【作者】 蔡为荣；

【导师】 汤坚；

【作者基本信息】 江南大学 ， 食品科学， 2008， 博士

【摘要】 米邦塔仙人掌(Opuntia Milpa Alta)为仙人掌科(Cactaceae)仙人掌属(Opuntiaficus-indica)。以该仙人掌(上海奇丰农艺园产)为原料,对其多糖的提取、分离纯化,活性多糖的结构表征和抗凝血活性进行了研究,主要研究结果如下:分析了原料米邦塔仙人掌茎的化学成分:水分95.7%,粗蛋白0.73%,粗脂肪0.17%,粗纤维0.77%,还原糖0.11%,多糖0.86%,灰分0.88%。比较了胶体磨均质水提仙人掌粗多糖与传统水提仙人掌粗多糖的属性,结果表明高速短时胶体磨均质可使仙人掌浆液粘度下降34%,而在粒径及分布、多糖红外光谱特征与传统水提粗多糖无统计意义上差异。在此基础上,以粗多糖得率为响应,Box-Benhnken中心组合试验及响应面分析,确定了粗多糖提取的最佳工艺参数:提取温度86.1℃、提取时间3.61h、水料比3.72:1。粗多糖(CP)得率为0.694%。紫外、红外与HPGPC法显示粗多糖经DEAE-Sepharose CL-6B及Sepharose CL-6B层析柱纯化得到三个均一多糖组分WSP1、WSP2a和WSP3,三者分子量分别为2.32×10~6、1.26×10~6和7.92×10~6Da。WSP2a为CP主要组分(45.21%),系由阿拉伯糖、半乳糖、木糖、鼠李糖、和少量甘露糖、葡萄糖构成。经过单糖组成和FT-IR分析推测WSP2a是一种鼠李半乳聚糖,并采用FT-IR法测得WSP2a的酯化度为41.9%。采用多糖部分酸水解和甲基化分析的化学方法,结合GC-MS、NMR和2D NMR技术对仙人掌的多糖WSP2a及其部分酸水解多糖进行结构分析,结果表明,WSP2a主链由→4)-α-D-GalpA(1→2,4)-β-L-Rhap(1→-构成。支链一为含有末端半乳糖α-D-Galp(1→,通过(1→4)键连结在→2,4)-α-L-Rhap(1→的C-4位上;支链二为由两个相连接的半乳糖→6)-β-D-Galp(1,3→6)-β-D-Galp(1,3→为核心的支链构成,末端阿拉伯糖α-L-Arap(1→,通过(1→3)键连在半乳糖C-3位上,β-D-Galp(1→通过(1→4)键连接于主链上→2,4)-β-L-Rhap(1→的C-4位;末端木糖α-L-Xylf(1→,通过(1→3)键连接于次主链上。首次采用原子力显微镜对CP、WSP2a高级结构进行了观察,发现CP缠绕成股,股径约150～200 nm,长约0.7-1.5μm;WSP2a直径10-40 nm,长约0.05-0.7μm不等,高度约为15-20 nm。选用活化部分凝血活酶时间(APTT)作CP、WSP1、WSP2a和WSP3的抗凝血活性体外初筛指标,PT和TT监测指标进一步显示WSP2a具有良好的抗凝血活性。WSP2a体内检测高剂量时能够显著延长APTT和TT,表明其作用途径为内源性和共同凝血途径。通过阳性对照,初步探讨了WSP2的抗凝机制为,WSP2与纤维蛋白原结合,以及与ATⅢ结合形成复合物,激活抗凝血酶ATⅢ,灭活凝血酶,阻止凝血酶和纤维蛋白原的结合,延缓纤维蛋白的生成速率。更多还原

【Abstract】 Opuntia Milpa Alta is the edibable Opuntia ficus indica variety belonging to the Cactaceae family.The objectives of the current project were to extract,isolate and purify the polysaccharides with anticoagulant activity from O.ficus indica,and to study the structure and functional properties of the bioactive polysaccharide.Chemical composition analysis revealed that Opuntia Milpa Alta contained 0.73%rude protein,0.17%crude fat,0.11%reducing sugar,0.86%polysaccharide,0.88%ash,95.7% moisture,0.77%crude fiber.Size distribution and features in FT-IR were compared between polysaccharides by aqueous extraction and polysaccharides by homogenize-assisted aqueous extraction,which indicated no significant.The intrinsic viscosity of polysaccharides by homogenize-assisted aqueous extraction reduced 34%.Hence,homogenize-assisted aqueous extraction was used to obtain the crude polysaccharides and the extraction procedure was optimized using Box-Behnken design and Surface Response Methodology(RSM).The obtained optimal parameters of the extraction procedure of CP were:temperature 86.1℃, time 3.61 h,and water to solid ratio 3.72,CP yield 0.694%.WSP1,WSP2a & WSP3 fraction were obtained from the crude polysaccharides(CP) following DEAE-Sepharose CL-6B and Sepharose CL-6B.The three fractions were identified by gel filtration chromatography on Sepharose CL-6B column and HPSEC as homogeneous in molecular weight,their molecular weights were 2.32x106Da,1.26x106 Da,and 7.92x106 Da,respectively.WSP2a consisted mainly of arabinose,galactose,xylose,rhamnose,along with low amount of mannose and glucose,was regard as a rhalnnogalacturonan confirmed by proportion of monosaccharide and FT-IR spectra.The degree of esterification(DE) value of WSP2a was 41.9%based on a FT-IR method.Structure characterization of WSP2a was carried out using partial acid hydrolysis and methylation analysis,combined with GC-MS and 1D & 2D NMR spectroscopy techniques. The results indicated that the configuration of sugar constituents were mainlyα-D-GalpA,β-L-Rhap,α-D-Galp,α-L-Arap andα-L-Xylf.The backbone of WSP2a-28H was found to consisting of a→4)-β-D-GalpA(1→2,4)-α-L-Rhap(1→.The galactose side-chain are 1,4-1inked and are attached mainly to C-4 of the rhamnose units in the backbone.The other side-chain consisting of two 1,6-β-linked galactosyl residues,attached to the backbone by the linkages of 1,4-D-Galp via the O-4 position of the rhamnose residues.T-L-Arafor T-L-Xylf were situated as the nonreducing terminals,which were linked to the O-3 position of the 1,6-D-Galp units.Through AFM observations of CP and WSP2a,it was found that CP and WSP2a have strand-like structure,with diameter about 150~-200 nm,10-40 nm,respectively,average length of CP and WSP2a was respectively about 0.7-1.5 gm,0.05-0.7 gm.Actived partial thromboplastin time(APTT),Prothrombin time(PT) amd Thrombin time (TT) were chosen as screening targets for Anticoagulation activity of CP,WSP1,WSP2a,and WSP3 in vitro,and APTT of WSP2a was prolonged remarkably comparing with normal saline. APTT and TT of WSP2 were prolonged while PT did not prolonged in vivo.The results showed that WSP2 with anticoagulant activity by way of intrinsic pathway and common pathway.Compared with positive control,WSP2 in combination with fibrinogen and ATⅢ, but did not like heparin and prompted AT thrombinⅢrapidly,resulted in fibrinogen inverting Thrombin slowly.更多还原