發(fā)布時(shí)間：2018-06-12 09:47

  本文選題：新疆蕪菁 + 多糖��； 參考：《南京農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：蕪菁(Brassica rapa L.),屬于十字花科(Cruciferae)蕓苔屬(Brassica)草本植物,十字花科主要由340個(gè)屬和3700個(gè)種組成,其中蕓苔屬是十字花科最大、分布最廣并被廣泛食用的一類(lèi)植物。流行病學(xué)研究表明食用蕓苔屬蔬菜與減少心血管疾病、癌癥、老年癡呆癥及白內(nèi)障等慢性病密切相關(guān),新疆蕪菁是一種藥食兼用的植物,地下根及種子為其藥用部分。多糖在生物體生長(zhǎng)和發(fā)育的過(guò)程中起著非常重要的作用,由于具有抗氧化、抗腫瘤、抗菌、免疫調(diào)節(jié)及抗缺氧等不同的生物活性而引起科研工作者越來(lái)越多的關(guān)注。因此,本論文以新疆蕪菁為原料,對(duì)新疆蕪菁多糖的分離純化、結(jié)構(gòu)表征及生物活性進(jìn)行研究。主要研究結(jié)果如下:1.新疆蕪菁多糖提取條件優(yōu)化在單因素實(shí)驗(yàn)基礎(chǔ)上,選擇提取溫度、提取時(shí)間及液料比3個(gè)參數(shù),利用響應(yīng)面法中的Box-Behnken design實(shí)驗(yàn)(BBD)進(jìn)行優(yōu)化,得出最佳的新疆蕪菁粗多糖提取條件為:提取溫度93 ℃,液料比75 mL/g,提取時(shí)間4.3 h,提取次數(shù)3次,預(yù)測(cè)得率為21.89% ±0.26。在該最優(yōu)提取條件下進(jìn)行驗(yàn)證實(shí)驗(yàn),新疆蕪菁多糖得率為21.48 ±0.41%,與預(yù)測(cè)值基本相符。2.新疆蕪菁多糖的分離純化及結(jié)構(gòu)表征新疆蕪菁粗多糖經(jīng)過(guò)DEAE-纖維素-52與Sephadex G-100凝膠色譜分離純化后,得到三種純化組分BRP-1-1、BRP-2-1及BRP-2-2。理化分析表明:粗多糖中蛋白質(zhì)及總酚含量較低,分別為1.01 ±0.03%及0.22±0.01 (mgGAE/100mg),經(jīng)過(guò)分離純化后,純化組分中的蛋白質(zhì)及總酚全部被去除,BRP-2-2中糖醛酸的含量最高,達(dá)到15.82 ±1.05%。采用ICP-AES法對(duì)新疆蕪菁多糖及原料進(jìn)行無(wú)機(jī)元素分析表明:新疆蕪菁原料與粗多糖中K、Ca、Mg和Na四種元素含量較高;BRP-1-1與BRP-2-1相似,K、Ca、Mg和Zn四種元素的含量較高;而B(niǎo)RP-2-2中K、Ca和Mg三種元素的含量較高。通過(guò)HPGPC分析表明:BRP-1-1、BRP-2-1及BRP-2-2三種純化組分的純度都很高,分子量分別為1510, 1110及838 kDa;紅外光譜分析表明三種純化組分都是吡喃糖,BRP-1-1中含有β-構(gòu)型的糖苷鍵,BRP-2-1及BRP-2-2中同時(shí)含有α-構(gòu)型糖苷鍵及β-構(gòu)型糖苷鍵;單糖組成分析表明:BRP-1-1由甘露糖、鼠李糖、葡萄糖、半乳糖及阿拉伯糖五種單糖組成,其摩爾比為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種單糖組成,其摩爾比為4.53:20.71:3.39:4.97,BRP-2-2的單糖組成最簡(jiǎn)單,由鼠李糖與半乳糖醛酸兩種單糖組成,其摩爾比為1.27:54.92;結(jié)合紅外光譜、單糖組成、f窕癗MR技術(shù)對(duì)新疆蕪菁多糖三種純化組分進(jìn)行結(jié)構(gòu)解析,推測(cè)BRP-1 -1結(jié)構(gòu)式如下:推測(cè)BRP-2-1的結(jié)構(gòu)式如下:推測(cè)BRP-2-2的結(jié)構(gòu)式如下:→4)-α-D-GalpA-(1→6)-α-D-GalpAc3Ac-(1→2)-α-D-Rhap-(1→2)-β-D-Rhap-(1→3.新疆蕪菁多糖的免疫調(diào)節(jié)活性研究通過(guò)體外細(xì)胞模型和體內(nèi)免疫抑制小鼠模型研究新疆蕪菁多糖的免疫調(diào)節(jié)活性。體外細(xì)胞模型結(jié)果表明:BRP-2-2能夠提高RAW264.7細(xì)胞的增殖能力、酸性磷酸酶活性和中性紅TQ噬能力,同時(shí)還可以促進(jìn)NO、TNF-α、IL-1β以及IFN-γ的釋放量;Cy誘導(dǎo)的免疫抑制小鼠模型結(jié)果表明:BRP-2-2能提高小鼠的脾臟指數(shù)、胸腺指數(shù)、血清中溶菌酶活性及溶血素水平,同時(shí)還能夠增強(qiáng)T-AOC、CAT、GSH-Px和T-SOD的酶活性,降低MDA的含量,從而恢復(fù)由Cy誘導(dǎo)引起的BALB/c小鼠氧化應(yīng)激損傷。上述實(shí)驗(yàn)結(jié)果表明新疆蕪菁多糖可以促進(jìn)機(jī)體的先天性免疫和適應(yīng)性免疫,具有一定的免疫調(diào)節(jié)活性。4.新疆蕪菁多糖的腸道微生態(tài)調(diào)節(jié)作用研究通過(guò)體外模擬胃腸液消化研究新疆蕪菁多糖在胃腸液消化過(guò)程中的穩(wěn)定性。結(jié)果表明:隨著消化時(shí)間的延長(zhǎng),BRP-2-1及BRP-2-2的分子量逐o8減小,而還原糖含量逐漸升高;其次,通過(guò)測(cè)定體外厭氧發(fā)酵過(guò)程中新疆蕪菁多糖的消化降解、SCFA含量及腸道菌群變化評(píng)價(jià)其益生活性。結(jié)果表明:隨著發(fā)酵時(shí)間的延長(zhǎng),粗多糖、BRP-2-1及BRP-2-2的分子量都會(huì)減小,同時(shí)還會(huì)伴隨有小分子物質(zhì)的出現(xiàn);三種多糖樣品都能增加腸道中的總菌數(shù)量、雙歧桿菌屬(Bifdobacteriumgenus)和乳桿菌屬-乳酸球菌屬(Lactobacillus-Entgrococcup )細(xì)菌的數(shù)量;同時(shí)還能增加乙酸、丙酸及丁酸的含量,減小乳酸的含量,以上實(shí)驗(yàn)結(jié)果表明新疆蕪菁多糖可以調(diào)節(jié)腸道菌群組成、改善腸道環(huán)境,對(duì)宿主健康產(chǎn)生有益的影響,具有潛在的益生活性。5.硫酸酯化新疆蕪菁多糖對(duì)H2O2誘導(dǎo)損傷PC12細(xì)胞的保護(hù)作用采用氯磺酸-吡啶法對(duì)BRP-2-2進(jìn)行硫酸酯化修飾,并利用響應(yīng)面法(RSM)對(duì)修飾條件進(jìn)行優(yōu)化。得出最優(yōu)的BRP-2-2硫酸酯化修飾條件為:反應(yīng)溫度61℃,吡啶/氯磺酸體積比為8,反應(yīng)時(shí)間3 h,在該最優(yōu)條件下S-BRP-2-2的取代度為1.02± 0.08。H2O2是體外主要的活性氧產(chǎn)生來(lái)源,經(jīng)常被用于作為體外細(xì)胞氧化損傷模型的誘導(dǎo)劑。研究表明:S-BRP-2-2在低濃度時(shí)無(wú)細(xì)胞毒性,且當(dāng)濃度為200μg/mL時(shí)對(duì)H202誘導(dǎo)的PC12細(xì)胞氧化損傷模型的保護(hù)作用最好,其還可以提高T-AOC、CAT和SOD的酶活力,降低MDA含量及LDH的酶活力,以上實(shí)驗(yàn)結(jié)果表明:S-BRP-2-2對(duì)H202誘導(dǎo)的PC12細(xì)胞氧化損傷模型具有較好的保護(hù)作用。6.新疆蕪菁多糖的流變學(xué)研究采用流變儀對(duì)新疆蕪菁粗多糖的流變學(xué)性質(zhì)進(jìn)行研究,結(jié)果表明新疆蕪菁粗多糖的濃度越大,溫度越低,其表觀粘度就越高,而且隨著剪切速率的增大,表現(xiàn)出明顯的剪切變稀現(xiàn)象,具有典型的弱凝膠的性質(zhì)。另一方面,隨著CaCl2、蔗糖和NaCl濃度的增加,新疆蕪菁粗多糖的表觀粘度也增加,同樣具有典型的弱凝膠性質(zhì);pH值對(duì)粗多糖的表觀粘度有非常顯著的影響,特別是當(dāng)pH值大于10.5時(shí),新疆蕪菁粗多糖的表觀粘度顯著增加,具有典型的凝膠性質(zhì)。
[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.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：Q946.3
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本文編號(hào)：2009254