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Neuroprotective activity of matricaria reticutta linn. Against alzheimer disease and parkinson’s disease in rat model


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NEUROPROTECTIVE ACTIVITY OF MATRICARIA RETICUTTA Linn.

AGAINST ALZHEIMER DISEASE AND PARKINSON’S DISEASE IN RAT MODEL

Protocol of Dissertation Submitted

By

Mr. KUSHWAHA JYOTI SHRIHEMRAJ

To

Rajiv Gandhi University of Health Sciences



Bangalore, Karnataka.

Under the guidance of

Mr. V.M. Chandrashekhar

Associate Professor





Department of Pharmacology

HANAGAL SHRI KUMARESHWAR COLLEGE OF PHARMACY

BAGALKOT-587101, KARNATAKA

(2011-2012)

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA-BANGALORE
ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECT FOR

DISSERTATION


1.

Name of the candidate and address




KUSHWAHA JYOTI SHRIHEMRAJ

Department of Pharmacology,

H.S.K. College of Pharmacy,

B.V.V.S campus

Bagalkot-587101,Karnataka.



2.

Name of institution


H.S.K. College of Pharmacy,

B.V.V.S campus

Bagalkot-587101,Karnataka.




3.

Course of study and subject



Master of Pharmacy in Pharmacology




4.

Date of admission to course



20 June 2011






5.

Title of topic : Neuroprotective activity of Matricaria recutita Linn. against



Alzheimer disease and Parkinson’s disease in rat model.






6.

7.

8.



BRIEF RESUME OF INTENDED WORK
6.1 NEED FOR THE STUDY
Alzheimer’s disease (AD) is a devastating neurodegenerative disorder manifested by deterioration in memory and cognition, impairment in performing activities of daily living, and many behavioural and neuropsychiatric illnesses1. Alzheimer’s disease is the most common form of dementia in the old age. The percentage of persons with Alzheimer disease increases by a factor of 2 with every 5 years of age, so 1% of 60 year old and 30% of 85 year old have the disease. By 2050, the number of cases in US is predicted to rise to 13.2 million2. One of the pathological characteristics of AD is the progressive deposition of insoluble amyloid β protein (Aβ) as a form of senile plaques3. AD is characterised pathologically by the presence of both extracellular amyloid-beta (Aβ) plaques and intraneuronal neurofibrillary tangles (NFTs), composed of the microtubule-associated protein tau, which occur mainly in regions of the brain involved in learning and memory4.

Parkinson disease is an idiopathic, slow progressive, degenerative CNS disorder characterized by slow decreased movement of muscular rigidity, resting tremor, and postural instability. Etiology of parkinson are of two types primary parkinson’s disease caused due to pigmented neurons of substantia nigra, locus caeruleus and other brain stem dopaminergic cell group are lost. Secondary parkinson disease, results from loss of or interference with the action of dopamine in basal ganglia due to other idiopathic degenerative diseases. It affects about 1% of those ≥ 40yrs old. The mean age of onset is about 57 yr 5.

At the present state of knowledge of treatment of Alzheimer disease and Parkinson’s disease not adequate and allopathic drugs for treatment of alzheimer and parkinson’s disease are not sufficient and not much benefited. In this concern, natural products (medicinal plants based products) probably represent an ideal source to develop safe and effective agents for management of alzheimer and parkinson’s disease6. The flavonoids7, Gingko biloba 8, Ginseng 9 have reported successfully recovery from Alzheimer disease and parkinson’s disease. Author has fascinated with this background and the present study will be purpose to know the safe and potential neuroprotective effect of Matricaria recutita studies will be carried out against alzheimer disease and parkinson’s disease in pre-clinical models.



6.2 REVIEW OF LITERATURE
Plant Profile

Title of the plant : Matricaria recutita Linn.

Family : Asteraceae (Compositae)

Synonyms : Matricaria, German Chamomile, Sweet False Chamomile’s.

Habitat : German Chamomile is annual herb originally from Europe which has to the wild and now naturalized in almost every continent. It can now be found growing along fence rows and in sunny open field from Southern Canada to Northern U.S. West to Minnesota.

Chemical constituents:

The plant contains Terpenoids like α-Bisabolol, α-Bisabolol oxide A and B, Chamazulene, Sesquiterpenes. Flavonoids like Apigenin, Luteolin, Quercetin, Coumarins, Umbelliferone and others are Anthemic acid, Choline, Tannin and Polysaccharides.



Medicinal uses :

Matricaria recutita Linn. has been used medicinally for thousands of years and is widely used in Europe. It is a popular treatment for numerous ailments including sleep disorders, anxiety, digestion, intestinal conditions, skin infections, inflammations (including eczema), wound healing, infantile colic, teething pains and diaper rash. In the USA, Chamomile is the used externally for wounds, ulcers, eczema, gout, neuralgia, rheumatic pain, hemorrhoids and leg ulcers etc 10.

Pharmacological actions :

Matricaria recutita is the potentially used as an antioxidant 11, neuroprotective12, diabetic complications13, cardiac effects 14, antimicrobial activity 15, anti proliferative and apoptic activity16. Cardiovascular effects as increase atrial rate, relaxed thoracic aorta17, hemodynamic effects and anti ulcer activity18. Chamomile’s main active constituents are apigenin and bisabolol. Apigenin have cardio protective activity 19, 20.

6.3 OBJECTIVE OF STUDY

The traditionally well known Matricaria recutita (M.R) will be extracted by using non-polar and polar solvents and extract will be used for the present study with following objectives.



  1. Neuroprotective activity of M.R extracts against colchicine induced Alzheimer disease models.

  2. Neuroprotective activity of M.R extracts against 6-OHDA induced Parkinson’s disease models.


MATERIALS AND METHODS:

    1. Source of data: All the data will be collected from the animal experiments and standard parameters for the study. The selection of doses will be based on earlier literature acute toxicity studies 21.

    2. Materials:

Chemicals : 6-OHDA, 2-Thiobarbituric acid, Trichloroacitic acid, colchicine.

Animals : Sprague dawley rats of either sex.

Instruments : Surgical nylon thread, Surgical microscope, Refrigerator centrifuge,

UV-Spectrophotometer.


    1. Method:

      1. Colchicine induced Alzheimer model :

The animal were divided into different groups:

Group- I : Normal animals receive normal saline. (n=8).

Group-II : Sham receives 3µl of 0.2% L-ascorbate saline orally. (n=8).

Group-III : Control animals receive colchicine in 0.2% L-ascorbate saline. (n=8).

Group-IV : Effect of low dose of M.R extract in colchicine induced Alzheimer disease. (n=8).

Group-V : Effect of moderate dose of M.R extract in colchicine induced Alzheimer disease. (n=8).

Group-VI : Effect of high dose of M.R extract in colchicine induced Alzheimer disease. (n=8).


      1. 6-OHDA induced Parkinson’s disease:

Group- I : Normal animals receive normal saline. (n=8).

Group-II : Sham receives 3µl of 0.2% L-ascorbate saline orally. (n=8).

Group-III : Control animals receive 6-OHDA. (n=8).

Group-IV : Effect of low dose of M.R extract in 6-OHDA induced Parkinson’s disease. (n=8).

Group-V : Effect of moderate dose of M.R extract in 6-OHDAInduced Parkinson’s disease. (n=8).

Group-VI : Effect of high dose of M.R extract in 6-OHDA induced Parkinson’s disease. (n=8).


7.4 Methods:

7.4.1 Preparation of plant extract:

The plant will be authenticated and collected from NBRI, Lucknow in ideal conditions will be air dried under the shade and powdered to a fine texture of uniform size by passing through the sieve # 44. The powder collected will be extracted with polar and non polar solvent and the extract obtained will be used for the present study.



7.4.2 Colchicine induced Alzheimer disease:

7.4.2.1 Animals: Young male Sprague dawley rats (180–200 g) will be procured from central animal house, H.S.K College of Pharmacy, Bagalkot. Animals will be acclimatized to laboratory conditions at room temperature prior to experimentation. Following surgery, animals will be kept under standard conditions of a 12-hour light/12-dark cycle with food and water ad libitum in groups of 2, in plastic cages with soft bedding. All the experiments will be carried out between 9.00 AM and 3.00 PM. The protocol was approved by the Institutional Animal Ethics Committee of H.S.K College of Pharmacy, Bagalkot and carried out in accordance with the CPCSEA Guidelines for the use and care of laboratory animals.

7.4.2.2 Surgery and intracerebroventricular administration of Colchicines. Animal will be anesthetized with thiopental sodium (45 mg/kg) and positioned in a stereotaxic apparatus. The head was positioned in a frame and a midline sagittal incision made in the scalp. Two holes were drilled in the skull for the placement of the injection cannula into both the lateral cerebral ventricles. Co-ordinates for the intracerebroventricular (ICV) cannula implantation were 0.8mm posterior to bregma, 1.8mm lateral to the sagittal suture, and 3.6mm beneath the cortical surface. The scalp was then closed with a suture. Gentamicin (5mg/kg, IP) was applied to the surgical area in order to prevent sepsis. Animals will be housed in a group of two with soft bedding. Special care of the animals was taken during the postoperative period to provide food and water inside the cage of rats. Rats will be infused ICV with either artificial cerebrospinal fluid (ACSF; in mmol/l: 147 NaCl, 2.9 KCl, 1.6 MgCl2, 1.7 CaCl2, and 2.2 dextrose) or 15 μg colchicines dissolved in ACSF. Solution (5 μL) will be injected using a hamilton microsyringe positioned in the injection cannula and the syringe will be kept in place for 2 minutes in order to allow for the diffusion of the injected volume and prevents pressure-induced damage.

7.4.2.3 Behavioural Assessment

7.4.2.3.1 Assessment of Cognitive Performance

Elevated Plus Maze Paradigm. The elevated plus maze consisted of two opposite black open arms (50 × 10 cm), crossed with two closed walls of the same dimensions with 40 cm high walls. The arms will be connected with a central square of dimensions 10 × 10 cm. The entire maze was elevated to a height of 50 cm from the floor. Acquisition of memory was tested on day 13 after colchicine administration. Animal will be placed individually at one end of the open arm facing away from the central square. The time taken by the animal to move from the open arm to the closed arm was recorded as the initial transfer latency (ITL). Animal will be allowed to explore the maze for 20 seconds after recording the ITL and then returned to the home cage. If the animal did not enter the enclosed arm within 90 seconds, it was guided on the back into one of the enclosed arm and the ITL was given as 90 seconds. Retention of memory was assessed by placing the rat in an open arm and the retention latency was noted on day 14 and day 21 of ITL and was termed as the first retention transfer latency (1st RTL) and second retention transfer latency (2nd RTL), respectively 22.

Spatial Navigation Task. The acquisition and retention of a spatial navigation task will be evaluated by using Morris water maze23. Animals will be trained to swim toward a visible platform in a circular pool (180 cm in diameter and 60 cm in height) located in a test room. In principle, rats can escape from swimming by climbing onto the platform and over time the rats apparently learn the spatial location of the platform from any starting position at the circumference of the pool. Thus the platform offers no local cues to guide the escape behavior of the rats. The only spatial cues are those outside of the tank primarily the visual cues. The pool was filled with water (28 ± 2C) to a height of 40cm, a movable circular platform(9 cm diameter),mounted on a column, was placed in a pool 2 cm above the water level during the acquisition phase. A similar platform was placed in the pool 2 cm below the water level for the maze retention phase. The water was made opaque by adding a nontoxic dye. Four equally spaced locations around the edge of the pool (N, S, E, and W) will be used as starting points and this divided the pool into four equal quadrants.

(1) Maze acquisition phase (training). Animals will be received a training session consisting of 4 trials on day 13. In all 4 trials, the starting position was different. A trial began by releasing the animal into the maze facing towards the wall of the pool. The latency to find the escape platform was recorded to a maximum of 90 seconds. If the rat did not escape onto the platform within this time, it was guided to the platform and was allowed to remain there for 20 seconds. The time taken by rat to reach the platform was taken as the initial acquisition latency (IAL).

(2) Maze retention phase (testing for retention of the learned task. Following 24 hour (day 14) and 8 days (day 21) after IAL, the rat will be released randomly from one of the edges facing the wall of the pool. The time taken to find the hidden platform was recorded and termed as first retention latency (1st RL) and second retention latency (2nd RL) on day 14 and day 21 following central administration of colchicines, respectively.
7.4.2.3.2 Assessment of Gross Behavioural Activity.

Gross behavioural activity will be observed on days 1, 7, 14, and 21 following ICV colchicine injection. Animal will be placed in a square (30 cm) closed arena equipped with infrared light-sensitive photocells using digital photoactometer.The animals will be observed for a period of 5 minutes and the values were expressed as counts/5 min 24.



7.4.2.4. Dissection and Homogenization. On day 24, after behavioural assessments, animals will be scarified by decapitation prior to deep anesthesia. The brains will be removed, forebrain was dissected out, and cerebellum was discarded. Brain put on ice and rinsed with ice-cold isotonic saline. A homogenate (10 % w/v) was prepared in 0.1M phosphate buffer (pH 7.4). The homogenate will be centrifuged at 10,000 g for 15 minutes and aliquots of supernatant was separated and used for biochemical estimation.

7.4.2.5. Biochemical Tests. The following biochemical tests will be performed measurement of lipid peroxidation25 and estimation of Reduced glutathione26, nitric acid27, Superoxide Dismutase activity 28, Catalase activity 29, Acetyl cholinesterase (AChE) activity 30 and Protein estimation 31.

7.4.3 6-OHDA induced Parkinson’s disease: To determine at which doses the extract shows maximum activity against parkinson’s disease. The different groups of rats will be separately treated with different doses of extract (low, moderate and high) according to body weight for a week before to 6-OHDA lesion. The striatal 6-OHDA lesions were performed by using 28-gauge cannula was inserted vertically into the striatum to a depth of 5.5 mm. At a rate of 1µl/min, 3.5 µl of 6-OHDA in 0.9% normal saline containing 0.2% ascorbic acid was injected. The same treatment will be continued after the lesion for a week32.

7.4.3.1 Behavioural assessment: Deficits in forepaw adjusting steps in this PD rat model provided a simple and consistent behavior phenomenological similar to akinesia in PD. After the 6-OHDA lesion, rats will be held by the rear part of the torso and placed on the surface of treadmill so that its weight was on one forepaw. The treadmill will be set to move at a rate of 90 cm/12s in the direction opposite to the weight-bearing forepaw resulting in the outward lateral shifting of the torso relative to the weight-bearing forepaw. The number of adjusting steps, defined as the movement of weight-bearing forepaw towards the torso to compensate for the outward lateral movement of the body was counted. Each stepping test consisted of five trials for each forepaw, alternating between forepaws, and each trial lasted 12s. The average of the five trials for each forepaw will be used for analysis. Adjusting steps will be tested in each group of animals at the beginning of the experiment and 2, 4, 6 and 8 weeks after the lesion33.

7.4.3.2 Effect of extract on biochemical profile: The 10% homogenated brain tissue, in cold phosphate buffer (pH 7.4). The homogenate will be centrifuged at 10,000 rpm for 20 min at 4ºC and the supernatant will be used for estimation of biochemicals as per the section 7.4.2.6.

7.4.4 Histopathological examination: Two animals from each will be sacrificed on the day of blood withdrawal and brain will be isolated and tissue will be immersed in 10 % formalin solution for histopathological studies. Sample will be embedded in paraffin sectioned and stained with haemotoxollin and eosin34.

7.4.5 Statistical analysis: All the data will be expressed in mean ± SEM. The significance of differences in mean between control and treated animals for different parameters determined by one way ANOVA followed by Turkey’s multiple comparison test. Significance for difference between groups were evaluated for student’s t-test to come to final conclusion.

7.5 Does the study require any investigations or interventions to be conducted on patients or other humans/animals? I so please describe briefly.

Yes, the above study requires to be carried out in rats. So for this study Sprague dawley rats will be

used.


7.6 Has ethical clearance been obtained from your institution in case of 7.2 and 7.3?

Yes, the study is cleared from Institutional Animal Ethics Committee. The copy is enclosed with this protocol.

References:

  1. Cummings JL. Alzheimer’s disease. N Engl J Med.2004;351:56-67.

  2. Hebert LE, Scherr PA, Bienias JL, Bennett DA, Evans DA. Alzheimer disease in the US population: prevalence estimates using the 2000 Census. Arch Neurol.2003;60:1119-1122.

  3. Wirths O, Multhaup G, Bayer TA. A modified beta-amyloid hypothesis: intraneuronal accumulation of the beta-amyloid peptide—the first step of a fatal cascade. J Neurochem.2004;91:513–520.

  4. Kerr F, Augustin H, Piper MDW, Gandy C, Allen MJ, Lovestone S, Partridge L. Dietry restriction delays aging, but not neuronal dysfunction, in Drosophila models of Alzheimer’s disease. Neurbiol Age.2011;32:1977–1989.

  5. Pereira Mark H, Beers MD, Berkow R. The merck manual 17th edition Merck Research Labs.1999;1417-1466.

  6. Marry AKK, Lloyd YY, Brian KA, Robin LC, Wayne AK. Applied therapeutics the clinical use of drug. WoltersKlawer / Lippincott Williams and Wilkins 9th edition.2009:53-61.

  7. Dajas F, Rivera –Megret F, Blasina F. Neuroprotective by Flavonoids. Brazil J Med Biol Res.2003;36:1613-1620.

  8. Kato Lee EJ, Chen HY, Chen TY, Ayoub IA, Maynard KI. Acute administration of Ginkgo biloba extract (Egb 761) affords Neuroprotection against permanent and transient focal cerebral ischemia in Sprague-Dawley rat. J Neurosci.2002;68:636-645.

  9. Shah ZA, Gilani RA, Sharma P, Vohora SB. Cerebroprotective effect of Korean ginseng tea against global and focal models of ischemia in rat. J Ethnopharmacol.2005;101:299-307.

  10. Newel CA, Anderson LA, Phillipson JD, Herbal medicines. A guide for health care professionals. London, Pharmaceutical press.1996;9:296.

  11. Pereira RP, Faachinetto R, de Souza Prestes A, Puntel RL, Santos da Silva GN, Heinzmann BM, Burger ME, Morel AF, Morsch VM, Rocha JB. Antioxidants effects of different extracts from Melissa officinalis, Matricariarecutita and Cymbopogoncitratus. Neurochem Res.2009; 34(5):973-983.

  12. Chandrashekhar VM, Ranpariya VL, Ganapathy S, Parashar A, Muchandi AA. Neuroprotective activity of Matricaria recutita Linn against global model of ischemia in rats. J Ethanopharmacol.2010;127:645-651.

  13. Kato A, Minoshima Y, Yamamoto J, Adachi I, Watson AA, Nash RJ. Protective effects of dietry Chamomile tea on diabetic complication. J Agric Food Chem.2008;56(17):8206-8211.

  14. Lawrence G, Raman Reddy CV, Robert FG. Cardial effects of Chammomile tea. J Clin Pharmacol.1973;13:475-479.

  15. Nogueira JC, DinizMde F, Lima EO. Anti microbial activity of plants in acute otitisexterna Braz J Otorhinolarygol.2008;74(1):118-24.




  1. Srivastava JK, Gupta S. Antiproliferative and apoptic effects of chamomile extract in various human cancer cells. J Agric Food Chem.2007;55(23):9470-9478.

  2. Lorenzo PS, Rubio MC, Medina JH. Involvement of monoamine oxidase and noradrenaline uptake in the positive chronotropic effects of apigenin in rat atria. Euro J Pharmacol.1996; 312:203-207.

  3. Ko FN, Huang TF, Teng CM. Vasodilatory action mechanism of apigenin isolated from apium graveollens in rat thoracic aorta. Biochem Biophys Acta.1991;14:1115(1):69-74.

  4. Gould L, Reddy CVR, Gomprecht RF. Cardiac effects of chamomile tea. J Clin Pharmacol New Drugs.1973;13:475-479.

  5. Tamasdon S, Cristea E, Mihele D. Action upon gastric secretion of Robiniae Flores, chamomillae Flores and strobulilupuli extracts. Faracia.1981;29:71-75.

  6. Joshi PV, Shirkhedkar A, Prakash K. Antidiarrheal activity, chemical and toxicity profile of Berberis aristata. Pharm Biol. 2010;123.

  7. Sharma AC, Kulkarni SK, Evaluation of learning and memory mechanisms employing elevated plus-maze in rats and mice. Progress in Neuro-Psychopharmacology and Biological Psychiatry.1992;16(1):117–125.

  8. Frautschy SA, Hu W, Kim P. Phenolic anti-inflammatory antioxidant reversal of Aβ-induced cognitive deficits and neuropathology. Neurbiol Age.2001;22(6)993–1005.

  9. Reddy DS, Kulkarni SK. Possible role of nitric oxide in the nootropic and antiamnesic effects of neurosteroids on aging- and dizocilpine-induced learning impairment. Brain Res.1998;799(2):215–229.

  10. Wills ED. Mechanisms of lipid peroxide formation in animal tissues. Biochem J.1966;99(3):667–676.

  11. Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophy.1959;82:48670–48677.

  12. Green LC, Wagner DA, and Glogowski J. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. The J BiolChem.1982;193:265–275.

  13. Kono Y. Generation of Superoxide radical during auto-oxidation of hydroxylamine and an assay for Superoxide dismutase. Arch BiochemBiophy.1978;186:189-195.

  14. Luck H. Catalase, in Methods of Enzymatic Analysis. H. U. Bergmeyer, Academic Press, New York, NY, USA. 1971:885-893

  15. Ellman GL, Courtney KD, Andres V, and Featherstone RM. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol.1961;7(2):88–90.

  16. Gornall AG, Bardawill CT, and David MM. Determination of serum proteins by means of Biuret reaction. The J BiolChem.1949;177:751–766.

  17. Kim MS, Lee JI, Lee WY, Kim SE. Neuroprotective effect of Ginkgo biloba L. extract in rat model of parkinson’s disease. Phytother Res.2009;18:663-666.




  1. Chang JW, Wachtel SR, Young D, Kang UJ. Biomedical and anatomical characterization of forepaw adjusting steps in rat models of parkinson’s disease studies on medial forebrrain bundle and striatal lesions. Neurosci.1999;88:617-628.

  2. Carbonell L.F, Salazai F.J, Garcia Estan J, Ubeda M, Quesada T. Normal homodyna mice parameters in conscious rats. Rev Exp Physiol.1985;41:437-442.




9


SIGNATURE OF THECANDIDATE




KUSHWAHA JYOTI SHRIHEMRAJ


10


REMARKS OF THE GUIDE



Matricaria recutita Linn has been mentioned as very good medicinal plant in various literatures. The present study will be evaluated for its neuroprotective activity scientifically was recommended


11




NAME AND DESIGNATION OF THE GUIDE

Mr. V.M. Chandrashekhar

Associate Professor



12


SIGNATURE







13


CO-GUIDE







14


SIGNATURE







15


HEAD OF THE DEPARTMENT



Dr. I.S. MUCHANDI


16


SIGNATURE







17


REMARKS OF THE PRINCIPAL




18


NAME OF THE PRINCIPAL



Dr. I.S. MUCHANDI


19


SIGNATURE







OFFICE OF THE INSTITUTIONAL ANIMAL ETHICS COMMITTEE (IAEC)

HANGAL SHRI KUMARESHWAR COLLEGE OF PHARMACY,

BAGALKOT-587101, KARNATAKA

REG NO.821/01/a/CPCSEA,Dated:6th AUG 2004 UNDER THE RULES 5(a) OF THE

“BREEDING OF AND EXPERIMENTS ON ANIMAL (control and supervision)

RULES 1998”

Ref: HSK CP/IAEC, Clear/2011-12/1-8

CERTIFICATE

This is to certify that Mr. Kushwaha Jyoti ShriHemraj, A student of first M.Pharm is permitted to carry out experiments on animal for the dissertation / thesis work entitled as “Neuroprotective activity of Matricaria recutita Linn. against Alzheimer disease and Parkinson’s disease in rat model.” as per details mentioned and after observing the usual formalities lay down by IAEC as per provision made by CPCSEA.



Animal house in charge Chairman

Form B

See rule [6 (a) and 8 (a)]

PART A

1]

Name and address of the establishment:


H.S.K.COLLEGE OF PHARMACY

BAGALKOT, KARNATAKA.

2]

Date and registration Number of the

Establishment:



821 /01/a CPCSEA


3]

Name, address and registration NO. of the

Breeder from whom acquired and the date of

Acquisition:

OFFICE OF CPSEA,

MINISTRY OF ENVIRONMENT AND

FOREST, 3RD SEAWARD ROAD,

VALMIKINAGAR,THRIRUVANMIYUR,

CHENNAI-600041.


4]

Place where the animals are presently kept:


ANIMAL HOUSE

H.S.K.COLLEGE OF PHARMACY,

BAGALKOT, KARNATAKA.


5]

Place where experiment is to be performed:


DEPARTMENT OF PHARMACOLOGY

H.S.K.COLLEGE OF PHARMACY,

BAGALKOT, KARNATAKA.


6]

The date on which the experiment is to be commence and the duration of the experiment :






The protocol form for the research proposal - PART B in the case of experiments using other than non-human primate animals for a few projects, PART C for use of non-human primate for new projects and PART D for use of non-human primate for extension of ongoing projects-should be duly filled, signed and annexed with this form.

Dated: Signature

Place: (NAME AND DESIGNATION)


PART –B
Protocol form for research proposal to be submitted to the committee on use of small animals / animals other than non-human Primate in biomedical research for ONGOING / NEW PROJECTS.

1.

Project title:

“Neuroprotective activity of Matricaria recutita, Linn. against Alzheimer disease and Parkinson’s disease in rat model.”


2.

Investigation (S):

Designation



Mr. Chandrashekhar V.M.

Associate professor

3.

Department(S):


Department of Pharmacology

H.S.K.College of Pharmacy,

Bagalkot, Karnataka.

4.

(a) Funding source (S) : if any

----


(b) Are sufficient funds available for purchase and maintenance of the animals



Yes



(c)

Duration of present project:




(1) Number of months:

6 months



(2) Date of start of the project:

( Experiment)



15 April 2012



(3) Date of termination of the project:

15 October 2012



5.

Date by which approval is needed in case the project is to be funded by outside agency (If less than six weeks from the date of admission, please justify below).

----




6.

Summary of project briefly summarize in laymen’s term the background, the objective and the experimental approach

(a)Background:

Enclosed



(b)Objectives

Enclosed



(c)Experimental procedure:

Enclosed



7.

(a)

Name of species

Sprague- Dawley rats.



Age

Sex

Weight

4-8 weeks


Either sex


200-250 g



(b)

Rationale for selection

Approximate number of animals required during the first 12 months.

96


Justification of number (define treatment group and number per group)

Twelve groups, each group containing eight animals

Number of animals housed per week

30


8.

List all invasive Non-Surgical Animals Procedures and Potentially stressful Non-invasive procedures to be used (example: IM injection, foot pad injection, venapunctures).

Invasive surgical animal procedures.



Procedure and approximate frequency:

Enclosed



Anaesthetic and /or Analgesic and dosage:

Ketamine hydrochloride 45 mg/kg, i.p.



Test substance injected and /or applied:

Test substance will administer orally.





9.

Does the protocol prohibit the use of anaesthetic and analgesic for the conduct of painful procedures?

No


10.

With surgical procedure/Experimental procedure be performed?
Yes



(a)Will the animal be sacrificed after surgery?
Yes


(b)Give anticipated post-operative survival time:
NO

11.

Will hazardous agent such as radio isotopes, carcinogens, radiation exposure, microbial and parasitic agent be administered to animals?
No.

INVESTIGATOR SIGNATURE

DATE:




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