Enrichment And Purification Process Of Total Glycosides,Iridoids And Saponins In Mongolian Medicine Chana-4 By Macroporous Resin And Its Quality Control

Objective In this study,the Mongolian medicine Chana-4 will be used as a guide,and the effective fraction containing total glycosides,iridoids and saponins will be prepared by macroporous resin technology,and quality control,pharmacodynamic studies will be carried out on it,in order to prepare the hepatoprotective effective parts of Mongolian medicine Chana-4 with definite curative effect and stable,controllable quality.Methods 1.Using UV-Vis spectrophotometry at the maximum absorption wavelengths of 228 nm,271 nm,and 589 nm,respectively,by examining the linear relationship,precision,stability,recovery,to establish the content determination method of total glycosides,iridoids and saponins in Mongolian medicine Chana-4.The contents of total glycosides,iridoids and saponins in Chana-4 raw powder and 70%ethanol extract will determined.2.Taking the content of total glycosides,iridoids,and saponins as the investigation index,the reflux extraction process of Mongolian medicine Chana-4 alcohol extract was optimized by single factor experiment combined with orthogonal experiment design.3.On the basis of determining the extraction process,taking the adsorption performance and desorption performance of macroporous resins on total glycosides,iridoids and saponins as the investigation index,static adsorption-desorption experiments will be used to investigate 4 different types of macroporous resins.4.Using the HPLC method,complete the research on the characteristic map of the effective parts of Chana-4,the chromatographic conditions are:chromatographic column Agilent Eclipse XDB-C₁₈（4.6 mm×250 mm,5μm）;the flow rate is 1 m L/min;the column temperature is 30℃;the detection wavelength is254 nm;the injection volume is 10μL;Acetonitrile（B）-0.1%phosphoric acid water（A）was used as the mobile phase for gradient elution.And by comparing the chromatographic peak retention time of the sample and the reference substance,the measured data were imported into the similarity calculation software（version 2012）of traditional Chinese medicine chromatographic fingerprints to determine their common components.Using UPLC-Q-Orbitrap-MS technology,with Waters ACQUITY UPLC HSS T3 C₁₈（2.1 mm×100 mm,1.8μm）as the chromatographic column;with 0.1%formic acid aqueous solution（B）-0.1%formic acid acetonitrile（A）for gradient elution;the column temperature was 30°C,the injection volume was 5μL,and the flow rate was 0.2 m L/min.Mass spectrometry conditions:electrospray ionization ion source（HESI）,scanning range m/z 100～1500,detection in positive and negative ion modes,respectively,capillary voltage 3.2 k V,MS resolution 70000,MS/MS resolution 17500,The positive spray voltage was 3.2 k V,the negative spray voltage was 3.0 k V,the capillary temperature was 320°C,and the assist gas flow rate was 15 Arb.The identification of unknown compounds used compound discovery 3.3,mz Cloud and mz Vault databases（https://www.mzcloud.org/）to analyze the chemical components of the effective parts of the Chana-4.Based on the HPLC method,by examining the linear relationship,precision,stability,and recovery rate,the simultaneous determination of the content of four index components,such as gentiopicroside,paeoniflorin,liquiritin,and ammonium glycyrrhizinate in the effective parts of Chana-4 was established.5.Finally,ninety mice were randomly divided into nine groups and each group contained ten mice:control group,model group,Hu Gan Pian group（HGP,0.6 g/kg）,CNST-Low group（CNST-L,0.43 g/kg）,CNST-Middle group（CNST-M,0.86 g/kg）,CNST-High group（CNST-H,1.72 g/kg）,CNSE-Low group（CNSE-L,0.215 g/kg）,CNSE-Middle group（CNSE-M,0.43 g/kg）,CNSE-High group（CNSE-H,0.86 g/kg）.The model group was injected intraperitoneally with D-Gal N（400 mg/kg）daily for 8 days.The treatment group was given intraperitoneal injection of D-Gal N（400 mg/kg）every day,and at the same time,the corresponding drug was given by intragastric administration,the model was established for 8 days,and the drug was administered continuously for14 days.After the administration,all mice were fasted for 12 hours,and the eyeballs were removed to take blood and liver tissue,the blood was centrifuged at 3000 r/min for 10 minutes,and the serum was taken,the automatic biochemical analyzer was used to detect ALT and AST in serum according to the instructions of the kit,to investigate the effect of the effective parts of Chana-4 on the activity of ALT and AST in mouse serum caused by D-Gla N.Results 1.The contents of total glycosides,iridoids and saponins in Chana-4were 29.25 mg/g,35.69 mg/g,and 21.72 mg/g,respectively.The contents of total glycosides,iridoids and saponins were 115.58 mg/g,223.23 mg/g,and 103.79 mg/g,respectively.2.According to the results of single factor screening,adopt 3 factors and3 levels of orthogonal experimental design,investigate the three factors of concentration multiple,solvent multiple,and extraction time,taking the content of total glycosides,iridoids and saponins as indicators,the optimal conditions for the extraction process were determined as follows:5 times the amount of 80%ethanol,reflux extraction for 3 h;under this condition,the total glycosides,iridoids and saponins contents are:123.88 mg/g,211.47 mg/g,111.34 mg/g.3.In the static adsorption-analysis experiment of macroporous resin,it was found that the AB-8 resin has better adsorption and desorption properties for Chana-4.Through the dynamic adsorption-analysis experiment,the optimal purification process was obtained as follows:the loading concentration is 0.032 g/m L,the diameter-to-height ratio of the loading resin is1:4,the loading volume is 0.61 BV,the loading p H is 4,the loading flow rate is 0.5 BV,the elution volume is 3.67 BV,and the elution flow rate is 0.5 BV,and the elution concentration is 90%.4.Take 10 batches of effective parts of Chana-4,inject samples according to the chromatographic conditions of the characteristic chromatogram,and establish the characteristic chromatogram of the effective parts of Chana-4,by comparing the chromatographic peak retention times of samples and reference substances,five common components including gentiopicroside,paeoniflorin,liquiritin,ammonium glycyrrhizinate and piperine were determined.Based on UPLC-Q-Orbitrap-MS liquid mass spectrometry technology,33 kinds of chemical components were preliminarily identified from the effective parts of Chana-4,mainly including flavonoids and their glycosides,iridoids,pentacyclic triterpenes,monoterpene glycosides;In the content determination results of the index components,the contents of four index components including gentiopicroside,paeoniflorin,liquiritin,and ammonium glycyrrhizinate were 78.95±1.27 mg/g,47.79±0.36 mg/g,22.34±0.48 mg/g,48.62±1.31 mg/g,respectively.5.The results of pharmacodynamic experiments showed that the effective parts of Chana-4 can significantly reduce the increase of ALT and AST levels in mouse serum caused by D-Gla N,and has a good hepatoprotective effect.Conclusions 1.Based on the extraction process research and macroporous resin adsorption technology,this subject carried out enrichment and purification process research on the effective parts of total glycosides,iridoids and saponins in the Mongolian medicine Chana-4 at the same time,for Mongolian medicine Chana-4 total glycosides,iridoids and saponins effective parts,explore their high content,high purity optimization technology,the total content of the three increased from 2%to 4%in the original raw powder to 80%.2.At the same time,HPLC and UPLC-Q-Orbitrap-MS liquid mass spectrometry technology were used to establish the characteristic map of the effective parts of Chana-4 and the determination method of the index component content,and analyze the main chemical components contained in it,a total of 33chemical components were preliminarily identified,so that a comprehensive quality control was carried out on the hepatoprotective effective parts of the Mongolian medicine Chana-4.3.Pharmacodynamic experiments have proved that the effective fraction of Chana-4 prepared in this project has a good hepatoprotective effect.