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A reliable high performance liquid chromatography with pre-column derivatization method was developed for the determination of the mannuronic acid (M)/guluronic acid (G) ratio of propylene glycol alginate sodium sulfate (PSS). The hydrolysis conditions of PSS were investigated by four degradation methods based on the degree of destruction of M and G, and the chromatographic separation conditions were also optimized. A satisfactory resolution of M and G was achieved with a KP-C18 column using 0.1 mol/L phosphate buffer (pH 7.0)-acetonitrile (83/17, v/v) as a mobile phase, after PSS was hydrolyzed with 0.1 mol/L sulfuric acid and labeled with 1-phenyl-3-methyl -5-pyrazolone. The M/G ratio of PSS determined by this method was in good accordance with that obtained by the (1)H NMR method with a desulfurization strategy. Our method is rapid, sensitive, accurate and reproducible. The limit of detection was found to be 0.25 μg/mL for M and 0.40 μg/mL for G., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Three derivatives of polymannuronic acid (PM) were prepared by chemically polyanionic modification. PM was substituted with phosphate (OPO3(2-)), H-phosphonate (OPO2H(-)) and sulfate (OSO3(-)) groups with the degree of substitution (DS) of 0.41, 1.42 and 1.04, respectively. The structures of all PM derivatives were characterized by FT-IR, (31)P NMR and (13)C NMR spectroscopy. The weight average molecular weight (Mw) was determined by high performance gel permeation chromatography (HPGPC). The antioxidant activities of PM and its derivatives were evaluated in vitro. The results indicated that both phosphate and H-phosphonate groups improved the hydroxyl radical scavenging activity, while sulfate group enhanced the superoxide radical scavenging activity. However, all of the derivatives scavenged DPPH less effectively than PM. The mechanism for how the different anionic substituent groups influenced the antioxidant activities was discussed., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Sulfated polymannuroguluronate (SPMG), a marine sulfated polysaccharide, has entered the Phase II clinical trial in China as the first anti-acquired immune deficiency syndrome (AIDS) drug candidate obtained from marine organisms. To determine the binding site(s) (receptors) of SPMG in lymphocytes mediating its anti-AIDS activities, fluorescein-5-isothiocyanate (FITC)-labeled SPMG was used to investigate SPMG binding to lymphocytes. Flow cytometry (FCM) and fluorescence microscopy analysis showed that the SPMG binds to lymphocytes in a rapid, specific, reversible, and saturable fashion. Several SPMG binding proteins were purified by affinity chromatography from lymphocyte membrane preparations. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis revealed that a 55kDa lymphocyte membrane protein is CD4. To characterize the SPMG and CD4 interaction, inhibition assay and surface plasmon resonance (SPR) assay were carried out. SPMG bound to CD4 in a multivalent fashion with specificity. The binding of SPMG to human lymphocyte CD4 was competitively inhibited by human soluble CD4 (hsCD4). Likewise, the binding between hsCD4 and immobilized SPMG was blocked by excess free SPMG. These results indicate that CD4 is one of the specific SPMG binding sites (receptors) in lymphocytes. The interaction between SPMG and CD4 may provide a mechanistic explanation of the immunopotentiating and anti-AIDS activities of SPMG in human immunodeficiency virus (HIV) infected individuals.

Alginate that was purified from the fermentation solution of marine bacteria Vibro sp. 510 under specific reaction conditions was hydrolyzed by alginate lyase. Seven oligosaccharides, including di-, tri- and tetrasaccharides, were isolated through low-pressure, gel-permeation chromatography (LP-GPC) and semipreparative strong-anion exchange (SAX) fast-protein liquid chromatography (FPLC). The oligosaccharide structures were elucidated based on ESIMS and 2D NMR spectral analysis. The hydrolytic specificity of this alginate lyase to alginate is discussed., (Copyright 2004 Elsevier Ltd.)

Anti-proliferation action and enhancement of endothelial cell immunity and related mechanisms by marine sulfated polymannuroguluronate (SPMG) were investigated in the present studies. Endothelial cell proliferation was evaluated by MTT assay. Intercellular adhesion molecule-1 (ICAM-1) expression was analyzed by flow cytometry. The interaction of SPMG with basic fibroblast growth factor (bFGF) was evaluated by surface plasmon resonance. Results showed that SPMG exhibited a significant inhibitory effect against proliferation in both normal human umbilical vein endothelial cells (HUVEC) and bFGF-treated HUVEC, the action of which was completely abrogated by bFGF antibody. SPMG exerted high affinity to bFGF in a multivalent pattern, characterized by one molecule SPMG binding to 3 - 4 molecules of bFGF. Moreover, SPMG enhanced ICAM-1 expression in HUVEC and prevented and restored bFGF-treated downregulation of ICAM-1 expression in HUVEC, the expression of which was not counteracted by bFGF antibody. In conclusion, this is the first report demonstrating that SPMG exerted an anti-proliferation effect dependent on the bFGF-regulated pathway and afforded upregulatory activity on ICAM-1 expression regardless of the involvement of bFGF.