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Estrogen Bioactivity in Fo-Ti and Other Herbs Used for Their Estrogen-Like Effects as Determined by a Recombinant Cell Bioassay

University of California–San Diego, San Diego, California 92123

Address all correspondence and requests for reprints to: Karen Oerter Klein, M.D., Pediatric Endocrinologist, Children’s Hospital and Health Center, 3020 Children’s Way–MC 5103, San Diego, California 92123.

	Abstract

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One of the most important issues in women’s health concerns the risks and benefits of estrogen replacement therapy. Continual uncertainty and lack of consensus regarding estrogen replacement therapy has driven many women to seek alternative sources of estrogen, including herbal remedies. We adapted a recombinant cell bioassay to measure estrogen bioactivity in herbs. We studied, in vitro, estrogen bioactivity in red clover, dong quai, black cohosh, soy, licorice, chaste tree berry, fo-ti, and hops. Soy, clover, licorice, and hops have a large amount of measurable estrogen bioactivity, as suspected, based on previous reports using other methods. We discovered surprisingly high estrogen activity in extracts of fo-ti not previously reported. Chaste tree berry, black cohosh, and dong quai did not have measurable activity with this method. We also discovered that removal of a glycone group from soy increases its estrogen bioactivity significantly. We conclude that this recombinant cell bioassay for estradiol can be used to measure bioactivity in herbal products. The preparations of fo-ti studied had estrogen activity of 409 ± 55 pmol/liter estradiol equivalents per microgram of herb, which is 1/300th the activity of 17ß-estradiol. Clinical studies are underway to determine the estrogen bioactivity in women using dietary supplements containing these herbs.

	Introduction

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ONE OF THE most important issues in women’s health concerns the risks and benefits of estrogen replacement therapy. However, continual uncertainty and lack of consensus of standard estrogen replacement therapy has driven many women to seek alternative sources of estrogen, including herbal remedies (1). There are several herbs used in the available preparations, of which the most common include: red clover, dong quai, cohosh, soy, licorice, and chaste tree berry. Despite considerable history of herbs used for their estrogen-like effects, only recently have there been reports of measurement of estrogenic activity in some of these herbs (2, 3, 4, 5, 6, 7, 8, 9). In addition, there have not been many controlled studies of the effect of these preparations in women. There remains no consensus regarding the use, the dosing, or the long-term safety of these supplements.

Previously, we have demonstrated the use of a recombinant cell bioassay for estradiol to assess the very low levels of estradiol in the serum of children and postmenopausal women (10, 11, 12, 13, 14, 15, 16). This assay measures estrogen activity through binding of estrogen receptor (ER) in a yeast system transformed with a plasmid coding estrogen response elements and a plasmid coding for ER (17). Using an adaptation of this methodology, we have attempted to assess the bioactivity of estrogen in the following herbs: red clover (Trifolium pretense L.), dong quai (Angelica sinensis), black cohosh (Cimicifuga racemosa), soy, licorice (Glycyrrhiza glabra), chaste tree berry (Vitex agnus-castus L.), fo-ti (Polygonum multiflorum), and hops (Humulus lupulus L.).

	Materials and Methods

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Herb extraction

Chaste tree berry, black cohosh, red clover, dong quai, fo-ti, licorice, hops, soy, and soy aglycone (SoyA) were analyzed for estrogen activity as follows. Dried herb extracts were obtained from Natural Alternative International (San Marcos, CA) before compounding into dietary supplement capsules. Sixty milligrams of each herb were used for extraction, with the exception of soy and SoyA, for which 3 mg were used. All herbs were extracted with 2 ml methanol, except for licorice, which was extracted with 2 ml ether. Herb and solvent were vortexed for 2 min and centrifuged at 3000 rpm for 10 min. Supernatant was removed and filtered through a 0.45-µm glass syringe filter and stored at room temperature for use.

Estrogen assay

The bioassay for estradiol was described previously in detail (17). Estrogen activity is defined by ER binding in a genetically engineered yeast system. Briefly, it uses a strain of Saccharomyces cerevisiae that is transformed with two plasmids. One plasmid contains the human ER complementary DNA, and the other contains an estrogen response element upstream of the yeast iso-1-cytochrome C promoter fused to the structural gene for ß-galactosidase. The transformed yeast is grown in selective media in the presence of herb extract. ß-galactosidase activity is assayed and converted to estradiol equivalent units by linear interpolation from a standard curve constructed by adding known amounts of estradiol to assay media. The sensitivity of the bioassay was 0.02–2 pg/ml (0.07–7 pmol/liter) during the time of the study.

Statistics

Each herb extract was analyzed in triplicate and repeated over three consecutive assays. Estradiol equivalents were calculated by interpolation from a standard curve constructed in assay media with known amounts of 17ß-estradiol. (Fig. 1). Therefore, the results are expressed in estradiol equivalents in picomoles per liter per microgram of dry herb extract. All data are expressed as the mean ± SD. Comparisons between groups were made using a multivariate ANOVA with post hoc correction (least statistical difference). Differences were considered significant at P < 0.05.

View larger version (23K): [in this window] [in a new window]   FIG. 1. Standard curve constructed in assay media with 17ß-estradiol standards. Interpolation of unknown represented. Unknown depicted here could represent any of the herbs studied, because all were extracted to fall in this portion of the standard curve.

	Results

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View larger version (18K): [in this window] [in a new window]   FIG. 2. Estrogen activity in five of the herbs. The bars represent mean ± SE. The bars with different letters are significantly different from each other. P < 0.001 for SoyA; P < 0.05 for other herbs. The conversion factor to metric units is 1/3.671.

	Discussion

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Using an ultrasensitive bioassay, we have measured estrogen bioactivity in several herbs commonly used for their estrogen-like effect, including soy, clover, licorice, and hops. Removal of a glycone group in soy, SoyA, significantly increased the estrogen activity. In addition, we discovered that fo-ti contained a large amount of estrogen activity that has not been described previously. The estrogen bioactivity of fo-ti was found to be approximately 1/300th that of estradiol itself.

Since the initial identification of estrogen activity in plant extracts in 1926, numerous plants have been described with estrogenic properties (18, 19). In the early 1980s, detection of plant estrogens was first demonstrated in human urine using gas chromatography-mass spectrometry and nuclear magnetic resonance, which clearly showed that diet may be a source of estrogen (20, 21). Subsequent efforts to quantify estrogen bioactivity in plants and herbal preparation have been limited by inadequate methodological techniques, variation in biopotency among plant products, and different measures of relative estrogen bioactivity. Recently developed techniques have permitted detection of specific quantities of estrogen and estrogen bioactivity in plant-derived products, which are commercially available to the consumer. Estrogenic activity has been demonstrated previously in some of these herbs using other techniques. Competitive binding for ER in an endometrial cell line was exhibited by extracts of red clover, hops, and chaste tree berry, whereas dong quai and licorice had weak binding affinity, and black cohosh showed no binding affinity (5). Glabridin, the major isoflavone in licorice, was found to have estrogenic properties, as measured by competition for ER in human breast cancer cells (T47D) (6). Licorice and red clover extracts acted like estrogen agonists by stimulating cell proliferation in breast cancer cell lines (MCF-7 and T47D). Dong quai and black cohosh had little ER binding in this system. Estrogenic activity in hops has been described using a human endometrial cell line (Ishikawa Var I) (7) and using a radioreceptor assay with MCF7 cells (8). A similar yeast system was used to show estrogen activity in red clover extracts, which the present study confirms (9). By comparison, Amato et al. (3) suggested that growth of MCF-7 cells may have been independent of estrogenic activity in dong quai and ginseng. These herbs showed no estrogenic activity in a gene expression system using HeLa cells cotransfected with an estrogen-dependent reporter plasmid and human ER or ERß (3). In addition, exposure to black cohosh or licorice failed to stimulate growth of MCF-7 cells and had no effect on HeLa cells.

Herbal preparations are notoriously variable from batch to batch, which leads to difficulties in standardizing products. Our results confirm this variability in the two lots of soy tested. The bioassay used in this study can be used to test herb batches before product compounding to increase standardization of these products. In addition, herb products used in future clinical studies will first be tested in vitro for estrogen activity. The present study reports the ability to measure estrogen activity using a yeast bioassay and the relative amounts of activity in several herbs. It does not address the range of variability possible for each herb.

The present study confirms estrogen bioactivity in soy, clover, licorice, hops, and fo-ti. Our results are consistent with the relatively lower amounts of estrogenic activity in licorice and hops as reported previously. The lack of measurable estrogen bioactivity in black cohosh may explain the failure of this herb to exhibit ER binding or bioactivity in endometrial and breast cell culture systems. Estrogen activity in fo-ti was equivalent to that observed for soy and red clover. This herb has not been studied previously by any system, and our finding suggests that fo-ti may be another candidate for alternative medicine therapy in the menopause. The finding that the removal of a glycone group from soy significantly increased its estrogen bioactivity suggests a possible benefit of this procedure in products manufactured for menopausal dietary supplementation. However, the present study does not address the efficacy or the safety of such products in women. Clinical studies are necessary to test the implications of these in vitro findings.

	Acknowledgments

 
We thank Drs. John Wise and Zimin Liu (Natural Alternatives International, San Marcos, CA) for supplying herb products and financial support for this study.

	Footnotes

 
This work was supported in part by a gift from Natural Alternatives International.

Abbreviations: ER, Estrogen receptor; SoyA, soy aglycone.

Received February 28, 2003.

Accepted May 28, 2003.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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21. Setchell KDR, Borriello SP, Kirk DN, Axelson M 1984 Non-steroidal estrogens of dietary origin: possible roles in hormone dependent disease. Am J Clin Nutr 40:569–578[Abstract/Free Full Text]

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Klein, K. O. Articles by Chang, R. J. Search for Related Content PubMed PubMed Citation Articles by Klein, K. O. Articles by Chang, R. J. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB ESTRADIOL Medline Plus Health Information Herbal Medicine