Planta Med 2013; 79 - PK48
DOI: 10.1055/s-0033-1352308

Enzyme-linked immunosorbent assays for quality control of miroestrol and deoxymiroestrol in Pueraria candollei

G Yusakul 1, O Udomsin 1, T Juengwatanatrakul 2, H Tanaka 3, C Chaichantipyuth 4, W Putalun 1
  • 1Khon Kaen University, Faculty of Pharmaceutical Sciences, Khon Kaen 40002, Thailand
  • 2Ubon Rajathani University, Faculty of Pharmaceutical Sciences, Ubon Ratchathani 34190, Thailand
  • 3Kyushu University, Graduate School of Pharmaceutical Sciences, Fukuoka 812 – 8582, Japan
  • 4Chulalongkorn University, Faculty of Pharmaceutical Sciences, Bangkok 10330, Thailand

Miroestrol (ME) and deoxymiroestrol (DME) are the most potent estrogenic compounds in Pueraria candollei, which has been applied for long time in Thai folk medicine. The clinical trials showed that menopausal symptoms were declined significantly after treatment with different doses of P. candollei var. mirifica, whereas no significant side effects were observed. Therefore, standardized plant materials of ME and DME are necessary to researches and industrial production involved with P. candollei. Indirect competitive enzyme-linked immunosorbant assays (ELISAs) for determination of ME and DME were developed and validated by using polyclonal antibodies (PAbs) from rabbits. The PAb against ME recognized specifically to ME, which exhibited cross-reactivity to DME and isomiroestrol with 6.68% and 1.05% respectively. Similarly PAb against DME exhibited very low cross-reactivity to ME and isomiroestrol with 1.26% and 0.42% respectively. The linearity range of ELISAs for both ME and DME were 0.73 – 3,000 ng/ml, which the coefficient of variation (CV) were less than 5%. The percentages of recovery were 98.80 – 104.37% and 99.82 – 102.58% for ME and DME, respectively in P. candollei samples Validated ELISAs were comparable with HPLC method in samples with high ME and DME contents, which coefficient of determination were 0.9996 and 0.9993, respectively. In additional, ELISAs could analyze in the samples undetectable by HPLC. The ELISAs was applied for ME and DME determination in Pueraria spp. The developed ELISAs were high performance for ME and DME determination, which could be applied for P. candollei involved researches of pharmacology and dosage form development. With simple and inexpensive procedure of ELISA, it could be applied for raw material and final product quality control during production process of industrial level.