Optimization Of Extraction Technology Of Effective Constituents From Rhizoma Smilacis Glabrae And Effect Of Intestinal Flora On Its Metabolism

Objective:Taking Rhizoma Smilacis Glabrae and the five dihydroflavonol compounds in R.Smilacis as the research object,the traditional ethanol extraction process and the new cloud point extraction process were compared.At the same time,fingerprint combined with a variety of chemometrics methods were used to screen the characteristic chemical components of R.Smilacis,distinguish different sources of medicinal materials and comprehensively evaluate the quality of Chinese herbal medicine.Using astilbin and its three isomers（neoastilbin,isoastilbin,neoisoastilbin）and engeletin,the relationship between intestinal flora and metabolism of effective components in R.Smilacis in vitro was studied based on the material basis of reducing uric acid in R.Smilacis.Methods:（1）The contents of neoastilbin,astilbin,neoisoastilbin,isoastilbin and engeletin in R.Smilacis were determined by HPLC.Taking the contents of five chemical components as evaluation indexes,the weight coefficients of each index were determined by analytic hierarchy process,and then the effects of extraction time,solid-liquid ratio and ethanol concentration on the process were investigated by orthogonal design.（2）Based on the ethanol extraction process,the surfactant was used as the extraction solvent instead of ethanol.The cloud point extraction method was used to optimize the extraction process on the basis of single factor experiment and response surface analysis.The effects of surfactant concentration,solid-liquid ratio,salt ion concentration and equilibrium time on the total content of five effective components in R.Smilacis were investigated,and the best cloud point extraction conditions were finally selected.（3）HPLC method,similarity analysis（SA）,cluster analysis（HCA）,principal component analysis（PCA）and chemometrics were used for analysis.Taking astilbin peak as reference peak,the HPLC fingerprints of 16 batches of samples were drawn.The similarity evaluation analysis was carried out by using the similarity evaluation software of chromatographic fingerprints of traditional Chinese medicine（2012.130723 Edition）.The common peaks were determined and the similarity was calculated.The cluster analysis and principal component analysis were carried out by SPSS 22.0 software,and then the effective parts of R.Smilacis were separated by D101 macroporous resin.（4）A HPLC method for the determination of effective constituents of R.Smilacis in intestinal flora was established.The effective constituents of R.Smilacis and the fecal bacteria liquid of normal rats were co-cultured in 37℃anaerobic environment in vitro by the method of fecal incubation in vitro.The contents of five effective components in the fecal bacteria liquid were analyzed and compared in 48 h.Results:（1）The optimal extraction conditions of R.Smilacis by ethanol were as follows:R.Smilacis powder passed through 60 mesh sieve,soaked in10 times of 60%ethanol for 8 h,ultrasonic extraction twice,30 min each time.（2）The results showed that the optimum extraction conditions of cloud point extraction technology of R.Smilacis were as follows:R.Smilacis powder passing through 60 mesh sieve,Genapol X-080 surfactant with mass concentration of 37.5 g·L^-1,solid-liquid ratio of 1:16,ultrasonic extraction for 30 min;the optimum enrichment conditions were as follows:Na Cl concentration of 0.6 mo L·L^-1,water bath temperature of 60℃,equilibrium time of 43 min.（3）There were 9 common peaks in the HPLC chromatograms of 16 batches of R.Smilacis from different areas in China,and the similarity of 16 batches of R.Smilacis was more than or equal to 0.90;the relative retention time RSD values of 9 common peaks were less than 1.0%,and the relative peak area RSD values of each common peak were larger.The results of cluster analysis are basically the same.The cumulative variance contribution rate of three principal components in the sample was 90.074%.After separation and purification by D101 macroporous resin,30%ethanol elution component was the effective part of R.Smilacis.（4）A HPLC method was established for the determination of neoastilbin,astilbin,neoisoastilbin,isoastilbin and engeletin in enterobacterial solution.The method was proved to be accurate and reliable with good specificity,precision,stability and repeatability.After 48 hours,the total contents of neoastilbin,astilbin and neoastilbin decreased by about 50%.Conclusion:The ethanol extraction and cloud point extraction conditions of R.Smilacis were optimized,and the best extraction process was selected.Then the quality control of R.Smilacis from different areas in China was carried out by HPLC combined with chemometrics method,and the main pharmacodynamic components of R.Smilacis were analyzed and evaluated.Finally,the metabolic effects of different sites of intestinal bacteria on R.Smilacis in vitro was preliminarily discussed.It provides a theoretical basis for the quality evaluation and development of R.Smilacis,and also provides a certain reference for the follow-up study of interaction between active components and intestinal flora in R.Smilacis.