Molecular-weight fractionation, preliminary structural characterization, bioactivity, and ICP-MS-based biodistribution of Grifola frondosa polysaccharide-derived fractions

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Introduction Grifola frondosa is an edible and medicinal mushroom rich in bioactive polysaccharides, but the relationships among molecular-weight distribution, compositional and linkage features, biological activity, and in vivo distribution remain incompletely characterized. Methods Polysaccharides from G. frondosa were extracted, purified, and separated into three molecular-weight fractions: PP-1 (>10 kDa), PP-2 (1-10 kDa), and PP-3 (< 1 kDa). The fractions were characterized by total sugar, acidic sugar and protein contents, monosaccharide composition, apparent molecular-weight distribution, and methylation analysis. Their preliminary bioactivities were evaluated using S180, HepG2, and RAW 264.7 cell-based assays. PP-3 was subsequently labeled with fluorescein isothiocyanate followed by iodination, and iodine content was quantified using inductively coupled plasma mass spectrometry (ICP-MS) to evaluate its preliminary in vivo distribution. Results Among the three fractions, PP-3 showed the highest sugar content and a relatively simple detected monosaccharide profile under the present Glc/Gal-standard-based analysis, with methylation analysis suggesting abundant terminal Glcp residues and limited internal glucose linkages. PP-3 exhibited the strongest preliminary in vitro antiproliferative activity against S180 sarcoma and HepG2 hepatocellular carcinoma cells under the present screening conditions. It also enhanced RAW 264.7 macrophage proliferation and increased nitric oxide, TNF-α, IL-1β, and IL-6 production. The ICP-MS method showed good linearity, precision, stability, reproducibility, and recovery. After oral administration in rats, iodine-labeled PP-3-derived material showed limited systemic absorption, with the highest blood absorption rate observed at 2 h, and iodine signal was mainly detected in the liver and kidney. Discussion Because ICP-MS detects iodine rather than the intact carbohydrate backbone, the biodistribution findings should be interpreted as preliminary tracer-based distribution data for iodine-labeled PP-3-derived material rather than a full pharmacokinetic profile of the native PP-3 fraction. Overall, this study identifies PP-3 as a low-molecular-weight carbohydrate/oligosaccharide-rich fraction with the strongest preliminary in vitro bioactivity among the tested G. frondosa fractions and provides an ICP-MS-based strategy for tracing iodine-labeled PP-3-derived material, supporting further investigation of mushroom-derived polysaccharide-derived fractions for functional-food and natural-product applications.

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