Novel vanillin-derived Schiff Bases: Synthesis, characterization, antibacterial activity, enzyme inhibition, antioxidant activity, anti-inflammatory activity on LPS-induced RAW264.7 macrophage cell line, and In Silico studies

• Novel Vanillin-Derived Schiff Bases (Vanillin SB-1 and SB-2) were synthesized and structurally characterized. • Vanillin SB-1 demonstrated significant acetylcholinesterase (AChE) inhibition. • Vanillin SB-1 and Vanillin SB-2 reduced pro-inflammatory cytokine levels (NFκB, IL-6, and TNF-α) and reactive oxygen species (ROS) in LPS-stimulated RAW264.7 macrophages. • Vanillin SB-1 and Vanillin SB-2 showed antibacterial activity . In this comprehensive study, we investigated the cytotoxic and anti-inflammatory effects of two new vanillin Schiff Base (SB) compounds at various concentrations (1–100 μM) on RAW264.7 macrophage cells stimulated with LPS (μg/mL for 24 h). The levels of NFκB, IL-6, and TNF-α in LPS-stimulated RAW 264.7 macrophage cells treated with vanillin SB compounds (5–50 μM) for 24 h were measured using the ELISA method. ROS levels in all groups were determined using a DCFDA kit. In silico studies were also carried out on the synthesized compounds, and their antibacterial ( against S. aureus, E. faecalis, P. aeruginosa, and Escherichia coli) and antioxidant activities were investigated. The results showed vanillin SB treatment affected cell viability, increased NFκB expression, and significantly reduced the production of proinflammatory cytokines and ROS levels ( p < 0.05). Vanillin SB-1 and 2 inhibited acetylcholinesterase enzyme. IC 50 and MIC values of vanillin SB-1 and 2 compounds were determined as 26.83 mM, 69.34 mM, and 62.5, 31.25 µg/mL, respectively. The MBC value of both compounds against E. faecalis was found to be 250 µg/mL. In silico docking revealed binding affinities of -6.086 and -6.060 kcal/mol for 2AZ5, -3.754 and -3.728 kcal/mol for 4ZS7, -5.258 and -7.726 kcal/mol for 2RAM, -9.405 and -9.319 kcal/mol for 1EVE (with tacrine at -7.746 kcal/mol), and -5.496 and -5.610 kcal/mol for 3G7B.