桑叶多糖结构特征及其对α-葡萄糖苷酶活性的抑制作用

Study on the structure of mulberry leaf polysaccharide and its inhibitory activity of α-glucosidase

采用水提醇沉法制备得到桑叶多糖(MLP),进一步经分离和纯化得到4个桑叶多糖纯化组分(MLP-1、MLP-2、MLP-3、MLP-4)。采用苯酚-硫酸法、凝胶色谱(GPC)、气相色谱(GC)、红外光谱(FT-IR)分别对桑叶多糖的含糖量、分子量、单糖组成、化学结构进行表征,并深入研究了桑叶多糖对α-葡萄糖苷酶的抑制作用及抑制动力学。结果表明,4个多糖的含糖量(质量分数)分别为88.46%,78.18%,69.42%和67.35%。GPC分析表明,4个多糖组分都含有两个或两个以上的峰,分子质量分别为6.14,8.08,10.25和2 017.55 ku。GC分析表明,4个多糖组分均由鼠李糖、阿拉伯糖、木糖、甘露糖、葡萄糖、半乳糖6种单糖组成,但具有不同的单糖物质的量比。FT-IR分析表明,MLP及其4个多糖纯化组分具有典型的多糖特征吸收峰,含有吡喃糖环结构。α-葡萄糖苷酶抑制试验表明,仅MLP与MLP-1对α-葡萄糖苷酶具有抑制作用,其中MLP-1对α-葡萄糖苷酶的抑制效果明显;而MLP-2、MLP-3和MLP-4对α-葡萄糖苷酶的抑制率均为0。α-葡萄糖苷酶抑制动力学分析表明,MLP-1对α-葡萄糖苷酶活性的抑制作用属于非竞争型可逆抑制,MLP-1和对硝基苯-α-D-吡葡萄糖苷(PNPG)分别独立地与酶的不同部位相结合,PNPG浓度的改变对抑制程度无影响。研究结果探明了桑叶多糖中降血糖活性的主要成分,可为桑叶多糖的开发利用提供理论依据。

In this study, mulberry leaf polysaccharide(MLP) was prepared by the water extraction and alcohol precipitation. Furthermore, four mulberry leaf polysaccharide components(MLP-1, MLP-2, MLP-3 and MLP-4) were separated and purified by the polyamide resin, S-8 macro-porous adsorption resin and ion exchange column chromatography. The polysaccharide content, molecular weight, monosaccharide composition and chemical structure of mulberry leaf polysaccharide were characterized by the phenol-sulfuric acid method, gel chromatography(GPC), gas chromatography(GC) and infrared spectroscopy(FT-IR). Finally, the inhibitory effect and mechanism of mulberry leaf polysaccharide on α-glucosidase were studied. The results showed that the polysaccharide contents of MLP-1, MLP-2, MLP-3 and MLP-4 were 88.46%, 78.18%, 69.42% and 67.35%, and the yields were 0.25%, 0.65%, 1.43% and 1.78%, respectively. The GPC analysis showed that four polysaccharide components contained two or more peaks, which indicated that the polysaccharide components purified by DEAE-52 cellulose still contained a small amount of impurity. The molecular weights of the four polysaccharide components were 6.14, 8.08, 10.25 and 2 017.55 ku, respectively. The GC analysis showed that the four purified components were composed of rhamnose, arabinose, xylose, mannose, glucose, and galactose, but with different molar ratios of monosaccharides. Galacturonic acid and uronic acid were not detected in all polysaccharide fractions. The FT-IR analysis showed that MLP and four purified components had typical characteristic absorption peaks of polysaccharides and contained pyranose ring structure. The inhibition of α-glucosidase activity showed that MLP and MLP-1 had inhibitory effect on α-glucosidase activity, and MLP had inhibitory effect on α-glucosidase activity with ICvalue of 0.503 8 mg/mL, while MLP-2, MLP-3 and MLP-4 had zero inhibition rate on α-glucosidase activity. The inhibition kinetics of MLP-1 showed that the inhibition of α-glucosidase activity by MLP-1 was non-competitive reversible inhibition, and the Michaelis constant Kwas 1.394 mmol/L. MLP-1 inhibited the activity of α-glucosidase by binding to essential groups outside the active center of the enzyme and the change of substrate concentration had no effect on the degree of inhibition. The results would be helpful to understand the main components of polysaccharide of hypoglycemic activity in mulberry leaves and promote the efficient development and utilization of polysaccharide resources in mulberry leaves.