Wang et al. BMC Complementary and Alternative Medicine (2018) 18:107
https://doi.org/10.1186/s12906-018-2170-4

RESEARCH ARTICLE

Open Access

The in vitro estrogenic activity of the crude
drugs found in Japanese herbal medicinesprescribed for menopausal syndrome wasenhanced by combining them
Zeyun Wang, Seiji Kanda*, Takaki Shimono, Dambajamts Enkh-Undraa and Toshimasa Nishiyama

Abstract
Background: Japanese herbal medicines can be used as alternatives to estrogen therapy and are sometimes prescribedfor menopausal syndrome because they have fewer side effects and are associated with better compliance than estrogentherapy, but little is known about the pharmacological mechanisms of such treatments. This study aimed to explore themechanisms responsible for the estrogen-like effects of five widely prescribed Japanese herbal medicines (unkeito,
kamishoyosan, nyoshinsan, keishibukuryogan, and tokishakuyakusan).
Methods: We evaluated the estrogenic activity of these five Japanese herbal medicines and their metabolites
using an estrogen receptor (ER)-dependent cell proliferation bioassay and an ER-dependent reporter assay. We
also investigated the estrogenic activity of the crude drugs within the medicines and attempted to detect intercrude drug synergistic effects using the ER-dependent reporter assay.
Results: We found that unkeito, kamishoyosan, and nyoshinsan exhibited estrogenic activity, and they displayedstronger estrogenic activity after being metabolized. Then, we focused on investigating the estrogenic activity of
the crude drugs present within unkeito. We found that glycyrrhizae radix, cinnamomi cortex, evodiae fructus, andzingiberis rhizoma demonstrated ERβ-dependent estrogenic activity. The combined use of evodiae fructus andglycyrrhizae radix, or evodiae fructus and cinnamomi cortex produced synergistic ERβ-dependent estrogenic activity.
Conclusion: It was suggested that unkeito, kamishoyosan, and nyoshinsan exert estrogenic activity, and hence, might
be useful for treating menopausal syndrome. Furthermore, synergistic estrogenic effects were detected between someof the crude drugs present within unkeito.
Keywords: Estrogenic activity, Japanese herbal medicines, Metabolism, Synergistic effectBackground
The menopause is a biological stage in a woman’s life
when her ovarian function diminishes and eventuallyceases. It involves the cessation of both egg maturationand sex hormone (principally estrogen and progesterone)secretion. Many women experience menopausal syndrome, which can cause hot flushes, night sweats, menstrual irregularities, vaginal dryness, etc., during themenopause and perimenopause [1–3]. Estrogen therapy is
widely used to treat menopausal syndrome. The biological* Correspondence: kandas@hirakata.kmu.ac.jp
Department of Public Health, Kansai Medical University, 2-5-1, Shin-machi,
Hirakata-shi, Osaka 573-1010, Japan

activity of estrogen is mediated by its binding to estrogen
receptors (ER); i.e., ERα and ERβ, which are encoded bydifferent genes and exhibit tissue-type- and cell-typespecific expression. The activation of each subtype leads
to different effects. In addition, ERα is associated with certain types of estrogen-dependent tumors, such as breast[4], endometrial [5], and ovarian cancer [6]. On the other
hand, it has been found that ERβ expression can inhibit
motility and cell invasion in breast [7], prostate [8], andovarian cancer cells [9]. For example, estrogen increased
the proliferation of breast cancer cells in the presence ofERα, but this was inhibited in the presence of ERβ [10].
Therefore, while estrogen therapy might have rapid effects© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Wang et al. BMC Complementary and Alternative Medicine (2018) 18:107

on menopausal syndrome, it might also increase the riskof estrogen-dependent tumors.
Herbal medicines are derived from various plants andhave been prescribed to treat many diseases for thousands of years. It was previously reported that phytoestrogens, such as daidzein and daidzin, can be found inpuerariae radix [11], and the glabridin present in licoriceroots was suggested to be a useful treatment for postmenopausal women because of its estrogenic activity[12–14]. Herbal medicines have recently been used as analternative to estrogen therapy for menopausal syndrome, but little is known about the pharmacological activity of such medicines. In this study, we evaluated the
estrogenic activity of five widely used Japanese herbal
medicines (unkeito [UKT], kamishoyosan [KSS], nyoshinsan [NSS], keishibukuryogan [KBG], and tokishakuyakusan [TSS]), in order to explain their pharmacologicalactivity against menopausal symptoms.
We examined the estrogenic activity of these five Japanese herbal medicines using a combination of an ERdependent cell-MtT/Se cell proliferation bioassay and ayeast two-hybrid reporter assay. The MtT/Se cell line wasestablished from rat pituitary cells [15], and both ERα andERβ are expressed in the cytoplasm around the nuclei ofthese cells [16]. It was reported that this cell line responds
well (and dose-dependently) to 17β-estradiol (E2) stimulation, and the E2-induced proliferation of MtT/Se cells wasdose-dependently inhibited by tamoxifen [15]; therefore,
an assay based on MtT/Se cell proliferation was developed
to evaluate the estrogenic activity of test substances [16].
In addition, a yeast-based estrogen screening test based on
the yeast two-hybrid system was used to clarify the estrogenic activity of Japanese herbal medicines. This system
was constructed by inserting the ligand-binding domain(LBD) of the human estrogen receptor and its coactivator
into yeast cells, which allowed the specific binding of ligands to the ER-LBD. Estrogenic activity was then quantitatively evaluated based on the expression level of areporter gene encoding the β-galactosidase enzyme [17].
Like all drugs, the chemical structures and pharmacological activity of Japanese herbal medicines might be altered by the enzymatic systems found in living organisms.
For example, the estrogenic activity of the phytoestrogenrich plant Pueraria mirifica has been found to increase significantly after being metabolized [18]. Therefore, themetabolic modulation of estrogenic activity must be considered in any assessment of the impact of Japanese herbal
medicines. In this study, we also investigated the estrogenic activity of Japanese herbal medicines after they had
been metabolized by the rat liver S9 fraction.
Herbal medicines are composed of a mixture of several
crude drugs. In order to study the pharmacological activityof herbal medicines, we first need to confirm which of thecrude drugs present within them are responsible for such

Page 2 of 12

activity. We assessed the estrogenic activity of the crude
drugs using the same protocol as was used to study theJapanese herbal medicines. We also conducted an experiment to detect inter-crude drug synergistic effects.

Methods
Medicines and extraction

Japanese herbal medicines; i.e., UKT (unkei decoction,
TJ-106), KSS (kamishoyo powder) (TJ-24), NSS (nyoshin
powder, TJ-67), KBG (keishibukuryo pills, TJ-25), andTSS (tokishakuyaku powder, TJ-23), were obtained from
Tsumura Corporation (Tokyo, Japan) in the form ofdried powder extracts.
Each herbal medicine extract powder was manufactured
as an aqueous extract containing processed raw materials
in the ratios shown in Table 1. The qualities of these raw
materials were tested as set out in the JapanesePharmacopoeia. Each herbal medicine was extracted withpurified water at 95°C for 1 h. The extract solution was
separated from the non-soluble waste and concentrated
by removing water under reduced pressure. Spray-drying
was used to produce a dried extract powder. The same extraction procedure was applied to each crude drug.
The active ingredient of each powdered medicine was
extracted by mixing 1 g of powder with 10 ml of 99.5%
ethanol and rotating the mixture for 4 h in our laboratory. The mixture was then filtered through a 0.45-μm
filter membrane. The solvent was evaporated to dryness
at room temperature in a vacuum, and the residue wasdissolved in dimethyl sulfoxide (DMSO) (Wako Chemical, Japan). The concentration of each extract was adjusted with DMSO as required for each assay.

Metabolic activation

In this study, in vitro metabolic activation via theaddition of the rat liver S9 fraction (Sigma Aldrich, SaintLouis, USA) was used to metabolize the examined Japanese herbal medicines. Metabolic activation was induced
using an S9 mixture containing 0.5 mg/ml of the S9
fraction in 100 mM potassium phosphate buffer(pH 7.4), 3.3 mM MgCl2 (Wako Chemical, Japan), andan NADPH-generating system [1.3 mM NADP (Sigma
Aldrich, USA), 3.3 mM glucose 6-phosphate (Oriental
Yeast, Tokyo, Japan), and 0.4 units/ml of glucose-6phosate dehydrogenase (Oriental Yeast, Tokyo, Japan)].
Each 200-μl sample of Japanese herbal medicine dissolved in DMSO was incubated with 800 μl of the S9
mixture at 37°C for 1 h or with 800 μl of the negative
mixture, which was composed of the abovementioned
S9 mixture minus the S9 fraction, as a negative control.
The metabolized Japanese herbal medicines were storedat − 80°C until use.

Wang et al. BMC Complementary and Alternative Medicine (2018) 18:107

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Table 1 The mass ratio of crude drugs in the Japanese herbal medicines
herbalsimplified
form

Kamishoyosan

Keishibukuryogan

Nyoshinsan

Tokishakuyakusan

Unkeito

KSS

KBG

NSS

TSS

UKT

alismatis rhizoma

arangelicae acutilobae radix

aar

arecae semenasasini corii collasaccatractylodis lanceae rhizomaalr

3bupleuri radixbr3

caryophylli floscf

cinnamomi cortex

cccnidii rhizoma

cr

4
3

3

3

2
3

ctr

1

cer

3

ef
gf
gra
gr

1.5

menthae herbamh1

moutan cortex

mc

2

3

3

3

or
pr

persicae semenpspinelliae tuber

ptporia sclerotium

pstsaussureae radix

srscutellariae radix

scr

zingiberis rhizoma

zr

2

1

glycyrrhizae radix

paeoniae radix2
3

2

ginseng radix

ophiopogonis radix

4

1

cyperi rhizoma

gardeniae fructus

323

coptidis rhizoma

evodiae fructus

3

2

2

2

1

2

2
4
4

2

3
4
3

3

4
1
2

1

1

The mass ratio of crude drugs in the Japanese herbal medicines was provided to us by Tsumura Corporation. In order to simplify the name of herbal medicinesand crude drugs in text, we made the simplified forms in the table

Cell culture and maintenance of the MtT/se cells

Rat mammotropic pituitary tumor cells, MtT/Se cells,
were donated by the Riken Bioresource Center cell bankin Japan. The MtT/Se cells were routinely cultured in
Dulbecco’s modified Eagle’s medium (DMEM)/HamF12(1∶1) medium (Nacalai Tesque, Japan) containing 2.5%
fetal bovine serum (FBS) (HyClone, USA), 10% horse
serum (HS) (Invitrogen, USA), and 1 × 10− 9 M of estradiol (E2) (Wako Chemical, Japan). The cells were incubated at 37°C in a humidified atmosphere of 5% CO2
and 95% air.
ER-dependent cell proliferation bioassay

Before the cell proliferation bioassay, the MtT/Se cells
were maintained for 4 days in routine medium from which
E2 had been removed, as described above. In the proliferation assay, the MtT/Se cells were placed in phenol red-free
DMEM/HamF12 (1:1) medium (Nacalai Tesque, Japan)
containing 2.5% charcoal dextran-treated FBS (HyClone,

USA) and 10% HS at a density of 1 × 105 cells/ml, before
being seeded at 95 μl/well in 96-well microplates. Then,the cells in each well were incubated with 5 μl of each test
substance for 6 days. In order to clarify whether the herbal
medicines induced cell proliferation through ER-mediatedmechanisms or other general mitogenic pathways, ICI182,780, an ER antagonist (Abcam, UK), was added as acompetitor. Cells that were incubated with DMSO alone
were used as a control, and the final concentration of
DMSO in all wells was 0.1%. Six days later, cell proliferation was measured with a modified MTT assay based on
WST-8, a highly water-soluble tetrazolium salt (Dojindo,
Japan). The amount of formazan dye formed by metabolically active cells was quantified by measuring the absorbance of each well at 450 nm. The estrogenic activity ofeach test substance was evaluated based on the cell proliferation that occurred in its presence, which was calculated
by comparing the associated A450nm value with the A450nm
value of the control, DMSO.

Wang et al. BMC Complementary and Alternative Medicine (2018) 18:107

Cell proliferation (%) = (A450nm test / A450nm DMSO) × 100%.
ER-dependent reporter assay (yeast two-hybrid assay)

In this study, we used human ERα (hERα) and hERβ
yeast cells carrying the LBD of hERα or hERβ and thecoactivator transcriptional intermediary factor 2 (TIF2)(the cells had been transfected with two expression plasmids, pGBT9-ERLBD and pGAD424-TIF2) [19].
The yeast cells were pre-incubated and shaken overnight at 30°C in selective medium (SD medium lacking
tryptophan and leucine). A 100-μl sample of the overnight
yeast cell culture was added to 400 μl of fresh medium
containing 5 μl of one of the test substances. After being
incubated for 4 h at 30°C, the yeast cells were collected bycentrifugation and resuspended in 250 μl Z-buffer [0.1 M
sodium phosphate (Wako Chemical, Japan), 10 mM KCl(Wako Chemical, Japan), 1 mM MgSO4 (Wako Chemical,
Japan)]. Then, 150 μl of the cell suspension were added toa 96-well microplate, and the cell density of each well wasmeasured based on its A600nm value. Next, the cell walls of
the remaining 100 μl of yeast cells were digested enzymatically by incubating them with 100 μl of 2 mg/ml Zymolyase 20 T (Nacalai Tesque, Japan) at 37°C for 30 min.
Then, the 200-μl lysate was mixed with 40 μl of 4 mg/ml
o-nitrophenyl-β-D-galactopyranoside (ONPG) (Sigma
Aldrich, USA) and incubated at 30°C for 1 h, before the
enzymatic reaction was stopped by the addition of 100 μlof 1 M Na2CO3 (Wako Chemical, Japan). Then, the yeast
debris was removed by centrifugation, and the absorbanceof the supernatant was measured at 450 nm. βgalactosidase activity was calculated using the following
equation:
β-galactosidaseactivity ðunitsÞ
¼ 1000  A450nm =ð60 min  0:1ml  A600nm Þ, where A600nm represents the cell density at the start of
the assay, A450nm represents the absorbance of the supernatant at the end of enzymatic reaction, 60 min is the duration of the enzymatic reaction, and 0.1 ml is the volumeof the yeast cell culture used in the enzymatic reaction.
The ERα estrogenic activity of the tested Japanese herbal
medicines was evaluated based on the β-galactosidase activity of hERα yeast cells, and their ERβ-dependent estrogenic activity was assessed based on the β-galactosidase
activity of hERβ yeast cells. REC10 (the concentration that
produced an activity level equivalent to 10% of the maximum activity of E2) was used to evaluate the estrogenic
activity of the Japanese herbal medicines.
Combination study of synergism and synergy quotient
calculations

As shown in Table 1, Japanese herbal medicines are composed of various crude drugs, and we tried to identify

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pairs of crude drugs that had synergistic effects. In the
combinatorial study, each pair of crude drugs was added
to yeast cells in a 1:1 ratio. The combinatorial study was
performed to detect synergistic crude drug effects on
ERβ-dependent estrogenic activity compared with the activity seen during the administration of each drug alone.
The synergism quotient (SQ) was calculated by subtracting the baseline values (the β-galactosidase activity
values of DMSO) from all treatments and then dividing
the activity value of the combined treatment by the sumof the activity values for the individual treatments. A SQof > 1.0 indicated a synergistic effect [20, 21].
3D-high-performance liquid chromatography (HPLC)
analysis

In this study, 3D-HPLC analyses of the herbal medicines
were performed by Tsumura Corporation to identify
each of their components.
The HPLC apparatus consisted of a Shimadzu LC-10A(analysis software: CLASS-M10A ver. 1.64, Tokyo, Japan)
equipped with a multiple wavelength detector (wavelength
range: 200–400 nm; Shimadzu SPD-M10AVP diode array
detector) and an autoinjector (Shimadzu CTO-10 AC).
The HPLC conditions were as follows: column, TSK-GEL
80TS octadecylsilyl column (internal diameter: 250 ×
4.6 mm; TOSOH, Tokyo, Japan); eluent, (A) 0.05 M
AcONH4 (pH 3.6), (B) 100% CH3CN. A linear gradient of
90% A and 10% B changing over 60 min to 0% A and100% B was used. Once a B concentration of 100% had
been achieved, it was maintained for 20 min (temperature,
40°C; flow rate, 1.0 mL/min).
Statistical analysis

All experiments were conducted at least three times foreach sample, and all data shown represent the mean ±standard deviation (SD). The Kolmogorov-Smirnov test
was used to confirm that all of the data for each sample
exhibited a normal distribution. All statistical analyses
were carried out using the Student’s t-test in SPSS,
version 15.0. P-values of < 0.05 were considered to bestatistically significant.

Results
The estrogenic activity of Japanese herbal medicines andtheir metabolites in an ER-dependent cell proliferation
bioassay.
The estrogenic activity of each Japanese herbal medicine was evaluated using an MtT/Se cell proliferation
assay. The cell proliferation that was induced by E2 plateaued at 10− 7 M, and the mean cell proliferation activity
level was 322% higher than the activity induced by
DMSO. UKT, KSS, and NSS induced significantly increased cell proliferation at final concentrations of
50 μg/ml and 100 μg/ml. However, neither TSS nor KBG

Wang et al. BMC Complementary and Alternative Medicine (2018) 18:107

induced significant increases in cell proliferation at concentrations of 50 μg/ml or 100 μg/ml (Fig. 1a). When thecells were treated with 10 nM of ICI, the proliferation induced by UKT, KSS, NSS, and E2 was inhibited (Fig. 1b).
These results showed that UKT, KSS, and NSS induced

Fig. 1 The cell proliferation activity induced by Japanese herbal
medicines. a MtT/Se cells were incubated with 50 μg/ml (whitecolumns) or 100 μg/ml of each Japanese herbal medicine (blackcolumns) or 10− 7 M of E2 for 6 days. Cell proliferative activity isshown as a percentage compared with that induced by DMSO
alone in the MTT assay. **significantly increased compared withthe activity induced by DMSO, p < 0.01. b Cells were treated with 10 nM
ICI alone or a combination of 10 nM ICI and 100 μg/ml of UKT, KSS, or
NSS or 10− 7 M E2 for 6 days. White columns: ICI(−)test substance(−);
blank columns: ICI(−)test substance(+); gray columns: ICI(+)test
substance(−); diagonal columns: ICI(+)test substance(+). **significantly
inhibited the induction of cell proliferation compared with herbal
medicines or E2 alone, p < 0.01; *p < 0.05. c The cell proliferationinduced by metabolized Japanese herbal medicines. The Japanese
herbal medicines were treated with the rat S9 fraction, and theproliferative activity induced by each Japanese herbal medicine(100 μg/ml, black columns) was compared with that induced by theunmetabolized medicine (white columns). *significantly increased
compared with the activity of the unmetabolized medicine, p < 0.05

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proliferation through ER-mediated mechanisms and not
via other general mitogenic pathways. Thus, these threeherbal medicines displayed estrogenic activity, and KSS
exhibited the strongest activity in this bioassay.
In order to study the effects of the examined Japanese
herbal medicines after they had been metabolized, we
evaluated their estrogenic activity after they had been
pre-treated with rat liver S9 extract in vitro. Metabolized
UKT and NSS induced significantly greater cell proliferation than unmetabolized UKT/NSS at a final concentration of 100 μg/ml (Fig. 1c). The estrogenic activity of
KSS also increased after it was treated with S9, but thedifference was not significant. These results showed thatthe estrogenic activity of UKT and NSS increased about
1.2-fold after they were treated with S9. TSS and KBG
did not show any estrogenic activity before or after they
were metabolized.
Estrogenic activity of Japanese herbal medicines andtheir metabolites in a yeast two-hybrid assay.
Next, we evaluated the ERβ-dependent estrogenic activity of the five examined Japanese herbal medicines.
We analyzed the ERβ-dependent estrogenic activity ofeach medicine using a reporter gene-based yeast twohybrid assay at final concentrations of 0.1 mg/ml to
10 mg/ml (Fig. 2a). Marked β-galactosidase activity was
induced by UKT, KSS, and NSS, and the activity increased dose-dependently. These results suggested that
UKT, KSS, and NSS exhibited estrogenic activity, which
agrees with their estrogenic effects in the MtT/Se cellproliferation assay. On the other hand, TSS and KBG
demonstrated low ERβ-dependent estrogenic activity,
even at higher concentrations. In addition, E2 induced
ERβ-dependent estrogenic activity at concentrations ranging from 1 × 10− 4 M to 1 × 10− 11 M, and this activity
plateaued at an E2 concentration of 1 × 10− 6 M. The
REC10 values of UKT, KSS, and NSS were 0.7 mg/ml,
2.2 mg/ml, and 4.5 mg/ml, respectively. These results
suggested that UKT, KSS, and NSS induced marked
ERβ-dependent estrogenic activity. UKT had the lowest
REC10 value of the five examined Japanese herbal medicines, which showed that it produced the strongest ERβdependent estrogenic activity. On the other hand, noneof the five examined Japanese herbal medicines displayed
ERα estrogenic activity in the yeast two-hybrid assay(data not shown).
The three herbal medicines that showed estrogenic activity in the yeast two-hybrid assay had their estrogenic
activity assessed after being metabolized. The herbal
medicines were pre-treated with a rat liver S9 mixture
that included metabolic enzymes, and their estrogenic
activity was compared with the estrogenic activity of theunmetabolized herbal medicines using the yeast twohybrid assay. The activity levels of the metabolized products are shown in Fig. 2b. The three herbal medicines

Wang et al. BMC Complementary and Alternative Medicine (2018) 18:107

Fig. 2 The ERβ-dependent estrogenic activity of the Japanese herbal
medicines and the metabolized herbal medicines in the yeast twohybrid assay. a Yeast cells that had been transfected with hERβ wereincubated with 0.1 mg/ml to 10 mg/ml of UKT (◊), KSS (□), TSS (○), NSS(▲), or KBG (■) for 4 h at 30°C. ERβ-dependent estrogenic activity wasevaluated based on the β-galactosidase activity measured at 450 nm
using ONPG as a substrate. b The Japanese herbal medicines (1 mg/ml)
were treated with the rat S9 fraction, and the ERβ-dependent
estrogenic activity of the metabolized herbal medicines(black columns) was compared with that of the unmetabolized medicines(white columns) using the yeast two-hybrid assay. **significantly increased
compared with the activity of the unmetabolized medicine, p < 0.01

that exhibited estrogenic activity in the yeast two-hybrid
assay also showed significantly elevated ERβ-dependent
estrogenic activity after being treated with the rat liver
S9 fraction. These results showed that metabolizing
UKT, KSS, and NSS with the rat liver S9 fraction
increased their ERβ-dependent estrogenic activity. On
the other hand, no significant differences between the
ERα-dependent estrogenic activity of the metabolized andunmetabolized medicines were seen for any of the five examined Japanese herbal medicines (data not shown).
Estrogenic activity of the crude drugs found in UKT

In order to clarify which crude drugs were responsible
for the pharmacological activity of the herbal medicines,
we focused on UKT. As shown above, both UKT and its
metabolites exhibited strong estrogenic activity in boththe cell proliferation bioassay and the yeast two-hybrid
assay. Therefore, we investigated the estrogenic activity

Page 6 of 12of the crude drugs present within UKT using the cellproliferation bioassay and yeast two-hybrid assay.
In the cell proliferation bioassay, it was found that glycyrrhizae radix, cinnamomi cortex, and evodiae fructus
all induced significantly greater cell proliferation than
DMSO at a final concentration of 10 μg/ml (p < 0.01,
Fig. 3a). Among these three crude drugs, glycyrrhizae
radix also induced significantly greater proliferative activity than DMSO at a concentration of 5 μg/ml. We
also investigated the ERβ-dependent estrogenic activityof the crude drugs present within UKT using the yeast
two-hybrid assay. As a result, glycyrrhizae radix, cinnamomi cortex, zingiberis rhizoma, and evodiae fructus
were found to exhibit significantly greater activity than
DMSO at final concentrations of 0.5 mg/ml and 0.1 mg/
ml (Fig. 3b). The activity levels of glycyrrhizae radix andevodiae fructus remained high at a concentration of
0.5 mg/ml, and glycyrrhizae radix even demonstrated
strong activity at a concentration of 0.1 mg/ml. None ofthe other crude drugs displayed significant estrogenic activity in the cell proliferation bioassay or yeast twohybrid assay. These results suggest that the strong estrogenic activity of UKT can be attributed to the crude
drugs it contains; i.e., glycyrrhizae radix, cinnamomi cortex, zingiberis rhizoma, and evodiae fructus, especiallyglycyrrhizae radix.
Synergistic effects of the crude drugs during combination
treatment

As described above, glycyrrhizae radix exhibited thestrongest estrogenic activity of all the crude drugs in
UKT and is present in all three of the Japanese herbal
medicines that demonstrated estrogenic activity in the
MtT/Se cell proliferation bioassay and ERβ-dependent
estrogenic activity in the yeast two-hybrid assay; i.e.,
UKT, KSS, and NSS. In order to detect inter-crude drug
synergistic effects on ERβ-dependent estrogenic activity,
the combined effects of 0.1 mg/ml glycyrrhizae radix
and 0.1 mg/ml of one of the other crude drugs present
in UKT were examined, and then SQ were calculated.
The β-galactosidase activity of glycyrrhizae radix, evodiae fructus, and glycyrrhizae radix+evodiae fructus areshown in Fig. 4a, and the SQ for glycyrrhizae radix+evodiae fructus was calculated according to the method described above. The activity of glycyrrhizae radix+evodiae
fructus was significantly greater than the sum of the activity levels of these two crude drugs alone (p < 0.01),
which resulted in it having an SQ of 1.91 (Fig. 4a). Furthermore, we examined the contribution of such synergistic effects to ERβ-dependent estrogenic activity by
combining evodiae fructus with other crude drugs. Accordingly, the β-galactosidase activity of evodiae fructus,
cinnamomi cortex, and evodiae fructus+cinnamomi cortex were assessed, and we found that evodiae fructus

Wang et al. BMC Complementary and Alternative Medicine (2018) 18:107

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Fig. 3 The estrogenic activity of the crude drugs present in UKT. a MtT/Se cells were incubated with each of the crude drugs found in UKT at aconcentration of 1 μg/ml (white columns), 5 μg/ml (gray columns), or 10 μg/ml (black columns), and the cell proliferation induced by treatment
with these crude drugs or 10− 7 M of E2 for 6 days was evaluated. Cell proliferative activity is shown as a percentage compared with that induced
by DMSO alone in the MTT assay. **significantly increased compared with the activity induced by DMSO, p < 0.01. b The β-galactosidase activityof all 12 of the crude drugs present in UKT was evaluated at concentrations of 0.1 mg/ml (white columns) and 0.5 mg/ml (black columns). The
data for the crude drugs that did not display significant activity in the yeast two-hybrid assay are not shown. **significantly increased comparedwith the activity of DMSO, p < 0.01

+cinnamomi cortex exhibited significantly greater activity than the sum of the activity levels of evodiae fructus
and cinnamomi cortex alone (p < 0.01). The evodiae
fructus+cinnamomi cortex combination had an SQ of
2.0 (Fig. 4b). We continued examining the contributionof such synergistic effects to ERβ-dependent estrogenic
activity by combining cinnamomi cortex with the othercrude drugs present in UKT. However, no other synergistic effects were found. These findings suggest that theglycyrrhizae radix+evodiae fructus and evodiae fructus+cinnamomi cortex combinations have synergistic effects on the estrogenic activity of UKT.

Discussion
Japanese herbal medicines are often prescribed formenopausal syndrome, which is caused by the cessationof both egg maturation and sex hormone (principally estrogen and progesterone) secretion. Therefore, we examined the pharmacological activity of Japanese herbal
medicines that are used to treat menopausal syndrome,
especially their estrogenic activity. In this study, the estrogenic activity of Japanese herbal medicines was evaluated using a combination of an ER-dependent cellproliferation bioassay and an ER-dependent reporter
assay. ER-dependent bioassays based on MtT/Se cells,

Fig. 4 Synergistic effects between the crude drugs found in UKT according to the yeast two-hybrid assay. a The synergistic effects of combining
glycyrrhizae radix with other crude drugs on ERβ-dependent estrogenic activity. b The synergistic effects of combining evodiae fructus with othercrude drugs on ERβ-dependent estrogenic activity. The β-galactosidase activity of the crude drugs alone and various pairs of crude drugs were
evaluated at concentrations of 0.1 mg/ml (black columns). **significantly increased compared with the sum of the activity levels of the two crude
drugs alone, p < 0.01. SQ values were calculated to determine whether each combination had synergistic effects, as described in the materials
and methods. The data for the combinations that did not have significant synergistic effects in the yeast two-hybrid assay are not shown

Wang et al. BMC Complementary and Alternative Medicine (2018) 18:107

which express receptors for both endogenous ERα andERβ [16], can be utilized to determine the overall effectsof signaling mediated by these two receptors. In order toclarify the estrogenic responses initiated by ERα or ERβ,
we used an ER-dependent yeast two-hybrid reporter
assay. As described in the results, UKT, KSS, and NSS
increased the proliferation of MtT/Se cells, and this activity was markedly inhibited by ICI, an ER antagonist.
These results suggest that the effects of UKT, KSS, and
NSS on the proliferation of MtT/Se cells are directly mediated by the ER.
On the other hand, in the yeast two-hybrid assay UKT,
KSS, and NSS exhibited ERβ-dependent estrogenic activity. Furthermore, a previous study involving a luciferase
assay also showed that UKT and KSS displayed such activity [22]. However, Watanabe et al. reported that they could
not detect any agonistic activity of UKT or NSS towards
hER [23], but they used aqueous solutions of herbal medicines. As E2 is soluble in fat, we extracted the herbal medicines using ethanol and prepared DMSO solutions of the
medicines. Thus, the different extraction solvents used
might explain the abovementioned discrepancies.
In a previous study, Pueraria mirifica, a phytoestrogenrich traditional herbal plant, bound more strongly to
ERβ than ERα [18], and a herbal medicine composed of
18 different crude drugs displayed slightly higher affinity
for ERβ [24]. UKT, KSS, and NSS are mixtures of various
traditional herbal plants, and therefore, we examined
whether these Japanese herbal medicines are also rich in
phytoestrogens.
Phytoestrogens are naturally occurring chemicals ofplant origin, which have the ability to cause estrogenic
and/or antiestrogenic effects due to their structural similarity to the human hormone E2 [25]. The most common phytoestrogens can be divided into four kinds ofcompound, isoflavones, coumestans, prenylflavonoids,
and lignans, all of which possess phenolic and hydroxyl
moieties [25]. As was shown in the 3D-HPLC patterns
provided by the Tsumura Corporation, isoliquiritigenin
and glycycoumarin, which were detected in all three of
the Japanese herbal medicines that exhibited estrogenic
activity in the present study (UKT, KSS, and NSS); liquiritigenin, which was detected in UKT and KSS; and skullcapflavone II, which was only detected in NSS, also
possess phenolic and hydroxyl moieties (Fig. 5). Liquiritigenin and isoliquiritigenin have been found in the licorice species Glycyrrhiza glabra, Glycyrrhiza uralensis,
and Glycyrrhiza inflata [26] and licorice root extracts that
displayed estrogenic activity [27]. The isoliquiritigeninfound in Glycyrrhiza glabra exhibits stronger docking
energy when it binds to ERβ than when it binds to ERα[28]. In addition, previous studies have reported thatliquiritigenin demonstrated greater selective affinity forERβ than for ERα [29–31]. Isoliquiritigenin is the precursor

Page 8 of 12

chalcone of liquiritigenin, and a previous study detected asignificant reduction in isoliquiritigenin content and acorresponding increase in liquiritigenin formation in cellbased assays, which suggested that the conversion of isoliquiritigenin to liquiritigenin via cyclization might occur
under physiological conditions [30]. Glycycoumarin, which
was detected in UKT, KSS, and NSS, is a member of thecoumarin family that exhibits estrogenic activity [32], andit has also been found to be an estrogen agonist [33]. As
shown in the abovementioned 3D-HPLC analysis, thestructure of glycycoumarin is similar to that of genistein,which has a linear and polar structure. ERβ has a flexible
binding pocket [34], and it can accept a linear group andtolerate the resultant polarity [35]. Therefore, glycycoumarin might also have greater selective affinity for ERβ.
Little is known about the estrogenic activity of skullcapflavone II, which is present in NSS, but skullcapflavone II is amember of the flavone family, which is the most common
type of phytoestrogen. Based on the above points, it is suggested that the estrogenic activity of UKT, KSS, and NSS isdue to constituents with common phytoestrogen structures. In the present study, these Japanese herbal medicines
demonstrated strong ERβ-dependent estrogenic activity,
but none of them displayed ERα estrogenic activity in theyeast two-hybrid assay.
We also investigated the estrogenic activity of Japanese
herbal medicines after they had been metabolized using
the rat liver S9 fraction. UKT, KSS, and NSS exhibited
greater ERβ-dependent estrogenic activity after they had
been metabolized. Due to the very low abundance andgreat variety of Japanese herbal medicine metabolites, we
did not identify the metabolites produced by treating theherbal medicines with the rat liver S9 fraction using 3DHPLC analysis in the current study. However, as shown in
the abovementioned 3D-HPLC patterns of UKT, KSS, and
NSS, the structures of the constituents within the oval
boxes were based on a sugar or a methyl group bound to aliquiritigenin or an isoliquiritigenin. It might be possible toconvert these structures into liquiritigenin or isoliquiritigenin through previously proposed metabolic pathways[36–38] (Fig. 6), and as described above liquiritigenin andisoliquiritigenin have been shown to be active estrogenic
constituents. Therefore, this provides a possible explanation for the increases in the estrogenic activity of UKT,
KSS, and NSS seen after they were treated with the S9 fraction. On the other hand, TSS, KBG, and their metabolites,
which do not contain the most common phytoestrogenstructures (data not shown), did not exhibit any estrogenic
activity in the MtT/Se cell proliferation bioassay or theyeast two-hybrid assay. Thus, we consider that these two
herbal medicines might exert pharmacological effects
against menopausal syndrome via other mechanisms.
Herbal medicines are mixtures of several crude drugs.
In order to clarify which crude drugs were responsible

Wang et al. BMC Complementary and Alternative Medicine (2018) 18:107

Page 9 of 12

Fig. 5 3D-HPLC patterns of three Japanese herbal medicines and glycyrrhizae radix. The 3D-HPLC patterns of UKT (a), KSS (b), NSS (c), and glycyrrhizae
radix (d) are shown. The constituents in the quadratic boxes possess phenolic and hydroxyl moieties, and those in the oval boxes might be convertible
into active estrogenic constituents; i.e., liquiritigenin or isoliquiritigenin, through metabolic reactions

for the pharmacological activity of the examined herbal
medicines, we focused on UKT and investigated the estrogenic activity of the crude drugs present within it and thesynergistic effects of pairs of these crude drugs. The proportions of the crude drugs in UKT ranged from 3.7% to15% (Table 1). Therefore, in this study we evaluated theFig. 6 A proposed metabolic pathway. R: a sugar group or a methyl group

estrogenic activity of the crude drugs at 10% of the tested
concentrations of the Japanese herbal medicines, and glycyrrhizae radix exhibited the strongest estrogenic activityof all the crude drugs found in UKT.
As discussed above, UKT, KSS, NSS and their metabolites
possess some constituents with common phytoestrogen

Wang et al. BMC Complementary and Alternative Medicine (2018) 18:107

structures. We found that these constituents were mostly
derived from the common crude drug glycyrrhizae radix(Fig. 5), which is a rich source of flavonoids [39, 40], andprevious studies have shown that it exhibits estrogenic activity [14, 27, 41]. Furthermore, other crude drugs (cinnamomi cortex, evodiae fructus, and zingiberis rhizoma)
displayed ERβ-dependent estrogenic activity. It was reported that cinnamomi cortex demonstrated anti-allodynic
activity [42]; evodiae fructus suppressed the proliferation ofhuman epithelial ovarian cancer cells [43]; and zingiberis
rhizoma induced nitric oxide production, which inhibitedtumor cell growth [44]. As far as we know, this was thefirst study to examine the estrogenic activity of these crude
drugs. During the analysis of the main constituents of these
crude drugs, we did not detect common phytoestrogenstructures; i.e., phenolic or hydroxyl moieties. A previous
review examined ERβ ligands [45], and some of the ligands
did have not phenolic or hydroxyl moieties. Therefore, we
speculate that previously unknown phytoestrogen constituents that do not have common phytoestrogen structures,
but possess affinity for ERβ, are present in cinnamomi cortex, evodiae fructus, and zingiberis rhizoma. However, zingiberis rhizoma did not increase the proliferation of MtT/
Se cells in the current study.
The in vivo activity of estrogen was investigated in aprevious review, which demonstrated that it involves anarray of factors and is controlled by complex signaling
pathways [46]. In the present study, an MtT/Se cell bioassay was used to investigate the overall effects of herbal
medicines, which might also involve complex signaling
pathways. Zingiberis rhizoma showed estrogenic activity,
but did not have estrogenic effects on MtT/Se cells.
This study is the first to detect inter-crude drug synergistic effects on ERβ-dependent estrogenic activity.
Herbal medicines are made from raw plant materials,
and their compositions are complex. In addition, even if
the same crude drugs are present in different herbal
medicines their ratios can vary. Therefore, we combined
pairs of crude drugs in 1:1 ratios. This is an easy way ofcomparing the effects of combinations of crude drugs. We
found that evodiae fructus had synergistic effects whencombined with glycyrrhizae radix, and evodiae fructus also
had synergistic effects when combined with cinnamomi
cortex. Evodiae fructus exhibited 78-fold higher activity
when its concentration was increased 5-fold. Hence, in
addition to the classical ligand-binding pattern in which
one ligand binds to one LBD [34, 38, 47], we speculatethat one ligand can bind to more than one LBD through abridge-bond effect. Such mechanisms could be involved in
the abovementioned synergistic effects. However, thisissue needs to be clarified in future studies. In addition,
several reports have shown that the unliganded ER may betranscriptionally activated by certain post-translational
modifications [48]. In MCF-7 breast cancer cells, it wasPage 10 of 12

shown that the phosphorylation of a kinase was sufficient
to allow it to directly bind to ER and activate them [49].
This mechanism might also be responsible for some synergistic effects. The combination of crude drugs can affectcell homeostasis, activate some unliganded ER, and havesynergistic effects. Synergistic effects might explain why
herbal medicines, which have been prescribed for thousands of years, work better than the administration of
their crude drug constituents alone.

Conclusions
UKT, KSS, and NSS exhibited estrogenic activity in thisstudy, and their activity increased further after they were
metabolized. Such activity might be the key to their efficacy against menopausal syndrome. Glycyrrhizae radix isa crude drug that is found in all three of these Japanese
herbal medicines, and it also displayed strong estrogenic
activity. By analyzing the main constituents of these threeherbal medicines, we found that some of their constituents possessed common phytoestrogen structures; i.e.,
phenolic and hydroxyl moieties. In addition, we demonstrated for the first time that cinnamomi cortex, evodiae
fructus, and zingiberis rhizoma all display ERβ-dependent
estrogenic activity. The abovementioned crude drugs
might contain some new phytoestrogen constituents that
do not possess common phytoestrogen structures, but exhibit affinity for ERβ. We also identified synergistic effects
between evodiae fructus and glycyrrhizae radix, and between evodiae fructus and cinnamomi cortex for the firsttime. Synergistic effects between crude drugs increase theefficacy of herbal medicines. However, the mechanisms responsible for these effects remain unknown.
Abbreviations
DMEM: Dulbecco’s modified Eagle’s medium; DMSO: dimethyl sulfoxide;E2: 17β-estradiol; ER: estrogen receptor; FBS: fetal bovine serum; HPLC: highperformance liquid chromatography; HS: horse serum; LBD: ligand-binding
domain; ONPG: o-nitrophenyl-β-D-galactopyranoside; SQ: synergism
quotient; TIF2: transcriptional intermediary factor 2
Acknowledgments
We are grateful to Dr. Satoshi Takatori for providing the hERα and hERβ yeast
strains. We would like to thank the Tsumura Corporation for providing the 3DHPLC patterns of UKT (unkeito), KSS (kamishoyosan), NSS (nyoshinsan), and kanzo(glycyrrhizae radix). We thank Dr. Pheophet Lamaningao and Andrew Waleluma
for their helpful technical advice and discussions throughout the experiments.
We also thank Yasuko Nishimura for her kind secretarial assistance.
Funding
Not applicable to this submission.
Availability of data and materials
All data generated or analyzed during this study are included in thispublished article.
Authors’ contributions
SK and ZW designed this study. ZW performed all the experiments, analyzed
the data, and wrote the manuscript. TS and DEU discussed the results. SK
and TN supervised the entire study and assisted in the revision of themanuscript. All of the authors have read and approved the final manuscript.

Wang et al. BMC Complementary and Alternative Medicine (2018) 18:107

Ethical approval and consent to participate
Not applicable to this submission.
Consent for publication
Not applicable to this submission.
Competing interests
The authors declare that they have no competing interests.

Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims in publishedmaps and institutional affiliations.
Received: 16 May 2017 Accepted: 15 March 2018

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