Evaluation of Total Phenolic Content, Antioxidant Potential, Cytotoxicity and Anti-inflammatory Activity of <i>Stylosanthes fruticosa</i> Leaf Extract

Background: Stylosanthes fruticosa is a relatively unexplored fodder shrub from the legume family Fabaceae. Aim: This study focuses on estimating the phenolic content of various solvent-based extracts from S. fruticosa leaves and evaluating their antioxidant, anticancer, and anti-inflammatory potential. Methods: The total phenolic content was estimated using a standard method with gallic acid as the positive control. The inhibition of lipid peroxidation was examined using goat liver tissue to assess MDA levels and the in vitro cytotoxicity of S. fruticosa extracts was studied against HepG2 cells by MTT assay. In vitro anti-inflammatory potential of the extracts was assessed using RBC membrane stabilization assay. Screening of phytocompounds in the plant extract was done using LC-MS/MS and the identified compounds were subsequently used for in silico docking studies against pro-inflammatory markers IL6 and TNF-α. Results: Among various extracts, ethanol-based extraction showed the highest phenol concentration. The ethanol extract of S. fruticosa exhibited higher antioxidant potential (85%), followed by hexane extract (46%). The hexane and ethyl acetate extracts showed better lipid peroxidation inhibition than ethanol extract. In vitro cytotoxic studies revealed that ethyl acetate extract showed higher level of toxicity (67.59%) against hepatocarcinoma cells when compared to hexane (41.27%) and ethanol extracts (-0.7%). In the membrane stabilization study the ethyl acetate extract of S. fruticosa was more effective in preventing membrane damage of RBC. Further analysis with LC-MS/MS identified six flavonoid-based phenolic compounds within the ethyl acetate extract, which were docked against TNF-α and IL6 to evaluate their anti-inflammatory potential. Major Findings: Datiscin emerged as a compound with the strongest interactions with both TNF-α and IL6, suggesting it to be a key factor in the anti-inflammatory effects observed in S. fruticosa. These findings offer a basis for further in vivo and molecular mechanistic studies.