Perillaldehyde, a Promising Antifungal Agent Used in Food Preservation, Triggers Apoptosis through a Metacaspase-Dependent Pathway in Aspergillus flavus

In the present study, we provide detailed insights into perillaldehyde (PAE)'s mechanisms of action on Aspergillus flavus and offer evidence in favor of the induction of an apoptosis-like phenotype. Specifically, PAE's antifungal mode of action was investigated through the detection of mitochondrial membrane potential (Mt Delta psi) and phosphatidylserine (PS) exposure, as well as intracellular Ca2+ level, reactive oxygen species accumulation, and metacaspase activation. This was done by way of fluorometry, measuring DNA fragmentation, and condensation by fluorescent microscopy. Furthermore, we searched for phenotypic changes characteristic of apoptosis by transmission electron microscopy and flow cytometry, determining the amount of cytochrome c released using Western blotting. Results indicated that cultivation of A. flavus in the presence of PAE caused depolarization of Mt Delta psi, rapid DNA condensation, large-scale DNA fragmentation, and an elevation of intracellular Ca2+ level. The percentage of early apoptotic cells with exposure of PS were 27.4% and 48.7%, respectively, after 9 h incubations with 0.25 and 0.5 mu L/mL of PAE. The percentage of stained cells with activated intracellular metacaspases exposed to PAE at concentrations of 0.25 and 0.5 mu L/mL compared with control subjects were increased by 28.4 +/- 3.25% and 37.9 +/- 4.24%, respectively. The above results has revealed that PAE induces fungal apoptosis through a caspase-dependent mitochondrial pathway. In all, our findings provide a novel mechanism for exploring a possible antifungal agent used in food preservation.