The protective effects of oxyresveratrol imine derivative against hydrogen peroxide-induced cell death in PC12 cells

Article abstract of DOI:10.3109/10715762.2012.762769

Oxyresveratrol (2',3,4',5-tetrahydroxystilbene) is a naturally occurring ingredient found in mulberries that shows potential as an antioxidant, anti-inflammatory, and neuroprotective agent. This study was performed to identify materials similar to oxyresv

Full text of DOI:10.3109/10715762.2012.762769

Free Radical Research, March 2013; 47(3): 212–218
© 2013 Informa UK, Ltd.
ISSN 1071-5762 print/ISSN 1029-2470 online
DOI: 10.3109/10715762.2012.762769
ORIGINAL ARTICLE
The protective effects of oxyresveratrol imine derivative against
hydrogen peroxide-induced cell death in PC12 cells
J. Hur1, S. Kim2, P. Lee3, Y. M. Lee1 & S. Y. Choi1
1Korea Food Research Institute, Seongnam, South Korea, 2College of Pharmacy, Seoul National University, Seoul, South Korea,
and 3Department of Natural Medicine Resources, Semyung University, Jecheon, South Korea
Abstract
Oxyresveratrol (2′,3,4′,5-tetrahydroxystilbene) is a naturally occurring ingredient found in mulberries that shows potential as an
antioxidant, anti-inflammatory, and neuroprotective agent. This study was performed to identify materials similar to oxyresveratrol that may
have more effective antioxidant properties. We synthesized a stilbene analog referred to as Compound 1 (2′,3,4′,5-tetramethoxystilbene);
a benzamide analog referred to as Compound 2 ((2,4-dimethoxyphenyl)-3,5-dimethoxybenzamide); and three imine analogs referred to
as Compound 3 (3,5-dimethoxybenzylidene)-(2,4-dimethoxyphenylamine), Compound 4 ((4-methoxybenzylidene)-(3-methoxyphenyl)
amine), and Compound 5 ((4-methoxybenzylidene)phenylamine). The cytoprotective effects of these compounds were subsequently
evaluated using hydrogen peroxide-treated PC12 cells. The cytoprotective effects of the imine analogs were greater than the effects
of oxyresveratrol and the other analogs at concentrations of 200 μM. The Compound 3, which is the most effective imine analog of
oxyresveratrol, exhibited these cytoprotective effects against hydrogen peroxide-induced oxidative stress through the regulation of
heme oxygenase-1 (HO-1) expression and the translocation of nuclear factor E2-related factor 2 (Nrf2). Our results suggest that imine
analogs of oxyresveratrol may be useful agents in reducing neuronal oxidative damage.
Keywords: oxyresveratrol, derivative, anti-oxidant, cytoprotection, heme oxygenase
Introduction
(Keap1) in the cytoplasm. However, under stress condi-
tions, Nrf2 is released and translocates to the nucleus
[16,17]. In addition, Yu et al. have reported that mitogen-
activated protein kinase (MAPK) involves the modulation
of ARE-mediated gene expression such as Nrf2 [18].
Natural plant derivatives, such as curcumin and sesamine,
have been reported to upregulate Nrf2 expression, subse-
quently increasing HO-1 enzyme expression [17,19].
For the identification of a new class of antioxidative
agents, we examined the cytoprotective effects of resvera-
trol, oxyresveratrol, and five synthetic oxyresveratrol
derivatives (Compounds 1–5) on PC12 cells exposed to
hydrogen peroxide. Compounds 1–3 (stilbene, benzamide
and imine analogs, see Figure 1) all possess four methoxy
groups at the same position, as well as a transformed con-
nection chain between the two benzene rings; Compounds
4 and 5 are also imine analogs. In addition, we also exam-
ined the inhibitory activity on intracellular reactive oxy-
gen species (ROS) generation via HO-1 and Nrf2 signaling
in MAPK dependent manners.
Increases in oxidative stress are linked to cardiovascular
disease, aging, and many neurodegenerative diseases [1].
Resveratrol (trans-3,4’,5-trihydroxystilbene) is a compo-
nent of red wine that reduces the risk of cardiovascular
disease and most likely underlies the phenomenon known
as the “French paradox” [2–4]. The physiological benefits
of resveratrol are most likely due to its antioxidative and
free radical scavenging activities [5,6]. In addition,
resveratrol has exhibited direct neuroprotective effects
in vitro against neuronal cell death induced by hydrogen
peroxide (H2O2)-induced oxidative stress or other oxida-
tive agents [7–10].
Oxyresveratrol is a naturally occurring analog of hydrox-
ystilbene, which has one more hydroxy group than res-
veratrol. Oxyresveratrol has recently been reported to be a
more effective tyrosinase inhibitor and antioxidative agent
than resveratrol [11,12]. Moreover, oxyresveratrol strongly
prevents neuronal apoptotic cell death in an animal model
of cerebral ischemia [13–15]. Therefore, we proposed that
a search for new antioxidative materials among oxyres-
veratrol derivatives might reveal other neuroprotective
agents that are more potent than oxyresveratrol.
Materials
Heme oxygenase-1 (HO-1) is one of the phase II anti-
oxidant enzymes regulated by transcriptional factor nuclear
factor E2-related factor 2 (Nrf2). Under unstressed condi-
tions, Nrf2 binds to kelch-like ECH-associated protein 1
Generals
UV spectra were obtained using a Molecular Devices
E09090 microplate reader (Sunnyvale, CA) TLC and
Correspondence: Sang Yoon Choi, PhD, Korea Food Research Institute, 516 Baekhyeon-dong, Bundang-gu, Seongnam 463-746, South
Korea. Tel: ϩ82-31-780-9307. Fax: ϩ82-31-709-9876. E-mail: sychoi@kfri.re.kr
(Received date: 24 September 2012; Accepted date: 25 December 2012; Published online: 18 January 2013)

C y t o p r o t e c t i v e e ff ects of oxyresveratrol imine derivative 213
OH
(A)
OH
HO
HO
OH
OH
OH
Resveratrol
Oxyresveratrol
OCH3
OCH3
O
H3CO
H3CO
N
H
OCH3
OCH3
OCH3
OCH3
Compound 1
Compound 2
R6
R5
(B)
O
H
H2N
R6
R5
R1
R2
R1
N
Reflux
+
Recrystallization
R4
R2
R4
R3
R3
a
a
R1
OCH3
H
R2
R3
OCH3
H
R4
OCH3
H
R5
H
R6
OCH3
H
Compound 3
Compound 4
Compound 5
H
OCH3
OCH3
OCH3
H
H
H
H
H
Figure 1. The chemical structure of resveratrol, oxyresveratrol, Compounds 1 and 2 (A) and synthesis of compounds 3–5 (B).
column chromatography were carried out on precoated
silica gel F254 plates (Merck, Whitehouse Station, NJ)
and Silica gel 60 (Merck, 70–230 mesh). All other
solvents were of analytical grade. Solvents for chromato-
graphy and organic synthesis were used after further
distillation.
Cell culture
PC12 rat pheochromocytoma cells were obtained from the
American Type Culture Collection (Manassas, VA, USA)
and maintained in Dulbecco’s modification of Eagle
medium (DMEM) supplemented with 10% fetal bovine
serum, 5% horse serum, 2 mM glutamine, and 1% of pen-
icillin-streptomycin (Gibco-BRL, Gaithersburg, MD).
Cells were cultivated in 5% CO2 at 37°C incubator. The
cells were grown on culture dishes precoated with poly-
D-lysine (50 μg/ml).
Chemicals and synthesis
Resveratrol was purchased from Sigma (St. Louis, MO).
Oxyresveratrol and compounds 1–4 were synthesized as
previously reported [20,21]. Compound 5 ((4-methoxy-
benzylidene)phenylamine) was synthesized using the fol-
lowing method: p-anisaldehyde (1.2 ml, 10.0 mmol) and
aniline (910 μl, 10.0 mmol) were dissolved in methanol
(50 ml), and the reaction mixture was refluxed for 12 h.
The product (27 mg) was recrystallized at –20°C.
Exposure to H2O2 and cytoprotective effect
PC12 cells were pretreated with the test samples for
90 min before exposure to H2O2 (300 μM). After a 24-h
incubation, the lactate dehydrogenase (LDH) levels in the
culture supernatants were measured with a modification
of the method reported by Koh and Choi [22]. Briefly,
30 μl of substrate (1.4 mM β-nicotinamide adenine dinu-
cleotide in 0.75 mM pyruvate) and 30 μl of medium were
mixed in a 96-well plate. After a 30-min incubation at
37°C, 30 μl of 2,4-dinitrophenylhydrazine (1 mM in 1 N
HCl) was added to the mixture and incubated for 20 min
at room temperature. Subsequently, 100 μl of 0.4 N NaOH
was added to each well, and the UV absorption at 405 nm
was measured.
(4-methoxybenzylidene)phenylamine (Compound 5)
1
Yellowish solid; H-NMR (CDCl3, 300 MHz) δ: 8.39
(1H, s), 7.87 (1H, t, Jϭ2.7 Hz), 7.84 (1H, t, Jϭ2.7 Hz),
7.38 (2H, m), 7.21 (3H, m), 7.00 (1H, t, Jϭ2.7), 6.97
(1H, t, Jϭ2.7 Hz), 3.88 (3H, s). 13C-NMR (CDCl3,
75 MHz) δ: 162.24, 159.68, 152.35, 131.98, 130.50,
ϩ
129.26, 129.08, 125.54, 120.85, 114.17, 55.41. MS (EI )
ϩ
m/z 211(M , 100), 195(6), 167(24), 77(47).

214 J. Hur et al.
Cytoprotective effects (%)
20 µM
2 μM
200 µM
Resveratrol
Oxyresveratrol
Compound 1
Compound 2
Compound 3
Compound 4
Compound 5
4.9 5.1
4.8 6.2
0.5 4.0
0.9 3.4
9.3 5.6
9.6 4.8
6.1 4.9
29.8 4.5
33.1 5.8
3.1 4.6
–16.7 4.4
–9.6 6.3
0.6 2.7
5.5 4.9
0.2 1.9
29.9 5.6
25.3 6.8
14.6 5.4
81.2 6.1
70.5 7.0
63.7 5.5
Figure 2. The cytoprotective effects of resveratrol, oxyresveratrol, and Compounds 1–5 against hydrogen peroxide-induced injury in PC12
cells. The graph shows the effect of the 200 μM compounds under control conditions and when 300 μM H2O2 was added. The viability of
***
the untreated cells was set to 100%. Data are presented as the mean Ϯ S.E. of three independent experiments.
pϽ0.001 compared with
the control.
Extracellular hydrogen peroxide-reducing activity
dichloro-2-hydroxybenzenesulfonic acid was added,
mixed, and incubated for 15 minutes at room temperature
for color development. The absorbance was measured at
520 nm.
Hydrogen peroxide-reducing activity was determined by
the method of catalase assay kit (Sigma). A 25 μl of
200 mM H2O2 solution was added to 75 μl test samples
in 50 mM potassium phosphate buffer (pH 7.0), and the
mixtures were shaken vigorously. After an incubation for
5 min at room temperature, 900 μl of 15 mM sodium azide
in water was added to the mixture. To 10 μl of the reaction,
1 ml of the 150 mM potassium phosphate buffer (pH 7.0)
containing 0.25 mM 4-aminoantipyrine and 2 mM 3,5-
Intracellular ROS generation
Intracellular ROS generation was measured by fluorescent
microscopy using cells stained with the ROS-sensitive
fluorescent dye 2’,7’-dichlorofluorescein diacetate
(DCF-DA, Sigma). PC12 cells were incubated with H2O2
Figure 3. Changes in extracellular hydrogen peroxide levels by resveratrol, oxyresveratrol and Compound 3. H2O2-reducing activity was
*
measured using a catalase assay kit (Sigma CAT100). Data are presented as the mean Ϯ S.E. of three independent experiments. pϽ0.05
compared with the control.

C y t o p r o t e c t i v e e ff ects of oxyresveratrol imine derivative 215
Figure 4. The inhibitory effect of Compound 3 on intracellular ROS production in PC12 cells. The ROS levels were then analyzed by
fluorescent microscopy (Nickon Eclips Ti) using cells stained with the ROS-sensitive fluorescent dye 2’,7’-dichlorofluorescein diacetate
(DCF-DA). A, Control; B, Hydrogen peroxide; C, Hydrogen peroxide and 200 μM Compound 3. Scale barϭ100 micrometers. DAPI was
used for nuclei marker.
Results
for 1 h with or without Compound 3 (200 μM). PC12
cells were washed with PBS and incubated with 3 μM
DCF-DA for 30 min at 37°C. DCF-DA positive cells
were counted from ten randomly chosen microscopic
fields (Nickon). These ten measurements averaged and
regarded as a data point. Fluorescence was observed
using a fluorescent microscope (Nickon, Eclips Ti
microscopy) at 485–535 nm and DAPI dye was used for
nucleus marker.
Cytoprotection against H2O2-induced Injury
To measure the cytoprotective effect of Compounds 1–5
against oxidative stress, we used PC12 rat adrenal pheo-
chromocytoma cells. It is generally accepted that these
cells are similar to dopaminergic neuronal cells and are
quite sensitive to heavy metal ions and free radical attack
[9]. For the experiments, we pretreated PC12 cells with
Compounds 1–5 (Figure 1), resveratrol or oxyresveratrol
for 90 min and then exposed them to H2O2 for 24 h. The
parent compound oxyresveratrol exhibited 33.1% of its
protective effect at 20 μM. However, at a concentration
of 200 μM, oxyresveratrol did not reduce the H2O2-
induced oxidative cell injury (Figure 2). In contrast, the
same dose of imine analog Compounds 3, 4, and 5 medi-
ated 81.2, 70.5, and 63.0% reductions, respectively. The
stilbene (Compound 1) and benzamide (Compound 2)
analogs did not exhibit significant cytoprotective effects
on 300 μM H2O2-induced injury at any concentration
we tested (Figure 2). These results demonstrate that
compared to oxyresveratrol, imine analog Compounds 3,
4, and 5 more effectively protect PC12 cells from H2O2-
induced cytotoxicity at higher concentrations.
Western blot analysis
The mitochondrial and cytosolic components were
isolated using a nucleic/cytosolic fractionation kit (Bio-
Vision, Mountain View, CA) according to the manufac-
turer’s instruction. An equal amount of protein from
each sample was separated by SDS-poly-acrylamide gel
electrophoresis (10% gel) and transferred to Hybond
ECL nitrocellulose membrane. The membranes were
blocked with 5% skim milk and incubated with rabbit
anti-Nrf2 (1:1000 dilution; Abcam, Cambridge, UK),
rabbit anti-HO-1(1:2000 dilution; Enzo life science,
Farmingdale, NY), anti-Erk, phosphor-Erk, JNK, phos-
phor JNK antibody (1: 1000 dilution; Cell signaling
Technology, Danvers, MA), mouse beta-actin (1:4000
dilution; Santa-Cruz Scientific, Santa Cruz, CA), and
HRP-conjugated secondary antibodies (1:1000 dilu-
tion; Cell signaling Technology), this step was followed
by enhanced chemiluminescence detection (Bio-Rad,
Hercules, CA).
Extracellular H2O2-reducing activity
We used a catalase assay to ascertain the extracellular
H2O2 decomposing and scavenging ability of Compound
3. Although a difference in mechanical properties between
decomposing and scavenging activity existed, both the
activities commonly could have led to a reduction of the
amount of H2O2. Our results demonstrated that resveratrol
and oxyresveratrol reduced the H2O2 level in a dose-
dependent manner (Figure 3). However, Compound 3 did
not exhibit H2O2-reducing activity at the same dose.
Statistical analysis
All data were presented as the mean Ϯ SE from three
independent experiments. Statistical comparisons
between the different treatments were conducted with a
Student’s t-test.

216 J. Hur et al.
Figure 5. Compound 3 induced Nrf2 and HO-1 expression in MAPK dependent manner. PC12 cells were treated with Compound 3
(20, 200 μM) for 16 h. The effects of Compound 3 on the expression of HO-1 in the whole fraction and of Nrf2 (NRF2(N)) in the nuclear
fraction (A, B); The effects of Compound 3 on Erk and JNK phosphorylation (C, D). The results shown are representative of three
*
**
independent experiments. pϽ0.05, pϽ0.01 compared with the control.
Inhibition of ROS generation in PC12 cells
in Figure 5A and B). PC12 cells treated with Compound
3 showed significant increases in HO-1 and Nrf2 protein
levels by over 2.0 and 80 fold, respectively, when treated
with 200 μM of Compound 3. These results suggest that
the antioxidative effects of Compound 3 may be medi-
ated through the upregulation of HO-1 and Nrf2. To
investigate the upstream pathway, we examined the acti-
vation of Erk and JNK, which have protective role in
oxidative stress. As shown Figure 5C and D, Compound
3 increases the phosphorylation of Erk and JNK, how-
ever, p38 is not phosphorylated by Compound 3 at16 h
incubation (data not shown).
Because Compound 3 did not cause extracellular H2O2-
reducing activity, we examined the effect of Compound
3 on ROS generation in PC12 cells. The excessive pro-
duction of ROS may lead to oxidative stress, loss of cell
function, and ultimately apoptosis [23]. To investigate
whether Compound 3 inhibits intracellular ROS in PC12
cells, we measured the level of ROS in PC12 cells treated
with H2O2 by using the fluorescent dye DCF-DA. Fewer
DCF-DA positive cells showed in the non-treated control
cells (Figure 4A) However, H2O2-treated PC12 cells
showed a 2-fold increase in ROS production (Figure 4B).
In contrast, PC12 cells treated with 200 μM of Com-
pound 3 exhibited significantly reduced DCF fluores-
cence (Figure 4C).
Discussion
Oxidative stress increases oxidative damage that plays a
role in neurodegenerative statements [24]. Recently, it has
been reported that many antioxidants may attenuate neu-
rodegenerative diseases [25]. In this study, we demon-
strated for the first time that oxyresveratrol derivatives,
such as Compound 3, have antioxidant properties on
H2O2-induced PC12 cell damage. Because Compounds
1–5 are modifications of the oxyresveratrol structure,
we compared the antioxidant effects of Compounds 1–5
Upregulation of HO-1 expression and Nrf2 translocation
in a MAPK dependent manner
To investigate the mechanism of ROS inhibition in PC12
cells, we examined the expression of HO-1 and Nrf2.
HO-1, which is regulated by Nrf2, is an important pro-
tein that provides a cellular defense against oxidative
stress. The protein levels of HO-1 and Nrf2 are shown

C y t o p r o t e c t i v e e ff ects of oxyresveratrol imine derivative 217
Figure 6. Scheme of the intracellular ROS inhibition mechanism by Compound 3. Compound 3 inhibited intracellular ROS generation via
HO-1 and Nrf2 signaling in MAPK dependent manners. The Erk and JNK pathways are involved in Compound 3-induced HO-1
expression.
to those of oxyresveratrol using cell-based and H2O2
reducing assays. Our results demonstrate the superior
cytoprotective effects of imine analog Compounds 3–5
compared to oxyresveratrol. As shown in Figure 2, Com-
pounds 3, 4, and 5 more effectively prevented the H2O2-
mediated cell death of PC12 cells compared to
oxyresveratrol. Compound 3 was most effective among
them. Moreover, PC12 cells treated with Compound 3
showed a reduction of intracellular ROS production. To
confirm the intracellular ROS inhibition effect of Com-
pound 3, we measured the ROS using DCF-DA. Our data
indicate that Compound 3 effectively blocked H2O2
released from the cells (Figure 4). However, Compound 3
did not affect extracellular H2O2-reducing activity. These
results suggest that the cytoprotective effects of Com-
pound 3 were not due to direct H2O2-reducing abilities but
result from inhibition of intracellular ROS production.
HO-1 is regarded as an important enzyme against
oxidative stress and a therapeutic enzyme in neurodegen-
erative disease [26]. In addition, many researches have
reported that MAPK and other protein kinase is an impor-
tant pathway for Nrf2 activation which related to HO-1
[27,28]. To investigate the intracellular signaling mecha-
nism of Compound 3, we measured the expression of
HO-1 and Nrf2 expression in PC12 cells treated with
Compound 3. As we expected, Compound 3 increased the
expression levels of these two proteins in the cytoplasm
and nucleus, respectively. Moreover, our results showed
that the Erk and JNK pathways are involved in Com-
pound 3 induced HO-1 (Figure 6). However, other kinase
study which is related the nrf2 translocation remains to be
elucidated.
In the present study, we are the first to report that oxy-
resveratrol imine derivatives can effectively protect cells

218 J. Hur et al.
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Declaration of interest
[15] Chao J, Yu MS, Ho YS, Wang M, Chang RC. Dietary oxyres-
veratrol prevents parkinsonian mimetic 6-hydroxydopamine
neurotoxicity. Free Radic Biol Med 2008;45:1019–1026.
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et al. Involvement of heme oxygenase-1 induction via Nrf2/
ARE activation in protection against H2O2-induced PC12 cell
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Bioorgan Med Chem 2011;19:1959–1965.
The authors report no declarations of interest. The authors
alone are responsible for the content and writing of
the paper.
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