In this study, Spirulina platensis (S.p.) polysaccharide (PSP) was obtained by ultrasonic-microwave-assisted extraction (UMAE) and purified by an aqueous two-phase system (ATPS). Two different methods were applied to purified Spirulina platensis (S.p.) polysaccharide (PSP), respectively, due to PSP as a complex multi-component system. Three polysaccharide fractions (PSP-1, PSP-2, and PSP-3) with different acidic groups were obtained after PSP was fractionated by the diethyl aminoethyl (DEAE)-52 cellulose chromatography, and two polysaccharide fractions (PSP-L and PSP-H) with different molecular weight were obtained by ultrafiltration centrifugation. The chemoprotective effects of PSP in cyclophosphamide (Cy) treated mice were investigated. The results showed that PSP could significantly increase spleen and thymus index, peripheral white blood cells (PWBC), and peripheral blood lymphocytes (PBL). The in vivo immunostimulatory assays demonstrated that PSP could in dose-dependent increase of TNF-α, IL-10, and IFN-γ production in sera. The in vitro immunostimulatory assays showed that PSP and its fractions (PSPs) could evidently enhance the proliferation of splenocytes and RAW 264.7 cells and increase the productions of nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin 6 (IL-6). PSPs could also enhance phagocytic activity of RAW 264.7 cells. The acidic polysaccharide fractions of PSP-2, PSP-3, and PSP-L with small molecular weight had the higher immunostimulatory activity. Signaling pathway research results indicated that PSP-L activated RAW264.7 cells through MAPKs, NF-κB signaling pathways via TLR4 receptor.

To address the structure–activity relationship of Chlamys farreri polysaccharides on their immunostimulatory efficacy, two polysaccharides (CFP-1 and CFP-2) were extracted from Chlamys farreri by hot water extraction, and separated through column chromatography. The isolated CFPs were chemically analyzed to clarify their physicochemical characteristics and cultured with murine macrophage RAW264.7 cells, in order to evaluate their immunostimulatory efficacy. Despite the fact that both CFP-1 and CFP-2 were mainly comprised of glucose lacking the triple-helix structure, as revealed through preliminary physicochemical analyses, obvious differences in regard to molecular weight (Mw), glucuronic acid content (GAc) and branching degree (BD) were observed between CFP-1 and CFP-2. In in vitro immunostimulatory assays for macrophage RAW264.7 cells, it was demonstrated that CFP-2 with larger Mw, more GAc and BD could evidently promote phagocytosis and increase the production of NO, IL-6, TNF-α and IL-1β secretion, by activating the expression of iNOS, IL-6, TNF-α and IL-1β genes, respectively. Hence, CFP-2 shows great promise as a potential immunostimulatory agent in the functional foods and nutraceutical industry, while CFP-1, with lower molecular weight, less GAc and BD, displays its weaker immunostimulatory efficacy, based on the indistinctive immunostimulatory parameters of CFP-1.