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Vasodilatory effects of mulberry leaf extract (MLE) and its possible underlying mechanisms on porcine basilar artery (PBA) were investigated by measuring changes in isometric tension in vitro. MLE slightly relaxed 60 mM KCl pre-contracted PBA rings (9.4% at 540 μg/mL). A treatment with MLE induced extensive relaxation under pre-contraction with 0.1 μM U-46619 (EC50 = 49.4 μg/mL). Neither endothelial denudation nor treatment with atenolol (10 μM) affected MLE-induced relaxation. Relaxation induced by MLE (540 μg/mL) was significantly attenuated by cimetidine (10 μM). MLE inhibited angiotensin I-induced contraction in endothelium-intact PBA rings (EC50 = 25 μg/mL), similar to captopril. Therefore, the possible mechanisms underlying MLE-induced relaxation in PBA might be mediated through: (1) functional antagonism of thromboxane A2 receptors; (2) suppression of angiotensin I-induced contraction; (3) Ca2+ influx inhibition; (4) stimulation of H2-receptors. Moreover, MLE contained significant amounts of nitrite, nitrate and several phenolic compounds, possibly associated with MLE-induced relaxation.