Spinach ( Spinacia oleracea L.) Extract and Its Bioactive Constituent, Linoleic Acid, Improved Working, and Aversive Memory in Trimethyltin-Injected ICR Male Mice

Spinacia oleracea (spinach) has been proposed as a functional food candidate for cognitive health, yet the active constituent(s) responsible for cholinesterase-modulating effects remain unclear. Here, we screened the acetylcholinesterase (AChE)-inhibitory activity of S. oleracea ethanolic extract and identified a candidate bioactive constituent using bioactivity-guided fractionation coupled with chromatographic and mass spectrometric analyses. An active silica-gel fraction was subjected to high-performance liquid chromatography and gas chromatography–mass spectrometry, and linoleic acid was identified in the active fraction, with its chromatographic identity further supported by coinjection analysis. Linoleic acid also exhibited measurable AChE-inhibitory activity in a tacrine-equivalent calibration assay. Preventive efficacy was evaluated in a trimethyltin chloride (TMT; 2.5 mg/kg, i.p.)-induced neurotoxicity model in male Institute of Cancer Researchmice. Mice received S. oleracea extract (400, 800, or 1200 mg/kg/day) or linoleic acid (10, 20, or 40 mg/kg/day) for 3 weeks prior to TMT challenge. Both the extract and linoleic acid ameliorated TMT-induced deficits in Y-maze spontaneous alternation and passive avoidance latency. The linoleic acid intervention also reduced brain AChE activity and lipid peroxidation, indicating attenuation of cholinergic dysfunction and oxidative stress. Serum AST and ALT activities were not significantly altered, indicating no detectable evidence of hepatocellular injury based on these markers under the tested conditions. Collectively, these findings support the potential of S. oleracea extract as a cognition-oriented functional material and identify linoleic acid as a candidate bioactive constituent warranting further investigation in TMT-induced cognitive impairment.