Anticonvulsant and behavioral effects observed in mice following treatment with an ester derivative of ferulic acid: Isopentyl ferulate

Article abstract of DOI:10.1016/j.cbi.2015.10.003

The objective of this study was to evaluate the potential anticonvulsant effect of isopentyl ferulate, a new ester derived from ferulic acid in mice (Mus musculus) subjected to two models of induced seizures. According to the results obtained, the IF at d

Full text of DOI:10.1016/j.cbi.2015.10.003

Accepted Manuscript
Anticonvulsant and behavioral effects observed in mice following treatment with an
ester derivative of ferulic acid: Isopentyl ferulate
Keylla C. Machado, George Laylson S. Oliveira, Kátia C. Machado, Md. Torequl
Islam, Antonio Luiz G. Junior, Damião P. De Sousa, Rivelilson M. Freitas
PII:
S0009-2797(15)30082-X
10.1016/j.cbi.2015.10.003
CBI 7486
DOI:
Reference:
To appear in: Chemico-Biological Interactions
Received Date: 27 May 2015
Revised Date: 4 September 2015
Accepted Date: 5 October 2015
Please cite this article as: K.C. Machado, G.L.S. Oliveira, K.C. Machado, M.T. Islam, A.L.G. Junior, D.P.
De Sousa, R.M. Freitas, Anticonvulsant and behavioral effects observed in mice following treatment
with an ester derivative of ferulic acid: Isopentyl ferulate, Chemico-Biological Interactions (2015), doi:
10.1016/j.cbi.2015.10.003.
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Graphical abstract

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Anticonvulsant and behavioral effects observed in mice following treatment with
an ester derivative of ferulic acid: Isopentyl ferulate
Keylla C. Machadoa, George Laylson S. Oliveirab, Kátia C. Machadob, Md. Torequl
Islamb, Antonio Luiz G. Junior b, Damião P. De Sousac, Rivelilson M. Freitasa,b,*
aPost-Graduation Program in Pharmacology, Federal University of Piauí, CEP 64.049-
550, Teresina, Piauí, Brazil.
bLaboratory of Research in Experimental Neurochemistry of Post-Graduation Program
in Pharmaceutical Sciences, Federal University of Piauí, CEP 64.049-550, Teresina,
Piauí, Brazil.
cDepartment of Pharmaceutical Science, Federal University of Paraíba, CEP 58.051-
900, João Pessoa, Paraíba, Brazil.
Corresponding Author:
*Programa de Pós-graduação em Ciências Farmacêuticas/ Universidade Federal do
Piauí, Ininga, Teresina, CEP 64.049-550, Piauí, Brazil. Phone/Fax: 55-86-3215-5870.
E-mail: rivelilson@pq.cnpq.br

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Abstract
The objective of this study was to evaluate the potential anticonvulsant effect of
isopentyl ferulate, a new ester derived from ferulic acid in mice (Mus musculus)
subjected to two models of induced seizures. According to the results obtained, the IF at
doses of 25, 50 and 75 mg/kg (i.p.) showed protective effect against induced seizures by
pilocarpine (400 mg/kg, i.p.) and pentylenetetrazole (70 mg/kg, i.p.). In the two animal
models of seizures, the pretreatment of the IF (25, 50 and 75 mg/kg) with flumazenil
blocked the anticonvulsant effect, suggesting that the mechanism of action of this ester
derived of ferulic acid may be related to activity in the benzodiazepine-binding site of
the GABAA receptor (γ-aminobutyric acid, type A). In addition to the anticonvulsant
effect, behavioral changes as neurotoxicity indication were assessed by using the rota
rod and open field tests. The results obtained showed that the IF (25, 50 and 75 mg/kg)
does not induce significant changes in locomotor activity and motor coordination when
compared with the control group, unlike the results presented by diazepam. Thus, these
results demonstrate a new pharmacological knowledge of IF with potential application
against epileptic seizures. However, further studies are needed to elucidate other
neurobiological mechanisms underlying epilepsy.
Keywords: Anticonvulsant effect; epileptic seizures; isopentyl ferulate; locomotor
activity; motor coordination.

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1. Introduction
Epilepsy is a severe neurological disorder characterized mainly by recurrent
seizures due to an abnormal neuronal hyperexcitability in the brain and according to the
World Health Organization, affects approximately 50 million people worldwide [1,2].
Even with the significant advances made by several research groups for the treatment of
epilepsy, approximately 30% of patients treated with antiepileptic drugs showed
recurrence of uncontrolled seizures [3]. Furthermore, the main problems associated with
antiepileptic drugs (e.g. - carbamazepine, ethosuximide, phenobarbital, phenytoin,
primidone, and valproate) involve to exhibit some side effects such as cognitive
dysfunction, ataxia, sedation, hypersensitivity or worsening of seizures [4, 5].
Way forward, there is a need for the development of new research with the
objective of evaluating new molecules with a better therapeutic effectiveness than
antiepileptic drugs already existing. The natural products derived from plants represent
a great opportunity for the discovery of new substances with potential therapeutic
interest for the treatment of epilepsy [6, 7]. Among the groups of substances from
natural origin with potential antiepileptic effect, the essential oils play an important role
in scientific research due to their high level of bioactive components, by which can be
detached the derivatives of phenylpropanoid and terpenoid [8].
The phenylpropanoids are a group of compounds derived from the carbon
skeleton of phenylalanine, which have various neuropharmacological activities [9, 10,
11]. Among these compounds, stands out the ferulic acid that presents several
pharmacological activities such as neuroprotective [12], antidiabetic [13], anticancer
[14] anti-inflammatory [15], antidepressant [16, 17] and mainly antioxidant [18]. In
addition, the development of their derivatives that comprise similar chemical structure
are promising in relation to a range of new pharmacological properties on the central

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nervous system, which may be related to neuroprotective action [19, 20, 21]. Despite its
promising pharmacological activities, few researches have directed a pharmacological
study on phenylpropanoids for the treatment of epilepsy.
Thus, the present study has the objective of providing information on potential
anticonvulsant effect and the possible mechanism of action of Isopentyl ferulate (IF), a
ester derivative of ferulic acid in mice subjected to two seizure models (Pilocarpine-
induced seizures and Pentylenetetrazole-induced seizures). This compound was also
tested for the muscular relaxation effect and locomotor activity using the rota rod test
and open field test, respectively.
2. Materials and methods
2.1. Reagents and drugs
Polyoxyethylene sorbate (Tween 80), pilocarpine, pentylenetetrazole and
flumazenil were obtained from Eg Sigma Chem Ex. Co. St. Louis, Missouri, USA.
Diazepam was purchased from Union Chemical (Brazil). All the other chemicals were
of the analytical grade.
2.2. Preparation of the substance
Isopentyl ferulate (IF, Fig. 1) has a molecular formula of C15H20O4, refractive
index of 1.544 ± 0.02, surface tension of 40.3 ± 3.0 dyn/cm and density of 1.104 ± 0.06
g/cm3. The process of developing the product consists of a reaction of esterification of
ferulic acid [22, 23]. The process of esterification of ferulic acid consists in a stirred
mixture of ferulic acid (5 mmol) in isoamyl alcohol (200 ml). Ethanol was added to
concentrated sulfuric acid (0.067 mL, 1.25 mmol) and the reaction mixture was
subjected to reflux for 3 hours in a 500 ml flask. After cooling to 25 °C, ethyl acetate
was added and the solution was washed with water and brine. The ethyl acetate fraction

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was dried over MgSO4, filtered, and concentrated under reduced pressure. The residue
was purified by chromatography on a silica gel column using 20% ethyl acetate in
hexane to produce isopentyl ferulate (yield 55%) [24]. Subsequently, the IF was
emulsified with 0.05% Tween 80 dissolved in 0.9% saline and administered via
intraperitoneal (i.p.) in doses of 25 mg/kg (IF 25), 50 mg/kg (IF 50) and 75 mg/kg (IF
75).
Fig. 1. Chemical structure of isopentyl ferulate (3-methylbutyl (E)-3-(4-hydroxy-3-
methoxyphenyl)prop-2-enoate (1 and 1A).
2.3. Animals and evaluation of behavioral seizure
Male Swiss (Mus musculus) adult mice (25–30 g; 2 months-old) were used in
this study (Central Animal Laboratory of the Federal University of Piaui). Animals were
housed in cages (35 cm L × 52 cm W × 17 cm H) with free access to food (Purina®
pellets) and water and were kept under standard artificial light–dark cycle (lights on at

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07:00 a.m.) with controlled temperature (25 ± 2°C). The animals were acclimated for
three days before the experiments and were housed in groups during the experiments.
Animals were tested during the light period and observed in a closed room with
controlled temperature (25 ± 2°C).
Protocol and procedures were approved by the Ethics Committee in Animal
Experimentation of UFPI (CEEA/UFPI - authorization number: #030/13). All protocols
were designed aiming to reduce the number of animals used to a minimum, as well as to
minimize their suffering. The experiments were performed according to the Guide for
Care and Use of Laboratory of US Department of Health and Human Services,
Washington, DC (1985).
To investigate the effects of IF, an acute treatment to the test animals received
by intraperitoneally. Vehicle group was treated with 0.05% Tween 80 dissolved in 0.9%
saline (0.1 mL/kg, negative control). IF 25, IF 50 and IF 75 groups were treated with
emulsified IF in the vehicle mentioned at doses of 25, 50 and 75 mg/kg, respectively.
The doses used were based on a preliminary pharmacological screening protocol [25]
with different doses of the IF administered to mice. At all doses used, no signs of acute
toxicity or behaviors suggestive of neurotoxicity were observed (data not shown).
Diazepam (DZP) group was treated with diazepam (2 mg/kg, positive control)
emulsified in vehicle. The selection of the dose of diazepam was based on previous
studies [26, 27].
In turn, to clarify the mechanism of action of IF, other groups were treated with
flumazenil (FLU), DZP and IF at a dose of 75 mg/kg and associations. The FLU group
was treated with flumazenil (5 mg/kg) emulsified vehicle. The DZP + FLU group was
pretreated with flumazenil (5 mg/kg) and, after 15 min treated with DZP (2 mg/kg). The

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IF 75 + FLU group (n = 8) was pretreated with FLU (5 mg/kg) and, after 15 min, treated
with IF 75.
2.4. Pilocarpine-induced seizure
This model was developed by Turski et al. [28]. Briefly, after 30 min from the
doses of 25, 50 and 75 mg/kg (i.p.), all the groups received pilocarpine (400 mg/kg,
i.p.). Direct observation was made for 4 h to monitor latency to the first seizure (tonic-
clonic seizures, with or without raising) and the number of animals that seized and/or
death [29, 30, 31].
2.5. Pentylenetetrazole-induced seizure
Pentylenetetrazole (PTZ) was used to induce clonic convulsions [32]. Briefly,
after 30 min from the doses of 25, 50 and 75 mg/kg (i.p.), all groups received PTZ (70
mg/kg, i.p.) and animals were observed for 4 h to monitor the same parameters of the
previous test [7].
2.6. Open Field Test
The open-field apparatus Archer [33], was made of acrylic (transparent walls
with black floor, 30 x 30 x 15 cm) divided into nine squares of equal area. After 30 min
of treatments with vehicle (0.05% Tween 80 dissolved in 0.9% saline, i.p.), DZP
(2 mg/kg; i.p.) or IF (25, 50 and 75 mg/kg, i.p.), the number of squares crossed with
four paws (spontaneous locomotor activity) was measured during 5 min. Furthermore, it
was observed the frequency of self-cleaning behavior (grooming) and number of
surveys (rearing) for 5 minutes [25].
2.7. Rota-Rod Test

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The rota rod test was used for the evaluation of motor coordination and muscle
relaxation produced by drugs in animals [34]. After 30 min of treatments with vehicle
(0.05% Tween 80 dissolved in 0.9% saline, i.p.), DZP (2 mg/kg; i.p.) or IF (25, 50 and
75 mg/kg, i.p.), mice were placed with the four paws on a bar of 25 mm of diameter at a
rotation speed of 17 rpm for a period of 3 min for each animal. The time of permanence
on the bar (second) and the number of falls was measured with three renewals at most.
2.8. Statistical analysis
The results were expressed as mean ± standard error of mean (SEM). Statistical
analysis was performed using one-way ANOVA for multiple comparisons and followed
by Student–Newman–Keuls as post hoc test by GraphPad Prism (version 6.0; GraphPad
San Diego, California, USA. copyright © 1994-1999). Chi square test was used to
analyse percentage of animals with seizures and mortality rate. Differences were
considered statistically significant when p<0.05.
3. Results
After administration of pilocarpine and pentylenetetrazol, the tested animals
(n=8) showed peripheral cholinergic signs (miosis, piloerection, chromodacryorrhea,
diarrhea and orofacial movements), and stereotyped movements (grooming and
rearing), which persisted until seizures (Table 1 and 2).
3.1. Anticonvulsant effect in pilocarpine-induced seizures
IF effects in pilocarpine-induced seizures in mice are presented in Table 1. The
groups of animals treated with IF 25, IF 50 and IF 75 showed an increased in latency to
first seizure of 159.6%, 189% and 245.8% when compared with the vehicle group,
respectively (p<0.05). The results of the IF 75 and DZP were reversed when pretreated
with the FLU.

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The percentages of the number of animals with seizures were 75%, 50% and
25% for the IF 25, IF 50 and IF 75 groups when compared with the vehicle group,
respectively (p<0.05). In the pilocarpine-induced seizure model, the IF 25, IF 50 and IF
75 significantly reduced the percentage of mortality when compared with the vehicle
(p<0.05).
Table 1. Effects of IF (25, 50 and 75 mg/kg, i.p.) on pilocarpine-induced seizure test in
mice.
Treatments
Vehicle
DZP
Dose (mg/kg)
Latency (s)
11.46 ± 0.89
31.11 ± 1.69a
11.84 ± 1.43
29.76 ± 0.98a
33.12 ± 1.59a
39.63 ± 1.36a,g,h
11.54 ± 1.87c
11.39 ± 1.16d
Seizures (%)
100
Death animals (%)
-
2
100
25b
100
75b
50b
25b
100e
75f
25b
FLU
5
100
IF
25
75b
50
50b
75
25b
FLU + DZP
FLU + IF
5 + 2
5 + 75
100e
75f
Values are the mean ± S.E.M. for 8 mice (per group). ap<0.05 (ANOVA followed by t-Student-Neuman-
b
Keuls as post hoc test), when compared with vehicle; p<0.05 (χ2 test), significantly different from
c
vehicle; p<0.05 (ANOVA followed by Student–Neuman–Keuls as post hoc test), significantly different
from the DZP group; dp<0.05 (ANOVA followed by Student–Neuman–Keuls as post hoc test),
significantly different from the IF 75 group; ep<0.05 (χ2 test), significantly different from the DZP group;
g
fp<0.05 (χ2 test), significantly different from the IF 75 group; p<0.05 (ANOVA followed by Student–
Neuman–Keuls as post hoc test), significantly different from the IF 25 group; hp<0.05 (ANOVA followed
by t-Student–Neuman–Keuls as post hoc test), significantly different from the IF 50 group. IF = Isopentyl
ferulate (25, 50 and 75 mg/kg, i.p.); DZP = Diazepam (2 mg/kg, i.p.); Vehicle = pilocarpine (400 mg/kg,
i.p.).
3.2. Anticonvulsant effect in pentylenetetrazole-induced seizures

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The evaluation of IF effects in PTZ-induced seizures in mice is shown in Table
2. The IF 25, IF 50 and IF 75 groups showed an increase of 96.5%, 96.7% and 269% in
latency to first seizure when compared with the vehicle group, respectively. Similarly to
the previous test, the pretreatment with FLU reversed the anticonvulsant effects in
animals treated with DZP and IF 75.
The percentages of the number of animals with seizure were 75%, 50% and 50%
for the IF 25, IF 50 and IF 75 groups when compared with the vehicle group,
respectively (p<0.05). In the PTZ‐induced seizure model, the IF 25, IF 50 and IF 75
significantly reduced the percentage of mortality when compared with the vehicle
(p<0.05).
Table 2. Effects of IF (25, 50 and 75 mg/kg, i.p, IF) on PTZ-induced seizure test in
mice.
Treatments
Vehicle
DZP
Dose (mg/kg)
Latency (s)
103.25 ± 1.78
885.27 ± 2.91a
101.89 ± 1.67
202.92 ± 1.18a
203.16 ± 1.79a
381.25 ± 1.82a,g,h
105.78 ± 2.55c
Seizures (%) Death animals (%)
-
2
100
00b
100
00b
FLU
5
100
75b
100
75b
IF
25
50
50b
50b
75
50b
50b
FLU + DZP
FLU + IF
5 + 2
5 + 75
100e
100f
100e
100f
109.67 ± 3.08d
a
Values are the mean ± S.E.M. for 8 mice (per group). p<0.05 (ANOVA followed by Student-Neuman-
b
Keuls as post hoc test), when compared with vehicle; p<0.05 (χ2 test), significantly different from
c
vehicle; p<0.05 (ANOVA followed by Student–Neuman–Keuls as post hoc test), significantly different
from the DZP group; dp<0.05 (ANOVA followed by Student–Neuman–Keuls as post hoc test),
significantly different from the IF 75 group; ep<0.05 (χ2 test), significantly different from the DZP group;
g
fp<0.05 (χ2 test), significantly different from the IF 75 group; p<0.05 (ANOVA followed by Student–
Neuman–Keuls as post hoc test), significantly different from the IF 25 group; hp<0.05 (ANOVA followed

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by t-Student–Neuman–Keuls as post hoc test), significantly different from the IF 50 group. IF = Isopentyl
ferulate (25, 50 and 75 mg/kg, i.p.); DZP = Diazepam (2 mg/kg, i.p.); Vehicle = Pentylenetetrazole (70
mg/kg, i.p.).
3.3. Effect of isopentyl ferulate on locomotor activity
The group treated with DZP showed a reduction of 38%, 58.5% and 54% in the
number of crossing, rearings and groomings, respectively, when compared with the
vehicle (p<0.05) (Fig. 2A, 2B, 2C). However, the mice treated with IF 25, 50 and
75 mg/kg did not present any difference in number of crossing, rearings and groomings
when compared to the vehicle (p>0.05).
Fig. 2. Effects on the number of crossing (A), rearings (B) and groomings (C) in the
open field test. Values are expressed as mean ± S.E.M. for the number of crossing,
rearings and groomings of 8 mice (per group) used in the experimental protocols. The
differences in the experimental groups were determined by analysis of variance
(ANOVA) followed by Student-Neuman-Keuls as post hoc test. IF = Isopentyl ferulate

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(25, 50 and 75 mg/kg, i.p.); DZP = Diazepam (2 mg/kg, i.p.); Vehicle = 0.05% Tween
80 dissolved in 0.9% saline. ap<0.05, compared to the vehicle group.
3.4. Effect of isopentyl ferulate on motor coordination test
The animals treated with DZP 2 mg/kg reduced 32.58% the time of permanence
on the swivel bar when compared to the vehicle (p<0.05), while the IF 25, IF 50 and IF
75 mg/kg had no statistical difference when compared to the vehicle (p>0.05) (Fig. 3A).
Considering the number of falls, the mice tread with DZP 2 mg/kg had this parameter
increased by 129.31% when compared to the vehicle (p<0.05), while the IF 25, IF 50
and IF 75 mg/kg did no statistical difference when compared to the vehicle (p>0.05)
(Fig. 3B).
Fig. 3. Effects of IF at doses of 25, 50 and 75 mg/kg in mice on the remaining time on
the rotating bar (A) and number of falls (B) on rota rod test.Values are expressed as
mean ± S.E.M. of the length of stay on the rotating rod and the number of falls (n = 8
mice per group). Differences between experimental groups were determined by analysis
of variance (ANOVA) followed by Student-Neuman-Keuls as post hoc test. Isopentyl
ferulate (25, 50 and 75 mg/kg, i.p.); DZP = Diazepam (2 mg/kg, i.p.); Vehicle = 0.05%
Tween 80 dissolved in 0.9% saline. ap<0.05 compared to vehicle group.
4. Discussion

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The demand for more effective, safer and cheaper therapeutic alternatives has
lead to decrease in the use of medicinal plant combinations and an increase in the use of
isolated and/or synthetic therapeutic substances. For this reason, different constituents
found in essential oils are synthesized and used as raw material for research of their
pharmacological activities [35, 36]. To our knowledge this is the first time, we are
demonstrating that IF exerts significant anticonvulsant effects in two separate models of
seizure.
The experimental procedure of seizure induced by pilocarpine in rodents (Rattus
norvegicus and Mus musculus) is one of the main model used to study epilepsy and still
reproduces the main characteristics of temporal lobe epilepsy in adult humans [37, 38].
In the present study, after the administration of pilocarpine 400 mg/kg, a cholinergic
muscarinic agonist, the animals showed behavioral changes that have evolved for
installation of seizures including clonic movements of the upper extremities and which
was observed in 100% of animals. In the same group, the seizures progressed to the
development of status epilepticus in 100% of animals with the survival rate by 0%.
When treated with isopentyl ferulate, an increase in the time of installation of the
first seizure, as well as the reduction in the number of animals with seizures and the
number of dead animals was observed. Similar result was obtained in the study
conducted by Santos et al. [39], in which it was demonstrated that the caffeic acid
(phenylpropanoid) has anticonvulsant effect in the pilocarpine-induced seizures in
Wistar rats. Despite the differences between the experimental procedures, the IF when
compared with the caffeic acid proved to be more effective at reducing the number of
animals that presented seizures, as well as in the percentage of mortality.
Pentylenetetrazole-induced seizure model is another experimental procedure
which is widely used for epilepsy study. In this model, PTZ induces convulsions by

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inhibiting the γ-aminobutyric acid (GABA) pathway in the central nervous system via
the inhibition of GABAA receptor [40, 41]. The IF at low doses attenuated the onset of
PTZ-induced seizures, decreased the number of seizures and the number of dead
animals. DZP acts directly by activation of GABAA receptors and therefore is highly
effective in this animal model of seizures, as observed in the present study (Table 2).
Similar result was demonstrated in the study conducted by Nugroho et al. [42]
with the caffeic acid (10-20 mg/kg) in PTZ-induced seizures (70 mg/kg, i.p.). The result
obtained for the IF is also according to the study conducted by Coelho et al. [43], in
which it was demonstrated that the rosmarinic acid (ester of caffeic acid) at low dose
increased the latency to first seizure and decreases the number of PTZ-induced seizures
(50 mg/kg) in mice. It is important to highlight that the ester of caffeic acid (2 mg/kg)
showed a better anticonvulsant effect at a lower dose than caffeic acid (4 mg/kg), which
may suggest that the ester compounds are promising anticonvulsants.
When the results obtained by Coelho et al. [43] are compared with the results
obtained in the present study, the IF was more effective in reducing the number of
animals with seizures. In addition, the caffeic acid and rosmarinic acid were tested in
three doses and only the intermediate dose was effective in reducing seizures, while the
IF showed a dose dependence of the anticonvulsant effect.
The results obtained for IF was similar to the study conducted by Ilhan et al.
[44], in which it was demonstrated that caffeic acid phenethyl ester at a dose of 100
ꢀmol/kg (i.p.) administered in mice showed a high anticonvulsant effect and prevented
oxidative stress induced by PTZ.
This study also evaluated the possible effect of the IF on the GABAergic system
during the induced seizures by pilocarpine and PTZ, since this system is directly
involved in control of neuronal activity. It was demonstrated that the pretreatment of

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mice with flumazenil, a GABAergic antagonist, reversed significantly the
anticonvulsant action of DZP and the IF at a dose of 75 mg/kg, suggesting that this ester
derived from ferulic acid can act on modulatory site of benzodiazepine in GABAA
receptor (γ-aminobutyric acid, type A) in the brain of mice (Fig. 4). Even being
observed a possible action on GABAA receptor, the development of future research is
needed to determine if the antiseizure effect of the IF was mediated by a direct
interaction in the same site which acts the DZP or at other allosteric sites of GABAA
receptor (Fig. 4).
Fig. 4. Possible action mechanism of IF in the GABAA receptors. The arrow with a solid
line indicates that the Diazepam modulates GABAA receptors (αγ) and the arrow with
dotted line indicates that the protective effect of the IFF against seizures may be related
to a possible modulation of GABAA receptors (αγ).
A study conducted by Yoon et al. [10] demonstrated that the sinapic acid
(phenylpropanoid) may act on the GABAA receptor. Kim et al. [45] demonstrated that
the anticonvulsant effect of this phenylpropanoid was blocked by the treatment with

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FLU, thus suggesting the possible activation of GABAA receptors, as also observed for
the IF.
The ferulic acid is a powerful antioxidant with neuroprotective properties in
several models of studies [12], but its anticonvulsant effect has not been reported in
previous studies. But analyzing the studies of Zhang et al. [16], it was demonstrated that
the ferulic acid (25 and 50 mg/kg) did not show affect the locomotor activity (number of
crossings) of mice in open field test. Using another experimental model (YLS-1ª Multi-
autonomous Activity Instrument), Tu et al. [46], has shown that the ferulic acid
significantly reduced the locomotor activity at doses of 15 and 30 mg/kg. Thus, only the
study of Zhang et al. [16], is in agreement with the results obtained for the IF, in which
it was demonstrated that there were no significant changes to the dose of 25, 50 and 75
mg/kg in groomings, rearings, and crossings, indicating the absence of changes in
locomotor activity.
Additionally, this ester derivative of ferulic acid did not cause deficits of the
motor coordination and muscle relaxation activities in mice by rod route test when
treated in doses that produced an anticonvulsant effect, unlike observed for DZP. Thus,
the observed anticonvulsant effect of IF may not be due to its muscle relaxation effects.
5. Conclusion
In conclusion, this is the first study that demonstrates a protective effect of
isopentyl ferulate against induced seizures in mice and that this ester derived from
ferulic acid may act through the GABAergic system. In addition, the neurotoxicity
study demonstrates that there were no changes in motor coordination and locomotor
activity, which is commonly seen in antiepileptic drugs such as diazepam. Thus, these
results demonstrate a new pharmacological knowledge of isopentyl ferulate with

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potential application against epileptic seizures. However, further studies are needed to
elucidate other neurobiological mechanisms underlying epilepsy.
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgements
The authors are grateful to the researchers of the Laboratory of Research in
Experimental Neurochemistry (LAPNEX/UFPI), FAPEPI and CAPES for materials,
equipments, scholarships and collaborations.
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Highlights
ꢀ Isopentyl ferulate (IF) acts as an anticonvulsant agent in Pilocarpine-induced
seizure.
ꢀ IF acts as an anticonvulsant agent in Pentylenetetrazole-induced seizure.
ꢀ
ꢀ
The anticonvulsant effects may be related to the activation of GABAA receptors.
IF produced no impairment in locomotor activity and motor coordination.

ACCEPTED MANUSCRIPT
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