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J BIOCHEM MOLECULAR TOXICOLOGY
Volume 00, Number 0, 2015
Fucoxanthin Inhibits the Inflammation Response in Paw
Edema Model through Suppressing MAPKs, Akt,
and NFκB
Jun-Hui Choi,1Na-Hyun Kim,1Sung-Jun Kim,2Hyo-Jeong Lee,1and Seung Kim1
1Department of Bio-Health Science, Gwangju University, Gwangju 503-703, Republic of Korea; E-mail: seungk@gwangju.ac.kr
2Department of Biomedical Science, Chosun University, Gwangju 501-759, Republic of Korea
Received 15 June 2015; revised 10 August 2015; accepted 27 August 2015
ABSTRACT: Undaria pinnatifida is a well-known
traditional Korean food with a variety of biologi-
cal activities. Carrageenan (carr) is commonly used
to induce paw edema in animal models. This study
was designed to elucidate the processes underlying
the anti-inflammatory effect of fucoxanthin isolated
from the sporophyll of U. pinnatifida in carr-induced
paw edema in ICR mice. Fucoxanthin significantly de-
creased carr-induced increased nitric oxide levels in
the plasma of mice with carr-induced paw edema.
Fucoxanthin protected catalase (CAT) and superoxide
dismutase (SOD) activity against disruption in mice
with carr-induced paw edema. In addition, fucoxan-
thin repressed carr-induced activation of inducible
nitric oxide synthase, cyclooxygenase-2, and nuclear
factor kappa B, as well as carr-induced phosphoryla-
tion of mitogen-activated protein kinase, extracellular
signal-regulated kinase, c-Jun N-terminal kinase, p38,
and protein kinase B/Akt. These results suggest that
fucoxanthin may have therapeutic potential as a treat-
ment for patients with inflammatory diseases. C2015
Wiley Periodicals, Inc. J Biochem Mol Toxicol 00:1–9,
2015; View this article online at wileyonlinelibrary.com.
DOI 10.1002/jbt.21769
KEYWORDS: Undaria pinnatifida (Harvey) Suringar;
Anti-inflammation; Fucoxanthin; Carrageenan;
Mitogen-activated protein kinases
Correspondence to: Seung Kim.
Contract Grant Sponsor: Gwangju University, Republic of Korea
(in 2014).
Contract Grant Number: 2014G04.
Contract Grant Sponsor: Leaders in INdustry-university Co-
operation (LINC) Project, Ministry of Education, Republic of Korea
(grant number: 15A15621561)
C2015 Wiley Periodicals, Inc.
INTRODUCTION
Acute inflammation is a response in injured
tissue that is characterized by increased blood flow,
increased temperature, redness, swelling, and pain
[1, 2]. Inflammatory responses are mediated by a
variety of cytokines. During inflammatory responses,
proinflammatory mediators are released, including
interleukin-1 (IL-1), IL-6, IL-12, tumor necrosis factor,
interferon gamma, cyclooxygenase-2 (COX-2), and
inducible nitric oxide synthase (iNOS) [3]. Nitric oxide
(NO) is a proinflammatory free radical produced by
NO synthase from L-arginine, oxygen, and nicoti-
namide adenine dinucleotide (reduced). After activa-
tion by cytokines, iNOS and NOS2 are produced and
subsequently generate NO to mediate host defense pro-
cesses. However, pathogenic NO overproduction and
iNOS overexpression in injured tissue are pathogenic
[4]. iNOS and COX-2 are primary inflammatory
response genes that are responsible for elevated levels
of NO and prostaglandins in injured tissue during
inflammatory responses [5]. Upregulated expression
of iNOS and COX-2 has been reported in rodents with
carrageenan (carr) induced paw inflammation [6]. Sys-
temic administration of inhibitors of iNOS and COX-2
has been found to inhibit carr-induced paw edema
[7, 8]. Mitogen-activated protein kinases (MAPKs)
act as important inflammatory mediators. The three
major MAPK cascades are mediated by extracellular
signal-regulated kinase (ERK), c-Jun N-terminal kinase
(JNK), and p38. In addition, the Akt pathway regulates
inflammatory responses, cellular activation, and apop-
tosis [9]. Because inactivation of signaling by MAPKs
and Akt inhibits production of inflammatory mediators
(NO and prostaglandin E2), these kinases are potential
targets for anti-inflammatory therapeutics [10–13].
The protective activities of natural products
against pathological changes associated with acute
1
2CHOI ET AL. Volume 00, Number 0, 2015
inflammation are usually assessed using animal
edema models. Anti-inflammatory studies using
the carr-induced paw edema model have been
reported by Chang et al. [14]. Carrs are sulfated
polysaccharides that are extracted from red seaweeds.
Carr induces acute inflammation, beginning with
migration of phagocytes, followed by a burst of free
radical production and the release of inflammatory
mediators [15].
Marine algae have been used as a food source
by humans for millennia and are an abundant source
of nutrients, dietary fiber, and bioactive compounds.
Edible seaweed, Undaria pinnatifida, is a popular and
important economic brown alga cultivated in many
countries, but particularly in China, Japan, and Korea.
Around 50% of wet seaweed produced in Korea is U.
pinnatifida, which, together with Gracilaria,Laminaria,
and Porphyra spp., constitutes 93% of the total algal
mass cultivated for nutritional purposes [16].
Fucoxanthin, a major marine carotenoid, and fu-
coidan, a group of sulfated polysaccharides containing
fucose, are major components of U. pinnatifida.Sulfated
fucoxanthin and fucoidan from U. pinnatifida possess
several biological activities, including antithrombotic
[17], antiangiogenic [18], antiviral [19], antitumor [20],
and anti-inflammatory effects [21]. This study reports
the isolation of fucoxanthin from U. pinnatifida and the
results of an investigation of the involvement of MAPK
signaling, Akt signaling, nuclear factor kappa B (NFκB)
signaling, iNOS expression, and COX-2 expression in
the anti-inflammatory effects of fucoxanthin.
MATERIALS AND METHODS
Materials
Silica gel 60 (70-230 mesh) and precoated silica gel
60 F254 TLC plates (0.25 mm) were purchased from
Merck Sharp & Dohme (Darmstadt, Germany). Highly
porous synthetic resin Diaion HP-20 was purchased
from Mitsubishi (Tokyo, Japan). p-ERK, ERK, p-JNK,
JNK, p-p38, and p38 antibodies were obtained from
Santa Cruz Biotechnology (Santa Cruz, CA). p-Akt
and Akt antibodies were obtained from Cell Signaling
Technology (Beverley, CA). Anti-actin antibodies were
purchased from Biomeda (Foster City, CA). WEST-
ZOL plus was obtained from INTRON Biotech (Seong-
nam, Korea). Bicinchoninic acid (BCA) (PubChem CID:
71068) protein assay kits were obtained from Pierce
(Rockford, IL). Indomethacin (PubChem CID: 3715)
was purchased from Sigma-Aldrich (St. Louis, MO).
Other reagents used were of analytical grade and ob-
tained from commercial sources.
Plant Material
Fresh sporophylls of U. pinnatifida were collected
from Wando-Island, Republic of Korea, in the month of
December and authenticated by Professor Myung-Kon
Kim, Department of Bio-Food Technology, Chonbuk
National University, South Korea. A voucher specimen
was deposited at Chonbuk National University. The
sporophylls were dried at room temperature and
ground into a fine powder.
Isolation of Primary Compounds from
Acetone–Methanol Extracts of U. pinnatifida
Sporophylls
Samples were kept at –20°C and protected from
light prior to use. The powdered sporophylls of U. pin-
natifida (200 g) were extracted with acetone–methanol
(70:30, v/v) (1 L) at room temperature. After filtration,
the solvent was evaporated to give an acetone–
methanol extract (8.8 g). The acetone–methanol extract
was dissolved in a solution of n-hexane–acetone
(75:25, v/v) and subjected to porous synthetic resin
(Diaion HP-20) column chromatography (3.5 cm ×
40 cm) on silica gels (150 g) at various n-hexane–
acetone ratios (all v/v, 500 mL): 75:25, 70:30, 60:40, and
50:50. One hundred 20-mL fractions were collected.
Fractions 30–32 (n-hexane–acetone eluate, 70:30, v/v)
were subjected to rechromatography (3.5 ×40 cm) on
silica gel (150 g) and eluted with n-hexane–acetone
(70:30, v/v). The active fraction of the acetone–
methanol extract of U. pinnatifida was freeze-dried
and stored at –20°C. Silica gel 60 (70-230 mesh) and
precoated silica gel 60 F254 TLC plates (0.25 mm)
were purchased from Merck Sharp & Dohme. Highly
porous synthetic resin Diaion HP-20 was purchased
from Mitsubishi. Other reagents were of analytical
grade and obtained from commercial sources.
Instrumental Analysis
Ultraviolet (UV) absorption spectra were recorded
using a Beckman DU-70 spectrophotometer (Fullerton,
CA, USA). Positive and negative ion LC–API–MS spec-
trometry was performed using an Agilent 1100 HPLC
system (Agilent Technologies, Palo Alto, CA). 1HNMR
and 13C NMR spectra were recorded using a JEOL FT-
NMR spectrometer (Jeol, Tokyo) at 600 MHz and 150
MHz in acetone-d6.
Paw Edema Model
The carr-induced hind paw edema model was
used to assess the anti-inflammatory activity of U. pin-
J Biochem Molecular Toxicology DOI 10.1002/jbt
Volume 00, Number 0, 2015 ANTI-INFLAMMATORY POTENTIAL OF FUCOXANTHIN 3
natifida extract [22]. After a 1-week adaptation period,
male ICR mice (20–25 g) were randomly assigned to
six groups (n=10): carr only, carr +fucoxanthin (4 and
8 mg/kg), carr +indomethacin (4 and 8 mg/kg), and
saline-treated groups. The animals were intraperi-
toneally administered fucoxanthin (4 and 8 mg/kg),
indomethacin (4 and 8 mg/kg), and saline 30 min
prior to injection of 50 μL of 1% carr into the right
hind paw. Paw thickness was measured 0, 1, 2, 4, and
6 h after carr injection using calipers. After 6 h, the
animals were sacrificed and paw tissue was excised,
rinsed in ice-cold normal saline, immediately placed in
1 volume of cold normal saline, and homogenized at
4°C. The resulting tissue homogenate was centrifuged
at 12,000 ×gfor 5 min. The supernatant of the hind
paw tissue homogenate was stored at –20°Candsub-
sequently used for the assessments of catalase (CAT)
and superoxide dismutase (SOD) activity, as well as for
Western blot analysis. In addition, a blood sample was
collected from the heart of each mouse and centrifuged
at 1500 ×gfor5minat4°C. The resulting plasma was
aliquoted, stored at –20°C, and subsequently subjected
to measurement of NO content.
Determination of CAT and SOD Activity in
Paw Tissue
The assessment of CAT activity was based on the
rate of H2O2reduction [23]. CAT activity was expressed
as units per gram of protein. In brief, the reduction of
10 mM H2O2in 20 mM of phosphate buffer (pH 7.0)
was monitored by measuring the absorbance at 240 nm.
CAT activity was calculated using a molar absorption
coefficient and expressed as nanomoles of dissipating
hydrogen peroxide per milligram protein per minute.
SOD activity was assessed by measuring the
progress (change in optical density per minute at
480 nm) of a reaction initiated by epinephrine bitartrate
(3 mM) as described by Misra and Fridovich [24]. SOD
activity was expressed as units per milligram of pro-
tein. The change in optical density per minute at the
point when the transformation of epinephrine into
adrenochrome by SOD was inhibited by 50% was taken
as the enzyme unit. A calibration curve was prepared
using 2–100 units of SOD.
Effect of Fucoxanthin on Plasma NO
Content
NO production was assessed indirectly by mea-
suring nitrite levels in plasma using a colorimetric
method based on the Griess reaction [14]. Serum
samples were diluted four times with distilled water
and deproteinized by adding 1/20 volume of 1 M zinc
sulfate to a final concentration of 50 mM. After cen-
trifugation at 10,000 ×gfor 5 min at room temperature,
100 μL of the supernatant of each sample was applied
to a microtiter plate well, followed by the addition
of 100 μL of Griess reagent (1% sulfanilamide and
0.1% N-1-naphthylethylenediamine dihydrochloride
in 2.5% polyphosphoric acid). After 10 min of color
development at room temperature, the absorbance was
measured at 540 nm with a UV–visible spectropho-
tometer. After a standard curve was generated with
sodium nitrite, the concentration of nitrite was deter-
mined by measuring the absorbance of each sample at
540 nm.
Western Blotting
Tissue samples were washed with ice-cold
phosphate-buffered saline (PBS) and lysed in RIPA
buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1
mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium
deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM
β-glycerophosphate, 1 mM Na3VO4,and1μg/mL
leupeptin). After homogenization, the homogenates
were incubated for 30 min and centrifuged at 17,700
×gfor 15 min. Protein concentrations were de-
termined by the BCA method using bovine serum
albumin as the standard. Equal amounts of tissue
extracts were separated by electrophoresis using a
10.5% SDS-polyacrylamide gel and transferred to
polyvinylidene difluoride membranes. After block-
ing at room temperature in 5% nonfat dry milk
with Tris-buffered saline/Tween-20 (TBST) buffer
(10 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween
20, pH 7.5) for 2 h, the membranes were incubated with
primary antibodies against iNOS (1:2000 dilution),
COX-2 (1:1000 dilution), ERK (1:1000 dilution), p-ERK
(1:1000 dilution), JNK (1:1000 dilution), p-JNK (1:1000
dilution), p38 (1:1000 dilution), p-p38 (1:1000 dilution),
NFκB (1:1000 dilution), nuclear factor of kappa light
polypeptide gene enhancer in B-cells inhibitor, alpha
(IκBα) (1:1000 dilution), Akt (1:2000 dilution), p-Akt
(1:2000 dilution), or actin (1:2500 dilution) overnight at
4°C. The membranes were washed three times in TBST
buffer and incubated with horseradish peroxidase
conjugated secondary antibodies for 2 h at room tem-
perature. To reveal the reaction bands, the membranes
were reacted with the WESTZOL (plus) Western Blot
Detection System (Intron Biotechnology, Sungnam, Ko-
rea) and exposed to X-ray film (Fujifilm, Tokyo, Japan).
Statistical Analysis
Each experiment was performed at least in trip-
licate. Data are expressed as mean ±SD. Statistical
J Biochem Molecular Toxicology DOI 10.1002/jbt
4CHOI ET AL. Volume 00, Number 0, 2015
FIGURE 1. Isolation of fucoxanthin. The main peak (A) and chemical structure (B) of fucoxanthin.
significance was assessed with one-way ANOVA fol-
lowed by the Bonferroni post hoc test for multiple
group comparisons. Differences with p-values less than
0.05 were considered statistically significant.
RESULTS
Isolation and Identification of Fucoxanthin
The active fraction of the acetone–methanol
extract of U. pinnatifida waselutedwithn-hexane–
acetone (70:30, v/v) to give the main compound
(105 mg) (Figure 1A) as a powder with UV (acetone)
λmax 446.5 nm (Figure 2A). The chemical structure of
the main compound was determined according to its
LC–API–MS, 1H NMR, and 13C NMR spectra. LC–
API–MS (positive mode): m/z 659 [M]+, 660 [M +H]+,
and 661 [M +2H]+. From these results, the molecular
weight of the main compound was estimated at 659
(Figure 2B). 1H NMR (600 MHz, acetone-d6): δ1.52 (1H,
m, CH2), 3.70 (1H, m, CH), 1.52 (1H, m, CH2), 3.80 (1H,
d, J=18.54, CH2), 7.38 (1H, d, J=10.98, CH), 6.65 (1H,
m, CH), 6.86 (1H, m, CH), 6.5 (1H, d. J=11.76, CH),
6.67 (1H, m, CH), 0.95 (3H, s, CH3), 0.84 (3H, s, CH3),
1.21 (3H, s, CH3), 1.99 (3H, s, CH3), 2.03 (3H, s, CH3),
1.99 (1H, m, CH2), 5.25 (1H, m, CH), 1.52 (1H, m, CH2),
6.10 (3H, s, CH), 6.20 (1H, d, J=11.20, CH), 6.66 (1H, m,
CH), 6.38 (1H, d, J=14.86, CH), 6.35 (1H, d, J=11.56,
CH), 6.76 (1H, t, J=12.74, 12.74, CH), 1.10 (3H, s, CH3),
1.33 (1H, s, CH3), 1.41 (3H, s, CH3), 1.86 (1H, s, CH3),
2.00 (1H, s, CH3), 2.09 (1H, s, CH3) (Table 1). The 13C
NMR chemical shifts (150 MHz, acetone-d6)areshown
in Table 1. From these results, the active compound was
identified as all-trans-fucoxanthin [25–27] (Figure 1B).
Fucoxanthin Reduces Edema Size in the
Carr-Induced Paw Edema Model
To determine whether fucoxanthin inhibits the
inflammatory activity of carr in vivo, mice were
pretreated with fucoxanthin or indomethacin and
injected with carr in the paw. Carr administration
induced paw edema (Figure 3A). However, 8 mg/kg
fucoxanthin significantly suppressed the development
of paw edema induced by carr 4 and 6 h after injection.
The inhibitory effect of fucoxanthin was similar to that
of indomethacin.
Fucoxanthin Protects against Carr-Induced
Decreases in CAT and SOD Activity in Paw
Tissue
Six hours after carr was administered, the activities
of antioxidant enzymes CAT and SOD were assessed in
paw tissue. CAT and SOD activity levels in paw tissue
were decreased by carr injection. However, CAT and
SOD activity levels in carr-treated mice were increased
by 84.4% and 85.8%, respectively, by the treatment with
8 mg/kg fucoxanthin (Table 2). In addition, adminis-
tration of 8 mg/kg indomethacin increased CAT and
SOD activity levels by 75.5% and 97.9%, respectively.
Fucoxanthin Reduces Plasma NO Content
in Mice with Carr-Induced Paw Edema
This study evaluated the inhibitory activity of
fucoxanthin on NO production. Blood samples were
collected from mice following carr injection and cen-
trifuged at 1500 ×gfor5minat4°C, after which plasma
nitrite concentration was determined using Griess
J Biochem Molecular Toxicology DOI 10.1002/jbt
Volume 00, Number 0, 2015 ANTI-INFLAMMATORY POTENTIAL OF FUCOXANTHIN 5
FIGURE 2. The molecular characteristics of fucoxanthin. Ultraviolet (UV) absorption spectra were recorded on a Beckman DU-70 spectropho-
tometer. Positive and negative-ion LC–API–MS spectrometry was performed using an Agilent 1100 HPLC system.
reagent by measuring the absorbance of each sample
at 540 nm. The treatment with 4 and 8 mg/kg fucoxan-
thin decreased plasma nitrite content in mice with carr-
induced edema (Figure 3B). NO production in mice
with carr-induced edema was inhibited by 32% follow-
ing treatment with 8 mg/kg fucoxanthin and by 28%
following treatment with 8 mg/kg indomethacin. The
suppressive effects of fucoxanthin and indomethacin
on plasma nitrite content were dose dependent.
Fucoxanthin Inhibits Expression of iNOS
and COX-2 in Paw Tissue from Mice with
Carr-Induced Paw Edema
We examined whether the inhibitory effect of
fucoxanthin on carr-induced paw edema was asso-
ciated with decreased iNOS and COX-2 expression.
Protein levels of iNOS and COX-2 were measured by
Western blotting. As shown in Figure 4A, following
carr treatment for 6 h, iNOS and COX-2 protein
levels were increased significantly (1.52- and 5.21-
fold, respectively). However, iNOS expression was
remarkably inhibited in carr-treated mice that were
pretreated with fucoxanthin or indomethacin (by 50%
following treatment with 8 mg/kg fucoxanthin and by
95% following treatment with 8 mg/kg indomethacin).
In addition, COX-2 expression was inhibited in mice
pretreated with fucoxanthin or indomethacin (by
100% following treatment with 8 mg/kg fucoxanthin
and by 55% following treatment with 8 mg/kg
indomethacin.
Fucoxanthin Inhibits Carr-Induced JNK,
p38, and ERK Phosphorylation
To elucidate the anti-inflammatory mechanisms of
fucoxanthin, the effects of fucoxanthin on MAPKs ERK,
JNK, and p38, which are upstream signaling molecules
involved in inflammatory reactions in response to carr-
induced paw edema, were evaluated by Western blot
analysis. As shown in Figure 4B, carr strongly activated
ERK1/2, JNK, and p38 (shown in their phosphorylated
J Biochem Molecular Toxicology DOI 10.1002/jbt
6CHOI ET AL. Volume 00, Number 0, 2015
TABLE 1. 13CNMRand1H NMR Analysis
Carbon No. 13C NMR (ppm) DEPT 1H NMR (ppm) (J =Hz)
135.92C
248.65CH
21.52 (1H, m)
3 62.26 CH 3.70 (1H, m)
442.45CH
2
566.05C
666.94C
740.98CH
23.80 (1H, d, J=18.54)
8 198.47 C
9 134.35 C
10 139.84 CH 7.38 (1H, d, J=10.98)
11 124.62 CH 6.65 (1H, m)
12 145.23 CH 6.86 (1H, m)
13 136.14 C
14 136.72 CH 6.5 (1H, d. J=11.76)
15 130.40 CH 6.67 (1H, m)
16 28.18 CH30.95 (3H, s)
17 25.06 CH30.84 (3H, s)
18 21.23 CH31.21 (3H, s)
19 12.12 CH31.99 (3H, s)
20 13.07 CH32.03 (3H, s)
135.40 C
246.33 CH21.99 (1H, m)
368.08 CH 5.25 (1H, m)
445.98 CH21.52 (1H, m)
571.37 C
6117.70 C
7202.19 C
8102.62 CH 6.10 (3H, s)
9133.13 C
10128.67 CH 6.20 (1H, d, J=11.20)
11126.53 CH 6.66 (1H, m)
12137.17 CH 6.38 (1H, d, J=14.86)
13138.16 C
14132.74 CH 6.35 (1H, d, J=11.56)
15133.30 CH 6.76 (1H, t, J=12.74, 12.74)
1631.07 CH31.10 (3H, s)
1729.32 CH31.33 (1H, s)
1832.46 CH31.41 (3H, s)
1914.32 CH31.86 (1H, s)
2013.30 CH32.00 (1H, s)
21170.31 C
2221.64 CH32.09 (1H, s)
forms: p-ERK1/2, p-JNK, and p-p38). Fucoxanthin
significantly inhibited carr-induced phosphorylation
of ERK1/2, JNK, and p38. The treatment with 4 and
8 mg/kg fucoxanthin inhibited p-ERK1/2 expression
by 94.2% and 100%, respectively. The treatment with
4 and 8 mg/kg indomethacin inhibited p-ERK1/2
expression by 56.6% and 100%, respectively. The
treatment with 4 and 8 mg/kg fucoxanthin inhibited
p-JNK expression by 98.2% and 100%, respectively. The
treatment with 4 and 8 mg/kg indomethacin inhibited
p-JNK expression by 57.1% and 84.6%, respectively.
The treatment with 4 and 8 mg/kg fucoxanthin inhib-
ited p-p38 expression by 29.8% and 94.1%, respectively.
The treatment with 4 and 8 mg/kg indomethacin inhib-
FIGURE 3. Effect of fucoxanthin on paw edema induced by car-
rageenan in mice. Mice were pretreated intraperitoneally with fu-
coxanthin or indomethacin for 30 min, followed by injection of
0.5 mg/paw carrageenan for 6 h. Paw thickness was measured at
0, 1, 2, 4, and 6 h after carrageenan injection using calipers (A). The
nitrite concentration in the plasma of mice with carrageenan-induced
paw edema was determined by the Griess reaction. Results are ex-
pressed as mean ±SD of three independent experiments. #p<0.05,
in comparison with saline-treated control mice; *p<0.05, in compar-
ison with the carrageenan-treated group (without fucoxanthin and
indomethacin).
TABLE 2. Alterations of Antioxidant-Associated Parameter:
CAT and SOD Activity in Carr-Induced Paw Edema
CAT SOD Activity
Experimental Group (U/mg Protein) (U/mg Protein)
Control 4.65 ±0.50 46.35 ±2.41
Carrageenan (carr) 2.28 ±0.21 13.52 ±0.95
Carr-Indo 4 mg/kg 3.46 ±0.28 40.51 ±2.13
Carr-Indo 8 mg/kg 4.07 ±0.33 45.63 ±1.88
Carr-Fuco 4 mg/kg 3.34 ±0.31 23.18 ±2.02
Carr-Fuco 8 mg/kg 4.28 ±0.45 41.65 ±1.27
Each value represents the mean ±SD for three determinations.
ited p-p38 expression by 94.6% and 100%, respectively.
These results indicate that fucoxanthin inhibited
inflammation in mice with carr-induced paw edema
by modulating activation of MAPK pathways.
J Biochem Molecular Toxicology DOI 10.1002/jbt
Volume 00, Number 0, 2015 ANTI-INFLAMMATORY POTENTIAL OF FUCOXANTHIN 7
FIGURE 4. Inhibitory effects of fucoxanthin on carrageenan-
induced expression of iNOS, COX-2, and MAPKs in paw tissue.
Protein levels of iNOS, COX-2, and MAPKs were measured by
immunoblotting. Actin was used as an internal loading control. 1,
saline-treated group; 2, carrageenan-treated group (0.5 mg/paw);
3, carrageenan +indomethacin (4 mg/kg) treated group; 4, car-
rageenan +indomethacin (8 mg/kg) treated group; 5, carrageenan
+fucoxanthin (4 mg/kg) treated group; 6, carrageenan +fucox-
anthin (8 mg/kg) treated group. The intensity of each band was
estimated by densitometric analysis. Results are expressed as mean
±SD of three independent experiments. #p<0.05, in comparison
with saline-treated control mice. *p<0.01, in comparison with the
carrageenan-treated group (without fucoxanthin and indomethacin).
Fucoxanthin Attenuates Carr-Induced
Activation of Akt and NFκB Signaling
To further clarify the anti-inflammatory mecha-
nisms of fucoxanthin, the effects of fucoxanthin on
Akt and NFκB signaling during inflammatory reac-
tions in response to carr-induced paw edema was
tested by Western blot analysis. As shown in Figure 5,
carr activated Akt and blocked NFκBandIκBαsig-
naling (5.08-fold (p-Akt/Akt), 3.83-fold (NFκB), and
4.76-fold (IκBα), respectively). Fucoxanthin inhibited
activation of Akt by carr (Figure 5A). The treatment
with 8 mg/kg fucoxanthin decreased p-Akt expression
by 52.3% in comparison with that of the mice treated
with carr only, which had a significantly stronger (3.98-
fold) inhibitory effect than that of indomethacin. NFκB
activation involves translocation of the p65 subunit of
FIGURE 5. Inhibitory effects of fucoxanthin on carrageenan-
induced Akt, cytosolic IκBα,andNFκB activation in paw tissue.
Paw tissue extracts were prepared and analyzed by immunoblot-
ting using Akt, IκBα,andNFκB antibodies. 1, saline-treated group;
2, carrageenan-treated group (0.5 mg/paw); 3, carrageenan +in-
domethacin (4 mg/kg) treated group; 4, carrageenan +indomethacin
(8 mg/kg) treated group; 5, carrageenan +fucoxanthin (4 mg/kg)
treated group; 6, carrageenan +fucoxanthin (8 mg/kg) treated group.
The intensity of each band was estimated by densitometric analysis.
Results are expressed as mean ±SD of three independent experi-
ments.
NFκB. Therefore, we investigated whether fucoxanthin
inhibits translocation of NFκB p65 protein during the
inflammatory response to carr. Western blot analysis
showed that NFκB p65 was located in the cytosol in
samples from the untreated group. In samples from
mice treated with carr only, cytosolic NFκB p65 and
IκBαprotein levels were reduced by 73.9% and 79%,
respectively, but these changes were not observed in
carr-treated mice that were also treated with fucoxan-
thin (Figure 5B).
DISCUSSION
In South Korea, almost all mothers are given U.
pinnatifida soup during the first month after birth, be-
cause it is believed to aid postnatal convalescence and
cleanse the blood [28]. Traditional medicinal prepara-
tions containing U. pinnatifida have been used to treat
lumps, swelling, hypertension, hemorrhoids, urinary
problems, and cancer in Donguibogam (Korean), Chi-
nese, Kampo (Japanese), and ayurvedic medicinal prac-
tices [29–32]. Fucoxanthin, a major marine carotenoid,
and fucoidan, a group of sulfated polysaccharides con-
taining fucose, are major components of U. pinnat-
ifida. Fucoxanthin possesses stroke-preventative [33],
J Biochem Molecular Toxicology DOI 10.1002/jbt
8CHOI ET AL. Volume 00, Number 0, 2015
antitumor [34], antiobesity [35], and anti-inflammatory
properties [29].
Carr-induced paw edema is an established model
of edema that is used to screen anti-inflammatory
drugs. Intraplantar injection of carr induces inflam-
matory responses, including paw edema and neu-
trophil infiltration [36]. The carr-induced inflammatory
response involves neutrophil infiltration and produc-
tion of neutrophil-derived free radicals [37]. In addi-
tion, many studies have demonstrated that the inflam-
matory response induced by carr is associated with free
radical generation and NO [14]. We found that fucox-
anthin inhibited iNOS and COX-2 protein expression
in mice with carr-induced paw edema, suggesting that
fucoxanthin inhibits the primary initiating steps in in-
flammatory signaling pathways. Moreover, we found
that plasma NO content was increased slightly 6 h after
carr injection. However, the treatment with 8 mg/kg
fucoxanthin or 8 mg/kg indomethacin decreased the
plasma NO content of carr-treated mice. The release
of inflammatory mediators is known to be involved in
activation of downstream signaling molecules, includ-
ing MAPKs and NFκB, in carr-induced paw edema
[5]. The ERK, JNK, and p38 pathways regulate a vari-
ety of biological activities, including inflammation and
cell death. To investigate the involvement of MAPK
signaling pathways in the inflammatory response to
carr-induced paw edema and the protective effect of
fucoxanthin, we evaluated the activities of ERK, JNK,
and p38. Consistent with previous findings [5], we
observed that carr induced phosphorylation of ERK,
JNK, and p38 in the paws of treated mice; how-
ever, fucoxanthin inhibited carr-induced activation of
ERK, JNK, and p38. This result might indicate that
fucoxanthin-mediated inhibition of MAPK activation
is one of the mechanisms underlying its inhibitory
effects on iNOS and COX-2 expression. Akt is a ser-
ine/threonine kinase that plays a substantial role in
many essential cellular processes, including apoptosis
and cell survival [9]. In dorsal horn neurons, phos-
phatidylinositol 3-kinase and Akt were activated by
injection of carr into the paw, which also induced pain
behavior by initiation of a parallel spinal intracellu-
lar cascade [38]. We examined the involvement of Akt
signaling in the anti-inflammatory effects of fucoxan-
thin in tissue from mice with carr-induced paw edema.
Western blot analysis indicated that p-Akt expression
was inhibited by fucoxanthin in paw tissue from mice
with carr-induced edema.
Many conditions that activate NFκB are known to
cause oxidative stress by increasing production of re-
active oxygen intermediates such as superoxide and
H2O2[39]. In addition, it has been reported that antiox-
idants inhibit NFκB activation and block production of
inflammatory cytokines by suppressing ROS genera-
tion [40]. Being a potent antioxidant, fucoxanthin effec-
tively inhibited the decrease in CAT and SOD activity
induced by carr and attenuated carr-induced degrada-
tion of cytosolic NFκB, p65, and IκBαin paw tissue.
Taken together, our results indicate that fucoxanthin
has anti-inflammatory properties and demonstrate its
therapeutic potential.
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