Structural elucidation and optimization of an immunomodulatory acidic polysaccharide from Amomum tsao-ko: Activity evaluation and degradation-analysis-modification paradigm

A novel acidic polysaccharide, ATP-4, was isolated from Amomum tsao-ko and demonstrated immunomodulatory potential in the immunosuppressed mouse model and cellular assays. Structural characterization via monosaccharide analysis and gel permeation chromatography revealed ATP-4 as a heteropolysaccharide (4.23 × 10⁴ Da), composed of Rha, GalA, Glc, Gal, Xyl, and Ara in a molar ratio of 9.03:52.37:7.89:12.34:6.47:11.89. Integrated analysis of conventional techniques (PMP-HPLC, GC–MS, and NMR) and UPLC-Orbitrap-MS/MS profiling of free radical-degraded components (AO-1/2/3) elucidated its unique repeating units, which are characteristic of a novel polysaccharide assembled through specific patterns between the HG regions and branched domains such as GRP, RGP, etc. In vitro and in vivo studies demonstrated ATP-4's dual mechanisms: direct immune cell activation and indirect gut microbiota modulation. Notably, active structural domains AO-2 (the nonlinear branched region of ATP-4) significantly induced cytokine production at a lower concentration, guiding activity-directed enzymatic modification of ATP-4. The optimized derivative, ATP-4e, maintained a nonlinear branching structure while demonstrating enhanced immunoregulatory capacity, improved conformational flexibility, and superior physicochemical properties compared to the native ATP-4. This study establishes an integrated degradation-analysis-modification paradigm for polysaccharide research and provides a methodological framework for elucidating structure-activity relationships of traditional Chinese medicine polysaccharides.