Bioactive‐Guided Isolation and Optimization of Luffa Acutangula Nanoemulsion for In vitro, In vivo, and In silico Cholinesterase Inhibition for Alzheimer's Disease Management

ABSTRACT Alzheimer's disease (AD) progressively impairs memory and cognition. Luffa acutangula (LA), rich in triterpenoids, fatty acids, and iridoid glycosides, exerts anti‐Alzheimer's effects by inhibiting acetylcholinesterase (AChE). This study aims to isolate bioactive compounds from LA fruits, formulate and optimize a nanoemulsion to improve brain targeting, and assess its anti‐Alzheimer efficacy through in vitro, in vivo, and in silico approaches. LA was extracted using a 1:1 hydro‐ethanol mixture and subsequently underwent chromatographic isolation to yield four major constituents: oleanolic acid, stearic acid, cucurbitacin H, and acutoside C. A multiple nanoemulsion (containing a mixture of isolated constituents) was formulated using Box–Behnken design and characterized for particle size, viscosity, entrapment efficiency, and FTIR compatibility. In vivo studies were conducted in scopolamine‐induced memory‐impaired mice using Hebb–William's Maze (HWM) and Cook's pole climbing tests. AChE activity was biochemically assessed, followed by histopathological brain analysis. Molecular docking was used to predict ligand‐AChE binding affinities. The optimized L. acutangula loaded multiple nanoemulsion (LAMN) exhibited an average particle size of 142.1 nm, viscosity of 60 cP, and entrapment efficiency (EE) of 96.8 %. It decreased learning scores in the HWM and shortened latency time in Cook's pole‐climbing test in memory‐impaired mice. AChE levels were significantly reduced in LAMN‐treated groups, correlating with histological evidence of hippocampal protection. Molecular docking revealed strong AChE binding, particularly by oleanolic acid (−10.5 kcal/mol), comparable to donepezil (−10.7 kcal/mol). LAMN offers a promising phytopharmaceutical intervention for AD, with multitargeted neuroprotective effects mediated through AChE inhibition and improved drug delivery across the blood–brain barrier. The findings support further clinical development of LA‐based nanoformulations for AD management.