Miroestrol is a chromene with potent estrogenic activity present in , commonly known as White Kwao Krua. Although this compound is only present in low amounts in the plant, it plays an important role in the estrogenic action of products. As a tool for further studies about the efficacy and safety of as a phytoestrogenic supplement, we generated a novel monoclonal antibody against miroestrol. This anti-miroestrol monoclonal antibody was used to develop an immunoassay for the determination of miroestrol content, which can be used for quality control purposes of. The developed ELISA against miroestrol has a calibration range of 10-780 ng/mL miroestrol, a limit of detection of 3.5 ng/mL, and a limit of quantitation of 12.2 ng/mL. According to the validation analysis, the established ELISA is precise, accurate, specific, and sensitive for miroestrol detection in plants. Furthermore, the anti-miroestrol monoclonal antibody was used to prepare an immunoaffinity column for the isolation of miroestrol from the tuberous root of . The column provides a simple procedure for miroestrol isolation, with a capacity of 3.91 µg of miroestrol per 1 mL of immunogel.

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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).

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Pueraria candollei or White Kwao Krua (Leguminosae) is an indigenous plant in Thailand which has long been used in Thai traditional medicine. The tuberous root of this plant is widely used for rejuvenation, particularly in elder women. Among the bioactive compounds in P. candollei, miroestrol and puerarin exhibit estrogenic activity. This study aims to develop an immunochromatographic strip (ICS) with a colloidal gold-based detection system for the simultaneous detection of miroestrol and puerarin in a one-step analysis. The developed method is sensitive and specific for the detection of miroestrol and puerarin in raw materials and marketed products. The detection limits of miroestrol and puerarin were 0.15 and 4.5 μg, respectively. In addition, the results from the developed ICS were confirmed with an enzyme-linked immunosorbent assay and presented a good correlation between these two methods. This is the first report on the development of an ICS that can detect miroestrol and puerarin in one step. The developed ICS provides a simplified method for the detection of miroestrol and puerarin in P. candollei and Pueraria spp.