MULTI-TARGET IN VITRO ENZYME INHIBITION (5-LOX, COX-2, ACHE/BCHE), ANTI-INFLAMMATORY PROTEIN PROTECTION, AND IN SILICO COMPUTATIONAL VALIDATION OF NIGELLA SATIVA FOLIUM HYDROETHANOLIC EXTRACT

Abstract

Nigella sativa L. seeds are extensively studied for their bioactive compounds, whereas its polyphenol-rich foliar biomass remains largely underutilised. This study investigated the in vitro anti-inflammatory, antioxidant, and enzyme-inhibitory activities of a crude hydroethanolic Nigella sativa leaf extract (NSLE) using animal-free experimental and computational approaches. Shade-dried leaves were macerated in 70% ethanol. Total phenolic content (TPC) and total flavonoid content (TFC) were determined colourimetrically. Anti-inflammatory activity was determined using heat-induced egg albumin and bovine serum albumin (BSA) denaturation assays. Enzyme inhibition was evaluated against 5-lipoxygenase (5-LOX), cyclooxygenase-2 (COX-2), acetylcholinesterase (AChE), and butyrylcholinesterase (BChE). Antioxidant activity was explored using DPPH, ABTS, and FRAP assays. Molecular docking of thymoquinone, rutin, quercetin, and kaempferol was performed against AChE (PDB: 4EY7), COX-2 (PDB: 5KIR), and 5-LOX (PDB: 6N2W). NSLE yielded 12.84% (w/w), with TPC and TFC values of 71.28 ± 2.14 mg GAE/g and 36.85 ± 1.62 mg QE/g, respectively. In the BSA denaturation assay, the IC₅₀ was 114.6 ± 4.2 µg/mL, compared with 78.2 ± 2.8 µg/mL for aspirin. Concentration-dependent inhibition was observed for 5-LOX (IC₅₀: 42.18 ± 1.95 µg/mL), COX-2 (68.34 ± 2.45 µg/mL), AChE (84.12 ± 3.10 µg/mL), and BChE (108.50 ± 4.20 µg/mL). DPPH and ABTS IC₅₀ values were 38.64 ± 1.42 and 29.15 ± 1.12 µg/mL, respectively. Rutin and quercetin exhibited docking energies of -8.2 to -9.6 kcal/mol. These findings suggest that N. sativa foliage may be a promising source of bioactive compounds with antioxidant, anti-inflammatory, and enzyme-inhibitory properties, warranting further investigation.