Metabolism Studies On Traditional Chinese Medicine Monomer Component In Vitro By LC-MS Technique

Radix saposhnikoviae is originated from Umbelliferae Plants Saposhnikovia divaricata(Turcz) Schischk not bloting root. It is warm in nature and sweet and pungent in taste. It has been widely used for treating pyrexia, rheumatism, headache and convulsion in Chinese clinical practice.Studies show that prim-O-glucosylcimifugin and cimifugin are main active ingredients in Radix Saposhnikoviae, which have a strong anti-inflammatory and antipyretic analgesic physiological activity.Genistin, is widely found in soybeans, locust and genistein. It has weak estrogenic activity, antioxidant activity, anti-hemolytic activity and antifungal activity, which has been used for treatment and prevention of leukemia,osteoporosis, stomach cancer, breast cancer and prostate cancer.In this study, incubating with liver microsomes, major phase I, phase II metabolites of prim-O-glucosylcimifugin and cimifugin were studied and compared to investigate biotransformation pathways in hepatic drug enzyme action by UHPLC-Q-TOF-MS. Incubations with UGT enzymes were conducted to identify the individual UGT enzyme involved in the biotransformation of cimifugin. HPLC-MS was used to investigate the enzyme kinetics of cimifugin, optimize the reaction conditions and parameters. The effect of cimifugin on rat CYP3 A isoform provides a reference for further research of drug interactions. The kinetics studies of genistin in rat liver microsomes optimize the reaction conditions, and calculate the kinetic parameters of the enzymatic reaction.Part one Metabolic studies of prim-O-glucosylcimifugin in human and rat liver microsomesObjective: Incubation samples with human and rat liver microsomes were analysed respectively for metabolites to summarize the metabolic rulesof prim-O-glucosylcimifugin and clarify its pharmacological mechanism.Methods: Phase I incubation: Samples with human and rat liver microsomes and NADPH reduction system were analysed respectively by UHPLC-Q-TOF-MS to characterize the phase I metablites structure.Phase II incubation: Samples with human and rat liver microsomes and UDPGA, alamethicin and Mg Cl2 were analysed respectively by using UHPLC-Q-TOF-MS to characterize the phase II metablites structure.Results: 5 metabolites were both found in the incubation with human and rat liver microsomes. No glucuronidation metabolites were found in the samples.Conclusion: Prim-O-glucosylcimifugin could metabolize in vivo and convert into many metabolites. The main metabolic pathways of prim-O-glucosylcimifugin were hydroxylation and hydrolysis reaction.Part two Metabolic studies of cimifugin in human and rat liver microsomesObjective: Incubation samples with human and rat liver microsomes were analysed respectively for metabolites to summarize the metabolic rules of cimifugin and presume the drug metabolism rules. Recombinant UGTs were analysed to identify the specific UGT enzymes involved in the biotransformation of cimifugin.Methods: Phase I incubation: Samples with human and rat liver microsomes and NADPH reduction system were analysed respectively by UHPLC-Q-TOF-MS to characterize the phase I metablites structure.Phase II incubation: Samples with human and rat liver microsomes,UDPGA, lamethicin and Mg Cl2 were analysed respectively by UHPLC-Q-TOF-MS to characterize the phase II metablites structure.Incubation samples with recombinant UGTs enzymes were detected for metabolites.Results: 9 phase I metabolites and 2 phase II metablites were found in the incubation with human liver microsomes. 11 phase I metabolites and 2phase II metablites were found in the incubation with human livermicrosomes.It was found in the recombinant UGTs enzymes experiment that UGT1A1, UGT1A9, UGT2B4, UGT2B7 catalyzed the glucuronidation of cimifugin.Conclusion: Cimifugin metabolic transformation occurred in human and rat liver microsomes. The different content of enzymes in human and rat may lead to discrepancy in metabolite species.The result of recombinant UGTs enzymes experiment shows that the UGT1A1, UGT1A9, UGT2B4, UGT2B7 take part in generating the glucuronidation metabolites of cimifugin.Part three The enzyme kinetics of cimifugin in rat liver microsome and inhibition effect on rat CYP3 A isoformObjective: To study the cimifugin enzymatic kinetics in rat liver microsome and effect on rat CYP3 A isoform by HPLC-MS.Methods: The enzymatic kinetics study: A HPLC-MS method was established for quantitative analysis of cimifugin with sulfamethoxazole as internal standard. Separation on a Diamonsil C18 column(150 mm×4.6 mm, 5μm) was achieved by methanol and 0.1% aqueous formic acid, gradient elution. The mass spectrometer was operated in the postive mode in MRM mode. Graph Pad Prism 5.0 software was used to calculate the enzyme kinetics parameters Vmax and Km.The experiment of effects on rat CYP3 A isoform: Rat liver microsomes,probe drugs and cimifugin at various concentration were cultured together for5 min. After the incubation, the concentration of metabolites was determined to acess the activities of enzymes. IC50 value of cimifugin was calculated by Graph Pad Prism 5.0.Results: The optimal incubation time in rat liver microsomes is 30 min,the optimal protein concentration is 1 mg·m L-1, the optimal substrate concentration is 25 μmol·L-1. The enzyme kinetics parameters of cimifugin were as follows: Km=32.60±2.52 μmol·L-1, Vmax=0.2731±0.0074μmol·(min·mg protein)-1. The activity of rat liver microsome enzyme CYP3Awas inhibited by cimifugin in vitro with IC50 value of 131.7μmol·L-1.Conclusion: The method was simple and reliable for the vitro metabolism research of cimifugin. Cimifugin shows no inhibition on CYP3 A in rat liver microsomes in vitro.Part four Analysis of the metabolites of genistin and enzyme kinetics of genistin in rat liver microsomeObjective: To characterize the metabolism of genistin and study its enzymatic kinetics in rat liver microsome by HPLC-MS.Methods: Genistin was incubated with rat liver microsomal incubation system. HPLC-MS method was used to characterize the metabolites. A metabolite generation method was established for quantitative analysis of genistein with sulfamethoxazole as internal standard. The enzyme kinetic parameters Vmax and Km were calculated by the Graph Pad Prism 5.0software.Results: The metabolite in vitro incubation system was identified as genistein. The optimal time in rat liver microsomes is 40 min, the optimal protein concentration is 1 mg·m L-1, the substrate concentration is 25 μmol·L-1.The enzyme kinetic parameters of genistein were as follows: Vmax =0.1042±0.0033 μmol·(min·mg pro)-1, Km = 28.96±2.80 μmol·L-1.Conclusion: The results indicated that genistin can be metabolited as the form of hydroxylation in rat liver microsome. Metabolite generation method is a reliable and simple method for determination of kinetic parameters of hepatic microsomal enzymes, and enzyme kinetic parameters obtained of genistin provided important parameters for further study.