Abstract: Objective: To separate and purify low-molecular-mass fractions from the chemical degradation products of the sulfated polysaccharide ascophyllan extracted from the brown seaweed Ascophyllum nodosum and to analyze the monosaccharide composition and in vitro immunostimulatory activity of each of these fractions for the purpose of providing useful information for the studies on the biological activities and structural characteristics of ascophyllan and other seaweed polysaccharides. Methods: The degraded products of ascophyllan, prepared separately by treatment with hydrochloric acid or hydrogen peroxide, were fractionated sequentially by ultrafiltration and Sephadex G-50 gel filtration chromatography into four low-molecular-mass fractions (HCl-F1, HCl-F2, H2O2-F1 and H2O2-F2). The monosaccharide composition of each fraction was determined by high-performance liquid chromatography (HPLC) with pre-column ethyl-p-aminobenzoate (ABEE) derivatization and some of their chemical groups were analyzed. Their weight-average molecular masses were evaluated by high-performance size exclusion chromatography (HPSEC), and their in vitro immunostimulatory activities were investigated by using mouse RAW264.7 macrophages as a model. Results: All these compounds were heteropolysaccharides with significant differences in their monosaccharide composition and sulfation level. The weight-average molecular masses of HCl-F1, HCl-F2, H2O2-F1 and H2O2-F2 were estimated to be 4.80, 4.20, 5.30 and 2.30 kDa, respectively. The in vitro immunomodulatory activity revealed that HCl-F1, HCl-F2, and H2O2-F2 significantly induced the activation of RAW264.7 macrophages to produce immune factors such as nitric oxide (NO) and tumor necrosis factor-a (TNF-a) in the test concentration range (0–200 mg/mL), whereas the ability of H2O2-F1 to induce the production of NO and TNF-a in RAW264.7 cells was significantly lower than that of any other polysaccharide fraction. Conclusion: All the low-molecular-mass polysaccharide fractions (HCl-F1, HCl-F2 and H2O2-F2) derived from ascophyllan possessed potent immunostimulatory activity, with HCl-F1 and H2O2-F2 being much stronger than ascophyllan. Taken together, the immunostimulatory activity is likely determined by a combination of monosaccharide composition and sulfate content.

Key words: ascophyllan, low-molecular-mass fractions, separation and purification, chemical composition, in vitro immunostimulatory activity