Characterization Of Human Metabolic Pathways Of Alisol B 23-acetate And Evaluation Of Pharmacological Activities Of Its Hydrolysate

Objective: The phase I metabolic pathways of alisol B 23-acetate were studied in vitro and in vivo experiments,to clarify its main metabolic reaction types and metabolic enzymes,as well as the agonistic effects on nuclear receptors and the inhibitory effects of metabolites on drug-metabolizing enzymes.To lay the foundation for metabolic research and clinical application of A.orientaleMethods:1.Identification of phase I metabolites of alisol B 23-acetate:Rats were given a certain dose of alisol B 23-acetate solution by gavage administration and orbital blood was collected,and the metabolites of alisol B 23-acetate in rats’ plasma were detected by LC-TOF-MS/MS system after administration.Phase I metabolites of alisol B 23-acetate in human liver microsomes(HLMs)and NADPH generating system in a 200 μL classical incubation system in vitro.The samples were analyzed by LC-TOF-MS/MS system,and the fragments of the metabolites were analyzed by MS and MS/MS to predict the possible structures of their phase I metabolites.2.Main metabolic reaction types and metabolic enzymes of alisol B 23-acetate:The eleven CYP isoforms were incubated with alisol B 23-acetate in the NADPH generation system at 37 °C for 1 hour with or without corresponding inhibitors,and the reaction was terminated with ice acetonitrile.The supernatant was collected by centrifugation for LC-TOF-MS/MS analysis to investigate the CYP metabolic pathways by comparing the MS/MS fragmentation information of the metabolites.The hydrolysis pathways of alisol B 23-acetate were studied by measuring the reaction phenotype and analyzing the chemical inhibition type of alisol B 23-acetate,and the enzyme sources of hydrolyzed alisol B 23-acetate were determined and its enzymatic kinetic characterization was determined.3.Study on the agonistic effect of alisol B 23-acetate and its hydrolysate alisol B on nuclear receptors:Study on the agonistic effect of alisol B 23-acetate and alisol B on Nrf2、Ah R、PPARα、PPARβ、PPARγ、FXR nuclear receptors at the cellular level.Molecular docking simulation was used to speculate the mechanism of its agonistic effect on FXR.4.Inhibitory effects of alisol B on drug-metabolizing enzymes:Based on the probe reaction,LC-MS/MS and microplate reader were used to quantitatively determine the inhibitory effects of alisol B on eight common CYP isoforms,carboxylesterases,and UGT.The inhibition kinetics of the enzymes with a strong inhibitory effect was analyzed,and the binding modes of alisol B and the enzymes were predicted by a molecular docking simulation.Results:1.Identification of phase I metabolites of alisol B 23-acetate: A total of 6 phase I metabolites were obtained by in vitro experiments,and the structures of some metabolites were predicted by using the methods of mass spectrometry breaking rule and standard substance comparison,while the remaining phase I metabolites could only be predicted as possible metabolic sites.2.Main metabolic reaction types and metabolic enzymes of alisol B 23-acetate:Through enzyme reaction phenotypes and chemical inhibition assays,it was determined that alisol B 23-acetate was hydrolyzed to form alisol B under the action of h CES1 A,h CES2 A,h Buch E,and h SA.Under the action of CYP3A4 and CYP3A5,alisol B(M1)underwent hydroxylation at the 1-position C atom to form M2 and further dehydrated to form M3 and M2 also dihydroxylation to form M6,alisol B 23-acetate was hydroxylated to form M5,and ketogenic reaction to form M4.3.Study on the agonistic effect of alisol B 23-acetate and its hydrolysate alisol B on nuclear receptors: The agonistic effects of alisol B 23-acetate and alisol B on Nrf2、Ah R、PPARα、PPARβ、PPARγ、FXR were studied,respectively.The results showed that alisol B 23-acetate and its hydrolyzed product alisol B had the same intensity of the agonistic effect on FXR at low concentration.Molecular docking mode results showed that alisol B23-acetate and alisol B bond to Trp469 and His447 to stimulate the FXR,respectively.The above results revealed the mechanism of its agonistic effect on FXR.4.Inhibitory effects of alisol B on drug-metabolizing enzymes: Alisol B can not only be metabolized by CYP enzymes but also inhibited the activities of some CYP enzymes.Alisol B had a weak inhibitory effect on CYP1 A,CYP2A6,CYP2D6,and CYP2E1,but a strong inhibitory effect on CYP3 A,CYP2C19,CYP2C9,and CYP2C8.Alisol B has a strong inhibitory effect on h CES2 A,a weaker inhibitory effect on h CES1 A but no inhibitory effect on UGT1A1.Alisol B inhibited the activities of CYP3A4 and h CES2 A in a non-competitive and mixed-type inhibition manner,respectively,and the binding mode of alisol B to the enzymes was speculated by molecular docking simulation.Conclusion:Alisol B 23-acetate produced 6 phase I metabolites in HLMs,and the enzymes involved in phase I metabolism was identified in the following experiments.It was found that alisol B was the substrate and inhibitor of some CYP isoforms.The agonistic effects of alisol B 23-acetate and alisol B on FXR provide a theoretical basis for their pharmacological studies on glucose and lipid metabolism,hepatoprotection,and resistance to non-alcoholic fatty liver disease.The above results suggested that attention should be paid to the risk of drug-drug interaction when alisol B 23-acetate was used in combination with other drugs metabolized by CYP3 A and CYP2C19.