Antifungal ternary mixture targeting fungal viability and virulence: In vitro and in vivo evaluation.

BACKGROUND: Medicinal plants remain a primary reservoir of specialized metabolites for addressing fungal resistance. Combining plant-derived compounds with conventional antifungal drugs represents a promising strategy to improve antifungal efficacy while reducing fungal virulence. PURPOSE: This study evaluated the antifungal activity, mechanism of action, anti-virulence properties, and in vivo efficacy of a ternary mixture composed of Zuccagnia punctata hexane extract, Gaillardia megapotamica methanol extract, and itraconazole. METHODS: Building on previous optimizations, antifungal activity was assessed by broth microdilution against clinically relevant fungi. Mechanistic studies included ergosterol binding and sorbitol protection assays. Anti-virulence effects were evaluated in Candida albicans by analyzing adhesion, morphogenesis, lytic enzyme secretion, and biofilm formation. In vivo efficacy was investigated using the Caenorhabditis elegans infection model. RESULTS: The ternary mixture exhibited potent antifungal activity against a broad spectrum of fungi, including Candida, Cryptococcus, Aspergillus, and dermatophytes, outperforming its individual components. Mechanistic assays suggested that antifungal activity was mainly associated with the G. megapotamica extract through interaction with ergosterol in the fungal membrane without affecting the cell wall. The mixture also significantly inhibited key virulence traits of C. albicans and increased the survival of infected C. elegans. CONCLUSIONS: These findings demonstrate that combining plant-derived extracts with itraconazole represents a promising multi-target antifungal strategy. The results support the exploration of ethnopharmacologically relevant plants as sources of synergistic partners capable of enhancing antifungal efficacy while attenuating fungal virulence.