【作者】 乔德亮；

【导师】 曾晓雄；

【作者基本信息】 南京农业大学 ， 食品科学， 2009， 博士

【摘要】 三角帆蚌(Hyriopsis cumingii Lea)俗称河蚌、珍珠蚌等,为我国特有的淡水贝类,具有多种应用价值。其分布范围广泛、自然资源丰富,如何科学合理地开发利用这一宝贵资源是一个值得重视的研究课题。三角帆蚌自古以来就是药食同源动物之一。目前对三角帆蚌多糖(HCPS)的研究较少,而且不够深入。本论文对HCPS进行了较系统的研究,包括提取条件优化、分离纯化、理化性质、抗氧化活性、免疫调节活性以及结构等方面。主要结果如下：1三角帆蚌多糖提取条件优化运用热水浸提、乙醇沉淀、Sevag法脱蛋白、乙醇再沉淀的方法提取获得了三角帆蚌多糖,提取得率1.30-3.60%之间。选择提取温度、提取时间、水料比及提取次数4个因素分别进行单因素实验,在单因素实验基础上,选择提取温度、提取时间及水料比3个参数进行三因素三水平的Box-Behnken实验(BBD),对此3个参数的实验组合进行优化。得出三角帆蚌多糖的最优提取条件为提取温度80℃、提取时间4.5 h、水料比8ml/g、提取次数2次。选用5次优化的提取条件和5次随机设定的提取条件,对模型方程的适用性进行验证。结果表明实验值与模型方程的预测值间存在很好的相关一致性,说明优化出的提取条件是切实可行的。2三角帆蚌多糖分离纯化运用DEAE-纤维素-52和葡聚糖G-100色谱柱对HCPS粗品进行初步分离和进一步纯化,得到HCPS-1、HCPS-2和HCPS-3三个组分。三个组分HCPS-1、HCPS-2和HCPS-3的得率分别是26.91%、30.18%和3.93%。采用HPLC法对HCPS-1、HCPS-2和HCPS-3的纯度进行进一步鉴定,证明HCPS-1、HCPS-2和HCPS-3三者都是均一的多糖组分。3三角帆蚌多糖基本理化性质运用化学的、物理的方法分别测量分析了三角帆蚌粗多糖及其纯化产品HCPS-1、HCPS-2和HCPS-3的基本理化性质。对于粗HCPS、HCPS-1、HCPS-2和HCPS-3:总糖含量分别是76.43%、98.88%、96.60%和80.06%,蛋白质含量分别是1.36%、未检出、未检出和9.42%,糖醛酸含量分别是13.17%、16.66%、16.37%和17.25%,硫酸基含量分别是1.38%、0.38%、0.60%和6.29%,相对粘度分别是1.18、1.16、1.16和1.31。粗HCPS由6种单糖组成,鼠李糖、阿拉伯糖、岩藻糖、甘露糖、葡萄糖、半乳糖摩尔百分含量分别是5.71%、2.21%、1.69%、3.40%、82.21%和4.78%;HCPS-1由2种单糖组成,阿拉伯糖、葡萄糖摩尔百分含量分别是2.12%和97.88%；HCPS-2仅由1种单糖葡萄糖组成；HCPS-3由5种单糖组成,鼠李糖、岩藻糖、甘露糖、葡萄糖、半乳糖摩尔百分含量分别是13.80%、4.50%、7.70%、64.92%和9.07%。HCPS-1、HCPS-2和HCPS-3的相对分子质量分别是432.2、457.9和503.1 kDa。粗HCPS、HCPS-1、HCPS-2和HCPS-3含有多糖类物质的特征吸收峰、含有羧基的吸收峰、含有吡喃糖环的吸收峰。HCPS-3比粗HCPS、HCPS-1及HCPS-2含有较强的蛋白氨基吸收峰。HCPS-3比粗多糖、HCPS-1及HCPS-2含有较强的硫酸基特征吸收峰。4三角帆蚌多糖抗氧化活性运用化学方法分别测量分析了粗HCPS、HCPS-1、HCPS-2和HCPS-3的体外抗氧化活性。结果表明：粗HCPS、HCPS-1. HCPS-2和HCPS-3在体外能够清除过氧化氢(H2O2)、清除超氧阴离子自由基(O2·)、清除DPPH自由基、螯合亚铁离子(Fe2+)、还原铁离子(Fe3+),具有一定的体外抗氧化作用。HCPS-3比粗HCPS、HCPS-1和HCPS-2具有较高的体外抗氧化活性。运用动物模型实验研究分析了粗HCPS的体内抗氧化活性。结果表明：应用低剂量粗HCPS处理可克服D-半乳糖诱导的氧化损伤；应用高剂量粗HCPS处理可以显著地抑制肝脏和血清中脂质过氧化产物(MDA)的生成,显著地提高肝脏和血清中超氧化物歧化酶(SOD)活性、过氧化氢酶(CAT)活性、谷光甘肽过氧化物酶(GSH-Px)活性以及非酶性抗氧化能力(TAOC),且这种变化与粗HCPS呈现剂量依赖关系。粗HCPS具有一定的体内抗氧化作用。5三角帆蚌多糖免疫调节活性运用细胞模型实验研究比较了粗HCPS、HCPS-1、HCPS-2和HCPS-3的体外免疫调节活性。结果表明：粗HCPS、HCPS-1、HCPS-2和HCPS-3在体外能够显著地促进脾细胞增殖、提高腹腔巨嗜细胞酸性磷酸酶活性、增强腹腔巨嗜细胞吞噬中性红能力,具有一定的体外免疫增强作用。HCPS-3比粗HCPS、HCPS-1和HCPS-2具有较高的体外免疫增强活性。运用动物模型实验研究分析了粗HCPS的体内免疫调节活性。结果表明：应用低剂量粗HCPS处理可以使因环磷酰胺(CPA)诱导产生的免疫功能低下得以恢复；应用高剂量粗HCPS处理可以显著地提高脾脏指数、胸腺指数、迟发型超敏反应(DTH)的耳肿胀度、血清中溶菌酶(LZM)的含量和活性,且这种变化与粗HCPS呈现剂量依赖关系。粗HCPS具有一定的体内免疫增强作用。6三角帆蚌多糖结构傅立叶变换红外光谱(FTIR)、核磁共振(NMR)技术研究表明：HCPS-1. HCPS-2和HCPS-3三个组分的糖环形式均为吡喃糖环；HCPS-1、HCPS-2的糖苷键构型均为α型,HCPS-3的糖苷键构型既有α型又有β型。高碘酸氧化、Smith降解、甲基化反应、气相色谱／质谱联用(GC/MS)以及NMR技术研究表明：HCPS-1、HCPS-2和HCPS-3三个组分中葡萄糖的连接方式都是以1→4糖苷键为主,存在1→4,6和1→3,4分支以及一些末端糖基(1→)；HCPS-1中的阿拉伯糖和HCPS-3中的鼠李糖、岩藻糖、甘露糖、半乳糖绝大部分或全部都是以1→3糖苷键为主,穿插在葡萄糖主链和支链中。刚果红实验研究表明HCPS-1、HCPS-2和HCPS-3三个组分均不具有三股螺旋结构。稀碱水解反应研究表明HCPS-3上肽链与糖链的连接键型为O-型糖肽键。酸水解-柱前衍生化-HPLC法测量分析表明：HCPS-3中共检测出13种氨基酸,它们分别是天冬氨酸、谷氨酸、丝氨酸、甘氨酸、苏氨酸、丙氨酸、缬氨酸、酪氨酸、甲硫氨酸、苯丙氨酸、异亮氨酸、亮氨酸、赖氨酸,其中甘氨酸含量最高,占总氨基酸质量百分比为25.18%,苯丙氨酸含量最低仅为3.13%。更多还原

【Abstract】 Hyriopsis cumingii, commonly called pearl mussel, belongs to freshwater bivalve mussel and has many applying value. Although Hyriopsis cumingii is endemic to China, there are extensively distribution and abundant resources of Hyriopsis cumingi in China. Thus, how to develop and utilize this precious resource scientifically and reasonably become a project deserving to research deeply.Hyriopsis cumingii has been one of the animals used as medicine and food simultaneously since ancient times. However, little information about Hyriopsis cumingii polysaccharides (HCPS) is available at present. In this paper, HCPS was studied systemically, including optimization of extraction parameters, isolation and purification, physicochemical characterization, antioxidation activity, immunomodulation activity and structure of HCPS. Main results are listed as follows:1 Optimization of extraction parameters of HCPSHCPS was extracted by using methods of hot water lixiviation, ethanol precipitation, Sevag’s deproteination and ethanol precipitation again. Extraction yield of HCPS was in the range of 1.30-3.60%.Extraction temperature, extraction time, ratio of water to raw material and extraction times were selected in single-factor experiments. Based on the single-factor experiments, combination of the extraction parameters (time, temperature and ratio of water to raw material) was optimized by using three-factor-three-level Box-Behnken design (BBD). The optimum conditions were extracting temperature 80℃, extracting time 4.5 h, ratio of water to raw material 8 ml/g and extraction times 2.Five optimized extraction conditions and five other extraction conditions were selected to certify applicability of model equation. There was no significant difference between experimental value and estimated value from the model equation. The optimized extraction condition was practicable.2 Isolation and purification of HCPSThe crude HCPS was separated firstly through DEAE-cellulose 52 chromatography column and further purified through chromatography column of Sephadex G-100. Three fractions, named as HCPS-1, HCPS-2 and HCPS-3, were obtained. The recovery rates of HCPS-1, HCPS-2 and HCPS-3 based on the amount of crude HCPS were 26.91%,30.18% and 3.93% respectively.Purity of HCPS-1, HCPS-2 and HCPS-3 was further confirmed by using HPLC, and results showed that HCPS-1, HCPS-2 and HCPS-3 were purified polysaccharides respectively.3 Basic physicochemical characterization of HCPSBasic physicochemical characterization of crude HCPS, HCPS-1, HCPS-2 and HCPS-3 were determined by using the methods of chemistry and physics.As to crude HCPS, HCPS-1, HCPS-2 and HCPS-3, polysaccharides content was 76.43%,98.88%,96.60% and 80.06% respectively. Protein content was 1.36%, not detected, not detected and 9.42% respectively. Uronic acid content was 13.17%,16.66%, 16.37% and 17.25% respectively. Sulfuric radical content was 1.38%,0.38%,0.60% and 6.29% respectively. Relative viscosity was 1.18,1.16,1.16 and 1.31 respectively. The crude HCPS was composed of rhamnose, arabinose, fucose, mannose, glucose and galactose with a molar ratio of 5.71:2.21:1.69:3.40:82.21:4.78. HCPS-1 was composed of only arabinose and glucose with a molar ratio of 2.12:97.88. HCPS-2 was composed only of glucose. HCPS-3 was composed of rhamnose, fucose, mannose, glucose and galactose with a molar ratio of 13.80:4.51:7.70:64.92:9.07. The relative molecular weight of HCPS-1, HCPS-2 and HCPS-3 was 432.2kDa,457.9kDa and 503.1kDa respectively.In FTIR spectrum, characteristic absorptions of polysaccharides, carboxyl group and pyranose ring were observed in crude HCPS, HCPS-1, HCPS-2 and HCPS-3. Absorption peaks of amino group and sulfuric acid radicals in HCPS-3 were stronger than that in crude HCPS, HCPS-1 or HCPS-2.4 Antioxidant activities of HCPSAntioxidant activities in vitro of crude HCPS, HCPS-1, HCPS-2 and HCPS-3 were measured by using the chemical methods. Results showed that crude HCPS, HCPS-1, HCPS-2 and HCPS-3 could scavenge H2O2, scavenge free radicals of O2·and DPPH·, chelate Fe2+ and reduce Fe3+ in vitro. Crude HCPS, HCPS-1, HCPS-2 and HCPS-3 had some antioxidant activities in vitro. HCPS-3 exhibited much higher antioxidant activities than crude HCPS, HCPS-1 and HCPS-2.Antioxidant activities in vivo of crude HCPS were determined by using the animal model experiments. Results indicted that administration of low dose crude HCPS could overcome oxidant injury induced by D-Gal, and treatment of high dose crude HCPS could inhibit significantly the formation of MDA, enhance significantly the activities of antioxidant enzymes (SOD, CAT and GSH-Px) and increase significantly the value of TAOC in livers and serums in a dose-dependent manner. Crude HCPS could exhibit some antioxidant activities in vivo.5 Immunomodulating activities of HCPSImmunomodulating activities in vitro of crude HCPS, HCPS-1, HCPS-2 and HCPS-3 were evaluated by using the cell model experiments. Results showed that crude HCPS, HCPS-1, HCPS-2 and HCPS-3 could promote proliferation of spleen cell, enhance activity of acid phosphatase in peritoneal macrophages and increase ability of devouring neutral red of peritoneal macrophages in vitro. Crude HCPS, HCPS-1, HCPS-2 and HCPS-3 had some immunoenhancing activities in vitro. HCPS-3 exhibited much higher immunoenhancing activities than crude HCPS, HCPS-1 and HCPS-2.Immunomodulating activities in vivo of crude HCPS were measured by using the animal model experiments. Results showed that administration of low dose crude HCPS could recovery weakened immune function induced by CPA, and treatment of high dose crude HCPS could increase significantly the index of spleen and thymus, the swelling rate of ear in DTH and the concentration and activity of LZM in serums in a dose-dependent manner. Crude HCPS could exhibit some immunoenhancing activities in vivo.6 Structure of HCPSSpectra of FTIR and NMR implied that there were pyranose rings in HCPS-1, HCPS-2 and HCPS-3. HCPS-1 and HCPS-2 showed feature of a-glycosidic bond, and HCPS-3 exhibited character of bothα-andβ-glycosidic bond.Results of periodate oxidation, Smith degradation, methylation analysis, GC/MS and NMR suggested that Glc in HCPS-1, HCPS-2 and HCPS-3 were linked to produce Glc backbone chain mainly by 1→4 glycosidic bond. Glc backbone chain were branched by 1→4,6 and 1→3,4 glycosidic bond in HCPS-1, HCPS-2 and HCPS-3. Ara in HCPS-1 and Rha, Fuc, Man and Gal in HCPS-3 were linked to Glc backbone chain or side chain mainly by 1→3 glycosidic bond.Congo red experiments suggested that there were not three-strand spiral structure in HCPS-1, HCPS-2 and HCPS-3. Linkage-bond between sugar chain and peptide chain, detected by using the method of alkaline hydrolysis, was O-glycopeptide linking bond in HCPS-3. Thirteen species of amino acid (Asp, Glu, Ser, Gly, Thr, Ala, Val, Tyr, Met, Phe, Ile, Leu and Lys) were detected by applying the method of acid hydrolysis-derivation-HPLC in HCPS-3. Content percentage of Gly to total amino acid was highest (25.18%), and that of Phe was lowest (3.13%).更多还原