Formulation Strategy of <i>Cymbopogon</i> spp. Essential Oils to Reduce Irritation of <i>Cinnamomum Burmani</i> Essential Oil While Preserving Anti‐ <i>Streptococcus mutans</i> Activity

ABSTRACT Dental caries, a prevalent global health issue, arises from ecological dysbiosis of the oral biofilm, with Streptococcus mutans ( S. mutans ) being a primary cariogenic pathogen. Although Cinnamomum burmani essential oil (CBEO) possesses strong antimicrobial properties, its clinical utility is constrained by irritancy and cytotoxicity. This study therefore aimed to enhance antibacterial efficacy against S. mutans and reduce cytotoxicity by combining CBEO with three Cymbopogon spp. essential oils: C. nardus (CNEO), C. citratus (CCEO), and C. khasans (CKEO). The chemical compositions of the essential oils were characterised by GC–MS. Optimal combination ratios were screened via the agar diffusion method, and the minimum inhibitory/bactericidal concentrations (MIC/MBC) were determined using the microbroth dilution method. Antibacterial, anti‐biofilm, and acid‐production‐inhibiting effects were evaluated through time‐kill assays, biofilm staining, and pH measurements, respectively. Virulence gene expression was assessed by qRT‐PCR, and cytotoxicity was determined via MTT assay on RAW 264.7 cells. GC–MS analysis revealed distinct chemical profiles: CBEO was predominantly (E)‐cinnamaldehyde (60.98%), whereas CNEO, CCEO and CKEO were rich in citronellal, α‐citral and geraniol, respectively. Synergistic antibacterial effects were observed for the combinations CNEO:CBEO (2:3), CCEO:CBEO (1:1), and CKEO:CBEO (1:4). At sub‐inhibitory concentrations, these combinations dose‐dependently inhibited biofilm formation (73.25%–91.25%), maintained a near‐neutral pH, and downregulated the expression of key virulence genes associated with adhesion ( gbpB, spaP ), exopolysaccharide synthesis ( gtfB, gtfC, gtfD ), acid tolerance ( brpA, relA ) and global regulation ( vicR ). Furthermore, the combination formulas significantly reduced cytotoxicity against RAW 264.7 cells compared to CBEO alone. In conclusion, the combination of Cymbopogon spp. essential oils with CBEO demonstrates a multitargeted synergistic effect against S. mutans and is associated with reduced cytotoxicity at the tested concentrations, suggesting potential for improved biosafety. These findings provide preliminary evidence for a promising formulation strategy, warranting further investigation to elucidate the underlying mechanisms and confirm safety in more complex models.