Neuropharmacological effects of carvacryl acetate on δ-aminolevulinic dehydratase, Na+, K+-ATPase activities and amino acids levels in mice hippocampus after seizures

Article abstract of DOI:10.1016/j.cbi.2014.12.001

Epileptic syndromes are highly prevalent neurological conditions and can often be disabling. In order to find an alternative for treatment, this study evaluated anticonvulsant effects of carvacryl acetate (CA), a derivative of monoterpene carvacrol, after seizures induced by pilocarpine (P400), picrotoxin (PIC) or pentylenetetrazol (PTZ). We also analyzed the CA effects on Na⁺, K⁺-ATPase and δ-aminolevulinic acid dehydratase (δ-ALA-D) activities in hippocampus mice after seizures induced by P400, PIC or PTZ. In addition, glutamate, δ-aminobutyric acid (GABA), glutamine and aspartate levels in mice hippocampus treated with CA after seizures induced by P400, PIC or PTZ were also measured. CA produced anticonvulsant effects against seizures induced by P400, PIC or PTZ, and its effects were reversed by flumazenil, suggesting that action mechanism can be mediated by GABAergic system. CA increased GABA levels, but did not alter glutamate and aspartate concentrations in mice hippocampus after seizures induced by P400, PIC or PTZ when compared with seizures induced by P400, PIC or PTZ (p < 0.05), respectively, as well as decreased glutamine content in mice hippocampus after seizures induced by PIC when compared with seizures induced by PIC (p < 0.05). In addition, CA also increased Na⁺, K⁺-ATPase and δ-aminolevulinic acid dehydratase activities after seizures induced by P400, PIC or PTZ when compared with seizures induced by P400, PIC or PTZ (p < 0.05), respectively. This study demonstrated that CA could be a future therapeutic option for treatment of epilepsy, with a multifactorial brain action mechanism.

Full text of DOI:10.1016/j.cbi.2014.12.001

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Contents lists available at ScienceDirect
Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint
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Neuropharmacological effects of carvacryl acetate on d-aminolevulinic
dehydratase, Na+, K+-ATPase activities and amino acids levels in mice
hippocampus after seizures
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b
b
Lúcio Fernandes Pires a, , Luciana Muratori Costa , Antonia Amanda Cardoso de Almeida ,
⇑
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Oskar Almeida Silva b, Gilberto Santos Cerqueira c, Damião Pergentino de Sousa d,
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Rosana Martins Carneiro Pires e, Prabodh Satyal f, Rivelilson Mendes de Freitas a,b,
⇑
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a Postgraduate Program of Pharmacology, Federal University of Piauí, Teresina, PI, Brazil
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b Department of Biochemistry and Pharmacology, Laboratory of Experimental Neurochemistry Research, Center of Pharmaceutical Technology, Federal University of Piauí, Teresina,
PI, Brazil
c Department of Physiology and Pharmacology, Faculty of Medicine, Federal University of Ceará, Fortaleza, CE, Brazil
d Department of Pharmaceutical Science, Federal University of Paraíba, Campus I, João Pessoa, PB, Brazil
e Postgraduate Program of Food and Nutrition, Federal University of Piauí, Teresina, PI, Brazil
f Chemistry Department, University of Alabama in Huntsville, Huntsville, AL 35899, USA
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a r t i c l e i n f o
a b s t r a c t
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Article history:
Received 24 May 2014
Received in revised form 24 November 2014
Accepted 1 December 2014
Available online xxxx
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Epileptic syndromes are highly prevalent neurological conditions and can often be disabling. In order to
find an alternative for treatment, this study evaluated anticonvulsant effects of carvacryl acetate (CA), a
derivative of monoterpene carvacrol, after seizures induced by pilocarpine (P400), picrotoxin (PIC) or
pentylenetetrazol (PTZ). We also analyzed the CA effects on Na+, K+-ATPase and d-aminolevulinic acid
dehydratase (d-ALA-D) activities in hippocampus mice after seizures induced by P400, PIC or PTZ. In
addition, glutamate, d-aminobutyric acid (GABA), glutamine and aspartate levels in mice hippocampus
treated with CA after seizures induced by P400, PIC or PTZ were also measured. CA produced anticonvul-
sant effects against seizures induced by P400, PIC or PTZ, and its effects were reversed by flumazenil, sug-
gesting that action mechanism can be mediated by GABAergic system. CA increased GABA levels, but did
not alter glutamate and aspartate concentrations in mice hippocampus after seizures induced by P400,
PIC or PTZ when compared with seizures induced by P400, PIC or PTZ (p < 0.05), respectively, as well
as decreased glutamine content in mice hippocampus after seizures induced by PIC when compared with
seizures induced by PIC (p < 0.05). In addition, CA also increased Na+, K+-ATPase and d-aminolevulinic
acid dehydratase activities after seizures induced by P400, PIC or PTZ when compared with seizures
induced by P400, PIC or PTZ (p < 0.05), respectively. This study demonstrated that CA could be a future
therapeutic option for treatment of epilepsy, with a multifactorial brain action mechanism.
Ó 2014 Published by Elsevier Ireland Ltd.
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Keywords:
Anticonvulsant
Acetate
Carvacryl
Epilepsy
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Mouse
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1. Introduction
common serious neurological disorder [1]. In spite of a large arse-
nal of antiepileptic drugs, approximately 30% of patients are refrac-
tory to treatments [2]. Benzodiazepines are often used acutely in
emergency units to control seizures, but, chronically, might
develop physical and chemical dependence [3].
Seizures induced by pilocarpine [4–6], pentylenetetrazole [7] or
picrotoxin [8,9] have been widely investigated to test new anticon-
vulsant agents. Thus, this study evaluated anticonvulsant properties
of carvacryl acetate (CA), an acetylated derivative of carvacrol,
which has already showed anticonvulsant effects [10].
CA has already suggested a Central Nervous System (CNS) action,
as anxiolytic-like properties, probable mediated via GABAergic sys-
tem. Thus, the authors tested if the probable anticonvulsant effects
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Epilepsy is characterized by recurrent spontaneous seizures
caused by abnormal discharges and corresponds to the most
Abbreviations: CA, carvacryl acetate; DZP, diazepam; FLU, flumazenil; GABA,
d-aminobutyric acid; PIC, picrotoxin; PTZ, pentylenetetrazol; P400, pilocarpine;
d-ALA-D, d-aminolevulinic acid dehydratase.
⇑
Corresponding authors at: Laboratório de Pesquisa em Neuroquímica
Experimental, Universidade Federal do Piauí-UFPI, Campus Universitário Ministro
Petrônio Portella, Curso de Farmácia, Bairro Ininga, Teresina, PI, CEP 64.049-550,
Brazil. Tel.: +55 86 3215 5870.
E-mail addresses: lucio-pires@uol.com.br (L.F. Pires), rivelilson@pq.cnpq.br
(R.M. de Freitas).
http://dx.doi.org/10.1016/j.cbi.2014.12.001
0009-2797/Ó 2014 Published by Elsevier Ireland Ltd.
Please cite this article in press as: L.F. Pires et al., Neuropharmacological effects of carvacryl acetate on d-aminolevulinic dehydratase, Na+, K+-ATPase activ-
ities and amino acids levels in mice hippocampus after seizures, Chemico-Biological Interactions (2014), http://dx.doi.org/10.1016/j.cbi.2014.12.001

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of CA could be mediated via GABAergic system. For this purpose, flu-
mazenil was used as a GABAergic antagonist to clarify the probable
action mechanism of action anticonvulsant [4,11].
O
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Regarding that the pathophysiology of epilepsy may be corre-
lated with impairments in d-ALA-D and Na+, Ka+-ATPase function,
we also analyzed if CA must improve those enzyme functions after
seizures induced by pilocarpine, pentylenetetrazole or picrotoxin.
It is important to note that some molecules derived from plants
have already suggested not only anticonvulsant effects, but also
antioxidant properties. Similarly, CA produced antioxidant effect
in previous study [17]. These findings may, therefore, confer an
advantage to use derivatives of natural products rather than drugs
which have only anticonvulsant effects [4,5,7,9,10].
For the purposes, we evaluated carvacryl acetate effects after
seizures induced by P400, PTZ [9], and PIC in mice, as well as, we
measured the amino acid levels (glutamate, GABA, aspartate and
glutamine) in mice hippocampus after seizures induced by P400,
PTZ or PIC [18,19].
These three epilepsy models were applied in order to increase
sensitivity to suggest clinical anticonvulsant activity, which was
characterized by latency to first seizure, number of seizures and
mortality rate. These models were also used to detect alterations
in amino acid levels and enzymatic functions (Na+, K+-ATPase
and d-aminolevulinic acid dehydratase (d-ALA-D) [4–9]. In this
study, for example, there were occasions in which only one of these
models showed sensitivity to detect amino acids alterations.
Fig. 1. Chemical structure of carvacryl acetate (ethyl 5-isopropyl-2-methyl-
phenyl).
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(vehicle) and administered intraperitoneally (i.p.) at dose
100 mg/kg. This dose was selected in accordance with CA doses
used in previous studies conducted in our laboratory [11,17,22].
Note that doses inferior of 100 mg/kg were tested in three epilepsy
models, but they were not statistically significant when compared
with negative controls in this study (data not shown).
The other chemicals used in these experiments were obtained
from Sigma Chemical Co. (St. Louis, MO, USA). All doses were
administered at 10 ml/kg and injected i.p.
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2.2. Animals
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Male Swiss albino mice (25–30 g; 2 months old) were used in
experiments. They were maintained at a controlled temperature
(25 2 °C) and randomly placed in acrylic cages (maximum of six
animals per cage) and 12/12 h light–dark cycle (light phase:
6–18 h), with free access to water and food (PurinaÒ). Different
groups were used for each test. All experiments were conducted
between 8:00 and 10:00 AM, in a room with no noise.
The animals were observed during 24 h under similar ambient
conditions. All experiments were previously approved by Ethics
Committee on Animal Experimentation of Federal University of
Piauí (# 013/2011).
In epilepsy models, seizures were identified according with pre-
vious experiments in our laboratory, which reproduces behavioral
and electroencephalographic alterations which are similar to those
of human temporal lobe epilepsy. Behavioral alterations were clo-
sely observed during 24 h, and included akinesia, ataxic lurching,
peripheral cholinergic signs (miosis, piloerection, chromodacryor-
rhea, diarrhea and masticatory automatisms), tremors, staring
spell, facial automatisms, stereotyped movements (continuous
sniffing, paw licking, rearing and wet dog shakes that persisted
for 10–15 min) and motor limbic seizures, which develop progres-
sively within 1–2 h into status epilepticus [15,23,24].
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2. Material and methods
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2.1. Preparation of carvacryl acetate
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CA is chemically defined as ethyl 5-isopropyl-2-methyl-phenyl,
obtained with 98% purity, molar mass of 192.26 g/mol, refractive
index of 1497, boiling point of 94.56 °C at 760 mmHg; 48,414 kJ/
mol of vaporization enthalpy. Its color is yellow–green. It has a
pungent and astringent flavor, with a characteristic odor like oreg-
ano (Origanum vulgare L.). CA is found in liquid state at ambient
temperature, with density of 0.994 0.06 g/cm3. It was obtained
by carvacrol acetylation in which acetic anhydride was used as
acylating agent and pyridine as catalyst. There were added carva-
crol (5 g; 0.033 mol), pyridine (7.5 ml) and acetic anhydride
(12.5 ml), in a flask (50 ml) equipped with magnetic stirrer, cou-
pled to a Friedrich condenser in an inert atmosphere. Then, the
solution was subjected to magnetic stirring and under constant
reflux for 24 h [11].
Reaction mixture was poured into ice water (60 ml). The reac-
tion product was extraction in a dropping funnel and chloroform
was used as solvent extractor (3 times of 60 ml). Chloroform
phases were combined and washed with saturated copper sulfate
(3 times of 60 ml) and was also washed with water (3 times of
60 ml) and dried with Na2SO4 anhydrous. Subsequently, the sol-
vent was evaporated in a rotary evaporator. Reaction product
was subjected to column chromatography using silica gel as sta-
tionary phase and a mixture of hexane and ethyl acetate (95:5),
as mobile phase. There were obtained 4.779 g (0.025 mol) of carv-
acryl acetate with 76% yield [20,21].
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2.3. Pilocarpine-induced seizures
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Groups were used as follows: group 1 – vehicle (0.05% Tween
80 dissolved in distilled water, i.p., negative control; n = 10); group
2 – vehicle (i.p.) and, 30 min later, administration of P400 (i.p.l
n = 10; P400 group); group 3 – CA 100 mg/kg i.p. (CA100) and,
30 min later, administration of P400 (i.p., n = 10, CA100 plus
P400); group 4 – CA100 and, 30 min later, administration of vehicle
(i.p., n = 10, CA100 group); group 5 – diazepam 5 mg/kg (i.p., DZP5)
and, 30 min later, administration of P400 (i.p., positive control,
n = 10, DZP5 plus P400 group); group 6 – vehicle (i.p.) and,
30 min later, administration of DZP 5 mg/kg (n = 10; DZP5 group);
group 7 – Flumazenil 5 mg/kg (i.p. FLU5) and, 15 min later, DZP5
was administered. And thirty minutes after DZP5 administration,
P400 was administered (i.p., n = 10; FLU5 plus DZP5 plus P400
group); group 8 – FLU5 and, 15 min later, CA100 was administered.
And thirty minutes after CA administration, P400 was adminis-
tered (i.p., n = 10, FLU5 plus CA100 plus P400 group); and group
9 – vehicle (i.p.) and, 30 min later, they received administration
of flumazenil 5 mg/kg (n = 10; FLU5 group) [6].
The confirmation of chemical structure of CA (Fig. 1) was per-
formed by infrared spectroscopic data, 1H NMR and 13C NMR DEPT:
IR (4000–400 cmÀ1): 3050; 2950; 2850; 1750; 1500; 850. 1H NMR
(200 MHz, CDCl3): 7.20 (d, J = 7.80 Hz, 1H); 7.00 (d, J = 7.80 Hz, 1H);
6.90 (s, 1H); 2.95–2.75 (m, 1H); 2.30 (s, 3H); 2.15 (s, 3H); 1.26 (d,
J = 6.80 Hz, 6H); 13C NMR DEPT (50 MHz-CDCl3): 169.1; 149.1;
147.9; 130.7; 127.0; 124.0; 119.6; 67.3; 33.4; 23.7; 20.6; 15.6.
Subsequently, CA was emulsified with 0.05% Tween 80 (Sigma
Chem. Co., St. Louis, Missouri, USA), dissolved in distilled water
Please cite this article in press as: L.F. Pires et al., Neuropharmacological effects of carvacryl acetate on d-aminolevulinic dehydratase, Na+, K+-ATPase activ-
ities and amino acids levels in mice hippocampus after seizures, Chemico-Biological Interactions (2014), http://dx.doi.org/10.1016/j.cbi.2014.12.001

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2.4. Pentylenetetrazol-induced seizures
and number of entries with four legs on each square (spontaneous
locomotor activity) was measured during 5 min.
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PTZ (60 mg/kg; i.p.) was used to induce tonic–clonic seizures
[22]. Groups were used as follows: group 1 – vehicle (0.05% Tween
80 dissolved in distilled water, ip, negative control, n = 10); group 2
– vehicle (i.p.) and, 30 min later, administration of PTZ (i.p., n = 10,
PTZ group); group 3 – CA100 and, 30 min later, administration of
PTZ (i.p., n = 10, CA100 plus PTZ group); group 4 – CA100 and,
30 min later, administration of vehicle (i.p., n = 10, CA100 group);
group 5 – DZP5 and, 30 min later, administration of PTZ (i.p.; posi-
tive control, n = 10, DZP5 plus PTZ); group 6 – vehicle (i.p.) and,
30 min later, administration of DZP5 (n = 10, DZP5 group); group
7 – Flumazenil 5 mg/kg (i.p., FLU5) and, 15 min later, DZP5 was
administered. And thirty minutes after DZP5 administration, PTZ
was administered (n = 10; FLU5 plus DZP5 plus PTZ group); group
8 – FLU5 and, 15 min later, CA100 was administered. And thirty
minutes after CA administration, PTZ was administered (n = 10;
FLU5 plus CA100 plus PTZ group); and group 9 – vehicle (i.p.)
and, 30 min later, they received administration of flumazenil
5 mg/kg (n = 10, FLU5 group).
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2.6.2. Rota rod test
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In rota rod test, we evaluated motor coordination and muscle
relaxation produced by drugs in animals [31]. The mice were
trained before the experiment to acquire the capacity to remain
for 180 s on a diameter rod, rotating at 17 rpm [29]. Two or three
trials were sufficient for the animals to learn this task. The com-
pounds were tested alone in mice to assess the ability to reproduce
the performance on the next morning. DZP1 (1 mg/kg; i.p.), CA
(100 mg/kg, i.p.) or vehicle (0.05% Tween 80 dissolved in distilled
water, i.p.) was administered in each of the experimental groups
(n = 10). Then, animals were placed in the four paws on the rotat-
ing bar, which has 2.5 cm in diameter is 25 cm high from the floor,
and observed for 3 min. The ability of mice to remain on the rota
rod for 180 s and number of falls (up to three drops) were recorded.
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2.7. Evaluation of anticonvulsant effects
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2.5. Picrotoxin-induced seizures
After seizures induced by P400, PTZ or PIC, the animals were
placed in chambers of 30 Â 30 cm and observed for 24 h to record
the latency to first seizure (tonic–clonic seizures during 30 min
intermittently), number of animals, which had seizure and number
of animals which died after seizures.
Phenotypes of animals that survived 24 h after administration
of P400, PTZ or PIC were selected. According with protocols
described in previous studies conducted by our group, the animals,
which survived were euthanized by decapitation and their brains
were dissected on ice to remove the hippocampus of both hemi-
spheres for determination of enzymatic activities and amino acid
levels [4,14].
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This method has already been previously described [24–26].
Groups were used as follows: group 1 – vehicle (0.05% Tween 80
dissolved in distilled water, i.p., negative control, n = 10); group 2
– vehicle (i.p.) and, 30 min later, administration of PIC (i.p.,
n = 10, PIC group); group 3 – CA100 and, 30 min later, administra-
tion of PIC (i.p., n = 10, CA100 plus PIC group); group 4 – CA100
and, 30 min later, administration of vehicle (i.p., n = 10, CA100
group); group 5 – DZP5 and, 30 min later, administration of PIC
(i.p., positive control, n = 10, DZP5 plus PIC group); group 6 – vehi-
cle (i.p.) and, 30 min later, administration of DZP5 (n = 10, DZP5
group); group 7 – FLU5 (flumazenil 5 mg/kg, i.p.) and, 15 min later,
DZP5 was administered. Thirty minutes after DZP administration,
PIC was administered (i.p., n = 10, FLU5 plus DZP5 plus PIC group);
group 8 – FLU5 and, 15 min later, CA100 was administered. And
30 min after CA administration, PIC was administered (i.p.,
n = 10; FLU5 plus CA100 plus PIC group); and group 9 – vehicle
(i.p.) and, 30 min later, they received administration of flumazenil
5 mg/kg (i.p., n = 10, FLU5 group).
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2.8. Preparation of synaptic plasma membrane of mice hippocampus
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Hippocampi were homogenized in 1:10 volume (w/v) of 0.32 M
sucrose solution containing 5.0 mM HEPES and 0.1 mM EDTA, pH
7.4. Synaptic plasma membranes were prepared according to the
method described by Jones and Matus (1974) [32]. These mem-
branes were isolated using a discontinuous density gradient of
sucrose, consisting of successive layers of 0.3; 0.8 and 1.0 M. After
centrifugation at 69,000Âg for 2 h, the fraction between 0.8 and
1.0 M sucrose interface were used for the preparation of the
enzyme membrane.
Due to be recognized as an antiepileptic drug, DZP was used as a
positive control to compare with CA [3]. FLU was used as a GABAer-
gic antagonist drug to test if CA exerted their action mechanism by
GABAergic system [11,27].
2.9. Determination of Na+, K+-ATPase activity
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2.6. Locomotor activity and motor coordination
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Vehicle (0.05% Tween 80 dissolved in 0.9% saline) was used as
negative control and diazepam 1 mg/kg (i.p.; DZP1), was used as
positive control. CA (100 mg/kg; i.p.) formed the experimental
group. The spontaneous locomotor activity of animals was exam-
ined in a cage of 50 cm  50 cm  50 cm, 30 min after drugs
administration [28]. Behavioral tests were performed (n = 10 mice
per group) to evaluate its effects on spontaneous locomotor activ-
ity (number of squares entered during 5 min) in open-field test
[29] and motor coordination (ability of the mice to remain on the
rota rod during 180 s and number of falls – up to three falls – were
registered) in rota rod test [7].
The reaction mixture for the assay of Na+, K+-ATPase contained
5.0 mM of MgCl2, 80.0 mM of NaCl, 20.0 mM of KCl and 40.0 mM of
Tris–HCl, pH 7.4 in a final volume of 200 ml. The reaction was
started by addition of ATP to a final concentration of 3.0 mM. Con-
trols were conducted using the difference between the two sam-
ples, as described by Wyse et al. [33]. The released inorganic
phosphate (Pi) was measured by the method described by Chan
et al. [34]. The specific activity of the enzyme was expressed as
nmol of Pi released per minute per mg of protein.
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2.10. Determination of d-ALA-D activity
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2.6.1. Open-field test
The activities of d-ALA-D in mice hippocampus from all groups
were measured by the rate of product formation of porphobilino-
gen [35]. An aliquot of 200 ll of synaptic plasma membranes
obtained from each sample was incubated for 3 h at 37 °C. Enzy-
matic reaction was initiated by addition of substrate (d-ALA-D) to
a final concentration of 2.2 mM in a medium containing 45 mM
The open-field arena was made of acrylic (transparent walls and
black floor, 30 Â 30 Â 15 cm) divided into nine squares of equal
areas [30]. Groups (n = 10) were treated with vehicle (0.05% Tween
80 dissolved in distilled water, i.p.), DZP1 (1 mg/kg; i.p.) or CA
(100 mg/kg; i.p.). Mouse was placed individually into the apparatus
Please cite this article in press as: L.F. Pires et al., Neuropharmacological effects of carvacryl acetate on d-aminolevulinic dehydratase, Na+, K+-ATPase activ-
ities and amino acids levels in mice hippocampus after seizures, Chemico-Biological Interactions (2014), http://dx.doi.org/10.1016/j.cbi.2014.12.001

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Table 1
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phosphate buffer, pH 6.8. Reaction was stopped by adding 250 ll of
Carvacryl acetate effects on pilocarpine-induced seizures in mice.
trichloroacetic acid10% with 10 mM of HgCl2 and reaction product
was determined using modified Ehrlich’s reagent at 555 nm. Reac-
tion was typically linear with respect to protein concentration and
time of incubation. Enzyme activity was expressed as nmol of PBG/
mg of protein/h. Protein concentration was measured by the
method described by Lowry et al. [36].
Treatments
Latency to first seizure (min) % of seizures % of death
Vehicle
P400
CA100 + P400
CA100
DZP 5
–
0
100
60d
0
0
100
60d
0
8.36 0.34
36.83 2.08a
–
–
0
0
DZP 5 + P400
FLU 5
31.62 1.04a
–
40d
0
40d
0
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2.11. Evaluation of amino acids levels in mice hippocampus
FLU 5 + DZP 5 + P400 8.74 0.42b
FLU 5 + CA100 + P400 8.62 0.47c
100e
100f
100e
100f
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Animals which survived in the period of 24 h during the seizure
experimental protocols were euthanized by decapitation for
removal of the hippocampus to assess the amino acids levels
(GABA, glutamate, glutamine and aspartate).
Hippocampi were sonicated in HClO4 for 30 s and centrifuged
for 15 min in a refrigerated centrifuge at 15,000 rpm. Supernatant
was separated and filtered through a membrane filter (Millipore,
0.2 mM) and combined with a solution of pre-column derivatiza-
tion, to obtain fluorescence in a ratio of 1:1. One minute after the
Values are mean S.E.M. to 10 mice per group.
a
p < 0.001 when compared with P400 group.
p < 0.001 when compared with DZP 5 + P400.
b
c
p < 0.05 when compared with CA100 + P400 (Fisher’s test followed by t-Stu-
dent–Newman–Keuls as post hoc test).
d
p < 0.001 when compared with P400.
p < 0.001 when compared with DZP 5 + P400.
p < 0.05 when compared with CA100 + P400 (Fisher’s test). Subtitles: CA:
e
f
carvacryl acetate; P400: pilocarpine 400 mg/kg i.p.; DZP: diazepam; FLU:
flumazenil.
start of this association, a rate of 20 ll was removed and injected
into the HPLC equipment for chemical analysis.
For amino acid analysis, CLC-ODS column (M) with a length of
15 cm in diameter and 4.6 mm particle diameter of 3 microns Shi-
madzu, Japan was used. The mobile phase was used in a gradient
using two phases: A – NaH2PO4 (50 mM) and methanol (20% v/v)
at pH 5.5, B – Pure methanol (100%). Aspartate, glutamate, gluta-
mine and GABA were detected using a fluorescence detector
(Model RF-535 from Shimadzu, Japan) with wavelengths of EX-
Wavelength (370 nm) and MS-wavelength (450 nm). The chro-
matograms were recorded and quantified by a computer using
software from Shimadzu.
358
359
360
361
362
363
364
365
366
30% in CA100 + PTZ when compared with 100% of PTZ
(p < 0.0001). An increase of 345.96% on latency for first seizure
for the CA100 + PTZ was observed when compared with PTZ
(p < 0.0001). In FLU + CA100 + PTZ group, there was a reversal of
the three measured parameters when compared with CA100 + PTZ.
The percentage of deaths on FLU + CA100 + PTZ was 100%; the per-
centage of seizure was 100% and the latency for first seizure was
reduced by 77.38% when compared with CA100 + PTZ
(p < 0.0001) (Table 2).
The amount of amino acid levels was calculated by comparing
the peak height obtained from the means of the patterns and the
results were expressed in lmol/g of tissue [19].
367
3.3. Carvacryl acetate effects after picrotoxin-induced seizures
368
369
370
371
372
373
374
375
376
377
378
In CA100 + PIC, there was a reduction in number of deaths (50%
of mice died when compared with 100% in PIC group (p < 0.001)).
There was a reduction in percentage of seizures to 50% in
CA100 + PIC when compared with 100% in the PIC group
(p < 0.001). An increase of 163.67% on latency for first seizure in
CA100 + PIC was observed, when compared with PIC (p < 0.001).
In FLU + CA100 + PIC, there was a reversal of three measured
parameters when compared with CA100 + PIC (p < 0.001). Percent-
age of deaths in FLU + CA100 + PIC was 100%; the percentage of sei-
zure was 100% and the latency for first seizure was reduced by
61.83% when compared with CA100 + PIC (p < 0.001) (Table 3).
334
2.12. Statistical analyzes
335
336
337
338
339
The data obtained on latency to first seizure and neurochemical
changes were evaluated by Fisher’s test followed by t-Student–
Newman–Keuls as post hoc test. Percentage of tonic–clonic limbic
seizures and deaths were evaluated by Fisher’s test. Differences
were considered statistically significant when p < 0.05.
340
341
3. Results
3.1. Carvacryl acetate effects after pilocarpine-induced seizures
Table 2
Carvacryl acetate effects on pentylenetetrazol-induced seizures in mice.
342
343
344
345
346
347
348
349
350
351
352
353
In CA100 + P400 group, the number of mice which died was
reduced in 60% when compared with P400 group (p < 0.001). A
reduction in percentage of seizures of 60% in CA100 + P400, when
compared with P400 group (p < 0.001) and an increase of
340.55% in latency to first seizure in CA100 + P400 when compared
with P400 (p < 0.001, Table 1).
In addition, FLU + CA100 + P400 group had a reversal of these
three parameters when compared with CA100 + P400. The percent-
age of mice which died and the number of animals which had sei-
zures in FLU + CA100 + P400 group were 100% and latency for first
seizure was reduced by 76.59% when compared with CA100 + P400
(p < 0.05) (Table 1).
Treatments
Latency for the first seizure % of
% of
death
(min)
seizures
Vehicle
PTZ
CA100 + PTZ
CA100
–
0
100
30d
0
0
100
30d
0
77.34 1.65
344.91 11.90a
–
DZP 5
DZP 5 + PTZ
FLU 5
FLU 5 + DZP 5 + PTZ
FLU 5 + CA100 + PTZ
–
0
0
795.12 17.67a
–
30d
0
30d
0
77.25 1.70b
78.03 1.75c
100e
100f
100e
100f
Values are mean S.E.M. to 10 mice per group.
a
p < 0.0001 when compared with PTZ.
p < 0.0001 when compared with DZP5 + PTZ.
b
c
p < 0.0001 when compared with CA100 + PTZ (Fisher’s test followed by t-Stu-
dent–Newman–Keuls as post hoc test).
354
3.2. Carvacryl acetate effects after pentylenetetrazol-induced seizures
d
p < 0.0001 when compared with PTZ.
p < 0.0001 when compared with DZP5 + PTZ.
p < 0.0001 when compared with CA100 + PTZ (Fisher’s test). Subtitles: CA:
355
356
357
In CA100 + PTZ group, number of deaths was decreased (30% of
the mice in this group died when compared with 100% in PTZ
(p < 0.0001)). There was a reduction in percentage of seizures for
e
f
carvacryl acetate; PTZ: pentylenetetrazole; DZP: diazepam; FLU: flumazenil.
Please cite this article in press as: L.F. Pires et al., Neuropharmacological effects of carvacryl acetate on d-aminolevulinic dehydratase, Na+, K+-ATPase activ-
ities and amino acids levels in mice hippocampus after seizures, Chemico-Biological Interactions (2014), http://dx.doi.org/10.1016/j.cbi.2014.12.001

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Table 3
A
Carvacryl acetate effects on picrotoxin-induced seizures in mice.
4
3
2
1
0
Treatments
Latency for the first seizure % of
% of
(min)
seizures deaths
a
Vehicle
PIC
CA 100 + PIC
CA 100
DZP 5
DZP 5 + PIC
FLU 5
FLU 5 + DZP 5 + PIC
FLU 5 + CA 100 + PIC
–
0
100
50d
0
0
100
50d
0
479 6.97
1263 10.12a
–
–
0
0
1288 9.81a
–
30d
0
30d
0
482 6.35b
483 7.14c
100e
100f
100e
100f
Values are mean S.E.M. to 10 mice per group.
a
p < 0.001 when compared with PIC.
p < 0.0001 when compared with DZP5 + PIC.
b
c
p < 0.001 when compared with CA100 + PIC (Fisher’s test followed by t-Student–
Newman–Keuls as post hoc test).
B
d
p < 0.0001 when compared with PIC.
p < 0.0001 when compared with DZP5 + PIC.
p < 0.0001 when compared with CA100 + PIC (Fisher’s test). Subtitles: CA:
e
180
f
a
160
140
120
100
80
carvacryl acetate; PIC: picrotoxin; DZP: diazepam; FLU: flumazenil.
379
380
In all experiments, none mice treated with CA alone showed
significant behavioral alteration when compared with vehicle.
60
381
3.4. Carvacryl acetate effects on mice subjected on open field test
40
382
383
384
385
386
387
The mice subjected to DZP1 (DZP1: 35.75 2.66) treatment
(n = 10) showed a reduction of 53.76% in number of square entries
when compared with negative control (vehicle: 79.00 2.09,
p < 0.0001). However, CA100 group (CA100: 73.75 1.74) had no
significant change in spontaneous locomotor activity when com-
pared with negative control (p > 0.05) (Fig. 2).
20
0
Fig. 3. Carvacryl acetate effects on number of falls (A) and on time spent on the
rotating bar (B) on rota rod test. Values represent the mean S.E.M. of number of
falls and the time spent on the rotating bar (n = 10 per group). ap < 0.0001 when
compared with negative control (vehicle); (Fisher’s test followed by t-Student–
Newman–Keuls as post hoc test). CA100: carvacryl acetate (100 mg/kg, i.p.); DZP:
diazepam (1 mg/kg, i.p.).
388
3.5. Carvacryl acetate effects on mice subjected on rota rod test
389
390
391
392
393
394
395
396
397
398
Fig. 3A shows that DZP1 group (n = 10; DZP1: 2.7 0.21) had
number of falls increased by 123% when compared with negative
control (vehicle: 1.20 0.29; p < 0.0001). CA100 group (n = 10)
did not alter this parameter when compared with negative control
(CA100 1.60 0.16, p > 0.05).
DZP1 group (n = 10, DZP1: 149.10 1.33) had the time spent on
rotating bar reduced by 18.27% when compared with negative con-
trol (vehicle: 176.60 0.98, p < 0.0001) while CA100 group (CA100:
169.50 1.20) had not statistical significance when compared with
negative control (p > 0.05) (Fig. 3B).
399
400
3.6. Carvacryl acetate effects on d-ALA-D hippocampal activity after
seizures
401
402
403
404
405
406
P400, PTZ and PIC groups reduced in 33%; 40.15% and 47.8% d-
ALA-D activity when compared with negative control, respectively
(p < 0.0001). In CA100 + P400 or PIC or PTZ groups, there was
increases of 47.35%; 57.24% and 83.38% in this parameter when
compared with P400, or PTZ, or PIC alone (p < 0.0001), respectively
(Fig. 4).
3.7. Carvacryl acetate effects on Na+, K+-ATPase activity after seizures
407
408
409
410
411
412
413
P400, PTZ and PIC groups showed reductions of 37%; 32.7% and
39.8%, respectively, on Na+, K+-ATPase activity when compared
with negative control (p < 0.0001). In CA100 + P400 or PTZ or PIC
groups, there was increases of 30.7%; 28% and 32.6% on this param-
eter when compared with P400 or PTZ or PIC alone (p < 0.0001),
respectively (Fig. 5).
414
3.8. Carvacryl acetate effects on amino acids levels after seizures
Fig. 2. Carvacryl acetate effects on number of squares entered in open field test.
Values represent the mean the standard error of the means (S.E.M.) number of
squares entered of mice (n = 10 per group) used in experiments. ap < 0.0001 when
compared with negative control (vehicle) (Fisher’s test followed by t-Student–
Newman–Keuls as post hoc test). CA100: carvacryl acetate (100 mg/kg, i.p.); DZP:
diazepam (1 mg/kg, i.p.).
415
416
417
418
419
P400, PTZ and PIC groups showed no statistical difference in
GABA concentrations when compared with negative control
(p > 0.05). However, CA100 + P400 had an increase in GABA by
78.25% when compared with P400 group (p < 0.001). CA100 + PTZ
increased GABA by 51.43% when compared with PTZ (p < 0.05).
Please cite this article in press as: L.F. Pires et al., Neuropharmacological effects of carvacryl acetate on d-aminolevulinic dehydratase, Na+, K+-ATPase activ-
ities and amino acids levels in mice hippocampus after seizures, Chemico-Biological Interactions (2014), http://dx.doi.org/10.1016/j.cbi.2014.12.001

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100
75
50
25
0
d
b
c
a
a
a
Vehicle
CA100
P400
CA100 + P400
PTZ
CA100 + PTZ
PIC
CA100 + PIC
Fig. 4. Carvacryl acetate effects on d-aminolevulinic acid dehydratase (d-ALA-D) activity in mice hippocampus after pilocarpine-, picrotoxin- or pentylenetetrazoleinduced
seizures. Differences were determined by Fisher’s test followed by t-Student–Newman–Keuls as post hoc test. ap < 0.0001, when compared with negative control; bp < 0.0001,
when compared with P400 group; cp < 0.0001, when compared with PTZ group. dp < 0.0001, when compared with PIC group. CA100: carvacryl acetate (100 mg/kg, i.p.); P400:
Pilocarpine (400 mg/kg, i.p.); PTZ: pentylenetetrazole (60 mg/kg, i.p.); PIC: picrotoxin (7.5 mg/kg, i.p.).
1250
c
b
d
1000
750
500
250
0
a
a
a
Vehicle
CA100
P400
CA100 + P400
PTZ
CA100 + PTZ
PIC
CA100 + PIC
Fig. 5. Carvacryl acetate effects on Na+, K+-ATPase activity in mice hippocampus after pilocarpine-, picrotoxin- or pentylenetetrazole-induced seizures. Differences were
determined by Fisher’s test followed by t-Student–Newman–Keuls as post hoc test. ap < 0.0001, when compared with negative control; bp < 0.0001, when compared with P400
group; cp < 0.0001, when compared with PTZ; group. dp < 0.0001, when compared with PIC group. CA100: carvacryl acetate (100 mg/kg, i.p.); P400: Pilocarpine (400 mg/kg,
i.p.); PTZ: pentylenetetrazole (60 mg/kg, i.p.); PIC: picrotoxin (7.5 mg/kg, i.p.).
420
421
422
423
424
425
426
427
428
429
430
431
432
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
471
472
CA100 + PIC increased GABA content in 103% when compared with
PIC group (p < 0.001) (Fig. 6A).
(Sections 3.1–3.3). Similarly with DZP, these effects were also
inhibited by FLU. According with scientific literature, FLU is used
as a GABAergic antagonist and does not induce seizures when
administered alone. These findings are in accordance with our
results (Tables 1–3). Using these references, we can propose that
CA exert anticonvulsant effect via GABAergic system [4,27]. This
effect may also be correlated, at least in part, with the anxiolytic-
like effect of CA in another study conducted in our laboratory [11].
In the results, it should be noted that some animal groups had a
discrepancy between the percentages of seizures and the percent-
ages of deaths (Tables 1–3). It proposes that a direct relationship
between these two parameters is not mandatory, meaning that
CA could reduce the percentage of seizures, but not reduce the per-
centage of deaths proportionally.
Considering the open-field test, DZP impaired locomotor activ-
ity, reflected by reduction in number of squares entered. It also
reduced motor coordination, verified by increase in number of falls
and reduced time spent in the rotating bar in rota rod test [40].
However, CA100 did not change the number of squares entered
in open field test. In trota rod test, the number of falls and the time
spent on rotating bar were not significant changed (Sections 3.4
and 3.5). These findings propose that CA at dose 100 mg/kg did
not present sedative action, nor muscle relaxing effect.
There was no statistical significance when glutamate and aspar-
tate concentrations in P400, PTZ and PIC groups were compared
with negative control. CA100 + P400 or PTZ or PIC neither had sta-
tistical significance when compared with respective positive con-
trols (p > 0.05) (Fig. 6B and C).
Additionally, there was no statistical significance on glutamine
concentrations in P400, PTZ and PIC groups when compared with
negative control, as well as when CA100 + P400 and CA100 + PTZ
were compared with their positive controls. CA100 + PIC decreased
by 42.8% glutamine concentration when compared with PIC
(p < 0.05) (Fig. 6D).
433
4. Discussion
434
435
436
437
438
439
440
441
442
443
444
Benzodiazepines are one of the pharmacology option precon-
ized in epilepsy treatment. GABAergic agonists, diazepam, act as
an allosteric modulator of GABAA receptor, potentiating the c-ami-
nobutyric acid effect when bound to this receptor [37]. Thus, in this
study, DZP was used as standard drug for comparison with CA
effects after seizures induced by P400, PTZ or PIC.
Prior administration of DZP prevented the onset of seizures,
increased latency for first seizure and reduced number of deaths,
as expected. FLU reduced its anticonvulsant effect (Sections 3.1–
3.3) [38,39]. CA also reduced number of seizures, deaths and
increased latency for first seizure in all the three epilepsy models
Muscle relaxing effect and reduction in motor coordination
indicate in locomotor activity retardation, which is one of the main
adverse effects observed during treatment with benzodiazepine.
Decreases in the number of crossings on open field test suggest a
muscle relaxing effect. And increases in number of falls, as well
Please cite this article in press as: L.F. Pires et al., Neuropharmacological effects of carvacryl acetate on d-aminolevulinic dehydratase, Na+, K+-ATPase activ-
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A
C
2.0
4
c
a
b
1.5
3
2
1
0
1.0
0.5
0.0
D
B
3
4
3
2
1
0
2
1
0
c
Fig. 6. Carvacryl acetate effects on d-aminobutyric acid (GABA) (A), Glutamate (B), Aspartate (C) and Glutamine (D) levels in mice hippocampus after pilocarpine-, picrotoxin-
or pentylenetetrazole-induced seizures. Differences were determined by Fisher’s test followed by t-Student–Newman–Keuls as post hoc test. ap < 0.001, when compared with
P400 group; bp < 0.05,when compared with PTZ group. cp < 0.05, when compared with PIC group. CA100: carvacryl acetate (100 mg/kg, i.p.); P400: Pilocarpine (400 mg/kg,
i.p.); PTZ: pentylenetetrazole (60 mg/kg, i.p.); PIC: picrotoxin (7.5 mg/kg, i.p.).
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
498
499
500
501
502
503
504
505
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522
as decreases in time spent in rotating bar in rota rod test suggest a
muscle relaxing effect and a reduction in the motor coordination
[7,40]. Considering the findings of Sections 3.4 and 3.5, CA showed
a potential to be more tolerated than DZP, the standard drug to
treat seizures, since CA did not significantly change the parameters
analyzed in open field and rota rod tests.
In neurochemical assays, we observed that CA100 improved d-
ALA-D and Na+, K+-ATPase activities, which was previously reduced
by P400, PTZ or PIC (Sections 3.6 and 3.7). d-ALA-D is a sulfhydryl-
containing enzyme essential for aerobic organisms that catalyzes
the synthesis of tetrapyrrolic compounds such as billins and
hemes. d-ALA-D contains sulfhydryl (ÀSH) groups and zinc, which
are essential for its activity. Therefore, d-ALA-D is inhibited by sub-
stances that compete with zinc and/or that oxidize the ÀSH groups
and is sensitive to situations associated with oxidative stress. Fur-
thermore, enzyme inhibition can lead to accumulation of substrate
5-aminolevulinate in the blood, which in turn can intensify oxida-
tive stress by generating carbon-centered reactive species or by
releasing iron from proteins such as ferritin [41].
antioxidant activity and by oxidative inactivation of Na+, K+-ATPase
[42–45].
Thus, considering the findings of Sections 3.6 and 3.7, we can
propose that, in addition to increase latency to first seizure, reduce
the number of seizures and the number of deaths, CA also sug-
gested a capacity to improve these two enzymatic functions, which
may be involved in pathophysiology of epilepsy.
Regarding amino acid levels, it is reported that an extracellular
aspartate and glutamate accumulation space can lead excitotoxic-
ity. However, some antiepileptic drugs inhibit these excitatory
amino acids and increase GABAergic action. Additionally, hippo-
campal glutamine levels could be increased in some epilepsy mod-
els, whereas levetiracetam reduces this increase [46–49]. CA
demonstrated a similar tendency to those antiepileptic drugs, since
increased GABA level in P400, PTZ and PIC models and reduced glu-
tamine content in PIC model (Fig. 6).
However, that tendency was not observed for all the other
amino acids concentration. In some occasions, only one epilepsy
model had sensibility to detect alterations in some amino acid
level, but the other epileptogenic substance did not. As cited above,
although reduction on glutamine levels by CA100 is consistent
with antiepileptic drug properties [47], this finding existed only
for PIC model. It justifies, at least in part, why we applied three epi-
lepsy models, considering that P400 and PTZ had not sensibility to
detect these changes.
Na+, K+-ATPase is present at high concentrations in brain cellu-
lar membranes, consuming about 40–50% of ATP generated in this
tissue. It is a membrane bound enzyme known to play a pivotal
role in cellular ionic gradient maintenance and is particularly sen-
sitive to reactive species. The overproduction of free radicals may
lead to the development of epileptic focus by disruption of
Please cite this article in press as: L.F. Pires et al., Neuropharmacological effects of carvacryl acetate on d-aminolevulinic dehydratase, Na+, K+-ATPase activ-
ities and amino acids levels in mice hippocampus after seizures, Chemico-Biological Interactions (2014), http://dx.doi.org/10.1016/j.cbi.2014.12.001

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It has been published that glutamate, aspartate and glutamine
578
References
levels are increased during seizures [46,47], instead the results
obtained in this study were different, showing no increase in these
amino acid levels in seizures induced by P400, PTZ and PIC when
compared with negative control.
However, it seems there is not a complete consensus about
these amino-acid levels in epilepsy models. On studying frontal
and temporal foci, Van Gelder and co-workers found a decrease
in aspartate in all the examined areas, and a decrease in glutamate
levels in excitable regions [48]. Conversely, another study reported
a 28% increase in aspartate concentration in surgical specimens
from patients with temporal epilepsy [48,49].
Nevertheless, there are a greater number of studies showing the
tendency of animal in epilepsy models suffer an increase in GABA
levels when treated with anticonvulsant agents, which is in accor-
dance with our results [46–50].
The addition of an ester group to carvacrol theoretically could
confer CA an additional lipophilic property, which would allow
an easier penetration on blood–brain barrier and exerts its possible
therapeutic potential more safely and effectively. However, there
are not any study comparing these parameters between carvacrol
and CA [51]. Additionally, it is not possible to compare anticonvul-
sant activity of CA with carvacrol yet, since the CA doses used in
our experiment were different from carvacrol doses used by Quin-
tans-Júnior et al., 2010 [10].
This study showed significant anticonvulsant effects of CA at
dose 100 mg/kg. Beyond this anticonvulsant effect, CA improved
Na+, K+-ATPase and d-ALA-D enzymatic activities. These findings
can be correlated with the antioxidant effect suggested previously,
in which CA at different concentrations and doses tested, including
at dose 100 mg/kg, reduced lipid peroxidation content and nitrite
levels (in vitro and in vivo) and hydroxyl radical formation
in vitro, as well as increased reduced glutathione levels and
improved glutathione peroxidase and catalase activities in vivo
[17]. Taken all these findings together, CA could suggest multifac-
torial action mechanisms that can contribute to their anticonvul-
sant effects.
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560
5. Conclusions
561
Although the anticonvulsant activity of CA was not superior to
diazepam in this study, but CA did not alter locomotor activity
and suggested that improve Na+, K+-ATPase and d-aminolevulinic
acid dehydratase activities. Some amino acid concentrations in
mice hippocampus after seizures induced by P400, PTZ or PIC,
which were altered by seizures, were also regulated after CA treat-
ment. In summary, CA anticonvulsant property seems to be multi-
factorial, demonstrating a significant potential in epilepsy models.
562
563
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566
567
568 Q2
569
570
Conflict of Interest
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We would like to thank the National Council of Technological
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ities and amino acids levels in mice hippocampus after seizures, Chemico-Biological Interactions (2014), http://dx.doi.org/10.1016/j.cbi.2014.12.001

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ities and amino acids levels in mice hippocampus after seizures, Chemico-Biological Interactions (2014), http://dx.doi.org/10.1016/j.cbi.2014.12.001