In vitro evaluation of the osteogenic associated cellular response and antibacterial activities of Vitex negundo leaf aqueous extract

Plant-derived bioactive compounds have emerged as promising candidates for enhancing osteoblast function and bone formation because of their osteogenic potential and with minimal cytotoxicity. This study investigated the osteogenic associated activity and antibacterial properties of Vitex negundo leaf aqueous extract (VNL-AE) using osteoblast-like SaOS-2 cells and pathogenic bacteria. LC/MS analysis was employed to profile the phytochemicals of VNL-AE, revealing the presence of 31 highly active phytocompounds. VNL-AE exhibited marked antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa , with minimum inhibitory concentrations (MIC) of 25 and 100 µg/mL and minimum bactericidal concentrations (MBC) of 50 and 200 µg/mL, respectively, along with significant inhibition of biofilm formation (P < 0.0001). Concentration-dependent effect on cell viability of SaOS-2 cells was observed in cytotoxicity assay. VNL-AE enhanced cell viability at concentrations up to 200 µg/mL, whereas higher concentrations (≥ 300 µg/mL) reduced viability, with an IC₅₀ of approximately 500 µg/mL after 72 h of exposure. Increased alkaline phosphatase activity was observed in dose-dependent manner (2.41 ± 0.03 in control vs. 2.58 ± 0.04 at 200 µg/mL) with enhanced mineralized matrix formation (0.44 ± 0.01 in controls vs. 3.14 ± 0.11 at 200 µg/mL; P < 0.0001) was observed. Quantitative PCR analysis showed significant up-regulation of osteogenic markers RUNX2 and BGLAP at 100 and 200 µg/mL. Additionally, VNL-AE preserved normal nuclear morphology, and increased the S-phase cell population from 21.16% in the control to 25.22% at 200 µg/mL. Overall, these finding suggest that VNL-AE may influence osteogenic associated response and cellular redox status in SaOS-2 in vitro model while exhibiting antibacterial activity. Further studies using primary human osteoblasts, mesenchymal stem cells, and appropriate in vivo models are necessary to validate these observations and elucidate the underlying mechanisms and translational relevance of VNL-AE.