POTENCY EVALUATION OF COMBINED THERAPY OF LOSARTAN

www.wjpps.com Vol 6, Issue 8, 2017.

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Attuluri et al. World Journal of Pharmacy and Pharmaceutical Sciences

POTENCY EVALUATION OF COMBINED THERAPY OF LOSARTAN

AND ANANAS COMOSUS DURING ISOPRENALINE MEDIATED

CARDIAC DYSFUCNTION IN RATS

1Sushma Attuluri*,

2Syed Inayath Hussain,

3Dr. Mohsina Hassan,

4Urooj Fatima,

5Syeda Amtul Aleem Afifa,

6Amtul Aziz Afia,

1Assistant Professor, Department of Pharmacology, Nizam Institute of Pharmacy, Deshmukhi

(V), Pochampally (M), Behind Mount Opera, Yadadri Bhuvanagiri (Dist)-508284,

Telangana, India. 2Department of Pharmacology, Nizam Institute of Pharmacy, Deshmukhi (V), Pochampally

(M), Behind Mount Opera, Yadadri Bhuvanagiri (Dist)-508284, Telangana, India. 3Department of Pharma. D, Nizam Institute of Pharmacy, Deshmukhi (V), Pochampally (M),

Behind Mount Opera, Yadadri Bhuvanagiri (Dist)-508284, Telangana, India. 4Assistant Professor, Department of Pharmacology, Nizam Institute of Pharmacy, Deshmukhi

(V), Pochampally (M), Behind Mount Opera, Yadadri Bhuvanagiri (Dist)-508284,

Telangana, India. 5Department of Pharmacology, Nizam Institute of Pharmacy, Deshmukhi (V), Pochampally

(M), Behind Mount Opera, Yadadri Bhuvanagiri (Dist)-508284, Telangana, India. 6Assistant Professor, Department of Pharmacology, Nizam Institute of Pharmacy, Deshmukhi (V),

Pochampally (M), Behind Mount Opera, Yadadri Bhuvanagiri (Dist)-508284, Telangana, India.

ABSTRACT

The present study was carried out to explore the cardioprotective

potency of pineapple (ananas comusus) with losartan in

isoprenaline(ISO) induced acute and chronic myocardial damage in

rats. The purpose of the study research was to uncover the potency of

anti inflammatory activity of pineapple on necrotic myocardium, in

combination with angiotensin blocker losartan(LTN). In acute model,

Sprague Dawley rats were pre-treated with losartan(LTN) (30 mg/kg,

ten days, po), low,intermediate and high doses of pineapple (100, 250

and 500 mg/kg, 30 days, po) and subsequently subjected to ISO

administration (150 mg/kg, s.c) for two consecutive days. The

influence of prophylactic treatment was analysed by quantification of

biomarkers, antioxidants and histopathological observations. LDH and

CK-MB activities were studied in serum and heart tissue homogenate

(HTH) where as SOD, catalase and TBARS were estimated in HTH.

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 6.647

Volume 6, Issue 8, 1352-1387 Research Article ISSN 2278 – 4357

Article Received on

14 June 2017,

Revised on 05 July 2017, Accepted on 26 July 2017,

DOI: 10.20959/wjpps20178-9750

*Corresponding Author

Sushma Attuluri

Assistant Professor,

Department of

Pharmacology, Nizam

Institute of Pharmacy,

Deshmukhi (V),

Pochampally (M), Behind

Mount Opera, Yadadri

Bhuvanagiri (Dist)-

508284, Telangana, India.


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There was significant elevation in LDH and CK-MB gactivities in HTH of all treated grups

as compared to ISO control.SOD and catalase activities were also significantlt increased in

treated groups. In chronic model, ISO (3 mg/kg/day, s.c) was administered to Sprague

Dawley rats treated with low,intermediate and high doses of pineapple (100, 250 and 500

mg/kg, p.o. thirty days) and LTN (30 mg/kg, p.o. ten days). Through retro orbital route blood

is withdrawn for LDH and CK-MB estimations. Antioxidants were estimated by biochemical

method and histopathological studies were carried out to support biochemical studies. Prior

treatment of animals with Pine apple and LTN resulted in elevation of biomarker activities in

heart tissue homogenate and decline in serum. The SOD and Catalase activities were

significantly elevated with depletion of TBARS and histological scores in groups subjected to

prophylactic treatment. Among various treatments, high dose of Pine apple and LTN was

found to be most effective. From the present results it may be concluded that high dose of

Pine apple (500 mg/kg) possess cardioprotective efficacy when given prophylactically in

experimental animals. However, further studies should be carried out to explore the

ultrastructural changes and underlying mechanisms of protection when these substances are

used together.

KEYWORDS: Isoprenaline, Losartan, Pine Paple, Myocardial Damage, LDH, CK-MB,

SOD, Catalase, TBARS.

INTRODUCTION

The treatment of diseases with medicines of plant origin is an integral part of many cultures

throughout the world.[1,2]

Natural drugs are gaining greater acceptance from the public and

the medical profession due to greater advances in understanding the mechanism of action by

which herbs can positively influence health and quality.[3]

When herbs were combined with

pharmaceutical agents, posing the possibility of potential interaction between the two groups

of substances.[4]

Although modern drugs are effective in preventing the cardiovascular

disorders, their use is often limited because of their side effects and adverse reactions.[5]

Hence it is imperative to promote credible research on the safety and efficacy of combined

herb-drug treatment for variety of ailments including cardiovascular diseases.[6]

Ananas

comosus belongs to the family Bromeliaceae, commonly called as pineapple.[7]

Pineapple

mainly contains enzyme complex called protease (bromelain), which contains peroxidase,

acid phosphate, several protease inhibitors and organically bound calcium which is found to

have cardioprotective activity.[8]

It is mainly reported in treatment of Rheumatoid


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arthritis[9-12]

, Cancer[13-16]

and Gastro intestinal disturbances.[17,18]

And found to have actions

like immunogenicity, platelet aggregation, wound healing[19-25]

, antioxidant, antimicrobial

and antidiarrheal. Its anti-inflammatory activity is helpful in the treatment of angioedema.[26]

Bromelain on long term administration was found to have anti hypertensive effect.[27]

Bromelain have the protective action on the myocardial ischemia-reperfusion by the up

regulation of survival kinases known to attenuate the process of apoptosis particularly the

serine or threonine kinase Akt is well established to play an important role in endothelial and

cardiomyocyte cell biology that activates an antiapoptotic or prosurvival signaling

cascade.[28]

Losartan is a competitive and selective Angiotensin II receptor antagonist, that is, it blocks

the action of Angiotensin II on ATI Receptor[29]

and lowers the blood pressure.[30]

It isolated

perfused rat heart, losartan reduced the duration of ventricular fibrillation induced by

reperfusion.[31]

However at high doses losartan has side effects[32]

on all over body systems

like allergic reactions, irregular heartbeat, fever, chills, or persistent sore throat; mental or

mood changes; muscle pain or cramps; one-sided weakness; persistent headache; severe or

persistent stomach pain (with or without nausea or vomiting); shortness of breath, severe

nausea or vomiting; symptoms of liver problems (eg, dark urine, loss of appetite, pale stools,

stomach pain, yellowing of the eyes or skin); symptoms of low blood pressure (eg, fainting,

severe dizziness, light-headedness); tremor; unusual bruising or bleeding; unusual sweating;

unusual tiredness or weakness; vision changes. So, necessity to be used along with other safe

cardioprotective regimen. As pineapple has the great benefits of providing protection to

myocardium and losartan is potent cardioprotective, it is of interest to determine the role of

combined therapy of fresh pineapple juice at different doses and losartan during isoprenaline

induced acute and chronic cardiac dysfunction in rats.

OBJECTIVES

Objective of study: The objective of the present study will be to evaluate the potency of

combined therapy of losartan and Ananas comosus during isoprenaline mediated cardiac

dysfucntion in rats.

SPECIFIC OBJECTIVES

1) To collect and authenticate the cloves of Ananas comosus.

2) To prepare and carryout phytochemical evaluation of Ananas comosus homogenate.


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3) To standardized acute and chronic models of isoprenaline for induction of myocardial

damage.

4) To select the dose of losartan.

5) To study the biochemical and antioxidant profile in serum and heart tissue homogenate

upon chronic administration of Ananas comosus homogenate with or without losartan.

6) To explore the role of losartan in presence/absence of Ananas comosus during isoprenaline

induced acute myocardial derangement in rats.

7) To study the benefits of adding Ananas comosus to cardioprotective therapy of losartan in

isoprenaline mediated chronic myocardial damage in rats.

METHODOLOGY

Experimental Animals

Sprague Dawley rats of either sex weighing 150-200 gm and Male Swiss albino mice

weighing between 25-30 gm were housed at 25° ± 5°C in a well-ventilated animal house

under 12:12 h light dark cycle. Institutional Animal Ethics Committee approved the

experimental protocol (KCP/IAEC-74/2011-12). The animals were maintained under

standard conditions in an animal house as per the guidelines of Committee for the Purpose of

Control and Supervision on Experiments on Animals (CPCSEA).

Preparation of Ananas comosus homogenate and Dose selection

Pineapple fruits will be purchased from the local fruit market. The fruit will be peeled, sliced

and ground into a paste and suspended in distilled water. Three different concentrations of the

pineapple will be selected based on OECD guidelines[121]

and will be administered within 30

min of preparation. The dose selections of pineapple for isoproterenol model were done based

on acute toxicity studies, carried out according to OPPTS (Office of Prevention, Pesticide and

Toxic Substance) following the limit test procedure.[122]

The animals were fasted over night

prior to the studies. Mice were divided into three groups of three each. Test dose of 2 g/kg

body weight, 3 g/kg body weight and 5 g/kg body weight were given orally to all group of

mice. Mice were observed for 72 hours for mortality. 1/10th,

1/25th

and 1/50th

of the

maximum safe dose corresponding to 500 and 250 mg/kg and 100mg/kg body weight were

selected as high, intermediate and low doses respectively.

Apparatus and chemical used

Analytical balance (Schimadzu, Japan), Autoanalyser (Qualigens, Mumbai), Centrifuge,

Colorimeter, Students Physiograph (INCO, India), Spectophotometer, CK-MB (Crest


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Biosystems, Coral Clinical Systems, Goa, India), LDH kits (Crest Biosystems, Coral Clinical

Systems, Goa, India), DL- Isoproterenol hydrochloride (Sigma Aldrich, U.S.A), losartan (Sun

Pharmaceuticals Industries), Copper sulphate, Disodium hydrogen orthophosphate (Merck

Specialities Private Limited, Mumbai, India), EDTA (Nice Chemicals Pvt Ltd, Cochin,

India), Ethanol (Hong yang chemical corp. China), Heparin (Gland Pharmaceutical Ltd,

Hyderabad, India), Hydrogen peroxide, Hydroxylamine HCL, Ketamine (Prem

Pharmaceutical Ltd, Indore, India), Malondialdehyde (MDA), N- Butanol, Nitro blue

tetrazolium (NBT) (Loba Chemicals, Mumbai, India), Phenol reagent, Phosphoric acid,

Potassium dihydrogen orthophosphate, Sodium carbonate (Merck Specialities Private

Limited, Mumbai, India), Sodium chloride (Merck Specialities Private Limited, Mumbai,

India), Sodium hydroxide (Merck Specialities Private Limited, Mumbai, India), Standard

bovine albumin, Sucrose (S D Fine Chemicals, Mumbai, India), Thiobarbituric acid, Xylazine

(Indian Immunological, Guntur, India).

Phytochemical estimations of the Almond[123-125]

Bitter almond was subjected to qualitative analysis to investigate the presence of various

phytochemical constituents such as alkaloids, carbohydrates, glycosides, phytosterols,

proteins, amino acids, saponins, tannins and flavonoids.

Test for Alkaloids

1. Hager’s Test: Extract was treated with Hager’s reagent (saturated picric acid solution).

Formation of a yellow coloured precipitate indicates the presence of alkaloids.

2. Mayer’s Test: Extract was treated with Mayer’s reagent (potassium mercuric iodide

solution). Formation of a cream coloured precipitate indicates the presence of alkaloids.

3. Dragendroff’s Test: Extract was treated with dragendroff’s reagent (potassium bismuth

iodide solution). Formation of orange brown precipitate indicates the presence of alkaloids.

4. Wagner’s Test: Extract was treated with Wagner’s reagent (iodine potassium solution).

Formation of reddish brown precipitate indicates the presence of alkaloids.

Test for carbohydrates

1. Molisch Test: Extract was treated with molisch reagent (α-naphthol in 95% ethanol) and

few drops of sulphuric acid were added through the side of the test tube. Appearance of violet

ring at the junction indicates the presence of carbohydrates.


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2. Fehling’s Test: A small portion of the extract was treated with felhing’s reagent A

(Copper Sulphate in water) and felhing’s reagent B (sodium potassium tartarate) and heated

in a water bath. Formation of red colour precipitate indicated the presence of reducing sugars.

3. Barfoed’s Test: Extract was treated with barfoed’s reagent (copper acetate in water and

glacial acetic acid), and heated in a water bath. Red coloured precipitate indicates the

presence of sugars.

4. Benedictt’s Test: Extract was treated with benedictt’s reagent (copper sulphate + sodium

citrate + sodium carbonate in water), and heated for 10 minutes. Red coloured precipitate

indicates the presence of sugars.

Test for Proteins and amino acids

1. Millon’s Test: Extract was treated with millons’s reagent (mercuric nitrate in nitric Acid).

Red colour indicates the presence of proteins.

2. Biuret Test: Extract was treated with sodium hydroxide. Copper sulphate solution was

added drop wise. Violet colour is produced which indicates the presence of proteins.

3. Ninhyrin Test: Extract was treated with ninhydrin reagent and ammonia and heated.

Violet colour indicates the presence of proteins.

Test for Steroids, Triterpenoids and Cardiac Glycosides

1. Salkowski Test: To the solution of extract in chloroform, a few drops of sulphuric acid

were added and the moisture was shaken and allowed to stand for some time. Red colour is

produced in the chloroform layer, which indicates the presence of steroids.

2. Liebermann-Burchard Test: Small portion of extract was dissolved in chloroform. To this

1ml acetic anhydride and then 2ml concentrated sulphuric acid was added through the side of

the test tube. A reddish violet colour at the junction of two liquids indicates the presence of

steroids, triterpenoids and cardiac glycosides.

3. Baljet Test: To the solution of extract in water a few drops of sodium picrate reagent was

added. Formation of yellow colour indicates the presence of cardiac glycosides.

4. Keller Killani’s test: A portion of extract was treated with 1ml of glacial acetic acid and a

few drops of ferric chloride were added to it. To this mixture 2ml concentrated sulphuric

acid, carefully through the sides of the test tube. A reddish brown colour is formed at the

junction of two layers and bluish green colour in the upper layer which indicates the presence

of deoxy sugars in the carbohydrates.


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Test for Tannins

1. Ferric Chloride Test: Extract was treated with ferric chloride solution. Formation of blue

colour indicates the presence of tannins.

2. Lead Acetate Test: Extract was treated with lead acetate solution; yellow precipitate

indicates the formation of tannins.

Test for Saponins

1. Froth test: Diluted 1ml of the extract with distilled water to 20ml and shaken in a

graduated cylinder for 15min. One-centimeter layer of foam indicates the presence of

saponins.

2. Haemolysis test: 2ml of 1% Nacl was mixed with 2ml of extract,to this 5 drops of blood

was added. Haemolysis indicates the presence of saponin.

Test for Flavonoids

1. Ferric Chloride Test: Extract was treated with few drops of ferric chloride solution.

Formation of blackish blue colour indicates the presence of flavonoids.

2. Lead Acetate Test: Extract was treated with lead acetate solution; yellow precipitate

indicates the formation of flavonoids.

Experimental Models

The cardioprotective role of combined therapy of losartan with Ananas comosus will be

determined in isoprenaline induced ischemia-reperfusion rats. The Sprague dawley rats of

either sex will be divided into following groups consisting of six animals each:

1. Group-I- animals kept as control without pretreatment.

2. Group II- ISO control.

3. Group-III- Ananas comosus (30 days oral treatment).

4. Group-IV- Ananas comosus (30 days oral treatment).

5. Group-V- Ananas comosus (30 days oral treatment).

6. Group-VI- Losartan (30mg/kg, orally for ten days).[126]

7. Group-VII- Ananas comosus (30 days oral treatment) + Losartan (10 days oral).

8. Group-VIII- Ananas comosus (30 days oral treatment) + Losartan (10 days oral).

9. Group-IX- Ananas comosus (30 days oral treatment)+ Losartan (10 days oral).


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Isoprenaline induced acute myocardial necrosis in rats

Male Sprague Dawley rats were treated for 30 days as per the above protocol. At the end of

the treatment period, isoprenaline (150 mg /kg, s.c)[127]

was administered to all the animals

(except the normal control) for two consecutive days. Forty eight hours after the first dose of

ISO, the animals were anesthetised with ketamine (75mg/kg, i.p) and xylazine

(8mg/kg, i.p)[128,129]

and blood was withdrawn by retro-orbital puncture. Serum was separated

by centrifugation for the estimation of biomarkers (LDH, CK-MB. Thereafter the animals

were sacrificed; three hearts were used for the preparation of homogenate to estimate

biomarkers (LDH and CK-MB) and antioxidants (SOD and Catalase). Remaining three hearts

were embedded in 10%v/v formalin solution in saline for histological examination.

The parameters estimated were

1. LDH and CK-MB activity in serum and heart tissue homogenate.

2. SOD, Catalase and TBARS activity in heart tissues homogenate.

3. Histopathological Studies: Scoring in myocardial tissue was determined from H-E

transverse stain.

Isoprenaline induced chronic myocardial necrosis in rats.

Female Sprague Dawley rats were treated with pineapple for 30 days, 10 days of losartan and

incorporation of losartan in the last week of 30 days of pineapple treatment in their respective

groups, animals in all groups except group I will receive isoprenaline 3 mg/kg/day

subcutaneously.[130]

At the end of the treatment period, the animals were anesthetised with

ketamine (75mg/kg, i.p) and xylazine. (8mg/kg, i.p) and blood was withdrawn by retro-

orbital puncture. Serum was separated by centrifugation for the estimation of biomarkers

(LDH, CK-MB). ECG recordings were made for each animal using lead II method.

Thereafter the animals were sacrificed; three hearts were used for the preparation of

homogenate to estimate biomarkers (LDH and CK-MB) and antioxidants (SOD and

Catalase). Remaining three hearts were embedded in 10%v/v formalin solution in saline for

histological examination.

The parameters estimated were

1. LDH and CK-MB activity in serum and heart tissue homogenate.

2. SOD, Catalase and TBARS activity in heart tissues homogenate.

3. Histopathological Studies: Scoring in myocardial tissue was determined from H-E

transverse stain.


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Note

1. Animals were injected ISO approximately the same time each day.

2. Animals were kept under cool conditions, to prevent death of the animals due to

hyperthermia and respiratory failure.

Preparation of Heart Tissue Homogenate[129]

The hearts removed after the ECG recording was made free of the adjacent vessels and fatty

tissue mass with the help of scissors. Hearts were then cut open, rinsed with saline (0.9%

NaCl) and dried using filter paper. The weight of the heart was recorded. Then the heart

homogenate was prepared in ice cold 0.25M sucrose solution using a mortar and pestle. The

homogenate thus obtained was centrifuged at 5000 rpm for 15 min. The supernatant was

decanted and used for the estimation of CK-MB, LDH, SOD, catalase and TBARS.

Procedure for Estimation of Biomarkers

Biomarkers like CKMB and LDH were estimated using CKMB (NAC act) Kit and LDH

(P-L) Kit respectively.

Estimation of CK-MB

CK is a dimeric molecule composed of M and B subunits which are immunologically

distinct. It exists as CK-MM, CK-MB and CK-BB isoenzymes form and found in muscles,

myocardial cells and brain respectively. Rise in the CK-MB level is an appropriate indicator

of MI as its level does not increase in chest pain caused by angina or pulmonary embolism.

The levels return to normal, in case there is no further myocardial damage.[131]

PRINCIPLE

CK-M fractions of CK-MM and CK-MB in the sample are completely inhibited by an anti

CK-M antibody in the reagent. The activity of CK-B fraction is measured by the CK (NAC

Act.) method.

SAMPLE MATERIAL

Serum (should be free from haemolysis) and heart tissue homogenate.

PROCEDURE

System Parameters

Reaction: U.V kinetic Interval: 60 sec

Wavelength: 340 nm Sample Volume: 0.05 ml

Blank: Distilled Water Reagent Volume: 1.00ml


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Delay time: 60sec Read Time: 180sec

Linearity: 100U/L

No. Of read: 4 Units: U/L

For the sample start assays 0.8 ml of enzyme reagent (L1) and 0.05 ml of sample

(serum/homogenate) are mixed in a clean dry Eppendorffs tube and incubated for 5 minutes

fallowing 0.5ml of starter reagent (L2) was added. Then the mixture was shaken and fed into

the auto analyser.

Estimation of LDH

LDH is found in many body tissues particularly heart, liver, skeletal muscles, kidney and

erythrocytes. LDH is found in the form of isoenzyme. Levels of LDH increase in myocardial

infarction, pulmonary diseases, hepatic diseases, haemolytic anaemia, renal diseases and

muscular dystrophy.

PRINCIPLE

Lactate dehydrogenase catalyses the reduction of pyruvate with NADH to form NAD. The

rate of oxidation of NADH to NAD is measured as a decrease in absorbance which is

propotional to the LDH activity in the sample.

Lactate

Pyruvate + NADH + H+

Lactate + NAD+

PROCEDURE

System Parameters

Reaction U.V Kinetics Interval 60sec

Wavelength 340nm Sample Volume 10µl

Blank Distilled Water Reagent Volume 10µl

Factor 8095

Delay Time 60sec React. Slope decreasing

Read Time 180sec Linearity 2000 U/L

No. Of Read 4 Units U/L

For the substrate start assay 0.8 ml of buffer reagent (L1) and 0.5 ml of sample

(serum/homogenate) were incubated for 1 minute. Then 0.2ml of starter reagent (L2) was

added. Thereafter the mixture was shaken and fed into auto analyser.

Note

RBC’s have a very high LDH content and hence haemolysed samples should not be used.


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Enzyme oxidants

Superoxide Dismutase (SOD)[132]

PRINCIPLE

Estimation of SOD was done by detecting O2- by oxidation of hydroxylamine hydrochloride

yielding nitrite, which was measured colorimetrically at 560 nm.

O2-

is also detected during auto oxidation of hydroxylamine at pH 10.2, Nitro blue

tetrazolium is reduced and nitrite is produced in the presence of EDTA, which can be

detected colorimetrically.

SOD acts in following way

O2- + O2

- + 2H

+ H2O2 + O2

Reagents needed

1. Sodium carbonate solution: 1.06 gm of sodium carbonate was dissolved in 100 ml of

distilled water.

2. Nitro Blue Tetrazolium (NBT): 10 mg NBT was dissolved in 100 ml water.

3. EDTA solution: 37 mg di sodium EDTA salt was dissolved in 100 ml of distilled water.

4. Hydroxylamine Hydrochloride: 16.5 mg of hydroxylamine hydrochloride was dissolved

in 100 ml distilled water.

PROCEDURE

1. To 100µl of 10% heart tissue homeogenate in 0.25 M sucrose, a mixture containing 1 ml

sodium carbonate, 0.4 ml NBT and 0.2 ml EDTA was added and zero minute reading was

taken at 560 nm.

2. By addition of 0.4 ml of 1 mM hydroxylamine hydrochloride, the reaction was initiated

3. The reaction mixture was incubated at 250C for 5 minutes, the reduction of NBT was

measured at 560 nm.

4. Without the tissue homogenate, parallel control was also made in same manner.

One enzymatic unit o SOD is equal to the amount in the form of protein in 100µl of 10%

tissue homogenate required to inhibit the reduction of 24 mM NBT by 50% and is expressed

as units/mg of protein.

The concentration of the enzyme is calculated using following formula[133]

Unit/mg= 1000

µgm of enzyme resulting in ½ max. inhibition


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Estimation of total proteins

Protein estimation is based on the formation of a coloured complex of proteins with Folin

catechu reagent, which was measured colorimetrically at 610 nm. Bovine albumin was used

as a standard.

Reagents

1. Alkaline reagent: 2 gm of Sodium carbonate was dissolved in 100 ml of 0.1N NaOH

solution.

2. Alkaline mixture: To 100 ml of alkaline reagent, 1 ml of 4 % aqueous copper sulphate

solution was added. This was prepared freshly.

3.Phenol reagent (Folin Ciocalteau reagent) (Loba chemie, Mumbai, India): Commercially

available reagent was diluted by adding equal volume of distilled water before use and stored

in the refrigerator.

4. 0.1N NaOH solution.

5. 90 % alcohol.

6. Preparation of standard bovine albumin solution: 8 mg (0.08 %) of standard bovine

albumin (S.D. fine chemicals, Mumbai, India) was dissolved in 10ml of distilled water.

PROCEDURE

1. To 0.1 ml of heart homogenate, 0.9 ml of 90 % alcohol was added and centrifuged at 3000

rpm for 10 minutes. The supernatant was discarded and precipitate was dissolved in 1 ml of

0.1N NaOH.

2. 0.05 ml of the above solution was taken in a test tube, to which 4 ml of freshly prepared

alkaline mixture was added and allowed to stand for 10 minutes.

3. To the above reaction mixture 0.4 ml of phenol reagent was added and allowed to stand for

further 10 minutes for the reaction to complete.

4. The absorbance was measured at 610 nm using distilled water as blank.

5. 0.1 ml of standard bovine solution was taken in a test tube and processed as mentioned in

step 2 and 3.

The amount of protein was calculated using the formula

Protein = O.D of sample X Final Volume X Dilution Factor X Concentration of Std

(Mg/ml) O.D of Standard


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Catalase[134]

PRINCIPLE

This enzyme is found in the peroxisomes and converts hydrogen peroxide to water and

oxygen.

2H2O2 2H2O + O2

ROOH + AH2 H2O + ROH + A

Catalase was estimated by determining the decomposition of H2O2 at 240 nm in an assay

mixture containing phosphate buffer (0.25 M, pH 7).

Reagents

1. Phosphate buffer (0.25 M, pH 7): 3.22 gm potassium hihydrogen orthophosphate and 7.268

gm disodium hydrogen orthophosphate was dissolved in 1000 ml distilled water.

2. Hydrogen peroxide (0.042 ml = 12.3 M): 0.34 ml of 30% hydrogen peroxide was diluted to

100 ml with distilled water.

PROCEDURE

1. To 100µl of 10% tissue homogenate in 0.25 M sucrose, 1.9 ml phosphate buffer was added

and absorbance was measured at 240 nm.

2. 1 ml hydrogen peroxide was added to above reaction mixture and it was allowed to stand

for 1 minute. The absorbance was measured at 240 nm using phosphate buffer as blank

solution.

One international unit of catalase utilised is that amount which catalyzes the decomposition

of 1mM hydrogen peroxide per minute at 370 C and expressed in terms of units/mg of

protein. Catalase activity can be calculated using the following formula.

Units/mg = Absorbance240 nm X 1000

43.6 X mg of enzyme/ml reaction mixture

Thiobarbituric acid reactive substances[135,136]

PRINCIPLE

Plasma concentration of thiobarbituric acid reactive substance is the index of oxidative stress

and lipid peroxidation. Elevated levels of TBARS are associated with increased risk of

cardiovascular diseases. Oxidative stress during reperfusion leads to lipid peroxidation.

Direct measurement of liberated ROS is difficult due to their instability, malondialdehyde


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(MDA), a stable lipid peroxidation end-product, is frequently used as a marker of ROS

production and measured at 532 nm.

Reagents

1. Phosphoric acid (1%): 1ml phosphoric acid was diluted to 100 ml with distilled water.

2. Thiobarbituric acid (TBA) (0.6%): 0.6 gm Thiobarbituric acid was dissolved in 100 ml of

distilled water.

3. N-butanol.

4. Malondialdehyde (MDA).

PROCEDURE

1. 3ml phosphoric acid and 1ml of 0.6% TBA was added 0.5ml of heart tissue homogenate.

This mixture was boiled over the water bath for 45 minutes.

2. Upon cooling, 4 ml n-butanol was added to the above mixture and mixed for 1 minute

followed by centrifugation at 20000 rpm for 20 minutes.

3. The organic layer formed, was transferred to a fresh test tube and the absorbance was

measured at 532nm.

Procedure for standard curve

1. Primary stock: 100mm MDA was prepared by dissolving 3.135 gm MDA in 100ml of

distilled water.

2. From the primary stock solution 1,2,3 and 4 ml was taken and diluted to 10 ml to get the

concentration of 10,20 30 40 mm of MDA.

3. 0.5ml of solution was taken from each of the above concentration and steps 1-3 were

repeated without the addition of heart homogenate.

4. Graph was plotted between absorbance Vs concentration and amount of TBARS in heart

homogenate was read by interpolation method.

Histopathological examination

Heart tissue obtained from all groups were washed immediately with saline and then fixed in

10% v/v formalin solution in saline. The ventricular mass from the left ventricular location

was sectioned from heart in order to obtain 0.4 cm thick transverse section and dehydrated

with alcohol fallowed by embedding with paraffin wax. These sections were stained with

hematoxylin and eosin (H&E).[136]


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The myocardial damage was determined by giving scores depending on the intensity as

follows; no changes-score 00; mild-score 01 (focal myocytes damage or small multifocal

degeneration with slight degree of inflammatory process); moderate-score 02 (extensive

myofibrillar degeneration and/or diffuse inflammatory process); marked-score 03 (necrosis

with diffuse inflammatory process).[80]

Statistical analysis: The statistical significance was assessed using One-way Analysis of

variance (ANOVA) followed by Tukey multiple comparison tests. The values were expressed

as mean ± SEM where P<0.05 was considered significant.

RESULTS

Preparation of Ananas comosus homogenate

Ananas comosus fruits were peeled, sliced, grounded in to paste and suspended in distilled

water. three different concentrations of ananas comosus homogenate were prepared, 0.1g/ml,

0.25g/ml and 0.5g/ml, corresponding to 100mg/kg body weight, 250mg/kg body weight and

500mg/kg body weight of animal.

Phytochemical investigation

Table. 1: Phytochemical investigation of various compound.

SL NO TEST INFERENCE RESULT

1. Test for alkaloids

1a Hager’s test Yellow colour - _

1b Mayer’s test Cream precipitate - _

1c Dragendroff’s test Orange precipitate _

1d Wagner’s test Red-brown precipitate _

2. Test for carbohydrates

2a Molisch’s test Violet colour +

2b Fehling’s test Brick red colour +

2c Barfoed’s test Red colour +

2d Benedict’s test Red colour +

3. Test for steroids, triterpenoids and glycosides

3a Liebermann-Burchard test Reddish- violet colour +

3b Salkowski test Red colour +

3c Baljet’s test Orange colour +

3d Keller-Killani test Red colour +

4. Test for saponins

4a Froth test 1 cm foam +

4b Haemolytic test No precipitate +

5. Test for tannins

5a Ferric chloride test Blue colour +

5b Lead acetate test Yellow colour +


1367


6. Test for proteins and Amino acids

6.a Millon’s test Red colour +

6.b Biuret test Violet colour +

6.c Ninhydrin test Violet colour +

7. Test for flavanoids

7.a Ferric chloride test Blackish red colour +

7.b Lead acetate test Yellow precipitate +

Toxicity studies: Acute toxicity studies carried out according to OPPTS guidelines[122]

,

demonstrated that extract was not toxic at doses 2 g/kg body weight, 3 g/kg body weight and

5 g/kg body weight P.O, Hence 1/10th

, 1/25th

and 1/50th

of maximum safe dose corresponding

to 500mg/kg, 250 mg/kg and 100mg/kg orally were selected as high, intermediate and low

doses respectively.

Isoprenaline induced myocardial infarction acute model

Effect on haemodynamic parameters

A) Endogenous biomarker enzymes

Effect on CK-MB and LDH activity: Administration of ISO through subcutaneous route

significantly elevated the CK-MB and LDH levels in ISO control but decreased in all pre-

treated groups compared to NC. A significant fall in the serum CK-MB and LDH was found

in all treated group compared to ISO control. LTN treatment alone and combination showed

significant reduction of CK-MB and LDH activity in serum. ACHD+LTN combination was

showing better effect in the reduction of CK-MB and LDH levels than LTN alone.

In HTH all the treated groups were showing significant decline in the CK-MB and LDH

levels compared to NC. There was a significant rise in the CK-MB and LDH levels in all

treated groups compared to ISO control. Concurrent administration of LTN with different

doses of AC was found to have significant effect on CM-MB and LDH levels compared to

LTN alone. (Table:2).

Table 2: Effect on CK-MB and LDH activities in serum and heart tissue homogenate

(units/gm) against isoprenaline induced acute myocardial damage.

TREATMENT CKMB ACTIVITY LDH ACTIVITY

SERUM (Unit\Lit) HTH (Unit\gm) SERUM (Unit\Lit) HTH (Unit\gm)

NORMAL 193.1±4.956

66.65±4.110

23.5±4.817 9.2±1.081

ISO CONTROL 1023.86±8.903

10.58±1.76c

989.56±9.193c

3.633±0.2906c

ACLD 709.86±6.55cf

26.4±1.47cf

68.88±2.128cf

5.835±0.198cd

ACID 650.716±11.9cf

52.68±2.031cf

40.3±1.143af

6.0±0.1807ce

ACHD 608.01±12.8cf

53.58±1.73cf

38.15±1.47f

6.433±0.1901cf


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LTN 509.86±10.036cf

31.05±1.003cf

29.3±1.0833cf

6.71±0.2330bf

ACLD+LTN 373.4±`9.82cfl

50.5±1.445cfil

21.466±0.833f

7.25±0.2062afl

ACID+LTN 281.6±9.247cfil

60.5±0.6414fij

15.17±0.651fj

8.11±0.1167fl

ACHD+LTN 269.63±11.03cfil

62.38±1.008fik

13.09±0.981fj

8.5±0.1713fl

All values are mean ± SEM, n=6, aP<0.05,

bP <0.01,

cP <0.001 when compared to normal

control; dP <0.05,

eP <0.01,

fP <0.001 compared to ISO control,

gP <0.05,

hP <0.01,

iP

<0.001 compared to ACLD/ACID/ACHD respectively, jP <0.05,

kP <0.01,

lP <0.001

compared to LTN. ACLD (ananas comosus low dose 100mg/kg), ACID (ananas comosus

intermediate dose 250mg/kg), ACHD (ananas comosus highdose 500mg/kg) and LTN

(30mg/kg).

B) SOD, Catalase AND TBARS

Effect on SOD and catalase: SOD and catalase activities were reduced significantly in the

ISO control compared to NC where as all the pre treated groups were showed significant rise

in the SOD and catalase activities compared to ISO control. LTN alone and combinations

with different doses of AC were found to have significant effect on reversing the ISO induced

elevation of SOD and catalase.

Effect on TBARS: Significant elevation of TBARS levels were found in ISO control and

decline in all treated groups compared to NC. All the treated groups were showing a

significant fall in TBARS activity compared to ISO control. ACLD, LTN and ACID+LTN

were found to have significant fall in TBARS levels.(table:3).

Table. 3: Effects on SOD, CATALASE and TBARS activity in heart tissue homogenate

against isoprenaline induced acute myocardial damage.

TREATMENT SOD (Units/mg

protein)

CATALASE

(Units/mg protein)

TBARS

(ng/mg protein)

Histological

score

NORMAL 6.75±0.38 17.15±0.4463 19.6±1.67 0

ISO CONTROL 0.303±0.021c

6.3±0.3393cf

27.8±3.59a

3

ACLD 1.383±0.1662cd

8.667±0.1909cf

2.3±0.34cf

2

ACID 2.88±0.088cf

9.416±0.1778cf

13.8±0.93be

2

ACHD 3.3±0.1633cf

10.66±0.2539cf

14.0±0.17cf

2

LOSARTAN 5.3±0.3260cf

10.61±0.3422cf

9.8±0.25cf

1

ACLD+LTN 5.61±0.1493afi

11.766±0.3422cfi

13.7±0.75cfl

2

ACID+LTN 6.7±0.2366fik

13.21±0.1922cfil

9.9±8.75d

2

ACHD+LTN 7.233±0.2060fil

13.21±0.1922cfil

10.2±0.20cfi

1


bP <0.01,


control; dP <0.05,

eP <0.01,


gP <0.05,

hP <0.01,

iP


kP <0.01,

lP <0.001


1369




(30mg/kg).

Effect on histological score: Isoprenaline injections caused necrosis of cells with

degeneration of myofibril and increased interstitial space. In ISO induced acute M.I. by

administration of isoprenaline for two consecutive day’s myocardial integrity was disturbed

evident with increased interstitial space and necrosis of cells with degeneration of myofibril

(Fig. 5.2.3). Pre-treatment of ACLD, ACID and ACHD with or without Carvedilol to rats

before subjected to Isoprenaline induced myocardial damage showed significant fall in

histological scores compared to ISO control.

Groups treated with ACLD, ACID and ACHD has shown less diffuse necrosis, mild

inflammation and fibrosis. Recovery from necrosis, mild inflammation with less interstitial

space and least multifocal degeneration was observed in the groups treated with LTN and

different doses of AC combinations.

Histopathological scores

0-normal cardiac muscle architecture,

1-mild focal necrosis/degeneration,

2-mild diffuse/moderate necrosis+/- mild inflammation,

3-moderate diffuse necrosis+/- mild inflammation and fibrosis,

4-sever necrosis+/-inflammation+ /-fibrosis.

Haematoxylin and eosin (H&E) stained section of heart in Isoprenaline induced acute

myocardial damage. Photographed at magnification 400X.

Fig.5.4.3a Heart tissue of Normal group. Fig.5.4.3b Isoprenaline Treatedgroup.


1370


Fig.5.2.3c ACLD (100 mg/kg) Treated Group Fig.5.2.3d ACID (250 mg/kg) Treated Group

Fig.5.2.3e ACHD (500mg/kg) Treated Group. Fig.5.2.4f losartan (30mg/kg) Treated Group.

Fig.5.2.3.g ACLD (100mg/kg)+LTN (30mg/kg) Fig.5.2.3.h ACID(250mg/kg)+LTN

treated group 30(mg/kg) treated group

Fig.5.2.3i ACHD (500mg/kg) +LTN (30mg/kg) treated group.

Isoprenaline induced chronic myocardial infarction model

The biomarkers such as LDH and CK-MB were estimated in serum and homogenate while

SOD, Catalase and TBARS estimated in heart tissue homogenate.


1371


Effect on haemodynamic parameters

A) Endogeneous biomarkers

The LDH and CK-MB activities were significantly increased and decreased in serum and in

heart tissue homogenate (HTH) respectively in ISO treated group when compare to Normal

group. Groups treated with ACLD,ACID, ACHD, LTN, ACLD+LTN,ACID+LTN and

ACHD+LTN showed significant fall in LDH and CK-MB activities in serum and elevation in

HTH compared to ISO control.(Table:4).

Table 4: Effects on LDH and CKMB level in serum and heart tissue homogenate against

Isoprenaline induced chronic myocardial infarction.

Treatment Ckmb Activity LDH Activity

Serum (Unit\Lit) HTH(Unit\gm) SERUM (Unit\Lit) HTH (Unit\gm)

NORMAL 161.94±5.2 75.33±1.4 29.9±0.764 16.88±6.4354

ISO CONTROL 451.833±6.571cf

7.15±0.32cf

767.91±6.89cf

2.566±0.152c

ACLD 269.483±11.936cf

36.3±1.429cf

593.7±1.926cf

6.766±0.091a

ACID 243.45±4.505cf

53.7±0.756cf

432.05±6.144cf

9.15±0.152

ACHD 228.66±2.535cf

60.46±3.99cf

406.23±2.35cf

10.816±0.453

LOSARTAN 210±1.597bf

62.33±1.26cf

360.58±4.46cf

11.48±0.364

ACLD+LTN 190.86±2.69fi

61.766±0.368cfij

268.75±18.46cfil

13.46±0.360d

ACID+LTN 175.70±3.203fij

67.433±0.6561bfij

258.86±6.71cfil

14.933±0.168e

ACHD+ LTN 163.833±3.477fij

73.11±0.808fjil

240.88±5.87cfil

14.966±0.320e


bP <0.01,


control; dP <0.05,

eP <0.01,


gP <0.05,

hP <0.01,

iP


kP <0.01,

lP <0.001



(30mg/kg).

B) SOD, Catalase AND TBARS

Effect on SOD and Catalase

In ISO control group there were decreased in SOD and Catalase activity when compared to

normal control. All treated groups were found to have significant effect on increase in SOD

and catalyse values. ACID+LTN and ACHD+LTN groups were having more significant

effect on SOD and catalase values.


1372


Effect on TBARS

A significant elevation in TBARS levels were found in ISO control compared to normal

control. All the treated groups were found to have significant effect on TBARS values.

ACLD, AClD+LTN treated group was found to more fall in TBARS level.

Effect on histological score

Administration of ISO caused necrosis of cells with degeneration of myofibril and increased

interstitial space. In ISO induced chronic M.I. by administration of Isoprenaline for 21 days

(3mg/kg/day) myocardial integrity was disturbed evident with increased interstitial space and

necrosis of cells with degeneration of myofibril (Figure 5.5.3). Pre-treatment of ACLD,

ACID and ACHD with or without LTN to rats before subjected to Isoprenaline induced

myocardial damage showed significant fall in histological scores compared to ISO control.

ISO treated group shows moderate diffuse necrosis+/- mild inflammation and fibrosis,

Groups treated with ACLD, ACID and ACHD has shown less diffuse necrosis, mild

inflammation and fibrosis as compare to ISO group. Group treated ACLD, ACID and ACHD

with LTN showed more effective than individual treatment. ACLD+LTN showed significant

fall in TBARS levels. (Table no. 5).

Table no. 5: Effects on SOD, Catalase and TBARS activity in heart tissue homogenate

against isoprenaline induced chronic myocardial damage.

TREATMENT SOD CATALASE TBARS Histologi

cal score

NORMAL 6.91±0.174

15.64±6.19

17.8±0.72 0

ISO CONTROL 0.571±0.030c

5.81±0.30c

30.9±4.1b

3

ACLD 2.946±0.2810cf

7.233±0.170c

10.3±1.6f

2

ACID 3.683±0.2242cf

8.416±0.132cf

13.6±0.34f

2

ACHD 3.766±0.189cf

9.033±0.289cf

12.9±3.29f

1

LOSARTAN 3.466±0.162cf

9.98±0.284cf

12.8±0.129f

1

ACLD+LTN 4.166±0.221cfi

11.93±0.287fil

10.9±1.89f

2

ACID+LTN 6.566±0.108fil

14.9±0.262fil

8.8±0.9f

0

ACHD+LTN 6.56±0.136fil

14.86±0.217afil

9.1±2.38f

0


bP <0.01,


control; dP <0.05,

eP <0.01,


gP <0.05,

hP <0.01,

iP


kP <0.01,

lP <0.001



(30mg/kg).

••

•••♦


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Histopathological scores

0-normal cardiac muscle architecture,

1-mild focal necrosis/degeneration,

2-mild diffuse/moderate necrosis+/- mild inflammation,

3-moderate diffuse necrosis+/- mild inflammation and fibrosis,

4-severe necrosis+/-inflammation+/-fibrosis.

Fig.5.5.3a Heart tissue of Normal group. Fig.5.5.3b Isoprenaline treated group.

Fig.5.5.3c ACLD(200 mg/kg) Treated group. Fig.5.5.3d AID(500 mg/kg) Treated group.

Fig5.5.3.e ACHD(500mg/kg) treated group Fig.5.5.3e Losartan (30 mg/kg) Treated group


1374


Fig.5.5.3f ACLD (100mg/kg)+LTN30mg/kg) Fig5.5.3gACID (250mg/kg)+LTN Treated

group (30mg/kg) treated group

F.5.5.3h ACHD (500mg/kg)+LTN(30mg/kg) treated group.

Figure 5.5.3 Hematoxylin and eosin (H&E) stained sectioned of heart in IRI induced

myocardial damage in rats. Photographed at magnification 400X.

DISCUSSION

The present investigation was to elucidate the role of AC during myocardial dysfunction and

metabolic derangement induced by ISO in rat heart and to explore its pharmacodynamic

interaction with conventional cardioprotective drug LTN. The findings of investigation

revealed the beneficial role of Ananas Comosus (AC) when treated concurrently with LTN in

conditions of anticipated cardiac injury. ISO, a synthetic cathecholamine and β-adrenoceptor

stimulant, is known to cause myocardial damage at higher concentration.[96]

Disturbances in

coronary microcirculation, anoxia and formation of cathecolamine oxidative products apart

from intracellular calcium load have been postulated to induce cardiac toxicity.[137]

ISO

administration results in increase in calcium uptake and energy consumption leading to call

death.[138]

Different biomarkers enzymes elevation in serum is due to leakage of enzymes

from the heart as a result of ISO- induced necrosis.[105]

ISO has increases the production of

cytotoxic free radicals through its auto-oxidation. It has been suggested that the oxidative

products of cathecolamines produce changes in the myocardium by stimulating lipid

peroxidation and cause irreversible damage to the myocardial membrane this alters

membrane permeability, leading to the loss of function and integrity of myocardial


1375


membranes. Hence, leakage of endogeneous biological markers and free radical formation

are attributed for myocardial distress in post isoprenaline administration.[86]

In acute model, administration of two high doses of ISO in consecutive days was found to

induce myocardial damage which is eveident from our observation.[138]

In chronic model,

administration of ISO (3mg/kg/day) ledas to cardiac hypertrophy with increased collagen

content. In the chronic phase of ISO infusion, increased ROS play important roles in

extracellular matrix biosynthesis, which may lead to the alteration of wall stiffness and affect

cardiac function by involving in the progression of cardiac hypertrophy and heart failure.[130]

Ananas comosus, contains an enzyme complex called Bromelain, contains peroxidase, acid

phosphate, several protease inhibitors and organically bound calcium which is found to have

cardioprotective activity.[2]

Bromelain has the capacity to reduce angina[139]

, exert

antihypertensive action[140]

, and significantly reduce the incidence of coronary infarct when

administered with potassium and magnesium orotate.[141]

Bromelain decreases the severity of

angina pectoris and the infarct size.[44, 45]

Bromelain decreases platelet aggregation[46,43]

, is an

effective fibrinolytic agent[47]

, and inhibits thrombus formation.[48]

Bromelain have the

protective action on the myocardial ischemia-reperfusion by the upregulation of survival

kinases known to attenuate the process of apoptosis particularly the serine or threonine kinase

Akt is well established to play an important role in endothelial and cardiomyocyte cell

biology that activates an antiapoptotic or prosurvival signaling cascade.[28]

Apoptisis is main

reason that contributes to many cardiac dysfunctions and it is the dominant form of

myocardial cell death in the infarct area, bromelain acts as anti apoptotic by phosphorylating

the protein kinases like Akt and FOXO protiens which inturn linked to the reduction of the

apoptotic process.[49]

Angiotensin receptor blockers (ARB) displace angiotensin II from the

angiotensin I receptor and produce their blood pressure lowering effects by antagonizing

angiotensin II induced vasoconstriction, aldosterone release, catecholamine release, arginine

vasopressin release, water intake and hypertrophic response. LTN is a competitive and

selective Ang II receptor antagonist. It has selective inhibition of angiotensin II by

competitive antagonism of the angiotensin II receptors, has been spectualted to reduce

adverse effects and possibly improve clinical efficacy.[30]

The presence of diagnostic biomarker enzymes like CKMB and LDH in heart tissue

homogenate (HTH) is indicative of myocardial integrity and their release in serum signifies

myocardial injury. In our study, there was substantial fall and rise in activities of marker


1376


enzymes in HTH and serum respectively upon ISO administration. These effects could be

secondary events following ISO induced lipis peroxidation of cardiac membranes, with a

consequent increase in enzyme leakage from cardiac myocytes.[106]

Pretreatment with ACLD,

ACID, ACHD, LTN or combination with LTN with different doses of AC prevented the

inclination of CK-MB and LDH in serum. Our biochemical findings were supported by the

improvement in histopathological architechture of heart tissues in all treated groups with

maximal improvement in the combination group, suggesting that these drugs may have a

potential protective effect against ISO induced cardiac damage. It can be speculated that the

damage caused to myocardium during ISO might be irreversible due to release of free

radicals and hence we also measured the endogeneous anti oxidant activities in HTH. Our

findings on antioxidant estimation explain the well established assumption of involvement of

OFRs in myocardial injury.[142]

Among number of OFRs associated with myocardial

contractile and rhythmic disturbances, contribution of superoxide to myocardial damage is

believed to be the highest and this radical is combaed by elevated activities of endogeneous

anti oxidant enzyme – the superoxidase (SOD). In addition to this, measurement of catalase

activity was carried out as elevation in SOD dimutes superoxide but results in accumulation

of H2O2 which could further precipitate the myocardial infarction. Pretreatment of animals

with AC (100mg/kg, 250mg/kg and 500mg/kg) alone or along with LTN produced

remarkable elevation in SOD and catalase activities when compared to ISO control indicating

cardioprotective effect.

AC decreases the apoptosis of the cardiac myocytes, which inturn decreases the release of

biomarkers from the myocyte in to the serum, and thus retains the antioxidant activity of the

myocytes, and protects the myocardium from cell death. The cardioprotective and antiapoptic

activity of ananas comosus was already reported in earlier studies. The potential of protective

effect may be due to the rich source of Bromelain present as a main constituent. AC

cardioprotection with or without LTN by inhibiting the apoptosis process of the myocytes.

CONCLUSION

From the results it may be concluded that three doses of AC (100, 250 and 500 mg/kg)

possess cardioprotective efficacy when given prophylactically against ISO induced acute and

chronic myocardial necrosis in rats. In both the models high dose was found more effective.

the manifestations induced by isoprenaline mediated stress were further aleviated with the

introduction of conventional cardioprotective drug Losartan. The efficacy of Ananas comosus


1377


could be attributed to presence of complex enzyme bromelain, which is responsible for the

increase in the biomarkers in heart homogenate, while the ARB blocking property of losartan

could be responsible for its cardioprotective nature. However for the interaction it could be

synergetic effect of Ananas comosus and losartan for cardioprotection. Further studies should

be carried out, to understand mechanism of interaction between Ananas comosus and

Losartan with extensive evaluation of histological and ultra structural changes.

SUMMARY

The current investigation dealt with the pharmacodyanmic interaction of pineapple juice with

losartan on experimentally induced myocardial infarction (MI) in rats. The study was carried

out using two different animal models like ISO induced acute and chronic myocardial

damage in Sprague dawley rats. Myocardial damage was either induced by administration of

isoprenaline (ISO) 150mg/kg s.c for two consecutive days or by daily dosage of 3mg/kg s.c

for 21 days. Animals were treated with pineapple juice orally for 21days and for interactive

groups last 10 days with LTN for both the models. Three doses of AC showed the significant

protection against acute and chronic administration of ISO induced myocardial damage by

indicating a significant decrease in biomarkers such as LDH and CKMB in serum and

increase in heart homogenate with respect to ISO control.

In this study high, medium and low dose of AC treatment along with conventional drug LTN

inclined the activity of antioxidants with decline in TBARS activity in HTH indicating the

structural integrity and protection to the myocardium. The cardioprotection offered by AC

could be attributed to the presence of enzyme complex bromelain that shows anti apoptic

activity effectby inhibiting the progressed cell death of myocytes. The protective activity of

AC was further augemented when used along with LTN. Above findings were further

conformed by decrease in the histological scores.

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