红松松子壳多糖的分离纯化、结构表征及其抗氧化活性研究

发布时间：2018-06-22 21:15

本文选题：红松松子壳多糖 + 分离纯化　；参考：《吉林大学》2017年硕士论文

【摘要】：多糖又称多聚糖,由10个以上单糖通过糖苷键连接而成,广泛存在于生物体中,在生命活动中具有重要作用。多糖具有抗肿瘤、抗病毒、抗氧化和抗菌等生物活性。红松松子壳多糖(Pinuskoraiensis Pinon Shell Polysaccharides,PPSP)是从中国东北地区经济型林木红松松子壳废弃物中提取而来,因其具有较强的抗病毒、抗肿瘤等生物活性而备受关注。本研究首先通过超声辅助热水浸提法提取PPSP,将纯化后的PPSP-b采用离子交换层析和葡聚糖凝胶层析法进行分离,然后通过紫外可见光分光光度计和化学方法检测PPSP及其主要多糖组分PPSP2-2和PPSP3-1的理化性质。本实验中,超声辅助热水浸提法提取PPSP条件:提取温度为80℃,浸提时间为40 min,浸提次数为2次,固液比为1:3。结果表明,在此条件下多糖提取率为2.37±0.23%,糖含量为25.78%;PPSP经过木瓜蛋白酶结合Sevag法脱蛋白,蛋白去除率为90.87%,多糖保留率为76.41%;PPSP-b经过DEAE-52离子交换层析后分离得到PPSP1、PPSP2和PPSP3,因PPSP1含量低,本实验将PPSP2和PPSP3进一步经过葡聚糖凝胶层析分别得到PPSP2-1、PPSP2-2和PPSP3-1、PPSP3-2,又因PPSP2-1和PPSP3-2含量较少,最终将PPSP2-2和PPSP3-1作为主要研究对象。理化性质的检测结果表明,PPSP、PPSP2-2、PPSP3-1均以多糖成分为主。然后本研究对PPSP2-2和PPSP3-1的一级结构和空间结构进行测定,刚果红实验结果表明,PPSP2-2分子内存在三股螺旋构象,而PPSP3-1分子内不存在三股螺旋构象;高效液相色谱图分析表明,PPSP2-2单糖组成及其摩尔比为Man:Rha:Glc A:Gal A:Glc:Gal:Xyl:Ara:Fuc=7.3:12.1:3.6:11.8:11.0:20.2:10.5:18.4:5.1;PPSP3-1单糖组成及其摩尔比为Man:Rha:Glc A:Gal A:Glc:Gal:Xyl:Ara:Fuc=4.6:5.6:2.9:44.9:23.8:9.1:2.5:5.2:1.3,PPSP2-2和PPSP3-1分子量分布相对均一,且分子量都较小,分别为1.89×103 Da、2.84×103Da;红外光谱结果显示,PPSP2-2和PPSP3-1均是酸性多糖,且都含有吡喃糖。最后,本研究对PPSP对小鼠抗氧化活性的影响进行检测,以不同剂量PPSP对小鼠连续灌胃21 d,检测小鼠肝脏和血清中的超氧化物歧化酶(super oxide dismutase,SOD)、过氧化氢酶(catalase,CAT)、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)活力和丙二醛(malondialdehyde,MDA)含量。结果表明,在灌胃不同剂量的PPSP后,小鼠的抗氧化作用均有所提高,且呈现出一定的剂量效应。与对照组相比,PPSP高剂量组(400mg/(kg·d))能极显著提高小鼠血清和肝脏中的SOD、CAT和GSH-Px活性(P0.01),并极显著降低MDA含量(P0.01);PPSP中剂量组(200mg/(kg·d))能够极显著提高(P0.01)小鼠肝脏组织中的SOD活力,并显著提高(P0.05)小鼠血清中的GSH-Px活力,同时显著降低小鼠血清和肝脏中的MDA含量(P0.05)。综上所述,本研究通过分离纯化PPSP,得到四种不同多糖组分,其中PPSP2-2和PPSP3-1是其主要组成部分,PPSP2-2主要由Gal、Ara、Rha、Gal A、Glc、Xyl单糖组成,分子量为1.89×103 Da;PPSP3-1主要由Gal A、Glc、Gal等单糖组成,分子量为2.84×103Da;通过红外检测,PPSP2-2和PPSP3-1是含有吡喃糖的酸性多糖。PPSP具有体内抗氧化生物活性,且呈现出剂量效应。
[Abstract]:Polysaccharide, also called polysaccharide, is made up of more than 10 monosaccharides through glucoside bonds. It is widely found in organisms and plays an important role in life activities. Polysaccharides have biological activities, such as anti-tumor, antiviral, antioxidant and antibacterial. Pinuskoraiensis Pinon Shell Polysaccharides (PPSP) is from the northeast of China. In this study, PPSP was extracted by ultrasonic assisted hot water extraction, and the purified PPSP-b was separated by ion exchange chromatography and glucan gel layer analysis, and then UV visible light was used. The physicochemical properties of PPSP and its main polysaccharide components PPSP2-2 and PPSP3-1 were detected by photometer and chemical method. In this experiment, the ultrasonic assisted hot water extraction method was used to extract the PPSP conditions: the extraction temperature was 80 C, the extraction time was 40 min, the extraction times were 2 times, and the solid-liquid ratio was 1:3.. The extraction rate of polysaccharide was 2.37 + 0.23% and the sugar content under this condition. For 25.78%, PPSP was deproteinized by papain combined with Sevag, the removal rate of protein was 90.87%, the retention rate of polysaccharide was 76.41%, and PPSP1, PPSP2 and PPSP3 were separated after DEAE-52 ion exchange chromatography. Because of low PPSP1 content, PPSP2 and PPSP3 were further obtained by glucan gel chromatography, respectively. PSP3-2, also due to the low content of PPSP2-1 and PPSP3-2, finally took PPSP2-2 and PPSP3-1 as the main research object. The results of physical and chemical properties showed that PPSP, PPSP2-2 and PPSP3-1 were dominated by polysaccharide components. Then the first order structure and spatial structure of PPSP2-2 and PPSP3-1 were measured. The results of Congo red experiment showed that PPSP2-2 molecular memory was in memory. In three strands of spiral conformation, there is no three strand conformation in PPSP3-1, and high performance liquid chromatography analysis shows that the composition of PPSP2-2 monosaccharide and its mole ratio are Man:Rha:Glc A:Gal A:Glc:Gal:Xyl:Ara:Fuc=7.3:12.1:3.6:11.8:11.0:20.2:10.5:18.4:5.1, and the composition of PPSP3-1 monosaccharide and its mole ratio are Man:Rha:Glc A:Gal A:Glc:Gal:Xyl:Ara:Fuc= The molecular weight distribution of 4.6:5.6:2.9:44.9:23.8:9.1:2.5:5.2:1.3, PPSP2-2 and PPSP3-1 is relatively uniform, and the molecular weight is smaller, 1.89 * 103 Da and 2.84 x 103Da respectively. The infrared spectrum shows that both PPSP2-2 and PPSP3-1 are acid polysaccharides and contain Piran. Finally, the study on the effect of PPSP on the antioxidant activity of mice is not used. The same dose of PPSP was administered to mice continuously for 21 d, and the activity of superoxide dismutase (super oxide dismutase, SOD), catalase (catalase, CAT), glutathione peroxidase (glutathione peroxidase, GSH-Px) and the content of malondialdehyde (malondialdehyde, etc.) in the liver and serum of mice were detected. In contrast to the control group, the PPSP high dose group (400mg/ (kg. D)) could significantly increase the SOD, CAT and GSH-Px activity (P0.01) in the serum and liver of mice, and significantly reduced the MDA content (P0.01) in the mice, and the PPSP dose group (200mg/ (200mg/)) could significantly improve the small dose. The activity of SOD in rat liver tissue, and significantly increase the GSH-Px activity in the serum of (P0.05) mice, and significantly reduce the MDA content in the serum and liver of mice (P0.05). To sum up, this study obtained four different polysaccharide components by separating and purifying PPSP, in which PPSP2-2 and PPSP3-1 are its main components, PPSP2-2 mainly from Gal, Ara, Rha, Rha. Al A, Glc, and Xyl monosaccharides are composed of 1.89 x 103 Da; PPSP3-1 is mainly composed of Gal A, Glc, Gal and other monosaccharides, and the molecular weight is 2.84 x 103Da.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R914

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