Study On Hepatoprotective Activity And The Metabolomics Of Erzhi Pills

1 Literature researchThrough consulting relevant ancient and modern literature,the origin of Erzhi pills,pharmacological effects,clinical applications,metabolomics and its application in traditional Chinese medicine research were reviewed and analyzied.2 Pharmacodynamic study on Erzhi pills of hepatoprotective activity componentsPolyamide column chromatography was used to separate the ethyl acetate of Erzhi Pills.Respectively,30%ethanol,50%ethanol,70%ethanol,95%ethanol was used to elution in order to obtain four hepatoprotective active ingredient groups,named EZW-1,EZW-2,EZW’3,EZW-4.2.1 Protective effect of active ingredients group in liver protection from Erzhi Pill on hepatocyte injury induced by CCl₄ or H₂O₂.L-O2 cells,the hepatocyte line,were incubated,and then injured by CCl₄ or H₂O₂.The levels of AST,ALT,MDA,SOD and GSH-Px in cultural supernatants were detected.Cell viability was assayed by MTT reduction method.The CCl1-induced elevation of MDA content in hepatocytes,increased AST and ALT levels in the supernatant of hepatocytes,and loss of SOD and GSH-Px activities were remarkably attenuated by EZW-1,EZW-2,EZW-3,EZW-4(0.32～40 mg·L^-1).EZW-2 is the best.The H₂O₂-induced elevation of ALT and AST levels in the supernatant of hepatocytes,increased MDA content of hepatocytes,and loss of SOD and GSH-Px activities were significantly attenuated by EZW-1,EZW-2,EZW-3,EZW-4(0.32～40 mg·L^-1).EZW-2 is the best.EZW-2,EZW-3 directly protects hepatocytes from in vitro injury induced by CCl₄ or H₂O₂.This might be associated with its anti-oxidative activity.2.2 Experimental study of the active ingredients group in liver protection from Erzhi Pill on acute hepatic injury induced by CCl₄ in mice.Fifteen Kunming mice were randomly divided into fifteen groups:the normal group,the model group,bifendate group(150 mg·kg^-1),EZW-1 of high,medium and low dose group(19.8,13.2,6.6 g·kg^-1),EZW-2 of high,medium and low dose group(19.8,13.2,6.6 g·kg^-1),EZW-3 of high,medium and low dose group(19.8,13.2,6.6 g·kg^-1),EZW-4 of high,medium and low dose group(19.8,13.2,6.6 g·kg^-1).The treatment groups were orally administered once per day for 7 d separately,whereas the normal and model groups were orally administered with saline.Except normal rats,all the other mice were injected intraperitoneally CCl₄ 20 mL·kg^-1 once.The rats were sacrificed 16 h after CCl₄ administration.Serum and liver samples were collected for analysis.The acute hepatic injury model was prepared by CCl₄ injected intraperitoneally.Then,the therapeutic effects of EZW-1,EZW-2,EZW-3,EZW-4 on the model were evaluated by the activity determination of serum alanine aminotransferase and aspirate aminotransferase（ALT and AST）,superoxide dismutase（SOD）and the content of malondialdehyde（MDA）in liver,and the hepatic pathohistological changes following the treatment-The activities of ALT and AST and the MDA content in liver was significantly increased and the activity of SOD was largely inhibited in the animals of modeling group.Following the treatment with AIEP,ALT and AST activities and MDA content were significantly reduced and SOD activity was obviously increased in the mice of treatment group.Furthermore,EZW-1,EZW-2,EZW-3,EZW-4 could ameliorate the hepatic pathological changes.EZW-2 is the best.EZW-2,EZW－3 have protective effects on acute hepatic injury induced by CCl₄ in mice,and are the effect of the liver protecting active sites.3 Metabolomics study of Erzhi Pills on acute hepatic injury induced by CCl₄ in rats3.1 Urine metabolomics study of Erzhi Pills on acute hepatic injury induced by CCl₄ in Rats.To observe urinary metabolic changes of acute hepatic injury induced by CCl₄ in rat and the regulating action of Erzhi Pills（EZW）on abnormal metabolism,and to find the potential biomarkers.To explore mechanisms of Erzhi Pills in treatment of acute hepatic injury induced by CCl₄.Methods Rat models of acute hepatic injury was established with CCl₄,and rats were divided into 4 groups,control group,model group,preventive treatment group and treatment group.Endogenous metabolites of four groups were determined with RRLC-Q-TOF/MS.Pattern recognition methods including PCA,PLS-DA,OPLS-DA were applied to analyze multivariate data,and T－test was used in significant statistical analysis.In pattern recognition,model group could be distinguished from 3 other groups.Twenty-seven ions which had a significant contribution to the classification from rat urine metabolic profiles of model group were screened further,fourteen potential metabolic biomarkers were identified qualitatively.These endogenous substances of the treatment group rats in vivo had varying degrees of recovery,and seven metabolic pathways were identified using ingenuity pathway analysis（IPA）·A metabolomic feature network of EZW to protect against acute hepatic injury induced by CCl₄ about urine was constructed.Changed metabolities can be certainly recovered by Erzhi Pills,and the treatment effect of Erzhi Pills can be connected with the regulation of 7 related metabolic pathways.3.2 Bile metabolomics study of Erzhi Pills on acute hepatic injury induced by CCl₄ in Rats.To observe urinary metabolic changes of acute hepatic injury induced by CCl₄ in rat and the regulating action of Erzhi Pills（EZW）on abnormal metabolism,and to find the potential biomarkers.To explore mechanisms of Erzhi Pills in treatment of acute hepatic injury induced by CCl₄.Methods Rat models of acute hepatic injury was established with CCl₄,and rats were divided into 4 groups,control group,model group,preventive treatment group and treatment group.Endogenous metabolites of four groups were determined with RRLC-Q-TOF/MS.Pattern recognition methods including PCA,PLS-DA,OPLS-DA were applied to analyze multivariate data,and T-test was used in significant statistical analysis.In pattern recognition,model group could be distinguished from 3 other groups.Twenty-six ions which had a significant contribution to the classification from rat urine metabolic profiles of model group were screened further,nine potential metabolic biomarkers were identified qualitatively.These endogenous substances of the treatment group rats in vivo had varying degrees of recovery,and four metabolic pathways were identified using ingenuity pathway analysis（IPA）.A metabolomic feature network of EZW to protect against acute hepatic injury induced by CCl₄ about bile was constructed.Changed metabolities can be certainly recovered by Erzhi Pills,and the treatment effect of Erzhi Pills can be connected with the regulation of 4 related metabolic pathways.IPA is an efficient procedure for drug target identification.It can guide testable predictions,provide insights into drug action mechanisms and enable us to increase research productivity.