Effect of Pueraria candollei var. mirifica and miroestrol
on ovariectomized-induced emotion deficit and osteoporosis in ICR mice

Name of dispatched researcher（派遣研究者名）

Ariyawan Tantipongpiradet

Master degree student

Instructor（指導教官，研究指導者）

Yaowared Chulikhit

Assistant Professor

Affiliation of Instructor（所属）
Host Collaborator（受入研究者）

Faculty of Pharmaceutical sciences,
KhonKaen University
Kinzo Matsumoto

教授

Abstract
Estrogen is an important steroid hormone which produced from female ovary. Estrogen take an actions
via bind to estrogen receptors (ER), ERα and ERβ that distribute in whole body. Recently estrogens were
found to effect on various systems especially in central nervous system (CNS) and musculoskeletal system.
Deprivation of estrogens can cause depression and cognitive impairment and osteoporosis, a burden symptom
in most of menopausal woman. Signal transducer and activator of transcription 3 (STAT3) which located inbrain area can activated by estrogens to control of cognitive function. Ovariectomized (OVX) mice is theanimal model use to represent estrogen deficiency conditions that can express the mood disorder and bone loss.
Pueraria candollei var. mirifica (PM) known to be the popular herbs used as folk medicine for rejuvenating
effects. PM contains various phytoestogens such as miroestrol (MR). The aim of this study was to investigate
the protective effects of PM and MR on ovariectomy induced mood disorder and bone loss. OVX exhibits morenumber of immobility time in TST and FST, decrease ERβ and STAT3 mRNA expression in hippocampus andfrontal cortex and also caused significantly decrease uterine weight and volume, femur and tibia dry weight,
ash weight and calcium content compared with sham group. Giving hormone replacement therapy (HRT)normalized mood disorder and bone loss from estrogen deficiency as same as treated with PM extract and MR.
This suggest that PM extract and MR has protective effects on ovariectomize induced depression and bone loss.
The mechanism of action involved activation of estrogen receptor by estrogen-like structure of phytoestrogen.
Our findings raise the possibility that MR and Pueraria candollei var. mirifica, the plant of origin of MR, may
have a beneficial effect on depression and osteoporosis in which menopause/ovariectomy are implicated as risk
factors.
Backgrounds
Menopause is defined by permanent cessation of the primary functions of the human ovaries. Emotional
disorders such as postpartum and postmenopausal depression are the most common forms of mental illness
associated with decreased serum levels of ovarian hormones [1]. High severity of depressionig can be amajor cause of suicidal thinking. Estrogen deficiency is considered as the main determinant for bone loss inpostmenopausal women [2]. Osteoporosis is caused by an imbalance in the normal bone remodeling process,in which there is excessive osteoclast resorption and adequate new bone formation by osteoblasts reduction.
Hormone replacement therapy (HRT) has proven to be efficacious in depression and preventing bone loss and－ 58 －

side effects and are suitable for long-term use.
Phytoestrogens and many other estrogen-like compounds in plants, due to fewer side effects,
have been massively proposed to prevent menopause-related cognitive decline. Pueraria candollei var.

reducing the incidence of skeletal fractures in postmenopausal women. However, long-term HRT increases themirifica: PM ), an cancer,
indigenous
herb belongs
to theknown to be thehighrisk of (KwaoKrueKao
breast cancer, endometrial
thromboembolic
eventsandFamily
vaginalLeguminosae
bleeding [3]. is
Traditional
medicines have been used from long days in prevention and treatment of postmenopausal symptoms. Since

popular herbs used as folk medicine for increasing appetite, enlarging breasts, improving hair growth, andthese medicines are prepared from medicinal plants they have fewer side effects and are suitable for long-other[4]. other
The estrogen-liketuberous rootof Pueraria
candollei
contains
term
use.rejuvenating
Phytoestrogenseffects
and many
compounds
in plants,
due to var.
fewermirifica
side effects,
have various
been massively proposed to prevent menopause-related cognitive decline. Pueraria candollei var. mirifica

compounds such as miroestrol, deoxymiroestrol, daidzein, genistin, and puerarin. Miroestrols are an(KwaoKrueKao : PM ), an indigenous herb belongs to the Family Leguminosae is known to be the popular herbs

isoflavonoid
compoundwhich hasthe chemicalsimilar tohair
femaleestrogenic
used
as folk medicine
for increasing
appetite,enlargingstructurebreasts, improving
growth,and otherhormones,
rejuvenatingestradiol
effects [4]. The tuberous root of Pueraria candollei var. mirifica contains various compounds such as miroestrol,[5]. It exhibited an estrogenic activity 0.25 times that of 17β-estradiol by a vaginal cornification assay [6].

deoxymiroestrol, daidzein, genistin, and puerarin. Miroestrols are an isoflavonoid compound which has thechemical structure
similar to female
estrogenic hormones,
estradiol[5]. It exhibitedan estrogenic
activity
0.25cognitive
To delineate
the relationships
betweenestrogendeficiency,
depression,andtimes that of 17β-estradiol by a vaginal cornification assay [6].

impairment, we first examined the effects of chronic miroestrol administration as hormone replacement
To delineate the relationships between estrogen deficiency, depression, and cognitive impairment,

wetherapy
first examinedthe effects ofchronic andmiroestrol
administration
as hormone mice
replacement
therapyof
onmenopause.
theon the depressive
behavior
osteoporosis
in ovariectomized
as the model
depressive behavior and osteoporosis in ovariectomized mice as the model of menopause. Moreover, the effectMoreover, the effect of miroestrol on depression and osteoporosis-related gene expression will beof miroestrol on depression and osteoporosis-related gene expression will be examined to clarify the molecularmechanism
phytoestrogen.
examinedoftothis
clarify
the molecular mechanism of this phytoestrogen.

Fig.1 Chemical structure of miroestrol

Materials and Methods

3

Plant material preparation
Tuberous root bark of P. candollei var. mirifica (PM) was collected in Ubon Ratchathani, Thailand andwas identified by Dr. Thaweesak Juengwatanatrakul, Faculty of Pharmaceutical Sciences, Ubon Ratchathani
University, Ubon Ratchathani, Thailand. Ten kilograms of dried tuberous root bark of PM was powdered andextracted by maceration method with hexane 3 × 20 l, and the product of maceration was extracted with ethyl
acetate 3 × 20 l. The ethyl acetate crude extracts were combined, evaporated, and fractionated by column
chromatography (Silica gel with hexane: ethyl acetate 3:1). The fractionated samples were combined andevaporated at 60 °C. The residual (PM) was stored at -20 oC until use.
Miroesterol was isolated from PM by high-performance liquid chromatography (HPLC). The HPLC
separation was performed using a C18 reverse phase column and a mobile phase consisting of 16% acetonitrile:water with a flow rate at 45.0 ml/min. The UV detection wavelength was set at 205 nm.
Animals
Forty female ICR mice weighing 20-25 g were obtained from National Laboratory Animal Centre,
Mahidol University, Nakhon Pathom, Thailand. At all times, the mice were housed on wood chip bedding in
stainless steel cages with water and commercial mouse diet supplied ad libitum and acclimated for at least 7－ 59 －

days in housing with a 12-hour dark/light cycle under controlled temperature (22°C ± 2°C) and humidity (45%
± 2%) before dosing. The animal experiment in this study had been approved by Animal Ethics Committee ofKhon Kaen University (AE 01/2556), and were conducted in accordance with the Guide for the Care and Use ofLaboratory Animals, Khon Kaen University, Thailand. All experiments were performed during 08:00 a.m. to 4.00
a.m. and each animal was used only once.
Surgical procedure
Animals were divided into OVX and Sham group. OVX group were ovariectomized by the surgicalremoval of both ovaries under anesthesia with Nembutal® (Pentobarbital sodium) 60 mg/kg, i.p.. Sham operation
was performed as the same conditions with ovaries intact. After a 3-day recovery period,the animals weredivided into seven groups: (1) sham, (2) ovariectomy (OVX), (3) ovariectomy + 1 μg/kg 17β-estradiol, (OVX +E2),(4) ovariectomy + 0.1 mg/kg miroestrol (OVX + MR 0.1), and (5)ovariectomy + 1 mg/kg miroestrol (OVX
+ MR 1),(6) ovariectomy + 2.5 mg/kg PM extract (OVX + PM 2.5), and (7)ovariectomy + 25 mg/kg PM extract(OVX + PM 25). The drugs were given daily (during 08.00 and 12.00 a.m.) for eight weeks.
Tail suspension test(TST)This test was carried out according to the method of Steru [7] TST was developed as a rodent screening
test for potential (human) antidepressant drugs. The mice were individually suspended of the tail suspension test
box, 60 cm above the surface of table. After treatment administration, each mouse was suspended on the edgeof the table 60 above the floor by adhesive tape placed approximately 1 cm from the tip of the tail. Immobility
duration was recorded for the last 4 min during 6 min period. Mice were considered immobile when they hanged
passively and completely motionless [8].
Forced swimming test (FST)This test demonstrates normal swimming and floating ability. FST used to examine ‘learned
helplessness’ [9] Mice were placed individually in transparent glass cylinder (12 cm in diameter, height 25 cm)
which was filled with water to a height of 10 cm of water at 25oC. Twenty-four hours before the test, pre-test
session for 15 minutes. In the test session, each mouse was administration with treatment 1 hour before test,
Immobility time were recorded during 5 minutes period.
Serum corticosterone
Blood samples were immediately collected after decapitation and centrifuged at 3000 rpm, 4 oC for
15 min to isolate serum from blood and store serum at -20 oC until use. The serum corticosterone level was
determined using Corticosterone (CORT) ELISA kit (Assay pro LLC. St. Charies, MO) follow ELISA kit
instruction.
Uterus weight and volume
Uterus was immediately removed from mouse body after decapitation without uterine fate and pieces ofoviduct and measure weight with 4-digit weighing machine, volume with venire.
Dry weight, ash weight and calcium content in femurs and tibias－ 60 －

Right femurs and tibias were dried in an oven at 60 °C after removed all tissues and wash with water.
The bones were put in porcelain cups, weighed by using 5-digits weighing machine, and then incinerated ina furnace at 800 °C 8 hours. The bone was then powdered and ash weight was determined by using 5-digits
weighing machine. The ash was dissolved in 15 ml of 6N HCl for 30 minutes before diluted to 1:100 with
distilled water. The calcium content was measured by an Atomic absorption spectrophotometer at 422.67 nm.
Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR)
Total DNA extraction
Total RNA from hippocampus and frontal cortex were extracted by using the TRIzol ® reagent.
Hippocampus and frontal cortex were homogenized with 1 ml of TRIzol® reagent. After homogenization,
0.2 ml of chloroform was add and mixed the sample vigorously for 15 seconds then incubated them at room
temperature for 2 to 3 minutes. The samples were centrifuged at 12,000 x g for 5 minutes at 4oC. Following
the centrifugation, the mixture was separated into lower red, phenol-chloroform phase, an interphase, and acolorless upper aqueous phase. RNA remains exclusively in the aqueous phase. Upper aqueous phase wastransfer carefully into fresh tube without disturbing interphase. The volume of the aqueous phase was measure(The volume of aqueous phase is about 60% of the volume of TRIzol® reagent used for homogenization). The
aqueous phase was mixed with 0.5 ml of isopropyl alcohol for precipitate the RNA. The sample was incubatedat 15 to 30 °C for 10 minutes and centrifuged at 12,000 x g for 10 minutes at 4 °C. The RNA precipitate, often
invisible before centrifugation, form a gel-like pellet on the side and bottom of the tube. The supernatant ascompletely removed. Then the RNA pellet was washed with 1 ml of 75%ethanol. The sample was mixed by
vortexing and centrifuged at 7,500 x g for 5 minutes at 4°C. After that, This step was repeated again. Then all
leftover ethanol was removed by air-dry or vacuum. It was important not to let the RNA pellet dried completely
as this will greatly decreased its solubility. The RNA was dissolve in 40 μl RNase free water by passing solution
a few times through a pipette tip. After that, the RNA was kept in -20°C refrigerator as stock RNA.
Estimation of RNA concentration
Stock RNA 1 μl was diluted with 99 μl of RNase free water. The sample concentration of total RNA
was determined by using microcuvette in the absorbance at 260 nm and 280 nm. The A260/A280 ratio shouldbe above 1.6 for purity. The convention apply that 1 OD at 260 nm equals to 40 μg/ml RNA. The RNA wascalculated and adjusted to 0.5 μg/μl by RNase free water and approximate final volume was 60 μl.
Reverse transcription
RNA sample (0.5 μg/ μl) 4 μl was pipette to nuclease-free microcentrifuge tube. The master mix1 was
prepared from 1 μl oligo (dT) 12-18, 1 μl 10 mM dNTP Mix (10 nM each dATP, dGTP ,dCTP and dTTP atneutral pH) and RNase free water adjust to 12 μl. The master mix 1 12 μl was added into microcentrifuge tube.
The mixture was heated to 65 °C for 5 min and quick chill on ice. Then 8 μl of master mix2 [4 μl 5X FirstStand Buffer, 2 μl 0.1 M DTT, 0.2 μl RNase inhibitor, 0.8 μl RNase free water] was added. The tube was gently
mixed and incubate at 37 °C for 2 min. One microlitter (200 units) of M-MLV RT was added, and mix bypipetting gently up and down. The tube was incubated at 37 °C for 50 min and inactivate the reaction by heating
at 70 °C for 15 min. Reverse transcription-polymerase chain reaction (RT-PCR) Total RNA from hippocampus
and frontal cortex were extracted. PCR amplification was carried out using gene-specific PCR primer setsas
follows: β-actin: 5’-AAC GGT CTC ACG TCA GTG TA-3’(sense) and5’-GTG ACA GCA TTG CTT CTG－ 61 －

TG-3’(antisense); ER-β: 5’- CAG TAA CAA GGG CAT GGA AC -3’(sense) and5’- GTA CAT GTC CCA CTTCTG AC -3’(antisense); STAT3: 5’- CAC TAA CAA GGG CAT GGA AC -3’(sense) and5’- GTA CAT GTC
CCA CTT CTG AC -3’(antisense) .PCR was performed with a preheating cycle at 95°C for 2 min, denaturation
at 95°C for 30 sec, annealing at 60°C for 1 min, extension at 72 °C for 6 min. Aliquots of PCR products wereanalyzed by gel electrophoresis with 38% polacrylamide and 2% bispolyacrylamide gel stained in Novel juice®,
photographed under UV light and analyzed by GeneSnap and ImageJ software.
Statistical analysis
Data were expressed as the mean ± SD for each group. The significant differences between SHAM
)RUP（様式２－２）

and OVX group were analyzed using t-test. The significant differences between OVX and treatment groups
Resultspost hoc Tukey’s test. The SigmaStat 3.5 (SYSTAT Software
were analyzed using one-way ANOVA andeffect of PM extract
depressive-likebehavior
Inc.,Richmond, CA, USA) was used. The The
differences
withor miroestrolp-valueadministration
less thanon0.05
were considered
statistically
To determine whether miroestrol modulates the depression in an estrogen deprivation model ofsignificant.

mice, the depressive-like behavior of OVX mice were elucidated in the tail suspension test (TST) and forced
swimming test (FST), respectively. OVX mice received once daily administration of vehicle corn oil,

Results17β-estradiol, PM extract or miroestrol for 8 weeks before starting the behavioral experiments. The

The effect of PM extract or miroestrol administration
onshowed
depressive-likebehavior
vehicle-treated OVX micesignificantly increase
immobility time than the sham-operated group,
indicating depressive-likebehavior caused
OVX. 17β-estradioland miroestrol-treatedgroupsTo determine whether miroestrol modulates
the depression
in anbyestrogendeprivation
model of OVX
mice,
the
showed significantly improved depressive-like behavior in the test compared with the vehicle-treateddepressive-like behavior of OVX mice were
elucidated in the tail suspension test (TST) and forced swimming

test (FST), respectively. OVX micereceived once daily administration ofcontrol OVX mice (Fig. 2A, 2B).

Immobility time (second)

vehicle corn oil, 17β-estradiol, PM
extract or miroestrol for 8 weeks before
starting the behavioral experiments.
The vehicle-treated OVX mice showedsignificantly longer immobility

#

250

A)

200

150

*

0

miroestrol-treated OVX groups showedsignificantly improved depressive-likebehavior in the test compared with thevehicle-treated control OVX mice (Fig.
2A, 2B).

*

*

*

Sham

VehicleEstradiol

PM 2.5

PM25

MR0.1

MR1

OVX
)RUP（様式２－２）

#

300

B)

Immobility time (second)caused by OVX. 17β-estradiol- and*50time than the sham-operated group,
indicating depressive-like behavior

*

100

250
200
150

*

100

*

50
0

Changes in uterus weight and volume after

Sham

Vehicle8

Estradiol

PM 2.5

PM25

MR0.1

MR1

OVX

PM extract or miroestrol administration.

Fig 2. Effects of PM extract or miroestrol and 17β-estradiol (E2) on OVX-induced depressive-like behavior in

The OVX group showed atail suspension test (A) and forced swimming test (B). Each column represents the mean ± S.E.M. (n=10–12).

significant decrease in uterus weight#P<0.05 vs. sham. *P<0.05, ** P < 0.001 vs. vehicle-treated OVX groupand volume compared with the shamChanges in uterus weight and volume after PM extract or miroestrol administration.

group (Table 1). Hormone replacement therapy of OVX mice with 17β-estradiol for 8 weeks significantly
The OVX group showed a significant decrease in uterus weight and volume compared with theincreased both the uterus weight and volume. Similarly, PM extract or miroestrol treatment significantly
sham group (Table 1). Hormone replacement therapy of OVX mice with 17β-estradiol for 8 weeks

increased the uterus weight and volume in OVX
animals.
significantly increased both the uterus weight and volume. Similarly, PM extract or miroestrol treatment
significantly increased the uterus weight and volume in OVX animals.

－ 62 －Group

Table 1
Uterus weight

Uterus volume(g/kg body weight)(cm3/kg body weight)

The OVX group showed a significant decrease in uterus weight and volume compared with thesham group (Table 1). Hormone replacement therapy of OVX mice with 17β-estradiol for 8 weeks
significantly increased both the uterus weight and volume. Similarly, PM extract or miroestrol treatment
significantly increased the uterus weight and volume in OVX animals.
)RUP（様式２－２）

Effects of PM extract or miroestrol

Table 1

Group
Group

UterusweightweightUterusUterusvolume
volumeUterus(g/kgbody
bodyweight)
weight)(g/kg

3/kg body weight)(cm3/kgbody weight)(cm

7.4223 ±± 0.66
0.78*
5.3728**4.50564.4961 ±± 0.28
0.6

OVX + MR 1
Shamadministration on dry weight, ashweight and calcium content inEach datumOVX
represents the means ± SEM(n=5–6#P<0.05
vs. #sham. *P<0.05,± 0.423.0115± 0.56in# each animal group). 1.3418

femurs and tibias**P<0.001 vs.
vehicle-treated
OVX group 4.9181 ± 0.71**
OVX
+ E2

4.5268 ± 0.36**

The effect of PM and MR on

6.3510 ± 0.58*

4.0661 ± 0.27**

femurs is consistent with the effect

OVX + PM 2.5* dry weight, ash weight**
Effect of PM
extractor miroestrol administration
calcium
content in
OVX+ PM 25
6.5633 ± 0.59on
5.5860and± 1.12)RUP（様式２－２）

femurs and OVX
tibias+ MR 0.1

5.6207 ± 0.58**

4.5169 ± 0.17**

9
The effect of PM and MR on femurs is consistent
with the effect on tibias. Ovariectomy was foundGroup
Uterus weight
Uterus volume

to cause significantly loss in dry weight, ash weight and calcium content as 3compared to sham operated(g/kg body weight)(cm /kg body weight)
group (Table 2 and 3). Dry weight was significantly increasedin PM and miroestrol treated groups
compared
OVX + MR 1
7.4223 ± 0.78*
4.5056 ± 0.28**
with OVX group. Ash weight and calcium content was significantly increased by both PM 2.5 and PM 25
Each datum represents the means ± SEM (n=5–6 in each animal group). #P<0.05 vs. sham. *P<0.05,
compared with the OVX mice. For MR treated groups, only MR1 significantly increased ash weight and**P<0.001 vs. vehicle-treated OVX groupcalcium content.

on tibias. Ovariectomy was found tocause significantly loss in dry weight,
ash weight and calcium content ascompared to sham operated group(Table 2 and 3). Dry weight wassignificantly increased in PM andmiroestrol treated groups compared

Table 2 Effect of PM and MR on dry weight, ash weight, and calcium content in femurs.
Effect of PM extract or miroestrol administration on dry weight, ash weight and calcium content inwith OVX group. Ash weight andfemurs and tibias

calcium content was significantly

Treatment
Dry weight (mg)Ash weight (mg)Calcium content (mg)The effect of PM and MR on femurs is consistent with the effect on tibias. Ovariectomy was foundincreased by both PM 2.5 and PM

to cause significantly loss in dry weight, ash weight and calcium content as compared to sham operated
SHAM
51.80 ± 3.37
29.90 ± 2.30
4.90 ± 0.34

25 compared with the OVX mice.

#

#

#

group
(Table 2 and 3). Dry weight
was±significantly
increasedin PM
and miroestrol treatedgroups
OVX
45.57
3.47
24.71
± 3.21
4.28± 0.28compared**
2.5 Ash weight and53.18± 1.38content
30.98 ± 1.49
± 0.18
withOVXOVX+ PM
group.
calcium
was significantly
increasedby both 4.89
PM 2.5and* PM 25

33.53 ± 4.19 *5.06 ± 0.38 *OVX + PM 25
56.25 ± 5.48 *compared with the OVX mice. For MR treated groups, only MR1 significantly increased ash weight and30.15 ± 1.94
4.41 ± 0.22
OVX + MR 0.1
54.65 ± 2.42 *calcium content.
31.23 ± 2.78 *4.95 ± 0.34 *54.68 ± 4.33 *OVX + MR 1
Table 2 Effect of PM and MR on dry weight, ash weight, and calcium content in femurs.
Each datum represents the means ± SEM (n = 5-6 in each animal group). #P<0.05 vs. sham. *P<0.05 vs. vs.
)RUP（様式２－２）

vehicle-treated OVX group.
Treatment
Dry weight (mg)Ash weight (mg)Calcium content (mg)Table 3 Effect of PM and MR on dry weight, ash weight, and calcium content in tibias
SHAM

51.80 ± 3.37

29.90 ± 2.30#

OVX
Treatment
OVX + PM 2.5

45.57 ± 3.47
Dry weight (mg)53.18 ± 1.38 *OVX + PM 25
SHAM
OVX + MR 0.1
OVX
OVX++PM
MR2.5
1
OVX

)RUP（様式２－２）

4.90 ± 0.34#

4.28 ± 0.28
Calcium content (mg)4.89 ± 0.18 *56.25 ±± 1.46
5.48 *
45.14

*
33.53
27.38 ±± 4.19
1.16

*
5.06
4.07 ±± 0.38
0.11

54.65±±1.262.42 *
39.51##

30.15 ±± 0.94
1.94
23.69

54.68 ± 4.33***

31.23 ± 2.78 **46.76 ± 2.36

significantly increased ash weight andcalcium content.
PM extract and miroestrol
administration reduced serum
corticosterone level in OVX#

24.71 ± 3.21
Ash weight (mg)30.98 ± 1.49 *10

For MR treated groups, only MR1##

decreased serum corticosterone level##

as compared to the sham-operated

4.95 ± 0.34 **group (Fig.3). Serum corticosterone

4.41
3.56 ±± 0.22
0.10

28.30 ± 1.68

Ovariectomy significantly

3.98 ± 0.16** = 5-6 in each29.85** #P<0.05 vs. sham.
Each
datum
represents
the
means
(n
animal± 2.61
group).
+ PMor25miroestrol
49.25 ±± SEM
3.46reduce
0.16 ** vs. vs.
PMOVX
extract
administration
serum corticosterone
level in OVX 4.13 ±*P<0.05

OVX + MR 0.1OVX group. 44.44 ± 1.65 *26.75 ± 1.23
3.86 ± 0.16
vehicle-treated
The effect of PM and MR on serum corticosterone. Ovariectomy significantly decrease as
28.13 ± 1.29 *4.04 ± 0.27 *OVX + MR 1
45.32 ± 1.59 *compared to sham operated group (Fig.3). Serum corticosterone significantly decreased in PM and MREach datum represents the means ± SEM (n=5–6 in each animal group). ##P<0.05 vs sham. *P<0.05,
treated groups compared with OVX group.
**P<0.001 vs. vehicle-treated OVX group.

significantly decreased in PM and MR
treated groups compared with OVXgroup.

10

Corticosterone (ng/ml)

Effects of PM extract and miroestrol#

30

on the expression levels of ERβ and

25

20

STAT3 mRNA down-regulated in a

15

OVX model

10

The mRNA expression levels

5
0

**

SHAM

**

**

Vehicle Estradiol PM2.5

PM25

**

MR0.1

**

MR1

OVX

were evaluated by RT-PCR. Intensityof genes expression was measured
using ImageJ and calculated into

Figure. 3 Effects of PM and MR on OVX-induced elevation of serum corticosterone level.

%relative expression. Administration

Each data column represents the mean ± S.E.M. (n=3–5). #p<0.005 vs. sham and **p<0.001 vs.

of 17β-estradiol, PM and miroestrolvehicle-treated OVX group.

－ 63 －

Effects of PM extract and miroestrol on the expression levels of ERβ and STAT3 mRNA down-regulated ina OVX model)RUP（様式２－２）

led to activation of ERβ and STAT

A)
Frontal Cortex

STAT3

STAT3
ERβ
β-actin
Vehicle

E2

MR0.1

¤

*

80
60
40
20
0

Sham Vehicle

E2

MR0.1

MR1

PM2.5

120

PM25

**

100

*

80

140

*
*

*

100

*

*

80
60
40
20
0

Sham Vehicle

E2

MR0.1

**

**

**

MR1

PM2.5

PM25

to sham group (p<0.001). The resultof ERβ and STAT mRNA after OVX

Sham

Vehicle

E1

MR0.1

MR1

PM2.5

PM25

ERβ mRNA expression on Frontal Cortex
#

¤

**

120

**

*

100

*

80

mice received 17β-estradiol (p<0.05).
Discussion

¤

140

*

The present study
demonstrated that miroestrol

60

ameliorated estrogen depletion-

40
20
0

oil in hippocampus when comparedshowed significantl increase in expression

20

160

There is significantly decreased ERβmRNA expression of OVX + corn

40

C2)

¤

¤

120

PM25

OVX

ERβ mRNA expression on Hippocampus
#

PM2.5

60

0

Relative ERβ mRNA Expression level

Relative ERβ mRNA Expression level

160

MR1

#

140

OVX

C1)

MR0.1

STAT3 mRNA expression on Frontal Cortex

160

*

¤

100

E2

OVX

#

*

VehicleB2)

STAT3 mRNA expression on Hippocampus

120

and frontal cortex as show in fig.4.
Sham

PM25

Relative STAT3 mRNA Expression level

Relative STAT3 mRNA Expression level

PM2.5

MR1

OVX

B1)
140

mRNA expression in hippocampus

ERββ-actin
Sham

160

Hippocampus

Sham

Vehicle

OVX

E2

MR0.1

MR1

PM2.5

PM25

OVX

Fig. 4. PM extract- and miroestrol-induced reversal of down-regulated expression of ERβ and STAT3 mRNA
expression in OVX animals. (A) Typical photo indicate effects of administration of 17β-estradiol (E2, 1µg/kg/day) Pueraria mirifica crude extract (PM 2.5 and 25 mg/kg/day) and miroestrol (MR 0.1 and 1
mg/kg/day) on relative STAT3 and ERβ mRNA expression in hippocampus and frontal cortex. (B1-B2)

induced depressive-like behavior
and bone loss suggested that theseeffects of miroestrol are attributable
to the serum corticosterone, estrogen
receptor-mediated facilitation of ERβ

Quantitative comparison expression levels of STAT3 and (C1-C2) ERβ against the expression level ofand STAT3 gene transcription in thehousekeeping gene, β-actin. Results are presented as the mean ± S.E.M (n=5-6). Statistical analysis wasbrain and calcium content in bone.

undertaken by one-way ANOVA followed by the Turkey test. #P<0.001 vs. sham; *P<0.05, **P<0.001 vs.

Our findings raise the possibility

vehicle-treated OVX group; ¤P<0.001 vs. a low dose of each test drug.

that MR and Pueraria candollei var.

13

mirifica, the plant of origin of MR,

may have a beneficial effect on depression and osteoporosis in which menopause/ovariectomy are implicated asrisk factors.
Estrogens or estradiol is protective against ischemic brain damage and essential for growth anddifferentiation of the uterus, cervix, vagina, breast, and some mammary tumors, to promotion of this effectestrogen need to bind to estrogen receptors which acts as a transcription factor that binds to a sequence known
as estrogen response element (ERE) to induce gene transcription. Pueraria mirifica can give the characteristic
compounds are deoxymiroestrol and miroestrol which is the very potent phytoestrogen and believed to be themiracle plant to use in post-menopause women. Deoxymiroestrol is not stable compounds, it can be lose in theprocess of extraction so miroestrol is the candidate to use in this study. Estrogen receptor β (ERβ) is the specificreceptor for mediated anxiety behavior by the activation of ERβ can decreases anxiety-related behavior, whichis the problem of post-menopausal women. In estrogen deficiency condition, ERβ mRNA is down-regulated(Shima N. et. al., 2003) and can be normalized when giving hormone replacement therapy. Estrogen reducesbone turnover by decreasing RANKL, which a key stimulator of osteoclast-induced bone resorption. It also
increases OPG, which works against RANKL, to induce bone formation. Previous studies showed that estrogen
deficiency induced by ovariectomy lead to increasing RANKL and decreasing OPG, which indicate bone
resorption. Treatment with PM extract and MR decreased the RANKL/OPG ratio by lowering level of RANKL(Udomsuk et al., 2012a; Tiyasatkulkovit et al., 2014). The results suggesting that their prevention effects on－ 64 －

ovariectomy induced bone loss may involve decreasing osteoclast-mediated bone resorption.
In conclusion, Pueraria mirifica crude extract and miroestrol are significantly increased uterus weightand volume, decrease serum corticosterone and immobility time of TST and FST, increase calcium content infemurs and tibias and also up-regulated the expression of ERβ and STAT3 mRNA as similar as giving synthetics
estrogen in estrogen deficiency condition model due to the similar structure between phytoestrogen andestradiol. These can indicated that PM crude extract and miroestrol has estrogenic activity to use as hormone
replacement therapy so PM crude extract and miroesterol may use as promising compound for alternative choice
in hormone replacement therapy.
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