Enantiospecific hepatotoxicity of usnic acid in vitro, and the attempt to modify the toxic effect

Usnic acid, a chiral secondary lichen metabolite, is known for its diverse biological activities, yet concerns about its hepatotoxicity limit its therapeutic potential. Notably, the enantiospecific effects of its two forms, (+)- and (−)-usnic acid, remain underexplored. This study aimed to compare the hepatotoxic potential of the enantiomers of usnic acid in wide range of concentration to HepG2 cells, by evaluating their impact on cell viability, membrane integrity, and mitochondrial function. Additionally, we investigated whether hepatotoxicity could be mitigated by co-treatment with known hepatoprotectants: silybin, squalene, and N-acetylcysteine. Our results demonstrated for the first time that (−)-usnic acid exhibited significantly greater hepatotoxicity than its (+)-enantiomer, particularly after 24–48 h of exposure (IC₅₀: 16.0 vs. 28.2 µg/mL at 48 h). This enantiospecific toxicity was confirmed by LDH leakage and mitochondrial membrane depolarization, with more pronounced effect induced by (−)-usnic acid. Co-treatment with hepatoprotectants partially alleviated toxicity at 48 h, with squalene and N-acetylcysteine showing better protective effects than silybin. However, the protective effects diminished after 72 h. These findings underscore the importance of enantiospecific analysis in natural compound safety assessments and suggest that the hepatotoxicity of usnic acid is influenced by more than just mitochondrial uncoupling. Further in vivo studies and mechanistic analyses are necessary to evaluate the clinical potential of usnic acid and to identify strategies for safe therapeutic use.