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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-Daspartate 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. Spatialmemory 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
1. Neelam V, Sandeep C, Bhatia MS, Tandan OP. Event related evoked potentials
in dementia role of vitamin E. Ind J Physiology and Pharmacology
2002;46(1):61-8.
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.