Coptisine And Epiberberine,the Characteristic Constituents Of Rhizoma Coptidis,inhibit Helicobacter Pylori:susceptibility And Mechanism

ObjectivesIn this study,we comparatively investigated the anti-llelicobactor pylori effects and urease inhibitory activities of five major protoberberine alkaloids in Rhizoma Coptidis,namely berberine,coptisine,palmatine,jateorhizine and epiberberine,to verify the bioactive ingredients and clarify the underlying mechanism.Methods1.H.pylori inhibition assay of five alkaloids in Rhizoma CoptidisThe Agar dilution method was carried out to determine the minimum inhibitory concentration(MIC)of five alkaloids in Rhizoma Coptidis,clarithromycin and metronidazole on H.pylori.2.Urease inhibition assay of five alkaloids in Rhizoma CoptidisThe urease activity was measured by the modified Berthelot method.And the inhibitory effect of five alkaloids in Rhizoma Coptidis and acetohydroxamic acid on HPU and JBU was obtained by measuring the half inhibitory concentration(IC50).3.Inhibition mechanism of coptisine and epiberberine against ureaseBased on HPU and JBU,kinetic research and urease inhibition site analysis,including molecular docking study,alkaloid-thiol-urease interaction test,protection experiment and reactivation assay of alkaloid-suppressive urease,were used to explore the potential inhibition mechanism of coptisine and epiberberine against urease.Results1.H.pylori inhibition assayResults indicated that MIC values of five tested alkaloids against H.pylori ranged between 25 and 100 ?g/mL.Among the five test compounds,coptisine had more potent inhibitory effect against H.pylori,followed by berberine,epiberberine,palmatine and jateorhizine.2.Urease inhibition assayResults suggested that the five tested alkaloids served as dose-dependent inhibitors of urease with IC50 values ranging between 2.96 and 4631.98 ?M for HPU and 2.23->10,000?M for JBU,respectively.Remarkably,epiberberine was found to be the most effective inactivator against HPU and JBU,which was more potent than the standard urease suppressant,acetohydroxamic acid.The inhibitory effect of palmatine was second only to epiberberine,followed by coptisine and jateorhizine.Berberine was proved to be the weakest urease inhibitor.3.Inhibition mechanism of coptisine against ureaseKinetic analysis demonstrated that the type of coptisine against both urease were mixed inhibition type.Urease inhibition site analysis results indicated that Ni2+ and sulfhydryl groups were the possible acting site for coptisine against HPU,while only the sulfhydryl groups for JBU.The suppression of both ureases by coptisine was proved to be invertible since urease activity could be recovered by dithiothreitol.4.Inhibition mechanism of epiberberine against ureaseKinetic research showed that the type of epiberberine against HPU was uncompetitive,while epiberberine competitively inhibited JBU.Urease inhibition site analysis results suggested that the sulfhydryl groups of urease was possibly responsible for the suppression of epiberberine against both ureases.ConclusionIn this study,we found that five tested alkaloids in Rhizoma Coptidis could effectively inhibit H.pylori growth and urease activity.However,the inhibitory effects of alkaloids against H.pylori and urease were not consistent.The inhibitory effect against H.pylori was coptisine>berberine>palmatine,epiberberine and jateorhizine,while the suppression action against both urease was epiberberine>palmatine>coptisine>jateorhizine>berberine.Moreover,there were differences of the urease inhibition mechanism between coptisine and epiberberine.Coptisine was proved to be the reversible mixed-type inhibitor targeting thiol groups and Ni2+ of urease,while epiberberine,possible binding to the sulfydryl groups of urease,was the invertible uncompetitive inhibitor for HPU and competitive inhibitor for JBU.These results suggested that coptisine and epiberberine inhibited urease activity by binding its active site,and urease played an important role in the development of H.pylori infection.