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328
Journal of Pharmacological Sciences
©2009 The Japanese Pharmacological Society
Full Paper
J Pharmacol Sci 111, 328 – 337 (2009)4
Antioxidant, Free Radical–Scavenging, and NF-κB–Inhibitory Activities
of Phytosteryl Ferulates: Structure–Activity Studies
Md. Shafiqul Islam1, Hiroshi Yoshida2, Naoaki Matsuki2, Kenichiro Ono2, Reiko Nagasaka3, Hideki Ushio3,
Ying Guo4, Toshiyuki Hiramatsu4, Takamitsu Hosoya4, Takahisa Murata1, Masatoshi Hori1,*,
and Hiroshi Ozaki1
1Department of Veterinary Pharmacology, 2Department of Veterinary Clinical Pathobiology,
Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
3Department of Food Science and Technology, Tokyo University of Marine Science and Technology,
Minato-ku, Tokyo 108-8477, Japan
4Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology,
4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan
Received May 12, 2009; Accepted September 26, 2009
Abstract. Some of the pharmacological properties of phytosteryl ferulates may be linked to
their antioxidant potential. In this study, 2,2-diphenyl-1-picrylhydrazyl (DPPH), electron spin
resonance (ESR), and thiobarbituric acid–reactive substances (TBARS) assays demonstrated that
phytosteryl ferulates such as cycloartenyl ferulate (CAF), 24-methylenecycloartanyl ferulate (24-
mCAF), and β-sitosteryl ferulate (β-SF) and ferulic acid (FA) each exerted strong free radical
scavenging and antioxidation of lipid membrane, which were comparable to α-tocopherol.
However, the sterol moiety alone, such as cycloartenol (CA), had neither activity. Since, the
reactive oxygen species (ROS) production in the cell complex defense mechanism cannot be
ruled out with the cell free system, we measured ROS production in NIH 3T3 fibroblast cells
induced by H2O2. CAF and ethyl ferulate (eFA) greatly decreased the ROS level in this system.
CA also significantly inhibited the ROS level, suggesting that CA could inhibit ROS production
in living cells. Besides these, CAF, 24-mCAF, β-SF, as well as eFA and CA, all these chemicals
significantly inhibited the NF-κB activity as analyzed by measuring translocation of NF-κB p65
in LPS-stimulated RAW 264.7 macrophages. These observations revealed that phytosteryl
ferulates are responsible for the antioxidant and anti-inflammatory activity via ROS scavenging
and inhibition of ROS production.
Keywords: phytosteryl ferulate, cycloartenyl ferulate, NF-κB, antioxidant,
reactive oxygen species (ROS)
Introduction
There has been considerable public and scientific
interest in the field of free radical chemistry and biology.
During the past 30 years, the fields of free radical
chemistry and biology have risen from relative obscurity
to become mainstream elements of biomedical investi-
gation and pharmaceutical development (1). Mitochon-
dria are an important source of reactive oxygen species
(ROS), and uncontrolled and/or sustained increase in
ROS production by mitochondria causes oxidative
damage to biological molecules, which could be impli-
cated in the pathogenesis of cancer, diabetes mellitus,
atherosclerosis, neurodegenerative diseases, rheumatoid
arthritis, ischemia/reperfusion injury, obstructive sleep
apnea, and other diseases (2). Therefore, the antioxidant
therapies could provide a potential means to treat condi-
tions in which the formation of reactive oxygen species
exceeds the capability of natural protective mechanisms
(3).
Identification of oxidant and antioxidant bioactive
compounds is important, not only for predicting and
*Corresponding author. ahori@mail.ecc.u-tokyo.ac.jp
Published online in J-STAGE on November 27, 2009 (in advance)
doi: 10.1254/jphs.09146FP
Structure–Activity of Steryl Ferulates 329
reducing health risks, but also for evaluating possible
combinatory beneficial effects of bioactive ingredients
in phyto-therapeutic formulations (4). Polyphenols have
attracted attention as a functional food with various
bioactivities including anticancer, antimutagenic, anti-
microbial, and antiviral activities (5). Caffeic acid, one
of the most commonly occurring phenolic acids in fruits,
grains, and dietary supplements, has shown strong anti-
oxidant efficacy against oxidative damage (6); and gallic
acid, one of the major constituents of grape seed
extracts, exhibited anticancer efficacy against human
prostate cancer cells and transgenic adenocarcinoma of
mouse prostate via a strong suppression of cell cycle
progression and cell proliferation and an increase in
apoptosis (7). Moreover, curcumin, the medicinal con-
stituent of turmeric (Curcuma longa rhizomes) has
received much attention recently as a promising dietary
supplement for its anti-inflammatory, anti-angiogenic,
and anti-oxidant effects; wound healing effects; and
anti-cancer effects (8).
Chemopreventive and chemoprotective phytochemi-
cals and phytonutrients can alter or correct undesired
cellular functions caused by abnormal pro-inflammatory
signal transmissions mediated by NF-κB (9). This key
regulator of inflammation as well as transcription factor
exists mainly as a heterodimer comprised of subunits
of the Rel family p50 and p65, which is normally seques-
tered in the cytosol as an inactive complex due to its
binding with inhibitors of κB (IκBs) in unstimulated
cells; activation of NF-κB involves the phosphorylation
of IκBs, and the resulting free NF-κB is then translocated
to the nucleus, where it binds to κB-binding sites in the
promoter regions of target genes and regulates the
expression of crucial mediators including pro-inflamma-
tory cytokines, chemokines, adhesion molecules, cyclo-
oxygenase (COX)-2, and inducible nitric oxide synthase
(iNOS) involved in chronic inflammatory disease and
cancer (10). It is well documented that ROS activate NF-
κB, which leads to the generation of pro-inflammatory
cytokines and inducible enzymes, such as COX-2 and
iNOS, in leukocytes and macrophages. Conversely, the
pro-inflammatory cytokines cause oxidative stress by
promoting the release of ROS by immune cells and also
by non-immune cells. Thus inflammation and oxidative
stress are involved in the spiraling vicious inflammatory
cycle (11, 12).
Rice bran is a component of raw rice that is obtained
when it is removed from the starchy endosperm in the
rice milling process. Rice bran oil derived from rice
bran has been found to possess promising health-related
benefits in the prevention of different diseases, including
cancer, hyperlipidemia, fatty liver, hypercalciuria, kidney
stones, and heart disease (13). Rice bran oil is a rich
source of γ-oryzanol (γ-ORZ), which contains a number
of phytosteryl ferulates such as 24-methylenecycloartanyl
ferulate (24-mCAF), cycloartenyl ferulate (CAF),
campesteryl ferulate, β-sitosteryl ferulate (β-SF), and
campestanyl ferulate; and the mixture of these is called
as γ-ORZ (14). It is of particular importance that γ-ORZ
exhibits antioxidant properties in in vitro systems (15).
The antioxidant potency of γ-ORZ makes it a good
candidate for pharmaceutical drugs, cosmetic formula-
tions, and health food (16).
Notably, we have recently reported that a phytosteryl
ferulates mixture, γ-ORZ, and its component CAF
inhibited NF-κB activity in dextran sulfate–induced
colitis in mice (17) and macrophages (18). In addition,
trans-ferulic acid (FA) and γ-ORZ also ameliorate
ethanol-induced liver injury in mice through enhancing
superoxide dismutase activity (19). Therefore, it seems
reasonable to assume that γ-ORZ components could
also be used for pharmaceutical purposes. Antioxidant
activity of steryl ferulates (γ-ORZ) has been shown to be
similar to that of non-esterified FA, indicating that the
FA moiety is responsible for the antioxidant properties
(20). The aim of this study was to investigate the
structure–activity relationships of FA and sterol moieties
of phytosteryl ferulates in relation to antioxidant and
NF-κB activities in a cell-free system and living cultured
cells.
Materials and Methods
Drugs and reagents
Drugs and reagents used in the experiment were,
2-thiobarbituric acid, 2',7'-dichlorodihydrofluorescein
diacetate (DCFH-DA), 2,2-diphenyl-1-picrylhydrazyl
(
DPPH), and
α
-tocopherol (Sigma-Aldrich, Tokyo); 2,2'-
azobis(2-methylpropionamidine) dihydrochloride, ferulic
acid (FA,
trans
-4-hydroxy-3-methoxycinnamic acid), and
ethyl ferulate (eFA, ethyl trans-4-hydroxy-3-methoxy-
cinnamate) (Wako Chemical, Osaka). γ-ORZ was puri-
fied from Japanese rice, Koshi-hikari, as reported earlier
(21).
CAF, β-SF, and 24-mCAF were purified from γ-ORZ
by repeated recrystalization as reported earlier (19).
Cycloartenol (CA) was prepared by alkaline hydrolysis
of CAF.
Determination of free radical–scavenging ability with
DPPH assay
The free radical–scavenging capacity of the phyto-
steryl ferulates, sterol, and FA were determined by using
DPPH according to the previously described procedure
(22). Antioxidant solution to be tested was added in a
final concentration of 1 – 60 μM to the reaction mixture.
MS Islam et al330
Absorbance of the DPPH-solution (0.45 mg/L in metha-
nol) was recorded at 517 nm using a spectrophotometer
(V-550; JASCO, Tokyo). α-Tocopherol was used as a
positive control.
Determination of antioxidation activity of lipid
membrane with thiobarbituric acid–reactive substances
(TBARS) assay
Modified thiobarbituric acid–reactive assay was used
to measure the potential antioxidant capacity using egg
yolk homogenates as lipid rich media. Malondialdehyde,
a secondary end product of the oxidation of polyunsatu-
rated fatty acids, reacts with two molecules of thiobarbi-
turic acid, yielding a pinkish red chromogen with an
absorbance maximum at 532 nm (23). Briefly, 0.1 mL of
10% (w/v) egg yolk homogenate and 1 – 60 μM of
sample to be tested was added to a test tube. A 0.01-mL
aliquot of 2,2'-azobis(2-methylpropionamidine) dihydro-
chloride
solution (0.07 M) in water was added to induce
lipid peroxidation. Then 0.3 mL of 20% acetic acid
(pH 3.5) and 0.5 mL of 0.8% (w/v) thiobarbituric acid
in 1.1% (w/v) sodium dodecyl sulphate solution was
added and the mixture adjusted to a final volume of
1.0 mL by adding distilled water. The resulting mixture
was vortexed and then heated in a water bath at 95°C
for 60 min. After cooling, 1.0 mL of 1-butanol was added
to each tube, extensively vortexed, and centrifuged at
1,200 ×g for 10 min. Absorbance of the organic upper
layer was measured using a spectrophotometer (V-550
or EMC-418; JASCO, Tokyo) set at 532 nm. All the
values were calculated on the basis of the percentage in
antioxidant index (AI %):
AI % =(1 −AT/AC)×100
Where AC is the absorbance value of the fully oxidized
control and AT is the absorbance of the test sample.
Cell culture and treatment
The NIH 3T3 (mouse embryonic fibroblast cell line)
cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% fetal bovine
serum (FBS), 100 U/mL penicillin, and 100 μg/mL
streptomycin. Cells were seeded at 1 ×106cells/mL and
grown at 37°C in 5% CO2 for 2 – 4 days in DMEM
supplemented with 10% fetal bovine serum. Treatment
with test compounds and H2O2 were carried out under
serum-free conditions.
Measurement of intracellular ROS with DCFH-DA assay
Intracellular ROS levels were measured by the
dichlorofluorescein assay, as described by T Teissier
et al. (24). Briefly, NIH 3T3 cells were pretreated with
10 μM CAF, eFA, and CA overnight followed by 2 h of
100 μM H2O2 stimulation. Afterwards, the cells were
washed twice with calcium-magnesium–free phosphate-
buffered saline (PBS) and incubated with 100 μM
DCFH-DA containing medium for 30 min. Medium was
removed; cells were washed with calcium-magnesium
free PBS and then solubilized in 1 mL of 1 M NaOH.
2',7'-Dichlorodihydrofluorescein (DCF) fluorescence
was read at λexc =485 nm and λem =530 nm using a
photospectrofluorometer (EMC-418, JASCO).
Immunohistochemical analysis of NF-κB p65 nuclear
translocation in RAW 264.7 macrophages
Immunofluorescence was employed to detect trans-
location of the p65 component of NF-κB to the nucleus
as descried by Lebron et al. (25). Briefly, RAW 264.7
macrophages were seeded onto sterile 22 ×22-mm glass
cover slips in 6-well tissue culture plates and pretreated
with 10 μM CAF, 24-mCAF, β-SF, eFA, or CA over-
night, followed by LPS for 2 h prior to fixation with
3.7% paraformaldehyde for 30 min at room temperature.
After fixation, the cells were subsequently perme-
abilized with 0.2% Triton X-100 for 30 min at room
temperature and rinsed with PBS. Blocking of non-
specific binding sites was performed with 3% goat
serum along with 1% BSA in PBS for 60 min at room
temperature. Rabbit polyclonal IgG against NF-κB p65
(Santa-Cruz Biotechnology, Tokyo), diluted 1:200 in
PBS containing 3% goat serum and 1% BSA, was added
and incubation carried out for 90 min at room tempera-
ture in a humidified chamber. The cover slips were
washed extensively and incubated for 60 min with a
1:500 dilution of Alexa Fluor 568–conjugated anti-
rabbit antibody. Nuclear staining was performed with
4',6-diamidino-2-phenylindole (DAPI, 0.1
µ
g/mL; Sigma-
Aldrich, Tokyo). Following extensive washing, the
cover slips were mounted on slides and analyzed by
confocal fluorescence microscopy (C1 Plus; Nikon,
Tokyo). Randomly, five fields of each slide were
counted to obtain the percentage of activated and non-
activated cells.
Hydroxyl radical–scavenging activity measured by
electron spin resonance spectroscopy (ESR)
The ESR spectra were recorded on a JEOL Model
JES-FA100 ESR spectrometer (JEOL, Tokyo) in accor-
dance with the previous study by Matsuki et al. (26).
Hydroxyl radical (⋅OH)–scavenging activity was deter-
mined by quantification of 5,5-dimethyl-1-pyrroline N-
oxide (DMPO)-OH spin trap adducts resulting from the
reaction of DMPO with the radicals generated by the
Fenton reaction. The experimental parameters were as
follows: temperature, ambient; microwave powder,
1.00 mW; modulation frequency, 100 kHz; modulation
width, 0.16 mT; sweep width, 5.0 mT; sweep time,
Structure–Activity of Steryl Ferulates 331
30.0 s; center field, 335.070 mT; and time constant,
0.03 s. A 40-μl aliquot of DMPO (1/10 diluted with ultra
pure water, UPW) was mixed with 76 μl of 0.2 mM
FeSO4⋅7H2O, 134 μl of UPW, and 0.4 μl of vehicle
(90% ethanol and 10% dimethyl sulfoxide mixture) or
40 mM stock chemical solution to make the final
concentration 40 μM each. The mixture was stirred,
added with 150 μl of 1 mM H2O2, and again stirred.
The solution was then transferred to a capillary tube
placed in the cavity of the ESR spectrometer for mea-
surement. After 5 min, the ESR signal was taken to mea-
sure the inhibition of ⋅OH radical by chemicals. The
inhibition was determined by the ratio of the peak height
of the second signal from the DMPO-OH spin adduct.
Statistical analyses
Numerical data are expressed as the mean ±S.E.M.
Statistical evaluation was carried out by Student’s t-test
and, where appropriate, one-way ANOVA; Tukey’s
multiple comparison test was applied.
Results
FA and sterol moieties in phytosteryl ferulates
In the early period, γ-ORZ was originally considered
as a single compound but later it has been characterized
as a mixture of FA esters of several sterols and triterpene
alcohols called α, β, and γ-ORZ, of which γ-ORZ has
been the most commonly mentioned compound. All
components of γ-ORZ contain one unit of FA, that is, a
FA moiety, and the residual moiety is a triterpene
alcohol or sterol similar to cycloartenol. According to
the nomenclature of the sterol moieties, steryl ferulates
differ principally in 1) presence or absence of a double
bond between C-5 and C-6; 2) the number of methyl
groups attached to C-4 classified as 4-desmethylsterols;
normal phytosterols without methyl group at C-4 posi-
tion; 3) 4-monomethyl; 4) 4,4-dimethylsterols, also
known as triterpene alcohols; and 5) the structure of
the side chain attached to the tetracyclic ring system
(27). To investigate the structure–activity relationship
of phytosteryl ferulates in relation to antioxidant, free
radical–scavenging, and anti-inflammatory activity, we
selected 4,4-dimethylsterol ferulates such as CAF and
24-mCAF; a 4-desmethylsterol ferulate such as β-SF; a
sterol such as CA; and non-sterols such as FA and eFA
(Fig. 1).
Effects of phytosteryl ferulates and sterol on DPPH free
radical–scavenging ability
To evaluate the structural and functional relationship
of FA and sterol moieties in phytosteryl ferulates for
free radical–scavenging ability, we investigated CAF,
24-mCAF, β-SF, FA, and CA (1 – 60 μM) in comparison
with that of α-tocopherol. The bleaching of DPPH
absorption is considered the representative capacity of
test compounds to scavenge free radicals independently
from any enzymatic activity. Our results demonstrated
that all phytosteryl ferulates such as CAF, 24-mCAF,
and β-SF and also FA strongly scavenged the free
radical dose-dependently (comparable to α-tocopherol);
however, CA, the sterol moiety alone, completely failed
to scavenge the free radical (Fig. 2a). Rice bran phyto-
steryl ferulates contain one unit of FA acid and the
residual moiety is triterpene alcohol or a sterol similar to
CA (Fig. 1). The 4-hydroxyl group of FA is an active
unit in the free radical–scavenging and antioxidant
reactions. Therefore, the hydroxyl group of phytosteryl
ferulates (equivalent to the 4-hydroxyl group of FA)
plays a pivotal role in the free radical–scavenging reac-
tion.
Effects of phytosteryl ferulates and sterol on cholesterol
oxidation accelerated by 2,2'-azobis(2-methylpropiona-
midine) dihydrochloride
Free radical scavengers are considered to be inhibitors
of lipid peroxidation. Therefore, we next evaluated the
structure–activity relationship of FA and sterol moieties
of phytosteryl ferulates using CAF, 24-mCAF, β-SF,
FA, and CA (1 – 60 μM) by estimating the free malon-
dialdehyde by the TBARS assay. As we expected,
phytosteryl ferulates (CAF, 24-mCAF, β-SF, and FA)
exhibited strong antioxidant potency (comparable to α-
tocopherol), whereas, sterol CA completely failed to
exhibit any antioxidant potency in the cholesterol oxida-
tion system (Fig. 2b). In accordance with the DPPH
assay, the FA moiety of the phytosteryl ferulates might
be responsible for these compounds having an anti-
oxidant potency like FA does.
Antioxidative effect of phytosteryl ferulates and sterol on
H2O2-induced oxidative stress in NIH 3T3 fibroblast
cells
To investigate the pharmacological action of CAF,
ethylester FA, and CA against oxidative stress, we
investigated H2O2-induced oxidative stress in NIH 3T3
cells using the latent fluorescent probe DCFH-DA. The
non-fluorescent molecule DCFH-DA freely permeates
into the cells. After incorporation into the cells, its
acetate moiety is rapidly hydrolyzed with esterase
followed by oxidation by cellular oxidative substances
affording fluorescent DCF. Therefore, DCFH-DA reveals
the intracellular production of redox-active substances
to investigate oxidative damage in intact cells (28).
Expectedly, 10 μM CAF and eFA exhibited strong anti-
oxidant potency; surprisingly, however, 10 μM CA, a
MS Islam et al332
sterol moiety alone, also significantly (P<0.05) sup-
pressed the H2O2-induced oxidative stress (Fig. 3).
Effects of phytosteryl ferulates and sterol on LPS-
induced NF-κB p65 translocation in RAW 264.7 macro-
phages
Degradation of cytosolic IκBα enables NF-κB sub-
units to migrate to the nuclear compartment, where they
promote gene transcription (25). Since the p65 subunit
has been demonstrated to exert critical activity in the
transcription of many inflammatory genes, we investi-
gated the effects on LPS-induced translocation of NF-κB
p65 in RAW 264.7 macrophages. Our results demon-
strated that LPS (10 ng/mL) exhibited NF-κB p65 trans-
Fig. 1. Structural nomenclature of phytosteryl ferulate showing ferulic acid and the sterol moiety (IUPAC-IUB 1989) (I),
cycloartenyl ferulate (CAF,
principal components of
γ
-oryzanol), 24-methylenecycloartanyl
ferulate (24-mCAF), and β-sitosteryl
ferulate (β-SF) showing ferulic acid and sterol moieties (II – IV); cycloartenol (CA) showing side chains and the tetracyclic ring
system (V); α-tocopherol (VI); ferulic acid (FA) (VII); and ethyl ferulate (eFA) (VIII).
Structure–Activity of Steryl Ferulates 333
location from the cytoplasm into the nuclei of the
macrophages. According to the results in the cell based-
oxidative stress assay (Fig. 3), CAF, 24-mCAF, β-SF,
CA, or eFA at 10 μM each significantly inhibited nuclear
translocation of NF-κB p65 in macrophages (Figs. 4: a
and b).
Effects of phytosteryl ferulates and sterol on the inhibi-
tion of hydroxyl radical
The
⋅
OH-scavenging activities of rice bran constituents
purified from γ-ORZ were assessed by ESR spectros-
copy. The ESR-spin trapping technique is widely used
and is a precise method for the measurement of ⋅OH-
scavenging activity. In Fig. 5, the relative signal inten-
sity of DMPO-OH spin trap adducts resulting from the
reaction of DMPO with the radicals generated by the
Fenton reaction in the presence or absence of phytosteryl
ferulates, sterol, or α-tocopherol have been demon-
strated. The height of the second peak of the spectrum
represented the relative amount of DMPO-OH adduct.
Addition of the phytosteryl ferulate compound: CAF,
24-mCAF, or β-SF; FA, or α-tocopherol, at 40 μM each,
significantly (P<0.05 and P<0.01) decreased the amount
of DMPO-OH adduct. It is to be noted that, β-SF and
FA strongly (P<0.01) decreased the DMPO-OH adduct.
On the other hand, the sterol compound CA did not
decrease the DMPO-OH adduct significantly (Fig. 5).
Discussion
There is emerging interest in the use of naturally
occurring antioxidants for the management of a number
of pathophysiological conditions, most of which involve
free radical damage (29). Dietary antioxidant is one of
the defense mechanisms protecting the body against the
damaging effects of ROS and may reduce the risk of
Fig. 2. Free radical–scavenging and anti-
oxidant potency of phytosteryl ferulates
and sterol in solution. a) Radical-scaveng-
ing ability of CAF, 24-mCAF, β-SF, FA,
and CA (1, 20, 40, and 60 μM) in compari-
son with α-tocopherol. Scavenging activity
was measured using the 2,2-diphenyl-1-
picrylhydrazyl assay (see Materials and
Methods). b) Antioxidant potency of CAF,
24-mCAF, β-SF, FA, and CA (1, 20, 40,
and 60 μM) in comparison with that of α-
Toco. Antioxidant potency was evaluated
by estimating the free malondialdehyde
using the TBARS assay (see Materials
and Methods).Values are expressed as the
mean ±S.E.M. from three individual
experiments. α-Toco, α-tocopherol; CAF,
cycloartenyl ferulate; 24-mCAF, 24-
methylenecycloartanyl ferulate; β-SF, β-
sitosteryl ferulate; FA, ferulic acid; CA,
cycloartenol.
MS Islam et al334
coronary heart disease, cancer, and other aging-associated
diseases (30). To explore the mechanism underlying the
antioxidant, free radical, and anti-inflammatory activity,
we investigated the effects of phytosteryl ferulates on
inflammation-related macrophages, fibroblasts, and
chemical reactions.
In this study, we initially measured antioxidant and
free radical–scavenging activities using TBARS and
DPPH assays, respectively. The results demonstrated
that CAF, 24-mCAF, and β-SF and also FA had strong
free radical–scavenging and antioxidant potencies, which
were comparable to those of α-tocopherol. However, the
sterol moiety, like CA, had neither antioxidant nor free
radical–scavenging activity. It is known that the anti-
oxidant activity of phenols depends on the electronic and
steric effects of the ring, substituents, and the strength of
hydrogen-bonding interactions between the phenol and
the solvent (31). Therefore, it is suggested that the
common FA-derived structure of CAF, 24-mCAF, and
β-SF with a phenolic hydroxyl group under the similar
situation exhibited both antioxidant potency and scav-
enging activity (Fig. 2: a and b). To confirm the ROS-
scavenging activity of γ-ORZ and the related com-
pounds, we further analyzed ROS-scavenging activity
by using ESR. Our results indicated that phytosteryl
ferulates compounds such as CAF, 24-mCAF, β-SF, and
FA significantly decreased the amount of DMPO-OH
adduct (Fig. 5), suggesting direct evidence that these
Fig. 3. Antioxidative effects of CAF, eFA and CA on H2O2-induced
oxidative stress in NIH 3T3 fibroblast cells. Cells were pretreated
with 10 μM CAF, eFA, or CA overnight followed by 100 μM H2O2
stimulation for 2 h. Afterwards, the cells were washed twice with
calcium-magnesium free PBS and incubated with 100 μM DCFH-
DA–containing medium for 30 min. Medium was removed; the cells
were washed with calcium-magnesium free PBS and then solubilized
in 1 mL of 1 M NaOH. DCF fluorescence was read at λexc =485 nm
and λem =530 nm. The data represent the mean ±S.E.M. (n =5–8).
*P<0.05, compared to the control and #P<0.05, compared to the
treatment groups. CAF, cycloartenyl ferulate; eFA, ethyl ferulate;
CA, cycloartenol; AFU, arbitrary fluorescent unit.
Fig. 4. Effects of phytosteryl ferulates and sterols on NF-κB p65
nuclear translocation in RAW 264.7 macrophages. a) Inhibitory
effect of CAF, 24-mCAF, β-SF, CA, and eFA, 10 μM each, on NF-
κB activation in LPS-stimulated RAW 264.7 macrophages. The
anti-NF-κB p65–bound cells were incubated with Alexa Flour 568–
conjugated anti-rabbit IgG and analyzed using a fluorescence micro-
scope. Experiments were repeated three times and similar results
were obtained. Arrows indicate the nuclear translocation of NF-κB
p65. b) Graphical representation of % NF-κB–activated cells per
field. Values are expressed as percent mean ±S.E.M. from three
individual experiments. **Significantly different between LPS and
LPS + treatment group. CAF, cycloartenyl ferulate; 24-mCAF, 24-
methylenecycloartanyl ferulate; β-SF, β-sitosteryl ferulate; CA,
cycloartenol; eFA, ethyl ferulate.
Structure–Activity of Steryl Ferulates 335
compounds can scavenge hydroxyl radicals.
It has been reported that rice bran extract significantly
inhibits xanthine oxidase activity and formation of
superoxide anions, scavenges hydroxyl radicals in a cell-
free system (32), and enhances superoxide dismutase
(SOD) activity in a cell-based system by using RAW
264.7 macrophages stimulated with LPS (18). In the
present study, we also determined effects of phytosteryl
ferulates compounds in a cell-based ROS production
system. It was found that CAF and eFA, a membrane
permeable FA, greatly inhibited the ROS production in
NIH 3T3 fibroblast cells induced by H2O2 (Fig. 3). This
result supports our data obtained in the cell-free systems
and also indicates the possibility that γ-ORZ potentially
protects activities of glutathione, SOD, catalase, and
glutathione peroxidase, similar to the action of FA (33).
Further investigation is required to clarify this point.
In the cell-free system to measure antioxidant and free
radical–scavenging activities using TBARS and DPPH
assays, the sterol moiety of γ-ORZ, CA, had no anti-
oxidant and free radical–scavenging activities. To support
these data, we also confirmed that CA did not affect the
amount of DMPO-OH adduct in the ESR assay system.
However, it is notable that CA significantly inhibited the
intracellular ROS production in living cells stimulated
with H2O2. Sultana et al. (34) reported that topical appli-
cation of CA significantly inhibits epidermal ornithine
decarboxylase activity, DNA synthesis, lipid peroxida-
tion, xanthine oxidase activity, and protects glutathione
and phase II metabolizing enzymes in benzoyl peroxide
and UVB radiation–induced tumor promotion and oxi-
dative stress in murine skin. Several lines of evidence
suggest that cholesterol protects the phospholipids
bilayer from oxidative stress in living cells (35). Intra-
cellular ROS production is a complex enzymatic pheno-
mena involving a number of events including NAD(P)H
oxidase, xanthine oxidase, and the mitochondrial respi-
ratory chain (36). There are numerous sources of ROS
production in the cell complex defense mechanism that
cannot be ruled out with chemical reactions in solution.
During the course of radical oxidation, cholesterol
may play an important role in lipid bilayer stabilization
as cholesterol-modified cell membrane provoked the
effects on peroxidation, with corresponding increases in
oxidative damage in the cell, possibly as a consequence
of lipid bilayer destabilization (37). As CA, the sterol
moiety of γ-ORZ, has a similar chemical structure to
cholesterol, it will be possible to induce radical-scaveng-
ing ability in living cells. Further investigations are
required to clarify this point.
Recently, great attention has been focused on the
relationship between NF-κB and ROS. Reactive oxygen
species are involved in the initiation of the NF-κB–
activating cascade to trigger a series of inflammatory
genes (38). Inflammatory stimuli such as LPS triggers
ROS production via the activation of the NADPH-
oxidase system in macrophages (39). In addition, there is
evidence that an exogenous oxidant such as H2O2
induces NF-κB activation in some cell lines (40), and
high concentration of antioxidants abolish NF-κB
activation in response to LPS or hypoxia, suggesting
that ROS is involved in NF-
κ
B activation (11). However,
the relationship between NF-κB activation and ROS is
still controversial (11, 12, 41). In this study, we finally
analyzed NF-κB activity by measuring translocation of
NF-κB p65 in LPS-stimulated RAW 264.7 macro-
phages. It is to be noted that 10 μM α-tocopherol
partially inhibited NF-κB p65 nuclear translocation in
macrophages stimulated with higher concentration of
LPS (1 μg/ml) (data not shown), indicating the possi-
bility that LPS-stimulation may produce ROS, and α-
tocopherol may scavenge generated ROS. In this cell
system, we found that CAF, 24-mCAF, and β-SF as
well as eFA and CA significantly inhibited the NF-κB
activity (Fig. 4: a and b). The NF-κB–inhibitory activity
of CA could not be explained precisely, but several lines
of scientific evidence suggest that CA, the free triterpene
alcohol, has strong anti-inflammatory potency, inhibits
leucocyte infiltration against carrageenan-induced peri-
tonitis in mice, and inhibits phospholipase A2 (42).
Similarly, Akihisa et al. (43) also reported that CA acts
as a strong anti-inflammatory compound against 12-O-
tettradecanoylphorbol-13-acetate–induced inflammation
Fig. 5. Hydroxyl radical–scavenging potency of phytosteryl feru-
lates and sterol. Relative signal intensity of DMPO-OH spin trap
adducts resulting from the reaction of DMPO with the radicals
generated by the Fenton reaction was evaluated by ESR assay.
Comparison of the ·OH-scavenging ability of CAF, 24-mCAF, β-SF,
FA, CA, and α-toco at 40 μM concentration (details described in
Materials and Methods). Each point represents the mean ±S.E.M.
*P<0.05 and **P<0.01: significantly different from the vehicle-
treated control (n =3). α-Toco, α-tocopherol; CAF, cycloartenyl
ferulate; 24-mCAF, 24-methylenecycloartanyl ferulate; β-SF, β-
sitosteryl ferulate; FA, ferulic acid; CA, cycloartenol.
MS Islam et al336
in mice. The CA-induced inhibitory action of NF-κB
p65 nuclear translocation may be partially contributed
by the antioxidant potency of CA, similar to the mecha-
nism of cholesterol.
Together with our previous report demonstrating an
anti-inflammatory effect of phytosteryl ferulates on the
intestinal inflammation model (17) and ethanol-induced
liver injury model (19), the present study suggested that
the modulation of cellular signaling involved in the
chronic inflammatory response by anti-inflammatory
phytochemicals may comprise a rational and pragmatic
strategy in molecular target–based chemoprevention.
In conclusion, our results suggest that phytosteryl
ferulates are responsible for the antioxidant and anti-
inflammatory activity in living cells via ROS scavenging
and inhibition of ROS production. We found that not
only the FA but also the sterol moiety exerts antioxida-
tive and anti-inflammatory activity in the complex cell
system.
Acknowledgments
This work was supported by Grants-in-Aid for Scientific Research
from the Ministry of Education, Culture, Sports, Science and Tech-
nology of Japan (to M.H., No. 18380173 and No. 21380178) and the
Yakult Bioscience Foundation.
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