Вы находитесь на странице: 1из 29

Accepted Manuscript

Antidepressant-like effect of gallic acid: Dual involvement of


serotonergic and catecholaminergic systems

Özgür Devrim Can, Nazlı Turan, Ümide Demir Özkay, Yusuf


Öztürk

PII: S0024-3205(17)30473-3
DOI: doi: 10.1016/j.lfs.2017.09.023
Reference: LFS 15348
To appear in: Life Sciences
Received date: 28 June 2017
Revised date: 12 September 2017
Accepted date: 19 September 2017

Please cite this article as: Özgür Devrim Can, Nazlı Turan, Ümide Demir Özkay, Yusuf
Öztürk , Antidepressant-like effect of gallic acid: Dual involvement of serotonergic and
catecholaminergic systems, Life Sciences (2017), doi: 10.1016/j.lfs.2017.09.023

This is a PDF file of an unedited manuscript that has been accepted for publication. As
a service to our customers we are providing this early version of the manuscript. The
manuscript will undergo copyediting, typesetting, and review of the resulting proof before
it is published in its final form. Please note that during the production process errors may
be discovered which could affect the content, and all legal disclaimers that apply to the
journal pertain.
ACCEPTED MANUSCRIPT

Antidepressant-like effect of gallic acid: dual involvement


of serotonergic and catecholaminergic systems

Özgür Devrim Can*, Nazlı Turan, Ümide Demir Özkay, Yusuf Öztürk

PT
Anadolu University, Faculty of Pharmacy, Department of Pharmacology, 26470 Eskişehir,
TURKEY

RI
SC
NU
MA
E D
PT

* Corresponding author:
CE

Assoc. Prof. Dr. Özgür Devrim Can

Anadolu University, Faculty of Pharmacy, Department of Pharmacology, 26470 Eskisehir, TURKEY


AC

Tel. : +90 222 3350580 / 3749

Fax number : +90 222 3350750

e-mail address : ozgurdt@anadolu.edu.tr


ACCEPTED MANUSCRIPT

Abbreviations:

AMPT, α-methyl-para-tyrosine methyl ester; ANOVA, one-way analysis of variance; 5-HT,


serotonin; i.p., intraperitoneally; MFST, modified forced swimming test; PCPA, p-
chlorophenylalanine methyl ester; p.o., per oral; S.E.M, standard error of mean; s.c.,
subcutaneously; TST, tail suspension test.

PT
RI
SC
NU
MA
E D
PT
CE
AC
ACCEPTED MANUSCRIPT

Abstract

AIMS: This study was planned to examine the antidepressant potency of gallic acid (30 and
60 mg/kg), a phenolic acid widely distributed in nature, together with its possible underlying
monoaminergic mechanisms.

MAIN METHODS: Antidepressant-like activity was assessed using the tail suspension
(TST) and the modified forced swimming tests (MFST). Locomotor activity was evaluated in
an activity cage.

PT
KEY FINDINGS: Administration of gallic acid at 60 mg/kg reduced the immobility duration
of mice in both the TST and MFST without any changes in the locomotor activity. The anti-

RI
immobility effect observed in the TST was abolished with pre-treatment of p-

SC
chlorophenylalanine methyl ester (an inhibitor of serotonin synthesis; 100 mg/kg i.p.
administered for 4-consecutive days), ketanserin (a 5-HT2A/2C antagonist; 5 mg/kg i.p.),
ondansetron (a 5-HT3 antagonist; 0.3 mg/kg i.p.), α-methyl-para-tyrosine methyl ester (an
NU
inhibitor of catecholamine synthesis; 100 mg/kg i.p.), phentolamine (non-selective alpha-
adrenoceptor antagonist; 5 mg/kg i.p.), SCH 23390 (a dopamine D1 antagonist; 0.05 mg/kg
MA

s.c.), and sulpiride (a dopamine D2/D3 antagonist; 50 mg/kg i.p.). However, NAN 190 (a 5-
HT1A antagonist; 0.5 mg/kg i.p.) and propranolol (a non-selective β-adrenoceptor antagonist;
2 mg/kg i.p.) pre-treatments were ineffective at reversing the antidepressant-like effects of
D

gallic acid.
E
PT

SIGNIFICANCE: The results of the present study indicate that gallic acid seems to have a
dual mechanism of action by increasing not only serotonin but also catecholamine levels in
CE

synaptic clefts of the central nervous system. Further alpha adrenergic, 5-HT2A/2C and 5-
HT3 serotonergic, and D1, D2, and D3 dopaminergic receptors also seem to be involved in
this antidepressant-like activity.
AC

Keywords: Gallic acid; modified forced swimming test; serotonergic system; monoaminergic
system; tail-suspension test
ACCEPTED MANUSCRIPT

1. Introduction

Gallic acid (3,4,5-trihydroxybenzoic acid) is a naturally occurring compound widely


distributed in nature as a free molecule or as a part of ester derivatives or polymers [1]. This
phenolic acid has been reported to be present in fruit, seeds, leaves, bark, peel, root, or stigma
of various pharmacologically active plants, such as Acacia confusa Merr [2]. Ajuga bracteosa
[3], Allium cepa [4], Crocus sativus [5], Phragmanthera austroarabica [6], Phyllanthus
emblica L. [7], Psidium guajava [8], Terminalia catappa Linn [9], and Olea europaea [10], as

PT
well as numerous foodstuffs such as raspberries, blueberries, strawberries, black and red
currants, grapes (red and white wine), green and black tea, oat flour and some rice varieties

RI
[1].

To date, numerous studies have been conducted to search for possible pharmacological

SC
effects of gallic acid. So far, this phenolic acid has been demonstrated to have the following
types of effects: antioxidant [5,11-13], free radical scavenging [7,13], anti-apoptotic [10],
NU
anticancer [14], chemopreventive [13,15], wound healing [16], antibacterial [17], anti-HIV
[18], hepatoprotective [2], antiallergic [19], anti-inflammatory [11,13,2021], antinociceptive
MA

[22], anti-hypertensive [23], anti-atherogenic [24], anti-colitic [25], appetite suppressant [26],
anti-obesity [27-30], metabolic syndrome preventing, and antidiabetic [27,28,31-33].

Today, gallic acid has attracted a great deal of attention due to its potential efficacy in
D

the central nervous system. Beneficial effects of this compound against neurotoxicity and
E

neurodegeneration have been demonstrated in different experimental models, such as


PT

scopolamine-induced amnesia [34], kainic acid-induced excitotoxicity and status epilepticus


[35], trimethyltin-induced hippocampal degeneration and emotional instability [36], 6-
CE

hydroxydopamine injection (full nigral lesion, animal model of Parkinson's disease)-induced


memory deficit and cerebral oxidative stress [37], beta-amyloid neurotoxicity [21],
AC

intracerebroventricular streptozotocin injection (experimental model of sporadic Alzheimer’s


disease)-induced cognitive impairment and cerebral oxidative stress [38], bilateral common
carotid artery occlusion-induced ischemia/reperfusion and cognitive deficits [20,39,40], and
traumatic brain injury-induced behavioural, electrophysiological, and inflammatory disorders
[20].

Even though there have been numerous studies reporting the neuroprotective capacity
of gallic acid, only a limited number of preclinical studies have suggested a potential efficacy
of this phenolic acid on emotional disorders such as anxiety [36,41-43] and depression [36,44-
46]. Although antidepressant-like activity of gallic acid has been previously demonstrated by
ACCEPTED MANUSCRIPT

a few preclinical studies, the possible monoaminergic mechanisms underlying this effect have
not yet been elucidated. Therefore, we planned to clarify the promising contributions of the
serotonergic and catecholaminergic systems to the antidepressant-like effects of gallic acid in
the present study.

2. Materials and methods

2.1. Animals

Tests were performed with adult male BALB/c mice, weighing 30–35 g, obtained from

PT
the Anadolu University Research Unit for Experimental Animals. Mice were housed under
standard laboratory conditions. Temperature (25 ± 1°C), light (lights on 08:00–20:00 h) and

RI
sound levels were not changed during the course of experiments. The animals had ad libitum

SC
access to food and water except during the test sessions. Experimental protocols and
procedures of this study were approved by the Local Ethical Committee on Animal
Experimentation of Anadolu University, Eskişehir, Turkey.
NU
2.2. Drugs and administrations
MA

α-methyl-para-tyrosine methyl ester (AMPT), fluoxetine hydrochloride, gallic acid,


NAN 190 hydrobromide, ondansetron hydrochloride, p-chlorophenylalanine methyl ester
(PCPA), phentolamine hydrochloride, propranolol hydrochloride, reboxetine mesylate
D

hydrate, R(+)-SCH-23390 hydrochloride, and sulpiride used in the experiments were acquired
E

from Sigma-Aldrich (St. Louis, MO, USA), while ketanserin tartrate was purchased from
PT

Tocris Cookson (Ballwin, MO, USA).

All of the drugs, except AMPT, were dissolved in physiological saline (NaCl, 0.9%).
CE

AMPT solution was prepared using saline containing 10% Tween 80. Vehicle treatments to
the appropriate control groups were carried out simultaneously.
AC

Gallic acid (30 and 60 mg/kg by oral gavage [p.o.]) was administered three times at
24, 5, and 1 h before test sessions [47]. Reboxetine (20 mg/kg p.o.) [48] and fluoxetine (30
mg/kg p.o.) [49], conventional antidepressants, were used as positive controls. All other drugs
were administered intraperitoneally (i.p.), at a volume of 10 ml/kg body weight, except
SCH23390, which was administered subcutaneously (s.c.).

2.3. Behavioural tests

2.3.1. Modified forced swimming test


ACCEPTED MANUSCRIPT

The modified forced swimming test (MFST) was conducted as described previously
[50]. In this test, the mice were forced to swim in a Plexiglas cylinder (height: 30 cm,
diameter: 12 cm), which was filled with water to a height of 20 cm. The temperature of the
water was adjusted to 25 ± 1 ◦C. In the “pre-test session” of the experiment, mice were
allowed to swim in the cylinder for 15 min. Twenty-four h later, in the “test session”, each
mouse was re-exposed to the water for 5 min. Total durations of climbing (upward-directed
movements with forelegs above the water level), swimming (horizontal movement on the
surface of the water), and immobility (only movements necessary to keep the head above the

PT
water) behaviours over 5-s intervals were recorded. The water was changed between the
individual mice to avoid the influence of alarm substances.

RI
2.3.2. Tail suspension test

SC
The tail suspension test (TST) was performed as described previously [51]. Briefly
here, each mouse was suspended 50 cm above the floor using adhesive tape, positioned about
NU
1 cm from the tip of the tail. The total duration of immobility, which can be defined as
motionless hanging without any struggling movements, was recorded during a test period of 6
MA

min. Mice that climbed up their tail during the tests were excluded from the experiments.

2.3.3. Activity cage test


D

The locomotor activity of each mouse was assessed in an activity cage apparatus (No.
E

7420; Ugo Basile, Varese, Italy), containing two pairs of 16 infrared photocells 3 cm and 12
PT

cm above the floor. Interruptions of light beams to the photocells during vertical and
horizontal movements of the mouse were automatically recorded and documented for 6 min
CE

[52]. Between tests, the device was carefully cleaned with ethanol to remove any residue or
odour from the former mouse.
AC

2.4. Mechanistic studies

We additionally investigated the monoaminergic mechanisms mediating the


antidepressant effect of gallic acid using various pharmacological agents. The mechanistic
studies were conducted with the effective dose of 60 mg/kg.

To address the role of catecholaminergic system in the antidepressant-like effect of


gallic acid, mechanistic studies were conducted with AMPT (inhibitor of tyrosine
hydroxylase), phentolamine (non-selective alpha-adrenoceptor antagonist), propranolol (non-
selective β-adrenoceptor antagonist), SCH 23390 (dopamine D1 receptor antagonist), and
sulpiride (dopamine D2/D3 receptor antagonist). For AMPT (100 mg/kg, i.p.), mice were pre-
ACCEPTED MANUSCRIPT

treated with AMPT or vehicle (saline with 10% Tween 80) 4 h prior the administration of
physiological saline or gallic acid. Sixty min later they were tested in the TST [53,54]. For
further antagonistic studies, in different experimental groups, mice were pre-treated with
phentolamine (5 mg/kg, i.p.) [55], propranolol (2 mg/kg, i.p.) [56], SCH 23390 (0.05 mg/kg,
s.c.) [57], sulpiride (50 mg/kg, i.p.) [58], or vehicle (saline) 15 min prior to the saline or gallic
acid treatments. The TST was performed 60 min after these oral administrations of saline or
gallic acid.

PT
To investigate the probable contribution of the serotonergic system to the
antidepressant-like effect of gallic acid, mechanistic studies were conducted with PCPA

RI
(inhibitor of tryptophan hydroxylase), NAN 190 (5-HT1A receptor antagonist), ketanserin (5-
HT2A/2C receptor antagonist), and ondansetron (5-HT3 receptor antagonist). For PCPA,

SC
mice were pretreated with PCPA or vehicle (saline) daily for 4 consecutive days. Twenty-four
h after the fourth injections, mice were administered either physiological saline or gallic acid
NU
by oral gavage. 60 min later they were tested in the TST. For further antagonistic studies, in
different experimental groups, mice were pre-treated with NAN 190 (0.5 mg/kg, i.p.) [59],
MA

ketanserin (1 mg/kg, i.p.) [60], ondansetron (0.1 mg/kg, i.p.) [61], or the vehicle (saline) 15
min prior to the saline or gallic acid treatments. The TST was performed 60 min after these
oral administrations of saline or gallic acid.
D

All doses used in the present study were chosen according to the literature as well as
E

the authors’ previous experimental practices.


PT

2.5. Data analysis


CE

The data used for statistical analysis were acquired from eight animals for each group.
For statistical assessment, GraphPad Prism 5.04 (GraphPad Software, San Diego, CA, USA)
was used. Data obtained from the MFST, TST, and activity cage test were analysed using
AC

one-way analysis of variance (ANOVA) followed by Tukey’s test. Moreover, data obtained
from the mechanistic studies (for the PCPA, AMPT, phentolamine, propranolol, SCH 23390,
NAN 190, ketanserin, sulpiride, and ondansetron experiments) were evaluated using a two-
way ANOVA followed by the Bonferroni post hoc test.

Results are presented herein as mean ± S.E.M, and differences were considered
statistically significant when their p-value was less than 0.05. The same statistical analysis
programme was used to create figures.

3. Results
ACCEPTED MANUSCRIPT

3.1. Assessment of antidepressant-like activity in tail suspension test

Data obtained from the TST show that fluoxetine (30 mg/kg), reboxetine (20 mg/kg),
and gallic acid (60 mg/kg) significantly shortened the duration of immobility of mice, with
respect to their control groups (F[4,35] = 10.49, P < 0.001; Fig. 2). Gallic acid was ineffective
when administered at 30 mg/kg.

3.2. Assessment of antidepressant-like activity in modified forced swimming test

In the MFST, administration of gallic acid at 60 mg/kg significantly shortened the

PT
immobility time (F[4,35] = 10.35, P < 0.001; Fig. 3A) while increasing the durations of
swimming (F[4,35] = 15.09, P < 0.001; Fig. 3B) and climbing behaviours (F[4,35] = 10.68, P

RI
< 0.001; Fig. 3C). Fluoxetine and reboxetine also decreased the immobility time, as expected

SC
(Fig. 3). Gallic acid was ineffective when administered at 30 mg/kg.

3.3. Assessment of locomotor activity in activity cage test


NU
In the activity cage test, gallic acid administration did not alter the total number of
horizontal (F[2,21] = 1.797, P = 0.1904; Fig. 4A) or vertical (F[2,21] = 0.7209, P = 0.4980;
MA

Fig. 4B) movements.

3.4. Assessment of participation of monoaminergic mechanisms to the antidepressant-like


activity of gallic acid
D

Fig. 5A displays the influence of AMPT pre-treatment on the anti-immobility effects


E

of gallic acid in the TST. A two-way ANOVA exhibited main effects of the pre-treatment
PT

(F[1,28] = 11.92, P < 0.01) and the treatment (F[1,28] = 8.225, P < 0.01) on immobility
duration. There was a significant interaction between the factors of pre-treatment and
CE

treatment (F[1,28] = 15.40, P < 0.001). Post hoc analyses showed that pre-treatment of
animals with AMPT was effective in reversing the antidepressant-like efficacy of gallic acid
AC

(P < 0.001). AMPT treatment alone did not significantly affect the immobility time.

Fig. 5B presents the results of pre-treatment with phentolamine on the anti-immobility


effects of gallic acid. There were significant main effects of pre-treatment (F[1,28] = 4.257, P
< 0.05) and treatment (F[1,28] = 28.24, P < 0.001) and a significant pre-treatment × treatment
interaction (F[1,28] = 12.44, P < 0.01). The influence of the propranolol pre-treatment on the
anti-immobility effects of gallic acid is demonstrated in Fig. 5C. Two-way ANOVA exhibited
main effect of the treatment (F[1,28] = 116.0, P < 0.001) but not of the pre-treatment (F[1,28]
= 0.1393, P > 0.05) on the duration of immobility; however, no significant pre-treatment ×
ACCEPTED MANUSCRIPT

treatment interaction was detected (F[1,28] = 3.331, P > 0.05). Results of the Bonferroni test
showed that pre-treatment of the mice with phentolamine was effective to prevent the anti-
depressant-like effect of gallic acid (P < 0.001). However, the propranolol pre-treatment was
not able to antagonize the anti-immobility effects of gallic acid.

Fig. 5D demonstrates the influence of pre-treatment with SCH 23390 on the anti-
immobility effects of gallic acid. There were significant effects of pre-treatment (F[1,28] =
4.503, P < 0.05) and treatment (F[1,28] = 42.84, P < 0.001) and a significant pre-treatment ×

PT
treatment interaction (F[1,28] = 11.17, P < 0.01). Fig. 5E presents the results of pre-treatment
with sulpiride on the anti-depressant-like effect of gallic acid. Two-way ANOVA exhibited

RI
significant differences of pre-treatment (F[1,28] = 5.068, P < 0.05) and treatment (F[1,28] =
18.91, P < 0.001), and a significant pre-treatment × treatment interaction (F[1,23] = 9.261, P

SC
< 0.01). Post hoc analyses showed that pre-treatment of the mice with SCH 23390 (P < 0.01)
and sulpiride (P < 0.01) effectively abolished the anti-depressant-like effect of gallic acid.
NU
None of the antagonists altered the immobility duration of the animals when they were
administered alone.
MA

Fig. 6A displays the influence of PCPA pre-treatment on the anti-immobility effect of


gallic acid. Two-way ANOVA exhibited a main effect of the pre-treatment (F[1,28] = 4.277,
P < 0,05) and the treatment (F[1,28] = 31.17, P < 0.001) on immobility duration. There was a
D

significant interaction between the factors of pre-treatment × treatment factors (F[1,28] =


E

6.083, P < 0,05). Post hoc analyses showed that the pre-treatment of animals with PCPA was
PT

effective in reversing the anti-depressant-like effect of gallic acid (P < 0.01). PCPA treatment
alone did not significantly change the immobility time.
CE

Fig. 6B presents the results of pre-treatment with NAN 190 on the anti-immobility
effect of gallic acid. Two-way ANOVA revealed a main effect of the treatment (F[1,28] =
AC

48.22, P < 0.001) but not of the pre-treatment (F[1,28] = 0.8696, P = 0.3590). Furthermore,
there was no significant pre-treatment × treatment interaction (F[1,28] = 0.3565, P = 0.5552).
Results of the Bonferroni test indicated that pre-treatment of the mice with NAN 190 was not
able to prevent gallic acid’s anti-immobility effect in the TST (P > 0.05).

The influence of the ketanserin pre-treatment on the anti-immobility effect of gallic


acid is demonstrated in Fig. 6C. Two-way ANOVA exhibited a major influence of the
treatment (F[1,28] = 5.714, P < 0.05) and pre-treatment (F[1,28] = 12.02, P < 0.01) on the
duration of immobility; moreover, a significant pre-treatment × treatment interaction was seen
(F[1,28] = 9.684, P < 0.01). Fig. 6D demonstrates the effect of pre-treatment of the mice with
ACCEPTED MANUSCRIPT

ondansetron on the anti-immobility effect of gallic acid. There were significant effects of pre-
treatment (F[1,28] = 5.820, P < 0.05) and treatment (F[1,28] = 37.19, P < 0.001) and a
significant pre-treatment × treatment interaction (F[1,28] = 5.535, P < 0.05). Post hoc
analyses revealed that the pre-treatment of mice with ketanserin (P < 0.001) and ondansetron
(P < 0.01) prevented the anti-immobility effect of gallic acid. None of the antagonists altered
the immobility time of the animals when they were administered alone.

4. Discussion

PT
Depression is a recurrent and common psychiatric disorder that affects more than 300
million people worldwide. The World Health Organization reports that incidences of

RI
depression and other mood disorders are increasing globally, and the treatment costs of these
diseases are quite high [62].

SC
Antidepressant pharmacotherapy together with simultaneous psychotherapy can help
NU
to alleviate symptoms of depression and prevent relapses [63]. However, currently prescribed
conventional antidepressant drugs (i.e. selective monoamine reuptake inhibitors, tricyclic
antidepressants, and monoamine oxidase inhibitors, etc.) have some disadvantages, such as
MA

prolonged onset of therapeutic effectiveness, high incidences of non-responding patients, and


significant unwanted adverse effects [64]. Therefore, discovering novel antidepressant drugs
as well as clarifying possible modes of actions are of great importance for providing new
D

options for pharmacotherapy of depression and other affective disorders.


E
PT

Based on this knowledge, we first investigated the therapeutic potency of gallic acid as
a natural antidepressant drug source. Then, we clarified the contribution of monoaminergic
CE

mechanisms, which are known to play vital roles in antidepressant effects, to this
pharmacological effect.
AC

The antidepressant-like effect of gallic acid was investigated using the MFST and
TST, the most widely used animal tests for assessing antidepressant-like effects [65,66]. The
MFST, a modified version of the Porsolt’s forced swimming test, is based on the tracking of
behaviours of rodent that is forced to swim in a narrow cylinder from which they cannot
escape. Unlike Porsolt’s test, the MFST takes into account not only passive immobility
behaviour of the animals but also active behaviours such as swimming and climbing [50,66].
The TST, another reliable method for evaluating antidepressant-like activity, shares the same
basis of “behavioural despair” with the MFST, but it differs in that immobility is induced by
suspending the animal by its tail [47].
ACCEPTED MANUSCRIPT

In the present study, data obtained from the MFST indicated that gallic acid,
administered at 60 mg/kg, induced a significant reduction in the immobility time of mice
compared to the untreated control group (Fig. 2A). The reference drugs fluoxetine (30 mg/kg)
and reboxetine (20 mg/kg) also decreased the duration of immobility behaviour, as expected.
Moreover, gallic acid treatment also increased the duration of swimming (Fig. 2B) and
climbing (Fig. 2C) behaviours. In the MFST, a decrease in the immobility time as well as
accompanying increases in swimming and climbing durations following gallic acid
administration demonstrate the antidepressant-like effect of this phenolic compound. In the

PT
TST, similar to the MFST, administration of gallic acid at 60 mg/kg decreased the immobility
time of mice with respect to the control animals (Fig. 3). The reference drugs fluoxetine (30

RI
mg/kg) and reboxetine (20 mg/kg) also shortened the duration of immobility behaviours.

SC
These data obtained from TST indicate the antidepressant-like effect of gallic acid and
confirm the results of the MFST tests conducted in the present study. Moreover, the findings
NU
from these two tests support the results of previous studies reporting an antidepressant-like
effect of gallic acid [36,44-46]. In the study of Chhillar and Dhingra [46], antidepressant-like
activity of gallic acid in unstressed and stressed mice was associated with its monoamine
MA

oxidase-A inhibitory activity, antioxidant capacity, and its lowering effect on plasma nitrite
and corticosterone levels.
D

The results of the activity cage tests indicated that administration of gallic acid at 30 or
E

60 mg/kg doses did not induce significant changes in the total number of horizontal (Fig. 4A)
PT

or vertical (Fig. 4B) movements of mice. This finding is quite important because it shows that
the antidepressant-like effect revealed in this study is not related to any increase in the
CE

locomotor activity of mice, since increased locomotor activity may cause false positive results
in the MFST or TST [67].
AC

After demonstrating the antidepressant-like effect of gallic acid, we performed several


mechanistic studies in order to clarify possible contributions of monoaminergic systems to
this pharmacological effect. For these additional experiments, we continued using the TST,
since it has been reported to have some additional advantages over the MFST such as the
absence of hypothermia risk, increased pharmacological sensitivity, and a quick return to
normal spontaneous activity at the end of the trial [47,67].

For the first part of the mechanistic studies, we searched for the involvement of the
catecholaminergic system in the antidepressant-like effect of gallic acid. We examined the
possible contribution of enhanced synaptic catecholamine levels to this effect by using
ACCEPTED MANUSCRIPT

AMPT, which is an inhibitor of tyrosine hydroxylase. It is well-known that pre-treatment with


AMPT induces significant decreases in brain noradrenaline (by 53%) and dopamine (57%)
levels of mice with no effect on the levels of serotonin [68,69]. In the present study, pre-
treatment of mice with AMPT (100 mg/kg, i.p) 4 h before the administration of gallic acid
successfully reversed the gallic acid-induced decrease in immobility in the TST (P < 0.001)
and abolished the antidepressant-like effects of this compound (Fig. 5A).

Due to the fact that both adrenergic and dopaminergic receptors may contribute to the

PT
mechanisms of antidepressive agents [70], we performed further mechanistic studies using
several specific receptor antagonist compounds. The possible involvement of alpha and beta

RI
adrenergic receptors to the antidepressant-like effect of gallic acid was investigated using
phentolamine and propranolol, respectively. Obtained data indicated that pre-treatment with

SC
phentolamine (Fig. 5B) was able to antagonize gallic acid-induced decreases in immobility
time (P < 0.001), but pre-treatment with propranolol was ineffective (P > 0.05; Fig. 5C). We
NU
also examined the probable participation of dopaminergic D1 and D2/D3 receptors in the
antidepressant-like effects of gallic acid by pre-treating animals with SCH 23390 and
MA

sulpiride, respectively, 60 min before the gallic acid administrations. Results of these
experiments showed that not only SCH 23390 (P < 0.01; Fig. 5D) but also sulpiride (P < 0.01;
Fig. 5E) pre-treatment blocked the antidepressant-like action of gallic acid by reversing the
D

shortened immobility times. Data obtained from these mechanistic studies indicate that
E

enhanced catecholamine levels in synapses are critical for the antidepressant-like effect of
PT

gallic acid together with the contribution of alpha adrenergic and dopaminergic (D1, D2 and
D3) receptors.
CE

For the second part of the mechanistic studies, we examined the contribution of the
serotonergic system to the antidepressant-like effect of gallic acid. We examined possible
AC

contributions of enhanced synaptic serotonin levels by using PCPA, which is an inhibitor of


tryptophan hydroxylase. It is well-known that pre-treatment with PCPA (100 mg/kg, i.p) daily
for 4 consecutive days ends the biosynthesis of serotonin and depletes the endogenous stores
(by 60–90%) of this neurotransmitter in the central nervous system of mice, without any
effects on central catecholamine levels [71,72]. In the present study, pre-treatment with PCPA
(100 mg/kg), successfully reversed the gallic acid-induced decrease in the immobility time of
mice (P < 0.01) and eliminated the antidepressant-like effects of this compound (Fig. 6A).

Since serotonergic receptors are known to potentially contribute to antidepressant


effects [70], we performed further mechanistic studies using several specific serotonin
ACCEPTED MANUSCRIPT

receptor antagonist compounds. Possible involvement of 5-HT1A, 5-HT2A/2C, and 5-HT3


receptors to the antidepressant-like effect of gallic acid were investigated using NAN 190,
ketanserin, and ondansetron, respectively. Obtained data indicated that pre-treatment with
ketanserin (P < 0.001; Fig. 6C) and ondansetron (P < 0.01; Fig. 6D) were able to eliminate the
gallic acid-induced decrease in immobility time, but pre-treatment with NAN 190 was
ineffective (P > 0.05; Fig. 6B). The findings from these experiments indicate that enhanced
serotonin levels in synapses are important for the antidepressant-like effect of gallic acid
together with the contribution of 5-HT2A/2C and 5-HT3 receptors.

PT
In summary, the results of the present study indicate that gallic acid seems to have a

RI
dual mechanism of action by increasing not only serotonin but also catecholamine levels in
the synaptic clefts of the central nervous system. Further, alpha adrenergic, 5-HT2A/2C and

SC
5-HT3 serotonergic, and D1, D2, and D3 dopaminergic receptors also seem to be involved in
this antidepressant-like activity. However, the potential roles of other pharmacological
NU
mechanisms which might contribute to the antidepressant-like effect of gallic acid, such as the
opioidergic, GABAergic, glutaminergic, and nitrergic systems, still remain to be elucidated.
MA

5. Conclusion

In conclusion, with a unique mechanism of action described for the first time in the
present study, gallic acid seems to be a beneficial antidepressant drug candidate for the
D

treatment of affective disorders. On the other hand, the results of this preclinical study need
E

further clinical validation to confirm their generalizability to patients with depressive


PT

disorders.
CE
AC
ACCEPTED MANUSCRIPT

References

[1] M. Daglia, A. Di Lorenzo, S.F. Nabavi, Z.S. Talas, S.M. Nabavi, Polyphenols: well
beyond the antioxidant capacity: gallic acid and related compounds as neuroprotective
agents: you are what you eat! Curr. Pharm. Biotechnol. 15 (4) (2014) 362–372.

[2] Y.T. Tung, J.H. Wu, C.C Huang, H.C. Peng, Y.L. Chen, S.C. Yang, S.T. Chang,
Protective effect of Acacia confusa bark extract and its active compound gallic acid against
carbon tetrachloride-induced chronic liver injury in rats, Food Chem. Toxicol. 47 (6)

PT
(2009) 1385–1392.

[3] K. Hafeez, S. Andleeb, T. Ghousa, K. Akhter, Phytochemical screening, alpha-glucosidase

RI
inhibition, antibacterial and antioxidant potential of Ajuga bracteosa extracts, Curr. Pharm.

SC
Biotechnol. (2017) [Epub ahead of print]

[4] B.N. Singh, B.R. Singh, R.L. Singh, D. Prakash, D.P. Singh, B.K. Sarma, G. Upadhyay,
NU
H.B. Singh, Polyphenolics from various extracts/fractions of red onion (Allium cepa) peel
with potent antioxidant and antimutagenic activities, Food Chem. Toxicol. 47 (6) (2009)
MA

1161–1167.

[5] E. Karimi, E. Oskoueian, R. Hendra, H.Z. Jaafar, Evaluation of Crocus sativus L. stigma
phenolic and flavonoid compounds and its antioxidant activity, Molecules 15 (9) (2010)
D

6244–6256.
E

[6] H.M. Aldawsari, E.G. Eid, T. Neamatallah, S.A. Zaitone, J.M. Badr, Anticonvulsant and
PT

neuroprotective activities of Phragmanthera austroarabica extract in pentylenetetrazole-


kindled mice, Evid. Based Complement. Alternat. Med. 2017 (2017).
CE

[7] W. Luo, M. Zhao,B. Yang, G. Shen, G. Rao, Identification of bioactive compounds in


Phyllanthus emblica L. fruit and their free radical scavenging activities, Food Chem. 114
AC

(2009) 499–504.

[8] K.C. Chen, C.M. Chuang, L.Y. Lin, W.T. Chiu, H.E. Wang, C.L. Hsieh, T. Tsai, R.Y.
Peng, The polyphenolics in the aqueous extract of Psidium guajava kinetically reveal an
inhibition model on LDL glycation, Pharm. Biol. 48 (1) (2010) 23–31.

[9] A.G. Terças, A.S. Monteiro, E.B. Moffa, J.R.A. Dos Santos, E.M. de Sousa, A.R.B. Pinto,
P.C.D.S. Costa, A.C.R. Borges, L.M.B. Torres, A.K.D. Barros Filho, E.S. Fernandes, C.A.
Monteiro, Phytochemical characterization of Terminalia catappa Linn. extracts and their
antifungal activities against Candida spp, Front. Microbiol. 8 (2017) 595.
ACCEPTED MANUSCRIPT

[10] Ö. Erol-Dayi, N. Arda, G. Erdem, Protective effects of olive oil phenolics and gallic acid
on hydrogen peroxide-induced apoptosis, Eur. J. Nutr. 51 (8) (2012 ) 955–960.

[11] H.M. Mohamed, S.M. Abd El-Twab, Gallic acid attenuates chromium-induced thyroid
dysfunction by modulating antioxidant status and inflammatory cytokines, Environ.
Toxicol. Pharmacol. 48 (2016) 225–236.

[12] A. Sarkak, H. Fathimoghaddam, S.M. Mansouri, M.S. Korrani, G. Saki, Y. Farbood,


Gallic acid improves cognitive, hippocampal long-term potentiation deficits and brain

PT
damage induced by chronic cerebral hypoperfusion in rats, Pak. J. Biol. Sci. 17 (8) (2014)
978–990.

RI
[13] O.R. Ogunsanwo, A.A. Oyagbemi, T.O Omobowale, E.R. Asenuga, A.B. Saba,

SC
Biochemical and electrocardiographic studies on the beneficial effects of gallic acid in
cyclophosphamide-induced cardiorenal dysfunction, J. Complement. Integr. Med. 2017 [in
NU
press]

[14] P.G. Anantharaju, P.C. Gowda, M.G. Vimalambike, S.V. Madhunapantula, An overview
MA

on the role of dietary phenolics for the treatment of cancers, Nutr. J. 15 (1) (2016) 99.
[15] A.A. Oyagbemi, O.T. Omobowale, E.R. Asenuga, A.S. Akinleye, R.O. Ogunsanwo,
A.B. Saba, Cyclophosphamide-induced hepatotoxicity in wistar rats: the modulatory role
D

of gallic acid as a hepatoprotective and chemopreventive phytochemical, Int. J. Prev.


E

Med.7 (2016) 51.


PT

[16] D.J. Yang, S.H. Moh, D.H. Son, S. You, A.W. Kinyua, C.M. Ko, M. Song, J. Yeo, Y.H.
Choi, K.W. Kim, Gallic acid promotes wound healing in normal and hyperglucidic
CE

conditions, Molecules.21 (7) (2016) E899.


[17] M.J. Rodríguez Vaquero, M.R. Alberto, M.C. de Manca de Nadra, Antibacterial effect of
phenolic compounds from different wines, Food Control 18 (2) (2007) 93–101.
AC

[18] Nutan, M. Modi, T. Goel, T. Das, S. Malik, S. Suri, A.K. Rawat, S.K. Srivastava, R.
Tuli, S. Malhotra, S.K. Gupta, Ellagic acid & gallic acid from Lagerstroemia speciosa L.
inhibit HIV-1 infection through inhibition of HIV-1 protease & reverse transcriptase
activity, Indian J. Med. Res.137 (3) (2013) 540–548.
[19] S.H. Kim, C.D. Jun, K. Suk, B.J. Choi, H. Lim, S. Park, S.H. Lee, H.Y. Shin, D.K. Kim,
T.Y. Shin, Gallic acid inhibits histamine release and pro-inflammatory cytokine production
in mast cells, Toxicol. Sci. 91 (1) (2006) 123–131.
ACCEPTED MANUSCRIPT

[20] A. Sarkaki, Y. Farbood, M.K. Gharib-Naseri, M. Badavi, M.T. Mansouri, A. Haghparast,


M.A. Mirshekar, Gallic acid improved behavior, brain electrophysiology, and
inflammation in a rat model of traumatic brain injury, Can. J. Physiol. Pharmacol. 93 (8)
(2015) 687–694.

[21] M.J. Kim, A.R. Seong, J.Y. Yoo, C.H. Jin, Y.H. Lee, Y.J. Kim, J. Lee, W.J. Jun, H.G.
Yoon, Gallic acid, a histone acetyltransferase inhibitor, suppresses beta-amyloid
neurotoxicity by inhibiting microglial-mediated neuroinflammation, Mol. Nutr. Food Res.

PT
55 (2011) 1798–1808.

[22] G. Trevisan, M.F. Rossato, R. Tonello, C. Hoffmeister, J.Z. Klafke, F. Rosa, K.V.

RI
Pinheiro, H.V. Pinheiro, A.A. Boligon, M.L. Athayde, J. Ferreira, Gallic acid functions as
a TPRA1 antagonist with relevant antinociceptive and antiedematogenic effects in mice,

SC
Naunyn-Schmiedeberg’s Archieves of Pharmacology, 387 (7) (2014) 679–689.
NU
[23] L. Jin, M.Q. Lin, Z.H. Piao, J.Y. Cho, G.R. Kim, S.Y. Choi, Y. Ryu, S. Sun, H.J. Kee,
M.H. Jeong, Gallic acid attenuates hypertension, cardiac remodeling, and fibrosis in mice
with NG-nitro-L-arginine methyl ester-induced hypertension via regulation of histone
MA

deacetylase 1 or histone deacetylase 2, J. Hypertens. 2017 [Epub ahead of print]


[24] T.T. Ou, M.C. Lin, C.H. Wu, W.L. Lin, C.J. Wang, Gallic acid attenuates oleic acid-
induced proliferation of vascular smooth muscle cell through regulation of AMPK-eNOS-
D

FAS signalling, Curr. Med. Chem.20 (31) (2013) 3944–3953.


E

[25] A.K. Pandurangan, N. Mohebali, M.E. Norhaizan, C.Y. Looi, Gallic acid attenuates
PT

dextran sulfate sodium-induced experimental colitis in BALB/c mice, Drug Des. Devel.
Ther. 9 (2015) 3923–3934.
CE

[26] Z. Glick, Modes of action of gallic acid in suppressing food intake of rats, J. Nutr. 111
(11) (1981) 1910–1916.
AC

[27] E.J. Bak, J. Kim, S. Jang, G.H. Woo, H.G. Yoon, Y.J. Yoo, J.H. Cha, Gallic acid
improves glucose tolerance and triglyceride concentration in diet-induced obesity mice,
Scand. J. Clin. Lab. Invest. 73 (8) (2013) 607–614.
[28] K.V. Doan, C.M. Ko, A.W. Kinyua, D.J. Yang, Y.H. Choi, I.Y. Oh, N.M. Nguyen, A.
Ko, J.W. Choi, Y. Jeong, M.H. Jung, W.G. Cho, S. Xu, K.S. Park, W.J. Park, S.Y. Choi,
H.S. Kim, S.H. Moh, K.W. Kim, Gallic acid regulates body weight and glucose
homeostasis through AMPK activation, Endocrinology. 156 (1) (2015) 157–168.
ACCEPTED MANUSCRIPT

[29] H. Makihara, T. Shimada, E. Machida, M. Oota, R. Nagamine, M. Tsubata, K. Kinoshita,


K. Takahashi, M. Aburada, Preventive effect of Terminalia bellirica on obesity and
metabolic disorders in spontaneously obese type 2 diabetic model mice, J. Nat. Med. 66 (3)
(2012) 459–467.
[30] Y. Oi, I.C. Hou, H. Fujita, K. Yazawa, Antiobesity effects of Chinese black tea (Pu-erh
tea) extract and gallic acid, Phytother. Res. 26 (4) (2012) 475–481.
[31] G.R. Gandhi, G. Jothi, P.J. Antony, K. Balakrishna, M.G. Paulraj, S. Ignacimuthu, A.
Stalin, N.A. Al-Dhabi, Gallic acid attenuates high-fat diet fed-streptozotocin-induced

PT
insulin resistance via partial agonism of PPARγ in experimental type 2 diabetic rats and
enhances glucose uptake through translocation and activation of GLUT4 in PI3K/p-Akt

RI
signaling pathway, Eur. J. Pharmacol. 15 (745) (2014) 201–216.

SC
[32] D.W. Huang, W.C. Chang, J.S. Wu, R.W. Shih, S.C. Shen, Gallic acid ameliorates
hyperglycemia and improves hepatic carbohydrate metabolism in rats fed a high-fructose
NU
diet, Nutr. Res. 36 (2) (2016) 150–160.
[33] H. Makihara, Y. Koike, M. Ohta, E. Horiguchi-Babamoto, M. Tsubata, K. Kinoshita, T.
Akase, Y. Goshima, M. Aburada, T. Shimada, Gallic acid, the active ingredient of
MA

Terminalia bellirica, enhances adipocyte differentiation and adiponectin secretion, Biol.


Pharm. Bull. 39 (7) (2016) 1137–11343.
D

[34] K. Nagpal, S.K. Singh, D.N. Mishra, Nanoparticle mediated brain targeted delivery of
gallic acid: in vivo behavioral and biochemical studies for protection against scopolamine-
E

induced amnesia, Drug Deliv. 20 (3–4) (2013) 112–119.


PT

[35] H.L. Huang, C.C. Lin, K.C. Jeng, P.W. Yao, L.T. Chuang, S.L. Kuo, C.W. Hou, Fresh
green tea and gallic acid ameliorate oxidative stress in kainic acid-induced status
CE

epilepticus, J. Agric. Food Chem. 60 (9) (2012) 2328–2336.


[36] M. Moghadas, M.A. Edalatmanesh, R. Robati, Histopathological analysis from gallic
AC

acid administration on hippocampal cell density, depression, and anxiety related behaviors
in a trimethyltin intoxication model, Cell. J. 17 (4) (2016) 659–667.

[37] M.T. Mansouri, Y. Farbood, M.J. Sameri, A. Sarkaki, B. Naghizadeh, M. Rafeirad,


Neuroprotective effects of oral gallic acid against oxidative stress induced by 6-
hydroxydopamine in rats. Food Chem. 138 (2–3) (2013) 1028–1033.

[38] M.T. Mansouri, B. Naghizadeh, B. Ghorbanzadeh, Y. Farbood, A. Sarkaki, K. Bavarsad,


Gallic acid prevents memory deficits and oxidative stress induced by
ACCEPTED MANUSCRIPT

intracerebroventricular injection of streptozotocin in rats, Pharmacol. Biochem. Behav. 111


(2013) 90–96.

[39] Y. Farbood, A. Sarkaki, S. Hashemi, M.T. Mansouri, M. Dianat, The effects of gallic
acid on pain and memory following transient global ischemia/reperfusion in Wistar rats,
Avicenna J. Phytomed. 3 (4) (2013) 329–340.

[40] M.S. Korani, Y. Farbood, A. Sarkaki, H. Fathi Moghaddam, M. Taghi Mansouri,


Protective effects of gallic acid against chronic cerebral hypoperfusion-induced cognitive

PT
deficit and brain oxidative damage in rats, Eur. J. Pharmacol. 733 (2014) 62–67.

[41] D. Dhingra, R. Chhillar, A. Gupta, Antianxiety-like activity of gallic acid in unstressed

RI
and stressed mice: possible involvement of nitriergic system, Neurochem. Res. 37 (3)

SC
(2012) 487–494.
[42] M.T. Mansouri, M. Soltani, B. Naghizadeh, Y. Farbood, A. Mashak, A. Sarkaki, A
NU
possible mechanism for the anxiolytic-like effect of gallic acid in the rat elevated plus
maze, Pharmacol. Biochem. Behav. 117 (2014) 40–46.
[43] K. Nagpal, S.K. Singh, D.N. Mishra, Optimization of brain targeted gallic acid
MA

nanoparticles for improved antianxiety-like activity, Int. J. Biol. Macromol. 57 (2013) 83–
91.
[44] K. Nagpal, S.K. Singh, D.N. Mishra, Nanoparticle mediated brain targeted delivery of
D

gallic acid: in vivo behavioral and biochemical studies for improved antioxidant and
E

antidepressant-like activity, Drug Deliv. 19 (8) (2012) 378–391.


PT

[45] S.F. Nabavi, S. Habtemariam, A. Di Lorenzo, A. Sureda, S. Khanjani, S.M. Nabavi, M.


Daglia, Post-stroke depression modulation and in vivo antioxidant activity of gallic acid
CE

and its synthetic derivatives in a murine model system, Nutrients. 8 (5) (2016) E248.
[46] R. Chhillar, D. Dhingra, Antidepressant-like activity of gallic acid in mice subjected to
AC

unpredictable chronic mild stress, Fundam. Clin. Pharmacol. 27 (4) (2013) 409–418.
[47] V. Castagné, P. Moser, S. Roux, R.D. Porsolt, Rodent models of depression: forced swim
and tail suspension behavioral despair tests in rats and mice, Curr. Protoc. Neurosci. 8
(8.10A) (2011).
[48] O.D. Can, I.B. Ismail, Y. Oztürk, N. Oztürk, I. Potoğlu-Erkara, G. Sagratini, M.
Ricciutelli,Vittori S., F. Maggi, New antidepressant drug candidate: Hypericum montbretti
extract, Nat. Prod. Res. 25 (2011) 1469–1472.
[49] L.G. Müller, L.A. Salles, A.C. Stein, A.H. Betti, S. Sakamoto, E. Cassel, R.F. Vargas,
G.L. von Poser, S.M. Rates, Antidepressant-like effect of Valeriana glechomifolia Meyer
ACCEPTED MANUSCRIPT

(Valerianaceae) in mice, Prog. Neuropsychopharmacol. Biol. Psychiatry. 10 (36) (1)


(2012) 101–109.
[50] J.F. Cryan, A. Markou, I. Lucki, Assessing antidepressant activity in rodents: recent
developments and future needs, Trends Pharmacol. Sci. 23 (5) (2002) 238–245.
[51] L. Steru, R. Chermat, B. Thierry, P. Simon, The tail suspension test: a new method for
screening antidepressants in mice, Psychopharmacology 85 (3) (1985) 367–370.

[52] C. Girish, V. Raj, J. Arya, S. Balakrishnan, Evidence for the involvement of the

PT
monoaminergic system, but not the opioid system in the antidepressant-like activity of
ellagic acid in mice, Eur. J. Pharmacol. 682 (2012) 118–125.

RI
[53] Ö.D. Can, Ü. Demir Özkay, U.İ. Üçel, Anti-depressant-like effect of vitexin in BALB/c
mice and evidence for the involvement of monoaminergic mechanisms, Eur. J.

SC
Pharmacol.699 (1–3) (2013) 250–257.
[54] S. Kwon, B. Lee, M. Kim, H. Lee, H.J. Park, D.H. Hahm, Antidepressant-like effect of
NU
the methanolic extract from Bupleurum falcatum in the tail suspension test, Prog.
Neuropsychopharmacol. Biol. Psychiatry 34 (2010) 265–270.
MA

[55] S. Schreiber, M.M. Backer, I. Herman, S. Shamir, T. Boniel, C.G. Pick, The
antinociceptive effect of trazodone in mice is mediated through both μ-opioid and
serotonergic mechanisms, Behavioural Brain Res. 114 (2000) 51–56.
D

[56] I. Yalcin, F. Aksu, S. Bodard, S. Chalon, C. Belzung, Antidepressant-like effect of


E

tramadol in the unpredictable chronic mild stress procedure: possible involvement of the
PT

noradrenergic system, Behav. Pharmacol. 18 (2007) 623–631.


[57] A.M. Bradford, K.M. Savage, D.N. Jones, M. Kalinichev, Validation and
CE

pharmacological characterisation of MK-801-induced locomotor hyperactivity in BALB/C


mice as an assay for detection of novel antipsychotics, Psychopharmacology 212 (2010)
AC

155–170.
[58] S. Gavello-Baudy, J. Le Merrer, L. Decorte, V. David, P. Cazala, Self-administration of
the GABAA agonist muscimol into the medial septum: dependence on dopaminergic
mechanisms, Psychopharmacology 201 (2008) 219–228.
[59] A.E. Gonçalves, C. Bürger, S.K. Amoah, R. Tolardo, M.W. Biavatti, M.M. de Souza,
The antidepressant-like effect of Hedyosmum brasiliense and its sesquiterpene lactone,
podoandin in mice: Evidence for the involvement of adrenergic, dopaminergic and
serotonergic systems, Eur. J. Pharmacol. 674 (2012) 307–314.
ACCEPTED MANUSCRIPT

[60] K. Yasuda, K. Suemaru, H. Araki, Y. Gomita, Effect of nicotine cessation on the central
serotonergic systems in mice: involvement of 5-HT(2) receptors, Naunyn Schmiedebergs
Arch. Pharmacol. 366 (2002) 276–281.
[61] M, Kesim, E.N. Duman, M. Kadioglu, E. Yaris, N.I. Kalyoncu, N. Erciyes, The different
roles of 5-HT(2) and 5-HT(3) receptors on antinociceptive effect of paroxetine in chemical
stimuli in mice, J. Pharmacol. Sci. 97 (2005) 61–66.
[62] World Health Organization: Facts sheet No 369 – Depression. Accessed on the 24th of

PT
May 2017. http://www.who.int/mediacentre/factsheets/fs369/en/
[63] G. Lee, H. Bae, Therapeutic effects of phytochemicals and medicinal herbs on

RI
depression. Biomed Res Int. 2017 (2017) 6596241.
[64] K.A. Paschos, S. Veletza, E. Chatzaki, Neuropeptide and sigma receptors as novel

SC
therapeutic targets for the pharmacotherapy of depression. CNS Drugs. 23 (9) (2009) 755–
772.
NU
[65] A. Can, D.T. Dao, C.E. Terrillion, S.C. Piantadosi, S. Bhat, T.D. Gould, The tail
suspension test, J. Vis. Exp. 59 (2012) e3769.
[66] D.A. Slattery, J.F. Cryan, Using the rat forced swim test to assess antidepressant-like
MA

activity in rodents, Nat. Protoc. 7 (6) (2012) 1009–1014.


[67] J.F. Cryan, C. Mombereau, A. Vassout, The tail suspension test for assessing
D

antidepressant activity: review of pharmacological and genetic studies in mice, Neurosci.


E

Biobehav. Rev. 29 (2005) 571–625.


[68] E. Widerlöv, T. Lewander, Inhibition of the in vivo biosynthesis and changes of
PT

catecholamine levels in rat brain after alpha-methyl-p-tyrosine; time- and dose-response


relationships, Naunyn Schmiedebergs Arch. Pharmacol. 304 (1978) 111–123.
CE

[69] A.J. Mayorga, A. Dalvi, M.E. Page, S. Zimov-Levinson, R. Hen, I. Lucki,


Antidepressant-like behavioral effects in 5-hydroxytryptamine(1A) and 5-
AC

hydroxytryptamine(1B) receptor mutant mice, J. Pharmacol. Exp. Ther. 298 (2001) 1101–
1107.
[70] K. Pytka, K. Podkowa, A. Rapacz, A. Podkowa, E. Żmudzka, A. Olczyk, J. Sapa, B.
Filipek, The role of serotonergic, adrenergic and dopaminergic receptors in antidepressant-
like effect, Pharmacol. Rep. 68 (2) (2016) 263–274.
[71] B.K. Koe, A. Weissman, p-Chlorophenylalanine: a specific depletor of brain serotonin, J.
Pharmacol. Exp. Ther. 154 (1966) 499–516.
ACCEPTED MANUSCRIPT

[72] J.P. Redrobe., M. Bourin, M.C. Colombel, G.B. Baker, Psychopharmacological profile of
the selective serotonin reuptake inhibitor, paroxetine: implication of noradrenergic and
serotonergic mechanisms, J. Psychopharmacol. 12 (1998) 348.

PT
RI
SC
NU
MA
E D
PT
CE
AC
ACCEPTED MANUSCRIPT

Figure Legends

Fig. 1. Chemical structure of gallic acid.

Fig. 2. Effects of the gallic acid (GA, 30 and 60 mg/kg) administration on the (A) immobility
time, (B) swimming time and (C) climbing time in the MFST. Values are given as
mean±SEM. Significance against control values, *p<0.05, **
p<0.01, ***
p<0.001, One way
ANOVA, post-hoc Tukey test, n=8.

Fig. 3. Effects of the gallic acid (GA, 30 and 60 mg/kg) administration on the immobility time

PT
of mice in the TST. Values are given as mean±SEM. Significance against control values,
***
p<0.001, One way ANOVA, post-hoc Tukey test, n=8.

RI
Fig. 4. Effects of the gallic acid (GA, 30 and 60 mg/kg) administration on the (A) number of

SC
horizontal activity and (B) number of vertical activity in the activity cage test. Values are
given as mean±SEM, One way ANOVA, post-hoc Tukey test, n=8.
NU
Fig. 5. Effects of pre-treatment of (A) AMPT (an inhibitor of catecholamine synthesis, 100
mg/kg, i.p), (B) phentolamine (non-selective alpha-adrenoceptor antagonist, 5 mg/kg, i.p.),
MA

(C) propranolol (non-selective beta adrenergic receptor antagonist, 2 mg/kg, i.p), (D) SCH
23390 (dopamine D1 receptor antagonist, 0.05 mg/kg, s.c) and (E) sulpiride (dopamine D2
and D3 receptor antagonist, 50 mg/kg, i.p) on gallic acid (60 mg/kg) induced decrease in
D

immobility time in the TST. Values are given as mean±SEM. Significance against vehicle
E

***
treated vehicle groups, p<0.001; Significance against gallic acid treated vehicle groups
PT

b
p<0.01, cp<0.001, Two way ANOVA, post-hoc Bonferroni test, n=8.

Fig. 6. Effects of pre-treatment of (A) PCPA (an inhibitor of serotonin synthesis, 100 mg/kg,
CE

i.p, for 4 consecutive days), (B) NAN-190 (a 5-HT1A receptor antagonist, 0.5 mg/kg, i.p), (C)
ketanserin (a 5-HT2A/2C receptor antagonist, 5 mg/kg, i.p), (D) ondansetron (a 5HT3
AC

selective receptor antagonist, 0.3 mg/kg, i.p) on gallic acid (60 mg/kg) induced a decrease in
immobility time in the TST. Values are given as mean±SEM. Significance against vehicle
** ***
treated vehicle groups, p<0.01, p<0.001; Significance against gallic acid treated vehicle
groups bp<0.01, cp<0.001, Two way ANOVA, post-hoc Bonferroni test, n=8.
ACCEPTED MANUSCRIPT

PT
RI
SC
NU
MA
D

Figure 1
E
PT
CE
AC
ACCEPTED MANUSCRIPT

PT
RI
SC
NU
MA

Figure 2
DE
PT
CE
AC
ACCEPTED MANUSCRIPT

PT
RI
SC
Figure 3 NU
MA
DE
PT
CE
AC
ACCEPTED MANUSCRIPT

PT
Figure 4

RI
SC
NU
MA
DE
PT
CE
AC
ACCEPTED MANUSCRIPT

PT
RI
SC
NU
Figure 5
MA
DE
PT
CE
AC
ACCEPTED MANUSCRIPT

PT
RI
SC
NU
MA

Figure 6
DE
PT
CE
AC