ABSTRACT Background: Management of cognitive disorders like dementia and Alzheimers disease has been challenging since no potential drug is available with proved efficacy. Some nootropic drugs like piracetam, aniracetam and cholinesterase inhibitors such as Donepezil® have found to exhibit severe toxic effects in elderly. Aim: In the present study, we assessed the nootropic potential of aqueous and methanol extract of Benincasa hispida on various behavioural models. Method: The young animals treated with aqueous and methanol extract (200 and 400 mg kg-1) showed dose-dependent reduction in transfer latency at both 9th and 10th day by elevated plus maze, morris water maze and object recognition task. Conclusion: From the results, it was concluded that methanolic extract of Benincasa hispida can be a useful memory restorative agent in the treatment of dementia.
How to Cite: Veni Bharti,
Surendra Kumar Sharma and
Sumitra Singh , 2013. Evaluation of the Memory and Learning Improving Effects of Benincasa hispida Seeds in Mice. Pharmacologia, 4: 249-253
INTRODUCTION Memory impairment is commonly seen by physicians in multiple disciplines
including neurology, psychiatry, medicine and surgery1.
Memory loss is often the most disabling feature of many disorders, impairing
the normal daily activities of the patients and profoundly affecting their families.
The key features of these dreaded disorders are memory impairments, deterioration
of language, visuo-spatial, motor, sensory abnormalities, gait disturbance and
seizures. There are around 30 million patients suffering from Alzheimers
Disease (AD) which is the major cause of dementia, all over the world2.
In India, AD patients are estimated to be around 3 million3.
Cognition is that operation of mind by means of which, we become aware of our
surroundings, objects and thoughts. Cognitive disorders such as delirium, dementia
and amnesic disorders are common in elderly individuals. Memory is vulnerable
to a variety of pathologic processes including neurodegenerative diseases, strokes,
tumors, head trauma, hypoxia, cardiac surgery, malnutrition, attention deficit
disorder, depression, anxiety, the side effects of medication and normal ageing4.
Presently, there are no satisfactory diagnostic procedures and therapeutic regimens
available for the management of these cognitive disorders. Despite the severity
and high prevalence of these diseases, Allopathic system of medicine is yet
to provide a satisfactory remedy. Therefore, neurobiologists all over the world
are looking for new directions and alternative strategies for managing cognitive
disorders. The most common cause of dementia in the elderly is probably Alzheimers
Disease (AD), a chronic, progressive disabling organic brain disorder characterized
by disturbance of multiple cortical functions, including memory, judgment, orientation,
comprehension, learning capacity and language5.
Nootropic agents like, Piracetam and Cholinesterase inhibitors like, Donepezil
are commonly used for improving memory, mood and behavior. However, the resulting
adverse effects of these drugs such as diarrhea, insomnia, nausea, bronchitis,
loose stools, muscular cramps and other known side effects6
have made their use limited and it is worthwhile to explore the utility of traditional
medicines in the treatment of various cognitive disorders. The present studies
deal with the evaluation of memory enhancement activity of ethanol and aqueous
extract of Benincasa hispida seeds.
MATERIALS AND METHODS Plant material: The plant Benincasa hispida (Thumb.)
Cogn. was collected from Hisar in the month of August 2011 and identified by
Dr. H.B. Singh, Head, Raw Materials, Herbarium and Museum Division, National
Institute of Science Communication and Information Resources (NISCIR), New Delhi,
vide reference No. NISCAIR/RHMD/Consult/-2011-12/1881/181 and voucher specimen
retained in the Pharmacognosy section of Department for the future reference.
Seeds were shade dried at 35±5°C and pulverized and used for carrying
the experimental work procedures pertaining biological evaluation.
Methanol extract: The air-dried crude drug (500 g) was pulverized and extracted with methanol using soxhlet apparatus for 16 h. Methanol removal carried out under pressure afforded a semi solid mass with a yield of 13.6% w/w. The extract was further used for the evaluation of memory enhancing activity.
Aqueous extract: The 500 g of coarse dried powder of seeds of plant Benincasa hispida (Thumb.) Cogn. was extracted with distilled water by cold maceration method for 72 h and filtered through muslin cloth. The filtrate was then concentrated to get thick extract.
Animals and chemicals used: The healthy albino mice of either sex, weighing 20-25 g, were used for the evaluation of memory enhancing activity. They were procured from Agronomy Department of Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar. The animals were housed under standard environmental conditions of temperature and humidity (25±0.50°C) and (12 h light/dark cycle) were utilized for the studies. The animals were fed with standard pellet diet and water ad libitum. The Institutional animal ethical committee (Guru Jambheshwar University of Science and Technology, Haryana, India) reference no.Ph/2010/250, dated 19/3/2010 approved the experimental protocol and care of laboratory animals were taken as per the guidelines of CPCSEA, Ministry of Forest and Environment, Government of India (Registration number 0436).
A total of 120 young male mice were employed in the present study. Each group
comprised of 6 mice:
Group I: Control group for young mice. Food was administered
orally for ten consecutive days. TL was recorded after 90 min of food administration
on 9th day and retention was examined after 24 h (i.e., on 10th day)
Group II: Positive control for young mice. Piracetam
(400 mg kg-1 i.p.) was injected to young mice for 10 consecutive
days. TL was recorded after 60 min of injection on 9th day and retention
was examined after 24 h (i.e., on 10th day)
Group III-VI: Aqueous and methanol extract (200-400
mg kg-1, respectively) were administered orally along with diet
for 10 consecutive days. TL was recorded after 90 min of food administration
on 9th day and retention was examined after 24 h (i.e., on 10th day)
Group VII: Scopolamine (0.4 mg kg-1 i.p.)
was injected on 9th day to young mice and TL was recorded 45 min after injection.
Retention was examined after 24 h (i.e., on 10th day)
Chemical used in the present study were Scopolamine (Hi-Media laboratories Pvt. Ltd., Mumbai, India) Piracetam, NootropilUCB India Pvt. Ltd., Vapi, India), normal saline, aqueous and methanolic extract of Benincasa hispida (Thumb.) Cogn. and Gum acacia. Solution of scopolamine and Piracetam was prepared in normal saline and injected intraperitoneal. All semisolid extracts of seeds of Benincasa hispida (Thumb.) Cogn. were suspended in gum acacia (2%) and prepared in distilled water. Piracetam and scopolamine were diluted in normal saline.
Behavioral methods for testing learning, memory and anti-anxiety activity Elevated plus maze (EPM): Elevated plus maize served as the exteroceptive
behavioral model to evaluate memory in mice. The procedure, technique and end
point for testing memory was followed as per the parameters described by the
investigators working in the area of psychopharmacology7,8.
The elevated plus maze for mice consisted of two open arms (16x5 cm2)
and two covered arms (16x5x12 cm3) extended from a central platform
(5x5 cm2) and the maze was elevated to the height of 25 cm from the
floor9. On the first day (i.e., ninth day
of drug treatment), each mouse was placed at the end of an open arm, facing
away from the central platform. Transfer latency was defined as the time (in
sec) taken by the animal to move from the open arm in to one of the covered
arms with all its four legs. TL was recorded on the first day (training session)
for each animal. The animal was allowed to explore the maze for another 2 min
and then returned to its home cage. Retention of this learned Task (memory)
was examined 24 h after the first day trial (i.e., tenth day, 24 h after last
dose). Significant reduction in TL value indicated improvement in memory.
Object recognition task (ORT): Object recognition task is a convenient
model used to evaluate the object recognition memory in mice. The observation
area consisted of a circular open field, 480 mm in diameter and the wall height
400 mm. Two different sets of objects (a cone, a ball, a cylinder etc.) made
of aluminum were used. All objects were available in triplicate. They could
not be displaced by the mouse nor could the mouse climb onto or hide under the
objects. The objects had no natural significance and they were never associated
with any kind of reinforcer. Two objects were presented in the first trial (T1)
and a third one in the recognition trial (T2) to prevent odor cues. Objects
were cleaned with tap water and detergents after each trial. During two consecutive
days, the mice were habituated to the apparatus and the testing procedure. They
were allowed to explore the empty apparatus twice for 3 min each day (one morning
and one afternoon session). Animal was placed into the apparatus, equidistant
from the two objects, facing the wall in front of the experimenter. Maximum
duration of exploration was allowed 3 min. Animals were trained in pairs of
two trials that were separated by a retention interval of 1 h. During the T1
the apparatus contained two identical objects, A1 and A2.
These objects were placed in a symmetrical position about 120 mm (with reference
to the centre of the object) away from the wall. During T2 the apparatus contained
two different objects, a copy of the familiar one A from T1 and
a novel object B. Mean time (in sec) exploring the familiar object
A and novel object B during T2 were measured. Discrimination Index (DI) is the
difference between the exploration time of novel object and familiar object.
Increase in DI indicates enhanced recognition memory. ORT allows the assessment
of acquisition, consolidation or retrieval of object information separately10.
Exploration is defined as directing the nose to the object at a distance of not more than 2 cm and/or touching the object with muzzle. Sitting on the object is not considered as exploratory behavior.
Morris water maze: Morris water maze for mice consisted of a circular pool (60 cm in diameter, 25 cm in height) filled to a depth of 20 cm with water maintained at 25°C. The water was made opaque with non-toxic white coloured dye. The dark was divided in to four equal quadrant with the help of 2 threads fixed at right angles to each other on the rim of the pool. A submerged platform (with top surface 6x6 cm and painted in white) was placed in sided target quadrants (Q4 in present study) of this pool 1 cm below surface of water. The position of platform was kept unaltered throughout the training period. Each animal was subjected to four consecutive trials each day with the gap of 5 min. for four consecutive days (stating from the 6th day of drug administration to 9th day), during which they were allowed to escape the hidden platform and to remain there for 20 sec. During the training session, the mouse was gently placed in the water between quadrants facing the wall of the pool with drop location changing for each trial and allowed 120 sec to submerged platform. If the mouse failed to find the platform with in 120 sec, it was guided gently to the platform and allowed to remain there for 20 sec. Each animal was subjected to training trial for four consecutive days, the starting point was changed with each exposure as mentioned below and target quadrant (Q4 in the present study) remained constant throughout the training period.
On 5th day (i.e., 10th day of drug administration), the platform was removed
and mouse was placed in any of 3 quadrant and allowed to explore the target
quadrant for 300 sec. Mean time spent in all quadrant Q1 Q2 Q3 was recorded.
The mean time spent in the target quadrant in search of missing platform mean
noted as index of retrieval or memory. The observer should stood at the same
position. Care was taken not to disturb the relative location of WM with respect
to other object in the laboratory11.
RESULTS Effect of Benincasa hispida (Thumb.) cogn. seed powder on transfer
latency using elevated plus maze: Transfer Latency (TL) of 9th day and 10th
day of drug treatment reflected improvement of both learning and memory. The
young animals treated with aqueous and methanol extract (200 and 400 mg kg-1)
showed dose-dependent reduction in TL of both 9th and 10th day, indicating significant
improvement in memory, when compared with control group. Scopolamine (0.4 mg
kg-1 i.p.) injected before training significantly increased (p<0.01)
the TL of 9th and 10th day indicating impairment in both learning and memory
(amnesia), respectively. The mice treated with extracts for 10 consecutive days
reversed successfully the amnesia induced by Scopolamine. Piracetam (used as
the positive control) at the dose of 400 mg kg-1, i.p. improved memory
(p<0.01) and reversed the amnesia induced by Scopolamine (Table
Effect of Benincasa hispida (Thumb.) cogn. seed powder on transfer latency using object recognition task: There was dose dependent significant increase in 10th day discrimination index for the aqueous and methanol extract of Benincasa hispida (200-400 mg kg-1) treated groups in comparison to control group. Aqueous and methanol extracts (200-400 mg kg-1) administered for 10 days reversed memory deficits due to Scopolamine induced amnesia. The results were comparable to Piracetam (400 mg kg-1 i.p.), a standard memory enhancer agent (Table 2).
Effect of Benincasa hispida (Thumb.) cogn. seed powder on transfer
latency using morris water maze: Time spent in the region that previously
contained the platform was recorded as Time Spent in Target Quadrant (TSTQ).
Seed extracts aqueous (200-400 mg kg-1) and methanolic (200-400 mg
kg-1) were administered orally for 10 days. Methanolic and aqueous
extract was significantly increased TSTQ, indicating significant improvement
Table 1: Effect of Benincasa hispida
(Thumb.) Cogn. seed extract on Transfer Latency (TL) of mice using Elevated
Plus Maze (EPM) model
Table 2: Effect of Benincasa hispida
(Thumb.) Cogn. seed extract on Transfer Latency (TL) of mice using Object
Recognition Task (ORT) model
Table 3: Effect of Benincasa hispida
(Thumb.) Cogn. seed extract on Transfer Latency (TL) of mice using Morris
Water Maize (MWM) model
Methanolic extract was more significant than aqueous extract. Piracetam (400
mg kg-1) was used as positive control and significantly increased
TSTQ on 10th day (Table 3).
DISCUSSION Anxiety is unpleasant feeling of apprehension or fearful concern. It can
be a normal, reasonable and expected response to a stressful situation or perceived
danger or it may be an excessive, irrational state that signifies a mental disorder.
Nootropics are a class of psychotropic agents with selective facilitatory effect
on integrative functions of the central nervous system, particularly on intellectual
performance, learning capability and memory12,13.
Piracetam, the first representation of a class of nootropic agents, has been
shown to improve memory deficits in geriatric individuals. Repeated injections
of piracetam had improved learning abilities and memory capacities of laboratory
animals14. The data shown by experimental
results underlined the importance of B. hispida methanol as well as aqueous
extract in preventing memory loss. New neurons are continuously being added
to certain areas of the brain, such as hippocampus and olfactory bulb in animals15,16,17
as well as humans. There is a possibility that methanol extract of B. hispida
for long periods not only arrests the neurodegenerative processes (responsible
for dementia) but also stimulated the process of neurogenesis The elevated plus
maze is a well established animal model for testing anxiolytic drugs18.
Methanol extract of B. hispida fruit has been proved to be effective
anixolytic agent in one study19. The results
indicates the reduction in Transfer Latency on 9th and 10th day in elevated
plus maze, while Time Spent in Target Quadrant was increased to a significant
level in Moris Water Maze model indicating methanol extract more potent than
that of aqueous extract by improving condition of amensia induced by Scopolamine.
Considering the lack and need of drugs with proven effectiveness in improving learning and memory, the specific memory improving effects of B. hispida reported here is of enormous interest and deserves further investigations using more experimental paradigms for further confirmation of memory improving potential of B. hispida in the treatment of various cognitive disorders.
Mesulam, M.M., 2000. Principles of Behavioral and
Cognitive Neurology. 2nd Edn., Oxford University Press, Oxford, UK., ISBN-13:
9780195134759, Pages: 574.
Hebert, L.E., P.A. Scherr, J.L. Bienias, D.A. Bennett and D.A. Evans, 2003.
Alzheimer disease in the US population: Prevalence
estimates using the 2000 census. Arch.
Neurol., 60: 1119-1122
Newman, M.F., J.L. Kirchner, B. Phillips-Bute, V. Gaver and H. Grocott et
al., 2001. Longitudinal assessment of neurocognitive
function after coronary-artery bypass surgery. N.
Engl. J. Med., 344: 395-402
Petersen, R.C., R.G. Thomas, M. Grundman, D. Bennett and R. Doody et
al., 2005. Vitamin E and donepezil for the treatment
of mild cognitive impairment. N.
Engl. J. Med., 352: 2379-2388
Rogers, S.H., M.R. Farlow, R.S. Doody, R. Mohs, L.I. Friedhoff and Donepezil
Study Group, 1998. A 24-week, double-blind, placebo-controlled
trial of donepezil in patients with Alzheimers disease. Neurology,
Reddy, R.S. and S.K. Kulkarni, 1998. Possible role
of the nitric peroxide in the nootropics and anti-amnesic effect of neurosteroid
on ageing and dizoclipine induced learning and impairment. Brain Res., 799:
Parle, M. and N. Bansal, 2011. Antiamnesic activity
of an ayurvedic formulation chyawanprash in mice. Evidence-Based Complementary
Altern. Med., 10.1093/ecam/neq021
Rahman, H., P. Muralidharan, D. Sivaraman and D. Saha, 2010. Continuous
sleep deprivation for 5 days produces loss of memory in mice and may be a
cause of Alzheimers disease. Ann.
Biol. Res., 1: 185-193
Kempermann, G., H.G. Kuhn and F.H. Gage, 1997. More
hippocampal neurons in adult mice living in an enriched environment. Nature,
Van Praag, H., G. Kempermann and F.H. Gage, 1999. .
Running increases cell proliferation and neurogenesis in the adult mouse dentate
Neurosci., 2: 266-270
Rhodes, J.S., H. van Praag, S. Jeffrey, I. Girard, G.S. Mitchell, T. Garland
Jr. and F.H. Gage, 2003. Exercise increases hippocampal
neurogenesis to high levels but does not improve spatial learning in mice
bred for increased voluntary wheel running. Behav.
Neurosci., 117: 1006-1016