Fermentation with aspergillus costiformis enhances the anti-migratory activity of Aquilaria sinensis leaves with possible involvement of MAPK-related pathways.

Aquilaria sinensis leaves (ALs) are an underutilized plant resource rich in polyphenols and flavonoids. Microbial fermentation has been proposed as an effective strategy to enhance the functional value of plant materials. However, its impact on the bioactivity and mechanisms of ALs remains unclear. This study aimed to evaluate the effects of fermentation with Aspergillus costiformis AHHC on the chemical composition and biological activities of ALs, and to elucidate the molecular mechanisms underlying the fermentation-induced functional changes. Non-targeted metabolomic analysis was performed to assess fermentation-induced metabolic alterations. The biological activities of fermented ALs (ALDT) and non-fermented ALs (ALT) extracts were evaluated using HeLa S3 cell-based assays. Network pharmacology and transcriptomic analyses were integrated to identify potential pathways and molecular targets, which were further validated by molecular analyses. The results revealed that fermentation significantly reshaped the metabolite profile of ALDT, particularly in flavonoid-related pathways. Compared with ALT, ALDT showed a stronger inhibitory effect on HeLa S3 cell migration while maintaining comparable anti-proliferative activities at higher concentrations. The integrated analyses indicated that fermentation-enriched metabolites were mainly associated with pathways related to cell migration and stress response, with prominent involvement of MAPK signaling. Molecular assays confirmed enhanced phosphorylation of MAPK components and increased expression of regulatory genes such as GADD45B and NR4A1 in response to ALDT treatment. Overall, fermentation with A. costiformis enhanced the anti-migratory activity of ALDT in HeLa S3 cells, with possible involvement of MAPK-related signaling pathways. These findings provide molecular-level evidence supporting microbial fermentation as a strategy to improve the functional value of this plant resource.