Study On The Bacteriostasis Of Combination Of Oleanolic Acid And Dimethyl Sulfoxide Against Enterococcus Faecalis In Vitro

Objective: Enterococcus faeealis(E.faecalis)is recognized as pathogenic bacteria with high detection rate in root canals infected secondly or with refractory periapical periodontitis.The root canal disinfection drugs frequently-used currently cannot achieve ideal effect to restrain E.faecalis growth in root canals safely and effectively.As a natural drug,oleanolic acid(OA)has good bacteriostatic action.It is of low toxicity,and can inhibit the growth of E.faecalis,which is used in the treatment of acute or chronic hepatitis in our country.As a kind of organic solvent,dimethyl sulfoxide(DMSO)has the ability to enhance the permeability of drugs,has effective effect on the penetration of biofilms,and has antibacterial effect itself.So it is the common excipient for OA.However,in the past studies about the antibacterial performance of OA failed to consider the antibacterial action of DMSO.We hypothesize OA may be an ideal root canal disinfection drug,and OA in combination with DMSO can improve the root canal therapeutic efficacy for the patients with acute or chronic hepatitis.In this experiment,OA is solubled in DMSO,by using multiple proportion dilution method,micrdilution checkerboard technique,plate colony-counting method and confocal laser scanning microscope(CLSM)after fluorescence staining,to investigate the bacteriostasis effect of OA combines with DMSO against pelagic E.faecalis.Then studying the effictiveness of drugs to inhibit the growth of E.faecalis in root canals of extracted teeth or in mono-biofilm to evaluate the mutual influence when the two drugs are used in combination,so as to test our hypothesis,and to explore the possibility of treating E.faecalis infected root canals,in order to provide theoretical basis and data support for further study of applying OA to the root canal theraphy.Methods: 1.MIC of determination of OA or DMSO by agar dilution method and liquid dilution method separately.The micrdilution checkerboard technique was applied to calculate the fractional inhibitory concentration index(FICI)value to evaluate the antibacterial effect of OA combined with DMSO.2.Modelling E.faecalis infected root canals in extracted single-canal permanent teeth.Samples were randomly divided into three groups: 1mg/ml OA+50% DMSO(experimental group,group A),2% chlorhexidine(CHX,positive control group,group B),deionized water(DIW,blank control group,group C).Intracanal medication in each groups,and sampling before treatment and after treatment 1 hour,24 hours and 7 days.By bacteria counting,compare the quantity change of bacteria and process statistical analysis.3.E.faecalis in the mono-biofilm was formed on cover glasses for 24 h.Samples were randomly divided into three groups: 1mg/ml OA+50% DMSO(experimental group),2%CHX(positive control group),DIW(blank control group).Intracanal medication in each groups for 30 min respectively.After acridine orange/ethidium bromide(AO/EB)fluorescent staining,samples were observed by CLSM.Results: 1.The MIC of DMSO against E.faecalis was 20%,while the MIC of OA was 100?g/ml.When combined,the MICs were 10%(DMSO)and 1.25?g/ml(OA).The value of FICI was 0.5125>0.5,which indicates additive joint action was existed between OA and DMSO against E.faecalis.2.Statistical analysis found that the bacteria amounts in three groups after treatment were reduced,the decrement of group A and B showed statistical significance(P<0.05),the decrement of group C did not prove a statistical significance(P>0.05)at 1h and 24 h,while it showed statistical significance(P<0.05)at 7d.Group A needed a little longer action time than group B,however,no viable E.faecalis can be detected when treatment maintained for 24 h or longer in both groups(P>0.05).After treatment for 1h or longer,the bacteria amounts in both group A and B were significantly different(P<0.05)when compared with group C.3.When observed by CLSM,the biofilms of experimental group and positive control group were mainly composed of dead bacteria,only scattered living bacteria can be found.The biofilms of blank control group was mainly composed of living bacteria,only tiny dead bacteria can be detected.Conclusion: OA combinating with DMSO can effectively inhibit the growth and bacterial activity of E.faecalis in planktonic condition,single biofilm and root canals,which may be an ideal root canal disinfection drug.If used in the clinic,they maybe can increase the cure rate of root canal retreatment and refractory periapical periodontitis.