An Experimental Study to Evaluate the Anti-inflammatory and  Immunmodulatory Effects of UNIM-352, a Polyherbal Preparation for Bronchial asthma

S. Guhathakurta<sup>1</sup>; K. Gulati<sup>1</sup>; N. Rai<sup>1</sup>; B.D. Banerji<sup>2</sup>; S. Shakir Jamil<sup>3</sup>; A. Ray<sup>1</sup>

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. Banerji²

, S. Shakir Jamil³

, A. Ray¹

1 Department of Pharmacology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India
2 Department of Biochemistry, UCMS & GTB Hospital, Delhi, India
3 Central Council for Research in Unani Medicine, New Delhi, India

Author

Correspondence author
Medicinal Plant Research, 2013, Vol. 3, No. 2 doi: 10.5376/mpr.2013.03.0002
Received: 14 Dec., 2012 Accepted: 21 Dec., 2012 Published: 27 Dec., 2012

This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Preferred citation for this article:

Guhathakurta S., et al., 2012, An experimental study to evaluate the anti-inflammatory and immunmodulatory effects of UNIM-352, a polyherbal preparation for bronchial asthma, Medicinal Plant Research, Vol.3, No.2 3-12 (doi: 10.5376/mpr.2013.03.0002)

Abstract

This study was conducted to assess the possible anti-inflammatory and immuno-modulatory effects of UNIM-352, a polyherbal Unani preparation, which was used for bronchial asthma in traditional system of medicine. In KLH immunized rats, UNIM-352 (200 mg/kg 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 that was used as reference standard for the experiment. 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 indicated that UNIM-352 might have anti-inflammatory and immunomodulatory properties that may be contributing to its beneficial effects in bronchial asthma.

Keywords

Polyherbal Unani preparation; Bronchial asthma; Anti-inflammatory; Immunomodulatory; UNIM-352

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 immu- nemodulation 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 hyper- sensitivity 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 pro- and 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 anti-inflammatory 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 800 mg/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 anti-infmmatory and immunomodulatory effects of the compound with a view to validate its use in bronchial asthma.

1 Results

1.1 Effect of UNIM–352 on cytokine levels

The effects of UNIM-352 (administered orally for 14 day) were evaluated on TNF-, IL-1β and IL-4 levels in blood and BAL fluid in KLH immunized rats. UNIM-352 (200 mg/kg 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 (400 mg/kg) on TNF-α levels in blood was comparable to that of prednisolone, whereas, in BAL fluid, the effects of UNIM-352 (400 mg/kg) were greater in magnitude (46% vs 21%) as compared to the comparator drug, prednisolone. These results are summarized in Figure 1.

Figure 1 Effect of UNIM-352 on TNF- a levels in (A) blood and (B) BAL fluid in KLH immunized rats

Assay for IL-1β showed that at both lower and higher doses of UNIM-352 (200 mg/kg 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.

Figure 2 Effect of UNIM-352 on IL-1β levels in (A) blood and (B) BAL in KLH immunized rats

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 mg/kg 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 (200 mg/kg) of UNIM-352 on IL-4 levels (P<0.05) and higher dose (400 mg/kg) (P<0.01) were significantly more than that of prednisolone in BAL fluid. These results are summarized in Figure 3.

Figure 3 Effect of UNIM-352 on IL-4 levels in (A) blood and (B) BAL in KLH immunized rats

1.2 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 5 mg/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 mg/kg 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<0.01 in 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.

Table 1 Effect of UNIM-352 on acute systemic anaphylaxis in ovalbumin immunized rats

1.3 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 UNIM-352 were even greater than prednisolone (P<0.01) on IgE levels. These results of UNIM-352 on serum IgE levels are summarized in Figure 4.

Figure 4 Effect of UNIM-352 on IgE levels in blood in KLH immunized rats

1.4 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.

Figure 5 Effect of UNIM-352 on DTH levels in KLH immunized and challenged rats

2 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 UNIM-352 were comparable to the corticosteroid, prednisolone (5 mg/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-αhave 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-αmay 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 thatUNIM-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 β-agonists. 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 demon- strated 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 (Kraneveldet 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 (5 mg/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 400 mg/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 comp- lement 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 pharma- cology 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.

3 Materials and Methods

3.1 Animals

Inbred Wistar rats (175~200 g) were used for the study. The animals were maintained under standard laboratory conditions of light-dark cycle (12 h light-12 h 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.

3.2 Assay for markers of inflammation and immunity

3.2.1Cytokine assay

The animals (n=6 per group) were immunized with Keyhole Limpet Hemocyanin (KLH) 1mg in 0.4ml PBS: FCA (1:1) subcutaneously (Institoriset al., 1995). These KLH immunized rats were treated orally for 14 days with UNIM-352 (200 mg/kg 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 immune- osorbent 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.

3.2.2 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 intraperi- toneal injection of ovalbumin (10mg per rat) adsorbed to 10μg of aluminium hydroxide. These rats were treated with UNIM-352 (200 mg/kg or 400 mg/kg) or prednisolone (5 mg/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) (Kwasniewskiet al., 1998). Rats remaining alive after the antigen challenge were counted to record the percentage of mortality due to anaphylactic shock. On the 14^th 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.

3.2.3 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 UNIM-352 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 450 nm and results were expressed in nanogram/mL.

3.3 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 mg/kg 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 plethys- mometer 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 evaluatedafter all treatments (Shanmugamet al., 2007).

3.4 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 mg/kg 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.

3.5 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.

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