Optimization and Metabolomic Profiling of Wolffia globosa Extract Reveal Multitarget Neuroprotective Activity Against...

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.