Cardio protective effect of Momordica cymbalaria Fenzl against experimental Myocardial injury induced by Isoproterenol
R Koneri, R Balaraman, K Vinoth, Hariprasad
antioxidant, cardioprotection, isoproterenol, myocardial ischemia
R Koneri, R Balaraman, K Vinoth, Hariprasad. Cardio protective effect of Momordica cymbalaria Fenzl against experimental Myocardial injury induced by Isoproterenol. The Internet Journal of Pharmacology. 2007 Volume 5 Number 2.
The pretreatment with ethanolic extract of
Animals develop 'infarct-like' lesions when injected with isoproterenol (ISO), a potent synthetic catecholamine. These lesions are morphologically similar to those of 'coagulative myocytolysis' (COAM) or myofibrillar degeneration, one of the findings in acute myocardial infarction in man 1 . Diabetic patients are more vulnerable to myocardial damage resulting in heart failure than nondiabetic patients 2 .It is suggested that heart failure subsequent to myocardial infarction may be associated with an antioxidant deficit, as well as increased myocardial oxidative stress 3 . Higher serum enzyme concentrations of aspartate transaminase (AST) and creatinine kinase (CK) act as markers and are associated with higher incidence of stroke after acute myocardial infarction 4 . Elevated serum uric acid lactate dehydrogenase (LDH), creatinine kinase
Momordica cymbalaria Fenzl (MC) (Cucurbitaceae) is a species found in the states of Karnataka and Andra Pradesh, India. The tuber is traditionally used as an abortifacient and for the treatment of diabetes mellitus 7 . We have reported its antiovulatory and abortifacient activity 8 . Fruit powder and extract of Momordica cymbalaria (MC) are reported to have antidiabetic and antihyperlipidemic activies in experimental diabetic models 9,10,11 . An antidiabetic and antihyperlipidemic effect of the plant may have a better therapeutic effect in cardioprotection. Many herbal extracts 12,13,14,15,16 and formulations 17,18,19 have been reported to have cardioprotective activity. However the effects of MC on myocardial infarction and cardioprotection have not been reported.
In the present study an attempt has been made to elucidate the effect of MC on ISO induced cardiac damage with reference to biochemical markers, antioxidant enzymes, lipid profile and histology.
Materials And Method
Twenty four male Wister rats weighing 120-150 g were purchased from NIMHANS (National Institute of Mental Health and Neuro Science) Bangalore. The animals were housed in polypropylene cages maintained in controlled temperature (27 ± 2°C) and light cycle (12h light and 12 h dark).They were fed with standard rat pellet diet (Amrut rat and mice feed, Pranav agro industries Ltd. Sangli, India) and water ad libitum. The animals were given a week's time to get acclimatized with the laboratory conditions. All the animal procedures were performed according to the CPCSEA (Chennai, India) norms. The IAEC (Institutional Animal Ethics Committee) approved the experimental procedures.
The fresh roots of MC were collected from Gadag district, Karnataka. The powdered roots soxhlet extracted with ethyl alcohol to get a yield of 14.1% w/w. Dried extract dissolved in distilled water was used for the study. Oral acute toxicity study was performed using the up and down procedure (OPPTS guidelines) 8 .
The rats were divided into four groups. Group I: control (distilled water p.o ), Group II: ISO (60mg/kg, s.c.) at an interval of 24 hours for two days 19 . Group III and IV rats were treated with ethanolic extract of roots of MC at a dose of 250 and 500 mg/kg p.o. respectively for 45 days followed by ISO (60mg/kg, s.c.).
Marker Enzymes in serum
Twelve hours after the second injection of ISO, the animals were sacrificed by cervical decapitation, blood was collected and the heart was dissected out. The serum was separated immediately by cold centrifugation and used for determination of myocardial infarction marker enzymes such as lactate dehydrogenase (LDH), creatinine kinase
Oxidative Stress in Heart tissue
Heart was immediately washed with ice-cold saline and a homogenate was prepared in 0.1 N Tris HCL buffer (pH 7.4). The homogenate was centrifuged and supernatant was collected which was used for the assay of lipid Peroxidation (LPO), reduced glutathione (GSH), catalase (CAT), and super oxide dismutase (SOD).
Estimation of lipid peroxidation
The extent of lipid peroxidation in tissues was assessed by measuring the level of malondialdehyde (MDA) as described by Wilbur 20 . Briefly 1 ml of trichloroacetic acid (TCA) 20% and 2 ml of thiobarbituric acid (TBA) 0.67% were added to 2 ml of homogenate supernant. The absorbance of the mixture was recorded at 530 nm and the values were expressed as ?M of MDA formed /mg of protein
Estimation of reduced glutathione activity
Reduced glutathione (GSH) in the rat hearts was assayed by the method previously described by Ellman 21 .Briefly 0.02mlof the homogenate supernant was added to 3ml of Ellman reagent. The samples were mixed and kept at room temperature for at least 1 hour. The changes in absorbance were read at 412 ?m. The amount of glutathione was expressed as µg of GSH/mg protein.
Estimation of SOD activity
The level of SOD was measured by the method of Kono 22 . Briefly 1.3 ml of solution A (0.1 nM EDTA containing 50 nM Na2CO3, pH 10.3),0.5 ml of solution B (90 M) nitro blue tetrazolium dye(NBT)) and 0.1 ml of solution C (20mM Hydroxylamine hydrochloride, pH6.0) were mixed and the rate of NBT reduction was recorded for 1 minute at 560?m. SOD activity was expressed as unit/mg protein change in optical density per min.
Estimation of Catalase activity
Catalase activity was estimated by determining the decomposition of H2O2 at 240 ?m in an assay mixture containing phosphate buffer as described by Hug O E Aebi 23 . The activity was expressed in units as µM of H2O2 consumed per min/mg of protein.
A portion of the heart was fixed in formalin (10%) and subjected to histopathology studies. The section of the heart was processed and embedded in paraffin wax. Sections of about 4-6 µm were made and stained with hematoxylin and eosin and photographed.
Mortality in the acute toxicity test was not seen in the limit test at the dose of 5000mg/kg. Therefore 1/10 th and 1/20 th of the dose were selected for the study
There was a significant elevation in the serum levels of marker enzymes AST, ALT, LDH, ALP, CPK and uric acid in ISO group when compared to control group (table 1). MC pretreatment followed by ISO at both doses significantly reduced the levels of the marker enzymes as well as uric acid when compared to isoproterenol administered rats. The effect was more prominent at the higher dose.
The serum lipid parameters, total cholesterol, triglyceride, HDL, LDL, & VLDL showed a significant elevation upon ISO treatment. MC pretreatment significantly prevented the elevation of lipid levels when compared to ISO treated rats. The effect was more prominent in 500mg/kg dose group rats (Table 2).
The level of reduced glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD) in the myocardium homogenate showed a significant increase while the malondialdehyde (MDA) which is the measure of lipid peroxidation (LPO) showed a significant decrease in ISO treated rats. The pretreatment with MC at 500 mg/kg followed by ISO significantly (P<0.001) changed these oxidative marker levels when compared to ISO treated rats (Table 3).
The cardiac sections of the ISO control group revealed degenerative changes in the muscle fiber, showing a coagulative necrosis characterized by more homogenous esocinophillic cytoplasm. The nuclei of myofibril revealed pyknotic nucleus. Interstitial edema was present in the connective tissue spaces. Pretreatment with MC showed a protective effect with normal myofibrillar structures with striations at 500mg/kg dose(Fig1).
Values expressed as mean ±SEM, n=6
††† P<0.001, †† P<0.01, † P<0.05 considered statistically significant as compared to group I.
***P<0.001,**P<0.01,*P<05 considered statistically significant as compared to group II.
MC fruit powder and extract were previously reported to have antidiabetic activity 9,10,11 . Diabetes is associated with a marked increase in the risk of coronary heart disease. It is recommended that patients with diabetes who do not have myocardial infarction should be treated as aggressively for cardiovascular risk factors as patients who had myocardial infarctions 24 . Hence antidiabetic medications with additional cardiovascular protective activity will have greater therapeutic advantage.
Reactive oxygen species (ROS) are formed at an accelerated rate in ISO treated myocardium. Cardiac myocytes, endothelial cells, and infiltrating neutrophils contribute to this ROS production and can lead to cellular dysfunction and necrosis 25 . 'Infarct-like' lesions are produced in the myocardium when injected with isoproterenol. These lesions are morphologically similar to those of 'coagulative myocytolysis' (COAM) or myofibrillar degeneration 1 . Milei J etal 26 suggested that myocardial necrosis induced by isoproterenol is probably due to a primary action on the sarcolemmal membrane, followed by stimulation of adenylate cyclase, activation of calcium and sodium channels, exaggerated calcium inflow and excess of excitation-contraction coupling mechanism leading to energy consumption and cellular death.
Free radicals generated by ISO 27 , initiate lipid peroxidation of the membrane bound polyunsaturated fatty acids, leading to impairment of membrane structural and functional integrity. The metabolic damage of myocardium results in increase in the concentration of the marker enzymes like LDH, CK-MB, AST, ALP and uric acid. This concurs with our findings also. The GSH, CAT, and SOD were decreased while LPO increased in the myocardial homogenate of ISO administered rats indicating oxidative stress. Pretreatment of MC 500mg/kg pretreatment significantly prevented the changes of serum myocardial infarction marker enzymes along with uric acid and oxidative stress marker enzymes.
Myofilamental alterations such as myocytosis and myofibrillar degeneration were reported in ISO treated rats 19 Cardiac sections of the group II showed infiltration of inflammatory cells continuity in the muscle fiber was lacking suggesting an irreversible cell injury. Rats pretreated with MC showed normal myofibrillar structures with striations and revealed a marked protection by the extract against myocardial necrotic damage.
Administration of ISO raised LDL cholesterol and decreased HDL cholesterol level in the serum. An increase in concentration of total cholesterol and LDL cholesterol, and a decrease in HDL cholesterol is associated with raised risk of myocardial infarction 6 .High level of circulating cholesterol and its accumulation in heart tissue are well associated with cardiovascular damage. Pretreatment with MC elevated HDL level and decreased LDL cholesterol level. There is a growing body of evidence from epidemiologic, clinical, and laboratory data indicating that elevated triglyceride levels are an independent risk factor for cardiovascular disease. Hypertriglyceridemic patients at a risk for cardiovascular disease often develop a lipoprotein profile characterized by elevated triglyceride, dense LDL, and low HDL cholesterol which causes myocardial membrane damage 28 .Hypertriglyceridamia seen in ISO treated rats is a condition observed in ischemic heart disease. Pretreatment with MC has prevented the elevation of triglycerides Cholesterol and LDL in serum, signifying that the myocardial membrane is intact and not damaged. Antihyperlipidemic, antioxidant and antidiabetic activity along with cardioprotective properties of Momordica cymbalaria can have increased therapeutic advantage of the plant.
Raju Koneri PhD Professor of Pharmacology Visveswarapura Institute of Pharmaceutical sciences Banashankari, BANGALORE E mail firstname.lastname@example.org