Experimental Study Of Inhibitory Effect Of The Grapefruit Seed Extract Mouthwash On The Streptococcus Mutans And Actinobacillus Actinomycetemcomitans In Vitro

Caries and periodontal disease are both infectious diseases which result from dental plaque. Streptococcus mutans (S. mutans) and Actinobacillus actinomycetemcomitans (Aa) paly an important role in the creation of oral plaque and they survive in oral cavity in the form of plaque biofilms. S.mutans is recognized as the main cariogenic bacteria. It has been reported that Aa is closely related to the occurrence of chronic periodontitis, especially related to localized aggressive periodontitis. Recently, it becomes one hot spot that scholars try to obtain active substances from natural resources instead of chemical drugs. Grapefruit-seed extract (GSE) is a natural plant extract, containing a great number of antioxidant polyphenols. It has been proved that GSE can inhibit the growth of bacteria, fungi, viruses and parasites, and it also has excellent biocompatibili- ty. GSE mouthwash all use raw material which is edible, and it does not include alcohol, chloride, iodine, tetracycline, metronidazole and other chemical ingredients.GSE mout- hwash would not produce oral mucosa irritation and make the surface of tooth and mucosa staining. Furthermore, it wouldn't cause the reduction of gustatory sensation, the secretion of saliva and make the mouth drying, burning pain, rash and other adverse reaction. It has be been found that GSE mouthwash has excellent ability of inhibiting plaque during the previous research. However, the mechanism is not fully understood. This paper is to investigate the anti-adhesive effect on S.mutans of GSE mouthwash, to measure its antibacterial activities of S.mutans and Aa, and also study the efficacy of GSE mouthwash on the vitality of the S.mutans biofilm observed by Confocal Laser Scanning Microscope(CLSM). The primary goal of this study is to explore the feasible mechanism of GSE mouthwash, which provide experimental evidence for it to be used in the clinical practice.This study has included three parts: Part I: In vitro study of Anti-bacteria adhesive effect of GSE mouthwash on S.mutans0bjective: To investigate the anti-adhesive effect of GSE mouthwash on S.mutans to the smooth glass surface. Methods: Different concentration of GSE mouthwash was added into the culture medium, S.mutans were cultivated with glass tube at 37℃for 48 hour.The adherent rates were determined by reading of the optical density. The adherent morphology of S.mutans in presence or absence of GSE mouthwash was observed by using a scanning electron microscope. Results: There were significant differences in adherence of S.mutans ATCC25175 cells to the smooth glass surface among seven doses with that of the control TPY media.The GSE mouthwash inhibited the adherence of S.mutans. The inhibition was dose-dependent. The difference of adherent morphol- ogy between GSE mouthwash and control media was noticeable. Conclusion: GSE mouthwash have the anti-adhesive effect on S.mutans to the smooth glass surface.PartⅡ: In vitro study of antibacterial activity of GSE mouthwash on S.mutans and AaObjective: To investigate the antibacterial activity of GSE mouthwash against Aa and S.mutans and to explore the potential mechanism. Methods: The minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) of the GSE mouthwash against S.mutans and Aa were detected using double dilution. And the cell wall alteration of S.mutans treated with GSE mouthwash or not were observed by using a scanning electron microscope(SEM). Results: The MIC and MBC determined for GSE mouthwash on Aa were 1:64 and 1:32.The MIC and MBC determined for GSE mouthwash on S.mutans were 1:32 and 1:16. SEM observations showed that both rupturing of the cell wall and releases of the intracellular components were observed in the GSE mouthwash-treated group. Conclusions: The GSE mouthwash can inhibit and kill the growth of Aa and S.mutans in vitro, it is suggested that GSE mouthwash has an antibacterial effect on the S.mutans by affecting the normal structure and function of the cell wall of S.mutans.PartⅢ: Efficacy of GSE mouthwash on the vitality of the S.mutans biofilm observed by CLSMObjective: To observe the efficacy of GSE mouthwash compared with Cetylpyridinium chloride on the S.mutans biofilms at in different time periods. Methods: S.mutans biofilms formed in vitro on glass slices in 3h,24h,48h respectively were acted on with the two mouthrinses for 1min, and were then stained with two fluorescent dyes, which selectively stain vital bacteria green and dead bacteria red. The changes of the vital bacteria percentage after treated with the two mouthrinses were then observed under CLSM. Results: Reductions of vital bacteria percentage were found in the outer layers of the 3, 24, 48h S. mutans biofilms and the middle layers of the 3 h S.mutans biofilms. However, there were no significant differences compared with Cetylpyridinium chlori- de in outer, middle and inner layers of S.mutans biofilms formed at different time periods(P>0.05). Conclusions: The two mouthrinses have a similar antibacterial effects on the outer layers bacteria of the S.mutans biofilms formed at different time periods, however, just affect the deep layers bacteria survival of the early(3h) S.mutans biofilms.