Liu Li-Na, Wang Ying, Jin Hong-Yu, Ma Shuang-Cheng, Liu Jia-Peng
Department of Institute for Control of Chinese Traditional Medicine and Ethnic Medicine, National Institutes for Food and Drug Control, Beijing 100050, China.
Huaan Magnech Bio-Tech Co., Ltd, Beijing 102200, China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2016 May 15;1021:122-128. doi: 10.1016/j.jchromb.2015.10.005. Epub 2015 Oct 22.
In the present study, the feasibility of immunoaffinity chromatography (IAC) as a purification technology for the analysis of bioactive components in Traditional Chinese Medicine (TCM) was evaluated. IAC was used to analyze hesperidin (HP) and narirutin (NR) in TCM preparations containing Citri reticulatae Pericarpium (CRP, Chenpi in Chinese). An IAC column for the specific extraction and enrichment of HP and NR from TCM preparations containing CRP was developed and characterized. After HP reacted with carbonyl diimidazole and coupled to protein, it was used to immune mice for the generation of antibody. Through cell fusion, cloning and screening, monoclonal antibody was obtained. The IAC column was constructed by covalently coupling specific monoclonal antibody against HP and NR to CNBr-activated Sepharose 4B and packed into a common solid phase extraction cartridge. The extraction conditions including loading, washing and eluting, as well as flow rate for the extraction of HP and NR were optimized. Under optimal conditions, the maximum capacity, extraction recovery rate and stability of IAC column was also characterized. Results revealed that the maximum capacity of IAC column for HP and NR was approximately 16μg and the relative binding capacity per 1mL of the column volume was 27μg. The extraction recovery rate of IAC column for HP and NR at three spiked levels was in the range of 94.05-109.15%. After the repeated application for 5 times, no significant loss of specific recognition was observed. Using high performance liquid chromatography (HPLC) as an effective analytic tool, HP and NR could be successfully separated via IAC column without the inference from impurities, suggesting that the extraction of HP and NR using the prepared IAC column is feasible. The application of IAC can solve the problem of quantitative analysis due to severe interference or low content. Furthermore, pretreatment methods in different matrixes can be unified. The IAC purification procedure can be used as an alternative effective analytical method for the pretreatment of bioactive components in TCM.
在本研究中，评估了免疫亲和色谱法（IAC）作为一种用于分析中药（TCM）中生物活性成分的纯化技术的可行性。IAC用于分析含有陈皮（Citri reticulatae Pericarpium，CRP，中文为陈皮）的中药制剂中的橙皮苷（HP）和柚皮芸香苷（NR）。开发并表征了一种用于从含有CRP的中药制剂中特异性提取和富集HP和NR的IAC柱。HP与羰基二咪唑反应并偶联到蛋白质上后，用于免疫小鼠以产生抗体。通过细胞融合、克隆和筛选，获得了单克隆抗体。IAC柱通过将针对HP和NR的特异性单克隆抗体共价偶联到溴化氰活化的琼脂糖凝胶4B上构建而成，并装填到普通的固相萃取柱中。优化了包括上样、洗涤和洗脱在内的提取条件以及HP和NR提取的流速。在最佳条件下，还表征了IAC柱的最大容量、提取回收率和稳定性。结果表明，IAC柱对HP和NR的最大容量约为16μg，每1mL柱体积的相对结合容量为27μg。IAC柱对HP和NR在三个加标水平下的提取回收率在94.05 - 109.15%范围内。重复使用5次后，未观察到特异性识别的明显损失。使用高效液相色谱（HPLC）作为有效的分析工具，HP和NR可通过IAC柱成功分离，不受杂质干扰，表明使用制备的IAC柱提取HP和NR是可行的。IAC的应用可以解决由于严重干扰或含量低导致的定量分析问题。此外，不同基质中的预处理方法可以统一。IAC纯化程序可作为一种替代的有效分析方法用于中药中生物活性成分的预处理。
