Kitisripanya Tharita, Inyai Chadathorn, Komaikul Jukrapun, Krittanai Supaluk, Juengwatanatrakul Thaweesak, Sakamoto Seiichi, Tanaka Hiroyuki, Morimoto Satoshi, Putalun Waraporn
Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, 40002, Thailand.
Research Group for Pharmaceutical Activities of Natural Products Using Pharmaceutical Biotechnology (PANPB), National Research University-Khon Kaen University, Khon Kaen, Thailand.
J Nat Med. 2017 Oct;71(4):659-664. doi: 10.1007/s11418-017-1096-3. Epub 2017 Jun 1.
White Kwao Krua (WKK)-derived products have been used worldwide as dietary supplements to relieve climacteric symptoms in menopausal women. Miroestrol is a unique chromene found in WKK tuberous roots that corresponds to the estrogenic activity of WKK. However, miroestrol naturally accumulates at low levels in WKK samples, which are difficult to detect. The development of a rapid and sensitive assay to detect miroestrol in numerous products derived from this plant would be a practical and useful method to guarantee the quality of raw materials. To allow rapid and easy qualitative detection of miroestrol, a lateral flow immunoassay (LFIA) using a colloidal gold-labeled monoclonal antibody (mAb) against miroestrol was developed. The qualitative LFIA was based on the competition of free miroestrol in the sample and immobilized miroestrol-conjugated proteins on the strip for a limited number of antibodies in the detection reagent. Anti-miroestrol mAb was colored by colloidal gold labels and used as the detection reagent in LFIA. Anti-mouse immunoglobulin G was used to indicate the functioning of the LFIA system. The detection limit of the LFIA was 0.156 μg of miroestrol. The LFIA was applied to determine the miroestrol content in WKK samples and products. The result was compared with the validated enzyme-linked immunosorbent assay (ELISA) and demonstrated a correlative outcome. This study shows that the developed LFIA is practical and suitable for detecting small amounts of miroestrol in WKK samples. This qualitative assay is more rapid in screening miroestrol in WKK samples (within 10 min) than conventional methods (ELISA and HPLC).
白高颗（WKK）衍生产品在全球范围内被用作膳食补充剂，以缓解绝经后女性的更年期症状。米罗雌醇是在WKK块根中发现的一种独特的色烯，它与WKK的雌激素活性相对应。然而，米罗雌醇在WKK样品中的自然积累水平较低，难以检测。开发一种快速灵敏的检测方法来检测源自这种植物的众多产品中的米罗雌醇，将是保证原材料质量的一种实用且有用的方法。为了能够快速简便地定性检测米罗雌醇，开发了一种使用针对米罗雌醇的胶体金标记单克隆抗体（mAb）的侧向流动免疫分析（LFIA）。定性LFIA基于样品中游离米罗雌醇与试纸条上固定的米罗雌醇偶联蛋白对检测试剂中有限数量抗体的竞争。抗米罗雌醇单克隆抗体用胶体金标记进行显色，并用作LFIA中的检测试剂。抗小鼠免疫球蛋白G用于指示LFIA系统的运行情况。LFIA的检测限为0.156μg米罗雌醇。将LFIA应用于测定WKK样品和产品中的米罗雌醇含量。将结果与经过验证的酶联免疫吸附测定（ELISA）进行比较，结果具有相关性。本研究表明，所开发的LFIA实用且适用于检测WKK样品中的少量米罗雌醇。这种定性分析方法在筛选WKK样品中的米罗雌醇时（10分钟内）比传统方法（ELISA和HPLC）更快。
