G Model

PHYMED-51628; No. of Pages 8 ARTICLE IN PRESS

Phytomedicine xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Phytomedicine
journal homepage: www.elsevier.de/phymed

Phloretin promotes osteoclast apoptosis in murine macrophages and

inhibits estrogen deficiency-induced osteoporosis in mice
Eun-Jung Lee 1 , Jung-Lye Kim 1 , Yun-Ho Kim, Min-Kyung Kang,
Ju-Hyun Gong, Young-Hee Kang ∗
Department of Food Science and Nutrition, Hallym University, Chuncheon, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history: Bone-remodeling imbalance induced by increased osteoclast formation and bone resorption is known to
Received 25 August 2013 cause skeletal diseases such as osteoporosis. The reduction of estrogen levels at menopause is one of the
Received in revised form 12 February 2014 strongest risk factors developing postmenopausal osteoporosis. This study investigated osteoprotective
Accepted 2 April 2014
effects of the dihydrochalcone phloretin found in apple tree leaves on bone loss in ovariectomized (OVX)
C57BL/6 female mice as a model for postmenopausal osteoporosis. OVX demoted bone mineral density
Keywords:
(BMD) of mouse femurs, reduced serum 17␤-estradiol level and enhanced serum receptor activator of NF-
Phloretin
␬B ligand (RANKL)/osteoprotegerin ratio with uterine atrophy. Oral administration of 10 mg/kg phloretin
Osteoclast apoptosis
Estrogen deficiency
to OVX mice for 8 weeks improved such effects, compared to sham-operated mice. Phloretin attenuated
Osteoporosis TRAP activity and cellular expression of ␤3 integrin and carbonic anhydrase II augmented in femoral
Ovariectomy bone tissues of OVX mice. This study further examined that osteogenic activity of phloretin in RANKL-
differentiated Raw 264.7 macrophages into mature osteoclasts. Phloretin at 1–20 ␮M stimulated Smac
expression and capase-3 activation concurrently with nuclear fragmentation of multi-nucleated osteo-
clasts, indicating that this compound promoted osteoclast apoptosis. Consistently, phloretin enhanced
bcl-2 induction but diminished bax expression. Furthermore, phloretin activated ASK-1-diverged JNK and
p38 MAPK signaling pathways in mature osteoclasts, whereas it dose-dependently inhibited the RANKL-
stimulated activation of ERK. Therefore, phloretin manipulated ASK-1-MAPK signal transduction leading
to transcription of apoptotic genes. Phloretin was effective in preventing estrogen deficiency-induced
osteoclastogenic resorption.
© 2014 Elsevier GmbH. All rights reserved.

Introduction
Osteoclasts specialized for bone resorption and osteoblasts
responsible for bone formation play a key role in bone turnover
(Proff and Römer 2009). There are multiple targets within osteo-
clasts for pharmacologic intervention to prevent bone loss. During
osteoclast formation, receptor activator of nuclear factor (NF)-␬B
ligand (RANKL) and macrophage colony-stimulating factor (M-CSF)
promote differentiation and modulate osteoclast survival (Blair
Abbreviations: CA, carbonic anhydrase; RANKL, receptor activator of nuclear
factor-␬B ligand; Smac, second mitochondrial-derived activator of caspase; TRAP,
tartrate-resistant acid phosphatase.
∗ Corresponding author at: Department of Food and Nutrition, Hallym University,
Chuncheon, Kangwon-do 200-702, Republic of Korea. Tel.: +82 33 248 2132;
fax: +82 33 254 1475.
E-mail address: yhkang@hallym.ac.kr (Y.-H. Kang).
1
These authors contributed equally to this work.
and Zaidi 2006; Cicek et al. 2011; Xing et al. 2005). Several drugs
act by targeting specific pathways within the osteoclastic cells
(Rejnmark and Mosekilde 2011). Synthetic bisphosphonates for the
antiresorptive treatment predominantly inhibit osteoclasts attach-
ment to bone matrix and induce apoptosis of mature osteoclasts
(Dominguez et al. 2011; Rejnmark and Mosekilde 2011). Accord-
ingly, potential agents targeting osteoclast apoptosis may display
favorable effects in combating resorptive bone diseases.
The reduction of estrogen levels at menopause is one of the
strongest risk factors for developing postmenopausal osteoporosis
(Fraser et al. 2011; Henriksen et al. 2011). Postmenopausal osteo-
porosis is characterized by low bone mass and microarchitectural
deterioration of bone tissue (Kanis 2002). Osteoporosis treatments
include assuring calcium and vitamin D as well as prescription
of medications such as bisphosphonates (Akesson 2003; Beard
2012; Dawson-Hughes and Bischoff-Ferrari 2007). Potential first-
line pharmacological therapies for postmenopausal osteoporosis
include agents improving bone strength and reducing osteoporotic

http://dx.doi.org/10.1016/j.phymed.2014.04.002
0944-7113/© 2014 Elsevier GmbH. All rights reserved.

Please cite this article in press as: Lee, E.-J., et al., Phloretin promotes osteoclast apoptosis in murine macrophages and inhibits estrogen
deficiency-induced osteoporosis in mice. Phytomedicine (2014), http://dx.doi.org/10.1016/j.phymed.2014.04.002
G Model
PHYMED-51628; No. of Pages 8 ARTICLE IN PRESS
2 E.-J. Lee et al. / Phytomedicine xxx (2014) xxx–xxx

Fig. 1. Chemical structure of phloretin (A), uterus transverse section (B), wet weight of uterine tissues (C) and serum 17␤-estradiol level (D) of OVX mice treated with
10 mg/kg phloretin daily for 8 weeks. Cross-sectional images of the uterine horn were obtained by staining with H&E and visualized under light microscopy. Magnification:
40-fold. Serum 17␤-estradiol level was determined by using assay kits. Values in bar graphs (means ± SEM, n = 9) not sharing a letter are different at p < 0.05.

fracture (Josse et al. 2013). Hormone replacement therapy may help
prevent adverse postmenopausal changes in bones (Eriksen 2012;
Tao et al. 2011). However, its long-term use is controversial.
Concerns pertaining to the risk of long-term hormone therapy
have prompted an increase in the use of natural alternatives (Bedell
et al. 2014). Phytoestrogens mainly belong to naturally occur-
ring nonsteroidal compounds that have estrogen-like biological
activity primarily through binding to estrogen receptors (Al-Anazi
et al. 2011; Turner et al. 2007). Numerous studies have focused
on revealing osteoprotective mechanism(s) of phytoestrogens. The
isoflavone formononetin prevents bone loss by reversing estro-
gen deficiency-induced detrimental bone biomechanical features
(Kaczmarczyk-Sedlak et al. 2013). Several polyphenols including
green tea (−)-epigallocatechin gallate (EGCG) prevent bone loss
(Hagiwara et al. 2011). This polyphenol inhibits osteoclast for-
mation and bone resorption (Kamon et al. 2009). Our previous
study revealed that phloretin had an antiosteoclastogenic activ-
ity by suppressing RANKL-induced osteoclast differentiation (Kim
et al. 2012). However, their action mechanisms for manipulating
bone-specific remodeling process remain unclear under in vivo
conditions.
Phloretin (Fig. 1A) is a natural dihydrochalcone present in apple
peels and displays anti-oxidative and anti-inflammatory activity
(Jung et al. 2009; Yang et al. 2011). Orally consumed phlorizin is
mainly converted into phloretin by hydrolytic enzymes in the small
intestine, and inhibits glucose absorption by the small intestine and
renal glucose reabsorption as a glucose transporter 2 inhibitor (Idris
and Donnelly 2009). This study investigated that phloretin would
help to antagonize osteoporosis due to estrogen deficiency. This
study evaluated the effects of phloretin on tartrate-resistant acid
phosphatase (TRAP) activity and osteoclastogenic marker induc-
tion in femoral bone tissues of ovariectomized (OVX) mice. Also
this study examined whether phloretin promoted osteoclast loss in
femoral bones of OVX mice and apoptosis of mature osteoclasts dif-
ferentiated from Raw 264.7 macrophages. The sequential molecular
events and signaling of osteoclast apoptosis induced by phloretin
were elucidated.
Materials and methods
Materials
Fetal bovine serum (FBS), penicillin–streptomycin, trypsin–
EDTA were purchased from Lonza (Walkersville, MD). Mini-
mum Essential Medium Alpha Medium (␣-MEM), Dulbecco’s

Please cite this article in press as: Lee, E.-J., et al., Phloretin promotes osteoclast apoptosis in murine macrophages and inhibits estrogen
deficiency-induced osteoporosis in mice. Phytomedicine (2014), http://dx.doi.org/10.1016/j.phymed.2014.04.002
G Model
PHYMED-51628; No. of Pages 8 ARTICLE IN PRESS
E.-J. Lee et al. / Phytomedicine xxx (2014) xxx–xxx 3

Table 1 800
Effects of phloretin on growth rate and feed intake in ovariectomized mice.
700 a
Sham control OVX OVX-MTE

serum OPG (pg/ml)

600
Initial BW (g) 22.89 ± 2.52 22.92 ± 3.15 22.35 ± 1.80 a,b
Final BW (g) 23.80 ± 2.31b 26.22 ± 2.64a 24.33 ± 2.00ab 500 b
ADG (mg/d) 23.19 ± 3.31c 62.65 ± 9.15a 42.26 ± 7.91b
ADFI (g/d) 3.85 ± 0.06 3.86 ± 0.06 3.73 ± 0.08 400
FER 0.59 ± 0.08c 1.62 ± 0.24a 1.17 ± 0.19b 300
Liver weight (g) 1.17 ± 0.08 1.14 ± 0.06 1.10 ± 0.05
LW/BW (%) 4.86 ± 0.27 4.38 ± 0.22 4.55 ± 0.30 200
Serum GOP (IU/L) 50.35 ± 1.94 50.25 ± 1.62 49.09 ± 2.19
Serum GPT (IU/L) 22.84 ± 1.31 21.96 ± 1.37 22.57 ± 2.56 100

Female C57BL/6 mice (11 weeks of age, 20–25 g) were surgically ovariectomized 0
and orally supplemented with 10 mg/kg phloretin daily for 8 weeks. Abbreviations:
BW, body weight; ADG, average daily gain; ADFI, average daily food intake; FER, 160
food efficiency ratio; OVX, ovariectomy; LW, liver weight. Values are means ± SEM a
140

serum RANKL (pg/ml)

of 9 mice. Respective values in the same row not sharing the same superscript differ,
p < 0.05. 120 a,b
100
b
modified eagle’s media (DMEM), and phloretin were purchased 80
from Sigma–Aldrich Chemicals (St. Louis, MO), as were all other
60
reagents, unless specifically stated elsewhere. Anti-mouse carbonic
anhydrase II (CAII) purchased from AbCam (Cambridge, UK). Anti- 40
bodies of mouse second mitochondrial-derived activator of caspase 20
(Smac) and mouse Bax were obtained from Santa Cruz Biotech-
0
nology (Santa Cruz, CA). Antibodies of mouse integrin ␤3, mouse
cleaved caspase-3, mouse phospho-ASK-1, mouse phospho-ERK, 45
mouse phospho-JNK and mouse phospho-p38 were purchased a
40
from Cell Signaling Technology (Beverly, MA). Mouse Bcl-2 anti-
35
serum RANKL/OPG
body was provided by BD Transduction Laboratories (Franklin b
Lakes, NJ), Horseradish peroxidase-conjugated goat anti-rabbit IgG 30
25 b
was provided by Jackson ImmunoReserach Laboratories (West
Grove, PA). RANKL was obtained from Peprotech (Rocky Hill, NJ). 20
Phloretin was dissolved in dimethyl sulfoxide (DMSO) for live 15
culture with cells; a final culture concentration of DMSO was <0.5%.
10
5
Animals and ovariectomy
0
sham phloretin
To investigate inhibitory effects of phloretin on osteoporotic control
activity in an estrogen-deficient animal model, this study OVX
introduced ovariectomical technique for mimicking estrogen
Fig. 2. Suppressive effects of phloretin on serum RANKL/OPG ratio in OVX mice. OVX
deprivation or senescent menopause. C57BL/6 mice (11 weeks of mice were orally administrated with 10 mg/kg phloretin daily for 8 weeks. Serum
age, 20–25 g) were provided by the Experimental Animal Cen- levels of OPG and RANKL were determined by using ELISA kits. Values in bar graphs
ter, Hallym University and kept on a 12 h light/dark cycle at (means ± SEM, n = 9) not sharing a letter are different at p < 0.05.
20–25 ◦ C with 60% relative humidity under specific pathogen-free
conditions. Mice were fed a non-purified diet (RodFeedTM, DBL,
Umsung, Korea) during the experimental period (8 weeks) with was observed in the average daily feed intake (ADFI) of each mouse
free access to water ad libitum at the animal facility of Hallym group. Accordingly, the food efficiency ratio (FER = ADG/ADFI) was
University. The animals were allowed to acclimatize for a week significantly higher than that of the other groups (Table 1). Ovariec-
before beginning experiments. All animal experiments were per- tomy did not affect the liver weight-to-BW ratio (LW/BW) as
formed in accordance with the University’s Guidelines for the Care compared to sham-operated mice.
and Use of Laboratory Animals approved by the Committee on Uterine tissues were obtained from three mice of each group.
Animal Experimentation of Hallym University (permission num- After being washed with saline, uterine tissues were fixed in 10%
ber: hallym 2011–24). For the ovariectomical surgery, 11 week-old neutral buffered formalin for 24 h. Tissues were stained using a
female animals were anesthetized using a ketamine/rompun cock- modified Harris hematoxylin and Shandon Instant eosin (H&E) for
tail (40 mg ketamine and 10 mg rompun/kg body weight) for the microscopic observation.
either a sham operation (Sham control) or bilateral oophorec-
tomy (ovariectomy, OVX). Mice receiving surgical OVX were orally Biochemical analyses
treated with 10 mg/kg phloretin once a day for 8 weeks (9 mice of
each group). After 8 weeks of treatment, blood samples and uterus Serum 17␤-estradiol levels were determined by using an
tissues were collected and serum samples obtained by centrifuga- enzyme-linked immunosorbent assay (ELISA) kit (Uscn Life Science,
tion (3000 rpm, 10 min) were stored at −70 ◦ C prior to analyses. Wuhan, China) according to the manufacturer’s instructions. Serum
After 8 weeks following ovariectomical surgery, the final body levels of OPG and RANKL were also measured with ELISA kits (R&D
weight (BW) and average daily gain (ADG) significantly increased Systems, Minneapolis, MN). Serum levels of glutamic oxaloacetic
in OVX mice, as compared to sham-operated control mice (Table 1). transaminase (GOT) and glutamlc pyruvic transaminase (GPT) were
The markedly enhanced ADG in OVX mice was significantly dimin- measured by using enzymatic kits (Asan Pharmaceuticals, Seoul,
ished by the supplementation of phloretin. No significant difference Korea) according to the manufacturer’s instructions. In this study,

Please cite this article in press as: Lee, E.-J., et al., Phloretin promotes osteoclast apoptosis in murine macrophages and inhibits estrogen
deficiency-induced osteoporosis in mice. Phytomedicine (2014), http://dx.doi.org/10.1016/j.phymed.2014.04.002
G Model
PHYMED-51628; No. of Pages 8 ARTICLE IN PRESS
4 E.-J. Lee et al. / Phytomedicine xxx (2014) xxx–xxx

Fig. 3. TRAP localization (A) in femoral bone tissue sections of OVX mice and inhibition of induction of ␤3 integrin and CAII in bone tissue by 10 mg/kg phloretin daily
for 8 weeks (B). Longitudinal femoral bone tissues were stained and the staining intensity was measured (means ± SEM, n = 3). Representative Western blot data of bone
tissues were obtained from three independent experiments, and ␤-actin protein was used as an internal control. The bar graphs (means ± SEM) in the right panels represent
quantitative results obtained from a densitometer. Values in bar graphs not sharing a letter are different at p < 0.05.

the serum GOT and GPT levels were not altered following the oral
administration of phloretin to OVX mice, indicating that phloretin
was not toxic to mice (Table 1).
The bone mineral density (BMD) and bone mineral content
(BMC) of mouse femoral bone tissues were determined by using
a PIXImus mouse densitometer (GE Lunar, Waukesha, WI). BMD
calculated by dividing BMC (mg) by the projected bone area (cm2 )
was assessed in the femoral regions.
Histological TRAP staining
Femoral bone tissues were decalcified in decalcifying solution
(Sigma–Aldrich Chemicals) and dehydrated in a graded series of
ethanol solutions for 18 h. For the histological staining of TRAP,
femoral bone tissues were then embedded in paraffin and cut
into 5 ␮m sections in thickness. The TRAP staining was con-
ducted by using leukocyte acid phosphatase kit (Sigma Chemicals).
The femoral tissue samples were incubated for 20 min in 50 mM
sodium acetate and 40 mM potassium sodium tartrate buffer (pH
5.0) and further incubated for 15 min in the same buffer con-
taining 2.5 mg/ml naphthol AS-BI phosphate and 0.5 mg/ml fast
garnet GBC. After each slide was mounted in VectaMount mounting
medium (Vector Laboratories, Burlingame, CA), images were taken
using an Axiomager optical microscope system (Zeiss, Germany).
The TRAP activity was measured by the image analysis program of
the microscope system.

Fig. 4. Acceleration of nuclear condensation and fragmentation by 1–20 ␮M phloretin in Raw 264.7 cells treated with 50 ng/ml RANKL for 5 d. Following Hoechst 33258 stain-
ing, nuclear fragmentation and condensation (arrowheads) were seen by fluorescence microscopy. Representative microphotographs were obtained from three independent
experiments. Original magnification of microscopic images (n = 3), 400×.

Please cite this article in press as: Lee, E.-J., et al., Phloretin promotes osteoclast apoptosis in murine macrophages and inhibits estrogen
deficiency-induced osteoporosis in mice. Phytomedicine (2014), http://dx.doi.org/10.1016/j.phymed.2014.04.002
G Model
PHYMED-51628; No. of Pages 8 ARTICLE IN PRESS
E.-J. Lee et al. / Phytomedicine xxx (2014) xxx–xxx 5

Fig. 5. Western blot data showing expression of bcl-2, bax and Smac, and activation of caspase-3. Cells were exposed to 50 ng/ml RANKL for 5 d with 1–20 ␮M phloretin (A
and B) and 20 ␮M estradiol (C). Cell lysates were subject to Western blot analysis with a primary antibody against bcl-2, bax, Smac or cleaved caspase-3. Representative blot
data were obtained from three experiments, and ␤-actin protein was used as an internal control. The bar graphs (means ± SEM) in the bottom panels represent quantitative
results of blots obtained from a densitometer. Values not sharing a common letter are different at p < 0.05.

Western blot analysis
The bone fragments were trimmed free of soft tissue and washed
to remove contaminants with PBS and incubated at 4 ◦ C overnight
in 1.2 M HCl to demineralize the bone tissue. The demineralized
bone tissue was washed with water and incubated for 72 h at 4 ◦ C
in 100 mM Tris lysis buffer (pH 7.4) containing 6 M guanidine–HCl,
and protease inhibitors cocktail. The supernatant (crude extract
proteins) was collected after centrifugation. The residue was fur-
ther extracted by repeating the above extraction procedure and
the collected supernatant was combined with the first supernatant.
Equal amounts of extract proteins were electrophoresed on 6–12%
SDS-PAGE gels and transferred onto a nitrocellulose membrane.
Nonspecific binding was blocked by soaking membranes in a TBS–T
buffer [50 mM Tris–HCl (pH 7.5), 150 mM NaCl and 0.1% Tween
20] containing 3% bovine serum albumin or 5% non-fat milk for
3 h. The membranes were incubated with rabbit anti-mouse integ-
rin ␤3 and rabbit anti-mouse CAII as a primary antibody. The
membranes were then incubated with goat anti-rabbit IgG con-
jugated to horseradish peroxidase as a secondary antibody. The
protein levels on gels were determined by using Supersignal West
Pico Chemiluminescence detection reagents (Pierce Biotechnology,
Rockford, IL), and Konica X-ray film (Konica, Tokyo, Japan). Incuba-
tion with monoclonal mouse antibodies of ␣-tublin or ␤-actin was
conducted for comparative control.
Osteoclast differentiation of Raw 264.7 cells
Murine macrophage Raw 264.7 cells (American Type Culture
Collection, Manassas, VA) were cultured in DMEM containing 10%
FBS, 2 mM glutamine, 100 U/ml penicillin and 100 ␮g/ml strepto-
mycin at 37 ◦ C in a humidified atmosphere of 5% CO2 in air. For the
osteoclast differentiation, Raw 264.7 cells were plated on 24-well
plates at the density of 1 × 104 cells/well, and cultured for 5 days in
␣-MEM containing 10% FBS and 50 ng/ml RANKL in the absence and
presence of 1–20 ␮M phloretin. Cell culture medium was changed
every 2 days.
Western blot analysis was also conducted with cell lysates pre-
pared from cultured Raw 264.7 cells. Equal amounts of lysate
proteins were electrophoresed on 6–15% SDS-PAGE gels and trans-
ferred onto a nitrocellulose membrane. The protein levels of
Smac, cleaved caspase-3, Bcl-2, Bax, phospho-ASK-1, phospho-ERK,
phospho-JNK, phospho-p38 and ␤-actin were measured by using
chemiluminescence detection reagents.
Nuclear morphology of osteoclasts
Nuclear morphology was examined by fluorescence microscopy
with an Axiomager optical microscope system equipped for fluores-
cence illumination. After the fixation of osteoclasts with ice-cold 4%
formaldehyde for 1 h, the nuclear stain Hoechst 33258 (Molecular
Probes, Eugene, OR) was added at a final concentration of 10 ␮g/ml
for 15 min to allow uptake and equilibration before microscopic
observation. The slides were mounted while wet in aqueous Vecta-
Mount mounting solution and fluorescent images were obtained.
Cells containing fragmented or condensed nuclei were considered
apoptotic.
Statistical analyses
The results were expressed as means ± SEM for each treatment
group in each experiment. Statistical analyses were performed
using Statistical Analysis Systems statistical software package (SAS
Institute, Cary, NC). Significance was determined by one-way
analysis of variance, followed by Duncan range test for multiple
comparisons. Differences were considered significant at p < 0.05.
Results
Effect of phloretin on ovariectomy-induced bone loss
There was a marked reduction of the uterus in size and wet
weight due to OVX, indicating that surgically induced estrogen
deficiency caused uterine atrophy (Fig. 1B and C). The oral admin-
istration of phloretin to OVX mice alleviated the atrophy, being
concomitant with increased wet weight of OVX mouse uterus. The
serum 17␤-estradiol level decreased in OVX mice by ≈50% due to

Please cite this article in press as: Lee, E.-J., et al., Phloretin promotes osteoclast apoptosis in murine macrophages and inhibits estrogen
deficiency-induced osteoporosis in mice. Phytomedicine (2014), http://dx.doi.org/10.1016/j.phymed.2014.04.002
G Model
PHYMED-51628; No. of Pages 8 ARTICLE IN PRESS
6 E.-J. Lee et al. / Phytomedicine xxx (2014) xxx–xxx

Table 2
Osteogenic activity of phloretin in ovariectomized (OVX) mice.

Sham control OVX OVX-phloretin

BMD 74.4 ± 2.0a 61.0 ± 0.8c 70.1 ± 1.0a

BMC 17.6 ± 0.4a 15.1 ± 0.2c 17.0 ± 0.4a
Bone area 0.237 ± 0.003 0.250 ± 0.004 0.243 ± 0.004

Female C57BL/6 mice (11 weeks of age, 20–25 g) were ovariectomized and orally
supplemented with 10 mg/kg MTE and silibinin daily for 8 weeks. Areal bone min-
eral density (BMD, mg/cm2 ), bone mineral content (BMC, mg) and bone area (cm2 )
were measured in femur bones by using a PIXImus mouse densitometer. Values are
means ± SEM of 9 mice. Respective values in the same row not sharing the same
superscript differ, p < 0.05.

the uterine loss for 8 weeks, whereas the treatment of phloretin

enhanced the reduced level by ≈35% (Fig. 1D).
The serum levels of OPG and RANKL were assessed following
surgical estrogen deprivation for 8 weeks. The serum OPG level
of OVX mice did not alter significantly, but the serum RANKL level
significantly increased, compared with that of sham-operated mice
(p < 0.05, Fig. 2). However, the 8 week treatment with phloretin post
OVX enhanced the serum OPG level significantly and decreased
the serum RANKL level. Accordingly, the serum RANKL/OPG ratio
declined in phloretin-treated OVX mice.
The estrogen deprivation caused significant loss of mouse
femoral BMD (Table 2). In contrast, the 8 week-administration of
10 mg/kg phloretin to OVX mice elevated BMD and BMC signifi-
cantly, compared with those of sham-operated mice (Table 2).

Inhibition of ovariectomy-induced osteoclastic activity by

phloretin

The TRAP staining showed larger osteoclasts (purple-stained)

in OVX mouse femora compared with those in the sham-operated
mice (Fig. 3A). At 8 weeks post OVX, many TRAP-positive osteo-
clasts were observed in OVX mice, while the osteoclastic TRAP
activity in femoral bones of phloretin-treated mice was signifi-
cantly attenuated (Fig. 3A).
This study further investigated whether phloretin attenuated
the induction of ␤3 integrin and CAII in OVX-challenged mice. CAII
is expressed in high amounts in resorbing osteoclasts and osteo-
clast precursors (Eriksen 2010). The surgical ovariectomy induced
the expression of ␤3 integrin in femur bones, which was sup-
pressed by oral administration of 10 mg/kg phloretin to OVX mice
(Fig. 3B). Likewise, the significant attenuation of CAII induction was
observed in femoral tissues of OVX-challenged mice orally treated
with 10 mg/kg phloretin. Accordingly, phloretin was effective in
retarding active bone resorption in a sealed compartment between
the ruffled border and the bone surface (Fig. 3B).
Promotion of osteoclast apoptosis by phloretin
This study examined whether phloretin promoted apoptotic
death of TRAP-positive multinucleated osteoclasts. Hoechst 33258
staining revealed that phloretin elicited nuclear condensation
and fragmentation of RANKL-differentiated osteoclasts in a dose-
dependent manner, indicating that phloretin promoted osteoclast
apoptosis (Fig. 4).
The anti-apoptotic bcl-2 was highly induced in RANKL-
differentiated osteoclasts, while phloretin dampened the bcl-2
induction dose-dependently (Fig. 5A). In contrast, phloretin
elevated the pro-apoptotic bax induction. Consistently, Smac
expression and caspase-3 activation were highly enhanced in
20 ␮M phloretin-treated mature osteoclasts (Fig. 5B and C). Like-
wise, ␤-estradiol at 20 ␮M induced the mitochondrial protein Smac
in differentiated osteoclasts (Fig. 5C). Phloretin like ␤-estradiol
appeared to potentiate osteoclast apoptosis mediated by RANKL.
Fig. 6. Phloretin inhibition of RANKL-induced activation of ASK-1, JNK, p38 and
ERK. Raw 264.7 cells were exposed to 50 ng/ml RANKL for 5 d in the absence and
presence of 1–20 ␮M phloretin. Cell extracts were Western blot analysis with a
primary antibody against phospho-ASK-1, phospho-JNK, phospho-p38, or phospho-
ERK. Representative blot data were obtained from three independent experiments,
and ␤-actin protein was used as an internal control. The bar graphs (means ± SEM,
n = 3) in the bottom panels represent quantitative results obtained from a densito-
meter. Means without a common letter differ, p < 0.05.
Activation of apoptotic MAPK pathway by phloretin
This study attempted to determine whether phloretin may
booster osteoclastic apoptosis in RANKL-mediated osteoclasts
through accelerating ASK-1-MAPK signaling cascades. When
phloretin was treated with mature osteoclasts exposed to RANKL,
the ASK-1-diverged JNK and p38 MAPK signaling pathways were
markedly enhanced (Fig. 6). In contrast, 1–20 ␮M phloretin dose-
dependently inhibited the activation of ERK enhanced by RANKL.
Accordingly, phloretin manipulated ASK-1-MAPK signal trans-
duction leading to transcription of gene proteins involved in
osteoclastic apoptosis.
Discussion
Bone constantly undergoes turnover and remodeling to secure
normal repair of skeletal damage and replacement of old bone
with new one. The elimination of old bone by osteoclasts

Please cite this article in press as: Lee, E.-J., et al., Phloretin promotes osteoclast apoptosis in murine macrophages and inhibits estrogen
deficiency-induced osteoporosis in mice. Phytomedicine (2014), http://dx.doi.org/10.1016/j.phymed.2014.04.002
G Model
PHYMED-51628; No. of Pages 8 ARTICLE IN PRESS
E.-J. Lee et al. / Phytomedicine xxx (2014) xxx–xxx 7

specialized for bone resorption is sequentially followed by the
formation of mineralized bone matrix by osteoblasts (Eriksen
2010; Proff and Römer 2009). The imbalance of bone remodel-
ing occurs due to bone resorption excessive for bone formation,
consequently leading to resulting in resorptive disorders such as
osteoporosis (Eriksen 2010). The form of osteoporosis most com-
mon in women after menopauses is referred to as postmenopausal
osteoporosis. The reduction of estrogen levels at menopause is
one of the strongest risk factors for developing postmenopausal
osteoporosis (Fraser et al. 2011; Henriksen et al. 2011). This
study mimicked menopause by eliminating the ovaries in mice,
thereby resulting in postmenopausal osteoporosis. The OVX in
mice led to the uterine atrophy and decreased serum 17␤-estradiol
level.
The estrogen deficiency incurs bone mass loss and microar-
chitectural deterioration in bone leading to an increased risk
of fracture (Kanis 2002). In the present study the TRAP activ-
ity in femoral bone tissues was highly enhanced following the
8 week-estrogen deprivation concomitantly with the induction
of ␤3 integrin and CAII responsible for bone resorption. It was
assumed that OVX increased the number of TRAP-positive multi-
nucleated osteoclasts and their resorptive activity in femoral bone
tissues. RANKL promotes the differentiation of osteoclast lineages
and their survival (Cicek et al. 2011). This study revealed that
the estrogen deficiency increased serum RANKL level, through
which many TRAP-positive osteoclasts were formed and activated
in femoral bone tissues. Accordingly, the inhibition of osteoclast
differentiation and activation would be a therapeutic strategy for
postmenopausal osteoporosis.
There are multiple targets within osteoclasts for the phar-
macologic interventions to prevent bone loss. Potential first-line
therapies for postmenopausal osteoporosis can be use of agents
that inhibit bone resorption, thereby improving bone strength
and reducing osteoporotic fracture (Akesson 2003; Josse et al.
2013). Hormone replacement therapy with estrogen and progesto-
gen or estrogen alone and selective estrogen receptor modulators
may help prevent adverse postmenopausal changes in bones (Tao
et al. 2011). However, potential risks of the long-term therapy
outweigh benefits. The natural compound phloretin antagonized
postmenopausal osteoporosis by encumbering osteoclastogenesis
due to surgical OVX. Oral administration of phloretin blunted the
osteoclast formation and activation by diminishing TRAP activ-
ity in the femoral bone tissues. BMD was enhanced in femoral
bones of OVX mice having received phloretin. Inhibiting RANKL
production of osteoblasts or antagonizing RANKL actions on osteo-
clasts might be an osteoprotective mechanism of phloretin against
excessive formation of osteoclasts. There are potential alterations
in the OPG/RANKL/RANK system in metabolic bone disorders
(Khosla 2001). The phloretin supplementation decreased serum
RANKL/OPG ratio, possibly blocking RANKL signaling. Encumber-
ing RANKL pathway might be a therapy to treat postmenopausal
osteoporosis (Wada et al. 2006).
Potential risks of hormone therapies have prompted an increase
in the use of complementary therapies to alleviate postmenopausal

Fig. 7. Schematic diagram showing anti-osteoporotic feature and mechanistic effects of phloretin on osteoclast apoptosis in murine macrophages and OVX mice. As depicted,
phloretin inhibits the direct apoptotic signaling cascades induced by RANKL and OVX. Arrows indicate activation or induction; ⊥ indicates inhibition or blockade.

Please cite this article in press as: Lee, E.-J., et al., Phloretin promotes osteoclast apoptosis in murine macrophages and inhibits estrogen
deficiency-induced osteoporosis in mice. Phytomedicine (2014), http://dx.doi.org/10.1016/j.phymed.2014.04.002
G Model
PHYMED-51628; No. of Pages 8 ARTICLE IN PRESS
8 E.-J. Lee et al. / Phytomedicine xxx (2014) xxx–xxx
osteoporosis (Banu et al. 2012; Bedell et al. 2014; Zhang et al.
2008). Phytoestrogens with estrogen-like biological activity have
provided practical hormone therapy alternative (Al-Anazi et al.
2011; Sunita and Pattanayak 2011). Formononetin prevents osteo-
clastogenesis by enhancing bone mechanical properties and bone
chemistry improvement during estrogen deficiency (Kaczmarczyk-
Sedlak et al. 2013). Other polyphenols including EGCG and
hydroxytyrosol prevent bone loss (Kaczmarczyk-Sedlak et al. 2013;
Kamon et al. 2009). The phloretin glucoside phloridzin plays a role
of phytoestrogen with estrogenic and antiestrogenic activity (Wang
et al. 2010). However, their action mechanisms for boosting bone
health remain unclear. We have previously found that phloretin
blunted RANKL-induced osteoclast activation and demineralization
of bone matrix by dampening the secretion of MMP-9 and cathep-
sin K though disturbing TRAF6-NF-␬B-NFATc1-MITF signaling (Kim
et al. 2012). Tissue specific therapeutic approaches and precise
mechanisms of phloretin targeting osteoporosis need to be estab-
lished.
This study attempted to explore the precise inhibitory
mechanism(s) by which phloretin abrogated postmenopausal
osteoporosis. The present study revealed that phloretin promoted
osteoclast apoptosis through triggering apoptotic signaling path-
ways. Phloretin inhibited the degradation of bone matrix occurring
in sealed lacunae beneath the ruffled border of osteoclasts (Kim
et al. 2012). Bisphosphonates inhibit osteoclast attachment to
bone matrix and induce apoptosis by targeting specific path-
ways within mature osteoclasts (Dominguez et al. 2011; Rejnmark
and Mosekilde 2011). Phloretin inflamed MAPK-dependent apop-
totic signaling of osteoclasts in response to RANKL stimulation.
Phloretin induced cellular Smac expression, bax expression and
caspase-3 activation in RANKL-differentiated mature osteoclasts
by firing ASK-1-responsive JNK-p38 MAPK signaling and deter-
ring ERK signaling. Accordingly, phloretin manipulating osteoclast
survival and apoptosis may display favorable effects in combating
postmenopausal osteoporosis. Since phloretin is known to exert
antioxidant activity (Yang et al. 2011), phloretin may affect the
redox status during osteoclast differentiation, which directly regu-
lates osteoclast apoptosis.
In summary, the current report demonstrated that phloretin
abrogated estrogen deficiency-induced osteoporosis and pro-
moted apoptosis in mature osteoclasts. The oral administration of
phloretin improved uterine atrophy and femoral BMD reduction
induced by OVX. Additionally, phloretin inhibited osteoclast
activation and bone resorption by diminishing femoral tissue
levels of ␤3 integrin and CAII elevated in OVX mice. Furthermore,
phloretin promoted apoptosis in RANKL-differentiated osteo-
clasts by promoting signaling pathway of ASK-1-JNK-p38 MAPK
(Fig. 7). Therefore, phloretin may serve as a modulator against
postmenopausal osteoporosis and pathological osteoresorptive
disorders in clinical point of view.
Funding sources
This work was supported by grant from the National Research
Foundation of Korea (2012R1A2A2A01012946), and by the Ministry
of Education, Science Technology and National Research Founda-
tion of Korea through the Human Resource Training Project for
Regional Innovation, Korea (2012H1B8A2026122).
Author contributions
E.-J.L., J.-L.K. and Y.-H.K. designed research; E.-J.L., J.-L.K. and Y.-
H.K. conducted research; E.-J.L., J.-L.K. and I.-H. C. analyzed data
and E.-J.L. and Y.-H.K. wrote the paper. Y.-H.K. had primary respon-
sibility for final content. All authors read and approved the final
manuscript.
Please cite this article in press as: Lee, E.-J., et al., Phloretin promotes osteoclast apoptosis in murine macrophages and inhibits estrogen
deficiency-induced osteoporosis in mice. Phytomedicine (2014), http://dx.doi.org/10.1016/j.phymed.2014.04.002
References
Akesson, K., 2003. New approaches to pharmacological treatment of osteoporosis.
Bull. World Health Organ. 81, 657–664.
Al-Anazi, A.F., Qureshi, V.F., Javaid, K., Qureshi, S., 2011. Preventive effects of phy-
toestrogens against postmenopausal osteoporosis as compared to the available
therapeutic choices: an overview. J. Nat. Sci. Biol. Med. 2, 154–163.
Banu, J., Varela, E., Fernandes, G., 2012. Alternative therapies for the prevention and
treatment of osteoporosis. Nutr. Rev. 70, 22–40.
Beard, M.K., 2012. Bisphosphonate therapy for osteoporosis: combining optimal
fracture risk reduction with patient preference. Curr. Med. Res. Opin. 28,
141–147.
Bedell, S., Nachtigall, M., Naftolin, F., 2014. The pros and cons of plant estrogens for
menopause. J. Steroid Biochem. Mol. Biol. 139, 225–236.
Blair, H.C., Zaidi, M., 2006. Osteoclastic differentiation and function regulated by old
and new pathways. Rev. Endocr. Metab. Disord. 7, 23–32.
Cicek, M., Vrabel, A., Sturchio, C., Pederson, L., Hawse, J.R., Subramaniam, M.,
Spelsberg, T.C., Oursler, M.J., 2011. TGF-␤ inducible early gene 1 regulates osteo-
clast differentiation and survival by mediating the NFATc1, AKT, and MEK/ERK
signaling pathways. PLoS ONE 6, e17522.
Dawson-Hughes, B., Bischoff-Ferrari, H.A., 2007. Therapy of osteoporosis with cal-
cium and vitamin D. J. Bone Miner. Res. 22 (Suppl. 2), V59L V63.
Dominguez, L.J., Di Bella, G., Belvedere, M., Barbagallo, M., 2011. Physiology of the
aging bone and mechanisms of action of bisphosphonates. Biogerontology 12,
397–408.
Eriksen, E.F., 2010. Cellular mechanisms of bone remodeling. Rev. Endocr. Metab.
Disord. 11, 219–227.
Eriksen, E.F., 2012. Hormone replacement therapy or SERMS in the long term treat-
ment of osteoporosis. Minerva Ginecol. 64, 207–221.
Fraser, L.A., Vogt, K.N., Adachi, J.D., Thabane, L., 2011. Fracture risk associated with
continuation versus discontinuation of bisphosphonates after 5 years of therapy
in patients with primary osteoporosis: a systematic review and meta-analysis.
Therapeut. Clin. Risk Manage. 7, 157–166.
Hagiwara, K., Goto, T., Araki, M., Miyazaki, H., Hagiwara, H., 2011. Olive polyphenol
hydroxytyrosol prevents bone loss. Eur. J. Pharmacol. 662, 78–84.
Henriksen, K., Leeming, D.J., Christiansen, C., Karsdal, M.A., 2011. Use of bone
turnover markers in clinical osteoporosis assessment in women: current issues
and future options. Womens Health 7, 689–698.
Idris, I., Donnelly, R., 2009. Sodium-glucose co-transporter-2 inhibitors: an emerging
new class of oral antidiabetic drug. Diabetes Obes. Metab. 11, 79–88.
Josse, R., Khan, A., Ngui, D., Shapiro, M., 2013. Denosumab, a new pharmacothe-
rapy option for postmenopausal osteoporosis. Curr. Med. Res. Opin. 29, 205–
216.
Jung, M., Triebel, S., Anke, T., Richling, E., Erkel, G., 2009. Influence of apple polyphe-
nols on inflammatory gene expression. Mol. Nutr. Food Res. 53, 1263–1280.
Kaczmarczyk-Sedlak, I., Wojnar, W., Zych, M., Ozimina-Kamińska, E., Taranowicz, J.,
Siwek, A., 2013. Effect of formononetin on mechanical properties and chemi-
cal composition of bones in rats with ovariectomy-induced osteoporosis. Evid.
Based Complement. Alternat. Med., 457052.
Kamon, M., Zhao, R., Sakamoto, K., 2009. Green tea polyphenol (−)-epigallocatechin
gallate suppressed the differentiation of murine osteoblastic MC3T3-E1 cells.
Cell Biol. Int. 34, 109–116.
Kanis, J.A., 2002. Diagnosis of osteoporosis and assessment of fracture risk. Lancet
359, 1929–1936.
Khosla, S., 2001. Minireview: the OPG/RANKL/RANK system. Endocrinology 142,
5050–5055.
Kim, J.L., Kang, M.K., Gong, J.H., Park, S.H., Han, S.Y., Kang, Y.H., 2012. Novel antios-
teoclastogenic activity of phloretin antagonizing RANKL-induced osteoclast
differentiation of murine macrophages. Mol. Nutr. Food. Res. 56, 1223–1233.
Proff, P., Römer, P., 2009. The molecular mechanism behind bone remodeling: a
review. Clin. Oral Investig. 13, 355–362.
Rejnmark, L., Mosekilde, L., 2011. New and emerging antiresorptive treatments in
osteoporosis. Curr. Drug Saf. 6, 75–88.
Sunita, P., Pattanayak, S.P., 2011. Phytoestrogens in postmenopausal indications: a
theoretical perspective. Pharmacogn. Rev. 5, 41–47.
Tao, M., Teng, Y., Shao, H., Wu, P., Mills, E.J., 2011. Knowledge, perceptions and infor-
mation about hormone therapy (HT) among menopausal women: a systematic
review and meta-synthesis. PLoS ONE 6, e24661.
Turner, J.V., Agatonovic-Kustrin, S., Glass, B.D., 2007. Molecular aspects of
phytoestrogen selective binding at estrogen receptors. J. Pharm. Sci. 96,
1879–1885.
Xing, L., Schwarz, E.M., Boyce, B.F., 2005. Osteoclast precursors, RANKL/RANK, and
immunology. Immunol. Rev. 208, 19–29.
Wada, T., Nakashima, T., Hiroshi, N., Penninger, J.M., 2006. RANKL-RANK signaling
in osteoclastogenesis and bone disease. Trends Mol. Med. 12, 17–25.
Wang, J., Chung, M.H., Xue, B., Ma, H., Ma, C., Hattori, M., 2010. Estrogenic and
antiestrogenic activities of phloridzin. Biol. Pharm. Bull. 33, 592–597.
Yang, Y.C., Li, C.K., Lin, A.H., Yeh, Y.W., Yao, H.T., Li, C.C., Liu, K.L., Chen, H.W., 2011.
Induction of glutathione synthesis and heme oxygenase 1 by the flavonoids
butein and phloretin is mediated through the ERK/Nrf2 pathway and protects
against oxidative stress. Free Radic. Biol. Med. 51, 2073–2081.
Zhang, D.W., Cheng, Y., Wang, N.L., Zhang, J.C., Yang, M.S., Yao, X.S., 2008. Effects of
total flavonoids and flavonol glycosides from Epimedium koreanum Nakai on
the proliferation and differentiation of primary osteoblasts. Phytomedicine 15,
55–61.