【作者】 朱彩平；

【导师】 张声华；

【作者基本信息】 华中农业大学 ， 食品科学， 2006， 博士

【摘要】 枸杞多糖作为天然多糖中的重要一员，在祖国的中医药中具有特殊的重要地位。本文在前人已有研究的基础上，继续开展枸杞多糖的提取、结构及生物活性的研究，并采用激光扫描共聚焦显微镜、流式细胞仪、原位末端标记等生物学前沿技术从细胞凋亡角度探讨了枸杞多糖的抗肿瘤作用机制，同时提出了枸杞多糖新的分离程序及简便准确测定枸杞子水提物中多糖含量的方法。主要研究结果如下： 1 枸杞多糖的特性及结构表征 作者提出了枸杞多糖新的分离程序：枸杞子通过粉碎、提取分离、真空浓缩、脱色、脱蛋白、凝胶渗透色谱、冷冻干燥、纯度监测等程序，获得组分单一的精制枸杞多糖，得率2.21％（W／W）。 经检测，上述分离程序得到的精制枸杞多糖中含中性糖45.09％、蛋白质6.98％、半乳糖醛酸46.76％。气相色谱测得枸杞多糖的中性糖由鼠李糖、阿拉伯糖、木糖、甘露糖、葡萄糖和半乳糖六种单糖组成，其摩尔比例为0.72∶5.82∶0.52∶0.26∶1∶4.48；由红外光谱图可知：枸杞多糖中含有-COOH、-OH和-NH₂或-NH-基团，糖链中含有β-D-吡喃葡萄糖、α-D-吡喃甘露糖、α-D-吡喃半乳糖；β-消去反应证明多糖链和氨基酸间为-O-型连接；刚果红实验表明枸杞多糖不具有三股螺旋结构；碘-碘化钾反应表明枸杞多糖存在较长的侧链和较多的分枝；经扫描电镜观察，枸杞多糖表面呈松散的碎片状聚集态。 2 枸杞子水提物中多糖含量的测定 本文首次提出测定枸杞子水提物中多糖含量的简便方法。以不同溶剂去除枸杞子水提物中的单糖、低聚糖、色素等杂质后，采用苯酚-硫酸法测定多糖含量，测定波长为490nm，多糖换算因子f=3.26；在7～49μg／mL范围内，其浓度与吸光度线性关系良好，R²=0.9984；多糖的平均回收率为99.74％，RSD为1.83％（n=6）；测定枸杞子水提物中多糖的含量为3.86％；该方法简便、准确、重现性好，也可作为其它水提物中多糖含量的测定方法。 3 枸杞多糖与人宫颈癌HeLa细胞凋亡的关系 枸杞多糖具有诱导细胞凋亡的作用。噻唑蓝（MTT）实验证明在添加有枸杞多糖6.25mg／L～100mg／L的培养液中，人宫颈癌HeLa细胞的增殖受到显著抑制；细胞形态学观察（普通光学显微镜、荧光显微镜、激光扫描共聚焦显微镜、透射电镜）发现，枸杞多糖可诱导HeLa细胞凋亡，细胞呈现典型的凋亡细胞形态；原位末端标记法表明枸杞多糖可使细胞凋亡指数显著提高。 流式细胞仪、激光扫描共聚焦显微镜检测发现，枸杞多糖能将人宫颈癌HeLa更多还原

【Abstract】 As a natural polysaccharide, Lycium barbarum polysaccharide （LBP） plays an important role in the Chinese traditional medicine. In this study, the extraction, structure and bioactivities of LBP were studied. And to further study the LBP’s function mechanism, laser scanning confocal microscope, flow cytometer and terminal deoxynucleotidyl transferase mediated dUTP nick end labeling （TUNEL） were performed in this paper. What’s more, a simple and reliable method was put forward to determine the content of polysaccharide in Lycium barbarum Extract. The main results are as follows:1 Characteristic and structure of LBPA new separate procedure was put forward in this study: Fructus lycii was chopped into pieces and extracted by different solvents, then the crude extract was decolored, removed protein and treated by gel chromatography, after have been vacuum concentrated and freeze-dried, a pure product of LBP was gained. The yield was 2.21%.With GC analysis the monosaccharides in LBP were composed of Rhamnose （Rha）, arabinose （Ara）, xylose （Xyl）, mannose （Man）, glucose （Glu）, galactose （Gal） with the molar ratio of 0.72: 5.82: 0.52: 0.26: 1: 4.48. The content of neutral sugar, galacturnic acid and protein in LBP was 45.09%, 46.76% and 6.98% respectively.According to the IR spectra, it contains -COOH group, -OH group, -NH₂ group, -NH-group, β-D-glucose, α-D-mannose and α-D-galactose. The results of β-elimination reaction indicated that the chain of polysaccharides and protein were connected by O-linked covalent bond. The Congo-red reaction and I₂-KI reaction indicated that no triple helix structure was occurred in LBP molecule with some long side chain and high branch chain. The images got by scanning electron microscope showed that the LBP was collected in fragment shape.2 Determination of Polysaccharide in Lycium barbarum ExtractFor the determination of polysaccharide in Lycium barbarum Extract, a simple and exact method was firstly put forward in this study. After the monosaccharide, oligosaccharides and pigment were removed from Lycium barbarum extract, which更多还原