Original Full Length Article
Ginsenoside Rh2 inhibits osteoclastogenesis through down-regulation of NF-κB,
NFATc1 and c-Fos
Long Hea,1, Junwon Leeb,1, Jae Hyuk Janga, Sung-Hoon Leeb, Mei Hua Nana, Byung-Chul Ohc,
Sang Gu Leea, Hong Hee Kimd, Nak Kyun Sounga, Jong Seog Ahna,⁎, Bo Yeon Kima,d,⁎
aKorea Research Institute of Bioscience and Biotechnology, Ochang, Cheongwon, South Korea
bDepartment of Biomedicinal Science and Biotechnology, Pai Chai University, Daejeon 302-735, South Korea
cDepartment of Molecular Medicine, Lee Gil Ya Cancer and Diabetes Institute, Gachon University of Medicine and Science, Incheon 406-840, South Korea
dSchool of Dentistry, Seoul National University, Yeongeon-dong, Jongno, Seoul 110-749, South Korea
a b s t r a c ta r t i c l e i n f o
Received 21 January 2012
Revised 12 March 2012
Accepted 17 March 2012
Available online 28 March 2012
Edited by: J. Aubin
Ginsenoside Rh2 is one of the most active components of red ginseng, controlling cancer and other metabolic
diseases including osteoclast differentiation. However, the molecular mechanism underlying the inhibition of
osteoclast differentiation by ginsenoside Rh2 remains poorly understood. In the present study, it was found
that ginsenoside Rh2 suppressed osteoclast differentiation from bone marrow macrophages (BMMs) treated
with receptor activator of nuclear factor κB ligand (RANKL) without any cytotoxicity. Ginsenoside Rh2 signifi-
cantly reduced RANKL-induced expression of transcription factors, c-Fos and nuclear factor of activated T-cells
(NFATc1), as well as osteoclast markers, TRAP and OSCAR. In defining the signaling pathways, ginsenoside Rh2
was shown to moderately inhibit NF-κB activation and ERK phosphorylation in response to RANKL stimulation
in BMM cells without any effect on p38 and c-Jun N-terminal kinase (JNK). Finally, ginsenoside Rh2 blocked
osteoporosis in vivo as confirmed by restored bone mineral density (BMD) and other markers associated osteo-
entiation in vitro and in vivo through the regulation of c-Fos and NFATc1 expressions, not excluding the
involvement of NF-κB and ERK. Ginsenoside Rh2 is also suggested to be developed as a therapeutic drug for
prevention and treatment of osteoporosis.
© 2012 Elsevier Inc. All rights reserved.
Human bone mass homeostasis is dynamically regulated by
coupled actions of osteoblasts (bone formation) and osteoclasts (bone
resorption), termed bone remodeling. Many osteopenia and patho-
logical diseases, including rheumatoid arthritis, lytic bone metastasis,
are characterized by progressive and excessive bone resorption by
osteoclasts, the specialized multinucleate cells (MNCs) formed from
mononuclear cells of hematopoietic origin .
There are many factors involved in osteoclast differentiation.
Receptor activator of NF-κB ligand (RANKL) is a tumor necrosis factor
(TNF) family member regulating differentiation, survival and activation
of osteoclasts. The receptor of RANKL (RANK) is expressed in osteoclast
precursors, and upon binding to RANKL, it initiates the recruitment
of TNF receptor-associated factor 6 (TRAF6) and induces signaling cas-
cades, c-jun N-terminal protein kinase (JNK) , p38 , extracellular
signal-related kinase (ERK)  andIκB kinase (IKK). NF-κB rapidly stim-
ulated through IKK1/2 pathway following ligand binding plays a pivotal
of NF-κB is associated with osteopetrosis [5,6]. The c-Fos component of
activator protein 1 (AP1) was also reported to block osteoclastogenesis,
consequently leading to the osteopetrosis . In addition, nuclear
factor of activated T cells (NFATc1) is another transcription factor in-
volved in RANKL-induced osteoclast differentiation. Dominant negative
NFATc1 blocked osteoclastogenesis, whereas over-expression of wild
type NFATc1 increased osteoclast formation from precursor cells .
Ginseng (Panax quinquefolius) is a popular herb in the world, with
ginsenoside Rh2 being one of its main components. It was reported
that ginsenoside Rh2 had a variety of biological activities including
anti-inflammation, anti-tumor and anti-diabetes activities in experi-
mental models [8–10]. In addition, ginsenoside Rh2 inhibited osteo-
clast formation without any cytotoxicity . However, the precise
molecular mechanism of anti-osteoporosis of ginsenoside Rh2
remains unknown, and the effect of ginsenoside Rh2 on pathological
bone destruction in vivo has not yet been well defined. In this
study, ginsenoside Rh2 was found to reduce the RANKL-induced
osteoclast differentiation through c-Fos and NFATc1 regulation. Oste-
oporosis was also potentially suppressed by ginsenoside Rh2 in vivo.
Bone 50 (2012) 1207–1213
⁎ Corresponding authors at: Korea Research Institute of Bioscience and Biotechnology
(KRIBB), Ochang, Cheongwon 363-883, South Korea.
E-mail addresses: firstname.lastname@example.org (J.S. Ahn), email@example.com (B.Y. Kim).
1The first two authors are equal contributors to this work.
8756-3282/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
Contents lists available at SciVerse ScienceDirect
journal homepage: www.elsevier.com/locate/bone
Hence, ginsenoside Rh2 could be developed as a good anti-
Materials and methods
Reagents and antibodies
α-MEM, fetal bovine serum and penicillin were purchased from
Invitrogen (Carlsbad, CA). Purified ginsenoside Rh2 was obtained
from Hongjiu Biotechnology (Jilin, China). TRAP staining solution
was from Sigma Aldrich (St. Louis, MO). Soluble human recombinant
M-CSF and mouse RANKL were purchased from PeproTech EC (London,
UnitedKingdom).Specific antibodies againstc-Fos, NFATc1andGAPDH
were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz,
CA). Specific antibodies against phospho-ERK, ERK, phospho-JNK,
JNK, phospho-p38, p38, phospho-IκB and IκB were from Cell Signaling
Technology (Danvers, MA).
BMM isolation and osteoclast differentiation
Mouse bone marrow cells were obtained from femurs and tibiae
of a 6-week-old ICR mouse and were incubated in α-MEM complete
media supplemented with 10% fetal bovine serum, 100 U/ml penicil-
lin in a 100 mm dish in the presence of M-CSF (30 ng/ml) for 3 days.
Adherent cells were used as bone marrow macrophages (BMMs) as
osteoclast precursors after non-adherent cells were removed. To gen-
erate osteoclasts, BMMs (4×104cells/well) were cultured in com-
plete medium containing M-CSF (30 ng/ml) and RANKL (25 ng/ml)
in a 48-well (1 ml/well) plate with or without ginsenoside Rh2. After
4 days, cells were fixed in 10% formalin for 10 min, permeabilized
with 0.1% Triton X-100, and then stained with tartrate-resistant acid
phosphatase (TRAP) using the Leukocyte Acid Phosphatase Assay Kit
Cell viability assay
Measurement of cytotoxicity was performed using a Cell Counting
Kit-8 (Dojindo Molecular Technology, Japan) according to the manu-
facturer's instructions. BMMs (1×103cells/well) were cultured with
or without ginsenoside Rh2 at various concentrations (0–50 μM) for
48 h in the presence or absence of M-CSF (30 ng/ml) and RANKL
(25 ng/ml) in 96-well plates (200 μl/well). After a one hour incuba-
tion of the cells in a medium containing 10 μl of CCK-8 solution,
0 μ μM 3 μ μM
12 μ μM 6 μ μM
TRAP (+) OC number
cell viability (%)
Fig. 1. Ginsenoside Rh2 inhibits osteoclast differentiation from bone marrow macrophage (BMM). A, Mouse bone marrow macrophage (BMM) cells were cultured with M-CSF
(30 ng/ml) and RANKL (25 ng/ml) at the indicated concentration of ginsenoside Rh2 for 1–4 days. After induction, cells were fixed and subjected to TRAP staining (left). TRAP-
positive, multinucleated osteoclast cell number was counted (right). B, BMM cells were stimulated with M-CSF alone or M-CSF and RANKL in the presence or absence of ginsenoside
Rh2 at the indicated concentrations for 3 days, followed by TRAP solution assay as described in Materials and methods. C, BMM cells were treated with M-CSF (30 ng/ml), RANKL
(25 ng/ml) and ginsenoside Rh2 at the indicated concentrations for 2 days. Cell viability was measured by CCK-8 solution kit as described in Materials and methods. All the bars
represent mean±SE from three independent experiments, and the significance was determined by student's t-test (*, Pb0.5; **, Pb0.1).
L. He et al. / Bone 50 (2012) 1207–1213
O.D. was read as 450 nm (650 nm reference) using 96-well plate
Western blot analysis
BMMs or osteoclasts were lysed in a buffer containing 50 mM
Tris–HCl, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM sodium
fluoride, 1 mM sodium vanadate, 1% deoxycholate and protease
inhibitor cocktail. The lysate was centrifuged at 15,000×g for
30 min and the protein concentration in the supernatant was mea-
sured. Equal amounts of proteins (30 μg) were subjected to 8–10%
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE) and immunoblotted with specific antibodies.
Quantitative PCR analysis
Total RNA was prepared using RNeasy Mini kit (QIAGEN, Valencia,
CA) according to the manufacturer's instructions, and cDNA was syn-
thesized from 3 μg of total RNA usingreverse transcriptase (Superscript
II Preamplification System; Invitrogen). Real-time PCR was performed
by CFX96™ real-time system using SYBR FAST KAPA iCycler qPCR
kit. The detector was programmed with the following PCR conditions:
40 cycles for 15 s denaturation at 95 °C and 1 min amplification at
0 d1 d2d3 d4
Relative mRNA expression
0 d1d2d3 d4
Relative mRNA expression
Relative mRNA expression
0 d1d2d3 d4
Relative mRNA expression
RANKL (25ng/ml) -123123
Rh2-- 3 6 12 (μM)
Fig. 2. Ginsenoside Rh2 inhibits RANKL-induced expression of c-Fos and NFATc1 in BMM. A, BMM cells were pretreated with ginsenoside Rh2 (12 μM) or vehicle (DMSO) in
the presence of M-CSF for 30 min and then were stimulated with RANKL (25 ng/ml) for various days. c-Fos and NFATc1 mRNA expressions were analyzed by real-time PCR with
β-actin as a reference gene. All the bars represent mean±SE from three independent experiments, and the significance was determined by student's t-test (*, Pb0.5; **, Pb0.1).
B, Cells were pretreated with ginsenoside Rh2 or vehicle (DMSO) in the presence of M-CSF for 30 min and then were stimulated with RANKL for 24 h. Cell lysates were prepared
for subjection to western blotting with specific antibodies. C, Cells were treated with M-CSF, RANKL and ginsenoside Rh2 as in (A) and were lysed at the indicated times for
immune-blot analysis with specific antibodies.
L. He et al. / Bone 50 (2012) 1207–1213
60 °C. All reactions were run in triplicates and were normalized to the
housekeeping gene β-actin. Relative differences in PCR results were
evaluated by the comparative cycle threshold method. The following
primer sets were used: mouse TNF-α: forward, 5′-GACGTGGAA-
GTGGCAGAAGAG-3′; reverse, 5′-TGCCACAAGCAGGAATGAGA-3′; mouse
ICAM-1: forward, 5′-GCCTAAGGAAGACATGATA-3′; reverse, 5′-CAAGAA-
GAGTTGGGGACAAT-3′; mouse c-Fos: forward, 5′-ACTTCTTGTTTCCGGC-
3′; reverse, 5′-AGCTTCAGGGTAGGTG-3′; mouse NFATc1: forward,
GAA-3′; and mouse β-actin: forward, 5′-TCTGCTGGAA GGTGGACAGT-3′;
reverse, 5′-CCTCTATGCC AACACAGTGC-3′. OSCAR: forward, 5′-CTG CTG
GTA ACG GAT CAG CTC CCC AGA-3′; reverse, 5′-CCA AGG AGC CAG AAC
CTT CGA AAC T-3; TRAP, forward, 5′-CTG GAG TGC ACG ATG CCA GCG
ACA-3′; reverse, 5′-TCC GTGCTCGGC GAT GGACCAGA-3′.
Nuclear fractionation and electrophoretic mobility shift assay (EMSA)
Cells grown in 100 mm dishes were lysed on ice for 15 min in a
hypotonic solution containing 10 mM HEPES-KOH (pH 7.8), 10 mM
KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.2 mM NaF, 0.2 mM Na3VO4,
0.1 mM phenylmethylsufonyl fluoride (PMSF), leupeptin (10 μg/ml),
1 mM dithiothreitol (DTT), and 0.15% NP-40. The lysate was centri-
fuged at 15,000 rpm for 2 min at 4 °C, and the resulting nuclear pellet
was resuspended in ice-cold high-salt buffer (50 mM HEPES-KOH pH
7.8, 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 0.2 mM NaF, 0.2 mM
Na3VO4, 0.1 mM PMSF, 1 mM DTT and 10% glycerol) and incubated
for 30 min at 4 °C with occasional vortex. The nuclear lysate was
then centrifuged at 15,000 rpm for 30 min at 4 °C, and the superna-
tant was immediately subjected to EMSA analysis. Oligonucleotide
containing NF-κB binding site (3.5 pmol) was incubated for 10 min
at 37 °C in 10 μl reaction mixture containing 10 μCi [γ-32P]ATP, 5 U
T4 polynucleotide kinase, and 1× kinase buffer (supplied with the
kinase). For EMSA assay, nuclear protein extract (10 μg) was incu-
bated for 30 min at room temperature in a final volume of 10 μl con-
taining 0.03 pmol of
reaction was terminated by the addition of electrophoresis sample
buffer, and the samples were fractionated on 5% non-denaturing
polyacrylamide gels in 0.5× Tris–boric acid–EDTA (TBE) buffer. The
gels were then subjected to autoradiography.
32P-end-labeled oligonucleotide. The binding
In vivo experiments
Eight week old C57BL/6 mice were obtained from The Jackson
Laboratory. Human recombinant RANKL was purchased from R&D
system. Osteoporosis model mice induced by RANKL were described
previously . More than 6 mice were examined for each group
of experiments. Ginsenoside Rh2 (3 mg/kg) or DMSO was injected
intraperitoneally 24 h before the first RANKL injection, and the mice
(n=6) were subsequently injected with ginsenoside Rh2 (3 mg/kg,
IP), DMSO, RANKL (0.5 mg/kg, IP) or phosphate buffered saline
(PBS) at 24 h intervals for 3 days.
Values are presented as the mean±S.D. values from three or more
experiments. Data were analyzed by Student's t-test for comparisons
between two mean values. A value of Pb0.05 was considered significant.
Ginsenoside Rh2 suppresses RANKL-induced osteoclast differentiation in
primary bone marrow cells
In order to determine the effect of ginsenoside Rh2 on osteoclast
differentiation, BMM cells were challenged with ginsenoside Rh2
followed by RANKL treatment. RANKL induced osteoclast differentia-
tion in the presence of M-CSF as revealed by the appearance of TRAP-
positive, multinucleated cells. Pretreatment of ginsenoside Rh2,
however, reduced osteoclast differentiation in a dose-dependent
manner (Figs. 1A and B) without any cytotoxicity to the cells even
at 50 μM (Fig. 1C).
Ginsenoside Rh2 negatively regulates RANKL-induced expression of c-Fos
To understand the inhibitory mechanism of ginsenoside Rh2
against osteoclastogenesis, expression of transcription factors c-Fos
and NFATc1 was analyzed. Quantitative PCR analysis indicated that
treatment of ginsenoside Rh2 prior to RANKL stimulation significantly
suppressed the transcriptional level of both c-Fos and NFATc1
(Fig. 2A). RANKL-induced expression of osteoclast-associated recep-
tor (OSCAR) and tartrate-resistant acid phosphatase (TRAP), two
other marker proteins in osteoclastogenesis, was also significantly
reduced in the presence of ginsenoside Rh2 (Fig. 2A). Accordingly,
western blot analysis also showed that RANKL-induced expression
of c-Fos and NFATc1 was significantly diminished by ginsenoside
Rh2 (Figs. 2B and C).
Ginsenoside Rh2 inhibits the RANKL-induced phosphorylation of ERK in
Enzymes associated with the RANKL-induced early signaling
pathways, including JNK, p38 and ERK, were examined to determine
whether they are involved in the inhibition of osteoclastogenesis
by ginsenoside Rh2. It was reported that phosphorylation of these
MAPK kinases could be observed 5 min after RANKL treatment to
BMM cells . It was interesting to note that RANKL-induced
ERK activation was only suppressed while p38, JNK and its down-
stream target c-Jun were not affected by ginsenoside Rh2 (Fig. 3).
Given that the activity of transcription factor c-Fos was regulated
by ERK , our results suggest that suppression of ERK phosphor-
ylation could contribute to the inhibition of RANKL-induced
RANKL(min)05 15 3005 1530
Fig. 3. Ginsenoside Rh2 down-regulates RANKL-induced ERK phosphorylation in BMM
cells. Cells were pretreated with ginsenoside Rh2 (12 μM) or vehicle (DMSO) for 6 h in
the presence of M-CSF (30 ng/ml) and were then stimulated with RANKL (100 ng/ml)
for the indicated times. The whole cell lysates were extracted and subjected to western
blotting analysis with antibodies to p-ERK, ERK, p-p38, p38, p-JNK, JNK and p-c-jun.
GAPDH served as a reference protein.
L. He et al. / Bone 50 (2012) 1207–1213
Ginsenoside Rh2 suppresses RANKL-induced NF-κB activation in BMM
NF-κB is essential for osteoclastogenesis and disruption of NF-κB
leads to an impaired osteoclast differentiation with an osteopetrotic
phenotype . In determining the association of NF-κB pathway
with the inhibition of osteoclast differentiation by ginsenoside Rh2,
it was found that IκBα phosphorylation by RANKL was reduced by
ginsenoside Rh2, with a slight restoration of IκBα level (Fig. 4A). It
was also of note that IκBα phosphorylation was reduced at 15 min
but increased again at 30 min after RANKL stimulation. In accordance
with IκBα phosphorylation and degradation, EMSA analysis further
showed that DNA binding activity of NF-κB binding in response
to RANKL was reduced by ginsenoside Rh2 (Fig. 4B). To see whether
ginsenoside Rh2 inhibits NF-κB-dependent transcription in BMM
cells, mRNA levels of two NF-κB target genes, tumor necrosis factor-
α (TNF-α) and inter-cellular adhesion molecule-1 (ICAM-1) which
contain NF-κB binding sites on their promoters, were measured by
real-time quantitative PCR after RANKL treatment. It was revealed
that pretreatment of ginsenoside Rh2 significantly reduced RANKL-
induced expression of these genes (Fig. 4C).
Ginsenoside Rh2 prevents bone destruction induced by RANKL in vivo
To address the effect of ginsenoside Rh2 in vivo, bone loss mouse
model was prepared by RANKL injection as previously described .
RANKL was intraperitoneally injected into 8-week-old female mice
every 24 h for 3 days. The mice were sacrificed 2 h after the third
injection for measurement of total femoral bone mineral density
(BMD) using a pDEXA® bone densitometer. BMD was markedly
decreased by RANKL injection in a dose-dependent manner. To inves-
tigate the effect of ginsenoside Rh2 on RANKL-induced osteoporotic
bone loss, ginsenoside Rh2 was intraperitoneally injected into the
mice 24 h before the first RANKL injection, and the mice (n=6) sub-
sequently received simultaneous injections of both ginsenoside Rh2
and RANKL every 24 h for 3 days. It was revealed that ginsenoside
Relative mRNA expression
05 15 30 05 15 30
0 15 30 15 30
Relative mRNA expression
Fig. 4. Effect of ginsenoside Rh2 on RANKL-induced NF-κB activation in BMM cells. A, Cells were pretreated with ginsenoside Rh2 (12 μM) or vehicle (DMSO) for 6 h in the presence
of M-CSF (30 ng/ml) and were then stimulated with RANKL (100 ng/ml) for the indicated times. Cytosolic fractions were prepared and subjected to western blotting with specific
antibodies. B, Cells were treated with ginsenoside Rh2 and RANKL as in (A) for the indicated times, and nuclear fractions were prepared for EMSA analysis as described in Materials
and methods. C, Cells were pretreated with ginsenoside Rh2 and RANKL as above for the indicated times, and TNF-α and ICAM-1 mRNA expressions were analyzed by real-time PCR
using β-actin mRNA as a reference gene. All the bars represent mean±SE from three independent experiments, and the significance was determined by student's t-test (*, Pb0.5;
L. He et al. / Bone 50 (2012) 1207–1213
Rh2 significantly restored BMD in RANKL-induced osteoporotic mice
(Fig. 5A). In addition, 2D micro-CT image also showed that RANKL-
induced bone destruction was significantly reduced by ginsenoside
Rh2 (Figs. 5B and C).
Bone resorption by osteoclasts is frequently caused by excessive
RANKL signaling which has been a valuable target for the treatment of
pathological bone loss. Various transcription factors are related with
osteoclast development, including PU.1, microphthalmia-associated
transcription factor, c-Fos, NF-κB and NFATc1. Each factor acts at differ-
of red ginseng belonging to the protopanaxadiol saponins, and exhibits
various beneficial impacts on cancer prevention [17,18] and some
metabolic diseases [10,19,20]. In the present study, ginsenoside Rh2
inhibited osteoclast differentiation from bone marrow cells induced by
RANKL. It was also found that RANKL-induced expression of c-Fos and
NFATc1 was significantly down-regulated by ginsenoside Rh2 (Figs. 2B
and C). These results indicate that c-Fos and NFATc1 are crucial for the
control of osteoclast differentiation, however, ERK and NF-κB could not
be excluded although they are moderately affected by ginsenoside
Rh2. Combined with the in vivo data, ginsenoside Rh2 could be a good
candidate for the treatment of osteoporosis.
The RANKL receptor, RANK, lacks intrinsic enzymatic activity in
its intracellular domain, but it transduces signaling by recruiting
adapters such as TRAF6 and further leads to the activation of
several signaling cascades [21,22]. Mitogen-activated protein kinases
(MAPK), JNK, ERK and p38, have been reported to be activated by
RANKL stimulation and associated with osteoclastogenesis . p38 is
important at the early stage of osteoclast generation, regulating the
microphthalmia-associated transcription factor , and dominant-
negative JNK prevented RANKL-induced osteoclastogenesis .
In comparison, ERK induced c-Fos for osteoclastogenesis , and
inhibition of ERK was shown to decrease osteoclast formation
[25,26]. In our study, ginsenoside Rh2 specifically down-regulated
ERK activation without affecting JNK, c-jun and p38 (Fig. 3). Given
that c-Fos was down-regulated by ginsenoside Rh2 (Figs. 2B and C),
it is suggested that the ERK pathway could be one of the efficient
targets for the control of osteoclastogenesis.
p50/p52 NF-κB double-knockout mouse exhibited severe osteope-
trosis, demonstrating that NF-κB signaling could play a crucial role in
osteoclast development [5,27]. It was also reported that NF-κB up-
regulated c-Fos expression during RANKL-induced osteoclastogenesis
. Moreover, inhibition of NF-κB suppressed NFATc1 expression
by RANKL  while NF-κB in combination with NFATc2 induced
NFATc1 expression . Based on the observation in our study
demonstrating that ginsenoside Rh2 inhibited RANKL-induced NF-
κB activation and osteoclast formation, it could be expected that the
down-regulation of NF-κB could also be a promising target for the
reduction of RANKL-induced c-Fos and NFATc1 expression during
An accurate balance between bone resorption (by osteoclasts) and
bone formation (by osteoblasts) maintains bone homeostasis.
Our data, combined with the previous observation of Kim et al. dem-
onstrating that ginsenoside Rh2 induced the mineralization and
differentiation of osteoblastic MC3T3-E1 cells through PKD/AMPK
pathways , suggest that ginsenoside Rh2 might have dual regula-
tory effects on both osteoclast and osteoblast although a more
detailed study is required for clear understanding of its effect on
Although the effect of ginsenoside Rh2 on bone-related disease
was exploited in this study, other components were also examined
to determine their anti-osteoporosis activity. Heat treatment of
ginseng led to an appearance of black ginseng containing various
saponin families different from those present in the normal ginseng
(data not shown). Among these components, some also showed
anti-osteoclast differentiation activity comparable to ginsenoside
Rh2. When normal and black ginseng extracts were compared for
their anti-osteoporosis activities, the latter showed increased activity
Tb. Th. (um)
Tb. Sp. (um)
Tb. N (1/mm)
Fig. 5. GinsenosideRh2inhibitsosteoporosisinbonedestructionmodelmice.A,Ginsenoside
Rh2 (3 mg/kg) was injected intraperitoneally 24 h before the first RANKL injection, and the
mice (n=6) subsequently received simultaneous injections of ginsenoside Rh2 (3 mg/kg,
IP) and RANKL (0.5 mg/kg, IP) every 24 h for 3 days. Total femoral bone mineral density
(BMD) was measured using pDEXA X-ray bone densitometer. BMD was calculated using
the bone mineral content (BMC) of the measured area. B, Tibiae from negative control
(NC), RANKL+DMSO and RANKL+ginsenoside Rh2 mice were examined under micro-
CT. Two-dimensional reconstruction of tibiae revealed increased bone mass in
ginsenoside Rh2 mice compared with control littermates. C, Histograms represent the
2D trabecular structural parameters in tibiae; bone volume per tissue volume (BV/TV),
trabecular separation (Tb.Sp.), trabecular thickness (Tb.Th.) and trabecular number
(Tb.N). All the bars are mean±SE of a representative experiment. The significance was
determined by ANOVA-test (*, Pb0.5; **, Pb0.1). (n=6).
L. He et al. / Bone 50 (2012) 1207–1213
to some extent. These studies indicate that change in saponin compo- Download full-text
sition in ginseng could affect the physiological function of ginsengs,
including its use as treatment for osteoporosis.
Even with lots of studies indicating that ginseng is effective for the
treatment of psychologic function, exercise performance, immune
function, diabetes as well as cancer [32–34], the underlying mecha-
nisms have not yet been clearly understood. A previous study
reported that ginsenoside Rh2 showed cytotoxic effect on cancer
cells through altering membrane permeability or inducing caspase-3
activity . Ginsenoside Rh2 has two isoforms, 20(S)-Rh2 and
20(R)-Rh2. 20(S)-Rh2 has a stronger anticancer activity compared
to 20(R)-Rh2 , due to the differential stereo-selective interactions
of (R) and (S) forms with the lipid membranes that might be related
with the cytotoxicity. In contrast, 20(R)-Rh2 was reported to be more
potent than 20(S)-Rh2 in the treatment of osteoclast formation .
Although anti-obesity effects of ginsenoside Rh2 is associated with
insulin secretion to lower plasma glucose level or activate AMPK
signaling pathway in 3T3-L1 adipocyte [19,20], further comprehen-
sive studies with the two isoforms of ginsenoside Rh2 will be
required for the development of an appropriate chemotherapeutic
or chemo-preventive drug.
osteoclastogenesis of bone marrow primary cells as well as osteo-
clastic bone destruction in vivo. The therapeutic effect of ginsenoside
Rh2 is associated with down-regulation of NF-κB and ERK, c-Fos and
NFATc1, leading to the lowered expression of TRAP and OSCAR.
Hence, it is strongly suggested that ginsenoside Rh2 could be devel-
oped as a potent anti-osteoporosis therapeutics.
This work was supported by the World Class Institute (WCI)
Program (WCI 2009-002), Global R&D Center (GRDC) Program of
of Education, Science and Technology (MEST), Technology Development
Program for Agriculture and Forestry, Ministry for Agriculture, Forestry
and Fisheries, and also supported by KRIBB Research Initiative
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