The Effects Of Pentamethylquercetin On Isolated Rat Aortic Rings And Its Mechanisms

Phytopolyphenol of flavonoids have always been highly concerned in the medicine kingdom. Quercetin (QUE) is one of the flavonoids which have been very well researched. Quercetin and its derivates are the most widely distributed flavonoids, generally in vegetables, fruit, dry fruits, drinks, Chinese herbal medicine. It is confirmed that they have a broad spectrum of pharmacological activities, including cardiotonic activities, vasodilatation, blood pressure reduction, anti-inflammatory, anti-oxidation and free radical scavenging, inhibition of platelet aggregation, protection against ischemia reperfusion injury, immunological enhancement, anti-tumor, anti-mutation, antibiosis, anti-virus, analgesia and so forth. However, they have a low bioavailability, short half life and low potency, which hampered their exploitation and utilization.Polymethoxylflavonoids are the methylation derivates of polyphenolflavonoid, which have obvious pharmacokinetics characteristics and pharmacodynamics advantages.However, there is no breakthrough in the extraction technology of natural polymethoxylflavonoids up to now. The yield of natural polymethoxylflavonoids is limited and the price is very high, which hindered the research. So we synthesized 3,3',4',5,7-pentamethylquercetin (PMQ) which is of high purity and low price. In this study, the effects of PMQ and QUE on isolated rat aortic rings and the possible underlying mechanisms involved in its vasodilation were investigated.Partâ… Effects of PMQ on Methoxamine Dose-response CurvesThe methods of isolated aortic rings are used to investigate the effect of PMQ and QUE on Methoxamine (METH) dose-response curves. The results showed that isolated rat aortic rings with intact endothelium, when pretreated with either PMQ or QUE, exhibited a dose-related decrease of contraction in response to METH. The pD′2 values for this effect were 5.62 and 4.78 for PMQ and QUE, respectively. The minimum concentration required for the effect is 1μmol·L^-1 for PMQ and 10μmol·L^-1 for QUE. While PMQ, at 10μmol·L^-1, shifted the EC50 for METH from 1.91μmol·L^-1 to 15.0μmol·L^-1 (P<0.05), in contrast, QUE shifted the EC50 for METH from 1.55μmol·L^-1 to 5.70μmol·L^-1 (P<0.05).Partâ…¡Effects of PMQ on endothelium-intact and endothelium-denuded rat aortic ringsPreparation of endothelium-intact and endothelium-denuded rat aortic rings, the endothelium-denuded rings were prepared using such method, gently rubbing the intimal surface of the vessels with the needles wrapped with cotton, successful removal of the endothelium was demonstrated by the lack of a relaxant response to acetylcholine(100μmol·L^-1) at the beginning of each experiment. The results showed that PMQ 3,10,30μmol·L^-1 can relax the endothelium-intact and endothelium-denuded rat aortic rings, and the Emax values (percentage of relaxation) of the relaxant effect of PMQ 3,10,30μmol·L^-1 for the intact and denuded rings pre-contracted with METH were significantly different (40.0%±4.2% vs 22.1%±3.8%, 78.0%±4.4% vs 62.7%±9.9%, 90.9%±3.4% vs 70.6%±6.4%, P<0.05, respectively), similarly, QUE 10, 30, 100μmol·L^-1 can relax the endothelium-intact and endothelium-denuded rat aortic rings, The Emax values (percentage of relaxation) of the relaxant effect of QUE 10, 30, 100μmol·L^-1 for the intact and denuded rings pre-contracted with METH were significantly different (43.2%±11.3% vs 27.0%±4.7%, 70.6%±4.9% vs 45.9%±3.5%, 97.0%±4.1% vs 56.4%±4.8%, P<0.05, respectively).Partâ…¢Analysis of the Mechanisms underlying the vasorelaxant action of PMQ on the isolated rat aortic rings1. Effects of PMQ(1¹00μmol·L^-1) on 30,80 mmol·L^-1 KCl-induced contraction The methods of isolated aortic rings are used. A sustained contraction in response to 30 and 80 mmol·L^-1 KCl was obtained and PMQ was applied cumulatively (1¹00μmol·L^-1). The results showed that PMQ can obviously relax 30 mmol·L^-1 KCl depolarized rings, but less effect on 80 mmol·L^-1 KCl depolarized rings, compare the PMQ-dose response curves, the latter was shifted upward and the maximal relaxation response was decreased (P<0.05).2. Effects of several K+ channel blockers on PMQ-induced relaxation The methods of isolated aortic rings are used, investigate the effect of ATP-sensitive K+ channel blocker (Glibenclamide), voltage-dependent K+ channel blocker (4-AP) and Ca-activated K+ channel blocker (TEA-Cl) on PMQ-induced relaxation, the preparations with endothelium were contracted with METH (30μmol·L^-1), 15min after being incubated with one of the following drugs: Glibenclamide (10 mmol·L^-1), 4-AP (1 mmol·L^-1), TEA-Cl (5 mmol·L^-1).The results showed that pre-incubation of the rings with TEA-Cl produced a rightward displacement of the concentration-response curves for PMQ, and can reduce the maximal relaxation of PMQ 10μmol·L^-1 from 54.1±5.5% to 21.8±4.7%. On the other hand, Glibenclamide and 4-AP did not alter the relaxant effect of PMQ.3. Effects of PMQ on the two components of METH-evoked contraction The methods of isolated aortic rings are used. After equilibrium in normal K-H solution, the preparations were washed with Ca2+ free K-H solution 4⁵ times and left for 20 min before application of METH (30μmol·L^-1), and the first contractile response is intracellular calcium-dependent component of METH-induced contraction, then recover its Ca2+ contraction 2.5 mmol·L^-1 (administration of 50μl 250 mmol·L^-1 CaCl2), and the second contractile response is extracellular calcium-dependent component. Compare the tension of 2 components before and after the administration of PMQ (calculate the ratio of intracellular calcium-dependent and extracellular calcium-dependent component).The results showed that 1, 3μmol·L^-1 PMQ can not inhibit the extracellular calcium-dependent component and intracellular calcium-dependent component of METH-induced contraction, and 10μmol·L^-1 PMQ can obviously inhibit the former (P<0.01), while 30μmol·L^-1 PMQ can inhibit both (P<0.01, P<0.05, respectively).In conclusion:1. It is concluded that PMQ and QUE exhibited a dose-related decrease of contraction in response to METH, and reduced the maximal contraction.2. PMQ and QUE can relax the endothelium-intact and endothelium-denuded rat aortic rings. PMQ was found to be a significantly more potent vasorelaxant than QUE.3. PMQ can obviously relax 30 mmol·L^-1 KCl depolarized rings, but less effect on 80 mmol·L^-1 KCl depolarized rings.4. Neither ATP-sensitive K+ channel nor voltage-dependent K+ channel contributed to the relaxant response of PMQ on isolated rat aortic rings. While the activation of Ca-activated K+ channel contributed to PMQ-induced relaxation.5. Besides, PMQ may directly inhibit both the extracellular calcium-dependent component and intracellular calcium-dependent component of METH-induced contraction,and the latter was more potent than the former.