| Objective:By optimizing the preparation technology of trichosanthis drop pills,and establishing the quality standard of trichosanthis drop pills,so as to study the pharmacodynamic effect of trichosanthis drop pills on myocardial ischemia reperfusion injury in rats and elucidate the mechanism of trichosanthis drop pills.Method 1:Taking the accumulative value of hardness and roundness as the final evaluation index,the orthogonal optimization experiment was designed according to several single factors,such as the quality ratio of the drug and the matrix,the ratio of the matrix,the drop preparation and condensation temperature,the choice of the drop head,the drop distance and the drop rate.Method 2:HPLC-DAD?high performance liquid chromatography-diode array detector?method was used to establish the chemical fingerprint of 10 batches of trichosanthis drop pills.Uracil,hypoxanthine,guanosine and adenosine in trichosanthis drop pills were quantitatively analyzed by standard curve method.Method 3:Will choose qualified by SPF SD rats were randomly divided into 6groups,each group of 6,the control group,model group were given saline,trichosanthis dropping pill group were 0.2 g·kg-1,1.0 g·kg-1,2.0 g·kg-1,compound Salvia miltiorrhiza dropping pill group 85.05 mg·kg-1,continuous dosing 7 d,the last for 1 hour in the left coronary artery ligation in rats after ligation 120 min after 30 min solution preparation of myocardial ischemia reperfusion model.The changes of ecg were recorded and the contents of creatine kinase-mb,troponin-t and Myogiobin in plasma were measured.The pathological changes of myocardial tissue were observed under light microscope.Method 4:Put in advance-80?of heart kept in a refrigerator,cut into the appropriate weight round bottom tube,add weight of x 8 RIPA cracking liquid?RIPA Lysis Buffer,tissue rapid pyrolysis liquid?,homogenate slurry machine,ice stand for two hours after the centrifugal?12000 RPM,15 min?,take supernatant,boil 15 to 20min,and 20?save,add protein sample Buffer on x 1/4,get the samples.According to the instruction manual with good separation,enrichment,pull out a comb after electrophoresis liquid put panels in electrophoresis apparatus?40 min?gel inside the tank filled with electrophoresis liquid,and join in each slot on the corresponding sample liquid?blank as the marker,in turn,compound danshen dropping pill group and snakegourd fruit dropping pill low concentration group,fructus dropping concentration group,fructus markedly high concentration group,filling group,filling group?,external tank 2 gels electrophoresis liquid,electrophoresis?80 V,30 min?,until it ran out of the red mark,change the electrophoresis conditions to?120 V,70min?.The film was transferred after the glue was cut and then closed with 5%skim milk powder.Remove the PVDF membrane,rinse with TBST once,wash off the milk,then put two pieces of PVDF membrane protein face to back into the general anti-diluent solution?1:300?,overnight.The membrane was removed from the primary antibody and washed with TBST for 3 times?5min/time?.The membrane was added into the diluent of the specified secondary antibody?GAPDH,JNK1,ERK1/2,P38??1:6000?and incubated for 2h.After incubation,clean with TBST once?10min?,put in the Alpha supersensitive fluorescence detector,and add the luminescent solution 600 ul to the PVDF film to obtain the strip.Results 1:The optimal preparation process of trichosanthis drop pills was as follows:the quality ratio of the drug and the matrix was 1:1.5(g·g-1),the ratio of the matrix was PEG 4000:PEG 6000=1:5(g·g-1),the drop rate was 20 d·min-1,and the drop spacing was 7 cm.Results 2:The similarity of fingerprints of 10 batches of trilosanthis drop pills was 0.99?Mark peak matching?and 0.97?full spectrum peak matching?.The RSD values of precision,stability,repeatability and recovery were 2.24%,3.55%,4.23%and 4.44%respectively,which could meet the requirements of analysis.The contents of uracil,hypoxanthine,guanosine and adenosine in trichosanthis drop pills were0.06%?0.03%?0.11%?0.17%respectively.Results 3:Compared with the sham operation group,the ST segment elevation in the model group was more than 0.2mV,indicating that the modeling was successful.Compared with the model group,trichosanthis drop pills can effectively reduce the elevation of ST segment of electrocardiogram?P<0.01?during myocardial ischemia reperfusion injury in rats,reduce the plasma CK-MB,MYO and cTNT content in rats?P<0.01?,and improve the pathological changes of myocardial cells.Compared with compound salviae miltiorrhiza drop pills,the effect of trichosanthis drop pills on the elevation of the ST segment of electrocardiogram in the process of myocardial ischemia reperfusion injury in rats was more significant?P<0.05 or P<0.01?,and the plasma CK-MB,MYO and cTNT contents in rats were more effectively reduced?P<0.05 or P<0.01?,and the pathological changes of myocardial cells were improved.Results 4:Compared with the model group,the trichosanthis drop pills showed dose-dependent up-regulation of phosphorylation expression of ERK1/2?P<0.05?,and dose-dependent down-regulation of phosphorylation expression of P38 and JNK1?P<0.05?,indicating that trichosanthis drop pills play an anti-myocardial ischemia reperfusion injury role by regulating the phosphorylation of P38,JNK1,ERK1/2proteins in the MAPK signaling pathway.Conclusion:The preparation technology oftrichosanthis drop pills is reasonable,and the preparation of trichosanthis drop pills conforms to the relevant regulations and requirements of Chinese pharmacopoeia 2015 edition.The chemical fingerprint characteristics of trichosanthes trichosanthis drop pills and the quantitative analysis method of four components can be used for the quality control of trichosanthis drop pills.The trichosanthis drop pills has a better effect of anti-myocardial ischemia reperfusion injury in rats,and its mechanism of action is to play an anti-myocardial ischemia reperfusion injury role by regulating the P38,JNK1,ERK1/2 proteins and their phosphorylated expressions in the MAPK signaling pathway. |
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