Indian Journal of Pharmacology Medknow Publications on behalf of Indian Pharmacological Society ISSN: 0253-7613 EISSN: 1998-3751 Vol. 37, Num. 5, 2005, pp. 300-303

Indian Journal of Pharmacology, Vol. 37, No. 5, September-October, 2005, pp. 300-303

Research Paper

Antihistaminic and antianaphylactic activity of HK-07, a herbal formulation

Gopumadhavan S, Rafiq Mohamed, Venkataranganna MV, Mitra SK

R&D Center, The Himalaya Drug Company, Bangalore-562 123
Correspondence Address:R&D Center, The Himalaya Drug Company, Bangalore-562 123 Email: research@himalayahealthcare.com

Date of Submission: 26-Mar-2005
Date of Decision: 11-Jul-2005
Date of Acceptance: 13-Jul-2005

Code Number: ph05078

Abstract

Keywords: Hypersensitivity, IgE, mast cell degranulation

Introduction

Allergy is one of the common diseases that affect mankind with diverse manifestations. The prevalence of allergy and asthma has risen in the recent years despite an improvement in the general health of the population.[1] Allergic diseases are responsible for significant morbidity and have severe economic impact.[2] Various epidemiological studies have identified the causes for an increase in the prevalence of upper and lower respiratory tract allergic diseases. Some of the postulated reasons are increasing environmental pollution[3] and increased predisposition of individuals producing excessive IgE through a major change in the gene pool, changing lifestyles, and an increasing awareness of the disorders.[4]
Intensive research during the last several decades has highlighted the role of lymphocytes, immunoglobulins, mast cells, and various autacoids in the etiopathogenesis of allergic conditions. Inspite of the voluminous literature on the subject, the treatment of allergic diseases continues to be far from satisfactory. The available treatment options for upper and lower respiratory tract allergic diseases have major limitations owing to low efficacy, associated adverse events, and compliance issues.[5]

Ayurveda, an Indian system of medicine, has described several drugs from indigenous plant sources for use in the treatment of bronchial asthma and allergic disorders. HK-07 is one such polyherbal formulation containing mainly the extracts of Curcuma longa, Zingiber officinale, Piper longum, Emblica officinalis, Terminalia belerica, Ocimum sanctum, Adhatoda vasica, and Cyperus rotundus . The dry rhizome of Curcuma longa contains curcumin, the main bioactive component, demethoxycurcumin, and bisdemethoxycurcumin. The traditional uses of turmeric or natural curcuminoids in folk medicine are multiple, and some are based on their antioxidant, antiinflammatory and antiallergic properties which have been confirmed by various experimental studies.[6] Curcumin is also found to be a potent blocker of nuclear transcription factor (NF)-kB,[7] which is linked to a variety of diseases including allergy and asthma.[8] Ocimum sanctum has been demonstrated to protect against histamine, as well as pollen-induced bronchospasm in guinea pigs and inhibited antigen-induced histamine release from sensitized mast cells[9] apart from its established antiinflammatory[10] and antioxidant[11] properties. Adhatoda vasica is documented for its potent antiinflammatory,[12] antiallergic, and antitussive activities.[13] Piper longum has been shown to reduce the passive cutaneous anaphylaxis in rats and protect guinea pigs against antigen-induced bronchospasm.[14] Emblica officinalis was found to exhibit antiinflammatory, antitussive and antioxidant activities.[15], [16] Terminalia belerica demonstrated potent antiperoxidative activity and inhibited lipid peroxide formation by scavenging hydroxyl and superoxide radicals in vitro .[17] Zingiber officinale has been found to exert anti-inflammatory activity and is reported to be a potent inhibitor of inflammatory mediators such as prostaglandins and leukotrienes.[18] Cyperus rotundus inhibited the nitric oxide and superoxide production in in vitro studies, which used murine macrophage cell lines.[19] HK-07 was found to be clinically effective and safe in children suffering from upper and lower respiratory tract infections.[20]

In the present study, the effect of HK-07, a polyherbal formulation was studied on the active anaphylaxis and mast cell stabilization in rats, and histamine-induced bronchospasm in guinea pigs.

Materials and Methods

Animals
Inbred Wistar rats (175-200 g) and guinea pigs (400-600 g) of either sex housed in standard conditions (temperature 22 ± 2 ^oC, relative humidity 60 ± 5% and 12 h light/dark cycle) were used. They were fed with standard pellet diet and water ad libitum . The Institutional Animal Ethics Committee approved the experimental protocol. All the animals received humane care according to the criteria outlined in the "Guide for the Care and Use of Laboratory Animals" prepared by the "National Academy of Sciences" and published by the "National Institute of Health".

Histamine and horse serum were procured from Sigma Chemicals and toluidine blue from Loba-Chemie, Mumbai. Elisa kit for IgE was supplied by Orion diagnostics, Espoc, Finland. All other chemicals and reagents were procured from Hi-Media Laboratories limited, Mumbai.

Active anaphylaxis
Twenty-eight rats were sensitized by injecting subcutaneously 0.5 ml of horse serum along with 0.5 ml of triple antigen containing 20,000 million Bordetella pertussis organisms (Serum Institute of India Ltd., Pune, India). The sensitized rats were divided into 4 groups of 7 each. Group I served as control and received water (vehicle). Groups II, III and IV were administered HK-07 at 125, 250, and 500 mg/kg respectively, orally, once a day for 14 days. On day 14, after 2 h of treatment, the rats were challenged with intravenous injection (tail vein) of 0.25 ml horse serum in normal saline. They were then observed for the onset of symptoms such as dyspnea and cyanosis, duration of the persistence of symptoms (min), and mortality. The severity of symptoms was scored.[21] The optimal pharmacological effective dose of HK-07, which is derived from this dose response study was used for the remaining studies.

Serum total IgE was quantified with an ELISA protocol according to the manufacturer′s instructions. Briefly, the plates were coated with affinity-purified rabbit anti IgE overnight at 4 °C and then blocked with 1% bovine serum albumin (BSA) in phosphate buffered saline (PBS) for 1 h at 37°C. The serum samples and appropriate dilutions of a standard IgE preparation were placed in the wells, and the plates were incubated for 3 h at 4 °C. Sample blank wells were treated similarly but without serum. The bound IgE was detected with polyclonal goat anti IgE antibodies (incubation for 1 h at 37 °C), followed by HRP-conjugated rabbit anti-goat antibodies (incubation for 1 h at 37 °C). The plates were developed by the addition of O-phenylene diamine and read in an ELISA (Anthos HT-II, USA) plate reader at 490 nm.

Mast cell stabilizing activity
Thirty-two rats were divided into four groups of eight animals in each group. Group I served as control and received vehicle (water). Group II (sensitized control group, received only water), Groups III (HK-07) and IV (prednisolone) were sensitized by injecting 0.5 ml of horse serum subcutaneously along with 0.5 ml of triple antigen containing 20,000 million Bordetella pertussis organisms (Serum Institute of India Ltd., Pune). Group III were administered HK-07 500 mg/kg, p.o., once a day for 14 days. Group IV were administered prednisolone (reference drug) 10 mg/kg, p.o., for the same duration. On day 14, the rats were sacrificed 2 h after the treatment and the intestinal mesentry was taken out for the study on mast cells. Mesentries along with intestinal pieces were excised and kept in Ringer Locke solution (NaCl 154, KCl 5.6, CaCl₂ 2.2, NaHCO₃ 6.0, glucose 5.55 mM/L of distilled water) at 37^oC. The mesenteric pieces were challenged with 5% horse serum for 10 min after which the mast cells were stained with 1.0% toluidine blue and examined microscopically for the number of intact and degranulated mast cells.[21]

Histamine-induced bronchospasm in guinea pigs
Bronchospasm was induced in guinea pigs by exposing them to 1% histamine aerosol under constant pressure (1 kg/cm²) in an aerosol chamber (24 x 14 x 24 cm) made of perplex glass. Of the two groups of six animals each, Group I served as control and Group II received HK-07 500 mg/kg, p.o., once a day for 5 days. The animals were exposed to 1% histamine aerosol under constant pressure (1 kg/cm²) in an aerosol chamber on day 0 without any treatment. The end point, preconvulsive dyspnea (PCD) was determined from the time of aerosol exposure to the onset of dyspnea leading to the appearance of convulsions.[22] As soon as PCD commenced, the animals were removed from the chamber and exposed to fresh air. This PCD was taken as day 0 value. On days 1 and 5, 2 h after the administration of the drug, the time for the onset of PCD was recorded as on day 0.

Statistical analysis
The results of various studies were expressed as mean ± SEM and analyzed statistically using one-way ANOVA, followed by Bonferroni′s multiple comparison post-hoc test 0 or c0 hi- square test or unpaired Student′s ′ t ′ test to find out the level of significance. P<0.05 was considered statistically significant. The analysis was performed using Graphpad Prism software package (Version 4.0).

Results

0Effect of HK-07 on anaphylactic shock-induced bronchospasm in sensitized rats
HK-07 protected the sensitized rats against anaphylactic shock in a dose-dependent manner. In control rats, intravenous antigen challenge (horse serum) caused shock in 100% of the animals, while in treated rats (500 mg/kg of HK-07), the onset of symptoms of shock was delayed (P <0.001), and symptoms were less severe (P <0.05) with reduced mortality (P <0.05) [Figure - 1]. HK-07 (500 mg/kg) also resulted in significant reduction of serum IgE levels (25.80 ± 4.85 ng/ml, P <0.001) as compared to sensitized controls (125.06 ± 9.66 ng/ml). Serum IgE levels in control group was 8.83 ± 0.84 ng/ml (P <0.001 as compared to sensitized control). HK07 showed optimal pharmacological effect at 500 mg/kg dose. Hence, this dose of HK-07 was used for the remaining studies.

Mast cell stabilizing potential of HK-07
Antigen challenge resulted in significant degranulation of the mesentric mast cells (approximately 88%, P <0.001). Pretreatment of sensitized animals with HK-07 at 500 mg/kg, p.o., for 2 weeks resulted in a significant reduction in the number of disrupted mast cells (P <0.001) when challenged with horse serum. The effect of HK-07 was also comparable with the reference drug prednisolone [Table - 1].

Effect on histamine-induced bronchospasm
HK-07 at 500 mg/kg, p.o., significantly prolonged the latent period of PCD (P <0.008) as compared to control, following exposure to histamine aerosols on day 5 [Figure - 2].

Discussion

Experimental animal model of asthma is characterized by allergen-induced immediate airway constriction and late airway reactivity to a pharmacological vasoconstrictor such as histamine and leukotrienes. Histamine is a central mediator in the pathogenesis of allergic and inflammatory disorders. In the present study, HK-07 prolonged the latent period of PCD in guinea pigs following histamine aerosol. This may be suggestive of an antihistaminic activity following treatment with HK-07. It also offered protection against anaphylactic shock-induced bronchospasm in rats.

Basophils, mast cells, and their preformed de novo synthesized mediators, play a pivotal role in the pathogenesis of allergic disorders. These molecules are potent vasoactive and bronchoconstrictor agents and they modulate local immune responses and inflammatory cell infiltration.[23], [24] Immunoglobulin E (IgE)-mediated mast-cell stimulation is an important initial event in the development of type I allergic reactions such as asthma and atopic disorders. Clinical studies have found a close association between asthma and serum IgE levels, as well as IgE-dependent skin test reactivity to allergens.[25] Antigen challenge, in sensitized animals, results in the degranulation of mast cells, which is an important feature of anaphylaxis. In the present study, HK-07 showed marked protection against the mast cell degranulation following antigen challenge in sensitized animals. Mast cell stabilizing activity of HK-07 may be attributed to the presence of herbal extracts, which are known for their mast cell stabilizing potential against antigen-antibody reaction and/or due to the suppression of IgE antibody production, which is responsible for degranulation mast cells.[9] This antianaphylactic and antihistaminic effect may be caused by the stabilization of the mast cell membrane, suppression of IgE, and inhibition of pathological effects induced by the release of inflammatory mediators in HK-07 treated animals.

All the above findings lend credence to the beneficial use of HK-07 in the treatment of asthma and related conditions. However, further studies with other experimental models, especially to explore the role of cytokines are warranted to substantiate the antiasthmatic and antiallergic activity of HK-07.

References

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