In recent years, the remarkable bioactivities and nutritional value of Dendrobium flexicaule flowers have become increasingly evident, underscoring their potential for broad applications. However, optimal extraction techniques and their health-promoting effects remain inadequately unexplored. This study investigated the neuroprotective effects of supercritical fluid-extracted DFF extract (DFFE), identified its key constituents, and elucidated underlying mechanisms via integrated in vitro and in silico approaches. Herein, the extract was obtained using supercritical fluid extraction (SFE), which demonstrated superior efficiency in preserving thermo-labile compounds and minimizing oxidative degradation compared to conventional ethanol reflux extraction, as evidenced by enhanced recovery of unsaturated fatty acids and sterols. Gas chromatography-mass spectrometry (GC-MS) analysis identified a total of 96 chemical constituents in DFFE. Notably, DFFE significantly mitigated glutamate-induced damage in PC12 cells by reducing LDH release, ROS generation, and enhancing antioxidant enzyme activity. Network pharmacology revealed linoleic acid, hexadecenoic acid, and pentadecanoic acid as core bioactive components, targeting PPARG, GSK3β, IL6, PTGS2, and MAPK3, which modulate Alzheimer’s and TNF signaling pathways. Molecular docking demonstrated stable interactions between most bioactive components and core targets through hydrogen bonds, with binding energies predominantly less than −5 kcal/mol. Ultimately, the extraction process was optimized using a decision tree model, which highlighted temperature and pressure as crucial parameters. Overall, these findings underscore DFFE’s potential as a neuroprotective agent for functional food or therapeutic development.
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