Structural characterization and immunostimulatory effects of polysaccharide AP-1 from ashwagandha (Withania somnifera) roots

Ashwagandha ( Withania somnifera ) is a well-known medicinal plant, with polysaccharides recognized as key bioactive constituents. In this study, we isolated a neutral polysaccharide, AP-1, from ashwagandha roots and elucidated its structure. AP-1 has a molecular weight of 9.21 kDa and is primarily composed of mannose, glucose, and galactose in a molar ratio of 0.1:1.5:1. Spectroscopic analyses revealed that AP-1's backbone consists of 1,4-α-Glc p and 1,3-α-Gal p , with branching at the C-6 of 1,4,6-α-Glc p and C-4 of 1,3,4-α-Gal p positions. The immunostimulatory activity of AP-1 was evaluated in murine macrophage RAW264.7 cells. AP-1 significantly enhanced macrophage immune responses, promoted M1-type polarization, and activated the NF-κB signaling pathway. It increased the production and mRNAs expression of pro-inflammatory mediators including nitric oxide, oxygen species, tumor necrosis factor, interleukins-1β and -6, and upregulated the M1 surface biomarker CD86. These findings highlight the potential of AP-1 as a promising bioactive ingredient for functional foods targeting immune enhancement.