ISSN
0974-3618 www.rjptonline.org
RESEARCH ARTICLE
Cardioprotective Effect of
Ethanolic Extract of Buchanania lanzan Spreng. Against Isoproterenol
Induced Myocardial Infarction in Rats:A Biochemical, Electrocardiographic
Evaluation
Hardik Joshi*, Manoj Pagare, Leena Patil and Vilasrao
Kadam
Bharati Vidyapeeth’s College of Pharmacy, C.B.D.
Belapur, Navi Mumbai 40061.
Corresponding author: joshihardik17@gmail.com
ABSTRACT:
The present study was designed to investigate the
cardioprotective effect of ethanolic extract of Buchanania lanzan Spreng.
(EEBL) against isoproterenol induced myocardial infarction in rats by studying
myocyte injury markers, antioxidant defense system, serum and
electrocardiographic changes. Wistar rats were randomly divided into five
groups, namely
control, EEBL control, isoproterenol control, 250 mg/kg EEBL+ isoproterenol and
500 mg/kg EEBL + isoproterenol treated group. EEBL treatment group received
EEBL (250 and 500 mg/kg/day, p.o) for 30 days. Myocardial infarction in rats was induced by isoproterenol
administration (200 mg/kg, s.c.) at an interval of 24 h on 29th and
30th day. On 30th day ECG, biochemical parameters were assessed.
Isoproterenol administration showed changes in ECG pattern, including
ST-segment elevation (diagnostic of myocardial infarction) increase in the
serum levels of cardiac injury markers (Creatine kinase-MB, lactate
dehydrogenase, aspartate transaminase and alanine transaminase), decreased
antioxidant defense system in the heart. EEBL pre-co-treatment prevented almost
all the parameters of isoproterenol induced myocardial infarction in rats.
Results of the present study suggest that EEBL has a significant effect on the
protection of the heart against isoproterenol induced myocardial infarction
through maintaining endogenous antioxidant enzyme activities.
KEYWORDS: Isoproterenol, Myocardial infarction, Buchanania
lanzan Spreng, Antioxidant, Electrocardiography
1. INTRODUCTION:
Myocardial
infarction (MI) and the resultant complication in cardiac function represent
the leading cause of morbidity and mortality in developed countries.1
Moreover, with advanced life style in developing countries, like India,
particularly in metropolitan cities, MI is making increasingly important
contribution to mortality statistics of such countries.2
Isoproterenol [1-(3,4-dihydroxyphenyl)-2-isopropylaminoethanol hydrochloride]
is a synthetic catecholamine and β-adrenergic agonist, which has been
documented to produce severe stress in the myocardium resulting in the
myocardial infarction, if administered in supramaximal doses.3 It
produces myocardial necrosis which caused cardiac dysfunction, increased lipid
peroxidation along with an increase in the level of myocardial lipids, altered
activities of the cardiac enzymes and antioxidants.4,5
The
pathophysiological and morphological aberrations produced in the heart of this
myocardial necrotic rat model are comparable with those taking place in human
myocardial infarction.3 Among the various mechanisms proposed to
explain the isoproterenol induced cardiotoxicity, generation of highly
cytotoxic free radicals through auto-oxidation of catecholamines has been
implicated as one of the important causative factors.
Buchanania
lanzan Spreng. belonging to family
Anacardiaceae, commonly known as Chironji. The leaves are reported to be valued
for their tonic, cardiotonic properties, their powder is a common medicine for
wounds and also used in the treatment of skin diseases6-10. A new
glycoside Myricetin 3’-Rhamnoside-3- Galactoside was identified from leaves of
chironji.11
The
present study was designed to study the effect of ethanolic extract of Buchanania
lanzan (EEBL) pretreatment on the MI induced by supramaximal doses of
isoproterenol. The present study also attempted to demonstrate the possible
mechanism of its therapeutic efficacy by studying the biochemical markers,
antioxidant defense system and electrocardiographic changes.
2.
MATERIALS AND METHODS:
2.1
Chemicals, Plant material and Extraction:
Isoproterenol hydrochloride was purchased from Sigma
chemical company. Fresh leaves of Buchanania lanzan Spreng. Procured
from uttan van-ausadhi sanshodhan sanstha, Bhayander, Thane, authenticated from
Department of life science, Ruia College, Mumbai. The leaves were shade dried.
Dried leaves were ground to coarse powder. Powder was extracted with ethanol,
which is further evaporated to dryness to obtain alcoholic extract.
2.2 Animals:
All
experiments and protocols (IAEC/PR/2010/1) described in present study were
approved by the Institutional Animal Ethics Committee (IAEC) of Pharmacology
Department, The Bharati vidyapeeth’s college of pharmacy and with permission
from Committee for the Purpose of Control and Supervision of Experiments on
Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of
India. Wistar rats were purchased from Haffkin institute and housed in-group of
3 animals and maintained under standardized conditions (12-h light/dark cycle,
24 ± 2 °C, 35 to 60% humidity) and provided free access to palleted diet and
purified drinking water ad libitum.
2.3.
Induction of experimental myocardial infarction:
Isoproterenol
was dissolved in normal saline and injected to rats (200 mg/kg, s.c.) at an
interval of 24 h for 2 days to induce experimental myocardial infarction.
Animals were sacrificed 48 h after the first dose of isoproterenol.
2.4
Experimental design:
After
acclimatization, the animals were randomly divided into the following groups
consisting of 6 rats each.
|
Group I
|
Vehicle Control: Distilled
water (3 ml/kg, p.o. for 28 days) and sterile water for injection
subcutaneously (s.c.) at an interval of 24 h for 2 days.
|
|
Group II
|
Negative Control:
Isoproterenol injection (200mg/kg, s.c) on day on 29th and 30th day
|
|
Group III
|
EEBL (Ethanolic Extract of
Leaves of Buchanania lanzan) (250 mg/kg, p.o. for 28 days) and
Isoproterenol injection (200mg/kg, s.c) on day on 29th and 30th day
|
|
Group IV
|
EEBL (500 mg/kg, p.o. for 28
days) and Isoproterenol injection (200mg/kg, s.c) on day on 29th and 30th
day
|
|
Group V
|
Extract Control: EEBL (500
mg/kg, p.o. for 28 days) and sterile water for injection subcutaneously
(s.c.) at an interval of 24 h for 2 days.
|
2.5.
Electrocardiography:
ECG
was recorded under mild anesthesia through Lead II position using BIOPAC mp30
data acquisition system (BIOPAC systems, santa Barbara, CA, USA). The changes
in heart rate, QT interval and ST interval were determined from ECG.
2.6.
Biochemical analysis:
After
recording the ECG, the animals were sacrificed and blood samples were
collected. Serum was separated from each sample and used for the biochemical
analysis. Immediately after sacrifice, heart tissues were excised in ice cold
condition.
2.6.1.
Biochemical parameters in serum:
The
collected serum was used for the estimation of cardiac marker enzymes
creatinine kinase-MB (CK-MB), lactate dehydrogenase (LDH), alanine transaminase
(ALT), aspartate transaminase (AST) using commercially available standard
enzymatic kits.
2.6.2.
Biochemical parameters in heart:
The
excised heart was then weighed and homogenized in chilled Tris buffer (10 mM,
pH 7.4) at a concentration of 10% (w/v). The homogenates were centrifuged at
10,000 × g at 4°C for 20 minutes using Remi C-24 highspeed cooling centrifuge.
The clear supernatant was used for the assays of malondialdehyde content as
indicator of LP12, endogenous antioxidant enzymes, SOD13,CAT14
and GSH15.
2.6.3.
Membrane bound enzymes:
The
sediment after centrifugation of tissue homogenate was resuspended in ice-cold
Tris buffer (10 mM, pH 7.4) to get a final concentration of 10% and was used
for the estimation of different membrane bound enzymes such as Na+K+ATPase16,
Ca2+ATPase17, Mg2+ATPase18and
total proteins.19
2.6.4.
Statistical Analysis:
Results
of all the above estimations were indicated in terms of mean±SEM. Difference
between the groups was statistically determined by analysis of variance (ANOVA)
followed by Dunnett's test.
3.
RESULTS:
3.1. Electrocardiographic Changes:
The ECG changes in all the groups are as shown in
figure 1 and Table 1. The isoproterenol administration significantly increases
ST and QT interval.
a) Normal Control
b) Isoproterenol control
c) 250mg/kg+Isoproterenol
d) 500mg/kg+Isoproterenol
e) Extract control
Fig.1. Effect of EEBL on the changes of ECG patterns
Table 1: Effect of administration of isoproterenol
alone and along with EEBL (30 days) on ECG
|
Treatment group
|
ST interval (ms)
|
QT interval (ms)
|
|
Group I
|
8.58±0.375
|
70.19±0.529
|
|
Group II
|
12.51±0.621#
|
83.07±0.818#
|
|
Group III
|
11.76±0.483*
|
78.50±1.09**
|
|
Group IV
|
9.91±0.431**
|
73.11±0.648***
|
|
Group V
|
7.98±0.217
|
70.96±0.649
|
All values are expressed as Mean±SEM (n=6). *p<0.05,
**p<0.01, ***p<0.001, ns-non significant as compared to Isoproterenol
control group using one way ANOVA followed by Dunnet’s test. #p<0.001
as compared to normal control group using unpaired t test.
3.2 Serum markers:
The levels of serum marker enzymes in all the groups
are given in Table 2. Isoproterenol administration significantly increases
serum levels of CK-MB, LDH, ALT and AST as compared to control rats.
Administration of EEBL significantly restores the marker levels towards normal
in a dose-dependant manner.
3.3 Biomarkers of the oxidative stress:
The levels of biomarkers of oxidative stress enzymes in
all the groups are presented in Table 3. Isoproterenol administration
significantly increases LP while there was a significant decrease in GSH, SOD
and CAT levels as compared to control rats. Administration of EEBL
significantly improves GSH, SOD and CAT levels after Isoproterenol
administration while LP level changes towards normal values in a dose-dependant
manner.
3.4 Membrane bound enzymes:
Isoproterenol damages cell membrane as evident from
significant decrease in the levels of membrane bound enzymes like Na+K+ATPase,
Ca2+ATPase and Mg2+ATPase as compared to control. EEBL
significantly improves the level of membrane bound enzymes.
Table 2: Effect of administration of isoproterenol
alone and along with chronic EEBL (30 days) on serum markers
|
Treatment group
|
Serum ALT level (U/L)
|
Serum AST level (U/L)
|
Serum LDH level (U/L)
|
Serum CK-MB level (U/L)
|
|
Group I
|
70.15±3.145
|
118.4±7.74
|
129.8±7.763
|
222.05±13.347
|
|
Group II
|
122.3±7.163#
|
238.1±9.19#
|
230.2±9.01#
|
499.08±19.077#
|
|
Group III
|
97.44±4.963**
|
200.4±10.89*
|
190.8±12.59*
|
418.36±18.705*
|
|
Group IV
|
94.73±4.540**
|
187.1±10.58**
|
185.4±9.56*
|
339.63±25.104***
|
|
Group V
|
75.41±2.580
|
118.2±7.21
|
126.2±8.98
|
229.04±22.739
|
All values are expressed as Mean±SEM (n=6). *p<0.05,
**p<0.01, ***p<0.001, ns-non significant as compared to Isoproterenol
control group using one way ANOVA followed by Dunnet’s test. #p<0.05 as
compared to normal control group using unpaired t test.
Table 3: Effect of administration of isoproterenol
alone and along with chronic EEBL (30 days) on biomarkers of the oxidative
stress in heart
|
Treatment group
|
SOD (U/mg protein)
|
CAT (U/mg protein)
|
GSH (μg of GSH/mg protein)
|
LP (nmoles of MDA/mg protein)
|
Na+/K+ ATPase
|
Mg+2 ATPase
|
Ca+2 ATPase
|
|
Group I
|
5.68±0.887
|
25.09±0.864
|
6.13±0.899
|
2.28±0.430
|
7.18±0.66
|
4.44±0.238
|
5±0.490
|
|
Group II
|
1.83±0.279#
|
13.37±1.109#
|
2.64±0.311#
|
6.83±0.854#
|
3.57±0.334#
|
2.05±0.385#
|
7.97±0.588#
|
|
Group III
|
4.22±0.510*
|
17.84±1.509 ns
|
4.95±0.695 ns
|
4.24±0.609*
|
5.81±0.769*
|
3.44±0.309*
|
6.25±0.332*
|
|
Group IV
|
4.45±0.462*
|
20.06±1.373 **
|
5.20±0.508*
|
3.43±0.369 **
|
6.85±0.626**
|
3.99±0.245***
|
6.07±0.332*
|
|
Group V
|
5.03±0.482
|
22.45±1.108
|
6.27±0.565
|
2.59±0.564
|
8.25±0.477
|
4.59±0.349
|
5.26±0.316
|
ATPase expressed as μmol of
inorganic phosphorous liberated/min/mg protein. All values are expressed as
Mean±SEM (n=6). *p<0.05, **p<0.01, ***p<0.001, ns-non significant as
compared to Isoproterenol control group using one way ANOVA followed by
Dunnet’s test. #p<0.05 as compared to normal control group using unpaired t
test.
4.
DISCUSSION:
EEBL was found to have in vitro antioxidant
activity.20 The objective of present work was confined to
investigate the in vivo pharmacological activity of crude plant extract
in stepwise manner in utmost possible pharmacologically valid model system.
Catecholamines are important regulators of myocardial contractility and
metabolism. However, it has been known for a long time that excess
catecholamines are responsible for cellular damage, observed in clinical
conditions like angina, transient myocardial hypoxia, acute coronary
insufficiency and sub endocardial infarct. Animals develop infarct like lesions
when injected with isoproterenol, a potent synthetic catecholamine. These
lesions are morphologically similar to those of ‘coagulative myocytolysis’ or
myofibrillar degeneration, one of the finding described in acute myocardial
infarction and sudden death in man. Several mechanisms of isoproterenol
induced myocardial infarction have been reported. Isoproterenol acts both on
β1 and β2 adrenoceptors, activation of which
leads to positive inotropic and chronotropic effects. Thus, isoproterenol
produces relative ischemia due to myocardial hyperactivity and coronary
hypotension. Other probable mechanisms include increased cyclic adenosine
monophosphate, increased intracellular Ca++ overload, depletion of
high energy phosphate stores and oxidative stress. Increased generation of
cytotoxic free radicals, due to the auto-oxidation metabolic products of
isoproterenol, is one of the well recognized mechanisms of isoproterenol
induced myocardial necrosis. Isoproterenol, upon auto-oxidation produces
quinones which react with oxygen to produce superoxide anion (O2−)
and H2O2. The production of superoxide radical results in
secondary formation of H2O2 and hydroxyl radical (•OH).21
The results obtained above
indicate that isoproterenol induces pathological changes in ECG and biochemical
markers, suggestive of cardiotoxicity and increase in free radical production.
Further results also lead us to believe that administration of EEBL improved
the ECG and biochemical marker levels indicating decrease in oxidative stress
as evident by increased levels of GSH, SOD and CAT with decreased production
of LP. The restoration of membrane bound enzymes like Na+K+ ATPase,
Ca2 +ATPase and Mg2+ATPase in EEBL treated rats is
indicative of membrane stabilising protective effect of EEBL. These protective
effects are also supported by the restoration of serum marker enzymes towards
normal levels.
We conclude that Buchanania
lanzan Spreng. may play an important role in protecting cardiac oxidative
stress, and further studies should be initiated to established the exact
mechanism of action and elaborative Phytochemical investigations must be
carried out to find the active constituents responsible for cardioprotective
action.
5.
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