JOURNAL OF FUNCTIONAL FOODS x x x ( 2 0 1 3 ) x x x –x x x

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Suppressive effect of carotenoid extract of Dunaliella salina

alga on production of LPS-stimulated pro-inflammatory
mediators in RAW264.7 cells via NF-jB and JNK inactivation

Deng-Jye Yanga,b, Jau-Tien Linc, Yi-Chen Chend, Shih-Chuan Liua,b, Fung-Jou Luc,
Tien-Jye Change, Meilin Wangf, Hui-Wen Line,*,1, Yuan-Yen Changf,*,1
a
School of Health Diet and Industry Management, Chung Shan Medical University, 110, Section 1, Jianguo N. Road, Taichung 402, Taiwan
b
Department of Nutrition, Chung Shan Medical University Hospital, 110, Section 1, Jianguo N. Road, Taichung 402, Taiwan
c
Department of Applied Chemistry, Chung Shan Medical University, 110, Section 1, Jianguo N. Road, Taichung 402, Taiwan
d
Department of Animal Science and Technology, National Taiwan University, 1, Section 4, Roosevelt Road, Taipei 106, Taiwan
e
Department of Veterinary Medicine, College of Veterinary Medicine, National Chung-Hsing University, 250, Kuo Kuang Road, Taichung 402,
Taiwan
f
Department of Microbiology and Immunology, and Institute of Microbiology and Immunology, Chung Shan Medical University, and
Department of Medical Education, Chung Shan Medical University Hospital, 110, Section 1, Jianguo N. Road, Taichung 402, Taiwan

A R T I C L E I N F O A B S T R A C T

Article history: The carotenoid extract from Dunaliella salina was used to evaluate the suppressive effects on
Received 18 September 2012 lipopolysaccharide (LPS)-induced pro-inflammatory mediators in RAW264.7 cells. The
Received in revised form extract composed all-trans forms of a-carotene (28.8 mg/g extract), b-carotene (471.1 mg/g
21 December 2012 extract), lutein (7.1 mg/g extract) and zeaxanthin (7.2 mg/g extract), 13- or 13 0 -cis-b-carotene
Accepted 3 January 2013 (12.1 mg/g extract), 9- or 9 0 -cis-a-carotene (19.1 mg/g extract) and 9- or 9 0 -cis-b-carotene
Available online xxxx (440.3 mg/g extract) dose-dependently reduced the production of interleukin (IL)-1b, IL-6
and tumor necrosis factor-a (TNF-a), the protein expression of inducible nitric oxide
Keywords: synthase (iNOS) and cyclooxygenase-2 (COX-2), and the secretion of nitric oxide (NO) and
Caroteinoid prostaglandin E2 (PGE2) in LPS-activated RAW264.7 cells. Its attenuation of LPS-induced
Dunaliella salina inflammatory responses was closely related to inhibition of the nuclear NF-jB p50 subunit
Pro-inflammatory mediator
translocation by blocking inhibitor of jBa (IjB) phosphorylation and degradation correlated
NF-jB
with suppressing IjB kinase (IKK) a/b phosphorylation, as well as down-regulation of the
JNK
c-Jun NH2-terminal kinase (JNK) activation.
 2013 Elsevier Ltd. All rights reserved.

1. Introduction
Dunaliella salina (Chlorophyceae) is a halophilic unicellular
microalga with a mucus surface coat (no cell wall) (Garcı́a-Gon-
zález, Moreno, Manzano, Florencio, & Guerrero, 2005). The alga
contains abundant carotenoids (especially all-trans-b-carotene
and 9- or 9 0 -cis-b-carotene) (Hu, Lin, Lu, Chou, & Yang, 2008; Lin
et al., 2010), and has been exploited as a health food product, a
pro-vitamin A supplement, a food coloring agent, and an addi-
tive to food and cosmetics (Edge, McGarvey, & Truscott, 1997).
Carotenoids are effective antioxidants, which can quench
singlet oxygen (1O2), (Foote & Denny, 1968), suppress lipid

* Corresponding authors. Tel.: +886 4 22840369x54 (H.-W. Lin), tel.: +886 4 24730022x12028; fax: +886 4 24727178 (Y.-Y. Chang).
E-mail addresses: d9138001@mail.nchu.edu.tw (H.-W. Lin), cyy0709@yahoo.com.tw (Y.-Y. Chang).
1
These authors contributed equally.
1756-4646/$ - see front matter  2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jff.2013.01.001

Please cite this article in press as: Yang, D.-J. et al., Suppressive effect of carotenoid extract of Dunaliella salina alga on production of LPS-stimulated pro-inflam-
matory mediators in RAW264.7 cells via NF-jB and JNK inactivation, Journal of Functional Foods (2013), http://dx.doi.org/10.1016/j.jff.2013.01.001
2 JOURNAL OF FUNCTIONAL FOODS
peroxidation (Burton & Ingold, 1984) and prevent oxidative
damage (Edge et al., 1997). Several studies indicated that
consumption of carotenoid-rich vegetables and fruits could
protect humans against cardiovascular disease, certain can-
cers and other degenerative diseases (Hu et al., 2008). Recent
investigations also demonstrated that carotenoids including
all-trans forms of b-carotene, lycopene, lutein, crocin (a repre-
sentative of carotenoid compounds in Gardenia jasminoides
Ellis) and astaxanthin had anti-inflammatory activities (Bai
et al., 2005; Ohgami et al., 2003; Rafi & Shafaie, 2007; Xu
et al., 2009).
Reports indicated that many constituents in algal samples
had anti-inflammatory effects such as oxygenated fatty acids
from red alga Gracilaria verrucosa (Dang et al., 2008), sulpholi-
pids from the red alga Porphyridium cruentum (Bergeä, Debiton,
Dumay, Durand, & Barthomeuf, 2002), crude polysaccharide
from marine brown alga Turbinaria ornata (Ananthi et al.,
2010), dieckol (a hexameric compound of phloroglucinol) from
marine brown alga Ecklonia cava (Jung et al., 2009) and lipid
extract from blue-green alga Nostoc commune var sphaeroides
Kützing (Park et al., 2008). There is, however, no thorough
report concerning anti-inflammatory property of algal carot-
enoid extract.
During inflammation, activated inflammatory cells (e.g.
eosinophils, macrophages, mononuclear phagocytes and
neutrophils) secrete excess levels of nitric oxide (NO), prosta-
glandin E2 (PGE2) and cytokines such as interleukin-1b (IL-1b),
interleukin-6 (IL-6) and tumor necrosis factor-a (TNF-a), which
cause cell and tissue damage and activate macrophages in
inflammation-associated diseases such as rheumatoid arthri-
tis, chronic hepatitis and so on (Yoon et al., 2009). NO and
PGE2 are important pro-inflammatory mediators produced
by inducible nitric oxide synthase (iNOS) and cyclooxygen-
ase-2 (COX-2), respectively (Huang et al., 2009; Lai, Lai, Kuo,
Ho, & Pan, 2011; Pan, Yang, Tsai, Sang, & Ho, 2009). COX-2
and iNOS expression are regulated by the transcription factor,
nuclear factor-jB (NF-jB). NF-jB existed in the cytoplasm as
inactive heterodimers composed of two subunits, P50 and
P65, and are bound to the inhibitor of jB (IjB). Upon stimula-
tion, IjB is phosphorylated and proteolytically degraded, and
then NF-jB is translocated from the cytosol to the nucleus,
and regulates gene transcription (Lau, Joseph, McDonald, &
Kalt, 2009; Park, Lee, Lee, & Kim, in press; Tak & Firestein,
2001).
Macrophages play a principal role in regulating several var-
ious immunopathological conditions in inflammatory process,
and inducing overproduction of the pro-inflammatory media-
tors (Yoon et al., 2009). Lipopolysaccharide (LPS), an endotoxin,
is the major component of the cell wall in
Gram-negative bacteria. It can stimulate a number of major
cellular responses, which play key roles in the inflammatory
pathogenesis (Jung et al., 2009). Because of reproducible re-
sponse of RAW264.7 to LPS, the murine macrophage cell line
is widely adopted for studies of inflammation (Lai et al., 2011;
Pan et al., 2009). According to recent reports indicated that
LPS-stimulated macrophages activate several intracellular sig-
naling pathways, including the NF-jB pathway and three
mitogen-activated protein kinase (MAPK) pathways: extracel-
lular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase
Please cite this article in press as: Yang, D.-J. et al., Suppressive effect of carotenoid extract of Dunaliella salina alga on production of LPS-stimulated pro-inflam-
matory mediators in RAW264.7 cells via NF-jB and JNK inactivation, Journal of Functional Foods (2013), http://dx.doi.org/10.1016/j.jff.2013.01.001
x x x ( 2 0 1 3 ) x x x –x x x
(JNK), and p38 (Guha & Mackman, 2001; Lai et al., 2011; Tang,
Chen, Daun, Ho, & Pan, 2011).
The comprehensive mechanisms for the anti-inflamma-
tory effects of the extract from D. salina are not explored
yet, and so do the effects of the extract on expression of
pro-inflammatory mediators in RAW264.7 cells. Although
the inflammatory responses induced by Herpes simplex-like
virus, i.e. Pseudorabies virus have been mentioned in our pre-
vious report (Lin et al., 2012), the inflammatory response in-
duced by bacterial toxin, i.e. LPS is different. Moreover, even
though it has been recently demonstrated that carotenoids
(b-carotene and lycopene) are able to inhibit the activation
of NF-jB induced by different inducers (Di Tomo et al., 2012;
Kaulmann, Serchi, Renaut, Hoffmann, & Bohn, 2011; Kim,
Seo, & Kim, 2011), those results only show that anti-inflam-
matory activity of b-carotene in endothelial cells (HUVECs,
human umbilical vein endothelial cells) and epithelial cells
(Caco-2 and AGS cells) are through the suppressing NF-jB
activation. Bai et al. (2005) also reported that b-carotene inhib-
its the production of LPS-induced inflammatory mediators by
blocking NF-jB p65 subunit activation and as a consequent
suppression of IjBa phosphorylation and degradation in
macrophages (RAW264.7 cells). These results only suggest
that b-carotene, probably due to its antioxidant activity,
inhibits the production of inflammatory mediators by block-
ing NF-jB activation and as a consequent suppression of
IKK activity and IjBa degradation. Currently, its molecular
mechanism has not been clearly defined in macrophages
(RAW264.7 cells). However, more and more evidence has sug-
gested the possible existence of other functional regulators in
LPS-stimulated signal transduction. Moreover, the D. salina
extract appears to act at multiple steps in the signaling
cascade underlying inflammation. Thus, in this paper, we
describe another potent mechanism underlying the anti-
inflammatory effect of D. salina extract.
For the first time, our results demonstrated that the algal
carotenoid extract inhibited the productions of NO, PGE2,
and pro-inflammatory cytokines (IL-1b, IL-6 and TNF-a) as
well as the protein expressions of iNOS and COX-2 estimated
with the LPS-stimulated RAW264.7 cells. The D. salina extract
exhibited anti-inflammatory activities through the inhibition
of NF-jB activation and JNK phosphorylation.
2. Materials and methods
2.1. Dunaliella salina sample
Spray dried powder of D. salina algae cultivated in Taiwan in
2011 was from Gong Bih Enterprise Co., Ltd. (Wunlin, Taiwan).
2.2. Chemicals and reagents
Solvents used for preparation of algal carotenoid extract
including acetonitrile (ACN), methanol (MeOH), methylene
chloride (CH2Cl2), ethanol (EtOH) and n-hexane were from
Merck Co. (Darmstadt, Germany). Distilled deionized water
(H2O) was prepared with Ultrapure water purification
system (Lotun Co., Ltd. Taipei, Taiwan). Potassium hydroxide
(KOH) was from Merck Co. (Darmstadt, Germany). All-trans
 JOURNAL OF FUNCTIONAL FOODS
forms of lutein (M.W. 569) and zeaxanthin (M.W. 569), lipo-
polysaccharide (LPS), glutamine, penicillin, streptomycin,
dimethyl sulphoxide (DMSO), Griess reagent (1% sulphanila-
mide, 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride
and 2.5% phosphoric acid), 3-(4,5-dimethylthiazol-2-yl)-2,
5-diphenyltetrazolium bromide (MTT), ethylenediaminetetra-
acetic acid (EDTA), leupeptin, phenylmethanesulphonyl
fluoride (PMSF), phosphate-buffered saline (PBS), sodium
chloride (NaCl), sodium nitrite (NaNO2), Triton X-100, Tris
and Escherichia coli LPS were from Sigma Co. (St. Louis, MO,
USA). All-trans forms of a-carotene (M.W. 537) and b-carotene
(M.W. 537) were from Wako Pure Chemical Industries, Ltd.
(Osaka, Japan). RPMI-1640 medium and fetal bovine serum
(FBS) were from Gibco/Invitrogen Co. (Carlsbad, CA, USA). En-
zyme immunoassay (EIA) kits for the measurement of TNF-a,
IL-1b and IL-6 were from R&D Systems (Minneapolis, MN,
USA). EIA kit for PGE2 measurement was from Cayman
Chemical (Ann Arbor, MI, USA). The specific antibodies for
iNOS, COX-2, phosphorylated IjBa (P-IjBa), IjBa, IjB kinase
(IKK) a/b, phosphorylated IKKa (Ser180)/IKKb (Ser181)
(P-IKKa/b), p65, p50 and b-actin were from Santa Cruz
Biotechnology Inc. (Santa Cruz, CA, USA). The horseradish
peroxidase-conjugated anti-goat or anti-rabbit IgG were from
Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Antibodies
against ERK, phosphorylated ERK (P-ERK), p38, phosphory-
lated p38 (P-p38), JNK and phosphorylated JNK (P-JUK) were
from Cell Signaling Technology, Inc. (Danvers, MA, USA).
Nuclear extraction kit (AY2002) was from Panomics, Inc.
(Redwood City, CA, USA).
2.3. Cell line
Murine macrophage RAW264.7 cells from the American Type
Culture Collection (ATCC, Manassas, VA, USA) were cultured
in RPMI-1640 medium supplemented with 2 mM glutamine,
antibiotics (100 units/mL of penicillin and 100 lg/mL of strep-
tomycin) and 10% heat-inactivated FBS and maintained at
37 C in a humidified incubator (Astek Co., Fukuoka, Japan)
containing 5% CO2.
2.4. Preparation of D. salina carotenoid extract
Carotenoid extract of D. salina was prepared according to the
method of Hu et al. (2008). The algal sample (10 g) was ex-
tracted for 2 h at room temperature in 250 mL of hexane/ace-
tone/ethanol (2:1:1, v/v/v) with a shaker. Saponification was
then performed through adding 10 mL of 40% methanolic
KOH at 25 C for 16 h. After filtering, the extract was trans-
ferred to a separatory funnel and washed with 250 mL of dis-
tilled water for 3 times. The solvent was evaporated to
dryness in a rotary evaporator (Panchun Scientific Co.,
Kaohsiung, Taiwan) to yield carotenoid extract. Composition
of carotenoids in the extract was measured by high perfor-
mance liquid chromatography (HPLC) with the conditions
established in our previous report (Hu et al., 2008): column,
YMC C30 (250 · 4.6 mm, 5 lm) (Waters Co., Milford, MA,
USA); mobile phase, MeOH–ACN–H2O (84/14/2, v/v/v)/CH2-
Cl2 = 75/25 (v/v); flow rate, 1 ml/min; detection, 210–650 nm
at a rate of 1.00 spectrum/s. The equipments were a Prime-
Line Gradient Model 500G HPLC pump system (Analytical
Please cite this article in press as: Yang, D.-J. et al., Suppressive effect of carotenoid extract of Dunaliella salina alga on production of LPS-stimulated pro-inflam-
matory mediators in RAW264.7 cells via NF-jB and JNK inactivation, Journal of Functional Foods (2013), http://dx.doi.org/10.1016/j.jff.2013.01.001
x x x ( 2 0 1 3 ) x x x –x x x 3
Scientific Instruments, Inc., El Sobrante, CA, USA) and a
S-3210 photodiode-array (PDA) detector (Schambeck SFD
GmbH, Bad Honnef, Germany). The extract was then dis-
solved in DMSO for experiment.
2.5. Cell viability assay
The cell viability was measured through blue formazan, the
metabolized product of MTT, which was active by mitochon-
drial dehydrogenases in live cells only (Alley, Scudiero, &
Monkds, 1988). RAW264.7 cells were plated at a density of
1.5 · 105 cells/well into 24-well plates, pre-incubated for 1 h
with various concentrations (0–25 lM) of D. salina carotenoid
extract (prepared according to the caroteniod contents), and
stimulated with or without 1 lg/mL of LPS in medium at
37 C for 24 h. DMSO concentration in each well was 0.0125%.
The cells were subsequently incubated in 1 mL of MTT solution
(5 mg/mL) at 37 C for 4 h. The formation of formazan was dis-
solved in isopropanol and measured at wavelength 540 nm
using a microplate reader (Multiskan Spectrum, Thermo Co.,
Vantaa, Finland).
2.6. Nitrite assay
NO concentrations in cultured medium were measured as ni-
trite by the Griess reagent (Yoon et al., 2009). RAW264.7 cells
were plated at a density of 1.5 · 105 cells/well into 24-well
plates, pre-incubated with various concentrations (0, 5, 10
and 25 lM) of D. salina carotenoid extract for 1 h, and stimu-
lated with 1 lg/mL of LPS in medium at 37 C for 24 h. DMSO
concentration in each well was 0.0125%. Each cultured med-
ium (100 lL, supernatant) was mixed with 100 lL of Griess re-
agent and incubated at room temperature for 10 min. The
absorbance of the mixture was determined at 540 nm using
a microplate reader. All measurements were performed in
triplicate. The nitrite levels were determined through a stan-
dard curve established with NaNO2. All-trans-b-Carotene was
used for comparison.
2.7. Determination of pro-inflammatory cytokines and
PGE2
RAW264.7 cells were plated at a density of 1.5 · 105 cells/well
in 24 well plates, pre-treated with various concentrations (0,
5, 10 and 25 lM) of D. salina carotenoid extract for 1 h, and
stimulated with 1 lg/mL of LPS in medium at 37 C for 24 h.
The levels of IL-1b, IL-6, TNF-a and PGE2 in cultured medium
(supernatant) were measured with enzyme-linked immuno-
sorbent assay (ELISA) according to the manufacturer’s proto-
cols. The absorbance was determined at 450 nm using a
microplate reader. All-trans-b-Carotene was used for
comparison.
2.8. Western blotting analysis
Western blotting was performed according to the report of
Rhee et al. (2007). RAW264.7 cells were plated at a density of
3 · 106 cells in 6-cm plates, pre-treated with various concen-
trations (0, 5, 10 and 25 lM) of D. salina carotenoid extract
for 1 h, and stimulated with 1 lg/mL of LPS in medium at
4 JOURNAL OF FUNCTIONAL FOODS
37 C for 24 h. The cells were subsequently washed with PBS,
collected, suspended in the lysis buffer (150 mM NaCl, 10 mM
Tris (pH 7.5), 5 mM EDTA, 1% Triton X-100) containing prote-
ase inhibitors (1 lg/mL leupeptin and 100 lg/mL PMSF) and
centrifuged at 12,000g at 4 C for 20 min to yield cell lysates.
The protein level in each sample was measured using a pro-
tein assay kit (Bio-Rad, Laboratories, Inc., Hercules, CA,
USA). The proteins (20 lg of RAW264.7 lysates) were separated
with 10% SDS polyacrylamide gels and transferred to polyvi-
nylidene difluoride (PVDF) membranes (NewTM Life Science
Product, Inc., Boston, MA, USA). The membranes were then
blocked in Tris-buffered saline (TBS)–Tween 20 solution con-
taining 5% non-fat dry milk and incubated sequentially with
primary antibody and horseradish peroxidase-conjugated
anti-goat or anti-rabbit IgG (Bio-Rad, Hercules, CA, USA).
The protein bands were then visualized with an enhanced
chemiluminescence (ECL) system (Amersham Biosciences,
Piscataway, NJ, USA). The protein band densities were quanti-
fied using Alpha Imager 2200 software (Alpha Innotech Co.,
San Leandro, CA, USA).
2.9. Statistical analysis
All assays were carried out in triplicate and the mean values
were calculated. The data were subjected to analysis of vari-
ance (ANOVA) and Duncan’s multiple range tests assess dif-
ferences between means. Differences were considered
significant at a level of p < 0.05
3. Results and discussion
3.1. Carotenoid composition of D. salina carotenoid extract
The yield of the D. salina extract was 30.28%. Table 1 shows
that all-trans-b-carotene and 9- or 9 0 -cis-b-carotene were the
major carotenoids in D. salina cultivated in Taiwan as that re-
ported by Hu et al. (2008) and Lin et al. (2010). The contents of
all-trans forms of a-carotene, b-carotene, lutein and zeaxan-
thin were 28.8, 471.1, 7.1 and 7.2 mg/g extract, respectively;
other components including 13- or 13 0 -cis-b-carotene, 9- or
9 0 -cis-a-carotene and 9- or 9 0 -cis-b-carotene were 12.1, 19.1
and 440.3 mg/g extract, respectively. Our data could also
corroborate the results of Ben-Amotz, Katz, and Avron
(1982), and Garcı́a-González et al. (2005); they found that D.
salina contained abundant all-trans-b-carotene and 9-cis-b-
Table 1 – Carotenoid composition of the D. salina extract.
Compound Content (mg/g extract)a
All-trans-lutein 7.1 ± 0.3
All-trans-zeaxanthin 7.2 ± 0.3
13- or 13 0 -cis-b-carotene 12.1 ± 0.4
All-trans-a-carotene 28.8 ± 1.2
9- or 9 0 -cis-a-carotene 19.1 ± 0.37
All-trans-b-carotene 471.1 ± 17.2
9- or 9 0 -cis-b-carotene 440.3 ± 12.4
Total amount 985.7
a tively suppressed the production of NO, PGE2 and iNOS pro-
All values are mean ± SD obtained by triplicate analyses.
Please cite this article in press as: Yang, D.-J. et al., Suppressive effect of carotenoid extract of Dunaliella salina alga on production of LPS-stimulated pro-inflam-
matory mediators in RAW264.7 cells via NF-jB and JNK inactivation, Journal of Functional Foods (2013), http://dx.doi.org/10.1016/j.jff.2013.01.001
x x x ( 2 0 1 3 ) x x x –x x x
carotene. The prepared algal extract was then dissolved in
DMSO for evaluation of anti-inflammatory capacity with
LPS-stimulated RAW264.7 cells.
3.2. Cytotoxicity of D. salina carotenoid extract on
RAW264.7 cells
In previous study, Eberlein et al. (2008) demonstrated that
DMSO (0.1–2.0% in cell medium) could significantly inhibit
TNF-a expression in hyaluronan fragment-stimulated macro-
phages and present a dose-dependent effect. In order to lower
DMSO interference to the assay for anti-inflammatory effect
of samples, various DMSO concentrations were estimated be-
fore experiment. The DMSO concentration, 0.0125% in the
medium of RAW264.7 cells, was used in the study; it could
provide good dissolution property, and did not influence
anti-inflammatory activity assay for the algal extract. MTT as-
say indicated that RAW264.7 cell proliferation was not signif-
icantly changed (p > 0.05) under our experimental conditions
as described in Section 2.5.
Bai et al. (2005) found that b-carotene did not affect growth
of RAW264.7 cells at concentrations up to 50 lM. The viability
of RAW264.7 cells was not affected by treatment with D. salina
carotenoid extract (5–25 lM) in the investigation.
3.3. Effect of D. salina carotenoid extract on LPS-
stimulated NO and PGE2 production, and iNOS and COX-2
protein expression in RAW264.7 cells
All-trans-b-carotene is a commercial carotenoid and also a
major component in the algal extract; the compound was
used for comparison in the experiments. Nitrite and PGE2 lev-
els in un-stimulated RAW264.7cells were almost undetect-
able; their levels were increased substantially after LPS
stimulation ( Fig. 1A and B). As RAW264.7 cells treated with
5, 10 and 25 lM of D. salina carotenoid extract in presence of
LPS, 55%, 76% and 96% for nitrite levels, and 28%, 42% and
84% for PGE2 levels were lowered, respectively. At 5 and
10 lM, the algal extract presented a significantly higher inhib-
itory activity for NO generation than all-trans-b-carotene
( Fig. 1A). The algal extract also had a significantly higher
inhibitory effect for PGE2 production than all-trans-b-carotene
at 10 lM (Fig. 1B).
Western blot analysis showed that the protein expression
of iNOS and COX-2 in RAW264.7 cells was highly induced in
the presence of LPS; their expression could be significantly
suppressed when the algal extract were added to the culture
media at the time of cell stimulation. The higher concentra-
tion of the algal extract was used, the higher suppressive ef-
fect was observed (Fig. 2).
Many carotenoids have anti-inflammatory activity. Bai
et al. (2005) reported that a dose-dependent decrease in iNOS
and COX-2 protein expression as well as NO and PGE2 produc-
tion was observed in RAW264.7 cells treated with b-carotene
in the presence of LPS. Rafi, Yadav, and Reyes (2007) found
that lycopene treatment decreased the LPS-induced iNOS pro-
tein in RAW264.7 cells but not affected the COX-2 protein.
Ohgami et al. (2003) illustrated that astaxanthin could effec-
tein in LPS-activated RAW264.7 cells in a dose-dependent
 JOURNAL OF FUNCTIONAL FOODS x x x ( 2 0 1 3 ) x x x –x x x 5

mock LPS LPS+b-carotene LPS+algal extract a d c b

Relative intensity (%) − 100 18 ± 2 29 ± 3 53 ± 3
a
80
A
iNOS
Nitrite (uM)

60 b
c
40
d -Actin
e
20
f LPS (1 µ g /ml) − + + + +
f f
0 Extract (µ M) 0 0 25 10 5

2500 a
B
PGE 2 (pg/ mL)

a d c b
2000 b c Relative intensity (%) − 100 41 ± 3 65 ± 5 91 ± 4
d d
1500

1000
COX-2
e e
500
f -Actin
0
LPS (1 µ g /ml) − + + + +
C
5 a
Extract (µ M) 0 0 25 10 5
IL-1B (pg/mL)

4
b
c Fig. 2 – Effects of D. salina carotenoid extract on LPS-
3

2
stimulated nitric oxide synthase (iNOS) and
d
e cyclooxygenase-2 (COX-2) protein expression in RAW 264.7
1 f f
f cells. Cells pre-treated with various concentrations (0, 5, 10
0
and 25 lM) of D. salina carotenoid extract for 1 h were

D stimulated with LPS (1 lg/mL) for 24 h. The protein levels of

a
1200
iNOS (A) and COX-2 were determine with Western blot
IL-6 (pg/mL)

900 analysis; the iNOS signal and COX-2 signal were normalized
600 to the b-actin signal. Data are the mean ± SD of three
300 b b separate experiments and expressed as the percentage of
c c c c c
the culture treated with LPS alone. Means with different
0
letters were significantly different at the level of p < 0.05
a when analyzed by ANOVA and Duncan’s multiple range test.
2000 b b c c E
TNF-a (pg/mL)

d d
1500

1000
500
0
LPS (1µ g/ml) + +
Sample ( µ M) 5 10
Fig. 1 – Effects of D. salina carotenoid extract on the
production of LPS-stimulated nitric oxide, prostaglandin E2
(PGE2), interleukin (IL)-1b, IL-6, and tumor necrosis factor
(TNF)-a in RAW 264.7 cells. Cells pre-treated with various
concentrations (0, 5, 10 and 25 lM) of D. salina carotenoid
extract for 1 h were stimulated with LPS (1 lg/mL) for 24 h.
The cultured media were used to determine the level of
nitrite by the Griess reagent to evaluate NO production (A);
PGE2 (B), IL-1b (C), IL-6 (D) and TNF-a (E) were measured by
enzyme-linked immunosorbent assay. Each bar represents
mean ± SD of three separate experiments. Means with
different letters were significantly different at the level of
p < 0.05 when analyzed by ANOVA and Duncan’s multiple
range test.
fashion. Xu et al. (2009) reported that crocin dose-depen-
dently inhibited the LPS-induced PGE2 in RAW264.7 cells by
inhibition of COX activity. Rafi et al. (2007) revealed that levels
of NO and iNOS protein in LPS-stimulated RAW264.7 cells
could be lowered after treatment with lutein.
Our results demonstrated that D. salina carotenoid extract
e could strongly inhibit production of NO and PGE2, and protein
expression of inflammation-related enzymes (iNOS and COX-
2) in LPS-stimulated RAW264.7 cells in a dose-dependent
+ − +
25 0 0 manner. These results suggest that inhibitory effects of the
algal extract on NO and PGE2 production are associated with
down-regulation of iNOS and COX-2 expression during the
activation of macrophages by LPS. The algal extract also pre-
sented stronger inhibitory capacities for LPS-stimulated NO
and PGE2 production in RAW264.7 cells than b-carotene.
3.4. Effect of D. salina carotenoid extract on IL-1b, IL-6,
and TNF-a production in RAW264.7 cells
Stimulation of RAW264.7 cells with LPS led to a substantial
increase in production of pro-inflammatory cytokines (IL-1b,
IL-6, and TNF-a). However, LPS-stimulated RAW264.7 cells
pre-treated with a higher concentration of D. salina carotenoid
extract had a greater reduction of pro-inflammatory cyto-
kines (Fig. 1C–E). At 5 and 10 lM, the algal extract showed a
significantly higher suppressive effect for LPS-induced IL-1b
production than all-trans-b-carotene ( Fig. 1C). Levels of IL-6
and TNF-a in LPS-stimulated RAW264.7 cells could also be
lowered by pre-treated with the algal extract in a dose-depen-
dent manner; the inhibitory activities of IL-6 and TNF-a
production for the algal extract were similar to those for all-
trans-b-carotene (Fig. 1D and E).

Please cite this article in press as: Yang, D.-J. et al., Suppressive effect of carotenoid extract of Dunaliella salina alga on production of LPS-stimulated pro-inflam-
matory mediators in RAW264.7 cells via NF-jB and JNK inactivation, Journal of Functional Foods (2013), http://dx.doi.org/10.1016/j.jff.2013.01.001
6 JOURNAL OF FUNCTIONAL FOODS
Bai et al. (2005) indicated that b-carotene effectively inhib-
ited LPS-induced IL-1b and TNF-a production in RAW264.7
cells. Our results could corroborate theirs; nevertheless, D.
salina carotenoid extract had higher effect for inhibition of
LPS-induced IL-1b production as compared with all-trans-b-
carotene. The algal extract significantly suppressed LPS-in-
duced IL-1b, IL-6 and TNF-a production, which illustrates that
it inhibits the initial phase of the LPS-stimulated inflamma-
tory response.
A dimer from the cytoplasm to the nucleus. In order to conclude
B Fig. 3A shows that LPS treatment induced high level of IjB
C
Fig. 3 – Effect of D. salina carotenoid extract on LPS-induced
IjB phosphorylation and degradation, translocation of
nuclear factor-jB (NF-jB) p50 and p65 subunits, and IjB
kinase (IKK) a/b phosphorylation in RAW264.7 cells. Cells
pre-treated various concentrations (0, 5, 10 and 25 lM) of D.
salina carotenoid extract for 1 h were stimulated with LPS
(1 lg/mL) for 24 h. The levels of IjB-a (A), IjB-a
phosphorylation (B), and nuclear translocation of NF-jB p50
(C) and p65 (D) subunits were determined with Western blot
analysis; each of their signals was normalized to the b-actin
signal. The levels phosphorylation of IKKa/b and IKKa/b (E)
were measured with Western blot analysis; the ratios of
immunointensity between the phosphorylated IKKa/b and
IKKa/b are shown. Data are the mean ± SD of three separate
experiments and expressed as the percentage of the culture
treated with LPS alone. Means with different letters were
significantly different at the level of p < 0.05 when analyzed
by ANOVA and Duncan’s multiple range test.
Please cite this article in press as: Yang, D.-J. et al., Suppressive effect of carotenoid extract of Dunaliella salina alga on production of LPS-stimulated pro-inflam-
matory mediators in RAW264.7 cells via NF-jB and JNK inactivation, Journal of Functional Foods (2013), http://dx.doi.org/10.1016/j.jff.2013.01.001
x x x ( 2 0 1 3 ) x x x –x x x
3.5. Effect of D. salina carotenoid extract on LPS-induced
IjB phosphorylation and degradation, translocation of NF-jB
p50 and p65 subunits, and IKKa/b phosphorylation in
RAW264.7 cells
NF-jB is a principal transcription factor, which regulates the
expression of pro-inflammatory mediators such as COX-2,
iNOS, IL-1b, IL-6, and TNF-a (Rhee et al., 2007). Henkel et al.
(1993) and Pan et al. (2009) indicated that the IjB (a cytoplas-
mic inhibitor of NF-jB) phosphorylation and degradation
were induced by inflammatory stimuli, which results in the
translocation of the activated p65/p50 (NF-jB subunit) hetero-
the mechanisms for the inhibition of LPS-stimulated expres-
sion of pro-inflammatory mediators, the effects of D. salina
carotenoid extract on IjB phosphorylation and degradation,
and translocation of p50 and p65 in RAW264.7 cells were
assayed.
phosphorylation; addition of D. salina carotenoid extract
dose-dependently reduced this LPS-induced phosphorylated
IjBa. LPS treatment also caused IjBa degradation significantly;
pre-treatment with the algal extract inhibited this degrada-
A
Fig. 4 – Effects of D. salina carotenoid extract on LPS-
activated mitogen-activated protein kinases (MAPKs) in
RAW264.7 cells. Cells pre-treated with various
concentrations (0, 5, 10 and 25 lM) of D. salina carotenoid
extract for 1 h were stimulated with LPS (1 lg/mL) for 24 h.
The levels of total or phosphorylated JNK (A), p38 (B) and
ERK (C) were measured with Western blot analysis; the
ratios of immunointensity between the MAPKs and the
phosphorylated MAPKs are shown. Data are the mean ± SD
of three separate experiments and expressed as the
percentage of the culture treated with LPS alone. Means
with different letters were significantly different at the level
of p < 0.05 when analyzed by ANOVA and Duncan’s multiple
range test.
 JOURNAL OF FUNCTIONAL FOODS
Fig. 5 – The possible role of D. salina carotenoid extract on production of pro-inflammatory mediators in LPS-stimulated
RAW264.7 cells.
tion in a dose-dependent fashion (Fig. 3B). Upon LPS stimula-
tion, most cytoplasmic p50 and p65 were translocated to the
nucleus. Through pre-treatment with D. salina carotenoid ex-
tract, the p50 levels in the nucleus were dose-dependently de-
creased ( Fig. 3C), but p65 levels did not show significant
changes (Fig. 3D). These data illustrated that the algal extract
mediated inhibition of the expression of pro-inflammatory
mediators was regulated by the NF-jB pathway in LPS-
stimulated macrophages.
Ghosh and Karin (2002) indicated that IKK phosphorylates
IjB to release NF-jB. Fig. 3E shows that LPS stimulation greatly
increased phosphorylation of IKK a/b; pre-treatment with D.
salina carotenoid extract dose-dependently inhibited this in-
crease. Therefore, inhibition of NF-jB activation for the algal
extract should be caused by reducing phosphorylation of
the IKK complex. LPS-stimulated.
Xu et al. (2009) reported that crocin prevented the LPS-in-
duced nuclear translocation of the NF-jB p50 and p65 sub-
units in RAW264.7 cells. Kim et al. (2008) indicated that
lutein inhibited LPS-induced NF-jB activation, which highly
correlated with its inhibitory effect on LPS-induced IKK a/b
activation, IjBa degradation, and nuclear translocation of
the NF-jB p65 subunit in RAW264.7 cells. Bai et al. (2005) dem-
onstrated that b-carotene inhibited IjBa phosphorylation and
degradation, and blocked nuclear translocation of NF-jB p65
subunit in LPS-stimulated RAW264.7 cells. Our results showed
that D. salina carotenoid extract effectively suppressed NF-jB
activation by blocking IKK a/b phosphorylation, IjBa phos-
phorylation and degradation, and nuclear p50 subunit trans-
location in LPS-stimulated RAW264.7 cells. These results
Please cite this article in press as: Yang, D.-J. et al., Suppressive effect of carotenoid extract of Dunaliella salina alga on production of LPS-stimulated pro-inflam-
matory mediators in RAW264.7 cells via NF-jB and JNK inactivation, Journal of Functional Foods (2013), http://dx.doi.org/10.1016/j.jff.2013.01.001
x x x ( 2 0 1 3 ) x x x –x x x 7
suggested that D. salina carotenoid extract lowered the
expression of pro-inflammatory mediators via down-
regulating the NF-jB pathway in stimulated macrophages.
3.6. Effect of D. salina carotenoid extract on LPS-induced
phosphorylation of MAPKs in RAW264.7 cells
Because MAPK pathways are involved in LPS-induced iNOS
and COX-2 expression in macrophages (Chen, Chen, & Lin,
1999), the effects of D. salina carotenoid extract on the LPS-
stimulated phosphorylation of JNK, p38, and ERK MAPKs in
RAW264.7 cells were estimated in the study. LPS treatment
markedly activated MAPKs including JNK, p38, and ERK
(Fig. 4A–C). Pre-treatment with the algal extract significantly
reduced LPS-induced phosphorylated JNK (Fig. 4A), but not
p38 or ERK.
Kim et al. (2008) demonstrated that RAW264.7 cells treated
with LPS resulted in considerable increases in ERK, JNK, and
p38 phosphorylation, and these circumstances were not
changed after co-treatment with lutein. Our results showed
that D. salina carotenoid extract availably attenuated
LPS-stimulated JNK phosphorylation in RAW264.7 cells; those
suggested that JNK signaling pathway should be an important
modulator in the algal extract-mediated suppression of
LPS-induced iNOS and COX-2 expression. Our work indicated
that the molecular mechanism for D. salina carotenoid
extract-mediated attenuation of LPS-induced inflammatory
responses in RAW264.7 cells should be related to down-regu-
lating IKK a/b phosphorylation, thus inhibiting IjBa phosphor-
ylation and degradation, and further p50 subunit
8 JOURNAL OF FUNCTIONAL FOODS
translocation into nuclear; the extract also suppressed the
LPS-induced activation of JNK which were summarized in
Fig. 5.
4. Conclusions
Dunaliella salina has great amounts of all-trans-b-carotene and
9- or 9 0 -cis-b-carotene, and other five carotenoids. According
to the evaluation of anti-inflammatory effect using LPS-stim-
ulated RAW264.7 macrophages, the algal carotenoid extract
could markedly lower production of IL-1b, IL-6 and TNF-a,
protein expression of iNOS and COX-2, and secretion of NO
and PGE2 in a dose-dependent manner. The results suggest
that carotenoids especially all-trans-b-carotene and 9- or
9 0 -cis-b-carotene are the important active ingredients in D.
salina, which can modulate inflammatory processes via
NF-jB and JNK inactivation. D. salina should have potential
to be exploited for the treatment of inflammation-associated
diseases.
Acknowledgment
This work was supported by Chun Shan Medical University
Taichung, Taiwan (Project No. CSMU 100-OM-A-047). We
thank Instrument Resource Center of Chung Shan Medical
University for chemiluminescence/ fluorescence imaging
analyzer.
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