Extraction, structural characterization, and potential immunologic adjuvant activity of polysaccharides from Polygonum cuspidatum Sieb.et Zucc against influenza virus

Background：Polysaccharides represent essential bioactive compounds derived from Polygonum cuspidatum Sieb.et Zucc . The present investigation aimed to enhance polysaccharide extraction efficiency, achieve purification, elucidate structural characteristics, and determine their potential application as immune adjuvants against influenza virus. Results：An acid-based extraction method was employed for isolating polysaccharides from P. cuspidatum, with optimized extraction parameters established at a liquid-to-solid ratio of 20 mL/g, extraction temperature of 84 °C, duration of 50 minutes, and HCl concentration at 0.1 M. The purified fraction obtained was designated as PCPs. Structural elucidation of PCPs involved comprehensive analytical approaches. Results indicated that PCPs comprised glucans featuring a backbone structure of →4)-α-D-Glcp-(1→ residues, branched by →4,6)-α-D-Glcp-(1→ to →6)-α-D-Glcp-(1→ side chains. The O-6 position was confirmed as the branching site, and reducing terminal residues included both →4)-α-D-Glcp and →4)-β-D-Glcp. To evaluate the immunological adjuvant properties of PCPs for H1N1 influenza vaccines, mice received immunizations containing PCPs mixed with inactivated H1N1 antigen. To comprehensively evaluate the immune-enhancing properties of PCPs, we assessed specific IgG antibodies and subclasses (IgG1 and IgG2a) in serum, activation of splenic lymphocyte-derived CD4+ and CD8+ T cells, differentiation of central memory T-cell subsets characterized by the CD44 ^high CD62 ^Lhigh phenotype, and Th1/Th2 cytokine secretion profiles. Compared with mice receiving no adjuvant or antigen only, the PCP-immunized group demonstrated markedly increased antibody production of IgG2a (indicative of a Th1-type response) and IgG1 (indicative of a Th2-type response), significantly enhanced proliferation of CD4+ T cells, elevated numbers of CD8+ central memory T cells, and heightened secretion of the cytokines IFN-γ, IL-2, IL-4, IL-6, and IL-10. Conclusion：Collectively, these observations reveal that PCPs effectively boost both cellular and humoral immune responses as vaccine adjuvants against H1N1, emphasizing their promising applicability in veterinary vaccine formulations for livestock and poultry.