本文選題：近江牡蠣多糖 + 純化�。� 參考：《廣州中醫(yī)藥大學(xué)》2017年博士論文

【摘要】：目的:近江牡蠣是我國南部沿海常見的養(yǎng)殖貝類。其殼為中藥材"牡蠣"的來源之一;其肉品質(zhì)鮮美、富含多種營養(yǎng)成分,且具有多種生物活性;尤其是近江牡蠣多糖具有抗氧化、生精、免疫調(diào)節(jié)等活性。因此如何合理、科學(xué)地開發(fā)近江牡蠣資源是一個有意義的課題。當(dāng)前關(guān)于近江牡蠣多糖的研究報(bào)道雖然不少,但不夠深入。因此本文擬以中醫(yī)理論為依據(jù),應(yīng)用現(xiàn)代技術(shù)手段,以近江牡蠣肉為原材料,系統(tǒng)研究近江牡蠣多糖分離、純化工藝及其理化性質(zhì),并對其進(jìn)行硒化修飾,得到硒化近江牡蠣多糖。研究近江牡蠣多糖體外抗氧化活性及其作用機(jī)制,進(jìn)而研究其對環(huán)磷酰胺致氧化應(yīng)激小鼠生精能力的影響,探討近江牡蠣多糖改善環(huán)磷酰胺致氧化應(yīng)激小鼠生精活力的作用機(jī)制。為近江牡蠣多糖相關(guān)藥物研究積累資料。為其開發(fā)成藥品及相關(guān)功能保健食品奠定理論基礎(chǔ)。方法:1.近江牡蠣多糖提取、分離及純化采用超聲輔助提取近江牡蠣肉中多糖,將近江牡蠣粗多糖用磁性殼聚糖微球(MCM)吸附去除蛋白質(zhì),再用DEAE-纖維素52及Sephadex G100柱分離純化粗多糖,得到純化多糖組分。2.近江牡蠣多糖理化性質(zhì)及結(jié)構(gòu)鑒定采用苯酚硫酸法測定總糖含量,考馬斯亮藍(lán)法測定蛋白質(zhì)含量,BaCl2-明膠法測定硫酸基含量,烏氏粘度計(jì)法測定多糖相對粘度,乙�；磻�(yīng)-GC法測定近江牡蠣多糖的單糖組成,HPLC測定純度及分子量,UV法測定吸收情況,FT-IR法測定官能團(tuán)及糖環(huán)形式,甲基化、GC-MS測定糖苷鍵連接位點(diǎn)、糖基鏈接次序及分支情況,NMR法測定異頭碳構(gòu)型、羥基取代情況及糖基鏈接次序,采用剛果紅法測定糖鏈?zhǔn)欠窬哂腥陕菪Y(jié)構(gòu)。3.近江牡蠣多糖硒化修飾通過BaCl2-HN03催化,采用Na2Se03硒化修飾近江牡蠣多糖,得到其硒化衍生物。4.ORP抑制H202誘導(dǎo)TM4細(xì)胞氧化應(yīng)激作用機(jī)制研究以小鼠睪丸Sertoli細(xì)胞(TM4細(xì)胞)為實(shí)驗(yàn)對象,采用H202建立TM4細(xì)胞氧化應(yīng)激模型,考察近江牡蠣多糖及其硒化衍生物的抗氧化能力,同時采用實(shí)時熒光定量PCR及Western Blot技術(shù)檢測TM4細(xì)胞中Nrf2基因表達(dá)水平和與其相關(guān)的上游調(diào)控因子及下游目標(biāo)蛋白的影響,來研究近江牡蠣多糖及其硒化衍生物的抗氧化活性作用機(jī)制。5.ORP抑制環(huán)磷酰胺所致小鼠生殖損傷及其作用機(jī)制將Balb/c小鼠腹腔注射環(huán)磷酰胺,建立環(huán)磷酰胺介導(dǎo)的雄性小鼠氧化應(yīng)激模型,考察ORPp及ORPse對氧化應(yīng)激小鼠的睪丸組織恢復(fù)能力、精子存活率、畸形精子率,采用實(shí)時熒光定量PCR及Western Blot技術(shù)檢測小鼠睪丸中Nrf2基因表達(dá)水平和與其相關(guān)的上游調(diào)控因子及下游目標(biāo)蛋白的影響來研究ORPp及ORPse拮抗環(huán)磷酰胺導(dǎo)致生殖損傷的作用機(jī)制。成果:1.近江牡蠣多糖提取、分離及純化(1)通過超聲輔助提取近江牡蠣臟器中多糖,其得率為9.74±0.55%。近江牡蠣粗多糖中蛋白質(zhì)含量8.62±0.23%。(2)將粗多糖進(jìn)一步用MCM吸附除蛋白,所合成的MCM材料粒徑為2-6μm,最佳除蛋白吸附溫度50℃,吸附反應(yīng)時間4h。該方法為物理吸附,吸附過程動力學(xué)和吸附等溫線分別可以用拉格朗日準(zhǔn)一級動力學(xué)方程及Freundlich方程擬合。此外,根據(jù)吸附方程求得△G及△Ea判斷,MCM吸附是自發(fā)、吸熱的物理吸附反應(yīng)。(3)近江牡蠣粗多糖經(jīng)DEAE-纖維素52柱按極性洗脫后再經(jīng)Sephadex G100篩分后,得到一個純化組分ORPp,ORPp得率為32.7%。2.近江牡蠣多糖理化性質(zhì)及結(jié)構(gòu)鑒定ORPP中總糖含量為88.7%,硫酸基含量為1.2%,蛋白質(zhì)含量0.17%,糖醛酸含量為8.59%,相對粘度為1.15。其相對分子質(zhì)量為656 kDa。ORPP由葡萄糖構(gòu)成。ORPP具有 D-Glc-(1→、→3)-D-Glc-(1 →、→4)-D-Glc-(1→及→3,4)-D-Glc-(1→結(jié)構(gòu),其分支率約為12.7。ORPp無三股螺旋結(jié)構(gòu)。3.近江牡蠣多糖硒化修飾通過BaCl2-HN03催化,采用Na2Se03硒化修飾近江牡蠣多糖,得到其硒化衍生物ORPse中Se元素含量為181.9±5.6μg/g。4.近江牡蠣多糖對TM4細(xì)胞中Nrf2/ARE通路的影響ORPp及ORPse均可以提高H202誘導(dǎo)的氧化應(yīng)激TM4細(xì)胞存活率,且細(xì)胞存活率與給藥劑量相關(guān)。表明ORPp及ORPse均可以提高TM4細(xì)胞抗氧化能力。通過實(shí)時熒光定量PCR技術(shù)及Western Blot技術(shù)分析,ORPP及ORPse可能通過激活Nrf2/ARE通路,增加Nrf2與Keap1解偶聯(lián)使其進(jìn)入胞核,激活A(yù)RE元件,促進(jìn)SOD、HO-1、NQO1等抗氧化酶及Ⅱ相解毒酶生成,從而提高TM4細(xì)胞抗氧化能力。5.近江牡蠣多糖對環(huán)磷酰胺介導(dǎo)的雄性小鼠生殖損傷的作用ORPp及ORPse可能作為誘導(dǎo)物,提高小鼠睪丸組織中Nrf2表達(dá)水平,激活Nrf2/ARE信號通路,有助于下游Ⅱ相解毒酶NQO1及抗氧化酶HO-1等目標(biāo)基因表達(dá),提高機(jī)體對外界抵抗能力,避免因環(huán)磷酰胺導(dǎo)致的氧化應(yīng)激損傷,緩解雄性小鼠生殖損傷,提高精子數(shù)量、精子存活率,降低畸形精子率。相同劑量下,ORPSe的生精活性顯著優(yōu)于ORPp。結(jié)論:本文以近江牡蠣多糖為實(shí)驗(yàn)對象,系統(tǒng)研究其分離、純化工藝,通過物理及化學(xué)方法研究其理化性質(zhì),通過建立H2O2誘導(dǎo)TM4氧化應(yīng)激模型研究其體外抗氧化活性及其作用機(jī)制,建立環(huán)磷酰胺誘導(dǎo)小鼠氧化應(yīng)激模型考察近江牡蠣多糖體內(nèi)生精活性及其作用機(jī)制。發(fā)現(xiàn)MCM材料是一種理想的除粗多糖中蛋白質(zhì)的材料,與酶法、凍融法除蛋白各有優(yōu)勢,適合用于需要同時保留活性蛋白及多肽的粗多糖除蛋白;硒化修飾可以將無機(jī)硒嫁接進(jìn)入多糖中形成有機(jī)硒多糖ORPSe,ORPSe是無毒物。ORPp及ORPSe體外抗氧化作用機(jī)制應(yīng)該是通過激活TM4細(xì)胞中Nrf2/ARE信號通路,促使SOD、HO-1、NQO1等抗氧化酶及Ⅱ相解毒酶生成,從而提高TM4細(xì)胞抗氧化能力。此外,ORPp及ORPSe可能通過激活小鼠睪丸Nrf2/ARE信號通路,提高睪丸對外界抵抗能力,避免因環(huán)磷酰胺導(dǎo)致氧化應(yīng)激損傷,緩解雄性小鼠生殖損傷,且ORPSe活性明顯優(yōu)于ORPp。
[Abstract]:Objective: the oyster is a common culturing shellfish in the southern coast of China. Its shell is one of the sources of the Chinese herbal medicine "Oyster". Its meat quality is delicious, rich in many nutrients and has many biological activities. Especially, the polysaccharides of the river oyster are antioxidation, spermatogenesis and immunization. Therefore, how to rationally develop the capital of the near river oyster is a reasonable and scientific way. The source is a meaningful subject. Although there are many reports on the polysaccharides of oysters in the near river, there are many reports, but they are not deep enough. Therefore, this paper, based on the theory of traditional Chinese medicine, uses modern technical means to systematically study the separation, purification and physicochemical properties of Polysaccharides from oysters, and to make selenium modification to them. The antioxidant activity and mechanism of Polysaccharide from oyster oysters were studied in vitro and its mechanism of action in vitro, and the effect of it on the spermatogenesis ability of cyclophosphamide induced oxidative stress in mice was studied, and the mechanism of improving spermatogenesis activity of cyclophosphamide induced oxidative stress in mice was discussed. Methods: 1. the polysaccharide extraction, separation and purification of Polysaccharides from the 1. River oyster were extracted, separated and purified by ultrasonic assisted extraction of Polysaccharides from the meat of the oyster. The crude polysaccharide of the oyster was adsorbed by magnetic chitosan microspheres (MCM), and then the DEAE- cellulose 52 and the Sephadex G100 column were used. The physicochemical properties and structure identification of polysaccharides of.2. near Jiang oyster were purified from the purified polysaccharide. The content of total sugar was determined by phenol sulfuric acid method. The content of protein was determined by Kaumas brilliant blue method, the content of sulphuric acid base was determined by BaCl2- gelatin method. The relative viscosity of polysaccharide was determined by the method of ulcometer viscometer, and the acetylation reaction -GC method was used to determine the polysaccharide of the river oyster. Monosaccharide composition, HPLC determination of purity and molecular weight, UV method for the determination of absorption, FT-IR method to determine the form of functional group and sugar ring, methylation, GC-MS determination of glucoside linkage site, glycosyl linking order and branches, NMR method for the determination of carbohydrate configuration, hydroxyl substitution and glycosyl linking order, using Congo red method to determine whether the sugar chain has three strands. The selenium modification of polysaccharides in the spiral structure of.3. oysters was catalyzed by BaCl2-HN03, and Na2Se03 selenide was used to modify the polysaccharides of the near river oyster. The mechanism of its selenide derivative.4.ORP to inhibit the oxidative stress of H202 induced TM4 cells was studied. The mice testis Sertoli cells (TM4 cells) were used as experimental object, and TM4 cell oxidative stress model was established by H202. The antioxidative ability of polysaccharides and their selenide derivatives was observed, and the expression level of Nrf2 gene in TM4 cells and the influence of upstream regulatory factors and downstream target proteins were detected by real time fluorescence quantitative PCR and Western Blot, to study the antioxidant activity of polysaccharides and their selenide derivatives in the near river oyster .5.ORP inhibited the reproductive damage of mice induced by cyclophosphamide and its mechanism of action, the Balb/c mice were intraperitoneally injected with cyclophosphamide, and a cyclophosphamide induced oxidative stress model in male mice was established. The ability of ORPp and ORPse to restore the testicular tissue of mice with oxidative stress, the survival rate of sperm, the rate of abnormal sperm, and real-time fluorescent quantitative PCR and Wes were used. Tern Blot technique was used to detect the expression level of Nrf2 gene in mouse testis and the influence of its upstream regulator and downstream target protein to study the mechanism of ORPp and ORPse antagonistic cyclophosphamide induced reproductive damage. 1. the extraction, separation and purification of Polysaccharides from the near river oyster (1) were extracted by ultrasonic assisted extraction of the viscera of the near river oyster. The yield of sugar is 9.74 + 0.55%., the protein content of crude polysaccharide in the near river oyster is 8.62 + 0.23%. (2), and the coarse polysaccharide is further adsorbed by MCM. The particle size of the synthesized MCM is 2-6 mu m, the best protein adsorption temperature is 50, and the adsorption time 4h. is physical adsorption. The kinetics of adsorption process and the adsorption isotherm can be used respectively. Garan quasi first order dynamic equation and Freundlich equation fitting. Furthermore, according to the adsorption equation to determine Delta G and delta Ea, MCM adsorption is a spontaneous and endothermic physical adsorption reaction. (3) a purified component ORPp, ORPp yield of 32.7%.2., is obtained after DEAE- cellulose 52 columns are eluted and then screened by Sephadex G100, and the ORPp yield is 32.7%.2.. The physical and chemical properties and structure of the polysaccharides of oysters were 88.7%, the content of sulphuric acid group was 1.2%, the content of the protein was 0.17%, the content of aluronic acid was 8.59%, the relative viscosity was 1.15. and the relative molecular weight was 656 kDa.ORPP, and the.ORPP had D-Glc- (1 -, - 3) -D-Glc- (1 -, 4) -D-Glc- (1 - and 3,4) -D-Glc- (1 >). Structure, its branching rate is about 12.7.ORPp no three strand helix structure.3. near river oyster polysaccharide selenide modification through BaCl2-HN03 catalysis, Na2Se03 selenide modification of near river oyster polysaccharide, the content of Se element in its selenide derivative ORPse is 181.9 + 5.6 mu g/g.4., the effect of ORPp and ORPse on Nrf2/ARE pathway in TM4 cells Increase the survival rate of oxidative stress TM4 cells induced by H202, and the cell survival rate is related to the dose of drug. It shows that both ORPp and ORPse can improve the antioxidant capacity of TM4 cells. ORPP and ORPse may be activated by activating Nrf2/ARE pathway through real-time quantitative quantitative PCR and Western Blot technology. Activating ARE elements and promoting the production of antioxidant enzymes and phase II detoxification enzymes such as SOD, HO-1, NQO1 and so on, thus enhancing the antioxidant capacity of TM4 cells,.5., ORPp and ORPse may be an inducer for the reproductive damage of cyclophosphamide induced male mice, and improve the Nrf2 expression level in the testis tissues of the mice and activate the Nrf2/ARE signaling pathway. It is helpful to the expression of target genes such as NQO1 and HO-1, which can improve the resistance of the body to the outside world, avoid the oxidative stress caused by cyclophosphamide, alleviate the reproductive damage of the male mice, increase the number of sperm, the survival rate of sperm, and reduce the rate of abnormal sperm. The spermatogenic activity of ORPSe is significantly better than that of the ORPp. knot at the same dose. In this paper, the polysaccharides of oysters were studied in this paper. The physicochemical properties of the polysaccharides were systematically studied. The physicochemical properties of the polysaccharides were studied by physical and chemical methods. The antioxidant activity in vitro and the mechanism of action were studied by establishing the H2O2 induced TM4 oxidative stress model. The oxidative stress model induced by cyclophosphamide was established to investigate the polysaccharide body of the near river oyster. It is found that MCM is an ideal material for protein removal from crude polysaccharide, and it has the advantages of enzyme method and freezing thawing method except protein. It is suitable for the removal of crude polysaccharide protein which needs to retain active proteins and peptides at the same time. Inorganic selenium can be grafted into polysaccharide to form organic selenium polysaccharide ORPSe, ORPSe The antioxidation mechanism of.ORPp and ORPSe in vitro is to enhance the antioxidant capacity of TM4 cells by activating the Nrf2/ARE signaling pathway in TM4 cells and promoting the production of SOD, HO-1, NQO1 and other antioxidant enzymes and II phase detoxification enzymes. In addition, ORPp and ORPSe may improve testicular resistance to the outside world by activating the testicular Nrf2/ARE signaling pathway in mice. Ability to avoid oxidative stress damage caused by cyclophosphamide, alleviate reproductive damage in male mice, and ORPSe activity is significantly better than ORPp.

【學(xué)位授予單位】：廣州中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R284;R285

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 柯珂;王俊;王一兵;莊軍蓮;何碧娟;;近江牡蠣中生物鋅的提取制備方法研究[J];食品研究與開發(fā);2015年14期

2 姜輝;鄧春華;商學(xué)軍;王忠;;維生素E在男性不育中臨床應(yīng)用專家共識(2014版)[J];中華男科學(xué)雜志;2015年03期

3 范巧云;李朝品;王克霞;;中華圓田螺多糖對小鼠酒精性肝損傷的保護(hù)作用[J];中國病原生物學(xué)雜志;2014年02期

4 劉小燕;李朝品;王克霞;;中國圓田螺多糖體外抗乙肝病毒的實(shí)驗(yàn)研究[J];中國中藥雜志;2013年06期

5 劉賀;郭曉飛;李君;朱丹實(shí);勵建榮;;大豆種皮多糖在甘露聚糖酶修飾下力學(xué)行為的演變[J];食品工業(yè)科技;2012年04期

6 范巧云;許禮發(fā);李朝品;王克霞;;扇貝多糖體外抗乙型肝炎病毒活性的研究[J];中國人獸共患病學(xué)報(bào);2011年04期

7 張軼;楊大林;韓杰;朱向玲;趙坤;趙萍;;磁性殼聚糖微球吸附馬鈴薯淀粉廢水中蛋白的應(yīng)用研究[J];食品工業(yè)科技;2010年09期

8 許子茂;李江濱;侯敢;;翡翠貽貝多糖對荷瘤小鼠的抗腫瘤和免疫功能調(diào)節(jié)作用[J];中國現(xiàn)代藥物應(yīng)用;2010年09期

9 陳艷輝;李超柱;吳磊;陳艷華;侯華新;張艷軍;黎丹戎;;廣西產(chǎn)牡蠣多糖的制備和抗腫瘤活性初步研究[J];中國現(xiàn)代醫(yī)學(xué)雜志;2010年07期

10 王紅連;張東升;張凌裳;杜郭君;張銀志;趙曉聯(lián);;硒化靈芝多糖制備及免疫學(xué)相關(guān)研究[J];食品科學(xué);2009年17期

相關(guān)博士學(xué)位論文 前4條

1 楊靜峰;基于牡蠣糖原硫酸酯結(jié)構(gòu)的多糖構(gòu)效關(guān)系研究[D];江蘇大學(xué);2013年

2 任香梅;硒對鎘致雄性小鼠生殖毒性的保護(hù)作用及其機(jī)理的研究[D];南京醫(yī)科大學(xué);2012年

3 蔣長興;青蛤多糖分離鑒定、硫酸酯化及其生物活性研究[D];南京農(nóng)業(yè)大學(xué);2011年

4 喬德亮;三角帆蚌多糖提取、純化、生物活性及其結(jié)構(gòu)[D];南京農(nóng)業(yè)大學(xué);2009年

相關(guān)碩士學(xué)位論文 前10條

1 欒曉紅;兩種海蟶多糖的提取、分離和結(jié)構(gòu)分析[D];中國海洋大學(xué);2015年

2 杜挺挺;厚殼貽貝糖胺聚糖結(jié)構(gòu)及抗腫瘤活性測定[D];浙江海洋學(xué)院;2015年

3 楊夢濤;納米硒—牡蠣多糖的制備及其抗氧化活性研究[D];中國海洋大學(xué);2014年

4 李世杰;陽春砂多糖的提取、純化及生物活性研究[D];廣州中醫(yī)藥大學(xué);2014年

5 樂小炎;烏賊墨多糖干預(yù)環(huán)磷酰胺介導(dǎo)睪丸氧化應(yīng)激損傷的Nrf2/ARE調(diào)控機(jī)制研究[D];廣東海洋大學(xué);2014年

6 蘇運(yùn)聰;扇貝多糖提取方式及活性的研究[D];河北科技大學(xué);2014年

7 孫昌華;環(huán)磷酰胺對SD大鼠生殖毒性研究[D];山東農(nóng)業(yè)大學(xué);2013年

8 黃瑩瑩;固定化酶制備牡蠣ACE抑制肽及抗氧化肽[D];哈爾濱工業(yè)大學(xué);2013年

9 邢曉旭;扁玉螺多糖的提取分離及結(jié)構(gòu)分析[D];中國海洋大學(xué);2013年

10 張艷磊;金釵石斛多糖對高糖誘導(dǎo)的大鼠腎系膜細(xì)胞氧化應(yīng)激及Nrf2表達(dá)的影響[D];遵義醫(yī)學(xué)院;2012年

，

本文編號：1819719