Study On The Protective Effect Of Alcoholic Liver Injury And Metabolomics Of Erzhi Pills

In recent years the diseases caused by the alcohol are serious,more and more researches are involved in the prevention and treatment of alcohol-induced liver damage.Erzhi pill is composed of Ligustri Lucidi Fructus and Eclipta Herb,which has the efficacy of protecting liver and kidney.There is no report of study of Erzhi pill preventing alcoholic liver injury so far.Quality control of Erzhi pill was established,the active ingredients were identified,and the effects of Erzhi pill preventing alcoholic liver injury was explored in the article.The research mainly includes the following sections:Part one:Literature reviewBy means of reviewing relative domestic and overseas documents,the progress of alcoholic liver injury and recent application process of metabonomics in liver and kidney injury research were reviewed.Part two:Simultaneous Determination of four compounds in Erzhi pills by HPLCAn HPLC method for simultaneous determination of nuzhenide,specnuezhenide,wedelolactone and oleanic acid in Erzhi pills was established.The components was separated on DIKMA C18(200 mm×4.6 mm,5 ?m)using acetonitrile and 0.1%phosphoric acid solution as the mobile phase;The flow rate was 1.0 ml/min and the volume of injection was 20 ?L;the column temperature was maintained at 30 ? and the detective wavelength was set at 215 nm.RESULT showed that there were good liner relationships between the peak area and concentration at ranges of 2.008?80.32 ?g mL-1(r=0.9996),5.872?234.88 ?g·mL-1(r=0.9997),0.9?36 ?g mL-1(r=0.9999),13.24?529.6 ?g mL-1(r=0.9996)for nuzhenide,specnuezhenide,wedelolactone and oleanic acid,respectively;the average recovery rates of nuzhenide,specnuezhenide,wedelolactone and oleanic acid were 99.25%,98.70%,96.23%and 101.45%,respectively,with RSD less than 3%.Part three:Analysis of the chemical composition of Erzhi pill and its Components by rapid-resolution liquid chromatography-time of flight mass spectrometryChemical constituents of Eezhi pill and its Components were analyzed by rapid-resolution liquid chromatography-time of flight mass spectrometry(RRLC-TOF/MS).15 of the major chemical constituents of Erzhi pill were identified by RRLC-TOF/MS,12 of the major chemical constituents of Ligustri Lucidi Fructus were identified,and 7 of the major chemical constituents of Eclipta Herb were identified.The compositions were mainly iridoids,triterpenoids and flavonoid.This study laid the foundation of material basis for future research.Part four:Protective effect of alcoholic liver injury of Erzhi pillAfter 7 days of protection by giving medicine in treatment groups,the model of alcoholic liver injury in rats was established by giving alcohol ig per day for 10 days.At the end of the study,all rats were sacrificed.The serum ALT,AST,TG,ADH and hepatic SOD,MDA were measured respectively.Hepatic histopathology was observed by microscope.The result showed that,combined with the control group,Serum levels of ALT,AST,ADH and TG of the model group increased significantly(P<0.01),indicating that the model was established successfully;and the increasing levels of ALT,AST,ADH of all the treatment groups were inhibited(compared with model group,P<0.05 or P<0.01).The TG levels of Positive group and EZW-H group showed significant difference,combined with the model group.Combined with the control group,hepatic levels of MDA and SOD in model group increased or decreased significantly(P<0.01),the increasing levels of MDA in EZW-H and EZW-Z groups were inhibited(compared with model group,P<0.05 or P<0.01).The decreasing levels of SOD in the positive groap,EZW-H and EZW-Z groups were inhibited(compared with model group,P<0.05 or P<0.01).The histopathological analysis suggested that all of the treatment groups could significantly ameliorate pathological changes of hepatocytes,among the total,EZW-H group showed best.Water decoction of Erzhi pill was extracted in order by different solvents.After drying,petroleum leve,acetoacetate and n-butanol fractions were obtained,and dissolved by water.70 male Wistar rats were randomly divided into seven groups.The model of alcoholic liver injury in rats was established by giving alcohol ig per day for 28 days.The serum ALT,AST,TG,ADH,serum endotoxin,tumor necrosis factor-?(TNF-?),interferon-y(IFN-y),interleukin-18(IL-18)were assayed.And hepatic SOD,MDA were measured respectively.Hepatic histopathology was observed by microscope.The result showed that,combined with the control group,Serum levels of ALT,AST,ADH,TG,serum endotoxin,TNF-?,IFN-? and IL-18 of the model group increased significantly(P<0.01),indicating that the model was established successfully;and the increasing levels of ALT,AST,ADH,TG,serum endotoxin,TNF-a,IFN-y and IL-18 of all the treatment groups were inhibited(compared with model group,P<0.05 or P<0.01).Combined with the control group,hepatic levels of MDA and SOD in model group increased or decreased significantly(P<0.01),the increasing levels of MDA in the positive group,petroleum leve and acetoacetate groups were inhibited(compared with model group,P<0.05 or P<0.01).The decreasing levels of SOD in the positive groap,acetoacetate,n-butanol and raffinate groups were inhibited(compared with model group,P<0.05 or P<0.01).The histopathological analysis suggested that all of the treatment groups could significantly ameliorate pathological changes of hepatocytes.Part five:Metabolomics study of Erzhi pill on acute hepatic injury induced by alcohol in Rat urine and bile.The endogenous metabolite changes after alcoholic liver injury were studied,to explore potential biomarkers for explaining the mechanism of the protective and therapeutic effects of Exzhi pill.Wistar rats were randomly divided into 3 groups as follows:control group,model group and EZP group,the model of alcoholic liver injury in rats was established by giving the Red Star Erguotou.The samples of rat urine and bile were detected by rapid-resolution liquid chromatography-time of flight mass spectrometry(RRLC-TOF/MS)using electrospray positive/negative monitoring mode.Orthogonal partial least squared discriminant analysis(OPLS-DA)and ANOVA were associated to analyze Multivariate data.The model group was distinguished from control group,and EZP group by pattern recognition.According to the numerous of VIP,the most differentiating components were generated,and then t test of significant statistical analysis was applied.11 biomarkers(Hydrocinnamic acid,Uridine and so on)were identified:in rat urine,which were recoveryed in EZP group.By IPA,We find four liver damage metabolic pathways.10 biomarkers(Bile acids,amino acids,lysophospholipids)were identified in rat bile,which were recoveryed in EZP group.By IPA,We find eight liver damage metabolic pathways,which may be related to the protective and therapeutic effects of Erzhi pill.Metabolomics can not only help to predict the occurrence of alcoholic liver disease,but also provide the basis for monitoring metabolites of disease state.