1种新型乌贼墨糖胺聚糖的分离纯化、理化性质及其抗氧化活性研究

Separation,purification and analyses on physicochemical properties,antioxidative activity of a novel glycosaminoglycans fromSepia esculentaink

目的从金乌贼墨(Sepia esculentaink)中提取其活性多糖成分,对其理化性质及体外抗氧化活性进行研究。方法依次采用两步酶解法提取乌贼墨粗多糖,并使用Q Sepharose 4Fast Flow阴离子交换层析柱对其进行分离纯化,获得主要组分。通过高效液相(HPLC)、紫外光谱(UV)、红外光谱(IR)、离子色谱法对乌贼墨多糖进行理化性质分析以及初步的结构研究,采用ABTS法对乌贼墨进行总体抗氧化活性研究。结果两步酶解后乌贼墨粗多糖的得率为3%,分离纯化后得到0.05、0.25、0.5mol/L NaCl 3个洗脱组分,分别命名为SE-1、SE-2、SE-3,其中以SE-2为最主要组分,得率为50%。SE-2为均一性多糖,分子量8.5kD,主要含有半乳糖胺(GalN)、岩藻糖(Fuc)、葡萄糖胺(GlcN)、乙酰氨基半乳糖(GalNAc)、葡萄糖醛酸(GlcUA)以及甘露糖(Man)等单糖;该多糖SE-2的总糖含量、蛋白质含量、硫酸根含量、氨基己糖含量和糖醛酸含量分别为54.20%、3.92%、7.74%、20.50%和9.86%;IR光谱进一步证明该糖为SE-2糖胺聚糖,构象为D-吡啶环结构,并有硫酸根的存在;SE-2对ABTS自由基的清除能力较强,其半数清除质量浓度为0.210mg/mL。结论金乌贼墨多糖SE-2是1种硫酸酯化的新型糖胺聚糖,具有较强的体外ABTS自由基的清除活性。

Objective To extract,isolate and purify polysaccharides fromSepia esculentaink and analyze physicochemical properties and antioxidative activity of the polysaccharides.Methods The crude polysaccharides were extracted through two-step enzymatic hydrolysis by papain and trypsin.The main component from the crude polysaccharides was fractionated by a Q Sepharose fast flow column.HPLC,HPGPC,UV spectrophotometer,IR spectrometry and ion chromatography were used to determinate the physicochemical and structural properties;ABTS was used to analyze the total antioxidative activity of the main polysaccharide.Results After enzymatic hydrolysis,the yield of the crude polysaccharide was 3%.Three components were eluted with a step-wise gradients of 0.05,0.25 and 0.5mol/L NaCl on Q Sepharose fast flow column,which were named SE-1,SE-2,SE-3respectively.While SE-2was the main component,the yield of which was 50%relative to the pure polysaccharides.SE-2was a homogeneous polysaccharide with a molecular weight of 8.5kD and was composed of galactosamine,fucose,glucosamine,N-acetylgalactosamine,glucuronic acid and mannose.The contents of total sugar,protein,sulfate,aminohexose and hexuronic acid of SE-2were 54.2%,3.92%,7.74%,20.5%and 9.86% respectively.In the IR spectrogram,it was shown the typical absorption of sugar residues,glycosaminoglycans and sulfate ester.SE-2showed strong scavenging ability on the ABTS free radicals.The half maximal inhibitory concentration（IC50）against ABTS free radicals was 0.21mg/mL.Conclusion The polysaccharide SE-2isolated fromSepia esculentaink was a novel sulfated glycosaminoglycans and had excellent antioxidative activity on ABTS free radicals.