| Marine-derived microorganisms can often yield various biologically active exopolysaccharides with novel functions and structures, and showed the enormous development prospects for the marine microorganisms. Therefore marine microorganisms exopolysaccharides as the new sources of bioactive substances, have caused an emerging interest in the biomedical area in recent years. In this paper, three crude exopolysaccharides LEPS, JEPS and WEPS extracted from the broth of a deep sea fungus Penicillium sp F23 (E148°44.8′, N19°24.1′, 5080m), marine-sponge fungus Epicoccum nigrum and marine sediment collected in Zhanjiang, and the total yield are 0.97g/L, 0.52 g/L and 0.72g/L, respectively. In addition, the isolation, structure characteristics and antioxidant activities of purified exopolysaccharides were also investigated.Eight exopolysaccharides LEPS1-1, LEPS1-2, LEPS-2, JEPS-1, JEPS-2 WEPS-1, WEPS-2 and WEPS-3 were successfully isolated from LEPS, JEPS and WEPS through ion-exchange chromatography and low-pressure gel permeation chromatography. They were analyzed by Shodex Ohpak SB-804 HQ collumn HPLC, and the result showed that they have good purity. Their chemical characteristics and structure features were investigated by a combination of chemical and instrumental analysis including acid hydrolysis, periodate oxidation-Smith degradation, methylation analysis, GC, GC–MS, HPGPC, FT-IR and NMR.The results indicated that LEPS1-1 was mainly composed of (1→2)-linked -β-D-mannopyranosyl and (1→6)-linked-β-D- mannopyranosyl in approximate molar ratio of 2:1. In addition, LEPS1-2 mainly possessed a backbone of (1→2)-linked -β-D-mannopyranosyl and (1→4)-linked-β-D-mannopyranosyl, and LEPS-2 comprised of (1→2)-linked-β-D-mannopyranosyl and (1→6)-linked-β-D- glucopyranosyl with the molar ratio of 1:1 and 2:1, respectively.The backbone of JEPS-1 was composed of (1→2)-linked-α-D-mannopyranosyl, (1→3)-linked-α-D-mannopyranosyl and (1→2,3)-linked-β-D-galactopyranosyl residues in the ratio of 2.96:2.86:1.00, and the side chains were (1→6)-linked-β-D-glucopyranosyl, (1→4)-β-D-glucopyranosyl and (1→3,6)-linked-β-D-glucopyranosyl. The backbone of JEPS-2 contained (1→4)-β-D-galactopyranosyl, (1→6)-β-D-glucopyranosyl and (1→3,6)-β-D-glucopyranosyl in the ratio of 1.97:2.09:2.02, branched (1→2)-β-D-glucopyranosy, (1→3)-α-D-mannopyranosyl and (1→2,3)-β-D-galactopyranosyl. WEPS-1 had a backbone of (1→2)-linked -α-D-mannopyranosyl, (1→3)-linked-α-D-mannopyranosyl and (1→2,3)-linked-α-D- mannopyranosyl residues in the about ratio of 5:5:3, and (1→)-linked -α-D-mannopyranosyl was distributed in branches. while WEPS-2 had a backbone consisting (1→2)-linked-α-D-mannopyranosyl, (1→6)-β-D-glucopyranosyl, (1→3)-β-D-glucopyranosyl, (1→2,3)-linked-α-D-mannopyranosyl, and (1→3,6)-α-D- mannopyranosyl in the ratio of 6.07:5.22:5.92:6.13:5.08. In addition, the backbones and branches of all polysaccharides terminated with terminal Man, Gal or Glc. Eight oligosaccharides fractions (F2-F9) with symmetrical sharp peaks from WEPS were obtained by depolymerization reactions using mild acid partial hydrolysis at 60°C for 3 h, and purified by on Bio-Gel P-4 column with on-line by a refractive index detector. In addition, the full exclusion fraction F was also repeated the same hydrolysis and purification assays, and obtained seven fractions (f2-f8). The symmetric IR spectroscopy indicated that the parent polysaccharide WEPS and these oligosaccharides fragments were structurally similar. Molecular weights of the purified sections were determined by ESI-MS, and the results showed the molecular weights of F2～F7 were 268 Da, 518 Da, 768 Da, 1018 Da, 1268 Da and 1518 Da, and f2～f4 were 268 Da, 518 Da and 768 Da, respectively. The result showed the polymerization degree of F2～F7 was one to six, respectively. Structure features of fractions F2 and F3 were investigated by instrumental analysis including ESI-CID-MS/MS and 600-MHz NMR spectroscopy (1HNMR, 13CNMR, 2D-COSY, HMBC, HMQC and DEPT), and confirmed the structure of the polysaccharide as the repeating unit ofα-2→8 linked 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN, which is synonymous with 3-deoxy-D-glycero-D-galacto-nonulosonic acid and deaminated neuraminic acid ). This is the first report of the presence ofα-2→8 polyKdn from the marine actinomycete THW-7. This can also have the important referenced value for the research of the marine microbial exopolysaccharide. Kdn is a group name for Neuraminic acid derivatives of sialic acid. Substantial evidence now shows that Kdn is a possible risk factor for tumor, cancer, diabetes, cardiovascular diseases, and their thrombotic complications. Moreover, it is believed that their evolvement may have stimulated evolution and rendered organisms less vulnerable to environmental attacks. However, disturbance of their metabolism may cause diseases. According to the research, sialylated compounds in breast milk could help infants keeping away from intestine infection and improve their digestion and intelligence. However, bovine milk, the substitute of breast milk, has much less sialic acid than breast milk actually.Furthermore, their antioxidant activity was investigated by various antioxidant assay in vitro systems, including DPPH radical scavenging, superoxide anion radical scavenging, hydrogen radical scavenging, inhibition against liposome peroxidation and reducing power. The results indicated that antioxidant activities of purification polysaccharides increased with increased amount of samples. Among these assays, JEPS-1 and JEPS-2 showed strong antioxidant, and LEPS1-1, LEPS1-2 and LEPS-2 showed the best effect on the scavenging of superoxide radicals; WEPS-1,WEPS-2 and WEPS-3 showed significant ability on lipid peroxidation with EC50 values 0.59, 0.43 and 0.34. LEPS-2, JEPS-2 and WEPS-3 exhibited equivalent or even higher antioxidant activities than Vc. One of the important factors is that LEPS-2, JEPS-2 and WEPS-3 exhibited the higher antioxidant activities providing with acid monosaccharide appeared to increase the antioxidant activity compared to which appear the free-radical-scavenging activity was partially related to monosaccharide constituents. Apart from this, the apparent discrepancies between the characteristic of and linkages can influence the outcome of their antioxidant activities. The antioxidant activities of the tested samples were not a function of a single factor but a combination of several factors. |
PDF Full Text Request