Cognitive-enhancing effect of quercetin in a rat model of Parkinson's disease induced by 6-hydroxydopamine.

Abstract

Oxidative stress has been reported to induce cognitive impairment in Parkinson's disease. This paper aimed to determine the effect of quercetin, a substance possessing antioxidant activity, on the cognitive function in a rat model of Parkinson's disease. Male Wistar rats, weighing 200-250 g, were orally given quercetin at doses of 100, 200, 300 mg/kg BW once daily for a period of 14 days before and 14 days after the unilateral lesion of right substantia nigra induced by 6-hydroxydopamine (6-OHDA). Their spatial memory was assessed at 7 and 14 days of treatment and neuron density was determined, malondialdehyde (MDA) level, the activity of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were evaluated at the end of the experiment. In addition, the activity of acetylcholinesterase (AChE) was also measured. It was found that all doses of quercetin enhanced spatial memory. Therefore, it is suggested that the cognitive-enhancing effect of quercetin occurs partly because of decreased oxidative damage resulting in increased neuron density.

Full text

Page 1

Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2012, Article ID 823206, 9 pages
doi:10.1155/2012/823206
Research Article
Cognitive-EnhancingEffectof Quercetinina Rat Model of
Parkinson’s Disease Inducedby 6-Hydroxydopamine
Napatr Sriraksa,1JintanapornWattanathorn,2SupapornMuchimapura,2
Somsak Tiamkao,3Kamoltip Brown,4andKowit Chaisiwamongkol4
1Department of Physiology and Graduate School (Neuroscience Program), Faculty of Medicine, Khon Kaen University,
Khon Kaen 40002, Thailand
2Department of Physiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand
3Department of Medicine, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand
4Department of Anatomy, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand
Correspondence should be addressed to Jintanaporn Wattanathorn, jinwat05@gmail.com
Received 3 March 2011; Revised 1 May 2011; Accepted 25 May 2011
Academic Editor: Jos´ e Luis R´ ıos
Copyright © 2012 Napatr Sriraksa et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Oxidative stress has been reported to induce cognitive impairment in Parkinson’s disease. This paper aimed to determine the effect
of quercetin, a substance possessing antioxidant activity, on the cognitive function in a rat model of Parkinson’s disease. Male
Wistar rats, weighing 200–250g, were orally given quercetin at doses of 100, 200, 300mg/kg BW once daily for a period of 14 days
before and 14 days after the unilateral lesion of right substantia nigra induced by 6-hydroxydopamine (6-OHDA). Their spatial
memorywasassessedat7and14daysoftreatmentandneurondensitywasdetermined,malondialdehyde(MDA)level,theactivity
of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were evaluated at the end of the experiment. In
addition, the activity of acetylcholinesterase (AChE) was also measured. It was found that all doses of quercetin enhanced spatial
memory. Therefore, it is suggested that the cognitive-enhancing effect of quercetin occurs partly because of decreased oxidative
damage resulting in increased neuron density.
1.Introduction
Parkinson’s disease (PD) is the second most common
neurodegenerative disease. To date, the precise mechanism
responsible for pathogenesis of PD is not well understood.
Accumulating lines of evidence have demonstrated that
oxidative stress plays a crucial role on the pathogenesis of
PD[1–5].Themotorimpairmentobservedinpatientsseems
to be the most notable point of concern when prescribing
medicine or planning treatment. In recent years, it was
indicated that nonmotor deficits became an important part
of patient management [6]. It has been known that patients
intheearlystagesofPDalreadyhaveimpairmentofcognitive
performance and this deficit in turn gives rise to a poor
qualityoflife[7,8]andeconomicburden[9].Recently,itwas
found that 6-hydroxydopamine (6-OHDA) was recognized
as a good model for early stages of PD, especially in
terms of emotional and cognitive deficits [10]. Although the
substantia nigra is the brain area that has been indicated
to be the most vulnerable to neurodegeneration in PD and
mediates the clinical manifestation (rigidity, resting tremor,
slowness of voluntary movement, and postural instability),
but other brain regions such as locus ceruleous, raphe
neuclei, nucleus basalis of Meynert, and the brain area that
responsible for cognition, hippocampus, were also affected.
Previous MRI studies clearly revealed that decreased hip-
pocampal volume accompanied the cognitive impairment
in PD patients [11–13]. Based on the role of oxidative
stress on the pathophysiology of PD, the neuroprotective
and cognitive enhancing effects of substances possessing
antioxidant activity have gained much attention.
Quercetin (QC) is a polyphenolic compound found in
common vegetables and fruits such as onions, broccoli, and
apples. Previous studies have demonstrated that quercetin

Page 2

2Evidence-Based Complementary and Alternative Medicine
0
1
2
3
4
5
6
7
Escape latency (s)
Control
Vehicle + 6-OHDA
QC100 + 6-OHDA
QC200 + 6-OHDA
QC300 + 6-OHDA
L-dopa + 6-OHDA
Vitamin C+ 6-OHDA
∗∗∗
∗∗∗
#
##
##
######
###############
7days after lesion 14days after lesion
Figure 1: Effect of quercetin on escape latency in the Morris water
maze test in an animal model of Parkinson’s disease induced by 6-
OHDA. Data were expressed as mean ± S.E.M. for 8 rats in each
group.∗∗∗P < 0.001 compared to control;#P < 0.05;##P < 0.01;
###P < 0.001 compared to the vehicle + 6-OHDA-treated group.
contains many good biological properties for human health
including antioxidant [14], anti-inflammation [15], and
anticancer [16] activities. Recently, it has been reported
that quercetin can pass through the blood-brain barrier
of in situ models [17]. In addition, quercetin exerts the
protectiveeffectinastrokemodelinducedbytransientglobal
ischemia[18].Quercetinsignificantlyprotectedtheneuronal
cells from the oxidative stress-induced neurodegeneration
in Alzheimer’s disease [19], decreased lipid peroxidation,
improved the activity of catalase and superoxide dismutase
[20] and also prevented glutathione depletion [21]. Previous
study showed that the EGb761, a standardized extract from
the herbal medicine Ginkgo biloba, contains a high amount
of quercetin and exhibits the neuroprotective effect against
oxidativedamageinducedby6-OHDA[22].Moreover,itwas
found that quercetin attenuated the neuronal death in the
hippocampus resulting in improved learning and memory in
arm maze test [23]. Therefore, these pieces of evidence point
out the possibility that quercetin might exert an influence on
the central nervous system.
Based on the antioxidant and anti-inflammatory actions
of QC, we hypothesized that QC might mitigate the neu-
rotoxicity and cognitive impairment in an animal model
of PD induced by 6-OHDA. To elucidate this issue, the
current study aimed to determine the effects of QC on
spatial memory and neuron density in the hippocampus of
an animal model of PD induced by 6-OHDA. In addition,
the possible underlying mechanism was also investigated.
2.MaterialsandMethods
2.1. Animals. Adult male Wistar rats (10 weeks old) were
obtained from the National Laboratory Animal Center,
Salaya, Nakhon Pathom. The animals weighted between 200
to 250grams at the beginning of experiment. They were
housed 5 per cage and maintained in 12:12 light:dark cycle
0
5
10
15
20
25
Retention time (s)
∗
∗∗
#
#
#
##
####
Control
Vehicle + 6-OHDA
QC100 + 6-OHDA
QC200 + 6-OHDA
QC300 + 6-OHDA
L-dopa + 6-OHDA
Vitamin C+ 6-OHDA
7days after lesion14days after lesion
Figure 2: Effect of quercetin on retention time in the Morris water
maze test in an animal model of Parkinson’s disease induced by 6-
OHDA. Data were expressed as mean ± S.E.M. for 8 rats in each
group.∗P < 0.05;∗∗P < 0.01 compared to control;#P < 0.05;##P <
0.01 compared to the vehicle + 6-OHDA-treated group.
and given access to food and water ad libitum. The exper-
iments were performed to minimize animal suffering in
accordance with the internationally accepted principles for
laboratory use and care of European Community (EEC
directive of 1986; 86/609/EEC). The experimental protocols
were approved by the Institutional Animal Care and Use
Committee.
2.2. Drugs and Chemicals. Quercetin was purchased from
Sigma-Aldridge Company. All chemical substances used in
this study were analytical grade. Both Levodopa (L-dopa),
a standard drug used for PD treatment, and vitamin C,
an antioxidant possessing the neuroprotective and cognitive
enhancingeffects,wereusedaspositivecontrolsinthisstudy.
Normalsalinesolution(NSS)wasusedasvehiclethroughout
the study. All administered substances were freshly prepared.
2.3. Experimental Protocol. The animals were randomly
divided into 7 groups (n = 8 animals/group) as described
below.
Group I: Control. Animals were not treated with any
drugs or injections.
Group II: Vehicle plus 6-OHDA injection. Animals were
treated with NSS once daily via oral route for 14 days before
and 14 days after the injection of 6-OHDA into the right
substantia nigra.
Groups III–V: Rats were treated with quercetin at various
doses ranging from 100, 200, and 300mg/kg BW once daily
via oral route for 14 days before and 14 days after the
injection of 6-OHDA into the right substantia nigra.
Groups VI–VII: Rats were orally treated with either
Levodopa (L-dopa; 5mg/kg BW) or vitamin C (200mg/kg)
once daily for 14 days before and 14 days after the lesion of
the right substantia nigra induced by 6-OHDA.
Each rat in all groups, except the control group, was
orally given the same volume of substance suspension for
14 days. The rats were then subjected to 6-OHDA injection

Page 3

Evidence-Based Complementary and Alternative Medicine3
0
10
20
30
40
50
60
70
80
90
Density of neuron (cells/225µm2)
∗∗∗
##
∗∗∗
∗∗∗
∗∗∗
##
∗∗∗ ∗∗∗
Vehicle + 6-OHDAQC100 + 6-OHDAQC200 + 6-OHDA
QC300 + 6-OHDAL-dopa + 6-OHDAVitamin C + 6-OHDA
Control
50µm
50µm
50µm
50µm
50µm
50µm
50µm
(a)
Vehicle + 6-OHDA QC100 + 6-OHDAQC200 + 6-OHDA
QC300 + 6-OHDAL-dopa + 6-OHDA Vitamin C + 6-OHDA
Control
∗∗
#
∗
##
∗
##
∗∗
#
##
∗∗∗
0
10
20
30
40
50
60
70
80
Density of neuron (cells/225µm2)
50µm
50µm
50µm
50µm
50µm
50µm
50µm
(b)
Control
Vehicle + 6-OHDA
QC100 + 6-OHDA
QC200 + 6-OHDA
QC300 + 6-OHDA
L-dopa + 6-OHDA
Vitamin C+ 6-OHDA
∗∗
##
∗∗∗
∗∗∗
∗∗∗
#
∗∗∗ ∗∗∗
Vehicle + 6-OHDA QC100 + 6-OHDAQC200 + 6-OHDA
QC300 + 6-OHDAL-dopa + 6-OHDA Vitamin C + 6-OHDA
Control
0
10
20
30
40
50
60
70
80
Density of neuron (cells/225µm2)
50µm
50µm50µm
50µm
50µm
50µm
50µm
(c)
130
135
140
145
150
155
160
Control
Vehicle + 6-OHDA
QC100 + 6-OHDA
QC200 + 6-OHDA
QC300 + 6-OHDA
L-dopa + 6-OHDA
Vitamin C+ 6-OHDA
Vehicle + 6-OHDAQC100 + 6-OHDA QC200 + 6-OHDA
QC300 + 6-OHDA L-dopa + 6-OHDAVitamin C + 6-OHDA
Control
∗∗
#
#
Density of neuron (cells/225µm2)
50µm
50µm
50µm
50µm
50µm
50µm
50µm
(d)
Figure 3: Effect of quercetin on neuron density in various subregions of the hippocampus (a = CA1; b = CA2; c = CA3; d = Dentate gyrus)
in an animal model of Parkinson’s disease induced by 6-OHDA. Scale bars 50µm, the magnification was 40X. Data were expressed as mean
± S.E.M. for 8 rats in each group.∗P < 0.05;∗∗P < 0.01;∗∗∗P < 0.001 compared to control;#P < 0.05;##P < 0.01 compared to the vehicle +
6-OHDA-treated group.

Page 4

4Evidence-Based Complementary and Alternative Medicine
Control
Vehicle + 6-OHDA
QC100 + 6-OHDA
QC200 + 6-OHDA
QC300 + 6-OHDA
L-dopa + 6-OHDA
Vitamin C+ 6-OHDA
0.01
0.02
0.03
0.04
0.05
0.06
0.07
AChE (µmole/min.mg protein)
0
#
Figure 4: Effect of quercetin on the activity of acetylcholinesterase
(AChE) in the hippocampus in an animal model of Parkinson’s
diseaseinducedby6-OHDA.Datawereexpressedasmean ±S.E.M.
for 8 rats in each group.#P < 0.05 compared to the vehicle + 6-
OHDA-treated group.
0
0.5
1
1.5
2
2.5
MDA (nmole/mg protein)
∗∗
#
Control
Vehicle + 6-OHDA
QC100 + 6-OHDA
QC200 + 6-OHDA
QC300 + 6-OHDA
L-dopa + 6-OHDA
Vitamin C+ 6-OHDA
Figure 5: Effect of quercetin on the level of malondialdehyde
(MDA) in the hippocampus in an animal model of Parkinson’s
diseaseinducedby6-OHDA.Datawereexpressedasmean ±S.E.M.
for 8 rats in each group.∗∗P < 0.01 compared to control;#P < 0.05
compared to the vehicle + 6-OHDA-treated group.
using the stereotaxic apparatus. The 6-OHDA infusion was
performed using a Hamilton syringe and rats were allowed
to recover after the lesion for 6 days. They were then tested
for spatial memory using Morris water maze test at 7 and 14
days after 6-OHDA injection. At the end of experiment, rats
weresacrificedandtheirbrainswereremovedandthedensity
of survival neurons, the activity of acetylcholinesterase
(AChE), and scavenger enzymes including superoxide dis-
mutase (SOD), catalase (CAT), and glutathione peroxidase
(GPx) in the hippocampus were determined. Moreover, the
malondialdehyde (MDA) level in the mentioned area was
also determined.
2.4. The Administration of 6-OHDA. 6-OHDA was prepared
as described previously by Ferro and coworkers [24]. Rats
Control
Vehicle + 6-OHDA
QC100 + 6-OHDA
QC200 + 6-OHDA
QC300 + 6-OHDA
L-dopa + 6-OHDA
Vitamin C+ 6-OHDA
0
2
4
6
8
10
12
14
16
18
20
SOD (units/mg protein)
##
##
##
Figure6:Effectofquercetinontheactivityofsuperoxidedismutase
(SOD) in the hippocampus in an animal model of Parkinson’s
diseaseinducedby6-OHDA.Datawereexpressedasmean ±S.E.M.
for 8 rats in each group.##P < 0.01 compared to the vehicle + 6-
OHDA-treated group.
Control
Vehicle + 6-OHDA
QC100 + 6-OHDA
QC200 + 6-OHDA
QC300 + 6-OHDA
L-dopa + 6-OHDA
Vitamin C+ 6-OHDA
0
0.5
1
1.5
2
2.5
3
GPx (units/mg protein)
∗∗
##
##
##
##
#
Figure 7: Effect of quercetin on the activity of glutathione
peroxidase (GPx) in the hippocampus in an animal model of
Parkinson’s disease induced by 6-OHDA. Data were expressed as
mean ± S.E.M. for 8 rats in each group.∗∗P < 0.01 compared to
control;#P < 0.05;##P < 0.01; compared to the vehicle + 6-OHDA-
treated group.
were anesthetized with 50mg/kg sodium thiopental (i.p.),
each animal was mounted on a stereotaxic stand, the skin
overlying the skull was cut to expose the skull, and the
coordinates for the substantia nigra par compacta (SNpc)
were accurately measured (anteroposterior −0.5mm from
bregma, mediolateral 2.1mm from midline, and dorsoven-
tral −7.7mm from the skull). The 6µg of 6-OHDA, which
dissolved in 2µL 0.2% ascorbic acid saline, were perfused
into SNpc through a 30-gauge stainless needle. After the
surgery, animals were allowed to recover from anesthesia and
then placed in their cages.
2.5. Morris Water Maze Test. The water maze consisted of
a metal pool (170cm in diameter × 58cm tall) filled with
tap water (25◦C, 40cm deep). The pool was divided into

Page 5

Evidence-Based Complementary and Alternative Medicine5
0
5
10
15
20
25
CAT (units/mg protein)
#
#
Control
Vehicle + 6-OHDA
QC100 + 6-OHDA
QC200 + 6-OHDA
QC300 + 6-OHDA
L-dopa + 6-OHDA
Vitamin C+ 6-OHDA
Figure 8: Effect of quercetin on the activity of catalase (CAT) in the
hippocampus in an animal model of Parkinson’s disease induced by
6-OHDA. Data were expressed as mean ± S.E.M. for 8 rats in each
group.#P < 0.05 compared to the vehicle + 6-OHDA-treated group.
6-OHDA injection
Oxidative stress
MDA
SOD, GPx, CAT
Quercetin
MDA
SOD, GPx, CAT
Neurodegeneration
Mitigates the
neurodegeneration
Improves spatial
working memory
Cognitive impairment
SNpc
Mesolimbic pathway
Reverses
Attenuates
Figure 9: Hypothetical diagram of the possible underlying mech-
anism for the cognitive enhancing effect of quercetin on an animal
model of Parkinson’s disease induced by 6-OHDA.
4 quadrants (NE, NW, SE, and SW) by two imaginary
lines crossing the center of the pool. The water surface was
covered with nontoxic milk. The removable platform was
placed below the water level at the center of one quadrant.
For each animal, the location of the invisible platform was
placed at the center of one quadrant and remained there
throughout training. The rats must memorize the platform
location in relation to various environmental cues because
the location of the escape platform in and outside the pool
is not visible. Each rat was gently placed in the water facing
the wall of the pool from one of the four starting points
(N, E, S, or W) along the perimeter of the pool, and the
animal was allowed to swim until it found and climbed
onto the platform. During training session, the rat was
gently placed on the platform when it could not reach the
platform in 60 seconds. In either case, the subject was left on
theplatformfor15secondsandthenremovedfromthepool.
The time for animals to climb on the hidden platform was
recorded as escape latency or acquisition time. In order to
determine the capability of the animals to retrieve and retain
information, the platform was removed 24hr later and the
ratwasreleased into the quadrantdiagonallyopposite to that
which contained the platform. Time spent in the region that
previously contained the platform was recorded as retention
time. In each trial, the animal was quickly dried with towel
before being returned to the cage [25].
2.6.
sodium pentobarbital (50mg/kg BW), the brain fixation was
carried out by transcardial perfusion with a fixative solution
containing 4% paraformaldehyde in 0.1M phosphate buffer
pH 7.3. After the perfusion, the brains were removed and
stored overnight in a fixative solution that is used for per-
fusion. They were then infiltrated with 30% sucrose solution
at, and kept at, 4◦C. The specimens were frozen rapidly and
the coronal sections at 30µM thick were prepared using
cryostat. All sections were rinsed in the phosphate buffer
and picked up on slides coated with 0.01% aqueous solution
of a high molecular weight poly L-lysine.
Histological Procedure. Following anesthesia with
2.7. Nissl Staining. The duplicate coronal sections of brains
were stained with 0.75% cresyl violet, dehydrated through
graded alcohols (70, 95, 100% 2x), placed in xylene, and
mounted using DPX.
2.8. Determination of Acetylcholinesterase, Superoxide Dismu-
tase, Glutathione Peroxidase and Catalase Activity, and the
Malondialdehyde Level. The rats were divided into various
groups as previously described in the experimental protocol.
After the last dose of administration, all rats were sacrificed.
The hippocampus of the lesion side was isolated and
prepared as a homogenate to determine the MDA level and
the activities of AChE, SOD, CAT, and GPx. MDA was
estimatedbydeterminingtheaccumulationofthiobarbituric
acid reactive substances (TBARS) [26] in the hippocampal
homogenate whereas the activities of acetylcholinesterase
[27], superoxide dismutase [28], glutathione peroxides [29],
and catalase [30] were determined using the colorimetric
method.
2.9. Statistical Analysis. Data are expressed as means ±
S.E.M. and were analyzed statistically by one-way ANOVA,
followed by post hoc (LSD) test. The results were considered
statistically significant at P value < 0.05.
3.Results
3.1. Morris Water Maze Test. Cognitive impairment is
commonly observed in Parkinson patients. Therefore, we
examined the effect of quercetin on spatial memory by using
the Morris water maze test and recorded escape latency
and retention time as the indices. The results were shown
in Figures 1 and 2. Figure 1 demonstrates that 6-OHDA