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

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 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

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

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* Corresponding author:
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Assoc. Prof. Dr. Özgür Devrim Can

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

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Tel. : +90 222 3350580 / 3749

Fax number : +90 222 3350750

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

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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.

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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.

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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-

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

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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
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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
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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
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gallic acid.
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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
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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.
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Keywords: Gallic acid; modified forced swimming test; serotonergic system; monoaminergic
system; tail-suspension test
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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

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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

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[1].

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

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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],
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anticancer [14], chemopreventive [13,15], wound healing [16], antibacterial [17], anti-HIV
[18], hepatoprotective [2], antiallergic [19], anti-inflammatory [11,13,2021], antinociceptive
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[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
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the central nervous system. Beneficial effects of this compound against neurotoxicity and
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neurodegeneration have been demonstrated in different experimental models, such as

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

[35], trimethyltin-induced hippocampal degeneration and emotional instability [36], 6-
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hydroxydopamine injection (full nigral lesion, animal model of Parkinson's disease)-induced

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

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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

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

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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.
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2.2. Drugs and administrations
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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
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hydrate, R(+)-SCH-23390 hydrochloride, and sulpiride used in the experiments were acquired
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from Sigma-Aldrich (St. Louis, MO, USA), while ketanserin tartrate was purchased from
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Tocris Cookson (Ballwin, MO, USA).

All of the drugs, except AMPT, were dissolved in physiological saline (NaCl, 0.9%).
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AMPT solution was prepared using saline containing 10% Tween 80. Vehicle treatments to
the appropriate control groups were carried out simultaneously.
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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

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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

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

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2.3.2. Tail suspension test

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

2.3.3. Activity cage test

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The locomotor activity of each mouse was assessed in an activity cage apparatus (No.
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7420; Ugo Basile, Varese, Italy), containing two pairs of 16 infrared photocells 3 cm and 12
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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
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[52]. Between tests, the device was carefully cleaned with ethanol to remove any residue or
odour from the former mouse.
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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-
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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.

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

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

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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
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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],
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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.
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All doses used in the present study were chosen according to the literature as well as
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the authors’ previous experimental practices.

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2.5. Data analysis

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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
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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
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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

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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

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

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

3.3. Assessment of locomotor activity in activity cage test

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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;
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Fig. 4B) movements.

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

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

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

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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

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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

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< 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.
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None of the antagonists altered the immobility duration of the animals when they were
administered alone.
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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
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significant interaction between the factors of pre-treatment × treatment factors (F[1,28] =

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6.083, P < 0,05). Post hoc analyses showed that the pre-treatment of animals with PCPA was
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effective in reversing the anti-depressant-like effect of gallic acid (P < 0.01). PCPA treatment
alone did not significantly change the immobility time.
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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] =
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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
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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

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

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

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Antidepressant pharmacotherapy together with simultaneous psychotherapy can help
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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
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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
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options for pharmacotherapy of depression and other affective disorders.

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

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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

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

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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
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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
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oxidase-A inhibitory activity, antioxidant capacity, and its lowering effect on plasma nitrite
and corticosterone levels.
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The results of the activity cage tests indicated that administration of gallic acid at 30 or
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60 mg/kg doses did not induce significant changes in the total number of horizontal (Fig. 4A)
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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
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locomotor activity of mice, since increased locomotor activity may cause false positive results
in the MFST or TST [67].
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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
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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

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

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

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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
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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
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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
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shortened immobility times. Data obtained from these mechanistic studies indicate that
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enhanced catecholamine levels in synapses are critical for the antidepressant-like effect of
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gallic acid together with the contribution of alpha adrenergic and dopaminergic (D1, D2 and
D3) receptors.
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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
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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
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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.

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

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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

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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
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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.
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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
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treatment of affective disorders. On the other hand, the results of this preclinical study need
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further clinical validation to confirm their generalizability to patients with depressive

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disorders.
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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

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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.

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

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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.
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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.),
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(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
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immobility time in the TST. Values are given as mean±SEM. Significance against vehicle
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***
treated vehicle groups, p<0.001; Significance against gallic acid treated vehicle groups
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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,
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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
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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.
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