POTENCY EVALUATION OF COMBINED THERAPY OF LOSARTAN
Transcript of POTENCY EVALUATION OF COMBINED THERAPY OF LOSARTAN
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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 +
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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
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
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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).
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.
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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.
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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
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
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.
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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
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).
••
•••♦
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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
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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
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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
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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
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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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