山茱萸籽多糖分离纯化、结构表征及抗氧化活性

Isolation,Purification,Structure Characterization and Antioxidant Activity of Cornus officinalis Seed Polysaccharides

为了对山茱萸籽多糖进行分离纯化、结构表征及抗氧化活性研究,本实验通过亚临界水萃取、体积分数30%H2O2脱色、Sevag法脱蛋白得到山茱萸籽多糖,并用DEAE-52纤维素层析法对其进行分离纯化得到了5个多糖组分,即COSP-1、COSP-2、COSP-3、COSP-4和COSP-5,并采用Sephadex G-100凝胶色谱法对主要多糖组分COSP-4进一步纯化。凝胶色谱和单糖组成表明,COSP-4是分子质量约为2.03×10^(4) Da的酸性均相多糖组分,由鼠李糖、葡萄糖醛酸、半乳糖醛酸、葡萄糖、半乳糖和木糖组成,物质的量之比为0.96∶0.18∶5.48∶0.28∶1∶10.70。甲基化反应和核磁共振表明COSP-4包含14种甲基化糖,残基同时包含了α构型糖基和β构型糖基,含量最高的单糖结构为1,4,5-三乙酰基-2,3-二甲基木糖。扫描电子显微镜观察表明COSP-4呈不规则碎片结构,松散多孔,类似海绵结构。抗氧化活性实验表明,COSP-4对DPPH自由基、羟自由基和ABTS阳离子自由基具有较强的清除能力。COSP-4对DPPH自由基、羟自由基和ABTS阳离子自由基的半抑制质量浓度分别为(1.72±0.14)、(1.48±0.17)mg/mL和(2.87±0.27)mg/mL。综上,本实验为进一步研究山茱萸籽多糖的构效关系及促进其应用提供参考。

In this study,polysaccharide was prepared from Cornus officinalis seeds by subcritical water extraction,30%(V/V)H2O2 decolorization,and Sevag deproteinization.Using DEAE-52 cellulose chromatography,the polysaccharide was fractionated into five fractions,namely COSP-1,COSP-2,COSP-3,COSP-4 and COSP-5.Sephadex G-100 gel chromatography was used to further purify the main fracion COSP-4.Gel chromatography and monosaccharide composition analysis showed that COSP-4 had a molecular mass of 2.03×10^(4) Da as an acidic homogeneous polysaccharide composed of rhamnose,glucuronic acid,galacturonic acid,glucose,galactose and xylose,with a molar ratio of 0.96:0.18:5.48:0.28:1:10.70.The methylation reaction and nuclear magnetic resonance(NMR)showed that COSP-4 contained 14 kinds of methylated sugars,as well asα-andβ-glycosyl residues.The most abundant monosaccharide in the polysaccharide was 1,4,5-tri-O-acetyl-2,3-di-O-methyl xylitol.Scanning electron microscopy(SEM)showed that COSP-4 had an irregular,fragmentary,loose and spongy structure.The antioxidant experiments showed that COSP-4 had strong scavenging capacity toward 1,1-diphenyl-2-picrylhydrazyl(DPPH),hydroxyl and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS)cation radicals with half maximal inhibitory concentrations(IC50)of(1.72±0.14),(1.48±0.17),and(2.87±0.27)mg/mL,respectively.These results will provide the basis for further research on the structure-activity relationship of Cornus officinalis seed polysaccharides and will promote their application.