Structural characterization, antioxidant and antitumor properties of an acidic polysaccharide from toad venom

As a valuable agricultural product derived primarily from cultivated toads, toad venom has been widely used for its significant antitumor properties in pharmacological formulations. However, the structural and biological characteristics of its major constituents, polysaccharides, remain poorly understood, limiting the development of innovative antitumor drugs. In this study, an extracellular acidic homogeneous polysaccharide (TvPs3-2, average molecular mass: 3.43 × 102 kDa) is isolated from toad venom. Compositional analysis by 1-phenyl-3-methyl-5-pyrazolone (PMP) pre-column derivatization high-performance liquid chromatography (PMP-HPLC) reveals that TvPs3-2 consists of mannose (Man), glucuronic acid (GlcA), glucose (Glc), galactose (Gal), arabinose (Ara), and fucose (Fuc) in a molar ratio of 9.30:26.52:17.26:2.20:8.40:36.31. Comprehensive structural characterization through methylation analysis combined with extensive nuclear magnetic resonance (NMR) spectroscopy indicates a possible backbone: → 4)-β-D-GlcAp-(1 → 3,6)-β-D-Galp-(1 → 3)-α-L-Fucp-(1 → 4)-α-D-Manp-(1 → 2)-β-D-Glcp-(1 → 2,4)-α-D-Glcp-(1 → 4)-β-D-Glcp-(1 → 2)-α-L-Fucp-(1 → 4)-β-D-GlcAp-(1 → , with multiple branched segments. Morphologically, TvPs3-2 exhibits interconnected bulges, undulating surfaces, and square/rhombic crystals while maintaining a triple-helix conformation. Additionally, TvPs3-2 exhibits good antioxidant activity, particularly against 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS, half maximal inhibitory concentration (IC50) = 0.36 mg/mL), alongside moderate 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl radical scavenging effects. Furthermore, TvPs3-2 demonstrates notable antiproliferative effects on lung (A549), liver (SK-Hep1), and colon (HCT116 and HT-29) cancer cell lines, with the highest efficacy against SK-Hep1 hepatoma cells (IC50 = 1.6 mg/mL), mediated by apoptosis induction, migration inhibition, and modulation of metabolic/oxidative stress pathways, while it demonstrates relatively low inhibitory activity against healthy H9C2(2-1) cells. This study comprehensively characterizes a novel acidic polysaccharide (TvPs3-2) from toad venom with remarkable antioxidant and antitumor activities, providing a molecular basis for its future application in anticancer drug development.