Inhibitory and preventive effects of *Arnebia euchroma* (Royle) Johnst. root extract on *Streptococcus mutans* and dental caries in rats

Dental caries, a prevalent oral health issue, is a chronic infectious disease primarily caused by bacterial biofilms, particularly *Streptococcus mutans* (*S. mutans*). Current treatments like chlorhexidine (CHX) have limitations due to side effects and emerging drug resistance. Therefore, there is a growing interest in exploring natural compounds for caries prevention and treatment. *Arnebia euchroma* (Royle) Johnst., a traditional herbal medicine, possesses anti-inflammatory, anti-cancer, and antibacterial properties. Previous research showed that it can inhibit the growth, acid generation, and sugar production of cariogenic bacteria, but its efficacy and safety in preventing caries *in vivo* were yet to be established. This study aimed to investigate the in vitro and in vivo anti-caries effects of AR extract, addressing a critical need for safe and effective alternative caries control strategies.

Extensive literature supports the global burden of dental caries, highlighting its significant impact on public health. *S. mutans*, a key cariogenic bacterium, is known for its ability to metabolize sucrose, produce acid, and form biofilms that contribute to enamel demineralization. Current clinical treatments including fluoride and chlorhexidine, while effective, suffer from drawbacks like side effects and the development of bacterial resistance. This necessitates a search for novel anti-caries agents with improved safety profiles and efficacy. Natural products have emerged as a promising area of research, offering a rich source of bioactive compounds with potential antimicrobial properties and reduced toxicity compared to synthetic alternatives. The medicinal properties of *Arnebia euchroma* (Royle) Johnst. are well-documented in traditional medicine, making it an attractive candidate for investigation as a potential anti-caries agent. However, prior to this study, there was a lack of comprehensive *in vivo* data to support its efficacy and safety in preventing dental caries.

The study employed a multifaceted approach encompassing both in vitro and in vivo methods. In vitro experiments assessed the antibacterial activity of AR extract against *S. mutans* using MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) assays. Biofilm formation and inhibition were evaluated using MBIC (minimum biofilm inhibition concentration) and MBRC (minimum biofilm reduction concentration) assays. Live/dead staining was used to visualize the effect on biofilm structure. The impact on *S. mutans* adhesion to saliva-coated hydroxyapatite (HAP), simulating the tooth surface, was determined using a fluorescence-based assay. In vivo studies utilized a rat caries model. SPF-SD rats were subjected to cariogenic diet and *S. mutans* inoculation, followed by topical application of AR extract at different concentrations. The anti-caries efficacy was evaluated using Keyes scoring, X-ray analysis, CLSM (Confocal Laser Scanning Microscopy), and saliva bacterial counts. The biosafety of AR extract was evaluated by monitoring rat body weight, oral mucosal histology, and organ coefficient.

In vitro studies revealed that AR extract effectively inhibited the growth of *S. mutans*, with a MIC₅₀ of 1.0 mg/mL and MBC of 8.0 mg/mL. It also significantly inhibited biofilm formation (MBIC₅₀ of 4.0 mg/mL) and reduced pre-formed biofilms (MBRC₅₀ of 8.0 mg/mL). Live/dead staining confirmed the disruption of biofilm structure. Importantly, AR extract notably reduced the adhesion of *S. mutans* to saliva-coated HAP, with a concentration of 1.0 mg/mL causing a 50% reduction in adhesion. In the rat caries model, topical application of AR extract significantly reduced the number of *S. mutans* in rat saliva. Keyes scoring, X-ray analysis, and CLSM imaging all demonstrated a significant reduction in caries incidence and severity in the AR extract treatment groups, comparable to the 0.12% CHX positive control group, particularly at higher concentrations (1 and 2 mg/mL). Importantly, AR extract exhibited excellent biocompatibility, with no significant adverse effects observed in the rats based on body weight, organ coefficient, and oral mucosal histology.

The findings strongly support the hypothesis that AR extract possesses significant anti-caries properties. The observed effects on *S. mutans* growth, biofilm formation, and adhesion *in vitro* are consistent with its potential to prevent and treat dental caries *in vivo*. The comparable efficacy to CHX in the rat model demonstrates the potential of AR extract as a safe and effective alternative to conventional anti-caries agents. The mechanism of action likely involves the disruption of *S. mutans* biofilm structure and inhibition of bacterial adhesion. The absence of significant toxicity in the rat model highlights the biosafety of AR extract. The study successfully bridged the gap between in vitro and in vivo studies, providing strong evidence for the clinical translation of AR extract as a novel anti-caries agent. The results are particularly relevant given the current limitations of existing treatments, including CHX.

This study demonstrated the strong anti-caries potential of *Arnebia euchroma* (Royle) Johnst. root extract. Both in vitro and in vivo findings indicate its efficacy in inhibiting *S. mutans* and preventing caries development, without significant toxicity. This warrants further research to explore the isolation and identification of the active compounds within AR extract for potential development into novel, natural-based oral health products.

The study primarily focused on *S. mutans*, while dental caries is a multi-species disease. Future research should examine the effect of AR extract on other cariogenic bacteria and the overall oral microbiome. The rat caries model, while widely used, may not perfectly replicate human caries. Larger-scale studies, including clinical trials, are needed to further validate these promising findings before widespread clinical application.