中成药中甘草成份的免疫学检测方法的研究(英文)

USING IMMUNOLOGICAL METHOD FOR DETECTION AND QUANTITATIVE MEASUREMENT OF THE CONTENTS OF GLYCYRRHIZAE RADIX COMPONENT COMPOSING TRADITIONAL CHINESE MEDICINES

通过 ELISA和免疫印迹法分析确定了甘草蛋白 ( GRP)的特异性 ,并建立 GRP的竞争型含量分析 ELISA法 .该 ELISA法显示了良好的特异性、再现性 ( c.v.=3.9% )和检测限 ( 4 μg· L- 1 ) ,证明 GRP为甘草的特异性成分 .用该法测定 GRP同 HPLC法测定甘草甜素在三种中成药中的含量相比呈现良好的相关性 .该法简便精确快速并可同时进行大样本分析 ,为中成药中甘草成份的分析提供一种新方法 .

Development of an immunological method to detection and quantitative measurement of the contents of Glycyrrhizae radix (GR) component composing traditional Chinese medicines (TCM). A characteristic protein, Glycyrrhizae radix protein (GRP), present in GR used as solid-phase for the development of an enzyme-linked immunosorbent assay (ELISA) for GRP in TCMs. The assay principle is based on competition between an analyte and the GRP for an antibody specific to GRP in anti-GR serum, followed by immunoreaction with horseradish peroxidase (HRP)-labeled goat anti-rabbit immunoglobulin. The ELISA was showed no cross-reactivity with any leguminosae crude drug other than GR, which suggested that GRP is a characteristic compound only present in GR. This ELISA detected GRP with excellent reproducibility (coefficient of variation= 3.9%), an EC 50 of 33 μg·L -1and a detection limit of 4 μg·L -1. The stability was confirmed that GRP existed in TCM stably by using the ELISA and western blot method, respectively. The utility of the method was demonstrated by measuring levels of GRP in GR extract and three kinds of TCMs. While the GRP contents were compared with glycyrrhizin, it showed a good correlation between them. Using GRP as a maker, the ELISA is simple, specific, accurate method, and can easily be used to analyze large numbers of specimens in parallel. It should thus show a new potential to study the GR-based TCM by immunological method.