A Reporter Gene Assay for the Detection of Phytoestrogens in Traditional Chinese Medicine
D. M. Miller-Martini, R. Y. K. Chan, N. Y. Ip, S. J. Sheu, and Y. H. Wong
Bupleurum & Peony Formula (Jia Wei Xiao Yao San) is a herbal formula which possesses a clinical history for the treatment of menopausal syndrome and menstrual irregularity. The present investigation reports the ability to monitor the formula’s phytoestrogen content that will allow for the implementation of a standardization protocol that is based on a quantifiable biological response. Utilizing an oestrogensensitive chimeric receptor/reporter gene element which has been stably transfected into HeLa cells, the botanical formula was shown to induce the expression of the reporter gene, luciferase, in a dose dependent manner. Pretreatment of the HeLa cells with the botanical formula produced a 5-fold increase in bioluminescence compared with the control. Additionally, our studies showed that the response of the cells, when challenged by the botanical formula, was oestrogen specific. Pretreatment of the cells with tamoxifen effectively blocked the activation of the chimeric oestrogen receptor by the botanical formula. The cell line provides a sensitive assay that can easily detect the presence of phytoestrogens in complex botanical formulas.
Copyright © 2001 Miller-Martini et al., Published by Phytotherapy Research,
John Wiley & Sons, Ltd.
2. Material and Methods
1. Preparation of stock solutions of Bupleurum & Peony Formula: Bupleurum & Peony Formula used in the present study was donated by the Sun Ten Pharmaceutical Co. Limited, Taiwan. 4% (w/v) stock solutions were prepared for several production lots of the Bupleurum & Peony Formula.
2. Chimeric constructs and host cell line
3. Pretreatment of the chimeric/reporter gene HeLa cell line with stock solutions
4. Luciferase reporter gene assay
1. Determination of the responsiveness of the chimeric receptor/reporter gene in stably transfected HeLa cells
2. Analysis of the phytoestrogen content of Bupleurum & Peony Formula
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