吴茱萸多糖的提取纯化与结构解析

Isolation,purification and structural characterization of polysaccharides from Euodiae fructus

以吴茱萸为原料,先对水提醇沉获得的吴茱萸粗多糖分离纯化再对纯化后的均一组分进行结构表征。首先,根据多糖保留率、脱色率和分子量变化等指标,确定最适合吴茱萸多糖(EFP)的脱色方法;其次,通过乙醇分级沉淀获得吴茱萸多糖纯化组分EFP-20。采用比色法、高效凝胶渗透色谱仪(HPGPC)、离子色谱(HPAEC-PAD)、甲基化处理结合气相色谱-质谱(GC-MS)和核磁共振(NMR)分析其化学组成、分子量和糖苷键类型等结构特征。结果表明,活性炭对吴茱萸多糖的脱色效果最好,其脱色率达68.74%,且中性糖、糖醛酸保留率分别为94.26%和91.79%。通过乙醇分级沉淀获得的多糖组分EFP-20分子量为1.78×10^(7) Da,主要由鼠李糖(20.83%)、阿拉伯糖(23.16%)、半乳糖(31.52%)和半乳糖醛酸(23.64%)组成。甲基化结果表明主要的糖苷键为T-Arap(20.99%)、1,2,4-Rhap(21.82%)、1,4-Galp(11.05%)、1,2-GalpA(14.33%)和1,4-GalpA(11.05%),结合核磁共振分析推断EFP-20是一种果胶,含有I型鼠李半乳糖醛酸聚糖型结构域,该结构域主链由α-1,4-GalpA和α-1,2,4-Rhap重复残基组成,分支点在C-4位。通过对吴茱萸多糖提取方法和结构特征的研究可作为重要中药资源及为相关功能活性产品的开发利用提供基础数据和理论支持。

Euodiae fructus was used as raw material to separate and purify the crude polysaccharides of Euodiae fructus extracted by hot water and ethanol precipitation,with the structure of purified components being characterized.Firstly,the decolorization method was determined based on the polysaccharide retention rate,decolorization rate and molecular weight distribution.Secondly,EFP was fractionated by gradient ethanol precipitation to obtain the purified polysaccharide fraction named EFP-20.Colorimetric assay,high-performance gel permeation chromatography(HPGPC),high performance anion exchange chromatography coupled with pulsed amperometric(HPAEC-PAD),methylation combined with gas chromatography-mass spectrometry(GC-MS)analysis and NMR spectroscopy were conducted to reveal the structural features including chemical composition,molecular weight and linkage patterns,etc.The result indicated that activated carbon had the best decolorization effect on Euodiae fructus rutaecarpa polysaccharides,with a decolorization rate of 68.74%,and the retention rates of neutral sugar and uronic acid were 94.26%and 91.79%,respectively.After gradient ethanol precipitation,the molecular weight of the obtained polysaccharide,EFP-20,was 1.78×10^(7) Da,mainly composed of Rha(20.83%),Ara(23.16%),Gal(31.52%)and GalA(23.64%).Methylation analysis indicated that the main glycosidic linkages included T-Ara p(20.99%),2,4-Rha p(21.82%),1,4-Gal p(11.05%)and 1,2-Gal p A(14.33%)and 1,4-Gal p A(11.05%).Combined with NMR analysis,EFP-20 was deduced as pectin,containing type I rhamnogalacturonan(RG-I)structures,with the major chain composed ofα-1,4-Gal p A andα-1,2,4-Rha p repeating unit and branched at C-4.This study,focusing on the isolation and structural characterization of the polysaccharide from Euodiae fructus,would provide theoretical basis and data for development and utilization of essential resources from Traditional Chinese Medicine and their related functional products.