Naturally Plant-Derived Compounds: Role in Bone Anabolism

14 Pages·2012·0.3 MB·English

Current Molecular Pharmacology, 2012, 5, 205-218 205
Naturally Plant-Derived Compounds: Role in Bone Anabolism
Marie-Noëlle Horcajada* and Elizabeth Offord
Nestle Research Center, Nutrition and Health, Vers Chez les Blanc, 1000 Lausanne 26, Switzerland
Abstract: From a nutritional point of view, several factors are involved in ensuring optimal bone health. The most
documented of these are calcium and vitamin D. However, it is now well acknowledged that some phytochemicals, also
known as phytonutrients, which are plant-based compounds that are present in our daily diet, can positively regulate a
number of physiological functions in mammalian systems involved in chronic diseases such as osteoporosis.
Indeed, emerging data in animal models of postmenopausal osteoporosis has shown that exposure to some of these
naturally plant-derived compounds (e.g. flavonoids) positively influences bone metabolism through preserved bone
mineral density. In vitro experiments with bone cells have reported cellular and molecular mechanisms of phytonutrients
involved in bone metabolism. Indeed, phytonutrients and especially polyphenols can act on both osteoblasts and
osteoclasts to modulate bone metabolism, a balance between both cell type activities being required for bone health
maintenance. To date, most studies investigating the effects of polyphenols on osteoblast cells have reported involvement
of complex networks of anabolic signalling pathways such as BMPs or estrogen receptor mediated pathways.
This review will report on the interaction between phytochemicals and bone metabolism in cell or animal models with a
particular focus on the molecular mechanisms involved in the bone anabolic response.
Keywords: Anabolism, bone, flavonoids, osteoblast, phytochemicals, signalling.
INTRODUCTION implicated in some of the positive links found between fruit
and vegetable intake and higher bone mineral density in
The importance of adequate calcium and vitamin D adults and children. [4, 5]. Nevertheless, no long-term
intakes for bone health is now well established. However, in intervention studies in humans have investigated the effect of
addition to essential macro and micronutrients, the human specific phytochemicals on the prevention of bone loss in
diet contains a complex array of non-nutrient natural postmenopausal women, except for phytoestrogens.
bioactive molecules that may influence and protect bone. However, some data in animal models of postmenopausal
Currently, most pharmacological approaches focus on osteoporosis are available, where consumption of some of
inhibiting bone resorption, with only one or two anabolic these naturally occurring plant-derived compounds has been
agents (strontium, PTH). Similarly, until recently, the main shown to positively influence bone anabolism through
effects of nutrition, in terms of calcium and vitamin D, have preserved bone mineral density. Moreover, in vitro
focussed on prevention of bone loss. However, recent experiments with bone cells have reported cellular and
research has shown potential anabolic effects of non-nutrient molecular mechanisms of phytonutrients involved in bone
bioactives [1]. metabolism. Indeed, phytonutrients and especially
polyphenols can act on both osteoblasts and osteoclasts to
Phytochemicals, also known as phytonutrients, are plant-
modulate bone metabolism.
based compounds that are present in our daily diet through
fruit and vegetables; beverages such as fruit juices, green or This review will focus on the interaction between
black tea, coffee; beans and grains. They are usually phytochemicals and bone metabolism in cell or animal
classified as: phenolics (flavonoids, phenolic acids, tannins, models. It will mainly address the action of polyphenols and
stilbenes, coumarins and lignans, carotenoids, phytosterols, more particularly flavonoids which represent the most
alkaloids, terpenes and sulfur-containing compounds studied sub-group of phytochemicals regarding human
(sulfides and glucosinolates). To date, between 5’000 and health. In addition, the few data available reporting an effect
10’000 phytochemicals have been identified in the food we of other phytonutrients or plant extracts on bone metabolism
consume. It has been estimated that the average diet contains will also be commented on. Finally, the potential
a daily dose of about 1.5 g [2]. mechanisms by which these plant compounds may modulate
osteoblastic function responsible for bone formation will be
It is now well acknowledged that these compounds can
discussed.
regulate a number of physiological functions in mammalian
systems and thus, they may offer beneficial effects in
slowing down or protecting against chronic diseases, BONE METABOLISM
including cardiovascular disease, cancer, as well as
osteoporosis [3]. Indeed, these compounds may be Normal bone consists of a hard outer shell (the cortex)
and an inner network of spicules (fibers), called trabeculae
(cancellous bone). The arrangement of compact and

*Address correspondence to this author at the Nestle Research Center, cancellous bone provides strength and density suitable for
Nutrition and Health, Vers Chez les Blanc, 1000 Lausanne 26, Switzerland; mobility and protection [6]. All bones consist of a solid
Tel: [phone]; Fax: [phone]; mineral phase in close relation with an organic matrix. The
E-mail: [email protected]
mineral phase is made up of crystalline hydroxyapatite

1874-4(cid:26)(cid:19)2/12 $58.00+.00 © 2012 Bentham Science Publishers
206 Current Molecular Pharmacology, 2012, Vol. 5, No. 2 Horcajada and Offord
containing calcium, phosphate and hydroxide, the organic osteonectin, osteopontin, osteocalcin, and bone sialoprotein
bone matrix comprises 95 % of Type I collagen and 5 % of was decreased despite the fact that the Runx2 mRNA level
non-collagenous proteins secreted by bone forming cells remained unchanged compared to wild-type mice. In
(osteopontin, osteocalcin, sialoproteins...). Some of these contrast, the expression of Osterix was diminished in
proteins organize collagen fibrils; others initiate Runx2/Cbfa1 null mice. These data suggested that Osterix is
mineralization and binding of the mineral phase to the matrix downstream of Runx2 during osteoblast differentiation [16].
[6]. During growth, bone develops by remodelling and On the other hand, BMP-2-induced Osterix expression can
replacing previously calcified cartilage (endochondral bone be independent of Runx2. Indeed, Osterix expression was
formation) or is formed without a cartilage matrix still induced by BMP-2 treatment in Runx2 null cells [17].
(intramembranous bone formation). In adults, the process is Furthermore, it has been shown that during osteogenic
stopped after closure of the growth plates. Peak bone mass is lineage progression, in addition to the BMP/Smad pathway,
attained in early adult life (20-30 years of age). However, IGF-l and MAPK signalling can mediate Osterix expression
bone is a dynamic tissue which is constantly remodeled [12].
throughout adulthood in order to ensure adaptation of the
AP-1 and NF-κB are also implicated in regulation of
skeleton to its functional needs, to repair microdamage and
genes involved in many important biological processes in
to maintain calcium homeostasis [7]. Indeed, nearly 4% of
osteoblasts as well as osteoclasts. ATF, Jun and Fos are part
the surface of trabecular bone is involved in active
of the dimeric transcription factor complex AP-1 and many
resorption, whereas 10 to 15% of trabecular surfaces are
bone-specific genes like alkaline phosphatise (ALP),
covered with osteoid tissue. Radioisotope studies indicate
osteocalcin and collagen I have AP-1 sites in their promoters
that around 18% of the total calcium in adult skeleton is
[18]. NF-κB, a dimeric transcription factor composed of Rel
deposited and removed each year [6]. Bone loss occurs in all
(cRel), RelA (p65), RelB, NF-κB1 (p50) and NF-κB2 (p52)
individuals after mid-life. The mechanisms of bone loss are
recognizes a common DNA sequence (κB site). AP-1 and
essentially due to changes in bone cell activity during bone
NF-κB transcription factors involved in MAPK signalling
remodelling [8] causing an imbalance between bone
pathways are regulated by different mechanisms, but it
resorption and formation. Estrogen has a central role to play
appears that they could be activated simultaneously by the
in normal physiological remodelling, and estrogen
same agents. Thus these transcription factors work together
deficiency after the menopause results in a remodelling
and may modulate the activity of each other [19].
imbalance [9], inducing bone mineral density loss leading to
bone disorders such as osteoporosis and increased fracture Expression of all these transcription factors is known to
risk. be modulated by several hormones (parathyroid hormone,
PTH; estrogens; glucocorticoids; 1,25-dihydroxy vitamin D)
The remodelling process is mainly due to various cell
[20] or signalling molecules belonging to the growth factors
types that play crucial roles in bone turnover [8]. The three
super family (bone morphogenetic proteins, BMPs;
main types of bone cells are: osteoblasts, osteocytes and
transforming growth factor β, TGFβ; insulin-like growth
osteoclasts, bone formation being supported by osteoblasts,
factor 1, IGF-1; fibroblast growth factor 2, FGF-2) [21].
while resorption by osteoclasts. Bone anabolism is a tightly
Various pathways such as BMP, TGF-β, IGF, FGF,
regulated process characterized by a sequence of events
Hedgehog, Wnt-LRP5 and MAPK have been indeed
starting by the commitment of osteoprogenitor cells from
implicated in bone metabolism [13, 14, 22-24] (Fig. 1).
mesenchymal cells, their differentiation into immature pre-
osteoblasts and then into mature functional osteoblasts [10]. In particular, several studies have shown that bone
Osteoblasts synthesize almost all constituents of the bone morphogenetic proteins (BMPs) induce osteoblast
matrix and direct its subsequent mineralization. Once a differentiation and play an important role in
phase of active bone formation is completed, the osteoblasts osteogenesis[26]. BMP regulatory molecules can transmit
do not become senescent but instead redifferentiate into signals through Smad-dependent and Smad-independent
osteocytes (or bone lining cells). In the process of pathways, including ERK, JNK, and p38 MAP kinase
osteoblastogenesis, different stages are involved including pathways [27]. Members of the BMP family can bind to
proliferation, extracellular matrix synthesis, maturation and various serine/threonine kinase receptors I and II which are
mineralization. Each stage is regulated by the coordinated necessary for signal transduction [28]. Moreover, cross-talk
expression of major transcription factors [10]. The most occurs between BMP and other signalling pathways such as
important transcription factors controlling osteoblast TGF-β, Notch, Wnt, STAT and MAPK [27]. Since BMPs
differentiation are Runx2 (also known as Cbfa1), Osterix, β- stimulate new bone formation [29], indicating a clear role in
catenin, ATF4, AP-1 and C/EBPs. regulation of osteoblast function, they represent molecular
targets for identification of new agents to prevent
Cbfa1 regulates the expression of most genes expressed
osteoporosis [1].
in osteoblasts [11-13]. Indeed, bone morphogenetic protein
(BMP) signalling, well known to be implicated in osteoblast To date, several sequential molecular markers are
differentiation, may up-regulate expression of Runx2. This available and currently used to study the influence of
factor is known to activate the transcription of various compounds on osteoblast developmental stages or functions
osteoblast-related genes (leading to osteoblast such as alkaline phosphatase (ALP) collagen type I (Col1),
differentiation) [14, 15]. Another important transcription osteopontin (OPN) and bone sialoprotein (BSP) being early
factor involved in modulating osteoblast function is Osterix, markers of the osteoblast phenotype while osteocalcin
mediated by multiple signalling pathways [12]. In Osterix- (OCN) is used as a marker of mineralization [30].
null mutant mice [16], expression of osteoblast markers like
Plant Bioactives and Bone Anabolism Current Molecular Pharmacology, 2012, Vol. 5, No. 2 207

Fig. (1). Regulation of osteoblast functions: signalling pathways involved (adapted from Tzreciakiewicz et al. [25]).
Finally, osteoblasts can also regulate osteoclastogenesis on the structural elements bound to these rings. Thus,
and thus play a direct role in regulation of osteoclast polyphenols have been classified as phenolic acids,
function. Osteoclasts derive from hematopoietic progenitors flavonoids, stilbenes, tannins, coumarins and lignans (Fig.
(monocyte/macrophage lineage) in the bone marrow [31] and 2). Moreover among flavonoids, six subclasses exist and
are responsible for bone resorption and therefore for normal share a common structure of two aromatic rings bound
skeletal development (growth and modelling). Macrophage together by 3 carbon atoms that form an oxygenated
colony-stimulated factor (M-CSF) produced by heterocycle. These are flavones, flavonols, flavanones,
osteoblasts/stromal cells is required for osteoclast formation isoflavones, flavanols (catechins and proanthocyanidins) and
and development [32-34] as is the OPG/RANKL/RANK anthocyanidins [38].
triade. Indeed, osteoprotegerin (OPG) is a member of the
The chemical structure of compounds is related to their
tumor necrosis factor (TNF) receptor family. This protein
biological activity and thus activation of different signalling
was characterized as an essential osteoclast differentiation
pathways could be involved [39] in their mode of action,
factor expressed by osteoblastic/stromal cells [35, 36].
studied in in vitro models. Knowledge on phenolic
Osteoblasts produce OPG which acts as a decoy receptor for
metabolites generated in an organism after consumption is
receptor activation of nuclear factor-kappaB (RANK) ligand
important to choose the appropriate molecule for these
(RANKL) and thereby neutralizes its function in
studies. Indeed, the compounds abundant in plants are not
osteoclastogenesis. Indeed, RANKL activates osteoclasto-
exactly the same as those found in plasma. Neither aglycones
genesis by binding to RANK while OPG may inhibit the
(except for green tea catechins) nor forms found in the diet
activation of osteoclasts and promote osteoclast apoptosis.
reach the blood and tissue. The compounds, after
It is very important to maintain the balance between these consumption, are metabolized by the organism and/or
two cellular processes (formation and resorption), an microflora enzymes to conjugates of glucuronate or sulfate
imbalance being responsible for decreased bone mineral (with or without methylation across the catechol functional
density leading to an increase in the risk of osteoporosis group) during absorption to the bloodstream and/or
during aging [37]. Actually, in the last decade, most research eventually eliminated [40]. The circulating forms may
on possible bone health benefits of phytochemicals or possess different biological properties within cells and
naturally plant-derived compounds has focused on the tissues compared to polyphenol aglycones. However, at the
polyphenols. This review will focus on their effects on bone present time, there are few studies that have used conjugated
anabolism. forms in osteoblastic cells, many metabolites not being
commercially available due to problems of isolation and
synthesis.
POLYPHENOLS AND BONE ANABOLISM
Concerning polyphenols, despite the large number of
Polyphenols can be divided into different groups molecules identified, to date, most research on possible bone
depending on the number of phenol rings they contain and health benefits has focused on the flavonoids sub-group.
208 Current Molecular Pharmacology, 2012, Vol. 5, No. 2 Horcajada and Offord

Fig. (2). Chemical structures of polyphenols.
Isoflavones lactating ovariectomized rats [45] and in elderly female rats
[46]. A recent study by Ha et al. [47] showed increased
Isoflavones are flavonoids also called phytoestrogens
trabecular bone areas within the tibia and in the lumbar
because of their weak estrogenic activity [2]. Like most
vertebrae of OVX rats fed formononetin, daidzein or
flavonoids, they are present in leguminous plants mainly as
genistein (1 and10 mg/kg/day) compared to controls without
glycosides. The primary isoflavones in soybeans are isoflavones. In a further study in recently weaned Sprague-
genistein and daidzein and their respective β -glycosides, Dawley rats, Gautam et al[48], showed by dynamic
genistin and daidzin. Much lower amounts of glycitein and histomorphometry of bone that rats fed cladrin for 30
its glycoside, glycitin, are present in soybeans. Intestinal
consecutive days exhibited increased mineral apposition and
microflora can convert daidzein into several different
bone formation rates compared with controls whereas
metabolites including the isoflavonoid equol (7-
formononetin had no effect. A further study investigated a
hydroxyisoflavan) [41], which is more estrogenic than its once daily oral (by gavage) treatment for 30 consecutive
precursor daidzein, as shown in many in vitro studies and in days to recently weaned female Sprague-Dawley rats with
animal models [42]. However only 30-40 % of the Western each of these compounds at 10.0 mg kg-1 day-1 dose.
population consuming soyfoods are “equol producers” [41].
Cajanin increased bone mineral density (BMD) at all skeletal
Cladrin and formononetin are two structurally related
sites studied, bone biomechanical strength, mineral
methoxydaidzeins found in soy food and other natural
apposition rate (MAR) and bone formation rate (BFR),
sources. A further methoxyisoflavone, isoformononetin, is compared with control. BMD levels at various anatomic
found in a stem-bark extract of Butea monosperma [43]. positions were also increased with isoformononetin
Most of the bone studies on soy isoflavones have been compared with control however, its effect was less potent
performed in ovariectomized (OVX) female rodents, a model than cajanin. Isoformononetin had no effect on the
which mimics postmenopausal osteoporosis. Convincing parameters of bone biomechanical strength although it
data showing the significant improvement of trabecular enhanced MAR and BFR compared with control.
and/or cortical bone mineral density after soy protein or Isoformononetin had very mild uterotrophic effect, whereas
isolated isoflavone-enriched soy extract supplementation has cajanin was devoid of any such effect. This data suggests
been reported [44]. A beneficial effect of genistein on that cajanin is more potent than isoformononetin in
calcium content in the femur has been also described in accelerating peak bone mass achievement [49]. Finally,
Plant Bioactives and Bone Anabolism Current Molecular Pharmacology, 2012, Vol. 5, No. 2 209
Butea extract and its acetone soluble fraction, were recently and daidzein) on bone components was completely
shown to be effective in preventing OVX-induced bone loss prevented in the presence of cycloheximide, an inhibitor of
and stimulating new-bone formation [43]. protein synthesis, suggesting that their effect is dependent on
osteoblastic protein synthesis.
However, many other studies found minimal or no effect
of purified isoflavones on bone parameters (bone mineral Numerous in vitro studies with human and animal
density and bone biomarkers of remodelling activity) of osteoblasts or osteoblast-like cell lines have also been carried
ovariectomized rats [50-54]. out to explore the action of soy isoflavones on bone
formation. Genistein and daidzein have been found to have a
The possible role of soybean isoflavones in bone
stimulatory effect, with concentrations of 10-6 and 10-5 M, on
metabolism in castrated male rodents has also been assessed
DNA content, on protein synthesis and on alkaline
[55-58]. In the male skeleton with androgen deficiency,
phosphatase in mouse osteoblast-like cells MC3T3-E1 [65-
isoflavones seem to exert modest beneficial effects on bone
67]. In addition, soy isoflavones (effective concentrations:
mineral density.
10-7 – 10-5 M) have been shown to improve not only
Similar mixed results have been found with isoflavones differentiation but also mineralization in osteoblast cells [68,
in human intervention studies. i.e. some studies found a 69, 70]. Genistein and daidzein (10-5M) were able to inhibit
positive effect of isoflavones on preservation of bone TNF-α induced apoptosis and modulate the production of
mineral density in postmenopausal women while others IL-6 and prostaglandin E2 in MC3T3-E1 osteoblastic cells
found no effect. This may be due to the different sources or [71], suggesting that soybean isoflavones may play an
doses of isoflavones used whether that be in a purified form important role in bone remodelling. Moreover, genistein has
or part of soy extracts. In addition, the role of the gut been found to stimulate the production of osteoprotegerin by
microflora in the metabolism of isoflavones (e.g. conversion human trabecular osteoblasts [72]. In the same way,
of daidzein to equol) is important and can partly account for daidzein, at a concentration of 1 nM, may exert its anti-
responders vs non-responders [59]. resorptive action by regulating runx2/Cbfa1 production and
by stimulating the secretion of osteoprotegerin and RANK-
The estrogenic properties of isoflavones were
ligand [61]. Alternatively, the anabolic effects of daidzein in
investigated by Chen and co-workers [60] in two hFOB
primary cultures of osteoblasts could be mediated by an
osteoblastic cell lines expressing different levels of estrogen
increased production of BMPs, in particular BMP2 [73].
receptors (ERs). The expression and the production of
osteclastogenesis-regulatory cytokines such as interleukin 6 Mechanistic studies in osteoblasts on the effects of the
(Il-6) and osteoprotegerin (OPG) were dependent on the methoxydaidzeins, cladrin and formononetin showed that
level of estrogen receptors. The human fetal osteoblastic cell cladrin, at as low as 10 nM, maximally stimulated both
lines hFOB1.19 (400 ER/nucleus) and hFOB/ER9 (8000 osteoblast proliferation and differentiation by activating the
ER/nucleus) were treated with 10 nM 17β-estradiol, 0.1-10 MEK-Erk pathway whereas formononetin maximally
nM genistein and daidzein and/or 10 µM ICI (inhibitor of stimulated osteoblast differentiation at 100 nM involving the
estrogen receptor). Il-6 protein production was decreased p38 MAPK pathway but with no effect on osteoblast
about 30-40% in hFOB cells and 40-60% in hFOB/ER9 cells proliferation [48].
by isoflavone treatments in comparison to the control cells
Although the bone anabolic effect of isoflavones has
(hOB/ER9). The effect of genistein and daidzein was slightly
been mainly attributed to their ability to bind estrogen
dose-dependent and was greater in cells that were most
receptor (ERβ) present in osteoblastic cells, recently, new
abundant in ERs (hFOB/ER9) [60]. Moreover, it has been
mechanisms of action are emerging. Indeed, it has been
shown that daidzein could enhance the cell content of
ERβ which seems to be involved in the daidzein action in postulated that genistein may regulate osteoblast
differentiation by modulating the NF-κB pathway [74].
osteoblasts [61]
Yamaguchi et al. examined the effect of 17β-estradiol (10-9
From a mechanistic point of view, it has been shown that to 10-7 M) and genistein (10-6 to 10-5 M) on basal and TNFα-
isoflavones have anabolic effects on bone cells by stimulated NF-κB activity in the preosteoblastic cell line
stimulating osteoblasts and decreasing osteoclast MC3T3. While 17β-estradiol had no effect on basal NF-κB
functions[62]. The anabolic effect of genistein on bone activity in MC3T3 cells, it significantly antagonized NF-κB
metabolism was investigated in tissue culture using femoral- activity induced by TNFα (1 or 10 ng/ml). By contrast,
metaphyseal tissues obtained from elderly female rats [63]. genistein (10-6 or 10-5 M) significantly increased NF-κB
The presence of genistein (10-6 and 10-5 M) was found to activity, and showed no antagonistic effects on TNFα-
induce a significant increase in calcium content, alkaline induced NF-κB promoter activity. These studies suggest that
phosphatase activity and DNA content in bone tissues. The the estrogenic compounds, 17β-estradiol and genistein,
effect of genistein (10-5 M) was equal to the stimulatory mediate very different actions on osteoblastic cells. While
effect of 17 β-estradiol (10-10 and 10-9 M). These findings 17β-estradiol may stimulate bone anabolism, in part, by
suggest that the effect of genistein may be partly mediated antagonizing TNFα-induced NF-κB activation, genistein not
through estrogen-like action. The effect of daidzein was only fails to prevent cytokine-induced NF-κB activation, but
similar to that observed with genistein at the same dose in directly promotes NF-κB activation in MC3T3 cells. These
the same culture model [64]. However, the anabolic effect of data suggest important mechanistic differences in the
daidzein (10-5 M) on bone components was not significantly mechanisms by which 17β-estradiol and genistein promote
enhanced by the combination with genistein (10-5 M). In osteoblast differentiation. This may be partly due to the fact
addition, the stimulatory effect of both isoflavones (genistein that 17β-estradiol binds to ERα whereas isoflavones bind to
210 Current Molecular Pharmacology, 2012, Vol. 5, No. 2 Horcajada and Offord
ERβ and thus different downstream pathways may be These molecules are ubiquitous and considered as the second
differentially affected by estradiol compared to most abundant natural phenolic compounds after lignans.
phytoestrogens. Dietary intake of proanthocyanidins has been largely
unknown because of the lack of accurate data for their food
Mechanistic studies with the methoxyisoflavones,
content.
formononetin and isoformononetin, have shown modulation
of non ER-related pathways. Indeed, isoformononetin
Catechins
exhibited potent antiapoptotic effect in addition to promoting
osteoblast differentiation that involved parallel activation of
Until now, the potential impact of catechins on bone
MEK-Erk and Akt pathways [49].
metabolism has been essentially investigated using in vitro
Taken together, these studies indicates that isoflavones models. It has been demonstrated that a pre-treatment with
may positively influence osteoblast function, probably not (+)-catechin in embryonic mouse calvaria was able to induce
only through estrogen-like action but also via interaction a resistance to the action of bone resorbing agents [80]. A
with other signaling pathways such as NF-κB or MEK-Erk direct stimulatory effect on osteoblast growth through a
pathways depending on the subclass or metabolite of significant elevation of alkaline phosphatase activity in
isoflavones, and this is translated in enhanced bone osteoblastic MC3T3-E1 cells has also been reported [81]. In
anabolism, as also shown in pre-clinical studies. these conditions, treatment with (+)-catechin also decreased
bone-resorbing cytokines (TNF-α and IL-6) production.
Lignans However, the mediators of (+)-catechin action on bone are
still unclear and the cellular mechanism remains to be
The term lignan is used for a diverse class of elucidated.
phenylpropanoid dimers and oligomers. Secoisolariciresinol
and matairesinol are two lignan dimers that are not Epigallocatechin Gallate (EGCG)
estrogenic by themselves, but are readily converted by gut
microflora to mammalian lignans, enterodiol and Several epidemiological studies have reported reduced
enterolactone, which are estrogenic [75]. Flaxseed is the risk of hip fractures or higher bone mineral density in
richest food source of lignans. These phytoestrogens are also habitual tea drinkers [82-84]. Despite numerous reports on
present in measurable concentrations in many cereals, pulses, the benefits of tea on human health [85], the osteogenic
fruit and vegetables commonly consumed in the Western effects of tea have rarely been investigated. Moreover, the
diet. effective components in tea and their mechanisms of action
on bone remodelling remain unclear. (-)-epigallocatechin
In 3-month old ovariectomized rats fed with a basal diet
gallate (EGCG) was found to induce apoptotic cell death in
supplemented with 10 % of flaxseed, no effect on bone
osteoclast-like cells, in a dose-dependent manner (12.5 to
mineral density was induced but a positive impact on
100 µM), after a coculture with primary mouse osteoblastic
trabecular architecture was shown [76]. However, in
cells for 24h [86]. EGCG was also able to induce an
ovariectomized mice with human MCF-7 breast tumor
inhibition of MMP-9 expression, a matrix metalloproteinase
xenografts, treatment with enterodiol and enterolactone had
implicated in the digestion of bone collagen by osteoblasts
no effect on femoral bone mineral density and biomechanical
[87]. In this study, at the same dose, it was also observed that
strength [77]. Interestingly, it was found that female rat bone
EGCG was able to impair osteoclast formation as shown by
could be more sensitive to the estrogen-like action of lignans
a reduced number of TRAP-positive multinucleated cells.
from flaxseed during early life when endogenous levels of
EGCG has also been demonstrated to have a stimulatory
sex hormones are low [78]. Molecular mechanisms of action
effect on osteogenesis of murine bone marrow mesenchymal
on osteoblast metabolism however remain to be elucidated.
stem cells. This green tea catechin after 48 hours treatment,
In one study, it was reported that lignans were able to
at concentrations of 1 µM and 10 µM, significantly up-
increase ALP activity and collagen synthesis in a study with
regulated the expression of transcription factors Runx2 and
primary mouse osteoblasts, suggesting an effect on
Osterix as well as osteoblast markers ALP and osteocalcin
differentiation, mediated by the estrogen receptor [79].
[88]. On the contrary, runx2 expression was decreased by
EGCG in the SaOS-2 osteoblast–like cell line while
Flavanols
mineralization was increased [89]. The effect of EGCG on
prostaglandin F (PGF )-stimulated VEGF (vascular
Flavanols exist in both the monomer form (catechins) 2α 2α
endothelial growth factor) synthesis via SAPK/JNK
and the polymer form (proanthocyanidins). The main
activation was studied in MC3T3-E1 cells [90]. Pre-
flavanols include catechin, epicatechin, gallocatechin and
treatment with 10 µM EGCG amplified PGF -induced
epigallocatechin. When esterified with gallic acid, 2α
phosphorylation of c-jun which is a part of the AP-1
epicatechin gallate and epigallocatechin gallate (EGCG) are
complex and well known as a downstream effector of
formed. Catechin and epicatechin are found in many types of
SAPK/JNK [90]. A recent study assessed the effect of EGC
fruit but also in red wine, green tea (more than 80 % of green
on osteoblastic activity and it was shown to promote
tea polyphenol are catechins) and chocolate, whereas
osteoblastic activity, through stimulation of ALP production
gallocatechin, epigallocatechin (EGC) and epigallocatechin
and mineralization, in the rat osteoblast-like sarcoma cell
gallate (EGCG) occur in certain seeds of leguminous plants,
line UMR-106 [91]. A further interesting effect of EGCG is
in grapes and above all in tea. Concerning
its ability to inhibit growth and proliferation of
proanthocyanidins, also known as condensed tannins, they
correspond to dimers, oligomers and polymers of catechins.
Plant Bioactives and Bone Anabolism Current Molecular Pharmacology, 2012, Vol. 5, No. 2 211
chondrosarcoma cells in vitro by inducing apoptosis, partly main conjugate, quercetin-3-Glucuronide (5µM) were shown
mediated by decreased expression of Bcl-2 and Bax [92]. to stimulate bone sialoprotein expression, a noncollagenous
protein of the extracellular matrix in the mineralized tissue,
Taking all the in vitro data together, it appears that
in rat osteoblast-like cell line ROS17/2.8 [106]. Finally, in
flavanols show a potential anabolic effect on bone cells.
the MC3T3-E1 murine pre-osteoblastic cell line, kaempferol
However, the concentrations used in these in vitro
(at 10 and 20 µM levels) promoted the differentiation and
experiments are quite high with respect to the known
mineralization [107].
concentrations (from hundreds nanomolar to a few
micromolar) of EGCG aglycone and its conjugates found in Taken together, these results strongly indicate that
human plasma [93]. Moreover, demonstration of in vivo flavonols should be considered as bioactive anabolic
efficacy is required. molecules for bone. However, caution is required when
interpreting results from in vitro experiments, as it is
Flavonols necessary to consider both the concentration and the form
metabolite of flavonoid studied.
Flavonols including quercetin, kaempferol and myricetin
are generally present at relatively low concentrations of ~
Flavanones
15-30 mg/kg fresh wt [94]. The most important sources are
onions, curly kale, leeks, broccoli, apples, blueberries [95]. In human foods, flavanones are found at small quantities
Flavonols are mostly present in glycosylated forms in foods. in tomatoes and certain aromatic plants, but they are present
The flavonoid quercetin is one of the most studied at significant concentrations only in citrus fruit [94]. Orange
polyphenols and many conjugate forms (plant conjugates as juice may contain between 200 and 600 mg hesperidin/L and
well as mammalian conjugates) have been identified. For 15-85 mg narirutin/L [108]. Like other flavonoids,
instance, in plants, quercetin forms the glycosides quercitrin flavanones occur mostly as glycosides (hesperidin, naringin
and rutin with rhamnose and rutinose respectively. and narirutin) that must be hydrolysed by the gut microflora
before absorption of the released aglycones in the colon. The
Increasing data from in vivo and in vitro studies show
main aglycones are naringenin in grapefruit, hesperetin in
that the flavonols, quercetin and kaempferol, are bioactive
oranges and eriodictyol in lemons. Glucuronides were shown
molecules which may be able to counteract the bone
to be the major forms of circulating mammalian metabolites
deleterious effect of estrogen deficiency occurring during
of flavanones found in plasma after ingestion of orange juice
menopause. Indeed, it has been shown that rutin or pure
[109].
quercetin inhibit ovariectomy-induced osteopenia in rodents
[96, 97] as well as kaempferol [98]. As a result of an investigation by Mühlbauer et al.,
oranges were shown to significantly inhibit bone resorption
In vitro, it has been demonstrated that both quercetin and
in young male rats fed with a semi-purified diet to which
kaempferol aglycones exert a potent inhibitory effect on
oranges were added at a dosage per rat of 1g/day [110].
osteoclastic bone resorption and apoptosis in a rabbit long
Hesperidin was shown in Chiba’s study to inhibit femoral
bone osteoclast model [99], probably via a mechanism
bone loss when administrated in 2 month-old ovariectomized
involving inhibition of NFκB and AP-1, transcription factors
mice [111]. Identically, consumption of hesperidin (0.5% in
highly related to osteoclastic differentiation [100]. However,
the diet) inhibited ovariectomy-induced bone mineral density
Pang et al. demonstrated that kaempferol, but not quercetin,
loss in female rats after 3 months duration [112]. Moreover,
dose dependently (5-20 µM) inhibited TNF-α-induced
improved bioavailability of hesperidin (through cleavage of
secretion of IL-6 and MCP-1 (monocyte chemoattractant
rhamnose) resulted in a higher bone sparing effect in rodents
protein-1) in mouse primary calvaria osteoblasts, while both
[113]. Moreover, hesperidin and naringin may be two of the
flavonols inhibited RANKL-induced formation of
components responsible for the findings by Deyhim et al.
multinucleated osteoclasts and expression of osteoclastic
that citrus juice modulates bone strength in male
differentiation markers: RANK and osteocalcin receptor
orchidectomized senescent rats [114]. The mechanisms of
[101]. It was consistently demonstrated that quercetin (1-10
action of hesperetin and one glucuronide form were assessed
µM) could accelerate TNFα-mediated apoptosis in MC3T3-
in primary osteoblast cells and it was demonstrated that the
E1 cells via activation of caspases and the JNK pathway
aglycone form was able to induce osteoblast differentiation
[102]. DNA binding activity of AP-1 and nuclear
by upregulating Runx2 and osterix, expression, including
translocation of c-jun protein were increased after treating
enhanced Smads protein phosphorylation, thus implicating
the MC3T3-E1 cells with TNFα and these effects were
BMP signalling [115, 116]. In the same way, an osteogenic
augmented in the presence of quercetin [102]. Furthermore,
effect of naringin on the collagen matrix of rabbit bones was
Notoya et al. reported that quercetin aglycone (1-10 µM)
associated with enhanced BMP-2 production by bone
inhibited the proliferation, differentiation and mineralization
forming cells [117]. Wong et al. investigated the effect of
of rat calvaria osteoblast-like cells [103], whereas an
naringin in UMR 106 osteoblastic cell line [118]. The
increase in alkaline phosphatase activity in human osteoblast
experimental group consisted of cells cultured with different
cells (MG-63) was reported with both quercetin and
concentrations of naringin (0.001 µM, 0.01 µM and 0.1 µM)
kaempferol at 10 and 50 µM [104]. The authors suggested
for 24h, 48h and 72h. Results for the naringin group showed
that this effect was mediated through ERK and the estrogen
a dose dependent increase in total protein content. Naringin
receptor pathway. However, recent findings reported that
at 0.1 µM significantly enhanced ALP activity by up to 20
quercetin at lower concentrations (2-10 µM) enhanced
%.
osteogenic differentiation via a mechanism independent from
estrogen receptor activation [105]. Indeed, quercetin and its
212 Current Molecular Pharmacology, 2012, Vol. 5, No. 2 Horcajada and Offord
The currently available data suggests that, flavanones expression in rat calvarial osteoblast-like cells [127].
exert interesting anabolic bone effects. A number of Similarly, Chang et al. examined the effect of piceatannol - a
preclinical studies have confirmed an effect on bone mineral natural stilbene occurring in the skins of grapes, rhubarb and
density. Indeed, after isoflavones, the flavanones are the sugar cane - on the proliferation and differentiation of two
subgroup of polyphenols with most in vivo preclinical human osteoblastic cell lines (hFOB and MG-63) [128]. This
evidence for improved bone metabolism. However, further study highlighted the hypothesis that the BMP-2 signalling
studies, particularly in humans would be helpful to confirm pathway could play an important role in piceatannol-
these effects and thus to emphasize the role of nutrition in mediated cell maturation and differentiation in MG-63 and
osteoporosis prevention. hFOB cells.
Results from many studies have shown that polyphenols
Flavones
are able to influence bone anabolism by modulating
osteoblast differentiation and mineralization. These effects
Flavones are much less common in fruit and vegetables.
were mostly mediated through an increase in BMPs
Only celery and parsley are edible sources of glycosides of
production which in turn could activate phosphorylation of
luteolin and apigenin, the two main flavones [94].
specific proteins (Smads, p38, ERK1/2) or through an
Few data are available on the potential impact of flavones estrogen receptor pathway. Taken together, we can conclude
on bone metabolism. Only one preclinical study showed that polyphenols can affect osteoblast function in vitro, by
bone-protective effects of apigenin, in estrogen-deficient interacting with the complex network of anabolic signalling
ovariectomized rats administered at a dose of 10 mg/kg three pathways.
times a week for 15 weeks fed [119]. Recently, the
physiological effects of apigenin on bone cells were studied
BONE ANABOLIC EFFECT OF OTHER PHYTO-
[120]. Firstly, in the MC3T3-E1 mouse calvarial osteoblast
CHEMICALS OR PLANT EXTRACTS
cell line, apigenin was shown to inhibit dose-dependently
(from 5 up to 20 mM) the tumor necrosis factor alpha (TNF-
The effects of phytonutrients on osteoblasts were mostly
α)- and interferon gamma (IFN-γ)-induced secretion of
studied using pure molecules. However, people do not
several osteoclastogenic cytokines. Similar results were
consume isolated molecules in their diet but rather fruit and
achieved with lower doses of apigenin (0.01µM) in a further
vegetables which are rich in many different phytochemicals.
study [121]. To our knowledge no other studies are available
It could be thus interesting to assess effects of phenolic
to conclude on apigenin’s mechanisms of action on bone
mixtures or plant extracts on bone metabolism. However, to
anabolisn.
assess molecular mechanisms of action of the active
compounds, in vitro studies with pure compounds are then
Stilbenes required.
Resveratrol, a natural stilbene present in various plant
Olive Extracts
species and relatively abundant in red wine (concentrations
up to 25 µmol/L [122]), has been of great scientific interest
Oleuropein, a polyphenol belonging to the secoiridoid
over the last years. Resveratrol has been shown to have an
class found in olives and their derivatives has been shown to
impact on bone metabolism. Given at a dose of 0.7 mg/kg of
have anti-inflammatory effects, notably by inhibiting the
body weight to ovariectomized rats, it could increase
lipooxygenase activity and the production of leucotriene B4
epiphysis BMD. Thus, in these conditions, trans-resveratrol
[129].
was able to prevent bone loss induced by estrogen
deficiency. This bone sparing effect could be mediated by The effect of oleuropein was investigated by Puel et al.
the antioxidant, the anti-inflammatory or the phytoestrogenic using a rat model of bone loss associating ovariectomy and
properties of resveratrol [123]. Further studies were acute inflammation [130]. All doses of oleuropein tested
undertaken to explore the effect of resveratrol in vitro. (2.5, 5, 10 and 15 mg/kg body weight per day) elicited
Mizutani et al. examined the effect of trans-resveratrol on protective effects on bone mass. It was hypothesized that
the proliferation and differentiation of osteoblastic MC3T3- oleuropein may exert its bone-sparing effect by modulating
E1 cells [124]. Resveratrol dose-dependently increased ALP inflammation rather than acting directly on bone metabolism.
activity (10-7 – 10-5 M) and DNA synthesis (10-9 – 10-7 M). It Indeed, neither oleuropein nor whole olive oil was able to
was also able to stimulate collagen synthesis. The affect bone mineral density in ovariectomised rats when
stimulatory effect of resveratrol on osteoblastic cells inflammation was not induced [131]. Very recently, the
functions was clearly blocked by the presence of the anti- effects of oleuropein on the processes of osteoblastogenesis
estrogen tamoxifen. These results suggest that the effect of in mesenchymal stem cells (MSCs) from human bone
resveratrol in increasing bone formation could be explained marrow have been studied. Enhanced differentiation of
by an estrogen-like action. Indeed, ICI completely abolished osteoblasts at concentrations between 10-6 and 10-4 M of pure
the resveratrol-induced phosphorylation of ERK1/2 and p38 oleuropein was observed. Gene expression of
in HBMSC cells, suggesting that estrogen receptors were osteoblastogenesis markers, RUNXII, osterix, collagen type
implicated in resveratrol MAPK activation [125]. I, osteocalcin, or alkaline phosphatase (ALP), was higher in
Resveratrol was able dose-dependently (25 µM-100 µM) to osteoblast-induced oleuropein-treated cells. Also, the ALP
promote the expression of osteoblast markers like activity and extracellular matrix mineralization were higher
osteocalcin and osteopontin in human bone marrow when oleuropein was present in the media [132].
mesenchymal stem cells [126] as well as osteocalcin mRNA
Plant Bioactives and Bone Anabolism Current Molecular Pharmacology, 2012, Vol. 5, No. 2 213
Extracts of Dried Plum Carotenoids
Prunes, the dried fruits of Prunus domestica L. are known Among all vitamins identified, most attention has been
to be rich in several phenolics including phenolic acid paid to vitamin A and carotenoids, with respect to their
derivatives, flavonoids and coumarins. The total content of potential effect on bone metabolism, except vitamin D.
phenolics has been reported to be 184 mg/100 g in dried Indeed, the action of retinol and β-carotene on bone cells
plums. The major components in these fruits are chlorogenic was investigated in vitro in the MC3T3-E1 cell line. Retinol
acid isomers (i.e. neochlorogenic acid, cryptochlorogenic induced differentiation of the MC3T3-E1 cells, by increasing
acid and chlorogenic acid which are ester of caffeic acid with alkaline phosphatase activity dose dependently, in a range
quinic acid). from 1 to 100 nM. Beta-carotene increased alkaline
phosphatase activity in a dose-related manner in a range
It was found that feeding rats with 1g/d of prunes
from 0.1 to 5 µM. The induction of differentiation of
inhibited bone resorption [110]. Deyhim et al. confirmed that
MC3T3-E1 cells by β-carotene was dose-dependent but was
increased consumption of dried plum may help in preventing
two orders of magnitude less active than that produced by
bone loss in an ovariectomized rat model of osteoporosis
retinoids. Within the MC3T3-E1 cells, part of the β-carotene
[133]. Even at doses as low as 5 %, prunes were able to
was effectively converted into retinol. Alpha-carotene,
restore femoral and tibiae bone density and to improve
canthaxanthin and lycopene also increased ALP activity and
trabecular micro architecture in comparison with
osteopontin mRNA expression, but less potently so than β-
ovariectomized controls. A more recent study was designed
carotene. Retinol and carotenoids were concluded to have a
to determine if dried plum could also prevent skeletal
direct stimulatory effect on the differentiation of osteoblasts
deterioration in a male osteoporosis model and to explore
at the physiological concentration [140, 141]. Similar effects
more precisely the mechanism of action of its food item
were achieved with retinoic acid [141]. The effect of
[134]. The results of this study demonstrate dietary
lycopene on osteoblast differentiation has also been shown to
supplementation with dried plum (doses of 5, 15 and 25 %)
be dependent on the stage of cell differentiation in SaOS-2
prevents the deterioration in bone mass and in both cortical
cells [142]. Moreover, the effect of β-cryptoxanthin on
and trabecular bone microstructure due to orchidectomy.
osteoblast function was also evaluated in some experiments.
These beneficial effects may be partly attributed to a
In MC3T3-E1 cells, β-cryptoxanthin was able to stimulate
decrease in osteoclastogenesis via down-regulation of
ALP activity at 1µM and stimulate expression of IGF-1 and
RANKL and a stimulation of bone formation via IGF-1. Bu
TGF-β1, suggesting that carotenoids could stimulate
et al. also obtained a significant effect on bone of dried plum
transcriptional activity of osteoblastic cells [143, 144].
at a high dose (25 % in the diet) in terms of bone mass,
Moreover, the stimulatory effect of this compound appeared
micro architecture and strength in osteopenic male rats [135].
to be mediated through induction of runx2 and α1 collagen
They also studied the influence of dried plum extract at
expression, this being upregulated by zinc [145]. Finally,
concentration of 2.5, 5 and 10 µg/ml on MC3T3-E1 cell
recently the same authors demonstrated that β-cryptoxanthin
under normal and inflammatory conditions [136]. This
was active on osteoblast via amplified TGFβ1-induced Smad
extract was able to stimulate ALP activity and mineralize
activation, suggesting specificity of action of TGFβ1
nodule formation. The plum extract was not able to alter the
pathway (involved in commitment of osteoblast
expression of the transcription factors Runx2 and Osterix nor
precursor)[146]. This could partly explain that β-
genes implicated in osteoclast regulation like RANKL and
cryptoxanthin, given orally for 3 months to ovariectomized
OPG. However, it could restore Runx2 and Osterix
rats (10µg/100g body weight) prevented bone mineral
expression in cells that had been pre-treated with TNFα
density loss in this animals [147] as well as lycopene at
(inflammatory condition) and as a result exhibited reduced
20mg/Kg diet [148].
Runx2 and Osterix expression. Consistent with pre-clinical
data, the extract also suppressed the TNFα-induced up- These data provide potential insight into carotenoids as
regulation of RANKL expression [136]. bone anabolic agents to be further studied.
Walnut Extract PERSPECTIVES AND CONCLUSIONS
Walnut extract (Juglans regia L.) and its major phenolic
Results from various studies dealing with phytonutrients
component ellagic acid [137] were evaluated on nodule
at nutritional and physiological levels provide promising
formation in the osteoblastic cell line KS483. Both walnut
evidence that flavonoids, stilbenes but also carotenoids are
extract (at 10-25 µg/ml) and ellagic acid (at 10-9 - 10-8M)
able to positively influence bone metabolism. Even though
induced nodule formation in KS483 osteoblasts. The present
all the studies reported here provide some evidence that
results suggest that the walnut extract has a potent
phytochemicals regulate various transcription factors
stimulatory effect on osteoblastic activity, an effect
involved in bone anabolic signalling pathways (e.g. BMPs,
mediated, at least in part, by its major component ellagic
MAPKs), molecular mechanisms of these compounds in
acid [138]. Moreover, it has been shown that inhibitors of
osteoblastic cells need to be further investigated. In
estrogen receptors decreased the activity of ALP and
particular, it is difficult to evaluate and compare results of
mineralization induced by ellagic acid in osteoblastic cells
compounds tested at different concentrations (some
[139]. Indeed, ellagic acid may act as a natural selective
physiological (1-10 uM) and some pharmacological (50 -100
estrogen receptor modulator (SERM) [139] and may act
uM) and in different osteoblast cell lines from rodent or
through estrogen receptors, but the molecular mechanisms
human origin. Validation of the results in human primary
are not well known and they need to be further investigated.
214 Current Molecular Pharmacology, 2012, Vol. 5, No. 2 Horcajada and Offord
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None declared.
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