Hydroalcoholic Extracts of Cucumis prophetarum L. Affect the Insulin Signaling Pathway in an In Vitro Model of Insulin-Resistant L6 Myotubes

Type 2 diabetes mellitus (T2DM) can be traditionally treated by edible and medicinal species rich in flavonoids and triterpenoids known for their metabolic benefits. <i>Cucumis prophetarum</i> L. has shown antioxidant and antidiabetic properties in decoction extracts. Since solvent polarity strongly influences the extraction of secondary metabolites, this study investigated the hydroalcoholic extracts of <i>C. prophetarum</i> L. to explore their chemical composition and insulin-sensitizing potential. Hydroalcoholic extracts from the leaf, stem, and root of <i>C. prophetarum</i> L. were analyzed by UV-Vis spectroscopy, ATR-FTIR, and UHPLC-ESI-QqTOF-MS/MS to profile their secondary metabolites. The insulin-sensitizing potential of each extract was assessed using an in vitro model of palmitic-acid-induced insulin resistance in L6 skeletal muscle cells, followed by Western blot analysis of key insulin-signaling proteins. Flavonoid glycosides such as apigenin-<i>C</i>,<i>O</i>-dihexoside, apigenin-malonylhexoside, and luteolin-<i>C</i>,<i>O</i>-dihexoside were abundant in leaf and stem extracts, while cucurbitacins predominated in the root. MTT assay confirmed that hydroalcoholic stem and root extracts of <i>C. prophetarum</i> L. were non-cytotoxic to L6 myotubes, whereas the leaf extract reduced viability only at higher concentrations. Oil Red O staining revealed a pronounced decrease in lipid accumulation following stem and root extract treatment. Consistently, the stem extract enhanced insulin signaling through the activation of the IRS-1/PI3K/Akt pathway, while the root extract primarily modulated the AMPK-mTOR pathway. Importantly, both extracts promoted GLUT4 translocation to the plasma membrane, highlighting their complementary mechanisms in restoring insulin sensitivity. Hydroalcoholic extracts of <i>C. prophetarum</i> L. alleviate insulin resistance through multiple molecular mechanisms, with bioactivity and composition differing markedly from previously reported in the decoctions, which highlight a promising source of insulin-sensitizing phytochemicals and underscore the importance of solvent selection in maximizing therapeutic potential.