Calcium antagonistic activity of Bacopa monniera in guinea-pig trachea.

OBJECTIVE: To demonstrate the calcium antagonistic property of ethanol extract of Bacopa monniera in guinea-pig trachea.
MATERIALS AND METHODS: The dose response curves of CaCl(2) (1 × 10(-5) to 1 × 10(-1) M) were constructed in the absence and presence of ethanol extract of Bacopa monniera (100, 500 and 700 μg/ml) or nifedipine (1 × 10(-6) M) in guinea-pig trachea in calcium free high K(+)-MOPS-PSS (3-(N-morpholino)-propanesulphonic acid physiological salt solution). The data was analyzed by ANOVA followed by least significant difference test or by Student's 't' test for unequal variance when appropriate. A probability of at least P < 0.05 was considered statistically significant.
RESULTS: The plant extract (500 and 700 μg/ml) significantly (P < 0.05) depressed and shifted the calcium concentration-response curves (1 × 10(-3)- 1 × 10(-1) M) to rightward similar to that of nifedipine.
CONCLUSIONS: Bacopa monniera extract exhibited calcium channel blocking activity in guinea-pig tracheal smooth muscles that may rationalize its relaxant action on guinea-pig trachea and its traditional use in respiratory disorders.

Introduction

Bacopa monniera (L.) Wettstein (Scrophulariaceae)[1] is a herbaceous plant found in wet and marshy areas of Indo-Pakistan. In folklore medicine it is used for the cure of various ailments including bronchitis, asthma, inflammation and nervine problems.[2] The plant is reported to have a broad spectrum of biological effects e.g., antioxidant,[3] anti-inflammatory,[4] anxiolytic,[5] hepatoprotective[6] and antidepressant activity.[7] Our earlier studies demonstrated that ethanol extract of B.monniera produced relaxant effect on blood vessels, trachea and intestinal tissues of rabbit and guinea-pig via a partial contribution of β-adrenoceptors and prostaglandins.[8] B.monniera also produced bronchodilation in anesthetized rats involving β-adrenoceptors dependent and independent mechanisms.[9] Later, studies carried on B.monniera in our laboratory clearly indicated that the spasmolytic effects of B.monniera extract on vascular and intestinal smooth muscles were predominantly due to inhibition of calcium channels (both voltage and receptor operated).[10] However, involvement of calcium channels in relaxant action of B.monniera on tracheal smooth muscle is not reported yet. Hence, the aim of present study was to observe the calcium antagonistic properties of B.monniera extract on isolated guinea-pig trachea.

Materials and Methods

Plant material and extraction

Bacopa monniera was collected during the summer from Karachi, Pakistan by Abrar Hussain. The botanical verification was done by Dr. Rubina Abid (Associate Professor) Department of Botany, University of Karachi. A voucher specimen was deposited at the Herbarium of this department under the reference number KUH-67276.

The whole plant material (air dried, five kg) was soaked in 95% ethanol (30 l) for eight days. The filtered solvent was evaporated by rotary evaporator and the resulting gummy material was transferred in an open Petri dish. It was then air dried in fume hood to remove residual ethanol. The crude ethanol extract of 259 g with a yield of 5.2% (w/w with reference to dried material) was obtained.[4]

Animals

Mongrel guinea-pigs (both sexes, weighing 400-800 g) bred in the animal house of H.E.J. Research Institute of Chemistry were kept in air conditioned rooms (23–25°C), lighting (12 h:12 h light–dark cycle) with free access to normal food and water. All animals received humane care and experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, published by the US National Institute of Health (NIH Publication, revised in1985).

Effect of Bacopa monniera extract on voltage-dependent calcium channels

The tracheal ring segments (2-3 mm) were prepared as described earlier.[11] To assess the effect of B.monniera extract on calcium influx, the response to various concentrations of calcium chloride was evaluated in trachea. The methodology was followed as described earlier.[1213] The bath solution (Krebs solution) was replaced by calcium free high K+-MOPS-PSS with the following composition (mM/L): NaCl 60.3, KCl 40.0, MgCl2 1.19, MOPS 10 and glucose 11.1. The pH of the solution (MOPS- PSS) was adjusted to 7.4 with NaOH. The tissues were initially incubated in the depolarized solution for 10-30 minutes followed by cumulative concentration-response curves to CaCl2 from 1 × 10-5 to 1 × 10-1 M representing the first concentration response curve (control). The time interval between each successive calcium additions was usually 4-40 minutes. After 30 minutes of tissue revival, nifedipine (1 × 10-6 M) or vehicle (0.0095% acetone or 0.35% dimethyl sulfoxide, DMSO) or extract (100, 500 and 700 μg/ml) was added to the bath for 10 minutes followed by a second cumulative concentration response curve to CaCl2. Each preparation was exposed to single concentration of either extract or nifedipine. To minimize photolysis, the experiments with nifedipine were performed in the dark.

Drugs and solubility

Nifedipine was purchased from Sigma Chemical Co., St. Louis, USA. Other chemicals, including the reagents used in the preparation of physiological solutions were of analytical grade. Both nifedipine and Bacopa monniera extract were dissolved in 95% acetone and 10% DMSO (20 mg/ml), respectively as stock solution. Control experiments showed that either acetone (0.0095%) or DMSO (0.35% final concentration in the organ bath) had no effect on either baseline or contractile responses to CaCl2 on trachea.

Data analysis

The experimental data were expressed as mean ± S.E.M. and the significance was analyzed by analysis of variance (ANOVA), followed by least-significant-difference (LSD) test to assess the significance of the differences between individual groups or by Student's t-test for unequal variance when appropriate.[14] A probability of at least P < 0.05 was considered statistically significant. The concentration of the agent producing 50% of the maximal contraction (EC50) in an individual experiment was determined graphically from concentration-response curve.

Results

The cumulative-response curves for CaCl2 alone and in the presence of different concentrations of B.monniera extract (500 and 700 μg/ml) and nifedipine (1 × 10-6 M) in trachea are depicted in [Figure 1]. The addition of calcium chloride caused concentration-dependent contraction with maximum tension of 640 ± 76 mg at 1 × 10-2 M. These responses of CaCl2 were reproducible over time, as three consecutive concentration-response curves obtained in control preparation of trachea remained unaffected during the course of the experiment. Pretreatment of the tissue with nifedipine or extract caused significant non-parallel and rightward shift to CaCal2 (1 ×10-3- 1 × 10-1 M) induced responses. The extract at 100 μg/ml caused non-significant reduction to CaCl2 response (not shown). At 500 μg/ml it caused 46 ± 17% reduction to CaCl2 however, almost complete inhibition (98 ± 1%) of maximum response to CaCl2 was evident at 700 μg/ml which was comparable to that (97 ± 2%) produced by nifedipine. The EC50 values of CaCl2 were significantly decreased by the extract at both doses (500 and 700 μg/ml) [Table 1]. The nifedipine abolished the CaCl2 curves completely in some experiments making it difficult to measure EC50 values.

Figure 1

Tension (mg) produced by cumulative concentrations of calcium chloride in the absence or presence of ethanol extract of B.monniera and nifedipine in guinea-pig trachea. Control (■), 500 μg/ ml(▲), 700 μg/ml (•) of extract and nifedipine (1 × 10-6 M, □). Values presented are mean ± S.E.M. at P < 0.05, using ANOVA followed by LSD test compared to control values. The inhibitory effect of extract on CaCl2 was long lasting and completely reversible within 1 h. On the other hand the effect of nifedipine was revived completely only at 1 × 10-2 M of CaCl2

Table 1

The EC50 values of CaCl2 in the absence and presence of nifedipine and ethanol extract of Bacopa monniera in guinea-pig trachea

Discussion

Calcium-induced contractions of K+-depolarized smooth muscles are produced as a result of Ca2+ flux; the inhibition of these contractions in such conditions is commonly accepted as a test for agents that act non-specifically by inhibiting Ca2+ participation in excitation-contraction coupling process.[1516] Likewise, in present investigation the tracheal preparations were pre-incubated in depolarizing medium as described by other workers.[1718] The B.monniera extract suppressed calcium chloride-induced responses non-competitively like nifedipine, a known calcium channel blocker[19] thus demonstrating interaction of calcium channels with its relaxant action in trachea. These results are in agreement with our previous reports which indicated the existence of calcium antagonism property in ethanol extract and various fractions isolated from B.monniera in intestinal and vascular tissues from rabbit and guinea-pig.[1020] However, the presence of calcium antagonism property in B.monniera in guinea-pig trachea is reported for the first time here. Indeed, guinea-pig trachea was the tissue showing complete and long lasting relaxation by ethanol extract of B.monniera in our previous studies.[8]

Supporting results were also demonstrated earlier where B.monniera, its various fractions and pure constituents showed more potency towards inhibition of bronchoconstriction (tracheal pressure) induced by carbachol as compared to that on cardiovascular parameters of anesthetized rats.[920] In addition to the involvement of calcium channels, other possible mechanisms for guinea-pig tracheal relaxation by B.monniera are the participation of prostaglandins and β-adrenoceptors.[89]

Thus it can be suggested that relaxant effect of B.monniera on trachea encompass multiple mechanisms with the major participation of voltage-operated calcium channels. Nevertheless, characterization of specific type of voltage-operated calcium channel (s) remains to be elucidated.