https://doi.org/10.52973/rcfcv-e33258
Received: 10/04/2023 Accepted: 11/05/2023 Published: 21/06/2023
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Revista Científica, FCV-LUZ / Vol. XXXIII, rcfcv-e33258, 1 – 6
ABSTRACT
Gallic acid is a phenolic compound found in many plant sources with
strong antioxidant activity. In this study, the bioactivity of Gallic acid
was investigated in Japanese quails induced by oxidative stress. The
study was performed on four groups of 40–day–old male Japanese
quail (Coturnix japonica). Oxidative stress was created for 1 week
by adding 0.5% hydrogen peroxide. The study was terminated by
administering 100 mg·kg
-1
body weight Gallic acid intraperitoneally.
Total antioxidant and total oxidant level analyzes from liver tissue
homogenates were performed using a ready–made commercial kit.
TNF–α levels from blood samples taken for anti–inammatory activity
were investigated by ELISA method. There were no statistically
signicant results on live weight gain between the experimental
groups and control group. However, Gallic acid in liver homogenates
together with H
2
O
2
increased total antioxidant state (TAS) compared
to H
2
O
2
application, while it decreased total oxidant state (TOS) in the
same groups. Moreover, while the oxidative stress index increased
in the H
2
O
2
group, it decreased signicantly in both the Gallic acid
and Gallic acid + H
2
O
2
groups. Gallic acid application also caused
regression in blood TNF–α expression levels, which were increased
by H
2
O
2
. In quails, Gallic acid showed antioxidant activity by increasing
TAS levels and decreasing TOS levels, providing a signicant decrease
in oxidative stress index. It also provided anti–inammatory activity
by suppressing TNF–a levels. However, advanced molecular analyzes
are needed to obtain more detailed information on the subject.
Key words: Gallic acid; growth performance; oxidative stress;
inammation; quails
RESUMEN
El ácido gálico es un compuesto fenólico que se encuentra en
muchas fuentes vegetales con fuerte actividad antioxidante. En este
estudio, se investigó la bioactividad del ácido gálico en codornices
japonesas inducidas por estrés oxidativo. El estudio se realizó en
cuatro grupos de codornices japonesas macho (Coturnix japonica) de
40 días de edad. Se creó estrés oxidativo durante 1 semana mediante
la adición de peróxido de hidrógeno al 0,5 %. El estudio nalizó con la
administración de 100 mg·kg
-1
de peso corporal de ácido gálico por vía
intraperitoneal. Los análisis del nivel de antioxidante total y oxidante
total de homogeneizados de tejido hepático se realizaron utilizando
un kit comercial ya preparado. Los niveles de TNF–α de muestras
de sangre tomadas para determinar la actividad antiinamatoria
se investigaron mediante el método ELISA. No hubo resultados
estadísticamente signicativos sobre la ganancia de peso vivo entre
los grupos experimentales y el grupo control. Sin embargo, el ácido
gálico en homogeneizados de hígado junto con H
2
O
2
aumentó el estado
antioxidante total (TAS) en comparación con la aplicación de H
2
O
2
,
mientras que disminuyó el estado oxidante total (TOS) en los mismos
grupos. Además, mientras que el índice de estrés oxidativo aumentó
en el grupo H
2
O
2
, disminuyó signicativamente, tanto en el grupo de
ácido gálico como en el de ácido gálico + H
2
O
2
. La aplicación de ácido
gálico también provocó una regresión en los niveles de expresión de
TNF–α en sangre, que aumentaron con H
2
O
2
. En codornices, el ácido
gálico mostró actividad antioxidante al aumentar los niveles de TAS
y disminuir los niveles de TOS, proporcionando una disminución
signicativa en el índice de estrés oxidativo. También proporcionó
actividad antiinflamatoria al suprimir los niveles de TNF–a. Sin
embargo, se necesitan análisis moleculares avanzados para obtener
información más detallada sobre el tema.
Palabras clave: Acido gálico; desempeño del crecimiento; estrés
oxidativo; inamación; codornices
Antioxidant and anti–inammatory activities of Gallic acid in Japanese
quails induced by oxidative stress
Actividades antioxidantes y antiinamatorias del ácido gálico en codornices japonesas inducidas
por estrés oxidativo
Mehmet Mustafa İşgör
1
* , Altuğ Küçükgül
1
, Sema Alaşahan
2
1
Hatay Mustafa Kemal University, Faculty of Veterinary, Department of Biochemistry. Antakya, Turkey.
2
Hatay Mustafa Kemal University, Faculty of Veterinary, Zootechnical department. Antakya, Turkey.
*Correspondence author: mmisgor@gmail.com
Gallic acid bioactivity Japanese quails / İşgör et al. _________________________________________________________________________________
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INTRODUCTION
Recently, especially after the limitation of the use of antibiotics,
the use of alternative natural compounds, which are abundant in
plants and fungi and have important biological activities, as natural
feed additives in poultry farming has been increasing. Polyphenols
constitute the most effective groups of phytochemicals added
to animal feeds as an alternative to antibiotics [1, 2]. Polyphenols
are dened as compounds containing at least one hydroxyl group
attached to a phenol ring in their chemical structure. The most widely
known of these compounds, which have a wide range of varieties,
are Caffeic acid, Ferulic acid, p–Hydroxybenzoic acid, Protocatechic
acid, Vanillic acid, Salicylic acid and Gallic acid [3, 4].
Gallic acid (3,4,5–Trihydroxybenzoic acid), an important member of
the tannins group of these phenolic compounds, attracts the attention
of researchers in scientific studies due to its strong antioxidant
bioactivity [5], although Gallic acid, or gallate, is found in many plants
[6] and in fungal species [7]. In addition to the antioxidant activity of
this compound, which was discovered many years ago, anticancer [8],
anti–HIV [9], antiulcerogenic [10], anti–inammatory [11], antimicrobial
[12] and antifungal [13] have also been reported in studies.
Oxidative stress basically develops when free radicals cannot be
adequately removed by antioxidant defense mechanisms. Normally,
there are antioxidant defense mechanisms in order to maintain the
oxidant–antioxidant balance in living things. When this defense
system, which includes enzymatic mechanisms such as superoxide
dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and
nonenzymatic mechanisms such as glutathione, vitamin C, vitamin E
and plant polyphenolic compounds, is weakened, the production and
accumulation of reactive oxygen species in cells and tissues increases
[14]. Oxidative stress is one of the main reasons for the deterioration
of animal health and reduced meat quality in poultry farming [15].
Phytochemicals found in plants, especially polyphenols, stand out as
the most important supplement compounds in coping with oxidative
stress thanks to their strong antioxidant activities [16]. Therefore, these
compounds have the potential to be used as feed additives to reduce
oxidative stress in poultry and increase productivity in animals [17].
This study was aimed to investigate some parameters of antioxidant
and anti–inammatory activities of Gallic acid in a hydrogen peroxide–
induced oxidative stress model in quails (Coturnix japonica).
MATERIALS AND METHODS
Ethical approval
This study was approved by Hatay Mustafa Kemal University Animal
Experiments Local Ethics Committee (Decision No: 2021/06–17).
Experimental design
The study was performed on 40–day–old male Japanese quail. The
quails were individually weighed on a precision scale (Ohaus NV622,
USA), and their average body weight was recorded and distributed
to the experimental groups (n=10).
In the study groups, the control group (C) was fed only commercial
starter feed, group I was administered commercial feed starter + Gallic
acid intraperitoneally (IP), group II was given commercial feed starter
and hydrogen peroxide (HP) added to drinking water, nally group III
was applied commercial feed starter + HP + Gallic acid IP. Oxidative
stress was created for 1 week by adding 0.5 % hydrogen peroxide
(Tekkim, Turkey) to the drinking water of quails in groups II and III.
Then, the study was terminated by administering Gallic acid (Sigma
Aldrich, EU,) dissolved in serum physiologic at a concentration of 100
mg·mL
-1
body weight intraperitoneally for 2 days to the experimental
groups I and III every other day.
In the study, quails were fed with commercial growth feed (TABLEI).
In the study, rst body weight and nal body weight, 10–day Body
Weight change rate (%) and Average Daily Feed Intake Amount g
were measured (n=10). Afterwards, tissue and blood samples were
taken and oxidative stress parameters and biochemical–molecular
analyzes were performed (n=5).
TABLE I
Nutrient composition of commercial grower feed
Nutrient values Amount (%)
Crude protein 20.0
Crude Fat 3.00
Crude ber 3.00
Crude ash 4.80
Lysine 1.12
Methionine 0.51
Calcium 0.880
Total phosphorus 0.440
Sodium 0.140
Vitamin–Mineral premix* 0.250
Metabolizable energy kcal·kg
-1
2859.3
*1 kg of the premix provided: 15.000.000 IU of Vitamin A, 5.000.000 IU of Vitamin D3,
100.000 mg of Vitamin E, 3000 mg of Vitamin K3, 5000 mg of Vitamin B1, 8.000 mg of
Vitamin B2, 60.000 mg of niacin, 15.000 mg of D–calcium pantothenate, 5000 mg of
Vitamin B6, 20 mg of Vitamin B12, 200 mg of D–biotin, 2000 mg of Folic acid,100.000
mg of Vitamin C, 0.02 mg of Cyanocobalamin, 74 mg of Mn (from MnO), 45 mg of Zn
(from ZnO), 4 mg of Cu (from CuO), 12.5 mg of Fe (from FeSO
4
), 0.3 mg of I (from KI),
0.15 mg of Se (from NaSe)
Determination of biochemical parameters
In the study, liver samples were taken from 5 quails from each group
for biochemical parameters. Total antioxidant status (TAS) and total
oxidant status (TOS) analyzes from tissue homogenates (Rel assay–
TR) were performed using a ready–made commercial kit, following
the methods of Kucukgul and Erdogan [18]. The Oxidative Stress
Index (OSI) in the experimental groups was calculated by the ratio of
these two values. In addition, Tumor necrosis factor alpha (TNF–α)
levels from blood samples taken for anti–inammatory activity were
investigated by ELISA method with commercial kits (Kit Origin).
Statistical analysis
Statistical analysis with the help of IBM SPSS 22 package
program, the comparison of the groups in terms of the examined
features was analyzed with One–way Anova. Differences were
determined by Duncan's test.
CGroup I Group II Group III
0,0
0,5
1,0
LiverTAS levels
1,0306
0,9748
0,6401
0,9971
FIGURE 1. Total antioxidant status in liver homogenates of experimental groups
(C: Control group, group I: only Gallic applied group, group II: only H
2
O
2
applied
group, group III: gallic acid+H
2
O
2
applied group)
______________________________________________________________________Revista Cientifica, FCV-LUZ / Vol. XXXIII, rcfcv-e33258, 1 – 6
3 of 6
RESULTS AND DISCUSSIONS
Studies on performance and egg quality of Gallic acid and its
derivatives in poultry (Gallus gallus domesticus), especially in layers,
are quite limited. However, when reviewing the literature, it is seen
that there are studies using grape (Vitis viniferas) seed extract gallic
acid abundant in the seeds and skin of the grape. For example, Abdel–
Wahab et al. [19] reported that grape seed polyphenols decreased total
cholesterol lipid levels, blood sugar and liver enzyme activities, and
increased glutathione peroxidase enzyme activity in Japanese quails,
especially in 10 to 38–day periods. Again, Silici et al. [20] reported that
the addition of 0.5, 1 and 1.5 % ground grape seed to quail compound
feeds did not have a negative effect on yield, hatchability and egg quality
of quails, and also increased feed eciency. Moreover, Abu Hafsa and
İbrahim [21] reported that with the addition of 20 g·kg
-1
grape seed
(GS) to the basal diet, the nal live weight and live weight gain of quails
increased, their feed conversion ratio increased, but feed intake was
not affected. However, they stated that meat signicantly increased
carcass physical and chemical composition properties, carcass yield,
and gizzard percentage. They reported that the addition of 40 g·kg
-1
GS signicantly reduced the percentage of abdominal fat in poultry. In
addition, in a study by Ao and Kim [22], it was reported that the addition
of grape seed extract increased feed eciency, increased antioxidant
enzyme activities and immunity, and improved meat quality and body
weight gain in Peking ducks (Anatidae).
In the present study, the live weight change rates of quails in the
experimental groups (group I, II and III) were found to be lower (4.45 %,
4.25 % and 2.69 % respectively) than the control group (10.52 %). Also,
according to the results obtained, feed conversion ratios between the
experimental groups and the control group were not found statistically
signicant. In addition, when the experimental groups were compared
among themselves, it was determined that the live weight gain rates
changed in parallel with the feed intake amount.
Oxidative stress, known as the disruption of the oxidant–antioxidant
balance between reactive oxygen species and the body's antioxidant
defense systems, constitutes one of the most fundamental problems
of modern poultry farming [23]. Increasing superoxide and hydroxyl
radicals as a result of the disruption of this balance cause serious
damage to lipid, deoxyribonucleic acid (DNA), protein and other cellular
components, leading to disruption of cell integrity and tissue damage
[24]. It is known that oxidative stress affects commercial poultry
production, growth performance, productivity and reproductive
performance of layer hens in poultry farming. Although there are
various strategies for the control of oxidative stress in poultry, natural
antioxidant compounds (polyphenols), which are non–toxic and do
not leave residues, have recently come into the focus of scientic
studies [25]. The health benets of plant phenolics are mainly due
to their antioxidant and anti–inammatory abilities [26]. Among
such plant phenolics, Gallic acid (a trihydroxybenzoic acid) found in
various foods and plants can be given as an example [27]. The curative
effects of Gallic acid on many metabolic diseases including obesity
have been reported [28, 29]. Moreover, several reviews have been
published focusing on the therapeutic potential of Gallic acid. In 2013
Locatelli et al. [30] studied alkyl esters of Gallic acid as anticancer
agents. Again, Choubey et al. [31] demonstrated the anticarcinogenic,
antimicrobial, antimutagenic and antiangiogenic properties of Gallic
acid and its esters.
When the liver total antioxidant capacity values were compared, TAS
value in group I (0.9748 ± 0.15944) was found to be 5 % lower than the
control group (1.0306 ± 0.12777), whereas in group II (0.6401 ± 0.09142)
decreased by 37 % (P<0.5). However, this value increased 55 % in
groupIII (0.9971 ± 0.13702) compared to group II (P<0.5) (FIG. 1). According
to the results that was obtained in the present study, the TAS levels of
Japanese quails decreased with the application of hydrogen peroxide,
while the application of hydrogen peroxide together with Gallic acid
prevented this decrease and increased the TAS value to the levels of
the control group. In a study, Samuel et al. [32] showed that while Gallic
acid decreased MDA, it increased plasma SOD and TAS signicantly.
Moreover, studies have reported that Gallic acid increases the activities
of enzymes such as SOD, GPx and CAT, and increases GSH and vitamin
C levels [33, 34]. Accordingly with the present study, it is thought that
Gallic acid caused an increase in TAS levels by regulating nonenzymatic
and/or enzymatic antioxidant mechanisms.
As another finding, when liver TOS values were compared,
it decreased by 32 % in group I (13.3718 ± 1.13462) compared to
the control group (19.6446 ± 2,41352) (P<0.5), whereas in group II
(15.0483 ± 0.81577) a 23 % decrease was observed (P<0.5). However,
it was determined that this value decreased by 14 % in group III
(12.8825 ± 2.12539) compared to group II (P<0.5) (FIG. 2). In addition,
liver OSI values decreased by 28 % in group I (13,7175) compared to
the control group (19.0613), while it increased by 23 % in group II
(23.5093). However, this value showed a 45 % regression in groupIII
(12,9200) compared to group II (FIG. 3). The use of Gallic acid with
H
2
O
2
has been shown to reduce liver TOS levels compared to hydrogen
TABLE II
Live weight values of experimental groups
Initial live
weight (g)
Final live
weight (g)
10–day live
weight change
rate (%)
Average Daily
Feed Intake
Amount (g)
Control 210.52 ± 8.10 232.66 ± 6.93 10.52 33.40 ± 1.80
Group I
(Gallic acid)
203.21 ± 5.88 212.26 ± 8.01 4.45 26.40 ± 4.80
Group II
(H
2
O
2
)
210,82±7.39 219.79 ± 9.24 4.25 36.40 ± 4.80
Group III
(Gallic + H
2
O
2
)
210.16 ± 9.08 215.81 ± 10.92 2.69 24.40 ± 0.80
P 0.878 0.216 0.191
CGroup I Group II Group III
0
5
10
15
20
25
LiverTOS values
19,6446
13,3718
15,0483
12,8825
CGroup I Group II Group III
0
5
10
15
20
25
LiverOSI values
19,0613
12,9200
23,5093
13,7175
CGroup I Group II Group III
0
50
100
150
SerumTNF-± levels
134,1212
126,8182
137,0303
127,4545
FIGURE 2. Total oxidant status of experimental groups in liver homogenates
(C: Control group, group I: only Gallic applied group, group II: only H
2
O
2
applied
group, group III: gallic acid+H
2
O
2
applied group)
FIGURE 3. Oxidative stress index values in liver homogenates of experimental
groups (C: Control group, group I: only Gallic applied group, group II: only H
2
O
2
applied group, group III: Gallic acid+H
2
O
2
applied group)
FIGURE 4. Effects of Gallic acid and Hydrogen peroxide on TNF–α translation
levels in serum samples (C: Control group, group I: only Gallic applied group,
group II: only H
2
O
2
applied group, group III: Gallic acid+H
2
O
2
applied group)
Gallic acid bioactivity Japanese quails / İşgör et al. _________________________________________________________________________________
4 of 6
peroxide. Moreover, when the OSI values are considered, hydrogen
peroxide increased the oxidative stress index, while the use of Gallic
acid alone or in combination with H
2
O
2
signicantly decreased the
OSI levels. In a recent study, Ignea et al. [35] reported that Gallic acid
GA increases the body's antioxidant capacity by directly neutralizing
reactive oxygen species ROS and free radicals, or both, and reducing
plasma malondialdehyde MDA levels. Jung et al. [36] reported that
chicken breast meat was significantly larger in the group fed a
mixture of 0.5 % and 1.0 % Gallic acid and Linoleic acid compared
to the control group. They also reported that a mixture of Gallic acid
and Linoleic acid were electron donors capable of neutralizing free
radicals in broiler breast meat samples. Samuel et al. [32] reported
that 100 mg/kg administration of Gallic acid signicantly increased the
broiler chest muscle mass compared to the control group, however,
there was a decrease in the jejunal crypt depth and an increase in
the villus width. Lee et al. [37] reported that the pH value of the
broiler leg meat improved and the water holding capacity of the thigh
increased in chickens fed with basal feed containing 1 % Gallic acid for
2 weeks. In the same study, it was shown that Gallic acid increased the
antioxidant capacity of muscle tissues and signicantly decreased
the thiobarbituric acid reactive substances (TBARS) value. Jung et al.
[38] showed that in chickens fed a mixture of Gallic acid and Linoleic
acid, it reduced the cholesterol level of eggs, and also increased the
antioxidant potential by reducing the TBARS level.
Inammation is a natural, protective response of the organism to
pathogens, chemical harmful stimuli or stress. As a physiological
process, the main purpose of inammatory responses is to induce
the process of eliminating pathogens and toxins and repairing
damaged tissue [39]. In the present study, blood TNF–α values
decreased by 5 % in group I (126.8182 ± 21.06224) compared to the control
group (134.1212 ± 21.14828) (P>0.5), while group II (137.0303 ± 23.78826)
showed an increase of 2 % (P<0.5). However, this value showed a 7 %
regression in group III (127.4545 ± 12.26777) compared to group II (P<0.5)
(FIG. 4). Oxidative stress is closely associated with inflammation
due to the fact that the NF–kB pathway, which is important in the
regulation of inammation, is activated due to the increase of reactive
oxygen species [40]. It has been reported that increased ROS may
cause nuclear factor kappa B (NF–κB) activation and expression of
inflammatory cytokines [41]. Therefore, according to the results
obtained, it is thought that Gallic acid may have shown this effect by
reducing ROS with its strong antioxidant property.
It was found that while H
2
O
2
induced blood TNF–α gene expression
levels of Japanese quails, Gallic acid signicantly suppressed the
expression levels of this gene. It was seen that there are studies
investigating the anti–inammatory potential of Gallic acid. In a study
investigating the effects of polyphenols in broilers, it was determined
that tea polyphenols (0.03–0.09 g·kg
-1
body weight) in the long term
were found to show anti–inammatory activity by reducing gene
expression levels of proinammatory cytokines such as IL–1β, IL–4,
IL–6, IL–10, TNF–α and IFN–γ [39].
CONCLUSIONS
In the present study, Gallic acid application did not show a
statistically significant change on live weight change rates of
______________________________________________________________________Revista Cientifica, FCV-LUZ / Vol. XXXIII, rcfcv-e33258, 1 – 6
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Japanese quails. However, it increased liver TAS levels suppressed
by hydrogen peroxide and decreased liver TOS levels in quails.
Considering the total OSI values, it can be said that it makes a
signicant contribution to suppressing oxidative stress. In addition,
it showed anti–inammatory activity by signicantly suppressing
TNF–α, one of the proinammatory cytokines. As a result, in terms
of its antioxidant and anti–inammatory activities, Gallic acid is
a compound that may have antioxidant and anti–inflammatory
potentials in Japanese quails. However, further and detailed studies
are needed to fully elucidate this argument.
Conict of interests
The authors of this study declare that there is no conict of interest
with the publication of this manuscript.
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