Herbex-kid (HK) at various doses in C 48/80 (Compound 48/ 80)-induced itching in Balb/C mice. Each bar represents mean of six mice each. ***a P 5 0.001 vs vehicle control; *b P 5 0.05, ***b P 5 0.001 vs C 

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... solution was replaced every 15 min intervals. Dose response curves were obtained for histamine acid phosphate at various concentrations. Later the Tyrode solution was replaced by HK prepared in Tyrode solution containing various concentrations (0.1, 1 and 10% v/v) and the contractile response of the ileum was obtained in the presence of histamine. Contact time between the agonist and the tissue was between 30 and 45 s. Experiments were carried out in triplicate ( n 1⁄4 3). The IC 50 was calculated for histamine, in the presence and absence of HK, at various concentrations (23). Results were expressed as mean Æ SEM. The intergroup variation was compared statistically using One way Analysis of Variance (ANOVA) followed by Dunnett’s multiple comparison test or with Tukey’s multiple comparison test wherever applicable. Statistical signifi- cance was considered at P 5 0.05. The analysis was carried out using GraphPad Prism software V.4. C48/80, a known mast cell degranulating agent, produced a significant ( P 5 0.001) increase in degranulation in rat mesenteric mast cells (92.7 Æ 1.3), when compared with the mesentery exposed to Ringer Locke’s solution alone (18.5 Æ 3.4). The C 48/80 treated mesentry when exposed to HK produced a dose dependent reduction in the number of degranulated mast cells. HK at 1.07 mg ml À 1 showed 74.0 Æ 3.5 number of degranulated mast cells per 100 mast cells counted, while 10.75 and 107.5 mg ml À 1 showed significant reduction ( P 5 0.01) in the number of degranulated mast cells namely, 25.2 Æ 4.6 and 10.7 Æ 3.9, in the presence of 0.8 m g ml À 1 C 48/80. Disodium cromoglycate (1 mg ml À 1 ), a known mast cell-stabilizing agent also brought significant ( P 5 0.01) reduction in degranulated mast cells (Fig. 1). In vehicle treated mice, systemic anaphylaxis was observed within 19.3 Æ 1.5 min. Oral pre-treatment with HK, 1 h before the administration of C 48/80, showed a dose-dependent increase in latency to death time. Only HK at 107.5 mg kg À 1 produced significant increase ( P 5 0.01) in latency to death time, namely, 49.5 Æ 9.6 min when compared with controls (Fig. 2). Fourteen days of treatment with HK to presensitized rats produced dose dependent reduction in the degranulation of mast cell. HK at both 10.75, 107.5 mg kg À 1 produced significant increase ( P 5 0.01) in the number of intact mast cells, namely, 58.9 Æ 3.7 and 68.1 Æ 2.8, when compared with vehicle control upon exposure to 0.5% horse serum, 30.1 Æ 4.4. Prednisolone (10 mg kg À 1 ), used as a positive control also produced significant ( P 5 0.01) increase in intact mast cells, 73.6 Æ 3.5 (Fig. 3). In passive anaphylaxis model, only HK at 107.5 mg kg produced significant ( P 5 0.01) increase in the number of intact mast cells, 64.2 Æ 2.7 when compared with control namely 40.8 Æ 4.5, while at all other doses, HK did not produce significant increase in the number of intact mast cells when compared with vehicle control (Fig. 3). The vehicle treated OVA sensitized mice, produced 17.3 Æ 2.8 number of sneezes and the time spent in nasal rubbing was 245.3 Æ 10.7 s. The onset of sneezing was at 156.5 Æ 48.3 s. Pre-treatment of HK to OVA sensitized Balb/C mice produced dose–dependent reduction in the number of sneezes and time spent in nasal rubbing. At all dose levels, HK produced significant reduction ( P 5 0.01) in time spent in nasal rubbing. HK at 1.07 mg kg À 1 showed 184.3 Æ 16.6 s ( P 5 0.05) of nasal rubbing while 10.75 and 107.5 mg kg À 1 treated animals showed significant reduction ( P 5 0.01) in the time spent in nasal rubbing, namely, 160.5 Æ 10.3 s, 84.7 Æ 15.5 s. Only HK at 107.5 mg kg À 1 showed significant reduction ( P 5 0.01) in number of sneezing, 4.5 Æ 0.8, when compared with control. Moreover, both the standard drug, cetrizine and the herbal formulation, HK, however, did not produce any significant changes in onset of sneezing (Table 1). In vehicle treated mice, intradermal injection of 20 m l of 100 m g of C 48/80 produced significant ( P 5 0.001) increase in the number of itches (181.7 Æ 14.1) and the time spent in itching (267.7 Æ 18.7 s) when compared with the vehicle treated mice injected with 20 m l of normal saline intradermally, namely, 13.0 Æ 6.0 s and 6.2 Æ 3.2 s. Pre-treatment with HK produced dose-dependent reduction in both these parameters. HK at 1.07 mg kg À 1 showed non-significant reduction in the numbers of itches (135.7 Æ 4.4) and significantly ( P 5 0.001) reduced the time spent in itching, namely, 165.3 Æ 4.4 s when compared with C48/80 control. HK at 10.75 and 107.5 mg kg À 1 produced significant reduction ( P 5 0.01) in time spent in itching, namely, 146.2 Æ 8.4 and 140.0 Æ 15.8, and the same dose levels also reduced the number of skin itches, namely, 124.5 Æ 3.6 and 118.0 Æ 20.6, when compared with C 48/80 control. The standard cyproheptadine at 1 mg kg À 1 also showed significant reduction in the number of itches ( P 5 0.05) and time spent in itching ( P 5 0.001) induced by C48/80 (Fig. 4). HK at concentrations of 0.1, 1 and 10% did not have any effect on histamine induced guinea pig ileum contraction (data not shown). Allergies are one of the common disorders affecting mankind. The basic components involved in Type I hypersensitivity allergic reactions are the mast cells and IgE antibodies (10). Presently most of the antiallergic agents are of H 1 -antagonist (24). Recently rupatadine dual histamine (H 1 ) and platelet activated factor (PAF) has been developed (25). It is becoming clear that antihistaminics alone do not provide the full therapeutic value for most of the Type I allergic reaction (26). From the histamine induced guinea pig ileum contraction, we conclude that HK does not possess prominent antihistaminic properties. Degranulation of mast cells, causes release of pre-formed and newly formed mediators leading to acute and late phase allergic reactions depending on the allergic disease (24). From the present study, HK possesses significant mast cell stabilization activity. Nevertheless certain types of allergic reactions like perennial rhinitis, atopic dermatitis and even in allergic asthma, several immune cells along with inflammatory mediators play an important role. By active and passive anaphylaxis model, it has been established that HK possess a certain degree of immunological property, which may be due to the herbs such as O. sanctum or A. chinensis which possess immunomodulatory properties (3,4). Allergic rhinitis is a very common condition, characterized by early and late phase immune response (27). Antihistamines are commonly used for the relief of allergic rhinitis which may be beneficial in relieving sneezing but not other symptoms of allergic rhinitis (26). In the present study, OVA sensitized rat animal model of nasal allergy was used, as this model closely corresponds to the allergic rhinitis in humans, involving both early and late phase of immune response (28). In the early phase, the principal component is mast cell and histamine and is characterized by sneezing, and the late phase is characterized by nasal rubbing, wherein other inflammatory components are involved (21). The OVA model for nasal allergy involves both these phases. HK significantly reduced the time spent in nasal rubbing (late phase) at all the three dose level employed, but only at high dose the number of sneezes (early phase) was reduced significantly. Based on these observations, HK’s involvement with the immunological component along with mast cell stabilization property would be the major feature in attenuating the allergic response without the involvement of histamine receptors. Based on the above fact, C48/80 induced itch model in mice, was, therefore employed, as antihistamines do not provide protection against C48/80-induced itching. Itching behavior is thus independent of histamine though mast cells are degranulated (29). Lipid mediators also do not play important roles and 5-HT seems to participate in induction of itching (30). From the results of this experiment, it was evident that of all the dose level employed, HK produced significant reduction in number and time spent in itching, suggesting that HK has potent antipruritic activity probably mediated by mast cell stabilization along with 5-HT antagonism. On the whole, the antiallergic property of HK could be attributed to the presence of immunomodulatory herbs such as A. chinensis (3), O. sanctum (4), P. longum (5) and antiallergic herbs such as B. monniera (31) and P. longum (32) used in the formulation, which further strengthens the concept of synergistic healing potential of polyherbal formulations (33). In conclusion, the antiallergic property of HK could be through the involvement of mast cell stabilization, immunomodulatory activity and 5-HT antagonism without the involvement of histamine receptors. From the encouraging results obtained from this study, further experiments are being conducted to evaluate the potential of HK in experimental allergic asthmatic model and its immunological ...

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... namely, 146.2 AE 8.4 and 140.0 AE 15.8, and the same dose levels also reduced the number of skin itches, namely, 124.5 AE 3.6 and 118.0 AE 20.6, when compared with C 48/80 control. The standard cyproheptadine at 1 mg kg À1 also showed significant reduction in the number of itches (P50.05) and time spent in itching (P50.001) induced by C48/80 (Fig. ...