The Metabolism Of Phillygenin In Liver Microsomes And Inhibitory Potency On CYP3A In Rat Liver Microsomes

Forsythia suspense(Thunb.) Vahl belongs to the family of Oleaceae is widely distributed in China, Korea, Japan and many European countries. The dry fruit of the plant, named “Lianqiao” in Chinese, is a famous traditional Chinese medicine due to the effective treatment of many diseases, such as acute nephritis, erysipelas, gonorrhea and ulcer. In present studies, a number of bioactive components including lignans, flavonoids, and so on has been found in the plant. Among them, phillygenin is a major ligan component exhibits significant biological activities such as antioxidant, reduce blood lipid and inhibit the oxidation of low density lipoprotein.A LC-MS method was established to characterize the metabolites of phillygenin in liver microsomal incubation system, and the enzyme kinetics of phillygenin metabolism and the effects of selective CYP450 inhibitors on the metabolism of phillygenin in liver microsomes of rat has been investigated. The metabolite selective probe substrate( 1-hydroxymidazolam) was detected to calculate the value of IC50 and it was used to assess the inhibitory potency of phillygenin on CYP3 A enzymes in rat liver microsomes. Part one Metabolism studies of phillygenin in rat liver microsomesObjective: A LC-MS/MS method was established to characterize the metabolites of phillygenin in rat liver microsomal incubation system.Methods: Rat liver microsomes were prepared by using ultracentrifugation method and a LC-MS/MS method was established to detected the metabolites in the incubation pool. The incubation samples were analysed in EMS-IDA-EPI and MRM-IDA-EPI modes for metabolites of phillygenin. The chromatography separations were performed on a C18 reversed phase LC column(Diamonsil C18, 250×4.6 mm, 5 μm). The mobile phase consisted of water-formic acid(100:0.1, v/v) and acetonitrile at the flow rate of 800 μL·min-1 and a gradient program was adopted. The mass spectral analysis was performed in a negative electrospray ionization mode, the turbo spray temperature was 650 ℃.Results: No glucuronidation metabolites were found in the samples after comparing with the blank sample and 13 phaseⅠmetabolites were detected in rat liver microsomal incubation system. The possible fragmentation pathways of phillygenin were speculated in a negative electrospray ionization mode.Conclusion: The LC-MS/MS method was suitable for the research of phillygenin in rat liver microsomes, which providing valuable reference for phillygenin study in pharmacokinetics. Part two Metabolism studies of phillygenin in human liver microsomesObjective: A UHPLC-Q-TOF-MS method was established to characterize the metabolites of phillygenin in human liver microsomal incubation system.Methods: The chromatography separations were performed on a C18 reversed phase LC column(Phenomenex Kinetex C18, 100×2.1 mm, 2.6 μm). The mobile phase consisted of water-formic acid(100:0.1, v/v) and acetonitrile at the flow rate of 400 μL·min-1 and a gradient program was adopted. The mass spectral analysis was performed in a positive electrospray ionization mode, the turbo spray temperature was 550℃. The full MS experiments were run with a scan range from m/z 100 to m/z 1000.Results: The possible fragmentation pathways of phillygenin were speculated in a positive electrospray ionization mode, and 8 metabolites were identified in human liver microsomal incubation system.Conclusion: The UHPLC-Q-TOF-MS method was very convenient and efficient for detecting the phillygein in human liver microsomes. No glucuronidation metabolites were detected in human liver microsomal incubation system. Part three Investigation of enzyme kinetics and reaction phenotyping ofphillygenin in rat liver microsomesObjective: To study the enzyme kinetics of phillygenin enantiomers in rat liver microsomes and get the parameters. The effects of selective CYP450 inhibitors on the metabolism of phillygenin in liver microsomes of rat has been investigated.Methods: Time, protein concentration of rat liver microsomes and the substrate concentration were detected as investigating objects to optimize the conditions of rat liver microsomal incubation system. The kinetic parameters were calculated by Lineweaver-Burk plot. The CYP450 isoforms involved in the metabolism of phillygenin in rat liver microsomes were investigated by selective CYP inhibitors.Results: The optimized reaction condition is that 30 μmol·L-1 substrate incubated with 1 mg·m L-1 protein for 20 mintutes. The enzyme kinetics parameters were as follow: Km=44.85 μmol·L-1, Vmax=0.4286 μmol·(min·mg protein)-1. It was found in the inhibition experiment that quinine, 4-Methylpyrazole, ticlopidine hydrochloride can significantly inhibit the metabolism of phillygenin.Conclusion: CYP450 enzymes incubation experiment showed that CYP2B、CYP2D and CYP2E1 were the major enzymes involved in the metabolism of phillygenin. There were no significant participation of CYP1A2, CYP2 C and CYP3 A to catalyze the metabolism. Part four Inhibitory potency of phillygenin on CYP3 A in rat livermicrosomesObjective: A probe substrate was used to assess the inhibitory potency of phillygenin on CYP3 A enzymes in rat liver microsomes.Methods: The probe substrate was incubated in rat liver microsomes with phillygenin of different concentrations. A LC-MS/MS method was established to detected 1-hydroxymidazolam which is the metabolite of selective probe substrate. The IC50 value was determined. Ketoconazole,a selective CYP inhibitor, was also incubated in rat liver microsomes with the probe substrate to validate the method.Results: The IC50 value of phillygenin for CYP3 A in rat liver microsomes was 92.68 mol·L-1. The IC50 of ketoconazole obtained good agreement with previously reported value in the literature.Conclusion: This probe substrate method can be utilized for the determination of the inhibition potential of phillygenin on CYP3 A in rat liver microsomes. The IC50 value of phillygenin indicated that phillygen showed minor inhibitory effect of CYP3 A in rat liver microsomes.