title: High performance enzyme-linked immunosorbent assay for determination of miroestrol, a potent phytoestrogen from Pueraria candollei.
abstract: Pueraria candollei associated preparation is widely applied in folk Thai medicine for rejuvenating purpose in aged people, which correlated with its pharmacological activities reported by pre-clinical and clinical trials. Therefore, standardized products of this plant are needed by consumers and health care personnel. Miroestrol, a potent and stable phytoestrogen in P. candollei, exhibited potential to be biomarker for quality control of P. candollei samples in research or industrial levels. Indirect competitive enzyme-linked immunosorbant assay (ELISA) for miroestrol determination was developed and validated by using polyclonal antibody from rabbit immunization. The polyclonal antibody recognized specifically to miroestrol, which exhibited cross-reactivity to deoxymiroestrol and isomiroestrol with 6.68% and 1.05%, respectively. The linearity range of measurement was 0.73-3000 ng mL(-1), which coefficient of variation (CV) of both intra- and inter-plate determination was less than 5%. With spiked samples of known amount miroestrol, the percentages of recovery were 98.80-104.37% and 98.31-106.69% in P. candollei and its involved product samples, respectively. Validated ELISA was comparable with published HPLC method (R(2)=0.9996) (Yusakul et al.) in samples with various miroestrol contents. For application, the P. candollei involved preparations contained miroestrol 0.695±0.037-12.108±0.285 μg g(-1) dry wt. The developed ELISA was high performance for miroestrol determination, which could be applied for P. candollei quality control in research fields and industrial productions.
authors.affiliation: Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand.
authors.forename: Gorawit
authors.initials: G
authors.lastname: Yusakul
authors.forename: Orapin
authors.initials: O
authors.lastname: Udomsin
authors.forename: Thaweesak
authors.initials: T
authors.lastname: Juengwatanatrakul
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Tanaka
authors.forename: Chaiyo
authors.initials: C
authors.lastname: Chaichantipyuth
authors.forename: Waraporn
authors.initials: W
authors.lastname: Putalun
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards; D048789:Phytoestrogens / Q000032:analysis*; D018517:Plant Roots / Q000737:chemistry; D029908:Pueraria / Q000737:chemistry*; D011786:Quality Control; D011817:Rabbits; D013256:Steroids / Q000032:analysis* / Q000276:immunology / Q000592:standards
chemical_list: D000906:Antibodies; D048789:Phytoestrogens; D013256:Steroids; C571572:miroestrol