| Objective: The present study aims to explore the metabolic mechanism of oleanolic acid(OA)and its metabolic toxicity.Methods: By using Q-Exactive high performance liquid chromatography coupled with an Orbitrap high resolution spectrometer,we analyzed the metabolites of oleanolic acid in human liver microsome incubation system with cytochrome P450 reaction factors.Specific chemical inhibitions of cytochrome P450 were used to explore the main metabolic pathway of OA.Metabolisms of OA were studied in liver microsomes of human,rat,mouse,pig,rabbit,cattle,and sheep,and comprehensively evaluated the differences between species.The primary hepatocytes were isolated and cultured to investigate the relationship between the metabolism and hepatotoxicity mechanism of OA.Results: 1.We observed six metabolites of OA in human liver microsome incubation system named as M-469,M-471,M-485-1,M-485-2,M-487-1,and M-487-2.Specific chemical inhibition indicated that that CYP3 A is the major isozyme responsible for OA metabolism.2.Moreover,we observed the absence of M-485-2 metabolite in liver microsome incubation system of pigs,rabbits,cattles,and sheep.However,SD rats and C57 mice showed the similar metabolic pattern with human.Regarding difference between mice and rats,mice is more active in catalysis of OA metabolism.The same as human,CYP3 A contributed most yield of OA metabolites in contrast to other P450 isozymes.3.As CYP3 A are identified as the major isozyme responsible for OA metabolism in human and mice.We investigated the cytotoxicity of OA with and without the co-incubation of ketoconazole(KET),a specific inhibitor of CYP3 A.In KET inhibited group,along with the decrease of OA metabolism,the cell survival rates also decreased.Conclusion: OA mainly has been metabolized by CYP3 A.The toxicity of OA would be increased when the activity of CYP3 A was inhibited.It indicated that the liver toxicity of OA could be reduced through metabolism by CYP3 A,... |
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