Neurochemistry International 90 (2015) 20e27

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Neurochemistry International
journal homepage: www.elsevier.com/locate/nci

Anticonvulsant properties of Euterpe oleracea in mice

Rogerio Souza-Monteiro a, Moise
Jose
s Hamoy b, Danielle Santana-Coelho a,
Gabriela P.F. Arrifano , Ricardo S.O. Paraense a, Allan Costa-Malaquias a,
a

Jackson R. Mendonça a, Rafael F. da Silva c, Wallena S.C. Monteiro c, Herve
Rogez d,

e
Luiz M. do Nascimento , Maria Elena Crespo-Lo
Diogo L. de Oliveira , Jose f
pez a, *
a
Laborato
rio de Farmacologia Molecular, Instituto de Ci^
gicas (ICB), Universidade Federal do Para
encias Biolo
(UFPA), Bel
em, Brazil
b
Laborato
rio de Farmacologia e Toxicologia de Produtos Naturais e, Instituto de Ci^
gicas (ICB), Universidade Federal do Para
encias Biolo
(UFPA), Bel

em,
Brazil
c
, Brazil
Petruz Fruity, Castanhal, Para
d
College of Food Engineering & Centre for the Valorization of Bioactive Compounds from Amazonia, UFPA, Bel
em, Brazil
e
Departamento de Bioquímica, Instituto de Ci^
sicas da Saúde (ICBS), Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Brazil
encias Ba
f
rio de Neuroquímica Molecular e Celular, Instituto de Ci^
Laborato
gicas (ICB), Universidade Federal do Para
encias Biolo
(UFPA), Bel
em, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Açai (Euterpe oleracea Mart.), a highly consumed fruit in Amazon, is from a common palm with
Received 14 April 2015 remarkable antioxidant properties. Because oxidative stress and seizures are intimately linked, this study
Received in revised form investigated the potential neuroprotective and anticonvulsant effects of commercial clarified açai juice
9 June 2015
(EO). EO did not alter spontaneous locomotor activity. Four doses of EO were sufficient to increase la-
Accepted 27 June 2015
Available online 2 July 2015
tencies to both first myoclonic jerk and first generalized toniceclonic seizure and significantly decrease
the total duration of tonic-clonic seizures caused by pentylenetetrazol administration. Also, electro-
cortical alterations provoked by pentylenetetrazol were prevented, significantly decreasing amplitude of
Keywords:
Seizure
discharges and frequencies above 50 Hz. EO was also able to completely prevent lipid peroxidation in the
Euterpe oleracea cerebral cortex, showing a potent direct scavenging property. These results demonstrate for the first time
Anticonvulsant that E. oleracea significantly protects against seizures and seizure-related oxidative stress, indicating an
Oxidative stress additional protection for humans who consume this fruit.
EEG © 2015 Elsevier Ltd. All rights reserved.
Amazon

1. Introduction mortality due to this pathology.

Seizures result from isolated responses in the brain to acute
neurological insults or alterations of homeostasis in acute diseases,
such as stroke and head trauma. Seizures are the major hallmark of
epilepsy, and the pathology is characterized by unprovoked and
spontaneous recurrent events. Clonic and tonic-clonic seizures are
considered the most serious types of epileptic episodes, definitively
affecting the quality of life of patients and significantly increasing
Abbreviations: EO, clarified açai (Euterpe oleracea) juice; ILAE, International
League Against Epilepsy; DM, dry matter; PTZ, pentylenetetrazol; DPPH,
1,1diphenyl-2-picrylhydrazyl; MDA, malondialdehyde; EEG, electroencephalogram;
ECoG, electrocorticogram.
* Corresponding author. Laborato
rio de Farmacologia Molecular, Instituto de
^ncias Biolo
Cie
gicas; Universidade Federal do Para
(UFPA), Rua Augusto Corre^a 01,
Campus do Guama
, 66075-110 Bele

m, PA, Brazil.

pez).
E-mail address: maria.elena.crespo.lopez@gmail.com (M.E. Crespo-Lo
Approximately 50 million people worldwide are affected by
epilepsy and approximately 80% of them are from developing
countries, such as Brazil (WHO, 2012). Major concerns in this pa-
thology are refractory epilepsy, as seizures are not controlled with
current anticonvulsant pharmacotherapy in nearly 30% of patients,
and difficulties accessing adequate treatment (Shneker and
Fountain, 2003). In developing countries, almost three out of four
patients do not receive this treatment (WHO, 2012). Therefore, new
proposals of therapy based on bioactive compounds in the diet or
plants become realistic alternatives to prevent, stop, or even
reverse the events surrounding seizures and epilepsy (Puttachary
et al., 2015; Sharma and Jain, 2014; Zhu et al., 2014).
One of the fruits highly consumed in the Amazon is açai, which
shows remarkable antioxidant properties (Gordon et al., 2012;
Kang et al., 2011). Açai, or açai drink, is made from the fruit of a
very common palm in the eastern Amazonian floodplains (Euterpe
oleracea Martius, family Arecaceae). Clarified açai is largely

http://dx.doi.org/10.1016/j.neuint.2015.06.014
0197-0186/© 2015 Elsevier Ltd. All rights reserved.
 J.R. Souza-Monteiro et al. / Neurochemistry International 90 (2015) 20e27
available on the international market (Bichara and Rogez, 2011)
which shows remarkable antioxidant properties.
Oxidative stress and seizures are intimately linked. In addition
to behavioral changes, seizures promote oxidative stress in both
animal models of seizures and patients with epilepsy (Aguiar et al.,
2012; Puttachary et al., 2015). Oxidative stress is an imbalance
between the generation of oxidant compounds and the activities of
antioxidant defense systems, resulting in an overproduction of
reactive oxygen species (Aguiar et al., 2012; Cardenas-Rodriguez
et al., 2013; Puttachary et al., 2015). In addition to contributing to
neuronal hyperexcitability and macromolecules (protein, DNA,
lipids) damage, oxidative stress plays a major role in refractory
epilepsy in experimental models and humans (Aguiar et al., 2012;
Cardenas-Rodriguez et al., 2013). During the last decade, different
antioxidants have been proposed as new therapeutic tools against
seizures and epilepsy with a focus on natural products because they
are naturally present in human food or folk medicine (Puttachary
et al., 2015). Thus, some flavonoids presented in açai, such as
wogonin, fisetin, vitexin, and rutin, have already showed their ef-
ficacy to prevent seizures in experimental models with PTZ (Zhu
et al., 2014).
Therefore, this study aimed to investigate the potential neuro-
protective and anticonvulsant effect of clarified açai (E. oleracea)
juice (EO) in an experimental model of seizures.
2. Materials and methods
2.1. Animals and ethical aspects
Male Swiss mice (25e35 g) were maintained in a controlled
environment (21 ± 2
C; 12 h light/dark cycle) with access to food
and water ad libitum. All experimental procedures were approved
by the Committee for Ethics in Experimental Research with Ani-
mals of the Universidade Federal do Par
a (license number BIO197-
14) and followed the guidelines suggested by the NIH Guide for the
Care and Use of Laboratory Animals. A total number of 68 animals
were used in the present study: 20 for open field test, 32 for
convulsive behavior and biochemical tests and 16 for electro-
corticographic recordings. Animals were randomly grouped and
the exact number of animals for each group can be found in the
legend of figures. Animals for electrocorticographic recordings
were submitted to surgical procedures before any treatment (see
section 2.6). No animal died naturally (without euthanasia) from all
treatments carried out in the present study. All efforts were carry
out to reduce the number of animals and to minimize their
suffering.
2.2. Clarified açai (E. oleracea Mart.) juice (EO)
Plants were identified as E. oleracea Martius (Arecaceae) by
comparing with a voucher specimen (#268513) deposited in the
Herbarium of Instituto Nacional de Pesquisas da Amazo ^ nia (INPA,
Brazil). Samples of clarified açai juice were kindly provided by
Amazon Dreams (Bele
m, Para
, Brazil). This juice was produced by a
patented process licensed by both Amazon Dreams and Uni-
versidade Federal do Para
(PI 1003060-3) that consisted of micro-
filtration and centrifugation of an açai juice prepared with fresh
fruits. Consequently, the final product contained no lipids, proteins,
of fibers (total solids < 1% DM) and constitute an ideal model for
evaluating the impact of phenolic compounds (>1400 mg gallic
acid equivalents/l).
2.3. Treatments
All animals were treated (10 ml/g body weight) with EO or saline
21
by gavage once a day for four days. One hour after the last dose of
EO or saline, a set of 20 animals (control and EO groups) were
evaluated with open field test to analyze possible alterations of
spontaneous locomotor activity due to EO treatment. At the same
time (also 1 h after the fourth dose of EO or saline), all the other
animals were injected intraperitoneally with only one dose of
pentylenetetrazol (PTZ; 60 mg/kg) or saline (Pires et al., 2015) and
behavioral analyses of seizures or electrocorticographic recordings
were carried out with continuous monitoring.
2.4. Open field test
Analysis of spontaneous locomotor activity with the open field
test was carried out between 8:00e12:00 a.m. The animals were
placed in a wooden box (with floor divided by black lines into 12
equal quadrants) and they were observed for 5 min. Number of
crossed quadrants (crossings) and fecal bolus and behaviors of
rearing and grooming were recorded as described elsewhere
(Rodrigues et al., 2012).
2.5. Behavioral analyses of seizure
After PTZ injection, animals were continuously monitored for
20 min. Latencies to first myoclonic jerk and first generalized ton-
iceclonic seizure, as well as the total duration of tonic-clonic sei-
zures, were recorded (Souza et al., 2013). The animals were then
sacrificed by cervical dislocation and the cerebral cortex homoge-
nized and stored at 4
C to analyze lipid peroxidation.
2.6. Surgical procedures
A different set of animals (n ¼ 16) was anesthetized with xyla-
zine (5 mg/kg i.p.) and ketamine (80 mg/kg i.p.) and immobilized in
a steriotaxic apparatus. After subcutaneous application of lidocaine
2%, electrodes (1 mm diameter) were implanted 1 mm posterior to
the bregma, ± 1 mm lateralelateral (both hemispheres). Electrodes
were fixed in the dura mater and isolated with dental autopoly-
merized acrylic. Animals were then monitored every 6 h and
allowed to recovery from surgery for 2 days, before treatment with
EO or saline as described above.
2.7. Electrocorticographic recordings
On the last day of treatment, the electrodes of each animal were
connected to a data acquisition system (high-impedance amplifier,
Grass Technologies, P511; coupled to an oscilloscope, Protek, 6510).
The recording electrode was located on the right side of the
hemisphere, and the electrode on the left side was used as a
reference. The whole experiment was performed in Faraday cages.
Data were monitored continuously with a range of 1 KHz (National
Instruments, Austin, TX) and analyzed using LabVIEW Express
software.
After 10 min of accommodation, the baseline of each animal was
recorded for 30 min and then PTZ (60 mg/kg i.p.) injected as
described above. Recording was continued for another 30 min.
Data were graphically expressed showing differences in poten-
tial between the two electrodes (reference and recording). Spec-
trograms were calculated using a Hamming window of 256 points
(256/1000 s), and each frame was generated with an overlap of 128
dots per window. For each frame, the power spectral density (PSD)
was calculated with the Welch average periodogram method. Fre-
quency histograms were generated with the PSD of the signal (with
1 Hz boxes).
22 J.R. Souza-Monteiro et al. / Neurochemistry International 90 (2015) 20e27
2.8. Assay of direct free radical scavenging
To evaluate the possibility of direct free radical scavenging by
EO, we used the protocol described by Gulcin et al. (2004). Briefly,
dilutions of EO (1:10, 1:100, 1:1000) were added to a solution of
40 mg/ml 1,1diphenyl-2-picrylhydrazyl (DPPH) in ethanol. After
30 min of incubation in darkness at room temperature, absorbance
was measured at 517 nm and the percentage of scavenged DPPH
molecules calculated according to the following formula:
% scavenged DPPH ¼ ½ðA0  A1 Þ=A0   100;
where A0 is the absorbance of the control group and A1 is the
absorbance in the presence of EO.
2.9. Quantitation of lipid peroxidation
Lipid peroxidation in the cerebral cortex samples was spectro-
photometrically assessed using malondialdehyde (MDA) as an in-
dicator (Esterbauer and Cheeseman, 1990). Briefly, after
centrifugation of the samples at 2500 g for 10 min, a solution
containing methanesulfonic acid and N-methyl-phenyl indole
(10.3 mM in acetonitrile) diluted in methanol (1:3) was added to
the supernatants and incubated for 40 min at 45
C. Absorbance
was measured at 570 nm and compared to standard concentrations
of MDA. The lipid peroxidation values were corrected for the pro-
tein concentration of each sample and expressed as nanomoles
MDA per milligram of protein.
2.10. Total protein content
Total protein content was determined in all samples as
described elsewhere (Bradford, 1976). Aliquots of the homogenates
were incubated with Bradford reagent (5% ethanol, 8.5% phosphoric
acid, 0.25% Coomassie Brilliant Blue G-250) for 2 min at room
Fig. 1. Treatment with clarified açai (E. oleracea) juice (EO) does not alter spontaneous locomotor activity. EO or saline (SAL) was given by gavage (10 ml/g per day) for 4 days. Data of
number of crossed quadrants (A, total crossings), rearing (B), grooming (C), and fecal bolus (D) are mean ± SEM (n ¼ 10). No statistical differences were detected.
temperature. The absorbance of each sample was measured at
595 nm and compared to standard solutions of bovine serum
albumin.
2.11. Statistical analysis
Statistical analysis was performed using the software Graph Pad
Prism (version 5.0). Initially, the Gaussian distribution of the data
was tested by the KolmogoroveSmirnov test. Non-parametric data
for behavioral parameters (latencies and duration of seizures) were
expressed as median ± interquartile range and analyzed using the
ManneWhitney test. Parametric data for open field test, lipid per-
oxidation, scavenging property, and electrocoricograms (ECoGs)
were analyzed by ANOVA followed by Tukey post hoc test when
appropriate, and they were expressed as mean ± standard error of
the mean (S.E.M). P < 0.05 was considered significant for all
analyses.
3. Results
3.1. Treatment with EO does not affect spontaneous locomotor
activity
No statistical differences between animals treated with saline or
EO were detected for all parameters analyzed in the open field test
(Fig. 1).
3.2. EO protects against behavioral changes caused by seizures
Only four doses of EO were sufficient to significantly increase
the latency to both first myoclonic jerk and first generalized tonic-
clonic seizure induced by PTZ (Fig. 2A and B). Treatment with açai
also decreased 61% of the time spent in tonic-clonic seizures
(Fig. 2C).
 J.R. Souza-Monteiro et al. / Neurochemistry International 90 (2015) 20e27 23

3.3. Treatment with EO significantly reduces electrical alterations

caused by seizures

Animals treated with saline had ECoGs indicating the cerebral

activity of environment exploration (irregular pattern of low am-
plitudes and more frequencies below 10 Hz; Fig. 3). The ECoGs of
animals treated with açai were very similar to those of controls
(Fig. 3), with low amplitudes and more frequencies below 10 Hz.
PTZ provoked a pattern of variable amplitudes with increasing
excitability and elicited discharges characteristic of convulsive ep-
isodes (Fig. 3). The spectrograms for these animals showed
increased frequencies reaching 50 Hz. Treatment with four doses of
EO and PTZ led to transitory increases in amplitude, but signifi-
cantly lower than those provoked by PTZ, pointing to partial inhi-
bition of PTZ effect (Fig. 3). The spectrograms of these latter animals
showed energy distributions with few frequencies above 10 Hz.
Regarding the amplitude distributions (Fig. 4), no significant
differences were found in the distributions of 1e50 Hz frequencies
between animals treated with saline solution or açai (Fig. 4).
However, PTZ caused significant increases in amplitudes with a
clearly altered distribution pattern, especially for 1e25 Hz fre-
quencies, but it was partially prevented by treatment with EO
(Fig. 4). Moreover, quantitation of the amplitudes for each group
revealed a significant increase with PTZ, which decreased in
approximately 50% by treatment with four doses of EO (Fig. 4).

3.4. EO has a high capacity for direct scavenging, preventing the

deleterious consequences of oxidative stress caused by seizures in
the cerebral cortex

The high antioxidant capacity of açai was detected by the DPPH

method (Fig. 5). The direct scavenger property of açai was so potent
that a dilution of 1:10 EO was sufficient to show a similar effect as
ascorbic acid (1 mg/ml). In addition, PTZ administration provoked a
significant increase in lipid peroxidation in the cerebral cortex
(Fig. 6), and this effect was completely prevented by treatment with
açai.

4. Discussion

This work demonstrated, for the first time, that E. oleracea juice
significantly protects against seizures and seizure-related oxidative
stress.
We used an experimental model of seizures with PTZ, a selective
blocker of the chloride channel coupled to the GABAA receptor
complex. PTZ treatment is an important model of myoclonic and
generalized tonic-clonic seizures and is currently considered the
gold standard for screening potential anticonvulsant compounds
(Loscher, 2011; Pahuja et al., 2012; Souza et al., 2013; Yuen and
Troconiz, 2015). Additionally, this model may predict more accu-
rately the physiological consequences of seizures in humans and
their responses to treatments when compared with other models
(Yuen and Troconiz, 2015).
In our model, commercial samples of EO were used to ensure
that the samples were indicative of normal human consumption.
Also, clarified juice was chosen because the clarification process
Fig. 2. Effects of clarified açai (E. oleracea) juice (EO) on behavioral alterations induced
eliminates all macronutrients (lipid, fiber, and protein), avoiding
by pentylenetetrazol (PTZ): latency to first myoclonic jerk (A), latency to first gener-
possible interference by these compounds in the results. Thus, EO is alized tonic-clonic seizure (B) and total time spent in generalized tonic-clonic seizures
basically composed of phenolic compounds, with anthocyanins (C). EO was given by gavage (10 ml/g per day) for 4 days. PTZ was given as 60 mg/kg i.p.
representing a major part of it. Analysis of the samples used in the Data are median and interquartile range (n ¼ 10). ***P < 0.001 and **P < 0.01 vs. PTZ.
present study revealed a content of 1662.15 mg gallic acid equiva-
lents/l of phenolic compounds, including 529 mg cyanide equiva- serious type of seizures (Fig. 2). Still, electrocorticographic re-
lents/l of anthocyanins. cordings showed that PTZ induced electrical alterations character-
Surprisingly, only four doses of EO were enough to significantly ized by increased amplitudes and that a previous ingestion of açai
increase latencies and strongly reduce the duration of the most prevented these increases in approximately 50% (Figs. 3 and 4).
24 J.R. Souza-Monteiro et al. / Neurochemistry International 90 (2015) 20e27

Fig. 3. Electrocorticograms (ECoGs). Animals were treated with saline (first line) or clarified açaí (E. oleracea) juice (10 ml/g per day by gavage) (second line) for 4 days and then
treated with pentylenetetrazol (60 mg/kg) (third and fourth lines, respectively). ECoGs at 20 min (left), amplification of a representative fragment of ECoGs (center), spectrograms of
frequencies (right).

Electrical recordings, in addition to behavioral evaluation, have
been a useful and objective tool for verifying alterations caused by
neuronal excitability in both epileptic patients and experimental
models. A recent report demonstrated the sensibility of this
method for seizures detection and characterization in the PTZ
model (Niknazar et al., 2013). This method takes advantages of
minimal encephalic damage and is sensitive enough to indicate
changes in the severity of crises (characterized by increased fre-
quencies and/or amplitude of waves) that cannot be quantified only
with the observation of behavior. In our work, açai partially pre-
vented the alterations caused by PTZ in ECoGs by significantly
decreasing both the frequency of discharges and mean wave
amplitude (Figs. 3 and 4).
These results are more remarkable when we consider that new
therapeutic tools are sometimes proposed as co-adjuvants in pol-
ytherapy to improve the effect of classical drugs, but their potency
alone is low; this was not the case with açai, because treatment
with only the fruit already showed a potent anticonvulsant effect.
Moreover, near of 30% of epileptic patients are affected by seizures
resistant to current anticonvulsant pharmacotherapy (WHO, 2012),
making it essential to find new alternatives, especially those that
are easy for isolated populations to access.
The EO dose used in this study (10 ml/g body weight, equivalent
to approximately 700 ml for a person of 70 kg) was slightly more
than the daily mean human consumption in several regions of
Brazil (e.g., in the North, it is not uncommon to find people who
consume 1 L/day) and the total concentration of phenolic com-
pounds and anthocyanins is largely representative of those usually
measured in açai juice. No changes in body weight (data not shown)
or spontaneous locomotor activity (Fig. 1) were detected in animals
treated with EO. Moreover, consumption of a similar amount of
clarified açai is apparently not toxic for humans (Mertens-Talcott
et al., 2008), and acute and subchronic treatments with higher
doses of açai pulp do not provoke genotoxic effects in rodents
(Ribeiro et al., 2010). Therefore, our results suggest that diet sup-
plementation with EO could have additional value in patients,
providing an extra anticonvulsant effect and probably allowing the
use of lower doses of classical anticonvulsant drugs, avoiding major
 J.R. Souza-Monteiro et al. / Neurochemistry International 90 (2015) 20e27
Fig. 4. Electrocorticographic amplitudes. The distribution of amplitudes (A and B) and
mean amplitudes (C) were electrocorticographically registered in animals treated with
saline (SAL) or clarified açai (E. oleracea) juice (EO, 10 ml/g per day by gavage for 4 days)
and then treated with pentylenetetrazol (60 mg/kg; PTZ and EO þ PTZ, respectively).
Data are mean ± SEM (n ¼ 8). *P < 0.001 vs. basal and EO groups.
Fig. 5. Direct scavenging activity of ascorbic acid (ASC) and increasing dilutions of
clarified açai (E. oleracea) juice (EO) against free radicals. ASC and EO were incubated
with a 1,1diphenyl-2-picrylhydrazyl (DPPH) solution. Data are mean ± standard de-
viation (n ¼ 4). ***P < 0.001 vs. all groups. *P < 0.05 vs. EO 1:10 and ASC 1 mg/ml.
25
Fig. 6. Effect of clarified açai (E. oleracea) juice (EO) on pentylenetetrazol (PTZ)-
induced lipid peroxidation of the cerebral cortex. EO was given by gavage (10 ml/g per
day) for 4 days. PTZ was given as 60 mg/kg i.p. Data are mean ± SEM (n ¼ 10).
***P < 0.001 and *P < 0.05 vs. all groups. MDA ¼ Malonaldehyde.
concerns about the numerous side effects of these drugs. Additional
studies are necessary to confirm this hypothesis about a possible
synergic effect.
The anticonvulsant effect of açai is particularly important for
isolated regions, such as the Amazon, where compliance with
chronic pharmacological treatment based on current anticonvul-
sant drugs is reduced due to socioeconomic factors (i.e., poverty,
isolation) that make it difficult to access health services. Thus, a
common palm widespread distributed in the Amazon (Bichara and
Rogez, 2011) could be an extremely useful tool for treating seizures,
especially in these populations.
What molecular mechanism underlies the potent anticonvul-
sant effect of açai? Although a more in depth study is necessary to
completely answer this question, the antioxidant capacity of açai
seems to be the first explanation of the anticonvulsant action,
because the high phenolic compounds content, such as flavones,
which are the main bioactive compounds present in açai (Dias et al.,
2013; Kang et al., 2010, 2011). Direct challenge with DPPH radical
revealed a scavenger effect of EO that is at least 10-fold more potent
than a solution of 1 mg/ml ascorbic acid (Fig. 5). Moreover, an EO
dilution of 1:100 demonstrated higher scavenger properties (91.4%
of inhibition of DPPH radicals) than 800 mM of Trolox (approxi-
mately 81% for the same assay, as described by Bonomo Lde et al.
(2014), a compound considered the gold standard in biological
assays evaluating antioxidant properties. These results confirm
previous studies demonstrating açaís exceptional antioxidant ca-
pacity (Bonomo Lde et al., 2014; Gordon et al., 2012; Kang et al.,
2010, 2011; Xie et al., 2011).
Recent studies support a major role of oxidative stress in the
development of epilepsy and epilepsy that is refractory to phar-
macological treatment (Aguiar et al., 2012; Cardenas-Rodriguez
et al., 2013; Puttachary et al., 2015; Shin et al., 2011). Moreover,
classical experimental models of seizures (PTZ, strychnine, and
picrotoxin) and epilepsy (pilocarpine and kainic acid) induce sei-
zures via different mechanisms, but share a common oxidative
stress pathway (Aguiar et al., 2012; Loscher, 2011).
The role of oxidative stress on seizures in humans has not been
fully elucidated and remains controversial (Aguiar et al., 2012;
Puttachary et al., 2015; Shin et al., 2011). The brain is extremely
vulnerable to oxidative damage, mainly because of weak antioxi-
dant defenses, the increased consumption of oxygen and high
oxidable compounds content compared to other organs
(Puttachary et al., 2015; Shin et al., 2011). Lipid peroxidation, one of
the most deleterious consequences of oxidative stress, is a cascade
26 J.R. Souza-Monteiro et al. / Neurochemistry International 90 (2015) 20e27
of biochemical events involved in non-specific oxidation of poly-
unsaturated fatty acids mediated by free radicals. A significant in-
crease in lipid peroxidation has already been shown in both
epileptic patients and experimental models of seizures and epi-
lepsy (Aguiar et al., 2012; Puttachary et al., 2015; Shin et al., 2011).
In our work, the administration of 60 mg/kg PTZ significantly
increased lipid peroxidation in the cerebral cortex (Fig. 6). These
results are in accordance with recent studies using the same dose of
PTZ and showing a 44e98% increase in lipid peroxidation
compared to control group (Chowdhury et al., 2013; Naziroglu
et al., 2013). Seizures induced by PTZ exacerbate oxidative stress
through the increased production of ROS and decreased activities of
antioxidant enzymes, such as catalase or superoxide dismutase
(Branco Cdos et al., 2013; Chowdhury et al., 2013; Kumar et al.,
2013; Rodrigues et al., 2012).
Interestingly, açai was able to completely prevent lipid peroxi-
dation in the cerebral cortex of animals receiving PTZ (Fig. 6).
Although treatment with açai did not completely eliminate sei-
zures, prevention of lipid peroxidation in the cerebral cortex may
lead to a significant reduction in subsequent seizures and their
deleterious consequences, an important concern in epilepsy
patients.
Although our results with the DPPH method and lipid peroxi-
dation point to that the scavenger capacity of açai must be a major
responsible for this protection (Figs. 5 and 6), other possible indi-
rect mechanisms cannot be discarded. Treatment with açai was
recently shown to modulate the expression of oxidative stress-
related genes, such as gsc-1 (the limiting enzyme of glutathione
synthesis), and transcription factors (especially those regulated by
the insulin/IGF-1 signaling pathway) affecting stress resistance
(Bonomo et al., 2014). The possible occurrence of these genetic
mechanisms plus the apparent absence of genotoxic effects of
subchronic treatment with açai (Ribeiro et al., 2010) may be addi-
tional support for the use of chronic treatment with açai in epilepsy
patients.
Which components of EO are responsible for eliciting these
possible mechanisms of protection? The relationship between
anticonvulsant and antioxidant activity has been shown by the
induction of anticonvulsant effects through the antioxidant actions
of plant extracts (Naziroglu et al., 2013; Pahuja et al., 2012;
Rodrigues et al., 2012). The anticonvulsant effects of medicinal
plants have been explained based on anxiolytic or sedative prop-
erties, the influence on receptors (GABAA, glutamate, etc.), and
modulation of neurotropic factors (Zhu et al., 2014). Although
additional behavioral studies are needed, treatment with EO may
not have sedative effects, as no differences were detected in the
open field test (Fig. 1).
The EO used in our work is an aqueous product characterized by
the absence of insoluble solids and lipids while maintaining a high
antioxidant capacity due to the presence of phenolic compounds,
such as anthocyanins, flavones, proanthocyanidins, and other fla-
vonoids. Cyanidin-3-rutinoside, cyanidin-3-glucoside, orientin,
homoorientin, and taxifolin deoxyhexose are the major flavonoids
present in açai (Dias et al., 2012, 2013; Gordon et al., 2012). Inter-
estingly, recent studies have demonstrated that the major absorp-
tion of anthocyanins occurs at the stomach level only 30 min after
consumption (Fernandes et al., 2014). The efficacy of flavonoids,
such as wogonin, fisetin, vitexin, and rutin (one of the most com-
mon flavonoids in plants and fruits), has already been shown in
experimental models with PTZ (Zhu et al., 2014).
Independent of the exact composition of EO, the present human
consumption of açai means that additional efforts must be dedi-
cated to analyzing the bioactive compounds responsible for these
in vivo effects. Although many in vitro studies have reported the
antiproliferative, anti-inflammatory and cardioprotective effects of
açai (Kang et al., 2011; Moura et al., 2012; Xie et al., 2011), in vivo
studies are still scarce.
5. Conclusions
Despite the other benefits of açai consumption, this work
demonstrated for the first time that clarified E. oleracea juice
significantly protects against seizures and seizure-related oxidative
stress in an in vivo model. The results suggest that regular intake of
açai juice may be an additional protective factor in epilepsy.
Conflicts of interest
Authors declare that no conflict of interests exists. The funders
had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript. The donations from
Amazon Dreams or Instituto Evandro Chagas do not imply any
competing interests and it does not alter the authors' adherence to
Neurochemistry International policies.
Acknowledgments
We thank Instituto Evandro Chagas (IEC, Brazil) and Amazon
Dreams (Para
, Brazil) for kindly providing the animals and the
samples of clarified açai juice for this study, respectively. This work
was supported by Conselho Nacional de Cie ^ncia e Tecnologia em
Pesquisa (CNPq, Brazil; grants numbers 478580/2012-6, 467143/
2014-5 and 447568/2014-0) and Pro
-Reitoria de Pesquisa da UFPA
(PROPESP, UFPA, Brazil). J.L.M. do Nascimento, D.L. Oliveira, H.
Rogez, M.E. Crespo-Lo
pez, R.S.O. Paraense and J.J. Mendonça thank
CNPq for their researcher fellowships. Also, J.R.S. Monteiro and
G.P.F. Arrifano thank Coordenaç~ ao de Aperfeiçoamento de Pessoal
de Nivel Superior (CAPES, Brazil) and A. Costa-Malaquias thanks
Fundaç~ ao de Amparo a  Pesquisa do Estado do Para
(FAPESPA,
Brazil), for their PhD fellowships.
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