近江牡蛎多糖的纯化、结构鉴定、硒化及其生物活性研究

发布时间：2018-04-29 11:25

本文选题：近江牡蛎多糖 + 纯化　；参考：《广州中医药大学》2017年博士论文

【摘要】：目的:近江牡蛎是我国南部沿海常见的养殖贝类。其壳为中药材"牡蛎"的来源之一;其肉品质鲜美、富含多种营养成分,且具有多种生物活性;尤其是近江牡蛎多糖具有抗氧化、生精、免疫调节等活性。因此如何合理、科学地开发近江牡蛎资源是一个有意义的课题。当前关于近江牡蛎多糖的研究报道虽然不少,但不够深入。因此本文拟以中医理论为依据,应用现代技术手段,以近江牡蛎肉为原材料,系统研究近江牡蛎多糖分离、纯化工艺及其理化性质,并对其进行硒化修饰,得到硒化近江牡蛎多糖。研究近江牡蛎多糖体外抗氧化活性及其作用机制,进而研究其对环磷酰胺致氧化应激小鼠生精能力的影响,探讨近江牡蛎多糖改善环磷酰胺致氧化应激小鼠生精活力的作用机制。为近江牡蛎多糖相关药物研究积累资料。为其开发成药品及相关功能保健食品奠定理论基础。方法:1.近江牡蛎多糖提取、分离及纯化采用超声辅助提取近江牡蛎肉中多糖,将近江牡蛎粗多糖用磁性壳聚糖微球(MCM)吸附去除蛋白质,再用DEAE-纤维素52及Sephadex G100柱分离纯化粗多糖,得到纯化多糖组分。2.近江牡蛎多糖理化性质及结构鉴定采用苯酚硫酸法测定总糖含量,考马斯亮蓝法测定蛋白质含量,BaCl2-明胶法测定硫酸基含量,乌氏粘度计法测定多糖相对粘度,乙酰化反应-GC法测定近江牡蛎多糖的单糖组成,HPLC测定纯度及分子量,UV法测定吸收情况,FT-IR法测定官能团及糖环形式,甲基化、GC-MS测定糖苷键连接位点、糖基链接次序及分支情况,NMR法测定异头碳构型、羟基取代情况及糖基链接次序,采用刚果红法测定糖链是否具有三股螺旋结构。3.近江牡蛎多糖硒化修饰通过BaCl2-HN03催化,采用Na2Se03硒化修饰近江牡蛎多糖,得到其硒化衍生物。4.ORP抑制H202诱导TM4细胞氧化应激作用机制研究以小鼠睾丸Sertoli细胞(TM4细胞)为实验对象,采用H202建立TM4细胞氧化应激模型,考察近江牡蛎多糖及其硒化衍生物的抗氧化能力,同时采用实时荧光定量PCR及Western Blot技术检测TM4细胞中Nrf2基因表达水平和与其相关的上游调控因子及下游目标蛋白的影响,来研究近江牡蛎多糖及其硒化衍生物的抗氧化活性作用机制。5.ORP抑制环磷酰胺所致小鼠生殖损伤及其作用机制将Balb/c小鼠腹腔注射环磷酰胺,建立环磷酰胺介导的雄性小鼠氧化应激模型,考察ORPp及ORPse对氧化应激小鼠的睾丸组织恢复能力、精子存活率、畸形精子率,采用实时荧光定量PCR及Western Blot技术检测小鼠睾丸中Nrf2基因表达水平和与其相关的上游调控因子及下游目标蛋白的影响来研究ORPp及ORPse拮抗环磷酰胺导致生殖损伤的作用机制。成果:1.近江牡蛎多糖提取、分离及纯化(1)通过超声辅助提取近江牡蛎脏器中多糖,其得率为9.74±0.55%。近江牡蛎粗多糖中蛋白质含量8.62±0.23%。(2)将粗多糖进一步用MCM吸附除蛋白,所合成的MCM材料粒径为2-6μm,最佳除蛋白吸附温度50℃,吸附反应时间4h。该方法为物理吸附,吸附过程动力学和吸附等温线分别可以用拉格朗日准一级动力学方程及Freundlich方程拟合。此外,根据吸附方程求得△G及△Ea判断,MCM吸附是自发、吸热的物理吸附反应。(3)近江牡蛎粗多糖经DEAE-纤维素52柱按极性洗脱后再经Sephadex G100筛分后,得到一个纯化组分ORPp,ORPp得率为32.7%。2.近江牡蛎多糖理化性质及结构鉴定ORPP中总糖含量为88.7%,硫酸基含量为1.2%,蛋白质含量0.17%,糖醛酸含量为8.59%,相对粘度为1.15。其相对分子质量为656 kDa。ORPP由葡萄糖构成。ORPP具有 D-Glc-(1→、→3)-D-Glc-(1 →、→4)-D-Glc-(1→及→3,4)-D-Glc-(1→结构,其分支率约为12.7。ORPp无三股螺旋结构。3.近江牡蛎多糖硒化修饰通过BaCl2-HN03催化,采用Na2Se03硒化修饰近江牡蛎多糖,得到其硒化衍生物ORPse中Se元素含量为181.9±5.6μg/g。4.近江牡蛎多糖对TM4细胞中Nrf2/ARE通路的影响ORPp及ORPse均可以提高H202诱导的氧化应激TM4细胞存活率,且细胞存活率与给药剂量相关。表明ORPp及ORPse均可以提高TM4细胞抗氧化能力。通过实时荧光定量PCR技术及Western Blot技术分析,ORPP及ORPse可能通过激活Nrf2/ARE通路,增加Nrf2与Keap1解偶联使其进入胞核,激活ARE元件,促进SOD、HO-1、NQO1等抗氧化酶及Ⅱ相解毒酶生成,从而提高TM4细胞抗氧化能力。5.近江牡蛎多糖对环磷酰胺介导的雄性小鼠生殖损伤的作用ORPp及ORPse可能作为诱导物,提高小鼠睾丸组织中Nrf2表达水平,激活Nrf2/ARE信号通路,有助于下游Ⅱ相解毒酶NQO1及抗氧化酶HO-1等目标基因表达,提高机体对外界抵抗能力,避免因环磷酰胺导致的氧化应激损伤,缓解雄性小鼠生殖损伤,提高精子数量、精子存活率,降低畸形精子率。相同剂量下,ORPSe的生精活性显著优于ORPp。结论:本文以近江牡蛎多糖为实验对象,系统研究其分离、纯化工艺,通过物理及化学方法研究其理化性质,通过建立H2O2诱导TM4氧化应激模型研究其体外抗氧化活性及其作用机制,建立环磷酰胺诱导小鼠氧化应激模型考察近江牡蛎多糖体内生精活性及其作用机制。发现MCM材料是一种理想的除粗多糖中蛋白质的材料,与酶法、冻融法除蛋白各有优势,适合用于需要同时保留活性蛋白及多肽的粗多糖除蛋白;硒化修饰可以将无机硒嫁接进入多糖中形成有机硒多糖ORPSe,ORPSe是无毒物。ORPp及ORPSe体外抗氧化作用机制应该是通过激活TM4细胞中Nrf2/ARE信号通路,促使SOD、HO-1、NQO1等抗氧化酶及Ⅱ相解毒酶生成,从而提高TM4细胞抗氧化能力。此外,ORPp及ORPSe可能通过激活小鼠睾丸Nrf2/ARE信号通路,提高睾丸对外界抵抗能力,避免因环磷酰胺导致氧化应激损伤,缓解雄性小鼠生殖损伤,且ORPSe活性明显优于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.

【学位授予单位】：广州中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R284;R285

【参考文献】