Aruna.P et al. / IJDFR volume 3 Issue 1, Jan- Feb. 2012 Available online at www.ordonearresearchlibrary.org

ISSN 2229-5054

Research Article

INTERNATIONAL JOURNAL OF DRUG FORMULATION AND RESEARCH

LEARNING AND MEMORY ENHANCING ACTIVITY OF VIGNA RADIATA LINN EXTRACT 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

ABSTRACT Dementia is an acquired global impairment of intellect, memory and personality or it is neurodegenerative disorder in which loss of memory is there. Many experimental models are currently available for the evaluation of agents that affects learning and memory processes. The present work was undertaken to assess the potential of Vigna radiata 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/kg and ageing- induced memory deficit in mice using the Radial arm maze and Morris water maze models. Piracitam (400 mg/ kg i.p.) was used as a standard nootropics agent. VR extract is significantly improved learning and memory 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.

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 person’s 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]. Alzheimer’s 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 patient’s 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 98

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

Aruna.P et al. / IJDFR volume 3 Issue 1, Jan- Feb. 2012 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 of 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 tight container for extraction. Method of extraction: Continuous hot percolation process. Requirements: Soxhelet apparatus, dried course powder of Vigna radiata Linn. Solvents: Petroleum ether 60-80 o c, Alcohol 99% v/v, Distilled water. a) Petroleum ether extract of Vigna radiata linn: The dried seeds of vigna radiata linn were extracted with petroleum ether 60-80 o c, 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: 99

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

Aruna.P et al. / IJDFR volume 3 Issue 1, Jan- Feb. 2012 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 brownish–black 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. [17]:

Animals

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 least 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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Aruna.P et al. / IJDFR volume 3 Issue 1, Jan- Feb. 2012 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 of 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 total 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) of 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 quad- rants 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 60’s 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. 101

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

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

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 successful 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 Dunnett’s t-test and values * p