Comparative Cytotoxicity Evaluation of a Propolis–Curcumin Mouthwash Using Danio rerio Embryos and Artemia salina Nauplii Models

Abstract

Background: Natural product–based mouthwashes are increasingly investigated as safer, multifunctional alternatives to conventional chemical formulations. Propolis and curcumin possess well-documented antimicrobial, anti-inflammatory, and antioxidant properties; however, their cytotoxic profile in combined formulations remains inadequately explored across biological models.

Aim: To comparatively evaluate the cytotoxic potential of a novel propolis–curcumin mouthwash using two ecotoxicological models: Danio rerio (zebrafish) embryos and Artemia salina (brine shrimp) nauplii.

Materials and methods: The mouthwash was formulated by combining ethanolic propolis extract with solubilized curcumin in a hydroalcoholic vehicle, stabilized with surfactants and adjusted to physiological pH. Zebrafish embryos at 6 hours post-fertilization and brine shrimp nauplii hatched within 24 hours were exposed to serial dilutions (0.1–10%) of the formulation. Mortality, developmental abnormalities, and motility were assessed at 24, 48, and 72 hours post-exposure. Median lethal concentration (LC50) values were calculated for both models.

Results: Minimal cytotoxicity was observed at concentrations ≤1% in both models. The LC50 value was 4.5% for zebrafish embryos and 5.2% for brine shrimp nauplii. Zebrafish embryos exhibited pericardial edema and delayed hatching at higher concentrations, whereas brine shrimp nauplii demonstrated reduced motility followed by lethality. Comparable LC50 values across models indicated cross-system consistency.
Conclusion: The propolis–curcumin mouthwash demonstrated low cytotoxicity at clinically relevant concentrations. The comparable LC50 values in both ecotoxicological models suggest acceptable safety margins, supporting its further development as a biocompatible adjunct in oral healthcare.