【作者】 王建华；

【导师】 张声华；

【作者基本信息】 华中农业大学 ， 农产品加工及贮藏工程， 2001， 博士

【摘要】 目的：提取、分离、纯化枸杞多糖（Lycium barbarrum polysaccharide,LBP），研究枸杞多糖亚级分的组成和结构特点及枸杞总多糖、枸杞多糖级分和亚级分的抗氧化、抗衰老活性，评价枸杞多糖口含片的延缓衰老和抗疲劳保健功能。 1 枸杞多糖的纯化和结构特点经纯化得到如下五个新的枸杞多糖缀合物： 枸杞经去脂溶物、水提取、Sevag法去蛋白质等步骤得枸杞总多糖（LBPt）：用DEAE-Cellulose柱层析并NaCl梯度洗脱、透析、冻干等步骤得4个枸杞多糖级分LBP1～LBP4；Sephadex G-200柱层析法分别分离4个级分可得到相应的枸杞多糖亚级分，研究了其中的LBP1a、LBP2a、LBP3a、LBP4a和LBP4b。用凝胶柱层析法和SDS-凝胶电泳法研究表明所得各亚级分为分子量均一体，它们的冻干品为白色或略显黄色的絮状固体。经检索5个亚级分为新的枸杞多糖缀合物。 用SDS-凝胶电泳法测得LBP1a、LBP2a、LBP3a、LBP4a和LBP4b分子量分别为32399、77482、38385、34841、13224，主要含糖、半乳糖醛酸和少量氨基酸，属于酸性糖蛋白类化合物。它们的中性糖含量分别为（％）：89.6、69.3、56.2、38.6和41.3，除LBP2a不含山梨糖外，它们都含有不同量的鼠李糖、山梨糖、木糖、阿拉伯糖、甘露糖、葡萄糖和半乳糖，LBP4b还含少量艾杜糖，据红外光谱分析中性糖构型可能有β-D-吡喃葡萄糖、α-D-吡喃甘露糖和α-D-毗喃半乳糖等；半乳糖醛酸分别占（％）6.4、23.8、35.4、55.0和54.1；蛋白质含量分别为（％）3.1、5.3、4.8、3.4和2.8，LBP4a和LBP4b分别有19和18种氨基酸组成。根据β-消除反应前后紫外吸收变化可推测它们的糖链和氨基酸问有-O-连接，根据氨基酸组成变化，LBP4a和LBP4b的糖链与氨基酸连接主要是-O-Ser。根据¹HNMR和13CNMR推测LBP4a中有α、β两种异头碳的糖基和1，4连接的α-D-半乳糖醛酸。 2 枸杞多糖的抗氧化作用 主要研究了枸杞总多糖、4个级分和1个亚级分LBP4a的抗氧化作用，包括对自由基的作用，体外和体内抗氧化效果。 2．1 枸杞多糖对自由基的作用 枸杞多糖具有清除羟自由基（·OH）的作用。在同浓度（200μg／mL）下不同枸杞多糖清除·OH活性大小为：LBPt>LBP3>LBP4>LBP2>LBP1。LBP1、LBP2、LBP3、LBP4和LBPt的·OH半清除率浓度(SO₅₀)分别约为：615μg／mL、513μg／mL、186μg／mL、408μg／mL和18μg／mL。亚级分LBP4a也可清除·OH，其半清除率浓度SC₅₀≈449μg／mL。 枸杞多糖对超氧阴离子自由基（·O₂^-）生成的影响因其浓度高低而起促进或抑制作用。在较低浓度下，LBP可促进·O₂^-的产生，并随LBP浓度增加，·O₂^-生成量增加，当高于某浓度时，随LBP浓度增加，促进作用呈下降趋势，以至达更高浓度时，LBP可抑制·O₂^-的产生，并随LBP浓度增加，抑制率升高。就其抑制效果来看，LBPt最好，LBP1最低，其它三种较为接近。 2.2构祀多糖体外抗氧化效果用小鼠肝、脑、心、肾等作材料研究表明，构祀多糖可抑制小鼠组织自发条件下和·OH诱导条件下的脂质过氧化作用，以肝为例，在自发条件下LBPI、LBPZ、LBP3、LBP4、LBPt和IJBP4a对肝组织丙二醛生成的半抑制率浓度分别约为（pg/mL）:2760、957、107一、2736、649和3397，在·OH诱导条件下则约为（pg/mL）:2445、1409、2485、 2447、1342、2054。 研究还表明，构祀总多糖和各级分可以抑制·OH诱导的小鼠肝线粒体肿胀，减少过氧化氢诱导的小鼠红细胞氧化溶血，抑制·OH诱导的小鼠肝线粒体膜流动性下降。所以，构祀多糖具有抑制脂质过氧化反应和保护细胞膜的作用。 2.3构祀多糖体内抗氧化效果用10 mg/kg·d和20mg/kg·d剂量的LBPt或LBP3分别灌喂小鼠31d，以灌喂水小鼠为对照，结果表明血清、肝、肾、脑、心等中的超氧化物歧化酶（S OD）活性高于对照，血清和肝组织中的谷肤甘肤过氧化物酶（GSH一Px）活性都有一定程度的提高，而血清、肝、’肾、脑、心等中的丙二醛含量以及心、脑组织中脂褐素含量都低于对照。说明，构祀多糖可提高小鼠体内的抗氧化能力，降低小鼠体内脂质过氧化水平。 3拘祀多糖及拘祀多糖口含片的延缓衰老作用以雄、雌果蝇分设4个剂量构祀多糖口含片组和1个对照组，高剂量组的雄、雌性果蝇平均寿命都显著高于对照，中、次高、高剂量组的雄性最高寿也显著高于对照，低组无显著影响。可见构祀多糖在一定剂量下可延长果蝇生存时间，而且对雄性作用效果好于雌性。 设构祀多糖口含片老龄小鼠（12月龄）三个剂量组，灌蒸馏水的老龄对照组和青年对照组。各口含片剂量组小鼠红细胞SOD活力均明显高于老龄对照组:各口含片组血清中MDA含量低于老年对照组，其中高剂量组显著降低，而且与青年对照组MDA含量相当。 造同剂量D一半乳糖衰老模型小鼠三组，其中两组分别灌喂10或20mg/kg·d剂量的LBPt，一组罐水作衰老对照组，另设正常小鼠对照。结果表明LBPt可抑制D一半乳糖所致的小鼠体内SOD和GSH一PX活性下降，以及减少D一半乳糖所致的体内丙二醛和脂褐素生成。 构祀多糖和构祀多糖口含片具有延缓衰老作用。 4拘祀多糖口含片的抗疲劳作用结果更多还原

【Abstract】 Fructus lycii, the fruit of Lycium barbarian L., is a well-known traditional Chinese medicine and food. The polysaccharides from it, called Lycium barbarum polysaccharides (LBP), play multiple roles in pharmacological and health care functions. In order to understand more about their structures and functions, the studies were conducted on the extraction, purification, and structural elucidation of them, and on the valuation of their functions with total LBP (LBPt), LBP fractions, LBP sub-fractions, and LBP oral tablets. The main results are as follows: 1 Extraction, purification, and structural characteristics of LBP sub-fractions.LBPt were obtained after a series of steps, such as eliminating lipids and pigments with petroleum ether, isolating monosaccharides and oligosaccharides with 80% ethyl alcohol, extracting LBP with hot water, and removing free proteins with Sevag’s method, and so on. Four LBP fractions, LBP1, LBP2, LBP3 and LBP4, were got by DEAE-Cellulose column with eluent of water, 0.05 mol/L, 0.1 mol/L, and 0.25 mol/L NaCl respectively. Five sub-fractions, including LBPla, LBP2a, LBPSa, LBP4a and LBP4b, were got from the corresponding LBP fractions by Sephadex G-200 column with water as eluent. All the sub-fractions are molecular weight (MW) homogeneous illustrated by the ways of SDS-gel electrophoresis, longer Sephadex G-200 column chromatography, and high performance gel permeation chromatography. They are all white cotton-like solids.MWs of LBPla, LBP2a, LBPSa, LBP4a and LBP4b are 32399, 77482, 38385, 3484 K 13224 respectively by SDS-gel electrophoresis. They are the acid giyco-protems composed of sugar, galacturnic acid, and amino acid. Their neutral monosaccharides contents are 89.6% x 69.3% v 56.2% , 38.6% and 4h3%. The monosaccharides identified by GC-MS are arabinose (Ara), galactose (Gal), glucose (Glu), mannose (Man), rhamnose (Rha), xylose (Xyt) and sorbitose (Sor), but LBP2a has no Sor, and LBP4b has a few amount of iditose (Idi). Some monosaccharides may be -D- glucose, a -D-mannose, and -D- gatactose according to their BR. spectra. The contents of galacturnic acid in them are 3.1%, 5.3%, 4.8%, 3.4% and 2.8%. The protein contents are 3.1%, 5.3%, 4.8%, 3.4% and 2.8%. There are 19 and 18 kinds of amino acid in LBP4a and LBP4b respectively. It suggested that there are -O-glycosidic linkages between sugars and amino acids in them according to the changes of UV absorption at 240 nm after -elimination reactions treated by 0.2 mol/L NaOH, and there are -O-Ser linkages in LBP4a and LBP4b by the amino acid content changes after the treatments of 0.2 mol/L NaOH-1 mol/L NaBH4. According to the spectra of ’HNMR and 13CNMR, LBP4a consists of -4 (- -D-GalpA (l- . The sub-fractions are new glycan-conjunctions. 2 Antioxidant effects of LBP 2. 1 Effects of LBP on free radicalsLBP has the scavenging effect on hydroxyl radical ( OH). The clearing activity from low to high are as LBPt > LBP3 > LBP4 > LBP2 > LBP I at the concentration of 200 g/mL of samples. The concentration of half OH scavenging rates (SC50) of LBP 1, LBP2, LBP3, LBP4 and LBPt are about 615 g/mL, 513 u g/mL, 186 u g/mL, 408 g/mL 18 P g/mL, respectively. SC50 of LBP4a is about 449 y g/mL.They show promoting or inhibiting effects on superoxide anion ( O2") generation in relation to their concentrations. At lower concentrations, O2~ production increased with LBP increasing to high concentrations, and then, O2~ production decreased. At higher concentrations, the O2" generations were inhibited. LBPt has the highest inhibiting activity, LBP1 is the lowest, and the others show little difference. 2.2 Antioxidant effects of LBP in vitroLBP1, LBP2, LBP3, LBP4, LBPt and LBP4a had the inhibiting effects on lipid peroxidation spontaneously or induced by OH with mice liver, brain, heart and kidney as the experimental materials in vitro. Their concentrations of half inhibition rates of malondialdehyde (MDA) in liver homogentes are 2760, 957, 1071, 2736, 649 and 3397 u g/mL at spontaneous condition, and 2445, 1409, 248更多还原