MYOCARDIAL POTENCY OF AN AQUEOUS EXTRACT OF HARUNGANA MADAGASCARIENSIS STEM BARK AGAINST ISOPROTERENOL-INDUCED MYOCARDIAL DAMAGE IN RATS

The present study was undertaken to evaluate the effects of Harungana madagascariensis on electrocardiographical, biochemical and histopathological changes in isoproterenol (ISO)-induced myocardial infarction in rats. Male Wistar albino rats were randomly divided and treated with the aqueous extract of Harungana madagascariensis stem bark (AEHM, 200 and 400 mg/kg per os), or normal saline or vitamin E for 7 days with concomitant administration of ISO (85 mg/kg, subcutaneously) on 8th and 9th days, at 24 h interval. The ISO injections to the rats caused cardiac dysfunction evidenced by a marked (P<0.01) elevation in ST-segment, a reduction in R wave amplitude (P<0.01), decrease in endogenous antioxidant reduced glutathione (GSH), increase in malondialdehyde (MDA), a lipid peroxidation marker, increase of cardiac marker enzymes lactate dehydrogenase (LDH), aspartate amino transferase (AST) and alanine amino transferase (ALT). All these changes in cardiac function as well as GSH, MDA and the enzymes (LDH, AST and ALT) were ameliorated when the rats were pretreated with AEHM. Additionally, the protective effects were strengthened by improved histopathological changes, which specify the protection of cardiomyocytes from the deleterious effects of ISO. This study demonstrates the cardioprotective effect of Harungana madagascariensis on isoproterenolinduced myocardial infarction in rats. The mechanism might be associated with the enhancement of antioxidant defense, reduction of lipid peroxydation and it is confirmed by amending electrocardiographic pattern, improvement of cardiac markers and less histopathological damages following ISO-induced myocardial infarction. It could provide experimental evidence to support the use of Harungana madagascariensis used in traditional medicine to treat cardiovascular disorders.


INTRODUCTION
According to the recent world health organization survey, an estimated 17.7 million people died from to cardiovascular disease (CVDs) in 2015, representing 31% of all global deaths. Of these deaths, an estimated 80% were due to myocardial infarctions (MI) and strokes 1 . According to the same survey, over three quarters of CVDs deaths occurred in low-and middleincome countries. MI is followed by several biochemical alterations, such as lipid peroxidation, free radical damage, hyperglycemia, hyperlipidemia, elevation in cardiac markers and pro-inflammatory cytokines leading to qualitative and quantitative alterations of myocardium 2 . Catecholamines at low concentrations are beneficial in regulating heart functions by exerting a positive inotropic action on the myocardium 3 , whereas high concentrations of catecholamines or chronic exposure to catecholamines over a prolonged period produces deleterious effects on the cardiovascular system 4 . Isoproterenol (ISO) is a ISSN: 2456- 8058 18 CODEN (USA): UJPRA3 synthetic catecholamine, a non-selective βadrenoreceptor agonist, which causes severe stress in the myocardium and produces infarct like lesions, when injected in rats 5 . The ISO model is a well standardized and most reliable model for assessing the cardioprotective activity of several drugs. Since its pathophysiological and morphological changes following ISO administration are comparable to those taking place in human MI 6 . Nowadays, a number of pharmacological interventions such as beta-blockers, angiotensine-converting enzyme inhibitors, antiplatelet agents, thrombolytics, calcium antagonists, nitrates, antioxidants have been shown to counteract the ill effect of myocardial ischemic injury and to reduce morbidity and mortality in patients with ischemic heart disease 7, 8 . However, their chronic usage is often associated with adverse effects 9 . Therefore, the development of new and safer drugs for the treatment and prevention of ischemic heart disease is still a major concern. There is increasing trend towards the application of herbal medicines to treat the cardiovascular diseases 10, 11 . Harungana madagascariensis is one of the most popular trees in the African traditional medicine system. It is used as an abortifacient and antiseptic, in the treatment of cardiovascular disorders, anemia, tuberculosis, fever, angina, diarrhea, dysentery, syphilis, gonorrhea, malaria, parasitic skin diseases and wounds, as a natural source of dermatological agents and cosmetics 12-16 . Its benefits have also been reported in liver diseases, diabetes, pancreatic and biliary problems 17, 18 . Biological studies on the barks or leaves of this plant revealed antihelminthiase properties 19 , anti-plasmodial 20 , antidiabetic 21 , antimicrobial activities 22 , analgesic and anti-inflammatory activities 23 . Some of the constituents and isolated compounds from H. madagascariensis includes flavonoids, alkaloids, saponins, terpenes, cardiac glycosides, and tannins 24 . A prenylated 1, 4anthraquinone isolated from the hexane extract of the stem-bark of H. madagascariensis possess alphaglucosidase inhibition and antioxidant activities 25 . In this context, an attempt has been made to investigate the effect of an aqueous extract of H. madagascariensis on maintaining the myocardial integrity in animals employing electrocardiographical, biochemical and histopathological parameters in ISO-induced myocardial infarction.

MATERIALS AND METHODS Plant material collection and extraction
Fresh H. madagascariensis stem barks were collected at Essezok, Mbalmayo (Center Region, Cameroon) in June 2016. The identification of the plant was done at the Cameroon National Herbarium where voucher sample were deposited under the registration number NO. 4224 HNC. Bark pieces were dried under room temperature and powdered with the help of electrical grinder. 500 g of powder was introduced into 3.5 L of distilled water and boiled for 20 minutes. The resulting decoction was filtered through Whatman paper No. 3 and further lyophilized. A crude brown extract powder (HM extract, 31.73g) was obtained, giving a yield of 6.35%.

Experimental animals
Male albino Wistar rats (150-200g) were obtained from the Animal House of the Faculty of Science at the University of Yaoundé I (Cameroon). They were kept at standard laboratory conditions under natural light and dark cycles, at constant room temperature (20±5°C) and were allowed to have standard food and tap water freely. This study was approved by the Cameroonian National Ethical Committee (Ref NO. FW-IRB00001954).

Drugs and chemicals
Isoproterenol hydrochloride was purchased from Sigma Aldrich, USA. Lactate deshydrogenase (LDH) kit for enzyme estimation was purchased from Hospitex Diagnostics. Aspartate amino transferase (AST) and alanine amino transferase (ALT) kits were from Fortress Diagnostics Biosystems. All chemicals used in the present study were of analytical grade.

Induction of experimental myocardial infarction
Isoproterenol was freshly dissolved in 0.9% saline and injected (85 mg/kg) subcutaneously to the rats for two successive days (on days 8th and 9 th respectively) at an interval of 24h. Animals were sacrificed 48h after the first injection of isoproterenol.

Experimental design
The animals were randomly divided into 7 groups consisting of 7 rats each. HM extract was dissolved in distilled water. Vitamin E was used as standard drug. Rats in group 1 (normal control) received distilled water (10 ml/kg) orally, for 9 days. Rats in group 2 (ISO control) received distilled water for 9 days and were injected isoproterenol (85 mg/kg, SC) on the 8th and 9 th days at an interval of 24 hour. Animals of groups 3 to 5 were pretreated with the aqueous extract of HM (200 and 400 mg/kg/day) or vitamin E (100 mg/kg/day) orally for 9 days and on the 8 th and 9 th days they received isoproterenol SC at an interval of 24 hour. Rats in groups 6 and 7 were treated with the aqueous extract of HM (400 mg/kg/day) and vitamin E (100 mg/kg/day) orally for 9 days and on the 8th and 9 th days they were injected saline (0.1ml/100g SC) at an interval of 24h. Changes in body weight in all groups were noted every 2 days during the experimental period.

Electrocardiogram measurement
Twenty four hours (24h) after the last administration of the drugs, the animals were anesthetized by intraperitoneal injection of urethane (15 mg/kg). Needle electrodes were inserted under the skin of the animals in lead II position. Electrocardiograh recordings were made using Biopac Student Lab Experiment system (BSL 3.7, USA).

Blood collection and assessment of cardiac hypertrophy
After recording the ECG, blood was collected from the abdominal aorta and allowed to clot for 1 h at room temperature. It serum was subsequently separated by centrifugation at 3000 rpm for 15 min at 4°C and stored at -20°C for biochemical assays. After the blood collection, the animals were euthanized. Their hearts were removed, rinsed in ice-cold normal saline and weighed. The wet heart weight to body weight ratio was calculated to assess the degree of myocardial weight gain.

Assay of cardiac marker enzymes
Activities of lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum were measured using commercial kits (from Hospitex Diagnostics for LDH and Fortress Diagnostics for AST and ALT).

Estimation of lipid peroxidation product and reduced gluthathione in myocardium
After weighing, the heart tissue was divided into two longitudinal parts. One part was homogenized in Mc Even physiological ice-cold solution (pH 7.4, 1:5 w/v). The homogenate was centrifuged at 3000 rpm for 30 min at 4°C and the supernatant was stored at -20°C for biochemical assays. Malondialdehyde (MDA), a thiobarbiturate reactive substance, was measured as a marker for oxidative stress in myocardial homogenates using trichloroacetic acid (TCA, 20%) and thiobarbituric acid (TBA, 0.67%) 26 . The level of reduced glutathione (GSH) was estimated as previously described 27 .

Histopathological examination
After weighing, the second part of the heart was fixed in 10% buffered formalin. The fixed tissues were embedded in paraffin, sectioned at 5 µm and stained with hematoxylin and eosin (Hand E). The sections were examined under a light microscope (Scientico STM-50) and photomicrographs were taken by a photomicroscope (Minisee 1.0) at x200 magnification.

Statistical analysis
Results are shown as mean ± SEM. The statistical comparisons among the groups were performed with tstudents test using Sigma Stat 3.5 statistical package. Mann Whitney post-test was employed to compare the mean values between the treated groups and the control. P-values less than 0.05 were considered as statistically significant.

Effect of Harungana madagascariensis on electrocardiogram
The Lead II electrocardiograms obtained from the animals are shown in Figure 1.  Figure 2A). However, treatment with all doses of H. madagascariensis resulted in a non-significant decrease in the ST-elevation as compared to the rats treated with isoproterenol alone ( Figure 2B).

Effects of Harungana madagascariensis on the heart weight to body weight ratio and body weight
The mean body weight of the rats at the end of the experiment in all experimental groups had no significant change ( Table 1). The heart weight and the ratio of heart weight to the body weight were increased significantly (P<0.05 and P<0.001 respectively) in ISO-administered groups when compared with control group. The extract of H. madagascariensis when given alone, significantly reduce the heart weight and the ratio of heart weight to body weight (P<0.001) as compared to the ISO-treated group.

Effect of Harungana madagascariensis on serum marker enzymes
As shown in Table 2, there was a significant rise observed in the levels of diagnostic marker enzymes (LDH (P<0.01), AST (P<0.05) and ALT (P<0.05)) in the serum of the ISO-treated rats. Pre-treatment with H. madagascariensis (200 and 400 mg/kg) as well as vitamin E (100 mg/kg) showed a significant reduction in the levels of all serum diagnostic marker enzymes compared to ISO group.

Effects of Harungana madagascariensis on lipid peroxidation and reduced glutathione level
To determine the lipid peroxidation, MDA levels were measured in myocardial homogenates. Heart MDA levels increased insignificantly in isoproterenol alone treated rats as compared to the control group (Table 3). Pre-treatment with the H. madagascariensis (200 and 400 mg/kg) extract induced a dose-dependent but nonsignificant decrease of MDA levels of myocardium. There was a significant (P<0.001) decrease in GSH level in the heart of ISO-treated rats as compared to the control group. Pre-treatment with H. madagascariensis (200 and 400 mg/kg) significantly increased (P<0.001) the myocardial GSH level. Heart tissues were stained with hematoxylin and eosin and visualized under light microscope at x200 magnification.

Histopathological examination of the cardiac tissue
In the control group, myocardial fibers were arranged regularly with clear striation, without any damage ( Figure 3A). Histopathological sections of the isoproterenol alone treated hearts displayed hypertrophy, degeneration of myocytes, infiltration of neutrophilic granulocytes and increased edematous inter-muscular space and myofibroblasts ( Figure 3B) myocardial injury evidenced by decreased myocytes degeneration as well as edema and minimal inflammation ( Figure 3C, D).

DISCUSSION
The purpose of this work was to evaluate the potential cardioprotective role of Harungana madagascariensis aqueous extract stem bark aqueous extract (AEHM) in isoproterenol-induced myocardial damage model in rats. ISO in high doses induces morphological and functional alterations in the heart which closely resembles local myocardial infarction-like pathological changes seen in human myocardial infarction 28 . It has been reported that auto-oxidation of excess catecholamines such as ISO results in free radical mediated peroxidation of membrane phospholipids and consequently leading to permeability changes in the myocardial membrane, intracellular calcium overload and irreversible damages 29 . Electrocardiogram (ECG) is considered the most important clinical tool for the diagnosis of myocardial infarction 30 . In the present study, subcutaneous injection of isoproterenol (85 mg/kg) for two consecutive days caused ST-segment elevation and Ramplitude depression. The elevated ST-segment reflects the potential difference in the boundary between ischemic and non-ischemic zones and a consequent loss of cell membrane function and the depressed R-amplitude might be due to the isoproterenol-induced myocardial edema 31 . Harungana madagascariensis (200 mg/kg) pre-treatment as well as vitamin E markedly inhibited isoproterenol-induced Ramplitude depression and amended the ST-segment elevation, indicating its protective effects on cell membrane function and electrical discharges.
In the present study, we have observed a significant increase in the heart weight and the ratio of heart weight to body weight in ISO-treated rats. The observed increase in the heart weight in ISO-induced rats might be due to the increased water content, edematous intramuscular space and extensive necrosis of cardiac muscle fibers followed by the invasion of damaged tissues by the inflammatory cells 31,32 . Pretreatment with the plant extract or vitamin E did not modify this increase. These results suggest that AEHM does not affect the gain or loss of weight of this organ. However, the short duration of the preventive treatment (seven days) could be responsible for the observed result. It would be wise to consider a longer duration in future experiments to better elucidate the effects of the plant extract on this parameter. Myocardium contains many diagnostic marker enzymes like lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Upon administration of isoproterenol, the oxygen demand of the heart increases with increase in ionotropic effect in the heart, resulting in prolonged ischemia and glucose deprivation. The cells are damaged with increased muscle contractility, which results in increasing the cell membranes permeability allowing cardiac enzymes to leak out into the bloodstream 33 . Increased activities of these marker enzymes in the serum are indicative of cellular damage and loss of functional integrity 34 . In the present study, the significant increase observed in the activities of LDH, AST, ALT in the serum of ISO-induced rats may be due to the leakage of them from the heart as a result of necrosis induced by ISO. The aqueous extract of H. madagascariensis seems to preserve the structural and functional integrity and/or permeability of the cardiac membrane and thus restricting the leakage of these indicative enzymes from the myocardium, as evident from the markedly blunted levels of these enzymes in the extract pre-treated groups when compared to the ISO-treatment group, thereby establishing the cardioprotective effect of the aqueous extract of H. madagascariensis. Malondialdehyde (MDA), a product of the reaction of polyunsaturated fatty acids with reactive oxygen species, is a biomarker of oxidative stress. Since the major constituents of biological membranes are lipids, their peroxidation can lead to cell damage and death 35  Glutathione (GSH) is a tripeptide which has a direct antioxidant function by reacting with superoxide radicals, peroxy radicals and singlet oxygen followed by the formation of oxidized GSH and other disulfides. It plays an important role in the regulation of variety of cell functions and in cell protection against free radical mediated injury 37, 38 . Depressed GSH levels may be associated with an enhanced protective mechanism to oxidative stress in myocardial infarction. In this study, ISO administration was found to reduce the levels of GSH. This observation concurs with several earlier findings 29,32,35 . Pre-treatment with H. madagascariensis (400 mg/kg) significantly improved the level of GSH. This points to the potential antioxidant and free radical scavenging activity of H. madagascariensis. In previous studies, H. madagascariensis has been described as an antioxidant and free radical scavenger 24, 39 . The current study shows the antioxidant activity of H. madagascariensis and endorses its cardioprotective effect mediated by its antioxidant effect in myocardium. Histopathological examination of myocardial tissue in the control rats illustrated clear integrity of the myocardial cell when compared to the hearts of ISO treated rats. ISO-induced rats showed separations of cardiac muscle fibers, edema and extensive infiltration of neutrophil granulocytes. Pretreatment with the aqueous extract of H. madagascariensis (200 and 400 mg/kg) considerably attenuated the edema, reduced inflammatory cell infiltration and preserved normal cardiac muscle fibers structure, further confirming the cardioprotective effect of H. madagascariensis.

CONCLUSION
In conclusion, our study reveals that pre-treatment of rats with the aqueous extract of H. madagascariensis exerts a remarkable protective potential against damages caused by isoproterenol-induced myocardial infarction. This cardioprotective effect could be associated with the enhancement of antioxidant defense, reduction of lipid peroxydation and is confirmed by amending electrocardiographic pattern, improvement of cardiac markers and less histopathological damage following isoproterenol-induced myocardial infarction. Although this study has provided a possible new therapeutic tool for myocardial infarction, more studies are required to elucidate the precise mechanism of H. madagascariensis in reversing the pathogenesis of myocardial infarction.