海参中单糖检测方法的建立及含量测定

Establishment and Determination of Monosaccharide in Sea Cucumber

中文关键词: 高效阴离子交换-脉冲安培检测法  海参多糖  单糖测定

英文关键词: HPAEC-PAD  Sea cucumber polysaccharide  Monosaccharide determination

中文摘要:

      本研究建立了高效阴离子交换–脉冲安培检测法(HPAEC-PAD)分析海参(Apostichopus japonicus)多糖的单糖组成及含量测定。通过比较3种蛋白去除法对海参多糖含量的影响，优化海参多糖纯化方法；比较淋洗液浓度对单糖分离效果的影响，优化海参中单糖分离的色谱条件。结果显示，Sevag法去蛋白的效果最好，在淋洗液为20 mmol/L NaOH的条件下分离岩藻糖、氨基半乳糖、氨基葡萄糖、半乳糖、甘露糖、阿拉伯糖、葡萄糖和乳糖；在淋洗液为160 mmol/L NaOH和200 mmol/L NaAC的条件下分离葡萄糖醛酸和半乳糖醛酸。各单糖标准曲线的线性相关系数不低于0.998，检出限为2.5~50 μg/L，加标回收率在83.6%~113.1%之间。该方法可测定海参多糖的中性糖、氨基糖和糖醛酸，可作为海参及其制品中的单糖分析及定量方法，为相关标准的制定提供参考。

英文摘要:

      High-performance anion-exchange chromatography with pulsed amperometric detector (HPAEC-PAD) was widely used to analyze monosaccharide in botanical and fungal polysaccharides, but lightly used in marine animal polysaccharides. A method has been developed for determining monosaccharide composition and content in sea cucumber polysaccharide by HPAEC-PAD. Purifying sea cucumber polysaccharide were optimized by comparing three deproteinization methods. Optimum chromatographic conditions for the separation of monosaccharide were optimized by comparing eluent concentration on the separation of monosaccharide. The results indicated that the reagent of Sevag removed micro-molecule polypeptide more effectively than others from sea cucumber polysaccharides. The concentration of eluent had significant effect on separating monosaccharide. Eight monosaccharide, such as Fuc, Gal, Ara, GalN, GlcN, Man, Glc, and Lac, were separated using 20 mmol/L NaOH as eluent. GlcUA and GalUA were separated by 160 mmol/L NaOH and 200 mmol/L NaAC as eluent. The linear coefficients of monosaccharide standard were above 0.998. The detection limit of each monosaccharide was between 2.5 and 50 μg/L, and the recovery rates ranged from 83.6% to 113.1%. The method determining neutral sugar, amino sugar and uronic acid in sea cucumber polysaccharides could be used as analysis and quantitative method of monosaccharide in sea cucumber and its products. The composition and content of monosaccharide were significant indices for determining the biological activity of sea cucumber. The established method could provide basic monosaccharide information and promote the polysaccharides research progress in sea cucumber. It could provide references for the establishment of sea cucumber standards, and expand the application of active substances in medicine and other fields.