基于分子修饰的粒毛盘菌多糖结构与生物活性研究

发布时间：2018-04-25 11:53

本文选题：粒毛盘菌多糖结构 + 分子修饰　；参考：《合肥工业大学》2017年硕士论文

【摘要】：本文研究了粒毛盘菌YM240胞外多糖的分离纯化、结构表征、羧甲基化修饰、硫酸化修饰,以及修饰前后的体外抗氧化和降血糖及对体内降血糖和降血脂活性。粒毛盘菌YM240发酵液经水提醇沉、脱色和脱蛋白后获得胞外多糖,再经DEAE-cellulose 52和Sephadex G-100色谱柱分离纯化,得单一组分LEP;HPLC、GC-MS、甲基化分析、FT-IR和1D/2D NMR等实验结果表明LEP的分子量为1.68×103 kDa,由摩尔比为16.3:1.0的甘露糖(Man)和半乳糖(Gal)构成;(1→3)-α-D-Manp、(1→3,4)-α-D-Manp和(1→3)-α-D-Galp构成LEP的主链;α-D-Manp-(1→2)-α-D-Manp-(1→3)-α-D-Manp-(1-构成支链,连接在主链上Manp的C-4位。对LEP进行化学修饰,获得取代度为0.360的羧甲基化多糖CLEP和取代度为0.144的硫酸化多糖SLEP。红外光谱和核磁共振碳谱分析结果表明,多糖的的羧甲基化和硫酸化修饰成功,羧基甲基-CH2COOH取代在(1→2)-α-D-Manp的C-3、(1→3)-α-D-Manp的C-4和(1→)-α-D-Manp的C-6上,而硫酸基-SO3H主要取代在(1→2)-α-D-Manp的C-4和(1→3)-β-D-Galp的C-6上。体外抗氧化实验表明,与LEP相比,CLEP和SLEP对DPPH自由基和羟基自由基的清除作用及还原力都显著增强。体外降血糖活性实验结果显示,CLEP和SLEP对α-葡萄糖苷酶和α-淀粉酶抑的制活性及葡萄糖扩散的抑制作用明显高于LEP。采用不同含量的LEP、CLEP和SLEP处理链脲佐菌素(STZ)及高脂饲料诱导的II型糖尿病小鼠(T2DM)。与LEP相比,CLEP和SLEP显著增加体重、脏器指数、肝糖原、葡萄糖耐量,明显降低空腹血糖水平(FBG)、空腹血清胰岛素(FINS)和糖化血红蛋白(HbA1c)以及血清甘油三酯(TG)、胆固醇(TC)和游离脂肪酸(FFA),CLEP的作用更显著。此外,CLEP和SLEP比LEP更明显地上调肝脏中的葡萄糖激酶(GK)和腺苷一磷酸激活蛋白激酶(AMPK),骨骼肌中的AMPK和葡萄糖转运蛋白4(GLUT4),脂肪组织过氧化物酶体增殖物激活受体(PPAR-γ),下调肝脏中葡萄糖-6-磷酸酶(G6P),并且CLEP比SLEP作用更明显。因此,CLEP和SLEP具有显著的降血糖和降血脂活性。
[Abstract]:In this paper, the isolation and purification, structure characterization, carboxymethylation modification, sulfation modification of extracellular polysaccharide of YM240 were studied. The in vitro antioxidation and hypoglycemia, hypoglycemic and hypolipidemic activities were studied before and after modification. Extracellular polysaccharides were obtained from YM240 fermentation broth by water extraction and alcohol precipitation, decolorization and deproteinization, and then purified by DEAE-cellulose 52 and Sephadex G-100 chromatographic column. A single component, LEPP-HPLCX GC-MS.Methylation analysis of FT-IR and 1D/2D NMR showed that the molecular weight of LEP was 1.68 脳 103kDa.The molecular weight of LEP was 1.68 脳 103kDa. the molecular weight of LEP was 1.68 脳 103kDa.The molecular weight of LEP was 16.3: 1.0 mannose) and galactose-a-D-Manp34- 伪 -D-Manp formed the main chain of LEP, 伪 -D-Manp-Manp-1- 伪 -D-Manp-1,3kDa-伪 -D-Manp-131- formed the branching chain of LEP. The C-4 bit of Manp attached to the main chain. Carboxymethylated polysaccharide (CLEP) with a degree of substitution of 0.360 and a sulfated polysaccharide with a degree of substitution of 0.144 were obtained by chemical modification of LEP. The results of IR and NMR showed that carboxymethylation and sulfation of polysaccharides had been successfully modified, and carboxymethyl CH2COOH was substituted on C-4 and C-6 of C-3DMAP and 伪 -D-Manp. The sulfate-SO3H was mainly substituted on C-4 and C-6 of 伪 -D-Manp and 尾 -D-Galp. The antioxidant activity of CLEP and SLEP on DPPH radical and hydroxyl radical was significantly enhanced compared with LEP in vitro. The results of hypoglycemic activity test in vitro showed that CLEP and SLEP inhibited the inhibitory activity of 伪 -glucosidase and 伪 -amylase and the inhibitory effect of glucose diffusion on 伪 -glucosidase and 伪 -amylase were significantly higher than that of LEP. Streptozotocin (STZ) and type II diabetic mice induced by high fat diet were treated with different contents of LEPP-CLEP and SLEP. Compared with LEP, CLEP and SLEP significantly increased body weight, organ index, liver glycogen, glucose tolerance, The effects of FBGN, fasting serum insulin (fins) and glycosylated hemoglobin (HbA1c), serum triglyceride triglyceride (TGN), cholesterol triglyceride (TC) and free fatty acid (FFAA) CLEP were significantly decreased. In addition, CLEP and SLEP upregulated the levels of glucokinase (GK) and adenosine monophosphate activated protein kinase (AMPK) in the liver, AMPK and glucose transporter 4 (GLUT4) in skeletal muscle, adipose tissue peroxisome proliferator activator receptor (PPAR- 纬), PPAR- 纬, respectively. The glucose-6-6 phosphatase G 6 PX in the liver was regulated, and the effect of CLEP was more obvious than that of SLEP. Therefore, CLEP and SLEP have significant hypoglycemic and hypolipidemic activities.
【学位授予单位】：合肥工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O636.1

【参考文献】