Abstract

Candida species are opportunistic fungi infecting humans and animals, with increasing incidence of non–Candida albicans Candida species (NCACs). This study evaluated the antifungal and antivirulence activities of Thai Piper betle leaf extract (Ethanolic P. betle extract; EPE) and its primary phenolic compounds, hydroxychavicol and eugenol, against six Candida strains isolated from animals. Antifungal efficacy was assessed using broth microdilution to determine minimum inhibitory (MIC) and minimum fungicidal (MFC) concentrations. Anti-virulence activities—biofilm formation, extracellular enzyme activity, and hyphal transition— were evaluated via standard assays. Cytotoxicity was examined in Vero cells using the MTT assay and phase-contrast microscopy. All compounds exhibited antifungal activity, with hydroxychavicol demonstrating the lowest MICs (0.008-0.256mg/mL) and consistent fungicidal activity, followed by EPE (0.016-0.256mg/mL) and eugenol (0.667-1.334mg/mL). Biofilm inhibition occurred only in C. krusei WU1, with hydroxychavicol achieving 76.93% reduction at 1/2MIC, followed by eugenol (74.36%) and EPE (69.34%). Enzymatic assays revealed selective inhibition of lipase activity—hydroxychavicol in C. albicans and EPE in C. krusei—while other enzymes were unaffected. Hyphal formation in C. albicans ATCC90028 was markedly suppressed by all compounds, particularly hydroxychavicol. Cytotoxicity profiling revealed that EPE maintained high Vero cell viability (≥98% viability at ≤1MIC; IC₅₀>2MIC), whereas hydroxychavicol and eugenol were cytotoxic at 2MIC but biocompatible at sub-MIC levels. These findings support the potential of P. betle extracts, especially hydroxychavicol, which possesses the strongest antifungal potency, while EPE demonstrated preliminary in vitro safety as an antifungal agent, supporting their potential as antifungal candidates for veterinary applications targeting Candida spp.