Naringin as a Non-antibiotic Agent for Multi-species Oral Biofilm Control: In Vitro Antimicrobial Mechanisms and In Vivo Safety in a Rat Caries Model

Jie Song铭岐 刘Yizinigaer YasenYing ZhaoZeyu Wu^*Jin Zhao^*

• First Affiliated Hospital of Xinjiang Medical University, Urumqi, China

Dental caries is closely linked to oral microbiota dysbiosis, while conventional antibiotic interventions easily disrupt oral commensal balance. Thus, there is an urgent need to develop natural-sourced non-antibiotic agents to restore oral cariostatic homeostasis by regulating cariogenic biofilms. This study investigated naringin as a non-antibiotic agent to evaluate its role in multi-species oral biofilm control and anti-caries potential. In vitro, naringin was tested against planktonic and biofilm forms of Streptococcus mutans, Streptococcus sanguinis, Streptococcus sobrinus (mono-species) and their 1:1:1 consortium (multi-species). Minimum inhibitory/bactericidal concentrations (MIC/MBC) and minimum biofilm inhibition/reduction concentrations were determined. Its effects on extracellular polysaccharide synthesis, acid production, and bacterial adhesion to hydroxyapatite (enamel mimic) were assessed. In vivo, Sprague-Dawley rats with multi-species-induced caries received topical naringin for 4 weeks (2×MIC, MIC, 1/2×MIC). Caries lesions were evaluated via Keyes scoring and micro-computed tomography. Oral microbiota balance, serum biochemistry, and histopathology were analyzed to assess safety. Naringin exhibited concentration-dependent antimicrobial activity: MICs were 1.00 mg/mL for S. mutans/S. sanguinis, 0.50 mg/mL for S. sobrinus, and 0.50 mg/mL for the consortium; corresponding MBCs were 2.00, 2.00, 1.00, and 1.00 mg/mL. At MIC, naringin reduced S. mutans EPS by 35%, maintained biofilm pH > 5.5 (critical enamel demineralization threshold), inhibited multi-species Hydroxyapatite (HAP) adhesion by 68.3%, and exhibited a potential selectivity by effectively suppressing cariogenic pathogens while exerting lesser impact on the commensal S. sanguinis, thereby contributing to microbiota stabilization. In vivo, naringin (MIC) reduced Keyes scores of rat molar smooth/sulcal surfaces by > 60%, preserved enamel integrity (micro-CT), and showed no organ toxicity or mucosal irritation. Collectively, Naringin, a non-antibiotic agent, effectively regulates multi-species cariogenic biofilms by targeting the "adhesion-biofilm-acid-EPS" cascade. Its natural origin, favorable in vivo safety, and ecological selectivity (protecting commensals) enable it to address oral dysbiosis (the root cause of caries), holding great potential for non-antibiotic caries prevention.