基于糖谱法的地黄药材及饮片定性定量分析

Qualitative and Quantitative Analysis of Rehmanniae Radix and Its Decoction Pieces Based on Sugar Spectrum

目的:以地黄中低聚糖类成分为研究对象,建立含量测定方法及薄层色谱法(TLC)鉴别方法,探究不同地黄药材及饮片中低聚糖类成分含量及分布规律。方法:收集鲜地黄和生地黄药材各10批,生地黄和熟地黄饮片各12批,采用硅胶G薄层板,以乙酸乙酯-水-无水甲酸-冰乙酸(12∶6∶5∶4)为展开剂,10%硫酸乙醇溶液为显色剂,建立地黄中果糖、蔗糖、棉籽糖、甘露三糖和水苏糖的TLC鉴别方法;采用高效液相色谱-蒸发光散射检测法(HPLC-ELSD),建立不同地黄药材及饮片中果糖、葡萄糖、蔗糖、蜜二糖、棉籽糖、甘露三糖和水苏糖7种糖类成分的含量测定方法。对不同地黄药材及饮片中7种糖类成分含量进行主成分分析(PCA)及偏最小二乘法-判别分析(PLS-DA),以变量重要性投影(VIP)值>1筛选差异性成分。结果:TLC鉴别结果显示,鲜地黄、生地黄药材和生地黄饮片样品在与水苏糖、棉籽糖和蔗糖对照品相应的位置上显现相同颜色的斑点,而熟地黄饮片在此基础上与甘露三糖和果糖对照品相应的位置上显示相同颜色的斑点。7种待测成分的方法学考察结果均符合含量测定的要求。鲜地黄、生地黄药材中仅检出葡萄糖、蔗糖、棉籽糖和水苏糖,其在鲜地黄药材中的平均质量分数分别为0.84%、4.62%、2.42%和57.90%,在生地黄药材中的平均质量分数分别为3.16%、9.36%、7.05%和38.10%。生地黄饮片中,果糖、葡萄糖、蔗糖、蜜二糖、棉籽糖、甘露三糖和水苏糖的平均质量分数分别为1.68%、4.27%、9.96%、0.53%、6.85%、3.05%和37.52%;熟地黄饮片中,上述7种糖的平均质量分数分别为10.62%、11.01%、1.25%、3.35%、1.12%、28.16%和6.39%。多元统计分析结果显示,糖类成分可明显区分鲜地黄、生地黄和熟地黄,三者的主要差异性成分分别为水苏糖、蔗糖、棉籽糖和甘露三糖。结论:地黄不同药材及饮片中糖类成分的含量及种类存在较大差异,且基于此建立的薄层色谱鉴别方法可用于区分生地黄和熟地黄,可为地黄质量标准提升和地黄不同炮制品中低聚糖开发及应用奠定基础。

Objective Taking the oligosaccharides in Rehmanniae Radix(RR)as the research object,the content determination method based on high performance liquid chromatography-evaporative light scattering detection(HPLC-ELSD)and thin layer chromatography(TLC)identification method were established to explore the content and distribution of oligosaccharides in different RR herbs and decoction pieces.Method A total of 10 batches of fresh and raw RR,12 batches of RR decoction pieces and Rehmanniae Radix Praeparata(RRP)were collected.A TLC identification method for fructose,sucrose,manninotriose,raffinose and stachyose in RR was established by using silica gel G thin-layer plates with ethyl acetate-water-anhydrous formic acid-glacial acetic acid(12∶6∶5∶4)as the developing agent and 10%sulfuric acid-ethanol solution as chromogenic agent.A HPLC-ELSD was used to determine the contents of fructose,glucose,sucrose,melibiose,raffinose,manninotriose and stachyose in different RR herbs and decoction pieces.Then principal component analysis(PCA)and partial least squares-discriminant analysis(PLS-DA)were used to analyze the contents of 7 kinds of saccharides in RR herbs and decoction pieces,and the differential components were screened with the value of variable importance in the projection(VIP)>1.Result The results of TLC identification showed that fresh RR,raw RR and its decoction pieces showed spots of the same color on the corresponding positions with the control products of stachyose,raffinose and sucrose,while the TLC of RRP showed spots of the same color at corresponding positions to manninotriose and fructose controls.The results of methodological investigations of 7 analytes met the requirements of determination.Only glucose,sucrose,raffinose and stachyose were detected in 10 batches of fresh RR and 10 batches of raw RR herbs,the average contents of which were 0.84%,4.62%,2.42%and 57.90%in fresh samples,while those were 3.16%,9.36%,7.05%and 38.10%in raw samples,respectively.In 12 batches of RR decoction pieces,the contents of the above seven sugars(fructose,glucose,sucrose,melibiose,raffinose,manninotriose and stachyose)were 1.68%,4.27%,9.96%,0.53%,6.85%,3.05%and 37.52%,respectively.In 12 batches of RRP,the contents of the above seven sugars were 10.62%,11.01%,1.25%,3.35%,1.12%,28.16%and 6.39%,respectively.The results of multivariate statistical analysis showed that fresh RR,raw RR and RRP could be distinguished from each other by the contents of the 7 sugars,and the main differential components were stachyose,sucrose,raffinose and manninotriose.Conclusion In terms of oligosaccharides,the contents and types of saccharides in different herbs and decoction pieces of RR are quite different,and the TLC identification method based on this can be used to distinguish raw RR from RRP,which can lay a foundation for improving the quality standard of RR and developing and applying oligosaccharides in different processed products of RR.