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Toxicology and Industrial Health
http://tih.sagepub.com/content/early/2013/01/28/0748233713475506
The online version of this article can be found at:
DOI: 10.1177/0748233713475506
published online 30 January 2013Toxicol Ind Health
El-Benna and Mohamed Amri
Abdellaziz Souli, Hichem Sebai, Latifa Chehimi, Kaïs Rtibi, Haifa Tounsi, Samir Boubaker, Mohsen Sakly, Jamel
Hepatoprotective effect of carob against acute ethanol-induced oxidative stress in rat
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Article
Hepatoprotective effect of carob
against acute ethanol-induced
oxidative stress in rat
Abdelaziz Souli
1,2
, Hichem Sebai
1,3
, Latifa Chehimi
3
,
Kaı
¨s Rtibi
1,2
, Haifa Tounsi
4
, Samir Boubaker
4
,
Mohsen Sakly
3
, Jamel El-Benna
5
and Mohamed Amri
2
Abstract
The present study was undertaken to determine whether subacute treatment with aqueous extract of carob
(Ceratonia siliqua L.) pods (AECPs) protects against ethanol (EtOH)-induced oxidative stress in rat liver.
Animals were divided into four groups: control, carob, EtOH and EtOH þcarob. Wistar rats were intra-
peritoneally pretreated with AECP (600 mg/kg body weight (bw)) during 7 days and intoxicated for 6 h by acute
oral administration of EtOH (6 g/kg bw) 24 h after the last injection. We found that acute administration of
EtOH leads to hepatotoxicity as monitored by the increase in the levels of hepatic marker aspartate amino-
transferase and alanine aminotransferase as well as hepatic tissue injury. EtOH also increased the formation of
malondialdehyde in the liver, indicating an increase in lipid peroxidation and depletion of antioxidant enzyme
activities as superoxide dismutase, catalase and glutathione peroxidase. Subacute carob pretreatment pre-
vented all the alterations induced by EtOH and returned their levels to near normal. Importantly, we showed
that acute alcohol increased hepatic and plasmatic hydrogen peroxide and free iron levels. The carob pre-
treatment reversed EtOH effects to near control levels. These data suggest that carob could have a beneficial
effect in inhibiting the oxidative damage induced by acute EtOH administration and that its mode of action may
involve an opposite effect on plasma and tissue-free iron accumulation. Indeed, carob can be offered as a food
additive to protect against EtOH-induced oxidative damage.
Keywords
Carob, EtOH, iron, liver, oxidative stress, rat
Introduction
Alcoholic liver disease (ALD) is characterized by mor-
phological changes ranging from hepatic steatosis,
inflammation and hepatic necrosis to progressive fibro-
sis (Younossi, 2008). Ethanol (EtOH) oxidation
generates toxic metabolites, free radicals and induces
a state of oxidative stress characterized by an enhance-
ment of lipid peroxidation and depletion of endogenous
antioxidant enzyme activities such as superoxide dis-
mutase (SOD), catalase (CAT) and glutathione perox-
idase, which contribute to the pathogenesis of ALD
(Samuhasaneeto et al., 2009). These free radicals are
capable of damaging many cellular components such
as DNA, protein and lipid (for review see Gate´etal.,
1999). However, several naturally occurring
antioxidant compounds were largely used to protect
1
Laboratoire de Nutrition et Physiologie Animale, Institut
Supe
´rieur de Biotechnologie de Be
´ja, Be
´ja, Tunisia
2
De
´partement des Sciences Biologiques, Laboratoire de
Neurophysiologie Fonctionnelle et Pathologies, Faculte
´des
Sciences de Tunis, Campus Universitaire El Manar II-2092 Tunis,
Tunisia
3
Laboratoire de Physiologie Inte
´gre
´e, Faculte
´des Sciences de
Bizerte, Jarzouna, Tunisia
4
Laboratoire d’anatomie pathologique humaine et expe
´rimentale,
Institut Pasteur de Tunis, Tunis, Tunisia
5
INSERM U773 Centre de Recherche Biome
´dicale, Faculte
´de
Me
´decine X. Bichat, Paris France
Corresponding author:
Hichem Sebai, De
´partement des Sciences de la Vie, Laboratoire
de Physiologie Inte
´gre
´e, Faculte
´des Sciences de Bizerte,
Zarzouna 7021, Tunisia
Email: sebaihichem@yahoo.fr
Toxicology and Industrial Health
1–9
©The Author(s) 2013
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DOI: 10.1177/0748233713475506
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at Scientific library of Moscow State University on January 5, 2014tih.sagepub.comDownloaded from
against liver diseases both in experimental and in clin-
ical situations.
The carob tree (Ceratonia siliqua L.) has been
widely cultivated in Mediterranean countries for years.
The carob fruit, brown pod 10–25 cm in length, con-
tains lots of polyphenols, especially highly condensed
tannins (Avallone et al., 1997; Makris and Kefalas,
2004). For this reason, carob extract has several bene-
ficial effects on health such us cholesterol-lowering
activities in humans suffering from hypercholesterole-
mia (Zunft et al., 2001, 2003) and antioxidant proper-
ties in different in vitro test systems (Haber, 2002;
Kumazawa et al., 2002). Recent studies discovered
that Tunisian leaf carob extract presents some ameli-
orative effects against CCl
4
-induced oxidative damage
in rats’ tissues (Hsouna et al., 2011).
Hence, the present study is aimed at investigating
the putative protective effect of aqueous extract of
carob pods (AECP) on hepatotoxicity induced by
acute EtOH treatment.
Materials and methods
Chemicals
2-Thio-barbituric acid (TBA) and butylated hydroxy-
toluene (BHT) were from Sigma chemicals Co.
(Sigma-Aldrich GmbH, Steinheim, Germany). All
other chemicals were of analytical grade.
Preparation of carob extract
Immature carob was cultivated from the region of
Tabarka (NW-Tunisia). Powder mixture containing
carob pulp (90%) and seeds (10%) was dissolved in
double distilled water and centrifuged 10 min at
10,000gto eliminate insoluble material. Supernatant
was aliquoted and stored at 80C until use.
Animals and treatment
Adult male Wistar rats (weighing 220–240 g, 15
weeks old and housed five per cage) were purchased
from Pasteur Institute of Tunis and used in accordance
with the local ethics committee of Tunis University
for use and care of animals in conformity with the
NIH recommendations. They were provided with
standard food (Almes, Tunisia) and water ad libitum
and maintained in animal house at controlled tem-
perature (22 +2C) with a 12-h light–dark cycle.
Rats were divided into 4 groups of 10 animals each:
control, carob, EtOH and EtOH þcarob. The animals
were intraperitoneally (i.p.) injected daily for 7 days
with vehicle (control, bidistilled water) or with
600 mg/kg body weight (bw) of AECP (injection vol-
ume was 1 mL/kg bw). Twenty-four hours after the
last carob injection, rats were intoxicated by a single
oral administration of EtOH (6 g/kg bw; Chen et al.,
2011) or control (NaCl 9%). After 6 h, the animals
were killed, the liver rapidly excised and homoge-
nized in in 50 mM potassium phosphate buffer (19.5
mM KH
2
PO
4
/30.5 mM K
2
HPO
4
, pH 7.4) for deter-
mining the biochemical parameters. After centrifuga-
tion at 10,000gfor 10 min at 4C, supernatant was
used for biochemical determination of protein, free
iron, hydrogen peroxide (H
2
O
2
), malondialdehyde
(MDA) and antioxidant enzyme activities. On the
other hand, blood was also collected in heparinized
tubes. After centrifugation at 3000gfor 15 min,
plasma was processed for free iron, H
2
O
2
and transa-
minases determinations.
Lipid peroxidation measurement
Liver lipid peroxidation was determined by MDA
measurement according to the double heating method
(Draper and Hadley, 1990). Briefly, aliquots from
liver tissue homogenates were mixed with BHT-
trichloracetic acid (TCA) solution containing 1%BHT
(w/v) dissolved in 20%TCA (w/v) and centrifuged at
1000gfor 5 min at 4C. Supernatant was blended with
0.5 N hydrochloric acid 120 mM TBA in 26 mM
Tris and then heated at 80C for 10 min. After cooling,
absorbance of the resulting chromophore was mea-
sured spectrophotometrically (DU 640B Spectrophoto-
meter, Beckman Coulter Inc., CA, USA) at 532. MDA
levels were determined using an extinction coefficient
for MDA-TBA complex of 1.56 10
5
M
1
cm
1
.
Antioxidant enzyme activity assays
SOD activity was determined using modified epi-
nephrine assay (Misra and Fridovich, 1972). At alka-
line pH, superoxide anion O
2
causes the
autoxidation of epinephrine to adenochrome; while
competing with this reaction, SOD decreased the ade-
nochrome formation. One unit of SOD is defined as the
amount of the extract that inhibits the rate of adeno-
chrome formation by 50%. Enzyme extract was added
in 2 ml reaction mixture containing 10 ml of bovine
CAT (0.4 U/ml), 20 ml epinephrine (5 mg/ml) and
62.5 mM sodium carbonate/bicarbonate buffer pH
10.2. Changes in absorbance were recorded at 480 nm.
CAT activity was assayed by measuring the initial
rate of H
2
O
2
disappearance at 240 nm (Aebi, 1984).
2Toxicology and Industrial Health
at Scientific library of Moscow State University on January 5, 2014tih.sagepub.comDownloaded from
The reaction mixture contained 33 mM H
2
O
2
in
50 mM phosphate buffer pH 7.0, and the CAT activity
was calculated using the extinction coefficient of 40
mM
1
cm
1
for H
2
O
2
.
GPx activity was measured by the procedure of
Flohe´ and Gu
¨nzler (1984). Briefly, 1 mL of reaction
mixture containing 0.2 mL of liver supernatant,
0.2 mL of phosphate buffer 0.1 M pH 7.4, 0.2 mL
of GSH (4 mM) and 0.4 mL of H
2
O
2
(5 mM) was
incubated at 37C for 1 min and the reaction stopped
by the addition of 0.5 mL TCA (5%, w/v). After cen-
trifugation at 1500gfor 5 min, aliquot (0.2 mL) from
supernatant was mixed with 0.5 mL of phosphate buf-
fer 0.1 M pH 7.4 and 0.5 mL 2,20-dithiobis-5,50-nitro-
benzoic acid (10 mM) and absorbance recorded at
412 nm. GPx activity was expressed as nanomoles
of GSH consumed per minute per milligram protein.
Assessment of liver function
Plasma aspartate aminotransferase (AST) and alanine
aminotransferase (ALT) were measured using com-
mercially available diagnostic kits supplied by Ran-
dox Laboratories (Ardmore, Northern Ireland, UK).
Histopathological analysis
Immediately after killing the rats, small pieces of liver
were harvested and washed with ice-cold saline. Tis-
sue fragments were then fixed in a 10%neutral-
buffered formalin solution, embedded in paraffin and
used for histopathological examination. Sections of
5mm thickness were cut, deparaffinized, hydrated and
stained with hematoxylin and eosin. The hepatic sec-
tions were examined in blind fashion in all treatments.
H
2
O
2
determination
The tissue H
2
O
2
level was performed according to
Dingeon et al. (1975). Briefly, in the presence of per-
oxidase, the H
2
O
2
reacts with p-hydroxybenzoic acid
and 4-aminoantipyrine, leading to a quantitative for-
mation of a pink-colored quinoneimine detected at
505 nm.
Iron measurement
Tissue and plasma nonheme iron were measured col-
orimetrically using ferrozine as described by Leardi
et al. (1998). Briefly, the iron dissociated from trans-
ferrin–iron complex by a solution of guanidine acetate
and reduced by ascorbic acid reacts with ferrozine to
give a pink complex measured at 562 nm.
Protein determination
Protein concentration was determined according to
Hartree (1972), which is a slight modification of the
Lowry method. Serum albumin was used as standard.
Statistical analysis
Data were analyzed by unpaired Student’s ttest or
one-way analysis of variance and were expressed as
means +SEM. Data are representative of 10 inde-
pendent experiments. All statistical tests were two
tailed, and a pvalue of 0.05 or less was considered
significant.
Results
Effect of carob on EtOH-induced liver injury
To test the in vivo effect of carob on EtOH-induced
liver injury, rats were treated with carob extract
(600 mg/kg bw) for 7 days then intoxicated by a single
dose of EtOH (6 g/kg bw). As shown in Figure 1,
plasma AST and ALT were increased in acute
EtOH-treated rats, while carob alone has no effect
in both parameters. Interestingly, when animals were
pretreated with carob before EtOH intoxication, the
activities of these enzymes were practically restored
and reached the normal values of control rats.
Effect of carob on EtOH-induced liver histology
damage
The histological study revealed that liver sections of
control rats showed hepatic lobules consisting of a
central vein surrounded by radiating hepatocytes
separated by irregular blood sinusoids (Figure 2(a)).
No histopathological changes could be observed in
the liver of rats treated with carob alone (Figure
2(b)). The liver of rats treated with EtOH alone
showed various histological changes as assessed by
microvacuolar steatosis and necrosis appearance (Fig-
ure 2(c)). Carob pretreatment greatly but partially
reduced the vacuolization process and hepatocellular
steatosis induced by EtOH treatment (Figure 2(d)).
Effect of carob on EtOH-induced liver
lipoperoxidation
We further looked at the effect of carob on EtOH-
induced liver lipoperoxidation. As expected, EtOH
administration (6 g/kg bw) significantly increased
the liver MDA level, and this effect was completely
Souli et al. 3
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reversed by carob (600 mg/kg bw) pretreatment
(Figure 3).
Effect of carob on EtOH-induced depletion of liver
antioxidant enzyme activities
We also studied the hepatic antioxidant enzyme activ-
ities (Figure 4). EtOH treatment significantly
decreased hepatic antioxidant enzyme activities as
SOD (a), CAT (b) and GPx (c). Carob per se signifi-
cantly ameliorated SOD activity but not CAT and
GPx activities. Pretreatment with carob significantly
reversed all EtOH-induced decrease in antioxidant
enzyme activities to near control.
Effect of carob on EtOH-induced liver and plasma
elevation of iron and H
2
O
2
Lipoperoxidation could be the result of an increase in
the production of oxidants such as H
2
O
2
and iron or
a decrease in antioxidant enzymes levels. We further
looked at the effect of carob on the levels of plasma and
liver H
2
O
2
and free iron levels. As expected, EtOH per
se significantly increased liver and plasma H
2
O
2
con-
tents (Figure 5). Pretreatment of animals with AECP
decreased the basal level of H
2
O
2
and dramatically
protected from EtOH-induced H
2
O
2
production. On
the other hand, acute EtOH treatment highly increased
the rate of free iron in liver and plasma (Figure 6).
Carob pretreatment abolished EtOH-induced increase
in liver and plasma free iron to near control levels.
Discussion
The present investigation revealed that acute adminis-
tration of EtOH (6 g/kg bw) for 6 h resulted in a clear
hepatotoxicity as evidenced by an increase in plasma
transaminases (ALT and AST), used as indexes of
liver injury, and tissue damages revealed by histologi-
cal observations. EtOH-induced liver toxicity was
also reflected by increased lipoperoxidation, H
2
O
2
and free iron levels as well as depletion of antioxidant
enzyme activities such as SOD, CAT and GPx.
Our data first showed that subacute (7 days) pre-
treatment with AECP (600 mg/kg bw) protected
against the increase in plasma transaminases (ALT and
AST) and attenuated EtOH-induced hepatic tissue
injury. The high level of ALT and AST in the plasma
compartment is a central indication of the degree of
damage to the liver caused by EtOH administration
(Bain, 2003; Chen, 2010; Valentine et al., 1990). We
suggest that carob extract prevents the activation of
Kupffer cells by decreasing the formation of inflamma-
tory and fibrogenic mediators. The same mechanism
was, in part, proposed by Zhong et al. (2003), explain-
ing the protective effect of polyphenols was extracted
from Camellia sinenesis against cholestasis-induced
liver fibrosis in rats.
Acute alcohol-induced oxidative stress was widely
documented in liver (Dey and Cederbaum, 2006;
Nencini et al., 2010; Zhao et al., 2010), kidney (Ibra-
him et al., 2012), heart (Kannan et al., 2004) and brain
(Zloch, 1994). EtOH administration provoked oxida-
tive imbalance through a number of pathways includ-
ing the generation of reactive oxygen species via
xanthine oxidase (Dinda et al., 1996) and an alteration
of defense mechanisms with decreased GPx activity
Figure 1. Subacute effect of AECP on acute EtOH-induced
changes in plasma AST (a) and ALT (b). Animals were pre-
treated for 7 days with AECP (600 mg/kg body weight (bw),
intraperitoneally) or vehicle (bidistilled H
2
O), challenged
with a single oral administration of EtOH (6 g/kg bw) or
NaCl 9%(control) for 6 h. Assays were carried out in tri-
plicate. *p< 0.05 compared to control group and
#
p< 0.05
compared to EtOH group. AECP: aqueous extract of carob
pod; EtOH: ethanol; AST: aspartate aminotransferase; ALT:
alanine aminotransferase.
4Toxicology and Industrial Health
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following selenium deficiency (Thuluvath and Triger,
1992).
Importantly, our data also showed that carob pre-
treatment abolished almost all parameters of EtOH-
induced liver dysfunction. Carob pretreatment pro-
tected against lipid peroxidation and depletion of anti-
oxidant enzyme activities. Previous studies have
shown the richness of polyphenols in carob fruit
(Papagiannopoulos et al., 2004) or leaf (Hsouna
et al., 2011). These molecules are the major source
of antioxidant ability of carob, by scavenging free
radicals (Kumazawa et al., 2002). EtOH-induced oxi-
dative stress and liver dysfunction have been shown to
be attenuated by resveratrol (Kasdallah-Grissa et al.,
2007), N-stearoylethanolamine (Hula et al., 2010),
curcumin (Nanji et al., 2003), folic acid (Lee et al.,
2011) and apocynin (Fan et al., 2012). Moreover, it
has been recently shown that leaf extract of C. siliqua
protects against CCl
4
-induced hepatic oxidative dam-
age in rats (Hsouna et al., 2011). However, to our
knowledge, our report is the first one to deal with the
protective effect of the fruit extract of C. siliqua
(carob) on acute EtOH-induced oxidative stress in rat
liver.
Our data also showed that subacute pretreatment
with AECP abolished acute EtOH-induced increase
in liver H
2
O
2
and free iron levels. Several studies
have demonstrated that either acute or chronic alcohol
determined an increase in nonheme iron in the liver
(Conde-Martel et al., 1992; Gonzalez-Reimers et al.,
Figure 2. Liver histology. Normal architecture in (a) control and (b) carob-treated animals. Various histological changes
as assessed by microvacuolar steatosis and necrosis appearance (c) and partially protected by carob pretreatment (d).
Figure 3. Subacute effect of AECP on acute EtOH-induced
changes in liver MDA level. Animals were pretreated for
7 days with AECP (600 mg/kg body weight (bw), intraper-
itoneally) or vehicle (bidistilled H
2
O), challenged with a sin-
gle oral administration of EtOH (6 g/kg bw) or NaCl 9%
(control) for 6 h. Assays were carried out in triplicate.
*p< 0.05 compared to control group and
#
p< 0.05 com-
pared to EtOH group. AECP: aqueous extract of carob
pods; EtOH: ethanol; MDA: malondialdehyde.
Souli et al. 5
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1996; Houze et al., 1991; Shahbazian et al., 1994).
Furthermore, both iron deficiency and iron excess can
lead to cellular dysfunction, and maintaining normal
iron homeostasis is therefore crucial (Andrews,
1999). Iron has long been implicated in the pathogen-
esis of chronic liver disease including ALD (Powell,
1975). A consensus has been that iron accumulated
in chronic liver inflammation and catalyzed hydroxyl
radical-mediated oxidative injury via its participation
in the Fenton pathway. In fact, administration of an
iron chelator ameliorated alcoholic liver injury in rats
(Sadrzadeh et al., 1994). We suggest that aqueous
extract of carob is capable of chelating free iron and
scavenging H
2
O
2
. The same mechanism was pro-
posed for other extracts rich in phenolic compounds
as grape seeds and skin extracts (Charradi et al.,
2011; Hamlaoui-Gasmi et al., 2011).
Figure 4. Subacute effect of AECP on acute EtOH -induced
changes in liver antioxidant enzymes SOD (a), CAT (b) and
GPx (c). Animals were pretreated for 7 days with AECP
(600 mg/kg body weight (bw), intraperitoneally) or vehicle
(bidistilled H
2
O), challenged with a single oral administration
of EtOH (6 g/kg bw) or NaCl 9%(control) for 6 h. Assays
were carried out in triplicate. *p< 0.05 compared to control
group and
#
p< 0.05 compared to EtOH group. AECP: aqu-
eous extract of carob pods; EtOH: ethanol; SOD: superox-
ide dismutase; CAT: catalase; GPx: glutathione peroxidase.
Figure 5. Subacute effect of AECP on acute EtOH-induced
changes in liver (a) and plasma (b) free iron. Animals were
pretreated for 7 days with AECP (600 mg/kg body weight
(bw), intraperitoneally) or vehicle (bidistilled H
2
O), chal-
lenged with a single oral administration of EtOH (6 g/kg
bw) or NaCl 9%(control) for 6 h. Assays were carried out
in triplicate. *p< 0.05 compared to control group and
#
p< 0.05 compared to EtOH group. AECP: aqueous
extract of carob pods; EtOH: ethanol.
6Toxicology and Industrial Health
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Hepcidin, an iron-shuttling protein, exerted a pivo-
tal role in the pathogenesis of iron overload (Papani-
kolaou et al., 2005). Alcohol downregulated hepcidin
expression in the liver, leading to an increase in duo-
denal iron transporter expression (Harrison-Findik
et al., 2006). It is tempting to speculate that EtOH and
AECP exerted opposite effects on hepcidin. However,
further work is needed to assess the effect of aqueous
extract of carob on iron-shuttling proteins as hepcidin
or lipocalin 2.
In conclusion, our findings clearly demonstrate that
administration of acute EtOH at 6 g/kg to rats induced
oxidative stress in the liver. Therefore, the observed
hepatoprotective and antioxidant activity of AECP
could be attributed to the presence of polyphenols that
can function as an effective free-radical scavenger
and free iron chelator.
Funding
Financial support of the Tunisian Ministry of Higher Edu-
cation and Scientific Research is gratefully acknowledged.
Authors’ Note
AS and HS contributed equally to this work.
Conflict of interest
The authors alone are responsible for the content of this
article.
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