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International Journal of
Molecular Sciences
Article
Anti-Osteoclastic Activity of Artemisia capillaris
Thunb. Extract Depends upon Attenuation of
Osteoclast Differentiation and Bone
Resorption-Associated Acidification Due to
Chlorogenic Acid, Hyperoside, and Scoparone
Sang-Hyun Lee, Jung-Yun Lee, Young-In Kwon and Hae-Dong Jang *
Department of Food and Nutrition, Hannam University, Daejeon 34430, Korea;
[email protected] (S.-H.L.); [email protected] (J.-Y.L.); [email protected] (Y.-I.K.)
* Correspondence: [email protected]; Tel.: +82-426-298-795
Academic Editor: Maurizio Battino
Received: 14 December 2016; Accepted: 26 January 2017; Published: 4 February 2017
Abstract: The present study attempts to elucidate the anti-osteoporotic activity of Artemisia
capillaris Thunb. in the form of anti-osteoclastic effect and responsible bioactive compounds.
The contents of chlorogenic acid, caffeic acid, hyperoside, isoquercitrin, isochlorogenic acid A,
and scoparone in Artemisia capillaris hydroethanolic extract (ACHE) were 38.53, 0.52, 4.07, 3.03,
13.90, and 6.59 mg/g, respectively. ACHE diminished osteoclast differentiation and bone resorption
due to chlorogenic acid, hyperoside, and scoparone. In addition, ACHE attenuated acidification
as well as reducing tumor necrosis factor receptor-associated factor 6 (TRAF6) expression and its
association with vacuolar H+ -adenosine triphosphatase (V-ATPase). Furthermore, chlorogenic acid,
hyperoside, and scoparone from A. capillaris abrogated the association of V-ATPase with TRAF6,
suggesting that the blockage of bone resorption by A. capillaris was partially mediated by reducing
acidification through down-regulating interaction of V-ATPase with TRAF6 due to scoparone as
well as chlorogenic acid and hyperoside. These results imply that the anti-osteoclastic effect of
A. capillaris through down-regulating osteoclast differentiation and bone resorption may contribute
to its anti-osteoporotic effect.
Keywords: Artemisia capillaris; osteoclast differentiation; bone resorption; acidification
1. Introduction
The bone remodeling cycle is an elaborately controlled process of bone metabolism, in which
osteoclasts resorb the mineralized matrix and osteoblasts form new bone matrix [1]. Accordingly, bone
mass depends upon an orchestrated balance between osteoclastic bone resorption and osteoblastic
bone formation. Osteoclast differentiation from precursors and tartrate-resistant acid phosphatase
(TRAP) activity belong to essential factors for bone resorption by osteoclasts. Osteoblast differentiation
and proliferation, alkaline phosphatase activity, and type I collagen synthesis are required for bone
formation by osteoblasts [2]. However, the imbalance between bone resorption and bone formation
can cause metabolic bone diseases such as osteoporosis and osteopetrosis [3].
Osteoclasts, bone-resorbing multinucleated cells, are differentiated by the fusion of their
mononuclear precursors, monocytes and macrophages, which is triggered by the receptor activator
of nuclear factor-κB ligand (RANKL) produced by osteoblasts [4–6]. Bone resorption requires that
osteoclasts have the ability to produce protons since an acidic pH is required for the solubilization
of the alkaline salts of bone matrix and the digestion of bone matrix by acid enzymes secreted by
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2 of 14
2 of 14
+ -adenosine
localized
on[7].
theThis
ruffled
border of osteoclasts,
which H
hydrolyzes
ATP
to produce protons
and extrudes
osteoclasts
acidification
is due to vacuolar
triphosphatase
(V-ATPase)
localized
them
into
extracellular
It was reported
that the interaction
of V-ATPase
necrosis
on the
ruffled
border ofspace.
osteoclasts,
which hydrolyzes
ATP to produce
protonswith
andtumor
extrudes
them
factor
receptor-associated
factor
6
(TRAF6)
is
required
for
its
activation
[8].
into extracellular space. It was reported that the interaction of V-ATPase with tumor necrosis factor
Artemisia capillaris
Thunb.
belongs
the genus
Artemisia
and[8].
has been widely used as an edible
receptor-associated
factor
6 (TRAF6)
is to
required
for its
activation
herbalArtemisia
medicine
in Asian
countries.
Many
have reported
that
A. widely
capillaris
extracts
or its
capillaris
Thunb.
belongs
to thestudies
genus Artemisia
and has
been
used
as an edible
bioactive
compounds
such
as caffeoylquinic
acids,
flavonoids,
andA.coumarins
[9–12] or
have
various
herbal medicine
in Asian
countries.
Many studies
have
reported that
capillaris extracts
its bioactive
biological
functions
including antioxidant
activity [13,14],
anti-inflammatory
anticompounds
such as caffeoylquinic
acids, flavonoids,
and coumarins
[9–12] haveactivity
various[15],
biological
microbiological
activity
[16],
anti-tumor
activity
[17],
cytoprotective
effects
[14],
hepatoprotective
functions including antioxidant activity [13,14], anti-inflammatory activity [15], anti-microbiological
effects
[18,19],
and anti-osteoporosis
[20]. The recent
demonstrated thateffects
the water
extract
activity
[16], anti-tumor
activity [17],effects
cytoprotective
effectsstudy
[14], hepatoprotective
[18,19],
and
of
A. capillaris suppressed
RANKL-induced
osteoclast differentiation
anti-osteoporosis
effects [20]. The
recent study demonstrated
that the water from
extractbone
of A. marrow
capillaris
macrophages
and bone resorption
by the attenuated
expression
of biomarkers
including cathepsin
K
suppressed RANKL-induced
osteoclast
differentiation
from bone
marrow macrophages
and bone
and
ATPasebyV(0)
domain (ATPv0d2)
Unfortunately,
therecathepsin
is no systematic
study on
antiresorption
the attenuated
expression[20].
of biomarkers
including
K and ATPase
V(0)the
domain
osteoclastic
effect
of bioactivethere
compounds
of A. study
capillaris
the down-regulation
of
(ATPv0d2) [20].
Unfortunately,
is no systematic
on thethrough
anti-osteoclastic
effect of bioactive
acidification.
compounds of A. capillaris through the down-regulation of acidification.
The
Theobjective
objectiveof
ofthe
the present
present study
study was
was to
to investigate
investigate the
the anti-osteoclastic
anti-osteoclasticeffects
effectsof
ofA.
A. capillaris
capillaris
and
responsible
bioactive
compounds
using
an
osteoclastic
cells
model
for
developing
functional
and
bioactive compounds using an osteoclastic cells model for developing functional food
food
the treatment
of bone
diseases
caused
by bone
in theintreatment
of bone
diseases
caused
by bone
loss. loss.
Results
2.2.Results
2.1.The
TheContent
ContentofofSix
SixMarker
MarkerCompounds
Compoundsin
inArtemisia
Artemisiacapillaris
capillaris Hydroethanolic
Hydroethanolic Extract
Extract (ACHE)
(ACHE)
2.1.
Chlorogenicacid,
acid,caffeic
caffeicacid,
acid, hyperoside,
hyperoside,isoquercitrin,
isoquercitrin,isochlorogenic
isochlorogenicacid
acidA,
A,and
and scoparone
scoparone
Chlorogenic
demonstratedin
inFigure
Figure11have
havebeen
beenreported
reportedas
assix
siximportant
importantconstituents
constituentsof
ofA.
A.capillaris
capillaris[11].
[11].Thus,
Thus,
demonstrated
they
were
identified
by
the
retention
time
and
ultraviolet
(UV)
spectrum
data
of
standard
substances
they were identified by the retention time and ultraviolet (UV) spectrum data of standard substances
andtheir
theircontents
contents were
were determined
determined by
and
by straight
straight calibration
calibrationwith
withaastandard
standardcurve.
curve.As
Asshown
shownininFigure
Figure2,
the
retention
time
of
chlorogenic
acid,
caffeic
acid,
hyperoside,
isoquercitrin,
isochlorogenic
acid
and
2, the retention time of chlorogenic acid, caffeic acid, hyperoside, isoquercitrin, isochlorogenicA,acid
scoparone
were
13.78,
14.78,
22.52,
22.88,
26.61,
and
27.39
min,
respectively.
The
content
of
chlorogenic
A, and scoparone were 13.78, 14.78, 22.52, 22.88, 26.61, and 27.39 min, respectively. The content of
acid, caffeicacid,
acid,caffeic
hyperoside,
isoquercitrin,
isochlorogenic
acid A, and acid
scoparone
ACHE were
chlorogenic
acid, hyperoside,
isoquercitrin,
isochlorogenic
A, andinscoparone
in
38.53,
0.52,
4.07,
3.03,
13.90,
and
6.59
mg/g,
respectively
(Table
1),
indicating
that
chlorogenic
acidthat
and
ACHE were 38.53, 0.52, 4.07, 3.03, 13.90, and 6.59 mg/g, respectively (Table 1), indicating
isochlorogenic
were the major
components
ACHE.
This resultin
appear
toThis
be consistent
with
chlorogenic
acidacid
andAisochlorogenic
acid
A were theinmajor
components
ACHE.
result appear
previous
study in
which
the content
acid Aofwas
43.14 mg/g followed
by 43.14
chlorogenic
to
be consistent
with
previous
studyofinisochlorogenic
which the content
isochlorogenic
acid A was
mg/g
acid
(21.06
mg/g),
hyperoside
(8.44
mg/g),
and
scoparone
(5.56
mg/g)
[11].
followed by chlorogenic acid (21.06 mg/g), hyperoside (8.44 mg/g), and scoparone (5.56 mg/g) [11].
Figure1.1.Chemical
Chemicalstructures
structuresof
ofsix
sixcompounds.
compounds.
Figure
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Figure 2. High performance liquid chromatography (HPLC) chromatograms of ethanol extract of
Figure 2. High performance liquid chromatography (HPLC) chromatograms of ethanol extract of
Artemisia capillaris. The detection wavelength was set at 255 nm for hyperoside and isoquercitrin, at
Artemisia capillaris. The detection wavelength was set at 255 nm for hyperoside and isoquercitrin, at
320 nm for chlorogenic acid, caffeic acid, and isochlorogenic acid, and at 340 nm for scoparone.
320 nm for chlorogenic acid, caffeic acid, and isochlorogenic acid, and at 340 nm for scoparone.
Table 1. The content of six marker compounds of hydroethanolic extract of A. capillaris. (n = 3).
Table 1. The content of six marker compounds of hydroethanolic extract of A. capillaris. (n = 3).
Compounds
Compoundsacid
Chlorogenic
Chlorogenic
acid
Caffeic acid
Caffeic
acid
Hyperoside
Hyperoside
Isoquercitrin
Isoquercitrin
Isochlorogenic
acid
Isochlorogenic
acid
Scoparone
Scoparone
(1)
Content (1) (mg/g)
(1) (mg/g)
Content
38.526 ± 0.927
38.526
0.927
0.515 ±±0.048
0.515 ±
0.048
4.072
± 0.220
4.072 ± 0.220
3.031 ± 0.148
3.031 ± 0.148
13.898
± 0.667
13.898 ±
0.667
6.589
± 0.193
6.589 ±
0.193
(1) Each
Each
value
is expressed
deviation
triplicate
experiments.
value
is expressedas
asmean
mean ±
± standard
standard deviation
inin
triplicate
experiments.
2.2. Suppressive
Suppressive Effect
Effect of
of ACHE
ACHE and
and Its
Its Six
Six Marker
Marker Compounds
Compounds on
on Osteoclast
Osteoclast Differentiation
Differentiation
2.2.
Tostudy
studythethe
anti-osteoporosis
of ACHE
through
the attenuated
bone resorption
by
To
anti-osteoporosis
effecteffect
of ACHE
through
the attenuated
bone resorption
by osteoclasts,
osteoclasts,
the
suppressive
effects
of
ACHE
on
RANKL-induced
osteoclast
differentiation
were
the suppressive effects of ACHE on RANKL-induced osteoclast differentiation were analyzed. RANKL,
analyzed.
RANKL,
is required of
formonocytes/macrophages
the differentiation of monocytes/macrophages
which
is required
forwhich
the differentiation
into osteoclasts, was usedinto
as
osteoclasts,
was
used
as
an
inducer
of
osteoclast
differentiation
from
RAW
264.7
[18]. induced
RANKL
an inducer of osteoclast differentiation from RAW 264.7 cells [18]. RANKL
(50 cells
ng/mL)
(50 ng/mL) induced
TRAP-positive
formation from
cells, which
was
TRAP-positive
osteoclast
formation osteoclast
from pre-osteoclastic
cells,pre-osteoclastic
which was visualized
by light
visualized by light(Figure
microphotography
ACHE at 1–20 attenuated
μg/mL dosemicrophotography
3A). However, (Figure
ACHE at3A).
1–20However,
µg/mL dose-dependently
the
dependently
attenuated
the
number
of
osteoclasts
(Figure
3B).
In
addition,
ACHE
at
μg/mL
number of osteoclasts (Figure 3B). In addition, ACHE at 1–20 µg/mL dose-dependently1–20
diminished
dose-dependently
TRAP
of osteoclasts
was up
significantly
(p RANKL
< 0.001)
the
TRAP activity ofdiminished
osteoclasts the
which
wasactivity
significantly
(p < 0.001)which
enhanced
to 172.1% by
enhanced
up
to
172.1%
by
RANKL
treatment
compared
to
the
control
group
(Figure
3C).
To
exclude
treatment compared to the control group (Figure 3C). To exclude the possibility that the inhibitory
the
possibility
that
the
inhibitory
effect
was
due
to
the
decreased
viability
and/or
the
proliferation
of
effect was due to the decreased viability and/or the proliferation of the pre-osteoclastic cells, cell
the pre-osteoclastic
cells, cell
was that
checked.
The
showed
that ACHE
diddoses
not affect
cytotoxicity
was checked.
Thecytotoxicity
results showed
ACHE
didresults
not affect
cell viability
at the
that
cell
viability
at
the
doses
that
effectively
suppressed
osteoclast
differentiation
(Figure
2D).
effectively suppressed osteoclast differentiation (Figure 2D).
To determine
determine which
which of
of the
the six
six marker
marker compounds
compounds of
of ACE
ACE contributed
contributed to
to inhibitory
inhibitory activity
activity of
of
To
ACHE ininRANKL-induced
RANKL-induced
osteoclast
differentiation,
TRAP was
activity
was atmeasured
at the
ACHE
osteoclast
differentiation,
TRAP activity
measured
the concentration
concentration
of
10
μM
at
which
scoparone
moderately
inhibited
RANKL-induced
osteoclast
of 10 µM at which scoparone moderately inhibited RANKL-induced osteoclast differentiation in our
differentiation
ourthe
previous
study [21];
the compounds
cell cytotoxicity
of six
compounds
wasThe
not potent
found
previous
study in
[21];
cell cytotoxicity
of six
was not
found
(Figure 3D).
(Figure
3D).
The
potent
inhibitory
effect
on
TRAP
activity
was
observed
in
chlorogenic
acid,
inhibitory effect on TRAP activity was observed in chlorogenic acid, hyperoside, and scoparone
as
hyperoside, and scoparone as compared with caffeic acid, isoquercitrin, and isochlorogenic acid at
10 μM (Figure 3E). On the other hand, isoquercitrin displayed the pro-osteoclastic effect in this
Int. J. Mol. Sci. 2017, 18, 322
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Int. J. Mol. Sci. 2017, 18, 322
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compared with caffeic acid, isoquercitrin, and isochlorogenic acid at 10 µM (Figure 3E). On the other
experiment.
Thus, displayed
this resultthe
suggests
that the effect
inhibitory
of ACHE
onthis
RANKL-induced
hand,
isoquercitrin
pro-osteoclastic
in thiseffect
experiment.
Thus,
result suggests
osteoclast
differentiation
may
be
attributed
to
three
compounds
such
as
chlorogenic
acid,
hyperoside,
that the inhibitory effect of ACHE on RANKL-induced osteoclast differentiation may be attributed
to
and scoparone.
three
compounds such as chlorogenic acid, hyperoside, and scoparone.
Figure 3. Cont.
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Figure
Inhibitoryeffect
effectofofhydroethanolic
hydroethanolicextract
extractof
ofA.
A.capillaris
capillaris and
and its six marker
Figure
3. 3.
Inhibitory
marker compounds
compounds on
(A) The
Thevisualized
visualizedtartrate-resistant
tartrate-resistantacid
acid
phosphatase
(TRAP)-positive
onosteoclast
osteoclast differentiation. (A)
phosphatase
(TRAP)-positive
multinucleated
osteoclasts
with
capillaris
hydroethanolic
extract
(ACHE)
treatment;
(B,C)
multinucleated
osteoclasts
with
A. A.
capillaris
hydroethanolic
extract
(ACHE)
treatment;
(B,C)
TheThe
counted
number
and TRAP
of TRAP-positive
multinucleated
ACHE
counted
number
and TRAP
activityactivity
of TRAP-positive
multinucleated
osteoclasts osteoclasts
with ACHE with
treatment;
(D) The cytotoxicity
of A.and
capillaris
its six compounds;
marker compounds;
The TRAP
activity
(D)treatment;
The cytotoxicity
of A. capillaris
its sixand
marker
(E) The(E)
TRAP
activity
of
multinucleated
osteoclasts
withwith
six marker
compounds
treatment.
RAW
264.7
cells
were
exposed
of multinucleated
osteoclasts
six marker
compounds
treatment.
RAW
264.7
cells
were
exposed
to receptor
activator
of nuclear
factor-κB
ligand
(RANKL;
50 50
ng/mL)
forfor
5 days
in in
thethe
absence
and
to receptor
activator
of nuclear
factor-κB
ligand
(RANKL;
ng/mL)
5 days
absence
and
presence
of ACHE.
After
5 days
in culture,
thethe
cells
were
fixed
andand
stained
using
a leukocyte
acid
presence
of ACHE.
After
5 days
in culture,
cells
were
fixed
stained
using
a leukocyte
acid
phosphatase
kit.kit.
TRAP-positive
multinucleated
osteoclasts
were
visualized
at 200-fold
magnification
phosphatase
TRAP-positive
multinucleated
osteoclasts
were
visualized
at 200-fold
magnification
under
light
microscopy.
TRAP-positive
multi-nucleated
osteoclasts
were
counted
and
TRAP
activity
under light microscopy. TRAP-positive multi-nucleated osteoclasts were counted and TRAP
activity
was
measured
at λat=λ405
nm.nm.
Data
areare
expressed
as as
percentages
of of
thethe
value
of of
cells
treated
with
was
measured
= 405
Data
expressed
percentages
value
cells
treated
with
RANKL
(means
± standard
deviations,
SD,
n =n 3).
Data
areare
expressed
as as
percentages
of of
thethe
values
of of
RANKL
(means
± standard
deviations,
SD,
= 3).
Data
expressed
percentages
values
untreated
cellscells
(means
± standard
deviations,
n = 3). Different
letters indicate
significant
untreated
(means
± standard
deviations,
n = 3). corresponding
Different corresponding
letters
indicate
## p < 0.01, ##### p < 0.001
###
differences
at
p
<
0.05
by
Duncan’s
test.
vs.
C.
C:
control,
which
was
not
was
significant differences at p < 0.05 by Duncan’s test. p < 0.01, p < 0.001 vs. C. C: control, which
treated;
TC: treated
control,control,
which was
treated
with RANKL;
CHA: chlorogenic
acid; CAA:
not treated;
TC: treated
which
was treated
with RANKL;
CHA: chlorogenic
acid;caffeic
CAA:acid;
caffeic
HY:acid;
hyperoside;
IQ: isoquercitrin;
ICA: isochlorogenic
acid; SC:acid;
scoparone;
NS: not significant.
HY: hyperoside;
IQ: isoquercitrin;
ICA: isochlorogenic
SC: scoparone;
NS: not significant.
Inhibitory
Effect
of ACHE
Three
Bioactive
Compounds
Bone
Resorption
of Osteoclast
2.3.2.3.
Inhibitory
Effect
of ACHE
andand
Three
Bioactive
Compounds
on on
Bone
Resorption
of Osteoclast
The
inhibitory
effect
ACHE
bone
resorption
was
determined
RANKL-differentiated
The
inhibitory
effect
of of
ACHE
onon
bone
resorption
was
determined
in in
RANKL-differentiated
osteoclasts
cultured
5 days
onplates
well coated
plates coated
with fluoresceinamine-labeled
chondroitin
osteoclasts
cultured
for 5for
days
on well
with fluoresceinamine-labeled
chondroitin
sulfate
sulfate
(FACS)
andphosphate
calcium phosphate
(CaP)
using
a bone resorption
assay kitBio,
(CosMo
Bio,
Tokyo,
(FACS)
and
calcium
(CaP) using
a bone
resorption
assay kit (CosMo
Tokyo,
Japan).
Japan).
FACS
bound
CaP was
released
from
the
CaP
into by
theosteoclastic
medium byresorption.
osteoclastic
The
FACS The
bound
to CaP
wasto
released
from
the CaP
layer
into
thelayer
medium
resorption.
Therefore,
bone
resorption
activity istoproportional
fluorescence
intensity
FACS in
Therefore,
bone
resorption
activity
is proportional
fluorescencetointensity
of FACS
in the of
medium.
the
medium.
Theon
resorbed
area
onvisualized
the plates was
visualized
200-fold under
magnification
under light
The
resorbed
area
the plates
was
under
200-foldunder
magnification
light microscopy.
microscopy.
RANKL
treatment
markedly
increased
thesize
number
and
size of bone
resorption
areas
RANKL
treatment
markedly
increased
the number
and
of bone
resorption
areas
(Figure 4A).
(Figure 4A).
theactivity
bone resorption
activity
significantly
(p <RANKL
0.001) (Figure
enhanced
Consistently,
theConsistently,
bone resorption
was significantly
(p was
< 0.001)
enhanced by
4B).by
4B).
In contrast,
ACHE at 1–20 μg/mL
dose-dependently
alleviated
bonests.
resorption
In RANKL
contrast,(Figure
ACHE at
1–20
µg/mL dose-dependently
alleviated
bone resorption
of osteocla
of osteocla sts.
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Figure
4. Inhibitory
Inhibitory effect
effectofofhydroethanolic
hydroethanolic
extract
capillaris
its bioactive
compounds
on
Figure 4.
extract
of of
A. A.
capillaris
andand
its bioactive
compounds
on bone
bone
resorption.
(A)
The
visualized
absorbed
area
with
ACHE
treatment;
(B)
The
fluorescence
resorption. (A) The visualized absorbed area with ACHE treatment; (B) The fluorescence intensity of the
intensity
thewith
absorbed
area with
with ACHE
treatment
ACHE
and (C)compounds.
three bioactive
compounds.
A assay
bone
absorbedof
area
treatment
andwith
(C) three
bioactive
A bone
resorption
resorption
assayusing
was aperformed
using
commercial
assay
RAW
264.7tocells
were(100
exposed
to
was performed
commercial
assaya kit.
RAW 264.7
cellskit.
were
exposed
RANKL
ng/mL)
RANKL
ng/mL)
forand
5 days
in the
absenceorand
of ACHE
or threeareas
compounds.
The
for 5 days(100
in the
absence
presence
of ACHE
threepresence
compounds.
The absorbed
on each plate
absorbed
areas at
on200-fold
each plate
were visualized
at 200-fold
magnification
under
lightwas
microscopy.
were visualized
magnification
under light
microscopy.
Fluorescence
intensity
measured
Fluorescence
intensity
was
measured
at
an
excitation
wavelength
of
485
nm
and
an
emission
at an excitation wavelength of 485 nm and an emission wavelength of 535 nm using a fluorometric
plate
wavelength
nm using
a fluorometric
plate
reader.
Data arecells
expressed
asstandard
percentages
of the
reader. Data of
are535
expressed
as percentages
of the
values
of untreated
(means ±
deviations,
values
untreated
cells (means ±letters
standard
deviations,
n = 3).differences
Different corresponding
indicate
n = 3). ofDifferent
corresponding
indicate
significant
at p < 0.05 byletters
Duncan’s
test.
### p < 0.001
### p < 0.001 vs. C. C: control, which was not treated;
significant
differences
p < 0.05which
by Duncan’s
test.
vs. C. C:atcontrol,
was not
treated;
TC: treated control, which was treated with
TC:
treated
which
wasHP:
treated
with RANKL;
CA: chlorogenic acid; HP: hyperoside; SP:
RANKL;
CA:control,
chlorogenic
acid;
hyperoside;
SP: scoparone.
scoparone.
Bone resorption can be achieved by multi-nucleated osteoclasts after the differentiation process.
Bone resorption can be achieved by multi-nucleated osteoclasts after the differentiation process.
Because the strong inhibitory effect on osteoclastic differentiation was found in three compounds such
Because the strong inhibitory effect on osteoclastic differentiation was found in three compounds
as chlorogenic acid, hyperoside, and scoparone, these three bioactive compounds were used for bone
such as chlorogenic acid, hyperoside, and scoparone, these three bioactive compounds were used for
bone resorption assay. To determine which of the three compounds of ACHE with potential antiosteoclastic activity could be related with the inhibitory activity of ACHE on bone resorption of
Int. J. Mol. Sci. 2017, 18, 322
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Int. J. Mol.assay.
Sci. 2017,
322
7 of 14
resorption
To18,determine
which of the three compounds of ACHE with potential anti-osteoclastic
activity could be related with the inhibitory activity of ACHE on bone resorption of osteoclasts, the
osteoclasts, the bone resorption assay was carried out using a bone resorption assay kit. Though all
bone resorption assay was carried out using a bone resorption assay kit. Though all of three compounds
of three compounds showed the inhibition effect on bone resorption (Figure 4C), the strongest
showed the inhibition effect on bone resorption (Figure 4C), the strongest inhibition effect was observed
inhibition effect was observed in scoparone. These data imply that scoparone as well as chlogenic
in scoparone. These data imply that scoparone as well as chlogenic acid and hyperoside may be major
acid and hyperoside may be major components of ACHE, contributing to its inhibitory activity on
components of ACHE, contributing to its inhibitory activity on bone resorption of osteoclasts.
bone resorption of osteoclasts.
2.4. Suppressive Effect of ACHE and Three Bioactive Compounds on the Acidification by Osteoclasts
2.4. Suppressive Effect of ACHE and Three Bioactive Compounds on the Acidification by Osteoclasts
The effect of ACE on acidification was examined by measuring pH since the acidic pH is essential
The effect of ACE on acidification was examined by measuring pH since the acidic pH is
for essential
bone resorption
ostaoclasts.
RANKL significantly
(p < 0.001)
the pHthe
of pH
a culture
for bone by
resorption
by ostaoclasts.
RANKL significantly
(p <diminished
0.001) diminished
of a
medium
as
compared
to
the
control
group,
but
ACHE
at
1–20
µg/mL
reversed
this
decrease
and
culture medium as compared to the control group, but ACHE at 1–20 μg/mL reversed this decrease
further
augmented
pH in pH
a dose-dependent
manner
(Figure
5A).5A).
In addition,
the
enhancing
and further
augmented
in a dose-dependent
manner
(Figure
In addition,
the
enhancingeffect
effectof
three
compounds
on
pH
is
clearly
shown
in
Figure
5B.
Furthermore,
scoparone
markedly
increased
pH
of three compounds on pH is clearly shown in Figure 5B. Furthermore, scoparone markedly increased
as compared
to
chlorogenic
acid
and
hyperoside,
which
is
in
line
with
the
result
of
their
suppressive
pH as compared to chlorogenic acid and hyperoside, which is in line with the result of their
effects
on bone effects
resorption.
suppressive
on bone resorption.
Figure 5. (A) Inhibitory effect of hydroethanolic extract of A. capillaris and (B) three bioactive
Figure 5. (A) Inhibitory effect of hydroethanolic extract of A. capillaris and (B) three bioactive
compounds on acidification. RAW 264.7 cells were exposed to RANKL (50 ng/mL) for 5 days in the
compounds on acidification. RAW 264.7 cells were exposed to RANKL (50 ng/mL) for 5 days in
absence and presence of ACHE. After 5 days, the pH of the differentiated medium was measured
the absence and presence of ACHE. After 5 days, the pH of the differentiated medium was measured
using a pH meter. Data are expressed as percentages of the values of untreated cells (means ± standard
using a pH meter. Data are expressed as percentages of the values of untreated cells (means ± standard
deviations, n = 3). Different corresponding letters indicate significant differences at p < 0.05 by
deviations, n = 3).##Different ###
corresponding letters indicate significant differences at p < 0.05 by Duncan’s
Duncan’s test. p < 0.01, p < 0.01 vs. C. C: control, which was not treated; TC: treated control, which
##
###
test. p < 0.01,
p < 0.01 vs. C. C: control, which was not treated; TC: treated control, which was
was treated with RANKL; CA: chlorogenic acid; HP: hyperoside; SP: scoparone.
treated with RANKL; CA: chlorogenic acid; HP: hyperoside; SP: scoparone.
Int. J. Mol. Sci. 2017, 18, 322
Int. J. Mol. Sci. 2017, 18, 322
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Down-Regulating Effect
Effect of
of ACHE
ACHE and
andThree
ThreeBioactive
BioactiveCompounds
Compoundson
onTRAF6
TRAF6Expression
Expressionand
andItsItsBinding
2.5. Down-Regulating
to
V-ATPase
Binding
to V-ATPase
RANKL binding to RANK localized on the membrane of precursor cells may recruit TRAF6 to
induce osteoclast differentiation and to enhance
enhance bone
bone resorption.
resorption. For efficient bone resorption by
osteoclasts, acidification is required and the primary mechanism responsible for this acidification is
V-ATPase
interaction of V-ATPase
V-ATPase which
which transports
transports H
H++ to extracellular resorption lacunae [7]. The interaction
with
ofof
nuclear
factor-κB
(RANK)
results
in its
[8].
with TRAF6
TRAF6recruited
recruitedby
bythe
thereceptor
receptoractivator
activator
nuclear
factor-κB
(RANK)
results
in activation
its activation
Therefore,
the the
effect
of ACHE
onon
expression
ofof
TRAF6
[8]. Therefore,
effect
of ACHE
expression
TRAF6and
andV-ATPase
V-ATPaseand
andtheir
their interaction
interaction was
investigated using western blot analysis and immunoprecipitation.
immunoprecipitation. RANKL significantly (p < 0.001)
enhanced the expression of both TRAF6 and V-ATPase
V-ATPase (Figure
(Figure 6A).
6A). However,
However, ACHE
ACHE at
at20
20µg/mL
μg/mL
significantly
significantly (p
(p < 0.001) diminished the TRAF6 expression enhanced by RANKL, but showed no effect
on V-ATPase
V-ATPase expression
expression increased
increased by
by RANKL.
RANKL. Furthermore, to determine whether V-ATPase binds
with
TRAF6
for
its
activation,
the
association
of TRAF6
withwith
V-ATPase
was investigated.
In anti-TRAF6
with TRAF6 for its activation, the association
of TRAF6
V-ATPase
was investigated.
In antiimmunoprecipitates,
RANKLRANKL
significantly
(p < 0.001)
augmented
V-ATPase
bound
to TRAF6,
and
TRAF6 immunoprecipitates,
significantly
(p < 0.001)
augmented
V-ATPase
bound
to TRAF6,
ACHE
at 20at
µg/mL
significantly
(p < (p
0.001)
abrogated
this this
increased
V-ATPase
(Figure
6B). 6B).
and ACHE
20 μg/mL
significantly
< 0.001)
abrogated
increased
V-ATPase
(Figure
Which of
of the
thethree
threecompounds
compoundscontributed
contributed
inhibitory
effect
on expression
of TRAF6
toto
thethe
inhibitory
effect
on expression
of TRAF6
and
and
its interaction
V-ATPase
was examined.
According
to Figure
6C,
the significant
difference
its interaction
withwith
V-ATPase
was examined.
According
to Figure
6C, the
significant
difference
in the
in
the protein
level
of TRAF6
and V-ATPase
not found
among
three
compounds
(Figure
protein
level of
TRAF6
and V-ATPase
was was
not found
among
three
compounds
(Figure
6C).6C).
In
In
addition,
anti-V-ATPaseimmunoprecipitates,
immunoprecipitates,RANKL
RANKLsignificantly
significantly(p(p << 0.001)
0.001) induced
induced the
addition,
in in
anti-V-ATPase
interaction V-ATPase with
with TRAF6.
TRAF6. The most potent inhibition effect on interaction V-ATPase with
TRAF6
followed
byby
hyperoside
andand
chlorogenic
acidacid
(Figure
6D). 6D).
This data
TRAF6 was
wasobserved
observedininscoparone,
scoparone,
followed
hyperoside
chlorogenic
(Figure
This
is
consistent
with the
result
the inhibitory
effect of three
from A. capillaris
data
is consistent
with
theofresult
of the inhibitory
effectcompounds
of three compounds
from on
A. acidification
capillaris on
by
osteoclasts.
these results these
strongly
suggest
that suggest
scoparone
well as chlorogenic
acidification
byConsequently,
osteoclasts. Consequently,
results
strongly
thatasscoparone
as well as
acid
and hyperoside
responsible
ACE for attenuating
association
V-ATPase
chlorogenic
acid andare
hyperoside
arecompounds
responsible of
compounds
of ACE forthe
attenuating
theofassociation
with
TRAF6. with TRAF6.
of V-ATPase
Figure 6. Cont.
Int. J. Mol. Sci. 2017, 18, 322
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Int. J. Mol. Sci. 2017, 18, 322
9 of 14
Figure 6.
6. Suppressive
Suppressiveeffect
effectofofhydroethanolic
hydroethanolicextract
extract
capillaris
and
three
bioactive
compounds
Figure
ofof
A.A.
capillaris
and
three
bioactive
compounds
on
+-adenosine
on
tumor
necrosis
factor
receptor-associated
factor
6
(TRAF6)
and
vacuolar
H
+
tumor necrosis factor receptor-associated factor 6 (TRAF6) and vacuolar H -adenosine triphosphatase
triphosphatase
(V-ATPase)
expression
and (A)
their
The hydroethanolic
effect of A. capillaris
(V-ATPase)
expression
and their
interaction.
Theinteraction.
effect of A. (A)
capillaris
extract
hydroethanolic
extract
(ACHE)
on
TRAF6
and
V-ATPase
expression
and
(B)
their
interaction.
(C) The
(ACHE) on TRAF6 and V-ATPase expression and (B) their interaction. (C) The effect
of three bioactive
effect of three
compounds
onexpression
TRAF6 and
V-ATPase
andRAW
(D) their
compounds
onbioactive
TRAF6 and
V-ATPase
and
(D) theirexpression
interaction.
264.7interaction.
cells were
RAW
264.7
cells
were
exposed
to
RANKL
(50
ng/mL)
for
5
days
in
the
absence
and
presence
exposed to RANKL (50 ng/mL) for 5 days in the absence and presence of ACHE (20 µg/mL)oforACHE
three
(20 μg/mL)
or three (10
bioactive
compounds
μM).
were
lysed
and cell
lysates
were
bioactive
compounds
µM). The
cells were(10
lysed
andThe
cellcells
lysates
were
incubated
with
TRAF6
or
incubated
with
TRAF6
or
V-ATPase
antibody
and
protein
A
beads.
After
washing
the
beads,
V-ATPase antibody and protein A beads. After washing the beads, precipitated proteins and cell
precipitated
proteins and
cell lysates
weresulfate-polyacrylamide
separated by sodium gel
dodecyl
sulfate-polyacrylamide
gel
lysates
were separated
by sodium
dodecyl
electrophoresis
(SDS-PAGE) and
electrophoresis for
(SDS-PAGE)
andproteins
immunoblotted
for theantibodies.
indicated proteins
specific
antibodies.
immunoblotted
the indicated
using specific
Data are using
expressed
as percentages
Data
expressed
as percentages
of the
of untreated
of
theare
values
of untreated
cells (means
± values
standard
deviations,cells
n = (means
3). ## p ±< standard
0.01, ### pdeviations,
< 0.001 vs.nC;=
## p < 0.01, ### p < 0.001 vs. C; *** p < 0.001 vs. TC. C: control, which was not treated; TC: treated
3).
*** p < 0.001 vs. TC. C: control, which was not treated; TC: treated control, which was treated with
control, which
was treated
with
RANKL;
CA: chlorogenic
acid;
HP:
RANKL;
CA: chlorogenic
acid;
HP:
hyperoside;
SP: scoparone;
NS:
nothyperoside;
significant. SP: scoparone; NS:
not significant.
3. Discussion
3. Discussion
The osteoclast responsible for bone resorption is a multinucleated cell differentiated from
The osteoclast
responsible
for monocytes
bone resorption
is a multinucleated
from
mononuclear
precursors
including
and macrophages
[3]. Thecell
lowdifferentiated
level of nontoxic
mononuclear
precursors
monocytes
and growth
macrophages
low level
of nontoxic
amount
of reactive
oxygen including
species (ROS)
with several
factors [3].
and The
cytokines
is induced
by the
amount
of
reactive
oxygen
species
(ROS)
with
several
growth
factors
and
cytokines
is
induced
by the
RANKL binding to the receptors [5]. This increase in ROS may play an important role as a secondary
RANKL
binding
to
the
receptors
[5].
This
increase
in
ROS
may
play
an
important
role
as
a
secondary
messenger in RANKL-mediated signaling pathways for osteoclast differentiation [6]. Previous reports
messenger
in RANKL-mediated
signaling
pathwaysameliorating
for osteoclast
differentiation
[6]. Previous
have
demonstrated
that potent flavonoid
antioxidants
osteoclastic
differentiation
have
reports
have
demonstrated
that
potent
flavonoid
antioxidants
ameliorating
osteoclastic
been also found in genistein [22], luteolin [23], baicalein [24], epigallocatechin-3-gallate
[25], and
differentiation
been study
also found
in genistein
[22], luteolin
[23],
baicalein
[24],
epigallocatechin-3fisetin
[4]. Our have
previous
confirmed
that scopoletin
with
strong
cellular
antioxidant
capacity
gallate
[25],
and
fisetin
[4].
Our
previous
study
confirmed
that
scopoletin
with
strong
cellular
reduces ROS production as superoxide anions to suppress osteoclastic differentiation from RAW
264.7
antioxidant
capacity
reduces
ROS
production
as
superoxide
anions
to
suppress
osteoclastic
cells [26]. In addition, the potent antioxidant activity of A. capillaris was recently reported [13,27].
differentiation
fromhave
RAW
[26]. In the
addition,
the potent
antioxidant
of A. capillaris
Thus,
these reports
led264.7
us tocells
investigate
inhibitory
effect of
A. capillarisactivity
on RANKL-induced
was
recently
reported
[13,27].
Thus,
these
reports
have
led
us
to
investigate
the
inhibitory
effect of A.
osteoclast differentiation.
capillaris
on
RANKL-induced
osteoclast
differentiation.
In the present study, the anti-osteoclastic effect of A. capillaris was confirmed by measuring
In the present
study,
the anti-osteoclastic
of is
A.consistent
capillaris was
measuring
the
the number
and TRAP
activity
of osteoclasts,effect
which
withconfirmed
the recentby
study
reporting
number
and
TRAP
activity
of
osteoclasts,
which
is
consistent
with
the
recent
study
reporting
the
the suppressive effect of A. capillaris on osteoclast differentiation [20]. This anti-osteoclastic effect of
suppressive
effect
A. capillaris
osteoclast
differentiation
[20]. This
anti-osteoclastic
effect of A.
A.
capillaris may
beofattributed
to on
three
bioactive
compounds such
as chlorogenic
acid, hyperoside,
capillaris
may be
attributed
to three
bioactive compounds
such as differentiation
chlorogenic acid,
and
and
scoparone
because
the potent
suppressive
effect on osteoclast
washyperoside,
found in them
scoparone because the potent suppressive effect on osteoclast differentiation was found in them
compared to caffeic acid, isoquercitrin, and isochlorogenic acid. Recently, it has been reported that
Int. J. Mol. Sci. 2017, 18, 322
10 of 14
compared to caffeic acid, isoquercitrin, and isochlorogenic acid. Recently, it has been reported that
scoprone can attenuate RANKL-induced osteoclastic differentiation through suppressing ROS production
and scavenging [21] and chlorogenic acid can inhibit RANKL-induced osteoclast differentiation [28],
implying that the anti-osteoclastic effect of A. capillaris may be attributed to the antioxidant activity of
three bioactive compounds including chlorogenic acid, hyperoside, and scoparone.
Excessive bone resorption by osteoclasts can result in osteoporosis in older post-menopausal
women. The inhibitory effects of natural polyphenols including silibinin and phloretin on bone
resorption have been also reported [29,30]. To check the possibility of A. capillaris in the treatment
of osteoporosis through regulating bone resorption, the suppressive effect of A. capillaris on bone
resorption by osteoclasts was observed in the current study. For efficient bone resorption, the
solubilization of bone mineral and the hydrolysis of organic bone matrix by enzymes are required,
which depends on the pH of the environment. The result that A. capillaris significantly augmented pH
lowered by RANKL treatment appears to be associated with decreased bone resorption. According to
Figure 4 and 5, the inhibitory effect of scoparone on bone resorption and acidification was more potent
than that of chlorogenic acid and hyperoside. In addition, the inhibitory effect of chlorogenic acid on
bone resorption was reported in the previous study [28]. Therefore, these data support that scoparone
as well as chlorogenic acid and hyperoside may be major active components of A. capillaris, attenuating
bone resorption of osteoclast through controlling acidification.
The primary cellular protein of the acidification by osteoclasts is known as V-ATPase which
hydrolyzes ATP to produce protons [7]. In addition, the interaction of V-ATPase with TRAF6 is
essential for its activation [8]. The present study revealed that A. capillaris markedly alleviated TRAF6
expression and the association of TRAF6 with V-ATPase compared to RANKL treatment. In addition,
although three bioactive compounds including chlorogenic acid, hyperoside, and scoparone displayed
the potent inhibitory effect on bone resorption in comparison with caffeic acid, isoquercitrin, and
isochlorogenic acid, they did not significantly affect the expression of either TRAF6 or V-ATPase.
In contrast, they vigorously interrupted the association of V-ATPase with TRAF6 compared to RANKL
treatment even though the underlying mechanism remains unclear. The possible explanation for the
reduced bone resorption of A. capillaris due to suppresssed acidification may be the reduced osteoclast
differentiation and/or the attenuated interaction of V-ATPase with TRAF6. Accordingly, this finding
indicates that scoparone, as well as chlorogenic acid and hyperoside, may be partially responsible for
the inhibitory effect of A. capillaris on bone resorption through disrupting the association of V-ATPase
with TRAF6.
4. Materials and Methods
4.1. Reagents and Cell Culture Materials
Neocuproine, Dulbecco’s modified Eagle’s medium (DMEM), minimum essential medium
alpha medium (α-MEM), fetal bovine serum (FBS), 2,5-diphenyltetrazolium (MTT), Triton X-100,
RANKL, sodium tartrate, p-nitro-phenylphosphate (PNPP), fluoresceinamine-labeled chondroitin
sulfate (FACS), phosphate-buffered saline (PBS, pH 7.4), ethylenediaminetetraacetic acid (EDTA),
dithiothreitol (DTT), phenylmethanesulfonyl fluoride (PMSF), dimethylsulfoxide (DMSO), chlorogenic
acid, caffeic acid, isoquercitrin, and isochlorogenic acid A were purchased from Sigma-Aldrich
(St. Louis, MO, USA). Hyeroside was purchased from Hwz Analytic GmbH (Ruelzheim, Germany).
Scoparone was obtained from Phytolab GmbH&Co (Vestenbergreuth, Germany). Antibodies and
protein A-agarose beads were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA),
including anti-TRAF6 (sc-7221), V-ATPase (sc-20946), and β-actin (sc-130656). A bone resorption assay
kit was purchased from CosMo Bio Co., Ltd. (Tokyo, Japan). MC3T3-E1 subclone 4 and RAW 264.7
cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).
Int. J. Mol. Sci. 2017, 18, 322
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4.2. Preparation of A. capillaris Hydroethanolic Extract (ACHE)
The aerial parts of A. capillaris Thunb. were purchased at Kyoungdong market, an herbal medicine
market in Seoul, and taxonomically identified by Young-Ho Kim who is a professor in the College
of Pharmacy, Chungnam National University, Daejeon, Republic of Korea. A voucher specimen
(CNU-10103) was deposited at the Herbarium of the College of Pharmacy, Chungnam National
University. The plants were cut in 1-cm lengths and were extracted with a 6-fold volume of 50%
ethanol at 60 ◦ C for 6 h. Then, a second extraction was done with a 5-fold volume of 50% ethanol at
50 ◦ C for 16 h. After the pooled extraction solution was concentrated under a vacuum and spray-dried,
it was kept at −20 ◦ C until used.
4.3. High Performance Liquid Chromatography (HPLC) Analysis
The HPLC analysis was carried out using a HP Agilient HPLC system (Santa Clara, CA, USA)
with a solvent delivery unit, an online degasser, a column oven, an autosampler and a photodiode
array (PDA) detector. The analytical column was C18 (21.5 × 300 mm, particle size 10 µM, Tosoh,
Tokyo, Japan) maintained at 40 ◦ C. The mobile phases were distilled water (A) and phosphoric acid in
60% acetonitrile (pH 2.4, B). The gradient flow rate was composed of 0%–10% B for 0–5 min, 10%–50% B
for 5–30 min, and 50%–100% B for 30–35 min. The flow rates and injection volumes were 1.0 mL/min
and 10 µL, respectively. The detection wavelength was 255 nm for hyperoside and isoquercitrin,
320 nm for chlorogenic acid, caffeic acid, and isochlorogenic acid A, and 340 nm for scoparone.
Six pure compounds such as chlorogenic acid, caffeic acid, hyperoside, isoquercitrin, isochlorogenic
acid A, and scoparone, were used as chemical markers. The identification of the compounds was
performed by comparison of the retention time and UV spectrum.
4.4. Cell Cytotoxicity by MTT Assay
RAW 264.7 cells were plated in 24-well plates at a density of 2 × 104 cells/mL in triplicate.
Cells were treated with RANKL (50 ng/mL) and increasing concentrations of ACHE (1–50 µg/mL),
or six compounds (1–20 µM) were added to DMEM supplemented with 10% (v/v) FBS and 1% (v/v)
antibiotics. After 5 days, MTT reagent was added to each well. The plate was incubated at 37 ◦ C
for 1 h. After removing the medium, the plate was washed twice with PBS. DMSO was then added
to dissolve the intracellular insoluble formazan. The absorbance was measured at 570 nm using an
enzyme-linked immunosorbent assay (ELISA) reader (Tecan, Salzburg, Austria), and the percentage
proliferation was calculated.
4.5. TRAP Staining and Activity
RAW 264.7 cells were plated in 96-well plates at a density of 1 × 104 cells/well. The cells were
treated with RANKL (50 ng/mL) and ACHE (1–20 µg/mL), or six compounds (10 µM) were added
to DMEM medium supplemented with 10% (v/v) FBS and 1% (v/v) antibiotics. The medium was
changed every 2 days. After 5 days, the cells were fixed in 3.5% formalin for 10 min and stained
with an acid phosphatase kit. The multinucleated osteoclast cells which were stanined with TRAP,
were observed at 200-fold magnification with a light microscopy. For measuring TRAP activity, the
cells was washed twice with ice-cold PBS and fixed in 3.5% formaldehyde and ethanol/acetone (1:1)
for 10 and 1 min, respectively. The dried cells were then incubated in 50 mM citrate buffer (pH 4.5)
supplemented with 10 mM sodium tartrate and 6 mM PNPP for 40 min. After the mixtures were
added to the new wells with an equal volume of 0.1 N NaOH, the absorbance was determined at
405 nm using an ELISA reader.
4.6. Bone Resorption Assay
A bone resorption assay kit (CosMo Bio, Tokyo, Japan) was used for bone resorption assay. In this
assay, phenol red-free DMEM was used to avoid the interruption of fluorescence measurement due to
Int. J. Mol. Sci. 2017, 18, 322
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phenol red. RAW 264.7 cells were seeded in FACS and CaP-coated 24-well plates (1 × 104 cells/well)
containing phenol red-free DMEM plus 10% FBS, and the medium was replaced with test sample (1 to
20 µg/mL ACHE or 10 µM six compounds) in DMEM containing 50 ng/mL RANKL. After 5 days,
100 µL of the medium and 50 µL resorption assay buffer (catalogue number CSR-BRA-B1, CosMo
Bio Co. Ltd) were transferred into the wells of a 96-well plate and mixed for 3 min under dark
conditions. A fluorometric plate reader (Tecan GENios, Salzburg, Austria) was used to measure the
fluorescence at an excitation of 485 nm and an emission of 535 nm. After washing the cells with
5% sodium hypochlorite, the resorbed areas on the plate were visualized at 200-fold magnification
under light microscopy.
4.7. pH Measurement
RAW 264.7 cells were seeded in 60 mm dishes (1 × 104 cells/dish) containing DMEM plus 10%
FBS, and the medium was replaced with test sample (1–20 µg/mL ACHE or 10 µM of six compounds)
in differentiation medium. The differentiation medium was changed every 2 days. After 5 days, the pH
of the differentiated media was measured using a pH meter (Thermo Scientific, Hudson, NH, USA).
4.8. Immunoprecipitation and Immunoblotting
Immunoprecipitation was performed according to the method of Cho et al. with a slight
modification [31]. RAW 264.7 cells were plated in 6-well plates a density of 1 × 104 cells/well. The cells
were treated with RANKL (50 ng/mL) and ACHE (1–20 µg/mL) or six compounds (10 µM) were
added to DMEM medium supplemented with 10% (v/v) FBS and 1% (v/v) antibiotics. The medium
was changed every 2 days. After 5 days, the cell were harvested using a cell scraper and centrifuged at
7500× g for 20 min. The cell pellets were lysed in NaCl EDTA Tris-nonyl phenoxypolyethoxylethanol
(NP)-40 lysis (NET-NL) buffer (1 M Tris at pH 7.5, 0.5 M EDTA, 1 M NaCl, 1 M DTT, 10% NP-40,
0.1 M PMSF, 10 mg/mL bovine serum albumin (BSA), and protease inhibitor cocktail). The cell lysates
were immunoprecipitated with 2 µg of TRAF6 and V-ATPase antibodies, and protein A–agarose beads
by overnight incubation. The immune complexes were then washed three times with NaCl EDTA
Tris-NP-40 washing (NET-NW) buffer (1 M Tris at pH 7.5, 0.5 M EDTA, 1 M NaCl, 1 M DTT, 10% NP-40,
and 0.1 M PMSF) and centrifuged at 570× g for 30 s. The immunoprecipitated proteins were mixed
with sample loading buffer, resolved by 8%–10% SDS-PAGE, and immunoblotted using anti-TRAF6,
anti-V-ATPase, and anti-β-actin antibodies. The proteins on nirocellulose membranes were detected
with a chemiluminescence kit (Intron Biotechnology, Seoul, Korea) and analyzed using a LAS4000
chemiluminescent image analyser (Fuji, Tokyo, Japan).
4.9. Statistical Analysis
All data are presented as mean ± standard deviation. Statistical analysis were done by the
statistical package for the social sciences (SPSS, Chicago, IL, USA) program. Student’s t-test was
used the parameters between two groups. One-way analysis of variance (ANOVA) and Duncan’s test
were used to compare the parameters among more than three groups and a p < 0.05 was considered
statistically significant.
5. Conclusions
The current report demonstrates that ACHE, an A. capillaris hydroethanolic extract, attenuated
osteoclastic effect including RANKL-induced osteoclast differentiation and bone resorption activity;
the responsible compounds for this effect were chlorogenic acid, hyperoside, and scoparone.
The blockage of bone resorption by A. capillaris was in part mediated by reducing acidification through
down-regulating the interaction of V-ATPase with TRAF6 due to scoparone as well as chlorogenic
acid and hyperoside. For these reasons, A. capillaris extract may have a high potential as an important
bioactive resource of functional food for the prevention or treatment of bone diseases caused by bone
loss. However, additional studies using in vivo models and clinical trials are required.
Int. J. Mol. Sci. 2017, 18, 322
13 of 14
Acknowledgments: This work was supported by a research grant from Hannam University, Dajeon, Korea
in 2016.
Author Contributions: Sang-Hyun Lee and Hae-Dong Jang designed the experiment. Sang-Hyun Lee performed
most parts of the experiments. Jung-Yuen Lee and Young-In Kwon carried out HPLC analysis. Sang-Hyun Lee
and Hae-Dong Jang wrote and revised the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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