低硫酸化杂多糖水解单糖高效液相色谱指纹图谱研究

HPLC fingerprints of monosaccharides from sulfated heteropolysaccharides by precolumn derivatization

低硫酸化杂多糖(UF)是从海洋褐藻中分离的一种单糖组成复杂的硫酸化杂多糖,在细胞和动物实验中均表现出显著的神经保护活性,对帕金森病具有显著作用。本研究采用水提-醇沉-DEAE-Sepharose Fast Flow交换层析柱制备UF,经三氟乙酸(TFA)水解和1-苯基-3-甲基-5吡唑啉酮(1-phenyl-3-methyl-5-pyrazalone,PMP)衍生化,通过对水解条件、衍生化试剂使用量、衍生时间的考察,确定最佳衍生条件,通过高效液相色谱(HPLC)法测定UF的单糖组成,并建立UF的指纹图谱。在UF水解单糖的指纹图谱中,共标示出8个特征共有峰,经鉴别主要色谱峰8个,分别是D-甘露糖、D-甘露糖醛酸、L-鼠李糖、D-葡萄糖醛酸、D-葡萄糖、D-半乳糖、D-木糖及L-岩藻糖,其中岩藻糖含量最高,其次是半乳糖。该方法操作简便、快速、灵敏度高、重复性好,可作为UF检查和鉴别的方法,建立实验室UF质量控制标准,为临床用UF质量标准的建立奠定基础。

Sulfated heteropolysaccharide(UF)is a novel marine polysaccharide extracted from Laminaria japonica,with a complex chemical composition and structure.UF showed a significant neuroprotective activity both in cell and animal studies.UF was extracted with hot distilled water and further fractionated by DEAE-Sepharose Fast Flow column chromatography.The optimal hydrolysis and PMP derivatization conditions were investigated.UF was hydrolyzed into monosaccharides by trifluoroacetic acid and derivated with 1-phenyl-3-methyl-5-pyrazolone and analyzed by HPLC to study the monosaccharide components and fingerprint of UF.Nine peaks were detected in the fingerprint,of which 8 were identified as D-mannose,D-Mannuronic acid,L-rhamnose,D-glucuronic acid,D-glucose,D-galactose,D-xylose,and L-fucose.Fucose content was the highest,followed by galactose content.The result shows that the method is convenient,sensitive,and rapid such that it can be applied for the quality control of UF.