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Abstract：
BACKGROUND: 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.
OBJECTIVE: 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.
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.
CONCLUSIONS: 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.
OBJECTIVE: 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.
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.
CONCLUSIONS: 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.
摘要：
背景：塔斯霍德勒斯。（Aspodelaceae），一个野生的，陆地,一年生无茎草本植物，在传统医学中广泛用于治疗高血压，糖尿病,动脉粥样硬化和循环问题。我们实验室先前的一项研究表明，tenuifolius在长期葡萄糖喂养的大鼠中具有降低血压和改善主动脉内皮功能障碍的有益作用。尽管Tenuifolus在体内降低血压并改善内皮功能，目前还没有关于其可能的作用机制的详细研究。
目的：本研究旨在为黄牛在高血压和循环系统问题中的传统使用提供药理基础和作用机制。我们探讨了Tenuifolus的血管舒张作用及其在猪冠状动脉环中的潜在血管舒张机制。
方法：通过浸渍工艺制备黄牛的甲醇水溶液粗提物，然后通过使用不同极性的溶剂进行活性指导分级分离。使用LC-DAD-MS进行植物化学研究。在金属丝肌电图仪中设置了猪远端冠状动脉的节段，以进行等距力测量。通过在存在或不存在细胞内信号传导级联的抑制剂的情况下测量在用血栓烷模拟物U46619预收缩后的色调变化,测试了毛虫种子的提取物/级分的血管扩张剂活性。
结果：毛毛虫的粗提物/部分在冠状环中产生剂量依赖性内皮非依赖性血管舒张反应，然而,A.tenuifolus（BS-AT）的丁醇部分产生最大的弛豫反应，在1mg/ml时具有100％的弛豫，因此确定了该部分的松弛机制。对BS-AT的松弛不受内皮去除的影响，用KCl预收缩，或者非选择性钾通道阻滞剂四乙基铵的存在，表明松弛是内皮非依赖性的，并且不涉及钾通道的激活。BS-AT（1mg/ml）抑制对钙的收缩反应，L型钙通道激活剂BAYK8664和离子霉素，表明它抑制钙诱导的收缩。在不存在细胞外钙的情况下，对BS-AT的松弛反应减弱。然而,用环吡嗪酸从细胞内储存中删除钙后，BS-AT的松弛也减少。与1mg/mlBS-AT孵育还可以抑制冠状动脉匀浆中肌球蛋白轻链的磷酸化。
结论：Tenuifolus的丁醇提取物通过抑制钙诱导的收缩，使猪冠状动脉产生大的非内皮依赖性舒张。这种效应似乎是钙流入的下游，可能通过抑制肌球蛋白轻链激酶。这项研究支持了先前的研究，这些研究表明tenuifolus可以降低血压。未来的研究将旨在确定这种反应背后的活性化合物。
目的：本研究旨在为黄牛在高血压和循环系统问题中的传统使用提供药理基础和作用机制。我们探讨了Tenuifolus的血管舒张作用及其在猪冠状动脉环中的潜在血管舒张机制。
方法：通过浸渍工艺制备黄牛的甲醇水溶液粗提物，然后通过使用不同极性的溶剂进行活性指导分级分离。使用LC-DAD-MS进行植物化学研究。在金属丝肌电图仪中设置了猪远端冠状动脉的节段，以进行等距力测量。通过在存在或不存在细胞内信号传导级联的抑制剂的情况下测量在用血栓烷模拟物U46619预收缩后的色调变化,测试了毛虫种子的提取物/级分的血管扩张剂活性。
结果：毛毛虫的粗提物/部分在冠状环中产生剂量依赖性内皮非依赖性血管舒张反应，然而,A.tenuifolus（BS-AT）的丁醇部分产生最大的弛豫反应，在1mg/ml时具有100％的弛豫，因此确定了该部分的松弛机制。对BS-AT的松弛不受内皮去除的影响，用KCl预收缩，或者非选择性钾通道阻滞剂四乙基铵的存在，表明松弛是内皮非依赖性的，并且不涉及钾通道的激活。BS-AT（1mg/ml）抑制对钙的收缩反应，L型钙通道激活剂BAYK8664和离子霉素，表明它抑制钙诱导的收缩。在不存在细胞外钙的情况下，对BS-AT的松弛反应减弱。然而,用环吡嗪酸从细胞内储存中删除钙后，BS-AT的松弛也减少。与1mg/mlBS-AT孵育还可以抑制冠状动脉匀浆中肌球蛋白轻链的磷酸化。
结论：Tenuifolus的丁醇提取物通过抑制钙诱导的收缩，使猪冠状动脉产生大的非内皮依赖性舒张。这种效应似乎是钙流入的下游，可能通过抑制肌球蛋白轻链激酶。这项研究支持了先前的研究，这些研究表明tenuifolus可以降低血压。未来的研究将旨在确定这种反应背后的活性化合物。
