Mechanism Of Yinchenhao Decoction On Promoting Bilirubin Metabolism Based On Transporters And Metabolic Enzyme

Objective:In this paper,the classic traditional Chinese medicine Yinchenhao Decoction?YCHD?was used as a model drug to establish a pharmacokinetic study method for the active constituents of traditional Chinese medicine compound with phase II glucuronic acid binding reaction as the main metabolic pathway.Based on the activity determination of key enzymes and transporters of bilirubin metabolism,the effects of YCHD,and its active constituents?genipin,emodin,etc.?on the metabolism of bilirubin were studied,reveals the correlation between choleretic effect of YCHD and glucuronidation process of active ingredients or the formation of glucuronic acid conjugates,thereby elucidating the material basis and mechanism of its liver protection.Methods:1.Based on the analytical technique UPLC-DAD method,14 kinds of medicinal ingredients in the YCHD?geniposidic acid,scandoside methyl ester,chlorogenic acid,genipin-1-?-D-gentiobioside,caffeic acid,geniposide,4-hydroxyacetophenone,crocin-I,crocin-II,aloe-emodin,rhein,emodin,chrysophanol,and physcion?were determinated,to lay the foundation for the in vivo metabolic process and efficacy of the main active ingredients of YCHD.2.Using UPLC/Q-TOF-MS technology,the prototype of medicinal ingredients and phase II metabolites in plasma,bile,feces and urine of rats with oral YCHD were identified.On this basis,UPLC-MS/MS analysis method and sulfatase enzymatic hydrolysis technique was adopted to investigate the difference of pharmacokinetics between the prototype of YCHD and the phase II metabolite in normal and cholestasis rats to study on the material basis of YCHD in the treatment of cholestasis.3.Using the way of prophylactic administration,and reference to the clinically recognized positive drug UDCA for the treatment of cholestasis,three oral doses?6.0,9.0,12 mg/kg?of YCHD on ANIT-induced cholestasis model rats were investigated.The expression of the drug-metabolizing enzyme UGT1A1 and other 9 transporters?MRP2,BSEP,OCT1,NTCP,MATE1,MDR1/Pgp,OATP1A1,OATP1A2,OATP1A4?in the liver of each group were studied by LC-MS/MS.Combined with serum ALT,AST,TBIL,DBIL,TBA content and liver pathological biopsy results,the comprehensive evaluation of the effect of YCHD on ANIT-induced cholestasis model rats was conducted.4.Establishing an in vitro glucuronic acid binding reaction system for bilirubin,and verifying the reaction system with the recognized bilirubin glucuronic acid binding inhibitors erlotinib,sorafenib,atazanavir,on the basis of this reaction system,the inhibition of bilirubin glucuronic acid binding by the main phase II metabolites of YCHD?genipin and emodin?in the body was investigated to verify its effect on the metabolism of bilirubin.5.The cholestasis model induced by ANIT by two oral doses?40,60 mg/kg?of genipin and emodin was investigated by means of preventive drug administration with reference to the clinically recognized positive drug UDCA for cholestasis.The therapeutic effect of YCHD on cholestasis rats was studied by determining the drug metabolism enzyme UGT1A1 and 9 other transporters?MRP2,BSEP,OCT1,NTCP,MATE1,MDR1/Pgp,OATP1A1,OATP1A2,OATP1A4?in the liver of each group,combined with the serum levels of ALT,AST,TBIL,DBIL,TBA,the effect on bile flow rate,and the results of liver pathological section,the effect of genipin and emodin on the ANIT induced cholestasis model rats was comprehensively evaluated.Results:1.The results showed that the linearity,precision,stability and sample recovery rate of UPLC-DAD quantitative method were all in accordance with the requirements.It can be used for the quantitative analysis of 14 active ingredients in YCHD,and the content of 14 components in YCHD?water decoction,1g crude drug/ml?was 42.6?g/m L of geniposidic acid,60.0?g/m L of scandoside methyl ester,280.1?g/m L of chlorogenic acid,and 2242?g/m L of geniposide,631.7?g/m L of caffeic acid,9274?g/m L of genipin-1-?-D-gentiobioside,79.7?g/m L of 4-hydroxyacetophenone,85.5?g/m L of crocin-I,0.8?g/m L of crocin-II,68.3?g/m L of aloe-emodin,260.0?g/m L of rhein,23.6?g/m L of emodin,10.8?g/m L of chrysophanol,and 2.3?g/m L of physcion.2.Seven prototype compounds in oral YCHD rats were detected by UPLC-Q/TOF-MS such as rhein,scandoside methyl ester,geniposide,genipin,caffeic acid,4-hydroxyacetophenone,emodin,and 19 metabolites,including 8 phase II metabolites,such as glucuronic acid conjugate of rhein,sulfuric acid conjugate of4-hydroxyacetophenone,and glucuronic and sulfuric acid combination of genipin,emodin,caffeic acid.Methodological validation of UPLC-MS/MS detection methods for 11 prototype compounds before enzymatic hydrolysis and 4 phase II metabolites after enzymatic hydrolysis,including specificity,linearity,precision,accuracy,extraction recovery,matrix and stability,the results are in line with the requirements.Using the above detection methods,the pharmacokinetics of eight active ingredient prototypes and four phase II metabolites after enzymatic hydrolysis in normal and cholestasis rats were investigated.The results showed that the pharmacokinetic behavior of phase II metabolites of rhein,emodin and genipin in normal and cholestasis rats was significantly different.In cholestasis rats,the AUC_?0-??values of rhein and emodin decreased by 74.7%and 64.4%compared with normal rats.K_a and K_e data showed that the absorption and metabolism of the rhein and emodin phase II metabolites in the cholestasis rats were significantly inhibited,similar to the metabolic behavior of the prototype drug.However,the AUC_?0-??and C_maxvalues of genipin increased by 241.1%and 97.6%,respectively,compared with normal rats.Overall,comparison of K_a and K_edata showed that phase II metabolites of genipin in cholestasis rats are more abundantly absorbed and metabolized than normal rats,exhibiting the same pharmacokinetic behavior as the prototypes of geniposidic acid and geniposide,but shows a clear difference with other phase II metabolites and prototype pharmacokinetics.3.The efficacy of YCHD in the treatment of cholestasis showed that compared with the normal control group,the ANIT model group showed significant pathological characteristics of cholestasis,including significantly elevated serum markers of ALT,AST,TBIL,DAIL,and TBA,hepatocyte damage observed by histopathological sections,decreased bile flow,and decreased expression of hepatic drug enzyme UGT1A1 and transporters MRP2,BSEP,OCT1,NTCP,MDR1,OATP1A1,OATP1A2,OATP1A4.In the drug-treated group,the 12.0 g/kg dose of YCHD had the most obvious therapeutic effects on cholestasis rats,such as significant reduction in ALT and AST,TBIL,DAIL,and TBA decreased by 26.1%,29.4%,and 16.6%,respectively;bile flow rate increased from 0.18±0.03?L/min/100 g in the model group to 0.48±0.02?L/min/100 g,and hepatocyte injury was also significantly reduced;for phase II metabolic enzyme UGT1A1,compared with the model group,increased by 2.4 times in the high-dose YCHD group,and MRP2,BSEP,OATP1A2,OATP1A4,OCT1,NTCP increased by 2.4,1.6,1.6,1.3,1.8,and 2.0 times,respectively it showed the best therapeutic effect,while the medium dose?9.0 g/kg?and the low dose?6.0 g/kg?showed a tendency to decrease with the decrease of the dose,indicating that the dose-dependent effect of YCHD on cholestasis.4.The results showed that the kinetics of TBG formation in the UGT1A1incubation system was in accordance with the Hill equation when the bilirubin concentration was 0.25-2.00?M,and the kinetics of TBG formation in the RLM incubation system was consistent with Michaelis-Menten equation at the bilirubin concentration of 0.25-2.00?M.Using this incubation system,the inhibitory effects of the active constituents of YCHD and western medicine on the in vitro glucuronic acid binding of bilirubin were studied.The results showed that the IC₅₀ of atazanavir,sorafenib and erlotinib on bilirubin glucuronic acid binding were 2.807,0.899,and3.599?M,respectively,revealing a very strong inhibitory effect;The inhibitory rate of bilirubin glucuronic acid binding was only 7.6%at the concentration of 100?M,and it is believed that genipin has almost no inhibition on the binding of bilirubin glucuronic acid;while emodin,which is also the active ingredient of YCHD,has an IC₅₀ of 25.745?M for inhibition of bilirubin glucuronic acid binding,showing a strong inhibitory effect.5.40 mg/kg genipin showed the most obvious therapeutic effect on cholestasis rats.The serum levels of ALT,AST,TBIL,TBA and DBIL were decreased by 51.5%,39.4%and 36.0%,26.7%,35.5%,respectively,bile flow rate increased from 0.36±0.21?L/min·100 g to 0.92±0.36?L/min·100 g within 6 h,and liver pathological sections of rats also showed significant improvement of hepatocyte damage.Compared with the model group,40 mg/kg of genipin also had the greatest improvement in the attenuation of metabolic enzymes and transporters caused by cholestasis,making UGT1A1,MRP2,OCT1,OATP1A4,NTCP,OATP1A1,MDR1,BSEP,OATP1A2 levels increased by77.7%,103.2%,180.6%,123.5%,82.4%,41.1%,65.9%,66.2%and 57.0%,respectively;60 mg/kg of genipin and 40 mg/kg of emodin could also reduces the serum index of cholestasis rats,increases bile flow rate,improves liver cell damage,enhances the expression of metabolic enzyme UGT1A1 and some transporters,and shows a certain therapeutic effect;while 60 mg/kg of emodin had no statistically significant improvement in the above indicators and was poorly treated for cholestasis.Conclusion:This study confirmed that Yinchenhao Decoction has a therapeutic effect on ANIT-induced cholestasis in rats,and its mechanism relates to the enhancement of the bilirubin II phase metabolism enzyme UGT1A1 and drug metabolism transporters MRP2,BSEP,NTCP,OCT1,MDR1,OATP1A1,OATP1A2,OATP1A4 in the liver that promotes the metabolism of bilirubin,and improvement of liver cell damage.Quantitative and qualitative studies showed that the main components of Yinchenhao Decoction are geniposide,chlorogenic acid,caffeic acid,4-hydroxyacetophenone,emodin,rhein and so on.These components mainly undergo phase II metabolism in rats.The glucuronic acid conjugate of rhein,the sulfuric acid conjugate of p-hydroxyacetophenone,and the glucuronic acid and sulfuric acid combination of genipin,rhein,emodin and caffeic acid were detected in rats.In the cholestasis rats,the excretion of geniposide,geniposide,and genipin II metabolites is weakened,while emodin can rapidly undergo phase II metabolism after entering the rat,and in the cholestasis rats,the degree of its phase II metabolites absorption decreased,the rate of absorption increased,and the duration of action was extended.Combined the influence of emodin and genipin on glucuronic acid metabolism of bilirubin in vitro?emodin has a strong inhibitory effect on bilirubin in vitro glucuronide binding,and genipin has little inhibitory effect on bilirubin glucuronic acid conjugation in vitro?and the efficacy of these two components on cholestasis confirmed that emodin can directly act on UGT1A1,induce the expression of UGT1A1 in the body,and increase the expression of transporters MRP2,NTCP,OCT1,OATP1A4,promote bilirubin metabolism and ultimately improve cholestasis symptoms.However,the enhancement of UGT1A1 expression by genipin is the result of indirect regulation,and it can also increase the expression of the transporters MRP2,NTCP,OCT1,OATP1A1,OATP1A2,OATP1A4,MDR1,BSEP,achieve the efficacy of promoting bilirubin metabolism to treat cholestasis.