Streptococcus mutans (S. mutans) is considered the main causative agent of dental caries. The study aims to evaluate the antimicrobial activity of a natural plant product, pure 4,5'’-dihydroxy-anthraquinone-2-carboxylic acid (Rhein) against S. mutans. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to determine the viability of immortalized human keratinocytes (HaCaT) following treatment with Rhein. Assay for antimicrobial activity and the time-killing test were performed to evaluate Rhein effects against planktonic S. mutans. The effect of different concentrations of Rhein on biofilm biomass and the metabolism of biofilm cells were evaluated through crystal violet and MTT assays. Further, Rhein-treated biofilms were viewed by confocal laser scanning microscopy. Rhein effects on acid production and acid environment tolerance were also assessed. The minimum inhibitory concentration (MIC) of Rhein, exerting bacteriostatic action on 90% of planktonic S. mutans (MIC90), was 5.69 µg/mL. MIC and sub-MIC concentrations of Rhein affected the metabolism of biofilm cells and disrupted biofilm biomass with minimal biofilm eradication concentrations (MBEC) inducing 50% (MBEC50) and 90% eradication (MBEC90) of 6.31 and > 50 µg/mL, respectively. Confocal images displayed a significant reduction in biofilm biomass following treatment with increasing concentrations of the compound. Rhein also reduced the virulence of the biofilm by affecting acid production and acid tolerance. Conversely, active concentrations of Rhein did not affect HaCaT cell viability. Together, these findings indicate that Rhein, a natural product that counteracts the virulence of S. mutans, may represent a novel therapeutic option for dental caries.
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