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Foeniculum vulgare Mill. Protects against Lipopolysaccharide-induced Acute Lung Injury in Mice through ERK-dependent NF-κB Activation

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Foeniculum vulgare Mill. (fennel) is used to flavor food, in cosmetics, as an antioxidant, and to treat microbial, diabetic and common inflammation. No study to date, however, has assessed the anti-inflammatory effects of fennel in experimental models of inflammation. The aims of this study were to investigate the anti-inflammatory effects of fennel in model of lipopolysaccharide (LPS)-induced acute lung injury. Mice were randomly assigned to seven groups (n=7~10). In five groups, the mice were intraperitoneally injected with 1% Tween 80-saline (vehicle), fennel (125, 250, 500µl/kg), or dexamethasone (1 mg/kg), followed 1 h later by intratracheal instillation of LPS (1.5 mg/kg). In two groups, the mice were intraperitoneally injected with vehicle or fennel (250µl/kg), followed 1 h later by intratracheal instillation of sterile saline. Mice were sacrificed 4 h later, and bronchoalveolar lavage fluid (BALF) and lung tissues were obtained. Fennel significantly and dose-dependently reduced LDH activity and immune cell numbers in LPS treated mice. In addition fennel effectively suppressed the LPS-induced increases in the production of the inflammatory cytokines interleukin-6 and tumor necrosis factor-alpha, with 500µl/kg fennel showing maximal reduction. Fennel also significantly and dose-dependently reduced the activity of the proinflammatory mediator matrix metalloproteinase 9 and the immune modulator nitric oxide (NO). Assessments of the involvement of the MAPK signaling pathway showed that fennel significantly decreased the LPS-induced phosphorylation of ERK. Fennel effectively blocked the inflammatory processes induced by LPS, by regulating pro-inflammatory cytokine production, transcription factors, and NO.
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183
Korean J Physiol Pharmacol
Vol 19: 183
189, March, 2015
http://dx.doi.org/10.4196/kjpp.2015.19.2.183
pISSN 1226-4512
eISSN 2093-3827
ABBRE VI ATI ON S: fennel, Foeniculum vulgare Mill.; LPS, lipopoly-
saccharide; BALF, bronchoalveolar lavage fluid; NO, nitric oxide;
ALI, acute lung injury; ROS, reactive oxygen species; MDA, malon-
dialdehyde; DEX, dexamethasone; LDH, lactate dehydrogenase;
H&E, hematoxylin and eosin; ECL, enhanced chemiluminescence;
ELISA, enzyme-linked immunosorbent assay.
Received December 31, 2014, Revised January 7, 2015,
Accepted January 7, 2015
Corresponding to: Geun Hee Seol, Department of Basic Nursing
Science, School of Nursing, Korea University, 145 Anam-ro, Seong-
buk-gu, Seoul 136-701, Korea. (Tel) 82-2-3290-4933, (Fax) 82-2-927-
4676, (E-mail) ghseol@korea.ac.kr
*The two authors equally contributed to this paper.
This is an Open Access article distributed under the terms of the
Creat ive Co mmons Attribut ion Non-Commerc ial Licens e (http://
creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial
use, distribution, and reproduction in any medium, provided the original work
is properly cited.
Foeniculum vulgare Mill. Protects against Lipopolysaccharide-induced
Acute Lung Injury in Mice through ERK-dependent NF-kB Activation
Hui Su Lee*, Purum Kang*, Ka Young Kim, and Geun Hee Seol
Department of Basic Nursing Science, School of Nursing, Korea University, Seoul 136-701, Korea
Foeniculum vulgare Mill. (fennel) is used to flavor food, in cosmetics, as an antioxidant, and to treat
microbial, diabetic and common inflammation. No study to date, however, has assessed the anti-inflam-
matory effects of fennel in experimental m odels of inflamm ation. The aim s of this study were to
investigate the anti- inflam matory effects of fennel in m odel of lipopolysaccharide (LPS )-induced acute
lung injury. Mice were randomly assigned to seven groups (n=710). In five groups, the mice were
intraperitoneally injected with 1% Tween 80-saline (vehicle), fennel (125, 250, 500μl/kg), or dexame-
thas one (1 m g/ kg ), followed 1 h l ater by intrat ra cheal ins till ation of L PS (1 . 5 m g/ kg ). In two grou ps ,
the mice were intraperitoneally injected with vehicle or fennel (250 μl/kg), followed 1 h later by intra-
tracheal instillation of sterile saline. M ice were sacrificed 4 h later, and bronchoalveolar lavage fluid
(BA LF) and lung tis sues were obtained. Fennel significantly and dose-dependently reduced LDH
activity and immune cell numbers in LPS treated m ice. In addition fennel effectively suppressed the
LPS-induced increases in the production of the inflamm atory cytokines interleukin-6 and tum or
necrosis fact or- a lpha , wit h 50 0 μl/kg fennel showing m axim al reduction. Fennel also significantly and
dose-dependently reduced the activity of the proinflamm atory mediator matrix metalloproteinase 9 and
the imm une m odulator nitric oxide (NO). Assessm ents of the involvement of the MA PK signaling
pathway showed that fennel significantly decreased the LPS-induced phosphorylation of ERK. Fennel
effectively blocked the inflam matory processes induced by LPS, by regulating pro-inflammatory cytokine
produ ction, tran script ion fact ors , and N O .
Key Words: ERK, Foeniculum vulgare Mill., LPS, TNF-α
INTRODUCTION
Acute lung injury (ALI), characterized by unbalanced in-
flammatory responses, is a leading cause of acute respira-
tory failure and multiple organ dysfunctions [1,2]. ALI is
associated with neutrophilic inflammation, which can be ac-
celerated by endotoxins such as lipopolysaccharide (LPS)
from Gram-negative bacteria [3]. Experimental models of
LPS-induced ALI have therefore been used to explore in-
flammatory responses in the lung. LPS-induced ALI has
been associated with the production of reactive oxygen spe-
cies (ROS) in alveolar macrophages and to involve NF-κB
signaling pathways, including the MAPK/JNK/p38/ERK
pathways [4].
Foeniculum vulgare Mill. (fennel) is used to flavor foods,
in cosmetics, and to treat microbial, diabetic and common
inflammation. Evaluation of fennel seed extracts using a
DPPH radical scavenging assay also indicated that fennel
may have antioxidant activity effect [5]. Oral administra-
tion of a methanolic extract of F. vulgare fruit decreased
malondialdehyde (MDA) level, suggesting that this extract
has inflammation-relieving effects in experimental animals
[6]. Moreover, fennel decreased ROS and MDA in mouse
tumor tissue [7]. Trans-anethole, the major component of
fennel, was found to reduce paw edema and inflammatory
pain [8], with the anti-inflammatory effects of trans-anet-
hole reported to derive from its regulation of NF-κB signal-
ing pathways [9].
Because fennel contains several components, which can
affect each other, there is a need to confirm that this essen-
tial oil shows consistent effects. To date, however, no study
has analyzed the anti-inflammatory effects of fennel in a
mouse model of LPS-induced ALI. This study therefore ex-
plored the anti-inflammatory effects of fennel in LPS-in-
184 HS Lee, et al
duced ALI in mice, and investigated the signaling pathways
involved.
METHODS
Animals and M aterials
Male BALB/C mice, aged five weeks and weighing 19 to
21 g, were obtained from Orient Bio (Sungnam, Korea) and
acclimatized to standard laboratory conditions for 3 to 5
days. All experimental procedures were conducted in ac-
cordance with guidelines relevant to the care of ex-
perimental animals, as approved by the Animal Research
Committee of Korea University (approval no. KUIACUC-
2012-181), informed by the Guide for the Care and Use of
Laboratory Animals published by the US National
Institutes of Health (NIH publication No. 85-23; revised
1996). Mice were randomly assigned to seven groups (n=7
10) and were anesthetized by intraperitoneal injection of
a mixture of 0.3 mg/kg tiletamine-zolazepam (Zoletil 50,
Virbac Laboratories, Carros, France) and 0.2 mg/kg xyla-
zine (Rompun, Bayer Korea, Ansan, Korea). In five groups,
the mice were intraperitoneally injected with 1% Tween
80-saline (vehicle), fennel (125, 250, 500μl/kg), or dex-
amethasone (DEX) (1 mg/kg), followed 1 h later by intra-
tracheal instillation of LPS (1.5 mg/kg). In the remaining
two groups, the mice were intraperitoneally injected with
1% Tween 80-saline (vehicle) or fennel (250μl/kg), followed
1 h later by intratracheal instillation of sterile saline. The
dose of fennel was based on a previous study of trans-anet-
hole, the main component of fennel [9]. The mice were sacri-
ficed 4 h later, and their bronchoalveolar lavage fluid
(BALF) and lung tissues were obtained. Lipopolysaccharide
(LPS, from E. coli 0.55:B5), Tween 80 and DEX were ob-
tained from Sigma-Aldrich (St. Louis, MO, USA). Pure fen-
nel essential oil was purchased from Aromarant Co. Ltd.,
Rottingen, Germany and came from locally cultivated
plants. The fennel essential oil that we used (batch No.
091119; Aromarant Co. Ltd) was analyzed by gas chroma-
tography/mass spectrometry (GC/MS). The main compo-
nents of fennel essential oil detected by GC/MS analysis
were 75.81% trans-anethole, 5.93% fenchonem, 5.82% limo-
nene, 4.30% methyl chavicol, 3.52% α-pinene and 0.39%
α-phellandrene.
Lactate dehydrogenase (LDH) assay
The activity of LDH, an enzyme used as a marker for
cytotoxicity, was measured using a commercial LDH assay,
according to the manufacturer’s instructions (Takara Bio
Inc., Otsu, Japan). BALF samples were mixed 11 with
freshly prepared reaction mixture and incubated in the
dark for 30 min at room temperature. Absorbance was
measured at 490 nm and at a reference wavelength of 620
nm using a microplate reader (BMG Labtech, Ortenberg,
Germany).
Cell counting
BALF samples were centrifuged at 500×g for 10 min at
4oC, and the sedimented cells were resuspended in PBS.
The cells were stained with Diff-Quick (International
Reagents Co., Kobe, Japan), and total and differential leu-
kocyte counts were determined using a Countess automated
cell counter (Invitrogen Life Technologies, Carlsbad, CA,
USA). Results are expressed as the number of each cell type
per milliliter of BALF.
Histopathology
Lung tissues were fixed in 10% paraformaldehyde, em-
bedded in paraffin, and cut into 4μm thick sections. The
sections were stained with hematoxylin and eosin (H&E),
and viewed under a light microscope (200×).
Enzyme-linked immunosorbent assay (ELIS A)
The concentrations of the inflammatory cytokines IL-6 and
TNF-α in BALF were measured using commercially avail-
able ELISA kits, in accordance to the manufacturer’s in-
structions (PeproTech, London, UK).
M easurem ent of nitric oxide (N O)
Since NO has a short half-life, we measured nitrite level,
an indirect measure of NO production [10]. The amount of
nitrite in BALF was measured using the Griess reaction.
Griess reagent included 0.1% N-(1-naphthyl) ethylenedi-
amine dihydrochloride and 1% sulfanilamide. Briefly, Griess
reagent was added to 100μl of BALF supernatant, and the
solutions were mixed and incubated for 10 min at room
temperature. Optical density at 540 nm was measured in
a microplate reader (BMG Labtech, Ortenberg, Germany).
Zy mographic analysis
The secretion of matrix metalloproteinase-9 (MMP-9)
protein was measured by gelatin zymography. A volume of
BALF sample was mixed with an equal volume of non-
reducing sample buffer, and the samples were electro-
phoresed in 8% sodium dodecyl sulfate polyacrylamide elec-
trophoresis gels (SDS-PAGE) containing 1 mg/ml gelatin.
The gels were washed with 2.5% Triton X-100 for 2 h and
subsequently incubated for 20 h at 37oC in 50 mM Tris-Cl
buffer (pH 7.4) containing 10 mM CaCl2 and 0.02% NaN3.
The gels were subsequently stained for 1 h with 0.5%
Coomassie Brilliant Blue G250 in 7.5% acetic acid/10%
propanol-2 and destained to visualize the protein bands.
Relative densities of MMP-9 were analyzed with Bio-Rad
Quantity One software (Bio-Rad, Hercules, CA, USA).
Extraction of lung nuclear proteins
Lung tissues obtained at sacrifice were immediately fro-
zen in liquid nitrogen, and 50 mg samples of frozen lung
tissue were subsequently homogenized with a Precellys 24
bead-based tissue homogenizer in 0.5 ml ice-cold buffer A
(10 mM HEPES with pH 7.9, 1.5 mM MgCl2, 10 mM KCl,
0.1 mM Na2EDTA, 0.5 mM DTT, 1% Nonidet P-40, 0.5 mM
PMSF, 0.5μg/ml leupeptin, 125μg/ml aprotinin, 25μg/ml
pepstatin A). Cell debris was removed by centrifugation at
2,000 rpm for 30 sec; the supernatants were incubated on
ice for 5 min and again centrifuged for 10 min at 5,000
rpm. Cytoplasmic proteins in the supernatant were col-
lected and the pellet was resuspended in 50μl of cold buffer
B (20 mM HEPES with pH 7.9, 1.5 mM MgCl2, 0.42 M
NaCl, 20% glycerol, 0.5 mM DTT, 0.5 mM PMSF, 0.5μg/ml
leupeptin, 125μg/ml aprotinin, 25μg/ml pepstatin A) and
incubated on ice for 30 min. The nuclear fraction was col-
Anti-inflammatory Effects of Foeniculum vulgare Mill. 185
Fig. 1. Effect of fennel on lactate dehydrogenase (LDH) activity in
BALF of LPS-treated mice. Mice were intratracheally administered
LPS (1.5 mg/kg) 1 h after intraperitoneal injection of 1% Tween
80-saline (vehicle), fennel (125, 250, 500μl/kg), or DEX (1 mg/kg).
The activity of LDH in BALF was measured to evaluate cell
damage. Data are reported as mean±S.E.M. (n=710 per group).
#
##p0.001 compared with vehicle group; **p0.01, ***p0.001
compared with the vehicle+LPS group.
Fig. 2. Effects of fennel on cell numbers in BALF of LPS-treated
mice. The numbers of total cells, neutrophils, macrophages, and
lymphocytes in BALF were analyzed. Data are reported as mean±
S.E.M. (n=710 per group). #p0.05, ##p0.01, ###p0.001
compared with the vehicle group; *p0.05, **p0.01, ***p0.001
compared with the vehicle+LPS group.
lected by centrifugation at 12,000 rpm for 2 min.
Western blot analysis
Lung tissue homogenate samples were separated on 10%
SDS-PAGE. The proteins were electrophoretically trans-
ferred onto nitrocellulose membranes, which were blocked
for 30 min at room temperature. The membranes were in-
cubated overnight at 4oC with primary antibodies to NF-κB
p65, Lamin B, IκB-α, GAPDH, p-ERK, ERK, p-p38, p38,
p-JNK, and JNK, followed by incubation with horseradish
peroxidase-conjugated secondary antibody for 1 h at room
temperature. Bands were visualized by enhanced chemilu-
minescence (ECL) reagents according to the manufacturer’s
protocol. Relative densities were analyzed using Bio-Rad
Quantity One software (Bio-Rad, Hercules, CA, USA).
Statistical analysis
All data were expressed as mean±S.E.M. and compared
using one-way analysis of variance, followed by the Tukey
HSD post hoc test. All statistical analyses were performed
using SPSS 20 software, with results considered statisti-
cally significant at p0.05.
RESULTS
Effect of fennel on LDH activity in BALF of mice with
LPS-induced ALI
The activity of LDH was significantly higher in BALF
of mice with LPS-induced ALI than in mice treated with
vehicle alone (63.02±27.28 U/L vs. 19.90±8.13 U/L, p0.001)
(Fig. 1), whereas pretreatment with DEX, which has been
shown to protect against LPS-induced ALI, significantly re-
duced LDH activity (25.66±4.35, p=0.004). Mice pretreated
with 125 (16.24±4.43, p0.001), 250 (11.07±2.21, p0.001),
and 500 (9.57±1.05, p0.001)μl/kg fennel, followed by
LPS, showed significantly decreased LDH activity com-
pared with mice treated with vehicle plus LPS. LDH level
was similar in mice treated with fennel (250μl/kg) and ve-
hicle without LPS.
Effect of fennel on inflamm atory cell count in BALF
Recruitment of excess numbers of inflammatory cells is
necessary for the pathogenesis of ALI. Compared with ve-
hicle alone, treatment with vehicle+LPS significantly in-
creased the numbers of total cells (8.84×105 cells/ml, p
0.001), neutrophils (3.67×105 cells/ml, p0.001), macro-
phages (2.84×105 cells/ml, p0.001), and lymphocytes
(2.44×105 cells/ml, p=0.005) in BALF (Fig. 2). In LPS-treat-
ed mice, pretreatment with fennel 125μl/kg (total cells,
6.49×105 cells/ml, p=0.204; neutrophils, 2.57×105 cells/ml,
p=0.099; macrophages, 1.47×105 cells/ml, p=0.001; lympho-
cytes, 1.64×105 cells/ml, p=0.236), 250μl/kg (total cells,
5.71×105 cells/ml, p=0.032; neutrophils, 2.16×105 cells/ml,
p=0.007; macrophages, 1.34×105 cells/ml, p0.001; lympho-
cytes; 1.95×105 cells/ml, p=0.769), and 500μl/kg (total cells,
2.41×105 cells/ml, p0.001; neutrophils, 0.85×105 cells/ml,
p0.001; macrophages, 0.74×105 cells/ml, p0.001; lym-
phocytes, 0.72×105 cells/ml, p0.001) significantly and
dose-dependently reduced the total numbers of cells, sim-
ilar to DEX, as well as decreasing the numbers of neu-
trophils, macrophages, and lymphocytes (Fig. 2).
Effect of fennel on lung histopathology of LPS -treated
mice
Hematoxylin and eosin (H&E) staining showed that LPS
treatment (Fig. 3B) was characterized by neutrophil se-
questration, infiltration around the pulmonary vessels, and
alveolar wall thickening in lung tissue compared with ve-
hicle (Fig. 3A). However neutrophil sequestration, infiltra-
tion around the pulmonary vessels, and alveolar wall thick-
ening were significantly alleviated by pretreatment with
500μl/kg fennel (Fig. 3C), as well as by DEX (Fig. 3D).
Effect of fennel on IL-6 and TN F-
α
in BA LF
LPS significantly increased the concentrations in BALF
of the inflammatory cytokines IL-6 (0.97±0.09 vs. 0.18±0.04
ng/ml, p0.001; Fig. 4A) and TNF-α (7.18±0.53 vs.
186 HS Lee, et al
Fig. 3. Effect of fennel on the histo-
pathology of lung tissues in LPS-
treated mice. Fennel (500μl/kg) or
DEX (1 mg/kg) was administered in-
traperitoneally to mice 1 h prior to
LPS treatment. Lung sections from
each group were stained with hema-
toxylin and eosin (H&E) (×200). (A)
Vehicle group, (B) Vehicle+LPS gro-
up, (C) Fennel+LPS group, (D) DEX+
LPS group.
Fig. 4. Effects of fennel on (A) IL-6 and (B) TNF-α expression in the BALF of LPS-treated mice. IL-6 and TNF-α in BALF were analyzed
by ELISA. Data are reported as mean±S.E.M. (n=710 per group). ##p0.01, ###p0.001 compared with the vehicle group; *p0.05, ***p
0.001 compared with the vehicle+LPS group.
0.10±0.01 ng/ml, 00.001; Fig. 4B) compared with vehicle.
Pretreatment with fennel 125μl/kg (IL-6, 0.76±0.10 ng/ml,
p=0.468; TNF-α, 5.27±0.30 ng/ml, p=0.010), 250μl/kg (IL-6,
0.77±0.07, p=0.517; TNF-α, 5.36±0.58 ng/ml, p=0.016), and
500μl/kg (IL-6, 0.58±0.11, p=0.017; TNF-α, 4.29±0.29
ng/ml, p0.001), however, significantly and dose-depend-
ently suppressed the production of IL-6 and TNF-α, with
500μl/kg fennel showing maximum reduction.
Effect of fennel on MM P-9 activity in LPS -treated mice
MMP-9, a representative proinflammatory mediator that
plays an essential role in lung inflammation, was analyzed
in BALF by gelatin zymography. BALF from mice treated
with vehicle+LPS showed a 10-fold increase in a gelati-
nolytic band at 92 kDa, the molecular weight of MMP-9
(p0.001), compared with vehicle-treated mice (Fig. 5).
Pretreatment with 250 and 500μl/kg fennel dose-depend-
ently reduced MMP-9 activity, and pretreatment with DEX
also reduced MMP-9 activity.
Effect of fennel on nitric oxide (N O) production in BALF
NO is a critical immune modulator in the proinflam-
Anti-inflammatory Effects of Foeniculum vulgare Mill. 187
Fig. 5. Effect of fennel on MMP-9 activity in LPS-treated mice.
Relative MMP-9 activity in BALF was analyzed by zymography
followed by scanning densitometry. Data are reported as
mean±S.E.M. (n=710 per group). ##p0.01, ###p0.001 compared
with the vehicle group; *p0.05, **p0.01, ***p0.001 compared
with the vehicle+LPS group.
Fig. 6. Effect of fennel on NO production in the BALF of
LPS-treated mice. NO concentrations in BALF were measured by
nitrite assays. Data are reported as mean±S.E.M. (n=710 per
group). ###p0.001 compared with the vehicle group; **p0.01,
***p0.001 compared with the vehicle+LPS group.
Fig. 7. Effect of fennel on NF-κB
activation in LPS-treated mice. Nu-
clear and cytosolic extracts in lung
tissue were fractionated and the ex-
pression of NF-κB p65 (A) and IκB-α
(B) proteins in nuclear and cytosolic
extracts, respectively, were assessed
by western blotting. Lamin B and
GAPDH were used as internal controls.
Data are reported as mean±S.E.M.
(n=710 per group). ##p0.01, ###p
0.001 compared with the vehicle group;
*p0.05, ***p0.001 compared with
the vehicle+LPS group.
matory cytokine response associated with ALI. Treatment
with LPS significantly enhanced the production of NO com-
pared with vehicle (2.98±0.45μM vs. 1.09±0.24μM, p=
0.001; Fig. 6). However, this increase was significantly and
dose-dependently reduced by pretreatment with fennel 125
μl/kg (0.52±0.27μM, p0.001), 250μl/kg (0.56±0.73μM,
p0.001), and 500μl/kg (0.67±0.23μM, p0.001).
Effect of fennel on activation of NF-
κ
B in LPS -induced
ALI mice
NF-κB activation was assessed by western blotting to
determine the anti-inflammatory pathways by which fennel
reduced LPS-induced ALI in mice. Treatment with ve-
hicle+LPS increased the level of expression of NF-κB p65
2.13-fold (p=0.007) compared with vehicle alone (Fig. 7A).
However, in LPS-treated mice, pretreatment with 500μl/kg
fennel reduced the expression of NF-κB p65 1.90-fold com-
pared with pretreatment with vehicle alone (p=0.019). Mice
treated with vehicle+LPS showed 4.05-fold lower IκB-α
expression compared with those treated with vehicle alone
(p0.001), whereas mice treated with 500μl/kg fennel plus
LPS showed 2.79-fold higher IκB-α expression compared
with those treated with vehicle+LPS (p=0.023) (Fig. 7B).
This finding indicated that fennel suppressed NF-κB acti-
vation by blocking IκB-α degradation.
Effect of fennel on the M APK signaling pathw ay
The effect of fennel on the MAPK signaling pathway was
analyzed to determine its anti-inflammatory mechanism of
action. LPS increased the levels of expression levels of phos-
pho-ERK (5.11-fold, p0.001) (Fig. 8A and 8B), phospho-p38
(1.27-fold, p=0.474) (Fig. 8C and 8D), and phospho-JNK
(1.97-fold, p=0.036) (Fig. 8E and 8F). In contrast, 250μl/kg
(2.86-fold, p=0.004) and 500μl/kg (2.07-fold, p=0.021) fen-
nel significantly reduced the level of LPS-induced ERK
phosphorylation.
DISCUSSION
Although inflammation is a normal immune reaction, un-
controlled inflammation can lead to organ dysfunction or
disease [11]. Clinical ALI involves neutrophilic inflam-
188 HS Lee, et al
Fig. 8. Effect of fennel on the MAPK
signaling pathway in LPS-treated
mice. Lung tissues were analyzed by
western blotting with antibodies to
p-ERK (A), p-p38 (C), and p-JNK (E),
and quantitative protein expression
was normalized to ERK (B), p38 (D),
and JNK (F), respectively. Data are
reported as mean±S.E.M. (n=710
per group). #p0.05, ###p0.001 com-
pared with the vehicle group; *p
0.05, **p0.01 compared with the
vehicle+LPS group.
mation and is a common complication of other conditions
[12]. Because LPS from Gram-negative bacteria evokes in-
flammatory responses and endotoxic symptoms [13], LPS
is used in experimental models of inflammation. In agree-
ment with previous findings, we found that LPS-treated
mice showed inflammatory responses, including elevations
in immune system cells and proinflammatory cytokines, as
well as alterations in lung histology. Fennel, which has
been shown to have anti-inflammatory effects, protected
mice against LPS-induced ALI. Fennel reduced lung dam-
age, the numbers of pro-inflammatory cells, and the pro-
duction of pro-inflammatory mediators induced by LPS.
NF-κB, an important transcription factor in inflam-
matory responses, has been shown to regulate the pro-
duction of pro-inflammatory cytokines [14]. Although NF-κB
activation is important in normal inflammatory responses,
its overproduction is closely associated with inflammatory
diseases, such as sepsis [14]. In the absence of stimuli, NF-
κB is located in the cytoplasm, where it binds to IκB-α
and remains inactive. Thus, regulating IκB-α may control
the NF-κB signaling pathway. Trans-anethole, the main
constituent of fennel, has been reported to reduce NF-κB
concentrations in mice with hepatic ischemia/reperfusion
injury [15], as well as to reduce NF-κB levels, while slight-
ly increasing IκB-α levels, in LPS-treated BALB/C mice
[9]. Similarly, we found that treatment with fennel not only
reduced p65 expression, but increased IκB-α level. Thus,
fennel may directly suppress NF-κB activation, perhaps
by enhancing the expression of its inhibitor, IκB-α.
Calcium signaling plays an important role in inflam-
matory conditions [16]. Administration of LPS has been
found to transiently elevate intracellular calcium level,
leading to ERK phosphorylation and the expression of TNF-α
[17]. Fennel was reported to significantly reduce the ex-
pression of TNF-α in response to S. aureus [18], suggesting
that it modulates intracellular calcium concentration.
Fennel induced the relaxation of guinea pig tracheal chains
via hyperpolarization, thus inhibiting calcium influx [19].
In addition, high doses of trans-anethole were reported to
modify calcium channels on isolated rat aortas [20], further
suggesting that the protective effect of fennel on TNF-α
and ERK expression was due, at least in part, to calcium
modulation.
The MAP kinase pathway, which includes ERKs, JNKs
and p38, is also involved in the endotoxic effects of LPS,
leading to inflammation. This pathway and TNF-α ex-
pression are both upstream and downstream of each other
[21]. NO is another important signaling molecule, which
regulates physiological functions, including vascular con-
traction, neuronal signal and inflammation [22]. NO may
derive from inducible NO synthase (iNOS) associated path-
ophysiological processes related to inflammation. Unlike
JNK and p38, ERKs negatively activate iNOS. We found
that fennel significantly reduced nitrate levels, suppressing
LPS-induced ERK expression but having no effect on JNK
or p38 levels.
Fennel contains mainly trans-anethole, limonene, and
anisole [18]. Trans-anethole was shown to have anti-inflam-
matory effects, substantially similar to those of fennel, on
pro-inflammatory cytokines, NO, and transcription factors
[9]. Moreover, d-limonene has shown anti-inflammatory ef-
fects in rat kidney by modulating NF-κB and iNOS [23].
Oral administration of limonene to rats suppressed both
NF-κB and IL-6 [24]. Taken together, these findings sug-
gest that limonene and trans-anethole, the main compo-
nents of fennel, are responsible for the anti-inflammatory
effects of fennel.
In conclusion, this study confirmed that fennel effectively
blocked LPS-induced inflammation, by regulating pro-in-
flammatory cytokines, transcription factors, and NO. These
findings suggest that fennel may have clinical activity in
mitigating inflammatory conditions.
Anti-inflammatory Effects of Foeniculum vulgare Mill. 189
ACKNOWLEDGEMENTS
This work was supported by the National Research
Foundation of Korea (NRF) grant funded by the Korean
government (MSIP) (No.2012R1A2A2A02007145).
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... The key element of fennel essential oil, trans-anethole, has demonstrated promising antiviral effects in opposition to the herpes virus (77). It can also improve the immune system and relieve inflammation, according to animalbased studies (78). ...
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... A study showed that fennel extract exhibited strong antiviral effects against herpes viruses and Para influenza type-3 (PI-3), which causes respiratory infections in cattle [9]. Trans-Anatole, the main component of fennel essential oil, has demonstrated powerful antiviral effects against herpes viruses [10].According to animal research, fennel may also boost your immune system and decrease inflammation, which may likewise help combat viral infections [11]. ...
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... Herpes viruses and parainfluenza type-3 (PI-3),were strongly inhibited by fennel extract and could reduce respiratory tract infections in cattle (Badgujar et al., 2014).Trans-anethole, the primary active ingredient of fennel essential oil, was found to possess powerful antiviral effects (herpes viruses). According to some studies done on animal research models, fennel has immuno-modulatory actions and helps to decrease inflammation, which may likely help to combat viral infections (Lee, et al, 2015). ...
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... Anti-inflammatory effects of fennel in the model of lipopolysaccharide (lps)-induced acute lung injury was determined. Fennel effectively blocked the inflammatory processes induced by LPS, by regulating pro-inflammatory cytokine production, transcription factors, and NO [34]. ...
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India has a rich source of tropical fruits containing edible seeds such as chia, hemp, sesame, pumkin, sunflower, mustard, nigella, guava, papaya, mangosteen, honeydew, pomegranate, fennel, fenugreek, cumin, sweet orange, cucumber, jackfruit, mango, melons, avocado and many more. These products such as the seed kernel, which constitutes about 10–35% of the weight, offer high nutritional value and therapeutic applications. This article explores the nutritional, medicinal, therapeutic applications, functional properties and bioactive constituents of the seeds of some fruits, which are analyzed for their functions and applications as sources of food value and bioactive phytochemical constituents. The seeds contain essential bioactive components such as alkaloids, carotenoids, flavonoids, glycosides, saponins, terpenoids, tannins, steroids and polyphenolic compounds and that exhibit excellent anti-inflammatory, antioxidant properties, anticancer, anti-diabetic, anti-hyperlipidemic, anti-obesity, neurological disorders, cardiovascular, skin diseases and chronic diseases. They have remarkable physicochemical properties and a high content of carbohydrates, fats, proteins, vitamins, and minerals. However extensive research activities can be carried out to determine the efficacy of the nutritional and bioactive components in different seed types, the bioavailability and potency. Extensive research with the seed parts can be investigated to identify the medicinal and functional potentials of these fruit seeds. This review gives an overview on the therapeutic applications and functional properties of seeds present in fruits, vegetables and medicinal plants. The medicinal and nutritional value, phytochemical composition, bioactive phytoconstituents, therapeutic activity, therapeutic applications and uses, proximate analysis, functional properties, analytical methods, spectroscopic methods and human clinical trials of some edible seeds are discussed in this review.
... [ 80,92] 6. Not reported [20,80,93] 7. Capsid protein disintegration. [96][97][98] 9. Inhibits herpes virus replication, inhibits HIV-1 LTR-directed gene expression, and inhibits transcription of HPV-18. ...
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Book
Plants are a fascinating group of plants that have been dominating the earth for 400 million years. During evolution, they have undergone series of evolutionary changes to suit themselves with the surrounding environment. These evolutionary changes not only included morphological changes to suit varied climatic conditions but also armed with intricate physiological changes to synchronize with the former and fortify better adaptability. These physiological changes of the plant later proved to be of immense help to the humans who evolved much later somewhere between 6 million to 2 million years ago. The physiological and biochemical evolution of the plants with the synchronous origin of various taxa resulted in the formation of numerous biochemical pathways producing a large number of secondary metabolites whose one primary aim is to protect the plants from herbivores and insect which in the due course of evolution became an integral part of the food chain. However, the secondary metabolites also proved to be of immense use to humans since antiquity who unknowingly since prehistoric times used plants for their food and medicine. It is only in the past hundred years or so, people became aware of the chemical constituent of the plants and started exploring their various beneficial properties. The agricultural activities also coevolved with human civilization and with the increase in population, higher yield along with protection of crops from pathogen attack became a necessity. This lead to the formulation of fertilizers which consequently paved the way for biofertilizers with a fewer side effects on humans and animals but with a more green approach towards fertility enhancement. With the advent of industrialization the menace of pollution cropped up and presently this pollution is encroaching soil water and air. This is having a deleterious effect on the ecosystem concerning human and animal health and also agricultural productivity. Thus keeping this in mind the scientific community was determined to remediate the polluted sites with the help of biological agents in which the plants and microbes played an important role. This provided major protection to agriculture from contamination thereby sustaining productivity. Thus, an attempt is made to highlight the progress and advances in the field of agriculture and plant science. Thus A handbook of Agricultural and Plant Sciences is an attempt to compile information related to the field of agriculture and plant science. The main purpose of the book is to provide relevant information to the readers on aspects largely cantered on plants. The book is divided into three sections namely agriculture and sustainable development, plants and microbes as nutraceutical agents, and medicinal potential of plants. Selected chapters in relevance to the sections have been accommodated to provide an overview. The first section deals with various aspects through which crops can be fortified through bio fertilization and also decontamination of polluted lands. The world population is presently stressing upon consumption of foods from natural sources as consumption of fast food with artificial agents is leading to the onset of several diseases. This has led to a group of foods that confers nutrition as well as a medicinal benefit at the same time. They are presently termed and considered nutraceuticals. The second section of the book deals with the nutraceutical potential of plants and microbes which are symbiotically associated with plants. The third section is also related to the second one concerning the medicinal importance. This section encompasses the medicinal importance of plants. Plants as antiviral agents have been accommodated because of the current pandemic situation. The section also contains a chapter on the ant diabetic potential of plants and also the medicinal importance of gymnosperms and bioactive potentials of bryophytes which adds up to the variation in chapters focusing on the medicinal aspect. The book is also accompanied by several tables within each chapter which gives a clear and systematic description of the theme that is discussed upon. The book is an academic venture and would benefit the scientific community and readers who are interested in the field of plant sciences.
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More than 199 countries worldwide are affected by a new coronavirus disease (COVID-19) caused by infection with SARS-CoV-2gh21. The transition from early symptoms to acute respiratory distress syndrome (ARDS) is most likely due to uncontrolled cytokine release. There is an urgent need to identify safe and effective drugs for treatment. Many drugs exhibit a promising inhibitory effect. However, the clinical use of some medications can cause serious side effects. We proposed that natural herbs could serve as a better therapeutic approach.
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COVID-19 is a severe respiratory disorder caused by the SARS COV-2 virus that involves limited innate immunity. Numerous publications have suggested that plants/minerals used in the traditional system of Ayurveda, has revealed much about the biology of COVID-19. One theory is that combination of anti viral, anti inflammatory, agents activating immune cells, herbs and metals may be helpful for severe acute respiratory syndrome coronavirus infection. Anti-viral drugs used for COVID-19 are those which block RNA synthesis and virus invasion, and bind to receptor proteins on the surface of cells, cell cycle protein, and physiological and pathological processes inhibitor. Anti-inflammatory drugs used for COVID-19 are those which controls transcription of DNA, cytokine production, break down the basement membrane, regulate outer mitochondrial membrane permeability, controlling the host cell life, stimulates activated B-cell and T-cell proliferation, virus dissemination, a slowdown of cell metabolism or secretion of cytokines. Drugs which is having role in the innate immunity, inhibits ROS, enhances cell lifespan, activates macrophages, physiological effects on cells activates the Lung resident immune cells. The focus of this review is to elucidate the Ayurvedic pharmacological properties with their therapeutic targets.
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Oxidation of biomolecules such as carbohydrates, proteins, lipids, and nucleic acids results in generation of free radicals in an organism which is the major cause of onset of various degenerative diseases. Antioxidants scavenge these free radicals, thereby protecting the cell from damage. The present study was designed to examine the free radical scavenging potential and oxidative DNA damage preventive activity of traditionally used spices Trachyspermum ammi L. (carom) and Foeniculum vulgare Mill. (fennel). The aqueous, methanolic, and acetonic extracts of T. ammi and F. vulgare seeds were prepared using soxhlet extraction assembly and subjected to qualitative and quantitative estimation of phytochemical constituents. Free radical scavenging potential was investigated using standard methods, namely, DPPH radical scavenging assay and ferric reducing antioxidant power assay along with the protection against oxidative DNA damage. The results stated that acetonic seed extracts (AAcSE and FAcSE) of both the spices possessed comparatively high amount of total phenolics whereas methanolic seed extracts (AMSE and FMSE) were found to have highest amount of total flavonoids. At 1 mg/mL concentration, highest DPPH radical scavenging activity was shown by FMSE (96.2%), AAcSE was recorded with highest FRAP value (2270.27 ± 0.005 μmol/L), and all the seed extracts have been shown to mitigate the damage induced by Fenton reaction on calf thymus DNA. Therefore, the study suggests that T. ammi and F. vulgare seed extracts could contribute as a highly significant bioresource of antioxidants to be used in our day-to-day life and in food and pharmaceutical industry.
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The binding of tumour necrosis factor α (TNFα) to cell surface receptors engages multiple signal transduction pathways, including three groups of mitogen-activated protein (MAP) kinases: extracellular-signal-regulated kinases (ERKs); the cJun NH2-terminal kinases (JNKs); and the p38 MAP kinases. These MAP kinase signalling pathways induce a secondary response by increasing the expression of several inflammatory cytokines (including TNFα) that contribute to the biological activity of TNFα. MAP kinases therefore function both upstream and down-stream of signalling by TNFα receptors. Here we review mechanisms that mediate these actions of MAP kinases during the response to TNFα.
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Aims: To further explore the anti-inflammatory properties of d-Limonene. Main methods: A rat model was used to compare evolution of TNBS (2,5,6-trinitrobenzene sulfonic acid)-induced colitis after oral feeding with d-Limonene compared to ibuprofen. Peripheral levels of TNF-α (Tumor Necrosis Factor alpha) were assessed in all animals. Cell cultures of fibroblasts and enterocytes were used to test the effect of d-Limonene respectively on TNFα-induced NF-κB (nuclear factor-kappa B) translocation and epithelial resistance. Finally, plasmatic inflammatory markers were examined in an observational study of diet supplementation with d-Limonene-containing orange peel extract (OPE) in humans. Key findings: Administered per os at a dose of 10mg/kg p.o., d-Limonene induced a significant reduction of intestinal inflammatory scores, comparable to that induced by ibuprofen. Moreover, d-Limonene-fed rats had significantly lowered serum concentrations of TNF-α compared to untreated TNBS-colitis rats. The anti-inflammatory effect of d-Limonene also involved inhibition of TNFα-induced NF-κB translocation in fibroblast cultures. The application of d-Limonene on colonic HT-29/B6 cell monolayers increased epithelial resistance. Finally, inflammatory markers, especially peripheral IL-6, markedly decreased upon OPE supplementation of elderly healthy subjects submitted or not to 56 days of dietary supplementation with OPE. Significance: In conclusion, d-Limonene indeed demonstrates significant anti-inflammatory effects both in vivo and in vitro. Protective effects on the epithelial barrier and decreased cytokines are involved, suggesting a beneficial role of d-Limonene as diet supplement in reducing inflammation.
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Background: Considering the key role of TF in coagulation of sepsis or acute lung injury (ALI), we investigated whether berberine (BBR) could inhibit TF expression and procoagulant activity and explored its possible mechanism. Methods: The effects of berberine on the expression, procoagulant activity of TF and related signal pathways induced by lipopolysaccharide (LPS) were observed in THP-1 cells. Results: Our results showed that berberine could inhibit LPS-induced TF activity and expression, and down-regulate NF-κB, Akt and MAPK/JNK/p38/ERK pathways. Conclusion: Berberine inhibits TF expression and related pathway, which provides some new insights on its mechanism for sepsis treatment.
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Aims Anethole, the major component of the essential oil of star anise, has been reported to have antioxidant, antibacterial, antifungal, anti-inflammatory, and anesthetic properties. In this study, we investigated the anti-inflammatory effects of anethole in a mouse model of acute lung injury induced by lipopolysaccharide (LPS). Main methods BALB/C mice were intraperitoneally administered anethole (62.5, 125, 250, or 500 mg/kg) 1 h before intratracheal treatment with LPS (1.5 mg/kg) and sacrificed after 4 h. The anti-inflammatory effects of anethole were assessed by measuring total protein and cell levels and inflammatory mediator production and by histological evaluation and Western blot analysis. Key findings LPS significantly increased total protein levels; numbers of total cells, including macrophages and neutrophils; and the production of inflammatory mediators such as matrix metalloproteinase 9 (MMP-9), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and nitric oxide (NO) in bronchoalveolar lavage fluid. Anethole (250 mg/kg) decreased total protein concentrations; numbers of inflammatory cells, including neutrophils and macrophages; and the inflammatory mediators MMP-9, TNF-α and NO. In addition, pretreatment with anethole decreased LPS-induced histopathological changes. The anti-inflammatory mechanism of anethole in LPS-induced acute lung injury was assessed by investigating the effects of anethole on NF-κB activation. Anethole suppressed the activation of NF-κB by blocking IκB-α degradation. Significance These results, showing that anethole prevents LPS-induced acute lung inflammation in mice, suggest that anethole may be therapeutically effective in inflammatory conditions in humans.
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D-limonene is a naturally occurring monoterpene and has been found to posses numerous therapeutic properties. In this study, we used D-limonene as a protective agent against the nephrotoxic effects of anticancer drug doxorubicin (Dox). Rats were given D-limonene at doses of 5% and 10% mixed with diet for 20 consecutive days. Dox was give at the dose of 20 mg/kg body weight intraperitoneally. The protective effects of D-limonene on Dox-induced oxidative stress and inflammation were investigated by assaying oxidative stress biomarkers, lipid peroxidation, serum toxicity markers, proinflammatory cytokines, and expression of nuclear factor kappa B (NFkB), cyclo-oxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) and Nitrite levels. Administration of Dox (20 mg/kg body weight) in rats enhanced renal lipid peroxidation; depleted glutathione content and anti-oxidant enzymes; elevated levels of kidney toxicity markers viz. kidney injury molecule-1 (KIM-1), blood urea nitrogen (BUN), and creatinine; enhanced expression of NFkB, COX-2, and iNOS and nitric oxide. Treatment with D-limonene prevented oxidative stress by restoring the levels of antioxidant enzymes, further both doses of 5% and 10% showed significant decrease in inflam-matory response. Both the doses of D-limonene significantly decreased the levels of kidney toxicity markers KIM-1, BUN, and creatinine. D-limonene also effectively decreased the Dox induced overexpression of NF-kB, COX-2, and iNOS and nitric oxide. Data from the present study indicate the protective role of D-limonene against Dox-induced renal damage.
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In this review we summarize recent major advances in our understanding on the molecular mechanisms, mediators and biomarkers of ALI and alveolo-capillary barrier dysfunction, highlighting the role of immune cells, inflammatory and non-inflammatory signaling events, mechanical noxae, and the affected cellular and molecular entities and functions. Furthermore, we address novel aspects of resolution and repair of ALI, as well as putative candidates for treatment of ALI, including pharmacological and cellular therapeutic means.
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The aim of this study was to investigate the hepatoprotective effect of anethole (trans-anethole, 1), a major component of Foeniculum vulgare, and its molecular mechanism during ischemia/reperfusion (I/R). Mice were subjected to 60 min of partial hepatic ischemia followed by 1 and 6 h of reperfusion. Compound 1 (50, 100, and 200 mg/kg) or the vehicle alone (10% Tween 80-saline) was orally administered 1 h prior to ischemia. After 1 and 6 h of reperfusion, serum alanine aminotransferase, tumor necrosis factor-α, and interleukin 6 levels significantly increased, but these increases were attenuated by 1. Nuclear translocation of interferon regulatory factor (IRF)-1, release of high mobility group box (HMGB) 1 into the extracellular milieu, and the interactions between IRF-1 and histone acetyltransferase p300 increased after I/R. These increases were attenuated by 1. Compound 1 suppressed increases in toll-like receptor (TLR) 4, myeloid differentiation primary response gene 88 protein expression, phosphorylation of p38, extracellular signal-regulated kinase, c-Jun N-terminal kinase, nuclear translocation of nuclear factor kappa B, and phosphorylated c-Jun. The present findings suggest that 1 protects against hepatic I/R injury by suppression of IRF-1-mediated HMGB1 release and subsequent TLR activation.
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Anethole has been reported to have antioxidant, antibacterial, antifungal, antiinflammatory, and anesthetic properties. In the present study, we evaluated the effects of anethole in two pain models of inflammatory origin: acute inflammation induced by carrageenan and persistent inflammation induced by Complete Freund's adjuvant. We evaluated the effects of anethole (125, 250, and 500 mg/kg) on the development of paw oedema and mechanical hypernociception. The liver was collected for histological analysis. Paw skin was collected to determine the levels of the cytokines tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-17 (IL-17), and myeloperoxidase activity. Blood was collected to assess alanine transaminase (ALT) and aspartate transaminase (AST). The chemical composition of star anise oil was determined by gas chromatography/mass spectrometry (GC/MS), showing a presence of anethole of 98.1 %. Oral pretreatment with anethole in mice inhibited paw oedema, mechanical pernociception, myelopewroxidase activity, TNF-α, IL-1β and IL-17 levels in acute and persistent inflammation models. Additionally, anethole treatment did not alter prostaglandin E(2)-induced mechanical hypernociception. Possible side effects were also examined. Seven-day anethole treatment did not alter plasma AST and ALT levels, and the histological profile of liver tissue was normal. The present study provides evidence of the antiinflammatory and analgesic activities of anethole in acute and persistent inflammation models.