| Objective:Da-Huang-Xiao-Shi Decoction(DHXSD)was the representative formula which was mainly used to treat jaundice caused by damp-heat pathogen.This study aimed to illustrate the pharmacokinetics of DHXSD and the hepatic and renal efflux mechanisms of berberine(one of the active compounds in DHXSD).These results could provide the basis for the further pharmacological studies of DHXSD.Methods:(1)A sensitive LC-LTQ-Orbitrap MS method was developed for simultaneously quantifying 21 compounds in the extract of DHXSD.(2)An LC-MS/MS method was developed for simultaneously quantifying multiple compounds of DHXSD in rat plasma.After oral administration of 2.4 g/kg DHXSD,the blood samples were collected at regular time.The plasma concentrations of target compounds were determined before and after β-glucuronidase/sulfatase hydrolysis.The pharamacokinetic parameters were calculated by WinNonlin software.(3)After oral administration of DHXSD,the prototypes and metabolites in mouse liver were qualitatively identified by LC-LTQ-Orbitrap MS.The concentrations of target compounds were determined before and after β-glucuronidase/sulfatase hydrolysis in DDC-induced cholestasis mouse liver and kidney after oral administration of 3.6 g/kg DHXSD for 4 weeks and 8 weeks.(4)Transfected cells,animal experiments and molecular docking were applied to elucidate the hepatic and renal efflux mechanisms of berberine.Results:(1)The developed LC-LTQ-Orbitrap MS method met the requirements of contents determination of extract and was successfully applied to quantify 21 compounds divided into 7 types in DHXSD.The content of berberine was highest in the extract of DHXSD,followed by geniposide.(2)The developed LC-MS/MS method met the requirements of the determination of biological samples.The method was successfully applied to quantify 13 prototypes and 12 glucuronide and sulfate metabolites after enzyme hydrolysis in rat plasma.Their pharmacokinetic parameters were obtained and presented various pharmacokinetic characteristics.The t1/2 of rhein,geniposide,and genipin 1-gentiobioside were 5.88 h,6.37 h,and 7.78 h,respectively whereas the other compounds had the elimination with a t1/2 of longer than 9 h.Compared with the plasma concentrations of other compounds,rhein and geniposide presented the higher plasma exposure.Besides,we found that the glucuronide and sulfate metabolites were important forms of phellodendrine and five anthraquinones in rat plasma.(3)After oral administration of DHXSD,36 compounds including prototypes and metabolites were identified in mouse liver.Besides,13 prototypes and 12 glucuronide and sulfate metabolites after hydrolysis were determined in DDC-induced hepatic cholestasis mouse liver and kidney.Geniposide,rhein and berberine were the three highest compounds in mouse liver and kidney.Moreover,the tissue/plasma concentration ratios of berberine were the highest.(4)P-glycoprotein and Multidrug and toxin extrusion protein 1 were found to be involved in the hepatic and renal efflux of berberine.Conclusion:This study illuminated the pharmacokinetics of DHXSD through analyzing the chemical constituents in the extract of DHXSD and the components in vivo after oral administration of DHXSD.Moreover,the hepatic and renal efflux mechanisms of berberine,one of the effective compounds of DHXSD,were revealed in this study. |
