Anti-histaminic, mast cell stabilizing and bronchodilator effect of hydroalcoholic extract of polyherbal compound- Bharangyadi.

Bronchial asthma is a chronic inflammatory disorder of the airways associated with reversible airway obstruction and increased airway responsiveness to a variety of stimuli. An intuitive inference from this definition is that a causal relationship may exist between airway inflammation and airway hyperresponsiveness. It can be say that "airway inflammation equal to airway hyperresponsiveness". Attachment of antigen antibody complex to the mast cell causes its disruption and release of inflammatory mediators such as histamine.To evaluate the efficacy of anti-asthmatic property of a drug, evaluation of anti-histaminic, mast cell stabilizing and bronchodilator property can be use as pharmacodynamic parameter. Bharangyadi is a polyherbal compound having Bharangi (Clerodendrum serratum), Sati (Hedychium spicatum) and Pushkarmoola (Inula racemosa) as ingredient herbs The present study aimed to evaluate the anti-asthmatic activity of an indigenous polyherbal compound Bharangyadi through various in-vitro & in-vivo experimental models.The results demonstrate that drug has potent histamine antagonism property with significant mast cell stabilizing and spasmolytic activity in the experimental animals. Compound 48/80, a potent mast cell degranulator, provoked 76% degranulation of mast cells in the control group. Ethanolic extract of Bharangyadi at the doses 500 and 1000 μg/ml protected from compound 48/80-evoked degranulation (P < 0.01) in dose dependent manner.Pre-treatment with Bharangyadi extract showed 80% & 86% protection from histamine induced bronchoconstriction in guinea pigs with 27.8% and 36.1% increase in preconvoulsion time (equal to standard drug). Screening of Histamine antagonism activity on guinea pig ileum showed that drug reduces the smooth muscle contraction in dose dependent manner. Increasing concentration of Bharangyadi extract with maximum dose of histamine (1.6μg) showed maximum inhibition at the dose of 50mg (99.78%). Inhibition of smooth muscle contraction by addition of drug in organ bath before adding histamine showed that drug has preventive type antagonism.

INTRODUCTION

Bronchial asthma is defined as a chronic inflammatory disorder of the airways in which many cells and cellular elements play a role. The chronic inflammation is associated with airway hyper-responsiveness that leads to recurrent episodes of wheezing, breathlessness, chest tightness, and coughing, particularly at night or in the early morning. In spite of superlative available conventional medicines, the prevalence of bronchial asthma is increasing drastically.[1-4] Recently, there has been a shift in universal trend from synthetic to herbal medicine, which we can say “Return to Nature.” Medicinal plants have been known for millennia and are highly esteemed all over the world as a rich source of therapeutic agents for the prevention of diseases and ailments. The polyherbal preparation used in this study contains Bharangi (Clerodendrum serratum), Sati (Hedychium spicatum), and Pushkarmoola (Inula racemosa). Bharangi is the most valuable herb to take internally in respiratory ailments and for all fevers in general. As Bharangi effectively liquefies the mucous, it is salutary in respiratory problems such as colds, bronchitis, bronchial asthma, and tuberculosis.[3] In such conditions, varied combinations of Bharngi are recommended. The rootstock of H. spicatum (Sati) is carminative, emmenagogue, expectorant, stimulant, stomachic, and tonic.[4] It is useful in the treatment of liver complaints and is also used in treating fevers, vomiting, diarrhea, inflammation, pains, and snake bite. In Ayurveda, I. racemosa is widely used for various disorders; it is mostly used in heart and respiratory disorders. Recent clinical studies also show that Pushkarmula (I. racemosa) is a potent coronary vasodialator agent and also has anti-inflammatory, anti-anginal, and anti-ischaemic properties.[5-7] It also acts as anti-microbial agent. Preliminary studies with the ethanolic extract of roots of I. racemosa exhibited anti-allergic and anti-asthmatic properties, the later being more pronounced.[89] Specific studies for bronchodilator properties on isolated trachea were performed and found it a potent bronchodilator. The extract also protected guinea pigs against various experimental asthma, plant pollen, etc. It possessed anti-histaminic as well as anti-5-HT activities, suggesting its use in bronchial asthma. Above all these drugs are indicated in bronchial asthma and are being practised in combination successfully since decades although their combination is not given in Ayurvedic texts. Thus it was decided to evaluate the anti-asthmatic property of drug on various experimental models.

MATERIALS AND METHODS

Plant material

The plants Clerodendrum serratum, H. spicatum, and I. racemosa were collected from local market of Varanasi [Table 1]. The identification of the drugs was done by Prof. A.K. Singh, Department of Dravyaguna, S.S.U., Varanasi (Identification number DG/AKS/604).

Table 1

Contents of Bharangyadi compound

Extraction of the plant material and sample preparation

Hydroalcoholic extraction (distilled water:ethanol = 2:1) of drug was carried out by hot percolation method through Soxhlet apparatus. Thereafter, extract was dried using rotary evaporator and dried extract was put to the process of standardization.[10] The percentage yield was noted.

Animals

Rats: Colony bred descendants of Charles–Foster strain procured from Animal Research Branch of the Institute of Medical Sciences, Banaras Hindu University, Varanasi. The animals were of either sex weighing 100 + 20 g.

Guinea pig: Local bred supplied by M/s Zoological Emporium, Institute of Medical Sciences, Banaras Hindu University, Varanasi, of either sex, weighing 300–450 g.

Drugs and solvents

Dexchlorpheniramine maleate (Schering), histamine dihydrochloride (Sigma), Kitotifen fumarate (Sigma), Mepyramine malate (Sigma), and Compound 48/80 (Sigma).

All drugs were dissolved in distilled water and desired concentrations were prepared.

Studies on isolated guinea pig ileum for anti-histaminic activity

• Overnight fasted guinea pigs of either sex weighing 400–600 g were killed using cervical dislocation method. The abdomen was quickly opened. In the right lower quadrant of the abdomen, a greenish sac-like structure, the cecum, was seen. This was gently turned outwards so as to expose its inner surface; this reveals a slender pinkish length of small intestine marked by a localized thickening in the wall—a Peyer's patch of lymphoid tissue. Using this patch as a landmark, the lower most 10 cm of ileum was removed from the abdomen and placed in a shallow dish containing warm Tyrode solution. Ileum lumen was cleaned by passing through warm 0.9% saline and then segments about 1 inch in length were made. The mesentric attachment and blood were carefully cleaned and the tissues were mounted in a thermostatically controlled Dale's organ bath (temp. 37+ 0.5°C) containing 20 ml Tyrode's solution under basal tension of 500 mg. The composition of solution in mM was NaCl, 137; CaCl2, 1.8; KCl, 2.7; glucose, 5.55; NaHCO3, 11.9; MgCl2, 1; NaH2PO4, 0.4. The solution was continuously bubbled with air. The responses to drug were recorded on a Student physiograph (BioDevices) using isotonic transducer, which exerted a basal tension equivalent to 500 mg load on tissue. The issue was allowed to equilibrate for 30 min, during which the bathing solution was changed every 10 min. Increasing concentration of histamine was added to the bath and the control cumulative concentration-response curve was constructed.

Studies on compound 48/80 induced rat mesentric mast

Cell degranulation: Rats were killed by cervical fracture and their abdomen was opened wide. Small intestine from jejunum to proximal 2/3 of ileum along with the entire mesenteric attachment was removed into a beaker containing Ringer-Locke's solution at 37° C, with continuous oxygenation through a capillary tube. Piece of suitable size to include 2–3 arcades of mesenteric vessels were made and the mesentery was then cut out in one piece by gentle dissection. Five to eight good mesenteric pieces can be obtained per animal. The mesentery 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: 0.1 ml of Ketotifen fumarate (10 μg/ml)

Petri dish no. 3: 0.1 ml of test agent in Tween-80 (Bharangyadi compound, 500 μg/ml)

Petri dish no. 4: 0.1 ml of test agent in Tween-80 (Bharangyadi compound, 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 20–25 min. After staining and fixation of mast cells, mesentery 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 and the treated groups were calculated by counting the number of degranulated mast cells from a total of at least 100 mast cells counted. Percent inhibition of mast cell degranulation for each treatment was calculated by using the following formula:

% inhibition of MCD = [1 - number of/total number of mast cells] × 100 degranulated mast cell

Composition of Ringer-Locke's solution was altered appropriately to suit the need of specific experiments. Such instances include experiments involving pH adjustment using additions of 0.1 N HCL or NaOH (universal pH indicator solution was resorted for making pH adjustment in aliquots of the altered medium) or various molar concentration constitution.

Bronchodilator effect

Studies on histamine-induced bronchospasm in guinea pigs

Guinea pigs of either sex weighing 350–500 g were selected and randomly divided into four groups each containing four animals. The drugs were dissolved in distilled water and administered orally through intubation canula. The single-dose treatments were given one and half an hour before the study. In control group 0.5% sodium carboxymethyl cellulose (CMC) was administered orally. The following schedule of treatment was administered:

Group I: (control) 0.5% CMC.

Group II: mepyramine melate (10 mg/kg = 0.1% solution) (standard)

Group III: alcoholic extract of Bharangyadi compound (200 mg/kg)

Group IV: alcoholic extract of Bharangyadi compound (500 mg/kg)

One and an half hour later, the animals were exposed to 0.2% histamine aerosol, and time for preconvulsion state (PCD) was noted for each animal (Sheth et al., 1972). The end point for PCD was determined from the time of aerosol exposure to the onset of dyspnea leading to the appearance of convulsions. As soon as PCD commenced, the animals were removed from chamber and placed in fresh air to recover. This time for PCD was taken as day 0 value. After 15 days of washout period, the animal of groups III and IV were again given the same schedule of drug and expose to histamine aerosol and time for PCD was noted. The % increase in time of PCD was calculated using the following formula:

Percentage increase in time of PCD = 1 – T1/T2 × 100

where T1 = time for PCD onset on day 0, T2 = time for PCD onset on day 15.

RESULTS

In vivo histamine-induced bronchospasm

Pretreatment with Bharangyadi extract produce significant (P < 0.001) protection from histamine-induced bronchospasm (80% and 86%) with 27.8% and 36.1% increase in preconvulsion time at a dose of 200 mg/kg and 500 mg/kg body weight of guinea pig.

In vitro histamine-induced guinea pig ileum contraction

Hydroethanolic extract of Bharangyadi compound showed significant inhibition in histamine-induced guinea pig ileum contraction in a dose-dependent manner [Figure 1]. Increasing concentration of Bharangyadi extract with maximum dose of histamine (1.6 μg) showed maximum inhibition at a dose of 50 mg (99.78%) [Figure 2]. Inhibition of smooth muscle contraction by addition of drug in organ bath before adding histamine showed that drug has preventive type antagonism.

Figure 1

Effect of increasing concentrations of dexchlorpheniramine (S) on the cumulative dose responses of histamine in the guinea pig ileum with comparison to control

Figure 2

% inhibition of contraction produced by maximum dose of histamine in the presence of different concentration of Bharangyadi extract

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 polyherbal compounds produced significant (P < 0.001) reduction in the Compound 48/80-induced mast cell degranulation in a dose-dependent manner [Figure 3]. The % inhibition of MCD was found to be 54.67% and 58.83% with 500 and 1000 μg/ml of Bharangyadi compound. Kitotifen fumarate, a known mast cell stabilizing agent, also brought significant (P < 0.001) reduction in degranulating mast cells.

Figure 3

Effect of polyherbal compounds on compound 48/80 induced rat mesentric mast cell degranulation. Each bar represents mean ± SEM (n = 6). *P < 0.001 as compared to positive control group (one-way ANOVA followed by Tukey's multiple range test)

DISCUSSION

The experimental study conducted to evaluate the anti-asthmatic effect of drug showed concentration and time-dependent antagonistic effects of the extract against the contraction induced by the standard spasmogens [Table 2]. The results of this study indicated a rightward shift in the log dose–response curve of histamine in the presence of the hydroalcholic extract of polyherbal compounds. The maximum effects of histamine induced contractions were inhibited in the presence of the extract. The EC50 obtained in the presence of the extract was not significantly different than the EC50 obtained from the standard spasmogens alone.

Table 2

Results obtained from guinea pig ileum preparation after treatment with Bharangyadi extract

The nonparallel rightward shift in histamine log dose–response curves obtained in the presence of the extract, with lowered maximum contraction effect to histamine would indicate a noncompetitive or an irreversible antagonistic effect of polyherbal compounds at histamine H1 receptors of guinea pig ileum (Linden et al., 1993). In this case the antagonist binds irreversibly to receptor site or to another site that inhibits response to the agonist and the antagonism is insurmountable no matter how much the agonist concentration is increased. Duration of the antagonist's action is dependent on the rate of turnover of receptor molecules.

The nonparallel rightward shift in histamine log dose–response curves with lowered maximum contraction indicated a noncompetitive inhibition by the extract, a typical characteristic of calcium antagonists (Janssen and Sims, 1993).

The properties of irreversible antagonists are markedly different from competitive antagonists. Antagonists that produce parallel rightward shifts of agonist dose–response curves with no alteration of the maximal response are classified as surmountable, while insurmountable antagonists depress the maximal response. Although the longevity of the antagonist receptor complex is quoted in many studies to explain insurmountable antagonism, slowly interconverting receptor conformations, allosteric binding sites, and receptor internalization have been evoked as alternative explanations. To complicate matters even further, insurmountable antagonism is not only drug-related but it may also depend on the type of tissues and species used (Vauquelin et al, 2002).

Irreversible receptor antagonists are chemically reactive compounds. These ligands first bind to the receptor. Following this binding step, the ligand then reacts with the functional groups of the receptor. The consequence of this chemical reaction is that the ligand becomes covalently bound to the receptor. Because a chemical bond is formed, an irreversible ligand does not freely dissociate from the receptor. It remains attached to the receptor for a long period of time. The synthesis of new receptor protein may be required to generate a receptor free of an irreversible blocker. Because the ligand is covalently bound to the receptor, the binding of agonists, and hence their pharmacologic activity, are blocked. Unlike competitive antagonists, the blocking activity of irreversible receptor antagonists cannot be overcome by increasing the agonist concentration. Because an irreversible receptor antagonist reduces the total number of active receptors, the maximal pharmacologic effect (Emax) is also decreased. The reduction in maximal agonist response without changing the EC50 is the hallmark of irreversible antagonists. The shape of the dose–response curve is also altered because of this decrease in maximal effect. The dose–response is shifted to the right and the maximal response is decreased (Linden et al., 1993).

In a situation where enough spare receptors are present, the presence of a noncompetitive antagonist will shift the dose–response of the agonist to the right without a change in the maximal response. Increasing the concentration of the noncompetitive antagonist, however, will eventually reduce the number of available receptors so that the maximal response cannot be obtained (Vauquelin et al, 2002).

The bronchodilator effect seen with polyherbal compounds (non-parallel shift in the log dose–response curve with decreased maximum response of the standard spasmogens) may not be mediated through the specific blocked of cholinergic or histaminergic receptors, rather through nonspecific inhibition like calcium antagonism (Gilani et al., 2005).

The extract reduced contraction of guinea pig ileum preparation induced by histamine. However, the exact mechanism of action of the extract is not yet known. It is generally accepted that an increase in concentration of cytoplasmic free Ca2+ in indispensable for smooth muscle contraction. One possible mechanism responsible for the functional antagonism effect of the extract might be explained by its calcium antagonistic effect (Janssen and Sims, 1993).

In airway smooth muscle, excitation–contraction coupling mechanism involves membrane depolarization via activation of large inward Cl and nonselective cation currents (Janssen and Sims, 1993; Wang, 1997). This depolarization activates voltage-dependent Ca2+ current, (Kotlikoff, 1988) which is sufficient for contraction. Ca2+ entry in airway smooth muscle may also involve nonselective cation channels and/or receptor-operated Ca2+ channels (Wang, 1997).

Studies have also shown that voltage-independent mechanisms seem to be most important for contraction in airway smooth muscle such that spasmogens could act by activation of phospolipase C and generation of inositol 1, 4, 5-triphosphate (IP3), which, in turn, triggers the release of Ca 2+ stored within the sarcoplasmic reticulum. The relative importance of these mechanisms seems to vary with different spasmogen (acetylcholine versus histamine) (Luke et al., 2001).

The above mechanisms could explain the effects observed with the extract studied. Other possible mechanisms responsible for the bronchodilator effect of the plant studied could include

Stimulation of inhibitory non adrenergic noncholinergic nerves system (NANC) or inhibition of stimulatory NANC (Linden et al., 1993)

Inhibition of phosphodiestrase enzymes (Van Amsterdam et al., 1989)

Opening of potassium channels (Buckle et al., 1993)

Stimulation of b-adrenergic receptors (Mohammad and Akram, 2004)

As the concentration of the extract was increased from 0.5 to 50 mg/ml, a more pronounced relaxant effect was observed indicating a dose dependent bronchodilatory effect of the extract.

Although the relaxant effect of the plant extracts was less potent than that obtained by dexchlorpheniramine, with increasing doses of the extract and/or isolation and purification of the active chemical components present in the extract, a comparable effect could be obtained.