4.2.2. Antispasmodic Effect of Crude Extract and Fractions

Experimental animals were euthanized by cervical dislocation. Antispasmodic activity of the crude extract and fractions was studied on the isolated ileum of mature albino Wistar rats as described by Gilani et al. [62]. The segmented ileum, measuring 2 cm in length, was suspended in a 10 mL tissue bath filled with Tyrode solution. The solution, composed of NaHCO₃ (11.90 mM), MgCl₂ (1.05 mM), KCl (2.68 mM), NaCl (136.9 mM), CaCl₂ (1.8 mM), glucose (5.55 mM), and NaH₂PO₄ (0.42 mM), was bubbled with carbogen gas at 37 °C. A constant resting tension of 1 g was applied to the tissues (ileum) throughout the experiment. Isometric contractions were recorded using force displacement transducers connected to a Power Lab Data Acquisition System (AD Instruments, Sydney, Australia) attached to a computer installed with labchart software (version 6). Tissues were equilibrated for a minimum of 30 min and stabilized with a sub-maximal concentration of acetylcholine (0.3 μM), which was washed off immediately and replaced with Tyrode solution before the start of the experiment. The spontaneous rhythmic contractions exhibited by the rat ileum under the above experimental conditions gave room for the direct testing of the antispasmodic activity without the use of any agonist. For determination of the antispasmodic effects of the extract and fractions, dosing of test drugs was carried out cumulatively by serial dilution of 300–3 µg/mL at 0.01–10 µg/mL at 3–5 min intervals. A high-potassium-ion concentration (high [K⁺]) (80 mM) was applied to depolarize the preparations as described by Farre et al. [63]; this was performed to assess whether the antispasmodic effect of the extracts was mediated through Calcium Channel Blockade (CCB). The addition of high [K⁺] to the tissue bath resulted in a sustained contraction. Relaxation of the ileum pre-contracted with [K⁺] by the extract was expressed as a percentage of the control response mediated by high [K⁺]. The assay was conducted in triplicates for each test sample. This assay was repeated for the determination of the antispasmodic potential of the isolated compounds.