Pre-clinical studies on the anti-caries potential of the aqueous extract of Malva sylvestris: evaluation of composition, tooth pigmentation, and cytotoxicity as well as antimicrobial and demineralization effects in vitro and in situ

Abstract

The aim of this proposal will be to evaluate the antimicrobial, anti-biofilm and anti-caries potential of Malva sylvestris extract on enamel and dentin, as well as its chemical composition, the capacity to change the tooth enamel color of and its cytotoxicity in human fibroblasts. This proposal will be performed in two phases, as follows: A) In vitro phase: Chemical fingerprint of the commercial extract of Malva sylvestris by UPLC-HRMS; Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) on microorganisms in planktonic phase (Streptococcus mutans, Lactobacillus casei and Candida albicans); Evaluation of the effect of experimental solutions on oral microcosm biofilm model, using bovine enamel and dentin samples. The experimental solutions that will be tested are: 1. Malva sylvestris (1-MIC); 2. Malva sylvestris (2-MBC); 3. Malva sylvestris (1) + 5% xylitol; 4. Malva sylvestris (2) + 5% xilitol; 5. Malva sylvestris (1) + 5% xylitol + Fluoride (NaF, 225 ppm F); 6. Malva sylvestris (2) + 5% xilitol + Fluoride (NaF, 225 ppm F); 7. 5% Xylitol; 8. Fluoride (NaF, 225 ppm F); 9. Malvatricin Plus® (positive control 2); 10. Periogard® (positive control 1) and 11. PBS (negative control). The anti-biofilm effect will be evaluated by Colony Forming Unit counting (Log10CFU/mL) and by microbiome analysis using 16S rRNA gene Illumina MiSeq. The effect on bacterial metabolites will be analyzed by UPLC-HRMS; while carious lesion development will be measured by Transverse Microradiography (TMR). Further, analysis of color alteration of tooth enamel using a digital spectrophotometer (Vita Easyshade®), and of the cytotoxic effect on human fibroblasts by MTT reduction assay and confocal microscopy will be done; B) In situ phase: a double-blind and crossover study will be performed with 15 participants who will wear the palatal appliance containing 4 bovine teeth samples (2 enamel and 2 dentin) protect by mesh, for 7 days, using 6 applications to 30% sucrose every day; the treatments will be carried out in three different phases for the following solutions: 1) The best solution containing Malva sylvestris determined in the 1st phase of the study; 2) Solution containing fluoride (225 ppm); and 3) Placebo solution (deionized water) (all 10 mL, 2x/day/1 minute). After 7 days, the biofilm from 2 samples will be removed and stored for analysis of microorganisms by CFU, while the biofilm from the 2 remaining samples will be transferred to a DNA/RNA protection solution (DNA/RNA shield, Zymo®) for microbiome analysis using 16S rRNA gene Illumina and the metabolites by UPLC-HRMS. The tooth demineralization will be evaluated by TMR. The data will be tabulated and submitted to statistical analysis using Graph Pad Software (San Diego, USA). It will be verified if the data have a normal and homogeneous distribution. After this verification, the most appropriate statistical test, parametric or non-parametric, will be applied to compare the different treatments. The significance level adopted in all tests will be 5%. (AU)