1892 Biol. Pharm. Bull. 32(11) 1892—1897 (2009) Vol. 32, No.

11

Estrogenic Activity of Xanthorrhizol Isolated from Curcuma xanthorrhiza

ROXB.
ANGGAKUSUMA,a YANTI,a,b Myoungsu LEE,a and Jae-Kwan HWANG*,a
a
Department of Biotechnology, Yonsei University; 134 Shinchon-dong, Seodaemun-gu, Seoul 120–749, South Korea: and
b
Faculty of Biotechnology, Atma Jaya Catholic University; Jl Jenderal Sudirman 51, Jakarta 12930, Indonesia.
Received May 13, 2009; accepted August 7, 2009; published online August 14, 2009

Plant-derived estrogen-like compounds, or phytoestrogens, are given much attention due to their potential
therapeutic use. In this study, xanthorrhizol, a natural sesquiterpenoid isolated from the rhizome of Curcuma
xanthorrhiza ROXB. (Zingiberaceae), was evaluated for its estrogenic activity. It has been known that compounds
acting as ligands for estrogen receptors (ERs) are considered to possess estrogenic activity. Therefore, the Gal-
4/ER transactivation assay in transiently transfected African green monkey kidney (COS-7) cells was used to ex-
amine the estrogenic activity of xanthorrhizol. Both subtypes of ERs, ERa and ERb , were involved in this assay.
Further transactivation assays and pS2 mRNA analysis were also conducted in estrogen receptor-positive human
breast cancer (MCF-7). Our results showed that xanthorrhizol significantly increased Gal-4/ER luciferase activ-
ity in a dose-dependent manner and induced the endogenous ER-estrogen response element (ERE) interaction in
MCF-7 cells. Xanthorrhizol also significantly enhanced the expression of the pS2 gene in MCF-7 cells. In con-
trast, treatment using ICI 182780, an ER antagonist, suppressed all activities induced by xanthorrhizol, indicat-
ing ER-dependant activities were involved. These results suggest that xanthorrhizol possesses estrogenic activity
and its estrogenic effects are mediated by estrogen-induced gene expression.
Key words xanthorrhizol; estrogenic activity; estrogen receptor; transactivation assay; COS-7 cell; MCF-7 cell

Plant-derived estrogenic compounds, known as phytoestro-
gens, have drawn attention due to their potential therapeutic
use. From epidemiologic data, Asian people, who frequently
consume phytoestrogen-rich diets such as soybean, have been
found to have a lower rate of osteoporotic fractures, cardio-
vascular diseases, postmenopausal symptoms (PMS), and
certain cancers than Western populations.1) These health ad-
vantages are notably decreased when Asians adopt a Western
lifestyle and eating habits. In other studies, phytoestrogens
have been thought to show beneficial effects in cardiovascu-
lar and Alzheimer treatments.2—5) Research involving phy-
toestrogens found largely in legumes, seeds, fruits, and veg-
etables has been extensively conducted.6—9) Red clover, the
first plant known for its estrogenic activities, contains high
amounts of formononetin and biochanin A.10) Both com-
pounds were found to interact with estrogen receptors (ERs)
due to their structural similarity to 17b -estradiol.11) Cur-
rently, there are four groups of phenolic compounds classi-
fied as phytoestrogens: isoflavones, stilbenes, coumestans,
and lignans.11) Based on current findings revealing several
new phytoestrogen candidates, it is believed that other, yet
unknown compounds might possess estrogen-like activities
with beneficial therapeutic potential.12—14)
Sesquiterpenoid xanthorrhizol, a major bioactive com-
pound isolated from Curcuma xanthorrhiza ROXB. commonly
known as Javanese turmeric, has often been cited for its great
potential in food and medical applications. Xanthorrhizol has
been shown to exert antioxidant,15,16) anti-inflammatory,16,17)
antibacterial,18) neuroprotective,19) nephroprotective,20) and
hepatoprotective activity.21,22) Furthermore, xanthorrhizol has
been shown to have efficacy as a tumor chemopreventive
agent. Xanthorrhizol was shown to suppress 12-O-tetrade-
canoylphorbol-13-acetate (TPA)-stimulated tumor promotion
in mouse skin cells22) and attenuate lung metastasis in an in
vivo model.23) In HeLa cervical cancer cells, p53 and bax-de-
pendent apoptosis were stimulated with xanthorrhizol treat-
∗ To whom correspondence should be addressed. e-mail: jkhwang@yonsei.ac.kr
ment24) while in MCF-7 breast cancer cells apoptosis was
modulated through bcl-2, p53, and poly(ADP-ribose)poly-
merase-1 (PARP-1).25) In another study using HepG2 he-
patoma cells, xanthorrhizol induced the cells to undergo
apoptosis via the mitochondrial pathway.26) In addition, it has
been reported that the combination of xanthorrhizol and cur-
cumin promoted apoptosis in MDA-MB-231 breast cancer
cells.27)
In the present study, using the Gal4/ERs ligand-binding
transactivation assay, we examined whether xanthorrhizol
might possess an estrogenic activity. It was hoped that this
assay would provide further information regarding xanthor-
rhizol and its benefit as a phytoestrogen. The assay was con-
ducted in transiently-transfected ER-negative COS-7 cells
using both subtypes of ERs, ERa and ERb , and ligand bind-
ing domains (LBD). To determine if xanthorrhizol could in-
duce endogenous ER-estrogen response element (ERE) inter-
action in cells, a further ligand binding assay was conducted
in ER-positive MCF-7 cells. In parallel, pS2 mRNA analysis
was performed to investigate whether xanthorrhizol could
promote ER target gene expression (pS2) in MCF-7 cells.
The ER antagonist, ICI 182780, was used as a method of
confirmation.
MATERIALS AND METHODS
Plant Material Dried rhizomes of C. xanthorrhiza
ROXB. were collected in Jakarta, Indonesia, and identified by
Dr. Nam-In Baek, Department of Oriental Medicinal Materi-
als and Processing, Kyunghee University (Yongin, Korea). A
voucher specimen (H015) has been deposited in the Depart-
ment of Biotechnology, Yonsei University (Seoul, Korea).
Xanthorrhizol isolated from C. xanthorrhiza was dissolved in
100% (v/v) dimethyl sulfoxide (DMSO).
Isolation of Xanthorrhizol Xanthorrhizol (Fig. 1) was
isolated from the ethyl acetate fraction of the methanol ex-
© 2009 Pharmaceutical Society of Japan
November 2009
Fig. 1. Structure of Xanthorrhizol
tract of C. xanthorrhiza ROXB., as described previously.18)
Briefly, the dried rhizomes of C. xanthorrhiza (100 g) were
ground and extracted with 75% MeOH (v/v; 400 ml). The
methanol extract was fractionated consecutively with ethyl
acetate, n-butanol, and water. The yield at each step was 4.8,
1.7, and 1.1 g, respectively. The ethyl acetate fraction was
separated chromatographically by applying the fraction to a
silica gel column (70—230 mesh, Merck & Co., Whitehouse
Station, NJ, U.S.A.) and eluting with an n-hexane–ethyl ace-
tate solution (10 : 1, v/v). The final product, xanthorrhizol,
was obtained as a single compound with a yield of 0.2 g.
Cell Cultures African green monkey kidney cells, COS-
7, and human breast cancer cells, MCF-7, were obtained
from the American Type Culture Collection (Manassas, VA,
U.S.A.). COS-7 cells were cultured in Dulbecco’s modified
Eagle’s medium (DMEM) and MCF-7 cells were cultured in
RPMI-1640 medium (Gibco, Rockville, MD, U.S.A.). In rou-
tine culture, both cell types were supplemented with 10%
fetal bovine serum (FBS) (Gibco, Rockville, MD, U.S.A.)
and antibiotics (100 U/ml penicillin A and 100 m g/ml of
streptomycin), and stored at 37 °C in a humidified incubator
containing 5% CO2. When cells reached about 80% conflu-
ence, the medium was changed to phenol red-free DMEM or
RPMI supplemented with 1% charcoal dextran-stripped FBS
(csFBS) (Thermo Fisher Scientific, Logan, UT, U.S.A.) for
2 d of hormone starvation. After hormone starvation, cells
were ready to use for assays.
Cytotoxicity of Xanthorrhizol on COS-7 and MCF-7
Cells Cell viability was determined by the MTT colorimet-
ric assay (3-[4,5-diethylthiazol-2-yl]-2,5-dipheniltetrazolium
bromide; Sigma-Aldrich, St. Louis, MO, U.S.A.) according
to the method of Mosmann. After hormone starvation, the
cells were seeded at a concentration of 4104 cells/ml in 96-
well plates (Microtest 96, Becton Dickinson Co., Franklin
Lakes, NJ, U.S.A.) and cultured in phenol red-free DMEM-
csFBS at 37 °C in a 5% CO2 atmosphere. After 24 h of
attachment, culture medium was discarded and the cells were
treated with various concentrations of xanthorrhizol or a
combination of xanthorrhizol and ICI 182780 (Sigma-
Aldrich, St. Louis, MO, U.S.A.) in 200 m l serum-free and
phenol red-free DMEM or RPMI. The COS-7 cells were
treated for 24 h and the MCF-7 cells were treated for 48 h.
After treatment, cells were incubated with 50 m l of MTT
(1 mg/ml) for 4 h at 37 °C. The insoluble formazan dye was
solubilized in 200 m l of DMSO and the spectrophotometric
absorbance was measured at 550 nm with a tunable mi-
croplate reader (Versa Max, Sunnyvale, CA, U.S.A.).
Transient Transfection and Luciferase Ligand Binding
Transactivation Assays COS-7 cells were seeded at con-
centration 1106 cells/ml in 6-well plates in phenol red-free
DMEM-csFBS. After seeding, a Gal4 DNA binding domain
(DBD)-ERa ligand binding domain (LBD) or Gal4 DBD-
ERb LBD plasmid, pFR-luciferase, and pFR-b gal reporter
plasmid were transfected into the cells using Lipofectamine
1893
and PlusTM Reagent (Invitrogen, Carlsbad, CA, U.S.A.). After
5 h transfection, the medium was changed to fresh medium
containing the indicated amount of experimental treatment.
In the MCF-7 experiment, the cells were seeded at concen-
tration 3105 cells/ml into 6-well plates in phenol red-free
RPMI-csFBS. An ERE-luciferase plasmid and pFR-b gal
plasmid were transfected into the cells using the previously
mentioned method. Medium replacement was done after 12 h
transfection. After 18 h incubation, cells were lysed and the
cell lysates were used in activities measurements. The rela-
tive luciferase unit (RLU) was measured using a Micro-
LumatPlus LB 96V luminometer (Berthold, Wildbad, Ger-
many) and the b gal activity was measured using the 2-nitro-
phenyl b -D-galactopyranoside (ONPG) method (Sigma-
Aldrich, St. Louis, MO, U.S.A.). Final results were obtained
from the RLU normalized against b gal activity. In this exper-
iment, the Gal4-ERa plasmid was a kind gift from Dr. Yoon
Kwang Lee (Kent State University, Kent, Ohio, U.S.A.) and
the Gal4-ERb and ERE-luciferase plasmids were kindly pro-
vided by Dr. Hinrich Gronemeyer (Institute of Genetics and
Molecular and Cellular Biology, Illkirch Cedex, France).
Both pFR reporter plasmids were purchased from Stratagene
(La Jolla, CA, U.S.A.).
Semi-quantitative RT-PCR for pS2 Expression MCF-
7 cells were grown in 6-well plates in phenol red-free RPMI-
csFBS. Cells were treated with the indicated amount of
reagent for 48 h. Total RNA from MCF-7 cells was extracted
using Trizol reagent (Invitrogen Corp., Carlsbad, CA,
U.S.A.) according to the manufacturer’s protocol and quanti-
fied spectrophotometrically at 260/280 nm. cDNA was syn-
thesized by using 1 m g total RNA and oligo dT reverse tran-
scriptase premix (Elpis-Biotech, Taejeon, Korea) in a 20 m l
total reaction volume. Reverse transcription was performed
as follows: 70 °C for 5 min, 42 °C for 60 min, and 94 °C for
5 min. The cDNA products were directly used in PCR reac-
tions conducted using Takara LA Taq polymerase (Takara
Bio Inc., Shiga, Japan). The primers used for pS2 and glycer-
aldehyde 3-phosphate dehydrogenase (GAPDH) were 5-
CACCATGGAGAACAAGGTGA-3 (pS2 forward), 5-CC-
GAGCTCTGGGACTAATCA-3 (pS2 reverse), 5-CAAT-
GACCCCTTCATTGACC-3 (GAPDH forward), and 5-
TGAGTCCTTCCACGATACCA-3 (GAPDH reverse) to
yield products of 323 and 425 bp, respectively. PCR con-
sisted of 25 amplification cycles of 1 min at 94 °C, 1 min at
annealing temperature, and 1 min at 72 °C in a thermal cycler
(Gene Amp PCR System 2700, Applied Biosystems, CA,
U.S.A.). The human GAPDH housekeeping gene was used as
an internal control. PCR products were separated elec-
trophoretically in a 2% agarose DNA gel and stained with
ethidium bromide. The stained gel was visualized by Gel-
Doc Quantity One software (Bio-Rad Laboratories, Hercules,
CA, U.S.A.).
Statistical Analysis Triplicate experiments were per-
formed throughout this study. All data are presented as the
meanstandard deviation (S.D.). The significant difference
between the control (DMSO) and treated groups were statis-
tically analyzed by the paired Student’s t-test (p0.05).
RESULTS
Cytotoxicity of Xanthorrhizol on COS-7 and MCF-7
1894 Vol. 32, No. 11

Cells The cytotoxicity of xanthorrhizol on COS-7 and

MCF-7 cells was determined by MTT assay. In the COS-7
assay, cells were treated with xanthorrhizol at concentrations
ranging from 1—50 m M for 24 h. At these concentrations,
xanthorrhizol did not affect the cell viability (Fig. 2A). Thus,
xanthorrhizol concentrations within this range were used in
further COS-7 transactivation assays. In contrast, in the
MCF-7 assay, cells demonstrated more sensitivity to xanthor-
rhizol than COS-7 cells. Xanthorrhizol at 1—7.5 m M resulted
in cell viability of more than 80% after incubation for 24 and
48 h (Fig. 2B). At 1 m M, xanthorrhizol slightly decreased cell
viability (13%) during a 48 h incubation. Therefore, we
used xanthorrhizol at concentrations of 0.5—5 m M for the as-
says involving MCF-7 cells.
Induction of Luciferase Activity by Xanthorrhizol in
Transiently Transfected COS-7 Cells In this study, ER-
negative COS-7 cells were transiently transfected with the
ERa and ERb reporter systems. As shown in Figs. 3A and B,
treatment of these cells with xanthorrhizol at various concen-
trations increased luciferase activity in a dose-dependent
manner. In ERa -transfected cells, 10 m M xanthorrhizol in-
duced luciferase activity by about 517% compared to control
treatment. Interestingly, luciferase activity was similar to that Fig. 2. Effects of Xanthorrhizol on COS-7 and MCF-7 Cell Viability
obtained with 5 m M estradiol (504%) (Fig. 3A). In ERb trans- COS-7 cells were treated with xanthorrhizol at concentrations of 1—50 m M for 24 h
fected cells, 10 m M xanthorrhizol induced luciferase activity (A) and MCF-7 cells were treated at concentrations of 1—25 m M for both 24 h and 48 h
(B). The cells treated with vehicle only (DMSO) were used as the control. Cell viability
by 832% compared with the vehicle (Fig. 3B). However, this was determined by the MTT assay. Data are shown as meanS.D. from triplicate ex-
luciferase activity was much lower than obtained with 5 m M periments. ∗∗ p0.01 compared with the control.
estradiol (5783%). Furthermore, to confirm that the agonistic
activity of xanthorrhizol was ER-mediated, ERa transfected

Fig. 3. Ligand Binding Activity of Xanthorrhizol in the Gal4/ERs Luciferase Transactivation Assays
COS-7 cells were transfected with the Gal4/ERa (A) or Gal4/ERb (B) fusion reporter system and treated with DMSO control, estradiol (5 m M), or xanthorrhizol (1, 5, 10 m M) for
18 h. (C) In the confirmation assay, transfected cells were co-treated with a combination of 5 m M xanthorrhizol and 1 m M ICI 182780. Final results were obtained from the RLU
which had been normalized against b gal activity. Cell viability for the co-treatment assay in COS-7 cells was also checked with 10 m M xanthorrhizol co-treated with various con-
centrations of ICI 182780 (1—25 m M). Cells treated with vehicle only (DMSO) were used as controls. Data are shown as meanS.D. from triplicate experiments. ∗ p0.05 com-
pared with the control.
November 2009 1895

cells were co-treated with 1 m M ICI 182780. These results
showed that the luciferase activity induced by 5 m M xanthor-
rhizol was completely diminished by ICI 182780 (Fig. 3C).
The quenching of luciferase expression was not a result of
reduced cell viability because co-treatment with both agents
did not cause COS-7 cell death (Fig. 3D). These results indi-
cate that the induction of luciferase activity is possibly medi-
ated through ER LBD-ligand interaction.
Effects of Xanthorrhizol on ERE Activation and pS2
mRNA Expression in MCF-7 Cells We investigated
whether xanthorrhizol activated endogenous ER in MCF-7
cells by transiently transfecting the cells with an ERE-lu-
ciferase reporter gene. As seen in Fig. 4A, xanthorrhizol in-
duced ERE-dependent luciferase activity by about 244% at
1 m M 24 h after treatment compared to controls. This result
meant that xanthorrhizol could activate the ER-ERE interac-
tion followed by expression of the target gene. In a parallel
assay, to show if the original ER gene target in MCF-7 cells
can also be induced by xanthorrhizol treatment, the level of
pS2 mRNA expression was assayed. As shown in Fig. 4B,
after 48 h sample treatment at 1 m M and 5 m M concentrations,
the cells increased expression of pS2 mRNA compared to
control treatment. This result also confirmed the previous
assay result. Finally, co-treatment of transiently transfected
MCF-7 cells with 1 m M ICI 182780 could completely abolish
both the luciferase activity and pS2 mRNA expression (Figs.
4C, D). Nevertheless, both of the activities were not abol-
ished due to the cell death. Since, as shown in Fig. 4E, the
cell viability under treatment of 1 m M xanthorrhizol com-
bined with 1 m M ICI 182780 was still remaining high
(80%) and similar with the 48 h treatment of 1 m M xanthor-
rhizol or ICI 182780 alone. This result implied that xanthor-
rhizol exerted estrogenic effects through ER binding to ERE.
DISCUSSION
Phytoestrogens, a term coined to describe plant-derived
chemicals that exert estrogenic activity, involve numerous va-

Fig. 4. Effects of Xanthorrhizol on ER Activation in MCF-7 Cells

(A) MCF-7 cells were transfected with the ERE-luciferase system and treated with vehicle, estradiol (1 m M), or xanthorrhizol (0.5, 1, 5 m M). (B) The confirmation assay where
transfected cells were co-treated with a combination of 1 m M xanthorrhizol and 1 m M ICI 182780. (C) The affect of xanthorrhizol on pS2 mRNA expression was assayed using semi-
quantitative RT-PCR. Cells were treated with vehicle, estradiol (1 m M) or xanthorrhizol (1, 5 m M). (D) The confirmation assay where transfected cells were co-treated with a combi-
nation of 1 m M xanthorrhizol and 1 m M ICI 182780. GAPDH mRNA was used as an internal control. (E) Cell viability for the co-treatment assay in MCF-7 cells was determined
using various concentration of xanthorrhizol (1, 5, 10 m M) co-treated with various concentrations of ICI 182780 (1—25 m M). Cells treated with vehicle were used as controls.
∗ p0.05 compared with the control.
1896
rieties of structurally diverse compounds and are grouped
into isoflavones, stilbenes, coumestans, and lignans. They
can regulate the actions of endogenous estrogens, usually by
binding to ERs.28,29) ER itself is a member of the nuclear re-
ceptor superfamily, which acts by regulating the transcription
process. The classical pathway of ER action involves ligand
binding to receptor in the nucleus, followed by dimerization
and binding to specific response elements known as EREs,
located in the promoter regions of target genes.30) In humans,
two distinct estrogen receptors, ERa and ERb , can be de-
tected in a broad spectrum of tissues. In some organs, both
receptor subtypes are expressed at similar levels, whereas in
others, one or the other subtypes predominates.31) Both ER
subtypes have functional differences, depending on the tissue
type. For example, ERb has been known for its potential as a
tumor suppression gene in breast tissue, while ERa plays an
important role in vessel wall pathophysiology in the cardio-
vascular system. ERa has also been known for playing an
important role in postpubertal bone elongation, while ERb
is responsible for repression of the ERa -mediated bone
growth-promoting effect. Furthermore, in adipocytes, ERa is
known to decrease adipose tissue mass by increasing lypoly-
sis.31) Both ER subtypes bind phytoestrogen, although their
binding patterns may vary and their affinities for phytoestro-
gen are 100- to 10000-fold lower than that of estradiol.32)
Based on recent reviews, phytoestrogens have frequently
been tested for their benefits. Phytoestrogens have been
known for their ability to improve bone mass and reduce car-
diovascular risk factors in postmenopausal woman.33) Re-
duced endometrial cancer risk has also been observed among
those taking phytoestrogen. Phytoestrogens may also act as
antioxidants and inhibit blood vessel growth, essential for
tumor expansion (a similar function was also proposed for
xanthorrhizol). In Alzheimer’s disease research, phytoestro-
gens showed beneficial effects on memory and central nerv-
ous system function.34) Furthermore, in recent studies, phy-
toestrogens have been assayed as cosmeceuticals as skin anti-
aging and anti-carcinogenesis agents.35,36)
In this study, using the Gal4 fusion reporter system, xan-
thorrhizol binding interaction to ERs was screened. From
our results, we found that xanthorrhizol could interact and
saturate the ER ligand binding domain. The saturation was
adequate to stimulate the luciferase signal in both ERa and
ERb systems. However, due to differences in the reporter
plasmids involved in this assay, the levels of luciferase signal
from both systems were different. The signals resulting from
xanthorrhizol treatment, if compared with estradiol, were far
lower in the ERb system than in the ERa system. In previous
studies, estradiol itself has been reported to have a lower
affinity to ERb than to ERa .37—39) Thus, it could be viewed
that xanthorrhizol might have a higher affinity for ERa be-
cause it stimulated luciferase expression in a dose-dependent
manner and reached the same level of luciferase signal pro-
duced by 5 m M estradiol at a concentration of 10 m M (Fig.
3A). Although xanthorrhizol’s luciferase signal in the ERb
assay was also dose-dependent, and significant relative to
controls, the signals were very low compared to that of estra-
diol (Fig. 3B). Finally, in the co-treatment assay using the ER
antagonist, ICI 182780, signal from the ERa system was
completely reversed without affecting the cell viability (Figs.
3C, D). These results imply that xanthorrhizol exerts its ef-
Vol. 32, No. 11
fects through the ER and follows the classical pathway of ER
activation.
To prove our hypothesis, xanthorrhizol was also tested in
MCF-7 cells, an estrogen positive cell line.28) Results showed
that, xanthorrhizol could enhance expression of both lu-
ciferase and pS2 mRNAs compared to controls. However,
both signals appeared weaker than that obtained with estra-
diol treatment (Figs. 4A, C). This was due to the fact that
xanthorrhizol, at the higher concentration range used previ-
ously in the MCF-7 assay (1—10 m M) exhibited strong cyto-
toxic activity.25) Finally, the up-regulated luciferase and pS2
expressions could be reversed by ICI 182780 co-treatment.
The complete reversal of both signals showed that xanthor-
rhizol exerted its estrogenic activity through the ER in MCF-
7 cells.
Our studies show that xanthorrhizol, isolated from C. xan-
thorrhiza ROXB. has estrogenic activity because it binds to
ERs and activates its target genes through the classical ER
pathway. This finding expands the potential uses for xanthor-
rhizol. From preliminary assays, we have determined that
xanthorrhizol does not interact with the glucocorticoid recep-
tor (data not shown). Therefore, we will explore if xanthor-
rhizol may also activate other steroidal hormone receptors
(androgen receptor (AR) and mineral receptors (MR)). Fur-
thermore, as a phytoestrogen, it will be determined if xan-
thorrhizol exhibits direct action on osteoblasts by inducing
mineralization and if it has non-genomic estrogenic activity.
Acknowledgements This work was supported in part by
the Yonsei Biomolecule Research Initiative of the two-step
Brain Korea 21 Project.
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