Study On In Vivo And In Vitro Metabolism And Pharmacokinetics Of Active Constituents In Dioscorea Bulbifera L.

Dioscorea bulbifera L.?DB?is traditional Chinese medicine,which displays multiple pharmacological properties,including antiviral,antitumor,anti-inflammation,and antimicrobial activities.DB is a toxic herbal medicine.It has high incidence of liver injury,which associated with long-term ingestion of DB.The safety of DB caused great concerns in clinic.Diosbulbin B?DIOB?and 8-epidiosbulbin E acetate?EEA?are the main constituents of DB.They play important roles in pharmacological effects and toxicity of DB.The recent studies indicated that the reactive metabolites of DIOB and EEA generated from CYP mediated metabolic activation were responsible for DB-induced hepatotoxicity.However,there still lack of systematic investigations in pharmacokinetics,metabolic elimination and kinetics of reactive metabolite formation.Hence,the present study is aimed to characterize the absorption,distribution,excretion and pharmacokinetics of DIOB and EEA after oral dose of ethanol extract of DB?EEDB?in rats.The liver microsomes and recombinant CYP enzyme incubation system were used to study the in vitro metabolic kinetics of the two active components,and also to explore the inter-species differences in their metabolic elimination and enzymatic kinetics.The findings of the study may help to enhance the understanding of the pharmacokinetics and pharmacological mechanisms of the drug,and also provide scientific basis and experimental data to guide further development of DB for clinical use.In the present study,a liquid chromatography-tandem mass spectrometry?LC-MS/MS?method was established to analyze the DIOB and EEA simultaneously,and was fully validated.The method was wide linear range,low matrix effect and high sensitivity,could satisfy the requirements of pharmacokinetics of DB.In rat pharmacokinetic study,rapid absorption and elimination of DIOB and EEA were observed after a single ig administration of EEDB(1 g·kg^-1).The peak time was both at 0.2 h,and the elimination half life(t_1/2)was 2.3 and 1.8 h,respectively.The absolute bioavailability of DIOB and EEA was calculated to be 23%and 30%,respectively.After an oral dose of EEDB,the total excretion of DIOB and EEA from urine and feces within 96 h was 0.21%and 1.41%.The accumulative bile excretion of DIOB and EEA in bile-duct cannulated rats within 72 h was 0.30%and 0.25%,suggesting that the two components were mainly excreted as metabolites.DIOB and EEA showed high permeability across the cell membranes,with the P_app?AP-BL?>10×10^-66 cm·s^-1 in Caco-2 cell monolayer model.DIOB and EEA could rapidly and widely distribute into rat tissues following an oral administration of EEDB.The tissue concentrations reached the peak level at 0.17 h.The exposure of the two compounds in liver,lung and kidney was significantly higher than those in other tissues and plasma.The protein binding of DIOB and EEA was in the range of26⁶7%,and the binding rate was higher in tissues than that in plasma.Higher free concentrations of the two components were measured in liver,lung and kidney.The unbound lung-to-plasma exposure ratio?Kp,AUC,?_u?of DIOB was 48⁴5,indicating the lung specific accumulation of the compound.DIOB and EEA underwent extensive phase I metabolic elimination in human and animal liver microsomes with significant inter-species differences.DIOB and EEA had higher intrinsic clearance in liver microsomes of rat,mouse and monkey,while lower in human and dog.Metabolism of DIOB and EEA was mediated by several CYP isoforms.CYP3A4 was the major isoform that had predominant contribution of68.55%and 82.36%for DIOB and EEA.When pretreated with CYP inhibitor ketoconazole,the plasma and tissue concentrations of DIOB and EEA were significantly increasd in rats.By using trapping agents,such GSH and NAL,several reactive metabolites of DIOB and EEA were detected in HLM and RLM.The enzymatic kinetics of the metabolite formation was evaluated.The CL_int of DIOB or EEA in RLM was noted to be significantly higher than that in HLM,indicating that the metabolic clearance of the parent compounds was quicker in RLM.However,the formation velocity of reative metabolites was higher in HLM.In summary,DIOB and EEA were absorbed rapidly in rats after an oral dose of EEDB.Liver,lung and kidney were the major target tissues of drugs with higher exposure levels determined.The elimination of DIOB and EEA from plasma was rapidly.The oral bioavailability was 23³0%.They were mainly excreted as metabolites after CYP3A mediated metabolism.The significant inter-species differences were observed in metabolic clearance of the parent compounds,and also in the formation kinetics of reactive metabolites.The inter-species diffrences should be take into account when extrapolation of animal data to human.The possible herb-drug interaction should also be mornitored in clinic.