Sakamoto Seiichi, Wada Shinji, Tanaka Hiroyuki, Morimoto Satoshi
Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University 3-1-1 Maidashi, Higashi-ku Fukuoka 812-8582 Japan
RSC Adv. 2018 May 14;8(31):17410-17416. doi: 10.1039/c8ra02922a. eCollection 2018 May 9.
Amarogentin (AG) is a naturally occurring secoiridoid glycoside produced mainly in the plant genera and . Extracts of these plants have a long history of use in Japan as bitter stomachics because of their strong bitterness. Because the AG content directly reflects the potential activity of the extract, the quality control of these plant extracts through the quantitative analysis of AG is important. In the present study, we aimed to develop a quantitative analysis of AG using a monoclonal antibody (MAb) against AG (MAb 1E9) in the plant family Gentianaceae. Surprisingly, production of MAb 1E9 was successfully achieved by immunizing AG-bovine serum albumin (BSA) conjugates into mice although the number of AG bound to BSA was only one. The characterization of MAb 1E9 revealed that it shows high specificity to AG, enabling the development of an icELISA for the specific determination of AG. In addition, the detectable concentration of AG in the developed icELISA ranged from 1.95 to 62.5 ng mL with a limit of detection of 1.28 ng mL, which is approximately 30-625 times higher in sensitivity compared with the conventional HPLC method. Validation analysis revealed that the icELISA using MAb 1E9 is precise (intra-assay variation <3.9%, inter-assay variation <8.8%) and accurate (recovery rates of spiked samples were between 91.0% and 106.4%). This method can be used for the quality control of plant extracts using AG as an index.
苦味龙胆酯苷（AG）是一种天然存在的裂环烯醚萜苷，主要在[具体植物属]植物中产生。由于其强烈的苦味，这些植物的提取物在日本作为苦味健胃剂有着悠久的使用历史。由于AG含量直接反映提取物的潜在活性，通过对AG进行定量分析来控制这些植物提取物的质量很重要。在本研究中，我们旨在开发一种利用抗AG单克隆抗体（MAb 1E9）对龙胆科植物中的AG进行定量分析的方法。令人惊讶的是，尽管与牛血清白蛋白（BSA）结合的AG数量只有一个，但通过将AG - BSA偶联物免疫小鼠，成功制备了MAb 1E9。对MAb 1E9的特性表征表明，它对AG具有高度特异性，从而能够开发一种用于特异性测定AG的间接竞争酶联免疫吸附测定（icELISA）。此外，所开发的icELISA中AG的可检测浓度范围为1.95至62.5 ng/mL，检测限为1.28 ng/mL，与传统的高效液相色谱法相比，灵敏度提高了约30 - 625倍。验证分析表明，使用MAb 1E9的icELISA具有精确性（批内变异<3.9%，批间变异<8.8%）和准确性（加标样品的回收率在91.0%至106.4%之间）。该方法可用于以AG为指标的植物提取物的质量控制。
