Endothelium-independent vasorelaxant effect of Asphodelus tenuifolius Cav. via inhibition of myosin light chain kinase activity in the porcine coronary artery

Abstract

Ethnopharmacological relevance: Asphodelus tenuifolius Cav. (Asphodelaceae), a wild, terrestrial, annual stemless herb, is widely used in traditional medicine for the treatment of hypertension, diabetes, atherosclerosis and circulatory problems. A previous research study from our laboratory revealed that A. tenuifolius has beneficial effects in reducing blood pressure and improves aortic endothelial dysfunction in chronically glucose fed rats. Despite the fact that A. tenuifolius reduces blood pressure and improves endothelial function in vivo, there are no detailed studies about its possible mechanism of action.

Aim of the study: This study was designed to provide pharmacological basis and mechanism of action for the traditional use of A. tenuifolius in hypertension and circulatory problems. We explored the vasorelaxant effect of A. tenuifolius and its underlying vasorelaxation mechanism in porcine coronary artery rings.

Materials and methods: Aqueous methanolic crude extract of A. tenuifolius was prepared by maceration process and then activity guided fractionation was carried out by using different polarity based solvents. Phytochemical studies were carried out using LC-DAD-MS. Segments of porcine distal coronary artery were set up in a wire myograph for isometric force measurements. Extract/fractions of A. tenuifolius seeds were tested for vasodilator activity by measurement of changes in tone after pre-contraction with the thromboxane mimetic U46619 in the presence or absence of inhibitors of intracellular signaling cascades.

Results: Crude extract/fractions of A. tenuifolius produced dose dependent endothelium independent vasorelaxant response in coronary rings, whereas, the butanol fraction of A. tenuifolius (BS-AT) produced the largest relaxation response with 100% relaxation at 1 mg/ml, therefore the mechanism of relaxation of this fraction was determined. The relaxation to BS-AT was unaffected by removal of the endothelium, pre-contraction with KCl, or the presence of the non-selective potassium channel blocker tetraethylammonium, indicating that the relaxation was endothelium-independent, and does not involve activation of potassium channels. BS-AT (1 mg/ml) inhibited the contractile response to calcium,the L-type calcium channel activator BAY K8664,and ionomycin, indicating that it inhibits calcium-induced contractions. The relaxation response to BS-AT was attenuated in the absence of extracellular calcium. However, relaxations to BS-AT were also reduced after deletion of calcium from intracellular stores with cyclopiazonic acid. Incubation with 1 mg/ml BS-AT also inhibited phosphorylation of myosin light chains in homogenates of coronary artery.

Conclusion: The butanol extract of Asphodelus tenuifolius produces a large endothelium-independent relaxation of the porcine coronary artery through inhibition of calcium-induced contractions. The effect appears to be downstream of calcium influx, possibly through inhibition of myosin light chain kinase. This study supports previous studies demonstrating that A. tenuifolius reduces blood pressure. Future studies will aim to determine the active compounds underlying this response.

Keywords: 1557; 912226; Alpha,beta-MeATP (PubChem CID: 9; Asphodelus tenuifolius; Bayk8644 (PubChem CID: 2303); Bradykinin (PubChem CID: 439201); Calcium influx; Coronary artery; Dicloromethane (PubChem CID: 6344); Ethyl acetate (PubChem CID: 8857); Forskolin (PubChem CID: 47936); Ionomycin (PubChem CID: 6; Methanol (PubChem CID: 887); Myosin light chain kinase; Nifedipine (PubChem CID: 4485); ODQ (PubChem CID: 1456); Tetraethylammonium (PubChem CID: 5413); U46619 (PubChem CID: 5311493); Vascular smooth muscle; Vasodilatation; Y27632 (PubChem CID: 448042); n-Butanol (PubChem CID: 263); n-Hexane (PubChem CID: 8058).