Handayani_2025_Mar.Drugs_23_

Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder characterized by cholinergic dysfunction, oxidative/nitrosative stress, and neuroinflammation. Marine green algae Caulerpa racemosa are rich in neuroactive lipids and fatty acid derivatives with reported antioxidant and anti-inflammatory properties. However, their integrated mechanistic potential against AD remains largely underexplored. This study aimed to elucidate the neuroprotective mechanisms of C. racemosa metabolites against AD using integrative metabolomics, network pharmacology, molecular docking, and in vitro validation assays. Untargeted LC-HRMS profiling was performed to identify major metabolites in the ethanolic extract of C. racemosa. Neuroprotective targets were predicted via TargetNet, STRING, and Cytoscape (MCODE, CytoNCA). Functional enrichment was conducted using KEGG, GO (BP, MF, CC), and ClueGO. Molecular docking (CB-Dock2) validated compound-target interactions with ACHE, CHRM1, NOS1, and NOS2. Antioxidant (DPPH) and cholinesterase (AChE/BChE) inhibitory activities were evaluated in vitro. Metabolomic profiling identified lipid-dominant metabolites-oleamide, hexadecanamide, palmitoyl ethanolamide, alpha-linolenic acid, alpha-eleostearic acid, and 9-oxo-octadecadienoic acid. Network analysis revealed key AD-related hubs (ACHE, CHRM1, NOS1, NOS2) enriched in cholinergic regulation, arachidonic-acid metabolism, oxidative stress response, and nitric oxide signaling. Docking showed moderate multi-target affinities (-6.0 to -8.4 kcal/mol), with alpha-linolenic acid, alpha-eleostearic acid, and oxidized C18 lipids exhibiting the strongest interactions-particularly with ACHE and NOS isoforms. In vitro assays showed moderate antioxidant activity (IC(50) = 120.97 +/- 10.93 microg/mL) and cholinesterase inhibition (AChE IC(50) = 136.48 +/- 1.70 microg/mL; BChE IC(50) = 145.98 +/- 3.28 microg/mL), aligning with predicted multi-target interactions. C. racemosa extract exhibits neuroprotective potential through a synergistic combination of cholinergic modulation, antioxidant activity, NOS-mediated nitrosative stress reduction, and suppression of arachidonic-acid inflammatory pathways. These findings support C. racemosa as a promising marine-derived multi-target candidate for AD intervention, warranting further mechanistic and in vivo evaluation.