目的 基于HPLC特征图谱,建立同时测定桑黄中原儿茶酸、原儿茶醛、咖啡酸、紫萁酮和牛奶树碱5种成分含量的一测多评方法(quantitative analysis of multi-components with a single-marker,QAMS),对不同来源的桑黄药材进行品质评价研究.方法 收集了 3个种源地共10个批次的桑黄药材样品,采用Wondasil C18 Superb色谱柱(4.6 mm ×250 mm,5 µm),以乙腈-0.1％甲酸水溶液为流动相,梯度洗脱,体积流量1.0mL·min-1,柱温25 ℃,进样量20 μL,采用变波长程序,建立内参物原儿茶醛与原儿茶酸、咖啡酸、紫萁酮、牛奶树碱的相对校正因子并进行了相对校正因子的耐用性考察.采用外标法(external standard method,ESM)和QAMS法分别测定不同来源共10批桑黄药材中该5种成分的含量以验证QAMS法的准确性.结果 采用SPSS与Origin Pro进行主成分分析、相似度分析和聚类分析.结果 显示,5种成分专属性、精密度、重复性、稳定性、线性范围(r2＞0.999 0)均良好、平均加样回收率99.74％～102.78％,RSD 1.34％～2.92％.以原儿茶醛为内参物,原儿茶酸、咖啡酸、紫萁酮、牛奶树碱的平均相对校正因子分别为0.851 9、1.446 9、1.615 7、0.774 7;t检验结果表明,ESM法与QAMS法测定结果差异不显著.通过统计学分析实现了数据集的可视化,聚类分析发现西藏和广东产地的桑黄样品呈现一定的地域差异,但也发现了源于广东和长白山的个别桑黄样品与源于西藏的桑黄样品聚为一类的现象,意味着不能仅仅依据产地评价桑黄品质优劣,因为可能存在基源不清或品种混淆的状况.不同桑黄样品中牛奶树碱、原儿茶醛、紫萁酮的含量差异较大,且此3类成分对主成分的贡献较多,可能为桑黄的主要质量标志物.结论 该研究基于HPLC指纹图谱建立的QAMS法简单、经济、实用,可以实现对桑黄中主要成分的快速、科学分析,也为其种质资源的品质评价提供了一定的科学依据.
Quality Evaluation of Sanghuangporus by HPLC Fingerprint Combined with Quantitative Analysis of Multi-Components with a Single-Marker Method
OBJECTIVE To establish a quantitative analysis of multi-components by a single-marker method(QAMS)for simul-taneous determination of protocatechuic acid,3,4-dihydroxybenzaldehyde,caffeic acid,osmundacetone,and hispidin in Sanghuang-porus based on HPLC fingerprint analysis of Sanghuangporus samples.METHODS The analysis was performed on an HPLC system equipped with Wondasil C18 Superb column(4.6 nm ×250 mm,5µm),the mobile phase consisted of acetonitrile and 0.1％formic acid solution,and gradient elution procedure was employed.The flow rate was set at 1.0 mL·min-1,column temperature was main-tained at 25℃,and the sample injection volume was 20 μL.The relative correction factors of protocatechuic acid,caffeic acid,osmun-dacetone and hispidin were calculated with 3,4-dihydroxybenzaldehyde as the internal reference,and the durability of the established method were also investigated.The contents of these five compounds in ten batches of Sanghuangporus from different producing areas or batches were determined by external standard method(ESM)and QAMS method,respectively.RESULTS SPSS,SIMCA and Origin Pro software were employed for principal components assay,similarity evaluation and cluster analysis.The specificity,precision,repeatability,stability and linear range(r2＞0.999 0)of the five components were all good.The average recovery was between 99.74％-102.78％and RSD value was between 1.34％-2.92％,respectively.Then 3,4-dihydroxybenzaldehyde was chosen as the internal reference for calculating the correction factors for the other four components,the average relative correction factors of protocate-chuic acid,caffeic acid,osmundacetone,and hispidin were 0.851 9,1.446 9,1.615 7,and 0.774 7,respectively.Student's t-test results showed that there was no significant difference between the data analyzed by ESM method and the data obtained from QAMS method.Through data visualization analysis,the cluster analysis indicated that samples from Tibet and samples from Guangdong were clustered in significantly different categories.However,there was still one batch Sanghuangporus sample from Guangdong and one batch from Changbai moutains showing more similarities to Tibet category.This indicated that there was a certain relationship between the quality of Sanghuangporus and the origins of Sanghuangporus samples,but there were still other factors affecting the quality of San-ghuangporus such as the confusing species or unclear basic sources.The contents of hispidin,3,4-dihydroxybenzaldehyde and osmun-dacetone in different samples showed significant differences,indicating that these three components might be the main quality markers of Sanghuangporus for giving more contributes to the principal components.CONCLUSION Based on HPLC fingerprint,the method of QAMS established in this study is a simple,economical and practical method with scientific and applicable characteristics for evalua-ting the quality of Sanghuangporus efficiently and scientifically,which can also provide a certain scientific basis for the quality evalua-tion of its germplasm resources.
Sanghuangporusfingerprint analysisquantitative analysis of multi-components with a single-markerexternal standard methodrelative correction factorquality evaluation
万方数据
维普
