Anti-Biofilm Activity of (+)-Endo-Borneol Against Streptococcus mutans: Experimental Evaluation, Virulence Gene Expression Analysis, and Molecular Docking

Streptococcus mutans is the primary etiological agent of dental caries due to its ability to form acidogenic biofilms on tooth surfaces. Natural monoterpenes have attracted considerable interest as potential antibiofilm agents for oral healthcare. The present study investigated the antibiofilm activity and possible mechanism of action of (+)-endo-borneol isolated from the essential oil of Achillea millefolium against S. mutans. The chemical composition of the essential oil was characterized by gas chromatography–mass spectrometry (GC–MS), and (+)-endo-borneol was isolated by chromatographic separation. Antibiofilm activity was evaluated using the crystal violet biofilm assay, while antimicrobial activity was determined by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays. The influence of subinhibitory concentrations of (+)-endo-borneol on the expression of the biofilm-associated genes gtfB and yycF was assessed by quantitative real-time PCR. Molecular docking was performed to investigate ligand–protein interactions, using a ligand geometry pre-optimized by density functional theory (DFT, B3LYP/6-31G**). The essential oil inhibited S. mutans biofilm formation by up to 98%, whereas isolated (+)-endo-borneol reduced biofilm biomass by 97–98% at concentrations of 2–10 mg/mL. The MIC and MBC values of (+)-endo-borneol were 2.5 and 5.0 mg/mL, respectively. Gene expression analysis demonstrated that subinhibitory concentrations of (+)-endo-borneol modulated the transcription of gtfB and yycF, indicating activation of bacterial regulatory responses. Molecular docking revealed favorable binding of (+)-endo-borneol to biofilm-related protein targets. These findings demonstrate that (+)-endo-borneol is a promising natural antibiofilm compound with potential application in the development of novel preventive and therapeutic oral healthcare products targeting S. mutans biofilms.