Xanthine Oxidase Inhibitory Potential of Achillea santolina Methanolic Extract with ROS Modulation in HepG2 Cells

Achillea santolina is traditionally utilized in herbal therapy; nonetheless, its xanthine oxidase inhibitory and cellular antioxidant properties are not extensively investigated. This work aimed to identify the phytochemical composition of the methanolic extract of A. santolina aerial parts via LC-MS and to assess its xanthine oxidase (XO) inhibitory activity and its modulatory effects on intracellular reactive oxygen species (ROS) in HepG2 cells. The methanolic extract underwent LC-MS analysis for phytochemical identification, and XO inhibitory activity was assessed spectrophotometrically using allopurinol as the reference medication. Intracellular ROS levels were determined using the DCFH-DA fluorescent assay following xanthine-induced oxidative stress. LC-MS profiling identified ten compounds representing 95.1% of the extract, with flavonoid glycosides (44.1%) as the predominant class. Rutin (18.6%) and luteolin-7-O-glucoside (15.2%) were the major constituents. The extract demonstrated concentration-dependent inhibition of XO, with an IC₅₀ value of 29.3 ± 0.4 µg/mL, compared to 2.1 ± 0.3 µg/mL for allopurinol. In HepG2 cells, xanthine significantly increased ROS production, while pre-treatment with the extract greatly diminished ROS levels in a dose-dependent manner, reaching approximately 75% inhibition at 50 µg/mL. The results demonstrate that the methanolic extract of A. santolina exhibits significant xanthine oxidase inhibitory and antioxidant properties, possibly due to its elevated flavonoid concentration, indicating its potential utility in addressing oxidative stress-related disorders. To the best of our knowledge, this study provides the first report on the combined xanthine oxidase inhibitory and intracellular ROS-modulating activities of A. santolina methanolic extract.