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Bone Sparing Effect of a Novel Phytoestrogen
Diarylheptanoid from
Curcuma comosa
Roxb. in
Ovariectomized Rats
Duangrat Tantikanlayapor n
1
, Patsorn Wichit
1
, Jittima Weerachayaphorn
1
, Arthit Chairoungdua
1
,
Aporn Chuncharunee
2
, Apichart Suksamrarn
3
, Pawinee Piyachaturawat
1
*
1 Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand, 2 Department of Anatomy, Faculty of Medicine, Siriraj Hospital, Mahi dol University,
Bangkok, Thailand, 3 Department of Chemistry, Faculty of Science, Ramkhamhaeng University, Bangkok, Thailand
Abstract
Phytoestrogens have been implicated in the prevention of bone loss in postmenopausal osteoporosis. Recently, an active
phytoestrogen from Curcuma comosa Roxb, diarylheptanoid (DPHD), (3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol, was
found to strongly promote human osteoblast function in vitro. In the present study, we demonstrated the protective effect
of DPHD on ovariectomy-induced bone loss (OVX) in adult female Sprague-Dawley rats with 17b-estradiol (E
2
,10mg/kg Bw)
as a positive control. Treatment of OVX animals with DPHD at 25, 50, and 100 mg/kg Bw for 12 weeks markedly increased
bone mineral density (BMD) of tibial metaphysis as measured by peripheral Quantitative Computed Tomography (pQCT).
Histomorphometric analysis of bone structure indicated tha t DPHD treatment retarded the ovariectomy-induced
deterioration of bone microstructure. Ovariectomy resulted in a marked decrease in trabecular bone volume, number
and thickness and these changes were inhibited by DPHD treatment, similar to that seen with E
2
. Moreover, DPHD
decreased markers of bone turnover, including osteocalcin and tartrate resistant acid phosphatase (TRAP) activity. These
results suggest that DPHD has a bone sparing effect in ovariectomy-induced trabecular bone loss and prevents
deterioration of bone microarchitecture by suppressing the rate of bone turnover. Therefore, DPHD appears to be a
promising candidate for preserving bone mass and structure in the estrogen deficient women with a potential role in
reducing postmenopausal osteoporosis.
Citation: Tantikanlayaporn D, Wichit P, Weerachayaphorn J, Chairoungdua A, Chuncharunee A, et al. (2013) Bone Sparing Effect of a Novel Phytoestrogen
Diarylheptanoid from Curcuma comosa Roxb. in Ovariectomized Rats. PLoS ONE 8(11): e78739. doi:10.1371/journal.pone.0078739
Editor: Brenda Smith, Oklahoma State University, United States of America
Received April 18, 2013; Accepted September 16, 2013; Published November 11, 2013
Copyright: ß 2013 Tantikanlayaporn et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by the Thailand Research Fund (TRF) through the Royal Golden Jubilee Ph.D. Program (grant PHD/0103/ 2549 to DT and PP),
the Strategic Basic Research Grant of TRF (to PP and to AS), the Office of the Higher Education Commission and Mahidol University under the National Research
and Universities Initiative, and The National Research Council of Thailand. The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have decl ared that no competing interests exist.
* E-mail: pawinee.pia@mahidol.ac.th
Introduction
Osteoporosis is a serious worldwide health problem that
primarily effect middle-aged and elderly women [1,2]. It is
characterized by reduced bone mass and the deterioration of bone
microarchitecture leading to increase the risk of bone fragility and
fracture [3]. An accelerated rate of bone resorption in menopausal
and post-menopausal women is associated with reduced levels of
the hormone estrogen [4]. Recently, efforts to reduce bone loss in
menopausal osteoporosis have been focused on compounds with
the potential to preserve bone mass through inhibition of
osteoclastic bone resorption or stimulation bone formation [5].
Among therapeutic agents, estrogen is the most effective
compound and is capable of limiting bone loss and reducing the
rate of bone fractures in postmenopausal women [6,7]. However,
long-term treatment with estrogen is limited due to its carcino-
genic risk and feminizing effects.
Phytoestrogens, non-steroidal plant-derived compounds with
estrogenic activity, have received increased interest as estrogen
alternatives to alleviate bone loss. Studies have suggested that a
diet rich in phytoestrogen may relieve menopausal symptoms and
protect against estrogen-associated diseases, including breast
cancers, cardiovascular diseases, and osteoporosis [8,9]. Isofla-
vones, such as genistein and daidzein the major phytoestrogens in
soybeans, are the most extensively studies phytoestrogens. These
compounds inhibit osteoclast bone resorption and suppress
osteoclast activity and survival in vitro [10,11]. In addition,
isoflavones have been identified as naturally occurring selective
estrogen receptor modulators (SERMs) and as bone-sparing agents
[12,13]. The known properties of phytoestrogens suggest that
these compounds may be alternatives to estrogen for preventing
and treating osteoporosis in postmenopausal women.
Curcuma comosa Roxb. (C. comosa), a plant in Zingiberaceae
family, has been widely used as a dietary supplement for relieving
postmenopausal symptoms in Thailand [14]. Consistent with the
presence of a phytoestrogen, hexane extract of C. comosa rhizomes
prevent bone loss in estrogen deficient mice [15]. Diarylheptanoid,
(3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol (hereafter DPHD), a
novel phytoestrogen isolated from C. comosa [16] has several
pharmacological properties including estrogenic-like activity
[17,18] and anti-inflammatory effects [19]. Recently, DPHD was
found to activate Wnt/b-catenin signaling and promote mouse
PLOS ONE | www.plosone.org 1 November 2013 | Volume 8 | Issue 11 | e78739
preosteoblastic (MC3T3-E1) cell proliferation through the estro-
gen receptor pathway [20]. Similarly, human osteoblast cell
differentiation and function were also enhanced upon DPHD
treatment [21] suggesting that DPHD may have a beneficial effect
in preventing bone loss in patients experiencing estrogen
deficiency.
The biological activities of DPHD appear to be selective with
anabolic effects predominantly on osteoblasts. We hypothesized
that DPHD may have a beneficial effect in preventing bone loss
due to estrogen deficiency. In the present study, we investigated
the bone sparing effect of DPHD in ovariectomized-rats that
exhibit estrogen deficiency. The effect of DPHD on bone mineral
density (BMD), changes to bone microarchitecture, and biochem-
ical markers of bone turnover were determined after a 12-week
course of treatment. Our analysis provides mechanistic insight into
the beneficial effects of the phytoestrogen DPHD in reducing bone
loss in estrogen deficient rats and suggests a potential clinical use
for DPHD in menopausal women.
Materials and Methods
The animal experimental protocol was approved by the
committee on Animal Care and Use, Faculty of Science, Mahidol
University (approval protocol number: MUSC-171). All animal
experiments were performed in accordance with the guidelines of
National Laboratory Animal Center, Mahidol University.
Chemicals and Plant Materials
Preparation of phytoestrogen diarylheptanoid (3R)-1,7-diphe-
nyl-(4E,6E)-4,6-heptadien-3-ol (DPHD) from C. comosa was
performed as previously described [16,21]. Rhizomes of C. comosa
were purchased from the Kampaengsaen district, Nakhon Pathom
province, Thailand. No specific permission is required for these
activities and the field study did not involve endangered or
protected species. Briefly, rhizomes were cut into small pieces,
dried and ground to powder then extracted with n-hexane in a
Soxhlet extractor. After removal of the solvent in vacuo, a pale
brown viscous oil was obtained. The DPHD was isolated from the
hexane extract as a major component (23.9%) by repeated silica
gel column chromatography. DPHD was eluted with hexane-
dichloromethane and each step utilized an increasing quantity of
the more polar solvent. The structure of DPHD was confirmed
and the absolute stereochemistry at the 3-position was determined
to be R by nuclear magnetic resonance and mass spectroscopy, the
same as that of DPHD previously isolated [16]. The purity of the
isolated material was assessed by TLC and NMR spectroscopy
and estimated to be 99% pure. The chemical structure is shown in
Figure 1A.
17b-estradiol (E
2
) and p-nitrophenyl phosphate were purchased
from Sigma-Aldrich Chemical Company (MO, USA). Methyl
methacrylate, 2-ethoxyethyl acetate and orange G were obtained
from Merck Company (Darmstadt, Germany). Haematoxylin,
fushin acid, and DePex mounting medium were purchased from
VWR International Ltd. (Poole, England). All compounds were
initially dissolved in 5% DMSO and diluted in olive oil to the final
doses.
Animals and Treatments
Eight-week-old female Sprague-Dawley rats, weighing 200–
220 g, were supplied by the National Laboratory Animal Centre
of Thailand (Salaya, Nakornpathom, Thailand). Animals were
housed in standard stainless steel cages under controlled condi-
tions: temperature at 2562uC, relative humidity of 50–60%, a 12-
h light/dark cycle, and allowed free access to food (rat pellets, C.P.
rat feed, Pokphand Animal Fed Co. Ltd., Bangkok, Thailand) and
water. Rats were randomly assigned to sham-operated control and
ovariectomized (OVX) groups. In OVX animals, both sites of
ovaries, which are the primary source of endogenous estrogen,
were removed under general anesthesia using pentobarbital
sodium (50 mg/kg Bw, i.p.). Animals were allowed to recover
from surgery for one week prior to use in experiments. Rats were
divided into six groups of six to eight animals each as follows: sham
operated control receiving vehicle (olive oil); OVX rats receiving
vehicle (olive oil, i.p.); OVX rats receiving DPHD at doses of 25,
50 and 100 mg/kg Bw (i.p.); OVX rats receiving 17b-estradiol (E
2
)
at a dose of 10 mg/kg Bw (s.c.) as a positive control. DPDH and E
2
were daily administered for 12 weeks and body weights were
recorded weekly. All rats were given subcutaneous injections of
10 mg/kg calcein, a fluorochrome bone marker, on Day 7 and
Day 1 before animals were sacrificed. At the end of treatments,
animals were euthanized with an overdose of sodium pentobar-
bital. Serum was collected and stored at 270uC until use and the
uterus was removed and weighed. Tibial bones were excised, kept
in saline-soaked gauze, covered with plastic and stored at 220uC
prior to analysis.
Measurement of Bone M ineral Dens ity (BMD)
The bone mineral density of left tibia was measured ex vivo by
peripheral Quantitative Computed Tomography (pQCT; XCT
Research SA
+
, Stratec Medizintechnik GmbH., Germany)
according to a previously protocol [22]. In brief, both the
trabecular and cortical bone density were scanned in cross-
sectional plane at metaphyseal sites of tibias. Proximal tibial
metaphysis was measured 2 mm below the growth plate. All bones
were scanned at 0.5 mm intervals using a voxel size of
0.09 mm60.09 mm60.09 mm. The trabecular bone was deter-
mined using contour mode 2 and peel mode 2 with a threshold
value of 720 mg/cm
3
. The cortical bone was determined using
separation mode 2 with a threshold value of 900 mg/cm
3
. All
parameters were analyzed using XCT-5.50E software (Stratec
Medizintechnik GmbH., Germany).
Bone Histomorphometric Analysis
All bone histomorphometries were conducted at the proximal
metaphyseal region of the right tibia. The adhering tissues and
bone marrow were removed from tibias followed by fixation for 3
days in 70% (vol/vol) ethanol, as previously described [23]. Bones
were then dehydrated in 95, and 100% (vol/vol) ethanol for 3 and
2 days, respectively, followed by embedding and undecalcification
in methyl methacrylate resin at 42uC for 48 h. To obtain 7 mm
and 12 mm thick sections, the embedded tibia was cut in
longitudinal section using a microtome (model RM2255; Leica,
Nussloch, Germany). The region of tibial studied was the
secondary spongiosa, the trabecular part of proximal tibia, at 1–
2 mm distal to the epiphysial plate and extending to 6 mm. The
7 mm sections were deplasticified in 2-ethoxyethyl acetate and
stained with Goldner’s trichrome then analyzed under bright field
microscopy. The structural variables were examined using the
histology section and parameters measured include trabecular
bone volume, normalized by tissue volume (BV/TV, %),
trabecular number (Tb.N, mm
21
), trabecular thickness (Tb.Th,
mm) and trabecular separation (Tb.Sp, mm). The 12 mm sections of
proximal tibia were left unstained to determine the mineral
apposition rate (MAR), an index of osteoblastic activity, calculated
by dividing the mean distance between double labels of the calcein
with time interval between the administration of the two labels.
Bone formation rate (BFR/TV) is another dynamic parameter
that is an index of bone turnover in general and bone formation in
Bone Sparing Effect of Curcuma comosa in OVX Rats
PLOS ONE | www.plosone.org 2 November 2013 | Volume 8 | Issue 11 | e78739
particular and allows for the determination of the age of bone [24].
All slides were analyzed under a light/fluorescent microscope
using a computer assisted Osteomeasure system (Osteometric,
Atlanta, GA), software version 4.1. Bone histomorphometric
parameters were reported according to the American Society for
Bone and Mineral Research Nomenclature Committee [25].
Serum Bone Biomarkers Assay
Tartrate-resistant acid phosphatase (TRAP) activity, a bone
resorption marker, was determined by using microplate assay
method. 4-nitrophenyl phosphate (4-NPP) was used as the
substrate according to the procedure of Lau et al. with modification
[26]. Serum was incubated for 30 min at 37uC with a substrate
solution consisting of 7.6 mmol/L 4-NPP in 100 mmol/L sodium
acetate buffer containing 50 mmol/L sodium tartrate (pH 5.5).
1 mmol/L NaOH was added to stop the reaction and the
absorbance at 405 nm was monitored to detect product formation.
Serum osteocalcin concentration, a bone turnover marker, was
measured using an enzyme immunoassay (EIA) kit specific for rat
osteocalcin (Biomedical Technology, Staughton, IN, USA).
Statistical Analysis
All data are expressed as means 6 SEM and were analyzed
using one-way analysis of variance (ANOVA) and Newman–Keuls
post-hoc test using SPSS for Windows, Version 17.0 (Chicago, IL,
USA). A non parametric Wilcoxon-type test for trend (Cuzick’s
Test for Trend) was employed for evaluation of the trend across
the groups. Differences were considered statistical significant at
p,0.05.
Results
Effects of Ovariectomy and DPHD Treatment on Body
Weight and Uterine Weight
All rats exhibited an increase in body weight during the 12
weeks of treatment, particularly in OVX rats. As shown in
Figure 1B, at the end of experiment, the body weight gain was
consistently highest in OVX control. However, the increases in
body weights of OVX rats was suppressed by treatment with E
2
(10 mg/kg Bw) to levels similar to the sham controls. Treatments of
OVX rats with DPHD at doses of 50 and 100 mg/kg BW also
significantly decreased body weight compared to OVX controls.
However, the effect of DPHD on body weight was not as
pronounced as that seen with E
2
(Figure 1B). These results indicate
that DPHD partially suppressed body weight gain in OVX rats.
The uterine weights of OVX rats was also changed but in this case
a significant decrease was observed when compared to sham
controls (p, 0.01). Uterine weight was increased in OVX rats
following treatment with estrogen and DPHD, though a significant
increase was only observed at 100 mg/kg Bw of DPHD (p,0.01)
(Figure 1C).
Figure 1. Estrogenic activity of DPHD compared to E
2
. Structure of the phytoestrogen diarylheptanoid DPHD, (3R)-1,7-diphenyl-(4E,6E)-4,6-
heptadien-3-ol, isolated from the rhizome of C. comosa (A). Effects of DPHD on body weight (B) and uterine weight (C) of sham-operated and
ovariectomized (OVX) rats receiving vehicle and various doses of DPHD (25, 50 and 100 mg/kg Bw) or 17b-estradiol (E
2
,10mg/kg Bw) for 12 weeks.
Results are expressed as the mean 6 SEM, n = 6–8. **p,0.01, significantly different from sham rats.
{
p,0.05 and
{{
p,0.01, significantly different from
OVX rats.
doi:10.1371/journal.pone.0078739.g001
Bone Sparing Effect of Curcuma comosa in OVX Rats
PLOS ONE | www.plosone.org 3 November 2013 | Volume 8 | Issue 11 | e78739
Effects of DPHD on ex vivo Bone Mineral Density (BMD)
Both total and trabecular bone mineral density (BMD) of tibial
metaphysis were markedly decreased in OVX rats (at 12 weeks)
compared to those of sham controls (Figure 2A and 2B,
respectively). E
2
treatment (10 mg/kg Bw) effectively prevented
the decreases in total and trabecular BMD. Treatments with
DPHD at doses of 25, 50, and 100 mg/kg Bw also prevented the
decrease in total and trabecular BMD compared to the OVX
group given the vehicle control. Similar to the effect observed for
body weight, treatment with DPDH did not restore BMD to the
level seen in the sham-operated group. Interestingly, DPHD had
no effect on the cortical BMD of tibial metaphysis though a
protective effect was observed with E
2
(Figure 2C). These findings
suggest that DPHD predominantly only protects against trabec-
ular bone loss, while E
2
effectively prevents the loss of both
trabecular and cortical bones.
Effects of DPHD on Bone cross Sectional Area and
Thickness
In Table 1, the total, trabecular and cortical bone cross sectional
areas (CSA) of tibia are shown. In OVX rats, total and trabecular
CSA of tibia were increased by 12% and 20%, respectively,
compared to sham controls. Treatment with E
2
, and DPHD at
doses of 50 and 100 mg/kg Bw prevented the increases in cross
sectional area. However, there was no significant change in
cortical area and thickness.
Effects of DPHD on Trabecular Bone Microarchitectural
Changes
Both static and dynamic changes in histomorphometry of the
proximal tibial metaphysis were evaluated. The growth plate and
spongiosa region of the proximal tibia of sham, OVX,
OVX+DPHD (100 mg/kg Bw), and OVX+E
2
(10 mg/kg Bw)
rats are shown in Figure 3A. Compared to the sham rats, a
decrease in trabecular bone and connectivity was observed in
OVX rats indicating that ovariectomy resulted in the deterioration
of trabecular bone microstructure. However, treatment with E
2
completely protected against this deterioration with partial
protection observed with DPHD treatment. Ovariectomy also
induced a marked decrease in the trabecular bone volume (BV/
TV) compared to that of the sham rats (73% reduction) (Figure 3C)
and again treatment with E
2
completely restored trabecular bone
volume to levels seen in the sham controls. All doses of DPHD
significantly increased BV/TV (Figure 3C) and trabecular number
(Tb.N) (p,0.05) (Figure 3D) in OVX rats but these values were
reduced compared to the sham and E
2
treated animals. DPHD
treatment also increased trabecular thickness (Tb.Th) in OVX rats
but significant difference was not observed at low dose of DPHD
(25 mg/Kg Bw)-treated group (Figure 3E). Trabecular separation
(Tb.Sp), another important structural index for static micro-
structural changes of bone, was markedly increased in OVX rats
compared to sham controls. E
2
treatment was capable of
significantly decreasing the separation of bone to the level seen
in the sham controls. The Tb.Sp in animals treated with DPHD
was also significantly reduced but were significantly higher than
that for the sham control group (Figure 3F). These results suggest
that DPHD treatment improved the connectivity of trabecular
bone in the ovariectomized rats though to a lesser degree than
treatment with E
2
.
The dynamic bone histomophometry was assessed using
fluorescence microscopy to monitor the uptake of calcein, a
fluorochrome bone marker (Figure 3B). Bone formation and
mineralization, expressed as mineral apposition rate (MAR), were
determined by the distance between two fluorochrome markers
given at different days and divided by the number of days between
administrations. This index reflects the activity of osteoblasts.
Compared to sham animals, MAR in OVX rats was significantly
increased from 2.8960.2 to 3.9860.1, indicating that ovariectomy
caused an increase in new bone formation leading to increase bone
turnover (Figure 3G).
Treatments with E
2
or DPHD at doses of 50, and 100 mg/kg
BW significantly decreased MAR to levels seen in the sham
controls. Bone formation rate (BFR), an index of bone turnover
provides the best correlation with the serum bone turnover
markers [24], and the bone formation rate per total volume (BFR/
TV) was significantly increased after ovariectomy (Figure 3H).
Figure 2. DPHD increases ex vivo bone mineral density (BMD),
as measured by pQCT. Total (A), trabecular (B), and cortical (C) BMD
of tibial metaphysis from sham-operated and ovariectomized (OVX) rats
receiving vehicle, DPHD (25, 50 and 100 mg/kg Bw) or 17b-estradiol (E
2
,
10 mg/kg Bw) for 12 weeks. Results are expressed as mean 6 SEM,
n=628. **p,0.01, significantly different from sham rats.
{
p,0.05 and
{{
p,0.01, significantly different from OVX rats.
doi:10.1371/journal.pone.0078739.g002
Bone Sparing Effect of Curcuma comosa in OVX Rats
PLOS ONE | www.plosone.org 4 November 2013 | Volume 8 | Issue 11 | e78739
Similar to MAR, the increase in BFR/TV in OVX animals was
effectively prevented by treatment with either E
2
or DPHD. The
reduction of MAR and BFR in DPHD treated rats indicated that
DPHD was capable of decreasing bone turnover rate in a similar
manner as E
2
.
Effects of DPHD Treatment on Biochemical Bone
Turnover Markers
To evaluate the effect of E
2
and DPHD treatments on bone
turnover in OVX rats, we measured the serum osteocalcin
concentration and tartrate-resistant acid phosphatase activity. As
shown in Figure 4A, the serum osteocalcin concentration in OVX
rats was significantly higher than that in sham animals and DPHD
treatment of OVX rats significantly reduced the serum osteocalcin
concentration. These results indicate that DPHD prevents the
ovariectomy-induced increase of bone turnover in rats. The TRAP
activity of osteoclast, an index of bone resorption, was 25% higher
in OVX rats compared to the sham group and DPHD treatment
restored TRAP activity to level similar to those of Sham and E
2
-
treated groups (Figure 4B). Since the decreases in bone turnover
and resorption markers are related to the suppression of bone
formation rate, these results suggest that DPHD decreased the
bone turnover rate by suppressing osteoclast activity in OVX rats.
Discussion
The present study has demonstrated for the first time of the
bone sparing effect of a novel diarylheptanoid phytoestrogen
(DPHD) isolated from C. comosa. In ovariectomy-induced osteo-
penia (OVX), a deterioration in trabecular bone microarchitecture
(12 weeks after ovariectomy) clearly led to the loss of bone mass in
rats. Treatments with DPHD effectively prevented the trabecular
bone loss and improved bone microstructure. Moreover, markers
of bone turnover, including osteocalcin and TRAP activity, were
decreased in DPHD treated animals. These results suggest that
DPHD provides a protective effect against OVX-induced bone
loss that is associated with decreased bone turnover through
suppressing bone resorption.
The integrity of skeletal is maintained through a bone
remodeling process that balances bone formation and bone
resorption [4] and estrogen plays an important role in the
maintenance of bone mass [27]. The rapid decline of estrogens in
postmenopausal women results in an imbalance in the bone
remodeling process leading to osteoporosis [28]. Mornitoring of
BMD is important for diagnosis and the treatment of osteoporosis
as decreased bone mass is a major characteristic of this disease. In
this study, decreased BMD in OVX rats, determined using pQCT
was observed only in the metaphysic of the tibia, which has a
greater proportion of trabecular bone in the proximal end.
Trabecular bone, a sponge-like bone found at the ends of long
bones and vertebrae, contains osteoblasts and osteoclast on its
surface and is more active in bone turnover and bone remodeling
compared to cortical bone [29,30]. Indeed, our analysis of OVX
rats showed only loss of trabecular BMD. This finding is consistent
with previous studies that report the loss of bone in adult OVX
rats was more prominent in trabecular than cortical bone [31].
However the loss of trabecular BMD in OVX rats was attenuated
by DPHD treatment. Consistent with reports that estrogen
decreases periosteal bone formation and radial growth [32],
OVX rats displayed an increase in cross sectional bone area
indicating that radial growth was increased. Similar to estrogen,
treatment with DPHD prevent the increase in bone area in OVX
animals. The improvement in bone measurements following
DPHD treatment may be partly attributed to its estrogenic like
activity, as evidenced by increased uterine weight in DPHD
exposed animals (Figure 1C) and our earlier study on uterotropic
activity of DPHD [17,18].
A rapid reduction in trabecular bone volume is known to occur
following ovariectomy and is associated with an increase in bone
turnover rate resulting from an excessive osteoclast activity [33].
Our analysis of the destruction of bone microarchitecture, another
important characteristic of osteoporosis, evaluated using bone
histomorphometry is consistent with an increased rate of bone
turnover in OVX rats. Ovariectomy also markedly decreased
static indices, including trabecular bone volume, thickness, and
number with an increase in trabecular separation. In addition to
direct effects on bone morphology, monitoring changes in
circulating bone biochemical markers can also reveal the status
of bone remodeling process [34]. These markers include
osteocalcin, an osteoblast-specific bone formation marker, and
tartrate-resistant acid phosphatase (TRAP) activity, an osteoclast-
specific bone resorption marker [34,35]. A dramatic increase in
serum osteocalcin and TRAP activity was observed 12 weeks
following ovariectomy, confirming that bone loss was due to an
increase in bone turnover rate. DPHD reduced these markers of
bone turnover in OVX rats suggesting that DPHD prevented
trabecular bone loss and micro-architecture deterioration by
suppressing the rate of bone turnover either by decreasing bone
resorption or increasing bone formation.
DPHD at doses of 25 and 50 mg/kg BW preserved bone mass
in OVX rats without showing an uterotrophic effect. These results
indicate that the beneficial effect of DPHD on bone is not limited
Table 1. Effect of DPHD on bone area and thickness of OVX rats.
Groups/Parameters Total CSA (mm
2
) Trebecular CSA (mm
2
) Cortical CSA (mm
2
) Cortical thickness (mm)
Sham 14.6160.38 7.8960.30 6.2160.14 0.6860.011
OVX + Vehicle 16.5060.50* 9.5160.49* 6.4160.11 0.7060.010
OVX + DPHD (25 mg/kg) 16.1560.42* 9.0360.40* 6.4560.12 0.7060.005
OVX + DPHD (50 mg/kg) 14.9960.39
{
8.3660.15
{
6.0260.12 0.6960.006
OVX + DPHD (100 mg/ kg) 14.7260.44
{
8.1260.39
{
5.9160.13 0.6960.005
OVX + E
2
(10 mg/kg) 14.7360.48
{
7.8260.51
{
5.9760.10 0.6860.007
Total, trabecular, and cortical cross sectional area (CSA) and cortical thickness were measured from sham-operated and ovariectomized (OVX) rats receiving vehicle,
DPHD (25, 50 and 100 mg/kg Bw) or 17b-estradiol (E
2
,10mg/kg Bw) for 12 weeks. Data are expressed as mean 6 SEM, n = 628.
*p,0.05, significantly different from sham rats.
{
p,0.05, significantly different from OVX rats.
doi:10.1371/journal.pone.0078739.t001
Bone Sparing Effect of Curcuma comosa in OVX Rats
PLOS ONE | www.plosone.org 5 November 2013 | Volume 8 | Issue 11 | e78739
to its estrogenic property but also mediate through other biological
effects of DPHD, such as an anti-inflammatory activity [19].
Inflammation is one of the causal factors of osteoporosis and
several cytokines, such as IL-1, M-CSF and RANKL, are involved
in the pathogenesis of osteoporosis. The role of these cytokines is to
activate osteoclast differentiation and bone resorption [36].
RANKL, a TNF family member, is synthesized by the osteoblast
and is an essential cytokines for activation of osteoclast formation,
function, and survival [37]. The interaction of RANKL and
RANK stimulates the osteoclastogenesis, the coupling process
between the osteoblast and osteoclast to control bone remodeling
[38]. Inhibiting the interaction of RANKL and RANK may have
benefits in the treatment of osteoporosis and DPHD treatment
reduces mRNA level of RANKL produced by osteoblast cells
during differentiation [21]. The inhibitory effect of DPHD on
RANKL may in turn attenuate the interaction of RANKL and
RANK and subsequently reduce the downstream inflammatory
cytokine induced osteoclastogenesis and bone resorption. Estrogen
deficiency in OVX rats is associated with the local disturbance of
cytokines in bone marrow, leading to an increase in osteoclast
numbers that ultimately penetrate trabecular bone and cause deep
resorption cavities [39]. Consequentially, trabecular bones are lost
Figure 3. Reversal of OVX induced bone microarchitectural changes by DPHD treatment. Representative 2D images of the proximal tibial
metaphysic (trabecular structure) of sham operated and OVX rats receiving vehicle, DPHD (DPHD 100 mg/kg Bw and 17b-estradiol (E
2
,10mg/kg Bw)
for 12 weeks. Samples were stained with Goldner’s trichrome for bright-field microscopy at a magnification of 2X showing the following: epiphysis,
epiphyseal plate (EP), trabecular bone (Tb), and cortical bone (Cor) (A). Fluorescence micrographs (calcein labeling) (B). Static parameters: trabecular
bone volume normalized by tissue volume (BV/TV, %) (C), trabecular number (Tb.N, mm
21
) (D), trabecular thickness (Tb.Th, mm) (E), and trabecular
separation (Tb.Sp, mm) (F). Dynamic parameters: mineral apposition rate (MAR) (G) and bone formation rate (BFR) (H). Data are expressed as the mean
6 SEM, n = 628. **p,0.01 significantly different from sham rats and
{
p,0.05 and
{{
p,0.01, significantly different from OVX rats.
doi:10.1371/journal.pone.0078739.g003
Bone Sparing Effect of Curcuma comosa in OVX Rats
PLOS ONE | www.plosone.org 6 November 2013 | Volume 8 | Issue 11 | e78739
and the remaining bones are less dense, thinner, and widely
separated [40]. Changes in cytokine levels in OVX rats may be
attenuated by DPHD treatment. If this is the case, then it suggests
that DPHD may suppress osteoclast activity. The inhibitory effect
of DPHD on both RANKL mRNA expression and interaction of
RANKL and RANK in osteoblast cells may in part account for
attenuation of bone turnover and preserving bone mass after
ovariectomy [21]. Pharmacokinetic analysis indicates that the
amount of DPHD used in treatment of OVX rats in the present
study would provide an effective concentration in the range similar
to that reported in the in vitro study [21,40]. However, the response
of cytokines to DPHD treatment in OVX rats has not been
investigated and any effect of DPHD on osteoclast cells and the
inflammatory system remains to be elucidated.
In conclusion, this is the first report on the effect of DPHD on
bone turnover and protection of trabecular bone loss in OVX rats.
Our results indicate that the novel phytoestrogen, DPHD, exhibits
low uterotrophic activity and has potential in clinical applications
for preserving bone mass and structure in postmenopausal
osteoporosis.
Acknowledgments
We thank Prof. Chumpol Pholpramool and Assoc. Prof. Dr. L. T. Jensen
for their critical reading of the manuscript. We also thank Prof. Suchinda
Malaivijitnond, Primate Research Unit, Department of Biology, Faculty of
Science, Chulalongkorn University and Prof. Yuzuru Hamada, Primate
Research Institute, Kyoto University, for allowing the use of peripheral
quantitative computed tomography (pQCT). We are also grateful to
COCAB for allowing the use of facilities for bone microstructure study.
Author Contributions
Conceived and designed the experiments: DT PP. Performed the
experiments: DT PW A. Chuncharunee. Analyzed the data: DT JW A.
Chairoungdua. Contributed reagents/materials/analysis tools: AS PP A.
Chuncharunee. Wrote the paper: DT PP JW.
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