Physicochemical characterization and immune activity of water-extract polysaccharides by stepwise ethanol precipitation from wild Cordyceps sinensis

Cordyceps sinensis is widely utilized in China as an edible and medicinal fungus for the treatment of immunodeficiency-related disorders. Evidence suggests that polysaccharides are the principal bioactive components responsible for its immunostimulatory effects. However, the physicochemical properties, bioactivities, and structure–function relationships of these polysaccharides remain inadequately elucidated. In this study, four distinct crude polysaccharides from wild Cordyceps sinensis (WCP) were isolated using stepwise ethanol precipitation at final concentrations of 20%, 40%, 60%, and 80% (v/v). These fractions were designated as WCP-20, WCP-40, WCP-60, and WCP-80, respectively. Results demonstrated that the molecular weight (MW) of WCP decreased as the ethanol concentration increased. High-MW fractions (WCP-20/-40) exhibited high glucose content, a partially triple-helical structure, levorotation (−), and greater thermal stability. In contrast, the low-MW fractions (WCP-60/-80) were enriched in galactose and mannose, exhibited a higher branching density, and dextrorotation ( +). Furthermore, this study revealed that WCPs activated macrophages by enhancing phagocytosis and stimulating the secretion of nitric oxide and interleukin-1 beta. These immunostimulatory effects were mediated through the MAPK/NF-κB signaling pathway. Specifically, WCP-20 triggered macrophage activation via the ERK/JNK/p65 pathway, with P38/ERK/JNK pathway for WCP-40, P38/ERK/JNK/p65 pathway for WCP-60, and JNK/p65 pathway for WCP-80. Correlation analysis revealed that the immunostimulatory effects of WCPs were closely linked to their monosaccharide composition and secondary structures. These findings established that the physicochemical properties of WCP were critical determinants of precise immune modulation. This study provided a foundational reference for developing precision polysaccharide-based immune-enhancing nutraceuticals.