Cardioprotective effect of Erythrina stricta leaves on isoproterenol-induced myocardial infarction in rat

The cardioprotective activity of Erythrina stricta leaves against isoproterenol- induced myocardial infarction was studied. Wistar albino rats were pretreated with leaf extract (200 mg/kg) daily for 28 days. After treatment, isoproterenol (8.5 mg/kg body weight, orally) was injected to rats at an interval of 24 hours for two days to induce myocardial injury. Cardioprotection was investigated by estimating the activities of serum aminotransferase , lactate dehydrogenase and creatinine kinase. Antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase, reduced glutathione and thiobarbituric acid reactive substances were determined. The activities of serum marker enzymes were increased significantly (p<0.05) in isoproterenol-induced rats. E. stricta leaf extract showed a decrease in serum enzyme levels and increase of antioxidant status. The results were confirmed by histopathological evidences. The present study concludes that E. stricta leaf extract has a prophylactic value in myocardial infarction.


Introduction
Acute myocardial infarction occurs when the blood supply to a part of the heart is interrupted.It is the leading cause of death all over the world.Isoproterenolinduced myocardial necrosis is a well established standard model to study the beneficial effect of drugs on cardiac dysfunction (Uma et al., 2008).Isoproterenol causes severe stress in myocardium and necrotic lesion in heart muscle.Loss of function and integrity of myocardial membranes are the outcomes of isoproterenolinduced myocardial injury involving change in membrane permeability alterations (Thippeswamy et al., 2009).
Erythrina stricta Roxb.(Papilionaceae), a medium sized deciduous tree used for various ailments in Indian system of medicine.The fresh juice of leaves used for the relief of earaches, toothaches, pain in joints, dysentery and antidote for snake bite.The plant contains alkaloids and non-alkaloidal constituents (Chopra, 1992).The leaves of the plant are found to possess rich anti-oxidant activity (Asokkumar et al., 2008).The present study was designed to evaluate the cardioprotective effect of ethanol extract of E. stricta leaves in isoproterenol-induced biochemical and histopathological changes using rats.adrenaline tartarate and tocopherol from S.D. Fine Chemicals Ltd.All chemicals were of analytical grade.

Animals
Wistar albino rats of either sex (150-200 g) were selected for the study.The animals were acclimatized to laboratory conditions for 5 days prior to experiments.They were housed in polypropylene cages and water ad libitum.The experimental protocol has been approved by the Institutional Animal Ethical Committee.

Plant material
The leaves of E. stricta were obtained from Kerala, India.The plant was authenticated by Mr. G.V.S. Murthy, Botanical Survey of India, Tamil Nadu Agricultural University Campus, Coimbatore and voucher specimen of the plant was kept in the college herbarium.

Plant extraction
E. stricta was shade dried and coarse powdered material (40 g) was extracted exhaustively with ethanol in a soxhlet apparatus.The extract was concentrated to a small volume using flash evaporator and further evaporated to dryness in a vacuum desiccator.The extracts obtained were in the form of thick paste due to the presence of resinous matter.Until the time of use the extract was stored in refrigerator.The yield of the extract was found to be 12% (w/w).

Acute toxicity study
Wistar albino rats (150-200 g) maintained under standard laboratory conditions was used.A total of five animals were used which received a single oral dose (2,000 mg/kg, body weight) of the extract.Animals were kept overnight fasting prior to drug administration.After the administration of the extract, food was withheld for further 3-4 hours.Animals were observed individually at least once during the first 30 min after dosing, periodically during the first 24 hours (with special attention during the first 4 hours) and daily thereafter for a period of 14 days.

Experimental protocol
Rats were divided into four groups of 6 animals each.Group I (control) rats were treated with 2% Tween 80 in water (10 mL/kg body weight, orally).Group II rats were treated with isoproterenol in 2% Tween 80 (10 mL/kg body weight orally).Group III rats were treated with E. stricta leaf extract (200 mg/kg body weight, orally) and Group IV rats were treated with αtocopherol (60 mg/kg body weight, orally).On 28th day, myocardial injury was induced to rats in Group II-IV by injection of isoproterenol (8.5 mg/kg body weight, subcutaneously) twice at an interval of 24 hours while normal control rats were given an equivalent volume of the vehicle (Prabhu et al., 2006;Rajadurai and Prince, 2005).
On completion of the experimental protocol, blood samples were collected by the retro-orbital plexus puncture method under light ether anesthesia and serum was separated by centrifugation and used for the biochemical estimations.The levels of creatine kinase, lactate dehydrogenase, serum glutamate oxaloacetate transaminase and uric acid were estimated.

Biochemical analysis
The activities of creatine kinase (CK), lactate dehydrogenase (LDH), and aminotransferase (AST) in serum were determined spectrophotometrically and the was measured at 340 nm and enzyme activity was expressed as U/L.Serum uric acid was measured at 546 nm.Animals were euthanized by phenobarbitone and hearts were removed, weighed and homogenized.The clear supernatant was used for of endogenous anti-oxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (Gpx), reduced glutathione (GSH) and thiobarbituric acid reactive substances (TBARS) by spectrophotometer (Budhwar and Kumar, 2005;Pari and Latha, 2004;Karthikeyan et al., 2003).

Histopathological studies
At the end of the study, all the rats were sacrificed by cervical decapitation and the hearts were dissected out, washed in ice cold saline.Then myocardial tissue was immediately fixed in 10% buffered neutral formalin solution.After fixation, tissues were embedded in paraffin and serial sections were cut and each section was stained with hematoxylin and eosin.The slides were examined under light microscope and photographs were taken.

Statistical analysis
Values are expressed as mean ± standard error mean (SEM) and analyzed using statistical package for social sciences (SPSS) version 10.0 using ANOVA followed by Dunnett's test.P values <0.05 were considered significant.

Results and Discussion
In acute toxicity study, it was found that the animals were safe up to a maximum dose of 2,000 mg/kg body weight.There were no changes in normal behavioral pattern and no signs and symptoms of toxicity and mortality were observed.
Isoproterenol-treated rats (Group II) exhibited significantly higher levels of serum myocardial injury marker enzymes such as serum CK, LDH and AST, uric acid levels when compared to control (Group I; Table I).Rat treated with E. stricta leaf extract (200 mg/kg) and αtocopherol (60 mg/kg) resulted in a significant decrease in those enzymes and levels of uric acid.The results indicate that E. stricta leaf extract has the tendency to reduce the elevated cardiac marker enzymes proving its cardioprotective effect.SOD and CAT are considered as primary enzymes since they are involved in the direct elimination of ROS.GSH also function as free radical scavenger in the repair of free radical induced biological damage.Decrease of these enzymes denotes the myocardial damage imposed by isoproterenol (Halliwell andGutteridge, 1985, Misra andFridovich, 1972).The level of these anti -oxidant enzymes such as CAT, GPx and SOD were found to be decreased after isoproterenol-induced myocardial infarction when compared with control (Table II).Rats pretreated with E. stricta leaf extract and α-tocopherol resulted in a significant elevation of the anti-oxidant enzymes in heart tissue.The level of GSH was found to be reduced in infracted rats.The rats treated with the extract and α-tocopherol showed an increase in levels of GSH when compared with isoproterenol-treated group and the results were found to be significant (p<0.01,p<0.05).The catecholamine-induced cardiotoxicity is due to the formation of free radicals and its oxidation products.Free radicals promoted lipid peroxidation by increasing membrane permeability ultimately leading to the development of MI (Trivedi et al., 2006).
In the present study, α-tocopherol was used as standard as it protects the myocardial membrane against the free radical damage by nature of its anti-oxidant ability (Velavan et al., 2009).
A significant elevation of tissue TBARS level was seen in isoproterenol-treated group.Administration of E. stricta leaf extract and α-toco-pherol for 28 days resulted in a significant reduction in TBARS and the levels were almost similar to normal control rats.It is well known that isoproterenol produces free radicals and these free radicals are involved in membrane damage, leading to elevated levels of TBARS (Ohkawa et al., 1979;Muralidharan et al., 2008).Treatment with E. stricta and α-tocopherol in isoproterenol-treated rats decreased the levels of TBARS and this action may be Values are expressed as mean ± SEM, n = 6 animals each; Group II was compared with Group I, III, and IV; a p<0.01, b p<0.05

Figure 1 :
Figure 1: Photomicrographs of heart sections of rat stained with hemotoxylin and eosin.A) Normal rats (Group I) showing normal myovcardial fibers and muscle bundles with normal architecture.B) Isoproterenol-treated rats (Group II) showing subendocardial infarction, atrophy, more diffuse esonophilia, edema and myocardial contraction band with vascular changes.C) Erythrina stricta leaf extract 200 mg/kg body weight treated rats (Group III) showing lesser necrosis and damage with normal architecture.D) Alphatocopherol-treated rats (Group IV) showing lesser focal myocarditis