Integrative Metabolomics, Pharmacoinformatics and Experimental Studies Reveal the Neuroprotective Potential of Caulerpa racemosa Metabolites Against Alzheimer’s Disease

Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder characterized by cholinergic dysfunction, oxidative/nitrosative stress, and neuroinflammation. Marine green algae <i>Caulerpa racemosa</i> 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 <i>C. racemosa</i> 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 <i>C. racemosa</i>. 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, α-linolenic acid, α-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 α-linolenic acid, α-eleostearic acid, and oxidized C18 lipids exhibiting the strongest interactions-particularly with ACHE and NOS isoforms. In vitro assays showed moderate antioxidant activity (IC₅₀ = 120.97 ± 10.93 µg/mL) and cholinesterase inhibition (AChE IC₅₀ = 136.48 ± 1.70 µg/mL; BChE IC₅₀ = 145.98 ± 3.28 µg/mL), aligning with predicted multi-target interactions. <i>C. racemosa</i> 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 <i>C. racemosa</i> as a promising marine-derived multi-target candidate for AD intervention, warranting further mechanistic and in vivo evaluation.