Study On The Effect Of Ginsenoside Rh2 Combined With Sodium Fluoride On Streptococcus Mutans And Biofilm

Background and Objectives:Streptococcus mutans（S.mutans）,as one of the cariogenic bacteria,has strong acid production and acid tolerance,and its extracellular polymer such as exopolysaccharides can contribute to the adhesion,colonization and aggregation of bacteria,thus contributing to the formation of biofilm.At present,sodium fluoride（NaF）has been proved to have a strong inhibitory effect on S.mutans,but improper use of NaF was prone to a series of adverse reactions such as dental fluorosis and induced the emergence of fluoride-resistant strains.Ginsenosides are the main active components of ginseng,and in recent years,it has been proved that ginsenosides had inhibitory effect on many kinds of bacterias.This paper studied the effects of single and combined application of G-Rh2 and NaF on growth,biofilm formation and metabolic activity,acid production,LDH activity and EPS production of S.mutans.To explore whether the combined application of G-Rh2 and NaF had synergistic inhibitory effect on S.mutans and its biofilm in vitro,so as to provide scientific basis for the development of more effective anti-caries agents.Methods:The half minimum inhibitory concentration(MIC₅₀)of G-Rh2 and NaF against S.mutans were measured by microdilution method,the MIC₅₀value of G-Rh2 combined with NaF on S.mutans were measured by chessboard microdilution method,and the fractional inhibitory concentration（FIC）value of graded bacteriostatic index was calculated to judge the combined effect of these two agents.The growth curves were used to detect the bacteriostatic effect of G-Rh2 and NaF alone or in combination on S.mutans.The effect of G-Rh2 and NaF on biofilm formation of S.mutans were determined by crystal violet staining test,the half minimum biofilm inhibition concentration(MBIC₅₀)were measured respectively,and the FIC index was also calculated.Methyl thiazolyl tetrazolium（MTT）assay was used to evaluate the effects of G-Rh2 and NaF alone or in combination on the metabolic activity of S.mutans biofilm.The changes of the number,morphology and biofilm structure of S.mutans were observed by Scanning Electron Microscope（SEM）,and the composition of living or dead bacterias in the biofilm of S.mutans were observed by Confocal Laser Scanning Microscopy（CLSM）.Phenol-sulfuric acid experiment was used to evaluate the effect of G-Rh2 and NaF alone or in combination on the contents of EPS in S.mutans biofilm.The acid production experiment was used to evaluate the effects of G-Rh2 and NaF alone or in combination on the acid production of S.mutans,and the effects of G-Rh2 and NaF alone or in combination on the LDH activity were evaluated by the method of oxidation of reduction coellzymeⅠ.The cell counting kit-8（CCK-8）assay was used to detect the cytotoxicity of G-Rh2 and NaF alone or in combination.Results:1.The MIC₅₀of G-Rh2 was 25 mg/L,and the MIC₅₀of NaF was 125 mg/L.When these two agents used in combination,the MIC₅₀of G-Rh2 and NaF were 5 and31.25 mg/L,respectively,and the FIC index was 0.45,less than 0.5.Compared with G-Rh2 or NaF used alone,the growth curves of S.mutans in combination groups were smoother.2.The MBIC₅₀of G-Rh2 was 22.5 mg/L,and the MBIC₅₀of NaF was 62.5mg/L.When these two agents used in combination,the MBIC₅₀of G-Rh2 and NaF were 4.5 and 15.625 mg/L,respectively,and the FIC index of S.mutans biofilm was0.45,less than 0.5.3.The metabolic activity of S.mutans biofilm in low concentration combined group(1/5 MBIC₅₀G-Rh2+1/4 MBIC₅₀NaF)was lower than that in 1/5 MBIC₅₀G-Rh2 group or 1/4 MBIC₅₀NaF group;the metabolic activity in high concentration combined group(MBIC₅₀G-Rh2+MBIC₅₀NaF)was lower than that in MBIC₅₀G-Rh2 group or MBIC₅₀NaF group.4.SEM images showed that compared with 1/5 MBIC₅₀G-Rh2 group and 1/4MBIC₅₀NaF group,the three-dimensional structure of S.mutans biofilm disappeared and the arrangement of bacterias were loosed in LC combined group.And compared with MBIC₅₀G-Rh2 group and MBIC₅₀NaF group,the biofilm structure disappeared,the number of bacterias decreased,and the morphology of some bacterias changed in HC combined group.5.CLSM images showed that compared with 1/5 MBIC₅₀G-Rh2 group and 1/4MBIC₅₀NaF group,the thickness of S.mutans biofilm in LC combined group decreased,the green signal decreased and the red signal increased.Compared with MBIC₅₀G-Rh2 group and MBIC₅₀NaF group,the bacterial density of HC combined group decreased significantly and almost no green signal.6.The production of EPS in LC combined group was less than that in 1/5MBIC₅₀G-Rh2 group or 1/4 MBIC₅₀NaF group,and the production of EPS in HC combined group was lower than that in MBIC₅₀G-Rh2 group or MBIC₅₀NaF group.7.TheΔp H value of LC combined group was lower than that of 1/5MBIC₅₀G-Rh2 group or 1/4 MBIC₅₀NaF group,and theΔp H value of HC combined group was lower than that of MBIC₅₀G-Rh2 group or MBIC₅₀NaF group.The acidogenic inhibition rate of the combination groups were higher than that of single agents groups,and the acidogenic inhibition rate of HC combined group was as high as 89.3%.8.The activity of LDH in LC combined group was lower than that in 1/5MBIC₅₀G-Rh2 group and 1/4 MBIC₅₀NaF group,and the activity of LDH in HC combined group was lower than that in MBIC₅₀G-Rh2 group and MBIC₅₀NaF group.9.The results of CCK-8 assay showed that when G-Rh2 was used alone,there were no obvious cytotoxicity in all groups.When NaF was used alone,the MBIC₅₀NaF group had inhibitory effect on L929 mouse fibroblasts,and the other groups had no obvious cytotoxicity.When combined with G-Rh2 and NaF,the MIC₅₀G-Rh2+MIC₅₀NaF combined group had inhibitory effect on cells,and the other combined groups had no obvious cytotoxicity.Conclusions:1.The combined application of G-Rh2 and NaF can synergistically inhibit the growth of S.mutans.2.The combined application of G-Rh2 and NaF can synergistically inhibit the formation of S.mutans biofilm and reduce its cariogenic virulences such as acid production and EPS production.3.The HC combined group,the LC combined group and the MIC₅₀G-Rh2+MIC₅₀NaF combined group had good biosafety.