EVALUATION OF CARDIOPROTECTIVE ACTIVITY OF AQUEOUS EXTRACT OF Garcinia indica Linn FRUIT RIND.

MASTER OF PHARMACY DISSERTATION PROTOCOL

SUBMITTED TO THE

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA, BANGALORE

BYPATEL DHRUV KAMLESHKUMAR

Under The Guidance of Mr. SUNIL KOSHY, M.PHARM

DEPARTMENT OF PHARMACOLOGY,SRINIVAS COLLEGE OF PHARMACY, MANGALORE – 574143

2012 – 2013(MID STREAM BATCH)

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES BANGALORE, KARNATAKA

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION

1.0 NAME AND ADDRESS OF

THE CANDIDATE

PATEL DHRUV KAMLESHKUMAR62,HARIOM SOC,BAVLA-382220DI-AHMEDABADGUJARAT

2.0 NAME OF THE

INSTITUTION

SRINIVAS COLLEGE OF PHARMACYVALACHIL,MANGALORE-574143

3.0 COURSE OF STUDY AND

SUBJECT

MASTER OF PHARMACY IN PHARMACOLOGY

4.0 DATE OF ADMISSION TO

COURSE

10/01/2013

5.0 TITLE OF THE TOPICEVALUATION OF CARDIOPROTECTIVE ACTIVITY OF AQUEOUS EXTRACT OF Garcinia indica Linn FRUIT RIND.

6.0 BRIEF RESUME OF THE INTENDED WORK :

6.1NEED FOR THE STUDY :

According to the findings of the National Vital Statistics Report (2008)

and the Morbidity and Mortality Weekly Report (2007) of the Centers for

Disease Control and Prevention (CDC), cardiovascular diseases including

myocardial infarction (MI) and the resultant complication in cardiac

function represent the leading cause of morbidity and mortality in

developed countries1,2. Moreover, with advanced life style in developing

countries, such as India, particularly in metropolitan cities, MI is making

increasingly important contribution to mortality statistics of such

countries3.

Although clinical care is improved, public awareness is raised

and health innovations are widely used, myocardial infarction remains the

leading cause of death worldwide4. It is the acute condition of myocardial

necrosis that occurs as a result of imbalance between coronary blood

supply and myocardial demand. Experimental and clinical studies have

shown that there is increased generation of reactive oxygen species such

as superoxide anion (.O-2) and hydroxyl radicals (.OH-) in heart failure,

which are involved in the formation of lipid peroxides, damage of cell

membrane, and destruction of antioxidative defense system 5,6.

Cell injury occurring after ischemia-reperfusion (I/R) in the heart is

attributed to necrosis caused by calcium overload, acidosis, and oxidative

stress. After I/R, myocardial cell die by necrosis and/or apoptosis. Free

radicals and antioxidants play an important role in cellular damage that

can cause atherosclerosis and myocardial infarction. Both neutrophils and

free radicals, such as superoxide anions, hydroxyl radicals and hydrogen

peroxide, can cause oxidative damage to cell lipids, proteins, and nucleic

acids. A consensus still exists that reactive oxygen species play an

important role in the pathogenesis of cardiac reperfusion injury.7 Because

of high incidence of morbidity, various drugs and regimes have been

advocated for the control of CVS diseases.Many new drugs have been

introduced which may demonstrate better efficacy but possess side

effects8. Recently attention has been focused towards herbal and mineral

preparations which are traditionally used as potential therapeutic agents

such as, Terminalia arjuna (arjun), Trigonella foenum-graecu(fenugreek),

Curcuma longa (turmeric), and Vitis vinifera (grapes) in the prevention

and management of cardiovascular diseases.

CRITERIA FOR SELECTION OF THE PLANT:

Fruits of Garcinia indica Linn was proved for its anti microbial activity,

antioxidant, anti-ulcer activity and anti-aglycation activity, anti-obesity

effects of Garcinia indica Linn fruits consists of phytochemical

constituents like the garcinol and isogarcinol were reported to show

antioxidant activity. In Ayurveda, Garcinia indica Linn was found in

many formulations used for cardioprotective action. In the present study,

cardioprotective effect of Garcinia indica will be assessed by using

different experimental models in animals.

6.2 REVIEW OF LITERATURE :

6.2.1

6.2.2

6.2.3 Name of the plant : Garcinia indica Linn is a plant native to certain

regions of India. The trees yield fruits annually in the summer season

during the months of March to May. The fruits are green when raw and

red to dark purple when fully ripe. They are used to prepare juice, pickles

and as acidulant in curries.

Family : : Clusiaceae (St John's Wort family)

Synonyms: Brindonia indica.

English : Goan garcinia, Kokum.

Hindi : Kokum,arathi,Bheranda,Bhiranda,Kokamba.

Tamil: Murgal, murgal-mara.

Malayalam:Kaattampi,Kokkam.

Kannada: Murgina, Punarpuli, Devana huli.

Oriya: Tintali .

Gujarati: Kokam .

Konkani: Bhirind, Kokam.

Sanskrit: Vrikshamia, Amlabija, Amlapura, Amlashaka .

Distribution:

Garcinia which is a large genus of polygamous evergreen trees and

shrubs native of Asia, Southern Africa and Polynesia (Anthony, 1997).

The genus Garcinia comprises of 200 old world tropical species of which

20 species are found in India. In India it is found in the states of Gujarat,

Goa, Orissa, Karnataka, Tamil Nadu and Kerala.

Plant Description:

Tree- Kokum is a tree with a dense canopy of green leaves and red-

tinged tender emerging leaves. The tree is large and handsome.

Leave- Elliptic, oblong or oblong-lanceolate, deep-green glossy

leaves, 5.5-8 cm long and 2.5-3 cm broad.

Flowers -Fleshy, Dark pink, Solitary or in spreading cluster, stalk

less stigma.

Fruits- The fruit is brownish or brownish-gray, marbled with

yellow, and is crowned by the 4-parted. There are from 6 to 8 seeds,

and the pulp is juicy, white, and delicious in taste and odor. It is

about the size of an orange. An average kokum tree bears hundreds of

fruits during march-april. When they are tender, they are green in color.

As they ripen, they get the beautiful purple color. The fruits are plucked

when they are ripened.

Chemical constituents 10 :

Kokum contains the following phytonutrients: protein, tannin, pectin,

sugars, fat.

Anthocyanins-Cyanidin-3-sambubioside, Cyanidin-3-glucoside

Fatty Acids -Palmitic, stearic, oleic, linoleic acid

Organic acids- Hydroxycitric acid (HCA)

Polyisoprenylated phenolics-Garcinol, isogarcinol

Anthocyanins are well known for their antioxidant, anti-inflammatory

and anti-carcinogenic activity.

Hydroxycitric acid (HCA) has gained much attention in recent years for

its pivotal role in fat/lipid metabolism, with implications for use in weight

loss.

Polyisoprenylated phenolics has gained much attention for antioxidant

activity.

Use 11 :

The nutraceutical studies on kokum indicated enormous medicinal

properties.

Anthelmetic,cardiotonic, curing piles ,dysentery, tumor

Pains,cardial problems.Antioxidative, chelating, free radical

scavenging, antiglycation, anticancer, anti-inflammatory, and

antiulcer activity,hypocholesterolaemic agent.

Ayurvedic medicine as it was traditionally used to treat sores,

dermatitis, diarrhea, dysentery, ear infection,cardioprotective and

7.0

to facilitate digestion.

Useful part : Fruit

6.3 OBJECTIVES OF THE STUDY :

1) Extraction of fruits of Garcinia indica Linn.

2) Preliminary phytochemical screening.

3) To study the cardioprotective activity of extracts using following

animal models.

a) Isoproterenol (ISO) induced myocardial necrosis in rats.

b) Ischemia-reperfusion induced (IRI) myocardial damage in

isolated rat heart.

4) Biochemical Estimation : Blood will be collected from the

animals and serum will be evaluated for

(a) LDH ( Lactate dehydrogenase )

(b) CK-MB ( Creatine phosphokinase-MB )

(c) Antioxidants ( Superoxide dismutase and Catalase )

5) Histopathological Examination : In all above models heart will

be isolated and examined for histopathological investigations.

6) In Isoproterenol (ISO) induced myocardial infarction model, ECG

changes will also be examined.

MATERIALS AND METHODS:7.1 SOURCE OF DATA:Experiment will be performed as described in the standard bibliography,

literatures and text books. The reputed journals and publications are

obtained from college library and through web search.

7.2 COLLECTION OF MATERIAL:

7.2.1 COLLECTION OF PLANT MATERIAL & EXTRACTION:

The Garcinia indica fruits will be collected from local region of

Mangalore district, and authenticated by a Taxonomist.

Fruits were cut open and the seeds were separated from the pulp.

Then the fruit rinds were allowed to dry in the shade. The fruits rinds

were cut into pieces and shade dried at room temperature. The dried fruits

were subjected to size reduction to a coarse powder by using mixer

grinder. The coarsely powdered form of shade dried fruit rinds was

placed in a conical flask containing distilled water and closed with cotton

plug for 7 days at room temperature. Then it was filtered using a piece of

clean, sterile, white cotton cloth and evaporated to dryness to yield

aqueous extract. The semisolid extract obtained was stored in an airtight

container in refrigerator for further use. The solution of aqueous extract

was prepared by using normal saline as solvent for experiment.

7.2.2 DRUGS AND CHEMICALS:

Isoproterenol, Heparine, Ketamine hydrochloride, Pentobarbitone,

Xylazine, carvedilol, LDH kits, CK-MB kits will be procured from Hi-

media suppliers. All other chemicals are of pure analytical grade will be

obtained from local suppliers.

7.2.3ANIMALS

Wistar rats (150-200 g) of either sex will be procured from Indian

Institute of Sciences. They will be maintained under standard conditions

(temperature 22 ± 2ºc, relative humidity 50±5% and 12 h light/dark

cycle).The animals will be housed in sanitized polypropylene cages

containing sterile paddy husk as bedding. They will have free access to

standard pellet diet and water ad libitum. The Institutional Animal Ethics

Committee approved the experimental protocol. All the animals received

human care according to the criteria outlined in the “Guide for the Care

and Use of Laboratory Animals” prepared by the “National Academy of

Sciences” and published by the “National Institute of Health”. All the

procedures will be performed in accordance with Institutional Animal

ethics committee constituted as per the direction of the committee for the

purpose of control and supervision of experiments on animals(CPCSEA),

under ministry of animal welfare division, Government of India, New

Delhi, India.

7.3 EXPERIMENTAL METHODS :

7.3.1.ISOPROTERENOL (ISO) INDUCED MYOCARDIAL

NECROSIS IN RATS12.

The albino rats (150-200 g) of either sex will be randomly divided into 5

groups of 6 each and assigned as below:

o Group-I- Normal control (Normal saline)

o Group-II - Toxic control (Isoproterenol: 85mg/kg)

o Group-III–Reference standard (carvedilol 1mg/kg)

o Group-IV Garcinia indica fruits extract (low dose:250mg/kg)

o Group-V- Garcinia indica fruits extract(high dose:500mg/kg)

Carvedilol will be administered to group III animals one week before

administration of Isoproterenol. All the animals in group IV & V will be

treated once daily post orally for 4 weeks. On 29th and 30th day after the

treatment, myocardial infarction will be induced in group II – group V

animals by sub cutaneous injection of Isoproterenol.

EVALUATION:

Symptoms and mortality in each group will be recorded and compared

with animals given with Isoproterenol alone. Forty-eight hours after the

first Isoproterenol administration, the rats will be sacrificed and

autopsied. The hearts will be removed and weighed and the frontal

sections will be embedded for histopathological examination.

BIOCHEMICAL ESTIMATION:

The collected blood as well as heart homogenate will be analyzed for

endogenous biological markers such as Lactate dehydrogenase (LDH),

Creatine phosphokinase-MB (CK-MB) and antioxidant enzymes like

Superoxide dismutase and Catalase.

7.3.2:ISCHEMIA-REPERFUSION INDUCED (IRI)

MYOCARDIAL DYSFUNCTION13

The albino rats (150-200 g) of either sex will be randomly divided into 5

groups of 3 each and assigned as below:

o Group-I-Normal control (saline)

o Group-II – Ischemia induced control

o Group-III – Ischemia induced + Reference standard (carvedilol

1mg/kg)

o Group-IV - Ischemia induced + Garcinia indica Linn extract (low

dose: 250mg/kg).

o Group-V- Ischemia induced + Garcinia indica Linn extract

(High dose: 500 mg/kg).

carvedilol will be administered to group III animals one week before

producing ischemia. All the animals in group IV & V will be treated

once daily post orally for 4 weeks. The heart will be excised from deeply

anesthetized rats (35 mg/kg sodium pentobarbitone, i.p) and perfused

with Kreb-Henseleit solution gassed with carbogen at 37o C and a

constant flow rate of 5ml/min by one-way circulation using modified

Langendorff setup. Measurement of contractile force will be done with

displacement transducer and recorded on Student physiograph. After a

short period of equilibrium (15 minutes), records will be taken for a

control period of 15 minutes, followed by 15 minutes ischemia and then

a washout period with Kreb-Henseleit solution. Recovery in terms of

inotropic and chronotropic effect will be studied and the extent of

cardioprotection due to Garcinia indica extract will be evaluated by

measuring the developed tension.

BIOCHEMICAL ESTIMATION:

The collected blood as well as heart homogenate will be analyzed for

endogenous biological markers such as Lactate dehydrogenase (LDH),

Creatine phosphokinase (CK) and antioxidant enzymes like Superoxide

dismutase and Catalase.

7.3.3 ECG STUDIES14.

The albino rats (150-200 g) of either sex will be randomly divided into 5

groups of 3 each and assigned as below:

o Group-I- normal control (saline)

o Group-II - toxic control (Isoproterenol: 85mg/Kg)

o Group-III – Reference standard ( carvedilol 1mg/kg).

o Group-IV – Garcinia indica fruit extract (low dose250mg/kg ).

o Group-V- Garcinia indica fruit extract (high dose500mg/kg).

carvedilol will be administered to group III animals one week before

administration of Isoproterenol. All the animals in group IV & V will be

treated once daily post orally for 4 weeks. On 29th and 30th day after the

treatment, myocardial infarction will be induced in group II – group V

animals by sub cutaneous injection of Isoproterenol.

After anesthetizing the rat with a combination of Ketamine hydrochloride

(75mg/kg, i.p) and Xylazine (8.0mg/kg, i.p), electrical leads will be

attached to the dermal layer of all the limbs and recording will be done

with the help of computerized ambulatory ECG system.

7.4 STATISTICAL ANALYSIS:

The data will be expressed as Mean value + SEM and will be analyzed by

the one-way ANOVA followed by the Dennett’s test.

7.5DOES THE STUDY REQUIRES ANY INVESTIGATIONS OR

INTERVENTIONS TO BE CONDUCTED ON PATIENTS OR

OTHER HUMANS OR ANIMALS? IF SO PLEASE DESCRIBE

BRIEFLY.

Yes. Study requires investigation on Wistar albino rats.

7.6HAS ETHICAL CLEARANCE BEEN OBTAINED FROM

YOUR INSTITUTION?

Yes. Ethical clearance has been obtained.

8.0 LIST OF REFERENCES:

1. Kung HC, Hoyert DL, Xu J, Murphy SL. Deaths: final data for

2005.Natl Vital Stat Rep 2008;56:1-120.

2. Burrows NR, Parekh S, Li Y, Geiss LS. Prevalence of self

reported cardiovascular disease among persons aged _35 years

with diabetes – United States, 1997–2005. MMWR.

2007;56:1129–1131. Available at:

http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5643a2.htm

Accessed 01 April2013 3:30 pm.

3. Levy RI, Feinleib M. Heart disease. In: Text book of

Cardiovascular Medicine. W.B. Saunders Company,

Philadelphia 1984; 2:1205-34.

4. Aronow WS. Epidemiology, pathophysiology, prognosis and

treatment of systolic and diastolic heart failure. Cardiol

2006;14:108-24.

5. Biase D, Pignatelli P, Lenti L, Tocci G, Piccioni F. Enhanced

TNF alpha and oxidative stress in patients with heart failure:

effect of TNF alpha on platelet O2 production. Thromb Haemost

2003;90:317-25.

6. Rajadurai M, Prince SM. Preventive effect of Naringin on

Isoproterenol-induced cardiotoxicity in Wistar rats: an in vivo

and in vitro study. Toxicology 2007;232:216-25.

7. AkcalıY, Ozturk F,Ceyran H, Narin F, Narin N, Akgun H,

Ceyran AB. The Effect of High Dose Melatonin on Cardiac

Ischemia reperfusion Injury. Yonsei Med J 2008;49(5).

8. Sakat SS, Wankhede SS, Juvekar AR, Mali VR, Bodhankar SL.

Antihypertensive effect of aqueous extract of Elaeocarpus ganitrus

Roxb. Seeds in renal artery occluded hypertensive rats.

International Journal of Pharm Tech Research, 2009 sep;1:779-82

9. Jai Ashish Tilak-Jain and Thomas Paul Asir Devasagayam:

Cardioprotective and Other Beneficial Effects of Some Indian

Medicinal Plants. J. Clin. Biochem. Nutr.2006,38;9-18

10. (Garcinia indica (Latin); Vrikshamia (Sanskrit)) A R T E M I S

[serial online] cited 2013 april 20] Available from:URL:

www.artemis-international.com/PDFs/Kokum.pdf.

11. Kirtikar & Basu, “Indian medicinal plants”, 2004; 1(2), 262.

12. Asdaq SMB, Inamdar MN, Asad M, Nanjundan PK. Interaction

of propranolol with garlic in Isoproterenol induced myocardial

infarction in rat. J PharmacolToxicol 2008;3(6):414-24.

13. Asdaq SMB, Inamdar MN. Pharmacodynamic interaction of garlic

withcaptopril in Ischemia-reperfusion induced myocardial damage

in rats. Phamacology online 2008;2:875-88.

14. Singh PN, Athar MS. Simplified calculation of mean QRS

vector(mean electrical axis of heart) of electrocardiogram. Indian J

Physiological Pharmacological 2003;47:212-6.

9. SIGNATURE OF THE CANDIDATE

10. REMARKS OF THE GUIDE Recommended and Forwarded

11. NAME AND DESIGNATION

11.1 GUIDEMR. SUNIL KOSHYAssociate ProfessorDepartment of PharmacologySrinivas College of PharmacyMangalore- 574 143

11.2 SIGNATURE

11.3 CO-GUIDE (IF ANY) Not Applicable

11.4 SIGNATURE

11.5 HEAD OF THE DEPARTMENT

MR. MOSES SAMUEL RAJANAssociate ProfessorDepartment of PharmacologySrinivas College of PharmacyMangalore- 574 143

11.6 SIGNATURE

12 12.1 REMARKS OF THE PRINCIPAL

12.2 NAME & SIGNATURE OF PRINCIPAL

DR. A. R. SHABARAYAPRINCIPAL,SRINIVAS COLLAGE OF PHARMACYMANGALORE- 574 143