Extraction And Isolation Of Red Ginseng Saponins And Their Protective Effect On Improving Cardiac Hypertrophy In Rats

Objective:Optimize the extraction and separation techniques for total saponins from red ginseng,in order to increase the purity and yield of the total saponins.Construct different methods for determining the individual saponins in the total saponins,investigate their saponin composition,establish a fingerprint of the total saponins,determine component stability,and explore their protective effects on rats with hypertrophic cardiomyopathy.This study aims to provide scientific data for the application of red ginseng.Methods:1.Take the absorbance value as the index to investigate the extraction solvent,extraction time,solvent multiple and extraction temperature,and on this basis,the response surface experiment is carried out to optimize the extraction process of red ginseng saponins;Furthermore,the above indicators were used to investigate and determine the effects of two factors,sample loading volume and elution multiplication,on the separation and purification of total saponins.2.The chromatograms and chromatographic data of 12 batches of red ginseng saponins were obtained by HPLC method,and the fingerprint of 12 batches of red ginseng saponins was analyzed by traditional Chinese medicine chromatographic fingerprint similarity evaluation software（2012 edition）,and the content of 12 saponins in red ginseng saponins was determined by HPLC.3.The red ginseng saponins extracted and isolated above were administered at high,medium and low doses of gavage,and after a total of 30 days of gavage administration to SD rats,isoprenaline was administered continuously for 11 days by isoprenaline back subcutaneous multi-point injection to establish a myocardial hypertrophy model,the last dose was anesthetized after the last dose,the levels of key cardiac enzymes（CK-MB,LDH,cTnT）and oxidative stress（MDA,SOD）markers in serum were measured using ELISA kits,the degree of myocardial hypertrophy and fibrosis in rats was observed by HE and Masson staining,and finally immunohistochemistry and Western were used The blot method detected the expression changes of apoptosis-related proteins Caspase-3,Bax and Bcl-2 in cardiomyocytes.Results:1.According to the results of single factor and response surface experiments,the extraction process of red ginseng saponin was 85%ethanol,40 times solvent,95℃,and extraction time 4 h.The separation method determined according to the univariate experiment was to extract the extract with a sample concentration of 0.139 g/m L,a loading volume of 125 m L,and an elution volume of 3 column volumes.The purity of the red ginseng saponin obtained after extraction and separation was about 80%,and the yield was5.84%.2.The fingerprint of 12 batches of red ginseng saponins was established,12 of them were identified.The contents of 12 ginsenosides（Rg₁,Re,Rf,Rg₂,Ro,Rb₁,Rc,Rb₂,Rb₃,Rd,Rg₃,Rh₂）in the total saponins of red ginseng were determined,among which the content of ginsenoside Rb₁was the highest,and the total content of 12 ginsenosides accounted for 69.93%of the total saponins.The similarity between 12 batches of red ginseng saponin samples and the control map was greater than 0.90.3.The in vivo experimental results showed that compared to the blank group,the expression levels of MDA,CK-MB,LDH,and cTnT in the model group increased,while SOD expression decreased.When compared with the ISO group,various doses of total ginsenosides from red ginseng showed different degrees of improvement,and the inhibitory effect on myocardial enzyme release was better in the medium and high dose groups than in the low dose group.HE and Masson results showed that compared to the model group,the low dose group of total ginsenosides from red ginseng did not have a significant effect on improving myocardial damage and fibrosis,but the medium and high dose groups significantly reduced the necrotic area and collagen fiber area,and the morphology of myocardial fibers and cells in the high dose group of total ginsenosides from red ginseng tended to be similar to that of the blank group.The results of immunohistochemistry and WB experiments showed that total ginsenosides from red ginseng promoted Bcl-2expression and inhibited Caspase-3 and Bax expression,especially in the regulation of Bax and Caspase-3 expression.The low dose group of total ginsenosides from red ginseng had almost no effect on inhibiting Bax expression,but had an improved effect on promoting Bcl-2 expression and inhibiting Caspase-3 expression.However,both the medium and high dose groups exhibited better intervention effects.Conclusion:1.The optimized extraction process for total saponins from red ginseng is more stable and yields a higher extraction rate.2.The established determination method for total saponins is accurate and reliable,and the purified total saponins from red ginseng have better quality and higher purity.3.Total saponins from red ginseng exert a protective effect on rats with hypertrophic cardiomyopathy,which is related to the inhibition of cell apoptosis.