Study On Methylation Of Phenolic Acids Including Baicalein And Rosmarinic Acid

Part one: Studies on the metabolism of baicalein in vitroObjective: Study the major metabolic pathways of baicalein that is the major active component of Chinese medicine Scutellaria baicalensis.To reveal the conjugation position of baicalein metabolites,the main enzymes and its isoforms involved in the metabolism,pharmacokinetics of baicalein,the metabolic differences among seven species.To evaluate the pharmacological activity of baicalein and metabolites,provide a reference for rational application of baicalein in clinic.Methods:(1)In vitro metabolism experiments: investigating the metabolism of baicalein in human liver S9,HLC,HLM,HIM,intestinal S9,14 recombinant UGT isoforms and experimental animal liver S9(Cy LS9,Rt LS9,Ms LS9,GLS9,DLS9,Ra LS9,PLS9).(2)Preparaing the related metabolites of baicalein using the biosynthesis method,isolated and purified the metabolites with the phytochemistry approaches,identified the metabolites by various methods including two-dimensional NMR and MS.(3)Evaluated its bioactivity variation during drug metabolism in LPS-induced RAW264.7 cell.Results: Metabolites of baicalein in human liver S9 catalyzed by COMT is 6-Omethyl baicalein(M-1: oroxylin A),baicalein methylation is mainly mediated by S-COMT.The metabolic characteristics in HLS9,HLC,HLM,HIS9 and S-COMT were followed the classic Michaelis Menten kinetics;the kinetic parameters(Vmax,Km,Ki and CLint)were analyzed systematically in various biological samples.Methylation of baicalein(oroxylin A)as the intermediate metabolite can be catalyzed to form 6-Omethyl-7-O-glucuronide baicalein by UGTs and later excreted in the urine(M-2:oroxylin A-7-O-?-D-glucuronide).UGT1 As family are main isform in glucuronidation according to chemical inhibition experiment.The analysis results indicated that5 glucuronidation of oroxylin A were followed classic Michaelis Menten kinetics in HLM,UGT1A10,1A9;yet were obeyed substrate inhibition kinetics in HIM,UGT1A1,1A3,1A7,1A8.The kinetic parameters(Vmax,Km,Ki and CLint)were obtained.The species difference studies showed that the glucuronidation showed perferred reaction rate in all species,and the methylation process showed obvious differences among species,kinetic study suggested that dynamic characteristics of monkeys is most similar to human liver reaction.We found M-1(oroxylin A)still showed potent anti-inflammatory activity,with IC50 value of 28 ?M,and the final product(oroxylin A-7-O-?-D-glucuronide)has no anti-inflammatory activity.Conclusion: In this research,we firstly reveals the two step process of baicalein in vitro metabolism including methylation and UDP-glucuronidation;S-COMT and UGT1 As guided the two metabolic processes.The study indicated that metabolism of COMT is slow and the enzyme's affinity is very poor.Moreover,the methylation of baicalein showed obvious differences among species;monkey may be used as a suitable animal model in related researches of pharmacology and toxicology.These findings may assist in selection of the suitable animal model for pharmacokinetic or toxicological studies of baicalin,and also provide useful guidance for clinical application.Part two: Studies on metabolism of rosmarinic acid in vitro and species differencesObjective: To explore the major metabolic pathways of rosmarinic acid in vitro.We also reveled the conjugation position of rosmarinic acid metabolites,the main enzymes involved in the metabolism,the metabolism differences among seven species.The drug-drug interactions(DDI)among rosmarinic acid and dopamine to provide some references for reasonable clinical application of rosmarinic acid.Methods:(1)In vitro metabolism experiments: investigation of the methylation metabolism of rosmarinic acid in human liver S9,HLC and experimental animal S9(Cy LS9,Rt LS9,Ms LS9,GLS9,DLS9,Ra LS9,PLS9).(2)Preparaing the related metabolites of rosmarinic acid using the biosynthesis method,isolated and purified themetabolites with the phytochemistry method,identified the metabolites by spectral methods including two-dimensional NMR and MS.(3)Evaluate the inhibition activity of rosmarinic acid on dopamine by using the in vitro incubation method,to confirm the related inhibition kinetic parameters.Results: The metabolites of rosmarinic acid is 3'(M-1)and 4'(M-2)methylation products.The results indicated that methylation of rosmarinic acid were followed substrate inhibition kinetics in human liver S9,HLC.Substrate inhibition kinetics were shown in the methylation of rosmarinic acid to generate M-1 in Cy LS9,DLS9,Rt LS9 and Michaelis-Menten model in Ms LS9,GLS9,Ra LS9,PLS9;Substrate inhibition kinetics were shown in the methylation of rosmarinic acid to generate M-2 in Cy LS9,DLS9,Rt LS9,PLS9 and Michaelis-Menten model in Ms LS9,GLS9,Ra LS9.We also systematically analyzed various kinetic parameters(Vmax,Km,Ki and CLint).The inhibition of rosmarinic acid on dopamine showed competitive inhibition,with Ki values of was 0.35 ?M.Conclusions: In present work,we firstly identified the structures of rosmarinic acid metabolites in vitro metabolism and investigatethed significant species difference;SD rats may be more suitable model for pharmacology and toxicology researches of rosmarinic acid.The metabolism of rosmarinic acid on dopamine showed competitive inhibition,which providing a reference for the future clinical trials.