Supplementary data tables for: Antimicrobial and anticaries effects of solutions containing Malva sylvestris combined or not with fluoride under a microcosm biofilm model

Malva sylvestris has demonstrated promising biological activity; however, the contribution of the plant itself, independent of xylitol or fluoride present in commercial formulations, remains unclear. This study evaluated the effects of different combinations of Malva sylvestris on microbial composition, colony-forming unit (CFU) counts, cytotoxicity, and tooth demineralization using a microcosm biofilm model. Enamel and dentin samples (n = 12 per group) were assigned to the following treatments: Malva sylvestris (25 mg/mL), Malva sylvestris + 5% xylitol, Malva sylvestris + fluoride (225 ppm), Malva sylvestris + xylitol + fluoride, xylitol, fluoride, Malva sylvestris in a commercial formulation (Malvatricin Plus®), chlorhexidine (0.12%), and PBS. Plant metabolites were extracted by percolation. Microcosm biofilms were developed according to Braga et al. (2021), and samples were treated for 1 minute daily from day 2 to day 5. CFU counts were performed for Streptococcus mutans / S. sobrinus, Lactobacillus spp., and Candida albicans. Biofilm composition was further analyzed by 16S rRNA gene sequencing (Illumina MiSeq) and QIIME, including relative abundance, diversity, and rank analyses. Cytotoxicity of Malva sylvestris (0.8–20 mg/mL) was evaluated using a human gingival fibroblast cell line. Tooth demineralization was quantified by transverse microradiography (TMR). Malva sylvestris at 20 mg/mL showed lower cytotoxicity than chlorhexidine (0.075 mg/mL). The combination of Malva sylvestris and xylitol reduced S. mutans counts in dentin biofilms compared with PBS (1 log₁₀/mL reduction; ANOVA, p = 0.03), but no significant effect was observed in enamel biofilms. Microbial communities in both enamel and dentin biofilms treated with Malva sylvestris were similar to the negative control. Although no direct antimicrobial effect was observed, the combination of Malva sylvestris, fluoride, and xylitol significantly reduced enamel demineralization compared with PBS (Kruskal–Wallis, p < 0.0001), with no effect on dentin demineralization. Overall, Malva sylvestris extract alone showed no antimicrobial or anticaries effects, with beneficial outcomes observed only when combined with fluoride and xylitol.