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Anti-Inflammatory Effects of Siegesbeckia orientalis Ethanol Extract in In Vitro and In Vivo Models

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This study aims to investigate the anti-inflammatory responses and mechanisms of Siegesbeckia orientalis ethanol extract (SOE). In cell culture experiments, RAW264.7 cells were pretreated with SOE and stimulated with lipopolysaccharide (LPS) for inflammatory mediators assay. In animal experiments, mice were tube-fed with SOE for 1 week, and s.c. injected with λ-carrageenan or i.p. injected with LPS to simulate inflammation. The degree of paw edema was assessed, and cytokine profile in sera and mouse survival were recorded. Data showed that SOE significantly reduced NO, IL-6, and TNF-α production in LPS-stimulated RAW264.7 cells. In vivo studies demonstrated that mice supplemented with 32 mg SOE/kg BW/day significantly lowered sera IL-6 level and resulted a higher survival rate compared to the control group ( P = 0.019 ). Furthermore, SOE inhibited LPS-induced NF-κB activation by blocking the degradation of IκB-α. The SOE also reduced significantly the phosphorylation of ERK1/2, p38, and JNK in a dose-dependent manner. In summary, the in vitro and in vivo evidence indicate that SOE can attenuate acute inflammation by inhibiting inflammatory mediators via suppression of MAPKs- and NF-κB-dependent pathways.
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Research Article
Anti-Inflammatory Effects of Siegesbeckia orientalis Ethanol
Extract in In Vitro and In Vivo Models
Yong-Han Hong,1Li-Wen Weng,2Chi-Chang Chang,2,3 Hsia-Fen Hsu,1Chao-Ping Wang,4
Shih-Wei Wang,2,5 andJer-YiingHoung
1,2
1Department of Nutrition, I-Shou University, Kaohsiung 82445, Taiwan
2Institute of Biotechnology and Chemical Engineering, I-Shou University, Kaohsiung 84001, Taiwan
3Department of Obstetrics and Gynecology, E-DA Hospital, I-Shou University, Kaohsiung 82445, Taiwan
4Division of Cardiology, E-DA Hospital, I-Shou University, Kaohsiung 82445, Taiwan
5Division of Allergy, Immunology, and Rheumatology, Department of Internal Medicine, E-DA Hospital, I-Shou University,
Kaohsiung 82445, Taiwan
Correspondence should be addressed to Jer-Yiing Houng; jyhoung@isu.edu.tw
Received  April ; Revised  July ; Accepted  July ; Published  August 
Academic Editor: Chia-Chien Hsieh
Copyright ©  Yong-Han Hong et al. is is an open access article distributed under the Creative Commons AttributionLicens e,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
is study aims to investigate the anti-inammatory responses and mechanisms of Siegesbeckia orientalis ethanol extract (SOE). In
cell culture experiments, RAW. cells were pretreated with SOE and stimulated with lipopolysaccharide (LPS) for inammatory
mediators assay. In animal experiments, mice were tube-fed with SOE for  week, and s.c. injected with 𝜆-carrageenan or i.p. injected
with LPS to simulate inammation. e degree of paw edema was assessed, and cytokine prole in sera and mouse survival were
recorded. Data showed that SOE signicantly reduced NO, IL-, and TNF-𝛼production in LPS-stimulated RAW. cells. In
vivo studies demonstrated that mice supplemented with  mg SOE/kg BW/day signicantly lowered sera IL- level and resulted
a higher survival rate compared to the control group (𝑃 = 0.019). Furthermore, SOE inhibited LPS-induced NF-𝜅B activation by
blocking the degradation of I𝜅B-𝛼. e SOE also reduced signicantly the phosphorylation of ERK/, p, and JNK in a dose-
dependent manner. In summary, the in vitro and in vivo evidence indicate that SOE can attenuate acute inammation by inhibiting
inammatory mediators via suppression of MAPKs- and NF-𝜅B-dependent pathways.
1. Introduction
Growing evidence suggests that systemic inammation is
associated with increased risk of chronic diseases [,].
e mechanisms of inammation may involve activation
ofmacrophagesandTlymphocytes,aswellastherelease
of proinammatory mediators, including tumor necrosis
factor- (TNF-) 𝛼, interleukin- (IL-) , IL-, nitric oxide (NO),
and prostaglandin E2(PGE2)thatamplifytheinammatory
activity []. Appropriate production of these mediators pro-
motes eective innate immune response; however, excessive
inammation may cause such conditions as chronic inam-
mation, sepsis, and even death [].
Notably, mitogen-activated protein kinases (MAPKs)
play an important role in the regulation of proinammatory
mediators on cellular responses [,]. Lipopolysaccharide
(LPS) stimulation on macrophages can mimic inammatory
responses []. e MAPK cascade, the key downstream
pathway for LPS-induced signaling events, leads to sev-
eral functional responses. Activated MAPKs are responsible
for phosphorylating and activating numerous transcription
factors, including ERK/, p, SAPK/JNK, and NF-𝜅B,
which then translocate into the nucleus of cells and induce
the transcriptional activation of various inammatory and
immune genes []. Beside LPS, sulfated polysaccharide and
carrageenan can also act as the proinammatory agent.
Recently, experimental evidence has shown that carrageenan-
activated inammatory cascades are related to generation of
reactive oxygen species and may be integrated at the level
of I𝜅B kinase (IKK) signalosome, leading to degradation of
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BioMed Research International
Volume 2014, Article ID 329712, 10 pages
http://dx.doi.org/10.1155/2014/329712
BioMed Research International
I𝜅B-𝛼and translocation of NF-𝜅Btonuclei[]. Accordingly,
modulating eectively aberrant production of proinamma-
tory mediators can reduce inammatory response. us,
analyzing the expression of proinammatory mediators may
facilitate identication of anti-inammatory substances.
e dietary application of natural products, including
food materials and Chinese medicinal herbs, has been pro-
posed that may prevent inammatory diseases [,]. Herba
Siegesbeckiae, one of common Chinese medicinal foods,
has been used to treat rheumatoid arthritis, malaria, and
snakebite based on its ability to dispelling wind, eliminating
dampness, and strengthening the sinews []. To date, three
originals, Siegesbeckia orientalis L. Siegesbeckia pubescens
Makino, and Siegesbeckia glabrescens Makino have been
identied. Among them, S. orientalis has been reported
to have antirheumatic [], antiallergic [], and immuno-
suppressive activities []. S. orientalis’s pure comp one nt,
kirenol, was also found to have a topical application on the
attenuation of skin inammation in murine models [].
However, S. orientalis via oral administration has not been
reported regarding its in vivo anti-inammatory activity and
related mechanisms. erefore, this study investigates the
anti-inammatory responses and their related mechanisms
in inammatory cells or mice pretreated with ethanol extract
from S. orientalis (SOE).
2. Materials and Methods
2.1. Reagents and Plant Materials. e reagents lipopolysac-
charide (LPS, E. coli serotype O:B), indomethacin (IND),
and ammonium pyrrolidinedithiocarbamate (PDTC),
obtained from Sigma-Aldrich (St. Louis, MO, USA), were
all of analytical grade and dissolved in phosphate buer
saline as a stock. e S. orientalis L. samples were purchased
from a local herbal store (Yuanshan Company, Kaohsiung,
Taiwan). e samples original was identied and its DNA
polymorphism had been reported []. In this study, the
samples were freeze-dried and then ground into powder. e
dried powder (. kg) was extracted with a -fold volume of
% ethanol by stirring at room temperature for  day. is
step was repeated three times. e extracted solutions were
collected and ltered through lter paper (Whatman number
; Whatman Paper Ltd., Maidstone,Kent, UK). e SOE
was acquired by removing solvent via a rotary evaporator
anddriedinafreezedryer.edryweightofthisextract
wasg,andtheyieldwas.%.eSOEwasdissolvedin
dimethyl sulfoxide (DMSO; the nal DMSO concentration
never exceeded .% in medium) for cell culture test and
was dissolved in sunower oil for tube feeding in the mice
experiment.
2.2. Cell Culture. e RAW. cells (Bioresource Col-
lection and Research Center; Hsinchu, Taiwan) were cul-
tured in Dulbecco’s modied Eagle’s medium (DMEM-
F) supplemented with % inactivated fetal bovine serum,
.% glutamine, % penicillin/streptomycin, .% sodium
bicarbonate, and pH .–.. e cells were cultivated in a
humidied incubator at Cwith%CO
2and % air.
In this experiment, the RAW. cells were seeded on 
cm dishes at a cell density of  ×5cells/mL for Western
blotting assay, or on -well plates at a cell density of  ×
4cells/well for culture medium test. e cells were then
pretreated with various concentrations of SOE for  h before
adding 𝜇g/mL LPS for the indicated assay. In the Western
blotting assay, the cell lysate was collected for detection
of target proteins aer  h (MAP kinase family) or  h
(iNOS) stimulation. In the culture medium test, culture
supernatants were harvested for analysis of the production
of proinammatory mediators aer stimulation for  h. e
cell viability was evaluated using the MTT method. e
medium solution was removed aer cultivation. An aliquot
of  𝜇L of DMEM medium containing  mg/mL of -
(,-dimethylthiazol--yl)-,-diphenyltetrazolium bromide
(MTT) was loaded to the plate. e cells were incubated
for  h, and then the medium solution was removed. An
aliquotof𝜇LofDMSOwasaddedtotheplate,whichwas
shaken until the crystals dissolved. e cytotoxicity against
cells was determined by measuring the absorbance of the
converteddyeatnminanELISAreader(Model,Bio-
Rad Laboratories, Hercules, CA, USA).
2.3. NO Determination. Griess reagent was freshly prepared
from reagents A (% sulfanilamide in .% phosphoric acid)
and B (.% N--naphthylethylenediamide dihydrochloride
in water) at a ratio of : . An equal volume of Griess reagent
was added to supernatants from cells treated with test samples
in a -well plate for min. Absorbance was measured by
an ELISA reader at  nm. e NO concentrations were
determined using a NaNO2standard curve.
2.4. Cytokine Production Assay. Production of cytokines
TNF-𝛼and IL- in cell supernatants and mice serum was
assayed using a commercial ELISA kit (eBioscience, Min-
neapolis, MN, USA). Briey, primary anti-IL- or TNF-𝛼
antibodies were coated onto -well plates. Aer overnight
incubation, plate wells were washed with washing buer
and blocked with blocking solution for  h. Aer washing,
diluted supernatants or sera were added to wells for  h
incubation. Next, wells were washed with washing buer
and biotin-conjugated anti-IL- or TNF-𝛼antibody was
added for  h. e wells were then washed and horseradish
peroxidase-conjugated streptavidin was added for  min,
washed, and incubated with tetramethylbenzidine (TMB;
Clinical Science Products, Manseld, MA, USA). Absorbance
was measured by an ELISA reader at  nm. Data were
calculated according to standard curves of cytokines.
2.5. Western Blot Analysis. e cells were trypsinized, washed
twice with phosphate buered saline (PBS), and lysed with
lysis buer (modied RIPA buer) at C. e pellet cel-
lular debris was removed by centrifugation at  rpm
for  min and the supernatants were then either analyzed
immediately or stored at C. Protein concentrations
were measured by BCA Protein Assay Kit (Pierce, Rock-
ford, IL, USA). Lysates in sample buer (% SDS, %
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glycerol,  mM Tris-base,  mM DL-dithiothreitol, and
.% bromophenol blue) were denatured at Cformin.
Equivalent amounts of protein ( 𝜇g) from total cell lysates
were subjected to SDS-polyacrylamide gel electrophoresis
(PAGE) and the proteins were transferred onto poly vinyli-
dene diuoride (PVDF) membrane (Millipore, Temecula,
CA, USA). Nonspecic binding was blocked by soaking the
membrane in Tris-buered saline (TBS,  mM Tris-base,
and  mM NaCl) containing % fat-free milk for  h. e
membrane was incubated with primary antibodies (anti-
p, anti-ASPK/JNK, anti-ERK, anti-I𝜅B-𝛼and anti-NF-𝜅B
at  :  in TBS; anti-actin at  :  in TBS) (Cell Signaling
Technology, Danvers, MA, USA) overnight at C. e
membrane was then incubated with a secondary antibody,
a goat antirabbit IgG, or goat antimouse IgG conjugated
to horseradish peroxidase. e protein levels were deter-
mined by using enhanced chemiluminescence (ECL) plus
western blotting detection reagents (Amersham Bioscience,
Uppsala, Sweden) and the bands intensities were scanned.
Densitometric analyses were conducted using the Quantity
One soware (Bio-Rad). Incubation with polyclonal mouse
antihuman 𝛽-actin antibody was performed for comparative
control.
2.6. Experimental Animals. Six-week-old female BALB/c
mice and -week-old female ICR mice were purchased
from the National Animal Center (Taipei, Taiwan). ese
mice were maintained in an air-conditioned room at 23 ±
2Conaregulatedhlight-darkcycle.eywerefeda
nonpuried diet (Lab Rodent Chow , Ralston Purina
Inc., St. Louis, MO, USA) for adaptation. At age of 
weeks, the mice were started on dietary treatment. Animal
care and handling conformed to the National Institutes of
Health’s Guide for the Care and Use of Laboratory Animals
[].
2.7. SOE Treatment Prior to 𝜆-Carrageenan-Induced Paw
Edema. To identify the eects of the SOE on local acute
inammation,  -week-old ICR mice were divided ran-
domly into four groups: the control group (𝑛=8), the
LSOE group (𝑛=8), the HSOE group (𝑛=8), and
the IND group (𝑛=4, positive control). e control and
the IND groups were tube-fed daily with  𝜇L sunower
oil, while the LSOE and the HSOE groups were tube-fed
daily with  and  mg SOE/kg BW in  𝜇L sunower
oil, respectively. All mice also had free access to chow diet
and water. ese oral doses of SOE were derived from the
eective dose of  𝜇g/mL in RAW. cells and primary
macrophages based on a previous report []. Aer tube
feeding with either sunower oil or SOE for  week,  𝜇Lof
% 𝜆-carrageenan (in saline) and  𝜇L saline was injected
subcutaneously (s.c.) into the right paw and le paw plantar
of each mouse, respectively. Mice in the IND group were
s.c. injected with  mg indomethacin/kg BW  h before 𝜆-
carrageenan challenge. Aer each 𝜆-carrageenan injection,
paw volume was measured at -hour intervals using a plethys-
mometer (Apelex , Massy, France). e stimulation index
(S.I.) was used to express the degree of murine paw edema,
which was calculated as
S.I.=thevolumeofrightpaw (carageenan injection)
the volume of le paw (PBS injection).
()
2.8. SOE Treatment Prior to LPS-Induced Systemic Inam-
mation. Forty-ve -week-old BALB/c mice were divided
randomly into four groups: the control group (𝑛=13), the
LSOE group (𝑛=12), the HSOE group (𝑛=12), and the
PDTC group (𝑛=8, positive control). All mice were fed chow
diet and supplemented with 𝜇L sunower oil daily. eir
experimental doses and treatment durations were the same as
thoseinthe𝜆-carrageenan experiment. Aer  week of tube-
feeding, all mice were injected intraperitoneally (i.p.) with
 mg LPS/kg BW to induce systemic inammation. Mice
in the PDTC group were i.p. injected with  mg PDTC/kg
BW, a dose with anti-inammatory eects,  h before LPS
challenge. Sera were collected at  and  h aer LPS challenge
for cytokine assay. e life spans of all mice were also
recorded.
2.9. Gas Chromatography-Mass Spectrometry. GC-MS anal-
ysis was performed using Varian -GC and -MS sys-
tem (Varian, Salt Lake City, UT, USA) with the electron
impact mode ( eV) injector and a Varian data system.
e GC column was VF- ms capillary column ( m ×
. mm, lm thickness . 𝜇m, FactorFour, USA). Injector
and detector temperatures were set at Cand
C,
respectively. Oven temperature was kept at Cformin,
then raised to Cbyarateof
C/min, kept at Cfor
min, and then raised to Cbyarateof
C/min. e
carrier gas was helium at a ow rate of mL/min. Diluted
samples of . 𝜇Lwereinjectedmanuallyandinthesplitless
mode. e percentages of the compounds were calculated
by the area normalization method. e components were
identied by comparison of their mass spectra with the NIST
MS . database (Gaithersburg, MD, USA). Caryophyllene
oxide, hexadecanoic acid ethyl ester, and caryophyllene
were purchased from Sigma-Aldrich. e compound ,,-
trimethyl--pentadecanone was purchased from Apollo Sci-
entic Co. (Stockport, Cheshire, UK).
2.10. High Performance Liquid Chromatography (HPLC) Anal-
ysis for Kirenol. e content of kirenol was determined
by HPLC (LC-AT, Shimadzu, Tokyo, Japan). e sample
was dissolved in methanol and ltered with a . mm
lter. e diluted sample was analyzed by an Ascentis
C column (number -U,  mm,  ×. mm;
Supelco, Bellefonte, PA, USA). e mobile phase con-
sisted of acetonitrile/methanol ( : , v/v) and water with
a linear gradient elution, – min for –% acetoni-
trile/methanol ( : , v/v) and – min for –% ace-
tonitrile/methanol ( : , v/v) at a ow rate of . mL/min.
e sample injection size was  𝜇L. e detection was
carried out at  nm. e residence time of kirenol was
. min.
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2.11. Statistical Analysis. Each experiment was performed at
least three times. e data are expressed as the means ±
SD. e signicant dierence compared to the control group
was statistically analyzed by Student’s t-test using the SAS
soware program (SAS/STATA version .; SAS Institute,
Cary, NC, USA). Statistical comparison between dierent
survival curves was analyzed by Cox’s proportional hazards
regression test (STATA version .; Stata Corp., TX, USA).
e relationship was analyzed by the simple correlation of the
SAS program. Statistical signicance is expressed as 𝑃 < 0.05.
3. Results
3.1. In Vitro Anti-Inammator y Eects of SOE. Notably, NO is
endogenously synthesized by inducible nitric oxide synthase
(iNOS) through activated NF-𝜅BandMAPKandisstrongly
related to inammatory responses. Because formation of
NO can induce inammation, this study rst determined
whether SOE suppresses NO generation in LPS-stimulated
RAW. cells. As presented in Figure (a), SOE suppressed
NO production dose-dependently. Compared to the Control,
NO production was inhibited by  ±% at the SOE concen-
tration of  𝜇g/mL. Next, the inhibitory eects of SOE on the
production of proinammatory cytokines were examined.
Experimental data shown in Figure (a) indicate that LPS-
stimulated IL- production was inhibited markedly by SOE
pretreatment in a dose-dependent fashion. Consistent with
the IL- result, SOE signicantly inhibited the production
of TNF-𝛼dose-dependently. e SOE signicantly reduced
iNOS protein expression (Figure (b)). ese data indicate
that the inhibitory eect of SOE on NO production is
related to its suppression on iNOS protein expression. e
cytotoxicity of SOE on LPS-induced RAW. cells was also
assessed using the MTT assay. Cell viability did not decrease
aer incubation for  h with SOE up to  𝜇g/mL, indicating
that SOE is not cytotoxic to cells within this concentration
range (Figure (c)).
3.2. In Vivo Eects of SOE on Inammatory Conditions.
Animal experiments were conducted to determine whether
-week gavage of SOE at the indicated dose ameliorates
𝜆-carrageenan-induced and LPS-stimulated inammation.
Experimental results shown in Figure (a) indicate that the
high dose of SOE (HSOE,  mg/kg BW/day) reduced the
degree of paw edema at  h aer 𝜆-carrageenan challenge.
Forsystemicinammation,onlyhalfofthecontrolgroup
mice survived at  h aer LPS challenge, but approximately
%ofmiceintheHSOEgroupwerealiveatthesame
time point. At  h aer LPS challenge, no mouse in the
control group survived, but around % of those in the HSOE
group survived (Figure (b)). e survival rate of the HSOE
group was signicantly higher than that of the control group
according to the COX proportional hazards regression test
(𝑃 = 0.019).
Our previous studies examined sera cytokine prole of
BALB/c mice with  mg/kg BW LPS challenge [,], which
is a similar animal model to this investigation. In those
studies, the life span was found to be negatively correlated
with sera IL- and TNF-𝛼level at the early stage (h) and the
late stage ( h) of the acute-inammation period. erefore,
this study examined the level of IL- and TNF-𝛼at  h and  h
aer LPS challenge. e HSOE group had signicantly lower
serum IL- levels at  and  h aer LPS challenge (Table ).
e PDTC group, a positive control group, had signicant
lower levels of cytokines than the control group.
3.3. In Vitro Eects of SOE on NF-𝜅BActivationandMAPK
Phosphorylation. As NF-𝜅B pathway is closely related to the
expression of iNOS and proinammatory cytokines, modu-
lation of SOE on NF-𝜅B activation was examined. Figure 
showsWesternblotresultsforLPS-inducedRAW.cells
under treatment with dierent SOE concentrations. e
SOE signicantly suppressed phosphorylation of I𝜅B-𝛼and
induced I𝜅B-𝛼expression in a dose-dependent manner. Con-
sistently, SOE reduced the degree of phosphorylation of NF-
𝜅B when treatment dose increased. ese analytical results
suggest that SOE inhibits LPS-induced NF-𝜅Bactivationby
blocking the degradation of I𝜅B-𝛼. To further investigate
whetherinhibitionofNF-𝜅B activation and inammatory
mediators by SOE is modulated by the MAPK pathway,
the eects of SOE on LPS-induced phosphorylation of
ERK/, p, and JNK were examined. e SOE signicantly
reduced phosphorylation of ERK/, p, and JNK in a dose-
dependent fashion (Figure ).
3.4. Chemical Compositions of SOE. e GC-MS analytical
results show that at least  compounds exist in SOE
(Figure ), of which a total of  constituents were identied
using mass spectrometry (Ta b l e  ). e mass spectra of
these compounds were matched with those found in the
NIST spectral database. e major compounds in SOE were
quantied as caryophyllene oxide (.%), [−]-spathulenol
(.%), and hexadecanoic acid ethyl ester (.%) based on
the results obtained from GC-MS analysis. Caryophyllene
oxide, hexadecanoic acid ethyl ester, caryophyllene, and
,,-trimethyl--pentadecanone were conrmed by com-
paring their mass spectral data with the NIST mass spectral
library and commercially available products. Inhibition of
NO production in LPS-induced RAW. cells shows that
the anti-inammatory eects of these four compounds were
insignicant (data not shown). erefore, these compounds
were not the bioactive anti-inammatory ingredients of SOE.
e content of kirenol, an ingredient isolated from
ethanolic extract of S. orientalis and was demonstrated to
exhibit signicant anti-inammatory activity [], in SOE
was determined by HPLC analysis. By matching the retention
time (RT = . min) with authentic standards, good linearity
was obtained (R2= .) and the quantity of kirenol was
determined as 4.2 ± 0.08 mg/g SOE.
4. Discussion
Inammation, a complex process, is regulated by various
immune cells and eector molecules, such as NO and proin-
ammatory cytokines. Inhibition of these mediators with
pharmacological modulators has been proved as an eective
BioMed Research International
0
2
4
6
8
10
12
0
20
40
60
80
100
120
Medium
Ctrl
IL-6 Nitrite
IL-6or TNF-𝛼production (ng/mL)
TNF-𝛼
SOE 10 𝜇g/mL
SOE 20 𝜇g/mL
SOE 30 𝜇g/mL
SOE 40 𝜇g/mL
SOE 50 𝜇g/mL
Nitrite production (𝜇M)
∗∗
∗∗
∗∗ ∗∗
∗∗
∗∗
∗∗
∗∗
∗∗
∗∗∗
(a)
0
20
40
60
80
100
120
LPS
SOE
iNOS
iNOS level (%)
𝛽-Actin
−+++++
∗∗
∗∗∗
∗∗∗
3020102.500
(b)
0 1020304050
Cell viability (%)
0
20
40
60
80
100
120
140
SOE conc. (𝜇g/mL)
(c)
F : Eects of SOE on proinammatory mediators production, iNOS expression, and cell viability in LPS-stimulated RAW.
macrophages. Cells were pretreated with  (control) or the indicated concentration of SOE for h and then stimulated with  𝜇g/mL LPS for
 h (iNOS) or  h (proinammatory mediators). e negative control (medium) is that cells were cultured with medium for the indicated
time. (a) Production of IL- and TNF-𝛼was measured by an ELISA kit. e nitrite concentration was analyzed using Griess reagent. (b)
e expression of the iNOS protein was determined by Western blotting analysis. e iNOS level was quantied by densitometric analysis
using the Quantity One soware (Bio-Rad). (c) Cell viability was determined by MTT assay. Bar values are means ±SD of three independent
experiments in these assays. A signicant dierence from the control (LPS alone) was indicated as 𝑃 < 0.05,∗∗𝑃 < 0.01,or∗∗∗𝑃 < 0.001 by
Student’s 𝑡-test.
therapeutic strategy for reducing inammatory reactions and
risk of inammatory diseases []. Macrophages are cru-
cial to host-defense against infections and in inammation
processes through the release of molecules such as NO, PGE2,
TNF-𝛼, and IL-. Overproduction of these mediators has
been implicated in several inammatory diseases and cancer
[]. us, inhibition of activation of these cells appears to
be an important target when treating inammatory diseases.
Stimulation of macrophages with LPS induces high produc-
tion of NO by iNOS and PGE2by cyclooxygenase- (COX-)
[]. erefore, a reagent that prevents the release of these
mediators or downregulates iNOS or COX- expression may
possess anti-inammatory activities.
S. orientalis, the most common used original of Herba
Siegesbeckiae, and its ethanol extract (SOE), which mim-
ics formulas in medicinal foods, were used to explore its
preventive eects against inammation. In cell culture test,
LPS induced-cellular production of IL-, TNF-𝛼,andNO
via iNOS activity was dose-dependently reduced by the SOE
in RAW. macrophages (Figure ). However, decreased
PGE2production and COX- expression were insignicant
within the tested SOE concentration range (data not shown).
Moreover,thecellviabilityroseslightlyastheSOEdose
increased (Figure (c)), implying that SOE exhibited signi-
cant anti-inammatory activity without causing cytotoxicity.
ese results imply that SOE has potential as an anti-
inammatory agent. With this in vitro inhibitory eect of
SOE on the production of proinammatory mediators, the in
vivo anti-inammatory potential of SOE was then evaluated.
First, a 𝜆-carrageenan-induced paw edema model, which
is considered as a highly sensitive tool for evaluating the
ecacy of acute inammation [], was adopted to assess
thepreventiveeectofSOEonlocalacuteinammation.
It has also been reported that this paw edema would be
BioMed Research International
Time aer carrageenan challenge (hr)
012345678910
Stimulation index
1.0
1.2
1.4
1.6
1.8
2.0
Control
Indomethacin
HSOE 32 mg/kgBW
LSOE 10 mg/kgBW
∗∗
∗∗
∗∗
∗∗ ∗∗
(a)
Time aer LPS challenge (hr)
12 16 20 24 28 32 36 40 44 48 52 56
Survival rate (%)
0
10
20
30
40
50
60
70
80
90
100
Control
PDTC
HSOE 32 mg/kgBW
LSOE 10 mg/kgBW
(b)
F : Eects of SOE pretreatment on 𝜆-carrageenan-induced paw edema and mouse survival in LPS-challenge model. (a) e ICR mice
tube-fed without (control or IND) or with the indicated dose of SOE ( or  mg/kg BW/day) for  days were s.c. injected with 𝜆-carrageenan
into right paw plantar, and the degree of mice paw edema was recorded. e positive control mice were s.c. injected with indomethacin
( mg/kg BW) at  h before 𝜆-carrageenan challenge. S.I. = the volume of right paw/the volume of le paw. A signicant dierence from
the control was indicated as 𝑃 < 0.05 or ∗∗𝑃 < 0.01 by Student’s 𝑡-test. (b) e BALB/c mice tube-fed without (control or PDTC) or with
the indicated dose of SOE ( or mg/kg BW/day) for  days were i.p. injected with LPS, and mouse survival was recorded. e positive
control mice were i.p. injected with PDTC ( mg/kg BW) at h before LPS challenge. e LSOE, HSOE, and PDTC positive control groups
had increased survival using the COX proportion hazards regression test (𝑃 = 0.027, ., and ., resp.).
T : Eects of SOE pretreatment on sera cytokine production in
LPS-challenged micea.
Group IL- (ng/mL) TNF-𝛼(ng/mL)
At  h aer LPS
challenge
Control  ± . ±.
LSOE  ±. . ±.
HSOE 205 ± 102. ±.
PDTC 202 ± 1171.47 ± 0.32
At  h aer LPS
challenge
Control  ± . ±.
LSOE  ±. 0.56 ± 0.26#
HSOE 81.3 ± 46.50.52 ± 0.35#
PDTC 27.9 ± 15.4∗∗ 0.25 ± 0.12
aSera at  h and  h aer LPS injection were collected for cytokines assay. e
cytokine production in serum was assayed by ELISA kits. Values are means
±SD. #0.05 < 𝑃 < 0.1,𝑃 < 0.05,or∗∗𝑃 < 0.01, signicantly dierent from
the control group analyzed by Student’s 𝑡-test.
ameliorated by reducing the levels of IL-, IL-, TNF-𝛼,and
NO []. In this study, SOE at a dose of  mg/kg BW/day
(HSOE) suppressed signicantly 𝜆-carrageenan-induced paw
edema (Figure (a)).Second,aLPS-challengemodel,which
mimics systemic endotoxemia, was applied to determine
whether SOE pretreatment could reduce systemic chronic
inammation. Previous studies had indicated that reduction
of sera TNF-𝛼and/or IL- levels in LPS-challenged mice
could benet their survival [,]. Our data show that
pretreatment with HSOE signicantly decreased the sera IL-
 level in mice but did not signicantly reduce the level of
sera TNF-𝛼(Table ). However, the HSOE mice had a higher
survival rate than the control mice (Figure (b)), implying
that reduction of IL- only remains useful in increasing mice
survival in LPS-induced systemic inammation. ese data
suggest that the oral administration of SOE has favorable
eects on prevention of local and systemic acute inamma-
tion by downregulating production of inammatory media-
tors.
e RAW. macrophage cell line has been used as
arapidin vitro screening method when studying anti-
inammatory agents [,,]. e cytokine prole in the
cell model in this study was resembled that in the LPS-
induced in vivo model. e SOE has similar signicant eects
on reduction of IL- expression in both in vitro and in vivo
studies. However, the eect of SOE on the reduction of the
TNF-𝛼level in cell cultures was signicant but insignicant
in mouse sera. is dierence is likely due to the SOE
dose used in the cell-model experiment and animal-model
experiment. e high dose of SOE of  𝜇g/mL in the cell-
modeltestinhibited%ofIL-productionand%ofTNF-
𝛼production (Figure ). In comparison, the high dose of SOE
of  mg/kg BW/day in the animal-model test inhibited %
BioMed Research International
0
20
40
60
80
100
∗∗
∗∗
∗∗
p-I𝜅B-𝛼/I𝜅B-𝛼level (%)
(a)
0
20
40
60
80
100
∗∗
p-NF-𝜅B/NF-𝜅B level (%)
(b)
LPS
0 0 30 40 50
I𝜅B-𝛼
NF-𝜅B
𝛽-Actin
SOE (𝜇g/mL)
40 kDa
39 kDa
65 kDa
65 kDa
43 kDa
−++++
p-I𝜅B-𝛼
p-NF-𝜅B
(c)
F : Eects of SOE on the expression of p-I𝜅B-𝛼and p-NF-𝜅B in LPS-stimulated RAW. cells. Cells were pretreated with the
indicated doses of SOE for  h and then stimulated with  𝜇g/mL LPS for h. Bar values are means ±SD of three independent experiments. e
electrophoresis experiment was repeated three times, and one representative result is shown here. A signicant dierence from the control
(LPS alone) was indicated as 𝑃 < 0.05 or ∗∗𝑃 < 0.01 by Student’s 𝑡-test.
T : Chemical compositions of SOE analyzed by GC-MS.
No. Component Rt (min)aR. match Percentage (%)b
-Oxabicyclo[,,]octane--ol .  .
 -tert-Butyl-,-dimethoxy-benzene .  .
Caryophyllene .  .
cis-𝛼-Bisabolene .  .
[]-Spathulenol .  .
Caryophyllene oxide .  .
cis-Lanceol .  .
[Z,Z,Z]-,,-Octadecatrienoic acid ethyl ester .  .
 ,,-Trimethyl--pentadecanone .  .
 Hexadecanoic acid ethyl ester .  .
aRetention time (min).
bRelative percentage calculated by integrated peak area.
of IL- formation and % of TNF-𝛼formation (Table ).
erefore, a higher dose of SOE in the animal-model test
would be worthy of further work.
Notably, NF-𝜅B plays an important role in the regu-
lation of cell survival genes and induction of the expres-
sion of inammatory enzymes and cytokines. erefore,
blocking the NF-𝜅B transcriptional activity in the nuclei of
macrophages may reduce the expression of iNOS, COX-,
and proinammatory cytokines and has been considered
to be an eective therapy for treating inammation-related
diseases []. Under unstimulated conditions, NF-𝜅Bisan
inactive complex bound to I𝜅B𝛼in cytosol. Aer stimulation
with LPS, NF-𝜅B is activated through phosphorylation and
degradation of I𝜅B𝛼by increasing IKK or Akt kinase activity
[]. Phosphorylation of NF-𝜅B, regulated by MAPK path-
way, plays a vital role in modulating transcriptional activity
BioMed Research International
0
20
40
60
80
100
∗∗
p-Erk/ERK level (%)
(a)
0
20
40
60
80
100
∗∗
∗∗ ∗∗
p-p38/p38 level (%)
(b)
0
20
40
60
80
100
p-JNK/JNK level (%)
(c)
Erk
p38
JNK
LPS
0 0 30 40
++++
𝛽-Actin
SOE (𝜇g/mL)
44 kDa
42 kDa
44 kDa
42 kDa
43 kDa
43 kDa
54 kDa
46 kDa
54 kDa
46 kDa
43 kDa
50
p-JNK
p-p38
p-Erk
(d)
F : Eects of SOE on the expression of p-Erk, p-p, and p-JNK in LPS-stimulated RAW. cells. Cells were pretreated with the
indicated doses of SOE for  h and then stimulated with  𝜇g/mL LPS for h. Bar values are means ±SD of three independent experiments. e
electrophoresis experiment was repeated three times, and one representative result is shown here. A signicant dierence from the control
(LPS alone) was indicated as 𝑃 < 0.05 or ∗∗𝑃 < 0.01 by Student’s 𝑡-test.
Intensity
1.25
1.00
0.75
0.50
0.25
0.00
010 3020 40
Time (min)
20.7
23.1
27.6
29.3
33.5
36.9
32.5
23.6 26.6
28.0
F : Gas chromatography-mass spectrometry prole of SOE.
of NF-𝜅B and is independent of I𝜅B𝛼proteins []. In this
study, Western blotting results show that SOE decreased
phosphorylated NF-𝜅B level but unaected total NF-𝜅Blevel
in LPS-stimulated RAW. cells. It was also observed that
SOE dose-dependently decreased p-I𝜅B𝛼/I𝜅B𝛼and SOE at
 𝜇g/mL signicantly increased I𝜅B𝛼level (Figure ). ese
suggest that regulatory eects of SOE on NF-𝜅Bactivationare
partly through modulating the degradation of I𝜅B𝛼.
e MAPKs are a family of serine/threonine kinases that
areinvolvedinavarietyofcellularprocesses.reeMAPK
molecules, ERK, p, and JNK, are activated in response
to certain extracellular stimuli such as LPS or carrageenan
challenge. ese kinases have dierent downstream targets
and mediate diverse cellular responses, including regulation
of apoptosis, proliferation, and inammation []. is study
shows that treatment by SOE signicantly inhibited LPS-
induced ERK/, p, and JNK phosphorylation in LPS-
stimulated macrophages (Figure ), which may contribute to
the inhibitory eect of SOE on the production of proinam-
matory mediators in LPS-induced macrophages.
In GC-MS analysis,  SOE constituents were identied
using mass spectrometry (Ta b l e  ). Among them,  com-
pounds, caryophyllene oxide, hexadecanoic acid ethyl ester,
caryophyllene, and ,,-trimethyl--pentadecanone, were
obtained from commercial sources. However, none of these
 compounds had a signicant inhibitory eect on NO
production in LPS-induced RAW. cells. Our laboratory
has separated SOE into several fractions using a partition
procedure with n-hexane, ethyl acetate (EA), and methanol.
e IC50 values of the SOE, n-hexane fraction, and EA
fraction on the inhibition of NO production in LPS-activated
BioMed Research International
macrophages were ., ., and . 𝜇g/mL, respectively,
while the methanol fraction was insignicant. Due to the
eectiveness of the EA fraction, the bioactive components
from this fraction will be screened and separated in our next
study.
5. Conclusions
In summary, in vivo evidence suggests that SOE has signif-
icant inhibitory eects on local and systemic acute inam-
mation, while in vitro data reveal that SOE could block
activation of NF-𝜅BandMAPKs,therebyinhibitingthe
induction of iNOS expression and the release of inam-
matory cytokines. Taken together, this study demonstrates
that SOE is a medicinal food material capable of preventing
inammation. Further studies are still needed to evaluate
the detailed molecular mechanisms and dene the main
bioactive phytochemicals in the SOE.
Conflict of Interests
e authors declare that there is no conict of interests
regarding the publication of this paper.
Acknowledgments
e authors would like to thank the nancial support of the
National Science Council, Taiwan (NSC --E--),
and E-Da Hospital, Taiwan (EDAHP).
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Supplementary resource (1)

... In terms of SG and SO, there is not any direct and scientific demonstration related to the RA. Hong et al. reported that SO attenuated l-carrageenan-induced paw edema and LPS-induced systemic inflammation in mice (Hong et al., 2014), which indicated the potential of SO on RA and valuable to be further investigated. Our previous works have made a systemic analysis on the chemistry for SH species (Tao et al., 2018;Tao et al., 2020) and identified some potential quality control markers for SO (Linghu et al., 2020b). ...
... Our previous works have made a systemic analysis on the chemistry for SH species (Tao et al., 2018;Tao et al., 2020) and identified some potential quality control markers for SO (Linghu et al., 2020b). Growing in vitro researches showing the anti-inflammatory effects of SO on macrophages (Hong et al., 2014;Guo et al., 2018;Zhang et al., 2019;Linghu et al., 2020b), which indicates that antiinflammation could be one of the anti-arthritic mechanisms of SO. An additional therapeutic and mechanical demonstration of SO on RA animal model and other potential target cells are essential to elucidate the anti-arthritic effects and mechanisms of SO comprehensively. ...
... An additional therapeutic and mechanical demonstration of SH on RA animal model is beneficial to contribute the rational use of this herb medicine. Hong et al. reported that SO attenuated lcarrageenan-induced paw edema and LPS-induced systemic inflammation in mice (Hong et al., 2014), which indicated the potential of SO on RA. At present study, we investigated the antiarthritic effects of SO extract (SOE) on collagen type II (C II)induced rheumatoid arthritis (CIA). ...
Article
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Excessive proliferation and inflammation of synovial fibroblasts accelerate and decorate the pathological process of rheumatoid arthritis (RA). Sigesbeckia orientalis L. (SO) is one of the main plant sources for Sigesbeckiae Herba (SH) which has been used traditionally in treating various forms of arthritis and rheumatic pain. However, the anti-arthritic mechanisms of SO are still not clearly understood. In this study, we investigated the therapeutic effects and the underlying mechanisms of SO against collagen type II (C II)-induced RA in rats as well as the interleukin (IL)-1β–induced human synovial SW982 and MH7A cells. For the in vivo studies, thirty-six Wistar male rats were randomly arranged to six groups based on the body weight, and then C II-induced to RA model for 15 days, followed by treatment with the 50% ethanolic extract of SO (SOE, 0.16, 0.78, and 1.56 g/kg) for 35 days. The results suggested that SOE significantly inhibited the formation of pannus (synovial hyperplasia to the articular cavity) and attenuated the cartilage damaging and bone erosion in the CIA-induced rats’ hind paw joints. Moreover, SOE decreased the production of C-reactive protein (CRP) in the serum and the expression of IL-6 and IL-1β in the joint muscles, as well as recovered the decreased regulatory T lymphocytes. The results obtained from the in vitro studies showed that SOE (50, 100, and 200 µg/ml) not only inhibited the proliferation, migration, and invasion of human synovial SW982 cells but also decreased the IL-1β–induced expression of IL-6 and IL-8 both in SW982 and MH7A cells. Besides, SOE reduced the expression of COX-2, NLRP3, and MMP9, and increased the expression of MMP2 in the IL-1β–induced SW982 cells. Furthermore, SOE blocked the activation of NF-κB and reduced the phosphorylation of MAPKs and the expression of AP-1. In conclusion, SOE attenuated the C II-induced RA through inhibiting of MAPKs/NF-κB/AP-1–mediated synovial hyperplasia and inflammation.
... Siegesbeckia orientalis (SO) has been documented to have therapeutic effects on wind-dampness, painful joints and bones, quadriplegia, traumatic bleeding and immune deficiency [24]. Recent literature reports that SO has anti-inflammation [25][26][27][28], anti-allergy [29], immunosuppression [29,30] anti-hyperuricemia [31], anti-diabetes [32,33], anti-bacterial [34,35] and anti-cancer properties [35][36][37][38][39], relieves rheumatoid arthritis [31,40] and knee osteoarthritis [41], attenuates postoperative cognitive dysfunction [42] and inhibits adipogenesis [43]. ...
... However, the expression of these three cytokines was significantly decreased when treated with SOE, which indicates that SOE exhibited an anti-inflammatory effect on the H 2 O 2stimulated HaCaT cells. Previously, Hong et al. [25] have demonstrated that SOE has an anti-inflammatory effect against acute inflammation in a mouse model induced by subcutaneous injection of λ-carrageenan or intraperitoneal injection of lipopolysaccharide (LPS), as well as in LPS-stimulated murine macrophage RAW264.7 cells. They also showed that SOE attenuated acute inflammation by suppressing inflammatory mediators through MAPKs-and NF-κB-dependent pathways. ...
Article
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Previous studies have demonstrated that Siegesbeckia orientalis (SO) has a suppressive effect on the growth and migration of endometrial and cervical cancer cells. The present study examined the effect of SO ethanolic extract (SOE) on the proliferation and migration of hepatocellular carcinoma (HCC) and examined the effects of SOE on non-cancerous cells using HaCaT keratinocytes as a model. The SOE effectively inhibited the proliferation of Hepa1-6 (IC50 = 282.4 μg/mL) and HepG2 (IC50 = 344.3 μg/mL) hepatoma cells, whereas it has less cytotoxic effect on HaCaT cells (IC50 = 892.4 μg/mL). The SOE treatment increased the generation of ROS in HCC, but decreased the expression of antioxidant enzymes such as superoxide dismutase, glutathione peroxidase and catalase. In contrast, it reduced intracellular ROS formation and upregulated the expression of the related antioxidant enzymes in the H2O2-stimulated HaCaT cells. The SOE intervention also down-regulated the anti-apoptotic Bcl-2 and the migration-related proteins including matrix metalloproteinases (MMPs) and β-catenin in the HCC, suggesting that SOE could promote HCC apoptosis and inhibit HCC migration. On the contrary, it reduced apoptosis and promoted the migration of the keratinocytes. Additionally, the SOE treatment significantly up-regulated the pro-inflammatory cytokines, including TNF-α, IL-6 and IL-1β, in Hepa1-6 and HepG2 cells. Conversely, it significantly decreased the expression of these cytokines in the H2O2-induced HaCaT cells. These findings indicated that SOE treatment can delay the progression of HCC by increasing oxidative stress, promoting inflammatory response, inducing cancer cell apoptosis and inhibiting their migration. It also has protective effects from pro-oxidant H2O2 in non-cancerous cells. Therefore, SOE may provide a potential treatment for liver cancer.
... Siegesbeckia orientalis linne is a folk herbal medicine used to treat waist and knee weakness, rheumatoid arthritis, bone pain, quadriplegia, traumatic bleeding, and immune deficiency. Previous studies have reported that S. orientalis ethanol extract (SOE) has significant anti-inflammatory, analgesic, and anti-hyperuricemic effects [19,20], immunosuppressive activity on mouse ovalbumin [21], and suppressive effects on the proliferation and metastasis of human cervical cancer cells and endometrial cancer cells [22][23][24]. ...
... More and more studies have shown beneficial effects of many plant extracts or phytochemicals on the prevention and treatment of diabetes [3,[14][15][16][17][18]28]. Previously, SOE was demonstrated to have anti-inflammatory, antioxidant activity and can inhibit the activity of α-glucosidase, pancreatic lipase and ACE and the formation of AGEs [19,25]. This study investigated the effects of SOE on cell proliferation, intracellular ROS generation, activity of the antioxidant enzymes, and insulin secretion in HG-stimulated RIN-m5f β-cells. ...
Article
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The glucotoxicity caused by long-term exposure of β-cells to high glucose (HG) conditions may lead to the generation of more reactive oxygen species (ROS), reduce the activity of antioxidant enzymes, cause cell damage and apoptosis, and induce insulin secretion dysfunction. Siegesbeckia orientalis linne is a traditional folk herbal medicine used to treat snake bites, rheumatoid arthritis, allergies, and immune deficiencies. In this study, we evaluated the protective effect of S. orientalis ethanol extract (SOE) on cell death and oxidative stress in RIN-m5f pancreatic β-cells stimulated by two HG concentrations (50–100 mM). In the cell viability assay, SOE could significantly increase the survival rate of pancreatic β-cells under HG-induced conditions. For the oxidative stress induced by HG condition, the treatment of SOE effectively reduced the ROS formation, increased the content of intracellular glutathione, and up-regulated the expression of antioxidant enzymes, catalase, superoxide dismutase, and glutathione peroxidase. As a result, the SOE treatment could decrease the glucotoxicity-mediated oxidative damage on RIN-m5F β-cells. Moreover, SOE had the function of regulating insulin secretion in pancreatic β-cells under different HG-mediated conditions. It could decrease the increasing intracellular insulin secretion under the low glucose concentration to normal level; while increase the decreasing intracellular insulin secretion under the relatively high glucose concentration to normal level. Taken together, this study suggests that SOE has a protective effect on pancreatic β-cells under the HG-stimulated glucotoxic environment.
... Currently, the officially authorized plant origins for SH include Sigesbeckia pubescens Makino (SP), S. glabrescens Makino (SG), and S. orientalis L. (SO). SO had been reported to attenuate λ-carrageenan-induced paw edema and LPS-induced systemic inflammation in mice [6]. SP had shown therapeutic effect on collagenase-induced osteoarthritis by inhibiting cartilage damaging in rabbits [7]. ...
... In summary, results from the in vivo and in vitro suggests the therapeutic effects of SG on RA, and the mechanisms would be the inhibition of synovial hyperplasia and inflammation through blocking NF-κB signaling and balancing immune system. In accordance with the previous in vivo reports showing the cartilage protection of SP [7], and the amelioration to paw edema by SO [6], our work further provided in vivo evidence for SG on RA treatment therefore suggesting the similarities in pharmacology and therapeutic effectiveness of the three species of SH (SP, SO, SG) in treating RA. (a, b). The images of the scratch wound were obtained with a microscope (d) and scratch wound changes were analyzed (c) after determining the wound size with Image J software ( # P < 0.05, ### P < 0.001 vs. Ctrl group; ** P < 0.01, *** P < 0.01 vs. IL-1β group; n = 3) Fig. 6 SGE reduced the production of IL-6 and IL-8 in SW982 cells. ...
Article
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Background: Sigesbeckia glabrescens Makino (SG) has been traditionally used for rheumatism and joint protection. However, the anti-arthritic effects and underling mechanisms of SG have not been demonstrated. In this study, we investigated the anti-arthritic effects and mechanisms of SG extract (SGE) on collagen-induced arthritic rats and interleukin (IL)-1β-stimulated human synovial SW982 cells. Methods: Rats were induced to arthritis by collagen for 15 days and then received SGE treatment for 35 days. The body weight and arthritis severity score of the rats were monitored weekly. At the end of the experiment, the radiographic and histological changes of rats' hind paw were obtained; the serum C-reactive protein was detected by enzyme-linked immunosorbent assay (ELISA); the expression levels of interleukin (IL)- 1β, IL6 and IL-10 in the joint muscles were determined by ELISA and immunohistochemical staining; and the level of regulatory T cells (Tregs) in the spleen was detected using flow cytometry. In addition, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and scratch wound healing assay were used to evaluate the proliferation of SW982 synovial cells. ELISA, western blot and immunofluorescence staining were used to investigate the anti-inflammatory mechanisms of SGE on IL-1β-induced SW982 cells and joint muscles of CIA rats. Results: SGE attenuated the collagen-induced hind paw swelling, cartilage damage and bone erosion. SGE inhibited the synovial hyperplasia to the articular cavity in the toe joint and ankle. Moreover, SGE decreased the production of C-reactive protein in serum and the expression of IL-6, IL-1β, cyclooxygenase-2 (COX-2) and phosphorylation of NF-κB p65 in the joint muscles. SGE also recovered the decreased Tregs. Results from the in vitro experiments showed that SGE not only inhibited the proliferation and migration of human synovial cell but also inhibited the IL-1β-induced expression of IL-6 and IL-8. Similarly, SGE inhibited the activation of NF-κB and the expression of COX-2. Conclusions: SGE attenuated the collagen-induced arthritis through inhibiting the synovial hyperplasia and inflammation.
... e residue was then dried in a freeze-dryer, from which 489 g of dry mass was obtained. e chemical composition of the SOE was previously reported [26,27]. ...
Article
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Diabetes in children and its complications are on the rise globally, which is accompanied by increasing in diabetes-related complications. Oxidative stress and inflammation induced by elevated blood sugar in diabetic patients are considered risk factors associated with the development of diabetes complications, including chronic kidney disease and its later development to end-stage renal disease. Microvascular changes within the kidneys of DM patients often lead to chronic kidney disease, which aggravates the illness. Sigesbeckia orientalis extract (SOE), reported to have strong antioxidative and excellent anti-inflammatory activities, is used in the modern practice of traditional Chinese medicine. Kidneys from three groups of control mice (CTR), mice with streptozotocin (STZ)-induced diabetes (DM), and mice with STZ-induced DM treated with SOE (DMRx) were excised for morphological analyses and immunohistochemical assessments. Only mice in the DM group exhibited significantly lower body weight, but higher blood sugar was present. The results revealed more obvious renal injury in the DM group than in the other groups, which appeared as greater glomerular damage and tubular injury, sores, and plenty of connective tissues within the mesangium. Not only did the DM group have a higher level of cytokine, tumor necrosis factor, and the oxidative stress marker, 8-hydroxyguanosine expression, but also factors of the nuclear factor pathway and biomarkers of microvascular status had changed. Disturbances to the kidneys in DMRx mice were attenuated compared to the DM group. We concluded that SOE is an effective medicine, with antioxidative and anti-inflammatory abilities, to protect against or attenuate diabetic nephropathy from inflammatory disturbances by oxidative stress and to cure vessel damage in a hyperglycemic situation.
Article
Ethnopharmacological relevance Herba Siegesbeckiae, mainly includes Sigesbeckia orientalis L, Sigesbeckiae pubescens Makino and Sigesbeckiae glabrescens Makino. Herba Siegesbeckiae, also known as ‘Xi-Xian Cao’ (Chinese: 豨莶草), has been regarded as an important traditional Chinese medicine since Tang dynasty. The dried aerial parts of Herba Siegesbeckiae are also being used as a herbal medicine in many countries such as Japan, Korea and Vietnam. In China, Herba Siegesbeckiae has been used for the treatment of rheumatic arthralgia with aching and weakness of loins and knees, as well as numbness of limbs. Aim of this review: The aim of this review was to provide critical analysis on the scientific evidence to support the traditional uses of Herba Siegesbeckiae. The information available on its in botanical characteristics, traditional uses, chemical constituents, pharmacological activities, clinical studies, toxicity and quality control was summarized to understand the current research and provided the leas for future study. Materials and methods The search terms “Herba Siegesbeckiae”, “Sigesbeckia orientalis”, “Sigesbeckia pubscens” and “Sigesbeckia glabrescens” were used to obtain the information from electronic databases such as Web of Science, China National Knowledge Infrastructure, PubMed, Google Scholar and SciFinder Scholar and other web search instruments (Springer, Yahoo search). The information provided in this review was based on peer-reviewed papers in English and Chinese. Besides, information was also collected from ancient documents. Result The studies showed that Herba Siegesbeckiae contains sesquiterpenoids, diterpenoids, flavonoids and organic acids, etc. Due to these constituents, it displayed numerous pharmacological activities, such as anti-inflammatory, antitumor, antiallergic, antioxidant, antithrombotic and antibacterial activities. In addition, it showed effects in protecting myocardial and cerebral ischemia injury. Conclusions According to its traditional uses, chemical constituents, pharmacological activities and clinic studies, Herba Siegesbeckiae is regarded as a promising medical plant with various chemical compounds and numerous pharmacological activities. However, fewer experimental studies were focused on toxicity and quantitative study of 3 species. It suggested that further in-depth study of toxicity and quality control were critical for future evaluation of drug efficacy and safety.
Article
Background: The management of acute inflammation, which arises from complex biological responses to harmful stimuli, is an important determinant in recovery of the system from an otherwise detrimental outcome such as septicemia. However, the side effects and limitations of current therapeutics necessitate the development of newer and safer alternatives. Mollugo cerviana is a common medicinal herb of the Indian subcontinent and has been traditionally used for its fever mitigating, anti-microbial and hepatoprotective action. We have already reported the rich presence of radical scavenging phytochemicals in the plant extracts and their strong antioxidant properties. Objective: In the present study, we have evaluated the anti-inflammatory effects of methanolic extract (ME) of the areal parts of M. cerviana in a lipopolysaccharide (LPS)-induced acute inflammatory cell culture model. Method: RAW 264.7 mouse macrophages cell were stimulated by the bacterial endotoxin LPS at a concentration of 1 µg/mL. Cytotoxicity and anti-inflammatory potential of ME were carried out. Results: M. cerviana extract concentration up to 150 µg/ml was found to be non-toxic to cells (MTT and NRU assay). LPS induces acute inflammation by binding to TLR-4 receptors, initiating downstream signalling cascade that result in pro-inflammatory cytokine secretion. Extract treatment at 100 µg/ml suppressed LPS-induced gene expression (qPCR) and secretion (ELISA) of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α, and the chemokine CCL2, indicating a dampening of the acute inflammatory cascade. LPS-induced elevation of ROS level (DCFDA method) was significant reduced by extract treatment. Nitric oxide production, as indicated by nitrite level, was significantly reduced post extract treatment. Conclusion: From this study, it is demonstrated that M. cerviana methanolic extract has potent anti-inflammatory effect in the in vitro acute inflammation model of LPS-stimulated RAW 264.7 cells. There is not reported study so far on the anti-inflammatory properties of M. cerviana in an LPS-induced acute inflammatory model which closely mimics a human bacteremia response. Hence, this study highlights the therapeutic potential of this extract as a source of anti-inflammatory lead molecules.
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Background Herba Siegesbeckiae (HS), the dried aerial parts of Siegesbeckia orientalis L., S. pubescens Makino, or S. glabrescens Makino, is traditionally used for treating chronic diseases in China. However, there is no information about the chronic toxicity of HS. The objective of this study is to evaluate the 24-week oral dosing toxicities of HS aqueous extract (HSE) in rats. Methods S. orientalis-originated HS was reflux-extracted with distilled water. Sprague–Dawley rats were randomly divided into four groups, with 10 males and 10 females in each group. The rats were intragastrically administered with HSE at 5, 1.67 and 0.56 g/kg (experimental groups) or an equal volume of distilled water (control group), 6 days a week, for 24 weeks. The high dose of HSE (5 g/kg) was its maximum tolerated dose. Body weight was recorded every 2 days during the experimental period. Chemical, hematological and histopathological parameters, as well as organ weights, were measured at the end of the experiment. Results Decreased body weight gain; increased liver and lung relative weights; histopathological alterations in liver and lung tissues; elevated serum levels of alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase were found after HSE treatments. In liver tissues, HSE treatment upregulated levels of three pro-inflammatory cytokines: IL-6, IL-1β and TNF-α. In lung tissues, HSE treatment caused oxidative stress and activated mitogen-activated protein kinases (MAPKs). Conclusion Long-term oral administration of HSE caused toxicities in rats evidenced by decreased body weight gain, as well as liver and lung damage. Treatment-induced oxidative stress, inflammation and MAPK activation are involved in HSE’s toxicities. Caution should be taken when using HS to treat chronic diseases.
Article
Sigesbeckia orientalis, more commonly referred to as Herba Sigesbeckiae or Xi Xian Cao in traditional Chinese medicine and hy thiêm in traditional Vietnamese medicine, is used in China and Vietnam to treat inflammatory diseases such as arthritis, rheumatism, and joint pain. In initial investigations, the dichloromethane extract from the aerial parts of S. orientalis showed distinct inhibitory effects on the release of interleukin-8 in human neutrophils. Therefore, the purpose of the present study was the phytochemical investigation of the bioactive dichloromethane extract and the in vitro analysis of the effects of the isolated compounds on interleukin-8, interleukin-1β, tumor necrosis factor-α, and monocyte chemoattractant protein 1 release, and surface expression of adhesion molecules (CD11a, CD11b, and CD62L) in lipopolysaccharide-stimulated human neutrophils to identify the active principle(s). The separation of the bioactive dichloromethane extract using various chromatographic techniques led to the isolation of nine compounds. Their chemical structures were elucidated from nuclear magnetic resonance and mass spectrometry data. One diterpene, 17(13 → 14)-abeo-ent-3S*,13S*,16-trihydroxystrob-8(15)-ene, was identified as a new natural product. Three germacranolide sesquiterpene lactones inhibited interleukin-8 production with IC50 values between 1.6 and 6.3 µM, respectively, and tumor necrosis factor-α production with IC50 values between 0.9 and 3.3 µM, respectively. Furthermore, they significantly inhibited interleukin-1β and monocyte chemoattractant protein 1 production and diminished the effects of lipopolysaccharide on the surface expression of the adhesion molecules CD11a, CD11b, and CD62L. These findings support the traditional use of S. orientalis in the treatment of inflammatory diseases.
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Sigesbeckiae Herba is a traditional Chinese herbal medicine and has been used for inflammatory diseases with a long history. The current official plant origins of Sigesbeckiae Herba include Sigesbeckia orientalis L. (SO), S. pubescens Markino (SP) and S. glabrescens Markino (SG). In this work, a systemic review about the scientific work for medicinal Sigesbeckiae Herba regarding their botanical descriptions, reported bioactive components, pharmacological activities and toxicities was performed. The results indicate that a total of 251 compounds including flavonoids, diterpenoids, sesquiterpenoids, triterpenoids, and sterols have been isolated from Sigesbeckia plants and reported to be characteristically distributed in SO, SP and SG. The extracts and pure compounds derived from Sigesbeckiae Herba are reported to present various pharmacological activities including anti-inflammation, immunomodulation, anti-cancer, and others. Although no significant side-effects and toxicities for Sigesbeckiae Herba have been reported in clinics, the potential pulmonary toxicity for the water extracts of SH are reported in experimental animals. In conclusion, Sigesbeckiae Herba is a valuable Chinese herbal medicine and has been investigated to present multiple pharmacological activities. The main pharmacological investigations support its traditional use for treating chronic inflammatory diseases. The available literature shows that much of the activity of Sigesbeckiae Herba can be attributed to diterpenoids, sesquiterpenoids and flavonoids. However, further investigations on the molecular mechanisms to pharmacological activities and the potential toxicities is recommended. Moreover, further investigations on the comparative comprehension among the three medicinal Sigesbeckia plants (SO, SP and SG) both in chemistry and pharmacology are also recommended for their precise applications in clinics.
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Neuropathic pain represents a major problem in clinical medicine because it causes debilitating suffering and is largely resistant to currently available analgesics. A characteristic of neuropathic pain is abnormal response to somatic sensory stimulation. Thus, patients suffering peripheral neuropathies may experience pain caused by stimuli which are normally nonpainful, such as simple touching of the skin or by changes in temperature, as well as exaggerated responses to noxious stimuli. Convincing evidence suggests that this hypersensitivity is the result of pain remaining centralized. In particular, at the first pain synapse in the dorsal horn of the spinal cord, the gain of neurons is increased and neurons begin to be activated by innocuous inputs. In recent years, it has become appreciated that a remote damage in the peripheral nervous system results in neuronal plasticity and changes in microglial and astrocyte activity, as well as infiltration of macrophages and T cells, which all contribute to central sensitization. Specifically, the release of pronociceptive factors such as cytokines and chemokines from neurons and non-neuronal cells can sensitize neurons of the first pain synapse. In this article we review the current evidence for the role of cytokines in mediating spinal neuron-non-neuronal cell communication in neuropathic pain mechanisms following peripheral nerve injury. Specific and selective control of cytokine-mediated neuronal-glia interactions results in attenuation of the hypersensitivity to both noxious and innocuous stimuli observed in neuropathic pain models, and may represent an avenue for future therapeutic intervention.
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