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Experimental Biology and Medicine 229:560-568 (2004)
© 2004 Society for Experimental Biology and Medicine

ORIGINAL RESEARCH ARTICLE

Mycotoxins in Root Extracts of American and Asian Ginseng Bind Estrogen Receptors {alpha} and ß

Sandra L. Gray*,{dagger},1, Brett R. Lackey{dagger}, Patricia L. Tate{ddagger}, Melissa B. Riley* and N. Dwight Camper*

* Department of Plant and Environmental Sciences, {dagger} Endocrine Physiology Laboratory, Department of Animal and Veterinary Sciences, and {ddagger} Department of Physics and Astronomy, Clemson University, Clemson, South Carolina 29634

1To whom requests for reprints should be addressed at Clemson University, Department of Animal and Veterinary Sciences, Poole Agricultural Building, Clemson, SC 29634. E-mail: sngry{at}clemson.edu


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Reference
 
The estrogenic activity of ginseng has been the subject of conflicting reports. Cell proliferation, induction of estrogen-responsive genes, and isolated cases of adverse reactions such as postmenopausal vaginal bleeding and gynecomastia have been reported after ginseng treatment. Other studies report antiproliferative effects with no induction of estrogen-responsive genes. We developed estrogen receptor (ER) {alpha} and ER{alpha} competitive binding assays using recombinant receptors and [3H]-17{alpha}-estradiol to detect phytoestrogens in extracts of Asian ginseng root (Panax ginseng C. A. Meyer) and American ginseng root (Panax quinquefolius L.). Root extracts contained substances that bound both receptor isoforms. These substances had a two to three times greater affinity for ER{alpha}. Significantly higher binding was found in methanol extracts than in hot water extracts. Subsequent analysis of the extracts revealed significant ER binding attributable to zearalenone, the estrogenic mycotoxin produced by several Fusarium species. The ER showed no binding affinity for Rb1 and Rg1, the major ginsenosides found in P. quinquefolius and P. ginseng, respectively. Thus, ginseng extraction methods, plant species tested, and mycotoxin contaminants may help to explain the disparate literature reports. The prevalence and health significance of fungal contamination in herbal products used for medicinal purposes should be further investigated.

Key Words: ginseng • estrogen receptor • zearalenone • phytoestrogen • herbal product


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Reference
 
Herbal remedies are used worldwide to alleviate symptoms, to treat illnesses, and to promote overall wellness. An estimated 60% of the world’s population and 40% of Americans use herbal remedies (1). However, use of medicinal herbs may be unsupervised and of unproved efficacy (2–7).

Ginseng is a popular herb with a long history of medicinal use; the Chinese have used ginseng for more than a thousand years to improve energy and longevity (8). In the United States, it ranks among the top five herbal products sold and is an ingredient in energy tonics and used as an immunostimulatory dietary supplement (9). Asian ginseng, Panax ginseng, is an official herbal remedy in the German Pharmacopoeia and is listed in the Commission E Monographs "as a tonic for invigoration and fortification in times of fatigue and debility, for declining capacity for work and concentration, also during convalescence" (10). American ginseng, Panax quinquefolius, has been recommended for alleviation of menopausal symptoms (8, 11–15). Ginseng is available in many forms, including capsules, powders, tinctures, teas, and whole roots. Commercial products, such as cosmetics, soaps, toothpaste, chewing gum, beverages, power bars, baby food, cigarettes, and coffee, may list ginseng as an ingredient.

Among the five major species of ginseng, P. ginseng, a native plant of Asia, and P. quinquefolius, native to North America, are used most frequently in herbal preparations (16). Historically, P. ginseng grew abundantly in China, Korea, and parts of Siberia. Today, the wild plant is rare, and most of the ginseng root is cultivated commercially. Similarly, P. quinquefolius initially grew wild in North America but is now cultivated for domestic use and for export.

Pharmacological actions of ginseng have been attributed to several active constituents, including the triterpene saponins. More than 30 unique triterpene saponins, named ginsenoside Rx according to their mobility on thin-layer chromatography plates, have been isolated from ginseng. Similarity of the ginseng molecule to the structure of some steroid hormones may contribute to the plant’s pharmacological actions (Fig. 1Go).



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  		Figure 1. Structures of estradiol-17ß, zearalenone, zearalenol, and the ginsenoside backbone. Rg1 and Rb1 are the major ginsenosides in P. ginseng and P. quinquefolius, respectively.

 
The endocrine activity of ginseng has been investigated. Panax quinquefolius may modulate insulin secretion to decrease postprandial glycemia in nondiabetic and type 2 diabetes mellitus patients (17, 18). Ginsenoside Rg1 is a functional ligand of the glucocorticoid receptor, synergistically activating gene transcription by cyclic AMP and downregulating glucocorticoid receptor content of cells (19, 20). Panax ginseng root extract bound to progesterone, mineral corticoid, and glucocorticoid receptors but not to androgen or estrogen receptors (ERs) isolated from rat uterine tissue (21). Patients treated with P. ginseng extracts had increased spermatozoa concentration and progressive motility, total and free plasma testosterone, dihydrotestosterone, follicle-stimulating hormone, and luteinizing hormone levels but decreased prolactin levels (22). Estradiol (E2) and P. quinquefolius extracts induced RNA expression of presenilin-2 (pS2) equally in ER-positive cells (23). Panax quinquefolius extract decreased the proliferation of the ER-positive breast cancer cell line, MCF-7, in one study (23), whereas a second study reported increased proliferation (24). Reports of estrogen-related sequelae from postmenopausal women after ginseng use include mastalgia, vaginal bleeding, and changes in the vaginal and cervical epithelium (25–28). In each case, symptoms ceased when use of the product was discontinued. Male gynecomastia has also been reported after ginseng use (29).

One method of ascertaining potential endocrine-modulating activity of a compound is competitive binding to hormone receptors. Although binding of ginsenosides to the glucocorticoid receptor has been examined, no studies have reported the competitive binding of P. quinquefolius and P. ginseng extracts to human ER{alpha} and ERß (20, 21). Investigating ginseng preparations for bioactive compounds capable of binding ER may explain estrogenic responses observed in individuals using ginseng.

Zearalenone (Fig. 1Go) is a widely distributed mycoestrogen produced by several species of Fusarium fungi. This mycotoxin contaminates food and agricultural commodities infected with the fungus. Zearalenone is rapidly transformed into {alpha}- and ß-zearalenol by 3{alpha}-hydroxysteroid dehydrogenase in the liver (30). These metabolites are more estrogenic than zearalenone, with {alpha}-zearalenol approximately three to four times higher than ß-zearalenol (31). The potency of zearalenone relative to E2 in the mouse uterotropic assay was approximately 0.001 (32). A bioassay based on the alkaline phosphatase gene induction by estrogens in the human endometrial Ishikawa cell line showed zearalenone and its metabolites were strongly estrogenic. In this study, {alpha}-zearalenol was as potent as ethinyl estradiol and diethylstilbestrol (33). In farm animals, zearalenone ingestion through contaminated feed is associated with decreased reproductive capacity and other hyperestrogenic conditions, such as vaginal swelling, enlargement of mammary glands, and testicular atrophy (34).

Herbal preparations, including ginseng extracts, are generally not screened for mycotoxins. The possibility exists that cultivation and storage of herbal products could result in Fusarium growth and subsequent zearalenone contamination. The goals of this study were to screen ginseng root extracts using a competitive binding assay for interaction with the ER and to elucidate the source of binding in the extracts using immunoassay and high-pressure liquid chromatography (HPLC).


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Reference
 
Plant Samples and Reagents.
Dried roots from 5-year-old plants (identified in these experiments as P. ginseng 1, P. ginseng 2, and P. quinquefolius 1) were obtained from commercial farms in Illinois and Indiana. Additionally, 20 roots of wild P. quinquefolius (approximately 5–9 years old and identified as P. quinquefolius 2) were collected in an intact, old growth forest at Table Rock Watershed, Oconee County, South Carolina. Wild-crafted plants were stored individually in unsealed plastic bags and allowed to air dry for 8 months at approximately 20°C.

All chemicals were from VWR International, Inc. (Atlanta, GA), Fisher Scientific (Atlanta, GA), or Sigma-Aldrich (St. Louis, MO) unless otherwise noted. Estradiol (2,4,6,7-3H[N]; 71 Ci/mM) (3H-E2) was purchased from NEN Life Sciences Products (Boston, MA). Human recombinant ER{alpha} and ERß were obtained from PanVera (Madison, WI), and ginsenoside Rg1 was purchased from Indofine (Belle Mead, NJ).

Preparation of Herbal Extracts.
Dried ginseng roots from each source were powdered (Mr. Coffee Grinder; Mr. Coffee, Minneapolis, MN) and stored separately in airtight glass containers at 4oC. Crude extracts of 1-g samples used for the competitive binding assays were extracted by using 10 ml of either deionized water (initially at 100oC) or 80% methanol (Mallinkrodt Laboratory Chemicals, Inc., Phillipsburg, NJ). Samples were mixed with solvent for approximately 8 hrs on a lateral shaker at room temperature and centrifuged at 1800 g for 15 mins, and the supernatant removed. The pellet was resuspended in 8 ml of solvent and mixed overnight in a lateral shaker. Supernatants from the two extractions were combined, placed in a Thermolyne Dri-Bath (VWR) at 40oC, and evaporated to dryness with filtered air. Samples were resuspended in 1 ml of ethanol and filtered using 0.45 µm of PVDF Acrodisc (Pall Gelman, Ann Arbor, MI). All dried samples were extracted and assayed three times.

Competitive Binding Assay.
Estrogen receptor binding studies were performed using the procedure of Obourn et al. with modifications (35). The assay binding buffer consisted of 10 mM Tris, pH 7.5, 10% glycerol, 2 mM dithiothreitol, and 0.1% bovine serum albumin. Varying concentrations of test samples were evaporated to dryness and reconstituted in 50 µl of binding buffer. Competition assay reactions included test samples evaporated to dryness and reconstituted in 50 µl of binding buffer, 50 µl of recombinant receptor {alpha} or ß, and 100 µl of 3H-E2 for a total incubation volume of 200 µl. Final concentrations in incubation volume were 4 nM ER{alpha} or ERß and 4.9 nM 3H-E2. Each extraction replicate was assayed in triplicate. Assays also included a standard curve using E2 concentrations at 0.0, 0.1, 0.2, 0.5, 1.0, 2.5, 5.0, 10.0, and 15 ng and nonspecific binding of 3H-E2. Nonspecific binding counts, determined by adding excess E2 (1 µg) to a set of assay tubes, were subtracted from total binding to determine specific binding. Standard concentrations (diluted in 100% ethanol) and plant extracts were added to assay tubes, evaporated to dryness under filtered air, and reconstituted in 50 µl of assay buffer. Incubations were conducted for 2 hrs at room temperature followed by 16 hrs at 4°C. Separation of bound and free steroid was performed by the addition of 1 ml of activated charcoal suspension (3%) in binding buffer. Samples were incubated for 5 mins on ice after charcoal addition and centrifuged for 10 mins at 1800 g. Supernatant was decanted into 7-ml scintillation vials, 4 ml of Ultima Gold (Packard, Meriden, CT) scintillation fluid was added, and samples were counted using a Beckman LS 1800 liquid scintillation counter (Irvine, CA). A five-parameter logistic standard curve was constructed using StatLIA Analysis software (Brendan Scientific, Carlsbad, CA). Concentration of test samples displacing approximately 50% 3H-E2 binding from each receptor was determined from the standard curve. The data represent the average ± SD of nine determinations.

Affinities of receptors and estrogenic ligands can be determined by measuring the equilibrium binding of the radioligand in the presence of different concentrations of the unlabeled ligand. These assays are conducted by determining the binding affinity of a test compound for the ER relative to a radiolabeled competitor with a known binding affinity. The plant tissue crude extracts may contain compounds that displace E2 from its receptor, but concentration of these compounds may be unknown. A convenient way of expressing the estrogenicity of such extracts is by calculation of estrogen binding equivalents (EBEs) in a 1-g sample. Different extract dilutions incubated with the radiolabeled E2 for receptor binding and EBEs are based on the extract dilution that displaces approximately 50% of the radioligand. Thus, EBEs represent a relative measure of ER binding potential by compounds from a 1-g plant tissue sample.

The ER{alpha} and ERß binding affinities to zearalenone and the metabolites {alpha}-zearalenol, ß-zearalenol, {alpha}-zearalanol, ß-zearalanol, and zearalanone were measured by assaying varying concentrations of each compound and determining the concentration required to displace 50% bound 3H-E2. Similarly, binding affinities for the estrogenic compounds 4-hydroxytamoxifen and diethylstilbestrol (DES) and the ginsenosides Rb1 and Rg1 were determined.

Fusarium Culture from Ginseng Roots.
Ginseng root sections were cultured for Fusarium according to the method of Nash and Snyder (36). Briefly, using aseptic techniques, 6-mm square sections were cut from root, dipped in 95% ethanol, and flame sterilized. Surface sterilized chips were transferred to 100 x 15-mm, plastic petri plates with potato dextrose agar (Difco Laboratories, Detroit, MI) and incubated for 8 days under normal laboratory lighting and temperature conditions. Fungal identification was made by culture pigmentation and microscopic morphologic analysis, including characteristic length and shape of the macroconidia, presence or absence of polyphialides, and number, shape, and arrangement of formation of microconidia. Three replicate plates were made for each root.

Enzyme-Linked Immunoabsorbent Assay (ELI-SA) Determination of Zearalenone.
Ginseng root samples were extracted and assayed using the Veratox Quantitative Zearalenone Test CD-ELISA (Neogen Corporation, Lansing, MI), a kit used commercially to detect food and feed possibly contaminated with zearalenone. Five grams of root sample was ground into a fine powder and extracted by vigorously shaking for 3–5 mins in 25 ml of 70% methanol and water. Aliquots of 100 µl from each sample were spin-filtered to remove particulate matter (0.45 µm of GHP Nanosep centrifugal device; Pall Gelman) and added to the microassay plates. Free zearalenone in the samples and controls competed for the antibody binding sites with enzyme-labeled zearalenone. Microassay plates were washed and substrate was added, which binds with the conjugate to produce blue color. Plates were read in a microwell reader using a 650-nm filter. Concentrations were read on a log-logit curve generated by the reader from a set of standards provided in the kit. Range of quantitation was 100–1000 ppb. Root samples were extracted and assayed in two separate experiments.

The HPLC Determination of Zearalenone.
The sample extraction method for HPLC analysis was adapted from the modified Association of Official Analytical Chemists (AOAC) official method for zearalenone (37). Briefly, 1-g, dry, powdered ginseng root samples were extracted in duplicate as previously described for the binding assay, except solvent was 70% methanol as recommended in the AOAC zearalenone extraction method. Supernatant was evaporated to dryness and resuspended in 20% methanol (8 ml). Samples were further purified using octadecyl (C18), disposable, 3-ml extraction columns (BAKERBOND SPE; J. T. Baker, Phillipsburg, NJ) with a vacuum manifold. The C18 columns were preconditioned by sequentially passing through 2 ml of methanol and 2 ml of water. Extracted test samples were aspirated through the conditioned column under vacuum (<0.8 cm [2 in.] Hg). The column was washed with 2 ml of 30% methanol and then dried under a vacuum for 3 mins. Zearalenone was eluted from the column by gently aspirating two 0.5-ml portions of 70% methanol. Eluates from the C18 column were evaporated to dryness, reconstituted in 1 ml of mobile phase buffer to a volume equivalent to 1 g/ml, and used for zearalenone determination. The HPLC method was adapted from Visconti and Pascale (38). A Hewlett Packard 1090 HPLC (Palo Alto, CA) with a 3-µm, 10cm x 4.6-mm C18 reverse phase column (Regis Technologies, Inc., Morton Grove, IL) was used for the analysis. The mobile phase consisted of acetonitrile-water-methanol (46:46:8) with a flow rate of 0.5 ml/min. The sample volume size injected into the HPLC for zearalenone determination depended on zearalenone concentration and varied from 10–100 µl. Standard curves were prepared by injecting standards (31, 62, 125, 250, 500, 1000, and 5000 ng/ml) at volumes equivalent to the test sample injection volume. Before injection, test samples and standards were filtered using 0.45 µm of Acrodisc LC13 PVDF (Pall Gelman). A diode array detector ({lambda} = 236 nm) was used for detection. Identification of zearalenone was determined by comparing retention times of test samples with those of the standard and comparison of spectra (170–400 nm) of test samples to those of standards. Concentrations of zearalenone were calculated by comparing test sample peak area with areas from the appropriate standard curve. Data represent the mean of two HPLC determinations using two different extractions.

Extracts of P. ginseng and P. quinquefolius were fractionated to determine the source of estrogenic activity. Approximately 0.25-ml fractions of eluant were collected during an HPLC gradient run beginning with 34:6:60 acetonitrile-methanol-water and ending with 60:10:30 acetonitrile-methanol-water. Samples were collected over 15 mins with a flow rate of 0.5 ml/min. Twenty-microliter aliquots of P. ginseng or P. quinquefolius (equivalent to 40 mg of dried root) were injected for each run. Fractions collected from 7 individual runs of P. ginseng runs were pooled for each collection time and each fraction assayed for estrogenic activity using the binding assay. The procedure was repeated using P. quinquefolius root. To determine elution patterns for the mycotoxin standards, zearalenone, {alpha}-zearalenol, and ß-zearalenol were added to a ginseng extract previously determined to have no ER binding for a final concentration of 20 µg/ml. Twenty-microliter aliquots of the spiked samples were run using the same HPLC and collection conditions. Individual fractions from samples and spikes were analyzed for ERß binding. Fractions were also collected from blanks for analysis.

All statistical analyses were conducted using SAS statistical software (SAS Institute Inc., Cary, NC). Analysis of variance was used to determine significant differences, and Pearson’s coefficient was used for correlation.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Reference
 
Affinity of Crude Plant Extracts and Compounds for ER{alpha} and ERß.
Extracts of P. ginseng and P. quinquefolius from the four different root sources bound to ER{alpha} and ERß in a concentration-dependent manner (Table 1Go). Overall, ERß bound methanol and water extracts with greater affinity (P = 0.05) than ER{alpha}. Methanol extracts of ginseng roots from one source, P. ginseng 2, bound ER{alpha} and ERß with significantly greater affinity than roots from the other sources. Water extracts of P. quinquefolius 1 had significantly higher binding to ER{alpha} than the other three sources yet had significantly lower binding to ERß when compared with P. ginseng 1 and P. ginseng 2.


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  		Table 1. ER{alpha} and ERß EBEs from Methanol and Water Extracts of Ginseng Root Using a Competitive Binding Assay
 
ER{alpha} and ERß binding affinity for zearalenone and some metabolites was examined. Based on an arbitrary relative binding affinity of 100 for ER{alpha} and ERß to E2, both ER{alpha} and ERß had less affinity for zearalenone, 4.2 and 6.6, respectively, than for E2 (Table 2Go). Data from the competitive binding tests showed the ER{alpha} affinities for zearalenone and its metabolites relative to E2 and some other estrogenic compounds were as follows: DES > {alpha}-zearalenol > E2 > 4-hydroxytamoxifen > {alpha}-zearalanol > ß-zearalanol > zearalanone > ß- zearalenol > zearalenone. ERß affinities of these same compounds were as follows: DES > {alpha}-zearalenol > E2 > 4-hydroxytamoxifen > {alpha}-zearalanol > ß-zearalanol > zearalanone > zearalenone. Affinity of the ER for the metabolite {alpha}-zearalenol is 15 times greater than affinity for zearalenone. The purified ginsenosides Rg1 and Rb1, the primary ginsenosides in P. ginseng and P. quinquefolius, respectively, had no detectable binding to either receptor in the binding assay.


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  		Table 2. The RBAs of ER{alpha} and ERß for Some Potentially Estrogenic Compounds Using a Competitive Binding Assay
 
Fusarium Culture from Ginseng Root.
Panax ginseng 1, P. ginseng 2, and P. quinquefolius 2, but not P. quinquefolius 1, were confirmed positive for Fusarium by identification of characteristic microconidia, macroconidia, and polyphialides (data not shown). Cultures were characteristically white with tinges of pink and yellow.

Zearalenone Concentrations in Ginseng Root Extracts Using ELISA.
Crude extracts of dried ginseng root from the four sources were screened for zearalenone content using CD-ELISA. All four ginseng root samples tested positive for zearalenone in the extracts. Zearalenone concentrations were highest in P. quinquefolius 2, the wild-crafted ginseng sample, at 680.1 ppb. Panax ginseng 1, P. ginseng 2, and P. quinquefolius 1 had zearalenone concentrations of 183.2, 386.1, and 177.4 ppb, respectively. Ginsenosides Rb1 and Rg1 did not bind to the antibody.

Zearalenone Concentrations in Ginseng Root Extracts.
Crude extracts of roots from each of the four sources were evaluated for zearalenone concentration using HPLC. The highest concentrations of zearalenone were found in extracts of P. ginseng 2 at 11.7 µg/g followed by P. ginseng 1 at 6.13 µg/g. Extracts of P. quinquefolius 2 had a concentration of 2.60 µg/g and P. quinquefolius 1 had the lowest concentration at 0.25 µg/g. Pearson’s correlation coefficient (r2) between binding affinities (EBEs) and zearalenone concentration in the root extracts was 0.62 for ER{alpha} ({alpha} = 0.0035) and 0.59 for ERß ({alpha} = 0.004) (Fig. 2Go).



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  		Figure 2. Estrogen binding equivalents in methanol extracts of P. ginseng (PG) and P. quinquefolius (PQ) from ER{alpha} and ERß competative binding assays and zearalenone concentrations determined from HPLC. Correlation (r2) with zearalenone concentration in corresponding extracts was 0.62 for ER{alpha} and ERß.

 
Estrogen Receptor Binding in HPLC Fractionated Ginseng Samples.
Spiked ginseng samples had HPLC retention peaks at 10.08, 7.75, and 6.70 mins for zearalenone, {alpha}-zearalenol, and ß-zearalenol, respectively (Fig. 3Go). The ERß binding assay detected peak binding activity from aliquots collected at times 10.6, 8.6, and 7.1 mins, indicating approximately a 0.5-min delay in collection time of eluted standards. The P. ginseng 2 extract had ERß binding peaks in fractions collected at 10.6, 8.6, and 6.6 mins (Fig. 4Go). Unexpectedly, binding peaks were also detected at 2.6, 4.1, and 5.6 mins. No binding was detected in fractionated samples of P. quinquefolius 1 or from the solvent blanks collected between runs.



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  		Figure 3. Estrogen binding equivalents (ng) in ERß binding assay from HPLC fractionation of a ginseng sample spiked with zearale-none (ZEA), {alpha}-zearalenol (alpha-z), and ß-zearalenol (beta-z). The ginseng sample used for the spiking matrix was previously determined to have no detectable ER binding or zearalenone as determined by HPLC. Vertical lines indicate HPLC retention times for respective compounds.

 


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  		Figure 4. Estrogen binding equivalents (ng) in ERß binding assey from HPLC fractionation of P. ginseng 2 extract. Vertical lines represent HPLC retention times for standards.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Reference
 
Increasingly, more patients are seeking alternative therapies (39–42). Women seeking treatments for relief of menopausal symptoms often use herbal products, and among these women ginseng is frequently used. Traditionally, ginseng is considered a safe herb with few adverse effects. However, estrogen-like effects after use of ginseng products have been reported (26–29). Our experiments revealed an estrogenic mycotoxin, zearalenone, in ginseng samples during ER assays. The discussion will center on interpreting the current results with respect to the relevant ginseng and mycotoxin literature.

Results from studies that investigated the estrogenic action of ginseng crude extracts and purified ginsenosides are conflicting. Although no significant binding of ginseng extracts to the ER have been previously demonstrated, studies have shown induction of the estrogen responsive protein pS2 in MCF-7 cancer cells and pS2 messenger RNA in S30 breast cancer cells, suggesting an ER-mediated response (9, 23, 43, 44). However, these studies reported either no change or decreased proliferation of ER-positive cells, suggesting the action of the extract is not singularly estrogenic. In contrast, a ginseng extract that failed to transactivate either ER{alpha} or ERß in a transient gene expression assay system and had no effect on uterine weight in vivo caused a 27-fold increase in MCF-7 cell proliferation (24). Effects of ginsenosides on estrogen-related cancer cell proliferation are similarly contradictory. Using various cancer cell lines, ginsenosides have been shown to exhibit antiproliferative activity (44–49). In contrast, it was recently reported that ginsenoside Rg1 extracted from Panax notoginseng stimulated MCF-7 breast cancer cell proliferation in a dose-dependent manner (12). Although Rg1 stimulated MCF-7 cell proliferation, it failed to displace 3H-E2 in a cell lysate, leading the authors to suggest stimulation occurred without direct interaction with ER. Rg1, a major active molecule from P. ginseng, was shown to be a functional ligand of glucocorticoid receptor, but there has been no evidence that the ginsenoside binds the ER (20). In another study, Rb1, the principal ginseno-side in P. quinquefolius, activated transcription of the estrogen-responsive luciferase reporter gene in MCF-7 cells and induced the estrogen-responsive gene c-fos, suggesting interaction of this ginsenoside with the ER (50).

Data from our study showed crude extracts of ginseng roots bound to ER{alpha} or ERß, but neither receptor type interacted with the ginsenoside compounds Rb1 or Rg1. This contradiction to previous studies that reported negative binding to ER may be related to the method used to obtain the extract, the concentration of extract tested, or receptor type used. In our study, both hot water and methanol solvents were used to extract compounds with different solubilities (Fig. 1Go). Zearalenone and its metabolites are more soluble in methanol; therefore, methanolic extracts of samples containing these potent compounds had greater estrogen binding than the hot water extracts. Although Lui et al. (44) reported no binding to ER{alpha} or ERß using an extract dilution equivalent to 1 µg of plant material, we detected receptor binding when using a more concentrated extract, equivalent to 1 mg of plant material. Furthermore, in our study, ERß had three to four times higher binding affinity than ER{alpha} for most ginseng extracts, suggesting that cell lines that express a high percentage of ERß may be more responsive than cells that express primarily ER{alpha}.

These experiments identified zearalenone or zearale-none-like compounds from P. ginseng and P. quinquefolius roots. Roots from three of the four sources cultured positive for Fusarium fungus, the only known natural source of zearalenone. Zearalenone in the extracts was detected using ELISA and HPLC. The root sample that cultured negative for Fusarium was the same sample showing the lowest zearalenone concentration using HPLC. Explanations as to why zearalenone was identified in a sample that cultured negative for Fusarium are as follows: (i) spores were nonviable before sterilization and the zearalenone persisted in the sample; and (ii) the sample was weakly infested, with the primarily surface-localized spores being killed by flame sterilization. Concentrations of zearalenone in some P. ginseng roots in the parts per million range (10 µg/g or 10 mg/kg) were measured using HPLC. In comparison, lower zearalenone concentrations detected by ELISA may result from using a nylon spin-filter to remove particulate matter. Visconti and Pascale (38) reported that up to 40% zearalenone concentration in corn extracts was lost due to filtration with a nylon spin filter. Additionally, the CD-ELISA, unlike HPLC, is an indirect method and can be subject to cross-reactivity and matrix effects and should be used only qualitatively as a preliminary screen.

A correlation greater than 50% between ER binding data and zearalenone concentration was found in the test samples, suggesting a substantial relationship between these factors. Also, {alpha}-zearalenol, a highly estrogenic metabolite of zearalenone, was identified in the root extract using HPLC. Results from fractionation of P. ginseng 2, the sample with highest EBE and zearalenone concentrations, confirm zearalenone, {alpha}-zearalenol, and ß-zearalenol in the extract. In contrast, no ER binding was detected in P. quinquefolius 1 fractions collected from HPLC. This sample had almost seven times less EBE concentration in the ER binding assay when compared with P. ginseng 2 and more than 10 times less zearalenone concentration as determined by HPLC. Perhaps fractionation of the sample diluted the estrogenic components of the extract beyond the range of the binding assay. In addition to the zearalenone and zearalenol binding peaks demonstrated in the fractionated P. ginseng 2, there were three unidentified binding peaks. Absorbance data on these peaks did not resemble zearalenone or the metabolites. Although ERß had three to four times higher affinity than ER{alpha} for most ginseng extracts, affinity of the two receptors for zearalenone and the metabolites was only slightly higher for ERß. Perhaps the xenoestrogen in the three unidentified peaks could account for the three to four times greater ERß binding affinity detected in the assays. The source of the unidentified xenoestrogen may be fungal or plant derived, and the possibility exists that this compound is the result of plant-fungal interaction (i.e., metabolic conversion).

The health consequences of zearalenone exposure in humans are uncertain. However, mice, rats, swine, and monkeys are susceptible to the estrogenic effects of zearalenone; hyperestrogenicity and severe reproductive and infertility problems can occur, especially in swine (32, 51–57). Zearalenone at concentrations of 0.3–30 µg/ml induced dose-dependent decrease in bovine oocyte maturation rate and chromatin abnormalities (58). Zearalenone possesses tumor-promoting activity similar to estrogen and may be associated with carcinogenesis (59–61). This mycotoxin has been detected in human endometrial adenocarcinoma tissue (62) and has been suggested as a contributing factor to outbreaks of precocious puberty in Puerto Rico (63). Zearalenone contamination of grains used for animal and human consumption is considered a health concern at the parts per billion level (37). Toxicity problems are seen in domestic animals, particularly swine and poultry, when ingesting feed contaminated with zearalenone at the parts per billion level (64, 65).

Ginseng is used medicinally at 0.5–4 g/d, a much lower consumption than would be expected from grains. However, as shown in the binding data and by others, the zearalenone metabolite {alpha}-zearalenol is as potent an estrogenic compound as E2. Certainly, levels of zearalenone in ginseng root at a parts per million concentration could present a potential health risk, particularly in consumers in whom estrogen exposure should be minimized.

There appears to be no uniform explanation for the contradictions in ginseng effects with respect to estrogen-related biological activity in individuals. Although commercial extracts of ginseng are usually standardized to a certain level of the saponin ginsenosides, this standardization does not reflect the concentration of ginseng root and other potentially biologically active components in the preparation, including zearalenone. Estrogenic responses reported after ginseng use may be attributed to biologically active compounds present in the root other than the ginsenosides. Herbal manufacturers often have proprietary methods for preparing their ginseng extracts, resulting in the inclusion or exclusion of potentially bioactive compounds.

In conclusion, the unexpected findings of zearalenone-like compounds in P. ginseng and P. quinquefolius extracts could explain the sporadic reports of estrogen toxicity after ginseng use. The presence of this mycotoxin in an herbal product should be investigated further to determine the source and prevalence of Fusarium fungi and zearalenone in ginseng root. Additionally, this finding raises the issue of mycotoxin testing for all herbal products. The methods described herein provide a valid approach for screening herbal products for ER binding and identification of zearalenone contamination.


   Acknowledgments
 
We thank Dr. Timothy Spira, botanist at Clemson University, for the generous gift of P. quinquefolius 2 samples; Dr. Bo Wang, Department of Plant Pathology and Physiology at Clemson University, for his expertise with Fusarium cultures and identification; and Dr. A. B. Bodine and Rebecca Rodgers, Animal and Veterinary Sciences Department at Clemson University, for help with mycotoxin analyses.


   Footnotes
 
Technical contribution No. 4950 of the South Carolina Agriculture Experiment Station.

Received for publication January 22, 2004. Accepted for publication March 3, 2004.


   Reference
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Reference
 

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