【作者】 孙海红；

【导师】 毛文君；

【作者基本信息】 中国海洋大学 ， 药物化学， 2009， 博士

【摘要】 海洋微生物由于其特殊的生存环境,具有产生结构新颖、功能独特的新型活性胞外多糖的潜力,是开发多糖类海洋新药的重要资源。近年来,海洋微生物胞外多糖作为活性物质的新来源,在生物医学领域正日益受到人们的关注。本论文以12种海洋微生物发酵液提取的胞外多糖为研究对象,并对其产率、理化性质、单糖组成和抗氧化活性进行研究,从中筛选出深海真菌Penicillium sp.、海绵共附真菌Epicoccum nigrum和海洋放线菌THW-7A胞外多糖,并进一步分离纯化,深入研究其理化性质、结构和抗氧化活性。以LEPS、JEPS和WEPS为研究对象,采用离子交换色谱和凝胶排阻色谱相结合对其进行分离纯化分别得到八种胞外多糖LEPS1-1、LEPS1-2、LEPS-2、JEPS-1、JEPS-2、WEPS-1、WEPS-2和WEPS-3。经Shodex OH pak SB-804柱高效液相色谱分析,证明八种多糖都具有较高的纯度。通过采用高碘酸氧化-Smith降解、甲基化等化学方法及现代仪器分析手段,包括IR、GC、HPGPC、HPLC、GC-MS、NMR等,分别对其结构进行了深入研究,确定了它们的基本化学结构特点。结果表明:LEPS1-1主链是以1→2, 1→6连接Man,其摩尔比近似为2:1,支链中主要为1→3连接的Glc,同时存在微量的1→2,4连接的Gal;LEPS1-2主链是1→2 Man和1→4 Man摩尔比近似1:1比例连接,支链是1→2,4 Gal为主,还含有较多的末端甘露糖和少量的1→6 Man、1→2 Gal;LEPS-2结构中主链是1→2 Man, 1→6 Glc,而其支链是以1→2,4 Gal和1→4 Man为主。JEPS-1主链主要为1→2 Man, 1→3 Man和1→2,3 Gal,支链为1→6Glc、1→4 Glc和1→3,6连接的Glc,同时存在大量1→Man。JEPS-2主链是1→4 Gal、1→6 Glc和1→3,6 Glc,支链是1→2 Glc,还含有较多的末端甘露糖和少量的1→3 Man、1→2,3 Gal。WEPS-1的主链主要为1→2Man、1→3Man和1→2,3Man,并且摩尔比近似等于5:5:3,同时含有大量的末端Man和少量的1→6甘露糖。而WEPS-2主链主要是1→2Man、1→6Glc、1→3Glc、1→2,3Man和1→3,6Man,支链主要是1→Man和1→Glc键型。WEPS-1和WEPS-2的结构相对简单,均是分支度很少的近似直链的结构。采用温和的0.01 mol L-1 HCl在60℃降解3小时作为降解条件制备WEPS-3系列寡糖,利用HPGPC-RID经Bio-Gel-P4凝胶柱对降解产物进行分离制备获得寡糖,在线示差检测到8个寡糖组分(F2～F9),并对全排阻部分在相同条件下降解和纯化得到7个寡糖组分(f2～f8)。将胞外多糖WEPS-3和获得的寡糖组分进行红外光谱分析,结果表明分离得到的寡糖与原料多糖的谱图十分相似,各种糖的特征吸收峰均未发生明显改变,并通过ESI-MS分析确定了各组分的分子量大小,测定结果为F2～F7的分子量分别为268 Da、518 Da、768 Da、1018 Da、1268 Da和1518 Da,f2～f4的分子量分别为268 Da、518 Da和768Da,结果证实F2～F7分别为聚合度1到6的糖单元,f2～f4分别为聚合度为1～3的糖单元。同时结合1H-NMR、13C-NMR、DEPT、1H 1H-COSY、HMQC、HMBC和ESI-CID-MS/MS等仪器分析手段对F2和F3片段进行结构和序列解析,结果表明胞外多糖WEPS-3为α-2→8连接的聚合度为25的脱氨基神经氨酸Kdn。这是首次报道从海洋放线菌THW-7A中获得多聚Kdn唾液酸,此研究对海洋微生物胞外多糖的研究具有重要的参考价值。Kdn是唾液酸中神经氨酸(Neuraminic acid)的衍生物。现代医学根据某些身体的化学水平来临床诊断和治疗疾病,Kdn被证明和多种疾病有关,例如肿瘤、癌症、糖尿病、心血管疾病等;有人甚至认为它们和进化有关,能增强生物体对环境的适应性。近期食品安全方面更进一步强调了唾液酸的作用。据研究,母乳中唾液酸化的成分能帮助婴儿抵抗肠道感染以及促进记忆和智力的发育。但是作为母乳替代品的牛乳里面的唾液酸含量要大大低于母乳。通过利用Fenton反应检测对羟基自由基、DPPH自由基、超氧阴离子自由基的清除能力以及抗脂质过氧化和还原能力等实验对八种胞外多糖的体外抗氧化活性进行了研究,并与阳性对照BHT和Vc进行比较。结果表明,JEPS-1和JEPS-2均表现出很强的抗氧化活性; LEPS1-1, LEPS1-2和LEPS-2对清除超氧阴离子和抗脂质过氧化的能力异常显著;WEPS-1、WEPS-2和WEPS-3抗脂质过氧化能力也非常强,其EC50值分别为0.59、0.43和0.34,并可与阳性对照Vc相比拟。酸性多聚糖的抗氧化活性均高于中性多聚糖,推断这可能与酸性多糖含有较多的带负电荷的糖醛酸有关,另外糖单元的类型和糖苷键的构型,多糖的取代基,多糖的空间结构,多糖的分子量大小等也是影响多糖抗氧化活性的主要因素。更多还原

【Abstract】 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.更多还原