison between the thickness obtained in CHT
treated group and control group was analyses using a Students group ‘t ’-test. A
5.3
Results
5.3.1 Consumption of water/CHT-containing water by guinea pigs
In order to determine the concentration of CHT to be used for the guinea
pigs, a pilot study was carried out to determine whether the guinea pigs would drink
CHT-containing water and if so the amount of water or CHT-containing water
which guineas pig consume in a day. CHT was given to 5 guinea pigs in drink
water (750mg/litre) and another 5 guinea pigs were given water only for a period of
2 weeks and the amount of consumption was recorded (Fig 5.3.1)
This experiment showed that there was no significant difference between the
amount of CHT-containing water and water only which guinea pigs drank everyday
(P > 0.05). Subsequent studies of CHT were based on a mean consumption of 200
CHT (ml) 250-1