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Aruna.Pet al. / IJDFR volume 3 Issue 1, Jan- Feb. 2012

98 Aruna.Pet al. / IJDFR volume 3 Issue 1, Jan- Feb. 2012

Available online at www.ordonearresearchlibrary.org ISSN 2229-5054

INTERNATIONAL JOURNAL OF DRUG FORMULATION AND RESEARCH

LEARNING AND MEMORY ENHANCING ACTIVITY OF VIGNA RADIATA LINNEXTRACT IN MICE USING SCOPOLAMINE INDUCED AMNESIA.

Bhandurge Aruna.P*, Bhandurge Sachin.P, Kshirsagar Sunil N, Pratapwar Atul. S.

Department of Pharmacology, Sudhakarrao Naik Institute of Pharmacy Pusad (M.S.) 445204 India

Received: 8 Dec. 2011; Revised: 12 Jan 2012; Accepted: 6 Feb. 2012 ; Available online: 5 Mar. 2012

INTRODUCTION

Learning may be defined as the ability to alter the behavior on the basis of experience and memory is the ability

to recall past events at the conscious and unconscious level. These two are obviously closely related to each

other and should be considered together [1].

The brain and the computer work in similar fashion to store day to day happenings, incidents and visuals. When

a persons brain is fresh he is able to store more information and remember the matter for long. An emotional

overload that in the modern language is sometimes called tension which may leads to loss of memory [2].

Alzheimers disease is a progressive neurodegenerative brain disorder that is slow in onset but leads to

dementia, unusual behavior, personality changes and ultimately death [3]. The

Personality distortions interfere with the patients professional life, social activities and relationships

[4].Nootropic agents such as Piracitam [5], pramiracetam, aniracetam [6] and choline esterase inhibitors like

Donepezil are being primarily used to improve memory, mood and behavior. However, the resulting adverse

effects associated with these agents have limited their use [7, 8]. Therefore, it is worthwhile to explore the

utility of traditional medicines for

Research Article

ABSTRACT

Dementia is an acquired global impairment of intellect, memory and personality or it is neurodegenerative disor

in which loss of memory is there. Many experimental models are currently available for the evaluation of agentsthat affects learning and memory processes. The present work was undertaken to assess the potential of Vignaradiata linn extract as a learning and memory enhancing agent in mice. Aqueous and ethanolic extract of dried

seeds of Vigna radiata linn 700mg/kg and 1000mg/kg ameliorated the amnesic effect of Scopolamine 0.4 mg/kgand ageing- induced memory deficit in mice using the Radial arm maze and Morris water maze models. Piracita

(400 mg/ kg i.p.) was used as a standard nootropics agent. VR extract is significantly improved learning andmemory in young mice when it was compared with the scopolamine induced group.

Key words: Dementia, Morris water maze, Radial arm maze, Scopolamine, Piracitam, Vigna radiate linn.
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the treatment of various cognitive disorders.

Green gram [Vigna radiata] belongs to the family Fabeaceae. It is a small herbaceous annual plant growing to a

height of 30 to 120 centimeters with a slight tendency to twining in the upper branches. Mung beans are a

dietary source of vitamin C, several B vitamins, calcium, fiber, iron, potassium, magnesium, and manganese.

Mung beans have been shown to have antibacterial [9, 10], hepatoprotective [11], antifungal, Nematicidal

Estrogen like effect [13], antidiabetics[14], anti-inflammatory, antioxidant [15], cytotoxic [16] and traditionally

it is also use as nervine tonic. But however, it has not been scientifically documented so far for its effect on

learning and memory. So the present study was therefore carried out for confirming veracity of aforementioned

traditional claim of using two animal models, namely Radial arm maze and Morris water maze.

Materials and Methods:

1) Selection of plant:

There are so many approaches for the selection of the plant material that may contain new biologically active

compounds. One of the approaches is the Dr. Dukes and phytochemical studies in which several number o

plants are there which shows anti amnestic activity. There have been claims that some traditional healers can

successfully treat amnesia using herbal drug. I under took the present study to evaluate the anti amnesic activity

of seeds of Vigna radiata linn based on the discussion with the traditional medical use in Maharashtra.

2) Collection and Authentication of the plant:The seeds of Vigna radiata ie mung beans were collected in the month of September 2010 from the Mahabej

Bhavan Washim District 444505, Maharashtra, India. The plant material were identified and authenticated by

professor and head of department Dr. A. Chaturvedi; Rastrasant Tukdoji Maharaj Nagpur University, Nagpur

India. A voucher specimen (No: 9593) was deposited at Herbarium, Department of Botany, RTM Nagpur

University Nagpur.

3) Preparation of crude drug for extraction:The seeds of Vigna radiata linn were collected from the Mahabeej Bhavan of Washim and authenticated by the

authorized person then it will be washed thoroughly and dried under shade then seeds were grind to flour with

the help of mechanical grinder. The powder was passed through the sieve No.40 and stored in an air tigh

container for extraction.

Method of extraction: Continuous hot percolation process.

Requirements: Soxhelet apparatus, dried course powder of Vigna radiata Linn.

Solvents: Petroleum ether 60-80o

c, Alcohol 99% v/v, Distilled water.

a) Petroleum ether extract of Vigna radiata linn: The dried seeds of vigna radiata linn were extractedwith petroleum ether 60-80

oc, upto 72 hrs. After completion of extraction, the solvent was recovered

and dark yellow colored oil was collected.

b) Ethanolic extract of Vigna radiata linn:

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The marc after petroleum ether extraction was dried and then extracted with ethanol 99% v/v (75-78), upto 72

hrs in soxhelet apparatus. Extract was evaporated under reduced pressure to obtain an average yield. The

solvent was then removed under reduced pressure which obtained a brownishblack colour sticky residue and

was used for identifying its activity on learning and memory activity.

c) Aqueous extract of Vigna radiata linn:Aqueous extract of vigna radiata was prepared by using maceration process by making a fine powder of seed

then soaked in water with a little quantity of chloroform for a period of ten days and then filtered with muslin

cloth and the extract was evaporated under reduce pressure collected into very fine powder by means of

evaporation and then the powdered drug was use for its learning and memory activity .Both the ethanolic and

aqueous extract were stored in tightly closed bottles.

d) Preparation of extract solution:Test solution (T.S) of ethanolic and aqueous extract were prepared by using Carboxy methyl cellulose and 0.5%

v/v tween 20 as a suspending agent in order to make a particular concentration.

Animals [17]

Swiss albino mice weighing -18 g (younger ones, aged 8 weeks and 25 g (older ones, aged 28 weeks) were used

in the present study. These mice were housed in standard laboratory conditions of temperature, relative

humidity and under standard environmental conditions (12h light and 12h dark) with adequate food and water

except for those, used for radial arm maze task performance .The mice were transferred to the laboratory at leas

one hour before the start of the experiment and all experiments were carried out form 8.00-11:00 am

Institutional Animal Ethical Committee approved the protocol. And care of animals was taken as per guidelines

of CPCSEA, Department of Animal Welfare, and Government of India.

Treatment schedule for Pharmacological studies:

Mice were divided into seven groups; each consisting of six mice for each experiments.

Group-I: Normal control group received only vehicle.

Group-II: Positive control group received only vehicle against scopolamine induced amnesia.

Group-III: Aqueous extract 700mg/kg p. o treated group against scopolamine induced amnesia.

Group-IV: Aqueous extract 1000mg/kg p. o treated group against scopolamine induced amnesia.

Group-IV: Ethanolic extract 700mg/kg p. o treated group against scopolamine induced amnesia.

Group-IV: Ethanolic extract 1000mg/kg p. o treated group against scopolamine induced amnesia.

Group-VII: Piracitam 400mg/kg i.p treated group against Scopolamine induced amnesia.

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I] Radial arm maze model:

Procedure:

The animals were trained for maze task performance by conducting once daily trial. It took 15 days to get the

animal completely trained. During training animals did not receive any drug treatment. The completely trained

animals were chosen for study. These animals were dosed once in a day with the respective drugs for ten days

along with daily training trial.

Evaluation of learning and memory enhancing activity by Radial arm maze model Construction of

Radial arm maze model:

A Radial arm maze is locally fabricated wooden radial arm maze elevated 50cm above the floor consisting o

an octagonal central hub 36cm in diameter with eight radial arms was used. Each arm 43 cm long, 15cm wide

with 12 cm sides, had small black plastic cups mounted at 30cm from the central hub [18].

Mechanical screening for memory in radial arm maze:

The mice were trained for radial maze task performance by conducting daily training trial which consisted of

two sessions wherein one food pellet was placed in a fixed arm and then in the variable arm to record the effect

of extract on spatial reference and spatial working memory respectively. Mice maintained at 85% of their tota

diet were placed individually in the central hub and were allowed to choose the arm freely to get the food with

upper cut off limit of 300 sec. The time taken by each mouse to find the food along with number of reentries

was considered to assess radial maze task performance. Mouse was considered to be learned when he found the

food with maximum one reentry for three consecutive days. The number of days required for making the mice

learned and the latency to find the food along with number of initial correct entries (i.e. before first reentry) o

learned mouse was recorded as the effect of the drug on learning and memory process. One-hour interval was

kept between the spatial reference and spatial working memory evaluation. The apparatus was cleaned with

damp cloth after each trial to avoid place preference and the influence of olfactory stimuli [19], [20], [21].

II] Morris water maze test model:

Construction of Morris water maze task

Morris water maze test was carried out in a circular pool (100cm in diameter and 50cm in height) made up of

opaque polyvinyl chloride filled to a depth of 30cm with water (25+ 20 c) containing milk. A plastic platform

(9cm diameter and 29cm height) was submerged in one of the pool quadrants located 2 cm below the water.

The Pool is fixed with filling and draining facilities and mounted on a frame work so that the water is as waist

level. The floor of the circular tank is marked off into four equal quadrants. The platform was located in the

same position on every trial. The first day of the experiment was dedicated to swimming training for 60s in the

absence of the platform. During the four consecutive days, the mice were given two trial sessions per day with

an inter-trial interval 20 min. Once the mouse found the platform, it was permitted to remain on it for10s.If the

mouse failed to find the platform within 90s, it was placed on the platform for 10s.

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Water maze represents a versatile tool in which a number of different tasks can be measured. The simplest

measure of performance is the latency to escape from the water onto the hidden platform [22].

Mechanical Screening for memory in Morris water maze model:

The animals for the experiments were pretreated by conducting at least one daily trial. Seven groups of animals

were made each group consist of six animals. Group I served as control which receive vehicle only, Group II

served as scopolamine induced group , Group III , IV,V, VI served as treatment control group and Group VII

served as standard group.

At the beginning of the trial animals are placed in the Morris water maze and allowed to swim freely and to seat

on the hidden platform. The trail was considered to be successful when mouse sit on the hidden platform within

three 90 seconds.

Time spent more than three minutes to find the hidden platform recorded as error. The percentage of successfu

mice was calculation as the index of Morris water maze task performance. On the 11th

day 60 minutes after the

last dose, animals of respective groups were subjected to scopolamine 0.4 mg/kg S.C. treatment for inducing

amnesia [23].

After 30 minutes each animal were subjected to Morris water maze task performance.

Results were shown in the table.

Statistical analysis

The data were statistically analyzed by Mean SD .The data were also analyzed by one way ANOVA followed

by Dunnetts t-test and values * p

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Table: 1 Effect of ethanolic and aqueous extract of Vigna radiata linn extract on learning and memory

retention against scopolamine induced amnesia by Radial arm maze model.

Values are mean SEM n=6, **p

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Figure: 1Effect of Vigna radiata linn extract and Piracitam in number of days

required to make mice learned using Radial arm maze model.

Figure: 2 Effect of Vigna radiata linn extract on Percentage Memory Retention

against Scopolamine Induced Amnesia by Radial Arm Maze Model in mice.

6.67 7.00

4.334 4.17

4.50 4.17

0

1

2

3

4

5

67

8

DaysRequired

Days Required

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Morris Water Maze Model

Table: 2 Effect of ethanolic and aqueous extract of Vigna radiata linn extract on learning and memory

retention against scopolamine induced amnesia by Morris water maze.

Group No. Average

bodyweight

in gm

Dose

(mg/kg)

Morris water maze performance

%memory

retention

Before scopolamine Afterscopolamine

Average

time(sec)

Average

daysrequired

Average time

(sec)(on 11

thday)

I Normal

control 20 20 59.2 7.5 76.6614.73 70.65

IIScopolamine

induce20 0.4 50.4 7.33 88.66 2.42 24.16

III TestAqVR 20 700 52.26 5 78.1615.11* 50.43

IV Test

(Aq.VR)20 1000

30.254..6 44.66 12.09*

52.34V Test

EthVR22 700

54.994.5 80.58.78** 53.63

VI Test

Eth VR20 1000 52.83 4.33 75.837.78** 56.46

VII Piracitam

(std.) 23 400 46.941 4 55.1615.96** 82.47

Values are mean SD, n=6 when compared with scopolamine induced group * p

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Morris Water Maze Model

Figure: 3 Effect of Ethanolic, aqueous extract of Vigna radiata linn and Piracitam for days required for learning

process in mice using Morris Water Maze Model.

Figure: 4 Effect of Vigna radiata linn Extract and Piracitam on Learning and Memory retention test in mice

using Morris Water Maze.

7.57.3

54.6 4.5 4.3

4

0

1

2

3

4

5

6

7

8DaysRequired

Days Required

70.65

24.16

50.43 52.3453.63

56.46

82.47

0

10

20

30

40

50

60

70

80

90

Percentage Memory Retention

Percentage

memory

retention

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terpenoids. Hence it was selected for pharmacological studies. And according to the literature survey these al

constituents are responsible for learning and memory activity.

The ethanolic and aqueous extracts were used for evaluation of activity in mice shows dose dependant activity

That means higher dose is more effective as compared to that of lower dose.

According to the pharmacological evaluation .Ethanolic extract is more effective as compared to that of aqueous

extract on facilitation of learning and memory activity against scopolamine induced amnesia.

Increased alertness is one of the properties of nootropic agent. In this investigation ethanolic extract is more

effective in accordance with the finding of neuropharmacological evaluation. The improved alertness may be

responsible to reduce the latency to find the food in radial arm maze, to locate the plat form in Morris water

maze.

Acknowledgement

I owe a great debt of gratitude to Dr. D. M. Sakarkar Sir, Principal, Mr. R. J. Mandade Sir and department o

pharmacology of Sudhakarrao Naik Institute of pharmacy, Pusad, for providing the necessary facilities.

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