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American Journal of Pharmacology and Toxicology
Year: 2009  |  Volume: 4  |  Issue: 2  |  Page No.: 29 - 37

Cardioprotective Effect of Cucumis trigonus Roxb on Isoproterenol-Induced Myocardial Infarction in Rat

B.S. Thippeswamy, S.P. Thakker, S. Tubachi, G.A. Kalyani, M.K. Netra, U. Patil, S. Desai, C.C. Gavimath and V.P. Veerapur    

Abstract: Problem statement: Cucumis trigonus Roxb fruit is used for various ailments in Indian traditional system of medicine such as anthelmintic, liver tonic, cardio tonic, appetizer, expectorant and intellect promoting. However, there is lack of information regarding the effect of Cucumis trigonus on the cardiac changes associated with ISO induced myocardial infarction. Approach: The present study was designed to evaluate the cardioprotective potential of ethanol extract of Cucumis Trigonus Roxb fruit (CTE) on the basis of electrocardiographic, biochemical and histopathological parameters in isoproterenol induced myocardial infarction in rats. Male albino sprague dawely rats were pretreated with CTE (75 and 150 mg kg-1) daily for a period of 14 days. After the treatment period, ISO (200 mg kg-1) was subcutaneously injected to rats at an interval of 24 h for two days to induce myocardial injury. After 48 h, rats were anaesthetized with anesthetic ether, electrocardiographic and the levels of biochemical and histological observations of the heart tissues were performed. Results: The activities of serum marker enzymes (ALT, AST, LDH and CPK) were increased significantly (p<0.05) in ISO-induced rats. In addition, it also exhibited Electrocardiographic (ECG) changes such as increase heart rate, reduced R-wave amplitude and ST-segment elevation. CTE at concentration of 150 mg kg-1, when administered orally showed a decrease in serum enzyme levels and the ECG changes brought to the near normal values. The observed results were further confirmed by histopathological findings. The histological sections obtained from ISO alone showed various degrees of focal lesions in many sections, consisting molted staining and fragmentation of muscle fibers with confluent retrogressive lesions were observed. Animals treated with CTE demonstrated marked improvement in ISO-induced alterations such as vacuolar changes, edema, capillary dilatation and leukocyte infiltration compared to ISO administered group. Conclusion: Our data showed that CTE (150 mg kg-1, p.o.) significantly restores most of the electrocardiographic, biochemical and histopathological parameters. The present study concluded that CTE may be therapeutic and prophylactic value in the treatment of myocardial infarction.

Group 1: Normal Control Rats treated with 1% Na CMC [~2 mL kg-1 day-1, p.o.]
Group 2: Rats treated with CTE (75 mg kg-1 day-1, p.o.)
Group 3: Rats treated with CTE (150 mg kg-1 day-1, p.o.)
Group 4: Rats treated with 1% Na CMC [~2 mL kg-1 day-1, p.o.] and then ISO (200 mg kg-1 day-1; for 2 days, s.c.)
Group 5: Rats pretreated with CTE (75 mg kg-1 day-1, p.o.) and then ISO (200 mg kg-1 day-1; for 2 days, s.c.)
Group 6: Rats pretreated with CTE (150 mg kg-1 day-1, p.o.) and then ISO (200 mg kg-1 day-1; for 2 days, s.c.)

Measurement of ECG: At the end of experimental period (after 24h of second ISO injection or 16th day of extract/vehicle treatment) the rats were anaesthetized with light anaesthetic ether and ECGs were recorded using computerized data acquisition system (Biopac MP 35, Santa Barbara, California). Recordings were made on the bi-polar standard lead-I, lead-II and lead- III. In all cases of myocardial infarction, Lead II show the clear, distinct individual waves than Lead I and III. Therefore, ECG was monitored on Lead II only.

Biochemical analysis: After recording the ECG, blood was collected from retro-orbital plexus, serum was separated and used for estimation of marker enzymes. The activities of aspertate Aminotransferase (AST) and alanine Aminotransferase (ALT) in serum were determined spectrophotometrically by the method of Mohur and Cook[14] and the absorbance was measured at 520 nm and enzyme activity was expressed as U L-1. The Lactate Dehydrogenase (LDH) activity in serum was assayed according to the method of King[15] and the absorbance was measured 520nm and the enzyme activity was expressed as U L-1. The Creatine Phosphokinase (CPK) activity in serum was determined by the method of Okinaka et al.[16] and the absorbance was measured at 640 nm and the enzyme activity was expressed as IU L-1.

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: The data were expressed as Mean ± SEM for six rats in each group. Statistical comparisons were performed by one-way ANOVA followed by Tukey’s post-test using GraphPad Prism version 4.0, USA.

RESULTS

Effect of cucumis trigonus roxb on different ECG parameters: Figure 1 shows the electrocardiographic pattern of control and experimental animals. Normal control and different doses of CTE (75 and 150 mg kg-1) alone treated rats showed a normal ECG pattern, where as animals treated with ISO alone showed significant elevation in ST segment, reduction in P wave, QRS complex and R-R interval. In addition there was an increase in heart rate, prolongation of QT interval and cardiac cycles compared to normal control animals. Pretreatment of CTE (75 and 150 mg kg-1) for 14 days and two doses of ISO (200 mg kg-1) administered rats exhibited normal ECG pattern with a slight elevation in ST segment. Furthermore, treatment also resulted in significant (p<0.001) increase in P wave, QRS complex and R-R interval, whereas heart rate, QT interval and cardiac cycle were maintained near to normal values. The data of the experimental animals such as P wave, QRS complex, QT interval, R-R interval, heart rate and cardiac cycle are shown in Table 1.

Effect of cucumis trigonus roxb on serum marker enzymes: ISO treated rats exhibited significantly (p<0.001) higher levels of serum myocardial injury marker enzymes such as AST (405±14.2 U L-1), ALT (131.6±9.78 U L-1), LDH (3310±160.7 U L-1) and CPK (1173±18.5 IU L-1) compared to normal control rats (Table 2).


Fig. 1: Effect of ethanol extract of Cucumis trigonus (CTE) on electrocardiographic pattern of (a): Normal control; (b): CTE (75 mg kg-1); (c): CTE (150 mg kg-1); (d): Isoproterenol (ISO: 200 mg kg-1) alone; (e): ISO+CTE (75 mg kg-1) and (f): ISO+ CTE (150 mg kg-1) in rats

Table 1: Effect of ethanol extract of Cucumis trigonus (CTE) on different ECG parameters in ISO-induced myocardial infarction in rats
Values are expressed as mean ± SEM for 6 animals in each group. The ECG parameters are expressed in seconds (sec) and the heart rate as Beats Per Minute (BPM). a: p<0.05, b: p<0.01 when compared with ISO group

Fig. 2: Effect of ethanol extract of Cucumis trigonus (CTE) on histopathological changes in rat myocardial tissue [H and E stain, X100]. (a): Normal control showing normal myocardium (b): CTE (75 mg kg-1) showing normal myocardium; (c): CTE (150 mg kg-1) showing normal myocardium (d): Isoproterenol (ISO: 200 mg kg-1) alone showing fragmentation of myocardial fibers and greater focal interstitial inflammatory response (e): ISO + CTE (75 mg kg-1) showing reduced focal interstitial inflammatory response (f): ISO + CTE (150 mg kg-1) showing reduced fragmentation of myocardial fibers and focal interstitial inflammatory response

The pretreatment of CTE (150 mg kg-1) for 14 days and ISO (200 mg kg-1, for 2 days) administration showed significant (p<0.05; p<0.001) reduction in all the tested diagnostic markers. Whereas, lower dose of CTE (75 mg kg-1) showed significant (p<0.05; p<0.01) reduction only in LDH and CPK levels. However there was no change in any of these marker enzyme levels in CTE (75 and 150 mg kg-1) treated alone group as compared to normal control group (Table 2).

Histopathological findings: Histopathological examination of myocardial tissue obtained from normal control animals and animals treated with CTE depicted clear integrity of myocardial membrane and an infiltration of inflammatory cells were not seen in these experimental groups (Fig. 2a-c). The histological sections obtained from the hearts of animals receiving ISO alone (Fig. 2d) shows various degrees of focal lesions in many sections consisting of molten staining, fragmentation of muscle fibers with confluent retrogressive lesions were observed.


Table 2: Effect of ethanol extract of Cucumis trigonus (CTE) on different biochemical parameters in ISO-induced myocardial infarction in rats
Values are expressed as mean ± SEM for 6 animals in each group. a: p<0.05; b: p<0.001; c: p<0.001 when compared to ISO alone

In addition marked sequestering mucoid edema and vacuolar changes along with hyaline necrosis were clearly visible in ISO treated rats. Pretreatment with CTE (75 and 150 mg kg-1, respectively) demonstrated marked improvement in ISO-induced alterations such as vacuolar changes, edema, capillary dilatation and leukocyte infiltration compared to ISO administered group (Fig. 2e and f).

DISCUSSION

Isoproterenol is well known cardiotoxic agent due to its ability it will destruct myocardial cells. As a result of this, cytosolic enzymes such as Lactate Dehydrogenase (LDH), transaminases (ALT, AST) and Creatine Phosphokinase (CPK) were released into blood stream and serve as the diagnostic markers of myocardial tissue damage[17,18]. The amount of these cellular enzymes present in blood reflects the alterations in plasma membrane integrity and/or permeability. Drug treatments such as naringin, Silibinin and squalene evidenced by a decline in lactate dehydrogenase, glutamic oxalacetic transaminase and creatine kinase levels indicated their membrane stabilizing action[17,19,20].

In the present study, ISO treated rats showed significant elevation in the levels of these diagnostic marker enzymes (AST, ALT, LDH and CPK). Moreover, elevated levels of these enzymes are an indicator of the severity of ISO-induced myocardial membrane necrosis, which is in line with an earlier report[17,18]. The prior administration of CTE (75 and 150 mg kg-1) showed significant reduction in ISO-induced elevated serum marker enzymes. This reduction in enzyme levels could be due to its action on maintaining membrane integrity thereby restricting the leakage of these enzymes. It is well known that isoproterenol-induced myocardial injury is mediated primarily via the β1-adrenergic receptor. Acute β-adrenergic receptor stimulation not only rapidly generates reactive oxygen species, but also depresses total cellular antioxidant capacity, downregulates copper-zinc superoxide dismutase enzyme activity, protein and mRNA and reduces glutathione level, leading to the loss of membrane integrity and inducing heart contractile dysfunction and myocyte toxicity finally producing myocardial necrosis[21,22]. In the present study, we found that CTE protected myocardium from isoproterenol-induced myocardial functional and structural injury via normalization levels of diagnostic marker enzymes.

It is reported that chronic treatment of N-acetyl-cysteine, S-allylcysteine and α-tocopherol ameliorates ISO-induced myocardial toxicity, resulted in stabilization of the cardiac mitochondrial membrane and lysosomal enzymes[23,24]. Furthermore, methionine an essential aminoacid is used as a supplement in health food and its deficiency has been associated with a variety of cardiac and vascular changes[25]. The similar classes of chemical constituents were reported from the title plant and which is also confirmed by performing preliminary phytochemical analysis of CTE. The observed myocardial protective effect of title plant could be due to the free radical scavenging activity of the extract in the presence of phytochemicals such as sterols and amino acids like arginine, luecine, lysine, cysteine cystine, methionine and phenylalanine[8]. These data further confirmed the cardioprotective action of CTE.

Electrocardiograph-abnormalities are the main criteria generally used for the definite diagnosis of myocardial infarction. ST-segment elevation was observed either in patient with acute myocardial ischemia[26] or in isoproterenol-induced myocardial infarction in rat[27]. The study shows significant alterations of ECG patterns were observed in ISO administered rats as compared to normal control rats. The characteristic findings were reductions in the P wave intensity, QRS complex, R-R intervals, QT interval and prolongation of cardiac cycle. We also observed a significant elevation in the ST segment and increase in heart rate. These alterations could be due to the consecutive loss of cell membrane in injured myocardium[28]. In the present study, we observed an elevation of ST-segments in isoproterenol-induced rat and pretreatment with CTE markedly inhibited isoproterenol-induced ST-segment elevation suggestive of its cell membrane protecting effects. The appearance of Q wave and ST segment elevation are some of the indicative signs of ischemia. In the present study we did not observe pathological Q wave due to conditions of ischemia. The prominent Q wave were seen only on severe ischemia, infarction and in patients with severe heart diseases. The consecutive loss of cellular membrane damage due to oxidative stress might be characterized by ST elevation[28,29]. CTE administration showed a protective effect against ISO-induced altered ECG pattern and eliminated the acute fatal complications by protecting the cell membrane damage.

Electrocardiograhic and biochemical findings were further confirmed by histopathological studies. Histopathological examination of myocardial tissue in control depicted clear integrity of the myocardial cell membrane. No inflammatory cells infiltration was seen in the rat heart of normal control. In ISO administered group, focal lesions in many sections consisting of moltled staining and fragmentation of muscle fibres with confluent retrogressive lesions, hyaline necrosis, sequestering mucoid edema were observed. Pretreatment with CTE demonstrated reversal of focal lesions, fragmentation of muscle fibres and retrogressive lesions with hyaline necrosis seen with ISO treated group. Inflammatory cells were seen with reduced density in CTE treated group confirming further the cardioprotective activity exerted by CTE. CTE (150 mg kg-1)-treated normal rats had no toxic effects on cardiac architecture. These data further confirmed the cardioprotective action of oral administration of CTE. Higher dose of isoproterenol induce sub-endocardial ischemia, hypoxia, necrosis and finally fibroblastic hyperplasia with decreased myocardial compliance and inhibition of diastolic and systolic function, which closely resembles local myocardial infarction-like pathological changes seen in human myocardial infarction[30]. In the present study, we found that CTE protected myocardium from isoproterenol-induced myocardial functional and structural injury. The data of the present study clearly showed CTE modulated most of the electrophysiological, biochemical and histopathological parameters were maintained to normal status in isoproterenol rats, suggesting the beneficial action of CTE as a cardioprotective agent.

CONCLUSION

These findings might be rational to understand the beneficial effects of ethanol extract of C. trigonus on cardioprotection against myocardial injury. The fruit was found to be most effective in the functional recovery of the heart and restoration of biochemical and histopathological alterations. Further isolation, characterization and purification of the active constituents and further experimentation would be necessary to elucidate the exact mechanism of action of Cucumis trigonus fruits.

ACKNOWLEDGEMENT

We thank Prof. A.D. Taranalli, Head, Department of Pharmacology, K.L.E.S’s College of Pharmacy J.N.M.C; Nehru Nagar; Belgaum; Karnataka for providing necessary facilities and help during the study.

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