The Antibacterial Mechanism Of Tea Polyphenols And Grapefruit-seed Extract Compounds Against Streptococcus Mutans: An In Vitro Study

Objective:Nowadays, tea polyphenols(TPs) and grapefruit-seed extract(GSE) are natural bacteriostats, which are normal in our reasearch. However, both of them have shortcomings when using alone. The TPs solution has certain bitter feeling when achieve the effective antibacterial concentration; and clinical study found that the antimicrobial effect of GSE is just the same as Cetylpyridinium Chloride, lower than chlorhexidine. This research is to explore the feasibility of anticariesaction when TPs combined with GSE, which is hoping to reduce the using dosage and side effects by the koinonia of the two bacteriostats. The dental caries is an infectious disease resulting in destruction of tooth structure. Streptococcus mutans(S.mutans) is the main cariogenic bacteria. This study is to prove the effects of TPs combined with GSE against the growth, adherence, extracellular polysaccharide and GTF activity of S.mutans, in order to explore their best concentrations and feasible cariostatic mechanism after they are combined, then can provide experimental proof for the clinical practice of the mixture.Methods:1.Effects of growth and acid production of S.mutans by the mixture of TPs with GSEMinimal inhibitory concentration (MIC) of alone and combined applications of TPs with GSE on growth of S.mutans (ATCC 25175) were measured by solution dilution and checkerboard methods. Fractional inhibitory concentration (FIC) was calculated accordingly to evaluate the combined effect of TPs with GSE; Theâ–³p H values were measured after combined application of TPs with GSE in the concentrations lower than MIC.2.Effect of adherence of S.mutans by the mixture of TPs with GSEThe 4 mixtures(1/2, 1/4, 1/8, 1/16MIC) of TPs with GSE in the concentrations lower than MIC were cultivated with S.mutans to measure the adherence ratio and the adherence inhibition ratio, then the morphology characteristic of the adherence on slide of S.mutans with different concentrations were obsereved by Scanning Electron Microscope(SEM).3. Effects of extracellular polysaccharide and GTF activity of S.mutans by the mixture of TPs with GSEThe 4 mixtures(1/2, 1/4, 1/8, 1/16MIC) of TPs with GSE in the concentrations lower than MIC were cultivated with S.mutans, the supernatant were divided into two batches after centrifugation. One batch of the solution was to obtain the crude GTF. Somogyi and bradford methods were used to measure the reducing sugar and the content of total protein of GTF, then specific activity was calculated. Another batch of solution was to measure the content of extracellular polysaccharide with anthrone method.Results:1.Effects of growth and acid production of S.mutans by the mixture of TPs with GSECompared with the application of TPs(MIC=2mg/ml) or GSE(MIC=46.88mg/L) alone, both of these two bacteriostats possess lower MICs when combined.For instance, TPs (1mg/ml) combined with GSE(37.5mg/L)could reach the MIC, with an FIC of 0.75; and theâ–³p H value of S.mutans decreased obviously when the concentration increased, all of the test groups had statistics differences compared with the control group(P<0.05).2.Effect of adherence of S.mutans by the mixture of TPs with GSEThe adherence ratios of S.mutans decreased and the adherence inhibition ratios of S.mutans increased when the concentration of the mixture increased, all of the test groups had statistics differences when compared with control group(P<0.05), and all of them had statistics differences when compared with each other(P<0.05). The photos of SEM showed 1/2MIC group had less amorphous matter between the bacterias than the blank group.3. Effects of extracellular polysaccharide and GTF activity of S.mutans by the mixture of TPs with GSEThe WIG and water-soluble polysaccharide of S.mutans decreased when the concentration of the mixture increased: the water-soluble polysaccharide of the test groups compared with the control group, only the highest concentration group had statistics difference(P<0.05), all of the WIG of the test groups had statistics differences (P<0.05) when compared with the control group. The GTF activity and compare energy of S.mutans decreased when the concentration of the mixture increased, all of the test groups had statistics differences when compared with the control group (P<0.05).Conclusion:1.Combined application of TPs and GSE has additive antibacterial effect on S.mutans. Compared with the application of TPs or GSE alone, combined application of TPs with GSE can inhibit the growth and acid production of S.mutans at lower concentrations.2.The mixture of TPs and GSE can effectively inhibit sucrose dependence adhesion of S.mutans, and it deduces that the mixture can inhibit the synthetizer of WIG to reduce sucrose dependence adhesion of S.mutans by SEM.3.The mixture of TPs and GSE can inhibit the WIG and GTF activity of S.mutans, but has no obvious inhibition of water-soluble polysaccharide of S.mutans.