Available online at www.ijntps.org | ISSN: 2277 – 2782 INTERNATIONAL JOURNAL OF NOVEL TRENDS IN PHARMACEUTICAL SCIENCES
RESEARCH ARTICLE
Memory and learning enhancing activity of different extracts of roots of Vetiveria zizanioides 1
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C. Velmurugan , SK Shajahan* , B.S. Ashok Kumar , S. Vijaya Kumar , R Anitha Priyadharshini 3 and Sujith Thomas 1
Department of Pharmacology, Sri Krishna Chaithanya College of Pharmacy, Madanapalle. Department of Pharmacognosy, Sri K.V. College of Pharmacy, Chickballapur, Karnataka. 3 Department of Pharmacology, Moulana College of Pharmacy, Kerala-679321. 2
Article Info Article history Received 25 Oct 2014 Revised 28 Oct 2014 Accepted 29 Oct 2014 Available online 30Dec 2014 Keywords Transfer latency, memory, learning, Vetiveria zizanioides, Alzheimer’s disease.
Abstract The present study was undertaken to investigate the effects of Vetiveria zizanioides on learning and memory in mice. Elevated plus maze were employed to test learning and memory. The dose of 500 mg/kg, p.o. of CEVZ, EAEVZ, EEVZ and AEVZ extracts were administered for 28 successive days in separate group of animals. The dose of 500-mg/kg p.o. of EAEVZ, EEVZ & AEVZ significantly improved learning and memory of mice by decreasing TL in EPM. The result shows CEVZ has no effect on learning and memory and EAEVZ reveal less significant than the EEVZ & AEVZ. Further more, this dose significantly reversed the amnesia induced by scopolamine (0.4 mg/kg, p.o.). To delineate the mechanism by which EA, E, & AEVZ exerts memory and learning, the effect of extracts on whole brain AChE activity was also assessed. Except CEVZ all other extracts decreased whole brain acetyl cholinesterase activity. Here, Piracetam (200 mg/kg, i.p) was used as a standard nootropic agent. Hence Vetiveria zizanioides appears to be a promising candidate for improving memory and it would be worthwhile to explore the potential of this plant in the management of dementia and Alzheimer’s disease.
INTRODUCTION Memory is the ability of an individual to record sensory stimuli, events, information etc., retain them over a short or long period of time and recall the same at a later date when needed [1]. Learning is the process of acquiring knowledge about the world and memory could be considered as the retention of the acquired knowledge, which can be retrieved as and when, required [2]. Poor learning abilities, impaired memory, lower retention and slow recall are the common problems in stressful situations. Moreover, age, stress and emotions are conditions that may lead to impaired learning, memory loss, amnesia, and dementia or to more ominous threats like Schizophrenia and Alzheimer’s disease [3]. Learning and memory are generated by an experience dependent and long-lasting modification of the central nervous system. In fact, reduced cholinergic activity in the brains of individuals with AD provides the rationale for the development of acetyl cholinesterase (AChE) inhibitors to treat the dementia associated with VOLUME 4 | NUMBER 6 | DEC | 2014
AD[4]. The commonly prescribed AChE inhibitors, such as Donepezil, Rivastigmine, and Galantamine were developed on this basis [5]. As memory involves many interwoven brain functions, there are several different types of memories and virtually any type of brain damage can result in one or other type of memory loss [6]. In recent years, efforts have been made to find new therapeutic agents for the prevention or treatment of memory disorders, such as the gradual impairment of memory in aging or in neurodegenerative pathology and even lifestyle factors, but an effective therapy for the resolution of the cognitive deficits is still needed. Moreover drugs that could not only treat but also prevent diseases of older adults are needed in the clinical practice. Both prevention and delay of these disorder onsets would have a large impact in terms of reducing both suffering and costs [7]. Through the ages, To whom correspondence should be addressed: SK. Shajahan E-mail:
[email protected]
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many medicinal herbs have been used to improve memory and cognitive function and to treat neurodegenerative diseases in traditional medicine [8]. Pharmacological effects of some plants have also been reported [9]. Ayurveda, the Indian system of medicine, is gaining greater attention and popularity in many parts of the world. The disease preventive and health promote approach of Ayurveda, which takes into consideration the whole body, mind and spirit while dealing with the maintenance of health promotions, now enjoys increasing acceptability [10]. Vetiveria zizanioides (Linn.) Nash, a member of the family Poaceae commonly known as the Khas-Khas, Khasor Khus grass in India [11]. An Overview on Vetiveria Zizanioides, shows the information about Uses of different parts such as roots, leaves and stem. Over 150 compounds have been isolated and characterized from Vetiver oil so far. A major portion of oil consist of sesquiterpenoide, hydrocarbons and their oxygenated derivatives, phytochemical screening of the powdered leaves shows the presence of alkaloids, flavonoids, tannins, phenols, terpenoids and saponins. The roots are aromatic, antifungal action, cooling, antiemetic, diaphoretic, hemostatic, expectorant, diuretic,
stimulant, hysteria ,insomnia, skin diseases, asthma, amentia, amenorrhea, antispasmodic ,kidney problems, gall stones, mosquito repellent and antioxidants [12]. Ayurveda claims that several plants, called as the "Medhya" plants (intellect promoting) herbs are beneficial in cognitive disorders [13]. Based on this the present study designed to evaluate the nootrophic activity of Vetiveria zizanioides (Linn.). MATERIAL AND METHODS Collection, Authentication and extracts preparationof the plant material The root of Vetiveria zizanioides had been collected from the field of tirumala forest, Chittoor District, Andhra Pradesh, India. The plant was identified and authenticated by the Botanist Dr. K. Madhava Chetty, Assistant Professor, Department of botany, Sri Venkateswara University, Tirupathi. Collected root of Vetiveria zizanioides dried under shade and ground into powder with mechanical grinder. The dried powder of root of Vetiveria zizanioides was defatted with petroleum ether. The defatted powder material (marc) thus obtained was successively extracted with solvent like Chloroform, Ethyl acetate, Ethanol and aqueous by maceration [14].
Table 1. Data showing the extractive values of dried root powder of Vetiveria zizanioides Plant name Part used Method of Solvent Colour of Nature of extraction extract extract Chloroform Green Semisolid Vetiveria Ethyl acetate Brownish green Semisolid zizanioides Root Maceration Ethanol Dark brown Semisolid aqueous Dark brown Semisolid Preliminary phytochemical investigation of various extracts of vetiveria zizanioides root The various extracts of Vetiveria zizanioides root was subjected to qualitative analysis for the various phytoconstituents [15]. Experimental Animals Swiss albino mice (20-25 g) of either sex and of approximate same age used in the present studies were procured from listed suppliers of Sri Venkateswara Enterprises, Bangalore, India. The animals were fed with standard pellet diet (Hindustan lever Ltd. Bangalore) and water ad libitum. All the animals were housed in polypropylene cages. The animals were kept under alternate cycle of 12 hours of darkness and light. The animals were acclimatized to the laboratory VOLUME 4 | NUMBER 6 | DEC | 2014
% yield of extract 5.56 9.43 14.86 13.45
conditions for 1 week before starting the experiment. The animals were fasted for at least 12 hours before the onset of each activity. The experimental protocols were approved by Institutional Animal Ethics Committee (SKCP/IAEC/PGCOL/13-14/05). Acute oral toxicity study Swiss albino mice with weight ranging (20-25 gm. female) were taken for the experiment. The animals were made into a group of 3 each, dose of extracts was given according to the body weight (mg/kg), starting dose of 5-5000 mg /kg was given to the first individual animal, no death was occurred, and higher doses were given to next group of animals. The observation shows the various extracts of root of Vetiveria zizanioides was screened for acute
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toxicity study by OECD guidelines 423 for determining the LD50. The results showed that LD50 th was found to be 5000mg/kg. 1/10 of LD50 is called as ED50. Therefore its ED50 was found to be 500mg/kg [16]. MEMORY AND LEARNING ACTIVITY Elevated plus maze The elevated plus maze served as the exteroceptive behavioral model (wherein the stimulus existed outside the body) to evaluate learning and memory in mice. The apparatus consisted of two open arms (16 cm × 5 cm) and two covered arms (16 cm × 5 cm ×12cm). The arms extended from a central platform (5cm×5cm) and the maze was elevated to a height of 25 cm from the floor. On the first the day, each mouse was placed at the end of open arm, facing away from central platform. Transfer latency (TL) was taken as the time taken by the mouse to move into any one of the covered arms with all its four legs. TL was recorded on the first day for the each animal. The mouse was allowed to explore the maze for another 2 min. and returned to its home cage. Retention of this learned task was examined 24 h after the first day trial [17].
administration on 28th day and again after 24 hr i.e. on 29th day. Group VI: EEVZ (500 mg/kg, p.o.) were administered orally to the young mice for 28 successive days. TL was noted after 60 min of administration on 28th day and again after 24 hr i.e. on 29th day. Group VII: AEVZ (500 mg/kg, p.o.) were administered orally to the young mice for 28 successive days. TL was noted after 60 min of administration on 28th day and again after 24 hr i.e. on 29th day. Group VIII: Piracetam (200 mg/kg, i.p.) was injected for 28 suc-cessive days to young mice. At 30 min after the injection of piracetam on the 28th day, scopolamine 0.4 mg/kg, i.p. was ad-ministered. TL was noted after 45 min of administration of scopolamine and again after 24 hr i.e. on 29th day. Group IX-XII: CEVZ, EAEVZ, EEVZ & AEVZ (500 mg/kg, p.o.) were administered orally to the young mice for 28 successive days and Scopolamine (0.4 mg/kg) was injected i.p. to young mice at 60 minutes after administration of extract on 28th day. TL was noted 45 minutes after scopolamine injection and after 24h (i.e. on 29th day).
Mice were divided into 12 groups and each group consisted of a 6 animals Group I: It represented the control group for young mice. Vehicle was administered orally for 28 successive days and transfer latency was measured after 60 min of administration on 28th and again after 24 hr i.e. on 29th day. Group II: It represented the positive control group for young mice. Piracetam (200 mg/kg i.p.) was injected to young mice for 28 successive days and transfer latency was measured after 30 min of administration on 28th day and again after 24 hr i.e. on 29th day. Group III: It represented the negative control group for young mice. Scopolamine (0.4 mg/kg) was injected i.p. to young mice and transfer latency was measured 45 min after injection on 28 th day and again after 24 hr (i.e. on 29th day). Group IV: CEVZ (500 mg/kg, p.o.) were administered orally to the young mice for 28 successive days. TL was noted after 60 min of administration on 28th day and again after 24 hr i.e. on 29th day. Group V: EAEVZ (500 mg/kg, p.o.) were administered orally to the young mice for 28 successive days. TL was noted after 60 min of
BIOCHEMICAL STUDIES Assay of Acetyl cholinesterase (AchE) activity in the brain After behavioural assessment on these tasks, the alteration in the AchE activity was evaluated in the whole brain. At the end of the study, mice were euthanized by decapitation, brains was removed, washed with ice-cold saline solution (0.9 % NaCl), weighed and stored at -80 ºC for the biochemical analyses. The tissues were homogenized with 0.1 M phosphate buffer saline at pH 7.4, to give a final concentration of 10 % w/v for the biochemical assays. For the determination of AchE level, frozen brain samples were homogenized in cold 0.1 Nperchloric acid. The esterase activity was measured by providing the substrate, acetylthiocholine (ATC). Thiocholine released by the cleavage of ATC by AchE is allowed to react with the -SH reagent 5,5’dithiobis-(2-nitrobenzoic acid) (DTNB), which is reduced to thionitrobenzoic acid, a yellow coloured anion with an absorption maxima at 420nm.The extinction co-efficient of the thionitro benzoic acid is 1.36 x104/mole/centimeter The concentration of thionitro benzoic acid detected using a UV spectrophotometer is then taken as a direct estimate of the AchE activity [18-19].
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Rate= Change in the absorbance/min X (5.74x10 ) Co Statistical Analysis One-way analysis of variance (ANOVA) followed by Dunnett’s method of multiple comparisons was employed using Graphpad Instat 5.0 software.
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