【作者】 李红燕；

【导师】 毛文君；

【作者基本信息】 中国海洋大学 ， 药物化学， 2011， 博士

【摘要】 宽礁膜是我国重要的经济海藻之一,隶属于绿藻门,绿藻纲,石莼目,礁膜科,礁膜属。目前,宽礁膜在我国人工育苗和栽培已获成功。本论文以我国人工养殖宽礁膜为研究对象,采用各种色谱分离技术,获得高抗凝血活性的宽礁膜多糖,采用化学和现代波谱方法对其结构进行研究,并对结构新颖的抗凝血多糖进行降解研究,建立可控酸降解法制备宽礁膜低分子量多糖片段及其寡糖的方法,通过核磁共振波谱和质谱技术,对获得的低分子量的糖基片段和寡糖的结构进行研究,并研究各种低分子量糖基片段对抗凝血活性的影响。研究结果如下: 1人工栽培宽礁膜藻体经冷、热水提取得到两种多糖冷水提多糖和和热水提多糖,相对于干燥藻体的得率分别为9.56%和39.1%。采用离子交换色谱和凝胶渗透色谱对热水提多糖进一步纯化,主要得到三种多糖MAS、MBS和MCS。通过化学方法对理化性质进行测定,结果表明,MAS、MBS和MCS总糖含量分别为74.1%、68.1%和65.3%;硫酸基含量为26.1%、28.5%和31.8%;糖醛酸含量为4.44%、3.94%和3.27%;蛋白质含量较低。高效凝胶渗透色谱法测得MAS分子量较高为512.5 kDa,而MBS和MCS分子量较低,分别为58.4 kDa和48.5 kDa;气相色谱法测得MAS、MBS和MCS以鼠李糖为主,还含有少量的葡萄糖、木糖、甘露糖和半乳糖等。经红外光谱、脱硫反应、甲基化反应和核磁共振波谱（1D,2D NMR）分析表明,MAS、MBS和MCS结构比较相似,主要由[→3)-α-L-Rhap-（1→]、[→2）-α-L-Rhap-（1→]和[→2,3）-α-L-Rhap-（1→]组成,但摩尔比例不同,硫酸基团主要位于[→3）-α-L-Rhap-（1→]的C-2位和[→2）-α-L-Rhap-（1→]的C-3位。多糖MAS、MBS和MCS中主要含有四种硫酸鼠李二糖结构单元: [→3）-α-L-Rhap（2SO₄）-（1→3）-α-L-Rhap→]、[→3)-α-L-Rhap（2SO₄）-（1→2）-α-L-Rhap→]、[→3)-α-L-Rhap-（1→2）-α-L-Rhap（3SO₄）→]和[→3)-α-L-Rhap（2SO₄）-（1→2,3）-α-L-Rhap→]。通过APTT、PT和TT对多糖MAS、MBS和MCS的抗凝血活性进行评价,得到抗凝血活性较好结构新颖的硫酸鼠李聚糖。2采用硫酸降解的方法,将多糖MAS降解为不同分子量的多糖。通过相对黏度法、高效薄层色谱法、聚丙烯酰胺凝胶电泳法和高效凝胶渗透色谱法对降解条件进行优化。采用0.1 mol/L H₂SO₄在40℃水解75 min和80℃水解60 min对多糖MAS进行降解,制备得到分子量在405.0⁵.1 kDa的七种多糖（MAS1^MAS7）。研究表明,七种多糖的理化性质、单糖组成和红外光谱特征均与母多糖MAS相似。以抗凝血活性高且温和的低分子量多糖MAS5为代表,通过甲基化反应和1D,2D NMR分析表明,MAS5由[→3)-α-L-Rhap-（1→]、[→2）-α-L-Rhap-（1→]和[→2,3）-α-L-Rhap-（1→]组成,三者摩尔比为4:1:1,硫酸基团位于[→3）-α-L-Rhap-（1→]的C-2位和[→2）-α-L-Rhap-（1→]的C-3位。MAS5结构与母多糖MAS相似,即酸降解后多糖主体结构没有发生改变。通过活化部分凝血酶时间（APTT）,对多糖MAS及其降解产物MAS1^MAS7的抗凝血活性进行了评价。结果表明,多糖随着分子量的减小,抗凝血活性减弱。分子量对宽礁膜多糖抗凝血活性具有较大的影响,其抗凝血活性依赖于一定长度的糖链。3多糖MAS通过0.1 mol/L H₂SO₄于80℃水解3 h,Bio-Gel P4凝胶渗透色谱分离纯化,得到寡糖组分O1^O8。通过ES-MS对寡糖组分的聚合度、单糖组成和纯度进行了分析。结果表明,硫酸鼠李寡糖聚合度（DP）在2⁹之间。采用1D,2D NMR对纯度较高的硫酸鼠李二糖结构进行研究,确定其结构为α-L-Rhap（2SO₄）-（1→3）-α-L-Rhap。通过对硫酸鼠李二糖ES-CID-MS/MS断裂碎片的分析和归属,建立了硫酸鼠李寡糖负离子模式下二级质谱序列分析方法,并将此方法用于聚合度为3⁹的硫酸鼠李寡糖的结构研究。结果表明,制备得到的硫酸鼠李寡糖均为[→3）-α-L-Rhap-(1→]连接的直链结构。这些硫酸鼠李寡糖为具有新型结构的海洋特征寡糖,目前国内外尚未有关硫酸鼠李寡糖的报道。本论文的研究成果,对宽礁膜多糖的研究和开发、对“海洋糖库”的建设以及发展具有我国特色的海洋药物具有重要的学术价值和经济意义。更多还原

【Abstract】 Marine green algae often contained bioactive substances with novel functions and structures because of their special living conditions, and the polysaccharides from marine green algae are of interest to new drug discovery. The sulfated polysaccharides from green algae Monostromaceae species show potent anticoagulant activity, and represent potential source to be explored. Until recently, few structural studies have been done on the polysaccharides from Monostromaceae.Monostroma latissimum is widely distributed in China, and has been used as fundamental source of food and drug in traditional Chinese medicine for thousands of years. Recently, the cultivation of marine green seaweed, Monostroma latissimum, was successfully obtained in China. Thus, the analyses of this species are very important in industrial application. In the present work, the heparinoid-active sulfated polysaccharides from the cultivation of Monostroma latissimum were isolated, and their structural characteristics were investigated by a combination of chemical and spectroscopic methods.The green alga, Monostroma latissimum from Yuhuan County in China, was extracted by cold （24°C） and hot （100°C） water to obtain two polysaccharides. The hot water extracted polysaccharide with higher yield and anticoagulant-active was further purified on Q-Sepharose Fast Flow ion-exchange and Sephacryl S-400/HR size-exclusion chromatography to generate three polysaccharides MAS, MBS and MCS of uniform size and charge. Chemical composition analysis indicated that MAS, MBS and MCS contained 74.1%, 68.1% and 65.3% total polysaccharides, with 26.1%, 28.5% and 31.8% sulfate, 4.44%, 3.94% and 3.27% glucuronic acid, and with a minor amount of protein. MAS, MBS and MCS were high rhamnose-containing sulfated polysaccharides with an average molecular weight of about 512.5 kDa, 58.4 kDa and 48.5 kDa, respectively. On the basis of methylation, one- and two-dimensional nuclear magnetic resonance （1D, 2D NMR） spectroscopic analyses, the polysaccharide chains of MAS, MBS and MCS were characterized to consist of [→3)-α-L-Rhap-（1→], [→2）-α-L-Rhap-（1→] and [→2,3）-α-L-Rhap-（1→] with different molar ratio, and the sulfate groups were substituted at C-2 of [→3）-α-L-Rhap-（1→] and C-3 of the [→2）-α-L-Rhap-（1→]. The disaccharide units in the structure of three polysaccharides were [→3）-α-L-Rhap（2SO₄）-（1→3）-α-L-Rhap→], [→3)-α-L-Rhap（2SO₄）-（1→2）-α- L-Rhap→], [→3)-α-L-Rhap-（1→2）-α-L-Rhap（3SO₄）→] and [→3)-α-L-Rhap（2SO₄）- （1→2,3）-α-L-Rhap→]. The polysaccharides had a high anticoagulant activity as evaluated by assays of the activated partial thromboplastin time and thrombin time. The investigation demonstrated that the polysaccharides appeared to be a sulfated rhamnan with different structural characteristics from other sulfated polysaccharides from Monostromaceae species, and could be a potential source of anticoagulant.According to the relative viscosity, high performance gel permeation chromatography, thin layer chromatography, polyacrylamide gel electrophoresis, the hydrolysis condition of the sulfated polysaccharide MAS was optimized. With the condition of 0.1 mol/L H2SO₄ at 40°C for 75 min and 80°C for 60 min, the polysaccharide MAS was degraded, and isolated to generate seven low molecular weight polysaccharides （MAS1^MAS7）, their molecular weights ranged from 405.0 kDa to 5.1 kDa. The physiochemical properties, monosaccharide composition and infra-red spectrum characteristics of the polysaccharides were all similar to those of the parent polysaccharide MAS. The structure of the polysaccharide MAS5 as representation was studied by methylation and 1D, 2D NMR spectroscopy, and the results showed that the structure consisted of [→3)-α-L-Rhap-（1→], [→2）-α-L-Rhap-（1→] and [→2,3）-α-L-Rhap-（1→] in the molar ratio of 4:1:1. The sulfate groups were substituted at C-2 of the [→3）-α-L-Rhap-（1→] and C-3 of the [→2）-α-L-Rhap-（1→], which is consistent with the parent polysaccharide MAS. The results indicated that the structure of the low molecular weight polysaccharides after mild acid hydrolysis was not destroyed. Base on the anticoagulant activity of APTT, the sulfated polysaccharides with different molecular weights showed different anticoagulant activities. The results suggest that the molecular size has a profound effect on the anticoagulant activity of the sulfated polysaccharides from Monostroma latissimum, and plays an important role in the anticoagulant action.The oligosaccharide fragments of the sulfated rhamnan MAS from Monostroma latissimum were obtained from partial depolymerization of the polysaccharide by mild acid hydrolysis, and their sequences were investigated with ES-MS. On the basis of negative-ion electrospray tandem mass spectrometry with collision-induced dissociation （ES-CID-MS/MS） and 1D, 2D NMR spectroscopy, the oligosaccharide R2S was characterized to be a monosulfated rhamnobiose and consisted ofα-L-Rhap-（2SO₄）-（1→3）-α-L-Rhap. The fragmentation pattern of the homogeneous disaccharide compositions in the product ion spectra was established, and was then applied to sequence determination of the other oligosaccharides. The results demonstrated that it was possible to derive the sequence of the sulfated rhamno-oligosaccharide directly from the glycosidic cleavage fragmentation in the product ion spectra, and negative-ion ES-CID MS/MS affords an efficient method for the sequence determination of oligosaccharides derived from the sulfated rhamnan. The results indicated that the linkage pattern of the sulfated rhamno-oligosaccharides are all [→3）-α-L-Rhap-(1→]. The structure of the sulfated rhamno-oligosaccharide was the first time to report.更多还原