Substance Of Polygonum Multiflorum Induced Hyperbilirubinemia Based On Bilirubin Metabolizing Enzyme And Transporters

Objective:In this study,Polygonum multiflorum（PM）and its active components were used as model drugs to establish natural inhibitors in vitro screening model of enzymes and transporters of bilirubin metabolism,which based on enzyme incubation and cell transport experiments.Combining the pharmacokinetic and pharmacodynamic experiments of PM in normal and pathological rat models,we investigated the correlation between the glucuronidation process of its active ingredients and its inhibitory effect on bilirubin metabolism,and to elucidate the material basis and mechanism of its adverse effects of hyperbilirubinemia.Methods:（1）Based on the analytical technique UPLC-DAD method,11 kinds of medicinal ingredients in PM（2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside,emodin,physcion,aloe-emodin,chrysophanol,rhein,emodin-8-O-β-D-glucoside,physcion-8-O-β-D-glucoside,resveratrol,gallic acid and catechin）were determinated,so as to provide foundation for further study on liver injury and pharmacokinetics of PM.（2）Different doses of aqueous extracts of PM（QE-PM）（6.3,12.6,25.2 g raw drug/kg）and alcohol extract of PM（AE-PM）（6.3,12.6,25.2,50.4 g raw drug/kg）were administered to normal rats for 60 consecutive days.The biochemical indices of ALT,AST,TBIL,DBIL,TBA,ALP andγ-GGT in serum and the expression levels of UGT1A1,the key enzyme in bilirubin metabolism,and nine transporters MRP2,BSEP,OATP1A1,OATP1A2,OATP1A4,NTCP,OCT1,MDR1 and MATE1 in liver were monitored,combined with the results of liver pathological sections,to evaluate the effects of QE-PM and AW-PM on liver injury in normal rats.（3）UPLC-MS/MS analysis method and sulfatase enzymatic hydrolysis technique was adopted to investigate the difference of pharmacokinetics between the prototype of AE-PM and the phase II metabolite in normal and pathological rats（ANIT and CCl₄）,and the expression levels of bilirubin metabolizing enzymes and transporters were analyzed in combination with pharmacokinetic parameters to study on the material basis of liver injury induced by PM.（4）The bilirubin in vitro glucuronidation reaction system and HEK-293T cell models overexpressing OATP1B1 and OATP1B3were established.Erlotinib,atazanavir,rifampicin,and cyclosporin A were selected as positive drugs for bilirubin in vitro glucuronidation and bilirubin uptake,respectively,to examine the inhibitory effect of the main active ingredient in PM on bilirubin glucuronidation and OATP1B1 and OATP1B3-mediated bilirubin uptake.Results:（1）All ingredients showed good linear relationship（R²≥0.9992）in the required concentration range,and the lowest limits of quantification were between 2.03 and 6.94μg/ml.The content of 11 active ingredients in of PM was 102.62 mg/g of2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside,31.94 mg/g of emodin,8.42 mg/g of physcion,1.44 mg/g of aloe-emodin,2.36 mg/g of catechin,8.98 mg/g of emodin-8-O-β-D-glucoside,1.24 mg/g of physcion-8-O-β-D-glucoside,5.16 mg/g of gallic acid,7.15mg/g of resveratrol,0.85 mg/g of chrysophanol,and 0.66 mg/g of rhein.The species and contents of each component in the 75%ethanol extract were significantly higher than those in the aqueous extract.（2）The results of liver injury by different extracts of PM showed that the expression levels of BSEP,MDR1 and OATP1A4 were up-regulated by1.3-,0.5-and 0.7-fold,respectively,in the high dose group（25.2 g/kg）of the QE-PM while all other indices did not show significant changes.The body weight growth of rats in AE-PM group C（25.2 g/kg）and group D（50.4 g/kg）was significantly inhibited,and the pathological sections of liver showed obvious pathological state.Moreover,the expression levels of ALT,ALP,TBA and DBIL in group D（50.4 g/kg）were increased by0.5-,0.7-,1.4-and 1.2-fold,but regarding the expression of metabolic enzymes and transporters,only oatp1a1,OATP1A2,and BSEP showed a trend of downregulation.That is,liver injury did not occur in normal rats with long-term administration of QE-PM at doses less than or equal to 25.2 g/kg,and liver injury occurred only with long-term administration of AE-PM at ultrahigh doses of 50.4 g/kg.（3）The methodological validation of the UPLC-MS/MS detection methods for 11 prototype compounds and 4phase II metabolites,including specificity,linearity,precision,accuracy,extraction recovery,matrix effect and stability,was performed,and the results met the requirements.The pharmacokinetics of 8 compound prototypes and 4 phase II metabolites in normal and pathological model rats were investigated using the established methods.The results showed that TSG（>80%）,emodin（>99%）and catechin（>70%）were overwhelmingly present in the animals as phase II metabolites,and resveratrol was converted into glucuronide and sulfate conjugates immediately after entering the body.Physcion(AUC（0-∞）and C_max values were 1.3 and 2.7 times higher,respectively,than in the normal group and are described similarly below),emodin（1.8 and 1.6 times）and its phase II metabolites（6.2 and 3.5 times）,emodin-8-O-β-D-glucoside（7.6 and 7.2 times）and catechin（0.12 and 1.8 times）in the CCl₄ pathological state exposure levels were higher.TSG（26.5 and 6.6-fold）and its phase II metabolites（30.43 and 20.17-fold）,aloe-emodin（11.7 and 1.2-fold）and gallic acid（2.3 and 0.4-fold）were higher in the cholestatic state.In contrast,the T_max values of all components in normal rats were smaller than in both pathological models.Correlation analysis of the AUC（0-∞）of 8 compound prototypes and 4 phase II metabolites with the expression levels of hepatic metabolic enzymes and transporters revealed that the AUC（0-∞）of TSG and aloe-emodin showed significant negative correlation with the expression levels of UGT1A1,BSEP,OATP1A4,OCT1,NTCP,MRP2 and MDR1.The AUC（0-∞）of emodin,physcion and resveratrol were positively correlated with the expression levels of OATP1A2,NTCP and MRP2.（4）The inhibition of the in vitro glucuronidation reaction of bilirubin by the main active ingredients in PM was investigated using a bilirubin in vitro glucuronidation reaction system.Resveratrol,TSG,emodin and aloe-emodin showed strong inhibition(IC₅₀<25μM)of in vitro glucuronide binding of bilirubin compared to atazanavir(IC₅₀=2.906μM)and erlotinib(IC₅₀=3.489μM).The methodologies for the determination of bilirubin in cellular samples was examined for specificity,linearity,precision,accuracy,extraction recovery,matrix and stability,and the results were satisfactory.HEK-293T cells overexpressing OATP1B1 and OATP1B3 were used for bilirubin uptake assay.The results showed that emodin,resveratrol and catechin had inhibitory effect on OATP1B1and OATP1B3-mediated bilirubin uptake at low concentrations（<15μM）compared with the positive drugs rifampicin(IC₅₀=20.35μM)and cyclosporine A(IC₅₀=0.893μM),but the inhibition was attenuated or even showed promotion with increasing concentrations.TSG had no significant effect on OATP1B1-mediated bilirubin uptake and inhibited OATP1B3 at low concentrations（<12.5μM）.Gallic acid promoted OATP1B1-mediated bilirubin uptake and inhibited OATP1B3.Conclusion:The main components in PM are TSG,emoodin,physcion,emodin-8-O-β-D-glucoside,gallic acid and resveratrol,the content of which is higher in AE-PM than QE-PM.AE-PM induces liver injury in normal rats when administered at ultra-high doses for a long time,and has certain effects on the expression levels of bilirubin metabolizing enzymes and transporters.TSG,emodin,catechin and resveratrol were mostly metabolized as glucuronide and sulfate conjugates in animals,thus affecting the bilirubin II phase metabolism process.The AE-PM showed different pharmacokinetics in normal and pathological rats,such as physcion,emodin and its phase II metabolites,emodin-8-O-β-D-glucoside and catechin showed higher exposure levels in the CCl₄ pathological state,and TSG and its phase II metabolites,aloe-emodin and gallic acid were exposed at higher levels in the cholestatic state.Meanwhile,the AUC（0-∞）of TSG and aloe-emodin were significantly correlated with the expression levels of UGT1A1,BSEP,OATP1A4,OCT1,NTCP,MRP2 and MDR1,and emodin,physcion and resveratrol were significantly correlated with OATP1A2,NTCP and MRP2.Bilirubin in vitro glucuronide-binding reactions with OATP1B1 and OATP1B3-mediated bilirubin uptake further indicated that TSG,resveratrol,emodin and catechin in PM do act on bilirubin phase II metabolizing enzyme UGT1A1 and bilirubin transporters OATP1B1 and OATP1B3,thereby causing liver injury.