Adaptogenic and Nootropic Activities Of

ADAPTOGENIC AND NOOTROPIC ACTIVITIES OF

PUNICA GRANTUM L(POMEGRANATE): AN EXPERIMENTAL STUDY IN RAT MODEL

SYNOPSIS FOR

M.PHARM DISSERTATION

SUBMITTED TO

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA

BY

RAMYA M.N

I M.PHARM

DEPARTMENT OF PHARMACOLOGY

VISVESWARAPURA INSTITUTE OF

PHARMACEUTICAL SCIENCES

BANGALORE-560070

(2011-2012)

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1.0 / NAME OF THE CANDIDATE
AND ADDRESS(IN BLOCK
LETTERS) / RAMYA M.N
# 53/3,1ST MAIN, B CROSS
NGEF LAYOUT, MALLATHAHALLI POST,
BANGALORE,
KARNATAKA-560056
2.0 /

NAME OF THE INSTITUTION

/

VISVESWARAPURA INSTITUTE OF

PHARMACEUTICAL SCIENCES

BANGALORE-560070

3.0 /

COURSE OF STUDY AND SUBJECT

/

MASTER OF PHARMACY IN

PHARMACOLOGY

4.0 /

DATE OF THE ADMISSION

/ 07/07/2010
5.0 /

TITLE OF THE TOPIC:

“ADAPTOGENIC AND NOOTROPIC ACTIVITIES OFPUNICA GRANTUM L (POMEGRANATE): AN EXPERIMENTAL STUDY IN RAT MODEL”
6.0
7.0
8.0 / BRIEF RESUME OF THE INTENDED WORK

6.1NEED FOR THE STUDY

Stress can be described as the sum total of all the reactions of the body to a stimulus, which disturb the normal physiological condition and result in a state of threatened homeostasis and has been defined as a nonspecific response of the body to any demand imposed on it1. In its most simplified sense, stress is what one feels when life's demands exceed one's ability to meet those demands. Stress is an internationally recognised phenomenon fortified by advancement of industrialisation in a demanding civilisation. In fact every individual is likely to face stressful situations in day to day life from headaches to heart disease and immune deficiency to digestive problems. Thus stress is a factor in many illness2.
Increased production of stress hormones and subsequent decrease in immune function appear to contribute to the stress induced decline in Health3.Similarly increased physical and psychological stress leads to increased incidence of amnesia. There is increasing evidence that Alzheimer's disease increases severe oxidative stress as a result of either beta amyloid mediated generation of free radicals or perturbed ionic calcium balance within neurons and their Mitochondria4.Supplementation with higher ascorbic acid and betacarotene was associated with better memory performance which indicates the role of potential antioxidants in brain aging and cognitive impairment5.Literature indicates that the role of free radicals in the pathogenesis of cancer, aging, Alzheimer's disease, diabetes and the compounds having capacity to scavenge free radicals has great potential in mitigation of these disorders.
Ayurveda is an ancient form of Indian medicine which deals with plants and plant products. This indigenous form of medicine uses the active ingredients present in plants for treating various diseases6. Since the introduction of adaptogens several plants have been investigated which were once used as tonics due to their adaptogenic and rejuvenating properties in Ayurvedic medicine7.
The drugs of plant origin are gaining increasing popularity and are Being investigated for remedies of a number of disorders including antistress (adaptogenic) activity8.Punica grantum L is one such plant which is extensively studied for its antistress properties.
The Pomegranate plant is a dried erect shrub of Punica grantum L belonging to family Punicaceae.Its fruit is known to be a rich source of bioactive ellagitannins. It has been used for centuries in ancient culture for medicinal purposes9.The phytochemical analysis of an ethanolic extract of Punica grantum L revealed the presence of a wide variety of constituents such as flavonids,glycosides,tannins, anthocyanins and ascorbic acid. Pomegranate fruit posses powerful antioxidant,antiflammatory,antidiabetic and neuroprotective activity10.
In the present investigation, the antistress activity of Punica grantum L (Standardized aqueous extract of peel and seed; ethanolic extract of peel and seed; Juice) procured from Green chem, Herbal extracts and formulations, Bangalore, India, will be evaluated in-vivo, by estimation of non invasive biochemical markers in normal and stress induced rat urine following a biochemical approach. Though Punica grantum L is widely studied for antistress activity, no work has been reported previouslyfor the above said parameter. Estimation of urinary levels of VMA and ascorbic acid metabolite which is an indirect biochemical index to represent the activity of sympathetic nervous system and hypothalamic-pituitary-adrenal (HPA) axis, a major pathway in stress response. Thus in present investigation the role of standardized extracts of Punica grantum L on sympathetic system and HPA axis will be determined in normal and stress induced rat.
The antistress and antioxidant activities will be correlated with the nootropic activity of the extract since the role of stress and free radicals have been implicated in the loss of memory, concentration and also in Alzheimer's disease11,12.
Several studies indicate that antioxidant activity of plants like Ocimum sanctum, Withania somnifera, Panax ginseng, etc was partly responsible for their antistress activity17. Thus antioxidant activity of the Punica grantum L extract was evaluated ex-vivo to support the antistress activity.
6.2 REVIEW OF LITERATURE
Kumar S et al.observed that Punica grantumL could be protective in the treatment of cognitive disorders such as dementia and alzheimer’s disease13.
Esch T et al. observed that the antistress and antioxidant activities were correlated with the nootropic activity of the extract since the role of stress and free radicals have been implicated in the loss of memory, concentration and also in alzheimer’s disease14.
Reddy MK et al. reported that ellagic acid, gallagic acid, punicalins and punicalagins polyphones present in Punica grantumL posses antioxidant activity9.
River C et al. observed that stressors are many and (e.g. psychological, physical, surgical, Trauma, strenuous exercise, undernutrition) activate a range of physiological system the most commonly studied are HPA axis and sympathoadrenal system15.
Rai Deepak et al. reported the sympathetic nervous system in response to stress results in the secretion of coticosterone from adrenal cortex and epinephrine from adrenal medulla. These hormones are released under stressed Conditions16.
Sreemantula S et al. observed thatvitis vinifera extract along with stress reversed the stress induced biochemical changes (Increase in urinary VMA and decrease in urinary ascorbic acid Levels) 17.
Kumar S et al.reported the CNS activities demonstrated by the ethanolic extract may be due to the presence of antioxidants present in the PunicagrantumL extract10.
Loren J.D et al.demonstrated that dietary supplementation of the mouse maternal diet with pomegranate juice,a food highly enriched in polyphenols,in the peripartum period provides significant neuroprotection from hypoxic-ischemic injury to neonatal Mice18.
Murthy C et al. concluded that the pomegranate extract containing ellagic acid and gallic acid act as a potent free radical scavenger, reducing the levels of hydrogen peroxide and super oxide anion and consequently lipid peroxidation and enzyme inactivation, restoring enzyme Activity19.
6.3 OBJECTIVE OF THE STUDY
To evaluate the antistress activity of Punica grantum Lin rat
Byestimation of non invasive biomarkers in urine of normal and stress induced rat
The antioxidant (lipid peroxidation inhibition) potential of the extract in both liver and brain homogenates by ex vivo will be done to support the antistress activity.
To evaluate the nootropic activityofPunica grantum Lusing conditioned avoidance response in rats.
MATERIAL AND METHODS
Pomegranate extracts:
Punica grantum L(Standardized ethanolic extract of peel and seed; aqueous extract of peel and seed; Juice) procured from Green chem, Herbal extract and formulations, Bangalore, India. It will be used for the study.
7.1 SOURCE OF DATA :
The data will be generated by performing experiments on animals The standard information is collected from various journals, standard text books available in library of Visveswarapura Institute of Pharmaceutical Sciences, Indian Institute of Science, RGUHS-digital library & from various standard websites.
Web sites:






METHOD OF COLLECTION OF DATA:
The data will be generated by performing the experiments using animal models like rats.
7.2 METHODOLOGY :
7.2.1 Acute toxicity tests20:
ANIMALS REQUIRED
a) Species : Swiss albino mice
b) Weight : 25-30g
c) Gender : either sex
d) Number : 06 mice
The acute toxicity will be determined on Swiss albino mice by fixed dose method of OECD Guide line no.420 given by CPCSEA. 6 mice will be administered test drug by oral route and mortality will be observed after 24 hr.
7.2.2 Forced Swim Endurance Model1:
ANIMALS REQUIRED
a) Species : Albino Wistar rat
b) Weight : 180-220 g
c) Gender : either sex
d) Number : 78 rats
Following groups will be made
To evaluate the aqueous and ethanolic extract of thePeel ofPunica grantum L for its antistress activity
Group I: animals (n=6) will be administered with vehicle
Group II: animals(n=6) will be administered lower dose of aqueous extract of Punica grantum L
Group III: animals(n=6) will be administered higher dose of aqueous extract
of Punica grantum L
Group IV: animals(n=6) will be administered lower dose of ethanolic extract
of Punica grantum L
Group V: animals(n=6) will be administered higher dose ethanolic extract
of Punica grantum L
To evaluate the aqueous and ethanolic extract of the Seed ofPunica grantum L for its antistress activity
Group I: animals (n=6) will be administered with vehicle
Group II: animals(n=6) will be administered lower dose of aqueous extract of Punica grantum L
Group III: animals(n=6) will be administered higher dose of aqueous extract
of Punica grantum L
Group IV: animals(n=6) will be administered lower dose of ethanolic extract
of Punica grantum L
Group V: animals(n=6) will be administered higher dose ethanolic extract
of Punica grantum L
To evaluate the Juice of Punica grantum L for its antistress activity
Group I:animals (n=6) will be administered with vehicle
Group II:animals (n=6) will be administered lower dose ofPunicagrantum
Group III :animals (n=6) will be administered higher dose ofPunica grantum
Procedure:
Rats of either sex weighing between 180–220gm will be divided into five groups (I, II, III, IV and V) each group containing six animals to evaluate the aqueous and ethanolic extract of the Peel and Seed of Punica grantum L for its antistress activity. Rats will be divided into three groups each group containing six animals to evaluate the Juice of Punica grantum L for its antistress activity. The 24hrurine sample from each group will be collectedinto two different beakers, one containing 5 ml of 10% oxalic acid for the spectrophotometeric determinationof ascorbic acid at 550nm and the other containing 0.5 ml of 6 N hydrochloric acidfor the determination of stress metabolites.
The experimental protocol will be divided into four phases:
In the first phase of the experiment, 24hr urine samples will be collected in all the five groups and subjected to analysis for VMAand ascorbic acid. The normal values will be recorded for Seven consecutive days.
In the second phase, after a recovery period of one week, the animals in each group will be subjected to fresh water swimming stressindividually. In this method, rats will be forced to swim until exhausted in a cylindrical vessel of 60 cm height and 45cm diameter containing water at room temperature (28°C). Water depth was always maintained at 40cm.The 24hr urinary levels of VMAand ascorbic acid under stressed conditions will be determined again as described above daily for Seven consecutive days.
The third phase of the experiment, after a recovery period of one week,the animalof Groups (II, III, IV and V) will be administered orallywith aqueous and ethanolic extract of peel and seed of Puica grantum L, Groups (II and III) will be administered orally with juice of Punica grantum L at two dose level dailyfor seven consecutive days,while Group I will serve as a control.The 24hr urine samples will be collected and the levels of VMAand ascorbic acid will be determined.
The final phase of the experiment after a recovery period of one week, the administration of Punica grantum L extracts will be done as mentioned in the third phase, one hour prior to the daily induction of stress for Seven consecutive days while Group Iserving as control. The 24hr urine samples will be collected and analyzed for VMAand ascorbic acid for Seven consecutive days to study the influence of Punica grantum Lextracts(Peel and Seed) and Juiceon the stress induced biochemical changes.
7.2.3Nootropic activity In Rat17.
ANIMALS REQUIRED
a) Species : Albino Wistar rat
b) Weight : 180-220 g
c) Gender : either sex
d) Number : 78 rats
Following groups will be made:
To evaluate the aqueous and ethanolic extract of the Peel of Punica grantum Lfor its nootropic activity
Group I: animals (n=6) will be administered with vehicle
Group II: animals (n=6) will be administered lower dose of aqueous extract of Punica grantum L
Group III: animals (n=6) will be administered higher dose of aqueous extract
of Punica grantum L
Group IV: animals (n=6) will be administered lower dose of ethanolic extract
of Punica grantum L
Group V: animals (n=6) will be administered higher dose of ethanolic extract
of Punica grantum L
To evaluate the aqueous and ethanolic extract of the Seed of Punica grantum L for its nootropic activity
Group I: animals (n=6) will be administered with vehicle
Group II: animals (n=6) will be administered lower dose of aqueous extract of Punica grantum L
Group III: animals (n=6) will be administered higher dose of aqueous extract
of Punica grantum L
Group IV: animals (n=6) will be administered lower dose of ethanolic extract
of Punica grantum L
Group V: animals (n=6) will be administered higher dose ethanolic extract
of Punica grantum L
To evaluate the Juice of Punica grantum L for its nootropic activity
Group I: animals (n=6) will be administered with vehicle
Group II: animals (n=6) will be administered lower dose ofPunicagrantumL
Group III : animals (n=6) will be administered higher dose ofPunica grantum L
Procedure:
After 60 minutes of oral administration, all the animals will be subjected to a training schedule individually by placing inside the Perspex chamber of the apparatus. After an accustomed period of five minutes to the chamber, a buzzer will be given followed by a shock through the grid floor. The rat had to jump on the pole to avoid foot shock. Jumping on the pole functionally terminates the shock and this will be classified as an escape while such jumping prior to the onset of the shock was considered as avoidance. The session will be terminated after completion of 60 trials with an interval of 20–30 seconds given for each trial. This procedure will be repeated at 24hr intervals until all groups reach 95 to 99% avoidance.
After attaining complete training of a particular group, the animals will be treated with a single dose of scopolamine butyl bromide (1 mg/kg body weight, i.p.), thirty minutes before the next day dosing. The training schedule will be continued further with the daily doses of the Punica grantum L extracts (Peel, Seed and Juice) and vehicle until they returned to normal level from scopolamine induced amnesia.
3) Anti oxidant activity21:
Rats weighing 150-200 g will be sacrificed by spinal traction and the whole brains and the livers will be isolated. The pooled brains and livers will be homogenized in four volumes of 40 mM Tris-HCl buffer (pH 7.0) using a tissue homogenizer. The antioxidant activity of Punica grantum L will be determined based on its ability to inhibit lipid peroxidation in homogenates of the liver and brain of rats. The reaction mixture (0.5 mL) containing rat liver homogenate (0.1 mL), KCl (30 mM), ascorbic acid (0.06 mM), and ferrous iron (0.16mM) and various concentrations of Punica grantum L extracts will beincubated for 1 h at 37’C. At the end of the incubation period 0.4 mL of the reaction mixture will be treated with 0.2 mL of sodium dodecyl sulphate (8.1%), 1.5 mL of thiobarbituric acid (0.8 %), and 1.5 mL of acetic acid (20% pH 3.5). The total volume will be then made up to 4 mL by adding distilled water and kept in an oil bath at 100’C for 1 hour. After this step the mixture will be cooled, 1 mL of distilled water and 5 mL of butanol pyridine mixture (15:1% v/v) will be added. Following vigorous shaking the tubes then will be centrifuged and the absorbance of the organic layer containing the chromophore was read at 532 nm. The percentage inhibition of lipid peroxidation by the Punica grantum Lextracts will be determined by comparing the absorbance values of the control andPunica grantum L extracts.
STASTICAL ANALYSIS
The results will be expressed as mean ± standard error of mean. Statistical analysis will be done using Students paired t-test. In all cases p < 0.05 was considered statisticallySignificant.
7.3 Does the study require any investigation or interventions to be conducted on patients or the human or animals? If so please describe briefly:
YES,Study requires investigation on animals. The effects of the drug will be studied on various parameters using rats as experimental animal model.
7.4 Has ethical clearance been obtained from your institution in case of 7.3?
Yes, Ethical clearance certificate will be attached along with the hard copy.
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18.Loren DJ, Seeram NP, Schulman RN and Holtzman DM. Maternal dietary
supplementationwith Pomegranate juice is neuro protective in an animal model of
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9.0 / SIGNATURE OF THE CANDIDATE
10. / REMARKS OF THE GUIDE
11. / NAME AND DESIGNATION
11.1 GUIDE / Mr. ANILKUMAR K.V
ASST. PROFESSOR
DEPT. OF PHARMACOLOGY
VIPS, BANGALORE-560 070
11.2 SIGNATURE
11.3 CO-GUIDE (IF ANY)
11.4SIGNATURE / .
11.5HEAD OF THEDEPARTMENT / Prof. RAMESH C
HEAD OF DEPARTMENT
DEPT. OF PHARMACOLOGY
VIPS, BANGALORE-560 070
11.6 SIGNATURE
12. / 12.1 REMARKS OF THE PRINCIPAL
12.2 SIGNATURE / Prof. Dr. D. H. HARISH KUMAR
PRINCIPAL
VIPS, BANGALORE-560 070