Apigenin and its derivative modulate nitric oxide production and interfere with TLR4-M2: In-vitro and in-silico insights

The imbalance in reactive oxygen/nitrogen species causes oxidative stress which contributes to chronic inflammation and diseases. In this study, the immunomodulatory potential of the natural product ‘Apigenin (4,5,7-trihydroxyflavone)’ (APG) and its derivative ‘Apigenin-7-O-β-D-(6″-p-coumaroyl)-glucopyranoside’ (APG-7) was elucidated through cell-based spectrophotometry, chemiluminescence, and fluorescent microscopy. Their effects were assessed on the production of superoxide anion, myeloperoxidase-dependent hypochlorite anion, intracellular oxidative stress and nitric oxide (NO). Moreover, their cellular toxicity was investigated on 3 T3 fibroblast cell line. APG significantly reduced superoxide anion (48.2 %) and hypochlorite production (IC₅₀ = 27.2 μg/mL), while APG-7 showed minimal activity in these assays. Both compounds inhibited NO production, with APG showing stronger inhibition (98 %) than APG-7 (55 %). However, APG was more cytotoxic (IC₅₀ = 4.5 μg/mL) as compared to APG-7 (∼25 μg/mL), indicating a safer profile of APG-7. NO is produced by LPS triggered activation of Toll-like receptor 4 (TLR4), therefore in-silico molecular docking and dynamics simulation were performed to deduce the binding affinity of APG and APG-7 at TLR4/MD-2 interface. Our in-silico findings suggest that both the compounds may target TLR4/MD-2 interface to inhibit the production of NO. Overall, the results support the immunomodulatory potential of APG and APG-7, warranting further investigation.