2001 JAFC Liu

2001 JAFC Liu
J. Agric. Food Chem. 2001, 49, 2472−2479
Evaluation of Estrogenic Activity of Plant Extracts for the Potential
Treatment of Menopausal Symptoms
Jianghua Liu,† Joanna E. Burdette,† Haiyan Xu,‡ Chungang Gu,† Richard B. van Breemen,†
Krishna P. L. Bhat,† Nancy Booth,† Andreas I. Constantinou,‡ John M. Pezzuto,†
Harry H. S. Fong,† Norman R. Farnsworth,† and Judy L. Bolton*,†
Department of Medicinal Chemistry and Pharmacognosy, Program for Collaborative Research in the
Pharmaceutical Sciences, UIC/NIH Center for Botanical Dietary Supplements Research,
College of Pharmacy, M/C 781, University of Illinois at Chicago, 833 South Wood Street,
Chicago, Illinois 60612, and Department of Surgical Oncology, University of Illinois at Chicago,
840 South Wood Street, Chicago, Illinois 60612
Eight botanical preparations that are commonly used for the treatment of menopausal symptoms
were tested for estrogenic activity. Methanol extracts of red clover (Trifolium pratense L.), chasteberry
(Vitex agnus-castus L.), and hops (Humulus lupulus L.) showed significant competitive binding to
estrogen receptors R (ERR) and β (ERβ). With cultured Ishikawa (endometrial) cells, red clover and
hops exhibited estrogenic activity as indicated by induction of alkaline phosphatase (AP) activity
and up-regulation of progesterone receptor (PR) mRNA. Chasteberry also stimulated PR expression,
but no induction of AP activity was observed. In S30 breast cancer cells, pS2 (presenelin-2), another
estrogen-inducible gene, was up-regulated in the presence of red clover, hops, and chasteberry.
Interestingly, extracts of Asian ginseng (Panax ginseng C.A. Meyer) and North American ginseng
(Panax quinquefolius L.) induced pS2 mRNA expression in S30 cells, but no significant ER binding
affinity, AP induction, or PR expression was noted in Ishikawa cells. Dong quai [Angelica sinensis
(Oliv.) Diels] and licorice (Glycyrrhiza glabra L.) showed only weak ER binding and PR and pS2
mRNA induction. Black cohosh [Cimicifuga racemosa (L.) Nutt.] showed no activity in any of the
above in vitro assays. Bioassay-guided isolation utilizing ER competitive binding as a monitor and
screening using ultrafiltration LC-MS revealed that genistein was the most active component of
red clover. Consistent with this observation, genistein was found to be the most effective of four red
clover isoflavones tested in the above in vitro assays. Therefore, estrogenic components of plant
extracts can be identified using assays for estrogenic activity along with screening and identification
of the active components using ultrafiltration LC-MS. These data suggest a potential use for some
dietary supplements, ingested by human beings, in the treatment of menopausal symptoms.
Keywords: Estrogen receptor; alkaline phosphatase; progesterone receptor; pS2; dietary supplement;
phytoestrogens; isoflavones
During the period of menopause and postmenopause,
many women experience one or more symptoms such
as hot flashes, depression, mood swings, sleeping disorders, vaginal dryness, and joint pain, largely due to a
lack of estrogens (1). Hormone replacement therapy has
helped to relieve menopausal symptoms; in addition, the
risk of osteoporosis, cardiovascular disease, dementia
from Alzheimer’s disease, and certain types of cancer
are reduced (2-5). Epidemiological data show that a diet
rich in phytoestrogens, such as those found in soy,
reduce the number of hot flashes and the incidence of
cancer in Oriental women (6). Since side-effects of
traditional estrogen replacement therapy include a
slight but significant increase in the risk of developing
breast and endometrial cancer (3, 7-10), women are
increasingly using herbal remedies as alternative therapy
* Author to whom correspondence should be addressed [fax
(312) 966-7107; e-mail judy.bolton@uic.edu].
† Department of Medicinal Chemistry and Pharmacognosy
and Program for Collaborative Research in the Pharmaceutical
‡ Department of Surgical Oncology.
Estrogen regulates gene expression by binding to
intracellular estrogen receptors (ER), which influence
the growth, differentiation, and function of many target
tissues. When estrogens bind to an ER, receptor dimerization occurs, which in turn binds to an estrogenresponsive element (ERE) in the DNA of estrogensensitive cells (14). Consequently, the ER-ERE complex
modulates the transcription of estrogen-regulated target
genes, such as the progesterone receptor (PR) and
presenelin-2 (pS2), and ultimately stimulates cell growth
and differentiation (15).
The differences between the two estrogen receptors
(ERR and ERβ) include tissue distribution and ligand
specificity (16, 17). In the midgestational human fetus,
ERR is most abundant in the uterus, and smaller
quantities have been detected in the ovaries, testes,
skin, and gut by semiquantitative reverse transcriptasepolymerase chain reaction (RT-PCR). In contrast, high
amounts of ERβ mRNA are present in fetal ovaries,
testes, adrenals, and spleen (18). Both ERR and ERβ
are coexpressed in the human central nervous system,
breast, cardiovascular tissue, and bone (19).
Black cohosh [Cimicifuga racemosa (CR)], red clover
[Trifolium pratense L. (TP)], hops [Humulus lupulus L.
10.1021/jf0014157 CCC: $20.00 © 2001 American Chemical Society
Published on Web 03/24/2001
Estrogenic Plants for Treatment of Menopause
J. Agric. Food Chem., Vol. 49, No. 5, 2001 2473
Table 1. ER Binding, AP Induction, PR and pS2 mRNA Expression, and Cytotoxicity of Methanol Extracts
ERR binding
IC50, µg/mL
ERβ binding
IC50, µg/mL
AP induction,
Ishikawa cells
IC50, µg/mL
PR expression,
Ishikawa cells
ratio of intensitya
Ishikawa cells
ED50, µg/mL
pS2 expression,
S-30 cells ratio
of intensityb
S-30 cells
ED50, µg/mL
dong quai
black cohosh
Asian ginseng
American ginseng
red clover
30 ( 0.4
5.6 ( 2.1
46 ( 3
27 ( 2.8
2.5 ( 0.6
64 ( 4
13.1 ( 6.1
1.0 ( 0.2
a Ratio of intensity/net intensity of PR band/net intensity of β-actin band. Extracts were tested at a concentration of 20 µg/mL. b Ratio
of intensity/net intensity of pS2 band/net intensity of β-actin band. Extracts were tested at a concentration of 20 µg/mL. c NA, not active
(IC50 > 50 µg/mL for ER binding; IC50 > 20 µg/mL for AP induction; no PR or pS2 expression at 20 µg/mL).
(HL)], and chasteberry [Vitex agnus-castus L. (VA)] are
four of the most frequently used herbs in Western
countries for menopausal symptoms or premenstrual
syndrome (PMS) (6, 20-22). Dong quai [Angelica sinensis (Oliv.) Diels (AS)] is a common Chinese herb used
for women’s health, and licorice (Glycyrrhiza glabra L.)
has been reported to have both estrogenic and antiestrogenic activities (23, 24). Several case reports implicate Asian ginseng [Panax ginseng C.A. Meyer (PG)] as
a possible candidate for the treatment of menopause (25,
26). In addition, the stimulation of the estrogen-sensitive gene, pS2, by North American ginseng [Panax
quinquefolius L. (PQ)] has been shown (27, 28).
In the present study using four in vitro assays, we
have systematically evaluated the estrogenic properties
of eight botanicals listed above. In addition, we have
examined the estrogenic activity of four isoflavones
known to be present in red clover (13, 29-31). These
data suggest that the combination of in vitro bioassays
for estrogenic activity along with isolation and characterization of the active components by LC-MS is a valid
approach for identification of plant extracts beneficial
in the treatment of menopausal symptoms.
Chemicals and Reagents. All chemicals and reagents
were from Fisher (Hanover Park, IL) or Sigma (St. Louis, MO)unless otherwise indicated. All media for cell culture were
purchased from Life Technologies (Grand Island, NY). Fetal
bovine serum (FBS) was from Atlanta Biologicals (Norcross,
GA). Genistein, daidzein, biochanin A, and formononetin were
purchased from Indofine Chemical Co. (Belle Mead, NJ). [3H]Estradiol (83 Ci/mmol) was obtained from NEN Life Science
Products (Boston, MA), and Cytoscint was purchased from ICN
(Costa Mesa, CA). Human recombinant ERR and ERβ were
purchased from Panvera (Madison, WI). Primers of PR, pS2,
and β-actin were obtained from Life Technologies.
Plant Material. A. sinensis (dong quai, roots) and T.
pratense (red clover, flowering aerial parts) were cultivated
at the University of Illinois Pharmacognosy Field Station
(Downer’s Grove, IL). C. racemosa (black cohosh, rhizomes and
roots) was collected in Rookbridge County, VA. V. agnus-castus
(chasteberry, berries) and G. glabra (licorice, roots) were
provided by Pharmavite (San Fernando, CA) and PureWorld
Botanicals (South Hackensack, NJ), respectively. H. lupulus
(hops, strobiles) was purchased from Hops Direct (Mabton,
WA). P. ginseng (Asian ginseng, roots) was obtained from the
Institute of Materia Medica, Chinese Academy of Traditional
Chinese Medicine (Beijing, China), and P. quinquefolius (North
American ginseng, roots) was a gift from Chai-Na-Ta Corp.
(Langley, BC, Canada). V. agnus-castus (chasteberry, fruits)
was a gift from PureWorld Botanicals.
Extraction and Fractionation. Plant materials (100 g)
were macerated in MeOH (600 mL) overnight. Following
filtration, the marcs were extracted twice with MeOH (600
mL), with gentle heating (<45 °C, 10 min). The extracts were
combined, and the solvent was removed in vacuo. Extracts of
the eight plants were initially tested in bioassays listed in
Table 1. The red clover fraction was redissolved in 30%
aqueous MeOH (600 mL) and partitioned against petroleum
ether (6 × 250 mL); residual MeOH was removed in vacuo
from the aqueous portion, and the latter was partitioned
against CHCl3 and BuOH successively (6 × 250 mL). Removal
of the solvent yielded the petroleum ether, CHCl 3, BuOH, and
H2O soluble fractions. The CHCl3 fraction was chromatographed on a silica gel (70-230 mesh) column and developed
successively with solvent mixtures of CHCl3/petroleum ether
(4:1, 480 mL), EtOAc (300 mL), CHCl3/MeOH (9:1, 190 mL),
and MeOH (80 mL). On the basis of their thin-layer chromatography (TLC) patterns, 14 subfractions were obtained from
the original crude chloroform extract.
Cell Culture Conditions. Ishikawa and S30 cell lines were
provided by Dr. R. B. Hochberg (Yale University, New Haven,
CT) and Dr. V. C. Jordan (Northwestern University, Evanston,
IL), respectively. Ishikawa cells were maintained in Dulbecco’s
Modified Eagle medium (DMEM)/F12 media with 10% heatinactived FBS, sodium pyruvate (1%), penicillin-streptomycin
(1%), and glutamax-1 (1%). One day prior to treating the cells,
the medium was replaced with phenol red-free, DMEM/F12
medium containing charcoal/dextran-stripped FBS to remove
estrogens. S30 cells were maintained in phenol-free minimum
essential medium (MEME) supplemented with 1% penicillinstreptomycin, 6 µg/L insulin, 500 mg/L G418 (geneticin disulfate salt), 1% glutamax, and 5% charcoal/dextran-stripped
ER Competitive Binding Assays. The procedure of
Obourn et al. (32) was used with minor modifications. Briefly,
24 h prior to the assay, a 50% v/v hydroxyapatite slurry was
prepared using 10 g of hydroxyapatite in 60 mL of TE buffer
(50 mM Tris-Cl, pH 7.4, 1 mM EDTA) and stored at 4 °C. The
ER binding buffer consisted of 10 mM Tris-Cl (pH 7.5), 10%
glycerol, 2 mM dithiothrietol, and 1 mg/mL bovine serum
albumin. The ERR and ERβ wash buffers contained 40 mM
Tris-Cl (pH 7.5), 100 mM KCl, 1 mM EDTA, and 40 mM TrisCl (pH 7.5), respectively. The reaction mixture consisted of 5
µL of test sample in DMSO, 5 µL of pure human recombinant
diluted ERR or ERβ (0.5 pmol) in ER binding buffer, 5 µL of
“Hot Mix” (400 nM, prepared fresh using 3.2 µL of 25 µM, 83
Ci/mmol [3H] estradiol, 98.4 µL of ethanol, and 98.4 µL of ER
binding buffer), and 85 µL of ER binding buffer. The incubations were carried out at room temperature for 2 h, then 100
µL of 50% hydroxyapatite slurry were added, and the tubes
were incubated on ice for 15 min with vortexing every 5 min.
The appropriate ER wash buffer was added (1 mL), and the
tubes were vortexed and then centrifuged at 2000g for 5 min.
The supernatant was discarded, and this wash step was
repeated three-times. The hydroxyapatite pellet containing the
ligand-receptor complex was resuspended in 200 µL of ethanol
and transferred to scintillation vials. Cytoscint (4 mL/vial) was
added, and the tubes were counted using a Beckman (Schaumburg, IL) LS 5801 liquid scintillation counter. The percent
inhibition of [3H]estradiol binding to each ER was determined
as follows: [(dpmsample - dpmblank)/(dpmDMSO - dpmblank) - 1]
2474 J. Agric. Food Chem., Vol. 49, No. 5, 2001
× 100. The binding capability (percent) of the sample was
calculated in comparison to that of estradiol (50 nM, 100%).
The data represent the average ( SD of three determinations.
Induction of Alkaline Phosphatase (AP) with Cultured Ishikawa Cells. The procedure of Pisha et al. (33) was
used as described previously. Briefly, Ishikawa cells (5 × 104/
well) were incubated overnight with estrogen-free media in
96-well plates. Test samples in DMSO were added, and the
cells in a total volume of 200 µL media/well were incubated at
37 °C for 4 days. For the determination of antiestrogenic
activity, 2 × 10-8 M estradiol was added to the media. Enzyme
activity was measured by reading the liberation of p-nitrophenol at 340 nm every 15 s for 16-20 readings with an ELISA
reader (Power Wave 200 microplate scanning spectrophotometer, Bio-Tek Instrument, Winooski, VT). The maximum slope
of the lines generated by the kinetic readings was calculated
using a computer program. The percent induction for determination of estrogenic activity was calculated as [(slopesample
- slopecells)/(slopeestrogen - slopecells)] × 100. For antiestrogenic
activity, the percent induction was determined as [(slopesample
- slopecells)/(slopeDMSO - slopecells)] × 100. The data represent
the average ( SD of triplicate determinations.
Cytotoxicity Assays. Ishikawa (15000 cells/well) and S30
cells (4000 cells/well) were preincubated in 96-well plates
overnight in estrogen-free media. The Ishikawa cells were
incubated with test samples for 4 days, and S30 cells were
incubated for 1 day. As an indication of cell viability, absorbance was measured at 515 nm on a microtiter plate reader
after the cells were fixed with 20% trichloroacetic acid (TCA)
and stained with 0.4% sulforhodamine B (SRB), and the bound
dye was liberated with 0.1 M Tris buffer (33, 34). The data
represent the average ( SD of triplicate determinations.
RT-PCR Analysis of PR and pS2 mRNA Expression in
Ishikawa and S30 Cell Lines. Ishikawa cells (2 × 105/well)
were preincubated overnight in estrogen-free media in a sixwell plate. Test samples in DMSO were added and incubated
at 37 °C for 4 days. S30 cells (4 × 104/well) were preincubated
overnight with estrogen-free media in 24-well plates, and then
the test samples were added and incubated at 37 °C for 24 h.
Total mRNA from both cell lines was extracted with TRIzol
reagent (Gibco, Grand Island, NY) following the manufacturer’s protocol, and RT-PCR was carried out using the SuperScript one-step RT-PCR system (Gibco) and a DNA thermal
cycler 480 (Perkin-Elmer, Foster City, CA). The primers used
for PR expression were 5′-CCATGTGGCAGATCCCACAGGAGTT-3′ (sense) and 5′-TGGAAATTCAACACTCAGTGCCCGG-3′ (antisense). The primers used for pS2 expression
were 5′-CATGGAGAACAAGGTGATCTG-3′ (sense) and 5′CAGAAGCGTG-TCTGAGGTGTC-3′ (antisense). The PCR products (5 µL) of PR (271 bp) and pS2 (365 bp) were separated by
electrophoresis in 1% agarose gels and visualized by staining
with ethidium bromide. The 621 bp sequence for β-actin was
used as an internal control for both PR and pS2. The sense
and antisense primers used for β-actin were 5′-ACACTGTGCCCATCTACGAGG-3′ and 5′-AGGGGCCGGACTCGTCATACT3′, respectively. The net intensity of the bands was measured
using Kodak Digital Science 1D software. The ratio of the
intensity of the target gene and the internal control of each
sample was calculated as shown in Tables 1 and 2.
Detection of ER Ligands in Red Clover Extracts Using
Ultrafiltration and LC-MS. Human recombinant ERβ (50
or 100 pmol) was mixed with the test sample in binding buffer
containing 50 mM Tris-Cl (pH 7.5), 10% glycerol, 50 mM KCl,
and 1 mM EDTA, in a total volume of 150 µL. After a 2 h
incubation at room temperature, the reaction mixture was
filtered through a Microcon YM-30 centrifugal filter (Millipore)
containing a regenerated cellulose ultrafiltration membrane
with a 30000 MW cutoff by centrifugation at 10000 rpm for 7
min at 4 °C. Unbound compounds were removed by washing
the filter three times by centrifugation with 150 µL aliquots
of ammonium acetate buffer at pH 7.5 at 4 °C. To disrupt the
ligand-receptor complex and release the bound ligands, 400
µL of MeOH/H2O (90:10) was added followed by centrifugation
at 10000 rpm for 10 min. The solvent in the ultrafiltrate was
removed under vacuum, and the ligands were redissolved in
Liu et al.
Figure 1. Structures of four major isoflavones in red clover
and estradiol.
60 µL of H2O/MeOH (80:20). Aliquots (10 µL) of this solution
were analyzed by using LC-MS, which consisted of a Waters
2690 HPLC system (Waters, Milford, MA) coupled to a
Micromass Quattro II electrospray triple quadrupole mass
spectrometer (Micromass, Manchester, U.K.). HPLC separations were carried out using a Micra (Northbrook, IL) C18
HPLC column, 4.6 × 21 mm, containing 1.5 µm nonporous
silica. The mobile phase consisted of H2O/MeOH (95:5, v/v)
containing 0.01% acetic acid (A) and MeOH containing 0.01%
acetic acid (v/v) (B), using linear gradients of 5-98% B (v/v)
over 20 min. The electrospray source was operated at 155 °C
in negative ion mode. Nitrogen was used as both nebulizing
and drying gas at flow rates of 20 and 450 L/h, respectively. A
control was used to correct for nonspecific binding of the
sample, in which ERβ was absent from the incubation solution.
Relative Affinity of Plant Extracts and Isoflavones for ERr and ERβ. Among the eight methanol
extracts tested, red clover, hops, and chasteberry showed
significant binding affinities with both ERR and ERβ
on the basis of their 50% inhibitory (IC50) values (Table
1). The order of binding potency was red clover . hops
> chasteberry, and their affinities for ERR and ERβ
were not significantly different. Dong quai and licorice
showed weak binding affinity (IC50 >50 µg/mL), whereas
Asian ginseng, North American ginseng, and black
cohosh displayed no binding (<20% at a concentration
of 200 µg/mL). Standards of isoflavones, known to be
present in red clover (see structures in Figure 1), were
tested with both ER receptor subtypes and exhibited
competitive binding potency following the order genistein
> daidzein > biochanin A > formononetin, based on
their IC50 values (Table 2). These four isoflavones
displayed higher affinity with ERβ compared to ERR,
consistent with previous studies (35).
Detection of ER Ligands in Red Clover by
a Combination of Ultrafiltration and LC-MS.
Since red clover showed the highest ER binding affinity
among the crude extracts tested, it was fractionated
with different solvents to help identify the active
compound(s). As shown in Table 3, the chloroform
fraction of red clover displayed the greatest potency
compared to other fractions. As a result, the chloroform
extract was analyzed using affinity ultrafiltration LCMS, which is a variation of pulsed ultrafiltation LCMS (36) developed by Wieboldt et al. (37). Unlike other
ultrafiltration LC-MS applications, this affinity method
was applied in the present study for the rapid screening
of botanical extracts for ligands to ERβ. Using ultrafiltration and LC-MS, daidzein (9.9 min retention time),
genistein (11.3 min), and biochanin A (14.9 min) were
identified as ERβ ligands in the chloroform extracts of
Estrogenic Plants for Treatment of Menopause
J. Agric. Food Chem., Vol. 49, No. 5, 2001 2475
Table 2. ER Binding, AP Induction, PR and pS2 mRNA Expression, and Cytotoxicity of Phytoestrogens in Red Clover
ERR binding
IC50, µM
ERβ binding
IC50, µM
AP induction
Ishikawa cells
IC50, µM
PR expression
Ishikawa cells, ratio
of intensitya
Ishikawa cells
ED50 µM
pS2 expression
S-30 cells, ratio
of intensityb
biochanin A
0.3 ( 0.01
17 ( 2.5
35 ( 1.4
104 ( 8.2
0.0065 ( 0.00058
0.018 ( 0.002
1.2 ( 0.0
4.1 ( 0.8
60 ( 7.1
0.0024 ( 0.00014
0.51 ( 0.1
1.2 ( 0.6
5.1 ( 0.4
12 ( 3.0
0.00014 ( 0.000014
47 ( 6.0
a Ratio of intensity/net intensity of PR band/net intensity of β-actin band. Compounds were tested at a concentration of 5 nM. b Ratio
of intensity/net intensity of pS2 band/net intensity of β-actin band. Compounds were tested at a concentration of 0.1 µM. c Milli.
Table 3. ER Binding, AP Induction, and Cytotoxicity of Red Clover Fractions and Subfractions
ERR bindinga
ERβ bindinga
AP inductionb
Ishikawa cells
Ishikawa cells
bound ligands detected by LC-MS
CHCl3 fraction 2
CHCl3 fraction 6
CHCl3 fraction 7
CHCl3 fraction 8
genistein, daidzein, biochanin A
genistein, daidzein, biochanin A
biochanin A
genistein, daidzein
a Percent inhibition at 200 µg/mL. b Percent induction at 20 µg/mL. c Percent cell survival at 20 µg/mL.
none detected. f IC50 ) 2.6 ( 0.1 µg/mL.
red clover on the basis of molecular weight, tandem
mass spectra, and HPLC retention time in comparison
with authentic standard compounds. The affinity of
genistein for ERβ was confirmed by the large enhancement of the LC-MS peak following affinity ultrafiltration
(solid line in Figure 2A) compared with that of a control
sample which did not contain ERβ (dashed line, Figure
2A). Of the 14 subfractions of the most potent chloroform
extract of red clover separated by column chromatography, the four fractions showing the highest competitive binding ability to ERβ were subjected to the
ultrafiltration and LC-MS ERβ binding assay. Genistein
was detected as the most ERβ-active component in
subfractions 6-8 (Table 3; Figure 2B). In addition to
genistein, daidzein was detected in fraction 8. Biochanin
A was the ERβ ligand detected in chloroform subfraction
2 (Table 3); however, this fraction (Table 3) and the pure
isoflavone (Table 2) were found to be cytotoxic with
Ishikawa cells.
AP Induction in Ishikawa Cells. Ishikawa is an
ER positive endomentrial adenocarcinoma cell line
derived from a glandular epithelial cell line. This cell
responds to estrogens and antiestrogens at concentrations approximating physiological levels (38). Induction
of AP activity in Ishikawa cells indicates an estrogenic
response, whereas inhibition represents an antiestrogenic effect (33). This cell line was used to investigate
the estrogenic or antiestrogenic effects of the test
samples, and PR expression was carried out to confirm
the results of AP induction. These two assays give
consistent results in comparison with ER binding data
in terms of the estrogenic activity of the test samples.
In Ishikawa cells, the red clover extract showed the
strongest AP induction ability with an IC50 value of 1.0
µg/mL (Table 1). Chloroform subfractions 6-8, which
showed the strongest ER binding affinity, also displayed
high AP induction in the Ishikawa cells, whereas
subfraction 2 appeared to be cytotoxic with these cells
(Table 3). Although the hops extract exhibited strong
cytotoxicity (Table 1), its estrogenic activity was still
detected with an IC50 value of 13.1 µg/mL. Chasteberry
displayed weak estrogenic activity (40%) at a concentra-
IC50 ) 1 ( 0.2 µg/mL
tion of 20 µg/mL, whereas the other plant extracts were
not active (Table 1). Genistein, daidzein, biochanin A,
and formononetin, which are all present in red clover,
exhibited AP induction activity with potency that
correlated with the ER binding assay, based on IC50
values (Table 2). None of the extracts or isoflavone
standards exhibited antiestrogenic activity (data not
Stimulation of PR mRNA Expression in Ishikawa Cells. Estradiol-mediated PR expression was not
observed in S30 cells, so experiments were conducted
using the Ishikawa cell line. PR expression, as measured
by RT-PCR, was significantly up-regulated by red clover,
hops, and chasteberry extracts at concentrations of 20
µg/mL (Table 1; Figure 3A). Dong quai and licorice
exhibited weak stimulation of PR expression at this
concentration; however, extracts of black cohosh and the
two ginseng species did not show activity. The four
isoflavones induced PR expression at concentrations of
5 nM (Table 2; Figure 3B). These results are consistent
with the ER binding data.
The chloroform subfractions of red clover demonstrated strong PR induction in comparison to the
petroleum ether, butanol, and H2O fractions at a
concentration of 20 µg/mL (data not shown). Chloroform
subfractions 6-8, which displayed high ER binding, also
displayed significant PR up-regulatory activity (data not
shown). Subfraction 2 was not tested in this assay due
to its toxicity with Ishikawa cells.
Stimulation of pS2 mRNA Expression in S30
Cells. The stimulation of pS2 expression in the estrogen
receptor-positive breast cancer cell line MCF-7 has been
reported previously (39-41). However, the expression
of the pS2 mRNA in MCF-7 was constitutive under our
experimental conditions, despite a change to estrogenfree media for 4 days (data not shown). In contrast,
Ishikawa cells did not show pS2 expression in incubations with estradiol (data not shown). S30 is a subclone
of the ER-negative MDA-MB-231 breast cancer cell line
that is stably transfected with ERR. We utilized this
cell line for pS2 expression because it was responsive
to estradiol; results were consistent with the other
2476 J. Agric. Food Chem., Vol. 49, No. 5, 2001
Liu et al.
Figure 3. Induction of PR mRNA expression in Ishikawa
cells: (A) methanol extracts (20 µg/mL) [1, control; 2, DMSO;
3, estradiol; AS, A. sinensis (dong quai); CR, C. racemosa (black
cohosh); GG, G. glabra (licorice); HL, H. lupulus (hops); PG,
P. ginseng (Asian ginseng); PQ, P. quinquefolius (North
American ginseng); TP, T. pratense (red clover); VA, V. agnuscastus (chasteberry)]; (B) phytoestrogens (5 nM) (1, control;
2, DMSO; 3, estradiol; 4, genistein; 5, daidzein; 6, biochanin
A; 7, formononetin).
Figure 2. Overlaid total ion chromatograms showing affinity
ultrafiltration and LC-MS screening results of (A) red clover
chloroform extract (10 µg/mL) and (B) one of its bioactive
subfractions (fraction 7, 20 µg/mL). The solid line (s) represents the experiment with ERβ (0.667 µM), and the dashed
line (- - -) indicates the control experiment without the receptor. By a combination of ultrafiltration and LC-MS, the
enhanced peaks of genistein (11.3 min), daidzein (9.9 min),
and biochanin A (14.9 min) were identified and confirmed as
active ligands in the chloroform extract. Genistein was the
active ligand in subfraction 7. See Table 3 for active ligands
identified in other bioactive subfractions of the red clover
chloroform fraction. The peak at 13.2 min is due to an impurity
in the water mobile phase collected during the equilibration
of the LC column. It serves as an internal standard for
normalizing the experiment with the control, because the
equilibration time of the LC column was the same in the LC
sequence before sample injection for both samples.
assays with the exception of data obtained with ginseng.
In S30 cells, all extracts except that of black cohosh
induced pS2 expression (Figure 4A) at a concentration
of 20 µg/mL. Interestingly, Asian ginseng and North
American ginseng did not show activity in the three
assays described above.
Similar to its PR induction in Ishikawa cells, the
chloroform fraction of red clover showed stronger pS2
expression than the petroleum ether, BuOH, and H2O
extracts at 20 µg/mL (data not shown). The purified
isoflavones from red clover also induced pS2 expression.
Expression induced by genistein and daidzein was
significantly stronger than that of biochanin A and
formononetin at a concentration of 0.1 µM (Figure 4B).
Zava et al. (42) previously reported the estrogenic and
progestin bioactivities of over 150 herbs including the
8 plants studied in this investigation. In their radioreceptor assay, red clover, licorice, and hops extracts were
reported to bind to the ER of MCF-7 cells. Red clover
also bound to the PR of the T47D cell line. Using cell
proliferation as an indicator of possible estrogenic
Figure 4. Induction of pS2 mRNA expression in S-30 cells:
(A) methanol extracts (20 µg/mL) [1, control; 2, DMSO; 3,
estradiol; AS, A. sinensis (dong quai); CR, C. racemosa (black
cohosh); GG, G. glabra (licorice); HL, H. lupulus (hops); PG,
P. ginseng (Asian ginseng); PQ, P. quinquefolius (North
American ginseng); TP, T. pratense (red clover); VA, V. agnuscastus (chasteberry)]; (B) phytoestrogens (0.1 µM) (1, control;
2, DMSO; 3, estradiol; 4, genistein; 5, daidzein; 6, biochanin
A; 7, formononetin).
activity, red clover, hops, and licorice extracts demonstrated growth that was significantly higher than that
of controls. Our results are consistent with these conclusions based on the estrogenic activities of red clover and
hops. However, in our tests, the licorice extract displayed only weak binding affinity to ER, weak stimulation of PR expression in Ishikawa cells, weak pS2
expression in the S30 cell line, and a lack of AP
induction in Ishikawa cells. Although different assays
and cell lines were used, further investigation of licorice
appears to be necessary to evaluate its potential estrogenicity.
Estrogenic Plants for Treatment of Menopause
Genistein, daidzein, biochanin A, and formononetin
have been implicated as causative for the estrogenic
activity of red clover (13, 29-31). Although many
flavonoids in red clover have been identified by using
LC-MS (43, 44), whether substances other than isoflavones contribute to its estrogenicity remains unclear.
The utilization of mass spectrometric characterization
combined with affinity enrichment of receptor ligands
from compound libraries or metabolite mixtures by
ultrafiltration assays has been reported previously (36,
37, 45-47). In this study, we applied this technique in
screening botanical extracts and successfully demonstrated that genistein likely plays the most important
role in terms of the estrogenic activity of red clover
followed by daidzein and biochanin A. Although abundant in red clover, formononetin had insufficient affinity
for ERβ to be detected in our affinity ultrafiltration LCMS assay.
The North American Menopause Society (NAMS)
recently published the results of a study concerned with
the therapeutic role of isoflavones in menopausal women
(48). As noted by NAMS, it is not clear whether the
observed health effects in humans are attributable to
isoflavones alone or to isoflavones plus other components in whole foods. Whereas a reduction in lowdensity lipoproteins and triglycerides and an increase
in high-density lipoproteins was associated with isoflavone ingestion, no differences in the incidence and
severity of hot flashes were observed between the
isoflavone recipients and the controls. Inadequate data
exist to establish the potential of isoflavones to modulate
breast and other hormone-dependent cancers, bone
mass, and vaginal dryness. Therefore, further work is
necessary to characterize the in vivo estrogenic activity
of isoflavones.
As currently reported, extracts of black cohosh displayed no estrogenic activity in the assays presented
here, which is consistent with previous results (21, 49).
Clinical trials with black cohosh have demonstrated a
significant reduction in serum lutenizing hormone (LH)
levels with women demonstrating climacteric symptoms; however, the extract had no effect on follicle
stimulating hormone (FSH) (50). These data indicate
that black cohosh may alleviate menopausal symptoms
by actions discrete from estrogen receptor regulation.
Chasteberry extract exhibited significant ER binding
and induced PR and pS2 mRNA expression, but no AP
induction activity was noted in Ishikawa cells. In
addition to the different sensitivities of the assays and
various targets detected in these assays, these results
may be due to the use of cell lines derived from different
tissues. Although hops extract was strongly cytotoxic
with Ishikawa cells, the estrogenic activity was still
detectable in the AP induction and PR expression
Extracts of Asian ginseng and North American ginseng mediated pS2 expression in S30 cells, but no ER
binding, AP induction, or PR expression in Ishikawa
cells. Stimulation of pS2 expression by American ginseng has been reported in MCF-7 cells, and this was
presumed to be partially mediated through the ER (27,
28). However, our data with S30 cells suggest that the
stimulation of pS2 expression by ginseng might not
occur through ER modulation. It is possible that the
constituents of ginseng modulate one or more elements
involved in ER function rather than directly through
J. Agric. Food Chem., Vol. 49, No. 5, 2001 2477
the ER. In a similar fashion, 3,3′-diindolylmethane
(DIM), a metabolite of indole-3-carbinol (I3C), was
shown to increase pS2 gene transcription in MCF-7 cells
without binding to the ER (51). This suggests a promoterspecific, ligand-independent activation of ER signaling
is a possible mechanism for natural modulation of ER
function. Several papers have suggested that the specific
down-regulation of pS2 expression is an early event in
sporadic late-onset Alzheimer’s disease (52) and may be
involved in the pathology of some cases of Alzheimer’s
(53). Therefore, extracts that can stimulate pS2 expression, such as ginseng, might benefit patients suffering
from Alzheimer’s disease.
Further studies are needed to fully understand the
mechanisms of dong quai, licorice, hops, chasteberry,
ginseng, and black cohosh. Alternative mechanisms
might involve receptors specific to other hormones or
neurotransmitters, such as luteinizing hormone release
hormone (LHRH), luteinizing hormone (LH), follicle
stimulating hormone (FSH), serotonin, and γ-aminobutyrate (GABA).
Of the eight plants tested, red clover extracts showed
the most consistent estrogenic effects in four different
in vitro assays. Hops extracts also displayed consistent
estrogenic potency, but it was found to be cytotoxic with
Ishikawa cells. Combined utilization of ultrafiltration
and LC-MS confirmed genistein was the most active
ERβ ligand in red clover, and this compound might be
responsible for AP induction, as well as PR and pS2
expression. Future studies utilizing affinity ultrafiltration LC-MS for ER binding as well as the four in vitro
bioassays will identify the estrogenic compounds in
other botanicals used for women’s health.
AP, alkaline phosphatase; DMEM, Dulbecco’s Modified Eagle Medium; E2, estradiol; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene glycol bis(βaminoethyl ether) tetraacetic acid; ELISA, enzymelinked immunosorbent assay; ER, estrogen receptor;
ERE, estrogen-responsive element; FBS, fetal bovine
serum; LC-MS, liquid chromatography-mass spectrometry; MEME, minimum essential medium; PE, petroleum ether; PMS, premenstrual syndrome; PR, progesterone receptor; pS2, presenelin-2; RT-PCR, reverse
transcriptase-polymerase chain reaction; SRB, sulforhodamine B; TCA, trichloroacetic acid; TLC, thin layer
We thank Dr. R. B. Hochberg of Yale University for
the Ishikawa cell line and Dr. V. C. Jordan of Northwestern University for the S30 cell line. We are grateful
to S. Schlecht, L. Chadwick, and D. Fabricant for the
extraction of the plants and to Steven Totura for
cultivation of red clover. Finally, we thank Mrs. Bambi
Teague at Blue Ridge Parkway National Park for
National Park Service Permits BLRI-00-030, BLRI-99028, Keith Langdon at Great Smoky Mountains National Park for National Park Service Permits GRSM00-096, GRSM-99-097, and Dr. Gwynn Ramsey and
Aubrey Neas for collection of plant material.
2478 J. Agric. Food Chem., Vol. 49, No. 5, 2001
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Received for review November 28, 2000. Revised manuscript
received February 12, 2001. Accepted February 12, 2001. This
research was supported by NIH Grant P50 AT00155, the Office
of Dietary Supplements (ODS), the National Institute of
General Medicine (NIGMS), the Office for Research on Women’s Health (ORWH), and the National Center for Complementary and Alternative Medicine (NCCAM).
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