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

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 IC₅₀ 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 (Fe²⁺ 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 (IC₅₀ = 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.