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

High Resolution Image Download MS PowerPoint Slide 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 2 CV = 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.