Purification, structural analysis and mechanism of murine macrophage cell activation by sulfated polysaccharides from Cystoseira indica

•Sulfated polysaccharides were isolated from Cystoseira indica.•Polysaccharides exerted high RAW264.7 macrophage stimulating activity.•RAW264.7 cells were activated through NF-κB and MAPK signaling pathways.•TLR4 was the major cell surface receptor interacting polysaccharides with RAW264.7 cells.•The main backbone of the polysaccharides was consisted of (1→3)-fucopyranosyl units. Sulfated polysaccharides were isolated and purified from the water extract of Cystoseira indica using DEAE Sepharose Fast Flow column to evaluate their structure and macrophage stimulating capacity. Crude and fractionated polysaccharides, CIF1 and CIF2, were mostly composed of neutral sugars (73.1%–78.6%) with relatively lower amounts of acidic sugars (1.3%–9.0%) and sulfate esters (6.9%–9.7%). The polymer chains of polysaccharides were mainly built of different levels of glucose (2.1%–30.8%), fucose (17.2%–24.4%), mannose (17.8%–20.6%) and galactose (16.7%–17.3%). The weight average molecular weight (Mw) of polysaccharides varied between 573.1 × 103 g/mol to 1146.6 × 103 g/mol. The CIF2 polysaccharide, as the most immunostimulating polysaccharide, remarkably induced the release of nitric oxide and inflammatory cytokines including TNF-α, IL-1β, IL-6 and IL-10 from RAW264.7 murine macrophage cells through NF-κB and PAMKs transduction signaling pathways via cell surface TLR4. The interconnections of sugars in CIF2 polysaccharide were complex with (1→3)-fucopyranose, (1→2,3,4)-glucopyranose, (→1)-galactopyranose, (→1)-xylopyranose, (1→2)-rhamnopyranose and (1→2,3)-mannopyranose units being the most predominant residues.