新疆芜菁多糖的分离纯化、结构表征及生物活性研究
发布时间：2018-06-12 09:47
本文选题：新疆芜菁 + 多糖 ； 参考：《南京农业大学》2016年博士论文
【摘要】：芜菁(Brassica rapa L.),属于十字花科(Cruciferae)芸苔属(Brassica)草本植物,十字花科主要由340个属和3700个种组成,其中芸苔属是十字花科最大、分布最广并被广泛食用的一类植物。流行病学研究表明食用芸苔属蔬菜与减少心血管疾病、癌症、老年痴呆症及白内障等慢性病密切相关,新疆芜菁是一种药食兼用的植物,地下根及种子为其药用部分。多糖在生物体生长和发育的过程中起着非常重要的作用,由于具有抗氧化、抗肿瘤、抗菌、免疫调节及抗缺氧等不同的生物活性而引起科研工作者越来越多的关注。因此,本论文以新疆芜菁为原料,对新疆芜菁多糖的分离纯化、结构表征及生物活性进行研究。主要研究结果如下:1.新疆芜菁多糖提取条件优化在单因素实验基础上,选择提取温度、提取时间及液料比3个参数,利用响应面法中的Box-Behnken design实验(BBD)进行优化,得出最佳的新疆芜菁粗多糖提取条件为:提取温度93 ℃,液料比75 mL/g,提取时间4.3 h,提取次数3次,预测得率为21.89% ±0.26。在该最优提取条件下进行验证实验,新疆芜菁多糖得率为21.48 ±0.41%,与预测值基本相符。2.新疆芜菁多糖的分离纯化及结构表征新疆芜菁粗多糖经过DEAE-纤维素-52与Sephadex G-100凝胶色谱分离纯化后,得到三种纯化组分BRP-1-1、BRP-2-1及BRP-2-2。理化分析表明:粗多糖中蛋白质及总酚含量较低,分别为1.01 ±0.03%及0.22±0.01 (mgGAE/100mg),经过分离纯化后,纯化组分中的蛋白质及总酚全部被去除,BRP-2-2中糖醛酸的含量最高,达到15.82 ±1.05%。采用ICP-AES法对新疆芜菁多糖及原料进行无机元素分析表明:新疆芜菁原料与粗多糖中K、Ca、Mg和Na四种元素含量较高;BRP-1-1与BRP-2-1相似,K、Ca、Mg和Zn四种元素的含量较高;而BRP-2-2中K、Ca和Mg三种元素的含量较高。通过HPGPC分析表明:BRP-1-1、BRP-2-1及BRP-2-2三种纯化组分的纯度都很高,分子量分别为1510, 1110及838 kDa;红外光谱分析表明三种纯化组分都是吡喃糖,BRP-1-1中含有β-构型的糖苷键,BRP-2-1及BRP-2-2中同时含有α-构型糖苷键及β-构型糖苷键;单糖组成分析表明:BRP-1-1由甘露糖、鼠李糖、葡萄糖、半乳糖及阿拉伯糖五种单糖组成,其摩尔比为0.58:1.14:1.65:12.39:23.72,BRP-2-1由鼠李糖、半乳糖醛酸、半乳糖及阿拉伯糖四种单糖组成,其摩尔比为4.53:20.71:3.39:4.97,BRP-2-2的单糖组成最简单,由鼠李糖与半乳糖醛酸两种单糖组成,其摩尔比为1.27:54.92;结合红外光谱、单糖组成、f窕癗MR技术对新疆芜菁多糖三种纯化组分进行结构解析,推测BRP-1 -1结构式如下:推测BRP-2-1的结构式如下:推测BRP-2-2的结构式如下:→4)-α-D-GalpA-(1→6)-α-D-GalpAc3Ac-(1→2)-α-D-Rhap-(1→2)-β-D-Rhap-(1→3.新疆芜菁多糖的免疫调节活性研究通过体外细胞模型和体内免疫抑制小鼠模型研究新疆芜菁多糖的免疫调节活性。体外细胞模型结果表明:BRP-2-2能够提高RAW264.7细胞的增殖能力、酸性磷酸酶活性和中性红TQ噬能力,同时还可以促进NO、TNF-α、IL-1β以及IFN-γ的释放量;Cy诱导的免疫抑制小鼠模型结果表明:BRP-2-2能提高小鼠的脾脏指数、胸腺指数、血清中溶菌酶活性及溶血素水平,同时还能够增强T-AOC、CAT、GSH-Px和T-SOD的酶活性,降低MDA的含量,从而恢复由Cy诱导引起的BALB/c小鼠氧化应激损伤。上述实验结果表明新疆芜菁多糖可以促进机体的先天性免疫和适应性免疫,具有一定的免疫调节活性。4.新疆芜菁多糖的肠道微生态调节作用研究通过体外模拟胃肠液消化研究新疆芜菁多糖在胃肠液消化过程中的稳定性。结果表明:随着消化时间的延长,BRP-2-1及BRP-2-2的分子量逐o8减小,而还原糖含量逐渐升高;其次,通过测定体外厌氧发酵过程中新疆芜菁多糖的消化降解、SCFA含量及肠道菌群变化评价其益生活性。结果表明:随着发酵时间的延长,粗多糖、BRP-2-1及BRP-2-2的分子量都会减小,同时还会伴随有小分子物质的出现;三种多糖样品都能增加肠道中的总菌数量、双歧杆菌属(Bifdobacteriumgenus)和乳杆菌属-乳酸球菌属(Lactobacillus-Entgrococcup )细菌的数量;同时还能增加乙酸、丙酸及丁酸的含量,减小乳酸的含量,以上实验结果表明新疆芜菁多糖可以调节肠道菌群组成、改善肠道环境,对宿主健康产生有益的影响,具有潜在的益生活性。5.硫酸酯化新疆芜菁多糖对H2O2诱导损伤PC12细胞的保护作用采用氯磺酸-吡啶法对BRP-2-2进行硫酸酯化修饰,并利用响应面法(RSM)对修饰条件进行优化。得出最优的BRP-2-2硫酸酯化修饰条件为:反应温度61℃,吡啶/氯磺酸体积比为8,反应时间3 h,在该最优条件下S-BRP-2-2的取代度为1.02± 0.08。H2O2是体外主要的活性氧产生来源,经常被用于作为体外细胞氧化损伤模型的诱导剂。研究表明:S-BRP-2-2在低浓度时无细胞毒性,且当浓度为200μg/mL时对H202诱导的PC12细胞氧化损伤模型的保护作用最好,其还可以提高T-AOC、CAT和SOD的酶活力,降低MDA含量及LDH的酶活力,以上实验结果表明:S-BRP-2-2对H202诱导的PC12细胞氧化损伤模型具有较好的保护作用。6.新疆芜菁多糖的流变学研究采用流变仪对新疆芜菁粗多糖的流变学性质进行研究,结果表明新疆芜菁粗多糖的浓度越大,温度越低,其表观粘度就越高,而且随着剪切速率的增大,表现出明显的剪切变稀现象,具有典型的弱凝胶的性质。另一方面,随着CaCl2、蔗糖和NaCl浓度的增加,新疆芜菁粗多糖的表观粘度也增加,同样具有典型的弱凝胶性质;pH值对粗多糖的表观粘度有非常显著的影响,特别是当pH值大于10.5时,新疆芜菁粗多糖的表观粘度显著增加,具有典型的凝胶性质。
[Abstract]:Brassica rapa L. (Cruciferae), belonging to the herbaceous plant of the Brassica (Brassica) of the family Cruciferae (Brassica). The family Cruciferae mainly consists of 340 genera and 3700 species, among which the Brassica is the largest, most widely distributed and widely eaten plant of the Cruciferae. Chronic diseases such as Alzheimer's disease and cataract are closely related, Xinjiang turnip is a kind of medicinal plant, root and seed are its medicinal parts. Polysaccharides play a very important role in the process of growth and development of organisms, which are induced by different biological activities such as antioxidation, anti-tumor, antibiosis, immunoregulation and anti hypoxia. In this paper, Xinjiang turnip was used as the raw material to study the separation, purification, characterization and biological activity of turnip polysaccharide in Xinjiang. The main results are as follows: 1. the extraction conditions of Xinjiang turnip polysaccharides were optimized on the basis of single factor experiment, and the extraction temperature, extraction time and liquid material ratio were selected as 3 parameters. By optimizing the Box-Behnken design experiment (BBD) in response surface method, the optimum extraction conditions of the crude polysaccharide of Xinjiang turnip were obtained: the extraction temperature was 93 C, the liquid ratio was 75 mL/g, the extraction time was 4.3 h, the extraction times were 3 times, the prediction rate was 21.89% + 0.26. under the optimal extraction condition, and the yield of the polysaccharide of the turnip was 21.4. The separation, purification and structural characterization of.2. Xinjiang turnip polysaccharides were basically in accordance with the predicted values. The crude polysaccharides in Xinjiang turnip were purified by DEAE- cellulose -52 and Sephadex G-100 gel chromatography. Three kinds of purified components were obtained, BRP-1-1, BRP-2-1 and BRP-2-2., which showed that the content of protein and total phenol in coarse polysaccharide was 1.01, respectively. 0.03% and 0.22 + 0.01 (mgGAE/100mg), after separation and purification, all the proteins and total phenols in the purified components were removed, and the content of aluronic acid in BRP-2-2 was the highest, reaching 15.82 + 1.05%.. The inorganic elements of Xinjiang turnip polysaccharides and raw materials were analyzed by ICP-AES method: the four elements of K, Ca, Mg and Na in the raw material of turnip in Xinjiang and the crude polysaccharides. The content of the four elements of K, Ca, Mg and Zn is higher, and the content of the three elements of K, Ca and Mg in BRP-2-2 is higher. The purity of the three components of the three kinds of purification components in BRP-2-2 are high, and the molecular weight is 1510, 1110 and 838 respectively. The FTIR analysis shows that the three components are all purified. BRP-1-1 contains glucoside bonds of beta configuration, BRP-2-1 and BRP-2-2 contain both alpha and beta glucoside bonds. Monosaccharide composition analysis shows that BRP-1-1 consists of mannose, rat Li Tang, glucose, galactose and Arabia sugar with five monosaccharides. The molar ratio is 0.58:1.14:1.65:12.39:23.72, BRP-2-1 is rhamnose, and galactose. Four monosaccharides of aluronic acid, galactose and Arabia sugar are composed of four monosaccharides. The composition of monosaccharides in BRP-2-2 is the simplest. The mole of rhamnose and galacturonic acid is composed of two monosaccharides. The molar ratio is 1.27:54.92; the structure solution of three kinds of purified components of turnip polysaccharide in Xinjiang by infrared spectroscopy, monosaccharide composition, and f? MR Technology The structure formula of BRP-1 -1 is as follows: speculating the structural formula of BRP-2-1 as follows: speculating the structure of BRP-2-2 as follows: - 4) - alpha -D-GalpA- (1 to 6) - alpha -D-GalpAc3Ac- (1 to 2) - alpha -D-Rhap- (1 to 2) - beta -D-Rhap- (1 to 3.) the immunomodulatory activity of the polysaccharide of Xinjiang turnip (1 to 3.) The immunoregulatory activity of cyanine polysaccharide in vitro showed that BRP-2-2 could improve the proliferation of RAW264.7 cells, acid phosphatase activity and neutral red TQ phagocytosis, and also promote the release of NO, TNF-, IL-1 beta and IFN- gamma. The result of Cy induced immunosuppressive mice showed that BRP-2-2 could improve the spleen of mice. Index, thymus index, serum lysozyme activity and hemolysin level, and can also enhance the activity of T-AOC, CAT, GSH-Px and T-SOD, reduce the content of MDA, and restore the oxidative stress injury induced by Cy induced BALB/c mice. The results showed that the Xinjiang turnip polysaccharide could promote the innate and adaptive immunity of the body. The study on the intestinal microecological regulation of a certain immunoregulatory activity.4. Xinjiang turnip polysaccharide, the stability of Xinjiang turnip polysaccharide in digestive process was studied by gastrointestinal digestion in vitro. The results showed that the molecular weight of BRP-2-1 and BRP-2-2 decreased with the prolongation of digestion time, while the reducing sugar content increased gradually. Secondly, by determining the digestive degradation, SCFA content and intestinal flora changes in Xinjiang turnip polysaccharide during the process of anaerobic fermentation in vitro, the results showed that the molecular weight of crude polysaccharides, BRP-2-1 and BRP-2-2 decreased with the prolongation of fermentation time, and also accompanied by the presence of small molecular substances; three kinds of Polysaccharides samples were all It can increase the number of total bacteria in the intestinal tract, the number of Bifdobacteriumgenus and Lactococcus lactis (Lactobacillus-Entgrococcup) bacteria, and also increase the content of acetic acid, propionic acid and butyric acid and reduce the content of lactic acid. The experimental results show that the polysaccharide of Xinjiang turnip can regulate the composition of intestinal flora and improve the composition of intestinal flora. The intestinal environment has a beneficial effect on the health of the host, with the potential probiotic.5. sulfated Xinjiang turnip polysaccharides to protect the H2O2 induced PC12 cells by using chlorsulfonic pyridine method to modify the BRP-2-2 by sulfation, and optimize the modification conditions by using the response surface method (RSM). The optimal BRP-2-2 sulfate is obtained. The modification conditions are: the reaction temperature is 61, the volume ratio of pyridine / chloro sulfonic acid is 8 and the reaction time is 3 h. Under this optimal condition, the degree of substitution of S-BRP-2-2 is 1.02 + 0.08.H2O2, which is the main source of active oxygen generation in vitro. It is often used as an inducer of the cell oxidative damage model in vitro. The study shows that S-BRP-2-2 has no cytotoxicity at low concentration. When the concentration is 200 g/mL, the protective effect of H202 induced PC12 cell oxidative damage model is best. It can also increase the enzyme activity of T-AOC, CAT and SOD, reduce the MDA content and LDH enzyme activity. The experimental results show that S-BRP-2-2 has a good protective effect on H202 induced PC12 cell oxidative damage model. The rheology study of polysaccharides uses rheometer to study the rheological properties of the crude polysaccharides in Xinjiang turnip. The results show that the greater the concentration of the crude polysaccharides in the turnip, the lower the temperature, the higher the apparent viscosity, and the obvious shear thinning phenomenon with the increase of the shear rate, and the characteristics of the typical weak gel. On the other hand, the characteristics of the shear thinning are obvious. With the increase of CaCl2, sucrose and NaCl concentration, the apparent viscosity of the crude polysaccharide in Xinjiang turnip also increased, and also had the typical weak gel properties. The pH value had a very significant effect on the apparent viscosity of the crude polysaccharides, especially when the pH value was greater than 10.5, the apparent viscosity of the crude polysaccharide in Xinjiang turnip increased significantly, and had a typical gel property.
【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q946.3
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本文编号：2009254
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