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Editor-in-Chief: John M. PezzutoPharm Biol, 2014; 52(12): 1558–1569
! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.908395
ORIGINAL ARTICLE
The protective effects of Cyperus rotundus on behavior and cognitivefunction in a rat model of hypoxia injury
Dhas Jebasingh1, Dhas Devavaram Jackson2, S. Venkataraman1, Ernest Adeghate3, and Bright Starling Emerald3
1Department of Pharmacology, CL Baid Metha Foundation for Pharmaceutical Education and Research, Thoraipakkam, Chennai, Tamil Nadu,
India, 2Padmavathi College of Pharmacy, Dharmapuri, Tamil Nadu, India, and 3Department of Anatomy, College of Medicine and Health Sciences,
United Arab Emirates University, Al Ain, United Arab Emirates
Abstract
Context: Hypoxia injury (HI) with its long-term neurological complications is one of the leadingcauses of morbidity and mortality in the world. Currently, the treatment regimens for hypoxiaare aimed only at ameliorating the damage without complete cure. The need, therefore, fornovel therapeutic drugs to treat HI continues.Objective: This study investigates the protective effects of the ethanol extract of Cyperusrotundus L. (Cyperaceae) (EECR), a medicinal plant used in Ayurvedic traditional medicineagainst sodium nitrite-induced hypoxia in rats.Materials and methods: We have evaluated the protective effect of 200 and 400 mg/kg of EECRagainst sodium nitrite-induced hypoxia injury in rats by assessing the cognitive functions,motor, and behavioral effects of EECR treatment along with the histological changes inthe brain. By comparing the protective effects of standard drugs galantamine, a reversiblecholinesterase inhibitor and pyritinol, an antioxidant nootropic drug against sodium nitrite-induced hypoxia in rats, we have tested the protective ability of EECR.Results: EECR at doses of 200 and 400 mg/kg was able to protect against the cognitiveimpairments, and the locomotor activity and muscular coordination defects, which are affectedby sodium nitrite-induced hypoxia injury in rats.Conclusion: Based on our results, we suggest that the medicinal herb C. rotundus possesses aprotective effect against sodium nitrite-induced hypoxia in rats. Further studies on theseprotective effects of EECR may help in designing better therapeutic regimes for hypoxia injury.
Keywords
EECR, galantamine, neuroprotection, pyritinol,sodium nitrite
History
Received 31 December 2013Revised 10 March 2014Accepted 23 March 2014Published online 15 July 2014
Introduction
Hypoxic injury (HI) is a life threatening condition in which
oxygen delivery is inadequate to meet the metabolic demands
of tissues. HI, with its long-term neurological complications,
is one of the leading causes of morbidity and mortality in
the world (Lawn et al., 2005; Rees et al., 2008). It is the
third most common cause of death next to coronary heart
disease and cancer worldwide. According to the World Heart
Federation, every year 6 million people die from stroke
(http://www.world-heart-federation.org/cardiovascular-health/
stroke). Death from hypoxia injury is projected to rise to
6.5 million by 2015 in the world (Strong et al., 2007). It was
suggested that the cause of death in more than 87% of
patients with hypoxia injury is due to cerebral ischemia
(Rosamond et al., 2008), which also leads to delayed neuronal
death resulting in significant morbidity with problems
of cognition, memory, and behavioral deficits (Volpe &
Petito, 1985).
Although our understanding of the cellular and biochem-
ical changes that occur after acute and chronic hypoxia has
increased significantly, the various categories of drugs
currently used to treat hypoxic brain injury including calcium
channel blockers (nifedipine), cholinesterase enzyme inhibi-
tors (galantamine and donepezil), nootropic agents (pirace-
tam), anti-epileptic agents (felbamate), and antidepressants
(fluoxetine) are aimed at slowing the progression without
cure. Hence, the search for an ideal drug to cure HI continues
and has also been extended to herbal drugs as a better
alternative to synthetic drugs.
Cyperus rotundus L. (Cyperaceae) is a well-known
Ayurvedic plant drug which has been shown to have anti-
inflammatory and wound healing (Puratchikody et al.,
2006), hepatoprotective (Kumar & Mishra, 2005), antidiar-
rheal (Uddin et al., 2006), and antioxidative (Yazdanparast &
Ardestani, 2007) activities. Studies have also shown that it
possesses antimalarial and antihyperlipidemic effects (Mengi
& Patel, 2008). Cyperus rotundus is also used to treat central
nervous system (CNS) disorders like loss of memory,
depression, Parkinson disease, and epilepsy (Lee et al.,
2010; Sharma et al., 2001). Although some of these properties
have been scientifically evaluated, the protective effect of
C. rotundus against HI is not well understood.
Correspondence: Dr. Bright Starling Emerald, Department of Anatomy,College of Medicine and Health Sciences, UAE University, PO Box17666, Al Ain, United Arab Emirates. E-mail: [email protected]
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In a recent study, we have evaluated the physiochemical
properties and toxicological effects of the ethanol extract
(EECR) of C. rotundus. It was found to have phenols, tannins,
glycosides, and flavonoids and safe up to 2000 mg/kg body
weight in Wistar rats (Jebasingh et al., 2012). The present
study evaluated the protective effect of C. rotundus against
sodium nitrite-induced hypoxia using EECR. By comparing
EECR with galantamine (a reversible cholinesterase inhibitor)
and pyritinol (an antioxidant nootropic), the drugs presently
used for the treatment of HI, we have assessed the learning,
memory, and behavioral deficits produced by sodium nitrite.
Materials and methods
Plant materials
The fresh tubers of C. rotundus were collected from
Kanyakumari district of Tamil Nadu during the months of
November and December 2008. The plant was identified and
authenticated by Prof. P. Jayaraman, Director, Plant Anatomy
Research Centre, Tambaram, Chennai, India, and a voucher
specimen (PARC/2008/140) was deposited at the Department
of Pharmacology, CL Baid Metha Research Foundation,
Chennai, for further reference.
Extraction and preparation of test sample
Freshly collected tubers of C. rotundus, were washed, shade-
dried, powdered, and soaked in chloroform for 48 h. The
resulting extract was filtered, distilled, and extracted
with chloroform. The resulting marc was extracted with
alcohol exhaustively and the EECR was prepared as a
suspension in 1% sodium carboxy methyl cellulose
(SCMC). It was analyzed by HPLC and found to have
13 peaks (Jebasingh et al., 2012).
Pharmacological study: animals
Inbred male Wistar rats weighing between 150 and 180 g
were received from the Committee for the Purpose of Control
and Supervision on Experimentation on Animals (CPCSEA)
approved animal house of Mohammed Sathak A. J. College
of Pharmacy, Chennai, India. All the experimental protocols
were approved by the Institutional Animal Ethical Committee
(IAEC) (Ref no. AJ/IAEC/10/05). Rats were housed at
25 ± 1 �C with a relative humidity of 55 ± 5% and were fed
with a standard pellet diet and water ad libitum. They were
maintained under a 12 h light/dark cycle. The animals were
acclimatized to laboratory conditions for 1 week prior to the
initiation of the study.
Grouping
The animals were weighed, numbered, and divided into five
groups of six. Sodium nitrite, which reduces the oxygen
carrying capacity of the blood by changing normal hemoglo-
bin to methemoglobin, was used to induce hypoxia. It was
given intraperitoneally (i.p.) daily for 30 d, at 60 mg/kg
with or without other drugs (Abdel-Baky et al., 2010).
The standard drugs galantamine, a reversible cholinesterase
inhibitor and pyritinol, an antioxidant nootropic drug, were
used as a positive control. Drugs were administered per
os (p.o.).
The animals were grouped as follows:
Group I animals received 1% SCMC at a dose of 10 ml/kg,
p.o. (vehicle control).
Group II animals received sodium nitrite 60 mg/kg, i.p.
(negative control).
Group III animals received pyritinol 100 mg/kg, p.o. +
galantamine 0.5 mg/kg, p.o. + sodium nitrite
60 mg/kg, i.p. (Dimitrova & Getova-Spassova,
2006) (positive control).
Group IV animals received EECR 200 mg/kg, p.o. + sodium
nitrite 60 mg/kg, i.p. (Jebasingh et al., 2012).
Group V animals received EECR 400 mg/kg, p.o. + sodium
nitrite 60 mg/kg, i.p. (Jebasingh et al., 2012).
We also tested the effect of EECR 200 mg/kg, p.o.; EECR
400 mg/kg; pyritinol 100 mg/kg, p.o, and galantamine 0.5 mg/
kg alone and did not see any significant change in brain
morphology, toxicity or behavior of rats (Jebasingh et al.,
2012).
The cognitive, behavioral, and physical effects of sodium
nitrite-induced hypoxia and the ameliorating effects of test and
standard drugs were evaluated in rats using the Cooks pole
climbing apparatus, Morris water maze, actophotometer,
rotarod, elevated plus maze, and two compartment passive
avoidance apparatus. The responses of the animals were
recorded on days 1, 10, 20, and 30 of the experiment unless
otherwise explained.
Assessment of learning and memory using Cook’spole climbing apparatus
The learning and memory of the animals were evaluated by
assessing the conditioned avoidance response using the
Cooks pole climbing apparatus (Cook & Weidley, 1957).
Male Wistar rats were trained in such a way that the animal
had to climb the pole (shock free zone) within 30 s to avoid a
shock. The shock was preceded by a buzzer that lasted for
15 s. The animals were trained to climb the pole at the sound
of the buzzer (conditioned avoidance response). At particular
intervals, 20 trials were given for each animal and the shock
avoidance and mistakes were recorded. Trained animals were
then treated with the SCMC, test drugs, or standard drugs and
the conditioned avoidance responses were assessed.
Assessment of retention of learned behavior using theTwo Compartment Passive Avoidance test
The retention of learned behavior was assessed using the Two
Compartment Passive Avoidance test (Elrod & Buccafusco,
1988). The apparatus consists of a square box with a floor
grid of 50� 50 cm and wooden walls of 35 cm height.
This box was illuminated with 100 watts bulb. In the center
wall, there was an opening of 6� 6 cm, which leads to a
small (15� 15 cm) dark compartment provided with an
electrifiable floor (Hugo Sachs Electronics, Baden-
Wurttemberg, Germany). The animals were trained by placing
them in the illuminated chamber facing away from the
entrance to the dark compartment. The latency to enter the
dark compartment was recorded and a 1 mA foot shock was
given for a period of 2 s when the rat stayed for more than
5 s in the dark chamber. Then the animal was returned to the
cage. All the animals were trained for a week before testing
with the drug. About 24 h after the trial period, each animal
DOI: 10.3109/13880209.2014.908395 Protective effects of Cyperus rotundus in hypoxia injury 1559
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was placed again in the illuminated chamber as before
for a maximum period of 180 s. The transfer latency of the
animals (in seconds) to re-enter the dark compartment was
assessed.
Assessment of anxiety like behavior using elevatedplus maze
Elevated plus-maze (Pellow et al., 1985) was used to assess
the anxiolytic effect of EECR. The elevated plus maze
apparatus consisted of a central platform (10 cm� 10 cm)
connected to two open arms (50 cm� 10 cm) and two covered
(enclosed) arms (50 cm� 40 cm� 10 cm) and the maze was
elevated to a height of 50 cm from the floor. During the
experiment, each rat was placed at the end of an open arm,
facing away from the central platform and the time (in
seconds) spend in the open or closed arms and the number of
entries into the open or closed arms with all its four legs were
noted for the 5 min observation period.
Assessment of spatial learning using Morris watermaze
The water maze test measures the spatial learning and
memory of previously trained animals, which have learned to
find a platform (Morris, 1984). It consists of a circular tank
(100 cm diameter and 20 cm depth) with a circular white
platform of 9 cm diameter hidden 2 cm below the water level.
The water at 23 �C was made opaque by powdered milk
during the experiment. The animal was left at one of the four
assigned pole positions and the time taken for the animal
to reach the platform was noted. Each animal received
four consecutive trials per day with an inter-trial interval of
6–10 min for 3 d. After the trial period, the platform was
removed and the experiment was repeated. On the day of
experiment, the animal was placed in one of the four assigned
polar positions and the time taken by the animal to reach the
platform in the first 60 s was recorded.
Assessment of motor skill learning using rotarod
The motor coordination was evaluated using a rotarod
apparatus (Caston et al., 1995; Lalonde et al., 1995). The
apparatus consists of a horizontal metal rod with grip attached
to a motor, whose speed can be adjusted. The rod is at a
height of 50 cm above the table in order to discourage the
animals from jumping off the roller. Before the experiment,
all animals in each group were habituated to balance for 180 s.
The rotarod speed of 20 revolutions per min (rpm) was used
for the experiment. The time each animal was able to balance
in the rotating rod with the prescribed speed up to 180 s
in each experiment was recorded. The experiments were
repeated five times in one session.
Assessment of locomotor activity usingactophotometer
Actophotometer records the locomotor activity of the
animal (Turner, 1965). The actophotometer operates on
photoelectric cells which are connected to a counter and a
count is recorded when the beam of light falling on
the photocell is cut off by the movement of an animal.
The animals were kept individually in the cage for 5 min and
activity scores of each group of animals were recorded on
days 1, 10, 20, and 30.
Assessment of brain histology
After the stipulated period of exposure to the drugs or the
vehicle, the animals were sacrificed with an overdose of
sodium pentobarbital and the brains were dissected out and
fixed with 4% para-formaldehyde. They were dehydrated
using graded alcohol and embedded in wax. About 5 mm
coronal section was taken at bregma-4.16 (Paxinos & Watson,
1986). Sections were stained with hematoxylin and eosin and
the morphology of the hippocampus, cortex, thalamus and the
cerebellum was analyzed by light microscopy. The extent of
neuronal damage was scored blindly in six different regions
within the hippocampus, cortex, thalamus, and the cerebellum
according to a previously described method (van den Tweel
et al., 2005). Scores were from 0 to 3: 0¼ 91–100% of
neurons damaged, 1¼ 51–90% of neurons damaged, 2¼ 11–
50% of the neurons damaged, 3¼ less than 10% neuronal
damage. Six sections/rat were scored, averaged, and the
scores of six rats/treatment group were added to obtain the
final score.
Statistical analysis
All experimental data were expressed as mean ± S.E.M of six
animals in each group. The statistical analysis was carried out
using a one-way ANOVA with the Bonferroni correction post
hoc. Difference in the values at p50.05 was considered as
statistically significant.
Results
Evaluation of learning and memory using Cook’spole climbing apparatus
Learning and memory were evaluated on days 1, 10, 20, and
30 using Cook’s pole climbing apparatus. On day 1, there was
no significant difference between the sodium nitrite-admin-
istered Group II animals compared with those of the vehicle
control Group I animals or with those of any drug-treated
animal groups (III, IV, and V) in terms of the conditioned
avoidance responses.
On day 10 onwards, there was a significant difference
between the sodium nitrite-administered Group II animals
compared with those of the vehicle control Group I animals in
terms of the number of conditioned avoidance responses
that decreased significantly (Figure 1A). The positive control
Group III animals, which received the combination of
pyritinol and galantamine, showed a significant improvement
in learning and memory when compared with Group II
animals (p50.001). No significant differences in conditioned
avoidance responses were observed between 200 and 400 mg/
kg of EECR treatment groups on days 10, 20, and 30 in
conditioned avoidance responses (Figure 1A). Interestingly
both 200 and 400 mg/kg of EECR treatment Groups IV and V
also showed significant improvement of learning and memory
when compared with Group II animals (p50.001), which
was comparable with those of the positive control Group III
animals (Figure 1A).
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Compared with day 1, all the groups except the sodium
nitrite-treated Group II animals showed an increase in their
conditioned avoidance responses at days 10, 20, and 30
(Figure 1B). Only the sodium nitrite-treated Group II animals
showed a non-significant decrease in their conditioned
avoidance responses (Figure 1B).
Evaluation of retention of learned behavior using theTwo Compartment Passive Avoidance (TCPA) test
The effects of EECR on retention of learned behavior were
assessed using the TCPA test and the results are depicted
in Figure 2. On day 1, the sodium nitrite-treated Group II
animals had a lower response (dark to bright chamber) when
compared with the vehicle-treated control Group I animals
(p50.01; Figure 2A).
The positive control Group III animals, which received the
combination of pyritinol and galantamine, showed a signifi-
cant improvement in their transfer latency when compared
with the sodium nitrite-treated Group II animals from day 1 to
day 30 (p50.05 on day 1 and p50.001 from day 10 onwards;
Figure 2A). The EECR-treated Groups (IV and V animals)
also showed a faster response when compared with the
sodium nitrite-treated Group II animals with no significance
variation in transfer latency between them from day 10
onwards (p50.001; Figure 2A).
Compared with day 1, all the groups except the sodium
nitrite-treated Group II animals showed a faster response at
days 10, 20, and 30 (p50.001; Figure 2B). The sodium
nitrite-treated Group II animals showed either no change or
a significant decrease in their retention of learned behavior
as seen by the increased latency at day 20 (p50.05;
Figure 2B).
Evaluation of animal anxiety like behavior usingelevated plus maze
Elevated plus maze was used to evaluate the behavioral
parameters such as anxiety and exploratory activity and the
results are shown in Figure 3. On day 1, the sodium nitrite-
treated Group II animals did not show any changes in the
anxiety level or exploratory activity when compared with all
other groups (Figure 3A). There was no significant difference
in the number of entries into the open or closed arms between
the sodium nitrite-treated Group II animals and the drug-
treated Groups III, IV, and V on day 1 (Figure 3C).
Figure 2. Effect of EECR on retention of learned behavior using Two Compartment Passive Avoidance (TCPA) test. (A) Comparison among day 1, day10, day 20, and day 30. (A(a)) Group I versus Group II, (A(b)) Group II versus Groups III, IV, and V, and (B) within each group. *Significantdifference, *p50.05, **p50.01, ***p50.001. Group I, vehicle control; Group II, sodium nitrite-treated animals (negative control); Group III,pyritinol, galantamine, and sodium nitrite (positive control); Group IV, EECR 200 mg/kg and sodium nitrite; Group V, EECR 400 mg/kg and sodiumnitrite. Values are expressed as mean ± SEM from six male animals in each group.
Figure 1. Effect of EECR on learning and memory using Cook’s pole climbing apparatus. (A) Comparison among day 1, day 10, day 20, and day 30.(A(a)) Group I versus Group II, (A(b)) Group II versus Groups III, IV, and V and (B) within each group. * Significant difference, *p50.05, **p50.01,***p50.001. Group I, vehicle control; Group II, sodium nitrite-treated animals (negative control); Group III, pyritinol, galantamine, and sodium nitrite(positive control); Group IV, EECR 200 mg/kg and sodium nitrite; Group V, EECR 400 mg/kg and sodium nitrite. Values are expressed as mean ± SEMfrom six male animals in each group.
DOI: 10.3109/13880209.2014.908395 Protective effects of Cyperus rotundus in hypoxia injury 1561
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Figure 3. Effect of EECR on anxiety like behavior using elevated plus maze (A–D). The results on time spent in open and closed arms are shown in(A) and (B), while the results of open and closed arm entries are shown in (C) and (D). Values are expressed as mean ± SEM from six male animals ineach group. (A) and (C) Comparison among day 1, day 10, day 20, and day 30. (A(a)) Group I versus Group II, (A(b)) Group II versus Groups III,IV, and V, and (B) within each group, day 1 versus 10, 20, and 30 d. (C(a)) Group I versus Group II, (C(b)) Group II versus Groups III, IV, and V.(D) Within each group, day 1 versus 10, 20, and 30th days. *Significant difference, *p50.05, **p50.01, ***p50.001. Group I, vehicle control;Group II, sodium nitrite-treated animals (negative control); Group III, pyritinol, galantamine, and sodium nitrite (positive control); Group IV, EECR200 mg/kg and sodium nitrite; Group V, EECR 400 mg/kg and sodium nitrite.
1562 D. Jebasingh et al. Pharm Biol, 2014; 52(12): 1558–1569
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Compared with the sodium nitrite-treated Group II
animals, all the other groups showed significant decreases
in their anxiety and an increase in their exploratory activity
from day 10 onwards (p50.001; Figure 3A). The number of
entries into open arm increased non-significantly in Group III
animals on day 10 while it increased significantly on days 20
and 30 (Figure 3C). Compared with the sodium nitrite-treated
Group II animals the EECR-treated Groups IV and V animals
showed significant increases in the number of entries from
day 10 onwards (p50.05, Group V, on day 10 to p50.001 on
day 30; Figure 3C).
Compared with day 1, the sodium nitrite-treated Group II
animals showed a significant reduction in the amount of time
spent in the open arm at days 10, 20, and 30 (p50.001;
Figure 3B).
A significant reduction in the number of entries into
the open arm from day 1 to day 30 was also seen (p50.01;
Figure 3D). The positive control Group III animals, which
received the combination of pyritinol and galantamine
showed a non-significant increase in the number of open
arm entries (Figure 3D).
The 400 mg/kg EECR-treated animals also showed a non-
significant increase in the number of open arm entries
from day 1 to day 30 (Figures 3B and D). The 200 mg/kg of
the EECR-treated Group IV animals showed a significant
increase in the number of open arm entries on days 10 and 20,
which decreased on day 30 (p50.01 on day 10 to p50.001
on day 20; Figure 3B and D).
Animals in the drug-treated Groups (III, IV and V) spent
almost the same time in the open arms on all days with a
slight decrease in days 20 and 30, although this is higher
compared with Group II animals (Figure 3B).
Evaluation of spatial learning using Morris water maze
Using the water maze, we have evaluated the effect of EECR
in spatial learning and the results are given in Figure 4.
On day 1, there was no significant difference between the
Figure 3. Continued.
Figure 4. Effect of EECR on spatial learning using Morris water maze. (A) Comparison among day 1, day 10, day 20, and day 30. (A(a)) Group Iversus Group II, (A(b)) Group II versus Groups III, IV, and V, and (B) within each group. *Significant difference, *p50.05, **p50.01, ***p50.001.Group I, vehicle control; Group II, sodium nitrite-treated animals (negative control); Group III, pyritinol, galantamine, and sodium nitrite (positivecontrol); Group IV, EECR 200 mg/kg and sodium nitrite; Group V, EECR 400 mg/kg and sodium nitrite. Values are expressed as mean ± SEM from sixmale animals in each group.
DOI: 10.3109/13880209.2014.908395 Protective effects of Cyperus rotundus in hypoxia injury 1563
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sodium nitrite-administered Group II animals compared with
those of the vehicle control Group I animals or with those
of any drug-treated animal Groups (III, IV, and V) in terms
of the time taken by the animals to identify the platform
(Figure 4A). From day 10 onwards, the time spend in
identifying the platform to rest increased significantly in the
sodium nitrite-administered Group II animals compared with
those of the vehicle control Group I animals (p50.001;
Figure 4A). Compared with the sodium nitrite-administered
Group II animals, animals in Groups IV and V treatment with
200 and 400 mg/kg of EECR had a faster response time from
day 10 onwards in identifying a platform to rest (p50.001),
which was comparable with the positive control Group III
animals which received a combination of pyritinol and
galantamine (Figure 4A).
Except for the sodium nitrite-treated Group II animals,
spatial learning ability also improved in all the animal groups
from day 1 to day 30 (p50.001; Figure 4B). In contrast,
the sodium nitrite-treated Group II animals took more time to
identify a platform from day 10 onwards, which increased
significantly at days 20 and 30 (p50.001) suggesting a
reduction in spatial learning ability (Figure 4B).
Evaluation of motor coordination by rotarodperformance test
We have evaluated the motor coordination ability of the
animals by assessing their ability to balance, using a rotarod,
which in turn depends on the skeletal muscle contraction/
relaxation state and the results are given in Figure 5. There
was no significant difference between the sodium nitrite-
administered Group II animals with those of the vehicle
control Group I animals or with those of any drug-treated
animal Groups (III, IV, and V) on day 1 (Figure 5A).
From day 10 onwards, there was a significant decrease
in the balancing time in the sodium nitrite-administered
Group II animals compared with those of the vehicle control
Group I animals (Figure 5A; p50.001) or the positive control
Group III animals, which received a combination of pyritinol
and galantamine or the 200 and 400 mg/kg of EECR
treatment Groups IV and V animals (p50.001; Figure 5A).
In the case of sodium nitrite-treated Group II animals,
there was a gradual decrease in the balancing time from day 1
onwards. There was a significant reduction in the balancing
time towards the end of day 30 (p50.001; Figure 5B).
The balancing time was not significantly different between
days 1 and 30 in the control Group I animals, in the positive
control Group III, and in the EECR-treated Groups IV and V
animals, although a non-significant reduction in balancing
time was seen towards day 30 (Figure 5B).
Evaluation of locomotor activity by actophotometer
The locomotor activity was assessed using an actophotometer
and the results are shown in Figure 6. There was no significant
difference between the sodium nitrite-administered Group II
animals compared with those of the vehicle control Group I
animals or with those of the 200 and 400 mg/kg of EECR
treatment Groups IV and V on day 1 in their locomotor
activity. The positive control Group III animals, which
received a combination of pyritinol and galantamine showed
an increase in their locomotor activity on day 1 compared
with the sodium nitrite-administered Group II animals
(p50.01; Figure 6A).
Compared with the sodium nitrite-administered Group II
animals, the drug-treated Group III, IV, and V animals showed
a significant increase in their locomotor activity on days
20 and 30 (p50.001; Figure 6A).
The sodium nitrite-administered Group II animals showed
a significant reduction in the locomotor activity, at days 20
and 30 (p50.001) compared with day 1, although there was
an increase at day 10 (p50.01). The locomotor activity was
unchanged in the control Group I between day 1 and day 30.
The locomotor activities were comparable at days 1 and 30
Figure 5. Effect of EECR on motor coordination using a rotarod. (A) Comparison among day 1, day 10, day 20, and day 30. (A(a)) Group I versusGroup II, (A(b)) Group II versus Groups III, IV, and V, and (B) within each group, day 1 versus 10, 20, and 30 d. *Significant difference, *p50.05,**p50.01, ***p50.001. Group I, vehicle control; Group II, sodium nitrite-treated animals (negative control); Group III, pyritinol, galantamine, andsodium nitrite (positive control); Group IV, EECR 200 mg/kg and sodium nitrite; Group V, EECR 400 mg/kg and sodium nitrite. Values are expressedas mean ± SEM from six male animals in each group.
1564 D. Jebasingh et al. Pharm Biol, 2014; 52(12): 1558–1569
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in the positive control Group III animals as well as in the
400 mg/kg of the EECR-treated Group V animals. The
200 mg/kg dose of EECR Group IV animals showed a slight
decrease in their locomotor activity at day 30 when compared
with day 1 (p50.05; Figure 6B).
Morphological evidence of the protective effect ofEECR in the brain
We have assessed the morphological changes in the cortex,
hippocampus, thalamus, and cerebellum of the brain of
sodium nitrite-administered Group II animals compared with
those of the vehicle control Group I, drug-treated positive
control Group III, and EECR-treated Groups IV, and V
animals (Figures 7 and 8). The cortex, hippocampus,
thalamus, and cerebellum of the brain from the sodium
nitrite-administered Group II animals showed significant
morphological changes with pyknotic and rounded nuclei
and with fragmented dead neurons (Figures 7B, G, L, Q, and
8). Vacuolization was also seen especially in the cortex,
thalamus, and the hippocampus (Figure 7B, G, and L).
The neuronal layer also shrank considerably in the cortex and
hippocampal regions (Figure 7B and G). The Purkinje cells
in the cerebellum were replaced with vacuoles (Figure 7Q).
There was a significant protection against these changes in
the pyritinol and galantamine-treated positive control
Group III animals (Figure 7C, H, M, and R). EECR-treated
Groups IV and V animals also showed protection against these
morphological changes in all examined (cortex, thalamus,
hippocampus, and cerebellum) brain regions (Figure 7D, E, I,
J, N, O, S, and T). Qualitatively, there was more protection
against the morphological changes in the 400 mg/kg EECR-
treated Group V animals (Figure 7E, J, O, and T) compared
with the sodium nitrite-treated animals (Figure 8). This was
comparable with the positive control Group III animals
(Figure 7C, H, M, and R). The cortex and the hippocampus
were even comparable with those of the vehicle control group
(Figures 7A and F and 8).
Discussion
An earlier study on the physiochemical characteristics and
toxicological effects of the ethanol extract of C. rotundus
showed that C. rotundus contains phenols, tannins, glycoside,
Figure 6. Effect of EECR on the locomotoractivity using an actophtometer.(A) Comparison among day 1, day 10, day20, and day 30. (A(a)) Group I versus GroupII, (A(b)) Group II versus Groups III, IV, andV, and (B) within each group, day 1 versus10, 20, and 30 d. *Significant difference,*p50.05, **p50.01, ***p50.001. Group I,vehicle control; Group II, sodium nitrite-treated animals (negative control); Group III,pyritinol, galantamine, and sodium nitrite(positive control); Group IV, EECR 200 mg/kg and sodium nitrite; Group V, EECR400 mg/kg and sodium nitrite. Values areexpressed as mean ± SEM from six maleanimals in each group.
DOI: 10.3109/13880209.2014.908395 Protective effects of Cyperus rotundus in hypoxia injury 1565
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and flavonoids and was safe even at a dose of 2000 mg/kg
body weight in Wistar rats (Jebasingh et al., 2012). In the
present study, we have evaluated the protective effect of
C. rotundus against the sodium nitrite-induced hypoxia injury
in rats. Short-term, long-term, and open field studies in rats
have shown that sodium nitrite induces learning, memory, as
well as behavioral deficits (Hlinak et al., 1990; Koziar et al.,
1994). The sodium nitrite-induced deficits in learning,
memory and behavior seen in our study correlated well with
earlier reports. We have used standard drugs galantamine
and pyritinol as the positive control (Group III) for the
comparison of the protective effect of EECR. Pyrinitol is a
well-known nootropic agent and was shown to affect the
behavior in correlation with the spatial memory in rodents
(Valzelli & Tomasikova, 1985). Galantamine is a reversible
acetyl cholinesterase inhibitor and has been shown to act as an
allosterically potentiating ligand on nicotine a4/b2 subtype
acetylcholine receptors (Barnes et al., 2000; Samochocki
et al., 2000). Animal studies have shown that galantamine
produces significant improvements in learning ability,
memory retention, and spatial-learning after ischemia
(Dimitrova & Getova-Spasssova, 2006; Iliev et al., 2000).
By comparing the protective effects of EECR-treatment with
that of standard drugs galantamine and pyritinol (positive
control), we were able to make a better correlation about the
protective effect of EECR.
The Cooks pole climbing apparatus, using the conditioned
avoidance responses, assesses the ability to acquire,
retain, and retrieve the learned responses from memory.
Accumulation of free radicals is said to affect the conditioned
avoidance responses in rodents (Sreemantula et al., 2005).
Our results suggest that there is a significant increase in the
conditioned avoidance responses in all the groups except the
sodium nitrite-administered Group II animals. The EECR-
treated animals in Groups IV and V showed an increase
in their conditioned avoidance responses, which is compar-
able with those of the vehicle control animals.
The elevated plus maze is used to assess anxiety responses
in rodents (Pellow et al., 1985). The behaviors that are
assessed here reflect the animal’s preference for an open
Figure 7. Morphological evidence of the protective effect of EECR in the brain. Representative photomicrographs of the cortex (A–E), hippocampus(F–J), thalamus (K–O), and cerebellum (P–T) of the brain sections from the different groups. A, F, K, and P are photomicrographs from Group I; B, G,L, and Q are photomicrographs from Group II; C, H, M, and R are photomicrographs from Group III; D, I, N, and S are photomicrographs from GroupIV; and E, J, O, and T are photomicrographs from Group V. Note the presence of significant number of intact neurons in the brain sections of EECR-treated Groups IV (200 mg/kg) and V (400 mg/kg) (D, E, I, J, N, O, S, and T). Group I, vehicle control; Group II, sodium nitrite-treated animals(negative control); Group III, pyritinol, galantamine, and sodium nitrite (positive control); Group IV, EECR 200 mg/kg and sodium nitrite; Group V,EECR 400 mg/kg and sodium nitrite. Arrows in Q indicate the Purkinje cells in the cerebellum replaced with vacuoles. Magnification scale bar 20mM.
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area as opposed to a protected place and their motivation to
explore a new environment and their anti-anxiety levels (Walf
& Frye, 2007). The sodium nitrite-administered Group II
animals had an increased anxiety as seen by their significantly
reduced time spend in the open arms. The EECR-treated
animals in Groups IV and V showed a significant reduction
in their anxiety level as seen by the increase in their time
in the open arm and the increase in the number of entries.
Moreover, the reduction in the anxiety exerted by the doses
of EECR was comparable with both the vehicle control and
the galantamine and pyritinol-treated positive control animals.
The anti-anxiety effects of EECR were further validated by
the fact that the number of entries into the open arm decreased
significantly after day 10 in the sodium nitrite-administered
Group II animals.
The Morris water maze assesses the learning and memory
deficits related to the hippocampus, striatum, basal forebrain,
cerebellum, and different neocortical areas (D’Hooge &
De Deyn, 2001). Different rodent models of ischemia such
as the focal, partial, and global cerebral ischemia using the
Morris water maze have shown that there is learning and
memory deficits and this is comparable with the learning
and memory deficit seen in the sodium nitrite-administered
Group II animals (Block, 1999; D’Hooge & De Deyn, 2001).
Earlier studies have also shown that cholinergic dysfunction
induced by choline uptake blockers impairs learning and
memory and cholinesterase inhibitors had shown to reverse
the effect (Hagan et al., 1989; Socci et al., 1995). The fact
that the EECR-treated animals in Groups IV and V showed
improvements in the Morris water maze performance suggests
that their spatial learning and memory are improved.
It has been shown that the corpus striatum is responsible
for controlling a range of motor and cognitive functions, and
the rotarod test assesses the motor performance related to
neuronal changes in the striatum in rodents (Hikosaka et al.,
1999; Lehericy et al., 2005; Poldrack et al., 2005). The motor
performance is also connected with movement as neuronal
structures innervate muscle fibers and the generated impulses
are transmitted to the muscle fibers to coordinate muscle
contraction and movement. Thus a reduction in the motor
activity is an indication of CNS depression (Rathor & Ram,
2010). Further, lack of muscular coordination is an indication
of abnormal muscle relaxation, which in turn leads to loss of
muscle grip (Jansen & Low, 1996a,b). In the present study,
although the locomotor activity did not change significantly
in the beginning in Group II animals, which were treated with
sodium nitrite, it started to decrease from day 20 onwards
suggesting that there is a motor impairment. The fact that the
EECR-treated animals in Groups IV and V showed improve-
ments in their locomotor activity suggested that EECR has the
potential to prevent the damaging effects of sodium nitrite-
induced hypoxia and restore the cortical and striatal inter-
action in the brain. Moreover, the ameliorating effect exerted
by the higher dose EECR was comparable to the vehicle
control as well as the galantamine and pyritinol-treated
positive control animals.
The actophotometer assesses the locomotor activity,
which is an index of mental alertness (Thakur & Mengi,
2005). Most of the drugs that have an effect on the CNS are
also said to influence the locomotor activity (Nehlig et al.,
1992; Walker et al., 1996). A similar damage to the brain may
reduce the motor activity. Our results have shown that there
is a significant reduction in the locomotor activity in the
sodium nitrite-administered Group II animals. The EECR-
treated animals in Groups IV and V showed no change in their
alertness behavior compared with those of the vehicle Group I
and positive control Group III animals.
The histological analysis showed that sodium nitrite-
induced hypoxia adversely affected the cortex, hippocampus,
thalamus, and cerebellum of the brain as there was an increase
in the number of pyknotic, shrunken neurons in these regions
with increased vacuolization. These brain regions are known
to play a significant role in the regulation and coordination
of movement and behavioral activities. Earlier studies on
hypoxia injury in different animal models reported these
changes and suggested that these morphological changes
occur because of the apoptosis of neurons or cell degeneration
(Cummings et al., 1984; Jensen et al., 1991). It was also
proposed that the cortex, hippocampus, and striatum are
particularly sensitive to hypoxia-induced damages
(Maiti et al., 2007; Nakajima et al., 2000; Ruan et al.,
2003). An increase in the oxidative stress is also shown to
be a reason for the changes in the morphology and cell
death during hypoxia (Maiti et al., 2006). The fact that
Figure 8. Neuronal damage was scored indifferent groups in the cortex, hippocampus,thalamus, and cerebellum at day 30 in sixdifferent areas for each tissue. Scores werefrom 0 to 3: 0¼ 91 to 100% of neuronsdamaged, 1¼ 51–90% of neurons damaged,2¼ 11 to 50% of the neurons damaged,3¼ less than 10% neuronal damage. Sixsections/rat were scored, averaged, and thescores of 6 rats/treatment group were addedto obtain the final score. Group I, vehiclecontrol; Group II, sodium nitrite-treated ani-mals (negative control); Group III, pyritinol,galantamine, and sodium nitrite (positivecontrol); Group IV, EECR 200 mg/kg andsodium nitrite; Group V, EECR 400 mg/kgand sodium nitrite. (a) Group I versus GroupII, (b) Group II versus Groups III, IV, and V.*Significant difference, *p50.05,**p50.01, ***p50.001.
DOI: 10.3109/13880209.2014.908395 Protective effects of Cyperus rotundus in hypoxia injury 1567
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EECR-treated Groups IV and V animals also showed protec-
tion against these changes in all of these brain regions
suggested that the medicinal herb, C. rotundus, has a
protective effect against sodium nitrite-induced hypoxia
injury.
Conclusion
Taken together, our study revealed that the traditionally used
medicinal herb C. rotundus has a protective effect against
the neurodegenerative changes produced by sodium nitrite-
induced hypoxia injury in rats. Further studies using different
model systems as well as hypoxic injury induced by different
methods will help us to clarify the protective effect of the
medicinal herb C. rotundus which may help in designing
better intervention strategies for hypoxia injury.
Declaration of interest
The authors declare that there are no conflicts of interests.
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