Optimization and Metabolomic Profiling of Wolffia globosa Extract Reveal Multitarget Neuroprotective Activity Against Alzheimer’s Disease with In Vitro, In Silico, and In Vivo Validation

Wolffia globosa, the world’s smallest flowering plant, is a sustainable protein- and phytochemical-rich food source with emerging neuroprotective potential. This study presents the first comprehensive characterization that integrates extraction optimization, multitarget bioactivity profiling, drug synergy assessment, high-resolution metabolomics, molecular docking, and in vivo validation in Drosophila melanogaster. Ethanol-based extraction was optimized using a Box–Behnken design and response surface methodology (RSM), yielding optimal conditions of 32% ethanol, 1:40 (g/mL) solid-to-solvent ratio, 60 °C, and 28 min. The optimized extract exhibited a total phenolic content (TPC) of 15.17 ± 0.27 mg GAE/g DW, a total flavonoid content (TFC) of 22.34 ± 1.71 mg QE/g DW, and an exceptional ORAC antioxidant activity of 4374.33 ± 395.65 µmol TE/g DW. Potent and selective BACE-1 inhibition was observed (IC50: 1.59 ± 0.11 mg/mL), alongside moderate AChE (IC50: 7.42 mg/mL) and BChE (IC50: 6.55 mg/mL) inhibition. Synergistic interactions with donepezil were confirmed by the Chou–Talalay combination index method, with combined AChE, BChE, and BACE-1 inhibitions reaching 60.3%, 77.0%, and 82.2%, respectively—substantially exceeding theoretical additive values. Multi-platform metabolite profiling by HPLC-QTOF-MS/MS and LC-ESI-MS/MS identified luteolin, apigenin, caffeic acid, isovitexin, schaftoside, and pinolenic acid as the principal bioactives. Molecular docking predicted favorable binding of luteolin (delta-G: −10.24 kcal/mol, AChE) and naringenin (delta-G: −8.76 kcal/mol, BACE-1) against crystal structures of human AChE (PDB: 7E3H), BChE (PDB: 4TPK), and BACE-1 (PDB: 6EQM). In vivo, the W. globosa extract significantly improved locomotor performance and suppressed brain BACE-1 activity in an Drosophila AD model over 28 days. These findings establish W. globosa as a multifunctional nutraceutical candidate for neurodegenerative disease prevention.