Objective To prepare different polysaccharide fractions of Xiaoyao San (逍遥散, XYS) by graded ethanol precipitation and to clarify the anti-inflammatory activity, structural characteristics, and in vivo absorption and distribution behavior of the major enriched fraction. Methods Different polysaccharide fractions of XYS (40%, 60%, and 80% ethanol-precipitated fractions) were obtained using a water extraction-ethanol precipitation procedure, and the primary enriched fraction was screened based on yield comparison. Based on the inflammation-depression correlation, an LPS-induced microglial inflammation model was established, and anti-inflammatory activity was evaluated by measuring inflammatory markers including TNF-α, IL-1β, IL-6, and iNOS. The composition and structural features of the target polysaccharide were characterized using UV-Vis spectroscopy, FT-IR, and NMR analyses. Fluorescently labeled polysaccharides were further prepared for in vivo pharmacokinetic studies in mice to systematically assess exposure levels and tissue distribution characteristics. Results Significant differences in yield were observed among polysaccharide fractions obtained at different ethanol precipitation ratios. The 60% ethanol fraction (XYSP-60) was identified as the major enriched fraction, with an extraction recovery of 37.5%. In the LPS-induced microglial inflammation model, XYSP-60 significantly downregulated the expression levels of TNF-α, IL-1β, IL-6, and iNOS, demonstrating clear anti-inflammatory activity. Structural characterization indicated that the polysaccharide mainly consisted of galacturonic acid, glucose, and fructose, with relatively stable structural features. Pharmacokinetic results showed that XYSP-60 exhibited high exposure in the intestine but limited entry into systemic circulation. Conclusion The 60% ethanol-precipitated fraction represents the principal enriched polysaccharide fraction of XYS, with stable structural characteristics and definite anti-inflammatory activity. Its in vivo behavior is characterized by high intestinal exposure and limited systemic absorption, suggesting that this fraction may serve as one of the key material bases underlying the anti-inflammatory effects of XYS.

Xiaoyao San  /  polysaccharides  /  material basis  /  fluorescent labeling  /  BV-2 cells