Comparative In-Vitro Evaluation of Probiotic Mouthwash Formulations with Cannabis Extracts and Propolis on TNF-α Reduction

Introduction Tumor necrosis factor-alpha (TNF-α) is a pro-inflammatory cytokine involved in the wound healing process. Elevated TNF-α levels contribute to chronic inflammation and impaired healing. Probiotics have demonstrated potential in reducing TNF-α, and both cannabis extract (CBD) and propolis possess anti-inflammatory properties. This study aimed to evaluate the effects of probiotic mouthwash formulations containing CBD, propolis, or both, on TNF-α secretion in vitro , with potential applications in managing oral inflammation. Methods Four probiotic mouthwash formulations were prepared: (1) probiotics with 1% propolis, (2) probiotics with 5% propolis, (3) probiotics with 1% CBD and 1% propolis, and (4) probiotics with 1% CBD and 5% propolis. Additional controls included propolis extracts at 1% and 5%, as well as a previously studied optimal formulation containing 10% probiotics and 1% CBD. TNF-α production was induced in LPS-stimulated THP-1 cells, and secretion levels were quantified using sandwich ELISA. Results All tested formulations significantly inhibited TNF-α secretion (>90%), with the highest inhibition (92.96%) observed in the formulation containing probiotics and 5% propolis. In contrast, the 10% probiotic + 1% CBD formulation and 1% CBD extract alone showed lower inhibition rates of 66.54% and 13.25%, respectively. Statistical analysis confirmed the superior efficacy of propolis-containing formulations ( p < 0.05). No cytotoxic effects were observed. Discussion The findings confirmed that propolis enhanced the anti-inflammatory efficacy of probiotic mouthwashes, likely through synergistic mechanisms involving flavonoids and polyphenols that suppress TNF-α by inhibiting the NF-κB pathway. In contrast, adding CBD did not yield additional benefit when combined with propolis. A concentration-dependent effect was observed, as 5% propolis yielded greater inhibition than 1%. Despite assay limitations and in vitro constraints, these results support the potential of probiotic–propolis mouthwashes as adjunctive therapies for inflammatory oral conditions. Conclusion Probiotic mouthwash formulations containing propolis, either alone or in combination with CBD, significantly suppressed TNF-α secretion more effectively than probiotic or CBD formulations alone. The formulation with 5% propolis demonstrated a significant anti-inflammatory effect, suggesting a promising role for propolis in oral healthcare applications.