LC–MS-Based Metabolomic Profiling of Excoecaria agallocha Leaf Extract: Integrated Antioxidant, Anti-Inflammatory, Cholinesterase Inhibitory and Neuroprotective Evaluation with AChE-Targeted Molecular Docking

Abstract

Excoecaria agallocha L. is a medicinally important mangrove species containing diverse secondary metabolites with potential antioxidant, anti-inflammatory, and neuroprotective activities. This study investigated the multifunctional biological potential of E. agallocha leaves using petroleum ether, chloroform, ethyl acetate, methanol, and aqueous extracts. Preliminary phytochemical screening revealed a solvent-dependent distribution of constituents, while total phenolic and flavonoid contents were quantified in the methanolic and aqueous extracts. Antioxidant activity was evaluated using DPPH radical-scavenging and ferric-reducing antioxidant power (FRAP) assays, while anti-inflammatory activity was assessed using protein-denaturation and COX-2 inhibition assays, with aspirin and celecoxib as reference standards, respectively. Cytotoxicity of the methanolic and aqueous extracts was evaluated in L929 normal fibroblasts, and neuroprotective activity was assessed in SK-N-SH neuroblastoma cells exposed to trimethyltin (TMT), with the aqueous extract administered 1 h before TMT exposure. Apoptotic changes were evaluated using acridine orange/ethidium bromide (AO/EB) dual-fluorescence staining. Acetylcholinesterase (AChE) inhibition was determined using galantamine as the reference inhibitor. LC–MS was employed for putative metabolite profiling of the aqueous extract, followed by AChE-targeted molecular docking of selected identified compounds against human acetylcholinesterase (AChE; PDB ID 7E3D) and ProTox-II-based in silico toxicity prediction. The aqueous extract showed the highest total phenolic (717.81 µg/g) and flavonoid (485.16 µg/g) contents compared with the methanolic extract (562.43 and 334.00 µg/g, respectively) and demonstrated the strongest DPPH radical-scavenging activity among the extracts (IC50 = 115.49 µg/mL) together with a marked ferric-reducing capacity. The aqueous extract also exhibited pronounced COX-2 inhibition (IC50 = 39.77 µg/mL), whereas the methanolic extract showed greater inhibition of protein denaturation (IC50 = 138.89 µg/mL). The aqueous extract displayed relatively low cytotoxicity toward L929 cells and markedly attenuated TMT-induced toxicity in SK-N-SH cells, with the greatest protection observed at the lowest pre-treatment concentration (20 µg/mL; 92.14 ± 1.77% cell viability versus 16.92 ± 1.55% with TMT alone). AChE inhibition by the aqueous extract increased concentration-dependently, with an IC50 of 55.64 µg/mL. Consistent with these findings, AO/EB staining demonstrated an increased proportion of viable cells and reduced apoptotic cell populations following aqueous-extract pre-treatment. LC–MS profiling revealed a chemically diverse array of putatively identified metabolites. AChE-targeted molecular docking putatively identified 6-hydroxy-2-(4-methoxyphenyl)-4H-chromen-4-one and daidzein showed comparatively favorable docking profiles together with Class 5 predicted toxicity. Collectively, the present findings demonstrate that E. agallocha leaf extracts possess measurable antioxidant, anti-inflammatory, cholinesterase-inhibitory, and neuroprotective-related activities under the experimental conditions investigated, with the aqueous extract showing comparatively pronounced activity. These findings provide preliminary experimental and computational evidence supporting further bioassay-guided isolation, experimental validation of individual compounds, mechanistic studies, and in vivo evaluation to determine the neuroprotective potential and biological relevance of E. agallocha.