Detailed elucidation of a glucuronoxylogalactoglucomannan from Auricularia auricula-judae via liquid chromatography-electrospray mass spectrometry and 1/2D-NMR spectroscopy.

This study reports the isolation and comprehensive structural elucidation of ME-3 (Mw = 7.0 × 104 g/mol), a novel glucuronoxylogalactoglucomannan (GXG'G"M) isolated from the Auricularia auricula-judae. The initial investigation of the structural parameter of ME-3 was undertaken, revealing the monosaccharide composition and O-acetyl content (12.83%). For an in-depth elucidation of its fine structure, the methylation and free-radical degradation products of ME-3 were examined using liquid chromatography-electrospray mass spectrometry (LC-ESI-MS/MS). To overcome the influence of O-acetyl groups and improve spectral resolution, high-resolution NMR analysis of the deacetylated product of ME-3 (dME-3) was thus performed at 333 K, including 1H/13C NMR, DEPT-135, 1H-1H COSY, 1D selective TOCSY, HSQC, H2BC, HMBC, and NOESY. Synthesizing LC-ESI-MS/MS and 1/2D-NMR data elucidated ME-3's repeating units and precise O-acetylation pattern. Importantly, this unique GXG'G"M structure correlated with significant immunomodulatory function, demonstrated by dose-dependent macrophage activation via TLR2/4-mediated NF-κB signaling. Collectively, these findings not only demonstrate the undeniable presence of the GXG'G"M type of polysaccharide, but also highlight its potential as a natural immunostimulant.