Download Anti inflammatory and immunmodulatory effects of

An experimental study to evaluate the anti-inflammatory and immunmodulatory effects
of UNIM-352, a polyherbal preparation for bronchial asthma
S. Guhathakurta1, K. Gulati1, N. Rai1, B.D. Banerji 2, S. Shakir Jamil 3, A. Ray 1*
Abstract
This study was conducted to assess the possible anti-inflammatory and immunomodulatory effects of UNIM-352, a polyherbal Unani preparation which is used for
bronchial asthma in traditional system of medicine. In KLH immunized rats, UNIM-352
(200 and 400 mg/kg, orally for 14 days) attenuated the levels of the proinflammatory
cytokines, TNF-α and IL-1β, and the Th2 cell derived cytokine, IL-4, in blood and BAL
fluids, as compared to vehicle controls. Studies with acute anaphylaxis revealed that
UNIM-352 prevented, antigen challenge induced mast cell degranulation in a dose related
manner and reduced anaphylactic mortality. At both dose levels, UNIM-352 was able to
significantly inhibit the serum IgE levels when compared to vehicle treated control group.
All these effects were comparable to prednisolone which was used as reference standard
for the experiments. In studies for cell mediated immunity, UNIM-352, at the dose level
studied, did not have any appreciable influence on the DTH assay as measured by change
in footpad thickness in immunized and challenged rats. These results indicate that UNIM352 may have anti-inflammatory and immunomodulatory properties which may be
contributing to its beneficial effects in bronchial asthma.
Key words Bronchial asthma; Anti-inflammatory; Immunomodulatory; UNIM-352
1. Background
Bronchial asthma is a chronic inflammatory disorder of the airways characterized by
reversible airway obstruction and airway hyperresponsiveness which involves complex
interactions between cellular and humoral elements. The pathophysiological changes
include infiltration of inflammatory cells into lung tissues, mucus overproduction, the
over expression of cytokines and chemokines. Inflammation and immunomodulation are
central to the disease and one of the aims of pharmacotherapy is to reverse and/or control
both processes. Atopic asthma, in particular, has a strong allergic component and is
characterized by an elevation of allergen specific IgE in the serum, increased numbers of
eosinophils, mast cells and T lymphocytes in the airways (Kim et al. 2002; Kraneveld et
al. 2006). Mast cell mediated hypersensitivity reactions to allergens are involved in the
pathogenesis of such asthma and their activation triggers the process of degranulation and
release of mediators like histamine and an array of inflammatory cytokines. Both proand anti-inflammatory cytokines determine the extent and progress of airway
inflammation and their interactions with modulators like histamine and leukotrienes
determine the severity of the disease (Withers et al., 1998; Mahajan and Mehta, 2006;
Steinke and Borish, 2001). Although corticosteroids are effective as nonspecific antiinflammatory agents, the side effects of corticosteroid treatment are of significant
concern (Akinbami and Schoendorf, 2002; Steurer-Stey et al. 2002).
The need for
effective and safe treatment of this disease is greater than ever. As a result alternative
forms of therapy are being increasingly considered and the use of this complementary
asthma therapy has grown substantially. Medicinal plants are rapidly emerging as
alternative/complimentary sources of pharmacotherapy in a variety of disease states and
their
reported
efficacy
and
safety
in
traditional
use
coupled
with
their
pharmacoeconomical viability has led to intensive research to scientifically validate their
effects (GINA guidelines, 2005; Dahanukar et al. 2000). Further, considering the
problems associated with conventional drug therapy in asthma there is a need to explore
the possible alternative strategies. UNIM-352 is a polyherbal, anti-asthmatic preparation
used in the traditional Indian Unani system of medicine consisting of the following
ingradients: Linum Usitatissimum Linn (Alsi), Trigonella foenum-graecum (methi),
Allium sativum Linn (seer), Apis mellifera Linn (chilbeenj), Honey (Asi), Caesalpinea
bondumello Fleming (Magz-e-Karanjwa) and Pongomia glabra Vent (Magz-e-Karanj).
Preclinical toxicological data showed that UNIM-352 was remarkably safe in tests of
both acute and chronic toxicity up to a dose of 800mg/kg p.o. Recent clinical studies have
also shown that UNIM-352 can reduce the frequency and intensity of asthmatic attacks
using modern evaluation techniques. However, the mechanism of action needs to be
elucidated and hence the present study was designed to evaluate the possible antiinfmmatory and immunomodulatory effects of the compound with a view to validate its
use in bronchial asthma.
2. Results
2.1 Effect of UNIM –352 on cytokine levels
The effects of UNIM-352 (administered orally for 14 day) were evaluated on TNF- α, IL1β and IL-4 levels in blood and BAL fluid in KLH immunized rats. UNIM-352 (200
and 400 mg/kg) reduced TNF- α levels, at both the dose levels, by 28% and 49% in
blood and by 28% and 46% in BAL, respectively. On the other hand, with prednisolone
pretreatment the TNF- α level were suppressed by 33% in blood and 20% in BAL.
Analysis of the data revealed that there were significant effects on the TNF- α levels [F
(3,23) = 8.9, for TNF- α in blood; and F (3,23) = 22.5, for TNF- α in BAL across all the
groups; P< 0.005 in both cases]. Further, inter group comparisons showed that there were
significant suppression of TNF- α levels at both lower and higher doses of UNIM-352
(P<0.05), as well as with prednisolone (P<0.01), when compared to vehicle treated
controls in blood. Similar significant differences were also seen between control and
UNIM-352 and prednisolone treated groups (P<0.01). The effect of higher dose of
UNIM-352 (400mg/kg) on TNF- α levels in blood was comparable to that of
prednisolone, whereas, in BAL fluid, the effects of UNIM-352 (400mg/kg) were greater
in magnitude (46% vs 21%) as compared to the comparator drug, prednisolone. These
results are summarized in Figure 1.
Assay for IL-1β showed that at both lower and higher doses of UNIM-352 (200 and 400
mg/kg) there were suppressions of IL-1β by 52% and 61% in blood and by 45% and 46%
in BAL respectively, as compared to controls, whereas, prednisolone induced
suppressions by 55% in blood and 40% in BAL. Analysis of the data revealed that
various drug treatments had significant effects on the cytokine IL-1β levels [F (3,23)
=18.8, for blood; and F (3,23) = 51.7, for BAL; P<0.001 in each case]. Further, inter
group comparisons revealed that the IL-1β levels were decreased significantly at both
lower and higher doses of UNIM-352 and prednisolone as compared to controls in blood
(P<0.01 in all cases). Similar reductions in IL-1β levels were seen in BAL fluid samples
after both doses of UNIM-352 and these were comparable with those of prednisolone
(P<0.01 in all cases). These results are summarized in Figure 2.
Results with IL-4 showed that at both lower and higher doses of UNIM-352 (200 and 400
mg/kg) suppressions in IL-4 levels were of 61% and 64% in blood and by 57% and 69%
in BAL fluid, respectively. On the other hand, the prednisolone treated group showed
suppression by 60% in blood and 41% in BAL, respectively. Analysis of the data
revealed that there were significant effects on the IL-4 levels [F (3,23) = 9.6, for IL-4 in
blood; and F (3,23) = 36.7, for IL-4 in BAL; P<0.001 in each case] in KLH immunized
rats by various drug treatments. Intergroup comparisons revealed that the IL-4 levels
were markedly decreased at both lower and higher dose of UNIM -352 (200 and 400
mg/kg), and prednisolone in blood (P<0.01 vs control), and similar dose related effects
were also seen in BAL fluid samples. The effects of lower dose (200mg/kg) of UNIM352 on IL- 4 levels (P<0.05) and higher dose (400mg/kg) (P<0.01) were significantly
more than that of prednisolone in BAL fluid. These results are summarized in Figure 3.
2.2 Effect of UNIM –352 on acute systemic anaphylaxis
In this experiment, the effects of UNIM-352 were assessed on acute systemic anaphylaxis
in ovalbumin immunized rats. The effect of the polyherbal agent was studied on
anaphylactic mortality and mast cell stabilization activity. Accordingly, the total number
of mast cells present in the rat mesentery after ovalbumin sensitization and challenge i.e.
the percentage of intact and degranulated mast cells (with extruded granules) were
counted following the antigen challenge. The vehicle treated control group of sensitized
animals when challenged with the antigen OVA expressed extensive degranulation upto
80%. Prednisolone, which was used as reference standard/ comparator or positive control
at 5mg/kg oral, was found to reduce degranulated mast cells to an extent of 24%.
Treatment with UNIM-352 at 200 and 400 mg/kg orally was also able to significantly
inhibit the mast cell degranulation i.e. to 35% and 30% respectively. Further, rats
surviving the antigen challenge were counted to record the percentage of mortality due to
anaphylactic shock and it was observed that there was no mortality (0%) in the groups
treated with both dose levels of UNIM–352 (200 or 400 mg/kg) and prednisolone (5
mg/kg), when compared to the 50% mortality seen in the vehicle treated control group.
Analysis of the data revealed that there were significant effects on the rate of mortality,
and intact/degranulated mast cells [F (3,39) = 317.4, P<0.005 for intact/degranulated
mast cells; and P<0.005 for mortality] in antigen sensitized and treated rats. Intergroup
comparisons showed significant effects of UNIM-352 on intact and degranulated mast
cells. For degranulated mast cells, the inter group comparisons were done with each of
the two doses of UNIM – 352 (200 and 400 mg/kg) (P<0.01in each case) and
prednisolone (P<0.01) as compared to vehicle treated controls. Similar significant
differences in the intact mast cells were seen after each of the two doses of UNIM-352
(P<0.01) and with prednisolone (P<0.01), as compared to vehicle treated control. Thus,
UNIM-352 treatment was comparable to prednisolone in inhibiting the mortality and
mast cell degranulation in antigen sensitized rats. The results of UNIM-352 on
parameters of acute systemic anaphylaxis are summarized in Table 1.
2.3 Effect of UNIM –352 on IgE levels
In this experiment, the effects of UNIM-352 were assessed on serum IgE levels in KLH
immunized rats. Results showed that UNIM-352 at both its dose levels (200 and 400
mg/kg oral) markedly reduced the serum IgE levels (as measured by ELISA method)
which is considered responsible for mediating the Type I hypersensitivity reaction seen
during acute systemic anaphylaxis, when compared to vehicle treated control group .
Percentage analysis of results revealed that UNIM-352 reduced the serum IgE levels at
both its lower and higher dose levels by 38% and 51% respectively. Interestingly,
prednisolone, which was used as a comparator drug/positive control for the experiment,
suppressed the IgE levels by 23%. Overall analysis of the data by ANOVA revealed that
there were significant effects on the IgE levels [F (3,23) = 86.2, P<0.001 for IgE], across
all groups. Intergroup comparisons revealed significant reductions of the IgE levels with
each dose of UNIM-352 and prednisolone as compared to vehicle treated control
(P<0.01, in each case). The reductions in IgE levels were more marked with the higher
dose (400mg/kg oral) of UNIM-352. The analysis of data showed that effects of UNIM352 were even greater than prednisolone (P<0.01) on IgE levels. These results of UNIM352 on serum IgE levels are summarized in Figure 4.
2.4 Effect of UNIM –352 on delayed type hypersensitivity (DTH) response
In this experiment, the effects of UNIM-352 (200 and 400 mg/kg) given orally for 14
days, were assessed on cell mediated immune responses as measured by the delayed type
hypersensitivity (DTH) response by using the footpad thickness test. KLH immunized
rats were challenged by antigen into the right paw whereas the left paw received saline.
The differences in paw volume were noted at 24h post challenge. The results showed that
UNIM-352 did not have any appreciable influence on the DTH response in this model.
Percentage analysis showed 10% and 26% enhancements of DTH response respectively
at both the dose levels of UNIM-352. Analysis of the data revealed no significant effects
[F (3,23) =1.2, P > 0.05, for DTH response] across the various groups tested. Further,
intergroup comparisons also did not reveal any appreciable differences between the
groups on paw edema, i.e. neither UNIM-352 (200 or 400 mg/kg) nor prednisolone were
able to influence the DTH response by any significant extent (P>0.05). The results of
UNIM-352 on DTH assay parameters are summarized in Figure 5.
3. Discussion
The chronic inflammatory process of asthma is associated with infiltration of
inflammatory cells into lung tissues, mucus overproduction, and the overexpression of
cytokines and chemokines. T-lymphocytes play a very important role in coordinating the
inflammatory response in asthma through the release of specific patterns of cytokines
which result in the recruitment of eosinophils and maintenance of mast cells in the
airways. These lymphocytes, present in increased number in the airways, further
orchestrate eosinophilic inflammation and IgE production by B lymphocytes. Studies in
animal models also indicate that T-cell derived cytokine production, rather than
eosinophil influx or IgE synthesis, is causally related to altered airway behavior
(Mojtabavi et al., 2002). IL-1, TNF-α and IL-4 are key cytokines which amplify the
inflammatory response in asthma. TNF-α is produced in increased amounts in asthmatic
airways and increased expression can further enhance the inflammatory process, which
can be linked to disease severity. Elevated levels of TNF-α have also been detected in
sputum, bronchoalveolar lavage fluid and biopsy samples from asthmatics. TNF-α is
released in allergic responses from both mast cells and macrophages via IgE-dependent
mechanisms, and elevated levels have been demonstrated in the bronchoalveolar lavage
(BAL) fluid of asthmatic subjects undergoing allergen challenge. Inhaled TNF-α
increases airway responsiveness to methacholine in normal and asthmatic subjects
associated with a sputum neutrophilia. Additional data indicate that TNF-α can
upregulate adhesion molecules, facilitate the immigration of inflammatory cells into the
airway wall and activate pro-fibrotic mechanisms in the sub epithelium. These data
further suggest that TNF-α plays a role in the initiation of allergic asthmatic airway
inflammation and the generation of airway hyper-reactivity (Thomas, 2001). The results
of our experiments show that treatment with the polyherbal preparation, UNIM-352, for
14 days, at both its dose levels (200 and 400 mg/kg oral), was effective in suppressing the
level of this cytokine in both blood and BAL fluid of KLH immunized rats. The
reductions in cytokine levels were more consistent in BAL fluid as compared to blood –
thus indicating the relative airway specificity of this drug effect. These effects of UNIM352 were comparable to the corticosteroid, prednisolone (5mg/kg oral, for 14 days),
which was used as a comparator drug, in a separate group of KLH immunized rats. The
data with TNF-α, in particular, is of great interest as this pleotropic cytokine has also
been implicated in a wide variety of inflammatory disorders and strategies to counteract
this cytokine by using drugs/biologics have been extremely successful. Drugs targeting
TNF-α have been developed to neutralize the deleterious effects of this inflammatory
cytokine and have proved to be safe and effective in the treatment of patients with
Rheumatoid Arthritis, Crohn’s Disease and Psoriasis, refractory to conventional
treatments. Biological therapies blocking TNF-α are likely to constitute a considerable
advance in the management of difficult cases of asthma that are particularly resistant to
typical treatment modalities (Russo and Polosa, 2005). Thus, drugs that have the ability
to neutralize the deleterious effects of TNF-α may also be useful in the management of
chronic severe asthma.
Our findings indicate that UNIM-352 also induced similar attenuating effects on IL-1β
levels in the blood and BAL, in KLH immunized rats. The pro-inflammatory properties
of IL-1β are known and IL-1β expression is increased in the airways of asthmatic
individuals and activates many inflammatory genes that are expressed in asthma. IL-1
receptor antagonist (IL-1Ra) administration, reduces airway hyper responsiveness
induced by allergens in mice, but human recombinant IL-1Ra (anakinra) is not effective
in the treatment of asthma. IL-1β also markedly activates macrophages from patients with
obstructive diseases like COPD to secrete inflammatory cytokines, chemokines, and
MMP9, and studies investigating the efficacy of an antibody that blocks IL-1β in patients
with COPD are currently in progress (Barnes, 2008).
Inflammation and immunity are very closely related processes with reference to the
respiratory tract, and recruitment of persistent inflammatory cells occurs due to the
release of TH-2 mediated cytokines (Kim et al., 2006). The Th2 derived cytokine, IL-4
plays an important pro-inflammatory function in asthma which includes induction of the
IgE isotype switch, expression of the vascular cell adhesion molecule-1 (VCAM-1),
promotion of eosinophil transmigration across endothelium, mucus secretion, and
differentiation of Th 2 lymphocytes leading to cytokine release (Mahajan and Mehta,
2006; Steinke and Borish, 2001; Moser and Fehr, 2002). In our study, UNIM-352
reduced levels of IL-4 in both blood and BAL fluid further suggesting that UNIM-352
may have both immunomodulatory and anti-inflammatory role in preventing airway
inflammation by significantly reducing the levels of the Th2 derived cytokine (IL-4) as
well the pro-inflammatory markers (TNF-α and IL-1β) - all of which play key roles in the
pathophysiology of allergic asthma. It is also interesting to note that these changes
induced by the polyherbal agent were comparable to that of prednisolone (the reference
drug) in the markers of inflammation and immunity in our experiments.
The mainstay of asthma therapy is inhaled corticosteroids, and the majority of symptoms
are controlled with inhaled corticosteroids alone or in combination with long-acting βagonists. However, 5% to 10% of the asthmatic population has severe refractory disease
that is resistant or poorly responsive to inhaled corticosteroid therapy. The observation of
reduced pro-inflammatory cytokines, TNF- α following UNIM-352, could be of
immense importance as reports have suggested that it plays an important role in
refractory asthma. Preliminary studies have demonstrated an improvement in asthma
quality of life, lung function, and airway hyper responsiveness and a reduction in
exacerbation frequency in patients treated with anti–TNF-α therapy (Brightling et al.,
2008).
Basophils and mast cells play a pivotal role in the pathogenesis of allergic disorders as
they release potent vasoactive and bronchoconstrictor agents which modulate local
immune responses and inflammatory cell infiltration (Kraneveld et al., 2002). The IgG
mediated formation of antigen-antibody complexes on the surface of mast cells and
basophils results in the release of inflammatory mediators of anaphylaxis such as
histamine, leukotrienes and prostaglandins (Dahanukar et al., 2002; Postma, 1995;
Shanmugam et al., 2007; Agarwal et al., 1999). The effect of UNIM-352 on the
degranulation of peritoneal mast cells in sensitized animals was studied and the results
showed that UNIM-352 significantly inhibited antigen challenge induced degranulation
of mast cells in a dose related manner and also protected the rats against mortality. These
effects of UNIM-352 were comparable to the standard anti-inflammatory (and also mast
cell stabilizing) drug prednisolone treatment (5mg/kg oral for 14 days). Our present study
highlights the protective effect of UNIM-352 on mast cell degranulation. It also suggests
that the polyherbal Unani preparation may ameliorate manifestations of anaphylaxis by
inhibiting mediator release from mast cells, and/or inhibiting IgE antibody production,
which is responsible for degranulation of mast cells.
Atopic or allergic asthma is characterized by the presence of IgE antibodies against
environmental allergens. An estimated two-thirds of asthma is allergic and greater than
50% of severe asthma has an allergic component. IgE production represents a feature of
the specific immune response orchestrated by the Th2 subset of CD4+ T cells. Increased
immunoglobulin (IgE) production in response to environmental allergens (atopy) is the
strongest detectable predisposing factor for the development of bronchial asthma. In our
study, UNIM-352 (200 and 400mg/kg) was effective in inhibiting the serum IgE levels
which was comparable to that of prednisolone. It is well known that the release of
mediators triggers the bronchoconstriction in allergic asthma and drugs which prevent
such degranulation induced mediator release from mast cells are effective prophylactic
agents in bronchial asthma. Since there is an association between increased IgE
production and development of allergic asthma, our present results with UNIM-352 on
IgE levels suggests that this polyherbal may also be producing its beneficial effect by
inhibition of IgE synthesis.
Cell mediated immune responses are implicated in bronchial asthma (Popa, 2002). In the
present experimental study the effects of UNIM-352 were evaluated in DTH model of
CMI immune response and results showed that it was not much influenced by either of
dose level, as no appreciable change in the paw volumes of rats were observed as
compared to control values. In view of this it appears that UNIM-352 does not have any
appreciable influence on the CMI response in this model.
In summary, the present study suggests that UNIM-352 may have considerable potential
as a drug for the treatment of allergic bronchial asthma. The results indicate that this
polyherbal agent may have a multi-target approach in inducing anti-inflammatory and
immunomodulatory effects which may complement each other to prevent and reverse the
changes associated with this obstructive airway disease. The study is of translational
value and especially relevant from the point of view that UNIM-352 has been used
extensively in traditional system of medicine (Unani) with beneficial effects. Our pilot
clinical trials with this compound have also indicated its possible use as an adjunct along
with conventional therapy in bronchial asthma (Gulati et al., 2010). The present
experimental study, by using the reverse pharmacology approach, has attempted to
validate the clinically observed effects by using modern methodology. Such integration
of traditional and modern medicinal concepts is of great relevance in today’s scenario for
promotion of interactions between the two medicinal systems in the interest of rational
drug development.
4. Materials and Methods
4.1 Animals
Inbred Wistar rats (175-200g) were used for the study. The animals were maintained
under standard laboratory conditions of light-dark cycle (12h light-12h dark) and
temperature (22 ± 2°C) and had free access to food and water. The animal care was as per
guidelines laid down by the Indian National Science Academy, New Delhi and the study
protocol was approved by the Institutional Animal Ethical Committee .
4.2 Assay for markers of inflammation and immunity
4.2.1Cytokine assay
The animals (n=6 per group) were immunized with Keyhole Limpet Hemocyanin (KLH)
1mg in 0.4ml PBS: FCA (1:1) subcutaneously (Institoris et al.,1995). These KLH
immunized rats were treated orally for 14 days with UNIM-352 (200 or 400 mg/kg)
whereas prednisonone (5 mg/kg) was given to a comparator group of rats. The animals
were then challenged with KLH and 24h later blood and BAL fluid samples were
collected by using standard experimental techniques. These blood and BAL samples were
processed and analyzed biochemically for estimation of levels of IgE and cytokines like
TNF- α, IL-1β, and IL-4 by ELISA using commercially available kits (Diaclone France).
Briefly, the cytokine assays were performed by using the solid phase sandwich enzyme
linked immunosorbent assay (ELISA). A polyclonal antibody specific for the cytokine in
question was coated on to the wells of the microtitre strips provided. Antigen and
biotinylated polyclonal antibody specific for the cytokine TNF- α, IL-1β, or IL-4 were
simultaneously incubated at room temperature for specified periods.
Then HRP
conjugated streptoavidin was added and incubated for 30min and then reacted with
chromogen substrate. A colored product was formed and the reaction was stopped. The
absorbance of the colored product was measured using the software based microplate
reader (ECIL) at 450 nm and results are expressed in picogram/ml.
4.2.2 Acute systemic anaphylaxis
The effects of UNIM-352 (200 or 400 mg/kg) was studied on mast cell degranulation in
albino rats of either sex which were divided into four groups (n=10 per group). Rats were
sensitized with an intraperitoneal injection of ovalbumin (10mg per rat) adsorbed to 10μg
of aluminium hydroxide. These rats were treated with UNIM-352 (200 or 400 mg/kg) or
prednisolone (5mg/kg) orally for 14 days. Fourteen days after the sensitization, the
animals were challenged with an intraperitoneal (i.p) injection of ovalbumin (1mg in 0.5
ml of isotonic saline) (Kwasniewski et al., 1998). Rats remaining alive after the antigen
challenge were counted to record the percentage of mortality due to anaphylactic shock.
On the 14th day after antigenic challenge, the mesentery was dissected away from the
small intestine and fragments of mesentery were fixed and stained with 0.2% toluidine
blue. Mesentery fragments were then mounted on a glass slide, care being taken not to
fold or stretch the tissue to assess mast cell degranulation by counting the % of cells with
extruded granules as well as those with intact cell membranes.
4.2.3 Serum IgE estimation
The animals were immunized with KLH (Okada et al., 2001) emulsified in Freund’s
Complete Adjuvant. After 14 days the animals were challenged with the antigen and the
blood samples were collected from the different treatment groups which received UNIM352 and prednisolone, in KLH immunized rats, and the samples were processed as per
standard methodology for the assay of the immunoglobulin (IgE) using commercially
available assay kit (Shibayagi Co., Ltd, Japan). The IgE assay was performed by enzyme
linked immunosorbent assay (ELISA) using the sandwich method. Briefly the samples
and biotin conjugated anti IgE antibody were incubated in monoclonal antibody coated
wells. After 2 hours of incubation HRP conjugated avidin was added and incubated for 1
hour. The HRP conjugated avidin was reacted with a chromogenic substrate reagent and
reaction was stopped. The absorbance of yellow product was measured using the
software based microplate reader (ECIL, India) at 450nm and results were expressed in
nanogram/ml.
4.3 Delayed Type Hypersensitivity (DTH) assay
The delayed type hypersensitivity (CMI) response was measured in pre immunized KLH
challenged rats for the footpad thickness test after prednisolone and UNIM-352 (200 and
400 mg/kg) treatments orally. After fourteen days of immunization, the animals were
challenged with KLH on the right hind foot pad. The contralateral paw received equal
volumes of saline. The thickness of the footpad was measured at 24 hr after the challenge
using a plethysmometer and data acquisition system (Ugo Basile, Italy). The difference in
the thickness of the right hind paw and the left hind paw was used as a measure of DTH
and the percentage response was evaluated after all treatments (Shanmugam et al., 2007).
4.4 Drugs and chemicals
UNIM-352 was a polyherbal, semisolid, preparation with the following ingredients:
Linum usitattissimum Linn (Alsi), Trigonella foenumgraecum (Methi), Allium sativum
Linn (Seer), Apis mellifera Linn (Chilbeenj), Honey (Asi), Caesalpinea Bondumello
Fleming (Magze-e-Karanjwa) and Pongomia glabra Vent (Magz-e-Karanj). The
standardized drug was provided by the Central Council for Research in Unani Medicine
(CCRUM), New Delhi, at the desired concentration and consistency, for purpose of
treatments to the animals for the various experiments planned for the study. UNIM-352
was dissolved in distilled water and administered orally to the rats at doses of 200 and
400 mg/kg. Ovalbumin, Freund`s Adjuvant, Keyhole Limpet Hemocyanin (KLH) and
prednisolone were procured from Sigma-Aldrich (USA). The ELISA assay kits for
cytokines (TNF- α, IL-1β, IL-4) were procured from Diaclone (France), whereas assay kit
for IgE was procured from Shibayagi, Japan. All other routine and standard laboratory
reagents were procured from Sisco Research Labs, New Delhi.
4.5 Statistical Analysis
The data was analyzed by using the Graph Pad PRISM (Version 4) statistical package.
The results are expressed as mean ± SEM. Data were analyzed using the one way
analysis of variance (ANOVA) followed by post hoc Newman-Keul’s Multiple
Comparison Test. Chi square test was used wherever appropriate. A p value of at least
0.05 was considered as the level of significance in all statistical tests.
Author’s contributions
S. Guhathakurta: acquisition and analysis of data, drafting of manuscript
K. Gulati : conception and design, analysis and interpretation of data, drafting and
critically revising the manuscript for intellectual content
N. Rai : acquisition and analysis of data
B.D. Banerji: conception and design
S. Shakir Jamil: conception and design
A. Ray : conception and design, analysis and interpretation of data, drafting and critically
revising the manuscript for intellectual content, final approval of version to be published
Acknowledgements
The financial support from Dept. of AYUSH, Ministry of Health and Family Welfare
(Govt. of India) is gratefully acknowledged.