Abstract
Background: Rind of Punica granatum is traditionally used in treatment of abdominal cramps and various GIT disorders. So far spasmolytic activity of rind of Punica granatum has been reported using in vitro model. However, its mode of action is not explored yet. Therefore, the current work describes the possible mode of action for spasmolytic activity of methanolic extract of rind of Punica granatum (Pg. Cr). Acute toxicity study is also performed to determine its safe dose range.
Methods: Rind of Punica granatum was subjected to shade drying. Shade dried materials were pulverized using conventional grinder. Grinded materials were macerated in commercial grade methanol. The extract of rind of P. granatum was concentrated using a rotary evaporator. Rabbits' jejunal preparations were mounted in organ bath containing 10 ml Tyrode's solution, constantly aerated with carbogen gas. Pg. Cr was tested on spontaneous rabbits' jejunal preparations in concentrations 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 5.0 and 10.0 mg/ml. Pg. Cr was also tested on KCl (80 mM)-induced contractions in rabbits' jejunal preparations. Since we observed spasmogenic activity for the first time, hence we also determined the effects of Pg. Cr in presence of atropine (0.03 mu M). Pg. Cr was also tested in presence of 0.03 mu M of loratadine HCl. Pg. Cr was also tested on barium chloride induced contractions. Calcium Concentration Response Curves (CCRCs) were constructed in the absence and presence of test samples of Pg. Cr in decalcified tissues to explore its possible mode of action. Acute toxicity screening was also performed to determine its safe dose range.
Results: Phytochemical screening revealed the presence of saponins, tannins, carbohydrates, proteins, flavonoids, saponins and steroids. However, Pg. Cr tested negative for alkaloids and triterpenoids. Pg. Cr was safe up to 100 mg/kg with its LD50 = 1305 mg/kg. Its respective EC50, in the absence and presence of atropine, were 9.7 +/- 0.3 and 3.12 +/- 0. 45 mg/ml. In the presence of 0.02 and 0.08 mu M of loratadine HCl, respective EC50 were 5.6 +/- 0.4 and 2.8 +/- 0.15 mg/ml. EC50 for relaxant effects on KCl-induced contractions was 8.6 +/- 1 mg/ml. In the presence of 0.3 mg/ml of Pg. Cr, a right shift was observed with EC50 (log [Ca++] M) = -1.8 +/- 0.09 vs. control EC50 -2.6 +/- 0.01. Similarly, EC50 for verapamil (0.1 mu M) was -2.4 +/- 0.011vs. control EC50= -2.4 +/- 0.01. The right shift of P. granatum resembled the right shift of verapamil suggesting for inhibition of voltage gated calcium channels.
Conclusions: P. granatum is safe up to 100 mg/kg. In low concentrations, P. granatum produced spasmogenic activity possibly through involvement of cholinergic and histaminergic receptors. The spasmolytic action may follow inhibition of the voltage gated calcium channels.