Optimization of an antifungal formulation based on eucalyptol, β-pinene, and α-terpinene against Candida albicans and Candida glabrata: A mixture design study.

This study aimed to optimize a natural antifungal formulation comprising eucalyptol, β-pinene, and α-terpinene against Candida albicans, and Candida glabrata, two clinically relevant opportunistic fungal pathogens. These three monoterpenes, commonly found in essential oils of medicinal and aromatic plants, are recognized for their broad-spectrum antimicrobial properties, including notable antifungal activity. Prior to formulation studies, the physico-chemical profiles of the compounds were characterized, and an in silico toxicological assessment was conducted using predictive tools. All molecules were found to be safe, showing no potential for organ toxicity or adverse toxicological endpoints. An augmented simplex-centroid mixture design with three components was applied to evaluate twelve distinct formulations, enabling the analysis of compositional effects on antifungal efficacy, as determined by minimum inhibitory concentration (MIC) values. The results demonstrated that the antifungal activity was formulation-dependent. For C. albicans, the MIC was significantly influenced by the individual contribution of eucalyptol (σ₁) and its binary interaction with β-pinene (σ₁₂) (p < 0.05). In contrast, for C. glabrata, the individual effects of all three components (σ₁, σ₂, σ₃) and their ternary interaction (σ₁₂₃) were statistically significant (p < 0.05). The fitted models showed high predictive accuracy, with determination coefficients (R