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. Cucumis prophetarum 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 C. prophetarum L. to explore their chemical composition and insulin-sensitizing potential. Hydroalcoholic extracts from the leaf, stem, and root of C. prophetarum 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-C,O-dihexoside, apigenin-malonylhexoside, and luteolin-C,O-dihexoside were abundant in leaf and stem extracts, while cucurbitacins predominated in the root. MTT assay confirmed that hydroalcoholic stem and root extracts of C. prophetarum 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 C. prophetarum 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.