ISSN   0974-3618                                                     



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:



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



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.



2.1 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



Group II



Group III



Group IV



Group V



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





Group II





Group III





Group IV





Group V





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








Group II








Group III


17.84±1.509 ns

4.95±0.695 ns





Group IV


20.06±1.373 **


3.43±0.369 **




Group V








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.



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.



1.        Chada SL. Urban-rural differences in prevalence of coronary heart disease and its risk factors. Curr Sci. 74; 1998:1069-73.

2.        Levy RI, Feinleib M. Heart disease. In: Brawnwald E, editor. Text book of Cardiovascular Medicine. 2nd ed. Philadelphia: W.B. Saunders Co; 1984.

3.        Rona, G. Catecholamine cardiotoxicity. J. Mol. Cell. Cardiol. 17;1985:291–306.

4.        Karthikeyan, K, et al. Efficacy of grape seed proanthocyanidins on serum and heart tissue lipids in rats subjected to isoproterenol-induced myocardial injury. Vascul. Pharmacol. 47;2007:295–301.

5.        Rajadurai, M, Prince, PSM. Preventive effect of naringin on lipid peroxides and antioxidants in isoproterenol-induced cardiotoxicity in Wistar rats: biochemical and histopathological evidences. Toxicology. 228;2006: 259–268.

6.        Chaudhary US, et al. Effect of water extract of the bark of Buchanania lanzan linn. On behaviour and chromatophores of a fresh water fish, Labeo rohita. J Environ Biol. 22(3);2001:229-31.

7.        Sengupta A, Roychoudhury SK. Triglyceride composition of Buchanania lanzan seed oil. J Sci Food Agric. 28(5);1977:463- 8.

8.        Kirtikar KR and Basu BD. Indian Medicinal Plants: Lalit Mohan Basu, Allahabad, 1964-1965.

9.        Dai Y, Antipruritic and antinociceptive effects of Chenopodium album L. in mice. J. Ethnopharmacol. 81(2);2002: 245-250.

10.     The wealth of india, A dictionary of Indian raw materials and industrial products raw materials. Vol-2: B, Revised edition, Publication and information directorate, CSIR hillside road, New delhi. 1989.

11.     Arya R, Myricetin 3’-Rhamnoside-3 galactoside from Buchanania lanzan (Anacardiaceae). Phytochemistry. 31(7);1992: 2569-2570,

12.     Ohkawa H, Ohishi N, Yagi K. Assay of lipid peroxidation in animal tissues by thiobarbituric acid reaction.  Anal Biochem.95(2); 1979:351-358.

13.     Sun M, Zigman S.  An improved spectrophotometric assay for SOD based on epinephrine auto-oxidation. Anal Biochem. 90 (1); 1978:81-89.

14.     Clairborne A. Catalase activity in: Greenwald R. CRS handbook of methods in oxygen radical research. CRS press.1985. p.283-284.

15.     Ellman GL. Tissue sulphydryl groups. Arch. Biochem. 82;1959:70-77.

16.     Bonting SL. Presence of enzyme system in mammalian tissues. Membrane and Ion Transport. London. Wiley. 1970. p. 257-63.

17.     Hjerten S, Pan H. Purification and characterization of two forms of a low affinity Ca2+ATPase from erythrocyte membranes. Biochim Biophys Acta. 728;1983:281-8.

18.     Ohnishi T, et al. A comparative study of plasma membrane Mg2+ATPase activities in normal, regenerating and malignant cells. Biochim Biophys Acta. 684;1982:67-74.

19.     Lowry OH, Rosenbrough NJ, Farr AC, Randell RJ. Protein measurement with folin-phenol reagent. J Biol Chem. 193;1975: 265-75.

20.     Joshi H, et al. In–Vitro Antioxidant Activity of Ethanolic Extract of Leaves of Buchanania lanzan Spreng. Research J. Pharm. and Tech. 4(6); 2011: 92-924.

21.     Patel V, et al. Cardioprotective effect of melatonin against isoproterenol induced myocardial infarction in rats: A biochemical, electrocardiographic and histoarchitectural evaluation. European Journal of Pharmacology. 2010; 644: 160–168




Received on 13.11.2011          Modified on 02.12.2011

Accepted on 13.12.2011         © RJPT All right reserved

Research J. Pharm. and Tech. 5(2): Feb. 2012; Page 263-266