A highly branched arabinogalactan from Codonopsis pilosula: Structural insights and hematopoietic-protective...

Polysaccharides from Codonopsis pilosula are widely recognized for their therapeutic potential, yet the structural and mechanistic basis of their hematopoietic activity remains largely unexplored. This study employed an integrated workflow combining polysaccharide profiling, bioactivity-guided isolation, progressive structural analysis, and mechanistic validation to identify CP2A, a homogeneous, ultrahigh molecular weight (1.29 × 10⁷ Da) arabinogalactan that demonstrated significant hematopoietic-protective effects in a TPEN-induced zebrafish model. Monosaccharide analysis revealed a galactose-to-arabinose molar ratio of 60.76: 39.24. Linkage analysis by GC-MS suggested a high proportion of branching residues and a scarcity of terminal residues, which we attribute to protein conjugation at non-reducing ends, a known feature of root-derived arabinogalactans. Multidimensional NMR and HILIC-MS/MS further confirmed the linkage and sequence information. Although CP2A's ultrahigh molecular weight restricts global molecular rotation, the observable ¹³C and 2D NMR signals arose from localized segmental motions of flexible repeating units, which provided sufficient mobility to permit structural assignment. Quantitative ¹³C NMR determined a residue ratio of 1: 3: 6: 3: 1, allowing the construction of a repeating unit, which finally elucidated a backbone of →4)-β-Galp-(1→3,6)-β-Galp-(1→ with ramified side chains at C3 per four residues composed of α-Araf-(1→, →5)-α-Araf-(1→, and →3,5)-α-Araf-(1→. Mechanistically, CP2A alleviated hematopoietic dysfunction by inhibiting TPEN-induced overactivation of the P53 pathway, including P21-mediated cell cycle arrest and proapoptotic factors. These findings identify CP2A as a structurally unique, highly branched, and functionally potent arabinogalactan with promising potential in hematopoietic disorder therapy.