Study On The Efficacy Difference And Related Substance Base Of Trichosanthis Fructus And Its Steamed Products

Objective:In this paper,we will make a comparative study on the efficacy difference and related substance base from two aspects: material basis and pharmacodynamics.To study the chemical constituents of Trichosanthis Fructus and its steamed products by infrared absorption spectrometry(IR),ultraviolet visible spectrophotometry(UV)and high performance liquid chromatography(HPLC),and to establish the chemical fingerprints of 10 batches of Trichosanthis Fructus and its steamed products.To study pharmacodynamics by using the rat myocardial ischemia reperfusion(I/R)model.To clearly define the difference in efficacy and related substances basis of Trichosanthis Fructus and its steamed products,and to provide a theoretical basis for the improvement of the method for the steaming of Trichosanthis Fructus,and also to guide the rational processing of Trichosanthis Fructus.Methods:1.Comparison of the spectroscopic studies of Trichosanthis Fructus and its steamed products: the chemical constituents of the qualified Trichosanthis Fructus were determined by IR,and the infrared spectra of the Trichosanthis Fructus and its steamed products were compared,and the effects of the steaming process on the main chemical composition categories of the Trichosanthis Fructus were analyzed,and the main components were deduced.Similarity evaluation of raw and infrared spectra and first derivative infrared spectra of Trichosanthis Fructus and its steamed products by using the similarity evaluation system for chromatographic fingerprint of Chinese medicine,and the common mode was established and the similarity was calculated.The principal component analysis(PCA)model and partial least squares discriminant analysis(PLS-DA)model were established by using the statistical software of SICAM-P 11.The score of scatter points and the value of variation importance(VIP)were obtained,and the contribution rate of each absorption peak to the difference of each sample of Trichosanthis Fructus was obtained.In accordance with the extraction process established in the early stage,the preparation of Trichosanthis Fructus and its steamed products for the test solution was prepared.The contents of total amino acids,total flavonoids and total saponins,reducing sugar and total sugar were determined by ultraviolet visible spectrophotometry.2.Study on HPLC fingerprint of Trichosanthis and its steamed products: 10 batches of Trichosanthis and its steamed products were selected and the HPLC-DAD was used to establish the fingerprint of Trichosanthis Fructus and its steamed products.The quantitative analysis of the characteristic components(5 hydroxymethyl furfural,vanillic acid,rutin,isoquercetin,quercetin and luteolin)was carried out and the method was carried out,and the verification of the methodology was carried out.Similarity evaluation of the HPLC fingerprints of Trichosanthis Fructus and its steamed products by using the similarity evaluation system for chromatographic fingerprint of Chinese medicine,and the common mode was established and the similarity was calculated.The principal component analysis(PCA)model and partial least squares discriminant analysis(PLS-DA)model were established by using the statistical software of SICAM-P 11.The score of scatter points and the value of variation importance(VIP)were obtained,and the contribution rate of the single chemical components to the difference between Trichosanthis Fructus and its steamed products was obtained.3.Study on the pharmacodynamic differences in myocardial ischemia reperfusion injury(MIRI)in rats of Trichosanthis Fructus and its steamed products:9 groups of SD rats were randomly divided into 9 groups(n=10):the sham operation group,the myocardial ischemia-reperfusion(I/R)model group,the Trichosanthis Fructus group and the steamed Trichosanthis group.A MIRI model was established by ligating the left anterior descending coronary artery(LAD)of 30 min and ligating 120 min.(1)The electrocardiogram(ECG)was continuously monitored by the standard II lead,taking ST segment elevation and cardiac cyanosis as the standard,the changes of electrocardiogram of myocardial ischemia reperfusion in each group were observed.(2)The biochemical indexes in plasma was determined by using the enzyme linked immunosorbent assay(ELISA)assay:the level of creatine kinase isoenzyme(CK-MB),myocardial myoglobin(MYO)and cardiac troponin T(c Tn T).(3)The area of heart ischemic risk area and infarct area in each group was compared according to the dyeing condition by double staining of Evans blue and chlorinated three phenyl tetrazolium chloride(TTC).(4)The pathological changes of myocardium were observed by hematoxylin eosin staining(HE)in the ischemic myocardium of rats,and the changes of myocardial tissue structure in rats were observed by light microscope.Results:1.(1)The results of spectroscopic studies:Compared with Trichosanthis Fructus,the contents of total amino acids and total flavonoids in the steamed Trichosanthis were reduced by 17.2% and 42.8%,respectively,and the content of total saponins,total sugar and reducing sugar increased by 4.3%,43.6% and 68.2% respectively,and the content of total sugar increased most,and the total content of total saponins changed the least.(2)The similarity of the original IR spectra of Trichosanthis and its steamed products is high,and the similarity of the first derivative infrared spectra is poor.It can be deduced from the original infrared spectrum that steaming method did not change the chemical composition too much.By calculating the VIP value of the peaks of each spectrum,7 VIP peaks are obtained,and the main spectral peaks of the variation between the Trichosanthis and their steamed products are found CC=? in 1456cm-1 and OC=? in 1726cm-1.2.Research results of chromatograph:(1)The characteristic fingerprint of Trichosanthis Fructus and its steamed products was established,and 6 characteristic peaks(5-hydroxymethyl furfural,vanillic acid,rutin,isoquercetin,quercetin and luteolin)were quantified.Compared with Trichosanthis Fructus,the content of 5-hydroxymethyl furfural in the steamed Trichosanthis was greatly increased,about 25 times that of the steaming,and the content of vanillin,rutin and isoquercetin increased by 26.3%,10%,25%,and the content of quercetin and luteolin decreased by 75.5%,35%,respectively.(2)There were 54 common peaks in the 10 batches of Trichosanthis and their steamed products,and the similarity between the 10 batches of Trichosanthis was 0.82 to 0.86,and the similarity between the 10 batches of the steamed Trichosanthis was 0.97 to 0.98.On the basis of the common model,the contents of the characteristic components of Trichosanthis and their steamed products were compared,and the changes of 5-hydroxymethyl furfural,vanilla acid,quercetin and luteolin were significant(P<0.01).3.Pharmacodynamic differences of Trichosanthis and their steamed products against MIRI:(1)Compared with the model group,Trichosanthis Fructus and its steamed products significantly inhibited the elevation of S-T segment during myocardial ischemia in rats(P<0.05 or P<0.01).Compared with Trichosanthis,the effect of Trichosanthis steamed products on the S-T segment elevation of electrocardiogram during myocardial ischemia injury in rats is poor.(2)Compared with sham operation group,the levels of plasma CK-MB,MYO and c Tn T increased significantly in model group(P<0.05 or P<0.01).Compared with the model group,the contents of CK-MB,MYO and c Tn T in the positive control group were significantly decreased,the difference was statistically significant(P<0.05 or P<0.01).Compared with Trichosanthis,the content of c Tn T in high and middle dose group of Trichosanthis was significantly increased(P<0.05 or P<0.01),and the content of MYO in high dose group and low dose group increased significantly.(3)Compared with the model group,the infarct size of the positive control group was significantly reduced(P<0.01).Compared with Trichosanthis steamed products,the high concentration of Trichosanthis could effectively reduce the myocardial infarction area(P<0.01),and had obvious protective effect on myocardial ischemia reperfusion in rats(P<0.05 or P<0.01),and was slightly worse than the compound salvia miltiorrhiza dropping pill group,but there was no statistical difference.(4)The results of light microscopy showed that compared with Trichosanthis,the arrangement of myocardial tissue in the steamed Trichosanthis was more evacuated,the muscle transverse fault was obvious,the inflammatory infiltration was more obvious than that in the non processed group,and the vacuolar change was significant.Conclusion:The chemical composition category of Trichosanthis and their steamed products have no obvious difference,and there are some differences in the content of chemical components,and the chemical composition of the steamed Trichosanthis is more concentrated.Trichosanthis Fructus and its steamed products have certain anti MIRI effects.Comparison of the same dose group phase,Trichosanthis Fructus is better than Trichosanthis steamed products and has a certain dose effect relationship.Therefore,although the Trichosanthis steamed products are easy to preserve and transport,their effects on MIRI decrease,and there are also differences in the material basis and pharmacodynamics.The results provide a theoretical basis for improving the steaming method of Trichosanthis,and also have guiding significance for promoting the rational processing of Trichosanthis Fructus.