Pharmacokinetic Study On Epimedium Flavonoids And Their Structure Modified Derivatives

Objective:To clarify the pharmacokinetic characteristics of epimedium flavonoids and the underlying mechanism.Methods:7-methyl-icaritin（7-MICT）,3,7-dimethyl-icaritin（3,7-DMICT）and 3,5,7-tri-methyl-icaritin（3,5,7-TMICT）obtained were by synthesis and purification.The LC-MS/MS methods for quantification of 7-MICT,3,7-DMICT,3,5,7-TMICT,ICT,baohuosideⅠ（BH）,and icariin（ICA）were established and validated.The pharmacokinetic behavior of rats was examined after oral and intravenous administration of equimolar 7-MICT,3,7-DMICT,3,5,7-TMICT,ICT,BH,and ICA,respectively.Then,the metabolism of each analyte in in-testinal contents,intestinal mucosal epithelium,and liver microsomes,the content of each analyte and their metabolites in the portal vein,as well as the distribution and excretion were examined to clarify the underlying mechanism responsible for the pharmacokinetic charac-teristics.Results:7-MICT,3,7-DMICT,and 3,5,7-TMICT were synthesized and purified with purity over 95%.The quantitative LC-MS/MS methods were established and fully validated.ICA contains rhamnose at the 3-position and glucose at the 7-position.After a single oral dose of ICA to rats,the unchanged ICA and the metabolite BH due to the loss of glucose at 7-site were found in systemic circulation with the value of(AUC_0-24,11.397±3.862μg/L*h)and(AUC_0-24,38.198±11.803μg/L*h),respectively.Afterβ-glucuronidase hydrolysis of plasma sample,the systemic exposure of BH was dramatically increased and its glycoside ICT was also found(AUC_0-24,1823.4±660.2μg/L*h),indicating that ICA underwent extensive hy-drolysis and glucuronidation at the 3-and 7-position.After administration of equimolar amounts of the ICA metabolite BH to rats,instead of the unchanged BH,the glucuronidation of BH and ICT were found in systemic circulation with the AUC_0-24 value of 95.11±12.95and 1777.7±548.89μg/L*h.Similar results were also found in rats treated with ICT.These data suggested that the epimedium flavonoids exert very poor pharmacokinetic characteris-tics owing to the extensive hydrolysis and glucuronidation at the 3-and 7-position.When administered with the methyl-ICT,their systemic exposure was found to be positively asso-ciated with the number and site of blocked hydroxy,correspondingly,the glucuronide me-tabolite was decreased after blocking the hydroxy moiety,which provides strong evidence that glucuronidation plays a vital role in the pharmacokinetics of epimedium flavonoids.When intravenous injection of the compound to rats,all the parent forms can be found in the systemic circulation,which indicated that the major metabolism of epimedium flavonoids may occur in the intestine or（and）the liver.In intestinal lumina incubation,ICA was found to be extensively hydrolyzed to BH and ICT,while the BH was converted to ICT,ICT,7-MICT,3,7-DMICT,and 3,5,7-TMICT are not hydrolyzed,Glucuronidation did not occur in intestinal lumina.In intestinal mucosal epithelial S9 incubation,ICA can also be hydrolyzed to form BH,BH,and ICT underwent extensive glucuronidation.The level of glucuronidation was dramatically reduced when 7-hydroxy motley was blocked,and no glucuronidation occurred when 3,5,7-hydroxy moiety was blocked.In rat hepatic portal vein plasma samples,a large amount of glucuronidation of BH and ICT was observed,while the level of glucuronidation was reduced when hydroxy moiety was blocked.The further glucuronidation in the liver caused the concentrations of ICA,BH,ICT,7-MICT,and 3,7-DMICT in the hepatic portal vein were higher than that in the systemic circulation.tissue distribution studies showed that ICT,BH and ICA were al-most undetectable in brain tissue,while the distribution of 7-MICT,3,7-DMICT and 3,5,7-TMICT was significantly increased.The excretion study revealed that the major excretion pathway was the glucuronide conjugates.Conclusion:The extensive hydrolysis and glucuronidation in the intestine and liver may be responsible for the low in vivo exposure of epimedium flavonoids.Blocking of the hy-droxy moiety can dramatically improve their pharmacokinetic characteristic.