Structural complexity and biofunctional properties of highly sulfated carrageenan-agaroid galactans from Calliblepharis fimbriata: Insights from selective chemical modifications.

Galactans isolated from Calliblepharis fimbriata were characterized using selective chemical modification approaches coupled with 1D and 2D NMR spectroscopy to elucidate their structural complexity and structure-activity relationships. The polysaccharides comprised mixed d and l-galactopyranosyl residues associated with ι-carrageenan (G4S - DA2S) and porphyran (G - L6S) motifs. 1H13C HSQC analysis of chemically desulfated derivatives revealed preferential removal of sulfate groups at C-4, while DA2S and L6S units remained largely preserved, yielding α-carrageenan (G - DA2S) segments. Alkali treatment induced partial cyclization of precursor residues, and the principal fraction exhibited a molar ratio of d-galactose, 3,6-anhydrogalactose, and sulfate ester of 1.00:0.04:0.36. Structure-activity relationship analysis suggested that sulfate substitution at C-2 and C-4 positions contributed to an enhanced anticoagulant activity, and high anionic moieties were further associated with increased inhibition of α-glucosidase (38.2-68.8%) and α-amylase (15.3-86%). Furthermore, the galactans exhibited non-cytotoxicity toward RAW264.7 macrophages at concentrations below 32 μg/mL and significantly enhanced (p < 0.05) phagocytic activity and nitric oxide production in a concentration-dependent manner, indicating an immunostimulatory potential through macrophage activation. Overall, these findings reveal the structural complexity of C. fimbriata galactans and establish clear links between their distinct sulfation patterns and biological functions, providing a basis for their potential biomedical and nutraceutical applications.