Content uploaded by Noureddine Djebli
Author content
All content in this area was uploaded by Noureddine Djebli on Nov 04, 2018
Content may be subject to copyright.
PHARMACOGNOSIE
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 l’aluminium 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
3
orally; and
the third treated group received pomegranate juice (v/v in
water) supplied in dark bottles for 4 h/day followed by
AlCl
3
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 L’aluminum (Al) est un élément omniprésent sur la
croûte terrestre et qui est à l’origine 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 l’eau pota-
ble, le groupe contrôle positif a été exposé quotidiennement
à 500 mg/kg d’AlCl
3
par voie orale et le troisième groupe a
reçu le jus de grenade (v/v dans l’eau) fourni dans des bou-
teilles sombres pendant quatre heures suivi par l’AlCl
3
àla
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 l’hippocampe ont été déterminées par
coloration à l’hématoxyline et à l’éosine, et l’accumulation
d’Al a été mesurée par absorption atomique au four graphite
avec correction de Zeeman. Le dépôt d’Al dans le cerveau a
provoqué une dégénérescence neuronale et une diminution
de la densité cellulaire avec l’installation d’un état d’anxiété,
de dépression et d’un déficit d’apprentissage, et de mémoire.
Le traitement au jus de grenade a atténué les altérations neu-
rocomportementales, a diminué la quantité d’Al dans le cer-
veau et a rétabli la structure histologique. HPLC-DAD a
révélé une gamme de molécules bioactives (à savoir l’acide
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 l’Al.
Mots clés Jus de grenade · HPLC-DAD · Aluminium ·
Neurotoxicité · Histopathologie
Introduction
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 : gadoucheleila@gmail.com
Phytothérapie
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 blood–brain 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 blood–brain barrier, and that
the potential for permeation is consistent with compound
lipophilicity.
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
3
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
cerebral.
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°06′04″N, 1°11′58″E, 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
3
500 mg/kg body weight (BW) orally in the drinking water
for 12 weeks.
Group 3: Treated with pomegranate juice (Al–J) 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
recorded.
Anxiety test: the “elevated plus maze (EPM)”test
The elevated plus maze (EPM), is an apparatus composed of
two arms—a 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
platform.
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
day.
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].
Results
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
blood–brain 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).
Discussion
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
during
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 blood–brain 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
3
alters spatial learning
ability [33]. Our results confirm those obtained by Sethi et al.
[34] who have demonstrated that administration of AlCl
3
(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
deposits)
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
3
-intoxicated rats’brain by
decreasing the aluminum accumulation and stimulating anti-
oxidant activities which reduces oxidative stress and brain
injuries.
Bihaqi et al. [38] found that AlCl
3
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 Anatomy–Pathol-
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
interest.
References
1. Willhite CC, Karyakina NA, Yokel RA, et al (2014) Systematic
review of potential health risks posed by pharmaceutical, occupa-
tional and consumer exposures to metallic and nanoscale alumi-
num, aluminum oxides, aluminum hydroxide and its soluble salts.
Crit Rev Toxicol 44:1–80
2. Yokel RA, Hicks CL, Florence RL (2008) Aluminum bioavail-
ability from basic sodium aluminum phosphate, an approved
food additive emulsifying agent, incorporated in cheese. Food
Chem Toxicol 46:2261–6
3. Exley C (2014) What is the risk of aluminium as a neurotoxin?
Expert Rev Neurother 4:589–91
4. Tüzmen MN, Yücel NC, Kalburcu T, et al (2015) Effects of cur-
cumin and tannic acid on the aluminum- and lead-induced oxida-
tive neurotoxicity and alterations in NMDA receptors. Toxicol
Mech Meth 25:120–7
5. Taïr K, Kharoubi O, Taïr OA, et al (2016) Aluminium-induced
acute neurotoxicity in rats: treatment with aqueous extract of
Arthrophytum (Hammada scoparia). J Acute Dis 5:470–82
6. Lakshmi BVS, Sudhakar M, Anisha M (2014) Neuroprotective role
of hydroalcoholic extract of Vitis vinifera against aluminium-
induced oxidative stress in rat brain. Neurotoxicology 41:73–9
7. Priyanka S, Jayantha Rao K, John Sushma N (2012) Therapeutic
effect of melatonin against aluminum-induced neurotoxicity in
cerebellum of albino mice. Toxicol Environ Chem 94:1422–32
8. Kennedy OD (2014) Polyphenols and the human brain: plant
“secondary metabolite”ecologic roles and endogenous signaling
functions drive benefits1,2. Adv Nutr 5:515–33
9. Aquilano K, Baldelli S, Rotilio G, et al (2008) Role of nitric
oxide synthases in Parkinson’s disease: a review on the antioxi-
dant and anti-inflammatory activity of polyphenols. Neurochem
Res 33:2416–26
10. Youdim KA, Dobbie MS, Kuhnle G, et al (2003) Interaction
between flavonoids and the blood-brain barrier: in vitro studies.
J Neurochem 85:180–92
11. Gomez-Pinilla F, Nguyen TTJ (2012) Natural mood foods: the
actions of polyphenols against psychiatric and cognitive disor-
ders. Nutr Neurosci 15:127–33
12. Viladomiu M, Hontecillas R, Lu P, et al (2013) Preventive and
prophylactic mechanisms of action of pomegranate bioactive con-
stituents. Evid Based Complement Alternat Med 789764
13. Fuster-Muñoz E, Roche E, Funes L, et al (2016) Effects of pome-
granate juice in circulating parameters, cytokines, and oxidative
stress markers in endurance-based athletes: a randomized con-
trolled trial. Nutrition 32:539–45
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)
intoxicated mice treated with pomegranate juice (v:v) for 90 days
(G × 400). (NC: normal cell; IF: inflammatory infiltrate; ND: neu-
ronal degeneration; V: vacuolization. AD: amyloid deposits)
8 Phytothérapie
14. Gong Q, Wu Q, Huang XN, et al (2005) Protective effects of
Ginkgo biloba leaf extract on aluminum-induced brain dysfunc-
tion in rats. Life Sci 77:140–8
15. Sáenz JCB, Villagra OR, Trias JF (2006) Factor analysis of
forced swimming test, sucrose preference test and open field
test on enriched, social and isolated reared rats. Behav Brain
Res 169:57–65
16. Porsolt RD, Bertin A, Jalfre M (1977) Behavioral despair in
mice: a primary screening test for antidepressants. Arch Int Phar-
macodyn Ther 229:327–36
17. Albrechet-Souza L, Oliveira AR, De Luca MC, et al (2005) A
comparative study with two types of elevated plus maze (trans-
parent vs. opaque walls) on the anxiolytic effects of midazolam,
one-trial tolerance and fear-induced analgesia. Prog Neuropsy-
chopharmacol Biol Psychiatry 29:571–9
18. Hogg S (1996) A review of the validity and variability of the
elevated plus-maze as an animal model of anxiety. Pharmacol
Biochem Behav 54:21–30
19. Morris R (1984) Developments of a water-maze procedure for
studying spatial learning in the rat. J Neurosci Methods 11:47–60
20. Gupta, V, Gill KD (2000) Influence of ethanol on lead distribu-
tion and biochemical changes in rats exposed to lead. Alcohol
20(1):9-17
21. Holcombe JA, Borges DL (2010) Graphite furnace atomic
absorption spectrometry. Encyclopedia of Analytical Chemistry
22. Mahdy KA, Farrag ARH (2009) Amelioration of aluminum tox-
icity with black seed supplement on rats. Toxicol Environ Chem
9:567–76
23. Harsha SN, Anilakumar KR (2013) Protection against aluminium
neurotoxicity: a repertoire of lettuce antioxidants. Biomed Aging
Pathol 3:179–84
23. Cao Z, Wang F, Xiua C, et al (2017) Hypericum perforatum
extract attenuates behavioral, biochemical, and neurochemical
abnormalities in aluminum chloride-induced Alzheimer’s disease
rats. Biomed Pharmacother 91:931–37
25. Shaw CA, Petrik MS (2009) Aluminum hydroxide injections lead
to motor deficits and motor neuron degeneration. J Inorg Bio-
chem 103:1555–62
26. Rebai O, Djebli NE (2008) Chronic exposure to aluminum chlo-
ride in mice: exploratory behaviors and spatial learning. Adv
Biomed Res 2:26–33
27. Lin SH, Chou ML, Chen WC, et al (2015) A medicinal herb,
Melissa officinalis L. ameliorates depressive-like behavior of
rats in the forced swimming test via regulating the serotonergic
neurotransmitter. J Ethnopharmacol 4:266–72
28. Xu J, Xu H, Liu Y, et al (2015) Vanillin-induced amelioration of
depression-like behaviors in rats by modulating monoamine neu-
rotransmitters in the brain. Psychiatry Res 28:509–14
29. Sharma DR, Wani WY, Sunkaria A, et al (2013) Quercetin pro-
tects against chronic aluminum-induced oxidative stress and
ensuing biochemical, cholinergic, and neurobehavioral impair-
ments in rats. Neurotox Res 23:336–57
30. Coleta M, Campos MG, Cotrim MD, et al (2008) Assessment of
luteolin (3′,4′,5,7-tetrahydroxyflavone) neuropharmacological
activity. Behav Brain Res 189:75–82
31. Aguirre-Hernández E, González-Trujano ME, Martínez AL, et al
(2010) HPLC/MS analysis and anxiolytic-like effect of quercetin
and kaempferol flavonoids from Tilia americana var. mexicana.
J Ethnopharmacol 127:91–7
32. Youdim KA, Qaiser MZ, Begley DJ, et al (2004) Flavonoid per-
meability across an in situ model of the blood-brain barrier. Free
Radic Biol Med 36:592–604
33. Cao Z, Yang X, Zhang H, et al (2016) Aluminum chloride induces
neuroinflammation, loss of neuronal dendritic spine and cognition
impairment in developing rat. Chemosphere 151:289–95
34. Sethi P, Jyoti A, Singh R (2008) Aluminum-induced electrophys-
iological, biochemical and cognitive modifications in the hippo-
campus of aging rats. Neurotoxicology 29:1069–79
35. Subash S, Braidy N, Essa MM, et al (2015) Long-term (15 mo)
dietary supplementation with pomegranates from Oman attenu-
ates cognitive and behavioral deficits in a transgenic mice
model of Alzheimer’s disease. Nutrition 31:223–9
36. Thenmozhi AJ, Raja TR, Janakiraman U, et al (2015) Neuropro-
tective effect of hesperidin on aluminium chloride induced Alz-
heimer’s disease in Wistar rats. Neurochem Res 40:767–76
37. Abdel Moneim A (2012) Evaluating the potential role of pome-
granate peel in aluminum-induced oxidative stress and histopath-
ological alterations in brain of female rats. Biol Trace Elem Res
150:328–36
38. Bihaqi SW, Sharma M, Singh AP, et al (2009) Neuroprotective
role of Convolvulus pluricaulis on aluminum induced neurotoxic-
ity in rat brain. J Ethnopharmacol 124:409–15
39. Sharma DR, Wani WY, Sunkaria A, et al (2016) Quercetin
attenuates neuronal death against aluminum-induced neurodegen-
eration in the rat hippocampus. Neuroscience 324:163–76
Phytothérapie 9
