Antifungal Activity and Potential Mechanism of N-Butylphthalide Alone and in Combination With Fluconazole Against Candida albicans

Abstract

<i>Candida albicans</i> is a common opportunistic fungal pathogen that may cause nosocomial fungal infections. The resistance of <i>Candida albicans</i> to traditional antifungal drugs has been increasing rapidly in recent years, and it brings a great challenge in clinical treatment. N-butylphthalide is originally extracted from the seed of <i>Apium graveolens</i> and is currently used for the treatment of ischemic stroke in the clinic. This study demonstrated that n-butylphthalide exhibited antifungal activity against <i>Candida albicans</i> with minimum inhibitory concentrations of 128 μg/ml; moreover, n-butylphthalide combined with fluconazole showed synergistic antifungal effects against resistant <i>Candida albicans</i>, resulting in a decrease in the minimum inhibitory concentrations of fluconazole from >512 to 0.25-1 μg/ml. Time-killing curves verified the antifungal activity in dynamic. Besides, n-butylphthalide exhibited anti-biofilm activity against <i>Candida albicans</i>, biofilms preformed <12 h with sessile minimum inhibitory concentrations of 128-256 μg/ml and synergism was observed when n-butylphthalide combined with fluconazole against resistant <i>Candida albicans</i> biofilms preformed <12 h, resulting in a decrease in the sessile minimum inhibitory concentrations of fluconazole from >1,024 to 0.5-8 μg/ml. Furthermore, <i>in vitro</i> antifungal effects of n-butylphthalide were confirmed <i>in vivo</i>. N-butylphthalide prolonged survival rate of larvae infected by <i>Candida albicans</i>, reduced the fungal burden in larvae and caused less damage to larval tissues. Notably, n-butylphthalide inhibited hyphal growth and induced intracellular reactive oxygen species accumulation and a loss in mitochondrial membrane potential, which was a potential antifungal mechanism. Besides, the synergistic effects between n-butylphthalide and fluconazole potentially relied on the mechanism that n-butylphthalide significantly promoted drug uptake, and suppressed drug efflux <i>via</i> down-regulating the drug transporter encoding genes <i>CDR1</i> and <i>CDR2</i>. These findings demonstrated the antifungal effects and mechanisms of n-butylphthalide against <i>Candida albicans</i> for the first time, which might provide broad prospects for the identification of new potential antifungal targets.