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Anti-Inflammatory and Antiosteoarthritis Effects of Saposhnikovia divaricata ethanol Extract: In Vitro and In Vivo Studies

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Saposhnikovia divaricata Schischkin has been used in traditional medicine to treat pain, inflammation, and arthritis. The aim of this study was to investigate the anti-inflammatory and antiosteoarthritis activities of Saposhnikovia divaricata extract (SDE). The anti-inflammatory effect of SDE was evaluated in vitro in lipopolysaccharide- (LPS-) treated RAW 264.7 cells. The antiosteoarthritic effect of SDE was investigated in an in vivo rat model of monosodium iodoacetate- (MIA-) induced osteoarthritis (OA) in which rats were treated orally with SDE (200 mg/kg) for 28 days. The effects of SDE were assessed in vivo by histopathological analysis and by measuring weight-bearing distribution, cytokine serum levels, and joint tissue inflammation-related gene expression. SDE showed anti-inflammatory activity by inhibiting the production of nitric oxide (NO), prostaglandin E 2 (PGE 2 ), tumor necrosis factor- α (TNF- α ), and interleukin-6 (IL-6) in LPS-induced RAW 264.7 cells. In addition, SDE promoted recovery of hind limb weight-bearing, inhibited the production of proinflammatory cytokines and mediators, and protected cartilage and subchondral bone tissue in the OA rat model. Therefore, SDE is a potential therapeutic agent for OA and/or associated symptoms.
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Research Article
Anti-Inflammatory and Antiosteoarthritis
Effects of Saposhnikovia divaricata ethanol Extract:
In Vitro and In Vivo Studies
Jin Mi Chun,1Hyo Seon Kim,1A Yeong Lee,1Seung-Hyung Kim,2andHoKyoungKim
3
1K-herb Research Center, Korea Institute of Oriental Medicine, 1672 Yuseong-daero, Yuseong-gu, Daejeon 34054, Republic of Korea
2Institute of Traditional Medicine and Bioscience, Daejeon University, Daejeon 34520, Republic of Korea
3Mibyeong Research Center, Korea Institute of Oriental Medicine, 1672 Yuseong-daero, Yuseong-gu, Daejeon 34054, Republic of Korea
Correspondence should be addressed to Ho Kyoung Kim; hkkim@kiom.re.kr
Received  November ; Revised  January ; Accepted  January 
Academic Editor: Ken Yasukawa
Copyright ©  Jin Mi Chun et al. is is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Saposhnikovia divaricata Schischkin has been used in traditional medicine to treat pain, inammation, and arthritis. e aim of
this study was to investigate the anti-inammatory and antiosteoarthritis activities of Saposhnikovia divaricata extract (SDE). e
anti-inammator y eect of SDE was evaluated in vitro in lipopolysaccharide- (LPS-) treated RAW . cells. e antiosteoarthritic
eect of SDE was investigated in an in vivo rat model of monosodium iodoacetate- (MIA-) induced osteoarthritis (OA) in which
rats were treated orally with SDE ( mg/kg) for  days. e eects of SDE were assessed in vivo by histopathological analysis
and by measuring weight-bearing distribution, cytokine serum levels, and joint tissue inammation-related gene expression. SDE
showed anti-inammatory activity by inhibiting the production of nitric oxide (NO), prostaglandin E2(PGE2), tumor necrosis
factor-𝛼(TNF-𝛼), and interleukin- (IL-) in LPS-induced RAW . cells. In addition, SDE promoted recovery of hind limb
weight-bearing, inhibited the production of proinammatory cytokines and mediators, and protected cartilage and subchondral
bone tissue in the OA rat model. erefore, SDE is a potential therapeutic agent for OA and/or associated symptoms.
1. Introduction
Osteoarthritis (OA) is the most frequent musculoskeletal
disorder and the most common degenerative joint disease in
the elderly []. OA is a condition caused in part by injury,
loss of cartilage structure and function, and dysregulation of
proinammatory and anti-inammatory pathways [, ]. It
primarily aects the articular cartilage and subchondral bone
of synovial joints and results in joint failure, leading to pain
upon weight-bearing including walking and standing [].
ere is no cure for OA, as it is very dicult to restore the
cartilage once it is destroyed []. e goals of treatment are
to relieve pain, maintain or improve joint mobility, increase
the strength of the joints, and minimize the disabling eects
of the disease. Pharmacological treatments of OA aim to
reduce pain in order to increase the patient’s joint function
and quality of life. Although cartilage destruction is the main
event in OA, the degradation of collagen is the fundamental
incident that determines the irreversible progression of OA
in association with inammation [, ]. Treatments with anti-
inammatory and chondroprotective activity are expected to
relieve pain and maintain matrix integrity in OA patients.
erefore, decreasing inammation will likely be bene-
cial in OA management. Recent studies suggest protective
roles for herbal resources on the progression of OA, in
terms of mitigating chondrocyte inammation and further
cartilage destruction, through their ability to interact with
joint-associated tissues, resulting in the mitigation of joint
pain [].
e root of Saposhnikovia divaricata Schischkin (Umbel-
liferae) has been widely used in traditional medicine for
the treatment of headache, pain, inammation, and arthritis
in Korea and China [, ]. e diverse pharmacologi-
cal eects of Saposhnikovia divaricata (SD) also include
anti-inammatory, analgesic, antipyretic, and antiarthritic
properties [, ]. A recent study demonstrated that SD
Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2016, Article ID 1984238, 8 pages
http://dx.doi.org/10.1155/2016/1984238
Evidence-Based Complementary and Alternative Medicine
chromone extract possesses potential antirheumatoid arthri-
tis eects in a mouse model of collagen-induced arthritis [];
however, few studies have been conducted to support the
anti-inammatory and antiarthritis activity of Saposhnikovia
divaricata extract (SDE).
erefore, the present study investigated the anti-inam-
matory and antiosteoarthritis activities of a % ethanol
extract of SD. First, the anti-inammatory eect of SDE
was evaluated in vitro in LPS-induced RAW . cells.
Next, the antiosteoarthritis eect of SDE was measured by
assessing weight-bearing distribution, degradation of artic-
ular cartilage, and inammatory responses in a rat model of
monosodium iodoacetate- (MIA-) induced OA.
2. Materials and Methods
2.1. Preparation of SDE. e rhizomes of SD were purchased
as a dried herb from Hanherb Co. (Guri, Korea). e
plant materials were conrmed taxonomically by Dr. Go-Ya
Choi of the Korea Institute of Oriental Medicine (KIOM).
A voucher specimen (number  SDE-) was deposited
in the Korean Herbarium of Standard Herbal Resources.
Dried rhizomes of SD (g) were extracted twice with
% ethanol (with a  h reux) and the extract was then
concentrated under reduced pressure. e decoction was
ltered, lyophilized, and stored at C. e yield of dried
extract from crude starting materials was .% (w/w).
2.2. Quantitative High-Performance Liquid Chromatography
(HPLC) Analysis. Chromatographic analysis was performed
with a HPLC system (Waters Co., Milford, MA, USA) and a
photodiode array detector. For the HPLC analysis of SDE, the
prim-O-glucosylcimifugin standard was purchased from the
Korea Promotion Institute for Traditional Medicine Industry
(Gyeongsan, Korea), and sec-O-glucosylhamaudol and 󸀠-O-
𝛽-D-glucosyl--O-methylvisamminol were isolated within
our laboratory and identied by spectral analyses, primarily
by NMR and MS.
SDE samples (. mg) were dissolved in % ethanol
( mL). Chromatographic separation was performed with an
XSelect HSS T C column (. × mm, 𝜇m, Waters
Co., Milford, MA, USA). e mobile phase consisted of
acetonitrile (A) and .% acetic acid in water (B) at a ow-rate
of . mL/min. A multistep gradient program was used as fol-
lows: % A ( min), –% A (– min), % A (– min),
and –% A (– min). e detection wavelength was
scannedatnmandrecordedatnm.einjection
volume was . 𝜇L. Standard solutions for the determination
of three chromones were prepared at a nal concentration of
. mg/mL (prim-O-glucosylcimifugin), . mg/mL (󸀠-
O-𝛽-D-glucosyl--O-methylvisamminol), and . mg/mL
(sec-O-glucosylhamaudol) in methanol and kept at C.
2.3. Evaluation of Anti-Inammatory Activity In Vitro
2.3.1. Cell Culture and Sample Treatment. RAW . cells
were obtained from the American Type Culture Collec-
tion (ATCC, Manassas, VA, USA) and grown in DMEM
medium containing % antibiotics and .% FBS. Cells
were incubated in a humidied atmosphere of % CO2at
C. To stimulate the cells, the medium was replaced with
fresh DMEM medium, and lipopolysaccharide (LPS, Sigma-
Aldrich Chemical Co., St. Louis, MO, USA) at  𝜇g/mL was
added in the presence or absence of SDE ( or  𝜇g/mL)
for an additional  h.
2.3.2. Determination of Nitric Oxide (NO), Prostaglandin E2
(PGE2), Tumor Necrosis Factor-𝛼(TNF-𝛼), and Interleukin-
6(IL-6)Production. Cells were treated with SDE and stim-
ulated with LPS for  h. NO production was analyzed by
measuring nitrite using the Griess reagent according to a
previous study []. Secretion of the inammatory cytokines
PGE2,TNF-𝛼, and IL- was determined using an ELISA
kit (R&D systems) according to manufacturer instructions.
e eects of SDE on NO and cytokine production were
determined at  nm or  nm using a Wallac EnVision™
microplate reader (PerkinElmer).
2.4. Evaluation of Antiosteoarthritis Activity In Vivo
2.4.1. Animals. Male Sprague-Dawley rats ( weeks old) were
purchased from Samtako Inc. (Osan, Korea) and housed
under controlled conditions with a -h light/dark cycle at
22 ± 2Cand55 ± 15% humidity. Rats were provided with
a laboratory diet and water ad libitum. All experimental
procedures were performed in compliance with the National
Institutes of Health (NIH) guidelines and approved by the
AnimalCareandUseCommitteeoftheDaejeonuniversity
(Daejeon, republic of Korea).
2.4.2. Induction of OA with MIA in Rats. e animals were
randomized and assigned to treatment groups before the
initiation of the study (𝑛=6per group). MIA solution
( mg/ 𝜇L of .% saline) was directly injected into the
intra-articular space of the right knee under anesthesia
induced with a mixture of ketamine and xylazine. Rats were
divided randomly into four groups: () the saline group with
no MIA injection, () the MIA group with MIA injection, ()
the SDE-treated group ( mg/kg) with MIA injection, and
() the indomethacin- (IM-) treated group (mg/kg) with
MIA injection. Rats were administered orally with SDE and
IM  week before MIA injection for  weeks. e dosage of
SDEandIMusedinthisstudywasbasedonthoseemployed
in previous studies [, , ].
2.4.3. Measurements of Hindpaw Weight-Bearing Distribution.
Aer OA induction, the original balance in weight-bearing
capability of hindpaws was disrupted. An incapacitance tester
(Linton instrumentation, Norfolk, UK) was used to evaluate
changes in the weight-bearing tolerance. Rats were carefully
placed into the measuring chamber. e weight-bearing force
exerted by the hind limb was averaged over a s period.
e weight distribution ratio was calculated by the following
equation: [weight on right hind limb/(weight on right hind
limb + weight on le hind limb)] × [].
2.4.4. Measurements of Serum Cytokine Levels. e blood
samples were centrifuged at , g for  min at C; then
Evidence-Based Complementary and Alternative Medicine
T : Real-time PCR primer sequences.
Gene Primer sequence
IL-𝛽Forward 󸀠-CCCTGCAGCTGGAGAGTGTGG-󸀠
Reverse 󸀠-TGTGCTCTGCTTGAGAGGTGCT-󸀠
IL- Forward 󸀠-TTCCTACCCCAACTTCCAATG-󸀠
Reverse 󸀠-ATGAGTTGGATGGTCTTGGTC-󸀠
TNF-𝛼Forward 󸀠-GACCCTCACACTCAGATCATCTTCT-󸀠
Reverse 󸀠-TGCTACGACGTGGGCTACG-󸀠
NOS-II Forward 󸀠-CTTTACGCCACTAACAGTGGCA-󸀠
Reverse 󸀠-AGTCATGCTTCCCATCGCTC-󸀠
COX- Forward 󸀠-TGGTGCCGGGTCTGATGATG-󸀠
Reverse 󸀠-GCAATGCGGTTCTGATACTG-󸀠
GAPDH Probe Applied Biosystems® Rat GAPD (GAPDH) Endogenous Control (VIC®/MGB Probe, E)
theserumwascollectedandstoredatCuntiluse.e
levels of IL-𝛽,IL-,TNF-𝛼,andPGE
2in the serum were
measured using ELISA kits from R&D Systems (Minneapolis,
MN, USA) according to manufacturer instructions.
2.4.5. Real-Time Quantitative RT-PCR Analysis. Tot al RNA
was extracted from knee joint tissue using the TRI reagent®
(Sigma-Aldrich, St. Louis, MO, USA), reverse-transcribed
into cDNA and PCR-amplied using a TM One Step RT PCR
kit with SYBR green (Applied Biosystems, Grand Island, NY,
USA). Real-time quantitative PCR was performed using the
Applied Biosystems  Real-Time PCR system (Applied
Biosystems, Grand Island, NY, USA). e primer sequences
and the probe-sequence are shown in Table . Aliquots of
sample cDNAs and an equal amount of GAPDH cDNA were
amplied with the TaqMan® Universal PCR master mixture
containing DNA polymerase according to manufacturer
instructions (Applied Biosystems, Foster, CA, USA). PCR
conditions were  min at C,  min at C,  s at C,
and  min at C for  cycles. e concentration of target
gene was determined using the comparative Ct (threshold
cycle number at cross-point between amplication plot and
threshold) method, according to manufacturer instructions.
2.4.6. Histopathological Analysis. Tissue specimens from the
kneejointofratswereremoved,xedin%formalin,
embedded in paran, and serially sectioned at  𝜇m. Tissue
sections were then stained with hematoxylin and eosin
(H&E) or Safranin O-fast green. Histological changes were
examined by light microscopy (Olympus CX/BX, Olym-
pus Optical Co., Tokyo, Japan) and photographed (Olympus
DP).
2.5. Statistical Analysis. All results are presented as the
mean ±standard deviation (SD). e statistical analysis was
performed using one-way analysis of variance (ANOVA),
Duncans multiple range test was performed to identify
signicant dierences between groups, and 𝑝values of <.
were considered to be statistically signicant.
3. Results
3.1. Chemical Prole of SDE. To identify and quantify the
levels of marker components in SDE, HPLC analysis was
performed. e chromatogram of the main components is
shown in Figure . e three components of SDE, prim-O-
glucosylcimifugin, 󸀠-O-𝛽-D-glucosyl--O-methylvisammi-
nol, and sec-O-glucosylhamaudol, were detected at approx-
imately ., ., and .min, respectively. e prim-O-
glucosylcimifugin content was the highest (.%), followed
by 󸀠-O-𝛽-D-glucosyl--O-methylvisamminol (.%) and
sec-O-glucosylhamaudol (.%).
3.2. Eect of SDE on NO, PGE2, TNF-𝛼,andIL-6Production
In Vitro. We examined the eects of SDE on the levels of
NO, PGE2,TNF-𝛼, and IL- in LPS-stimulated RAW .
cells. Cells were treated with SDE plus LPS or LPS alone for
 h. SDE signicantly inhibited the production of NO, PGE2,
TNF-𝛼,andIL-atarangeofor𝜇g/mL (Figure ).
In addition, SDE did not aect cell viability and was not toxic
to RAW . cells (data not shown).
3.3. Eect of SDE on Changes in Hindpaw Weight-Bearing
Distribution. Weight distribution was measured between
sensitized and contralateral hind limbs and used as an
index of joint discomfort in the arthritic knee. erefore,
we evaluated hindpaw weight-bearing using an incapacitance
tester for  days. e ratio of hindpaw weight distribution
between the right and le limbs was used to assess the
progression of OA []. e weight-bearing distribution of
the MIA group reduced rapidly and became signicantly
dierent from that of the saline group by day  post-MIA
injection and was maintained for at least  days. By contrast,
in the SDE- and IM-treated groups, these values were only
slightlydecreasedatdaycomparedwiththoseoftheMIA
group. Beyond that, there was full recovery and the balance
between both hind legs returned to normal in the SDE-
and IM-treated groups. ese results demonstrate signicant
recovery of hind limb weight-bearing in the SDE-treated
group (Figure ).
Evidence-Based Complementary and Alternative Medicine
(AU)
(min)
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80 1
2
3
(a)
(min)
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00
1
2
3
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
(AU)
(b)
F : HPLC chromatograms of standard compounds (a) and SDE (b). () Prim-O-glucosylcimifugin, () 󸀠-O-𝛽-D-glucosyl--O-
methylvisamminol, and () sec-O-glucosylhamaudol.
Nitric oxide (𝜇M)
### ∗∗
∗∗∗
SDE (𝜇g/mL) −−
LPS (1 𝜇g/mL) −+++
200 400
0.0
10.0
20.0
30.0
40.0
(a)
###
∗∗
∗∗∗
SDE (𝜇g/mL) −−
LPS (1𝜇g/mL) −++ +
200 400
0
200
400
600
800
1000
PGE2(pg/mL)
(b)
###
∗∗
SDE (𝜇g/mL)
LPS (1𝜇g/mL) −++ +
200 400
0
20000
40000
60000
80000
TNF-𝛼(pg/mL)
(c)
###
∗∗ ∗∗∗
SDE (𝜇g/mL) −−
LPS (1𝜇g/mL) −++ +
200 400
0
5000
10000
15000
20000
25000
IL-6(pg/mL)
(d)
F : Eects of SDE on the production of cytokines and inammatory mediators in LPS-stimulated RAW . macrophages. Cells were
treatedwithSDE(,,or𝜇g/mL) plus LPS ( 𝜇g/mL) or LPS alone for  h. (a) NO production was measured using the Griess reagent.
(bd)ProductionofPGE
2,TNF-𝛼, and IL- was measured by ELISA. Values are expressed as the means ±SD (𝑛=3). ###𝑝< 0.001 versus
untreatedLPSandSDE;𝑝< 0.05,∗∗𝑝< 0.01,and∗∗∗ 𝑝< 0.001 versus LPS alone.
3.4. Eect of SDE on Inammatory Cytokine Serum Levels.
Proinammatory cytokines have important roles in the main-
tenance of chronic inammation and tissue damage during
the progression of OA. erefore, we investigated the eects
of SDE on the serum levels of IL-𝛽,IL-,TNF-𝛼,andPGE
2
in the MIA-induced OA model. e serum levels of IL-𝛽,IL-
, TNF-𝛼,andPGE
2increased in the MIA group compared
with those in the saline group but were suppressed in the
SDE-andIM-treatedgroupscomparedwiththoseintheMIA
group (Figure ). ese results indicate that SDE might have
cartilage protection eects in the MIA model by regulating
inammatory cytokines.
3.5. Expression of Cytokine and Inammatory Mediator mRNA
in Knee Joint Tissues. We investigated the eects of SDE on
themRNAlevelsofCOX-,iNOS,IL-𝛽,IL-,andTNF-𝛼in
thekneejointtissuesofrats.Wealsoinvestigatedtheeects
of SDE on the mRNA levels of cytokines in the knee joint
tissues of rats. e expression of cytokine and inammatory
mediator mRNAs increased following MIA injection, but the
expression of the cytokine was attenuated in the SDE- and
IM-treated groups (Figure ). us, our results indicate that
SDE suppresses the expression of inammatory cytokines in
MIA-treated rats.
3.6.EectsofSDEonHistopathology. Following sacrice of
the rats, the knee joints were evaluated histologically for
severity of inammation, synovial hyperplasia, and bone
damage by H&E and Safranin O staining. e MIA group
exhibited histological changes indicative of severe arthritis,
with extensive inltration of inammatory cells into articular
tissues, exudation into the synovial space, synovial hyper-
plasia,andcartilageerosion;however,treatmentwithSDE
andIMinhibitedthedamageandsynovialhyperplasiain
Evidence-Based Complementary and Alternative Medicine
IM
SDE
MIA
Saline
###
### ###
∗∗
∗∗
∗∗∗
∗∗∗
0.0
20.0
40.0
60.0
Weight-bearing distribution (%)
714210
Days aer MIA induction
F : Eects of SDE on changes in hindpaw weight-bearing
distribution in MIA-induced OA in rats. e weight-bearing distri-
bution ratio was measured once a week for  days aer the injection
of MIA using an incapacitance tester, compared to that of the MIA-
induced group. ### 𝑝< 0.001 versus saline; 𝑝< 0.05,∗∗𝑝< 0.01,
and ∗∗∗𝑝< 0.001 versus MIA.
joints (Figure ). ese histological features show that SDE
attenuates the severity of MIA-induced OA in rats.
4. Discussion
e current available treatments for OA focus on target
symptom reduction, maintenance of joint mobility, and limi-
tation of the loss of functional capacity. Many studies suggest
that traditional herbal resources benet the management of
inammatory arthritis and may therefore benet OA [–
]. SD shows several medicinal therapeutic eects; however,
to date, no studies have been conducted to evaluate the
ecacy of SD for the treatment of OA. erefore, the present
study was conducted to evaluate the anti-inammatory
and antiosteoarthritic activities of SDE using LPS-induced
macrophages and a MIA-induced OA model.
Inammatory mediators play key roles in the progression
of cartilage destruction in OA []. e proinammatory
cytokines are deemed to display catabolic properties that
inuence the pathophysiological processes of OA []. Our
results show that SDE inhibits the production of NO, PGE2,
TNF-𝛼, and IL- in LPS-stimulated RAW . cells.
e antiosteoarthritic eect of SDE in MIA-induced
rats was evaluated by measuring weight-bearing distribu-
tion, inammatory cytokines, and mediators in serum,
inammation-relatedgeneexpressioninkneejoints,and
histopathological parameters. In this study, we also provide
evidence for an OA-related pain-relieving eect of SDE in the
MIAOAmodel.OApaincanbetriggeredbyjointmovement
and typically results in diminished use and reduced joint
mobility[].OurresultsshowthatSDEsignicantlyprotects
weight-bearing in MIA-induced OA in rats, suggesting that
SDEcouldbeusefulfortreatingOApain.
Saline MIA SDE IM
###
∗∗
∗∗∗
0
100
200
300
400
500
IL-1𝛽 (pg/mL)
(a)
Saline MIA SDE IM
##
0
20
40
60
80
100
IL-6(pg/mL)
(b)
Saline MIA SDE IM
##
0
20
40
60
80
TNF-𝛼(pg/mL)
(c)
Saline MIA SDE IM
###
∗∗
0
500
1,000
1,500
PGE2(pg/mL)
(d)
F : Eects of SDE on serum levels of cytokines and inam-
matory mediators in MIA-induced OA in rats. (a) Serum IL-𝛽,
(b) IL-, (c) TNF-𝛼,and(d)PGE
2levels were measured by ELISA.
##𝑝< 0.01 and ###𝑝< 0.001 versus saline; 𝑝< 0.05,∗∗𝑝< 0.01,
and ∗∗∗𝑝< 0.001 versus MIA.
e chondroprotective eects of SDE, via reduction of
inammation, have been established in rheumatoid arthritis
animal models []. Consistent with published work, the
present study demonstrated that SDE exerts chondropro-
tective eects in MIA-induced OA in rats by suppressing
the expression of inammatory cytokines and mediators in
serum and inammation-related genes in knee joints.
OA is a condition caused in part by injury loss of cartilage
structure and function and dysregulation of proinammator y
and anti-inammatory pathways [, ]. Inammation is an
important factor associated with the development and pro-
gression of OA. Catabolic and proinammatory mediators,
for example, cytokines, NO, and PGE2,areproducedby
inamed synovium and alter the balance of cartilage matrix
Evidence-Based Complementary and Alternative Medicine
Saline MIA SDE IM
##
0.0
0.5
1.0
1.5
iNOS expression (fold)
(a)
Saline MIA SDE IM
#
0.0
0.5
1.0
1.5
COX-2expression (fold)
(b)
Saline MIA SDE IM
#
0.0
0.5
1.0
1.5
IL-1𝛽 expression (fold)
(c)
Saline MIA SDE IM
##
∗∗ ∗∗
0.0
0.5
1.0
1.5
IL-6expression (fold)
(d)
Saline MIA SDE IM
#
0.0
0.5
1.0
1.5
TNF-𝛼expression (fold)
(e)
F : Eects of SDE on the expression of cytokines and inammatory mediators in the knee joint of rats with MIA-induced OA.
Expression of (a) iNOS, (b) COX-,(c) IL-𝛽, (d) IL-, and (e) TNF-𝛼mRNA was determined by real-time RT-PCR. #𝑝< 0.05 and ##𝑝< 0.01
versus saline; 𝑝< 0.05 and ∗∗𝑝< 0.01,versusMIA.
Saline MIA SDE (200 mg/kg) IM (2mg/kg)
(a)
Saline MIA SDE (200 mg/kg) IM (2mg/kg)
(b)
F : Histopathological features of the knee joint tissues of rats with MIA-induced OA. Representative photographs of knee joint tissues
stained with (a) H&E or (b) Safranin O-fast green (magnication, x).
Evidence-Based Complementary and Alternative Medicine
degradation and repair. ese processes will then exacer-
bate clinical symptoms and joint degradation in OA [].
erefore, inhibiting proinammatory cytokines could be an
important approach to managing OA. In this study, SDE may
inhibit inammatory reactions and partially prevent and slow
the progression of OA. In addition, we demonstrated that
SDE attenuates histological damage and synovial hyperplasia
in joints, compared with MIA group. ese results suggest
that SDE prevents the degradation of cartilage and cartilage
inammation, resulting in the prevention of OA progression.
SD contains major bioactive constituents, including
chromones such as prim-O-glucosylcimifugin, 󸀠-O-𝛽-D-
glucosyl--O-methylvisamminol, cimifugin, and sec-O-glu-
cosylhamaudol, which are usually obtained from the roots
of the plant [, , ]. Prim-O-glucosylcimifugin, the
chromone with the highest content in the roots of SD,
showed signicant anti-inammatory eects on LPS-
inducedinammatoryresponsesinRAW.cellsand
signicantly protected mice against LPS-induced acute lung
injury []. SD ethanol extract and chromones isolated
from SD showed potential anti-inammatory and protective
eects in LPS-activated RAW . cells [, ]. In the
present study, we conrmed the presence of the chromones,
which were prim-O-glucosylcimifugin, 󸀠-O-𝛽-D-glucosyl-
-O-methylvisamminol, and sec-O-glucosylhamaudol in
SDE, and this partially explains the anti-inammatory
activity of SDE.
5. Conclusions
In conclusion, SDE showed anti-inammatory activity by
inhibiting the production of NO, PGE2,TNF-𝛼,andIL-
in LPS-induced RAW . cells. SDE also attenuated joint
pain and stiness, inhibited the production of proinamma-
tory cytokines and mediators, and protected cartilage and
subchondral bone tissue in an OA rat model. erefore,
our results suggest that SDE may be a potentially suitable
therapeutic agent for OA and/or its associated symptoms.
Conflict of Interests
e authors declare that there is no conict of interests
regarding the publication of this paper.
Acknowledgments
is work was supported by Evaluation of Eectiveness
of Alternative Herbal Medicine Resources (K, K)
and Characteristic Analysis of Alternative Herbal Medicine
Resources (K) from the Korea Institute of Oriental
Medicine (KIOM). We thank Dr. Go-Ya Choi of KIOM for
thecriticalauthenticationofplantmaterialandforthehelpful
discussions.
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Supplementary resource (1)

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... The weight-bearing distribution of the sensitized and normal hind limbs was used as a surrogate marker for joint pain [18]. We evaluated the hind paw weight-bearing capacity using an incapacitance tester, and the hind paw weightdistribution ratio was used to assess OA progression [18,27]. Compared with the NC group, the OAC group exhibited a noticeably lower weigh-distribution ratio. ...
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Low-temperature stratification has a beneficial effect on releasing seed dormancy and germination. Taking Saposhnikovia divaricata (Trucz.) Schischk. seeds as the research object, the effects of low-temperature stratification on dormancy release and germination of S. divaricata seeds were studied. The morphological, physiological and biochemical indexes, hormone content and transcriptome of dormant seeds, seeds in the dormancy release period and germination period were discussed. The results showed that 4 °C low-temperature stratification treatment increased the contents of soluble sugar, gibberellin and indoleacetic acid, enhanced amylase and α-amylase activity, but decreased the contents of starch and abscisic acid. A total of 100,690 unigenes were obtained from S. divaricata transcriptome. RNA-seq analysis showed that compared with dormant seeds (CK), the number of up-regulated genes in seeds in the dormancy release period (S) and germination period (G) was 13,998 and 16,817, and the number of down-regulated genes was 9237 and 14,054, respectively. This study identified a large number of differentially expressed genes related to plant hormone signal transduction, starch and sucrose, plant energy metabolism pathway and hormones in low-temperature stratification reaction. The transcriptomic gene expression profiles present a valuable genomic tool to the molecular mechanisms of S. divaricata seed germination underlying low-temperature stratification, which can provide theoretical basis and practical guidance for cultivation and planting of S. divaricata.
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Skin graft surgery is a standard treatment that increases the survival rate of patients with burn injuries; however, it leaves many sequelae. Conventional external preparations for the treatment of burns also have various side effects. In this retrospective case study, we assessed the cases of four patients with topical third-degree burns who did not wish to undergo the skin graft surgeries recommended by medical doctors and were thus treated using traditional medicine alone. A Korean medicine doctor administered integrative traditional medicine treatment to the patients using acupuncture and herbal ointments. Analysis of the patients’ treatment photographs, quantitative evaluation indices, and vivid narratives suggested that their skin recovered well without adverse effects. Traditional Korean medicine treatment, including acupuncture and herbal ointment treatment, can be a new alternative therapeutic strategy for the treatment of patients with topical burns who do not want to undergo skin graft surgery or who have higher risks for poor surgical outcomes.
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Osteoarthritis (OA) is a multifactorial disease primarily noted by cartilage degradation in association with inflammation that causes significant morbidity, joint pain, stiffness, and limited mobility. Present-day management of OA is inadequate due to the lack of principal therapies proven to be effective in hindering disease progression where symptomatic therapy focused approach masks the actual etiology leading to irreversible damage. Here, we describe the effect of UP3005, a composition containing a proprietary blend of two standardized extracts from the leaf of Uncaria gambir and the root bark of Morus alba, in maintaining joint structural integrity and alleviating OA associated symptoms in monosodium-iodoacetate- (MIA-) induced rat OA disease model. Pain sensitivity, micro-CT, histopathology, and glycosaminoglycans (GAGs) level analysis were conducted. Diclofenac at 10 mg/kg was used as a reference compound. UP3005 resulted in almost a complete inhibition in proteoglycans degradation, reductions of 16.6% (week 4), 40.5% (week 5), and 22.0% (week 6) in pain sensitivity, statistically significant improvements in articular cartilage matrix integrity, minimal visual subchondral bone damage, and statistically significant increase in bone mineral density when compared to the vehicle control with MIA. Therefore, UP3005 could potentially be considered as an alternative therapy from natural sources for the treatment of OA and/or its associated symptoms.
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Introduction: Epigallocatechin 3-gallate (EGCG), a polyphenol present in green tea, was shown to exert chondroprotective effects in vitro. In this study, we used a post-traumatic osteoarthritis (OA) mouse model to test whether EGCG could slow the progression of OA and relieve OA-associated pain. Methods: C57BL/6 mice were subjected to surgical destabilization of the medial meniscus (DMM) or sham surgery. EGCG (25 mg/kg) or vehicle control was administered daily for four or eight weeks by intraperitoneal injection starting on the day of surgery. OA severity was evaluated by Safranin O staining and Osteoarthritis Research Society International (OARSI) score, and by immunohistochemical analysis to detect cleaved aggrecan and type II collagen, and expression of proteolytic enzymes matrix metalloproteinase (MMP)-13 and A Disintegrin And Metalloproteinase with Thrombospondin Motifs (ADAMTS5). Real-time polymerase chain reaction (PCR) was performed to characterize the expression of genes critical for articular cartilage homeostasis. During the course of the experiments, tactile sensitivity testing (von Frey test) and open field assays were used to evaluate pain behaviors associated with OA, and expression of pain expression markers and inflammatory cytokines in the dorsal root ganglion (DRG) were determined by real-time PCR. Results: Four and eight weeks after DMM surgery, the cartilage in EGCG-treated mice exhibited less Safranin O loss and cartilage erosion, and lower OARSI scores compared to vehicle-treated controls, which was associated with reduced staining for aggrecan and type II collagen cleavage epitopes, and reduced staining for MMP-13 and ADAMTS5 in the articular cartilage. Articular cartilage in the EGCG-treated mice also exhibited reduced levels of MMP-1, -3, -8, -13, ADAMTS5, interleukin (IL)-1β, and tumor necrosis factor (TNF)-α mRNA and elevated gene expression of the MMP regulator Cbp/p300 Interacting Transactivator 2 (CITED2). Compared to vehicle controls, mice treated with EGCG exhibited reduced OA-associated pain, as indicated by higher locomotor behavior (i.e. distance traveled). Moreover, expression of chemokine receptor (CCR2), and pro-inflammatory cytokines IL-1β and TNF-α in the DRG were significantly reduced to levels similar to sham-operated animals. Conclusions: This study provides the first evidence in an OA animal model that EGCG significantly slows OA disease progression and exerts a palliative effect.
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Background Houttuynia cordata Thunb. (Saururaceae) has been used in traditional medicine for treatment of inflammatory diseases. This study evaluated the anti-inflammatory effects of an ethyl acetate fraction derived from a Houttuynia cordata extract (HCE-EA) on the production of inflammatory mediators and the activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Methods To measure the effects of HCE-EA on pro-inflammatory cytokine and inflammatory mediator’s expression in RAW 264.7 cells, we used the following methods: cell viability assay, Griess reagent assay, enzyme-linked immunosorbent assay, real-time polymerase chain reaction and western blotting analysis. Results HCE-EA downregulated nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), and interleukin (IL-6) production in the cells, as well as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression. Furthermore, HCE-EA suppressed nuclear translocation of the NF-κB p65 subunit, which correlated with an inhibitory effect on IκBα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha) phosphorylation. HCE-EA also attenuated the activation of MAPKs (p38 and JNK). Conclusions Our results suggest that the anti-inflammatory properties of HCE-EA may stem from the inhibition of pro-inflammatory mediators via suppression of NF-κB and MAPK signaling pathways.
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Herbal remedies and dietary supplements have become an important area of research and clinical practice in orthopaedics and rheumatology. Understanding the risks and benefits of using herbal medicines in the treatment of arthritis, rheumatic diseases, and musculoskeletal complaints is a key priority of physicians and their patients. This review discusses the latest advances in the use of herbal medicines for treating osteoarthritis (OA) by focusing on the most significant trends and developments. This paper sets the scene by providing a brief introduction to ethnopharmacology, Ayurvedic medicine, and nutrigenomics before discussing the scientific and mechanistic rationale for targeting inflammatory signalling pathways in OA by use of herbal medicines. Special attention is drawn to the conceptual and practical difficulties associated with translating data from in-vitro experiments to in-vivo studies. Issues relating to the low bioavailability of active ingredients in herbal medicines are discussed, as also is the need for large-scale, randomized clinical trials.
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Natural products have always been a pivotal source of new drug development. Dry roots of Saposhnikovia divaricata (Turcz.) Schischk. (Umbelliferae) is a perennial herb and is also known as Bang Pung in traditional medicine. Numerous in vitro and in vivo studies have revealed the diverse pharmacological effects of S. divaricata and its role in the treatment of various diseases. This herb has exhibited significant inhibitory effects against inflammation and associated disorders. The present study explored the ethnopharmacological applications and molecular mechanisms behind the anti-inflammatory effects of S. divaricata herb and a single compound blockade of multi-signaling inflammatory cascades. Taken together, this review provides insight into the potential role of S. divaricata against various inflammatory diseases.
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Nuclear factor-kappaB (NF-κB) proteins constitute a family of transcription factors that are stimulated by pro-inflammatory cytokines, chemokines, stress-related factors and extracellular matrix (ECM) degradation products. Upon stimulation, the activated NF-κB molecules trigger the expression of an array of genes which induce destruction of the articular joint, leading to osteoarthritis (OA) onset and progression. Therefore, targeted strategies that interfere with NF-κB signalling could offer novel potential therapeutic options for OA treatment. In this review, we discuss the involvement of NF-κB in OA pathogenesis and how pharmacological inhibition of the NF-κB signalling pathway affects OA incidence and evolution.
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Ethnopharmacological relevance: Saposhnikovia divaricata (SD), called "Fangfeng" in China, is commonly used in clinical compound prescription for treatment of rheumatoid arthritis (RA), but its actions on RA have not been clarified. The present study aims to determine the anti-inflammatory activity of SD chromone extract (SCE), the major bioactive component of SD, on collagen-induced arthritis (CIA) rats, and elucidate its underlying mechanisms with regards to its molecular basis of action on human fibroblast-like synoviocytes derived from RA patients (HFLS-RA). Materials and methods: CIA model on rats was constructed by injection of bovine type II collagen. Rats were pre-treated with different dosages of SCE from 3 days before till 35 days after model building. The progression of CIA was evaluated by macroscopic scoring, X-ray observation and hematoxylin and eosin (HE) staining of paws. HFLS-RA were pre-treated with different concentrations of SCE prior to stimulation with 10 ng/ml of tumor necrosis factor (TNF) α. By radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), levels of interleukin (IL)-1β, IL-6, TNFα and prostaglandin E2 (PGE2) were quantified respectively. Nuclear factor (NF-κB) p65 expression and DNA-binding activity were tested by immunohistochemisty and electrophoretic mobility shift assay (EMSA) respectively. Phosphorylation of extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK) and p38 MAPKs were examined by immunohistochemisty staining and western blot analysis. Results: Histological examination and radiological observation demonstrated that SCE significantly reduced the inflammatory responses in the joints of CIA rats. SCE inhibited the production of TNFα, IL-1β, and IL-6 in the joint tissues and sera. The level of PGE2 in sera was also decreased by SCE. Moreover, SCE treatment in vivo was able to reduce protein level of NF-κB, the transcriptional factor closely related to the inflammatory process, in articular synovium and cartilage of CIA rats. In addition, SCE inhibited p-ERK, p-JNK and p-p38 expression, which were considered to be involved in the phosphorylation of transcription factor NF-κB and the transcription of pro-inflammatory factors. Further, SCE inhibited NF-κB DNA binding activity and attenuated the phosphorylation of ERK, JNK and p38 MAPKs, in a concentration-dependent manner in cultured HFLS-RA. Conclusion: These results highlight the anti-arthritic potential of SCE, and provide further evidence of the involvement of the NF-κB and MARKs inhibition in the effects of SCE.
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Prime-O-glucosylcimifugin is an active chromone isolated from Saposhnikovia root which has been reported to have various activities, such as anti-convulsant, anticancer, anti-inflammatory properties. The purpose of this study was to evaluate the effect of prime-O-glucosylcimifugin on acute lung injury (ALI) induced by lipopolysaccharide in mice. BALB/c mice received intraperitoneal injection of Prime-O-glucosylcimifugin 1h before intranasal instillation (i.n.) of lipopolysaccharide (LPS). Concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and interleukin (IL)-6 in bronchoalveolar lavage fluid (BALF) were measured by enzyme-linked immunosorbent assay (ELISA). Pulmonary histological changes were evaluated by hematoxylin-eosin, myeloperoxidase (MPO) activity in the lung tissue and lung wet/dry weight ratios were observed. Furthermore, the mitogen-activated protein kinases (MAPK) signaling pathway activation and the phosphorylation of IκBα protein were determined by Western blot analysis. Prime-O-glucosylcimifugin showed promising anti-inflammatory effect by inhibiting the activation of MAPK and NF-κB signaling pathway.
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Osteoarthritis is a condition caused in part by injury, loss of cartilage structure and function, and an imbalance in inflammatory and anti-inflammatory pathways. It primarily affects the articular cartilage and subchondral bone of synovial joints and results in joint failure, leading to pain upon weight bearing including walking and standing. There is no cure for osteoarthritis, as it is very difficult to restore the cartilage once it is destroyed. The goals of treatment are to relieve pain, maintain or improve joint mobility, increase the strength of the joints and minimize the disabling effects of the disease. Recent studies have shown an association between dietary polyphenols and the prevention of osteoarthritis-related musculoskeletal inflammation. This review discusses the effects of commonly consumed polyphenols, including curcumin, epigallocatechin gallate and green tea extract, resveratrol, nobiletin and citrus fruits, pomegranate, as well as genistein and soy protein, on osteoarthritis with an emphasis on molecular antiosteoarthritic mechanisms.
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Achyranthyes japonica Nakai (AJN) has been traditionally used to control pain and improve dysfunction in osteoarthritis (OA) patients. The objectives of the present study were to investigate anti-inflammatory and anti-osteoarthritis activities of fermented AJN (FAJN). Anti-inflammatory activity of non-fermented AJN (NFAJN) and FAJN was evaluated by in vitro assay using LPS-induced RAW 264.7 cells. In addition, their cartilage protective effects were also determined in vitro assay using SW1353 cell and in vivo model system using collagenase-induced arthritis (CIA) in rabbits. Moreover, we isolated and identified 20-hydroxyecdysone (20-HES) as a marker component in FAJN. FAJN showed stronger anti-inflammatory activity than NFAJN through inhibiting production of NO and PGE2 in LPS-induced RAW 264.7, and lowering levels of MMP-3 release in SW1353 cells treated with TNF-a. FAJN contained higher levels of 20-HES, as a marker component, than AJN. FAJN ameliorates the progress of OA by inhibiting local inflammation. It does this by regulating levels of TNF-a and IL-4, and protecting articular cartilage by preventing destruction of proteoglycan, collagens, and also preventing injury to chondrocytes. Therefore, FAJN is a potential therapeutic agent for reduction of cartilage damage that occurs in OA.