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Pomegranate juice attenuates neurotoxicity and histopathological changes of the nervous system induced by aluminum in mice


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Aluminum (Al) is an element with ubiquitous presence on the earth crust that may cause neuropathological, neurobehavioral, neurophysical, and neurochemical changes linked to its bioavailability. The purpose of the present study was to determine the neuroprotective potential of pomegranate juice on Al induced neurotoxicity. Three groups of 7 female albino Swiss mice were used: the control group received only drinking water; the positive control group was exposed daily to 500 mg/kg of AlCl3 orally; and the third treated group received pomegranate juice (v/v in water) supplied in dark bottles for 4 h/day followed by AlCl3 at a dose of 500 mg/kg orally for 20 h/day for 90 days. After 90 days, the mice were subjected to behavioral and memory tests. Cortex cerebral and hippocampus injuries were determined with hematoxylin and eosin staining and Al accumulation was measured by graphite furnace atomic absorption with Zeeman correction. The Al deposition in the brain caused neural degeneration and decreased cell density inducing a state of anxiety, depression, and a deficit of learning and memory. Pomegranate juice treatment attenuated neurobehavioral alterations, decreased Al in the brain and restored the histological structure. High-performance liquid chromatography with a diode-array detector (HPLC-DAD) revealed a range of bioactive molecules (i.e., gallic acid, quercetine, luteolin) in the pomegranate juice that may have neuroprotective value for the nervous disorders caused by Al intoxication.
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Pomegranate juice attenuates neurotoxicity and histopathological
changes of the nervous system induced by aluminum in mice
Le jus de grenade atténue la neurotoxicité et les altérations histologiques du système nerveux
induites par laluminium chez la souris
L. Gadouche · N. Djebli · K. Zerrouki
© Lavoisier SAS 2018
Abstract Aluminum (Al) is an element with ubiquitous
presence on the earth crust that may cause neuropathologi-
cal, neurobehavioral, neurophysical, and neurochemical
changes linked to its bioavailability. The purpose of the pres-
ent study was to determine the neuroprotective potential of
pomegranate juice on Al induced neurotoxicity. Three
groups of 7 female albino Swiss mice were used: the control
group received only drinking water; the positive control
group was exposed daily to 500 mg/kg of AlCl
orally; and
the third treated group received pomegranate juice (v/v in
water) supplied in dark bottles for 4 h/day followed by
at a dose of 500 mg/kg orally for 20 h/day for
90 days. After 90 days, the mice were subjected to behav-
ioral and memory tests. Cortex cerebral and hippocampus
injuries were determined with hematoxylin and eosin stain-
ing and Al accumulation was measured by graphite furnace
atomic absorption with Zeeman correction. The Al deposi-
tion in the brain caused neural degeneration and decreased
cell density inducing a state of anxiety, depression, and a
deficit of learning and memory. Pomegranate juice treatment
attenuated neurobehavioral alterations, decreased Al in
the brain and restored the histological structure. High-
performance liquid chromatography with a diode-array
detector (HPLC-DAD) revealed a range of bioactive mole-
cules (i.e., gallic acid, quercetine, luteolin) in the pomegran-
ate juice that may have neuroprotective value for the nervous
disorders caused by Al intoxication.
Keywords Pomegranate juice · HPLC-DAD · Aluminum ·
Neurotoxicity · Histopathology
Résumé Laluminum (Al) est un élément omniprésent sur la
croûte terrestre et qui est à lorigine des changements neuro-
pathologiques, neurocomportementaux, neurophysiques et
neurochimiques liés à sa biodisponibilité. Le but de la pré-
sente étude était de déterminer le potentiel neuroprotecteur
du jus de grenade contre la neurotoxicité induite par Al.
Trois groupes de sept souris albinos Swiss femelles ont été
utilisés ; le groupe témoin a reçu uniquement de leau pota-
ble, le groupe contrôle positif a été exposé quotidiennement
à 500 mg/kg dAlCl
par voie orale et le troisième groupe a
reçu le jus de grenade (v/v dans leau) fourni dans des bou-
teilles sombres pendant quatre heures suivi par lAlCl
dose de 500 mg/kg par voie orale pendant 20 heures/jour
pendant 90 jours. Après 90 jours, les souris ont été soumises
à des tests de comportement et de mémoire. Les lésions du
cortex cérébral et de lhippocampe ont été déterminées par
coloration à lhématoxyline et à léosine, et laccumulation
dAl a été mesurée par absorption atomique au four graphite
avec correction de Zeeman. Le dépôt dAl dans le cerveau a
provoqué une dégénérescence neuronale et une diminution
de la densité cellulaire avec linstallation dun état danxiété,
de dépression et dun déficit dapprentissage, et de mémoire.
Le traitement au jus de grenade a atténué les altérations neu-
rocomportementales, a diminué la quantité dAl dans le cer-
veau et a rétabli la structure histologique. HPLC-DAD a
révélé une gamme de molécules bioactives (à savoir lacide
gallique, la quercétine, la lutéoline) dans le jus de grenade
qui peut avoir un effet neuroprotecteur contre les troubles
nerveux provoqués par une intoxication chronique à lAl.
Mots clés Jus de grenade · HPLC-DAD · Aluminium ·
Neurotoxicité · Histopathologie
Considered as the third most abundant element in nature,
aluminum (Al) is present in airborne particulates [1] and
L. Gadouche (*) · N. Djebli · K. Zerrouki
Pharmacognosy Api-Phytotherapy Laboratory (LPAP),
Department of Biology, Faculty of Natural Sciences and Life,
University of Abdelhamid-Ibn-Badis, Mostaganem, Algeria
e-mail :
DOI 10.3166/phyto-2018-0016
may become bioavailable to humans from a variety of
sources [2].
Al is present in the human brain and may accumulate
there with age [3]. Exposure to bioavailable Al may cause
brain injuries [4].
Furthermore, Taïr et al. [5] reported that Al caused signif-
icant short-term and long-term memory disturbances, a
decrease in locomotor activity, a significant inhibition of
acetylcholinesterase activity in the brain, and a significant
depletion of antioxidant enzymes and of glutathione in rats.
Brain samples showed gross histopathological changes,
including neurodegeneration and vacuolated cytoplasm after
rats were treated with aluminum chloride [6]. Al acetate admin-
istration was found to produce a loss of morphology of cellular
structure, degenerated axons, degenerated Purkinje cells,
degenerated granular layer cells, vacuolation, and chromatin
condensation within the nucleus in the cerebellum of mice [7].
Diverse health benefits, including improved cardiovascu-
lar and brain function have been linked to the consumption
of dietary polyphenols derived from fruit and vegetables [8].
The neuroprotective effect of polyphenols resides in their
ability to cross the bloodbrain barrier and neutralize free
radicals and chelate transition metal ions [9]. Youdim et al.
[10] have demonstrated that flavonoids and some metabo-
lites are able to traverse the bloodbrain barrier, and that
the potential for permeation is consistent with compound
Furthermore, polyphenols possess properties that can bat-
tle oxidative stress and stimulate the activation of molecules
that aid in synaptic plasticity, thus affecting a broad range of
mechanisms in the brain that can assist in the maintenance of
cognitive and mental health, as well as recovery from neuro-
degenerative diseases [11].
Pomegranate juice is a functional food and nutraceutical
with strong biological properties which are related to the
presence of polyphenols such as ellagic acid, ellagitannins
(including punicalagins), punicic acid and other fatty acids,
flavonoids, anthocyanidins, anthocyanins, estrogenic flavo-
nols, and flavones [12].
The present study was designed to evaluate the potential
neuroprotective capacity of pomegranate juice against AlCl
induced neurotoxicity in terms of the development of anxi-
ety disorders, depression, and memory deficit in mice and
the deterioration of cells in their hippocampus and cortex
Materials and methods
Juice preparation
15 kg of Punica granatum fruits (Sefri Algerian variety)
were collected from local farms of the western region of
Chlef (36°0604N, 1°1158E, 90 m, Algeria). This species
has been identified and confirmed at the High National
School of Agronomic Sciences. The edible part of the pome-
granate (arils) was pressed using a food processor after
removing the seeds. The juice (43% yield) obtained was cen-
trifuged at 10,000 rpm for 15 min at 4 °C. The supernatants
were collected immediately, diluted with distilled water (v/v)
[13], and stored at 40 °C.
HPLC-DAD Analysis
The juice was analyzed using high performance liquid chro-
matography (HPLC). The system used is a 1100 series
HPLC apparatus with a quaternary pump, on-line degasser
and automatic injector and diode array detector (DAD).
The stationary phase used is a column (Hypersil BDS-
C18, 5 μm, 250 × 4.6 mm) at a temperature of 30 °C. The
mobile phase consists of two solvents: the first is water acid-
ified with acetic acid (0.2%) at pH = 3.1 (solvent A) and the
second is acetonitrile (solvent B). The two solvents are used
in a linear elution gradient for 30 minutes at 1 mL/min, start-
ing with 95% of solvent A and ending with 100% of solvent
B. The volume of the sample injected is 20 μL and detector
wavelength is fixed at 220 nm, 254 nm, 280 nm, 300 nm,
and 365 nm chosen according to the maximum absorbances
of the identified molecules.
The various constituents of the extract to be analyzed are
identified by comparing the retention times of the com-
pounds to be identified with standards that have been ana-
lyzed under the same operating conditions. The retention
time and maximum wavelengths of standards used are: gal-
lic acid (3.260 min), tannic acid (3.370 min), caffeic acid
(6.931 min), isovanillic acid (7.450 min), vanillin
(8.862 min), rutin (8.923 min), ferulic acid (9.266 min),
syringic acid (9.660 min), o-anisic acid (9.668 min),
3-hydroxy-4-methoxycinnamic acid (9.767 min), prunin
(10.383 min), m-anisic acid (11.865 min), luteolin
(12.713 min), quercetin (12.816 min), trans-cinnamic acid
(13.821 min), apigenin (14.497 min), isorhamnetin
(15.305 min), hesperidin (15.379 min).
Experimental animals
Twenty-one female albino mice of the Swiss strain (age =
four weeks, weight = 18.5 ± 1.98 g) were obtained from
Pasteur Institute of Algiers. The animals were kept under
standard conditions maintained at 25 ± 2 °C, 12 h light/
dark cycle, and given standard pellet diet and water provided
ad libitum. All the animals were housed in polypropylene
cages using paddy husk bedding. Principles of animal han-
dling were strictly adhered to and the handling of the animals
was made under the supervision of the animal ethics com-
mittee of the institute.
2 Phytothérapie
Experimental design
Animals were divided into three groups each containing
7 mice.
Group 1: Control (C) received drinking water for 90 days.
Group 2: Intoxicated (Al)administration of AlCl
500 mg/kg body weight (BW) orally in the drinking water
for 12 weeks.
Group 3: Treated with pomegranate juice (AlJ) diluted
with distilled water (v:v) supplied in dark water bottles
(renewed every 1 h) for 4 h/day followed by aluminum chlo-
ride at a dose of 500 mg/kg BW orally for 20 h/day for
90 days [14]. Weight gain and water intake were measured
daily and weekly for 12 weeks.
Behavioral and memory tests
Behavioral tests were carried out to determine motor, anxi-
ety, and depressive behavior. The open field and elevated
plus maze (EPM) were tests that was done in 20 min divided
into four phases of 5 min while the forced swimming test
was done in one phase of 5 min. Tests of memories were
done in 5 days where each phase lasted 5 min. 5 mice from
each experimental group were used for these tests at the rate
of one test per day and each mouse was tested individually.
Locomotor activity
The locomotor activity test was performed to measure the
motor functions of mice that tend to explore an enclosed
space. The apparatus (a cage) contains a platform divided
into 16 equal squares. Each mouse was placed individually
in the center of this platform and allowed to move freely
during 5 min of exploration [15]. The number of squares
visited was recorded for each mouse during a time of 5 min.
Forced swimming test
The forced swimming test is frequently used to examine
depressive behavior [16]. It consists of keeping the mouse
in a warm bath of 21 °C and a height of 16 cm so that the
mouse does not touch the bottom and does not use its lower
limbs to stand on the surface. The mouse will first struggle to
escape and then becomes immobile, just keeping its nose
above the water level, when it loses hope of escaping. The
mice were observed for 5 min and the immobility time was
Anxiety test: the elevated plus maze (EPM)test
The elevated plus maze (EPM), is an apparatus composed of
two armsa protected dark and enclosed environment and
an unprotected brightly lit, open and elevated environment
linked by a central platform allowing free access to both
arms. The test is based on the natural anxiety related behav-
ior of rodents to remain in shadow, close to walls, and to
avoid heights [17].
Mice were placed individually in the center of the maze
which it is elevated 50 cm above the floor level, facing one
of the open arms. The time spent in the open arms was
recorded for 5 min. Increased activity in the open arms was
indicative of less anxiety [18].
Morris water maze test
The Morris water maze is the most common test used to
evaluate cognitive functions related to memory and learning.
In test, the animal learns to swim in a water tank, and guided
by external cues finds (and climbs up to) a submerged plat-
form [19]. The test consists of two parts.
Spatial working memory visible platform: mice are
placed in a pool of water, where they must swim to a visible
Spatial memory reference hidden platform: Animals are
placed in a pool of water that is colored opaque, where they
must swim to a hidden escape platform. Time elapsed to
reach the platform is recorded. Duration of the experiment
was 5 min during 5 days for each mouse with one session per
Brain Al level
Al concentrations brains of each experimental group after
digestion in 5:1 nitric acid: perchloric acid, as described by
Gupta and Gill [20], were determined by graphite furnace
atomic absorption with Zeeman correction (Agilent 240
ZAA/GTA 120; based on the atomization. The operating
parameters were: wavelength: 309.3 nm; slit: 0.5 nm; lamp
current: 10.0; standard solution: 1,000 mg/L. The sample is
placed inside a graphite tube that is then resistively heated to
accomplish sample desolvation (for liquid samples), ashing
or charring (to decompose the sample and volatilize some of
the matrix), and finally atomization [21].
Statistical analysis
All data were expressed as the mean ± SE (standard error).
The statistical significance of differences between groups
was assessed with an analysis of variance followed by the
student t-test (AL vs. control and AL-J vs. control). Pvalues
less than 0.05 were considered as statistically significant.
Histological study
The cortex cerebral and hippocampus were dissected and
fixed in 10% formalin for 24 h. The specimens were
Phytothérapie 3
dehydrated in ascending grades of ethanol, cleared in xylene,
and embedded in paraffin wax. They were then sectioned at
5μm, and stained with hematoxylin and eosin for histopath-
ological examination [22].
The HPLC chromatogram of the pomegranate juice, in which
more than 53 distinct peaks were observed, was done;
18 peaks were identified using the standards (Fig. 1A). The
chemical formulas of identified compounds are listed in figure
1B. Our HPLC analysis allowed us to identify ten phenolic
acids (gallic acid, caffeic acid, vanillin, isovanillic acid, feru-
lic acid, syringic acid, o-anisic acid, 3-hydroxy-4-methoxy-
cinnamic acid), seven flavonoids (rutin, prunin, luteolin, quer-
cetin, apigenin, isorhamnetin, hesperidin, m-anisic acid, trans-
cinnamic acid) and a tannin (tannic acid).
The present study showed that the daily intake of alumi-
num chloride for 3 months induced neurological disorder,
cognitive function deficits, and neural loss. Table 1 shows
that positive control mice have a slow weight evolution
(P> 0.05) and significantly low water intake than the control
group (P< 0.05). Treated group had a weight gain similar to
the control group with moderate water intake (P>0.05).
As showed in figure 2A, chronic empoisoning with alu-
minum (500 mg/kg) increased significantly the number of
squares traveled by mice in all sessions compared with con-
trol group.
In the forced swimming test, the immobility time was
lower in the control mice and significantly higher in mice
poisoned by aluminum chloride. Fruit juice improves immo-
bility time in treated intoxicated groups (P< 0.05; Fig. 2B).
In the present study, the anxiolytic activity was examined
using EPM in mice. This test showed that mice exposed to
Fig. 1A HPLC-UV/DAD chromatogram of pomegranate juice recorded at 300 nm
Fig. 1B Chemical formulas of identified compounds in pomegran-
ate juice
4 Phytothérapie
aluminum spend less time in the open arm indicating
that these mice are anxious. Our data also show that the
level of anxiety was significantly reduced in poisoned
mice receiving pomegranate juice dietary supplementation
(P< 0.05, Fig. 2C).
The Morris water maze test was used to assess the cogni-
tive ability of mice. Each day, the time taken to reach the
platform was recorded to evaluate learning performance.
The mice had to learn the position of the hidden platform.
Al intoxicated animals showed a significant increase
(P< 0.05) in time taken to find the platform from the first
day until 5 days compared with the control and the treated
group. Learning poisoned mice that received fruit juice dur-
ing the 5 days spend less time to find the platform
(Fig. 2D1). The present study shows that chronic aluminum
intoxication of mice resulted in significantly decreased
learning performance in comparison with control group.
On the other hand, the chronic administration of pomegran-
ate juice during 90 days after weaning ameliorates signifi-
cantly the cognitive impairments of mice exposed to Al
(Fig. 2D2). Several mechanisms have been suggested to
explain the processes by which aluminum can cross the
bloodbrain barrier, access the brain tissue and accumulate
Table 1 Effect of aluminum chloride on water intake and body weight in mice after 12 weeks of experiment
Groups Body weight (g) Water intake (ml)
C Al Al-J C Al Al-J
Week 1 20.78 ± 1.31 17.23 ± 0.60 17.5 ± 0.53 212.77 ± 48.65 152.94 ± 37.94* 164.39 ± 32.43
Week 4 30.32 ± 2.74 26.95 ± 3.30 28.64 ± 2.91
Week 8 34.54 ± 2.87 31.48 ± 2.63 31 ± 0.58
Week 12 36.18 ± 3.09 31.57 ± 2.46 32.22 ± 0.94
C: control without any treatment; Al: aluminum exposed mice (500 mg/kg); Al-J: aluminum intoxicated mice treated with pomegran-
ate juice (Al-J); (Al vs. control); (Al-J vs. control); *P< 0.05
Fig. 2A Locomotor activity: (C) control without any treatment;
(Al) aluminum-exposed mice (500 mg/kg); and (Al-J) intoxicated
mice treated with pomegranate juice (v:v) for 90 days. Values rep-
resent the means of 5 experiments (Al vs. control); (Al-J vs. con-
trol); *P< 0.05
Fig. 2B Swimming test: (C) control without any treatment; (Al)
aluminum-exposed mice (500 mg/kg); and (Al-J) intoxicated
mice treated with pomegranate juice (v:v) for 90 days. Values rep-
resent the means of 5 experiments (Al vs. control); (Al-J vs. con-
trol); *P< 0.05
Phytothérapie 5
in it; these were confirmed by our graphite furnace atomic
absorption (GFAA) spectroscopic study that revealed a
highly significant content of Al in the brain of the intoxi-
cated group after 90 days of experimentation compared to
the control group (P< 0.05, Fig. 3).
Our histopathological findings showed serious alterations
in the hippocampus and cerebral cortex which related to alu-
minum accumulation. Histological examination of the ner-
vous system showed a high level of neural degeneration and
vacuolization in the cortex and hippocampus after aluminum
exposure in intoxicated group as compared to the control and
aluminum-treated group. This group also revealed inflam-
matory infiltrate and a decrease in cell density (Figs 4A,
4B). Neuronal damage in the mice treated with juice is
observed to be low. No sign of inflammatory infiltrate or
necrosis was observed in the hemotoxylin and eosin staining
in the hippocampus and cerebral cortex of mice. In the con-
trol group, the section was found to be intact and no neuronal
loss was observed (Figs 4A, 4B).
Pomegranate juice treatment during aluminum exposure pro-
tected the mice from the changes induced by aluminum.
Daily consumption of fruit juice significantly improved
locomotor activity in the last 15 min of this test. Our results
Fig. 2C Elevated plus maze (EPM): (C) control without any treat-
ment, (Al) aluminum-exposed mice (500 mg/kg); and (Al-J) intox-
icated mice treated with pomegranate juice (v:v) for 90 days.
Values represent the means of 5 experiments (Al vs. control);
(Al-J vs. control); *P< 0.05
Fig. 2D Morris water maze test. 1) Spatial working memory; 2) spatial memory reference: (C) control without any treatment; (Al)
aluminum-exposed mice (500 mg/kg); and (Al-J) intoxicated mice treated with pomegranate juice (v:v) for 90 days. Values represent
the means of 5 experiments (Al vs. control); (Al-J vs. control); *P< 0.05
6 Phytothérapie
do not agree with a large body of research which has showed
that aluminum exposure at different concentrations leads an
hypokinesia [23, 5,24]. Exposure to aluminum causes signif-
icant impairments in a number of motor functions and
increased apoptosis of motor neurons [25]. In addition to
the motor dysfunction, an increase in immobility time was
observed in animals exposed to Al.
This finding was reported by Rebai and Djebli [26],
who found that exposure to 50 mg/kg of AlCl
3 months increased immobility time in Al treated mice.
Antidepressant-like activity is related to immobility time in
the forced swimming test. Indeed, Lin et al. [27] found that
reduction of immobility time is an indicator of the improve-
ment of the depressed state, indicative of serotonergic neu-
rotransmission modulation. Pomegranate juice improves
depressive state of mice empoisoned by aluminum through
decreasing immobility time in the forced swimming test.
HPLC-DAD revealed the presence of vanillin, a phenolic
acid that may be a potential pharmacological agent for the
treatment of major depressive disorders by modulating
monoamine neurotransmitters, both serotonin and dopa-
mine, in brain tissue [28].
According to Sharma et al. [29], an increase in the open
arm activity reflects anti-anxiety behavior. In this context,
Coleta et al. [30] have shown that luteolin causes anxiolytic
effects through a gamma-aminobutyric acid-ergic (GABAer-
gic) mechanism. On the other hand, some authors have
reported that quercetin, a flavonoid that has been detected
in the chromatogram of pomegranate juice, may decrease
the level of anxiety [31,29]. Quercetin is able to traverse
the bloodbrain barrier and exert its neuroprotector effects
[32]. An improvement in the muscle coordination, cognition,
anxiety, locomotion, and initial exploratory patterns in
Al-treated rats were observed after administration of this fla-
vonoid [29].
In our study, the memory and learning abilities of mice
were significantly altered after a 90 day exposure to alumi-
num. These results indicate that AlCl
alters spatial learning
ability [33]. Our results confirm those obtained by Sethi et al.
[34] who have demonstrated that administration of AlCl
(50 mg/kg/day) in drinking water for 6 months results in
learning deficiencies in rats undergoing the Morris water lab-
yrinth. Pomegranate juice produced a strikingly significant
reduction in the time that mice needed to get to the hidden
and visible platform, which confirmed the higher acquisition
and memory level of the intoxicated treated group compared
with the intoxicated mice. This finding confirms the damage
reduction in the brain in the treated group compared to
the intoxicated group. Our results showed that dietary
Fig. 3 Atomic absorption spectroscopy of aluminum in brains
for the three experimental groups: (C) control without any treat-
ment; (Al) aluminum-exposed mice (500 mg/kg); and (Al-J) intox-
icated mice treated with pomegranate juice (v:v) for 90 days. (Al
vs. control); (Al-J vs. control); *P< 0.05
Fig. 4A Microscopic study of cerebral cortex performed by hae-
motoxylin and eosin staining showing histopathological changes
in the cerebral cortex in different groups: (C) control without
any treatment; (Al) aluminum-exposed mice (500 mg/kg); and
(Al-J) intoxicated mice treated with pomegranate juice (v:v)
for 90 days (G × 400). (NC: normal cell; IF: inflammatory infil-
trate; ND: neuronal degeneration; V: vacuolization; AD: amyloid
Phytothérapie 7
supplementation with pomegranate juice significantly attenu-
ated learning and memory deficits, improved motor coordina-
tion, and reduced anxiety [35]. Thenmozhi et al. [36] con-
cluded that hesperidin decreased memory loss and
attenuated acetylcholinesterase (AchE) activity and the amy-
loidogenic pathway induced by aluminum exposure.
However, Abdel Moneim [37] found that the pomegran-
ate peel protected the AlCl
-intoxicated ratsbrain by
decreasing the aluminum accumulation and stimulating anti-
oxidant activities which reduces oxidative stress and brain
Bihaqi et al. [38] found that AlCl
causes histopatholog-
ical lesions in the cerebral cortex including neuronal loss,
ghost cells hemorrhage, and vacuolated cytoplasm. Histo-
logical examination of the nervous system after aluminum
exposure by Sharma et al. [39] showed marked cell distor-
tion with high level of degeneration in the hippocampus.
Neurons were morphologically damaged (swollen cyto-
plasm or shrunken, darkened nuclei).
In conclusion, our experiments showed that pomegranate
juice improves learning and memory performances. This
fruit has shown an excellent anxiolytic and antidepressant
activity in neurobehavioral tests. A decrease of histological
alterations in the cerebral cortex and hippocampus attributes
to the juice a neuroprotective effect against the neurotoxic
effects of aluminum in mice neurons.
Acknowledgements I would like to sincerely thank Dr.
Medjamia for allowing me to work at the AnatomyPathol-
ogy Laboratory of Military hospital (HMRUO) and support-
ing me during my training. I would like to thank Mr. Yacine
Nait Bachir and Miss Labdi Aicha for their help.
Declaration of interest: The authors declare no conflicts of
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Fig. 4B Microscopic study of hippocampus performed by haemo-
toxylin and eosin staining showing histopathological changes
in the hippocampus in different groups: (C) control without any
treatment; (Al) aluminum-exposed mice (500 mg/kg); and (Al-J)
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Phytothérapie 9
... The rats were grouped and treated according to the following protocol: 1) Control group in which the rats were treated by vehicle (not with PSE) and saline was injected instead of scopolamine; 2) Scopolamine group in which the rats were treated with vehicle (not with PSE) and 2 mg/kg scopolamine (Sigma, St. Louis, USA) was injected (intraperitoneal (i.p.)) to induce learning and memory impairment (Sun et al. 2019;Hosseini et al. 2022); 3-5) Scopolamine-PSE 200, scopolamine-PSE 400, and scopolamine-PSE 800 groups in which the rats were orally gavaged 200, 400, or 800 mg/kg of PSE every day. PSE dosages were determined according to previous researches (Sarkari et al. 2015;Ahmad et al. 2014;Gadouche et al. 2018). The experiments lasted three weeks. ...
... For example, pomegranate extract ameliorated memory and learning ability measured via a novel object recognition test (Almuhayawi et al. 2020). Supplementation with pomegranate juice (500 mg/kg, given orally for 90 days) ameliorated learning and memory deficits in the MWM test in female albino Swiss mice exposed to AlCl 3 (Gadouche et al. 2018). Pomegranate benefits on learning and memory have been related to its antioxidant properties. ...
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Objective This study aimed to investigate the effects of pomegranate (Punica granatum L.) seed hydro-ethanolic extract (PSE) on cholinergic dysfunction, neuroinflammation, and oxidative stress in the scopolamine-induced amnesic rats. Methods The rats were given PSE (200, 400, and 800 mg/kg, gavage) for 3 weeks. In the third week, scopolamine was administered 30 min before the Morris water maze (MWM) and passive avoidance (PA) tests. Oxidative stress indicators, acetylcholinesterase (AChE) activity, and mRNA expression of necrosis factor (TNF)-α, interleukin (IL)-1β, AChE, and M1 acetylcholine receptor (CHRM1) in the brain, were measured. Results PSE reduced the time (maximum 173%) and distance (maximum 332%) required to reach the platform during MWM learning (P < 0.001). In the prob test (P < 0.001), it increased the target area time (maximum 44%) and distance (maximum 30%). PSE also increased delay and light time (maximums of 86 and 48%, respectively) (P < 0.001), while decreasing the time in dark region of PA (maximums 727%) (P < 0.001). PSE also reduced malondialdehyde and AChE in the cortex (maximum 168 and 171%, respectively) and hippocampus (maximum 151 and 182%, respectively) (P < 0.001). In the PSE-treated groups, the levels of thiol and superoxide dismutase were increased in the cortex (maximum 54 and 65%, respectively) and hippocampus (maximum 90 and 51%, respectively) (P < 0.001). TNF-α, IL-1β, and AChE expressions in the hippocampus were reduced by PSE (maximum 114, 137, and 106%, respectively, P < 0.01). Meanwhile, CHMR expression was increased (66%). Conclusion PSE successfully alleviated scopolamine-induced memory and learning deficits in rats which is probably via modulating cholinergic system function, oxidative stress, and inflammatory cytokines.
... In our memory study, mice treated with AlCl 3 and Dgalactose had significantly decreased memory and learning capacity (P < 0.01), which was associated with the presence of degenerating neurons in the cerebral cortex, hippocampus, and cerebellum, as well as histo-morphological abnormalities. These results and findings are in accordance with those reported by Chiroma et al., [4,22] Zerrouki et al., [23] Gadouche et al., [24] and Yang et al.. [25] The acetone fraction of P. edulis seeds (PEAS) results in a highly significant reduction in the number of repeated arms in mice treated with Alz-ED1 and Alz-ED2 as well as mice treated with Rivastigmine at 1 mg/kg when compared to Alzheimer's model mice (Alz) on various memory tests, including spatial working memory and the Morris water maze. ...
The stilbene-rich acetone fraction in high yield (6.6%, PEAS) of Passiflora edulis Sims was prepared and evaluated for neuroprotective activity in murine Alzheimer's disease model induced by aluminum chloride and D-galactose. The phytochemical and HPLC-DAD-MS analysis of the polyphenolic stilbene-rich acetone fraction showed that it contained different stilbenes including trans-piceatannol, scirpusins A-B and cassigarol E. The total phenolic content (TPC) of PEAS was 413.87 ± 1.71 mg GAE eqv/g. The neuroprotective activity of PEAS is typically presented in the Morris water maze-reference Spatial Memory test, where the Alzheimer's mice treated at 100 mg/kg (Alz-ED1) and 200 mg/kg (Alz-ED2) spent less than 47% and 66% of the time, respectively, than the Azheimer's model mice (Alz). Two simple stilbenes, trans-piceatannol and trans-resveratrol, showed selectively inhibitory activity in silico against AChE. Two stilbene dimers, cassigarol E and scirpusin A, exhibited low nanomolar inhibitory potential against AChE and BChE, significantly lower than those of the positive control, donepezil and tacrine. These findings suggest that the stilbenes from P. edulis seeds, particularly the stilbene dimers, warrant further investigation as potential neuroprotective candidates in the prevention of cognitive deficits associated with Alzheimer's disease.
... High-performance liquid chromatography with a diode array detector (HPLCDAD) revealed a variety of bioactive molecules (luteolin, quercetin, and gallic acid) contained in pomegranate. They conclude that eating pomegranate may be neuroprotective for neuropathy caused by Al poisoning (Gadouche et al., 2018). ...
... High-performance liquid chromatography with a diode array detector (HPLCDAD) revealed a variety of bioactive molecules (luteolin, quercetin, and gallic acid) contained in pomegranate. They conclude that eating pomegranate may be neuroprotective for neuropathy caused by Al poisoning (Gadouche et al., 2018). ...
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Age-related neurological disorders [AND] include neurodegenerative diseases [NDDs] such as Alzheimer's disease [AD] and Parkinson's disease [PD], which are the most prevalent types of dementia in the elderly. It also includes other illnesses such as migraine and epilepsy. ANDs are multifactorial, but aging is their major risk factor. The most frequent and vital pathological features of AND are oxidative stress, inflammation, and accumulation of misfolded proteins. As AND brain damage is a significant public health burden and its incidence is increasing, much has been done to overcome it. Pomegranate ( Punica granatum L .) is one of the polyphenol-rich fruits that is widely mentioned in medical folklore. Pomegranate is commonly used to treat common disorders such as diarrhea, abdominal pain, wound healing, bleeding, dysentery, acidosis, microbial infections, infectious and noninfectious respiratory diseases, and neurological disorders. In the current review article, we aimed to summarize the data on the pharmacotherapeutic potentials of pomegranate in ANDs.
... These results agree with Rashwan et al. (2018) and show that the open field test reflects behavior related to exploratory activity and anxiety that are expressed in AlCl3intoxicated rats by reduced horizontal and vertical exploratory activity, high latency, and high defecation. Exposition to AlCl3 induces significant alterations in a number of motor functions and an increase in motor neuron apoptosis (Gadouche et al., 2018). However, administration of probiotics resulted in a marked improvement in locomotor activity in both intoxicated and treated rats, as well as improvement in their exploratory and emotional states, suggesting that the probiotic helps support the nervous systems and may help release emotional blockages and bring a sense of balance. ...
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Purpose: Aluminum (Al) and its various forms are environmental xenobiotics that pose great threats to public health. The probiotic strain has been reported to have important protective effects against aluminum cytotoxicity. Therefore, the current study was designed to assess the disruption of neurobehavioral and hematobiochemical parameters caused by chronic exposure to AlCl3, and then explore the protective effects of the probiotic strain Lactobacillus rhamnosus against these adverse in male rats. Method: Thirty-two animals were gathered into four groups: The first group served as control. The second group received 100mg AlCl3/kg bw for 12 weeks. However, the third group was administered 10mL probiotic strain /Kg (108 CFU/ ml) bw/d daily for 4 weeks. Finally, group 4 received AlCl3 for 12 weeks, then the treatment was continued with probiotic for 4 weeks. Results: AlCl3 produced a decrease in body and brain weights with respect to their control. The results obtained also showed significant depression and anxiety-like behavior in the intoxicated animals, with a disturbance of the tested hematobiochemical parameters, particularly in renal and hepatic biomarkers. Overall, probiotics prevented enduring AlCl3-induced depression-like behavior in intoxicated rats. The results of the hematobiochemical, renal and hepatic analyses show a correction of the values after the administration of probiotics compared to those of intoxicated and untreated animals. Conclusion: The probiotic lactobacilli would be an alternative to reduce AlCl3 toxicity.
Background: Cognitive dysfunction such as dementia is the most common neurological disorder that affects memory, perception, learning, and problem-solving. Emerging evidence suggests that nutritional factors may prevent or accelerate the incidence of neurodegenerative diseases. Objective: This systematic review was designed to evaluate the association between pomegranate treatment and cognitive function. Methods: PubMed, Embase, Google Scholar, and Scopus databases were searched to extract original animal and human studies published until July 2021, without date restrictions. Firstly, 215 studies were retrieved via the search strategy. Irrelevant and duplicated studies were screened out and critical analysis was used to obtain data. The quality of the articles and the risk of bias were assessed by OHAT and Cochrane Collaboration's quality assessment tools. Results: Finally, 24 articles (20 animal and 4 randomized controlled trial studies) were included in this review. All included animal and human studies indicated that pomegranate treatment was positively associated with improving specific domains of cognitive function. Conclusions: Our findings demonstrated that pomegranate treatment can improve cognitive function. Therefore, including pomegranate intake during daily life may lower the risk of cognitive impairment at the population level.
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Entomopathogenic nematodes (EPN) of the families Steinernematidae and Heterorhabditidae were isolated from 57 of 91 localities in northern Poland. Of 489 soil samples collected in the field, EPNs were recorded in 27 per cent of them. Steinernema species were more frequent than Heterorhabditis: five of the family Steinernematidae and two of the family Heterorhabditidae were identified. Nematodes S. feltiae was the most frequently recorded species in different ecosystems. There were two rare of entomopathogenic nematodes species to the Polish fauna: S. silvaticum (only in natural ecosystems) and S. bicornutum (only in agrocoenoses). It seems that the presence of a suitable host in the environment is the most important factor for EPNs. Simple PCR-RFLP system was used to differentiate eight EPN species.
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The purpose of this study was to determine the neuroprotective potential of pomegranate peel methanolic extract (500 mg kg-1) on lead acetate (1000 ppm) induced neurotoxicity. After 12 weeks, the mice were subjected to behavioral tests. Brain injuries were determined with hematoxylin and eosin staining and lead accumulation was measured by graphite furnace atomic absorption with Zeeman correction. Lead exposure induced neurobehavioral alterations, reduced body growth, lead deposits in brain and histological change in the lead-treated group. Furthermore, co-administration of pomegranate extract with lead decreased locomotion, anxiety and depression in lead-exposed mice as indicated by the number of cells crossed by mice, the residence time in the dark compartment and the immobility time in forced swimming. Also, pomegranate extract improved weight loss and histological change of cortex cerebral and hippocampus by reducing the lead concentration in these sites. Pomegranate methanolic extract has neuroprotective effects against lead-induced neurological disorders probably by its various phytochemical components.
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Objective: To study the antioxidative and protective properties of aqueous extract of Hammada scoparia (H. scoparia) against the effects of sub-chronic aluminium (Al) intoxication on mnemonic process and some neurochemical markers. Methods: Al was administered intraperitoneally (50 mg/kg body weight, three times a week), and H. scoparia and malic acid were given orally by gavage at a daily dose (100 mg/kg body weight) to rats for 90 days. Results: Al caused significant short-term and long-term memory disturbances, a decrease in locomotor activity, a significant inhibition of acetylcholinesterase activity in brain and a significant depletion of antioxidant enzymes (catalase, glutathione reductase and glutathione peroxidase) and glutathione. It significantly increased lipid peroxidation levels in cerebrum and cerebellum. However, treatment with H. scoparia extract protected efficiently the neurological functions of intoxicated rats by considerably increasing antioxidants levels and decreasing production of thiobarbituric acid reactive substances by 4.26% compared to untreated group. We noted some controversial results with malic acid. It showed some positive results but it was not as efficient as H. scoparia extract. Current results were consistent with histopathological observations including neurodegeneration and vacuolated cytoplasm (spongiosis) in Al treated sections when H. scoparia and malic acid treated sections showed marked neuroprotection signs. Conclusions: This study strongly suggested that H. scoparia extract could possibly restore the altered neurological capacities and antioxidant power in rats, and it could even be a good alternative to chelating agents or other chemical medicines against Al-induced neurotoxicity.
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The present study was aimed to evaluate the protective effect of hesperidin (Hes) on aluminium chloride (AlCl3) induced neurobehavioral and pathological changes in Alzheimeric rats. Intraperitonial injection of AlCl3 (100 mg/kg body weight) for 60 days significantly elevated the levels of aluminium (Al), activity of acetylcholinesterase (AChE) and protein expressions of amyloid precursor protein (APP), β amyloid (Aβ1-42), β and γ secretases as compared to control group in hippocampus and cortex of rat brain. Hes administration orally along with AlCl3 injection for 60 days, significantly revert the Al concentration, AChE activity and Aβ synthesis-related molecules in the studied brain regions. Our results showed that aluminum exposure was significantly reduced the spontaneous locomotor and exploratory activities in open field test and enhanced the learning and memory impairments in morris water maze test. The behavioral impairments caused by aluminum were significantly attenuated by Hes. The histopathological studies in the hippocampus and cortex of rat brain also supported that Hes (100 mg/kg) markedly reduced the toxicity of AlCl3 and preserved the normal histoarchitecture pattern of the hippocampus and cortex. From these results, it is concluded that hesperidin can reverse memory loss caused by aluminum intoxication through attenuating AChE activity and amyloidogenic pathway.
Atomic absorption spectrometry (AAS) enjoys excellent elemental selectivity and widespread usage with documented methodologies for a wide range of samples and matrices. Replacing the traditional flame atomizer with a graphite furnace (or ‘electrothermal atomizer (ETA)’) adds significantly to the flexibility of AAS and provides significantly lower detection limits. The basic principles of atomic absorption (AA) and the use of a graphite furnace as a means of generating the free atoms that are used in the spectrometric analysis are presented in this article. We must begin by understanding the basic chemical and physical transformations that occur from the moment a sample is placed on the graphite surface until the atomic vapors generated by heating of the graphite furnace diffuse from the tube and the absorption signal terminates. The path leading to the production of free gas-phase atoms within the measurement volume within the graphite tube is a fascinating tale with many twists depending on sample and matrix. The chemical knowledge of various compounds has led to logical and innovative methodologies for particular analytes in specific matrices. All the efforts are in an attempt to permit interference-free analyses using simple aqueous standards in the preparation of calibration curves. Limits of detection in the parts per billion range (picogram, absolute), microsampling capabilities, and methods for solids and slurries analysis have made this a popular analytical technique for ultratrace metal analysis.
Alzheimer's disease (AD) is a progressive and ultimately fatal neurodegenerative diseases. Aluminum, a neurotoxic metal, is considered as the pathological hallmark and contributing factor of AD. Hypericum perforatum extract (HPE) is a neuroprotective agent that can prevent neurodegenerative pathologies through antioxidants, anti-inflammatory and regulating neurotransmitter release in animal model of neuropathy. The present study aimed to identify the potential neuroprotective of HPE on AlCl3-induced AD rats. Rats were treated with AlCl3 for 90 days to induce behavioral, biochemical, and neurochemical similar to AD. From 31th day, the rats were treated with HPE for 60 days. Our results showed HPE improved cognitive function in AlCl3-induced AD rats, and attenuated AlCl3-induced increase in acetylcholinesterase activity and glutamic acid level as well as decreased in noradrenaline and dopamine level. In addition, HPE reversed AlCl3-induced hippocampal pathology including amyloid-beta (Aβ) accumulation (elevated Aβ42 level and amyloid plaques), oxidative stress (increased reactive oxygen species level and thiobarbituric acid reactive substances level, decreased glutathione level and superoxide dismutase activity) and neuroinflammatory (increased mRNA expressions of Interleukin-1β, Interleukin-6, Tumor necrosis factor-α and major histocompatibility complex class II) in hippocampus of rats. Thus, HPE is conferred neuroprotection against AlCl3-induced AD like pathology.
Aluminum (Al) is present in the daily life of humans, and the incidence of Al contamination increased in recent years. Long-term excessive Al intake induces neuroinflammation and cognition impairment. Neuroinflammation alter density of dendritic spine, which, in turn, influence cognition function. However, it is unknown whether increased neuroinflammation is associated with altered density of dendritic spine in Al-treated rats. In the present study, AlCl3 was orally administrated to rat at 50, 150 and 450 mg/kg for 90d. We examined the effects of AlCl3 on the cognition function, density of dendritic spine in hippocampus of CA1 and DG region and the mRNA levels of IL-1β, IL-6, TNF-α, MHC II, CX3CL1 and BNDF in developing rat. These results showed exposure to AlCl3 lead to increased mRNA levels of IL-1β, IL-6, TNF-α and MCH II, decreased mRNA levels of CX3CL1 and BDNF, decreased density of dendritic spine and impaired learning and memory in developing rat. Our results suggest AlCl3 can induce neuroinflammation that may result in loss of spine, and thereby leads to learning and memory deficits.
Aluminum is a light weight and toxic metal present ubiquitously on earth, which has gained considerable attention due to its neurotoxic effects. It also has been linked ecologically and epidemiologically to several neurological disorders, including Alzheimer’s disease (AD), Parkinson’s disease (PD), Guamanian–Parkinsonian complex and Amyotrophic lateral sclerosis (ALS). The mechanism of aluminum neurotoxicity is poorly understood, but it is well documented that aluminum generates reactive oxygen species (ROS). Enhanced ROS production leads to disruption of cellular antioxidant defense systems and release of cytochrome c (cyt-c) from mitochondria to cytosol resulting in apoptotic cell death. Quercetin (a natural flavonoid) protects it from oxidative damage and has been shown to decrease mitochondrial damage in various animal models of oxidative stress. We hypothesized that if oxidative damage to mitochondria does play a significant role in aluminum-induced neurodegeneration, and then quercetin should ameliorate neuronal apoptosis. Administration of quercetin (10 mg/kg body wt/day) reduced aluminum (10 mg/kg body wt/day)-induced oxidative stress (decreased ROS production, increased mitochondrial superoxide dismutase (MnSOD) activity). In addition, quercetin also prevents aluminum-induced translocation of cyt-c, and up-regulates Bcl-2, down-regulates Bax, p53, caspase-3 activation and reduces DNA fragmentation. Quercetin also obstructs aluminum-induced neurodegenerative changes in aluminum-treated rats as seen by Hematoxylin and Eosin (H&E) staining. Further electron microscopic studies revealed that quercetin attenuates aluminum-induced mitochondrial swelling, loss of cristae and chromatin condensation. These results indicate that treatment with quercetin may represent a therapeutic strategy to attenuate the neuronal death against aluminum-induced neurodegeneration.
Ethnopharmacological relevance: Depression is a serious psychological disorder that causes extreme economic loss and social problems. However, the conventional medications typically cause side effects that result in patients opting to out of therapy. Lemon balm (Melissa officinalis L., MO) is an old and particularly reliable medicinal herb for relieving feelings of melancholy, depression and anxiety. The present study aims to investigate the antidepressant-like activity of water extract of MO (WMO) by evaluating its influence on the behaviors and the relevant neurotransmitters of rats performed to forced swimming test. Materials and methods: Two phases of the experiment were conducted. In the acute model, rats were administered ultrapure water (control), fluoxetine, WMO, or the indicated active compound (rosmarinic acid, RA) three times in one day. In the sub-acute model, rats were respectively administered ultrapure water (control), fluoxetine, or three dosages of WMO once a day for 10 days. Locomotor activity and depression-like behavior were examined using the open field test and the forced swimming test, respectively. The levels of relevant neurotransmitters and their metabolites in the frontal cortex, amygdala, hippocampus, and striatum were analyzed by high performance liquid chromatography. Results: In the acute model, WMO and RA significantly reduced depressive-like behavior but the type of related neurotransmitter could not be determined. The results indicated that the effect of WMO administration on the reduction of immobility time was associated with an increase in swimming time of the rats, indicative of serotonergic neurotransmission modulation. Chromatography data validated that the activity of WMO was associated with a reduction in the serotonin turnover rate. Conclusion: The present study shows the serotonergic antidepressant-like activity of WMO. Hence, WMO may offer a serotonergic antidepressant activity to prevent depression and to assist in conventional therapies.