Neurobiology of Learning and Memory 94 (2010) 293302

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Neurobiology of Learning and Memory

journal homepage: www.elsevier.com/locate/ynlme

Ameliorative effect of quercetin on memory dysfunction

in streptozotocin-induced diabetic rats
Pravinkumar Bhutada a,*, Yogita Mundhada a, Kuldeep Bansod a, Chetan Bhutada a, Santosh Tawari a,
Pankaj Dixit b, Dharmendra Mundhada a
a
Agnihotri College of Pharmacy, Pharmacology Division, Bapuji Wadi, Sindhi (Meghe), Wardha, 442 001 Maharashtra, India
b
College of Pharmacy, IPS Academy, Hukmakhedi, Knowledge Village, Rajendranagar, AB Road, Indore, 452 012 Madhya Pradesh, India

a r t i c l e i n f o a b s t r a c t

Article history: Diabetes-related cognitive dysfunction is a consequence of changes within the central nervous system
Received 18 February 2010 that are secondary to chronic hyperglycemia, oxidative stress, and cholinergic dysfunction, and probably
Revised 24 June 2010 therefore anti-diabetics, anti-oxidants, and acetylcholine esterase (AChE) inhibitors were found to have
Accepted 29 June 2010
benecial effects in animal models. Quercetin, a bioavonoid widely distributed in the plants is reported
Available online 8 July 2010
to be a potent anti-diabetic, anti-oxidant, AChE inhibitor, and memory enhancer. Therefore, we screened
its inuence against diabetes-induced cognitive dysfunction in streptozotocin-induced diabetic rats using
Keywords:
Morris water and elevated plus maze (EPM) paradigms. Thirty days after diabetes induction rats exhib-
Diabetes mellitus
Donepezil
ited marked and persistent hyperglycemia, weight loss, higher escape latency during training trials and
Elevated plus maze reduced time spent in target quadrant in probe trial in Morris water maze test, and increased escape
Morris water maze latency in EPM task. Treatment with quercetin (520 mg/kg, p.o., twice daily, 30 days) in streptozoto-
Open eld test cin-induced diabetic rats prevented the changes in blood glucose, body weight, and performance in Mor-
Quercetin ris water and elevated plus maze tasks. In another set of experiment, quercetin (40 mg/kg, p.o., twice
Vitamin C daily) treatment during training trials (3135 days) markedly decreased escape latency and increased
time spent in target quadrant during Morris water maze task. This treatment also decreased blood glu-
cose levels, but had no inuence on body weights. These effects were comparable to vitamin C
(100 mg/kg, twice daily, 30 days) and donepezil (3 mg/kg day 31day 35, during training trials), and
devoid of any motor decit and anxiety-like effect when tested in open eld test. In conclusion, quercetin
may provide a new potential option for prevention of the cognitive dysfunction in diabetes.
2010 Elsevier Inc. All rights reserved.

1. Introduction Kumagai, 1999; McCall, 2004). The cerebrovascular changes

Diabetes mellitus (DM) is the most common endocrine disorder
characterized by increased blood glucose levels resulting from
defective insulin secretion, resistance to insulin action or both.
DM is often associated with severe complications, and there is an
increasing appreciation that cognitive function declines in DM
(Reynolds et al., 2010; van den Berg, Kloppenborg, Kessels,
Kappelle, & Biessels, 2009; Wrighten, Piroli, Grillo, & Reagan,
2009). Diabetic children and adults exhibit reduced psychomotor
efciency, cognitive exibility, and rapid information-processing
(Brand, Biessels, de Haan, Kappelle, & Kessels, 2005; Rovet, 2000).
A growing body of literature suggest that diabetes-related cogni-
tive dysfunction is largely a consequence of changes within the
central nervous system (CNS) that are secondary to chronic hyper-
glycemia (Biessels, van der Heide, Kamal, Bleys, & Gispen, 2002;
* Corresponding author. Address: Agnihotri College of Pharmacy, Department of
Pharmacology, Wardha, 442 001 Maharashtra, India. Fax: +91 7152 232548.
E-mail address: psbhutada@live.com (P. Bhutada).
(Knopman et al., 2001; Mankovsky, Metzger, Molitch, & Biller,
1996; Wong et al., 2002), oxidative stress (Beckman & Ames,
1998; Rosen et al., 2001), increased advanced glycation end prod-
ucts (Vitek et al., 1994; Vlassara, 1997), and impairments in cere-
bral insulin signaling systems (Hoyer, 1998) are thought to be
the underlying causes for diabetic dementias. Moreover, anti-oxi-
dants (Hasanein & Shahidi, 2010), antihyperglycemics and insulin
sensitizing agents (Ryan et al., 2006) are reported to reduce cogni-
tive dysfunction in diabetic condition. However, at present, no spe-
cic treatments are available for the management and/or
prevention of cognitive dysfunction in DM (Biessels, Kerssen,
de Haan, & Kappelle, 2007).
Quercetin (3,30 ,40 ,5,7-pentahydroxyavone) is a bioavonoid
widely distributed in the plant kingdom. It is found in frequently
consumed foods, including apples, berries, onion, tea, red wine,
nuts, seeds, and vegetables that represent an integral part of the
human diet. Quercetin is reported to have many benecial effects
on human health, including cardiovascular protection, anticancer
activity, antiulcer activity, antiallergic activity, cataract prevention,

1074-7427/$ - see front matter 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.nlm.2010.06.008
294 P. Bhutada et al. / Neurobiology of Learning and Memory 94 (2010) 293302
antiviral activity, and anti-inammatory activity (Bronner &
Landry, 1985; de Whalley, Rankin, Hoult, Jessup, & Leake, 1990;
Kaul, Middleton, & Ogra, 1985). Quercetin also exhibits anti-dia-
betic activity in experimental animals (Sanders, Rauscher, &
Watkins, 2001; Vessal, Hemmati, & Vasei, 2003; Zunino, 2009).
Quercetin is an excellent free radical scavenging anti-oxidant
(Boots, Haenen, & Bast, 2008) and reported to reduce the risk for
oxidative stress related chronic diseases like diabetes (Hollman &
Katan, 1999; Skibola & Smith, 2000). In addition, quercetin is re-
ported to increase the insulin sensitivity (Kannappan & Anuradha,
2009). Quercetin is also reported to reduce diabetic complications,
like, neuropathy, nephropathy, retinopathy, heart diseases, and
depression (Anjaneyulu & Chopra, 2004a, 2004b; Anjaneyulu, Cho-
pra, & Kaur, 2003; Annapurna, Reddy, Akondi, & Rao, 2009; Kato
et al., 2008; Kumar, Annamalai, & Thakur, 2009; Ramana et al.,
2006; Valensi et al., 2005). Moreover, several experimental investi-
gations showed the potential of quercetin against cognitive decit
in various animal models (Kumar, Sehgal, Kumar, Padi, & Naidu,
2008; Liu, Yu, & Ning, 2006; Pu et al., 2007; Singh, Naidu, &
Kulkarni, 2003; Sun et al., 2007; Tota, Awasthi, Kamat, Nath, &
Hanif, 2010; Yao, Han, Zhang, & Yang, 2010). However, there are
no reports concerning the inuence of quercetin against diabe-
tes-induced cognitive dysfunction. Therefore, the present study
was designed to investigate the protective role of quercetin on cog-
nitive dysfunction in streptozotocin (STZ)-induced diabetic rats.
2. Materials and methods
2.1. Subjects
Adult male Wistar rats born and reared in the Animal House of
the Agnihotri College of Pharmacy, Wardha, from a stock originally
purchased from Shree Farms, Bhandara, India were used in the
present study. Young healthy male rats (200225 g) were group
housed (three per cage) and maintained at 23 2 C under
12:12 h light (08:0020:00 h)/dark cycle with free access to rodent
chow and tap water. The animal studies were approved by the
Institutional Animal Ethics Committee (IAEC), constituted for the
purpose of control and supervision of experimental animals by
Ministry of Environment and Forests, Government of India, New
Delhi, India. Animals were naive to drug treatments and experi-
mentation at the beginning of all studies. All tests were conducted
between 08:00 and 13:00 h.
2.2. Drugs and solutions
Quercetin, streptozotocin (SigmaAldrich Co., St. Louis, MO),
vitamin C (Sisco Research Laboratories, Mumbai, India) and
donepezil hydrochloride (Eisai, Mumbai, India) were used in pres-
ent study. All the drugs were dissolved in double distilled water
except streptozotocin, which was dissolved in sodium citrate buf-
fer (pH = 4.4). Drug solutions were prepared fresh and their doses
are expressed in terms of their free bases.
2.3. Experimental induction of diabetes
Diabetes was induced in rats by using an earlier reported meth-
od (Umathe, Kochar, Jain, & Dixit, 2009). In brief, STZ was dissolved
in 0.1 M sodium citrate buffer, pH 4.4 and administered at the dose
of 60 mg/kg through i.p. route. Streptozotocin-treated rats received
5% of glucose solution instead of water for 24 h after injection of
streptozotocin in order to reduce death due to hypoglycemic
shock. Blood samples were taken from the tail vein 48 h after
streptozotocin or vehicle injection to measure blood glucose levels.
Only animals with fasting blood glucose levels over 250 mg/dl
were considered diabetic and used for the further study.
2.4. Treatments schedule
2.4.1. Treatment 1
After diabetes induction, separate groups of rats (n = 12) were
orally administered quercetin (5, 10, or 20 mg/kg), vitamin C
(100 mg/kg), or vehicle twice daily (08:0020:00 h) for next
30 days (day 130), and at the end of 30 days rats were subjected
to Morris water or elevated plus maze test. Similar treatments
were given to control (non-diabetic) rats. The learning and mem-
ory was evaluated (day 3136 for Morris water maze test and
day 3132 for elevated plus maze test).
2.4.2. Treatment 2
In another set of experiment, 30 days after induction of diabe-
tes, rats (n = 6) were orally administered quercetin (10, 20, or
40 mg/kg), vitamin C (100 mg/kg), donepezil (3 mg/kg), or vehicle
twice daily (08:0020:00 h) during trials for next 5 days (day 31
35) by p.o. route. Similar treatments were given to control (non-
diabetic) rats. After these treatments, rats were subjected to Morris
water maze test. The learning and memory was evaluated during
day 3136.
2.5. Assessment of cognitive function
2.5.1. Morris water maze test
Cognitive function of rats was assessed by using Morris water
maze test as described earlier (Morris, Garrud, Rawlins, & OKeefe,
1982; Tiwari, Kuhad, Bishnoi, & Chopra, 2009a; Tiwari, Kuhad, &
Chopra, 2009b; Tuzcu & Baydas, 2006). The test apparatus was cir-
cular water tank (180 cm in diameter and 60 cm high) made up of
dark gray plastic that was partially lled with water (24 1 C).
Full cream milk (1.5 l) was used to render the water opaque. The
pool was divided virtually into four equal quadrants, labeled A
BCD. A platform (12.5 cm in diameter and 38 cm high) was
placed in one of the four maze quadrants (the target quadrant)
and submerged 2.0 cm below the water surface (see Fig. 1). The
platform remained in the same quadrant during the entire experi-
ment. The rats were required to nd the platform using only distal
spatial extra-maze cues available in the testing room. The cues
were maintained constant throughout the testing. The rats re-
ceived four consecutive daily training trials for 5 days (day 3135
after diabetes induction), with each trial having a ceiling time of
90 s and a trial interval of approximately 30 s. The rat had to swim
until it climbed onto the platform submerged underneath the
water. After climbing onto the platform, the animal remained there
for 20 s before the commencement of the next trial. The escape
platform was kept in the same position relative to the distal cues.
If the rat failed to reach the escape platform within the maximally
allowed time of 90 s, it was gently placed on the platform and al-
lowed to remain there for the same amount of time. The time to
reach the platform (latency in seconds) was measured.
To test possible decits in sensorimotor processes, rats were
tested in the water maze with a visible platform on a new location
on the nal day of training (Kamal, Biessels, Duis, & Gispen, 2000).
The test with the visual platform does not require special orienta-
tion (McNamara & Skelton, 1993) and was used to show possible
decits in sensorimotor processes. For the test, target platform
was placed inside the pool 1 cm above the water line. Rats were al-
lowed to swim for 60 s. Time to reach the platform was recorded as
escape latency. After completion of the last trial, rats were gently
dried with a towel, kept warm for an hour and returned to their
home cages.
 P. Bhutada et al. / Neurobiology of Learning and Memory 94 (2010) 293302
N possibly as a consequence of learning acquisition and retention. If
Platform
Quadrant B Quadrant A
W E
Quadrant C Quadrant D
S
Fig. 1. Schematic diagram of tank and site of the platform.
2.5.2. Memory consolidation test
A probe trial was performed wherein the extent of memory con-
solidation was assessed (Tiwari et al., 2009a, 2009b; Tuzcu & Bay-
das, 2006). The time spent in the target quadrant indicates the
degree of memory consolidation that has taken place after learn-
ing. The probe trial was conducted on day 36, wherein the individ-
ual rat was placed into the pool as in the training trial, except that
the hidden platform was removed from the pool. The time spent in
target quadrant was measured for 90 s. In probe trial, each rat was
placed at a start position directly opposite to where the platform
was located.
2.5.3. Elevated plus maze (EPM) test
Cognitive behavior was evaluated by using the EPM learning
task, which measures spatial long-term memory (Reddy &
Kulkarni, 1998). Transfer latency (the time in which the animal
moves from the open arm to the enclosed arm) was utilized as
an index of learning and memory processes. The principle in this
experiment is based upon the aversive behavior of rodents to open
and high spaces. The animals dislike open and high spaces and
move from them to an enclosed arm, the protected areas of the
maze.
The apparatus was designed as described earlier (Umathe, Bhut-
ada, Jain, Dixit, & Wanjari, 2008). In brief, rats were tested on an
elevated plus maze that consisted of two Plexiglas (painted black)
opposite facing open arms (50  10 cm) and two closed arms
(50  10  40 cm), connected by a central platform (10  10 cm).
The whole maze was raised 50 cm above the oor. Rats were tested
on the plus maze in a room with low, indirect incandescent light-
ing (40 lux) in a sound-attenuated chamber.
The procedure was performed as described earlier (Hlink &
Krejc, 1998, 2000, 2002). The animals were randomly assigned
to the different experimental and control groups. In the acquisition
session (on day 31), each rat was gently placed at the distal end of
an open arm of the apparatus facing away from the central plat-
form and the time it took to move from the open arm to either
of the enclosed arms (transfer latency) was recorded. Training (re-
peated exposure of animal to open arms) shortens this parameter,
295
the rat did not enter the enclosed arm within 90 s, it was excluded
from further experimentation. The criterion of an animals entry
into the enclosed arm was crossing with all four legs of an imagi-
nary line separating the enclosed arm from the central space. After
entering the enclosed arm, the rat was allowed to move freely in
the maze regardless of open and enclosed arms for 10 s. Then,
the rat was returned to its home cage. The retention session fol-
lowed 24 h after the acquisition session (on day 32). The rats were
put into the open arm and the transfer latency was recorded again.
The maze was cleaned after each rat.
2.6. Open eld test
Ten minutes after the EPM test on day 32, the rats were trans-
ferred to an open-eld apparatus, measuring 60  40  28 cm, with
the oor divided into 12 squares. The open eld session lasted for
5 min and during this time, an observer, recorded the number of
crossing (horizontal activity) and rearing (vertical activity) re-
sponses manually. The test was carried out to rule out the possibility
of motor disabilities and any inuence of anxiety on escape latency.
2.7. Evaluation of blood glucose levels and body weight
Blood glucose levels were measured with a portable glucometer
(Ascensia, Bayer, USA). In brief, blood sample was withdrawn from
the 6 h fasted rats using tail vein rupture method, and drop of
blood was placed on the glucometer strip loaded into the glucom-
eter for blood glucose determination. During the experiment, blood
glucose levels and body weights were veried in the interim (10,
20, 30 and 36 days after the beginning of treatment).
2.8. Statistical analysis
Results were expressed as mean S.E.M. The data were ana-
lyzed by two-way or one-way analysis of variance (ANOVA) fol-
lowed by Bonferroni and Tukeys multiple comparison test
respectively or Student-t test. Statistical signicance was consid-
ered at P < 0.05.
3. Results
3.1. Effect of quercetin on blood glucose levels and body weight
As shown in Table 1, 38 days after streptozotocin injection,
plasma glucose levels were highly elevated in diabetic rats as
compared to non-diabetic control rats. Also, there was a marked
decline in the body weights of streptozotocin-treated rats as com-
pared to age-matched control rats. Chronic quercetin (5, 10, and
20 mg/kg) treatment signicantly and dose dependently reduced
the blood glucose levels [F(6, 32) = 53.53, P < 0.0001] and increased
the body weight of diabetic rats [F(6, 32) = 18.71, P < 0.0001].
Chronic treatment with vitamin C (100 mg/kg) did not inuence
(P > 0.05) the blood glucose levels as well as the body weights as
compared to vehicle treatment in diabetic rats. Treatment with
quercetin (20 mg/kg) in non-diabetic animals had no signicant
inuence on blood glucose levels and body weight (P > 0.05) com-
pared to vehicle treatment in non-diabetic rats (Table 1).
Moreover, in the treatment schedule 2 [day 3135, quercetin
(20 and 40 mg/kg)] signicantly reduced the blood glucose levels
[F(6, 32) = 106.2, P < 0.0001] but did not inuence the body weight
of diabetic rats (P > 0.05) as shown in Table 2. During trials treat-
ment with quercetin (10 mg/kg) and vitamin C (100 mg/kg) had
no inuence (P > 0.05) on the blood glucose levels and the body
weight compared to vehicle treatment in diabetic rats. Further,
296 P. Bhutada et al. / Neurobiology of Learning and Memory 94 (2010) 293302

Table 1
Effect of chronic treatment with quercetin (30 days) on body weights and blood glucose levels (mean S.E.M. of 56 observations) in the different groups of rats at the onset and
at the end of the experiments.

Group Treatment Body weight (g) Blood glucose (mg/dl)

Onset of study End of study Onset of study End of study
ND control Vehicle (1 ml/kg) 217.0 3.39 251.8 10.46 102.83 4.37 111.6 5.35
ND + Que 20 Que (20 mg/kg) 216.17 4.69 247.17 7.19 106.33 3.18 103.33 5.33
D control Vehicle (1 ml/kg) 213.67 3.74 189.17 7.26* 104.17 4.80 401.0 10.71*
@ @
D + Que 5 Que (5 mg/kg) 218.0 4.59 256.8 6.30 102.17 5.07 281.6 20.65
@ @
D + Que 10 Que (10 mg/kg) 211.83 4.55 264.5 7.24 110.5 5.76 234.67 20.22
@ @
D + Que 20 Que (20 mg/kg) 218.33 2.12 271.67 8.29 101.17 3.51 195.17 13.92
D + Vit C Vit C (100 mg/kg) 221.6 1.29 190.2 9.03 102.83 5.00 365.0 25.75

ND control: non-diabetic control; ND + Que 20: non-diabetic quercetin (20 mg/kg) treated; D control: diabetic control; D + Que 5: diabetic quercetin (5 mg/kg) treated;
D + Que 10: diabetic quercetin (10 mg/kg) treated; D + Que 20: diabetic quercetin (20 mg/kg) treated; D + Vit C: diabetic vitamin C (100 mg/kg) treated.
*
P < 0.001 vs. non-diabetic control group;
@
P < 0.001 vs. diabetic control group (One-way ANOVA followed by Tukeys post hoc test).

Table 2
Effect of quercetin treatment during training trail (5 days) on body weights and blood glucose levels (mean S.E.M. of 56 observations) in the different groups of rats at the onset
and at the end of the experiments.

Group Treatment Body weight (g) Blood glucose (mg/dl)

Onset of study End of study Onset of study End of study
ND control Vehicle (1 ml/kg) 208.8 3.43 272.8 10.07 109.8 8.26 101.8 5.83
ND + Que 40 Que (40 mg/kg) 219.5 3.39 259.17 18.57 101.67 5.26 105.17 5.43
D control Vehicle (1 ml/kg) 218.33 3.82 173.17 6.68* 103.83 5.39 400.33 8.61*
D + Que 10 Que (10 mg/kg) 217.8 4.93 182.2 5.50 101.8 6.38 361.4 12.89
D + Que 20 Que (20 mg/kg) 215.0 3.42 179.33 7.96 99.67 5.87 329.83 19.12#
D + Que 40 Que (40 mg/kg) 218.33 2.12 179.33 4.75 102.33 4.80 314.33 12.83@
D + Vit C Vit C (100 mg/kg) 213.0 5.40 168.4 6.62 107.4 6.4 389.4 14.54
D + Donep Donep (3 mg/kg) 214.16 3.89 171.5 5.43

ND control: non-diabetic control; ND + Que 40: non-diabetic quercetin (40 mg/kg) treated; D control: diabetic control; D + Que 10: diabetic quercetin (10 mg/kg) treated;
D + Que 20: diabetic quercetin (20 mg/kg) treated; D + Que 40: diabetic quercetin (40 mg/kg) treated; D + Vit C: diabetic vitamin C (100 mg/kg) treated; D + Donep: diabetic
donepezil (3 mg/kg) treated.
*
P < 0.001 vs. non-diabetic control group.
#
P < 0.01 vs. diabetic control group (One-way ANOVA followed by Tukeys post hoc test).
@
P < 0.001 vs. diabetic control group (One-way ANOVA followed by Tukeys post hoc test).

donepezil (3 mg/kg) treatment during day 3136 did not inuence

(P > 0.05) the body weight as compared to vehicle treatment in
diabetic rats. Treatment with quercetin (40 mg/kg) in non-diabetic
animals had no signicant inuence (P > 0.05) on blood glucose
levels and body weight compared to vehicle treatment in
non-diabetic rats (Table 2).

3.2. Effects of quercetin on diabetes-induced cognitive dysfunction

3.2.1. Effects of chronic quercetin treatment on performance of Morris

water maze test
The cognitive function was assessed in the Morris water maze
test. The mean escape latency for the trained rats decreased over
the course of the 20 learning trials in all groups (Fig. 2). Control
diabetic rats (vehicle treated) exhibited signicantly (P < 0.05)
higher escape latency on day 2, 3, 4, and 5 (day 3235 after diabe-
tes induction) during training trials compared to non-diabetic
vehicle treated rats. Two-way repeat measure ANOVA revealed
that chronic quercetin treatment signicantly inuenced escape
Fig. 2. Effect of chronic treatment with quercetin (30 days) on the performance of
latency in diabetic rats; treatment effect [F(6, 128) = 9.73, spatial memory acquisition phase in Morris water maze. Each value represents
P < 0.0001] and time effect [F(4, 128) = 157.3, P < 0.0001]. Further, mean S.E.M. of 56 observations. P < 0.05, P < 0.01 and P < 0.001 vs. saline
post hoc test revealed that quercetin (10 and 20 mg/kg) signi- treatment in non-diabetic group. #P < 0.05, &P < 0.01 and @P < 0.001 vs. saline
treatment in diabetic group (two-way repeat measure ANOVA followed by
cantly (P < 0.05) decreased escape latency in diabetic rats com-
Bonferroni multiple comparison test). ND control: non-diabetic control; ND + Que
pared to vehicle treatment in diabetic rats on day 2, 3, 4 and 5. 20: non-diabetic quercetin (20 mg/kg) treated; D control: diabetic control; D + Que
Chronic treatment with vitamin C showed similar results. Treat- 5: diabetic quercetin (5 mg/kg) treated; D + Que 10: diabetic quercetin (10 mg/kg)
ment with quercetin (20 mg/kg) in non-diabetic animals had no treated; D + Que 20: diabetic quercetin (20 mg/kg) treated; D + Vit C: diabetic
signicant (P > 0.05) inuence on escape latency compared to vehi- vitamin C (100 mg/kg) treated.

cle treatment in non-diabetic rats (Fig. 2).

The mean latencies in all groups were similar in the rst trial, was unaffected by the hyperglycemia, whereas the STZ treated
which suggests that their motor performance (ability to swim) group used more time than controls in subsequent trials. All
 P. Bhutada et al. / Neurobiology of Learning and Memory 94 (2010) 293302
groups were submitted to a test of their ability to escape to a vis-
ible platform. The performance of all the groups in the trial with
the visible platform was not signicantly (P > 0.05) different (Sup-
plementary material). These observations suggest that there were
no any changes in sensorimotor function due to long-standing
STZ-induced diabetes.
The probe trial data of the Morris water maze study was ana-
lyzed as per the method reported by Bolding and Rudy (2006), as
this method is capable of expressing memory retention in terms
of selective search behavior, which specically correlates with
learning impairment. The data from probe trail is depicted in
Fig. 3AC, which provides three representations of selective perfor-
Fig. 3. Effect of chronic treatment with quercetin (30 days) on water maze
performance in probe trial, (A) probe trial performance as measured by comparing
time spent in the target quadrant with an average of time spent in all three non-
target quadrants, P < 0.001, @P < 0.01, vs. time spent in non-target quadrant in
respective group (two-way ANOVA followed by Bonferronis post hoc test); (B)
probe trial performance as measured by comparing the time each rat spent in the
target quadrant with the time it spent in its next most preferred quadrant,

P < 0.001, @P < 0.01, vs. time spent in next preferred quadrant in respective group
(two-way ANOVA followed by Bonferronis post hoc test); (C) difference score index
of selective search. This measure was obtained by subtracting the time each rat
spent in its next most preferred quadrant from the time it spent in the target
quadrant. Selective search is indicated by the magnitude of the difference score. A
score of 0 indicates no selective search. @P < 0.01 vs. non-diabetic control group;
#
P < 0.01, $P < 0.05, vs. diabetic control group (one-way ANOVA followed by Tukeys
post hoc test). Each bar represents mean S.E.M. of 56 observations. ND control:
non-diabetic control; ND + Que 20: non-diabetic quercetin (20 mg/kg) treated; D
control: diabetic control; D + Que 5: diabetic quercetin (5 mg/kg) treated; D + Que
10: diabetic quercetin (10 mg/kg) treated; D + Que 20: diabetic quercetin (20 mg/
kg) treated; D + Vit C: diabetic vitamin C (100 mg/kg) treated.
297
mance on the retention test. Fig. 3A is a standard measure and
compares time spent in the target quadrant against the average
time spent in other three quadrants. Two-way ANOVA indicated
a signicant preference for training quadrant [F(1, 64) = 104,
P < 0.0001] for various treatments [F(6, 64) = 2.255, P = 0.049].
Bonferronis post hoc test further indicated that the target quad-
rant preference was completely lost is diabetic animals (P > 0.05).
The treatment with quercetin (10, 20 mg/kg) signicantly pre-
vented the memory impairment as indicated by the increase in
the time spent in training quadrant (P < 0.001). These effects of
quercetin were similar to that shown by vitamin C treatment
(P < 0.01). However, per se quercetin was not able to inuence
the extent of selective search observed in non-diabetic control ani-
mals (P > 0.05).
However, it is reported that this standard measure of selective
search underestimates forgetting of place information e.g.,
although the time an individual rat spent in the training quadrant
might exceed chance (20 s), it might also have spent as much or
more time in another quadrant, in which case the individual rat
did not display selective search of the training quadrant. To con-
sider this outcome, the time each rat spent in the target quadrant
was compared with the time it spent in its other most preferred
quadrant (Fig. 3B).
Two-way ANOVA indicated a signicant difference between
time spent in training quadrant and the next preferred quadrant
[F(1, 64) = 65.44, P < 0.0001]. Bonferronis post hoc test further
indicated that there was no signicant difference between time
spent in target quadrant against that of next preferred quadrant
in diabetic animals (P > 0.05). The treatment with quercetin (10
or 20 mg/kg) signicantly prevented the memory impairment as
indicated signicant difference in the time spent in training quad-
rant against that of next preferred quadrant (P < 0.001). Interest-
ingly, vitamin C treatment was unable to show signicant
difference in selective search (P > 0.05). Similarly, per se quercetin
was not able to inuence the extent of selective search (P > 0.05).
Fig. 3C compares the groups using a difference score derived
from the data presented in Fig. 3B. An analysis of variance revealed
differences among the groups retention intervals [F(6, 38) = 4.698,
P = 0.0016]. Tukeys post hoc test indicated that the diabetes signif-
icantly impaired the memory retention as indicated by a signi-
cantly different (P < 0.01) lower score as compared to non-
diabetic control group. This impairment was signicantly pre-
vented by quercetin treatment at 10 mg/kg (P < 0.05), and 20 mg/
kg (P < 0.01) dose. Quercetin at 5 mg/kg had no inuence
(P > 0.05) in diabetic animals and at 20 mg/kg in non-diabetic ani-
mals. Vitamin C at the given dose had no inuence on the selective
search score (P > 0.05).
3.2.2. Effects of quercetin treatment during trials (treatment schedule
2) on performance of Morris water maze test
Two-way repeat measure ANOVA revealed that treatment with
quercetin and donepezil during training trials, signicantly inu-
enced the escape latency; treatment effect [F(7, 160) = 7.60,
P < 0.0001] and time effect [F(4, 160) = 159.4, P < 0.0001] (Fig. 4).
Further, post hoc test revealed that quercetin (40 mg/kg) and
donepezil (3 mg/kg), signicantly reduced (P < 0.05) escape latency
on day 5, whereas vitamin C (100 mg/kg) treatment failed to inu-
ence the same in diabetic rats (P > 0.05). Treatment with quercetin
(40 mg/kg) in non-diabetic had no signicant inuence (P > 0.05)
on escape latency as compared to vehicle treated rats.
The data from probe trail is depicted in Fig. 5AC. Two-way AN-
OVA indicated a signicant preference for training quadrant
[F(1, 80) = 47.76, P < 0.0001] for various treatments [F(7, 80) =
7.53, P = 0.0494]. Bonferronis post hoc test further indicated that
the target quadrant preference was completely lost is diabetic ani-
mals (P > 0.05). The acute treatment with quercetin (40 mg/kg) sig-
298 P. Bhutada et al. / Neurobiology of Learning and Memory 94 (2010) 293302
Fig. 4. Effect of quercetin treatment during training trials (5 days) on the
performance of spatial memory acquisition phase in Morris water maze. Each
value represents mean S.E.M. of 56 observations. P < 0.01 and P < 0.001 vs.
non-diabetic control group. #P < 0.05, &P < 0.01 and @P < 0.001 vs. diabetic control
group (two-way repeat measure ANOVA followed by Bonferroni multiple compar-
ison test). ND control: non-diabetic control; ND + Que 40: non-diabetic quercetin
(40 mg/kg) treated; D control: diabetic control; D + Que 10: diabetic quercetin
(10 mg/kg) treated; D + Que 20: diabetic quercetin (20 mg/kg) treated; D + Que 40:
diabetic quercetin (40 mg/kg) treated; D + Vit C: diabetic vitamin C (100 mg/kg)
treated; D + Donep: diabetic donepezil (3 mg/kg) treated.
nicantly prevented the memory impairment as indicated by the
increase in the time spent in training quadrant (P < 0.001). These
effects of quercetin were similar to that shown by donepezil treat-
ment (P < 0.01). However, per se quercetin was not able to inu-
ence the extent of selective search observed in non-diabetic
control animals (P > 0.05). Vitamin C treatment and quercetin
treatment in lower doses (10 and 20 mg/kg) had no inuence on
the selective search (P > 0.05).
Further, when the data was considered for next preferred quad-
rant, two-way ANOVA indicated a signicant difference between
time spent in training quadrant and the next preferred quadrant
[F(1, 80) = 10.12, P = 0.0021]. Bonferronis post hoc test further
indicated that there was no signicant difference between time
spent in target quadrant against that of next preferred quadrant
in diabetic animals (P > 0.05). The acute treatment with quercetin
(10, 20 and 40 mg/kg) or vitamin C or donepezil did not alter the
time spent in target quadrant as compared to that of next preferred
quadrant in diabetic rats (P > 0.05).
Finally, an analysis of variance revealed differences among the
groups retention intervals [F(7, 47) = 5.212, P = 0.0003]. Tukeys
post hoc test indicated that the diabetes signicantly impaired
the memory retention as indicated by a signicantly different
(P < 0.01) lower score as compared to non-diabetic control group.
This impairment was signicantly prevented by quercetin treat-
ment at 40 mg/kg (P < 0.05). Quercetin at 10, 20 mg/kg, vitamin C
and donepezil had no inuence (P > 0.05) in diabetic animals.
3.2.3. Effects of quercetin on performance of EPM test
The effects of various chronic treatments in diabetic and non-
diabetic rats on mean transfer latencies in the EPM test are pre-
sented in Fig. 6. One-way ANOVA revealed that chronic treatment
with quercetin (5, 10, and 20 mg/kg) and vitamin C (100 mg/kg),
had no effect on the transfer latencies of the rst day (acquisition)
compared to that of the vehicle treated diabetic animals (P > 0.05).
Student-t test revealed that transfer latency on day 2nd (retention)
signicantly reduced when compared with transfer latency on day
Fig. 5. Effect of quercetin treatment during training trial (5 days) on time spent in
target quadrant during probe trial in Morris water maze, (A) probe trial
performance as measured by comparing time spent in the target quadrant with
an average of time spent in all three non-target quadrants, P < 0.001, @P < 0.01, vs.
time spent in non-target quadrant in respective group (two-way ANOVA followed
by Bonferronis post hoc test); (B) probe trial performance as measured by
comparing the time each rat spent in the target quadrant with the time it spent in
its next most preferred quadrant, P < 0.001, @P < 0.01, vs. time spent in next
preferred quadrant in respective group (Two-way ANOVA followed by Bonferronis
post hoc test); (C) difference score index of selective search. This measure was
obtained by subtracting the time each rat spent in its next most preferred quadrant
from the time it spent in the target quadrant. Selective search is indicated by the
magnitude of the difference score. A score of 0 indicates no selective search.

P < 0.01 vs. non-diabetic control group; @P < 0.05, $P < 0.05, vs. diabetic control
group (One-way ANOVA followed by Tukeys post hoc test). Each bar represents
mean S.E.M. of 56 observations. ND control: non-diabetic control; ND + Que 40:
non-diabetic quercetin (40 mg/kg) treated; D control: diabetic control; D + Que 10:
diabetic quercetin (10 mg/kg) treated; D + Que 20: diabetic quercetin (20 mg/kg)
treated; D + Que 40: diabetic quercetin (40 mg/kg) treated; D + Vit C: diabetic
vitamin C (100 mg/kg) treated; D + Donep: diabetic donepezil (3 mg/kg) treated.
1st in non-diabetic vehicle treated rats (P = 0.0002), whereas there
was no difference in transfer latency tested on day 1 and 2 in vehi-
cle treated diabetic rats (P = 0.0821). Further, one-way ANOVA re-
vealed that quercetin and vitamin C treatment had signicant
inuence on transfer latency tested on day 2 (retention)
[F(6, 39) = 4.674, P = 0.0015]. Post hoc test revealed that quercetin
(10 and 20 mg/kg) and vitamin C (100 mg/kg) signicantly de-
creased transfer latency as compared to vehicle treated diabetic
rats on day 2 (P < 0.05), whereas quercetin (5 mg/kg) had no signif-
icant inuence on transfer latency. Treatment with quercetin
(20 mg/kg) in non-diabetic animals had no signicant inuence
 P. Bhutada et al. / Neurobiology of Learning and Memory 94 (2010) 293302 299

Fig. 6. Effect of chronic treatment with quercetin (30 days) on the performance of spatial memory acquisition and retention phase in EPM. Each bar represents mean S.E.M.
of 56 observations. P < 0.05, P < 0.001 vs. non-diabetic control rats during respective sessions (one-way ANOVA followed by Tukeys post hoc test); @P = 0.0002 vs. non-
diabetic control during acquisition (t-test). &P < 0.05, #P < 0.01 vs. diabetic control group during retention (one-way ANOVA followed by Tukeys post hoc test). ND control:
non-diabetic control; ND + Que 20: non-diabetic quercetin (20 mg/kg) treated; D control: diabetic control; D + Que 5: diabetic quercetin (5 mg/kg) treated; D + Que 10:
diabetic quercetin (10 mg/kg) treated; D + Que 20: diabetic quercetin (20 mg/kg) treated; D + Vit C: diabetic vitamin C (100 mg/kg) treated.

on transfer latency (P > 0.05) compared to vehicle treatment in the locomotor activity [F(6, 39) = 0.3452, P = 0.9076] and number
non-diabetic rats in both the sessions (Fig. 6). of rearing [F(6, 39) = 0.1353, P = 0.9907] (Fig. 7).

3.3. Effects of quercetin on locomotor activity in open eld test 4. Discussion

An animal model of DM has a great deal of potential to alter mo-
tor and anxiety-like behaviors that would alter escape latency.
Thus, the effects of quercetin on cognitive dysfunction in diabetic
rats may be related to motor disabilities and anxiety. To rule out
this possibility open eld study was carried out. One-way ANOVA
revealed that none of the treatment had signicant inuence on
Fig. 7. Effect of quercetin on locomotor activity and number of rearings in open
eld test. Each bar represents mean S.E.M. of 56 observations (One-way ANOVA
followed by Tukeys post hoc test). ND control: non-diabetic control; ND + Que 20:
non-diabetic quercetin (20 mg/kg) treated; D control: diabetic control; D + Que 5:
diabetic quercetin (5 mg/kg) treated; D + Que 10: diabetic quercetin (10 mg/kg)
treated; D + Que 20: diabetic quercetin (20 mg/kg) treated; D + Vit C: diabetic
vitamin C (100 mg/kg) treated.
The present study showed that experimental diabetes of
30 days impaired spatial learning and memory in the Morris water
maze task, which was substantiated by the results in the EPM task.
This impairment was avoided as well as reversed by treatment
with quercetin, a bioavonoid.
In the present study, the Morris water maze and elevated plus
maze test were used for the assessment of learning and memory.
Decreased escape latency in Morris water maze task in repeated
trials demonstrates intact learning and memory function. The
EPM test is suggested to be a simple method for the evaluation
of learning and memory processes. Since the animals are able to
remember the conguration of the open and enclosed arms, they
escape from the unsafe open arm more rapidly on the second trial.
It is possible to evaluate the fear motivated learning, which under-
lies the transfer latency procedure in this test. Shortened transfer
latency on second days trial in rats and mice is used as a parame-
ter for retention or consolidation of memory, while treatment of
drugs prior to rst day may also be utilized for acquisition related
action of drugs (Itoh, Nabeshima, & Kameyama, 1990; Sharma &
Kulkarni, 1992).
Morris water maze performance in diabetic rats was severely
impaired as compared with non-diabetic rats, conrming earlier
ndings (Biessels et al., 1998). The current study explored those
ndings demonstrating that diabetes reduced learning and mem-
ory performance. Furthermore, the present ndings indicate that
the impaired performance of diabetic rats is related to cognitive
impairment rather than to sensorimotor decits (Biessels et al.,
1998), since performance of diabetic rats were similar to non-dia-
betic rats in the task with the visible platform. In addition, in the
rst trial the mean escape latencies in all the groups were similar,
implying that their motor performance (ability to swim) was unaf-
fected by the persistent hyperglycemia and quercetin treatment.
Similarly, elevated plus-maze performance in diabetic rats was
also severely impaired. This test also revealed that diabetes re-
duced learning and memory performance. Further, open eld test
observations indicated that the impaired performance of diabetic
rats is related to cognitive dysfunction rather that thygmotaxic
behavior, since the number of locomotor counts and rearing of
300 P. Bhutada et al. / Neurobiology of Learning and Memory 94 (2010) 293302
diabetic rats were similar to non-diabetic rats ruled out such
possibilities.
Streptozotocin-induced diabetes is a well-documented model of
experimental diabetes. STZ produced signicant weight loss and
treatment with quercetin restored the same, which is in accor-
dance with earlier reports (Anjaneyulu & Chopra, 2004a, 2004b;
Hasanein & Shahidi, 2010; Vishwakarma, Sonawane, Rajani, &
Goyal, 2010). The benecial effects of quercetin observed in the
present study could be multivariate. It is reported that streptozoto-
cin-diabetes provides a relevant example of endogenous chronic
oxidative stress due to the resulting hyperglycemia (Low, Nickan-
der, & Tritschler, 1997). The roles of oxidative stress in nerve dam-
age have been studied extensively in experimental diabetes and
diabetic patients (Baynes, 1991). However, oxidative stress may
also contribute to the cognitive dysfunction during hyperglycemia.
Oxidative damage to the synapses in the rat cerebral cortex and
hippocampus is reported to contribute to the decit of cognitive
functions (Fukui, Onodera, Shinkai, Suzuki, & Urano, 2001; Gispen
& Biessels, 2000). Therefore, anti-oxidants might be of use in the
prevention of the neurodegeneration and cognitive dysfunctions
associated with diabetes (Hasanein & Shahidi, 2010). In present
study also chronic treatment with vitamin C protected the cogni-
tive dysfunction in diabetic rats. Interestingly, Tota et al. (2010) re-
ported that quercetin (510 mg/kg) dose dependently restored the
malondialdehyde and reduced glutathione levels in brain homoge-
nates of intracerebral STZ administered mice. Similar results were
observed by Kumar, Seghal, Naidu, Padi, and Goyal (2007) in col-
chicines-induced memory dysfunction in rats at higher doses
(2040 mg/kg). In one of the studies, quercetin (1050 mg/kg) also
reversed cognitive dysfunction and increase in thiobarbituric acid
reactive substances (TBARS) levels, and decline in forebrain total
glutathione, superoxide dismutase (SOD) and catalase levels by
chronic ethanol administration in mice (Singh et al., 2003). There-
fore in present study, quercetin might have protected diabetes-
associated cognitive dysfunction by reducing oxidative stress in
diabetic rats.
Most of the diabetic complications including cognitive impair-
ments are caused due to protracted hyperglycemia (Biessels
et al., 2007; Tuzcu & Baydas, 2006). Ryan et al. (2006) have re-
ported that antihyperglycemics and insulin sensitizers reduce cog-
nitive dysfunction in diabetic condition. In present study, quercetin
treatment signicantly reduced blood glucose levels. Earlier re-
ports also indicated anti-diabetic activity of quercetin via variety
of mechanisms (Coskun, Kanter, Korkmaz, & Oter, 2005; Vessal
et al., 2003). Therefore, the restoration of cognitive function
observed in diabetic rats may be due to the ability of quercetin
to reduce hyperglycemia.
Cholinergic neurotransmission plays a crucial role in regulating
learning, memory, and cortical organization of movement
(Mesulam, Guillozet, Shaw, & Levey, 2002). Literature has demon-
strated one of the most important mechanisms responsible for cor-
rect cholinergic function is performed by enzyme acetylcholine
esterase (AChE) that hydrolyses the acetylcholine (Appleyard,
1994). In addition to their role in cholinergic transmission, cho-
linesterases may also play a role during morphogenesis and neuro-
degenerative diseases (Layer, Alber, & Sporns, 1987; Reyes et al.,
1997). Interestingly, some studies have found an increased AChE
activity in brain of diabetics, which might be associated to cogni-
tive impairments (Kuhad, Sethi, & Chopra, 2008; Sanchez-Chavez
& Salceda, 2000; Schmatz et al., 2009). This is also supported by
our observation that donepezil, an AChE inhibitor, improved the
diabetes-induced cognitive dysfunction. Furthermore, the potent
inhibition of AChE by quercetin is widely reported (Tota et al.,
2010). Hence, the inhibitory inuence of quercetin against diabe-
tes-induced cognitive impairment might also involve AChE
inhibition.
An augmented activity of the hypothalamopituitaryadrenal
axis resulting in exaggerated glucocorticoid secretion is repeatedly
described in patients with DM and in animal models of diabetes
(Repetto et al., 2010; Reynolds et al., 2010). Recently, Stranahan
et al. (2008), reported that accelerated cognitive aging in diabetic
rats is prevented by lowering corticosterone levels. Moreover, it
is suggested that strategies targeted at lowering cortisol action
may be useful in ameliorating cognitive decline in individuals with
DM (Reynolds et al., 2010). Interestingly, quercetin is reported to
reduce the stress-induced elevation of corticosterone in rat brain
and serum (Haleagrahara, Radhakrishnan, Lee, & Kumar, 2009;
Kawabata, Kawai, & Terao, 2010). Therefore, the inhibitory inu-
ence of quercetin on diabetes-induced cognitive dysfunction may
also involve anti-stress effect i.e. modulation of corticosterone
levels.
In conclusion, quercetin treatment ameliorated cognitive dys-
function in the diabetic rats, which may nd clinical application
in treating neuronal decit in the diabetic patients. The protective
actions of quercetin on diabetic dysfunction may be attributed to
its multiple pleiotropic effects like anti-oxidant, antihyperglyce-
mic, and anticholinesterase activity.
Acknowledgments
The authors would like to thank Pandit shree Shankarprasadji
Agnihotri, President and Mr. Sachin Agnihotri, Chairman, Jai
Mahakali Shikshan Sanstha, Wardha, for funding the research
under the head of Excellence in Research and Academics.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.nlm.2010.06.008.
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