【作者】 王忠民；

【导师】 吴谋成； 尹经章；

【作者基本信息】 华中农业大学 ， 农产品加工及贮藏工程， 2002， 博士

【摘要】 近年来，发现了一些有临床价值的多糖及其衍生物类生化药物。陆续报道了有关活性多糖的提取、分离、鉴定的实验方法和研究成果。大量的药理及临床研究已表明：多糖类化合物是一种对正常细胞无毒副作用，能够激活免疫细胞、提高机体免疫功能的免疫调节剂，并且具有抗肿瘤、抗氧化、降血糖、降血脂等多种生物活性，越来越引起人们的极大重视，对多糖化合物的研究有了长足的进展。因此，寻找高效天然无毒副作用的多糖是保健食品功能因子的研究项目的重要开展领域之一。 有关葡萄多糖的研究尚未见过任何报道。葡萄是新疆维吾尔自治区的特产。本研究以新疆葡萄干为原料，利用现代的分离纯化和分析技术，对葡萄多糖（Vitis Linn Polysaccharide, VLP）的提取分离、纯化和葡萄多糖各级分的组成和结构特点进行了深入系统的研究。在此基础上，进一步对葡萄多糖粗品与各级分提高免疫功能、抗肿瘤、降血糖等活性功能进行了评价。为今后葡萄多糖的更深入研究与开发奠定了基础。 1、通过对葡萄多糖分离纯化的系统研究发现，葡萄干经水提醇沉，氯仿脱脂、Sevag法除蛋白，透析、冷冻干燥后，粗葡萄多糖的得率为8.68％。粗葡萄多糖经Sephadex A—25柱层析得四个级分的多糖，经纸层析、Sephadex G—200、聚丙烯酰胺凝胶电泳等方法鉴定和理化分析表明这四个级分的葡萄多糖均为均一多糖；不含游离糖、蛋白质、淀粉等物质。以粗多糖为起始原料计算，四个级分的纯品多糖得率分别为：VLP—1为25.32％，VLP—2为50.82％，VLP—3为7.55％，VLP—4为10.41％。 根据气相色谱分析表明，四个级分的多糖都含有不同量的阿拉伯糖、半乳糖、葡萄糖、甘露糖，VLP—2、VLP—3还含有鼠李糖和木糖。多糖构型中可能有β—D—吡喃葡萄糖。 VLP-1、VLP-2葡萄多糖富含苯丙氨酸、天冬氨酸、精氨酸、苏氨酸等氨基酸。 VLP-1、VLP-2的¹H-NMR和¹³C-NMR图谱分析证实了气相色谱分析结果，含有不同量的阿拉伯糖、半乳糖、葡萄糖、甘露糖、鼠李糖和木糖，红外光谱图谱的分析结果证实了α、β两种苷型均存在。 2、葡萄多糖的免疫功能研究 免疫是机体产生的一种保护性生理反应，是机体具有的对自我和异己抗原的认识或应答能力。免疫系统的存在及其功能的正常化是机体免疫功能稳定的基本保证。免疫调节功能是植物活性多糖最重要的功能，它与多糖的抗肿瘤、抗氧化、降血糖、降血脂等功能紧密相关。多糖的免疫调节功能实际上就是多糖作为抗原引起机体的免疫应答。实验结果显示: 多糖各剂量组对正常小鼠的淋巴细胞转化有明显的促进作用其中VLP一1在IOOmg/蛇·d剂量下作用最好，淋巴细胞转化率高达69.5013%。 各VLP一1剂量对正常小鼠免疫器官的增重均有明显作用，其中VLP一1在50mg/kg.d、100 mg/kg·d剂量下对胸腺、脾脏有极显著地增重作用，在25mg/kg .d剂量下极显著地增加胸腺重量，而对脾脏增重无影响;粗多糖仅在100mg/kg·d剂量下对脾脏增重有明显作用外，其余各剂量对正常小鼠胸腺、脾脏增重无影响。 葡萄多糖对DNCB诱导的迟发型超敏反应（DTH）有显著的促进作用，其中粗多糖在50mg/kg·d剂量下效果最明显，其次为剂量25mg/kg·d;各剂量对免疫器官均有显著的增重作用。葡萄多糖可以显著促进巨噬细胞的吞噬作用。由此可见，葡萄多糖能全面增强机体的细胞免疫和体液免疫功能、增加免疫器官重量。 3、葡萄多糖抗肿瘤研究 肿瘤研究的根本目的是降低死亡率及发病率，降低死亡率主要靠治疗，降低发病率主要靠预防。肿瘤的发生和机体的免疫状态密切相关，免疫功能缺陷或低下易诱发肿瘤。反之，通过刺激机体的免疫系统（如淋巴系统、免疫器官等），使机体的免疫功能得到恢复和提高，由机体本身的抵抗力间接去杀死肿瘤细胞。 对葡萄多糖抗肿瘤功能的实验研究中发现: 葡萄多糖个剂量组对荷瘤小鼠体重变化无明显影响。葡萄多糖各剂量组与对照组比较表明对5180实体瘤有一定的抑制作用，其中从瘤重、抑瘤率、瘤体比等几项指标综合反映，vLP一1（100mg/kg·d）、粗多糖（50mg/kg·d、100 mg/kg·d）、VLP一2（25 mg/kg·d、50 mg/kg·d）有明显的抑瘤效果，其中粗多糖抑瘤效果表现最佳。葡萄多糖对免疫脏器器官也有明显的影响，其中粗多糖（50mg/kg·d）、VLP一2（25 mg/kg·d、50 mg/kg·d）效果最明显，与抑瘤效果相一致的。 4、葡萄多糖降血糖作用的研究 糖尿病是一种常见的内分泌代谢病，胰岛素相对或绝对不足是引起糖、脂肪和蛋白质代谢紊乱的主要原因，其主要特征是高血搪和尿糖，可诱发冠心病和动脉粥样硬化，严重地危害人们的生命健康，开展寻找糖尿病治疗药物的研究是防治糖尿病及其血管并发症的重要研究内容之一。在葡萄多糖降血糖作用实验中发现:葡萄多糖及消渴丸对正常小鼠血糖有所降低，与对照组相比无显著差异，说明葡萄多糖的降血糖作用可能不是通过直接刺激胰岛p细胞分泌实现的，但能显著增加小鼠免疫器官重量;葡萄多糖对四氧峪陡糖尿病小鼠有明显的降血糖作用，各剂量均达到更多还原

【Abstract】 In recent years, some clinic-valuable polysaccharide and its derivative biochemical medicines have been found. It has already successively reported the methods about the extractionN isolation identification and the achievement.The study use Xinjiang currant as rough material, using polysaccharide abstraction depuration technique, instrumental analysis technique, race hygiene conservancy food function evaluate method, modern medicine analysis technique and so on, having done some deep systemic research on the extraction, isolation, identification, purification of VLP and the components and constructions features, then estimating the immunity function , repel tumor and decrease blood sugar research of VLP rough production, which give it the foundation for the further research. The researches about grape polysaccharides have not been reported.1 Through the research of VLP abstraction and purification, we found that VLP can be got by water extracting and alcohol precipitating, chloroform degreasing, eliminating albumen with the method of Sevag, dialysis and freeze drying from raisin, the rate is 8.68%. we got four fractions through the VLP Sephadex A-25 chromatography, it shows that the VLP of the four fractions are homogeneous after paper chromatography identification. It also shows that the four fractions are all homogeneous polysaccharides through Sephadex G-200 arbitration, acrylamide gel electrophoresis arbitration. Calculated with the rough polysaccharide as initiative material, the polysaccharide rates of the four fractions are as follows: VLP -1 is 25.32%,VLP-2 is 50.82%,VLP-3 is 7.55%,VLP-4 is 10.41%.According to Gas Chromatography infra-red spectra analysis, the four fractions all contain different quantities of arabinose , galactose, glucose and seminose, VLP-2 and VLP-3 also contain rhamnose and xylose, polysaccharide configuration may have B -D-pyran glucose.VLP-1 , VLP-2 contain abundant amino acid, such as Phe, Asp Arg and Thr. The analysis of 1H-NMR and 13C-NMR also infect the same results above.2 The study of the immunity function of VLPImmunity is a protective physiological reaction to the body, which has the knowing or respond ability to the self and others antigen. The existence of the immunity system and its functional normalization are the elemental guaranty of the body immunity function. Immunize adjusting function is the most important function of activity polysaccharide, which has a tight relationship with the function of repel tumor, oxidation prevention,decreasing blood sugar and adepts of polysaccharide. The immunization adjusting function is actually immunization respond caused by polysaccharide as antigen. The result of the experiment shows , polysaccharide dosage groups have apparent promoter actions to the transition of normal rats’ achroacyte. Among them VLP- 1 100 mg / kg d acts best, the transition rate of achroacyte reach 65.5013%, each VLP -1 dosage has apparent effects to the live weight gain of normal rats’ immunization organic, VLP-1 50 mg / kg d and 100 mg / kg d have sorely significantly live weight gain function to thymus and milt, when VLP-1 is 25 mg / kg d, the thymus weight increase sorely significantly while milt increase not too much. Except that 100 mg / kg d rough polysaccharide has apparently effects to the increase of milt, other dosages has no effects to normal rats’ thymus and milt, VLP has apparent facilitation to DTH. When rough polysaccharide is 50 mg / kg d its function is the most obvious, next is 25 mg / kg d; all dosages for immunity organs have substantial weights increase. VLP can prompt apparently the phagocytosis function of macrophage cells.From above we can see that VLP is able to increase all aspects of immunity function of cells and humors, increase the weights of immunity organs.3 The research of VLP repelling tumors.The fundamental object of tumor research is reducing the lethality and morbidity, reducing the lethality mainly depends on treatment, reducing morbidity hinges on prophylaxis. Tumors更多还原