[In vitro and in vivo experimental study on the anti-caries effect of ellagic acid from Xinjiang pomegranate peel].

Objective: To investigate the anti-caries effect of ellagic acid (EA) extracted from Xinjiang pomegranate peel. Methods: In vitro experiments: the broth microdilution method was used to determine the minimum reduction concentration (MIC₅₀) and minimum bactericidal concentration (MBC) of EA against planktonic states of Streptococcus mutans (Sm), Streptococcus sobrinus(Sso) and Streptococcus sanguinis (Ssa). Growth and acid production curves were plotted. The water-insoluble extracellular polysaccharide content was measured using the anthronesulfuric acid method. The minimum biofilm inhibitory concentration (MBIC₅₀) and minimum biofilm reduction concentration (MBRC₅₀) of EA against biofilms were determined by crystal violet staining. The effect of EA on acid production by mature biofilms was evaluated. Biofilm structure and bacterial viability were observed using scanning electron microscope (SEM) and live/dead bacterial staining, respectively. Confocal laser scanning microscope (CLSM) was used to analyze the inhibitory effects of EA on extracellular polysaccharides within Sm, Sso, and Ssa biofilms. The effects of EA on Sm, Sso, and Ssa adhesion to saliva-coated hydroxyapatite were also examined. In vivo experiments: forty-eight Sprague-Dawley (SD) rats were randomly divided into eight groups (6 rats per group) using a random number table: a caries model group (distilled water treatment), a solvent control group (negative control, EA solvent treatment), a chlorhexidine group (positive control, 0.12% chlorhexidine treatment), a sodium fluoride group (positive control, 1 000 mg/L sodium fluoride treatment), and three experimental groups (EA solution treatment) with high-(8 mmol/L), medium-(4 mmol/L), and low-dose (2 mmol/L) EA respectively, and a blank control group (no special intervention). Except for the blank control group, which was fed a normal diet, all other groups were fed a cariogenic diet. Rat caries models were established and treated accordingly. The rat teeth and oral mucosa were swabbed with sterile cotton swabs dipped in their corresponding drug solutions. The level of streptococci in rat saliva was detected using the dilution plate counting method. Subsequently, the severities of eight groups dental caries were evaluated using a combination of Keyes scoring and micro-CT analysis. Results: In vitro experiments results showed that the MIC₅₀ values of EA against planktonic states of three cariogenic bacteria: Sm, Sso, and Ssa, were 4.0, 2.0, and 1.0 mmol/L, respectively, while the MBC values were 8.0, 4.0, and 4.0 mmol/L, respectively. The MBIC₅₀ values against their biofilms were 8.0, 4.0, and 1.0 mmol/L, respectively. In contrast, EA exhibited limited efficacy in reducing mature biofilms of all three bacteria (MBRC₅₀≥32 mmol/L). EA disrupted biofilm structure in a concentration-dependent manner. With increasing EA concentrations, the morphology and architecture of monospecies biofilms formed by Sm, Sso, and Ssa, were increasingly severely damaged. Compared to the negative control, EA treatment groups significantly reduced the proportion of viable bacteria and markedly inhibited acid production, polysaccharide synthesis, and bacterial adhesion within the biofilms (all P<0.05). In vivo experiments results demonstrated that, compared to the dental caries model group (5.72±0.19), the salivary streptococcal counts were significantly reduced in the medium-and high-dose EA groups (2.65±0.05 and 3.22±0.11, respectively; both P<0.05). Furthermore, compared to the dental caries model group, the medium-and high-dose EA groups exhibited a significant reduction in the severity of caries lesions on both the smooth surfaces and fissures of the molars (P<0.05), with the high-dose group showing a particularly prominent protective effect (e.g., deep dentinal caries scores: model group: 3.67±0.98 vs high-dose EA group: 1.33±0.41, P<0.001). Critically, no related adverse effects were observed in any of the rat groups. Conclusions: The EA derived from Xinjiang pomegranate peel extract demonstrates significant anti-caries efficacy in both in vitro and in vivo experiments.