3D-QSAR Modeling of the Anti-Inflammatory Activity of 28 Rare Biflavones

Inflammation underlies a wide range of chronic diseases. Current treatments, such as nonsteroidal anti-inflammatory drugs, are effective, but they often result in adverse side effects. This has driven the search for alternative anti-inflammatory agents with improved safety profiles. Biflavones have emerged as promising candidates, yet existing studies primarily focus on naturally occurring biflavones that are limited to those with C3'-C8″ linkages. In this study, we explore the anti-inflammatory potential of 28 biflavones featuring the underexplored C2'-C6″ linkage. Using LPS-induced RAW 264.7 macrophage assays, biflavone 10 (synthesized from the flavones acacetin and 5,3',4'-trihydroxyflavone) inhibited nitric oxide production (20.94 ± 7.56%). A 3D-QSAR model was developed based on selected nitric oxide (NO) inhibition data (R ² = 0.960, R ²CV = 0.831, and Pearson's r = 0.950), revealing that hydrogen bond acceptor interactions are key determinants of bioactivity. The presence of HBAs at C-5 and C-7 of ring A1 enhanced activity, while their presence at C-4' of ring B2 reduced it. Molecular docking studies revealed a strong binding affinity between biflavone 10 and inflammation-related protein COX-2 (-6.881 kcal/mol). This prediction was validated by a COX-2 enzyme inhibitor assay; the biflavone 10 inhibits COX-2 activity in a dose-dependent manner. Molecular dynamics simulations confirmed the stability of the biflavone 10-COX-2 complex through stable root mean square deviation (RMSD) profiles, consistent hydrogen bonding, and minimal structural fluctuation. Together, this study provides a comprehensive assessment of the anti-inflammatory potential of structurally novel biflavones, offering mechanistic insight into their activity and guiding the rational design of biflavone-based therapeutics.