Life Sciences 147 (2016) 46–58

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Life Sciences

journal homepage: www.elsevier.com/locate/lifescie

Natural products for treatment of osteoporosis: The effects and

mechanisms on promoting osteoblast-mediated bone formation
Jing An ⁎, Hao Yang, Qian Zhang, Cuicui Liu, Jingjing Zhao, Lingling Zhang, Bo Chen
Translational Medicine Centre, Hong-Hui Hospital, Xi'an Jiaotong University College of Medicine, Xi'an 710054, China

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

Article history: Osteoporosis is a systemic metabolic bone disease characterized by a reduction in bone mass, bone quality, and
Received 31 August 2015 microarchitectural deterioration. An imbalance in bone remodeling that is caused by more osteoclast-mediated
Received in revised form 4 January 2016 bone resorption than osteoblast-mediated bone formation results in such pathologic bone disorder. Traditional
Accepted 13 January 2016
Chinese medicines (TCM) have long been used to prevent and treat osteoporosis and have received extensive at-
Available online 18 January 2016
tentions and researches at home and abroad, because they have fewer adverse reactions and are more suitable for
Chemical compounds studied in this article:
long-term use compared with chemically synthesized medicines. Here, we put the emphasis on osteoblasts, sum-
Icariin (PubChem CID): 5,318,997 marized the detailed research progress on the active compounds derived from TCM with potential anti-
Naringin (PubChem CID): 442,428 osteoporosis effects and their molecular mechanisms on promoting osteoblast-mediated bone formation. It
Genistein (PubChem CID): 5,280,961 could be concluded that TCM with kidney-tonifying, spleen-tonifying, and stasis-removing effects all have the
Puerarin (PubChem CID): 5,281,807 potential effects on treating osteoporosis. The active ingredients derived from TCM that possess effects on pro-
Psoralen (PubChem CID): 6199 moting osteoblasts proliferation and differentiation include flavonoids, glycosides, coumarins, terpenoids
Osthole (PubChem CID): 10,228 (sesquiterpenoids, monoterpenoids, diterpenoids), phenolic acids, phenols and others (tetrameric stilbene, an-
Costunolide (PubChem CID): 5,281,437
thraquinones, diarylheptanoids). And it was confirmed that the bone formation effect induced by the above nat-
Kirenol (PubChem CID): 15,736,732
ural products was regulated by the expressions of bone specific matrix proteins (ALP, BSP, OCN, OPN, COL I),
Vanillic acid (PubChem CID): 8468
Honokiol (PubChem CID): 72,303 transcription factor (Runx2, Cbfa1, Osx), signal pathways (MAPK, BMP), local factors (ROS, NO), OPG/RANKL sys-
tem of osteoblasts and estrogen-like biological activities. All the studies provided theoretical basis for clinical ap-
Keywords: plication, as well as new drug research and development on treating osteoporosis.
Osteoporosis © 2016 Elsevier Inc. All rights reserved.
Osteoblasts
Traditional Chinese medicines
Natural products
Molecular mechanism

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
2. Osteoblasts-mediated bone formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
3. Bone specific matrix proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
4. Modulatory effects of natural products on transcription factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
4.1. Transcription factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
4.2. Natural products from TCM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
5. Modulatory effects of natural products on OPG/RANKL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
5.1. OPG/RANKL system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
5.2. Natural products from TCM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Abbreviations: ALP, alkaline phosphatase; OCN, osteocalcin (bone gamma-carboxyglutamic acid protein, BGLAP); OPN, osteopontin (“bone-bridging” protein, secreted phosphoprotein
1, SPP1); BSP, bone sialoprotein; COL I, collagen type I; COL1A1, collagen type I, alpha 1; Runx2, runt-related transcription factor 2; Cbfa1, core binding factor α1; Osx, osterix; OPG, oste-
oprotegerin; RANK, receptor activator of NF-κB; RANKL, receptor activator of NF-κB ligand; MSCs, mesenchymal stem cells; BMSCs, bone marrow stromal cells; BMMSCs, bone marrow
mesenchymal stem cells; MAPKs, mitogen-activated protein kinases; ERK, extracellular signal-regulated kinase; JNK, c-Jun N terminal kinase; BMPs, bone morphogenetic proteins;
PKC, protein kinase C; BMD, bone mineral density; ERs, estrogen receptors; EREs, estrogen response elements; ROS, reactive oxygen species; C/EBP-β, CCAAT-enhancer-binding
protein-β; MDA, malondialdehyde; NO, nitric oxide; NOS, nitric oxide synthase; sGC, soluble guanylyl cyclase; cGMP, cyclic guanosine monophosphate; PKG, protein kinase-G; PI3K, phos-
phatidylinositol 3-kinase.
⁎ Corresponding author.
E-mail address: anjing198812@126.com (J. An).

http://dx.doi.org/10.1016/j.lfs.2016.01.024
0024-3205/© 2016 Elsevier Inc. All rights reserved.
 J. An et al. / Life Sciences 147 (2016) 46–58 47

6. Modulatory effects of natural products on MAPK pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

6.1. MAPK signaling pathways. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
6.2. Natural products from TCM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
7. Modulatory effects of natural products on BMP pathways. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
7.1. BMP signaling pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
7.2. Natural products from TCM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
8. Modulatory effects of natural products on ERs-mediated pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
8.1. ERs-mediated pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
8.2. Natural products from TCM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
9. Modulatory effects of natural products on oxidative stress-mediated pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
9.1. Oxidative stress-mediated pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
9.2. Natural products from TCM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
10. Modulatory effects of natural products on NO-mediated pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
10.1. NO-mediated pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
10.2. Natural products from TCM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
11. Conclusions and future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

1. Introduction
Bone, a highly mineralized connective tissue, is continuously
broken-down and re-formed in a process of turnover known as bone re-
modeling, which occurs through the interaction and balance between
bone forming cells, called osteoblasts, and bone resorbing cells, called
osteoclasts [1]. Bone remodeling is regulated by several systemic
hormones (e.g., parathyroid hormone, 1,25-dihydroxyvitamin D3, sex
hormones and calcitonin) and local factors (e.g., nitric oxide, prosta-
glandins, growth factors and cytokines) [2,3] This remodeling is respon-
sible for normal skeletal formation, skeletal functions and mineral
homeostasis. Deregulation in one of these processes can lead to bone
diseases such as osteoporosis.
Osteoporosis (OP) is a systemic metabolic bone disease that is char-
acterized by microarchitectural deterioration, low bone mass, and in-
creased risk of fractures, which is a prevalent disease that results from
an increase in bone breakdown relative to bone formation. The clinical
complications include fractures, disability and chronic pain. The causes
of osteoporosis are very complicated, and the disease itself is closely re-
lated to aging, endocrine disorders, calcium malabsorption, and limb
disuse, as well as immune, nutritional, and genetic factors [4]. Osteopo-
rosis falls into two categories: Postmenopausal osteoporosis (Type 1 os-
teoporosis) is the most common disease in women after menopause,
which is linked to an estrogen deficiency. Senile osteoporosis (Type 2
osteoporosis) is related to aging (N75 years) and results from a deficien-
cy in dietary calcium, Vitamin D decline, or increased activity of the
parathyroid glands [5].
Osteoporosis is a major global public health problem that is consid-
ered by the World Health Organization (WHO) to be an important
health issue secondary to coronary heart disease. It can occur at any
age and in any racial or ethnic group, affecting millions of people all
over the world, especially the aging population [6]. In America, it threats
an estimated 44 million people, or 55% of the people of 50 years of age
and older [7]. In Europe, osteoporosis related fractures account for
more disability-adjusted life years (DALYs) lost than common cancers
except for lung cancer [8]. In China, it is estimated that over 90 million
people are suffering from osteoporosis [9]. In addition, about 18% of
women and 6% of men worldwide will experience hip fragility fractures
resulting from osteoporosis [10]. Most cases of osteoporosis occur in
postmenopausal women due to the dramatic estrogen levels decline as-
sociated with menopause. About 40% of postmenopausal women are af-
fected by osteoporosis, and with an aging population, this number is
expected to steadily increase in the near future [11]. The economic bur-
den of osteoporosis is markedly increased with the expansion of aging
world population [12].
The ideal strategy for treating osteoporosis is to inhibit bone resorp-
tion by osteoclasts and/or increase bone formation by osteoblasts.
However, most of the current therapies for treating osteoporosis
focus on inhibiting bone resorption, and there are only few agents
available that promote bone formation. Major treatments currently
used for osteoporosis include hormone-replacement therapy (HRT)
[13], bisphosphonates [14], calcitonin, selective estrogen receptor
modulators (SERMs) like raloxifene and droloxifen [15], recombinant
human parathyroid hormone (rhPTH), Vitamin D analogs, and
ipriflavone [16,17]. The effect of these drugs in increasing bone mass
or recovering bone loss is relatively minor, probably no more than 2%
per year [18]. However, conventional drug therapies have both pros
and cons. For example, while estrogen replacement has produced posi-
tive results with respect to improved bone mineral density (BMD) and
reduced fracture incidence in early menopause, its prolonged use is re-
stricted because of potential complications such as breast cancer, uter-
ine bleeding, and cardiovascular events. One concern related to the
usage of bisphosphates is the complications of osteonecrosis of the
jaw (ONJ). The incidence of ONJ disease seems relatively low in patients
receiving oral bisphosphates for osteoporosis or Paget's disease and
considerably higher in patients with malignancy receiving high doses
of intravenous bisphosphates [19]. Despite an excellent safety profile
for parathyroid hormone (PTH), concerns do arise from its persistence
after discontinuation without sequential use of antiresorptive drugs
[20]. In addition, PTH is contraindicated for patients at risk for osteosar-
coma. The belief that combined use of both types of drugs may have a
synergistic effect on BMD is not fully supported by some observational
studies [21,22]. To date, most of the effective osteoporosis therapies re-
duce bone loss but do not restore lost bone mass and strength. It is de-
sirable, therefore, to have satisfactory bone building (anabolic) agents
that stimulate new bone formation and correct the imbalance of trabec-
ular microarchitecture characteristic of established osteoporosis, which
would create a new alternative for treating osteoporosis [23].
In recent years, there is a growing interest in the treatment of oste-
oporosis with plant-based therapies including traditional Chinese med-
icines (TCM), for which extensive experience has been accumulated
over thousands of years [24]. TCM have been widely used in clinical
practice to prevent and treat bone diseases in many countries of the
world, because they have fewer adverse reactions and are more suitable
for long-term use compared with chemically synthesized medicines.
TCM contain numerous chemical constituents, which usually exert
their therapeutic effects through multi-pathways and multi-targets,
these properties are in correspondence with the multi-factorial patho-
genesis of osteoporosis [25]. Natural products that derived from
TCM have been shown to be excellent and reliable sources for the
48 J. An et al. / Life Sciences 147 (2016) 46–58
development of new drugs, that through a combination of effective sin-
gle compounds or in combination with existing commercial drugs, such
as estrogen or growth hormone products used to prevent bone loss [26].
For centuries, TCM have been used all over the world for the treatment
of illness and disease, and it will undoubtedly continue to be used as a
cost-effective alternative to commercial pharmaceutical products.
There are a number of TCM that possess functions to ease the joints,
strengthen bones and muscles, and have been used for treatment of
bone injuries and bone related diseases, such as osteoporosis. As the
most common metabolic bone disease, the cause of osteoporosis is an
imbalance between the formation and resorption of the bone. At the cel-
lular level, communication and coupling between the main bone-cell
types, the bone-forming osteoblasts and the bone-degrading osteo-
clasts, constitute the smallest functional unit on bone metabolism. In
this paper, we focused on the osteoblasts, summarized the effect and
molecular mechanism on bone formation induced by natural products
derived from TCM. Many studies have shown that natural products
can improve osteoblasts proliferation and differentiation by regulating
the expression of bone specific matrix proteins, transcription factor, cy-
tokines, signal pathways, OPG/RANKL system and hormone-like biolog-
ical activities. This paper will provide the readers with the detailed
research progress on the active compounds from TCM with potential
of anti-osteoporosis effect and the pharmacological mechanism on pro-
moting osteoblasts-mediated bone formation from the above aspects.
2. Osteoblasts-mediated bone formation
Osteoblast cells play an important role in the bone formation, which
is composed of a three-step process: (i) proliferation—the increase of
pre-osteoblast number, (ii) differentiation—the process by which a
pre-osteoblast becomes a mature osteoblast, and (iii) matrix
mineralization—the formation of new bone matrix by the mature oste-
oblasts [27].
3. Bone specific matrix proteins
The osteoblast phenotype is specifically acquired in two stages. In
the first stage, cells proliferate and the matrix matures. Specific proteins
associated with the bone cell phenotype, such as ALP, can be detected
during cell proliferation and matrix maturation from the period
of 10–15 days. In the second stage of osteoblast phenotype
acquisition, the matrix becomes mineralized and late bone markers,
such as osteocalcin (OCN), are produced after 10–15 days and up to
25–30 days. Finally, multiple anabolic signaling pathways are positively
involved in controlling bone formation, such as the BMP, Wnt, and
Runx2 pathways [28].
Alkaline phosphatase (ALP) activity is the most widely recognized
biochemical marker for osteoblastic activity. It is the typical protein
product of an osteoblast phenotype and of osteoblast differentiation.
Composed of homodimeric metalloenzymes which contain zinc at the
active site [29], ALP hydrolyzes a variety of phosphate compounds,
such as ATP, releasing the products, specifically inorganic phosphate
(Pi), into the extracellular matrix [30]. As a result, ALP is associated
with Pi homeostasis. Also containing other Ca-dependent proteins, the
matrix vesicle uses the Pi to initiate crystal nucleation of extracellular
matrix calcium deposits, leading to the formation of hydroxyapatite
[31]. In general, the appearance of ALP activity is represented as an
early phenotypic marker for osteoblastogenesis [32]. Wennberg et al.
have demonstrated that osteoblasts obtained from ALP-deficient mice
differentiate normally but can't form mineralized nodules in vitro [33],
supporting the crucial role of ALP in this particular process.
A complicated network of various proteins is involved in the regula-
tion of osteogenesis. Bone sialoprotein (BSP) appears following ALP ex-
pression and localizes to the matrix of mineralization. BSP can promote
the nucleation of hydroxyapatite mineralization in vitro and increases
calcium incorporation and nodule formation [34]. Osteocalcin (OCN,
bone gamma-carboxyglutamic acid protein, BGLAP) appears on the ter-
minal of osteoblastic differentiation. Ducy et al. (1996) indicated that
OCN can bind to Ca2 + to regulate calcium ion homeostasis and
bone mineralization. Contradictorily, bone formation is even increased
in OCN-deficient mice [35]. These reports may imply that OCN is a
late osteoblastic marker but doesn't symbolize a degree of bone miner-
alization. The expression of BSP and OCN is representative of middle and
late stages of osteoblast differentiation phenotypic markers, respective-
ly. Osteopontin (OPN, “bone-bridging” protein, secreted phosphopro-
tein 1, SPP1), is one of the more abundant non-collagenous proteins in
bone matrix that is produced by osteoblasts and osteoclasts. Evidence
has suggested that OPN is a potent stimulator of the osteoclastogenesis
and resorptive activity of mature osteoclasts [36]. Although OPN does
not appear to be required for normal development of bones [37], the ab-
sence of OPN makes the animal less sensitive to ovariectomy- and
unloading-induced bone loss [38,39]. In addition, collagen type I (COL
I), an extracellular matrix protein, stimulates osteoblast adhesion and
differentiation [40]. The expressions of all these differentiation-related
genes can be regulated by several local and systemic factors, including
estrogen, growth factors and cytokines [41].
4. Modulatory effects of natural products on transcription factors
4.1. Transcription factors
Osteogenic differentiation of mesenchymal pluripotent cells is regu-
lated by various transcriptional factors, such as Runt-related transcrip-
tion factor 2/core binding factor α1 (Runx2/Cbfa1) and osterix (Osx).
Runx2/Cbfa1 and Osx are crucial for the regulation of genes responsible
for the production of bone specific matrix proteins, including ALP, COL I,
BSP, OCN, OPN and eventually stimulate mineralization of bone nodules
[42].
The transcriptional factor, core binding factor α1 (Cbfa1), is essential
for bone formation during embryogenesis and is a key mediator of bone
differentiation. Recent studies have shown that the expression of Cbfa1
correlates with the transition of osteoblasts from the proliferative to the
differentiated phenotype [43]. However, the over-expression of Cbfa1 in
cells of the osteoblastic lineage does not necessarily induce a substantial
increase in bone formation in the adult skeleton, but does express a pos-
itive effect on osteoclast differentiation in vitro and can dramatically en-
hance bone resorption in vivo through increased RANKL expression
[44].
Runx2 (Runt-related transcription factor 2) is the central control
gene of the osteoblast phenotype. Runx2 is an important osteogenic
transcription factor that can bind to the osteoblast-specific cis-acting el-
ement (OSE) 2 in the promoter region of osteogenic genes [45]. The im-
portance of Runx2 in osteoblast differentiation has been highlighted by
the evidence that disruption of one copy of the Runx2 gene can result in
cleidocranial dysplasia and that Runx2 gene knockout in mice prevents
osteoblast development [46].
Osterix (Osx, also known as Sp7) is a novel zinc finger-containing
osteoblast-specific transcription factor that is essential for osteoblast
proliferation, differentiation and bone formation. Osx proteins regulate
the expression of many specific osteoblast differentiation markers in-
cluding Runx2 and Osteonectin (ON). Genetic studies have indicated
that Osx acts as a downstream gene of Runx2 in the osteoblast differen-
tiation signaling pathways [47].
Mesenchymal stem cells differentiate into osteoblasts, adipocytes,
chondroblasts, myoblasts, and fibroblasts [48]. The nuclear receptor
PPAR-γ is the dominant regulator of adipogenesis, and CCAAT-
enhancer-binding protein-β (C/EBP-β) is the primary transcription
factor activated upon adipogenic induction. Activation of PPAR-γ and
C/EBP-β favors differentiation of mesenchymal stem cells to adipocytes
rather than to osteoblasts. In addition, PPAR-γ insufficiency leads to in-
creased osteoblast differentiation and higher trabecular bone volume
 J. An et al. / Life Sciences 147 (2016) 46–58
[49]. Thus, inhibition of adipocyte differentiation might induce mesen-
chymal stem cells to form osteoblasts.
4.2. Natural products from TCM
Icariin, the main active flavonoid glucoside isolated from Epimedii
Folium. Icariin treatment (0.1–100 nM) significantly increased COL I
and ALP mRNA expression of osteoblastic MC3T3-E1 cells in a dose-
dependent manner until 10 nM concentration whereas OPN expression
was marginally but significantly increased from 1 nM concentration on-
ward [50]. Naringin, dihydro-flavonoid, the main effective compound of
Drynariae Rhizoma. Neoeriocitrin, dihydro-flavonoid, a new compound
isolated from Drynariae Rhizoma. Neoeriocitrin more significantly im-
proved proliferation and ALP activity as well as up-regulated Runx2,
COL I and OCN expression in MC3T3-E1 cells by 56%, 37% and 14% re-
spectively than naringin [51]. Poncirin, flavanone glycoside, isolated
from the fruit of Poncirus trifoliata. Poncirin inhibited cytoplasm lipid
droplet accumulation and PPAR-γ and C/EBP-β mRNA expression in
the murine pluripotent mesenchymal cell line C3H10T1/2 cells. By con-
trast, poncirin enhanced the expression of Runx2 and transcriptional co-
activator with PDZ-binding motif (TAZ). It also enhanced ALP and OCN
mRNA expression, and increased mineral nodule formation in primary
bone marrow mesenchymal stem cells (BMMSCs) [52]. Quercetin and
Rutin are common flavonoids in fruits and vegetables. Both of them
were found to up-regulate the osteoblast differentiation of BMMSCs in
dose dependent manner. Quercetin and rutin increased ALP activity by
about 150 and 240% and demonstrated mineralization up to 110 and
200% respectively as compared to control. Further, both of them were
also found to increase the expression of OPN, Osx, Runx-2, OPG and
OCN [53]. Satue et al. reported that the certain doses of quercitrin and
taxifolin induced BSP and OCN mRNA expression, and decreased
RANKL gene expression in MC3T3-E1 cells [54]. Sweroside is a bioactive
herbal ingredient isolated from Fructus Corni. Sweroside significantly in-
creased the proliferation of human osteoblast-like MG-63 cells and rat
osteoblasts. Sweroside increased the ALP activity and OCN expression.
It also attenuated and inhibited apoptosis [55].
5. Modulatory effects of natural products on OPG/RANKL
5.1. OPG/RANKL system
Bone formation/resorption balance is governed by a wide variety of
cell signals, hormones, and growth factors that are essentially regulated
by the receptor activator of NF-κB ligand (RANKL) and its two receptors,
receptor activator of NF-κB (RANK) and osteoprotegerin (OPG) [56,57].
RANKL, a member of the TNF ligand family [57], is highly expressed on
the surface of bone marrow stromal cells (BMSCs), preosteoblasts and
T cells. When RANKL binds to RANK, osteoclast differentiation and func-
tion are enhanced [58]. OPG, produced by BMSCs and osteoblasts, is a
soluble decoy receptor for RANKL and prevents RANKL from binding
to, and activating, RANK on the surface of osteoclastic lineage cells [56,
57]. The relative ratio of RANKL to OPG is considered the critical deter-
minant and final step in the regulation of osteoclast biology and bone
resorption, and this ratio is modulated by various osteotropic hormones,
cytokines, and drugs [59].
5.2. Natural products from TCM
Echinacoside (ECH), a phenylethanoid glycosides isolated from
Cistanches Herba. ECH caused a significant increase in cell proliferation,
ALP activity, COL I contents, OCN levels and an enhancement of miner-
alization in osteoblastic MC3T3-E1 cells at the concentration range
from 0.01 to 10 nmol L−1. In addition, ECH enhanced the ratio of OPG/
RANKL [60]. Vanillic acid, a phenolic acid isolated from Sambucus
williamsii Hance, could stimulate proliferation, ALP activity as well as in-
creased Runx2, OCN and the ratio of OPG/RANKL mRNA expressions in
49
rat osteoblast-like UMR 106 cells [61]. Kirenol is a natural diterpenoid
compound isolated from Herba Siegesbeckia, including S. orientalis,
S. glabrescens, and S. pubescens. Kim's study demonstrated that kirenol
not only increased the expression of osteoblast differentiation markers,
such as ALP, COL I, and OPN, but also increased the expression of OPG/
RANKL ratio [62]. Studies have shown that honokiol [63], apocynin
[64], puerarin [65] icariin [66], baohuoside-I, epimedin B, and
sagittatoside A [67] significantly increased OPG release and decreased
the production of RANKL (P b 0.05).
6. Modulatory effects of natural products on MAPK pathways
6.1. MAPK signaling pathways
The mitogen-activated protein kinases (MAPKs) are the family of
secondary messengers that convey signals from the cell surface to the
nucleus in response to a wide range of stimuli, including hormones,
chemicals and stress. Three major families of MAPKs are extracellular
signal-regulated kinase (ERK), p38 kinase, and c-Jun N terminal kinase
(JNK) and each of these consists of their own subfamilies. MAPKs in-
volve in the regulation of many cellular physiological functions such
as proliferation, differentiation, inflammation, and apoptosis. Among
numerous transcription factors activated by MAPKs signaling, well rec-
ognized factors include c-fos, c-Jun, AP-1, CREB and NFAT. Many of these
transcription factors are directly involved in cellular proliferation in nu-
merous cell types [68].
Osteoblasts and adipocytes differentiate from a common precursor,
the pluripotent mesenchymal stem cells in bone marrow and regulation
of PPARγ2 activity has been shown to control the fate of these cells to-
wards osteogenesis or adipogenesis. Suppression of PPARγ2 activity is
associated with enhanced osteogenesis [69]. It has been reported that
ERK can directly result in the phosphorylation of PPARγ2 and reduces
the levels of PPARγ2 [70]. p38 MAPK plays a negative role in regulating
PPARγ2 transcriptional activities, inhibition or disruption of p38 leads
to increased PPARγ2 expression and transactivation [71].
Recently, many studies discussed the relationship between MAPK
family and Runx2/Osx. For instance, phosphorylation of p38 MAPK
induced the activation of Runx2 via TAK1 and MEK3 signal pathway
during early osteoblastic differentiation [72]. Furthermore, ERK1/2 me-
diated Runx2 phosphorylation and transcriptional activity in bone [73].
Previous studies also reported that p38 MAPK regulated osteoblastic dif-
ferentiation through Osx [74].
6.2. Natural products from TCM
Icariin enhanced MC3T3-E1 cell differentiation and mineralization.
Icariin treatment rapidly induced ERK and JNK activation but showed
no effect on activation of p38 kinase. Furthermore, Icariin-mediated ef-
fects on osteoblasts were dramatically attenuated by treatment with
specific inhibitors of MAPKs, U0126 (ERK inhibitor) and SP600125
(JNK inhibitor) [50]. Ugonin K is a flavonoid isolated from the roots
of Helminthostachys zeylanica. Ugonin K significantly increased the ALP
activity, expressions of BSP, OCN, and mineralization of MC3T3-E1 oste-
oblasts. The mRNA expressions of Runx2 and Osx were also up-
regulated. Both p38 and ERK participated in regulating ugonin K evoked
osteogenesis but p38 seemed to play a more important role [75].
Neobavaisoflavone (NBIF) is an isoflavone isolated from Psoralea
corylifolia. NBIF concentration-dependently promoted osteogenesis in
MC3T3-E1 cells. NBIF increased the phosphorylated level of p38. Appli-
cation of p38 inhibitor SB203580 repressed NBIF-induced activation of
ALP, the expression of COL I, OCN and BSP, and the matrix proteins min-
eralization, as well as abolished the stimulatory effect of NBIF on the ex-
pression of Runx2 and Osx [76]. Salvianolic acid B (Sal B), a phenolic acid
isolated from Salvia miltiorrhiza. Xu's study indicated that Sal B promot-
ed osteogenesis of human mesenchymal stem cells (hMSCs) through
activating ERK signaling pathway. Sal B supplementation (5 μM)
50 J. An et al. / Life Sciences 147 (2016) 46–58
increased the ALP activity, OPN, Runx2 and Osx expression and promot-
ed mineralization in hMSCs. Sal B promoted the phosphorylation of
ERK1/2, which in turn up-regulated the expression of Runx2, and when
the ERK signaling pathway was blocked, the anabolic effects of Sal B
were diminished [77]. Costunolide, one of the active sesquiterpene lac-
tones isolated from the roots of Saussurea lappa. Lee et al. reported that
the effect of costunolide in increasing cell growth might be partly medi-
ated from estrogen receptor (ER), PI3K, ERK, protein kinase C (PKC) and
mitochondrial ATP-sensitive K + channel; the effect on ALP activity
might be mediated from ER, ERK, p38, and PKC; the effect on collagen
synthesis might be mediated from PI3K, ERK, p38, JNK, and PKC; and
the induction of mineralization by costunolide is associated with in-
creased activation of ER and PI3K [78]. Imperatorin and Bergapten, two
furanocoumarins, isolated from Cnidium monnieri. Both of them en-
hanced the phosphorylation of SMAD (transcription factors activated
by TGF-β) 1/5/8, p38 and ERK. Pretreatment of osteoblasts with p38 in-
hibitor (SB203580) or MAPK inhibitor (PD98059) or transfected with
dominant negative mutant of p38 or ERK antagonized the elevation of
BMP-2 expression and ALP activity induced by imperatorin and
bergapten [79]. Kobophenol A, a tetrameric stilbene, is one of the major
active compounds from Caragana sinica Rhed. Kwak's study demonstrat-
ed that Kobophenol A enhances proliferation and ALP activity of human
osteoblast-like cells with activation of the p38 pathway [80].
7. Modulatory effects of natural products on BMP pathways
7.1. BMP signaling pathways
Bone morphogenetic proteins (BMPs), with more than 20 members,
belong to the TGF-β superfamily and are originally identified by their
unique ability to induce ectopic cartilage and bone formation in vivo.
BMPs play important roles in bone formation and bone cell differentia-
tion by stimulating ALP activity and synthesis of proteoglycan, collagen
type I, fibronectin and osteocalcin [2]. The action of BMPs is mediated by
heterotetrameric serine/threonine kinase receptors and the down-
stream transcription factors SMAD1/5/8 [81]. Once these transcription
factors are phosphorylated on serine residues, they form a complex
with a common mediator, SMAD4, and the complex is translocated
into the nucleus to activate the transcription of target genes and then
stimulates bone formation [82]. A genetic study using a combined link-
age and association analyses has linked the BMP-2 gene to a phenotype
of low bone mineral density (BMD) and high fracture risk [83]. Several
lines of evidence have demonstrated that BMP-2 induces or promotes
the expression of Runx2 and Osx [84], which are essential transcription
factors for osteoblast differentiation and bone formation [47].
Recent studies have demonstrated that BMP expression is activated
by canonical Wnt/β-catenin signaling in osteoblast cells and in bone
marrow stromal cells. It has indicated that the Wnt/β-catenin pathway
plays an important role in bone biology, particularly in bone mass acqui-
sition, remodeling, differentiation, and maintenance [85]. Cyclin D1, a
target gene of Wnt pathway, was reported to be up-regulated during
Wnt/β-catenin activation [86].
In addition, Ras activation has been shown to integrate the PI3K sig-
nal to Akt and MAPK for BMP-2 expression in osteoblast differentiation,
Ras is immediately activated by stimulus and then it subsequently in-
duces the activation of PI3K resulting in the activation of Akt and
MAPK (especially ERK) that are required for BMP-2 expression [87].
The mechanism by which Akt regulates BMP-2 transcription is not
clear, but one possibility may include the activation of NF-κB that has
been shown to regulate BMP-2 gene expression in chondrocytes [88].
7.2. Natural products from TCM
Icariin has been reported to enhance bone healing and reduce osteo-
porosis occurrence. Hsieh's study demonstrated that icariin may exert its
osteogenic effects through the induction of BMP-2 and NO synthesis,
subsequently regulating Cbfa1/Runx2, OPG/RANKL gene expressions.
Concurrent treatment involving the BMP antagonist (Noggin) or the
NOS inhibitor (L-NAME) diminished the icariin-induced osteoblasts pro-
liferation, ALP activity, NO production, as well as the BMP-2, SMAD4,
Cbfa1/Runx2, OPG, RANKL gene expressions [66]. Liang's research indi-
cated that icariin can modulate bone formation process via the BMP-2/
SMAD4 signal transduction pathway in human osteoblastic hFOB 1.19
cells [89]. Naringin, a polymethoxylated flavonoid, was shown to in-
crease BMP-2 expression and enhance osteogenic response via the
PI3K, Akt, c-Fos/c-Jun and AP-1-dependent signaling pathway. Naringin
enhanced ALP activity, OCN level, OPN synthesis and cell proliferation
in primary cultured osteoblasts. Naringin mediated the transcriptional
regulation of BMP-2 via phosphorylation of Akt and activation of the ac-
tivator protein-1 (AP-1) components c-Fos and c-Jun. It also enhanced
the binding of c-Fos and c-Jun to the AP-1 element on the BMP-2 pro-
moter. Transfection with dominant-negative mutant of p85 and Akt or
c-Fos and c-Jun antisense oligonucleotide inhibited the potentiating ac-
tion of naringin on BMP-2 production [90]. Psoralen, a coumarin-like de-
rivative extracted from Psoralea corylifolia. Psoralen up-regulated the
expression of BMP-2 and BMP-4 genes, increased the protein level of
phospho-SMAD1/5/8, and activated BMP reporter (12xSBE-OC-Luc) ac-
tivity in a dose-dependent manner, as well as enhanced the expression
of Osx, the direct target gene of BMP signaling. Deletion of the BMP-2
and BMP-4 genes abolished the stimulatory effect of psoralen on the ex-
pression of COL I, ALP, OCN and BSP in primary mouse calvarial osteo-
blasts [91]. Osthole, a coumarin-like derivative extracted from Fructus
Cnidii. Osthole activated Wnt/β-catenin signaling, increased BMP-2 ex-
pression, and stimulated osteoblast differentiation. Targeted deletion of
the β-catenin and BMP-2 genes abolished the stimulatory effect of
osthole on osteoblast differentiation [92]. Imperatorin and bergapten,
two coumarin derivatives, were shown to enhance ALP activity, COL I
synthesis and bone nodule formation in primary cultured osteoblasts.
Both imperatorin and bergapten increased mRNA levels of BMP-2, and
the BMP-2 antagonist noggin attenuated the increase of ALP activity
[79]. Kim's study demonstrated that kirenol is capable of promoting os-
teoblast differentiation in MC3T3-E1 cells through activation of the
BMP and Wnt/β-catenin signaling pathways. Kirenol treatment activated
the mRNA expression of BMP-2, Runx2, and Osx in MC3T3-E1 cells, as
well as up-regulated the mRNA expression of genes in the Wnt/β-
catenin signaling pathway related regulators, including LRP5, DVL2, β-
catenin, cyclin D1, and phosphorylated GSK3β [62]. Emodin is a naturally
occurring anthraquinone present in the roots and bark of numerous
plants of the genus Rhamnus. Lee et al. reported that the low concentra-
tion (5 μM) of emodin highly induced the mRNA expression of BMP-2 in
MC3T3-E1 cells. The up-regulation of BMP-2 could be mediated through
the activation of both Akt and MAPK by activating PI3K, because those in-
ductions by emodin were completely inhibited by the PI3K inhibitor,
LY294002 [93]. Salidroside (SAL), the glucoside-containing component
of Rhodiola rosea L. SAL increased the mRNA expressions of ALP, OPN,
Runx2 and Osx in either C3H10T1/2 or MC3T3-E1 cells. SAL also in-
creased the mRNA level of BMP-2, BMP-6 and BMP-7 and enhanced
the phosphorylation of SMAD 1/5/8 and ERK1/2. The osteogenic effect
of SAL was abolished by BMP antagonist noggin or by BMP receptor ki-
nase inhibitor dorsomorphin [94]. Acerogenin A, a diarylheptanoids iso-
lated from Acer nikoense Maxim. Acerogenin A treatment increased BMP-
2, BMP-4, and BMP-7 mRNA expression levels in MC3T3-E1 cells. Noggin
(a BMP antagonist) inhibited the acerogenin A-induced increase in the
OCN, Osx and Runx2 mRNA expression levels [95].
8. Modulatory effects of natural products on ERs-mediated
pathways
8.1. ERs-mediated pathways
Estrogens exert their physiological effects on target tissues by
interacting with estrogen receptors, which are members of the
 J. An et al. / Life Sciences 147 (2016) 46–58
superfamily of ligand regulated nuclear transcription factors. Estrogen
receptors (ERs) are a family of intracellular receptors, including estro-
gen receptor α (ERα) and estrogen receptor β (ERβ) [96]. Both recep-
tors have been identified in osteoblasts and osteoclasts as well as in
their precursors. Estrogens play an important role in coordinating activ-
ities of osteoblasts and osteoclasts in bone homeostasis and preventing
postmenopausal bone loss [97].
ERs stimulate classical effects by acting as nuclear transcription fac-
tors as well as non-classical effects by activating distinct cytoplasmic
protein kinase cascades. Typically, the majority of ER α and β are
found in the cytoplasm and nucleus, although small amounts are
expressed on cell membrane. In the classical pathway, ERs are activated
by directly binding to estrogen and subsequently by stimulating gene
transcription via interacting with estrogen response elements (EREs)
in the promoters of target genes [98]. Other transcription factors in nu-
cleus, that include NF-κB, AP-1 and SP-1, interact with the ligand–recep-
tor complex to influence gene transcription [99]. Such nuclear canonical
pathway mediates the long term actions of estrogen. Alternatively, es-
trogen can induce rapid effects through membrane-bound ERs. ERs
can be activated by MAPK via phosphorylation upon growth factor re-
ceptors activation in a ligand-independent manner. MAPK signaling
pathways involve the activation of membrane-associated receptor tyro-
sine kinases (e.g., IGF-IR and EGFR), Ras protein kinase, Raf mitogen-
activated protein kinase kinase (MEK/MAPKK) as well as downstream
extracellular signal-regulated protein kinase (ERK). [100].
ERα has been reported to be the major regulator mediating the ef-
fects of estrogen on bone metabolism. According to the classical mech-
anism, estrogen binds to ERα to form a receptor dimer, and then this
dimer is translocated from the cytoplasm to the nucleus. After binding
to its specific ERE, this ER-estrogen complex can initiate certain gene
expressions and control osteoblast differentiation [101]. Serine 118 is
reported to be the major phosphorylation site in ERα for ligand-
independent activation by the MAPK-mediated pathway. [100]. Multi-
ple lines of evidence suggest that activation of the tyrosine kinase c-
Src represents one of the initial steps in ERα-mediated cell signaling
[102].
Estrogen has an important role in the development and growth of
bones and later in the maintenance of bone mass. It is believed that
the major action of estrogen on the skeleton in vivo is through the inhi-
bition of bone resorption. Although some of the anti-resorptive effects
of estrogen are via direct actions on bone-resorbing osteoclasts, estro-
gen has also been shown to have indirect effects by regulating bone-
forming osteoblasts and bone marrow stromal cells. As one of these ef-
fects, 17β-estradiol has been suggested to protect osteoblasts from apo-
ptosis. Estrogen has been shown to protect calvaria-derived osteoblastic
cells from etoposide-induced apoptosis as well as to protect osteoblasts
from ethanol-induced bone loss [103,104].
8.2. Natural products from TCM
Icariin, baohuoside-I, epimedin B and sagittatoside A are four major
flavonoids isolated from Herba epimedii (HEP). Xiao's study showed
that all the four compounds significantly stimulated the cell prolifera-
tion rate, ALP activity and OPG/RANKL mRNA expression in rat
osteoblastic-like UMR-106 cells and their effects could be abolished by
co-incubation with 10−6 M ICI 182,780 (ER antagonist). None of the fla-
vonoids exhibited binding affinities towards ERα and ERβ. However,
sagittatoside A selectively activated ERE-luciferase activity via ERα. In
addition, icariin and sagittatoside A induced ERα phosphorylation at
serine 118 residue. The results indicated that all four flavonoids from
HEP stimulated ER-dependent osteoblastic functions in UMR-106 cells,
but only icariin and sagittatoside A appeared to exert their actions by
ligand-independent activation of ERα [67]. Song's study suggested
that treatment of osteoblasts with ICI 182,780 attenuated the stimulato-
ry effects of icariin on COL I, ALP and OPN mRNA expressions, associated
with suppression of ERK and JNK phosphorylation [50]. Ugonin K
51
induced increases in ALP activity, expressions of BSP, OCN, Runx2 and
Osx, and subsequent bone nodule formation in MC3T3-E1 cells were
inhibited by ICI 182,780. The raised c-Src phosphorylated level induced
by ugonin K was significantly attenuated by ICI 182,780. Application of
PP2 (c-Src specific inhibitor) repressed ugonin K-induced osteogenesis.
Ugonin K also increased the nuclear level of ERα protein. The results
demonstrated that ugonin K stimulated osteogenesis might act through
an ER dependent activation of a non-classical signaling pathway medi-
ated by phosphorylation of c-Src. Moreover, a transactivation potential
towards ERα through a classical pathway might not be precluded
[105]. Genistein enhanced the phosphorylation of ERK 1/2, p38 MAPK
and JNK 1/2 in MC3T3-E1 cells. Sequentially, it increased the transloca-
tion of NF-κB and c-Jun from the cytoplasm to the nucleus. Genistein in-
creased ERα mRNA expression and the amounts of cytosolic and nuclear
ERα in MC3T3-E1 cells and rat calvarial osteoblasts. And there are
several ERα-specific DNA-binding elements in the 5′-promoter regions
of the BMP-6, COL I and OCN genes. The results indicated that genistein
can induce ERα gene expression via the activation of MAPK/NF-κB/AP-1
and accordingly stimulates differentiation-related gene expressions and
osteoblast mineralisation [106]. Puerarin, the main isoflavone glycoside
found in the radix of Pueraria lobata (Willd.) Ohwi. Wang's study
showed that treatment with ICI 182,780 markedly abolished puerarin-
increased proliferation, ALP activity and COL I secretion in human oste-
oblastic MG-63 cells (P b 0.05). In addition, protective effect of puerarin
upon cisplatin-induced apoptosis was also significantly blocked by ICI
182,780 (P b 0.05). Using siRNA technology, it was demonstrated that
the effects of puerarin on proliferation, differentiation as well as
cisplatin-induced cell death are mediated by both ERα and ERβ [107].
Tiyasatkulkovit's study also showed that after exposure to ICI 182,780,
the puerarin-induced upregulation of ALP expression in UMR106 cells
was completely abolished [65]. Xiao et al. reported that co-treatment
of UMR 106 cells with ICI 182,780 abolished the stimulatory effects of
vanillic acid (VA) on osteoblast proliferation and ALP activity, but VA
failed to bind to ERα or ERβ and did not activate ERE-luciferase activity
via ERα or ERβ in UMR 106 cells. In contrast, VA induced the phosphor-
ylation of MEK 1/2, ERK 1/2 and ERα at serine 118 residue in UMR 106
cells. The results indicated that the bone sparing effects of VA involved a
ligand independent, ERE-independent and MAPK-mediated
nongenomic ER signaling pathway [61]. Resveratrol (RSVL), a polyphe-
nol mainly existing in red grapes and berries. Dai's study suggested that
treatment of human BMMSCs with ICI 182,780 attenuated the stimula-
tory effects of RSVL on cell growth, ALP activity, calcium deposition, and
the mRNA expressions of Runx2/Cbfa1, Osx and OCN, associated with
suppression of ERK 1/2 phosphorylation [108].
9. Modulatory effects of natural products on oxidative
stress-mediated pathways
9.1. Oxidative stress-mediated pathways
Oxidative stress, which is caused by excessive production of reactive
oxygen species (ROS) and insufficient antioxidant defense mechanisms,
has been shown to be a crucial pathogenic factor of osteoporosis [109].
Osteoporotic postmenopausal women exhibit a decrease in bone miner-
al density (BMD) that is related to higher oxidation of plasma lipids and
lower superoxide dismutase and catalase efficacies. In rat femurs, ovari-
ectomy (OVX) results in oxidative damage and decreases the capacity of
antioxidant defense mechanisms. Thus, oxidative stress might serve as a
major contributor to postmenopausal osteoporosis [110]. Both aging
and estrogen deficiency elevate the levels of ROS, and there is strong ev-
idence to indicate that the damage on bone mass due to estrogen loss
might be prevented by antioxidants.
Oxidative stress, results from deregulation of the intracellular reduc-
tion–oxidation balance, which is mainly manifested by superfluous su-
peroxide radical anion (O2−), H2O2 and the hydroxyl radical (OH.) in
cells. ROS, mostly derived from mitochondrial activity. A certain amount
52 J. An et al. / Life Sciences 147 (2016) 46–58
of oxidative stress is required by the mitochondrial respiratory chain
during the metabolic process, however, an over-generation of ROS
would cause lipid and protein oxidation and alter mitochondrial
and nuclear DNA integrity, which would lead to tissue damage. Under
certain pathological conditions, the dynamic balance between the gen-
eration and elimination of ROS may be broken and thus the intracellular
ROS levels increase significantly. It can be suggested that ROS produc-
tion is particularly involved in mineral tissue homeostasis and contrib-
utes mostly to bone remodeling by reducing bone formation and
promoting bone resorption [111,112].
Mitochondria not only produce ROS but are also the principal target
of ROS attacks. Impaired mitochondrial function can lead to increased
ROS generation and may increase oxidative stress if the antioxidant de-
fense mechanisms of the cells are overwhelmed. Increased oxidative
stress caused by mitochondrial dysfunction is considered the causal
link between elevated ROS and the major biochemical pathways postu-
lated to be involved in the pathogenesis of senile disorders. There are
two major sites of superoxide release in the respiratory chain, the first
being complex I, the second complex III. Antimycin A (AMA) is an inhib-
itor of mitochondrial electron transport via its binding to complex III,
has been used as a ROS generator in biological systems [113].
Hydrogen peroxide (H2O2) is favorable to serve as both an extra-
and an intercellular signal molecule. H2O2 is generated from nearly
all sources of oxidative stress. Because of its long half-life and the
ability to cross biological membranes, H2O2 has the characteristics
most suited to act both as an intra- and an intercellular signal. It
has been demonstrated that it plays a crucial role in postmenopausal
osteoporosis.
9.2. Natural products from TCM
2,3,5,40-Tetrahydroxystil bene-2-O-b-D-glucoside (TSG), a
polyhydrostilbenes derived from Polygonum multiflorum Thunb. TSG
might be effective in providing protection against osteoporosis associat-
ed with oxidative stress. TSG significantly increased cell survival, ALP ac-
tivity, calcium deposition, and the mRNA expressions of ALP, COL I and
OCN in the presence of H2O2 (P b 0.05). In addition, TSG decreased the
production of RANKL, IL-6, intracellular ROS and malondialdehyde
(MDA) of osteoblastic MC3T3-E1 cells induced by H2O2 [114].
Gastrodin, a glucoside isolated from Gastrodia elata, is a potent antioxi-
dant. Gastrodin inhibited H2O2-induced cytotoxicity in human BMMSCs,
attenuated H2O2-induced hBMMSC dysfunction, reduced lipid genera-
tion and inhibited hBMMSC adipogenic differentiation under oxidative
conditions. Gastrodin displayed good recovery effects on cellular ALP
activity, mRNA expressions of ALP, OCN, COL I, OPN and calcium miner-
alization which was suppressed by H2O2. Gastrodin showed protective
effects against osteoporosis linking to a reduction in ROS [115].
Albiflorin, a monoterpene isolated from Paeoniae Radix, was investigat-
ed for its ability to protect against antimycin A-induced osteoblast tox-
icity in the MC3T3-E1 cell line. The results suggested that albiflorin
enhanced mitochondrial function to suppress antimycin A-induced ox-
idative damage via the preservation of cytochrome c and cardiolipin.
Pretreatment with albiflorin before antimycin A reversed the loss of
cell viability, decreased apoptosis and lactate dehydrogenase release,
decreased ROS/RNS levels, and increased mitochondrial function com-
pared to antimycin A-treated cultures. Albiflorin reduced antimycin A-
induced mitochondrial cytochrome c loss and cardiolipin peroxidation,
conferring protection against ROS. In addition, albiflorin increased the
mineralization reduced by antimycin A [116]. Honokiol is a phenolic
compound isolated from the bark of Magnolia officinalis. Honokiol
caused a significant elevation of cell growth, ALP activity, collagen syn-
thesis, mineralization, glutathione content, and OPG release in the
MC3T3-E1 cells (P b 0.05). Moreover, honokiol significantly (P b 0.05)
decreased the production of osteoclast differentiation inducing factors
such as TNF-α, IL-6, and RANKL in the presence of antimycin A, which
inhibits mitochondrial electron transport and has been used as a ROS
generator [63]. Apocynin is a naturally occurring methoxy-substituted
catechol, experimentally used as an inhibitor of NADPH-oxidase.
Apocynin could protect osteoblastic MC3T3-E1 cells from mitochondrial
dysfunction-induced toxicity. Apocynin significantly (P b 0.05) in-
creased cell survival, calcium deposition, and OPG release and decreased
the production of ROS and osteoclast differentiation inducing factors
such as TNF-α, IL-6, and RANKL in the presence of antimycin A [64].
Ginsenoside-Rb2 (Rb2), a 20(S)-protopanaxadiol glycoside extracted
from ginseng, is a potent antioxidant. Huang's study indicated that the
potential anti-osteoporosis effects of Rb2 were linked to a reduction of
oxidative damage and bone-resorbing cytokines. Rb2 (0.1 to 10 μM)
promoted the proliferation, ALP activity, calcium mineralization and
mRNA expressions of ALP, COL1A1, OCN and OPN against oxidative
damage induced by H2O2 in MC3T3-E1 cells. Importantly, Rb2 reduced
the expression levels of RANKL and IL-6 and inhibited the H2O2-
induced production of ROS. The in vivo study indicated that the Rb2 ad-
ministered for 12 weeks partially decreased blood MDA activity and el-
evated the GSH activity in ovariectomized (OVX) mice [117].
Ophiopogonin D (OP-D), the chief active component isolated from
Radix Ophiopogon japonicus. OP-D increased osteogenic differentiation
by promoting calcium mineralization, ALP activity of MC3T3-E1 cells
and improved the mRNA expressions of OCN, COL1A1 and OPN. OP-D
suppressed ROS generation in both MC3T3-E1 and RAW264.7 cells. Fur-
ther research showed that the protective effects of OP-D against osteo-
porosis are linked to a reduction in oxidative stress via the FoxO3a-β-
catenin signaling pathway [118]. In addition, studies have shown that
treatment of osteoblast cells with kobophenol A resulted in improve-
ment of ROS scavenging activity [80].
10. Modulatory effects of natural products on
NO-mediated pathways
10.1. NO-mediated pathways
Nitric oxide (NO) is a short-lived signaling molecule generated from
L-arginine by nitric oxide synthase (NOS) isoenzymes. An accumulating
body of evidence has emerged to suggest that NO plays an important
role as a paracrine and autocrine mediator of bone metabolism and
bone cell activity in response to diverse stimuli [119]. The NO–sGC–
cGMP–PKG pathway, begins with activation of NOS, the enzyme that
synthesizes NO in cells. The increased NO levels activate the soluble
guanylyl cyclase (sGC), thus increasing the level of cyclic guanosine
monophosphate (cGMP) which acts to activate protein kinase-G
(PKG), a cGMP dependent protein kinase [120].
Serine/threonine protein kinase Akt, also known as protein kinase B,
is stimulated by a number of receptor tyrosine kinases and G protein-
coupled receptors through phosphatidylinositol 3-kinase (PI3K) [121].
In addition, it has been reported that Akt can phosphorylate eNOS, one
isoform of NOS [122]. Akt signaling has been reported to be involved
in osteoblast proliferation and skeletal development [123], and Akt is
upstream of NO pathway.
10.2. Natural products from TCM
Zhai et al. reported that the osteogenic effect of icariin involves the
PI3K–Akt–eNOS–NO–cGMP–PKG signal pathway. Icariin increased ALP
activity, NOS activity, iNOS and eNOS expressions, NO production, sGC
and cGMP contents and PKG expression besides the phosphorylation
of Akt. The addition of L-NAME, ODQ and PDE5 (the inhibitor of NOS,
sGC and cGMP) inhibited the icariin effects on above markers respec-
tively. The addition of LY294002 (the inhibitor of PI3K) decreased the
p-Akt level, NOS activity, eNOS expression and NO production, but had
no significant effect on iNOS expression. The addition of any of the
four inhibitors also abolished the osteogenic effect of icariin on rat
BMSCs as indicated by ALP activity, OCN synthesis, calcium deposition
 J. An et al. / Life Sciences 147 (2016) 46–58 53

Table 1
The category, source, structure, dose, cell lines and mechanism of 36 natural compoundsa on osteoblast-mediated bone formation.

Compound name Category Source Structure Dose Cell lines used Mechanism References
for in vitro
studiesb

Icariin Flavonoid glucoside Herba epimedii 10−7, 10−6, MC3T3-E1 cells ↑(ALP, COL I, bone nodule); [50]
10−5, 10−4, ER-mediated ERK and JNK
10−3 M signal pathway
10−8 M Primary ↑(BMP-2, NO, Cbfa1/Runx2, [66]
osteoblast cells OPG/RANKL)
10−5 M rBMSCs PI3K–AKT–eNOS–NO–cGMP– [124]
PKG signal pathway
10−12, 10−9, hFOB 1.19 cells ↑(BMP-2, SMAD4, Cbfa1/ [89]
10−6 M Runx2, OPG/RANKL)
10−8 M UMR-106 cells ↑(ALP, OPG/RANKL); ligand- [67]
independent activation of ERα
−10
Baohuoside-I, epimedin B Flavonoid glucoside Herba epimedii 10 , UMR-106 cells ↑(ALP, OPG/RANKL); [67]
Sagittatoside A 10−8 M ER-dependent osteoblastic
functions; Sagittatoside A:
ligand-independent activation
of ER α

Neoeriocitrin Dihydro-flavonoid Drynaria Rhizome 2 μg mL−1 MC3T3-E1 cells ↑(ALP, Runx2, COL I, OCN) [51]

Naringin Dihydro-flavonoid Drynaria Rhizome 2 μg mL−1 MC3T3-E1 cells ↑(ALP, Runx2, COL I, OCN) [51]
Citrus fruits 0.3, 1, 3, MC3T3-E1 cells; BMP-2; PI3K, Akt, c-Fos/c-Jun [90]
10 μM hOB; primary and AP-1 pathway
osteoblastic cells
Poncirin Flavanone glycoside Poncirus trifoliata 0.1, 1, C3H10T1/2 ↓(PPAR-γ, C/EBP-β); ↑(Runx2, [52]
10 μM cells; BMMSCs TAZ, ALP, OCN)

Rutin Flavonoid Fruits and vegetables 25, 50, BMMSCs ↑(ALP, OPN, Osx, Runx2, [53]
100 μM OPG, OCN)

Quercetin Flavonoid Fruits and vegetables 25, 50, BMMSCs ↑(ALP, OPN, Osx, Runx2, [53]
100 μM OPG, OCN)
200, 500 μM MC3T3-E1 cells ↑(BSP, OCN); ↓(RANKL) [54]

Taxifolin Flavonoid Fruits and vegetables 100, MC3T3-E1 cells ↑(BSP, OCN); ↓(RANKL) [54]
200 μM

Ugonin K Flavonoid Helminthostachys 0.5, 1, 5, MC3T3-E1 cells ↑(ALP, BSP, OCN, Runx2, Osx); [75,105]
zeylanica 10 μM p38 MAPK- and ERK- mediated
pathway; ER-dependent
non-classical Src pathway and
classical pathway
Neobavaisoflavone Isoflavone Psoralea corylifolia 1, 5, 10, MC3T3-E1 cells ↑(ALP, COL I, OCN, BSP, Runx2 [76]
15, 20 μM and Osx); p38-dependent
pathway
Genistein Flavonoid Sophora japonica L. 10 μM MC3T3-E1 cells; Induce ERα expression [106]
primary rat through MAPK/NF-κB/AP-1
osteoblast cells pathway
Puerarin Isoflavone glycoside Pueraria mirifica 0.1 μM MG-63 cells ER-dependent MEK/ERK and [107]
PI3K/Akt activation
0.1, 10, UMR 106 cells ↑(ALP, OPG/RANKL); [65]
1000 nM ER-dependent

Echinacoside Phenylethanoid Cistanche tubulosa 0.01, 0.1, 1, MC3T3-E1 cells ↑(ALP, COL I, OCN, [60]
glycoside (Schrenk) R. Wight 10 nM OPG/RANKL)
stems

(continued on next page)

54 J. An et al. / Life Sciences 147 (2016) 46–58

Table 1 (continued)

Compound name Category Source Structure Dose Cell lines used Mechanism References
for in vitro
studiesb

Sweroside Iridoid glycoside Fructus Corni 10−6– MG-63 cells; rat ↑(ALP, OCN); inhibit apoptosis [55]
10−1 g mL−1 osteoblast cells

Salidroside Phenylpropanoid Rhodiola rosea L. 0.5, 1, 5, 10, C3H10T1/2 ↑(ALP, OPN, Runx2, Osx, [94]
glycoside 50 μM cells; MC3T3-E1 BMP-2, BMP-6, BMP-7,
cells phosphorylation of SMAD
1/5/8 and ERK1/2); BMP
signaling

Gastrodin Phenolic glycoside Gastrodia elata 0.1, 1, 10 μM hBMMSCs ↑(ALP, OCN, COL I, OPN); ↓ROS [115]

Ophiopogonin D Steroidal saponin Radix Ophiopogon 1, 10, MC3T3-E1 ↑(ALP, OCN, COL1A1, OPN); [118]
japonicus 100 μM subclone 4 cells ↓ROS; the FoxO3a-β-catenin
signaling pathway

Ginsenoside-Rb2 20 Ginseng 0.1, 1, 10 μM MC3T3-E1 cells ↑(ALP, COL I, OCN, OPN); [117]
(S)-protopanaxadiol ↓(RANKL, IL-6, ROS)
glycoside

2,3,5,4′-Tetrahydroxystil Polyhydrostilbenes Polygonum 0.1, 1, 10 μM MC3T3-E1 cells ↑(ALP, COL I, OCN); ↓(RANKL, [114]
bene-2-O-β-D-glucoside (glycoside) multiflorum Thunb IL-6, ROS, MDA)
(TSG)

Imperatorin Furanocoumarins Cnidium monnieri 0.3, 1, 3, Primary ↑(ALP, COL I, bone nodule, [79]
(L.) Cuss 10 μM osteoblastic BMP-2, phosphorylation of
cells SMAD 1/5/8); p38 and
ERK-dependent pathway

Bergapten

Psoralen Coumarin-like Psoralea corylifolia 1, 10, Primary mouse ↑(ALP, COL I, OCN, BSP, Osx, [91]
derivative 100 μM calvarial BMP-2, BMP-4, phosphorylation
osteoblasts of SMAD 1/5/8); BMP signaling
Osthole Coumarin-like Cnidium monnieri 10, 50, Primary ↑BMP-2; Wnt/β-catenin [92]
derivative (L.) Cusson 100 μM calvarial signaling
osteoblasts

Costunolide Sesquiterpene Saussurea lappa 10 μg mL−1 MC3T3-E1 cells ER, PI3K, ERK, PKC, p38, JNK, [78]
lactones and mitochondrial
ATP-sensitive K+ channel

Albiflorin Monoterpene Paeonia albiflora Pall 0.01, 0.1, MC3T3-E1 cells protect against oxidative [116]
1 μM stress-mediated toxicity

Kirenol Diterpenoid Herba Siegesbeckia 40 μM MC3T3-E1 cells ↑(ALP, COL I, OPN, OPG/ [62]
RANKL, BMP-2, Runx2, Osx;
LRP5, DVL2, β-catenin,
CCND1); BMP and
Wnt/β-catenin pathways
Vanillic acid Phenolic acid Sambucus williamsii 10−10, UMR 106 cells ↑(ALP, Runx2, OCN, [61]
Hance 10−8 M OPG/RANKL); MAPK
(MEK/ERK)-mediated ER
signaling pathway
 J. An et al. / Life Sciences 147 (2016) 46–58 55

Table 1 (continued)

Compound name Category Source Structure Dose Cell lines used Mechanism References
for in vitro
studiesb

Salvianolic acid B Phenolic acid Salvia miltiorrhiza 5 μM hMSCs ↑(ALP, OPN, Runx2, Osx); ERK [77]
signaling pathway

Honokiol Phenolic Magnolia officinalis 0.01, 0.1, MC3T3-E1 cells ↑(ALP, COL I, OPG/RANKL, [63]
1 μM glutathione); ↓(TNF-α, IL-6,
mitochondrial electron
transport)

Resveratrol Polyphenol Red grapes and 10−8, 10−6, hBMMSCs ↑(ALP, Runx2/Cbfa1, Osx, [108]
berries 10−5 M OCN); ER-dependent ERK1/2
pathway

Apocynin Methoxy-substituted Picrorrhiza kurrooa 0.01, 0.1, MC3T3-E1 cells ↑(ALP, COL I, OPG/RANKL); [64]
catechol Royle ex Benth 1 μM ↓(ROS, TNF-α, IL-6)

Kobophenol A Tetrameric stilbene Caragana sinica Rhed 25, MG-63 cells ↓(NO-induced cell death); [125]
50 μg mL−1 regulate JNK, NF-κΒ and AP-1
signaling pathways
Human ↑(ALP); ↓(ROS); p38 pathway [80]
osteoblast-like
cells (MG-63,
HOS, SaOS-2)
Emodin Anthraquinone The genus Rhamnus 1, 2, 5 μM MC3T3-E1 ↑(BMP-2, ALP); PI3K, Akt and [93]
subclone 4 cells MAPK pathways

Acerogenin A Diarylheptanoids Acer nikoense Maxim 30 μM MC3T3-E1 cells; ↑(OCN, Osx, Runx2, BMP-2, [95]
RD-C6 cells; BMP-4, BMP-7)
primary
osteoblastic
cells
a
The 36 natural compounds include 14 flavonoids, 7 glycosides, 4 coumarins, 3 terpenoids, 2 phenolic acids, 3 phenols and 3 others.
b
Bone marrow mesenchymal stem cells (BMMSCs): multipotent stromal cells; C3H10T1/2: mesenchymal stem cells; hFOB 1.19: human osteoblastic cell line; MC3T3-E1 cells: oste-
oblast-like cell line; MG-63 cells: osteoblast-like cell line; UMR106 cells: osteoblast-like cell line.

and the number and areas of calcified nodules [124]. Hsieh's study
showed that 10− 8 M icariin increased the proliferation and matrix
mineralization of osteoblasts and promoted NO synthesis. L-NAME di-
minished the icariin-induced cell proliferation, ALP activity, NO produc-
tion, as well as the BMP-2, SMAD4, Cbfa1/Runx2, OPG, RANKL gene
expressions [66]. Kobophenol A (kob A), a tetrameric stilbene, is one
of the major active compounds from Caragana sinica Rhed. Sodium ni-
troprusside (SNP), a direct NO donor, can induce cell death and NO
production in MG-63 cells. Lee's study suggested that co-incubation of
kob A in SNP-treated MG-63 cells resulted in a significant protection
against NO-induced cell death. Furthermore, kob A inhibited SNP-
induced phosphorylation of JNK and c-Jun, and SNP-induced reduction
in NF-κΒ and AP-1 activities, implicated that protective effect of kob A
might occur through the regulation of JNK, NF-κΒ and AP-1 signaling
pathways [125].
11. Conclusions and future prospects
Cell molecular research on the pharmacological mechanisms behind
osteoblasts-mediated bone formation induced by natural products from
TCM has made great progress. According to the information collected in
this paper, it could be concluded that herbs with kidney-tonifying,
spleen-tonifying, and stasis-removing effects all have the potential ef-
fect on treating osteoporosis. As shown in Table 1, the active ingredients
from TCM that possess effects on promoting osteoblasts proliferation
and differentiation including flavonoids, glycosides, coumarins, terpe-
noids (sesquiterpenoids, monoterpenoids, diterpenoids), phenolic
acids, phenols and others (tetrameric stilbene, anthraquinones,
diarylheptanoids). And it is confirmed that the bone formation effect in-
duced by natural products was regulated by the expressions of bone
specific matrix proteins (ALP, BSP, OCN, OPN, COL I), transcription fac-
tors (Runx2, Cbfa1, Osx), signal pathways (MAPK, BMP), local factors
(ROS, NO), OPG/RANKL system of osteoblasts and estrogen-like biolog-
ical activities. It provided theoretical basis for clinical application, as
well as new drug research and development on treating osteoporosis.
Combined with modern scientific technologies, new developments
will be achieved in the fields of pharmacodynamic material basis, effect
targets and signaling pathways of TCM on the function of osteoblasts-
mediated bone formation in the future. Natural products on prevention
and treatment of osteoporosis possess the whole regulative effects and
the fewer adverse reactions, have received extensive attentions and re-
searches at home and abroad. It is desirable to have satisfactory anabolic
56 J. An et al. / Life Sciences 147 (2016) 46–58
agents that stimulate new bone formation and regulate the bone me-
tabolism balance, which would create a new alternative for treating
osteoporosis.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgments
The authors acknowledge financial support from the Youth Fund of
National Natural Science Foundation of China (No. 81403278).
References
[1] G. Karsenty, The complexities of skeletal biology, Nature 423 (2003) 316–318.
[2] M.R. Urist, Bone: formation by autoinduction, Science 150 (1965) 893–899.
[3] D.J. Hadjidakis, I. Androulakis, Bone remodeling, Ann. N. Y. Acad. Sci. 1092 (2006)
385–396.
[4] L.G. Raisz, Pathogenesis of osteoporosis: concepts, conflicts, and prospects, J. Clin.
Investig. 115 (2005) 3318–3325.
[5] B.L. Riggs, H. Wahner, E. Seeman, K. Offord, W. Dunn, R. Mazess, et al., Changes in
bone mineral density of the proximal femur and spine with aging, J. Clin. Investig.
70 (1982) 716–723.
[6] A. Leboime, C. Confavreux, N. Mehsen, J. Paccou, C. David, C. Roux, Osteoporosis
and mortality, Joint Bone Spine 77 (Suppl. 2) (2010) S107–S112.
[7] M.C.A. Buencamino, A.L. Sikon, A. Jain, H.L. Thacker, An observational study on the
adherence to treatment guidelines of osteopenia, J. Women's Health 18 (2009)
873–881.
[8] O. Johnell, J.A. Kanis, An estimate of the worldwide prevalence and disability asso-
ciated with osteoporotic fractures, Osteoporos. Int. 17 (2006) 1726–1733.
[9] C. Hu, C. Wang, L. Mo, C. Lin, L. Sun, A review on antiosteoporotic drugs, Chin. J.
Gerontol. 11 (2008) 2178–2180.
[10] P. Piscitelli, G. Iolascon, F. Gimigliano, M. Muratore, P. Camboa, O. Borgia, et al., In-
cidence and costs of hip fractures compared to acute myocardial infarction in the
Italian population: a 4-year survey, Osteoporos. Int. 18 (2007) 211–219.
[11] L. Melton, E. Chrischilles, C. Cooper, A. Lane, B. Riggs, Perspective. How many
women have osteoporosis? J. Bone Miner. Res. 7 (1992) 1005–1010.
[12] R. Burge, B. Dawson-Hughes, D. Solomon, J. Wong, A. King, A. Tosteson, Incidence
and economic burden of osteoporosis related fractures in the United States,
2005–2025, J. Bone Miner. Res. 22 (2007) 465–475.
[13] V. Beral, Million women study collaborators, breast cancer and hormone-
replacement therapy in the million women study, Lancet 362 (2003) 419–427.
[14] M.B. Zanchetta, M. Diehl, M. Buttazzoni, A. Galich, F. Silveira, C.E. Bogado, et al., As-
sessment of bone microarchitecture in postmenopausal women on long term bis-
phosphonate therapy with atypical fractures of the femur, J. Bone Miner. Res. 29
(2013) 999–1004.
[15] J. Pinkerton, S. Thomas, Use of SERMs for treatment in postmenopausal women,
J. Steroid Biochem. Mol. Biol. 142 (2014) 142–154.
[16] K. Cappuzzo, J. Delafuente, Teriparatide for severe osteoporosis, Ann.
Pharmacother. 38 (2004) 294–302.
[17] T. Ponnapakkam, R. Katikaneni, J. Sakon, R. Stratford, R. Gensure, Treating osteopo-
rosis by targeting parathyroid hormone to bone, Drug Discov. Today 19 (2013)
204–208.
[18] G.A. Rodan, T.J. Martin, Therapeutic approaches to bone diseases, Science 289
(2000) 1508–1514.
[19] S.K. Chair, D. Burr, J. Cauley, D.W. Dempster, P.R. Ebeling, D. Felsenberg, et al.,
Bisphosphonate-associated osteonecrosis of the jaw: report of a task force of the
American society for bone and mineral research, J. Bone Miner. Res. 22 (2007)
1479–1491.
[20] J.P. Bilezikian, Combination anabolic and antiresorptive therapy for osteoporosis:
opening the anabolic window, Curr. Osteoporos. Rep. 6 (2008) 24–30.
[21] D.M. Black, S.L. Greenspan, K.E. Ensrud, L. Palermo, J.A. Mcgowan, T.F. Lang, et al.,
The effects of parathyroid hormone and alendronate alone or in combination in
postmenopausal osteoporosis, N. Engl. J. Med. 349 (2003) 1207–1215.
[22] J.S. Finkelstein, A. Hayes, J.L. Hunzelman, J.J. Wyland, H. Lee, A.R.M. Neer, The effects
of parathyroid hormone, alendronate, or both in men with osteoporosis, N. Engl. J.
Med. 349 (2003) 1216–1226.
[23] A.V. Uihlein, B.Z. Leder, Anabolic therapies for osteoporosis, Endocrinol. Metab.
Clin. N. Am. 41 (2012) 507–525.
[24] Y. Liu, J. Liu, Y. Xia, Chinese herbal medicines for treating osteoporosis, Cochrane
Database Syst. Rev. (2014), http://dx.doi.org/10.1002/14651858.CD005467.pub2.
[25] H. Xu, D. Lawson, A. Kras, D. Ryan, The use of preventive strategies for bone loss,
Am. J. Chin. Med. (2005) 299–306.
[26] A. Maurice, Drug combination strategies for osteoporosis, Joint Bone Spine 73
(2006) 374–378.
[27] R. Marcus, S. Majumdar, The nature of osteoporosis, in: R. Marcus, D. Feldman, J.
Kelsey (Eds.), Osteoporosis, Seconded, Academic Press, San Diego 2001, pp. 3–17.
[28] R.T. Franceschi, C. Ge, G. Xiao, H. Roca, D. Jiang, Transcriptional regulation of oste-
oblasts, Ann. N. Y. Acad. Sci. 1116 (2007) 196–207.
[29] I. Bremner, J. Beattie, Copper and zinc metabolism in health and disease: speciation
and interactions, Proc. Nutr. Soc. 54 (1995) 489–499.
[30] J.R. Farley, S.L. Hall, M.A. Tanner, J.E. Wergedal, Specific activity of skeletal alkaline
phosphatase in human osteoblast-line cells regulated by phosphate, phosphate es-
ters, and phosphate analogs and release of alkaline phosphatase activity inversely
regulated by calcium, J. Bone Miner. Res. 9 (1994) 497–508.
[31] M. Balcerzak, E. Hamade, L. Zhang, S. Pikula, G. Azzar, J. Radisson, et al., The roles of
annexins and alkaline phosphatase in mineralization process, Acta Biochim. Pol. 50
(2003) 1019–1038.
[32] G.S. Stein, J.B. Lian, T.A. Owen, Relationship of cell growth to the regulation of
tissue-specific gene expression during osteoblast differentiation, FASEB J. 4
(1990) 3111–3123.
[33] C. Wennberg, L. Hessle, P. Lundberg, S. Mauro, S. Narisawa, U.H. Lerner, et al., Func-
tional characterization of osteoblasts and osteoclasts from alkaline phosphatase
knockout mice, J. Bone Miner. Res. 15 (2000) 1879–1888.
[34] J.A.R. Gordon, C.E. Tye, A.V. Sampaio, T.M. Underhill, G.K. Hunter, H.A. Goldberg,
Bone sialoprotein expression enhances osteoblast differentiation and matrix min-
eralization in vitro, Bone 41 (2007) 462–473.
[35] P. Ducy, C. Desbois, B. Boyce, G. Pinero, B. Story, C. Dunstan, et al., Increased bone
formation in osteocalcin-deficient mice, Nature 382 (1996) 448–452.
[36] K. Merry, R. Dodds, A. Littlewood, M. Gowen, Expression of osteopontin mRNA by
osteoclasts and osteoblasts in modelling adult human bone, J. Cell Sci. 104 (1993)
1013–1020.
[37] S.R. Rittling, H.N. Matsumoto, M.D. Mckee, A. Nanci, X.R. An, K.E. Novick, et al., Mice
lacking osteopontin show normal development and bone structure but display al-
tered osteoclast formation in vitro, J. Bone Miner. Res. 13 (1998) 1101–1111.
[38] H. Yoshitake, S.R. Rittling, D.T. Denhardt, M. Noda, Osteopontin-deficient mice are
resistant to ovariectomy-induced bone resorption, Proc. Natl. Acad. Sci. U. S. A. 96
(1999) 8156–8160.
[39] M. Ishijima, K. Tsuji, S.R. Rittling, T. Yamashita, H. Kurosawa, D.T. Denhardt, et al.,
Resistance to unloading-induced three-dimensional bone loss in osteopontin-
deficient mice, J. Bone Miner. Res. 17 (2002) 661–667.
[40] M. Mizuno, Y. Kuboki, Osteoblast-related gene expression of bone marrow cells
during the osteoblastic differentiation induced by type I collagen, J. Biochem. 129
(2001) 133–138.
[41] S. Mathews, R. Bhonde, P.K. Gupta, S. Totey, Extracellular matrix protein mediated
regulation of the osteoblast differentiation of bone marrow derived human mesen-
chymal stem cells, Differentiation 84 (2012) 185–192.
[42] E. Hinoi, S. Fujimori, L. Wang, H. Hojo, K. Uno, Y. Yoneda, Nrf2 negatively regulates
osteoblast differentiation via interfering with Runx2-dependent transcriptional ac-
tivation, J. Biol. Chem. 281 (2006) 18015–18024.
[43] J. Pratap, M. Galindo, S.K. Zaid, D. Vradii, B.M. Bhat, J.A. Robinson, et al., Cell growth
regulatory role of Runx2 during proliferative expansion of preosteoblasts, Cancer
Res. 63 (2003) 5357–5362.
[44] V. Geoffroy, M. Kneissel, B. Fournier, A. Boyde, P. Matthias, High bone resorption in
adult aging transgenic mice overexpressing cbfa1/runx2 in cells of the osteoblastic
lineage, Mol. Cell. Biol. 22 (2002) 6222–6233.
[45] R.T. Franceschi, G. Xiao, D. Jiang, R. Gopalakrishnan, S. Yang, E. Reith, Multiple sig-
naling pathways converge on the Cbfa1/Runx2 transcription factor to regulate os-
teoblast differentiation, Connect. Tissue Res. 44 (2003) 109–116.
[46] P. Ducy, R. Zhang, V. Geoffroy, A.L. Ridall, G. Karsenty, Osf2/Cbfa1: a transcriptional
activator of osteoblast differentiation, Cell 89 (1997) 747–754.
[47] K. Nakashima, X. Zhou, G. Kunkel, Z. Zhang, J.M. Deng, R.R. Behringer, et al., The
novel zinc finger-containing transcription factor osterix is required for osteoblast
differentiation and bone formation, Cell 108 (2002) 17–29.
[48] M.F. Pittenger, A.M. Mackay, S.C. Beck, R.K. Jaiswal, R. Douglas, J.D. Mosca, et al.,
Multilineage potential of adult human mesenchymal stem cells, Science 284
(1999) 143–147.
[49] D. Prusty, B.H. Park, K.E. Davis, S.R. Farmer, Activation of MEK/ERK signaling pro-
motes adipogenesis by enhancing peroxisome proliferator-activated receptor
gamma (PPARgamma) and C/EBPalpha gene expression during the differentiation
of 3T3-L1 preadipocytes, J. Biol. Chem. 277 (2002) 46226–46232.
[50] L. Song, J. Zhao, X. Zhang, H. Li, Y. Zhou, Icariin induces osteoblast proliferation, dif-
ferentiation and mineralization through estrogenre ceptor-mediated ERK and JNK
signal activation, Eur. J. Pharmacol. 714 (2013) 15–22.
[51] L. Li, Z. Zeng, G. Cai, Comparison of neoeriocitrin and naringin on proliferation and
osteogenic differentiation in MC3T3-E1, Phytomedicine 18 (2011) 985–989.
[52] H.Y. Yoon, S.I. Yun, B.Y. Kim, Q. Jin, E.R. Woo, S.Y. Jeong, et al., Poncirin promotes
osteoblast differentiation but inhibits adipocyte differentiation in mesenchymal
stem cells, Eur. J. Pharmacol. 664 (2011) 54–59.
[53] S. Srivastava, R. Bankar, P. Roy, Assessment of the role of flavonoids for inducing os-
teoblast differentiation in isolated mouse bone marrow derived mesenchymal
stem cells, Phytomedicine 20 (2013) 683–690.
[54] M.A. Satue´, M.D.M. Arriero, M. Monjo, J.M. Ramis, Quercitrin and taxifolin stimu-
late osteoblast differentiation in MC3T3-E1 cells and inhibit osteoclastogenesis in
RAW 264.7 cells, Biochem. Pharmacol. 86 (2013) 1476–1486.
[55] H. Sun, L. Li, A. Zhang, N. Zhang, H. Lv, W. Sun, et al., Protective effects of sweroside
on human MG-63 cells and rat osteoblasts, Fitoterapia 84 (2013) 174–179.
[56] W.S. Simonet, D.L. Lacey, C.R. Dunstan, M. Kelley, M.S. Chang, R. Lüthy, et al.,
Osteoprotegerin: a novel secreted protein involved in the regulation of bone den-
sity, Cell 89 (1997) 309–319.
[57] D.L. Lacey, E. Timms, H.L. Tan, M.J. Kelley, C.R. Dunstan, T. Burgess, et al., Osteopro-
tegerin ligand is a cytokine that regulates osteoclast differentiation and activation,
Cell 93 (1998) 165–176.
[58] T. Ikeda, M. Utsuyama, K. Hirokawa, Expression profiles of receptor activator of nu-
clear factor κB ligand, receptor activator of nuclear factor κB, and osteoprotegerin
messenger RNA in aged and ovariectomized rat bones, J. Bone Miner. Res. 16
(2001) 1416–1425.
 J. An et al. / Life Sciences 147 (2016) 46–58
[59] K. S, The OPG/RANKL/RANK system, Endocrinology 142 (2001) 5050–5055.
[60] F. Li, Y. Yang, P. Zhu, W. Chen, D. Qi, X. Shi, et al., Echinacoside promotes bone re-
generation by increasing OPG/RANKL ratio in MC3T3-E1 cells, Fitoterapia 83
(2012) 1443–1450.
[61] H. Xiao, Q. Gao, Y. Zhang, K.C. Wong, Y. Dai, X. Yao, et al., Vanillic acid exerts
oestrogen-like activities in osteoblast-like UMR 106 cells through MAP kinase
(MEK/ERK)-mediated ER signaling pathway, J. Steroid Biochem. Mol. Biol. 144
(2014) 382–391.
[62] M.B. Kim, Y. Song, J.K. Hwang, Kirenol stimulates osteoblast differentiation through
activation of the BMP and Wnt/β-catenin signaling pathways in MC3T3-E1 cells,
Fitoterapia 98 (2014) 59–65.
[63] E.M. Choi, Honokiol isolated from Magnolia officinalis stimulates osteoblast func-
tion and inhibits the release of bone-resorbing mediators, Int. Immunopharmacol.
11 (2011) 1541–1545.
[64] Y.S. Lee, E.M. Choi, Apocynin stimulates osteoblast differentiation and inhibits
bone-resorbing mediators in MC3T3-E1 cells, Cell. Immunol. 270 (2011) 224–229.
[65] W. Tiyasatkulkovit, N. Charoenphandhu, K. Wongdee, J. Thongbunchoo, N.
Krishnamra, S. Malaivijitnondc, Upregulation of osteoblastic differentiation marker
mRNA expression in osteoblast-like UMR106 cells by puerarin and phytoestrogens
from Pueraria mirifica, Phytomedicine 19 (2012) 1147–1155.
[66] T.P. Hsieh, S.Y. Sheu, J.S. Sun, M.H. Chen, M.H. Liu, Icariin isolated from epimedium
pubescens regulates osteoblasts anabolism through BMP-2, SMAD4, and Cbfa1 ex-
pression, Phytomedicine 17 (2010) 414–423.
[67] H.H. Xiao, C.Y. Fung, S.K. Mok, K.C. Wong, M.X. Ho, X.L. Wang, et al., Flavonoids
from herba epimedii selectively activate estrogen receptor alpha (ERα)
andstimulate ER-dependent osteoblastic functionsin UMR-106 cells, J. Steroid
Biochem. Mol. Biol. 143 (2014) 141–151.
[68] L. Chang, M. Karin, Mammalian MAP kinase signalling cascades, Nature 410 (2001)
37–40.
[69] T. Akune, S. Ohba, S. Kamekura, M. Yamaguchi, U. Chung, N. Kubota, et al., PPARγ
insufficiency enhances osteogenesis through osteoblast formation from bone mar-
row progenitors, J. Clin. Investig. 113 (2004) 846–855.
[70] E. Hu, J. Kim, P. Sarraf, B. Spiegelman, Inhibition of adipogenesis through
MAP kinase-mediated phosphorylation of PPARgamma, Science 274 (1996)
2100–2103.
[71] M. Aouadi, K. Laurent, M. Prot, Y.L.M. Brustel, B. Binétruy, F. Bost, Inhibition of p38
MAPK increases adipogenesis from embryonic to adult stages, Diabetes 55 (2006)
281–289.
[72] M.B. Greenblatt, J.H. Shim, W. Zou, D. Sitara, M. Schweitzer, D. Hu, et al., The p38
MAPK pathway is essential for skeletogenesis and bone homeostasis in mice,
J. Clin. Investig. 120 (2010) 2457–2473.
[73] C. Ge, G. Xiao, D. Jiang, R.T. Franceschi, Critical role of the extracellular signal-
regulated kinase-MAPK pathway in osteoblast differentiation and skeletal devel-
opment, J. Cell Biol. 176 (2007) 709–718.
[74] X. Wang, C.H. Goh, B. Li, p38 Mitogen-activated protein kinase regulates osteoblast
differentiation through osterix, Endocrinology 148 (2007) 1629–1637.
[75] C.H. Lee, Y.L. Huang, J.F. Liao, W.F. Chiou, Ugonin K promotes osteoblastic differen-
tiation and mineralization by activation of p38 MAPK- and ERK-mediated expres-
sion of Runx2 and osterix, Eur. J. Pharmacol. 668 (2011) 383–389.
[76] M.J. Don, L.C. Lin, W.F. Chiou, Neobavaisoflavone stimulates osteogenesis via p38-
mediated up-regulation of transcription factors and osteoid genes expression in
MC3T3-E1 cells, Phytomedicine 19 (2012) 551–561.
[77] D. Xu, L. Xu, C. Zhou, W.Y.W. Lee, T. Wu, L. Cui, et al., Salvianolic acid B promotes
osteogenesis of human mesenchymalstem cells through activating ERK signaling
pathway, Int. J. Biochem. Cell Biol. 51 (2014) 1–9.
[78] Y.S. Lee, E.M. Choi, Costunolide stimulates the function of osteoblastic MC3T3-E1
cells, Int. Immunopharmacol. 11 (2011) 712–718.
[79] C.H. Tang, R.S. Yang, M.Y. Chien, C.C. Chen, W.M. Fu, Enhancement of bone
morphogenetic protein-2 expression and bone formation by coumarin derivatives
via p38 and ERK-dependent pathway in osteoblasts, Eur. J. Pharmacol. 579 (2008)
40–49.
[80] J.H. Kwak, S.R. Lee, H.J. Park, H.E. Byun, E.H. Sohn, B.O. Kim, et al., Kobophenol A en-
hances proliferation of human osteoblast-like cells with activation of the p38 path-
way, Int. Immunopharmacol. 17 (2013) 704–713.
[81] T. Sakou, T. Onishi, T. Yamamoto, T. Nagamine, T.K. Sampath, P.T. Dijke, Localization
of Smads, the TGF-β family intracellular signaling components during endochon-
dral ossification, J. Bone Miner. Res. 14 (1999) 1145–1152.
[82] A. Nohe, E. Keating, P. Knaus, N.O. Petersen, Signal transduction of bone morphoge-
netic protein receptors, Cell. Signal. 16 (2004) 291–299.
[83] U. Styrkarsdottir, J.B. Cazier, A. Kong, O. Rolfsson, H. Larsen, E. Bjarnadottir, et al.,
Linkage of osteoporosis to chromosome 20p12 and association to BMP2, PLoS
Biol. 1 (2003), e69.
[84] M.H. Lee, Y.J. Kim, H.J. Kim, H.D. Park, A.R. Kang, H.M. Kyung, et al., BMP-2-induced
Runx2 expression is mediated by Dlx5, and TGF-beta 1 opposes the BMP-2-
induced osteoblast differentiation by suppression of Dlx5 expression, J. Biol.
Chem. 278 (2003) 34387–34394.
[85] T.F. Day, X. Guo, L.G. Beal, Y. Yang, Wnt/β-catenin signaling in mesenchymal pro-
genitors controls osteoblast and chondrocyte differentiation during vertebrate
skeletogenesis, Dev. Cell 8 (2005) 739–750.
[86] M. Shtutman, J. Zhurinsky, I. Simcha, C. Albanese, M. D'amico, R. Pestell, et al., The cy-
clin D1 gene is a target of the β-catenin/LEF-1 pathway, Proc. Natl. Acad. Sci. U. S. A.
96 (1999) 5522–5527.
[87] N. Ghosh-Choudhury, C.C. Mandal, G.G. Choudhury, Statin-induced Ras activation
integrates the phosphatidylinositol 3-kinase signal to Akt and MAPK for bone mor-
phogenetic protein-2 expression in osteoblast differentiation, J. Biol. Chem. 282
(2007) 4983–4993.
57
[88] J.Q. Feng, L. Xing, J.H. Zhang, M. Zhao, D. Horn, J. Chan, et al., NF-κB specifically ac-
tivates BMP-2 gene expression in growth plate chondrocytes in vivo and in a chon-
drocyte cell line in vitro, J. Biol. Chem. 278 (2003) 29130–29135.
[89] W. Liang, M. Lin, X. Li, C. Li, B. Gao, H. Gan, et al., Icariin promotes bone formation
via the BMP-2/Smad4 signal transduction pathway in the hFOB 1.19 human oste-
oblastic cell line, Int. J. Mol. Med. 30 (2012) 889–895.
[90] J.B. Wu, Y.C. Fong, H.Y. Tsai, Y.F. Chen, M. Tsuzuki, C.H. Tang, Naringin-induced
bone morphogenetic protein-2 expression via PI3K, Akt, c-Fos/c-Jun and AP-1
pathway in osteoblasts, Eur. J. Pharmacol. 588 (2008) 333–341.
[91] D.Z. Tang, F. Yang, Z. Yang, J. Huang, Q. Shi, D. Chen, et al., Psoralen stimulates os-
teoblast differentiation through activation of BMP signaling, Biochem. Biophys. Res.
Commun. 405 (2011) 256–261.
[92] D.Z. Tang, W. Hou, Q. Zhou, M. Zhang, J. Holz, T.J. Sheu, et al., Osthole stimulates os-
teoblast differentiation and bone formation by activation of b-catenin-BMP signal-
ing, J. Bone Miner. Res. 25 (2010) 1234–1245.
[93] S.U. Lee, H.K. Shin, Y.K. Min, S.H. Kim, Emodin accelerates osteoblast differentiation
through phosphatidylinositol 3-kinase activation and bone morphogenetic
protein-2 gene expression, Int. Immunopharmacol. 8 (2008) 741–747.
[94] J. Chen, N. Zhang, G. Mao, X. He, Y. Zhan, H. Deng, et al., Salidroside stimulates os-
teoblast differentiation through BMP signaling pathway, Food Chem. Toxicol. 62
(2013) 499–505.
[95] T. Kihara, S. Ichikawa, T. Yonezawa, J.-W. Lee, T. Akihisa, J.T. Woo, et al., Acerogenin
A, a natural compound isolated from Acer nikoense Maxim, stimulates osteoblast
differentiation through bone morphogenetic protein action, Biochem. Biophys.
Res. Commun. 406 (2011) 211–217.
[96] K.D. Wright, V. Cavailles, S.A. Fuqua, V.C. Jordan, J.A. Katzenellenbogen, K.S. Korach,
et al., International union of pharmacology. LXIV. Estrogen receptors, Pharmacol.
Rev. 58 (2006) 773–781.
[97] R.T. Turner, B.L. Riggs, T.C. Spelsberg, Skeletal effects of estrogen, Endocr. Rev. 15
(1994) 275–300.
[98] B.J. Cheskis, J.G. Greger, S. Nagpal, L.P. Freedman, Signaling by estrogens, J. Cell.
Physiol. 213 (2007) 610–617.
[99] M. Marino, M. Pellegrini, P.L. Rosa, F. Acconcia, Susceptibility of estrogen receptor
rapid responses to xenoestrogens: physiological outcomes, Steroids 77 (2012)
910–917.
[100] S. Kato, H. Endoh, Y. Masuhiro, T. Kitamoto, S. Uchiyama, H. Sasaki, et al., Activation
of the estrogen receptor through phosphorylation by mitogen-activated protein ki-
nase, Science 270 (1995) 1491–1494.
[101] C.J. Gruberemail, D.M. Gruber, I.M.L. Gruber, F. Wieser, J.C. Huber, Anatomy of the
estrogen response element, Trends Endocrinol. Metab. 15 (2004) 73–78.
[102] A. Migliaccio, G. Castoria, M. Domenico, A. Falco, A. Bilancio, F. Auricchio, Src is an
initial target of sex steroid hormone action, Ann. N. Y. Acad. Sci. 963 (2002)
185–190.
[103] A. Zallone, Direct and indirect estrogen actions on osteoblasts and osteoclasts, Ann.
N. Y. Acad. Sci. 1068 (2006) 173–179.
[104] S. Kousteni, T. Bellido, L.I. Plotkin, C.A. O'brien, D.L. Bodenner, L. Han, et al.,
Nongenotropic, sex-nonspecific signaling through the estrogen or androgen recep-
tors: dissociation from transcriptional activity, Cell 104 (2001) 719–730.
[105] C.H. Lee, Y.L. Huang, J.F. Liao, W.F. Chiou, Ugonin K-stimulated osteogenesis in-
volves estrogen receptor-dependent activation of non-classical Src signaling path-
way and classical pathway, Eur. J. Pharmacol. 676 (2012) 26–33.
[106] M.H. Liao, Y.T. Tai, Y.G. Cherng, S.H. Liu, Y.A. Chang, P.I. Lin, et al., Genistein induces
oestrogen receptor-α gene expression in osteoblasts through the activation of
mitogen-activated protein kinases/NF-κB/activator protein-1 and promotes cell
mineralisation, Br. J. Nutr. 111 (2014) 55–63.
[107] Y. Wang, W.L. Wang, W.L. Xie, L.Z. Li, J. Sun, W.J. Sun, et al., Puerarin stimulates pro-
liferation and differentiation and protects against celldeath in human osteoblastic
MG-63 cells via ER-dependent MEK/ERK and PI3K/Akt activation, Phytomedicine
20 (2013) 787–796.
[108] Z. Dai, Y. Li, L.D. Quarles, T. Song, W. Pan, H. Zhou, et al., Resveratrol enhances pro-
liferation and osteoblastic differentiation in human mesenchymal stem cells via
ER-dependent ERK1/2 activation, Phytomedicine 14 (2007) 806–814.
[109] S.C. Manolagas, From oestrogen-centric to aging and oxidative stress: a revised
perspective of the pathogenesis of osteoporosis, Endocr. Rev. 31 (2010) 266–300.
[110] O.F. Sendur, Y. Turan, E. Tastaban, M. Serter, Antioxidant status in patients with os-
teoporosis: a controlled study, Joint Bone Spine 76 (2009) 514–518.
[111] X. Bai, D. Lu, A. Liu, Z. Zhang, X. Li, Z. Zou, et al., Reactive oxygen species stimulates
receptor activator of NF-kappaB ligand expression in osteoblast, J. Biol. Chem. 280
(2005) 17497–17506.
[112] H. Ha, H.B. Kwak, S.W. Lee, H.M. Jin, H.M. Kim, H.H. Kim, et al., Reactive oxygen
species mediate RANK signaling in osteoclasts, Exp. Cell Res. 301 (2004)
119–127.
[113] K.H. Baek, K.W. Oh, W.Y. Lee, S.S. Lee, M.K. Kim, H.S. Kwon, et al., Association of ox-
idative stress with postmenopausal osteoporosis and the effects of hydrogen per-
oxide on osteoclast formation in human bone marrow cell cultures, Calcif. Tissue
Int. 87 (2010) 226–235.
[114] J.K. Zhang, L. Yang, G.L. Meng, J. Fan, J.Z. Chen, Q.Z. He, et al., Protective effect of
tetrahydroxystilbene glucoside against hydrogen peroxide-induced dysfunction
and oxidative stress in osteoblastic MC3T3-E1 cells, Eur. J. Pharmacol. 689 (2012)
31–37.
[115] Q. Huang, J. Shi, B. Gao, H.Y. Zhang, J. Fan, X.J. Li, et al., Gastrodin: an ancient
Chinese herbal medicine as a source for anti-osteoporosis agents via reducing re-
active oxygen species, Bone 73 (2015) 132–144.
[116] K.S. Suh, E.M. Choi, Y.S. Lee, Y.S. Kim, Protective effect of albiflorin against
oxidative-stress-mediated toxicity in osteoblast-like MC3T3-E1 cells, Fitoterapia
89 (2013) 33–41.
58 J. An et al. / Life Sciences 147 (2016) 46–58
[117] Q. Huang, B. Gao, Q. Jie, B.Y. Wei, J. Fan, H.Y. Zhang, et al., Ginsenoside-Rb2 displays
anti-osteoporosis effects through reducing oxidative damage and bone-resorbing
cytokines during osteogenesis, Bone 66 (2014) 306–314.
[118] Q. Huang, B. Gao, L. Wang, H.Y. Zhang, X.J. Li, J. Shi, et al., Ophiopogonin D: a new
herbal agent against osteoporosis, Bone 74 (2015) 18–28.
[119] S.J. Wimalawansa, Nitric oxide: novel therapy for osteoporosis, Expert. Opin.
Pharmacother. 9 (2008) 3025–3044.
[120] S. Gutiérrez, J.P. Petiti, L.D.V. Sosa, L. Fozzatti, A.L.D. Paul, A.M. Masini-Repiso, et al.,
17β-Oestradiol acts as a negative modulator of insulin-induced lactotroph cell pro-
liferation through oestrogen receptor alpha, via nitric oxide/guanylyl cyclase/
cGMP, Cell Prolif. 43 (2010) 505–514.
[121] L.C. Cantley, The phosphoinositide 3-kinase pathway, Science 296 (2002)
1655–1657.
[122] S. Dimmeler, I. Fleming, B. Fisslthaler, C. Hermann, R. Busse, A.M. Zeiher, Activation
of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation,
Nature 399 (1999) 601–605.
[123] S. Rokutanda, T. Fujita, N. Kanatani, C.A. Yoshida, H. Komori, W. Liu, et al., Akt reg-
ulates skeletal development through GSK3, mTOR, and FoxOs, Dev. Biol. 328
(2009) 78–93.
[124] Y.K. Zhai, X.Y. Guo, B.F. Ge, P. Zhen, X.N. Ma, J. Zhou, et al., Icariin stimulates the os-
teogenic differentiation of rat bone marrow stromal cells via activating the PI3K–
AKT–eNOS–NO–cGMP–PKG, Bone 66 (2014) 189–198.
[125] S.R. Lee, J.H. Kwak, D.S. Park, S. Pyo, Protective effect of kobophenol A on nitric
oxide-induced cell apoptosis in human osteoblast-like MG-63 cells: involvement
of JNK, NF-κB and AP-1 pathways, Int. Immunopharmacol. 11 (2011) 1251–1259.