Integrated In Vitro and In Silico Characterization of 5‐Hydroxyferulic Acid: Antioxidant, Anti‐Inflammatory, Anti‐Hemolytic, and Cytotoxic Potential

Abstract

ABSTRACT In the pursuit of novel antioxidant and anti‐inflammatory agents, we investigated 5‐hydroxyferulic acid (5‐OHFA), a hydroxylated derivative of the well‐known phenolic compound ferulic acid (FA). This study aimed to determine whether structural modification enhances the biological activity of FA. To this end, both compounds were subjected to a series of in vitro antioxidant assays (DPPH, ABTS, FRAP, and Fe(II)‐chelating) and anti‐inflammatory evaluations, complemented by in silico predictions. 5‐OHFA consistently exhibited superior antioxidant capacity, with significantly lower IC 50 values than FA based on literature‐reported values: 11.89 ± 0.20 versus 66 ± 2.3 µM (DPPH), 9.51 ± 0.15 versus 183.08 ± 2.30 µM (ABTS), 5.94 ± 0.09 versus 4.73 ± 0.14 µM (FRAP), and 36.31 ± 1.36 versus 270.27 ± 1.14 µM (Fe 2+ chelation). It also demonstrated stronger anti‐inflammatory potential in protein denaturation assays using egg albumin and bovine serum albumin (BSA). Although 5‐OHFA showed slightly greater hemolytic activity (IC 50 = 23.78 ± 1.48 µM) than FA, based on literature‐reported values (37.64 ± 2.01 µM), both remained within biologically acceptable limits. In silico analyses using SwissADME and ProTox III supported the experimental findings, predicting good oral bioavailability, high gastrointestinal absorption, mild blood–brain barrier permeability, and no significant toxicity for 5‐OHFA. Molecular docking studies further revealed stronger binding affinities of 5‐OHFA to key oxidative and inflammatory targets, including NADPH oxidase (2CDU), xanthine oxidase (1FIQ), 5‐lipoxygenase (3O8Y), cyclooxygenase‐2 (3LN1), myeloperoxidase (1DNU), and the EGFR enzyme's active pocket (1M17). Overall, the data suggest that 5‐OHFA possesses enhanced bioactivity relative to its parent compound FA, supporting its potential as a promising multifunctional candidate for pharmaceutical or nutraceutical development.