Noguchi Kanta, Imahori Daisuke, Kido Yusuke, Nuntawong Poomraphie, Tanaka Hiroyuki, Morimoto Satoshi, Sakamoto Seiichi
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
Faculty of Pharmaceutical Sciences, Sanyo-Onoda City University, Yamaguchi, Japan.
Phytochem Anal. 2023 Aug;34(6):652-660. doi: 10.1002/pca.3255. Epub 2023 Jun 24.
Hesperidin (hesperetin 7-rutinoside, HP), a flavonoid glycoside found in Citrus unshiu Marcowicz or Citrus reticulata Blanco (Rutaceae), has been reported to exert a variety of pharmacological effects. As the efficacies and qualities of their dried peel, Chinpi and its derived Kampo medicines can be evaluated by their HP contents, a method for HP detection must be developed.
To produce a specific monoclonal antibody against HP (mAb 5D12) to detect the HP contents in Japanese traditional medicines via indirect competitive enzyme-linked immunosorbent assay (icELISA).
BALB/c mice were immunised with many haptens of HP-bovine serum albumin (BSA) conjugates that were prepared using sodium periodate (NaIO ) to cause an immune response. In addition, conventional hybridoma techniques were utilised to generate mAb 5D12.
The detection range of HP by the mAb 5D12-based icELISA was 1.56-25.0 ng/mL, with a detection limit of 1.12 ng/mL. The maximum coefficient of variation, as evaluated from the intra- and inter-assays, was <10.0%, and the percentages of recovery, as determined by the spike-recovery tests, were 105%-115%. Moreover, the HP content, which was obtained from the developed icELISA, correlated well with that obtained via high-performance liquid chromatography-ultraviolet (HPLC-UV).
These validation analyses revealed that the established icELISA technique exhibited high precision and accuracy. Notably, this is the first report on the development of icELISA for the HP content-based quality control of Chinpi and its derived Kampo medicines.
橙皮苷（橙皮素7-芸香糖苷，HP）是一种在温州蜜柑（Citrus unshiu Marcowicz）或柑橘（Citrus reticulata Blanco，芸香科）中发现的黄酮糖苷，据报道具有多种药理作用。由于陈皮及其衍生的汉方药物的干燥果皮的功效和质量可以通过其HP含量来评估，因此必须开发一种HP检测方法。
制备一种针对HP的特异性单克隆抗体（mAb 5D12），通过间接竞争酶联免疫吸附测定（icELISA）检测日本传统药物中的HP含量。
用许多经高碘酸钠（NaIO）制备的HP-牛血清白蛋白（BSA）偶联物半抗原免疫BALB/c小鼠，以引起免疫反应。此外，利用传统的杂交瘤技术产生mAb 5D12。
基于mAb 5D12的icELISA对HP的检测范围为1.56 - 25.0 ng/mL，检测限为1.12 ng/mL。通过批内和批间测定评估的最大变异系数<10.0%，通过加标回收试验测定的回收率为105% - 115%。此外，通过开发的icELISA获得的HP含量与通过高效液相色谱-紫外（HPLC-UV）获得的HP含量相关性良好。
这些验证分析表明，所建立的icELISA技术具有很高的精密度和准确性。值得注意的是，这是关于开发icELISA用于基于HP含量的陈皮及其衍生汉方药物质量控制的首次报道。
