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July 2012 1137Biol. Pharm. Bull. 35(7) 1137–1144 (2012)
© 2012 The Pharmaceutical Society of Japan
Anti-inflammatory Effects of Violaxanthin Isolated from Microalga
Chlorella ellipsoidea in RAW 264.7 Macrophages
Waesarat Soontornchaiboon,a,# Seong Soo Joo,b,# and Sang Moo Kim*,a
a Department of Marine Food Science and Technology, Gangneung-Wonju National University; and b Department of
Marine Molecular Biotechnology, Gangneung-Wonju National University; 120 Gangneung Daehangno, Gangneung,
Gangneung 210–702, Republic of Korea. Received February 23, 2012; accepted April 23, 2012
Violaxanthin is a major carotenoid of microalgae Chlorella ellipsoidea and is also found in dark-green
leafy vegetables, such as spinach. In this study, the anti-inflammatory effect of violaxanthin isolated from C.
ellipsoidea was examined using lipopolysaccharide (LPS)-stimulated RAW 264.7 mouse macrophage cells.
In addition, the anti-inflammatory activity and mechanism of action of purified violaxanthin was assessed
using various assays, such as quantitative real-time polymerase chain reaction (PCR), Western blotting, and
electrophoretic-mobility shift assay (EMSA). The results of this combined analysis revealed that violaxanthin
significantly inhibited nitric oxide (NO) and the prostaglandin E2 (PGE2). Interestingly, violaxanthin effec-
tively inhibited LPS-mediated nuclear factor-κB (NF-κB) p65 subunit translocation into the nucleus, suggest-
ing that the violaxanthin anti-inflammatory activity may be based on inhibition of the NF-κB pathways. In
conclusion, violaxanthin of C. ellipsoidea holds promise for use as a potential anti-inflammatory agent for
either therapeutic or functional adjuvant purposes.
Key words anti-inflammation; Chlorella ellipsoidea; microalgae; violaxanthin; RAW 264.7 cell; nuclear
factor-κB p65
Inflammation is the normal physiological and immune
response to tissue injury and occurs when the human body
attempts to counteract potentially injurious agents, such as
invading bacteria, vir uses, and other pathogens.1) Among im-
mune cells, macrophages play important roles in inflammation
by overproducing inflammatory mediators, including nitric ox-
ide (NO) and prostaglandin E2 (PGE2), which are synthesized
by inducible nitric oxide synthase (iNOS) and cyclooxygenase
(COX-2), respectively.2) Since inflammatory mediators can
cause severe damage, such as sepsis and inflammatory dis-
eases, inhibiting NO and PGE2 production by blocking iNOS
and COX-2 at the molecular may be a useful strategy for the
treatment of acute or chronic inflammatory disorders.3,4)
Importantly, iNOS is highly expressed in macrophages and
can lead to organ destruction in some inflammatory and auto-
immune diseases.5) PGE2 is another important inflammatory
mediator and is produced from arachidonic acid metabolites
by the catalysis of cyclooxygenase-2 (COX-2).2,6) During
inflammation, macrophages play a central role in managing
many different immunopathological phenomena, including the
overproduction of pro-inflammatory mediators, such as NO
and PGE2. Experimentally, lipopolysaccharide (LPS) and pro-
inflammatory cytokines activate immune cells to up-regulate
inflammatory states, and these are therefore useful targets
in the development of anti-inflammatory agents and for the
exploration of molecular anti-inflammatory mechanisms.7,8) In
cell signal pathways, nuclear factor-κB (NF-κB) is known to
stimulate the expression of enzymes, such as inducible iNOS
and COX-2, and recent studies have identified that the toll-like
receptor 4 (TLR4) is a signal-transducing receptor for LPS.9)
LPS also directly activates the NF-κB pathway via TLR4 that
amplifies the inflammatory responses by establishing a posi-
tive autoregulator y loop.10)
A seawater microalga, Chlorella ellipsoidea, which contains
a high content of carotenoids, is commonly used as feed for
marine fishes. C. ellipsoidia, is photosynthetic organism con-
taining biologically active compounds, such as carotenoids,
phycobilins, fatty acids, polysaccharides, vitamins, and ste-
rols.11) The carotenoids from microalgae function as acces-
sory pigments in photosystems and str uctural components of
light harvesting complexes, as well as photoprotective agents,
which play roles in phototaxis.12,13) Various microalgae ac-
cumulate large quantities of carotenoids, which have been
exploited commercially, including β-carotene from Dunaliella,
astaxanthin from Haematococcus, and lutein from Chlorophy-
cean strains.14–16) C. ellipsoidea is abundant and commonly
used for marine fish and brine shrimp hatcheries in Japan and
Korea.17) There has been only one report that examined the
antiproliferative effect of carotenoids from C. ellipsoidea on
human colon cancer cells.18) The main carotenoid from ma-
rine C. ellipsoidea was found to be violaxanthin, which also
contains minor amounts of two xanthophylls, antheraxanthin
and zeaxanthin.18) The antioxidant potential of carotenoid is
primarily due to violaxanthin, which is present in orange
colored fruits and green vegetables.19) Violaxanthin from wa-
ter spinach (Ipomoea aquatica) was reported to have a more
potent scavenging ability than lutein and β-carotene in ABTS
radical-scavenging, inhibition of red blood cell hemolysis, and
inhibition of lipid peroxidation in liver.20) However, the poten-
tial anti-inflammatory effects of major carotenoids from C.
ellipsoidea have not yet been investigated.
The aim of this study was to investigate the effect of the
major carotenoid isolated from C. ellipsoidea on anti-inflam-
matory potencies by evaluating the inhibition of NO, PGE2
production, and the expressions of inducible nitric oxide syn-
thase (iNOS) and cyclooxygenase (COX-2) in LPS-stimulated
RAW 264.7 cells by deter mining the molecular mechanism of
violaxanthin action in LPS-induced NF-κB signaling pathway.
Regular Article
* To whom cor respondence should be addressed. e-mail: smkim@gwnu.ac.kr
The authors declare no conflict of interest.
# These authors contributed equally to this work.
1138 Vol. 35, No. 7
MATERIALS AND METHODS
Materials Dimethyl sulfoxide (DMSO), Griess reagent,
Celecoxib, and NG-Methyl-L-arginine acetate salt (L-NMMA)
were purchased from Sigma-Aldrich (MO, U.S.A.). Poly-
merase chain reaction (PCR) primers were purchased from Bi-
oneer (Deajeon, South Korea). All other chemicals used were
of analytical grade.
Preparation of the Microalgae Extract Chlorella ellip-
soidea was obtained from the Daesang company (Seoul, South
Korea). Chlorella cells were harvested from culture solution
by centrifugation at 7000
rpm for 10 min at 4°C and freeze-
dried in a vaccum freezer-dryer (Samwon Freezing Engineer-
ing, Seoul, South Korea). The dried sample was finely ground
in a mortar and stored at −80°C before extraction.
The Chlorella powder (4 g) was extracted with 200 mL of
90% ethanol for 30 min by ultrasound-assisted extraction at a
frequency of 35 kHz (Berlin, Germany) and room temperature,
which was repeated three times. The combined extracts were
filtered through Whatman No. 2 filter paper and saponified
by adding 6% KOH (w/v) (24 g), and then heated at 50°C for
30 min with shaking at 100 r pm. After cooling to room tem-
perature, the mixture was evaporated under vacuum at 40°C.
The ethanol extract was dissolved in distilled water (300 mL),
and then partitioned with ethyl acetate (300 mL) three times.
The concentrated ethyl acetate fraction (300 mL) was washed
several times with distilled water until the water phase was
colorless. After separation, the ethyl acetate fraction was
evaporated on a rotary evaporator at 40°C.
Isolation and Structural Identification of Violaxanthin
The ethyl acetate fraction was subjected to a silica gel col-
umn (2.0×60 cm), which was eluted with hexane (150 mL),
followed by hexane–acetone (7 : 3, v/v) (400 mL). The target
compound fractions were collected based on the spectral
characteristics of violaxanthin, which were examined over a
range of 350–550 nm using a spectrophotometer, by compar-
ing the wavelengths of maximum absorption and spectral
fine structural values (%III/II). In the epoxide test, 10 mL
of 0.1
M HCl was added to a 1 mL ethanolic carotenoid solu-
tion and the absorption maxima were determined using a
spectrophotometer. The purified compound was identified
by liquid chromatography/mass spectrometry (LC/MS) (HP-
1100MSD, Agilent Technologies, CA, U.S.A.) equipped with
an electrospray ionization (ESI). Mass spectra were acquired
over the m/z 400–700 scan range using a 0.1 unit step size.
Ten μL of isolated compound in methanol was injected into
the LC, Eclipse XDB-C18 column (5 µm, 150×46 mm i.d., Wa-
ters, MA, U.S.A.) and eluted using isocratic solvent system,
acetonitrile–methanol–dichloromethane (71 : 22 : 7, v/v), at a
flow rate of 0.5 mL/min. Finally, the structure of the isolated
compound was determined by spectroscopic methods, includ-
ing 1H-NMR and 13C-NMR. 1H and 13C-NMR were recorded
in chloroform-d1 (CDCl3) on a Bruker DRX-600 spectrometer
(Kalsruhe, Germany).
5,6 : 5′,6′-Diepoxy-5,5′,6,6′-tetrahydro- β-carotene-3,3′-
diol
1H NMR (600 MHz, CDCl3) δH 0.97 [ 2s, 6H, CH3
(16/16′)], 1.14 [ 2s, 6H, CH3 (17/17′)], 1.18 [ 2s, 6H, CH3
(18/18′)], 1.24 [m, 2H, Hax,-C (2/2′)], 1.60 [m, 2H, Hax-C
(4/4′)], 1.63 [m, 2H, Heq-C (2/2′)], 1.92 [2s, 6H, CH3 (19/19′)],
1.97 [2s, 6H, CH3 (20/20′)], 2.37 [ddd, 2H, Heq-C (4/4′),
J=14.2, 5, 1.4 Hz], 3.90 [m, 2H, H-C (3/3′)], 5.87 [2d, 2H, H-C
(7/7′), J=15.6 Hz], 6.18 [2d, 2H, H-C (10/10′), J=11 Hz], 6.25
[2d, 2H, H-C (14/14′), J=10 Hz], 6.28 [2d, 2H, H-C (8/8′),
J=15.1 Hz], 6.36[2d, 2H, H-C (12/12′), J=14.7 Hz], 6.58 [2dd,
2H, H-C (11/11′), J=15, 11.5 Hz], 6.62 [m, 2H, H-C (15/15′)]
;13C NMR(150 MHz, CDCl3) δC 35.4 (C-1,1′), 47.3 (C-2,2′),
64.4 (C-3,3′), 41.1 (C-4,4′), 67.1 (C-5,5′), 70.4 (C-6,6′), 123.9
(C-7,7′), 137.4 (C-8,8′), 134.4 (C-9,9′), 132.3 (C-10,10′), 124.8
(C-11,11′), 138.2 (C-12,12′), 136.5 (C-13,13′), 132.9 (C-14,14′),
130.3 (C-15,15′), 25.0 (C-16,16′), 29.7 (C-17,17′), 20.1
(C-18,18′), 12.9 (C-19,19′), 13.1 (C-20,20′).
Cell Culture Raw 264.7 (Mouse leukaemic mono-
cyte macrophage cell line) cells were grown in Dulbecco’s
modified Eagle’s medium (DMEM) (Hyclone, UT, U.S.A.)
supplemented with 10% fetal bovine serum (FBS), 4 mM/L
L-glutamate (Invitrogen, CA, U.S.A.), 100 U/mL penicil-
lin and 100 µg/mL streptomycin (Invitrogen). Cultures were
maintained under 5% CO2 at 37°C in tissue culture flasks. In
all experiments, the cells were grown to a confluency greater
than 90% and subjected to no more than 20 cell passages.
Cellular Cytotoxicity (Lactate Dehydrogenase) Assay
The cytotoxicity induced by violoaxanthin was quantitated
by measuring lactate dehydrogenase (LDH) release. The LDH
content was determined using a commercial non-radioactive
LDH assay kit, CytoTox 96® (Promega, WI, U.S.A.), which
was based on a coupled enzymatic reaction that converts tet-
razolium salt into a red formazan product. The increase in the
amount of formazan produced in the culture supernatant di-
rectly correlates with the increase in the number of lysed cells.
The formazan was quantified spectrophotometrically by mea-
suring its absorbance at 490 nm (Spectra Max 340, Molecular
Devices, CA, U.S.A.). Cytotoxicity in experimental samples
was determined as %LDH release compared with cells treated
with 1% Triton X-100.
Nitric Oxide Production in RAW 264.7 Cells RAW
264.7 cells (106 cells/mL) were seeded on a 24-well tissue
culture plate and pre-incubated at 37°C for 12 h to achieve
stable attachment. The wells were then washed with phosphate
buffered saline (PBS) after pre-incubation, refreshed with FBS
free DMEM containing lipopolysaccharide (LPS) (1 µg/mL) in
the presence of different concentrations of violaxanthin (10 to
60 µM) and incubated for 24 h. Nitric oxide (NO) production
was then monitored by measuring nitrite levels in the culture
media using the Griess Reagent at 540 nm (Sigma-Aldrich).
Table 1. Peak Identification of the Purified Compound from Chlorella ellipsoidea and Its Spectral Characteristics Following DAD
Retention time λmax
a) (nm) Hypsochromic shift
(nm) Epoxide test % III/IIb)ESI-MS
(positive, m/z)
Tentative identifica-
tion
4.084 417, 441, 470 380, 400, 426 Diepoxide 95 601.4 Violaxanthin
a) The wavelength of maximum absorption. A mobile phase of acetonitrile–methanol–dichloromethane (71 : 22 : 7, v/v) was used. b) Ratio of the height of the longest wave-
length absorption peak, designated III, and that of the middle absorption peak, designated II, taking the minimum between two peaks as baseline, multiplied by 100, in mobile
phase.
July 2012 1139
L-NMMA was compared as a positive control. The concentra-
tion of nitrite (μM) was calculated from a standard curve that
was established using known concentrations of sodium nitrite
dissolved in RPMI-1640 medium.
Prostaglandin E2 (PGE2) Assay The cells (106 cells/
mL) were incubated with LPS (1 µg/mL) in the presence of
violaxanthin at different concentrations (10 to 60 µM) for 24 h
in 24 well-plates. Celecoxib (3 µM) was used as a positive
control. The level of PGE2 in the supernatants from macro-
phage cultures was determined using a competitive enzyme
immunoassay kit (R&D System, MN, U.S.A.) according to the
manufacturer’s protocol.
Quantitative Real-Time Polymerase Chain Reaction
(PCR) Assay The RAW 264.7 cells were seeded at a density
of 5×106 cells/mL in a 6 well plate and pre-incubated for 12 h.
The cells were then treated with LPS (1 µg/mL) in the absence
or presence of vioaxanthin at different concentrations (10, 30,
60 µM) for 24 h. Total RNAs were prepared from cultured cells
using the Trizol method (Invitrogen). Complementary DNA
(cDNA) was synthesized from ribonucleic acid (RNA) by the
reverse transcription of 1 µg of total RNA using the Improm-II
reverse transcription system and oligo dT primers in a total
volume of 20 µL (Promega, WI, U.S.A.). PCR amplification
was performed using the primers described in Table 2 (Bi-
oneer, Deajeon, South Korea). Quantitative real-time PCR
(qPCR) reactions were run on a Rotor-Gene 6000 (Corbett
Research, Sydney, Australia) using SYBR Green PCR Master
Mix (Qiagen, CA, U.S.A.) in 20 µL reaction mixtures. Each
real-time-PCR master mix contained 10 µL of 2X enzyme
mastermix, 7.0 µL of RNase free water, 1 µL of each primer
(10 pmol each) and 1 µL of diluted template. PCR was per-
formed with an initial pre-incubation step for 10 min at 95°C,
followed by 45 cycles of 95°C for 15 s, annealing at 52°C for
15 s and extension at 72°C for 10 s. Melting curve analysis
was used to confirm formation of the expected PCR product,
and products from all assays were subjected to 1.2% agarose
gel electrophoresis to confirm that the products had the cor-
rect lengths. An inter-run calibrator was used, and a standard
curve was created for each gene to determine PCR efficien-
cies. Relative sample expression levels were calculated using
Rotor-Gene 6000 Series Software 1.7 (Corbett Research, Syd-
ney, Australia), and expressed relative to β-actin and corrected
for between run variability. Data for the experimental samples
were expressed as the percentage of the internal control gene.
Electrophoretic-Mobility Shift (EMSA) Assay (NF-κB
p65) Nuclear extracts for EMSA were prepared from RAW
264.7 cells using Nuclear Extraction Reagents (Pierce, IL,
U.S.A.) according to the manufacturer’s instructions. EMSA
was performed using the Panomics EMSA kit (Panomics, CA,
U.S.A.). Briefly, nuclear extracts containing equal amounts
of proteins for each sample were incubated with poly(dI-dC)
(1 µg/μL) for 5 min, followed by the addition of binding buffer
(20 mM N-(2-hydroxyethyl) piperazine-N´2-ethanesulfonic acid
(HEPES) pH 7.9, 1 mM dithiothreitol (DTT), 0.1 mM ethylene-
diaminetetraacetic acid (EDTA), 50 mM KCl, 5% glycerol and
200 µg/mL bovine serum albumin (BSA) and biotinylated
oligo (10 ng/μL). To control for the specificity of binding for
selected samples, a 5-fold excess of unlabeled oligo was added
prior to the addition of the biotinylated probe. Binding reac-
tion mixtures were incubated for 30 min at room temperature.
Protein–DNA complexes were separated on 6% non-dena-
turing polyacrylamide gels in 0.5×Tris–borate/EDTA buffer
(0.1
M Tris, 0.09 M boric acid containing 1 mM EDTA) at 4°C.
After electrophoresis, the gels were transferred to a positively
charged nylon transfer membrane (Amersham Bioscience, NJ,
U.S.A.). Transferred oligos were immobilized by UV cross-
linking for 10 min. To detect bound oligos, membranes were
blocked using blocking buffer (Panomics EMSA Gel-Shift
Kit). Streptavidin-HRPO was then added and blots were de-
veloped by chemiluminescent detection system according to
the manufacturer’s instructions (Panomics).
Western Blot Analysis RAW 264.7 cells were lysed in
1% radio immunoprecipitation assay (RIPA) buffer contain-
ing protease and phosphatase inhibitors (Roche, Mannheim,
Germany) and whole cell lysates were separated by 10%
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE). After electrophoresis, proteins were transferred
onto polyvinylidene fluoride (PVDF) membranes and the
membranes were blocked with 5% skim milk in Tris-buffered
saline solution containing 0.1% Tween-20. The membranes
were then immunoblotted with primary antibodies, anti-COX2
(Cell Signaling, MA, U.S.A.), anti-iNOS (Cell Signaling),
anti-extracelullar signal-regulated protein kinase, anti-phos-
pho-extracellular signal-regalated kinase (ERK)1/2 (Santa
Cruz Biotechnology, CA, U.S.A.), anti-phospho-inhibitor of
nuclear factor (I)κBα (Santa Cruz Biotechnology) and anti-
actin (Santa Cruz Biotechnology), followed by incubation with
horseradish peroxidase-conjugated anti-rabbit or anti-mouse
secondar y antibodies (Stressgen, CA, U.S.A.). Blots were de-
veloped using an enhanced chemiluminescence (ECL) solution
(Thermo Scientific, IL, U.S.A.)
Statistical Analysis Statistical comparisons between
the groups were performed using one-way analysis of vari-
ance (ANOVA) with a Dunnet’s post-hoc test, which were
performed using SPSS software (v.13) (IL, U.S.A.). Statistical
significance was set a priori at p<0.05.
RESULTS
Isolation and Structural Identification of Violaxanthin
Table 2. Primer Sequences Used for Real-Time RT-PCR
Gene Primer Amino acid sequences Product size (bp) Accession No.
iNOS 5′ Primer 5′- TGCCCCTGGAAGTTTCTCTT-3′252 M87039.1
3′ Primer 5′- ACTGCCCCAGTTTTTGATCC-3′
COX-2 5′ Primer 5′- CCCAGAGCTCCTTTTCAACC-3′241 NM_011198
3′ Primer 5′- AATTGGCACATTTCTTCCCC-3′
β-Actin 5′ Primer 5′- TACAGCTTCACCACCACAGC-3′
291 NM_007393
3′ Primer 5′- AAGGAAGGCTGGAAAAGAGC-3′
1140 Vol. 35, No. 7
The ethyl acetate fraction of C. ellipsoidea was further puri-
fied to isolate violaxanthin using silica gel and the target com-
pound fraction was collected based on the absorption spectra.
The visible spectrum characteristics (418, 442, 472 nm) and
spectral fine structural values (% III/II), are presented in Fig.
1A and Table 1, respectively, which were in agreement with
previous reports.21,22) In the epoxide test, a hypsochromic shift
of 40 nm was observed after the addition of HCl, which con-
firmed the presence of two epoxides. The peak purity of viola-
xanthin was determined to be 86% based on the chromato-
graphic purity (Fig. 1B). The mass spectrum corresponding to
the molecular weight of violaxanthin was 600 Da according to
m/z 601 [M+H]+ and fragments at m/z 583 [M+H−H2O]+ rep-
resented the elimination of one H2O (Fig. 1C). This compound
was identified as 5,6 : 5′,6'-Diepoxy-5,5′,6,6'-tetrahydro-β-
carotene-3,3′-diol or (all-E)-violaxanthin (Fig. 2) by comparing
its 1H-,13C-NMR with those found in the literature.23–25)
Effect of Violaxanthin on RAW 264.7 Cell Vability and
NO Production The cytotoxic effects of different concentra-
tions of violaxanthin (10–60 µM) on RAW 264.7 cells were de-
termined after incubation for 24 h. In these experiments, vio-
laxanthin did not affect the viability of RAW 264.7 at concen-
trations<100 µM. Hence, the inhibitory effect of violaxanthin
on the LPS-induced RAW 264.7 macrophages was deemed not
to be attributable to cytotoxicity. The effect of violaxanthin
on the cytotoxicity of R AW 264.7 cells was then investigated
over a broader range of concentrations (1 to 250 µM). Incuba-
tion of RAW 264.7 cells with violaxanthin produced a dose-
and time-dependent increase in cell cytotoxicity as measured
by LDH release. Figure 3A shows that violaxanthin concentra-
tions under 100 µM did not significantly increase LDH release
after exposure for up to 24 h, but a higher concentration
(250 µM) induced a significant increase in LDH release when
incubated for 24 h. The greatest cytotoxic effect (>90%) was
Fig. 1. Results of HPLC-ESI-MS
(A) Abs orbance spectr a of the purified compound peak recorded in th e high-perform ance liquid chromatography (HPLC) run with a diode array detector; (B), H PLC
profile of viola xanth in f rom C hlorella ellipsoidea; (C), Mass sp ectr um of violaxanthi n f rom C . elli psoidea.
Fig. 2. Chemical Structure of Violaxanthin
July 2012 1141
observed with 250 µM violaxanthin. Because the toxicity with
250 µM violaxanthin was so profound, higher concentrations
were not tested. Based on this result, a violaxanthin concen-
tration below 100 µM was used for further studies.
The potential anti-inflammatory properties of violaxanthin
in RAW 264.7 cells after 24 h of treatment with a mixture
of LPS (1 µg/mL) and violaxanthin (10 –60 µM) or LPS (1 µg/
mL) alone (positive control) was examined by adding the
Griess reagent to determine the concentration of nitrites (µM)
in cell super natants. Violaxanthin markedly inhibited the NO
production in LPS (1 µg/mL)-treated RAW 264.7 cells in a
dose-dependent manner (Fig. 3B) and this effect was maximal
at 60 µM (p<0.001).
Effect of Violaxanthin on LPS-Induced PGE2 Produc-
tion The effect of violaxanthin on PGE2 production was ex-
amined in RAW 264.7 macrophages. When the macrophages
were stimulated with LPS (1 µg/mL) for 24 h, the levels of
PGE2 production increased in the culture medium. Treatment
with violaxanthin at different concentrations significantly in-
hibited PGE2 production (Fig. 4). A dose-dependent effect of
violaxanthin was observed at 30 µM and maximized at 60 µM
(p<0.01 and p<0.001, respectively), which closely corre-
sponded to the positive control, cerecoxib.
Effect of Violaxanthin on the LPS-Induced iNOS and
COX-2 Expression To determine if violaxanthin inhibits
pro-inflammator y repertoires (iNOS and COX-2) at the gene
level, RAW 264.7 cells were stimulated by LPS for 24 h and
the expression of iNOS and COX-2 mRNAs was examined
in the presence of varying concentrations of violaxanthin
(10, 30, 60 µM). L-NMMA, an inhibitor of NO generation
Fig. 3. Effect of Violaxanthi n on LPS-Induced NO Production and Cy-
totoxicity in RAW 264.7 Cells
(A) RAW 264.7 cells were ex posed to viola xanth in (1, 10, 50, 100 and 250 µM) for
the indicated times. The concentrat ion depende nt a nd time dependent cytotoxicit y,
measu red as %LDH relea sed into cu lture supern atant, wa s compare d with cells
treat ed wit h 1% Triton X-100. (B) T he NO product ion was assayed fr om the culture
medium of cells stimulate d with LPS (1 µg/mL) in the pr esence of violaxa nthin for
24 h. T he ex periments were perfor med in t riplica te a nd the results are expressed a s
the mean±S.D. ** p<0.01, *** p<0.001 vs. LPS-treate d control group.
Fig. 4. Inhibitor y Effect of Violaxanth in on LPS-I nduced PGE2 Produc-
tion in RAW 264.7 Cells
PGE2 product ion was assayed fro m the cu lture med ium of c ells stimu lated
with LPS (1 µg/m L) i n the pr esence of violaxant hin for 24 h . Each value indicates
the mean±S.D. The experiments were performed in triplic ate and the results are
express ed as the mean±S.D. ** p<0.01 and *** p<0.001 vs. LPS-tr eated cont rol
group.
Fig. 5. Quantitative mR NA Analysis a nd Wester n Blot Profiles of iNOS
and COX-2
RAW 264.7 cells were stimulate d with LP S (1 µg/mL) in the absenc e or presence
of violaxanth in for 24 h. L-NMM A or Celecoxib were used as a positive control.
iNOS (A) and COX-2 (B) mRNA and protei n levels were analyzed using Rotor-
Gene 6000 Series Software 1.7 and 10% SDS-PAGE, respectively, as describ ed
in the Materials and Methods. For Wester n bot analysis, 60 µM violaxanth in was
treat ed. The experi ments were perfor med in t riplicate and the results are expresse d
as the mean±S.D. *** indicat es a sig nifica nt d iffere nce from the LPS -treat ed con-
trol group (p<0.001).
1142 Vol. 35, No. 7
from arginine, and Celecoxib, a selective COX-2 inhibitor,
were included for comparison.26, 27) The mRNA levels were
assessed using quantitative real-time PCR. As shown in Fig.
5A and 5B, RAW 264.7 cells were highly activated by LPS.
However, the mRNA expression levels for iNOS and COX-2
were significantly inhibited at violaxanthin concentrations
of 30 and 60 µM and the levels of inhibition were similar to
that produced by L-NMMA (50 µM) or Celecoxib (3 µM) treat-
ment. These results were also observed on the protein level by
Western blot analysis (upper right of each bar graph). These
findings clearly demonstrate that violaxanthin can effectively
control macrophage-derived pro-inflammatory repertoires af-
ter stimulation with LPS.
Inhibition of LPS-Induced NF-κB and ERK1/2 in RAW
264.7 Cells To confirm that violaxanthin might be correlated
with the inhibition of NF-κB and MAPK pathways, we exam-
ined translocation of NF-κB p65 in the cytosol by phosphory-
lation of IκB, followed by binding to specific DNA sequences
in the nucleus, and activation of ERK1/2. This can induce dis-
tinct transcriptional programs leading to macrophages activa-
tion trough LPS stimulation when NF-κB is translocated into
the nucleus, which allows it to bind to specific DNA sequenc-
es. Using immunoblot analysis of NF-κB p65 through the
EMSA supershift assay, we found that viola xanthin (60 µM)
effectively inhibited binding to specific NF-κB p65 DNA
sequences (Fig. 6A). Coincidently, IκBα, which binds to and
inhibits the transcriptional activity of NF-kB, and ERK1/2,
which is increased phosphorylation via LPS stimulation, were
remarkably attenuated in the presence of violaxanthin at 60 µM
(Fig. 6B). Interestingly, these results suggest that violaxanthin
might have an effective anti-inflammator y property in mac-
rophages that was comparable to the L-NMMA and celecoxib
controls.
DISCUSSION
In this study, violaxanthin was purified from C. ellipsoidea
and the anti-inflammatory effect of violaxanthin on LPS-
induced NO and PGE2 was examined in vitro. The mechanism
of this anti-inflammatory effect was also assessed by exam-
ining the expression of iNOS and COX-2 in LPS-stimulated
RAW 264.7 macrophages using qPCR and Western blot analy-
sis. In murine macrophage RAW 264.7 cells, which naturally
express TLR4 complexes, LPS induces iNOS transcription
and transduction with subsequent NO production.28,29) Further-
more, LPS stimulation is known to induce IκB proteolysis and
NF-κB nuclear translocation.30, 31) Therefore, RAW 264.7 cells
provide an excellent model for drug screening and subsequent
evaluation of potential inhibitors of the pathway leading to
iNOS induction and NO production. The reactive free radical
NO, which is synthesized by iNOS, is a major macrophage-
derived inflammatory mediator and has also been reported
to be involved in the development of inflammator y diseases.
Moreover, a large body of evidence suggests that prostaglan-
dins are involved in various pathophysiological processes,
including inflammation and carcinogenesis, and the inducible
cyclooxygenase isoform, COX-2, is mainly responsible for the
production of large amounts of these mediators. Based on this
information, we explored the anti-inflammatory activities of
S. micracanthum in LPS-induced NO and PGE2 production in
RAW 264.7 cells as well as NF-κB nuclear translocation. In
the present study, S. micracanthum downregulated the expres-
sion of iNOS and COX-2 proteins and RNA, indicating that
effects of S. micracanthum occur at the transcriptional level.
NO and PGE2 are produced by NOS and COX-2, respec-
tively, which are pro-inflammatory mediators involved in
the development of inflammatory human diseases.32) Com-
monly, NO plays an important role as an immune regulator
and neurotransmitter in a variety of tissues at physiological
concentrations.33) Overproduction of NO derived from iNOS
plays an important role in the pathogenesis of inflamma-
tion.34) On the other hand, PGE2 is a pleiotropic mediator
that causes pain, swelling, and stiffness.35) Hence, inhibitors
of NO and PGE2 production could be used as potential anti-
inflammatory agents. The results of this study demonstrated
that violaxanthin inhibited the production of NO and PGE2 in
a dose-dependent manner in RAW 264.7 cells. The effect of
violaxanthin on NO and PGE2 production was consistent with
the carotenoids, where β-carotene, lutein, and fucoxanthin
were shown to suppress NO production.36–38) Violaxanthin
concentrations of 30 and 60 µM significantly inhibited LPS-
induced NO production by RAW 264.7 cells. At 60 µM, viola-
xanthin significantly inhibited NO production by as much as
L-NMMA. Coincidently, iNOS mRNA and protein expression
were also significantly down-regulated. Moreover, violaxan-
thin reduced PGE2 production and expression of COX-2 at the
mRNA and protein level.
Fig. 6. Electrophoretic-Mobilit y Shif t Assay for NF-κB p65 and West-
ern Blot Profiles of Phosphorylated IκB and ERK
(A) LPS -stimu lated RAW 264.7 cells were t reated with violaxanthin (60 µM) and
L-NMM A (50 µM) for 6 0 m in, followed by preparation of nuclear extracts for EMSA
as described in t he Ma terial s an d Met hods. Lane 1, lab eled E MSA pr obe only w ith
untre ated sample. La ne 2, labeled EMSA probe w ith L-N MMA-t reated sample.
Lanes 3, lab eled EMSA probe with violaxanthi n. La ne 4, labeled EMSA pr obe wit h
LPS alone. (B) Analysis of phos phor-IκBα and -ER K in lysates of LPS-st imulated
RAW 264.7 cells after 6 h tr eatment . L-N MMA was used as a po sitive c ontrol.
July 2012 1143
TLRs are essential for innate host defense as well as for
controlling adaptive immune responses. Of these, TLR4 plays
an important role in activating NF-κB of LPS-stimulated
macrophages, followed by up-regulation of iNOS and COX-2
expression.39) At the molecular level, we investigated if viola-
xanthin appreciably suppressed the synthesis of iNOS and
COX-2 by inhibiting NF-κB nuclear translocation. NF-κB
activation can induce the overexpression of pro-inflammatory
genes in the nucleus, thereby initiating the inflammatory pro-
cesses. Interestingly, violaxanthin was shown to inhibit activa-
tion of NF-κB in LPS-activated RAW 264.7 cells in the gel
shift assay (EMSA). Coincidently, violaxanthin well inhibited
phosphorylation of ERK and IκBα, which induce the activa-
tion of LPS-dependent NF-κB and mitogen-activated protein
kinase (MAPK). These experimental findings suggest that
violaxanthin may exert its inhibitory effects on inflammation
by inhibiting the activation of NF-κB, followed by a reduction
in iNOS and COX-2 expression.
Several previous studies have reported that antioxidants,
such as β-carotene and astaxanthin, can inhibit NF-κB activ-
ity and suppress the expression of pro-inflammatory genes
as well as production of NO and PGE2, which might inhibit
NF-κB activity.36,40,41) These observations indicate that reactive
oxygen species are related to inflammatory gene expression
though the NF-κB signaling pathway. Recently, violaxanthin
was reported to have strong antioxidant properties in oxidative
stress and function as a chain breaking antioxidant in lipid
peroxidation.20) Therefore, the antioxidant ability of violaxan-
thin may be originated from inhibition of iNOS and COX-2
expression.
In summary, we suggested that LPS stimulated RAW 267.4
via the TLR4-ERK/ NF-κB in the pro-inflammatory signaling
pathway, and violaxanthin effectively inhibited these path-
ways. Taken together, our results revealed that violaxanthin
isolated from C. ellipsoidea can be a favorable candidate for
the treatment of inflammatory disorders, and the anti-inflam-
matory activity of violaxanthin may be based on its ability
to inhibit the expression of iNOS and COX-2 via inactivation
with NF-κB. Therefore, violaxanthin, a nonsynthetic natural
product, may serve as a safe and effective anti-inflammatory
agent, which could be used for therapeutic purposes.
Acknowledgements This research was supported by the
Grant of No. RTI05-01-02 from the Regional Technology In-
novation Program of the Ministry of Knowledge Economics
(MKE). W. Soontornchaiboon was the recipient of a graduate
fellowship provided by Brain Korea (BK21) program spon-
sored by the Ministry of Education, Science and Technology,
Republic of Korea.
REFERENCES
1) Henderson B, Poole S, Wilson M. Bacter ial moduli ns: a novel class
of vir ulence factors which cause host tissue pathology by inducing
cytokine synthesis. Microbiol. Rev., 60, 316–341 (1996).
2) Esposito E, Cuzzocrea S. The role of nitric oxide sy nthases i n lung
inflammation. Curr. Opin. Inve stig. Drugs, 8, 899 –909 (2007).
3) Stuhlmüller B, Unget hüm U, Scholze S, Martinez L, Backhaus M,
Kraetsch HG, Kinne RW, Burmester GR. Identification of known
and novel genes in activated monocytes from patients with rheuma-
toid a rth ritis. Arthritis Rheum ., 43, 775–790 (2000).
4) Hi rose M, Ishihara K, Saito A, Nakagawa T, Yamada S, Okuda K.
Expression of cytokines and inducible nitric oxide synthase in in-
flamed gingival tissue. J. Periodontol., 72, 590–597 (2001).
5) Blant z RC, Munger K. Role of nitr ic oxide in i nflammatory condi-
tions. Nephron, 90, 373 –378 (2002).
6) Mur akami A, Ohigashi H. Targeting NOX, INOS and COX-2 in in-
flammatory cells: chemoprevention using food phytochemicals. Int.
J. Cancer, 121, 2357–2363 (2007).
7) Zeilhofer HU, Brune K. Analgesic strategies beyond the inhibition
of cyclooxygenases. Trends Pharmacol. Sci., 27, 467–474 (2006).
8) Jachak SM. PGE synthase in hibitors as an alter native to COX-2
inhibitors. Curr. Opin. Investig. Drugs, 8, 411–415 (2007).
9) Itoh K, Udagawa N, Kobayash i K, Suda K, Li X, Takami M, Oka-
hashi N, Nishihara T, Takahashi N. Lipopolysaccharide promotes
the survival of osteoclasts via Toll-like receptor 4, but cy tokine pro-
duction of osteoclasts in response to lipopolysaccha ride is different
from that of macrophages. J. Immunol., 170, 3688–3695 (2003).
10) Hayden MS, Ghosh S. Signaling to NF-kappaB. Genes Dev., 18,
2195–2224 (2004).
11) Gouveia L, Raymundo A, Batista AP, Sousa I, Empis J. Chlorella
vulgaris and Haematococcus pluviales biomass as colouring and
antioxidant in food emulsions. Eur. Food Res. Technol., 222, 362–
367 (2006).
12) Hagen C, Braune W, Vogel K, Hä der PP. Functional aspects of sec-
ondar y carotenoids in Haematococcus lacust ris (Girod) ROSTAFINSK I
(Volvocales). V. Influences on photomovement. Plant Cell Environ.,
16, 991–995 (1993).
13) Taylor CB. Control of cyclic carotenoid biosynthesis: no lutein, no
problem! Plant Cell, 8, 1447–1450 (1996).
14) Raja R, Hemaiswarya S, Rengasamy R. Exploitation of Dunaliella
for β-carotene production. Appl. Microbiol. Biotechnol., 74, 517–523
(2007).
15) Rao AR, Sarada R, Ravishankar GA. Stabilization of astaxanthin
in edible oils and its use as an antioxidant. J. Sci. Food Agric., 87,
957–965 (2007).
16) Del Campo JA, Moreno J, Rodríguez H, Vargas M A, Rivas J, Guer-
rero MG. Carotenoid content of chlorophycean microalgae: factors
determining lutein accumulation in Muriellopsis sp. (Chlorophyta).
J. Biotechnol., 76, 51–59 (200 0).
17) Chihara M, Murano M. An Illustrated Guide to Marine Plankton in
Japan. Tokai Univer siry Press, Tokyo, p. 1574 (1997).
18) Cha KH, Koo SY, Lee DU. A ntiproliferative effects of carotenoids
extracted from Chlorella ellipsoidea and Chlorella vulgaris on
human colon cancer cells. J. Agric. Food Chem., 56, 10521–10526
(2008).
19) Kimura M, Rodriguez-Amaya DB. A scheme for obtai ning st an-
dards and HPLC quatificat ion of leafy vegetable carotenoids. Food
Chem., 78, 389–398 (2002).
20) Fu H, Xie B, Ma S, Zhu X, Fan G, Pan S. Evaluation of antioxidant
activit ies of principal carotenoids available in water spinch (Ipo-
moea aquatica). J. Food Compost. Anal., 24, 288–297 (2011).
21) Davies BH. Analysis of carotenoid pigments. Chem. Biochem. Plant
Pigments. 1965, 489–532 (1965).
22) Rodriguez-Garcia I, Guil-Guerrero JL. Evaluation of the anti-
oxidant activity of the three microalgal species for use as dietary
supplements and in the preservation of foods. Food Chem., 108,
1023–1026 (2008).
23) Acemoglu M, Uebelhar t P, Rey M, Eugster CH. Synthese von en-
antiomerenreinen Violaxa nthinen und verwa ndten Verbindungen.
Helv. Chim. Acta, 71, 931–956 (1988).
24) Märki-Fisher E, Buchecker R, Eugster CH, Englert G, Noack K,
Vecchi M. Absolute configuration of a nthra xanthin, ‘cis-arithra-
xanthin’ and of the stereoisomeric mutatoxanthins. Helv. Chim.
Acta, 65, 2198–2211 (1982).
25) Moss GP. Carbon-13 NM R spectra of carotenoids. Pure Appl.
Chem., 47, 97–102 (1976).
1144 Vol. 35, No. 7
26) Leiper J, Vallance P. Biological significance of endogenous methyl-
argin ines that inhibit nitric oxide synthases. Cardiovasc. Res., 43,
542–548 (1999).
27) Thun MJ, Henley SJ, Patrono C. Nonsteroidal anti-inflammatory
drugs as anticancer agents: mechanistic, pharmacologic, and clinical
issues. J. Natl. Cancer Inst., 94, 252–266 (2002).
28) Br unn GJ, Bungum MK, Johnson GB, Platt JL. Conditional signal-
ing by Toll-like receptor 4. FASEB J., 19, 872– 874 (2005).
29) MacMicking J, Xie QW, Nathan C. Nitric oxide and macrophage
function. Annu. Rev. Immunol., 15, 323–350 (1997).
30) Baeuerle PA, Henkel T. Function and activation of NF-kappaB in
the immune system. Annu. Rev. Immunol., 12, 141–179 (1994).
31) Baldwin AS. Control of oncogenesis and cancer therapy resistance
by the tra nscription factor N F-kappaB. J. Clin. Invest., 107, 241–246
(2001).
32) Guslandi M. Nitr ic oxide and inflammatory bowel diseases. Eur. J.
Clin. Invest., 28, 904–907 (1998).
33) Moncada S, Palmer R M, Higgs EA. Nitric oxide: physiolog y,
pathophysiology, and pharmacology. Pharmacol. Rev., 43, 109 –142
(1991).
34) Cherng SC, Cheng SN, Tarn A, Chou TC. Anti-inflam matory activ-
ity of c-phycocyanin in lipopolysaccha ride-stimulated RAW 264.7
macrophages. Life Sci., 81, 1431–1435 (2007).
35) Dinarello CA. Cytokines as endogenous pyrogens. J. Infect. Dis.,
179 (Suppl. 2), S294–S304 (1999).
36) Shao DZ, Lin M. Platonin inhibits LPS-induced NF-kappaB by pre-
venting activation of Akt and IKKbet a in human PBMC. Inflamm.
Res., 57, 601–606 (2008).
37) Bai SK, Lee SJ, Na HJ, Ha KS, Han JA, L ee H, Kwon YG, Chung
CK, Kim YM. β-Ca rotene inh ibits inflammator y gene expression in
lipopolysaccharide-stimulated macrophages by suppressing redox-
based NF-kappaB activation. Exp. Mol. Med., 37, 323–334 (2005).
38) Rafi MM, Shafaie Y. Dieta ry lutein modulates inducible nitric oxide
synthase (iNOS) gene and protein expression in mouse macrophage
cells (RAW 264.7). Mol. Nutr. Food Res., 51, 333–340 (2007).
39) Heo SJ, Yoon WJ, Kim KN, Ahn GN, Kang SM, Kang DH, Affan
A, Oh C, Jung W K, Jeon YJ. Evaluation of a nti-inflammatory effect
of fucoxanthin isolated from brown algae in lipopolysacchar ide-
stimulated R AW 264.7 macrophages. Food Chem. Toxicol., 48,
2045–2051 (2010).
40) Lee SJ, Bai SK, Lee K S, Nam koong S, Na HJ, Ha KS, Han JA, Yi m
SV, Chang K, Kwon YG, Lee SK, Kim YM. Astaxanthin inhibits
nitric oxide production and inflammatory gene expression by sup-
pressing I(κ)B kinase-dependent NF-kappaB activation. Mol. Cells,
16, 97–105 (2003).
41) Heiss E, Herhaus C, Klimo K, Bartsch H, Gerhäu ser C. Nuclear
factor κB is a molecular t arget for sulforaphane-mediated anti-
inflammator y mechanisms. J. Biol. Chem., 276, 32008–32015
(2001).