Isolation, structural characterization, and immunostimulatory activity of a polysaccharide from the stem of Trapa acornis Nakano on RAW264.7 macrophages

Trapa acornis Nakano, also known as “Nan-hu-ling” (NHL), is an important agricultural crop whose fruits are commonly consumed as food, while its stems are usually discarded as agricultural by-products. In the current investigation, a homogeneous polysaccharide (NHLP) with a weight-average molecular weight (Mw) of 62.0 kDa was isolated from the stem of NHL. Monosaccharide composition analysis revealed that NHLP was primarily composed of Gal (24.4%), Glc (18.9%), GalA (16.8%), Xyl (12.1%), and Rha (7.7%). Methylation analysis indicated that NHLP mainly contained residues of 4-GalpA, 4-Glcp, 4-Galp, 4-Xylp, 3,6-Galp, t-Araf, t-Galp, and t-Rhap. The linkage patterns of these residues were structurally resolved using one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy. In vitro assays demonstrated that NHLP stimulated RAW264.7 cells to produce nitric oxide (NO) in a dose-dependent manner, promoted the release of proinflammatory cytokines including interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α), and upregulated transcriptional profiles of genes involved in inflammatory responses (IL-6, IL-1β, TNF-α, COX-2, iNOS, and IκBα) and proteins (iNOS, COX-2, IL-1β, and TNF-α). This study clarified the proposed structure of the polysaccharide derived from T. acornis stem and its macrophage-activating effects, providing supportive evidence for its potential application in immune activation.