Epiberberine Biometabolic Reaction In Vivo And In Vitro

Epiberberine is one of the bioactive components isolated from Coptidis Rhizoma(Huanglian),which has been officially listed in the Chinese Pharmacopoeia and widely used in China for more than 2000 years.Pharmacological and clinical studies indicated that it possessed a wide range of pharmacological activities including have the protective effects on oxidative stress,inhibition of cellular peroxynitrite generation.Recently,the hypoglycemic activity of epiberberine has drawn much attention.Despite its important therapeutic value,there are no detailed studies on the metabolism of this compound,including elucidation of the structure of metabolites after intravenous administration of epiberberine and identification of the enzymes involved in drug metabolism in rat and human liver.In this study,there is a combined experimental methods of in vivometabolites and in vitro liver metabolism,from those two aspects to clarify the characterization of metabolites and identification of cytochrome P450(CYP)isoforms involved in the metabolism of epiberberine,which is the most effective ingredient in Huanglian(coptidis rhizoma),and to explore the inhibitory effect of epiberberine on five major CYP isoforms.The whole researchprocess will be summarized as follows:Part one:Literature ReviewThe new developments of study both in traditional Chinese medicine huanglian and epiberberine,as well as the study in the liver metabolism and drug metabolic interactionshave been surveyed and summarized.Part two:Experiment Studies1 LC-MS/MS for identification of in vivo metabolites of epiberberine1.1 Comparison ofmetabolitesafter intragastric and intravenous administration epiberberine in ratsThe in vivo metabolism of epiberberine was investigated using a highly specific and sensitive liquid chromatography-mass spectrometry(LC-MS/MS)method.In vivo samples including rat urine,feces,and biles were collected individually after intragastric administration of 50 mg/kg epiberberine to healthy rats.the metabolites were discovered by comparing the fullscan chromatograms of the test samples with the corresponding blanks.The structures of metabolites were identified by ESI-MS spectra and the product spectra of the corresponding adduct ions.four metabolites of epiberberine were identified in rat feces,three in bile,three in blood,seven phase I and five phase II in urine.Their structures were elucidated,separately.after intravenous administration of 10 mg/kg epiberberine,at least twelve,four and three metabolites were found in rat urine,feces,and biles,respectively.1.2Identification of Phase I metabolites of epiberberine in RLM and HLMIn vitro samples prepared by incubating epiberberine withRLM and HLM,respectively.As a result,two phase I metabolites III and IVwere detected in liver microsome incubation solution.1.3 Identification of Phase II metabolites of epiberberine in RLM and HLMEpiberberine was incubated with RLM and HLM,three phase ? metabolites X?XI and XII were detected in liver microsome incubation solution.1.4Identification of metabolites of epiberberine inintestinal floraEpiberberine was incubated with intestinal flora,onlyone metabolites? was identified in intestinal flora incubation solution and the main biotransformation reactions of epiberberine were the hydroxylation reaction,the methylation reaction,and the demethylation reaction of parent drug and its relative metabolites.Epiberberine was extensively metabolized in rat.The metabolites can be easily screened and identified by LC-MS/MS method.2 Identification of CYP450 isoforms involved in the metabolism ofepiberberineIn this part,the role that CYPs play in the metabolism of epiberberine were investigatied in liver microsomes from rat(RLM)and humans(HLM),as well as recombinant human CYPs.As a result,CYP2D6 was the primary recombinant human CYP in the metabolism of epiberberine,f-ollowed by CYP3A4 and 2E1.The metabolism of epiberberine in RLM and HLM was inhibited mainly by a CYP2D inhibitor,and moderately by inhibitors of CYP3A and 2E.CYP2D were den,onstrated to be major involved P450 isoiorms.3 The influence of epiberberine on rat liver enzyme activityThis experiment,a method of in vivo-inducing of rat liver microsomes was used to research the influence of epiberberine on the probes in vitro liver metabolism.Two groups of rats were respectively intragastric administered with the blank suspension and epiberbcrine suspension.After continuous administration for 7 days,microsomes of each group rats were prepared for the in vitro experiments of probes metabolism,and then,metabolism rate of all groups were calculated.Experimental results showed that:compared with blank control group,epiberberine group can obviously inhibit the activities of CYP 2D6(P<0.01)and CYP3A4(P<0.05),These data arc significantfor clinical application of epiberberine and herbscontaining epiberberine."Cocktail" method was imposed to simultaneously determine the substrate content of of CYP2D6,CYP3A4,CYPlA2,CYP2E1 and CYP2C9for evaluating the activity of CYP isoforms under different concentrations of epiberberine in RLM.Results showed that:epiberberine had no obvious inhibiting effect on the activities of CYP3A4,CY1A2,CYP2E1 and CYP2C9,but showed significant inhibition on CYP2D6 with IC50 value of 35.22?mol·L-1.Part 3 Summary and discussionTo analyze,discuss and summarize all results in the whole study,an comprehensive conclusion goes as following:characterization of the metabolites and CYP isoforms involved in the metabolism of epiberberine was investigated in vivo and in vitro experiment.At least twelve,four,three and three metabolites were found in rat urine,feces,blood and biles,respectively.Additionally,two unconjugated metabolites of epiberberine were produced by RLM,HLM,and recombinant human CYPs.CYP2D6 was the primary recombinant human CYP producing these metabolites,followed by CYP3A4 and 2E1.Epiberberine can obviously inhibit the activities of CYP2D6and CYP3A4.All the results were helpful for clinical application of epiberberine and Coptidis Rhizoma.