EVALUATION OF BEHAVIOURAL ACTIVITY OF Punica granatum L. FRUIT RIND IN RATS EXPOSED TO CHRONIC UNPREDICTABLE MILD STRESS

SYNOPSIS FOR

M.PHARM DISSERTATION

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SUBMITTED TO

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

KARNATAKA, BANGALORE.

BY

GUNNAM REDDY BUJJI REDDY

DEPARTMENT OF PHARMACOLOGY

NARGUND COLLEGE OF PHARMACY

BANGALORE-560 085

(2010-2012)

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA, BANGALORE.

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR PG DISSERTATION

1. / Name of the candidate and address(in block letters) /

GUNNAM REDDY BUJJI REDDY

NARGUND COLLEGE OF PHARMACY,
DATTATREYA NAGAR, II MAIN,100 FT RING ROAD, BSK III STAGE, BANGALORE-560 085
2. /

Name of the institution

/

NARGUND COLLEGE OF PHARMACY

3. /

Course of study and subject

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MASTER OF PHARMACY IN

PHARMACOLOGY

4. /

Date of the admission

/ 22-06-2010
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TITLE OF THE TOPIC:

EVALUATION OF BEHAVIOURAL ACTIVITY OF
Punica granatum L. FRUIT RIND IN RATS EXPOSED
TO CHRONIC UNPREDICTABLE MILD STRESS
BRIEF RESUME OF THE INTENDED WORK:
6.1 NEED FOR THE STUDY:
One potential negative outcome associated with chronic stress exposure is the development of mental illnesses including anxiety or affective disorders.1 The response to stress is non-specific and independent of the nature of the stress, as the stress induced state produced by diverse stressors is indistinguishable. A host of experimental stress situations are used to provoke measurable physiological responses.2 Currently different therapeutic regimens are employed to treat anxiety and depressive disorders; but their clinical uses are limited by their side effects such as psychomotor impairment, potentiating of other central depressant drugs and dependence liability. In search for new therapeutic product for the treatment of neurological disorder- medicinal plant research has also contributed significantly by demonstrating pharmacological effectiveness of different herbs in various animal models.3
Punica granatum L. (Punicaceae) commonly called pomegranate, has been used in the folk medicine of many cultures especially in the Middle East.4 Pomegranate and its derivatives have free radical scavenger and potent antioxidant activity. Reactive oxygen species (ROS) are highly reactive oxidizing agents belonging to the class of free radicals. However, the alterations in their synthesis stimulate the oxidation and DNA damage of cells. In general, antioxidants are compounds which dispose, scavenge, and suppress the formation of ROS and lipid peroxidation. Among the well known biological antioxidants, glutathione (GSH), glutathione peroxidase (GSH-Px), catalase(CAT), superoxide- dismutase (SOD) have a significant role as a suppressor or scavenger of free radicals. Conditions of stress precipitated glucose intolerance, immune-suppression, behavioural depression, cognitive deficits and male sexual dysfunction in rats.5 Hence the present study will be undertaken to investigate the potential role of methanolic extract of Punica granatum fruit rind in reversing the behavioural activity using different in vivo experimental models in rats namely open field test and light-dark model.
6.2 REVIEW OF LITERATURE:
In folk medicine pomegranate preparations of the dried pericarp and juice of the fruits are employed as an oral medication in the treatment of colic, colitis, leucorrhoea, menorrhagia, oxyuriasis, paralysis and rectocele. It is also used in external application to caked breast and to the nape of the neck in mumps and headache. The rinds of fruits are valued as astringents in diarrhoea and dysentery. A number of therapeutic actions of these materials have been described including vermifugal, taenicidal, astringent, antispasmodic, antihysteric, diuretic, carminative, sudorific, galactogogue and emmenagogue. Pomegranate peel is used for treating the infection of male or female sexual organs, mastitis, acne, folliculitis, piles, allergic dermatitis, tympanitis and for the treatment of oral diseases.6-8
·  P. granatum peel exhibited potent scavenging activity against superoxide anions.9
·  P.granatum fruit rind revealed the gastroprotective activity through antioxidant mechanism.10,11
·  Aqueous-ethanolic extract of P. granatum flowers led to significant blood glucose lowering effect in normal, glucose –fed hyperglycaemic and alloxan –induced diabetic rats.12
·  P. granatum seed extract treated animals showed significant inhibitory activity against castrol-oil induced diarrhoea.13
·  P. granatum fruit juice, peel and oil have shown to possess anti cancer-activities including interference with tumor, cell proliferation, cell cycle, invasion and angiogenesis. These components having clinical applications for treatment of inflammation.14
·  P.granatum fruit rind powder stimulates the cell mediated and humoral components of the immune system and acts as natural antioxidants.15
·  P.granatum extract (rind, seed and fruit) used in protecting skin against UVB – irradiation.16
6.3 OBJECTIVE OF THE STUDY:
·  To evaluate the behavioral activity of methanolic extract of P. granatum L. fruit rind in rat models exposed to chronic unpredictable mild stress using open field test and light-dark test.
·  To estimate the lipid peroxide content in rats brain and kidney.
MATERIAL AND METHODS:
7.1  SOURCE OF DATA:
Data will be obtained from the experimental work, which includes laboratory based animals studies and evaluation of various parameters.
7.2 METHOD OF COLLECTION OF DATA:
The data collected is based on animal experimentation as per the parameters studied under each animal model. The sampling procedure and collection of data is as follows.
METHODOLOGY:
Induction of stress :
Stress will be inducing by employing the Chronic Unpredictable Mild Stress (CMS) protocol. It is based on the modus operandi originally used by Pal and Dandiya17 with minor modifications. Stress regimen will be carried out with the following stressors: food deprivation for 24 h, day-night reversal, soiled bedding for 22 h, caged tilting (~45 degree inclined) for 22 h, crowded housing (10 animals per cage) for 12 h, exposure to a novel odor (household air freshener) for 12 h, restraint stress for 20 mins, cold stress 4-8o C and heat stress 38-39oC for 20 min and wet bedding (~150 ml water per cage) for 22 h.
OPEN FIELD TEST: 18
After induction of stress in rats with above stress regimen for 21 days and with simultaneous drug administration, animals will be subjecting to open field test to study the behavioral activity.
Model:
The open field apparatus consists of a square arena 68 x 68 cm with 45 cm high wall. The entire apparatus was painted black except for 6 mm white lines that divided the floor into 16 equal size squares. The apparatus was illuminated with a low intensity diffuse light (45 W) situated 45 cm above the floor level.
Procedure:
Each animal will be placed in the central square and observed for 5 min for the following behaviours recorded.
Ambulation: The number of grid lines crossed by rats with all the four paws.
Rearing: By counting the number of times the animal stood on its hind limbs.
Grooming: Number of times the animal made these responses viz. grooming of the face,
Licking and scratching the various parts of the body.
Defecation: The number of fecal boli excreted during the period and immobility period. Between tests, the apparatus was cleaned with 5% alcohol.
Evaluation:
·  Number of grid lines crossed by rats with all the four paws.
·  Number of times the animal stood on its hind limbs.
·  Number of times the animal made these responses viz. grooming of the face, Licking and scratching the various parts of the body.
·  Number of fecal boli excreted during the period and immobility period.
ANIMALS:
Species / Rats
Strain / Wistar
Age and sex / 1-2 months, either sex
Body weight / 180-200 g
No of animals in each group / N=8
No of groups / Five
Vehicle of herbal drugs / Distilled water
Vehicle of standard drugs / Distilled water
Water and food / Ad. Libitum
DRUGS AND TREATMENT:
GroupI : Vehicle control
Group II : Chronic unpredictable mild stress (CMS)
Group III : CMS + Diazepam 2 mg/kg
Group IV : CMS + Methanolic extract of P.granatum fruit rind (Low dose – 250 mg/kg)
GroupV : CMS+ Methanolic extract of P.granatum fruit rind (high dose – 500 mg/kg)
No. of days of drug treatment: 21days.
7.2.2. LIGHT AND DARK MODEL:19
After induction of stress in rats with above stress regimen for 21 days and with simultaneous drug administration, animals will be subjecting to light and dark test to study the behavioral activity.
MODEL: The testing apparatus consists of a light and a dark chamber divided by a photocell –equipped zone. A polypropylene animal cage, 44x21x21 cm, is darkened with black spray over one- third of it’s surface. A partition containing a 13 cm long x5 cm high opening separates the dark one third from the bright two thirds of the cage. The cage rests on animex activity monitor which counts total locomotor activity. As electronic system using 4 sets of photo cells across the partition automatically counts movements through the partition and clocks the time spent in the light and dark components.
PROCEDURE: Each animal will be placed into the light – dark cage and observed for 10 min. for their behavioral activity.
Evaluation:
·  Number of transitions between the light and dark compartments.
·  Initial latency to enter the dark compartment.
·  Total time spent in the light compartment.
ANIMALS:
Species / Rats
Strain / Wistar
Age and sex / 1-2 months, either sex
Body weight / 180-200 g
No. of animals in each group / N=8
No. of groups / Five
Vehicle of herbal drugs / Distilled water
Vehicle of standard drugs / Distilled water
Water and food / Ad. Libitum
DRUGS AND TREATMENT:
Group I : Vehicle control
Group II : Chronic unpredictable mild stress (CMS)
Group III : CMS + Diazepam 2 mg/kg
Group IV : CMS + Methanolic extract of P.granatum fruit rind (Low dose – 250 mg/kg)
Group V : CMS + Methanolic extract of P.granatum fruit rind (high dose – 500 mg/kg)
No. of days of drug treatment: 21 days
Estimation of Lipid Peroxide in rats brain and kidney :18
The lipid peroxidation in terms of thiobarbituric acid reactive substances (TBARS) was measured using the method of Ohkawa et al.20 Mixture of 500 µl of the supernatant, 200 µl of 8% sodium dodecyl sulphate, 1.5 ml of 20% acetic acid solution, 1.5 ml of 0.9% aqueous solution of thio- barbituric acid and 1.3 ml of distilled water were heated in boiling water bath for 30 min. after cooling, the red chromogen was extracted into 5 ml mixture of n-butanol and pyridine (15:1v/v). The organic layer was separated and the absorbance was measured at 532 nm. Lipid peroxide content was expressed as n mole of malondialdehyde (MDA)/mg tissue. The calibration curve was prepared by using 1,1,3,3-tetra ethoxy propane (TEP) as standard.
STATISTICAL ANALYSIS:
The data will be analyzed using analysis of variance (ANOVA) followed by the student- Newman-Keuls test for multiple comparisons.
7.3 Does the study require any investigation to be conducted on patients
Or other humans or animals? If so please describe briefly
Yes, The above study requires investigation on animals. The effect of drug will be studying on various parameters using rat model.
7.4 Has ethical clearance been obtained from your institution in case of 7.3?
Applied for the IAEC of Nargund college of pharmacy
REFERENCE:
1. Matuszewich L, Karney JJ, Carter SR, Janasik SP, O’Brien JL, Friedman RD. The delayed effects of chronic unpredictable stress on anxiety measures. Physiol Behav 2007;90:674-81.
2. Kirtikar KR, Basu BD. Withania somnifera dunal in Indian medicinal plants. Indian medicinal plants. 3rd ed. Delhi: Sri satguru publication; 2000. p. 2445-9.
3. Singh B, Saxena AK, Chandan BK, Gupta DK, Bhutani KK, Anand KK. Adaptogenic activity of a novel withanolide free aqueous fraction from the root of withania somnifera dun. Phytother Res 2001;15:311-8.
4. Gurib-Fakim A. Medicinal plants: traditions of yesterday and drugs of tomorrow. Mol Aspects Med 2006;27(1):1-93.
5. Turk G, Sonmez M, Aydin M, Yuce A, Gur S, Yuksel M et al. Effects of Pomegranate juice consumption on sperm quality, spermatogenic cell density, antioxidant activity, and testosterone level in male rats. Clin nutr 2008;27:289-96.
6. Khare CP. Indian medicinal plants. New York(NK): Springer; p. 527-8.
7. Ricci D, Giamperi L, Bucchini A, Fraternale D. Antioxidant activity of Punica granatum fruits. Fitoterapia 2006;77:310-2.
8. Prashanth D, Padmaja R, Samiulla DS. Effect of certain plant extracts on α-amylase activity. Fitoterapia 2001;72(2):179-81.
9. Saito K, Kohno M, Yoshizaki F, Niwano Y. Extensive screening for edible herbal extracts with potent scavenging activity against superoxide anions. Plant Food Hum Nutr 2008;63(2):65-70.
10. Ajaikumar KB, Asheef M, Babu BH, Padikkala J. The inhibition of gastric mucosal injury by Punica granatum L.(pomegranate) methanolic extract. J Ethnopharmacol 2005;96:171-6.
11. Alkofahi A , Atta AH. Pharmacological screening of the anti-ulcerogenic effects of some Jordanian medicinal plants in rats. J Ethnopharmacol 1999;67:341-5.
12. Jafri MA, Aslam M , Javed K, Singh S. Effect of Punica granatum Linn. (flowers) on blood glucose level in normal and alloxan-induced diabetic rats. J Ethnopharmacol 2000;70:309-14.
13. Das AK, Mandal SC, Banerjee SK, Sinha S, Das J, Saha BP et al .Studies on antidiarrhoeal activity of Punica granatum seed extract in rats. J Ethnopharmacol 1999;68:205-8.
14. Lansky EP, Newman RA. Punica granatum (pomegranate) and its potential for prevention and treatment of inflammation and cancer. J Ethnopharmacol 2007;109:177-206.
15.Ross GR, Selvasubramanian S, Jayasundar S. Immunomodulatory activity of Punica granatum in rabbits –a preliminary study. J Ethnopharmacol 2001;78(1):85-7.
16. Park HM , Moon E , Kim AJ, Kim MH ,Lee S ,Lee JB et al. Extract of Punica granatum inhibits skin photoaging induced by UVB irradiation. Int J Dermatol 2010;49(3):276-82.
17. Pal SN, Dandiya PC. Comparative study of diazepam, mapratroline, fluvoxiamine, trazodone and alprazolam in some animal models of depression. Indian J pharmacol 1993;25:204-8.
18. Nirmal J, Babu CS, Harisudhan T, Ramanathan M. Evaluation of behavioural and antioxidant activity of Cytisus scoparius link in rats exposed to chronic unpredictable mild stress. BMC Complement Altern Med 2008;8:15.
19.Vogel HG. Drug Discovery and Evaluation:Pharmacological Assays. 3rd Edition. Germany. Springer Verlag Berlin Heidelberg NY 2002;1: p. 622.
20.Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animals tissues by thiobarbituric acid reaction. Anal Biochem 1979;95:351-8.
9 / SIGNATURE OF THE CANDIDATE
10 / REMARKS OF THE GUIDE / RECOMMENDED
11 / NAME AND DESIGNATION
11.1:- GUIDE / Dr. H J. HRISHIKESHAVAN
PROFESSOR AND HEAD
DEPT. OF PHARMACOLOGY
NARGUND COLLEGE OF PHARMACY,
Bangalore-560085.
11.2:- SIGNATURE
11.3:- CO-GUIDE
11.4:- SIGNATURE
11.5:- HEAD OF THE DEPT / Dr. H J. HRISHIKESHAVAN
PROFESSOR AND HEAD
DEPT. OF PHARMACOLOGY
NARGUND COLLEGE OF PHARMACY,
Bangalore-560085.
11.6:- SIGNATURE
12 / REMARKS OF THE PRINCIPAL /
RECOMMENDED
12.1:- SIGNATURE

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