Polysaccharides are among the key bioactive components of Gynostemma pentaphyllum (Thunb.) Makino. In this study, a novel polysaccharide fraction, GPPA1-1, was isolated from G. pentaphyllum and purified using DEAE-Sepharose Fast Flow, Sephadex G-75, and Sepharose CL-6B column chromatography. GPPA1-1 was determined to have a molecular weight of 3.7 × 104 Da and was composed of mannose (Man), glucuronic acid (GlcA), rhamnose (Rha), glucose (Glc), galactose (Gal), and arabinose (Ara) in a molar ratio of 3.88:3.97:21.77:7.02:45.65:17.71. Comprehensive structural characterization was conducted using Congo red staining, FT-IR spectroscopy, Methylation analysis, and NMR analysis. The backbone of GPPA1-1 was identified as comprising →4)-α-Galp-(1→, →4)-α-Galp-(3,1→, →2)-α-Rhap-(4,1→, and →2)-α-Rhap-(1→, with branch chains containing α-Araf-(1→, →5)-α-Araf-(1→, →3)-α-Galp-(1→, and -α-GlcA-(1→. Biological assays demonstrated that GPPA1-1 effectively alleviates insulin resistance by inhibiting Galectin-3. This activity was confirmed through various experiments, including Galectin-3-mediated hemagglutination, Western blotting, CETSA, and glucose uptake assays. These findings highlight the potential of GPPA1-1 as a promising therapeutic agent for managing insulin resistance.
