The increasing resistance of Candida albicans biofilms underscores the urgent need for effective antifungals. This study evaluated the efficacy of zingerone and elucidated its mode of action against C. albicans ATCC 90028 and clinical isolate C1.

Minimum inhibitory concentrations (MICs) of zingerone were determined using CLSI methods against planktonic cells, biofilm formation, and yeast-to-hyphal transition. The mode of action was investigated through fluorescent microscopy, ergosterol assays, cell cycle analysis, and RT-PCR for gene expression.

Zingerone inhibited planktonic growth and biofilm formation at in C. albicans ATCC 90028 and clinical isolate C1 at 2 mg/mL 4 mg/mL and 1 mg/mL and 2 mg/mL respectively. Treatment with the MIC concentration caused significant cell cycle arrest at the G0/G1 phase, halting proliferation in both the strains. Propidium iodide Staining revealed compromised membrane integrity in both the strains. Also, acridine orange and ethidium bromide dual staining showed increased dead cell proportions in C. albicans ATCC 90028. RT-PCR studies showed downregulation of BCY1, PDE2, EFG1, and upregulation of negative regulators NRG1, TUP1 disrupting growth and virulence pathways. Zingerone induced elevated reactive oxygen species (ROS) levels, triggering apoptosis, evidenced by DNA fragmentation and upregulation of apoptotic markers. It also inhibited ergosterol synthesis in a concentration-dependent manner, crucial for membrane integrity. Importantly, zingerone exhibited minimal hemolytic activity. In an in vivo silkworm model, zingerone demonstrated significant antifungal efficacy, protecting silkworms from infection. It also modulated stress response genes, highlighting its multifaceted action.

In vitro and in vivo findings confirm the potent antifungal efficacy of zingerone against C. albicans ATCC 90028 and clinical isolate C1, suggesting its promising potential as a therapeutic agent that warrants further exploration.