Advances in Biological Research 5 (6): 304-308, 2011
ISSN 1992-0067
© IDOSI Publications, 2011
Evaluation of Mast Cell Stabilizing and
Anti-Anaphylactic Activity of Polyherbal Formulation
1
1
P.V. Gohil and 2A.A. Mehta
Department of Pharmacology, K.B. Institute of Pharmaceutical Education and Research,
Gandhinagar, Gujarat, India
2
Department of Pharmacology, L.M. College of Pharmacy, Ahmedabad, Gujarat, India
Abstract: Novel polyherbal formulation (PHE) containing mainly the ethanolic extract of Adhatoda vasica Nees.
(leaf), Clerodendrum serratum Linn. (root), Curcuma longa Linn. (rhizome), Solanum xanthocarpum Schrad
& Wendl. (fruit) and Piper longum Linn. (fruit). HPTLC analysis of the herbal extracts of PHE revealed presence
of 0.25%w/w vasicine, 3.4%w/w piperine, 1.56%w/w curcumin and 0.04%w/w solasodine. The mast cell
stabilizing and anti-anaphylactic property of PHE was investigated against compound 48/80-induced mast cell
degranulation as well as triple antigen-induced anaphylaxis in rats. PHE produced significant reduction in the
mortality of rats subjected to triple antigen-induced anaphylactical shock. PHE also depicted marked protection
of rat mesenteric mast cells from disruption by compound 48/80 in dose dependant manner. Our data suggest
anti-anaphylactic and mast cell stabilizing property of PHE.
Key words: Mast cells
Anaphylaxis
Immunoglobulin E
Triple antigen
Compound 48/80
allergic
disorders. PHE is one such polyherbal
formulation containing mainly the extract of
Adhatoda vasica Nees. (leaf), Clerodendrum serratum
Linn. (root), Curcuma longa Linn. (rhizome), Solanum
xanthocarpum Schrad & Wendl. (fruit) and Piper longum
Linn. (fruit). Adhatoda vasica Nees. is documented
for its potent anti-inflammatory [5], antioxidant [6],
anti-allergic, antitussive [7], anti-asthmatic [8],
bronchodilatory and smooth muscle relaxant activity
[9]. Clerodendrum serratum Linn. traditionally used for
the ailments such as asthma, bronchitis, body ache,
rheumatism, inflammation, ulcers, wounds, fever, cholera,
dropsy, tuberculosis, ophthalmia etc. [10]. Solanum
xanthocarpum Schrad & Wendl. is considered to be an
antispasmodic, diaphoretic, sedative, anodyne, antidotal,
antiseptic, antitussive, carminative, expectorant, febrifuge,
pectoral, preventative (cold) and tonic [11]. Curcuma
longa Linn. has been used for the treatment of various
diseases and disorders particularly for urticaria, skin
allergy, viral hepatitis, inflammatory conditions of joints,
sore throat and wounds [12]. Piper longum Linn. is
useful in asthma, tumors, spleen disorders, inflammations,
piles, tuberculosis etc. [13, 14]. Moreover, PHE is
reported to have anti-inflammatory and bronchodilatory
activity [15].
INTRODUCTION
Allergy is one of the common diseases that affect
mankind. The prevalence of allergy and asthma has risen
in recent years despite the general health improvement in
the population [1]. Intensive research during last several
decades has highlighted the role of lymphocytes,
immunoglobulin, mast cells and various autacoids in the
pathogenesis of asthma and other allergic conditions but
the fundamental immunologic components of
immediate hypersensitivity are the mast cells and
immunoglobulin E (IgE). Mast cells, the constituents of
virtually all organs and tissue are important mediators
of inflammatory responses, such as allergy and
anaphylaxis. Anaphylaxis is mediated by histamine
released in response to cross-linking of IgE bound to
FC RI on mast cells. Mast cell activation causes
process of degranulation that result in releasing of
mediators, such as histamine and an array of inflammatory
cytokines [2, 3]. The available treatment options for upper
and lower respiratory tract allergic diseases have major
limitations due to low efficacy, associated adverse events
and compliance issues [4]. Ayurveda, an Indian system of
medicine, has described several drugs from indigenous
plant sources in the treatment of bronchial asthma and
Corresponding Author: Anita A. Mehta, Department of Pharmacology,
L.M. College of Pharmacy, Ahmedabad-380 009, Gujarat, India.
Tel: +91-79-6302746, E-mails: dranitalmcp@rediffmail.com & dranitalmcp@gmail.com.
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Advan. Biol. Res., 5 (6): 304-308, 2011
In the present study, the effect of PHE, a polyherbal
formulation was studied against Compound 48/80-induced
mast cell degranulation as well as triple antigen-induced
anaphylaxis in rats.
Animal Ethical Committee as per the guidance of the
Committee for the Purpose of Control and Supervision
of Experiments on Animals (CPCSEA), Ministry of
Social Justice and Empowerment, Government of India.
Triple Antigen-Induced Anaphylaxis in Rats [16]:
Twelve Wistar albino rats of either sex weighing between
175-200 g were used for the study. All the rats were
sensitized by single subcutaneous injection of 0.5 ml
horse serum along with 0.5 ml of triple antigen containing
20 million Bordetella pertusis organisms. After
sensitization, the rats were divided into two groups of 6
animals in each. Rats of group-I received vehicle and
served as control. Rats of group-II received PHE
(200 mg/kg, per oral route) once a day for 10 days. On day
10, two hrs after treatment, the rats were challenged with
intravenous injection of 0.25 ml horse serum. The rats
were observed for: onset of symptoms such as dyspnoea
and cyanosis; duration of persistence of symptoms (min.);
death.
The severity score with respect to symptoms were
recorded using the method of Gupta et al. [12]: increased
respiratory rate - 2, dyspnoea for 10 min. - 4, dyspnoea
and cyanosis for 10 min. - 8 and collapse.
MATERIAL AND METHODS
Drugs and Chemicals: Horse serum and triple antigen
were procured from Animal Disease Investigation Office,
Mehsana, India and GlaxoSmithKline, Brentford, UK,
respectively. Compound 48/80 was procured from Sigma,
St. Louis, MO, USA and toluidine blue from SD Fine
Chemicals, Mumbai, India. Tween-80, acetone and xylene
obtained from Burgoyne Burbidges & Co. Mumbai, India.
Kitotifen fumarate was procured from Torrent Research
Centre, Ahmedabad, India.
Test Material: The plant materials (leaves of Adhatoda
vasica Nees. roots of Clerodendrum serratum Linn.
rhizomes of Curcuma longa Linn. fruits of Solanum
xanthocarpum Schrad & Wendl. and fruits of Piper
longum Linn.) were obtained from Government Ayurvedic
Udhyan, Gandhinagar, Gujarat, India. They were identified
and authenticated by the Department of Pharmacognosy,
K.B. Institute of Pharmaceutical and Education Research,
Gandhinagar, India. Voucher specimens (PH/508/002,
PH/508/003, PH/508/004, PH/508/005and PH/508/006) were
deposited at the Department of Pharmacognosy, K.B.
Institute of Pharmaceutical and Education Research,
Gandhinagar, India. The individual plants were evaluated
with regard to their standard specifications according to
the ‘Ayurvedic Pharmacopoeia of India’. Ethanolic
extracts were prepared by extracting each plant with
ethanol using Soxhlet extractor and each extract was
standardized to 0.25%w/w vasicine, 3.4%w/w piperine,
1.56%w/w curcumin and 0.04%w/w solasodine by HPTLC
method. PHE was prepared using ethanolic extract of
Adhatoda vasica Nees. (leaves), Clerodendrum serratum
Linn. (roots), Curcuma longa Linn. (rhizomes), Solanum
xanthocarpum Schrad & Wendl. (fruits) and Piper
longum Linn. (fruits) in proportion of 40%, 30%, 10%, 10%
and 10%, respectively [15].
Studies on Compound 48/80 Induced Rat Mesentric Mast
Cell Degranulation [17]: The animals were sacrificed and
the pieces of mesentry were collected in petri dish
containing Ringer Locke solution and then subjected to
the following treatment schedules.
Petri dish no. 1 - Ringer Locke solution (Positive
control)
Petri dish no. 2 - 0.1ml of Ketotifen fumarate (10
µg/ml)
Petri dish no. 3 - 0.1ml of test agent in Tween-80
(PHE, 500 µg/ml)
Petri dish no. 4 - 0.1ml of test agent in Tween-80
(PHE, 750 µg/ml)
Petri dish no. 5 - 0.1ml of test agent in Tween-80
(PHE, 1000 µg/ml)
Each petri dish was incubated for 15 min at 37°C.
Later Compound 48/80 (0.1 ml, 10 µg/ml) was added to
each petri dish and again incubated for 10 min. at 37°C.
After that, all pieces were transferred to 4% formaldehyde
solution containing 0.1% toluidine blue and kept a side for
20 to 25 min. After staining and fixation of mast cells,
mesentry pieces were transferred through acetone and
xylene two times and mounted on slides. All the pieces
were examined under the high power of light microscope.
Percent protection of the mast cells in the control group
Animals: Wistar albino rats (Zydus Cadila Limited,
Ahmedabad, India) of either sex were selected for the
study. All animals were housed at ambient temperature
(22±1°C), relative humidity (55±5%) and 12h/12h light
dark cycle. Animals had free access to standard pellet
diet and water given ad libitum. The protocol of the
experiment was approved by the Institutional (K.B.
Institute of Pharmaceutical Education and Research)
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Advan. Biol. Res., 5 (6): 304-308, 2011
and the treated groups were calculated by counting the
number of degranulated mast cells from total of at least
100 mast cells counted. Percent inhibition of mast cell
degranulation for each treatment was calculated by
following formula:
animals. PHE protected the sensitized rats against
anaphylactic shock. In control rats, intravenous antigen
challenge (horse serum) caused shock in 84% of the
animals characterised by symptoms of dyspnoea,
cyanosis and collapse, while in PHE (200 mg/kg) treated
rats, the onset of symptoms were delayed and symptoms
were less severe (p<0.001) with reduced mortality
(Table 1) .
1 - Number of
degranulated
mast
cell
× 100
% inhibition of MCD =
Total number of mast cells
Studies on Compound 48/80 Induced Rat Mesentric
Mast Cell
Degranulation:
Antigen challenge
resulted in significant degranulation of the mast cells.
Compound 48/80 (10 µg/ml), a known mast cell
degranulating agent, produced significant rat mesentric
mast cell degranulation (80.17±1.58). Prior exposure to
PHE produced significant (p<0.001) reduction in the
Compound 48/80-induced mast cell degranulation in dose
dependant manner (Figure 1). The % inhibition of
MCD was found to be 54.67%, 58.83% and 66.83% with
500, 750 and 1000 µg/ml of PHE, respectively (Table 2).
Kitotifen fumarate, a known mast cell stabilizing agent,
also brought significant (p<0.001) reduction in
degranulating mast cells.
Statistical Analysis: The results were expressed as mean
± standard error of mean. The significance was evaluated
by U npaired student ‘t test’ and One way
ANOVA,followed by Tukey’s multiple comparison test.
p<0.05 was considered statistically significant.
RESULTS
% mast cell degranulated
Triple Antigen-Induced Anaphylaxis in Rats: When
serum containing antigen was administered into sensitized
rats, it caused anaphylactic shock and death of the
Fig. 1:
90
80
70
60
50
40
30
20
10
0
Comp 48/80 induced mast cell degranulation
*
*
*
*
Comp 48/80
(10µg/ml)
Kitotifen
(10µg/ml)
PHE
(500µg/ml)
PHE
(750µg/ml)
PHE
(1000µg/ml)
Effect of PHE on Compound 48/80 induced rat mesentric mast cell degranulation
Each bar represents mean ± SEM (n= 6)
*p<0.001 as compare to positive control group (One way ANOVA followed by Tukey’s multiple range test)
Table 1: Effect of PHE on triple antigen induced anaphylaxis in pre-sensitized rats
Symptoms
----------------------------------------------------------------------------------------Treatment groups
Dose(p.o.)
Onset (min)
Duration (min)
Severity (score)
Control
3.5±0.5
32±1.83
10.67±0.85
PHE
200 mg/kg
4.83±0.83
10±1.29*
3.33±0.99*
Results are expressed in mean ± SEM (n=6).
*p<0.001 as compared to control group (Unpaired student ‘t test’).
Table 2: Effect of PHE on Compound 48/80 induced rat mesentric mast cell degranulation
Treatment groups
Concentration (µg/ml)
Positive control
Kitotifen
10
PHE
500
PHE
750
PHE
1000
Results are expressed in mean ± SEM (n=6).
*p<0.001 as compared to Positive control group (One way ANOVA followed by Tukey’s multiple range test).
306
Mortality(%)
83
17
% inhibition of degranulation
19.83±1.58
70.67±3.30*
54.67±2.18*
58.83±3.32*
66.83±2.19*
Advan. Biol. Res., 5 (6): 304-308, 2011
DISCUSSION
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Allergic manifestations include allergic rhinitis,
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