title: Enzyme-linked immunosorbent assay by enhanced chemiluminescence detection for the standardization of estrogenic miroestrol in Pueraria candollei Graham ex Benth.
abstract: Miroestrol (ME) is a potent phytoestrogen from the P. candollei tuberous root. It has been approved for use in clinical trials due to its beneficial effect on disorders associated with estrogen deficiency. To ensure medical efficacy and safety, high performance analytical methods for ME analysis are required to standardize products from the P. candollei root. An enhanced chemiluminescence enzyme-linked immunosorbent assay (ECL-ELISA) was developed and validated using a polyclonal antibody against ME and a chemiluminescent system of luminol-H2 O2 -horseradish peroxidase-4-(1-imidazolyl) phenol. The ECL-ELISA system exhibited linearity over a concentration range of 0.31-10.00 ng mL(-1) , for which the relative standard variation (%RSD) was less than 10% for both intra- and interplate determinations. The ECL-ELISA is reliable for the determination of ME as reflected by the high recovery percentage (101.22-103.06%). As a comparative analysis, the ME content in each sample determined by ECL-ELISA was correlated with high coefficients of determination with colorimetric ELISA (R(2)  = 0.998) and high performance liquid chromatography (HPLC) (R(2)  = 0.998) methods. The ECL-ELISA method could be applied to all of the commercial products containing P. candollei root, when the products contain between 0.706 ± 0.046 and 13.123 ± 0.794 µg g(-1) dry wt. of ME. This method is useful as a high performance analytical method for the quantity control of ME in raw materials and end products at both the research and industrial levels.
authors.affiliation: 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, 40002, Thailand.
authors.forename: Gorawit
authors.initials: G
authors.lastname: Yusakul
authors.affiliation: 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, 40002, Thailand.
authors.forename: Orapin
authors.initials: O
authors.lastname: Udomsin
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, 812-8582, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Tanaka
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, 812-8582, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Morimoto
authors.affiliation: Faculty of Pharmaceutical Sciences, Ubon Rachathani University, Ubon Ratchathani, 34190, Thailand.
authors.forename: Thaweesak
authors.initials: T
authors.lastname: Juengwatanatrakul
authors.affiliation: 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, 40002, Thailand.
authors.forename: Waraporn
authors.initials: W
authors.lastname: Putalun
mesh_terms: D002851:Chromatography, High Pressure Liquid; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards; D006735:Horseradish Peroxidase / Q000737:chemistry; D049449:Luminescence; D008165:Luminol / Q000737:chemistry; D018517:Plant Roots / Q000737:chemistry; D010946:Plants, Medicinal / Q000737:chemistry; D029908:Pueraria / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013256:Steroids / Q000032:analysis* / Q000276:immunology
chemical_list: D013256:Steroids; C571572:miroestrol; D008165:Luminol; D006735:Horseradish Peroxidase
keywords: Pueraria candollei; enhanced chemiluminescence; enzyme-linked immunosorbent assay; miroestrol