RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

BANGALORE, KARNATAKA

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECT DISSERTATION

1. / NAME OF THE CANDIDATE AND ADDRESS / BHADANIA MITALKUMAR BHARATBHAI
DEPT. OF PHARMACOLOGY
SET’s COLLEGE OF PHARMACY
S.R.NAGAR,
DHARWAD-580002
2. / NAME OF THE INSTITUTION / SET’s COLLEGE OF PHARMACY
S.R.NAGAR,
DHARWAD-580002
3. / COURSE OF STUDY AND SUBJECT / MASTER OF PHARMACY IN PHARMACOLOGY
4. / DATE OF ADMISSION OF COURSE / JUNE-2007
5. / TITLE OF THE TOPIC
“EVALUATION OF NOOTROPIC EFFECT OF MENTHOL, IT’S INFLUENCE ON BRAIN CHOLINERGIC SYSTEM AND AMINO ACID CONCENTRATION IN MICE”
BRIEF RESUME OF INTENDED WORK:
Dementia is a neurodegenerative disease characterized by a general decrease in memory, attention and cognitive functions. It is therefore important to develop adequate objective diagnostic tools to differentiate dementia and other neurodegenerative conditions1. Cognitive impairment is seen in a wide variety of neurodegenerative disorders. Working memory is a form of short-term memory with a limited capacity and an extremely rapid decay. Its impairment is more depictive of memory disorder in the Alzheimer’s dementia2. β-amyloid deposition is pathognomic for Alzheimer’s disease, but may occur in normal elderly people without apparent cognitive effect. Episodic memory impairment is an early and prominent symptom of Alzheimer’s disease3. Alzheimer’s disease (AD) is the most common cause of progressive cognitive decline and dementia in aged humans. The deposition of the β-amyloid peptide (Aβ) in extracellular neuritic plaques of Alzheimer’s disease patients is an early and invariant feature of this neurodegenerative disorder4.
Excitatory amino acids are very well known to modulate memory processes. Particularly important role in this respect is ascribed to the glutamate, N-methyl-D-aspartate receptor and its co-agonists like glycine and alanine. It has been recently reported that alanine can also act as a carrier of ammonia nitrogen between glutamatergic neurons and neighboring astrocytes, contributing to neurotoxicity5.
6.1 Need for the study:
Learning is a process of acquiring knowledge about the world and memory is its retrieval. This process is not an independent process, but is influenced or modified by the immune system. Dementia is a syndrome of failing memory and other intellectual functions with little or no disturbance in consciousness. Degeneration of the cerebral neurons is of one of the commonest and vital causes for dementia with increasing age, there by leading to deterioration in quality of life in elderly. A greater research is required in early diagnosis of the condition and development of newer effective drugs to prevent or halt the progression of the disease6.
The neuronal damage in the hippocampus is related to the accompanying memory dysfunction; since the hippocampus is well recognized as a structure participating in memory processes, and impairment to this brain area can produce sever amnesia. Spatial-memory deficits have been observed in many species with hippocampal damage. Disturbances of memory may be the consequence of seizure-induced bio-chemical changes in the brain, related to glutamate and aspartate release5.
Recently memory complaints and memory disorders are becoming more prevalent due to various factors such as natural (aging, physical, mental stress) , environmental (excess levels of CO, CO2 , Aluminum in foods) , iatrogenic ( electro convulsive shock therapy and use of certain CNS depressants) and diseases like Alzheimer’s disease, Brush field-Wyatt disease, and Huntington’s chorea7.
The present study has been designed to investigate the nootropic potential of menthol, 2-(2-Propyl)-5-methylcyclohexanol, a major phytochemical present in mentha species in mice.
6.2  Review of literature:
AD is a progressive neurodegenerative brain disorder that is slow in onset but leads to dementia, unusual behavior, personality changes and ultimately death. The personality distortions interfere with the patient’s professional life, social activities and relationships8.
Recent behavioral pharmacological and neurobiological studies have provided evidence for a cholinergic involvement in learning and memory. The cholinergic hypothesis claims that the decline in cognitive function in dementia is predominantly related to a decrease in cholinergic neurotransmission. Acetylcholinesterase inhibitors, which enhance the availability of acetylcholine in the synaptic cleft, able to reverse the scopolamine induced deficit9.
Menthol and many of its derivatives are reported to produce profound sensory and mental effects. Menthol can act directly on presynaptic Ca2+ stores of sensory neurons to release Ca2+, resulting in a facilitating of glutamate release and a modulation of neuronal transmission at sensory synapses10.
Ability of Menthol to chemically trigger cold-sensitive receptors in the skin is responsible for the well known cooling sensation that it provokes when inhaled, eaten, or applied to the skin. Menthol does not cause an actual drop in temperature. Mentha arvensis is the primary species of mint used to make natural menthol crystals and natural menthol flakes. This species is primarily grown in the Uttar Pradesh region in India.
6.3  Objective of the study:
The objectives of the proposed study are:
1. Effect of menthol on cognitive functions
2. Effect of menthol on the brain cholinergic system
3. Effect of menthol on the brain amino acid concentration
7. 0 MATERIAL AND METHODS:
7.1 Animals
Male Swiss mice having 18-35 g body weight will be used for the experiment.
7.2 Drugs and chemicals
1. Menthol (2-(2-Propyl)-5-methylcyclohexanol)
2. Piracetam (Nootropil® , UCB India Pvt. Ltd. , vapi ,Gujarat)
3. Scopolamine (Sigma Aldrich, Lt. Louis, MO,USA)
4. Diazepam (Valium®, Ranbaxy laboratories Ltd., Mumbai , India )
5. Phenytoin (Zydus Neurosciences , Ahmedabad, India )
MEMORY MODELS:
EXTEROCEPTIVE BEHAVIOR MODELS :
1.  Elevated Plus Maze:
Elevated plus maze served as the exteroceptive behavioral model to evaluate memory in mice. The procedure, technique and end point for testing memory as per the parameters described by the investigators working in the area of psychopharmacology11.
2. Morris Water maze :
The Morris water maze task is a sensitive model for studying the cognitive processes in rodents that not only reliably reflects the ability of the animal to learn the position of the hidden platform, but also reflects exploratory aspects of behavior. These rapid developments in the field of animal models of learning and memory processes may hopefully lead to an improved understanding of the pathophysiology of Alzheimer’s disease, and finally permit the rational designing of novel therapeutic strategies for distinct cognitive dysfunctions9.
INTEROCEPTIVE BEHAVIOR MODELS :
1.Scopolamine induced amnesia:
Scopolamine is a powerful muscarinic agonist capable of crossing blood brain barrier, acts both peripherally by blocking the receptors for acetylcholine at the synapse. It impairs cognitive functions. The dose of 0.4 mg/kg is approved to produce cognitive and memory changes without causing debilitating peripheral anticholinergic effect 12,13.
2. β-amyloid protein induced amnesia :
β- Amyloid (Aβ) deposition is pathognomic for Alzheimer’s disease (AD), but may occur in normal elderly people without apparent cognitive effect. Episodic memory impairment is an early and prominent sign of AD 3.
BIOCHEMICAL PARAMETERS :
1. Whole Brain AChE activity :
The whole brain AChE activity shall be assessed using the Ellman colorimetric method8.
2. Brain antioxidant activity :
Oxidative stress plays a primary role in the pathogenesis of AD14. Aging and cognitive decline thus appear to involve changes at multiple nodes within a complex regulatory network15. β-amyloidal induced reactive oxygen spicies ROS accumulation cause damages to neuronal membrane lipids, proteins, and nucleic acids, and ultimately leads to apoptosis which is believed to play a critical role in cell loss during progression of AD. Therefore, considerable efforts have been made to search for the antioxidants that reduced Aβ- induced oxidative stress AD14.
3. Determination of neurotransmitter concentration :
The various brain biogenic amines in discrete regions of the rat brain where estimated by the method of Wagner et al. Brains are rapidly removed and the cerebral cortex, cerebellum, hypothalamus, hippocampus, pons and corpus striatum shall be dissected on an ice-cold plate. Concentration of nor epinephrine (NE), epinephrine (E), dopamine, 5-hydroxytryptamine (serotonin,5-HT) are measured by high performance liquid chromatography (HPLC) couple with electrochemical detection (ECD)16.
4. Determination of brain amino acid concentration :
Whole Brain amino acids such as glutamate, N-methyl-D-aspartate, glycine, alanine shall be measured using HPLC5.
Source of data:
The sources of data are from the laboratory experiments, which involve
Evaluation of pharmacological activities on experimental animals.
7.3 Does the study require any investigations or intervention to be conducted on patients or other humans or animals? If so, please describe briefly.
Yes, the above studies require in vivo screening techniques on male mice.
7.4 Has ethical clearance been obtained from your institution in case of above 7.3?
The copy of ethical clearance certificate is enclosed.
8.0 LIST OF REFERENCES
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2.  Naveen K, Kohli K. Effect of Metoclopramide on scopolamine-induced working memory impairment in rat. Ind J pharmacol 2003;35:104-8.
3. Kerryn EP, Greg S, Victor LV, Steven NG, Simon AM , Paul M. β-amyloid
imaging and memory in non-demented individuals, evidence for preclinical
4.  Alzheimer’s Disease. Brain A J Neuro 2007;130(11):2837-4.
5.  Meziane H, Dodart JC, Mathis C, Little S, Clemens J, Paul SM. Memory enhancing effects of secreted forms of the β-amyloid precursor protein in normal and amnestic mice. Proc Natl Acad sci Neurobio USA 1998;95:12683-8.
6.  Janusz S, Agnieszka P, Kamilla BK, Anna S, Piotr M, Jerzy W. Effect of kindled seizures on rat behavior in water Morris maze test and amino acid concentrations in brain structures. Pharmacological reports 2006;58:75-82.
7.  Ashutosh A, Malini S, Bairy KL, Muddanna SR. Effect of Tinospora cordifolia on learning and memory in normal and memory deficit rats. Ind J Pharmacol 2002;34:339-49.
8.  Annapurna AV, Ramakrishna K. Muralikrishna , Krishna kumar, Prasad KB. Studies on nootropic activity of Ramipril and Losartan on Scopolamine–induced Amnesia in rats. Ind J Pharm sci 2004;66(1):31-5.
9.  Joshi H, Parle M. Brahmi rasayana improves learning and memory in mice. Evidence-based Complementary and alternative Medicine; 2006; 3(1):79- 85.
10.  Kulkarni SK, Hand Book of Experimental Pharmacology. New Delhi: Vallabh Prakashan; 2003.
11.  Kenzo T, Hong X, Jennifer L, Jianguo G. Menthol induced Ca2+ release from presynaptic Ca2+ stores potentiates sensory synaptic transmission. J Neurosci 2004; 24(3):762-1.
12.  Joshi H, Parle M. Nootropic activity of calyces of Hibiscus sabdariffa Linn. IranianJ Pharmacol Therapeutics 2006;5(1):1-10.
13.  Avadhesh C, Sharma, Kulkarni SK. Reversal of scopolamine and Dizocilpine
14.  induced memory dysfunction by angiotensin converting enzyme inhibitors in rats and mice. Ind J Pharmacol 1992;24:147-3.
15.  Joshi H, Parle M Evaluation of the antiamnesic effect of Phyllanthus amarus in mice. Colomb Med 2007;38:132-9.
16.  Moon H J, Xiang LP, Hyun YK, Eun JC, Seung HB, Sung WK. Reserveratrol oligomers from vitis amurensis attenuated β- amyloid induced oxidative stress in PC12 cells. Biol Pharm Bull 2007;30(6):1130-4.
17.  Wulf D, Hyman MS. Oxidative stress and aberrant signaling in aging and cognitive decline. J. Cognition 2007; 6:361-70.
18.  Rajan R, SheelaDevi R, James S, Manohar S. Noise-Stress-induced Brain neurotransmitter change and the effect of Ocimum sanctum (Linn) Treatment in Albino rats. J Pharmacol sci 2005;98:354-60.