Ryu et al , 2014

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ORIGINAL ARTICLE
Trans-anethole protects cortical neuronal cells against
oxygen–glucose deprivation/reoxygenation
Sangwoo Ryu • Geun Hee Seol • Hyeon Park •
In-Young Choi
Received: 20 January 2014 / Accepted: 3 April 2014 / Published online: 29 April 2014
� Springer-Verlag Italia 2014
Abstract Trans-anethole has been studied on pharma-
cological properties such as anti-inflammation, anti-oxi-
dative stress, antifungal and anticancer. However, to date,
the anti-ischemic effects of trans-anethole have not been
assessed. Therefore, we investigated the neuroprotection of
trans-anethole against oxygen–glucose deprivation/reox-
ygenation (OGD/R)-induced cortical neuronal cell injury,
an in vitro model of ischemia. The abilities of trans-ane-
thole to block excitotoxicity, oxidative stress and mito-
chondrial dysfunction were evaluated in OGD/R-induced
neurons. Trans-anethole significantly ameliorated OGD/R-
induced neuronal cell injury by attenuating the intracellular
calcium overload via the activation of NMDA receptors.
Trans-anethole also inhibited OGD/R-induced reactive
oxygen species overproduction, which may be derived
from the scavenging activity in peroxyl radicals, assessed
in an oxygen radical absorbance capacity assay. Further-
more, trans-anethole was shown to attenuate the depolar-
ization of mitochondrial transmembrane. These results
indicated that the neuroprotective effect of trans-anethole
on OGD/R-induced neuronal injury might be due to its
ability to inhibit excitotoxicity, oxidative stress and mito-
chondrial dysfunction. Considering these multiple path-
ways causing ischemic neuronal damage, the multi-
functional effect of trans-anethole suggested that it may be
effective in treating ischemic stroke.
Keywords Trans-anethole � Oxygen–glucose
deprivation/reoxygenation � Neuroprotection �
Excitotoxicity � Oxidative stress
Introduction
Trans-anethole-containing oils from fennel or anise have
been widely used in food flavors and traditional medicines.
Pure trans-anethole as one component has been recently
reported to have anti-inflammatory effects in various
models of inflammatory diseases [1–3]. Also, trans-ane-
thole has shown dose-dependent bimodal effects on con-
tractility of isolated rat aorta [4] and anti-oxidative activity
in a thiobarbituric acid reactive substances (TBARS) assay
[5]. However, the neuroprotective effect of trans-anethole
associated with cerebral ischemic injury and its underlying
mechanisms have still not been clarified.
Ischemic stroke, the leading cause of human death
worldwide, is primarily treated with tissue plasminogen
activator to re-canalize the occluded blood vessels, but this
has a narrow therapeutic window and only delays the
progression of hypoxia rather than preserving damaged
brain [6]. Ischemic brain damage is a consequence of
complicated pathological cascades including excitotoxic-
ity, oxidative stress and inflammation. Thus, a multi-
functional drug rather than a single-functional drug may be
required for effective neuroprotection against ischemic
stroke. Moreover, neuronal cells are more vulnerable to
injury than glial cells because they have higher energy
demands, less endogenous antioxidants and are susceptible
to excitotoxicity. Therefore, in the present study, we
S. Ryu and G. H. Seol contributed equally to this work.
S. Ryu � H. Park � I.-Y. Choi (&)
Department of Neuroscience, School of Medicine,
Korea University, Seoul 136-705, Republic of Korea
e-mail: iychoi@korea.ac.kr
G. H. Seol
Department of Basic Nursing Science, School of Nursing,
Korea University, Seoul 136-701, Republic of Korea
123
Neurol Sci (2014) 35:1541–1547
DOI 10.1007/s10072-014-1791-8
investigated whether trans-anethole could protect neuronal
cell injury in an in vitro model of cerebral ischemia, oxy-
gen–glucose deprivation/reoxygenation (OGD/R). Our
results demonstrated anti-ischemic effect of trans-anethole
in cortical neurons and its neuroprotective mechanisms
through anti-excitotoxic, anti-oxidative and mitochondrial
protective properties.
Materials and methods
Materials
5-(and-6)-chloromethyl-20,70-dichlorodihydrofluorescein
diacetate (CM-H2DCF-DA), Fluo-4 diacetate (Fluo-4-
AM), tetramethylrhodamine methyl ester (TMRM) and
B27 were obtained from Life Technologies Corporation
(NY, USA). Dulbecco’s modified Eagle medium
(DMEM) and fetal bovine serum (FBS) were pur-
chased from Thermo Fisher Scientific Inc. (MA, USA).
Trans-anethole, 2,20-azobis-(2-methylpropionamide)-
dihydrochloride (AAPH), 1,1-diphenyl-2-picrylhydrazyl
(DPPH) and all other drugs were purchased from Sigma-
Aldrich Co.
Primary cultures of neuron-enriched cortical cells
Rat cortical cells were prepared from 17 day-old Sprague–
Dawley rat embryos. Cortical cells (1.35 9 103 cells/mm2)
were cultured in DMEM supplemented with 10 % FBS,
1 % penicillin/streptomycin, 2 mM glutamine, 2 % B27,
30 mM HEPES and 5.5 lM b-mercaptoethanol. To reduce
glial proliferation, cells were treated with cytosine arabi-
noside 6 days after plating. The experiments were per-
formed on cultures 12–13 days after the initial plating.
Oxygen–glucose deprivation/reoxygenation (OGD/R)
and NMDA injury
Oxygen–glucose deprivation was induced by incubating
cells in a glucose-free DMEM (Sigma-Aldrich, St. Louis,
MO), within an anaerobic chamber (partial pressure of
oxygen \2 mmHg) for 1 h at 37 �C. OGD was terminated
by adding glucose (final concentration; 25 mM) under
normoxic conditions to allow reoxygenation (R). In con-
trast, the control group’s cells were continuously main-
tained in glucose-containing DMEM under normoxic
conditions.
To evoke excitotoxicity, cortical cells were treated with
100 lM NMDA for 10 min in EBSS containing 1.8 mM
CaCl2 and 25 mM glucose. After NMDA exposure, cells
were replaced with glucose-containing DMEM and incu-
bated for 8 h before the assessment of cell injury.
Where indicated trans-anethole or positive controls such
as MK801 or Trolox was applied immediately prior to the
initiation of OGD (or NMDA) and then maintained until
experiments were finished. They were used in the con-
centration of 10 lM except dose–response experiments.
Assessment of cell injury: LDH assay
Cell injury or death was assessed by morphological
examinations with a phase-contrast microscope (Leica,
Germany), and by measuring the amount of LDH released
into the culture medium using a diagnostic kit (Sigma-
Aldrich, Co.). The degree of cell injury was expressed as a
percentage of total LDH release, which was defined as the
amount released after repeated freeze/thaw cycles. Dose–
response experiments of trans-anethole in OGD/R or
NMDA model were performed between the ranges of 0.01
and 100 lM.
Measurement of intracellular calcium: Fluo-4-AM
assay
To detect intracellular calcium in OGD/R model, cells
were preloaded for 30 min, with 1 lM Fluo-4-AM in
DMEM (SigmaeAldrich, St. Louis, MO) containing
2.5 mM probenecid. After removing the loading medium,
cells underwent OGD/R. Immediately after the reoxygen-
ation, Fluo-4 fluorescence in three non-overlapping optical
regions (825 9 625 lm2) per sample was measured at
Ex488 nm/Em525 nm with a fluorescence microscope
(Leica, Germany) equipped with a digital camera. The
fluorescence intensity was quantified using an image ana-
lyzer (Saramsoft Co., Ltd., Korea) by a treatment-blinded
examiner.
In NMDA-treated cells, Fluo-4-AM was preloaded in
probenecid-containing EBSS. Fluo-4 fluorescence was
measured after 10 min of NMDA exposure. Detailed pro-
cess was the same with the measurement of intracellular
calcium in OGD/R model.
Measurement of intracellularoxidative stress:
CM-H2DCF-DA assay
The intracellular reactive oxygen species (ROS) level was
measured with CM-H2DCF-DA, which diffuses through
cell membranes and hydrolyzed by intracellular esterase to
its nonfluorescent CM-DCF-H. CM-DCF-H then reacts
with free radicals to form highly fluorescent CM-DCF.
After 3 h reoxygenation, cells were loaded with 1 lM CM-
H2DCF-DA in EBSS containing 2.5 mM probenecid for
10 min. After removing the loading medium, the DCF
fluorescence (Ex488 nm/Em525 nm) was measured and
quantified, as described in the method of Fluo-4-AM assay.
1542 Neurol Sci (2014) 35:1541–1547
123
Measurement of free radical scavenging activities:
oxygen radical absorbance capacity (ORAC) assay
and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay
In the ORAC assay, antioxidants (twofold serial diluted
from 12.5 lM) react to peroxyl radicals which were gen-
erated from 60 mM AAPH, in a competitive manner with 50
nM fluorescein. Fluorescence decay was measured every
5 min for 3 h at 37 �C using a fluorescence microplate
reader (at Ex 485 nm/Em 530 nm). Trolox equivalents (TE)
of trans-anethole were presented as its concentration, pro-
ducing the same net area-under the curve (AUC; net
AUC = AUCsample - AUCblank) of 50 lM Trolox.
In the DPPH reduction assay, antioxidants (0.001, 0.01,
0.025, 0.05, 0.1, 0.15, 0.2, 0.5, 1, 5 mM) were mixed with
23.6 lg/mL of DPPH, an organic nitrogen radical genera-
tor. After 30 min of incubation at 37 �C, the absorbance
was measured at 517 nm using a microplate reader
(Molecular Devices, USA). The scavenging activity of free
radicals was expressed as the percentage of maximum
inhibition [(Absmaximum - Abssample)/(Absmaximum -
Absminimum) 9 100], obtained from a standard curve gen-
erated using vitamin C.
Measurement of mitochondrial transmembrane
potential (MTP) in cells
The mitochondrial accumulation of TMRM is driven by
their membrane potential [7]. Cells were preloaded with 10
nM TMRM at 37 �C and then removed loading medium.
After OGD (1 h)/R (1.5 h), the TMRM fluorescence (at Ex
552 nm/Em = 570 nm) was measured and quantified, as
described in the method of Fluo-4-AM assay.
Statistical analysis
Independent samples were obtained from at least three
different primary cultures and two separate experiments (at
DIV 12 or 13). Sample size ‘‘n’’ was expressed as the
number of samples per group. The data were expressed as
mean ± standard deviation (SD) and analyzed for statisti-
cal significance using analysis of variance (ANOVA) fol-
lowed by a post hoc analysis using a Tukey’s test for
multiple comparisons. Otherwise, the data were expressed
as median and interquartile range (IQR: Q1–Q3) and ana-
lyzed by Kruskal–Wallis test followed by Mann–Whitney
U test. P values \0.05 were considered significant after a
Bonferroni correction.
Results
Trans-anethole inhibited OGD/R-induced cell death
in neuron-enriched cortical cells
We first evaluated whether trans-anethole protected neu-
ronal cells against OGD (1 h)/R (8 h). OGD/R-induced the
swelling or burst of the neuronal cell bodies and increased
a large amount of LDH release (Fig. 1a, b). The released
LDH primarily reflected neuronal injury under our exper-
imental conditions, because glia was resistant to OGD/R-
induced injury [8, 9]. Trans-anethole dose-dependently
reduced OGD/R-evoked LDH release (Fig. 1a, b). 10 lM
showed the significant decrease, compared with the drug-
untreated OGD/R group (36.3 ± 4.8 vs 63.8 ± 5.8,
**P \ 0.001).
Control OGD-R
OGD-R/TA OGD-R/MK
A B
Log [TA], µM
*
0
20
40
60
80
0.01 0.1 1 10 100
LD
H
 
re
le
as
e 
(%
 
o
f t
ot
al
) ControlOGD-R
MK, µM
***
***
0 10
Fig. 1 Trans-anethole attenuated OGD/R-evoked neuronal cell
injury. Cortical neurons were exposed to oxygen–glucose deprivation
(OGD, 1 h) and reoxygenation (8 h). Cells were pretreated with
trans-anethole [TA; at a 10 lM and b indicated concentrations] or
MK801 (MK, 10 lM) immediately before OGD/R. a Representative
phase-contrast images. Cell injury was assessed morphologically.
Scale bar 50 lm. b Trans-anethole inhibited OGD/R-evoked LDH
release. Cell death was assessed by measuring the activity of LDH
released from the culture medium 8 h after reoxygenation and
quantified as the percentage of total amount of cellular LDH. Data are
expressed as mean ± SEM and analyzed by one-way ANOVA
followed by Tukey’s post hoc test. n = 13. *P \ 0.05, ***P \ 0.001,
significantly different from the drug-untreated OGD/R group
Neurol Sci (2014) 35:1541–1547 1543
123
Trans-anethole reduced OGD/R-induced
the intracellular calcium overload
Overstimulation of glutamate receptors induces excessive
calcium influx, which is considered to be a primary trigger
for excitotoxic cascades during ischemic injury [10]. We
therefore examined whether trans-anethole reduced the
intracellular calcium overload, immediately after the end of
OGD exposure. As the most effective concentration,
10 lM of trans-anethole reduced cytosolic calcium over-
load, caused by OGD/R [Fig. 2; 194.3 (IQR: 168.2–212.7)
vs 100.6 (90.4–126.1), *P \ 0.05]. Furthermore, to verify
the anti-excitotoxicity of trans-anethole, we tested in
NMDA injury model. NMDA markedly increased the LDH
amount (56.6 ± 6.6), which was dose-dependently inhib-
ited by trans-anethole (Fig. 3a; 27.2 ± 4.4 at 10 lM,
**P \ 0.001). Trans-anethole also showed the decrease in
NMDA-induced calcium overload [Fig. 3b; 87.7 (IQR:
81.5–104.6) vs 209.2 (IQR: 181.5–258.5), **P \ 0.01].
Trans-anethole reduced OGD/R-induced ROS
overproduction and directly scavenged free
radicals in ORAC assay
The prolonged accumulation of calcium following the
activation of glutamate receptors increases the production
A 
Fl
uo
-4
 
(F
.
 
I.)
250
200
150
100
50
0 
Control - TA MK
OGD-R
* 
B 
Control OGD-R
OGD-R/TA OGD-R/MK
Fig. 2 Trans-anethole reduced OGD/R-induced intracellular calcium
influx. Cells were treated with trans-anethole (TA, 10 lM) or MK801
(MK, 10 lM), followed by OGD (1 h), and intracellular calcium
levels were measured immediately after. a Representative fluorescent
images. Scale bar 50 lm. b Quantification of Fluo-4 fluorescence
(fluorescence intensities; FI). n = 5. Horizontal bar, median; vertical
box, interquartile ranges (Q1–Q3); and whiskers, minimum/maxi-
mum. *P \ 0.05, **P \ 0.01, significantly different from the indi-
cated groups
Fl
uo
-4
 
(F
.
 
I.)
300
250
200
150
100
50
0
Control - TA MK
NMDA
**A B
0
20
40
60
80
0.01 0.1 1 10 100
LD
H
 
re
le
as
e 
(%
 
o
f t
ot
al
) Control
NMDA
Log [TA], µM MK, µM
0 
*** ***
*
10
Fig. 3 Anti-excitotoxicity of trans-anethole. Trans-anethole decreased
NMDA-induced neuronal cell death and calcium overload. a LDH
assay. Cells were treated with 100 lM of NMDA for 10 min and then
incubated for 8 h in the presence or absence of drugs [trans-anethole
(TA, indicated concentrations) or MK801 (MK, 10 lM)]. Data are
expressed as mean ± SEM and analyzed by one-way ANOVA
followed by Tukey’s post hoc test. n = 12. *P \ 0.05, **P \ 0.01,
***P \ 0.001, significantly different from the drug-untreated NMDA
group. b Quantification of Fluo-4 fluorescence. Cells were treated
with trans-anethole (TA, 10 lM) or MK801 (MK, 10 lM), followed
by NMDA (10 min), and then intracellular calcium was measured
immediately after. Horizontal bar, median; vertical box, interquartile
ranges (Q1–Q3); and whiskers, minimum/maximum. n = 5.
*P \ 0.05, ***P \ 0.001, significantly different from the indicated
groups
1544 Neurol Sci (2014) 35:1541–1547
123of free radicals, particularly following reperfusion (or
reoxygenation) after ischemia [11]. To evaluate the anti-
oxidant effect of trans-anethole in OGD/R-exposed cortical
cells, we measured the intracellular levels of free radicals.
Mostly neuronal cell bodies in OGD/R-exposed conditions
were brightly fluorescent. OGD/R-induced ROS overpro-
duction was significantly reduced by trans-anethole
(Fig. 4a, b; 74.45 (IQR: 56.4–84.3) vs 134.5 (IQR:
126.2–142.8), **P \ 0.01), to a similar degree as Trolox.
We tested the chemical scavenging effect of trans-ane-
thole, on hydrogen atom transfer using ORAC assay and on
electron transfer using DPPH assay [12]. In the ORAC
assay, trans-anethole delayed the AAPH-induced fluores-
cence decay of fluorescein (Fig. 4c, d), with 15.5 lM of a
Trolox equivalent (TE) (Fig. 4e). In contrast, trans-ane-
thole did not react with DPPH radicals (Fig. 4f), as pre-
viously studied [13]. Generally, many antioxidants that
react with peroxyl radicals may show little or no reaction
with DPPH radicals [12].
Trans-anethole attenuated the decrease in MTP
in OGD/R-induced cortical cells
Intracellular calcium overload and/or ROS overproduction
cause mitochondrial dysfunction such as the decrease in
MTP and the irreversible opening of mitochondrial per-
meability transition pore (MPTP) [14, 15]. Mitochondrial
injury is observed in numerous brain diseases including
cerebral ischemia/reperfusion [16]. Therefore, we exam-
ined that trans-anethole inhibits the mitochondrial depo-
larization caused by OGD/R. As time goes by, OGD/R
gradually induced the decrease in TMRM fluorescence.
The rapid decline of TMRM fluorescence was observed
between 1 and 2 h, which indicates the opening of MPTP
(data has not shown). At Fig. 5a, b, trans-anethole atten-
uated the decrease in TMRM fluorescence caused by OGD
(1 h)/R (1.5 h) (35.7 (IQR: 32.5–38.8) vs 68.6 (IQR:
62.3–80.1), **P \ 0.01).
bFig. 4 Anti-oxidative effect of trans-anethole. a, b Trans-anethole
decreased the ROS overproduction caused by OGD/R. Cells were pre-
treated with trans-anethole (TA, 10 lM), Trolox (T, 10 lM) or
MK801 (MK, 10 lM) right before OGD (1 h)/R (3 h). ROS
production was measured afterward. a Representative fluorescence
images. Scale bar 50 lm. b Quantification of CM-DCF fluorescence.
Horizontal bar, median; vertical box, interquartile ranges (Q1–Q3);
and whiskers, minimum/maximum. n = 5. **P \ 0.01, significantly
different from the indicated groups. c–f ROS scavenging effect of
trans-anethole (c–e ORAC assay; f DPPH assay). c, d AAPH-induced
fluorescence decay curve in the presence of different concentrations
of trans-anethole or Trolox (black square = 0 lM; white squar-
e = 1.5,625 lM; black up-pointing triangle = 3.125 lM; white up-
pointing triangle = 6.25 lM; black circle = 12.5 lM). The plots
present the average of four separate experiments. e Best-fit lines
between the net AUC (=AUCsample-AUCblank) and different concen-
trations of trans-anethole (TA) or Trolox. The data are expressed as
mean ± SEM and analyzed by one-way ANOVA followed by
Tukey’s post hoc test. n = 4. f DPPH reduction assay. Dose–response
curve of the DPPH inhibition in the presence of trans-anethole (TA)
or vitamin C (Vc). Data are expressed as mean ± SEM and analyzed
by one-way ANOVA followed by Tukey’s post hoc test. n = 4
A Control OGD-R
OGD-R/TA OGD-R/MK
180
150
120
90
60
30
0
CM
-D
CF
 
(F
.
I.)
-Control TA T MK
OGD-R
**
-20
0
20
40
60
80
100
120
D
PP
H
 
in
hi
bi
tio
n
 
(%
)
Log [Drug], mM
TA
Vc
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0 0.5 1 1.5 2 2.5 3
R
el
at
iv
e 
flu
or
es
ce
nc
e
Time (h)
t-anethole
0
30
60
90
120
0 3 6 9 12 15
n
et
 
A
UC
[Drug], µM
TA
Trolox
C
FE
0 0.001 0.01 0.1 1 10
B
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0 0.5 1 1.5 2 2.5 3
R
el
at
iv
e 
flu
or
es
ce
nc
e
Time (h)
Trolox
D
Neurol Sci (2014) 35:1541–1547 1545
123
Discussion
In the present study, we first demonstrated that trans-ane-
thole markedly reduced neuronal cell death induced by
OGD/R, an in vitro model of cerebral ischemia (Fig. 1).
Furthermore, we elucidated the neuroprotective mecha-
nisms of trans-anethole against OGD/R-induced cortical
neuronal injury. Previously, anise oil has been reported to
extend the latency, without changing the amplitude and
duration, of hypoxia-induced direct current depolarization
[17]. However, these results cannot be concluded to bring
about neuroprotection against hypoxia-induced neuronal
death and/or the anti-hypoxic/ischemic effects of trans-
anethole as a pure compound.
In cerebral ischemia and reperfusion, excitotoxicity,
oxidative stress and neuroinflammation play an important
role in neuronal cell death [18]. Excessive extracellular
glutamate in cerebral ischemic lesions leads to increased
calcium influx via the activation of glutamate receptors,
especially the NMDA receptor [10]. Our result showed that
trans-anethole significantly inhibited the OGD/R-induced
neuronal death and calcium overload through the activation
of NMDA receptor (Figs. 2, 3). These results showed the
anti-excitotoxicity of trans-anethole under ischemia/hyp-
oxic condition. Previously, trans-anethole was reported to
regulate neuronal excitability in a biphasic manner, at low
and high concentrations, through voltage-dependent cal-
cium channels (VDCCs) in snail F1 neurons and isolated
rat aortae [4, 19]. However, in the majority of rat cortical
cultures, the maximal activation of VDCCs induces much
lower calcium loading than what toxic NMDA receptor
activation does [20].
OGD/R has also been shown to induce oxidative stress
[8]. The present study showed that trans-anethole
attenuated the ROS overproduction in OGD/R-induced
neurons (Fig. 4a, b). These antioxidant effects might be
derived from the scavenging activity of peroxyl radicals,
tested in the ORAC assay (Fig. 4c–e). Nevertheless,
because MK801 markedly inhibited the OGD/R-induced
ROS overproduction (Fig. 4a, b), we suggest the impor-
tance of excitotoxicity as a prior cascade that causes neu-
ronal death, along with oxidative stress. Furthermore,
overloaded intracellular calcium and/or ROS under ische-
mic conditions induce mitochondrial depolarization which
could enhance secondary ROS overproduction via the
MPTP opening, causing irreversible cell death [14, 15].
Trans-anethole was shown to inhibit the mitochondrial
depolarization caused by OGD/R (Fig. 5). These results
indicate that neuroprotection of trans-anethole may be
related with mitochondrial protection from OGD/R-
induced damage, as well as anti-excitotoxicity and anti-
oxidative stress.
Following excitotoxicity and oxidative stress, neuroin-
flammation could exacerbate brain damage in the delayed
stage of ischemic stroke [21]. Previously, the anti-inflam-
matory effect of trans-anethole has been reported in
in vitro and in vivo models of inflammation [1–3]. Con-
sistent with previous studies, our preliminary data have
shown that trans-anethole inhibited monocyte chemoat-
tractant protein-1-induced microglial migration and
reduced OGD/R-induced nitric oxide production in cul-
tured pure microglia (data not shown). However, because
our present neuronal cell injury model was not related with
inflammatory response, we excluded anti-inflammatory
effects.
Conclusively, our results demonstrate the neuroprotec-
tive effect of trans-anethole in OGD/R-induced neuron-
enriched cultures through anti-excitotoxicity, anti-
A B
150
120
9060
30
0
TM
R
M
 
(F
.
I.)
Control - TA T MK
OGD-R
Control OGD-R
OGD-R/TA OGD-R/MK
**
Fig. 5 Trans-anethole attenuated the OGD/R-induced MTP depolar-
ization/MPTP opening. Cells were treated with trans-anethole (TA,
10 lM), Trolox (T, 10 lM) or MK801 (MK, 10 lM), followed by
OGD/R for 1.5 h. TMRM fluorescence was measured afterward.
a Representative images. Scale bar 50 lm. b Quantification of
TMRM fluorescence. Horizontal bar, median; vertical box, inter-
quartile ranges (Q1–Q3); and whiskers, minimum/maximum. n = 7.
**P \ 0.01, significantly different from the indicated groups
1546 Neurol Sci (2014) 35:1541–1547
123
oxidative stress and mitochondrial protection. These results
suggest trans-anethole as a neuroprotective agent to ame-
liorate ischemic neuronal damage. Nevertheless, to fully
understand the integrated neuroprotective effect/mecha-
nisms of trans-anethole, further studies should be extended
to both pre- and post-ischemic treatment in in vivo models.
Acknowledgments This research was supported by Grants from the
National Research Foundation of Korea (NRF) funded by the Korean
government (MEST) (No. 2012R1A2A2A02007145).
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123
	Trans-anethole protects cortical neuronal cells against oxygen--glucose deprivation/reoxygenation
	Abstract
	Introduction
	Materials and methods
	Materials
	Primary cultures of neuron-enriched cortical cells
	Oxygen--glucose deprivation/reoxygenation (OGD/R) and NMDA injury
	Assessment of cell injury: LDH assay
	Measurement of intracellular calcium: Fluo-4-AM assay
	Measurement of intracellular oxidative stress: CM-H2DCF-DA assay
	Measurement of free radical scavenging activities: oxygen radical absorbance capacity (ORAC) assay and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay
	Measurement of mitochondrial transmembrane potential (MTP) in cells
	Statistical analysis
	Results
	Trans-anethole inhibited OGD/R-induced cell death in neuron-enriched cortical cells
	Trans-anethole reduced OGD/R-induced the intracellular calcium overload
	Trans-anethole reduced OGD/R-induced ROS overproduction and directly scavenged free radicals in ORAC assay
	Trans-anethole attenuated the decrease in MTP in OGD/R-induced cortical cells
	Discussion
	Acknowledgments
	References