Antibacterial Effect Of Three Natural Plant Extracts In Pairs On Streptococcus Mutans: An In Vitro Study

Objective: Dental caries is a common and frequently occurring disease in human oral cavity.Streptococcus mutans,as the main cariogenic microorganism,plays a vital role in the occurrence and development of dental caries.Aloin,chlorogenic acid,and epigallocatechin gallate(EGCG)are three natural plant extracts,which have been proved to have antibacterial effects on Streptococcus mutans.The minimum inhibitory concentrations(MICs)of the mixtures composed of three single-component extracts compatible with each other(aloe-chlorogenic acid,aloin-EGCG,EGCG-chlorogenic acid)against Streptococcus mutans were determined by checkerboard dilution method.Classification inhibition concentration(FIC)index was calculated to determine the combined effect.The effects of two-fold dilution concentration gradient mixtures below the MIC on acid production,adhesion and biofilm formation of Streptococcus mutans were also determined,so as to explore their biological effects on Streptococcus mutans and provide experimental bases for their applications in the prevention of caries.Methods: MICs and MBCs of aloin,chlorogenic acid and EGCG against Streptococcus mutans were determined by micro-liquid dilution method.The MICs of three kinds of mixtures against Streptococcus mutans were determined by checkerboard dilution method.Select 5 concentration gradients(1/2,1/4,1/8,1/16,1/32MIC)that are lower than the MIC in the combined bacteriostatic experiment,each mixture and Streptococcus mutans were cultured at 37 ℃for 24 hours at constant temperature,measure the final p H value of the culture supernatant,and then calculate the p H value before and after the cultivation of Streptococcus mutans.The 24 h biofilm models of Streptococcus mutans were constructed in vitro,MTT method was used to determine the amounts of biofilm adhesion after the above-mentioned mixture were applied for 24 hours.The effects on biofilm activities and structures were observed by confocal laser scanning microscope(CLSM).All experimental datas were analyzed by SPSS 21.0 statistical software.Results: The MIC and MBC of aloin were 0.63mg/ml and 1.25mg/ml,respectively.The MIC and MBC of chlorogenic acid were 5mg/ml and 10mg/ml,respectively.The MIC of EGCG is 0.125mg/ml and MBC is 0.5mg/ml.The MIC of aloin-chlorogenic acid mixture was 0.31+2.5mg/ml.The MIC of aloin-EGCG mixture was 0.31+0.063mg/ml.The MIC of EGCG-chlorogenic acid mixture was 0.063+1.25mg/ml.The FIC indexes of the three mixtures were 1,1 and 0.75,respectively.They’re all additive effect.In acid production experiment,(35)p H values of Streptococcus mutans increased significantly with the decrease of mixtures concentration,and the differences were statistically significant(P<0.05).In the adhesion experiment,OD values decreased gradually when the concentration increased,and the differences were statistically significant(P<0.05).The result of CLSM observations showed that the biofilm became sparse after the treatment of each group of mixtures,the activities of biofilm were gradually reduced with the increases of the concentration of mixtures.Conclusion: Aloin combined with chlorogenic acid,chlorogenic acid combined with EGCG,and chlorogenic acid combined with EGCG all have additive effects on the in vitro antibacterial activity of Streptococcus mutans,which can effectively inhibit the growth,acid production and adhesion of Streptococcus mutans at a lower concentration than the single component drugs.Aloin-chlorogenic acid,aloin-EGCG and EGCG-chlorogenic acid,all three mixtures can be considered as mouthwashes to prevent dental caries.