The Effects Of Some Polyphenols Compounds And Their Polymethylether Derivates On The Normal And Oxidative Injured Rat Cardiac Muscles And Neonate Rat Myocardiocytes

Polyphenol compounds are widespread in many plants, and are active ingredients in the traditional Chinese drug, which are highly concerned in the medicine kingdom. The polyphenol compounds such as resveratrol(RES) and quercetin(QUE) are confirmed that they have a lot of biological activities, including blood pressure reduction, anti-oxidation, inhibition of lipid peroxidation, protection against ischemia-reperfusion injury, anti-cancer, anti-bacterial and so on. However, they have a low bioavailability, short half life and low potency, which hampered their exploitation and utilization.Polymethoxylflavonoids are the methylether derivates of polyphenolflavonoids, which have obvious pharmacokinetics characteristics and pharmacodynamics advantage. 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 pentamethyl-quercetin (PMQ), trans-trimethylresveratrol (trans-TMR) and cis-trimethylresveratrol (cis-TMR) that are high purity and low price. In this study, the effects of PMQ and QUE on physiological characteristics in normal or oxidative injury isolated rat cardiac muscle were investigated, and the anti-oxidation injury effect of QUE,PMQ,RES,trans-TMR and cis-TMR in neonate rat myocardiocytes were investigated.PartⅠThe Effects of PMQ on Physiological Characteristics in Isolated Rat Cardiac MusclesObjective: To study the influence of PMQ on the physiological characteristics and compare with quercetin. Methods: The effects of PMQ on contractile force, autorhyth- micity and effective refractory period (ERP) in isolated rat cardiac muscles were investigated. Results: (1) 0.1% DMSO has no obvious effects on above parameters. (2)Both PMQ and QUE at concentration of 3, 10, 30, 100μmol·L-1 can produce a positive inotropic effect in isolated rat papillary muscles, left atria and right atria (P<0.05); With the increased concentration, the positive inotropic effect increased. The positive inotropic effect of PMQ is weaker than the effect of QUE at the same concentration (P<0.05). (3) Both PMQ and QUE at concentration of 3, 10, 30, 100μmol·L-1 can increase the automatic contraction frequency of right atria. The positive chronotropic action was increased with the increased concentration. The positive chronotropic action of PMQ is weaker than the effect of QUE at the same concentration (P<0.05). (4) PMQ at concentration of 100μmol·L-1 can shorten ERP of left atria and papillary muscles (P<0.05); however QUE at concentration of 30, 100μmol·L-1 can extend the ERP of left atria and papillary muscles (P<0.05).PartⅡThe Anti-oxidative Injury Effects of PMQ in Isolated Rat Cardiac MusclesObjective: To investigate the effects of the anti-oxidative injury of PMQ and compare with quercetin. Methods: Inducing oxygen free radicals damage on rat isolated myocardial preparations by H2O2 (400μmol·L-1) and observing the anti-oxidative injury effects of PMQ and QUE. Results: H2O2 (400μmol·L-1) increased the contractile force at start and then decreased it and depressed the activity of SOD. QUE (30μmol·L-1) antagonized the negative inotropic effect of H2O2 in isolated left and right atria (P<0.01), but had no effect in isolated papillary muscles. PMQ (30μmol·L-1) antagonized the negative inotropic effect of H2O2 in isolated papillary muscles in some extent(P<0.05), but had no effect in isolated left and right atria. PartⅢThe Effects of Some Polyphenols Compounds and Their Polymethylether Derivates on the Oxidative Injury in Neonate Rat MyocardiocytesObjective: To investigate protective effects of QUE, PMQ, RES, trans-TMR and cis-TMR on hydroperoxide-induced myocardial injury. Methods: (1)Established the method to culture myocardiocytes of neonate rats; (2)prepared myocardiocytes-injury model induced by hydroperoxide; (3)determined cell viability by MTT. Results: After incubation with hydroperoxide (350μmol·L-1) for 12h, the viability rate of the myocardiocytes was significantly decreased (P<0.01). QUE,PMQ,RES,trans-TMR and cis-TMR all have the functions of anti-oxidative injury induced by hydroperoxide, and the minimal effective concentrations are 30,0.1,0.3,0.3 and 0.1μmol·L-1 respectively. The results suggest that myocardiocytes protective effect of PMQ, the polymethylether derivates of QUE, is more potent than QUE (about 2 order of magnitude). While the protective effect of trans-TMR is similar to RES,is slightly improved by cis-TMR.PartⅣThe Effects of PMQ on Intracelluar Free Calcium Concentration of Neonate Rat MyocardiocytesObjective: To observe the influence of PMQ on the intracelluar free calcium concentration in myocardiocytes. Methods: Intracelluar free calcium concentration([Ca2+]i) of neonate rat myocardiocytes were measured by fluorescent imaging Microscopy techniques. Results: (1) 0.1% DMSO has no obvious effects on [Ca2+]i. (2) 1μmol·L-1PMQ decreased the [Ca2+]i by 32±3%(P<0.01). However, the effect of 3, 10μmol·L-1PMQ on [Ca2+]i in myocardiocytes presented a rapid decline in short-term, then dramatically increased. The maximal decline amplitude were 48±1%, 42+3%(n=8,P<0.01) respectively and the peak increase were 42±7%,179±30% (n=8,P<0.01). (3)In rat myocardial cells oxidative damaged by H2O2, the [Ca2+]i showed a rapid increase forming a peak, then descended immediately, but still maintained a relatively high [Ca2+]i environment. (4) Myocardiocytes incubation 2h with 3μmol·L-1 PMQ before added H2O2 (350μmol·L-1), the dramatical increase of [Ca2+]i that induced by H2O2 was totally abolished.In conclusion:1. PMQ produced positive inotropic effect in isolated cardiac muscles, and can increase the autorhythmicity in right atria. The effect of PMQ is weaker than QUE at the same concentration on above parameters. QUE can extend the ERP, but PMQ has no obvious effects on ERP.2. PMQ (30μmol·L-1) can inhibit the negative force effect of H2O2 in papillary muscles caused dy H2O2 and increase the SOD activity in some extent.3.QUE,PMQ,RES,trans-TMR and cis-TMR could prevent the myocardial injury induced by H2O2. The protective effect of polymethylether derivates of polyphenols is significantly improved and PMQ is much more potent than QUE.4.Low concentration of PMQ (1μmol·L-1) reduced [Ca2+]i in myocardiocytes, high concentration of PMQ (3,10μmol·L-1) increased myocardiocytes [Ca2+]i. In H2O2 injured myocardiocytes, the [Ca2+]i increased. PMQ (3μmol·L-1) abolished the increase of [Ca2+]i in myocardiocytes.