In Vitro Metabolic Profiling of Periplogenin in Rat Liver Microsomes and its Associated Enzyme-kinetic Evaluation

Background: Periplogenin, an active ingredient in Cortex Periplocae, is widely noted for its multiple biological activities; however, the metabolism of this compound has been scarcely investigated. The present report proposed the in vitro metabolic profiling and reaction pathways of periplogenin in rat liver microsomes. Method and Results: The metabolites of periplogenin in rat liver microsomes were analyzed. Two main metabolites, namely 14-hydroxy-3-oxo-14β-carda-4, 20 (22)-dienolide and 5, 14-dihydroxy-3-oxo-5β, 14β-card-20(22)-enolide were identified by HPLC-MSn, 1H-NMR and 13C-NMR. HPLC method was established for the simultaneous determination of periplogenin and its related metabolites (M0, M1 and M2), which was performed on Waters ODS column with a methanol-water solution (53:47, v/v) as mobile phase and descurainoside as an internal standard at 220 nm. The linearity ranges of M0, M1 and M2 were 0.64-820.51, 0.68-864.86 and 0.64-824.74 μM respectively with the regression coefficient (R2) above 0.9995. The limits of quantitation for these metabolites (M0, M1 and M2) were 0.18, 0.22 and 0.15 μM respectively. The developed method was also accurate (with relative errors of -3.6% to 3.2%) and precise (with relative standard deviations below 7.9%). The recoveries of the three analytes were above 85.7% with stability in the range of -2.4% to 3.6%. The enzyme-kinetic parameters of periplogenin including Vmax (6.08 ± 0.19 nmol/mg protein/min), Km (288.62 ± 14.54 μM) and Clint (21 ± 1.0 μL/min/mg protein) were calculated using nonlinear regression analysis. Conclusion: These findings significantly highlighted the metabolic pathways of periplogenin and also provided some reference data for future pharmacokinetic and pharmacodynamic studies.