Studies On Anti-myocardial Ischemia Effects, And Their Mechanism Of Total Glycosides Of Ranunculus Japoniucus And Determination Of The Total Flavonoid Content In It

This thesis consists of three parts. The first part is the research on anti-myocardialischemic effects of total glycosides of Ranunculus japoniucus （TGRJ）. The second part isthe research on the effect of TGRJ on intracellular calcium dependent or extracellularcalcium dependent. The third part is the research on the method for determining totalflavonoids contents of TGRL.Part I Research on anti-Myocardial Ischemic Effect of TGRJ.Section I Protective Effect of TGRJ on Myocardial Ischemic-ReperfusionInjury in Isolated Rat HeartsObjectives: To study the protective effect of TGRL on myocardialischemic-reperfusion injury in isolated rats hearts. Methods: The SD rats were randomlydivided into normal control group, ischemia-reperfusion group, TGRJ in0.05,0.10,0.20mg/ml group. The ischemia-reperfusion injury model was built using Langendroff isolatedrats hearts perfusion system which had been ischemiaed for30min and reprimed for40min. The indexes of heart function such as heart rate（HR）, left ventricular systolicpressure（LVSP）, left ventricular systolic pressure and maximum rate of rise （+dp/dtmax）, leftventricular diastolic pressure and maximum rate of descent（-dp/dtmax） and coronaryflow（CF） before ischemia and later at1,5,10,20,30and40min after reperfusion wererecorded. The activity of LDH and CK at20,40min after reperfusion were determinated.The myocardial tissues were stained with chloride three phenyl tetrazole （TCC） and thepercentage of myocardial infarction area was calculated. Results: Compared to the control group, HR,LVDP, dp/dtmaxand CF of the I/R group were significantly decreased afterreperfusion1、5、10、20、30、40min, the LDH and CK levels were increased and the areaof myocardial infraction was58.78%. Conclusion: TGRJ has effects of lowering the heartrate and relaxing the coronary artery. Suggesting that it might be improved myocardialischemia and hypoxia by increasing the blood flow of coronary artery and alleviating themyocardial cell injury to protect the ischemia-reperfusion induced of myocardial injury.Section II Effect of TGRJ on Acute Myocardial Ischemia in ratsObjectives：To observed the effect of TGRJ on the ECG of acute myocardial ischemiainduced by pituitrin （Pit） in rats. Methods: Pit was injected into the rats through sublingualvein to build the acute myocardial ischemia model. Observing the peak of T-wave changingpercentage and the heart rate change of the model group and nifedipine group and50,150mg/kg dose groups of TGRJ at5s,15s,30s,60s,3min,5min and10min after injectingPit. Results: Compared to model group, TGRJ in150mg/kg dose group were reduced thechanges significantly of the peak of T-wave, inhibition of the slowing of heart rate causedby Pit. Conclusion: TGRJ have some protective effect of TGRJ on acute myocardialischemia induced by Pit in rats.Part II: Research on the Effect of TGRJ on Intracellular CalciumDependent/Extracellular Calcium DependentSection I The Study of the Effect of TGRJ on the Isolated Thoracic AortaRings in Rats by Intracellular Calcium Dependent/Extracellular CalciumDependentObjectives: To observed relaxing effects of TGRJ on isolated thoracic aorta rings inrats and discusses its influence on intracellular calcium or extracellular calcium. Methods:The effect of TGRJ （concentration is0.003,0.006,0.012mg/ml） on the contraction ofvascular smooth muscle induced by NE were observed using the method of isolatedthoracic aorta rings perfusion in rats. The same effect was observed under the condition ofincubation in Ca²⁺-free and provided activator Ca²⁺. Results: TGRJ can antagonize the effect of NE inducing the contraction of vascular smooth muscle. TGRJ has no effect on theNE mediated Ca²⁺release of vascular contraction, but has inhibited significantly on the NEmediated Ca²⁺influx of vascular contraction after provided activator Ca²⁺. Conclusion:The vascular relaxation caused by TGRJ is probably related to receptor-related Ca²⁺gateway and the inhibition of Ca²⁺influx.Section II The Study of the Effect of TGRJ on Vascular Smooth MuscleCells in Rats by Intracellular Calcium Dependent/Extracellular CalciumDependentObjectives: To observe the influence of TGRJ on vascular smooth muscle cells in ratsby intracellular calcium dependent and extracellular calcium dependent. Methods:â‘ Thecalcium in the vascular smooth muscle cells was marked with Fluo-3/AM fluorescenceprobe and observed the fluorescence intensity by inverted fluorescence microscope.â‘¡The intensity of calcium in cells was determinated and the influence of TGRJ on [Ca²⁺]ielevation induced by NE using fluorescence spectrophotometer.â‘¢The inhibitory effectof TGRJ on intracellular calcium was observed depending on the increase of [Ca²⁺]iinduced by NE in the case of the absence of extracellular Ca²⁺.â‘£In the case of normalcalcium, the influence of TGRJ on intracellular calcium concentration induced by NEfollowing the exhaustion of calcium-pool by using Thapsigargin （TpG） was observed. Theinhibitory effect of TGRJ on extracellular calcium depending on the increase of [Ca²⁺]iinduced by NE. Results:â‘ The qualitative analysis shows that TGRJ has reducedfluorescence intensity increase induced by NE.â‘¡The quantitative analysis shows that,TGRJ has inhibited intracellular calcium concentration induced by NE.â‘¢In the case ofCa²⁺-free, TGRJ has no effect on the NE mediated Ca²⁺release.â‘£In the case ofexhausting calcium-pool, TGRJ inhibites significantly on the NE mediated Ca²⁺influx.Conclusion: TGRJ inhibites on Ca²⁺influx, but it has no effect on Ca²⁺release. Part III: Research on the Method for Determination Total FlavonoidsContents of TGRJ.Objectives: To eatablish the method for determination total flavonoids content ofTGRJ and measure the total flavonoids. Method:The detection wavelength was determinate by full-wavelength scanning using rutin ascontrol. And the precision, stability, repeatability and recovery rate were investigated.Result: TGRJ and rutin have maximum absorptive length at360.5nm. By drawing standardcurve, the mass concentration of rutin has a good linear relationship with the absorbance inthe range of0.0081～0.0284mg/ml and r=0.9996. The RSD （%） of precision, stability,repeatability were0.22,0.34and2.73respectively. The total flavonoids content of TGRJwas44.15%. The average recovery rate is in the range of95%¹05%and RSD （%）=1.97.Conclusion: The method is convenient, precise and reliable, and it’s suitable indetermination of the content of total flavonoids in TGRJ.