Asperpentyn disrupts cell membrane integrity and 2,3-dihydrophomalactone potently inhibits aflatoxin B1 biosynthesis in Aspergillus flavus

Abstract Aflatoxin B 1 (AFB 1 ) is one of the most toxic mycotoxins produced by members of Aspergillus section Flavi during fungal growth and represents a serious threat to food safety and human health. In this context, the development of natural antifungal agents as safe alternatives has gained increasing attention. In this study, three secondary metabolites, (–)-asperpentyn, ( R )-phomalactone, and 2,3-dihydrophomalactone, isolated from the endophytic fungus Curvularia inaequalis , were evaluated for their antifungal and anti-aflatoxigenic activities against Aspergillus flavus . Their effects on fungal growth, biomass production, and AFB 1 biosynthesis were investigated using microdilution assays, dry biomass determination, thin-layer chromatography (TLC), and high-performance liquid chromatography (HPLC) analyses. Among the tested fungal metabolites, (–)-asperpentyn showed the lowest minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values, both at 0.312 mg/mL, whereas voriconazole exhibited lower MIC and MFC values of 0.125 mg/mL. (–)-Asperpentyn also increased propidium iodide uptake and reduced ergosterol content in a concentration-dependent manner, indicating membrane damage. ( R )-phomalactone showed moderate antifungal activity, with MIC and MFC values of 2.8 and 5.62 mg/mL, respectively. In contrast, 2,3-dihydrophomalactone showed limited inhibition of fungal growth (MIC > 2.8 mg/mL) but strongly inhibited AFB 1 biosynthesis. At 100 µg/mL, it completely inhibited AFB 1 production while reducing fungal biomass by only 15.9%, indicating that its anti-aflatoxigenic effect was not solely attributable to growth inhibition. These findings highlight 2,3-dihydrophomalactone as a promising lead for further investigation of natural inhibitors of aflatoxin biosynthesis, while (–)-asperpentyn showed antifungal activity associated with membrane damage.