基于Needs甲基化法枸杞多糖指纹图谱的建立

The Fingerprint of Lycium barbarum Polysaccharide was Established by Needs Methylation Method

目的多糖是枸杞中主要的活性成分,各产地间枸杞多糖的结构和含量均有差异。目前枸杞多糖的质量控制方法大多都通过单糖组成构建指纹图谱,但仅通过单糖指纹图谱对枸杞多糖进行质量控制,并不能系统地对不同产地的枸杞多糖进行质量控制。因此,我们在单糖指纹图谱的基础上,利用Needs甲基化法确定枸杞多糖的糖残基连接方式,建立Needs甲基化法枸杞多糖指纹图谱,实现对不同产地枸杞的质量控制。方法通过水提醇沉法获得枸杞多糖,并采用Needs甲基化、完全酸水解、硼氢化钠还原及乙酰化等方法结合GC-MS测定枸杞多糖的糖残基连接方式。结果将18批枸杞多糖的糖残基连接方式色谱图导入《中药色谱指纹图谱相似度评价系统》(2004版)构建出指纹图谱,并结合多种化学计量法评价不同产地间枸杞多糖的差异。相似度计算结果发现,除西藏自治区三批枸杞多糖相似度0.551-0.569,其余15批样品相似度均0.929以上。糖残基连接方式色谱图共标定了16个共有峰,指认了其中10个共有峰,分别为TArap、T-Araf、T-Xylp、1,2-Arap、1,3-Rhap、1,5-Araf、T-Glcp、T-Galp、1,4-Glcp、1,6-Galp。HCA、PCA和PLS-DA分析结果将18批次不同产地的枸杞多糖分为3类,并筛选出了3个标志性成分,分别为T-Araf、1,5-Araf和T-Glcp,以此来判断不同产地间枸杞多糖的差异性。结论首次建立了18批枸杞多糖的Needs甲基化法指纹图谱,为枸杞多糖质量控制提供实验数据参考,进一步证明了多糖在中药质量控制中的作用。

Objective Polysaccharide is a major active component in Lycium barbarum.the structure and content of polysaccharide are different among different producing districts.At present,most of the quality control methods of Lycium barbarum polysaccharide construct fingerprint from the aspect of monosaccharide composition,but the quality control of Lycium barbarum polysaccharide only by analyzing monosaccharide composition can not fully explain the relationship between structure and Lycium barbarum polysaccharide.Therefore,we established the Needs methylation method of Lycium barbarum polysaccharide fingerprint to achieve quality control of Lycium barbarum from different places by determining the sugar residue linkage mode of Lycium barbarum polysaccharide on the basis of monosaccharide composition.Methods Lycium barbarum polysaccharide was extracted through water extraction followed by alcohol precipitation,and the Needs methylation,complete acid hydrolysis,reduction by NaBH4,acetylation and other methods combined with GC-MS to determine the sugar residue linkage motif of Lycium barbarum polysaccharide.Result The chromatograms of 18 batches of Lycium barbarum polysaccharides were imported into the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System(2004 edition),combined with chemometrics to evaluate the differences of Lycium barbarum polysaccharide from different origin.The result of similarity showed that the similarity of three batches of Lycium barbarum polysaccharide from Tibet ranged from 0.551 to 0.569,others were more than 0.929.16 common peaks were marked in chromatogram of sugar residue linkage mode,and 10 of them was identified,they are T-Arap,T-Araf,T-Xylp,1,2-Arap,1,3-Rhap,1,5-Araf,T-Glcp,T-Galp,1,4-Glcp,1,6-Galp.The results of HCA,PCA and PLS-DA analysis divided 18 bathes of Lycium barbarum polysaccharide were divided into three categories,and three signature components were screened,which were T-Araf,1,5-Araf and T-Glcp,to judge the differences of Lycium barbarum polysaccharides from different places.Conclusion The Needs methylation fingerprint of 18 batches of Lycium barbarum polysaccharide was established for the first time.The establishment of the fingerprint can provide experimental data reference for the quality control of Lycium barbarum polysaccharide,and further prove the role of polysaccharide in the quality control of Traditional Chinese Medicine.