Biomedicine & Pharmacotherapy 83 (2016) 13271334

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The protective effect of Citrus limon essential oil on hepatotoxicity and

nephrotoxicity induced by aspirin in rats
Hafsia Bouzennaa,b,c,* , Sabah Dhibia,b , Noura Samouta,b , Ilhem Rjeibia,b ,
Hlne Talarminc , Abdelfattah Elfekia , Najla Hfaiedha,b
a
Laboratory of Environmental Physiopathology, Valorization of Bioactive Molecules and Mathematical Modeling, Faculty of Sciences Sfax, Road Soukra km 3.5,
PB no 1171-3000, Sfax, Tunisie
b
Laboratory Animal Eco Physiology, Faculty of Sciences of Gafsa, 2112, Tunisie
c
EA 1274, Facult de Mdecine et Sciences de la Sant, Universit de Bretagne Occidentale, 22 Avenue Camille Desmoulins, CS 93837, 29238 Brest Cedex 3,
France

A R T I C L E I N F O A B S T R A C T

Article history:
Received 24 May 2016 Citrus limon is a member of the large Rutaceae family characterized by its therapeutic proprieties and has
Received in revised form 10 August 2016 been widely used in traditional medicine to treat various diseases. This study investigates the protective
Accepted 15 August 2016 effect of Citrus limon essential oil against a high dose of aspirin-induced acute liver and kidney damage in
female Wistar albino rats. Twenty-eight adult female Wistar rats were divided into 4 groups of 7 each: (1)
Keywords: a control group; (2) a group of rats which was kept untreated for 56 days then treated with aspirin (A)
Aspirin (600 mg/kg) for 4 days; (3) a group fed with essential oil of Citrus limon for 56 days then (A) for 4 days; and
Citrus limon (4) a group of rats receiving essential oil of Citrus limon for 56 days, then given NaCl for 4 days. Estimations
Kidney
of biochemical parameters in blood were determined. Lipid peroxidation levels (TBARS), superoxide
Liver
dismutase (SOD), catalase (CAT), and glutathione peroxidas (GPx) activities in liver and kidney was
Oxidative stress
determined. A histopathological study was done. Under our experimental conditions, aspirin induced an
increase of serum biochemical parameters and it resulted in an oxidative stress in both liver and kidney.
This was evidenced by signicant increase in TBARS in liver and kidney by 108% and 55%, respectively,
compared to control. On the other hand, a decrease in the activities of SOD by 78% and 53%, CAT by 53%
and 78%, and GPx by 78% and 51% in liver and kidney, respectively. Administration of EOC to rats
attenuated the induced an effect of the high dose of aspirin induced in the afore mentioned serum
biochemical parameters. In conclusion, our data suggest that treatment with essential oil of Citrus limon
prevented the liver and kidney damage induced by aspirin.
2016 Elsevier Masson SAS. All rights reserved.

1. Introduction
Aspirin is one of the most highly consumed pharmaceutical
products in the world; it is widely used as a nonsteroidal anti-
inammatory drug. It has been used for the treatment of
rheumatoid arthritis and related diseases as well as the prevention
of cardiovascular thrombotic diseases [1]. It is rapidly absorbed
from the stomach and small intestine, primarily by passive
diffusion across the gastrointestinal tract, and hydrolyzed to
salicylic acid by esterases in the gastrointestinal mucosa and
plasma. Then, aspirin is widely distributed throughout the body
* Corresponding author at: Laboratory of Environmental Physiopathology,
Valorization of Bioactive Molecules and Mathematical Modeling, Faculty of
Sciences of Sfax, Road Soukra km 3.5, PB no 1171-3000, Sfax, Tunisia.
E-mail address: bouzenna.hafsia@hotmail.fr (H. Bouzenna).
with the highest concentrations in the plasma, liver, renal cortex,
heart and lungs. It is metabolized via conjugation in the liver to
form salicylic acid and several other metabolites [2]. However, the
use of aspirin is also associated with signicant morbidity and
mortality due to its adverse effects on multiple organ systems such
as liver, kidney, and stomach. Long-term therapeutic consumption
of aspirin is reportedly associated with the occurrence of
gastrointestinal ulcerations, nephrotoxicity, hepatotoxicity, and
even renal cell cancer [3].
Herbal medicines derived from plant extracts constitute an
indispensable part of the traditional medicine and are being
increasingly utilized to treat a wide variety of clinical diseases [4].
Also, the use of natural antioxidants can protect against the
harmful effect of free radicals in the human body [5]. In this
context, more attention has been paid to the protective effects of

http://dx.doi.org/10.1016/j.biopha.2016.08.037
0753-3322/ 2016 Elsevier Masson SAS. All rights reserved.
1328 H. Bouzenna et al. / Biomedicine & Pharmacotherapy 83 (2016) 13271334
natural antioxidants of the herbal medicine against drug-induced
toxicities [6].
Citrus limon is a medicinal plant of the family Rutaceae found in
Italy and other Mediterranean countries like Spain, Egypt, and
Turkey [7]. Lemons have many important natural chemical
components, including citric acid, ascorbic acid, minerals, avo-
noids and essential oils. The leaves of Citrus limon are used in folk
medicine for the treatment of obesity, diabetes, blood lipid
lowering, cardiovascular diseases, brain disorders, and certain
types of cancer [8].
Essential oil extracted from lemon peel has effects on the
behavioral response in rats and humans [9,10]. Its essential oil is
widely used in perfume, cosmetic and food industries. Hence, this
study is aimed to investigate the protective effect of essential oil of
Citrus limon (EOC) on the side effects of aspirin on liver and kidney
of female rats by measuring some physiological parameters and
determining some histopathological alterations.
2. Methods
2.1. Plant material
The aerial parts of Citrus limon were collected during October
and November 2013 from the region of Sidi Aich, a suburb of Gafsa
in the south west of Tunisia. A voucher specimen was identied by
botanists in the Faculty of Sciences of Gafsa (FSG), Tunisia. The
collected vegetative components were air-dried at room tempera-
ture (25  C) for 14 days in a well-shaded and naturally ventilated
space.
2.2. Extraction and isolation of essential oil
The essential oil sample was extracted from 50 g of Citrus limon
dried leaves by hydrodistillation after grounding and immersion in
500 mL of distilled water. The extraction process was carried out
for three hours. The oil was recovered from the extraction
apparatus using hexane. Anhydrous sodium sulphate was used
in order to the organic phase was dry the organic phase using until
the remaining water traces were completely drained. After
ltration, the solvent was evaporated and the essential oil was
kept at 4  C in a dark glass bottle until completion of the
phytochemical analyses [11].
2.3. Animals
Twenty-eight adult female Wistar rats weighing 150200 g
were brought up in the animal house of the Faculty of Sciences of
Sfax, Tunisia, upon approval by the Tunisian Ministry of Higher
Education and Scientic Research. The conditions of the animal
house meet the agreed international standards. The animals were
acclimatized to their new environment (a breeding farm) for two
days prior to experiment and kept under photoperiods (14 h light/
10 h dark) at 2122  C. Each group was housed in a spacious indoor
cage and the rats were allowed free access to food pellets obtained
from the Societ Industrielle de Conditionnement Optimis (S.I.C.
O), Sfax, Tunisia, and composed of corn, luzern, soybean, vitami-
nized mineral compounds, dicalcium phosphate, calcium carbon-
ate, salt-free methionine, trace elements (copper, iron, zinc,
manganese, cobalt, selenium, and iodine), and water ad libitum.
The animals were treated in compliance with the Tunisian code of
practice for the Care and Use of Animals for Scientic Purposes and
the European Convention for the Protection of Vertebrate Animals
used for Experimental and other Scientic Purposes (Council of
Europe No 123, Strasbourg, 1985).
2.4. Experimental design
The experimental rats were divided into four groups of seven
rats each. (C) was the control group, administered orally by gastric
intubation 0.9% NaCl everyday for 60 days.
(EOC) group consisted of rats given orally the essential oil of
Citrus limon (about 1.0% w/w with a dose of 1 mL/kg) [12] for 56
days and then given NaCL for 4 days.
(A) group consisted of rats treated with aspirin which was
suspended in normal saline and administered orally by gastric
intubation in acute dose 600 mg/kg (with DL50 = 5.3705 g/kg) [13];
it was administered three times a day each dose at an interval of 4 h
for 4 successive days. This high dose has been used in studies
investigating acute effects of aspirin in rats and mice. Such high-
dose therapy is being used to treat health problems such as
rheumatoid arthritis [3].
(EOC + A) group was made up of rats treated with essential oil of
Citrus limon for 56 days, then given aspirin for 4 days. At the end of
the experimental period, the rats in each group were rapidly
sacriced by decapitation in order to minimize the handling stress.
Blood serum was obtained by centrifugation (1500 x g, 15 min, and
4  C). Liver and kidney were removed, cleaned of fat, weighed and
stored at 80  C until use.
2.5. Determination of antioxidant enzyme activities
1 g of rat organs liver and kidney were shredded, immersed in
2 mL ice-cold Tris-buffered saline (TBS, pH = 7.4), and centrifu-
gated (5000 x g, 30 min, 4  C). Supernatants were collected and
used in order to determine: (a) Thiobarbituric acid reactive
substances (TBARS) according to Yagi [14]; (b) Glutathione-
peroxidase (GPx) according to the method described by Flohe
and Gunzle [15]; (c) superoxide-dismutase (SOD) using the
method of Misra and Fridovich [16] and (d) Catalase (CAT) using
Aebis method [17].
2.6. Serum marker assays
Serum glucose levels, total cholesterol, lactate dehydogenase
(LDH), alanin transaminase (ALT) as well as aspartate transaminase
(AST) activities, creatinine, urea, and proteins were determined
using kit methods (Spinreact).
2.7. Histopathological study of the liver and kidney
In order to conduct a histological study, parts of the
experimental rats' liver and kidney were dipped for 48 h into a
xative solution (solution 4% formaldehyde, in phosphate buffer,
pH = 7.6), dehydrated in ascending graded series of ethanol,
washed with toluene, embedded in parafn, and stained with
hematoxylin and eosin. The tissues were prepared, then observed
and examined under a light microscope.
2.8. Statistical analysis
Data are expressed as mean  SM standard error of the mean
(SEM). All the analyses were peformed using GraphPad Prism 6.0
for Windows (GraphPad Software, San Diego, CA). Statistical
signicance was determined by using one-way ANOVA and
subsequently performing a Tukey post hoc test. P < 0.05 was
considered statistically signicant.
 H. Bouzenna et al. / Biomedicine & Pharmacotherapy 83 (2016) 13271334 1329

Table 1
Ameliorative effect of citrus limon essential oil on aspirin-induced liver marker enzymes.

Parameters Control A EOC EOC + A

AST (U/L) 173.28  51.5 390.85  63.02*** 176.28  40.8+++ 190.14  11.72+++
ALT (U/L) 61.71  17.96 119.14  22.31* 65.42  4.15+++ 79.71  14.24+++
LDH(U/L) 848.42  291 1581.71  54,27** 831.85  193+++ 1088.28  139,38+++

The results are expressed as mean  S.E.M., n = 7 rats/group.

*
P  0.05 compared to control group (C).
**
P  0.01 compared to control group (C).
***
P  0.001 compared to control group (C).
+++
P  0.01 compared to (A)-treated group.

3. Results animals treated with essential oil of Citrus limon (EOC + A) (Figs. 1
and 2).
3.1. Hepatic biochemical variables
3.5. Histological examination
Tables 1 and 2 show that serum AST, ALT and LDH, cholesterol,
glucose activities in rats treated with aspirin (A) were signicantly 3.5.1. Histology of hepatic tissue
higher (390.85  63.02 U/L, 119.14  22.31 U/L and The histological prole of the liver sections in control (C) and
1581.71  54.27 U/L, 2.50  0.15 mmol/L, 11.66  0.57 mmol/L, re- (EOC) groups showed normal lobular architecture with central vein
spectively) than those in control group (C). The administration of and radiating cords of hepatocytes. The hepatocytes were
EOC in (EOC + A) group induced a signicant decrease in the levels polyhedral in shape and their cytoplasm was granulated. In
of the above mentioned markers (190.14  11.72 U/L, contrast, the histological outline of the liver section in the aspirin-
79.71 14.24 U/L and 1088.28  139.38 U/L, 1.46  0.32 mmol/L, treated group showed degenerations of hypatocytes, dilation of
7.6  0.48 mmol/L, respectively) when compared to the aspirin- sinusoids, increased leukocyte inltration, and congestion of the
treated group (A). However, there were no signicant changes in central portal vein compared with the control group (C). The
these parameters levels in the pretreated groups (EOC) compared (EOC + A) group preserved normal morphology, and showed
to control. normal architecture, of the liver (Fig. 3).

3.2. Renal biochemical variables 3.5.2. Histology of kidney tissue

Table 3 shows that the serum creatinine, protein and urea
activities were increased signicantly in the aspirin-treated rats
(70.75  13.25 mmol/L, 88.57  6.02 g/L and 14.31  2.5 mmol/L
respectively) compared to control (C). All these biomarkers were
signicantly decreased by administration of essential oil of Citrus
limon in (EOC + A) group (50.58  9.78 mmol/L, 55.42  8.1 g/L and
8.78  2.2 mmol/L) compared with group (A).
3.3. Lipid peroxidation levels in liver and kidney
Figs. 1 and 2 show that TBARS level, as an index of lipid
peroxidation, in both liver and kidney was increased in the (A)-
treated rats (by 108% and 55.7%, respectively) compared to the
control. A signicant decrease of TBARS level in the (EOC + A) group
occurred.
3.4. Antioxidant enzymes in liver and kidney
Activities of SOD, CAT, and GPx were found to be greatly reduced
in liver and kidney of the (A)-treated rats (by 78.77% and
53.84%, respectively) for SOD; ( 53.58% and 78.01%, respec-
tively) for CAT and ( 78.92% and 51.51%, respectively) for GPx
compared to control (C). These changes were corrected in the
Table 2
Protective effect of EOC on lipid prole and glucose level of aspirin-induced rats.
Parametrs Control A EOC
Cholesterol 1.50  0.39 2.05  0.48* 1.56  0.35+
(mmol/L)
Glucose 7.16  0.61 11.66  0.57*** 7.01  0.57+++
(mmol/L)
The results are expressed as mean  S.E.M., n = 7 rats/group.
*
P  0.05 compared to control group (C).
***
P  0.001 compared to control group (C).
+
P  0.05 compared to (A)-treated group.
+++
P  0.01 compared to (A)-treated group.
The histological outline of the kidney section of the (A)-treated
group proved signicant degenerative changes compared with the
control rats. The kidney section in the control and EOC-treated rat
kidneys revealed normal glomeruli and tubulointerstitial cells.
However, the kidneys of the (A)-treated animals showed histo-
patholgical damage; this group showed a signicant varying
glomerular and tubular degenerations (i.e. atrophy of the
glomerular and dilatation of urinary space). The (EOC + A) group
showed repaired architecture of the kidney (Fig. 4).
4. Discussion
The present study was conducted to document the ameliorative
effect of Citrus limon essential oil of against aspirin-induced
nephrotoxicity and hepatotoxcity. We believe that nonsteroid anti-
inammatory drugs are the most frequently prescribed therapeu-
tic agents, used for the treatment of rheumatic diseases, because
they perform analgesic, antipyretic, and anti-inammatory actions
[18].
Aspirin is one of nonsteroid anti-inammatory drugs also used
as an anticlotting agent that reduces the risk of clotting diseases
[19]. However, when it is administrated at high dose, it has life-
threating side effects.
Actually, our study demonstrated that the administration of
aspirin at 600 mg/kg dose (group A) to rats caused a signicant
damage in liver and kidney functions. The levels of serum glucose,
cholesterol, AST, ALT, LDH, creatinine, protein, and urea that were
signicantly increased compared to their corresponding values in
EOC + A
the control group (C). These ndings are in agreement with those
1.46  0.32+
reached by Gopi et al. [20]. They found that oral administration of
7.6  0.48+++ acetaminophen, analgesic and antipyretic drug as aspirin at a high
dose for 13 days in rats resulted in signicant elevation of total
cholesterol, blood urea nitrogen, serum creatinine, and aspartate
transaminase activity. Such high dose (500 mg/kg) of aspirin has
been reported to cause also damage in other organs;, it induced
gastric and duodenal ulcer in rats [21]. Then, compared with other
1330 H. Bouzenna et al. / Biomedicine & Pharmacotherapy 83 (2016) 13271334

Table 3
Effects of aspirin and Citrus limon essential oil on creatinine, protein, and urea levels in blood.

Parameters Control A EOC EOC + A

Creatinine (mmol/L) 47.57  3.06 70.75  13.25** 46,85  8.06++ 50.58  9.78++
Protein (g/L) 50.42  7.3 88,57  6.02*** 52.85  5.36+++ 55,42  8.10+++
urea (mmol/L) 8.12  2.7 14.31  2.5** 7.72  0.91+++ 8.78  2.20++

The results are expressed as mean  S.E.M., n = 7 rats/group.

**
P  0.01 compared to control group (C).
***
P  0.001 compared to control group (C).
++
P  0.01 compared to (A)-treated group.
+++
P  0.01 compared to (A)-treated group.

Fig. 1. Lipid peroxidation levels (TBARS) and activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) in the livers of the different groups. C:
control (normal) rats; (A): rats treated with aspirin, (EOC): Rats treated with essential oil of Citrus limon, (EOC + A): rats pretreated with EOC then treated with (A). Values
correspond to the mean of 3 measurements  SD. *P  0.05 compared to control group (C), **p  0.01 compared to control group (C); ++p  0.01, compared to group (A),
+P  0.05 compared to group (A). %: The percentage % of increase or decrease of TBARS o the antioxidant activity parameters (SOD, CAT, GPx) comp ared to control or aspirin-
treated group.

studies using the method of Merchant and Modi [3] found that
with the acute dose 600 mg/kg, aspirin treatment had damaging
effects; it reduced the circulating red cell counts, hemoglobin and
packed cell volume, and increased the mean cell hemoglobin
(MCH) as well as the mean cell volume (MCV).
This elevation could potentially be attributed to the release of
these enzymes from the cytoplasm into the blood circulation after
rupture of the plasma membrane and cellular damage. Serum AST,
ALT, and LDH are the most sensitive markers employed in the
diagnosis of hepatic damage because they are released into the
blood circulation after cellular damage [22]. Also, our results are
similar to those reported in the study of Abatan et al. [23] which
proved that nonsteroidal anti-inammatory drugs (NSAID) such as
aspirin, ibuprofen, and naproxen may alter the function of the
liver's, causing elevation of serum aspartate as well as alanine
aminotransferases and necrosis of hepatic cells.
In addition, the kidneys play an important role in the
elimination of toxic exenobiotics; they are more likely to be
exposed to toxic materials compared to other organs. Therefore,
our results showed that aspirin caused signicant elevations in the
serum levels of urea, protein, and creatinine when compared to the
normal rats (C), indicating that these drugs may adversely affect
renal function. This result is in agreement with the work of Aprioku
and Uche [24]. These effects can be explained by the renal
dysfunction associated with glomerular and tubular degenerations
caused by aspirin poisoning or salicylism. In fact, salicylates are
primarily excreted by the kidneys. Therefore, renal insufciency or
renal failure contributes to further accumulation of salicylate.
 H. Bouzenna et al. / Biomedicine & Pharmacotherapy 83 (2016) 13271334 1331

Fig. 2. Lipid peroxidation levels (TBARS) and activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) in the kidneys of the different groups.
C: control (normal) rats; (A): rats treated with aspirin, (EOC): Rats treated with essential oil of Citrus limon, (EOC + A): rats pretreated with EOC then treated with (A). Values
correspond to the mean of 3 measurements  SD. *P  0.05 compared to control group (C), **p  0.01 compared to control group (C); ++p  0.01, compared to group (A),
+P  0.05 compared to group (A). %: The percentage % of increase or decrease of TBARS ou the antioxidant activity parameters (SOD, CAT, GPx) compared to control or aspirin-
treated group.

Salicylates also uncouple oxidative phosphorylation at a cellular
level, producing an increased metabolic rate. This results in
increasing oxygen consumption, glucose utilization, and heat
production. Salicylates inhibit the Krebs cycle and alter lipid and
amino acid metabolisms, producing lactic acid and ketones
resulting from metabolic acidosis. They also interfere with
hemostasis by damaging hepatocytes and interfering with
prostaglandin synthesis. These effects cause hyperpnea, tachyp-
nea, tachycardia, fever, hemorrhage, and hypoglycemia, all of
which can subsequently develop into altered mental status and
seizures.
On the other hand, our study showed that the administration of
aspirin induced signicant increase of lipid peroxidation (TBARS)
in the liver and the kidney by about 108% and 55.17%, respectively,
compared to the control. The increase of TBARS was conrmed by
the peroxidative effect of aspirin. Our results conrmed recent
ndings [25] showing that aspirin induced an oxidative stress in
liver and kidney of rats by decreasing the activities of antioxidative
enzymes (SOD, CAT, GPx) when compared with normal control
rats. Bhattacharyya et al. [26] showed that aspirin induced an
oxidative stress in mice. The decrease in the level of SOD (the
enzyme responsible for the dismutation of superoxide to hydrogen
peroxide) led to excess superoxide radicals and organic peroxides
by generation of highly reactive entities, which results in an attack
on DNA, membrane lipids and other essential cell compounds [27].
Then, the decrease in CAT activity, i.e. the enzyme involved in the
decomposition of H2O2 to water and oxygen, leads to a reduction in
glutathione content as well as exacerbation of free radical
production [28].
The decrease in the levels of enzymatic antioxidants (SOD, CAT,
GPx) was observed in aspirin-treated rats and it could be due to
reduction in their biosynthesis or their excessive utilization in
trapping the free radicals generated.
Besides, the biochemical ndings are conrmed by histopatho-
logical observation of the liver and kidney. Based on our ndings,
the control group showed regular morphology in the liver and
renal tissue. However, the rats exposed to aspirin showed severe
alterations in both liver and kidney compared to the control.
Secondly, it has been reported that the natural antioxidants that
are present in herbs are responsible for preventing the harmful
consequence of oxidative stress. In this context, in the present
study, administration of essential oil of Citrus limon was found to
signicantly protect the rats from aspirin-induced hepaotoxicity
and nephrotoxicity. This ameliorating effect of Citrus limon
essential oil was shown by the decrease of levels of glucose,
cholesterol AST, ALT, LDH, creatinine, urea, and protein when
compared to the A-treated group. The decrease of the activities of
liver enzymes in blood was due to its ability to reduce free radical-
induced oxidative damage in the liver. These ndings are in
agreement with those reported by Jaiswal et al. [29] who showed
that administration of Citrus limon fruit extract ameliorated the
toxicity of carbofuran by restoring the levels of glucose,
cholesterol, AST, ALT, LDH to almost normal levels. The studies
conducted by Trovato et al. [30] demonstrated that the treatment
 1332 H. Bouzenna et al. / Biomedicine & Pharmacotherapy 83 (2016) 13271334

Fig. 3. Microscopic observations of rat liver sections (H & E, C, EOC, A and EOC + A: 400). (C) normal control groups showing normal parenchymal architecture, (EOP) Citrus
limon-treated group showing normal strutcture similar to control. (A) Aspirin-treated group showing diffuse central and peripheral necrosis and destruction of the lobular
architecture and (EOC + A) showing repairing of liver structure. : degenerations of hypatocytes, : dilation of sinusoids, : increased leukocyte inltration
and congestion of the central portal vein.

Fig. 4. Photomicrographs of rat kidney (H & E, C, EOC, A and EOC + A: 400): (C) normal control groups showing normal parenchymal architecture; (EOC) citrus limon-treated
group showing normal structure similar to control. (A) Aspirin-treated group showing degenerative changes, (EOC + A) showing repairing of architecture of the kidney.
: tubular degenerations, : Atrophy of the glomerular and dilatation of urinary space.
 H. Bouzenna et al. / Biomedicine & Pharmacotherapy 83 (2016) 13271334
of Citrus limon juice in rats reduced cholesterol, LDL, and
triglyceride levels by 49%, 50% and 51%, respectively.
In addition, the treatment of essential oil exerted a strong
protective effect on aspirin-induced oxidative stress, as revealed by
the decreased level of lipid peroxidation (TBARS), and enhanced the
enzymatic defense system (SOD, CAT and GPx). Our results are
conrmed by Campelo et al. [31] who showed that the essential oil of
Citrus limon exhibits an antioxidant action in preventing lip-
operoxidation. Also, these results are in agreement with results
found by Jaiswal et al. [29] who showed that the restoration of the
activity of the enzymes (SOD, CAT, GSH) to almost normal level was
due to the administration of Citrus limon extract in pesticide-treated
animals. They explained that the antioxidant present in the Citrus
limon fruit extract may help to trap the free radicals and offer
protection to enzymes.
Histopathological liver and kidney sections of the rats treated
with the Citrus limon essential oilshowed improved hepatocellular
and nephrocellar architecture with signs of recovery, indicating the
protective effect of the essential oil of Citrus limon.
This hepatoprotective and nephroprptevtive affects of Citrus
limon essential oil conrmed it had antioxidant as well as anti-
peroxidative property and potential role in defense against free
radicals. In accordance with these results, the protective effect of
essential oil of Citrus limon against aspirin may be attributed to the
presence of constituents of essential oil.
In fact, the chemical composition of essential oil of Citrus
limon is varied in several ndings such as those of Al-Jabri and
Hossain [32] who found that the main compounds in the oil
isolated by hydrosistilation from fruits and identied based on
gas chromatography (GC) retention time in Turkey were DL-
limonene (78.92%), a-pinene (5.08%), L-a-terpineol (4.61%),
b-myrcene (1.75%), b-pinene (1.47%) and b-linalool (0.95%).
However, in India, the predominant bioactive ingredients were
DL-limonene (53.57%), L-a-terpineol (15.15%), b-pinene (7.44%),
a-terpinolene (4.33%), terpinen-4-ol (3.55%), cymene (2.88%), and
E-citral (2.38%). These compounds could interact with the
reactive oxygen species (ROS) produced by aspirin which
induced aggressive oxidants.
Therefore, our results showed that the essential of Citrus limon
exhibited an excellent protective effect and may be considered as a
useful source of cellular defense agent in liver and kidney tissues
against aspirin.
5. Conclusion
In conclusion, aspirin induced damage in histo-morphological
changes in liver and kidney by increasing serum glucose,
cholesterol, AST, ALT, and LDH, as well aselevation in serum
creatine, protein and urea activities. But, the essential oil of Citrus
limon offered signicant protection against the toxicity of aspirin
by restoring these parameters to normal, which may be attributed
to its compositions and their antioxidant activities.
Conict of interest
The authors declare that there is no conict of interest.
Acknowledgements
This research was funded by the Tunisian Ministry of Higher
Education and Scientic Research, Tunis, Tunisia, through the
Integrated Physiology Laboratory, Faculty of Sciences of Gafsa,
Tunisia, and Laboratory of Environmental Physiopathology, Valo-
rization of Bioactive Molecules and Mathematical Modeling,
Faculty of Sciences Sfax, Tunisia. The authors would like to thank
Mr Zied Tlili, a teacher of English for Specic Purposes at the Higher
1333
Institute of Business Administration of Gafsa, Tunisia, for high-
quality language editing which signicantly contributed to the
completion of this work.
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