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CARDIOPROTECTIVE ROLE OF TERMINALIA CATAPPA. Linn AND CISSAMPELOS PAREIRA. Linn ON DOXORUBICIN

INDUCED CARDIAC TOXOCTIY IN RATSTHESIS

Submitted toJawaharlal Nehru Technological University, Anantapur,

In the partial fulfillment of the requirements for

the award of the degree of

MASTER OF PHARMACY

IN

PHARMACOLOGY

By

Y.LALITHA

[Reg.No.10AF1SO108]

Under the guidance of

V. JAYA SANKAR REDDY M.Pharm, (Ph.D)

Department of Pharmacology

KRISHNATEJA PHARMACY COLLEGE, TIRUPATHI, CHITTOOR (Dt)

SEPTEMBER -2012

AIM AND OBJECTIVE OF THE STUDY

The Goal of the present study is to investigate

• Cardiac protective activity of Terminalia catappa.L and Cissampelos pareira.L

against doxorubicin induced cardiac toxicity in rats.

The following objectives were set to achieve the goal of the present study

• To perform the acute toxicity studies of ethanolic extracts of Terminalia

catappa.L and Cissampelos pareira.L on mice.

• To investigate the effect of various doses of Terminalia catappa.L and

Cissampelos pareira.L on serum biomarkers (CK, LDH, SGOT, SGPT) in

normal and cardio toxic rats.

• To investigate the effect of various doses of Terminalia catappa.L and

Cissampelos pareira.L on antioxidant enzymes (SOD and catalase) in normal

and cardio toxic rats.

PLAN OF WORK

Plant selection and acquisition

Botanical identification

Drying of plant materials

Extraction of plant materials

Phytochemical profiling

Acute toxicological studies

Screening of cardio protective activity

INTRODUCTION

• Cardiovascular diseases remain the principal cause of death in

both developed and the developing countries.

• Myocardial infarction is a prominent implication of the

cardiovascular disease. According to WHO 16.7 million

people die each year owing to heart attacks.

• Traditionally, Terminalia catappa.Linn and Cissampelos

pareira.Linn were used as cardio-tonics and

pharmacologically both shown significant antioxidant

property, hence these plants were chosen to evaluate the

Cardio protective Activity.

• Doxorubicin, a successful antitumor drug also known for its

cumulative and dose dependent cardio toxicity. Hence Dox

has been chosen for the study to induce cardio toxicity.

MYOCARDIAL INFARCTION

• Myocardial infarction is an ischemic necrosis of a portion of the

myocardium due to sudden occlusion of a branch of coronary artery.

CLASSIFICATION:

. ST elevation MI(STEMI)

. Non- ST elevation (non-STEMI)

EPIDEMOLOGY:

• MI is a common occurrence in young man(40-60yrs) and in

women (60-85yrs).

• World wide more than three million people were suffering

with STEMI and more than four million people were suffering

with Non-STEMI.

• Blacks and whites are equally affected.

• Mortality estimates due to CVD vary widely by state, ranging

from 10% in Meghalaya to 49% in Punjab.

AEITOLOGY:

• Hypercholesterolemia

• Diabetes mellitus

• Smoking

• Stress

• Obesity

• Insufficient exercise

• Hypertension

SYMPTOMS:

• Pain

• Difficulty in breathing

• Dizziness

• Nausea

• Shortness of breath

• Mimics like MI

Pathophysiology:

Role of plaque in MI

DIAGNOSIS OF MYOCARDIAL INFARCTION

• The diagnosis of myocardial infarction can be made after

assessing patient's complaints and physical status.

• ECG changes, Coronary angiogram and levels of cardiac

markers help to confirm the level of infarction.

MYOCARDIAL INFARCTION COMPLICATIONS

• Arrhythmias

• Heart failure

• Pericarditis

• Emboli

PLANT PROFILE

Terminalia catappa.L Cissampelos pareira.L

TAXONOMY:

Kingdom :Plantae

Division :Magnoliophyta

Class :Magnoliopsida

Order :Myrtales

Family :Combrataceae

Genus : Terminalia

Species :catappa

TAXONOMY:

Kingdom : Plantae

Division :

Magnoliophyta

Class : Magnoliopsida

Family :

Menispermaceae

Genus : Cissampelos

Species : Pareira

Uses

Terminlia catappa.Linn

It has been used for dysentry,

rheumatoid arthritis, cough,

asthma, leprosy, nausea, eye

problems, liver problems and

STD.

It is used as cardiotonic, diuretic,

antipyretic, aphrodisiac and

antimicrobial agent.

Cissampelos pareira. Linn

It has been used for heart

problems,asthma,inflammat

ion, malaria, STD, snake

bite and Kidney problems.

It has been used as diuretic,

antiseptic and anti periodic

property.

REVIEW OF LITERATURETerminlia catappa. l

S.NO Authors & year Activity done1 B.V.Jawal et al.,(2012) Antioxidant potential

2 Abulmanzur et al.,(2011) Antimicrobial activity

3 A. Aimola et al., (2011) Erythropoiesis

4 Azrul et al.,(2011) Anthelmintic potential

5 Paul chidokachikezie (2011) Antipolymerization

6 Saheb L and SS Wadje.,(2011) Fungicide activity

7 Saroja M et al.,(2011) Antioxidant activity of phenolic fraction

8 H.V Annegowda et al.,(2010) Analgesic and antioxidant properties

9 Jing gao et al.,(2010)Kinoshita S et al.,(2007)

Hepatoprotective activity

10 Minakshi G Joshi et al., (2008) Antibiotic

11 Ahmed SA et al.,(2006) Antidiabetic activity

12 Fan YM et al., (2004) Phytochemical and anti-inflammatory activity

Cissampelos pareira.L

S.NO Authors & year Activity done1 Singh BK et al., (2012) Cardiac protective activity

2 Mahendra sigh Yadv etal.,(2011)

Hepatoprotective activity

3 PadminiShukla et al., (2011) In-vitro anthelmintic activity

4 Pramodine d et al., (2011) Memory enhancing activity

5 Surendran S et al., (2011) Hepatoprotective activity

6 Gupat M et al.,(2010) Antipyretic activity

7 Bafna A, Mishra S.,(2009) Immunomodulatory activity

8 Amresh G et al., (2008) Toxicological screening

9 Amresh G et al., (2007) Antinociceptive and antiarthritic

10 G. Amresh et al., (2011) Gastroprotective

11 AmreshG,Reddy GD et al., (2007)

Antioxidant activity

• MATERIALS:

Animals: Wistar rats(150-200gms)

Swiss albino mice(20-30gms)

S.NO Chemical Manufacture

1 Doxorubicin Celon

2 Normal saline Claris life sciences Ltd

3 CK-MB kit span diagnostics Ltd

4 SGOT kits span diagnostics Ltd

5 SGPT kits span diagnostics Ltd

6 Formalin Sd-fine chem. Limited

7 Potassium dihydrogen phosphate Merk specialities

8 Ethanol Changshuyangyuan chemicals

9 Disodium hydrogen phosphate Fisher scientific

• METHODS

Identification and authentification of plants

Collection of plant materials

preparation of plant materials for the extraction

Extraction procedure

Preliminary phytochemical studies

ACUTE TOXICITY STUDIES

The procedure was followed by using OECD 423 (Acute Toxic

Class Method).

The starting dose level of Terminalia catappa.L and

Cissampelospareira.L was 2000 and 5000mg/kg body weight p.o.

Dose was administered to overnight fasted mice`s. Food was

withheld for and further 3-4 hours after administration.

The body weight of the mice`s before and after administration were

noted.

the changes in the body weight were not so prominent.

The onset and signs of toxicity was also not observed. No mortality

was observed.

PHARMACOLOGICALSCREENING

GROUPING:

• Group I : Control (Normal control)

• Group II : Positive control(doxorubicin(15 mg/kg B.W, through I.P))

• Group III: Test I (Plant Extract-250mg/kg B.W of EETC+DOX)

• Group IV : Test II (Plant Extract-500mg/kg B.W of EETC+DOX)

• Group V : Test III(plant extract-250mg/kg B.W of EECP+DOX)

• Group VI : Test IV (Plant extract-500mg/kg BW of EECP+DOX)

• Group VII: Test V(DOX+Plant Extract-250mg/kg B.W of

EETC)

• Group VIII: Test VI(DOX+Plant Extract-500mg/kg B.W of

EETC)

• Group IX: Test VII(DOX+ Plant extract-250mg/kg B.W of

EECP)

• Group X: Test VIII(DOX+ Plant extract-500mg/kg B.W of

EECP)

• Rats were divided into ten groups each group contain six animals.

• Group I serves as normal which receives normal saline(5ml/kg

B.W) for 30 days and Group II serves as positive control which

receives doxorubicin 7.5mg/kg B.W in two equal injections at

27thand 29th day(15mg/kg B.W; I.P route)and normal

saline(1ml/kg B.W).

• Group III, Group IV, Group V and Group VI serves as test

which receives plant extracts of Terminalia catappa.L and

Cissampelos pareira.L at dose of 250 and 500 mg/kg B.W,

through oral route throughout the study period and also receives

doxorubicin (15 mg /kg B.W) at 27thand 29thday.

• Group VII, Group VIII, Group IX and Group X also serves as test

and receives doxorubicin(15 mg/kg B.W) first and then receives test

extracts at an dose of 250 and 500mg/kg B.W, through oral route

throughout the study period.

• After the study period animals were sacrificed by collecting blood

through cardiac puncture and collected blood was centrifuged and

serum was collected and biochemical analysis were performed for

serum levels CK-MB, LDH, SGOT, SGPT, SOD and Catalase.

• The hearts were isolated and placed in 10% formalin and

histopathological studies were performed.

RESULTS Phytochemical analysis:

+VE: Presence of compound

-VE : Absence of compound

S.NO COMPOUNDS

ETHANOLIC EXTRACT OF

TERMINALIA CATAPPA

ETAHANOLIC EXTRACT OF

CISSEMPELOS PAREIRA

1 Carbohydrates + +

2 Fixed oils + +

3 Alkaloids + +

4 Glycosides + +

5 Steroids + +

6 Flavonoids + +

7 Tannins + +

8 Phenolic compounds + +

9 Amino acids - -

10 Saponins + +

Acute toxicological study:

• From acute toxicity studies their was no mortality at a dose

of 5000mg/kg B.W.

Pharmacological screening: Effect of ethanolic extracts of Terminalia catappa. L and

Cissampelos pareira. L on serum biomarkers in normal and doxorubic in exposed rats

S.NO Groups CK-MB LDH SGOT SGPT

1 Group I 126.20±2.584 116.79±2.412 68.6±4.188 46.213±0.7026

2 Group II 246.52±6.609 234.76±4.889 161.98±0.7525 92.82±1.898

3 Group III 181.00±2.369*** 143.22±4.770*** 140.23±1.074*** 81.35±0.7794***

4 Group IV 152.80±7.061* 136.65±3.484*** 116.68±1.145*** 68.68±0.8520***

5 Group V 171.49±2.513*** 137.22±4.019*** 130.80+4.414*** 73.67±1.433***

6 Group VI 156.10±2.768** 120.49±5.220*** 87.53±6.7332*** 57.23±0.9472***

7 Group VII 223.28±8.677*** 198.25±3.146*** 178.51±2.785** 78.32±0.8053***

8 Group VIII 192.84±5.893*** 178.52±2.276*** 146.14±1.837* 69.21±1.686***

9 Group IX 178.12±3.503*** 147.22±2.742*** 126.61±4.753*** 68.88±1.879***

10 Group X 149.70±2.876* 133.86±2.883*** 88.248±3.664*** 51.84±1.501***

Effect of ethanolic extracts of EETC and EECP on antioxidant levels in doxorubicin treated rats hearts

All values were expressed as mean+ SEM; n= 6 observation, values were significant when compared with

positive control, * P<0.05,** P<0.01,*** P<0.001(one way ANOVA followed by Turkey`s test)

S.NO GROUPS SOD CATALASE

1 Group I 7.1±0.03890 11.24±0.1472

2 Group II 3.747±0.2449 5.07±0.1451

3 Group III 5.168±0.08392*** 7.75±0.2808***

4 Group IV 5.597±0.2800*** 9.12±0.1183***

5 Group V 4.980±0.2493** 8.30±0.2516***

6 Group VI 6.112±0.1657*** 10.250±0.07638***

7 Group VII 4.942±0.1241** 6.74±0.1700***

8 Group VIII 4.92±0.2554** 7.64±0.1148***

9 Group IX 4.97±0.1179** 8.54±0.2683***

10 Group X 5.900±0.3246*** 10.81±0.2629***

Effect of Terminalia catappa.L and Cissampelos pareira.L on serum CK, LDH, SGOT & SGPT levels in doxorubicin induced cardiac toxic rat

Histopathlogical studies

CONCLUSION

• The present study suggest that the Terminalia catappa.L and

Cissampelos pareira.L could be used as an antioxidant during or

after doxorubicin therapy plants shows a greater protective effect in

preventive as compared with curative model in rats.

• Further studies should be required for the detection, isolation and

structural elucidation of the chemical constituents and further

investigation should be required to find exact mechanism of action

for cardioprotective activity of Terminalia catappa.L and

Cissampelos pareira.L plants.