NOOTROPIC ACTIVITY OF CLITORIA TERNATEA.L ON SCOPOLAMINE INDUCED AMNESIA

About Authors:
Sharmila Shaik*, K. Anitha
Department of pharmacology, Krishna Teja Pharmacy College,
Chadhalawada Nagar, Renigunta Road, Tirupathi-517 507,
Andhra Pradesh, India.
*cherry.sharmilask@gmail.com

ABSTRACT :
Alzheimer’s disease (AD) is a progressive degenerative CNS disorder with slow onset with memory that leads to dementia. The drugs like barbiturates, alcohol, and diazepam will also impair learning and memory. Nootropic agents such as piracetam, pramiracetam, aniracetam, oxicetam and some of the herbal drugs are used to enhance cognitive function. The objective of the present study is to investigate the nootropic activity of methanolic extract of leaves of Clitoria ternatea.L (MELCT).Result showed that the MELCT improvement in the retention ability of normal and amnesic rats when compared to control and standard groups. It was suggested that in the MELCT could show beneficial improvement in cognitive disorders like Alzheimer’s disease and various neurogenerative disorders.

Reference Id: PHARMATUTOR-ART-1369

INTRODUCTION:
Nootropics are cognitive enhancers that are neuroprotective or extremely nontoxic.   Cognitive enhancers are drugs, supplements, nutraceuticals and functional foods that enhance concentration and memory (Lannic et al., 2008).Memory is ability of an individual to record event, information and retains them over short or long periods of time (Kulkarni S.K et al., 1993).The different conditions such as age, stress and emotion may lead to memory loss, amnesia, anxiety, high blood pressure, dementia to more threat like schizophrenia and Alzheimer’s disease (D. C. Mash et al., 1985).

The nootropic agents are clinically used in situations where there is organic disorder in learning abilities and for improving  memory ,mood and behavior, but the resulting side effects associated  with these agents have made their utility limited (dogpile.com). so, some of the herbal plants like ,prunus amygdalus(S.Kulkarni et al.,2011),Hibiscus Sabdariffa etc (Hanumathachar joshi ,2006) have been investigated for their effect on cognitive function  of the brain.

Clitoria ternatea.L (CT)is a herbaceous medicinal plant. It is commonly known as a shankupushpi and butterfly pea. It is a perennial twing herb, stems are more or less pubescent (Gomez SM  et al.,2003). The leaves are odd pinnately compound   with obovate in shape, entire margin, emarginated tip and symmetrical base, green in color, bitter in taste and characteristic odour (Hall TJ,1985). CT have reported a number of pharmacological activies such as antioxidant (A.P. Patil and V.R.Patil, 2011), antipyretic (Parimaladevi. B et al., 2004), anti inflammatory and analgesic (Parimaladevi .B et al., 2003), Anti inflammatory and analgesic (Parimaladevi et al., 2007),Nephroprotective(K..Sarumanthy et al.,2010),Larvicidal (Mathew N et al.,2009)  anti microbial (Mhaskar AV et al.,2010), proteolytic (ainouz IL et al.,1994), diuretic (Piala JJ et al., 1962), antihelmenthetic  (Nirmal et al 2008),Platelet aggregation  (Honda T  et al., 1991), Antidiarrhoeal (Nitin kumar upwar et al., 2010), Hepatoprotective (Ramalingam shanmugsundaram et al., 2010),Anti-diabetic (Daisy .p et al ., 2009). The present study was undertaken to investigate the effects of CT leaves on cognitive functions.

Material and Methods:
Plant material:
The leaves of Clitoria ternatea.Linn was collected from the hills of tirumala region, chittoor(dist).A.P, India. The plant was identified and authenticated by Dr.Madhava chetty, Department of botany, S.V. University, Tirupathi.

Preparation of extract:
The leaves of CT were collected, washed, cleaned and shade dried. The dried leaves were powdered with thehelp of mechanical mixer andpassed through a 40 – meshsieve to obtain coarse powder. The weighed quantity of coarsely powdered material was extracted with methanol by using soxhlet apparatus. After completion of extraction, it was undergone distillation under reduced pressure and the remaining solvent was removed by evaporation to dryness on a water bath. Residue was obtained and it was kept in a desiccators and used for further experiment (Treas and Evans, 2002). The percentage yield from 60gm was found to be 2.98%.

Phytochemical screening:
The presences of phytochemical constituents in the MELCT was tested by using the standard methods (Harbone JP,1973) .These standard methods revealed the presences of glycosides, flavonoids, steroids, alkaloids.

Evalution of nootropic ACTIVITY:
Requirements:
a. Animal:
Albino rats (150-200g) of either sex were used for the experiment. They were kept in the animal house in a controlled room temperature at 25+2 c, relative humidity 44-56% light and dark cycles of 10 and 14 hr, respectively for 1 week before the experiment.

b. Drugs and Chemical:
i.            Extract:  The MELCT was dissolved in 1 % tween 80 as a vehicle and administered P.O in a dose of 200mg/kg and 400mg/kg .
ii.            Standard drug: The piracetam was dissolved in 1% tween 80 and administered i.p in a dose of 200mg/kg (Hanumathachar joshi and Milind parle ,2006).
iii.            Scopolamine:

c. Acute Toxicity studies :
The Acute Toxicity studies was performed as per OECD 423 guidelines in order to establish the therapeutic index of a test drug. It was administered as 5,100,1000 up to 2000 mg/kg. It was observed that the test extract was not lethal to rats even at 2000mg/kg dose (OECD, 2002). Hence we selected 1/10^th of the doses (ie,200mg/kg and 400mg/kg).

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d. Experimental design:
Rats were divided in to 4 groups . Each group contains 6 rats and treated with the
following drug for seven successive days and 8^th day scopolamine was administered and evaluated the behavioral models.
Group 1: Control – Normal saline (1ml/kg)    
Group 2:  STD – 200mg/kg
Group 3:  Test-1 - 200mg/kg
Group 4: Test-2 - 400mg/kg.

i. Elevated Plus  Maze (EPM) :
The elevated plus maze served as the exteroceptive behavioral model to evaluate learning and memory in rats. This apparatus consist of two open arms(50 x 10 cm x 40 cm)  and two enclosed arms (50cm x 10 cm x 40cm), with an open roof, arranged such that the two open arms each other. The maze was elevated to a height of 50cm from the floor to measure the anxiety index in rats.
The rat was placed at the end of open arm facing away the central platform and measure the transfer latency (TL).TL was taken as the time taken by the  rat to move in to any one of the arms with all its four legs. Number of entries in to open and closed arms are evaluated. Scopolamine (0.4mg/kg, i.p.) was administered after 60 min of administration of the last dose of test drugs. After 30 mins, each rat was placed on the EPM and tested again for successful trail and evaluated the parameters.(Maribel Herrara Ruiz et al., 2005).

ii. Spatial Learning in Water Maze :
The water maze consists of large circular tank made of hard board or PVC. The pool was filled with water (20-22 c)to a depth of 0.3-0.4m. The floor of circular tank was marked off in to four equal quadrants arbitrarily designed north, south, east or west. An escaped platform was placed at the centre of the circular tank and covered with a coarse material that provides a good grip for the rat when climbing on a platform. The simplest measure of performances was the latency to escape from the water on to the platform. At the beginning trail animals were placed in the water maze and allowed to swim freely and to identify the escape platform. The trial was considered to be successful when rat identified the platform within three minutes .Time spent more than three minutes to identify the platform was recorded as error. The training should be conducted for before and ater 7^th & on 8^th  day scopolamine administration of the last dose of drug. After 30 min of scopolamine each rat was placed on the water maze and tested again for successful trail and evaluated the parameters. (Gerhard Vogel, 2002).

RESULTS:
1.    On 8^th day the effect of MELCT leaves on scopolamine induced Amnesia by Elevated plus maze method.

Values are expressed in mean + SEM, (n=6), when compared with control, *P<0.05, **P<0.01,      ***P<0.001 one-way ANOVA followed by Dunnet’s t – Test.

2.    On 8^th day effect of meLct leaves  on scopolamine induced amnesia by spatial learning in water maze method.

MELCT– Methanolic extract of Clitoria ternatea.Linn
Values were expressed in mean + SEM, (n=6), when compared with control, *P<0.05, **P<0.01,      ***P<0.001 one-way ANOVA followed by Dunnet’s t – Test.

Discussion:
Alzheimer’s diseases (AD) is a genetically heterogeneous neurodegerative disorder, which is slow onset with loss of memory which leads to dementia. AD is a neurodegerative disorder manifested by cognitive and memory deterioration progressive impairment of routine activity  of living and a variety of neuropsychiatric symptoms and behavioral disturbances.

(Hanumathacharjoshi, 2006).
Nootropics are the cognitive enhancers that are extremely nontoxic. These cognitive enhancers are the drugs ,  supplements and functional foods that improve the mental function (Lanni c et al., 2008). Various nootropic drugs such as piracetam, oxicetam, pramiracetam are used but due to its side effects they have limited utility. so, the herbal drugs are used such asPrunus  amygdalus (S.Kulkarni et al.,2011), Hibiscus Sabdariffa etc (Hanumathachar joshi ,2006) have been investigated for their effect on cognitive function  of the brain. Several models for amnesia are available, among them scopolamine induced memory deficits have been used in our study Clitoria ternatea.L is a perennial herb possessing many pharmacological activities such as antioxidant (A.P. Patil and V.R.Patil,2011), antipyretic (Parimaladevi. B et al., 2004), anti inflammatory and analgesic (Parimaladevi .B et al., 2003), Anti-inflammatory and analgesic (Parimaladevi et al., 2007), Nephroprotective (K.Sarumanthy et al.,2010), Larvicidal (Mathew N et al.,2009), anti microbial (Mhaskar AV et al.,2010), proteolytic (ainouz IL et al.,1994), diuretic (Piala JJ et al., 1962), antihelmenthetic  (Nirmal et al 2008), Platelet aggregation  (Honda T  et al., 1991), Antidiarrhoeal (Nitin kumar upwar et al., 2010), Hepatoprotective (Ramalingam shanmugsundaram et al., 2010), Anti-diabetic (Daisy .p et al ., 2009).

Results in  Elevated plus maze method  was - i) control Vs all the groups ie, control 24+ 0.3651, standard 18 + 0.3651**,test-I – 21.2+ 0.4773**,test-II- 19+0.3651**, the values were compared with control and Values are expressed in mean + SEM, (n=6), when compared with control, *P<0.05, **P<0.01, ***P<0.001 one way ANOVA followed by Dunnet’s t – Test. Results in spatial learning in water maze  was i) control Vs all the groups i.e., control 7.5+ 0.5000, standard 4 + 0.2582**,test-I – 4.8+ 0.3073**,test-II- 3.5+0.5477**, Values are expressed in mean +  SEM, (n=6), when compared with control, *P<0.05, **P<0.01, ***P<0.001 one way ANOVA followed by Dunnet’s t – Test , The MECT (200mg/kg, 400mg/kg) prevented the scopolamine induced amnesia in elevated plus maze method and spatial learning in water maze ,both piracetam and MECT meet major criteria for nootropic activity, namely improvement of memory in absence of cognitive deficit. Hence Clitoria ternatea.L may be useful as a nootropic agent in the treatment of various cognitive disorders.

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CONCLUSION:
Clitoria ternatea.L possess various pharmacological activities such as antioxidant, antimicrobial, proteolytic, Larvicidal, Hepatoprotective, nephroprotective, wound healing, antihelmenthetic, diuretic .In the present study, the methanolic extract of leaves of Clitoria ternatea.L showed a promising nootropic effect in scopolamine induced amnesia in rats. Hence oral administration of MELCT has the potential to improve the memory & learning of cognitive functions. Further the active principles of Clitoria ternatea.L leaves responsible for this property is too isolated phytochemically and studies with these purified constitutions are waiting to understand the complete mechanism of nootropic activity.

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