猪脑中单唾液酸四己糖神经节苷脂提取工艺的研究

发布时间：2018-03-04 06:11

本文选题：单唾液酸神经节苷脂　切入点：提取　出处：《西北大学》2014年硕士论文　论文类型：学位论文

【摘要】：单唾液酸神经节苷脂(Monosialoganglioside)位于生物体的细胞膜上,主要分布于哺乳动物的中枢神经系统。神经节苷脂作为一种特异性受体,与一些活性物质相互作用,可以促进大脑发育,调节神经系统功能,并且有再生神经组织的作用。临床研究表明,单唾液酸四己糖神经节苷酯(Monosialotetrahexosylganglioside, GM1)对神经细胞有定的保护修复作用,可以用于瘫痪、痴呆、生活能力丧失、老年痴呆、心脑血管意外创伤和帕金森等神经性疾病的治疗。目前的GM1提取分离及纯化工艺复杂,所得产品中也通常残留一些高分子物质杂质及其他神经节苷脂杂质。因此,建立一个方便、快捷的提取方法,获得高纯度的GM1,具有重要的社会意义和经济价值。本课题以鲜猪脑为原材料分离纯化得到GM1,提取分离过程包括丙酮粉的制备、神经节苷脂的萃取、弱阴离子交换层析及凝胶排阻层析,并用氨基键合柱和二醇基柱两种高效液相色谱法和染色法对得到的样品与Sigma公司生产的GM1标准品进行了对照。结果显示,本方法得到的样品与标准品的物理化学性质一致,样品纯度高且不含毒害物质。本论文的主要研究内容如下：①提取分离。将鲜猪脑在冷丙酮中匀浆处理,去除组织中包含的水分和极性水溶性小分子物质,得到丙酮不溶物。丙酮不溶物经一定比例的氯仿-甲醇-水抽提除去中性脂类,上层甲醇-水溶液经旋转蒸发得到粗提取的样品水溶液,粗提取样品产率约为3.36%±0.64%。将所得的样品水溶液脱盐后,进行DEAE Sepharose Fast Flow弱阴离子交换层析。平衡系统为pH7.420mmol/L Tris-HCl缓冲液,洗脱系统为0-70%1.0mol/L NaCl+pH7.420mmol/L Tris-HCl, GM1提取率约为(4.59±0.49)×10-2%。所得GM1样品进一步进行Sephacryl S-100HR凝胶排阻层析分离,洗脱系统为pH7.420.0mmol/L PBS缓冲液,得到纯品GM1,总提取率约为5.02mg/500g湿组织。②样品检测。将得到的GM1样品分别在氨基键合高效液相上进行纯度分析,并附以特征显色反应和二醇基柱高效液相检测,以Sigma公司GM1标准品为对照。结果表明,所得的GM1样品与标准品的物理化学性质一致,并具有较高的纯度。
[Abstract]:Monosialoganglioside (Monosialoganglioside) is located on the cell membrane of organisms and is mainly distributed in the central nervous system of mammals. Ganglioside, as a specific receptor, interacts with some active substances to promote brain development. Clinical studies have shown that Monosialotetrahexosylganglioside (GM1) has a definite protective and repair effect on nerve cells, which can be used for paralysis, dementia, and loss of life ability. Treatment of neuropathies such as dementia, cardio-cerebrovascular injury and Parkinson's disease. The current extraction, isolation and purification of GM1 are complicated, and some macromolecule impurities and other ganglioside impurities are usually retained in the obtained products. It is of great social significance and economic value to establish a convenient and fast extraction method to obtain high purity GM1.In this paper, GM1 was isolated from fresh pig brain, and the extraction and separation process included the preparation of acetone powder. Extraction of ganglioside, weak anion exchange chromatography and gel exclusion chromatography, Amino-bonded column and diol column were used to compare the obtained samples with the GM1 standard produced by Sigma. The results showed that the physical and chemical properties of the samples obtained by this method were consistent with those of the standard. The main contents of this thesis are as follows: 1. The fresh pig brain was homogenized in cold acetone to remove the water and polar water-soluble small molecule in the tissue. Acetone insoluble substance was obtained. Acetone insoluble substance was extracted by chloroform-methanol-water extraction to remove neutral lipids, and the upper methanol-water solution was rotated to obtain crude sample aqueous solution. The yield of crude extracted samples was about 3.36% 卤0.64. DEAE Sepharose Fast Flow weak anion exchange chromatography was performed after desalination of the sample solution. The equilibrium system was pH7.420mmol/L Tris-HCl buffer. The elution system was 0-70mol / L NaCl pH7.420mmol/L Tris-HCl, the extraction rate of GM1 was about 4.59 卤0.49 脳 10-2.The GM1 sample was further separated by Sephacryl S-100HR gel exclusion chromatography, and the elution system was pH7.420.0mmol/L PBS buffer. The purified GM1 was obtained and the total extraction rate was about 5.02 mg / 500g. The purity of the obtained GM1 samples was analyzed in amino bonded high performance liquid phase, followed by characteristic color reaction and high performance liquid phase detection on diol column. The results showed that the physical and chemical properties of the GM1 samples were consistent with those of the standard samples, and the purity of the GM1 samples was higher than that of the standard samples.
【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96

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