Impact of extraction techniques on the structural features and immunomodulatory activity of polysaccharides from...

Polysaccharides from Cremastra appendiculata (CAP) have reported immunomodulatory activity, yet how extraction method shapes their structure features and macrophage-stimulatory effects remains unclear. Seven methods, including hot water (HWE), acid-assisted (ACE), ultrasonic-assisted (UAE), microwave-assisted (MAE), enzyme-assisted (EAE), pressurized water (PWE), and deep eutectic solvent (DESE) extraction, were compared for their effects on CAP structure and in vitro immunomodulatory activity. All fractions were composed mainly of mannose and glucose and showed structural features consistent with a predominantly (1 → 4)-linked glucomannan framework. Comparative methylation analysis of DESE-P, ACEP, HWEP, and UAE-P confirmed shared predominant →4)-Manp-(1→ and →4)-Glcp-(1→ linkages, while revealing differences in minor terminal, 2-linked, 4,6-linked, and Galp-derived residues. Their weight-average molecular weights (2.67-15.77 × 10⁴ Da), degree of O-acetylation (25.56-47.64%), monosaccharide compositions, and chain conformations varied among extraction methods. In RAW264.7 macrophages, ACE-P produced the strongest increases in nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) without reducing cell viability. TAK-242 attenuated all three ACE-P-induced responses, C29 had no significant effect under the conditions tested, and BAY 11-7082 reduced NO and IL-6 but not TNF-α, supporting TLR4-associated activation and differential downstream regulation of these mediators. Exploratory across-fraction correlations suggested that higher activity co-occurred with higher carbohydrate content and lower molecular weight and O-acetylation, although UAE-P showed that low molecular weight alone was insufficient. These findings demonstrate extraction-dependent variation in CAP structure and macrophage-stimulatory activity and identify ACE-P as a candidate for further mechanistic and in vivo evaluation.