The Effect of Toothpastes Containing Natural Extracts on Bacterial Species of a Microcosm Biofilm and on Enamel Caries Development

Abstract

This study investigated the effects of herbal toothpaste on bacterial counts and enamel demineralization. Thirty-six bovine enamel samples were exposed to a microcosm biofilm using human saliva and McBain saliva (0.2% sucrose) for 5 days at 37 °C and first incubated anaerobically, then aerobically-capnophilically. The following experimental toothpaste slurries (2 × 2 min/day) were applied: (1) Vochysia tucanorum (10 mg/g); (2) Myrcia bella (5 mg/g); (3) Matricaria chamomilla (80 mg/g); (4) Myrrha and propolis toothpaste (commercial); (5) fluoride (F) and triclosan (1450 ppm F), 0.3% triclosan and sorbitol (Colgate^®, positive control); (6) placebo (negative control). The pH of the medium was measured, bacteria were analyzed using quantitative polymerase chain reaction, and enamel demineralization was quantified using transverse microradiography. The total bacterial count was reduced by toothpaste containing Myrcia bella, Matricaria chamomilla, fluoride, and triclosan (commercial) compared to the placebo. As far as assessable, Myrcia bella, Matricaria chamomilla, and Myrrha and propolis (commercial) inhibited the outgrowth of S. mutans, while Lactobacillus spp. were reduced/eliminated by all toothpastes except Vochysia tucanorum. Mineral loss and lesion depth were significantly reduced by all toothpastes (total: 1423.6 ± 115.2 vol% × μm; 57.3 ± 9.8 μm) compared to the placebo (2420.0 ± 626.0 vol% × μm; 108.9 ± 21.17 μm). Herbal toothpastes were able to reduce enamel demineralization.

Keywords: antimicrobial strategies; dental caries; oral biofilms; plants.

Figure 1

Experimental microcosm biofilm protocol as well as the correspondent treatments and the response variables applied to analyze the biofilm and the tooth.

Figure 2

The image shows the 24-well microtiter plates during the experiment (a) and after the removal of enamel specimens, leaving a print in the biofilm (b).

Figure 3

Boxplot of genome counts of total bacteria, Lactobacillus spp., and Streptococcus mutans present in microcosm biofilm after treatments. (a) Boxplot of genome counts of total bacteria present in microcosm biofilm after treatments (Kruskal–Wallis/ Dunn’ multiple comparison test, p < 0.0001). (b) Boxplot of genome counts of Lactobacillus spp. present in microcosm biofilm after treatments (Kruskal–Wallis/ Dunn’ multiple comparison test, p = 0.003). Lactobacillus spp. (n = 3 Vochysia tucanorum, n = 1 Myrchia bella, n = 6 placebo). (c) Boxplot of genome counts of Streptococcus mutans present in microcosm biofilm after treatments (Kruskal–Wallis/ Dunn’ multiple comparison test, p > 0.99). S. mutans (n = 2 Vochysia tucanorum, n = 1 F and triclosan toothpaste, n = 1 placebo). Genomes per microliter of samples were determined by qPCR. Different letters show significant differences among the treatments.

Figure 3

Boxplot of genome counts of total bacteria, Lactobacillus spp., and Streptococcus mutans present in microcosm biofilm after treatments. (a) Boxplot of genome counts of total bacteria present in microcosm biofilm after treatments (Kruskal–Wallis/ Dunn’ multiple comparison test, p < 0.0001). (b) Boxplot of genome counts of Lactobacillus spp. present in microcosm biofilm after treatments (Kruskal–Wallis/ Dunn’ multiple comparison test, p = 0.003). Lactobacillus spp. (n = 3 Vochysia tucanorum, n = 1 Myrchia bella, n = 6 placebo). (c) Boxplot of genome counts of Streptococcus mutans present in microcosm biofilm after treatments (Kruskal–Wallis/ Dunn’ multiple comparison test, p > 0.99). S. mutans (n = 2 Vochysia tucanorum, n = 1 F and triclosan toothpaste, n = 1 placebo). Genomes per microliter of samples were determined by qPCR. Different letters show significant differences among the treatments.

Figure 4

Representative TMR pictures (20×) of the artificial carious enamel lesions created using microcosm biofilm after treatment with different toothpastes. (a) Vochysia tucanorum; (b) Myrcia bella; (c) Matricaria chamomilla; (d) Myrrha and propolis toothpaste; (e) F and triclosan toothpaste (positive control); (f) Placebo (negative Control). The black arrow shows the initial caries lesions (darker in color). Placebo showed a more radiolucent area (darker) indicating more enamel demineralization compared to the other groups, which were more radiopaque (brighter), thus, less demineralized.