Study On The Material Basis And Mechanism Of Active Ingredients Of Tibetan Medicine Chrysoplenium Nudicaule Bunge Against Intrahepatic Chilestasis Injury

Objection: Clarify the material basis and effects of the characteristic Tibetan medicine Chrysosplenium nudicaule Bunge ethyl acetate extract againstα-naphthylisothicoyanate(ANIT)-induced intrahepatic cholestasis(IC)injury mechanism.Methods:(1)Eight SD rats were randomly divided into the blank group and the administration group,with four in each group.Chrysosplenium nudicaule Bunge ethyl acetate extract(15.75 g/kg)was orally administered to rats in the administration group,and 0.5% CMC-Na solution of the same volume was administered to rats in the blank group,which was administered for 3 days,2 times a day,and last time was 1 hour Blood was collected from the portal vein of the liver,and rat-containing plasma and blank plasma were prepared separately.UPLC ultra-high-performance liquid was used to analyze the treated rats’ drug-containing,blank plasma,and bare stem golden waist ethyl acetate extract to determine the rats.Transition components in plasm;(2)Under the guidance of UPLC,the prototype components in the ethyl acetate extract of Chrysosplenium nudicaule Bunge were prepared using traditional chemical methods,and the structure was identified based on physical and chemical properties and modern Popography technology;(3)A single oral administration of 0.5% CMC-Na solution of CN1 and CN2 in mice at a dose of 2000 mg/kg and a gavage volume of 40ml/kg.The daily activity of the mice and their weight were recorded.After the observation period,the mice were dissected,the organ index of the main organs of each group of mice was calculated,and the serum levels of ALT,AST,ALP,TP,ALB,and BUN in the mice were measured,assessing the acute toxicity of CN1 and CN2.(4)Establish a mouse model of acute liver injury induced by ANIT(60 mg/kg),measure the levels of ALT,AST,ALP,TBA,DBIL,and TBIL in mouse serum,and detect SOD,MDA,GDH-Px in mouse liver homogenate Contents;HE staining was used to observe the pathological status of liver tissue of mice;Western Blot method was used to determine the expression of FXR,SHP,CYP7A1,NTCP,NF-κB protein in liver tissue of mice in each group.Result:(1)Using UPLC technology to analyze the chromatograms of Chrysosplenium nudicaule Bunge ethyl acetate extract,drug-containing serum,and blank serum,7 blood transition components were found,4 of which were prototype components,and the other 3 transition components may be metabolites or organism Endogenous small molecules produced by stress.(2)UPLC fingerprint-guided separation obtained three flavonoids CNs 1-3from the ethyl acetate extract,of which CN1 is a new compound whose struct ural identity is 5,5′-trihydroxy-3,4′,6,7-tetramethoxyflavone-2′-O-D-glucoside,nam ed chrysosplenoside I.CN2 and CN3 were identified as 5,5′-dihydroxy-3,4′,7-tri methoxyflavone-2′-O-D-glucoside and 5-hydroxy-3,6,7,4′,5′-pentamethoxyflavone-2′-O-D-glucoside(chrysosplenoside H).(3)Compared with the mice in the control group,the mice of the same sex in the two monomer administration groups had no obvious poisoning and no death during the observation period,and there was no significant difference in the trend of weight gain;There were no significant differences in the indexes;there was no significant change in serum ALP,ALB,TP,and BUN levels.The AST levels of male mice in the CN2 administration group were significantly increased(P<0.01).The ALT levels and AST levels of female mice were significantly higher in individual mice.There was no significant difference in the ALT level of the mice in the CN1 administration group and the AST level in the male mice,but there was a significant increase in AST levels in individual mice in the female administration group,but the difference was not statistically significant.(4)The two monomers and each dose group can significantly reduce serum ALT,AST,ALP,TBA,DBIL,and TBIL levels(P<0.01 or P<0.05),and increase SOD and GSH-Px levels in liver tissues(P<0.01 or P<0.05),reducing MDA levels(P<0.01 or P<0.05),and in a dose-dependent manner;liver tissue sections showed that the mice in the administration group had less inflammatory infiltration of liver tissue and liver damage was improved;the administration group Mouse liver tissues up-regulated FXR,SHP,and NTCP protein expressions,and down-regulated CYP7A1 and NF-κB protein expressions.Conclusion: Tibetan medicine Chrysosplenium nudicaule Bunge’s material basis for anti-IC liver injury may be 7 transitional components in blood(4 original components,3 metabolites);3 of these compounds(CN1,CN2,CN3)were identified and identified using traditional chemical methods),Which lays the foundation for subsequent drug safety and efficacy evaluation;the preliminary safety evaluation results of the original components CN1 and CN2 suggest that the LD50 of these two monomers is 4-5 g/kg,which is a low-toxicity drug.However,taking large doses has certain damage to the liver,and the liver toxicity of CN1 is lower than that of CN2.The monomers CN1 and CN2 have obvious protective effects on acute IC mice induced by ANIT.The mechanism of action is to inhibit the synthesis and recovery of bile acids.The liver’s uptake function of bile acids,inhibits the body’s lipid peroxidation,and reduces the inflammatory infiltration of liver cells.It is an effective component of bare stem golden waist to resist intrahepatic cholestasis.