The aerial parts of O. basilicum were purchased before flowering in June from local market. The botanical identification was made by Dr. F. Fathiazad (Department of Pharmacognosy). A voucher specimen was deposited at the herbarium of the Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.
The fresh leaves (500 g) were chopped up and extracted with ethanol (96%; 2 L × 4) by maceration at room temperature and the solvent was removed at 40°C using a rotary evaporator. A greenish residue weighing 9.2 g was obtained and kept in air tight bottle in a refrigerator until use.
Determination of total phenolic compound
The total content of phenolic compounds was determined with the Folin-Ciocalteau reagent . The extract samples (0.5 ml of different dilutions) were mixed with Folin-Ciocalteu reagent (5 ml, 1:10 diluted with distilled water) for 5 min and 4 ml aqueous Na2CO3 (1 M) were then added. The mixture was allowed to stand for 15 min and the phenols were determined by colorimetry at 765 nm. The standard curve was prepared by 0, 50, 100, 150, 200, and 250 mg/ml solutions of gallic acid in methanol:water (50:50, v/v). Total phenolic contents were calculated as gallic acid equivalent per gram of the extract from a calibration curve preaperd with 0, 50, 100, 150, 200, and 250 μg/ml solutions of gallic acid in methanol.
Determination of total flavonoid content
The content of flavonoids was determined using colorimetric aluminum chloride method . Briefly, 0.5 ml solution of the extract were mixed with 1.5 ml of methanol, 0.1 ml of 10% aluminum chloride, 0.1 ml of 1 M potassium acetate and 2.8 ml of distilled water, and were left at room temperature for 30 min. The absorbance of the reaction mixture was measured spectrophotometrically at 415 nm. Total flavonoids contents were calculated as quercetin from a calibration curve.
A Preliminary HPTLC (high-performance TLC) analysis of the extract was performed on silica gel 60 F254 HPTLC plate (Merck) with ethylacetate, methanol, and water in the ratio of 100:13.5:10 as mobile phase and the spots were detected under UV . Subsequently, quantitative analysis of rosmarinic acid as the major compound in the extract was performed using a fully automated CAMAG’s TLC scanner 3 coupled with an automatic TLC sampler 4, an automatic developing chamber 2, and a TLC Visualizer (CAMAG; Switzerland). The winCATS (Planar chromatography manager) software was used for analyzing the results of the scan and image of the plate.
Determination of in vitro total antioxidant activity
1,1-diphenyl-2-picryl-hydrazil (DPPH; Sigma-Aldrich Quı′mica S.A., Madrid, Spain) was used to determine the free radical-scavenging potential of the ethanolic extract. IC50 (50% inhibitory concentrations) of the extract was calculated versus MeOH as a negative control and rutin was used as a positive control . Briefly, a stock solution of the extract was prepared in MeOH to achieve the concentration of 1 mg/ml. Dilutions were made to obtain concentrations of 5 × 10-2, 5 × 10-3, 5 × 10-4, 5 × 10-5, 5 × 10-6 and 5 × 10-7 mg/ml. Two ml of each solution was added to 2 ml of DPPH solution (in MeOH; 80 g/ml). The absorbance was measured at 517 nm after 30 min of reaction at 25°C. The experiments were performed in duplicate and the average absorption was noted for each concentration.
Male albino wistar rats (260-280 g) were used in this study. Rats were housed at constant temperature (20 ± 1.8°C) and relative humidity (50 ± 10%) in standard polypropylene cages, six per cage, under a 12-h light/dark cycle, and were allowed food and water freely. This study was performed in accordance with the Guide for the Care and Use of Laboratory Animals of Tabriz University of Medical Sciences, Tabriz-Iran (National Institutes of Health publication No 85–23, revised 1985).
Induction of acute myocardial infarction
Isoproterenol (Sigma Co; USA) was dissolved in normal saline and injected subcutaneously to rats (100 mg/kg) for two consecutive days at an interval of 24 h to induce acute myocardial infarction. Animals were sacrificed 48 h after the first dose of isoproterenol.
The animals were randomized into 6 groups consisting of 6 rats each. Rats in group 1 (normal control) received a subcutaneous injection of normal saline (0.5 ml) and were left untreated for the whole period of the experiment. In group 2 (sham) rats received a subcutaneous injection of normal saline and O. basilicum extract was given orally (40 mg/kg) two times per day for two days. Rats in group 3 (isoproterenol group) received a subcutaneous injection of isoproterenol (100 mg/kg) for two consecutive days at an interval of 24 h and normal saline as vehicle (0.5 ml) was given orally concurrent with and 8 h after each isoproterenol injection. Rats in groups 4 to 6 received a subcutaneous injection of isoproterenol (100 mg/kg) for two consecutive days at an interval of 24 h, the extract was suspended in saline and given orally (10, 20 and 40 mg/kg) concurrent with and 8 h after each isoproterenol injection.
Animals were anaesthetized by intraperitoneal injection of ketamin, xylazin, and acepromazin mixture 48 h after the first dose of isoproterenol. When the rats no longer responded to external stimuli, the systemic arterial blood pressure was recorded from a catheter inserted into the left carotid artery. A standard limb lead II ECG was monitored continuously throughout the experimental period. The mean arterial blood pressure was calculated from the systolic and diastolic blood pressure traces. The heart rate was calculated from the ECG. To evaluate the cardiac left ventricular function, a Mikro Tip catheter transducer (Millar Instruments, INC) was advanced into the lumen of the left ventricle. This helped to measure the left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), maximum and minimum rates of developed left ventricular pressure (LVdP/dtmax and LVdP/dtmin) and the rate of pressure change at a fixed right ventricular pressure (LVdP/dt/P) . All the parameters were continuously recorded using a Powerlab system (AD Instruments, Australia).
After the hemodynamic measurements, the animals were killed by an overdose of the anesthetic and the hearts were removed and weighed. The wet tissue to body weight ratios was calculated to assess the degree of congestion.
The hearts were fixed in 10% buffered formalin. The tissues were embedded in paraffin, sectioned at 5 μm and stained with hematoxylin and eosin (H&E) for evaluation of histology, and Gomeri trichrome for distinguishing muscle and interstitial connective tissue. Myocardial fibrosis and necrosis was evaluated in each section of the heart tissue using a morphometric point-counting procedure . Two persons graded the histopathological changes as 1, 2, 3, and 4 for low, moderate, high, and intensive pathological changes, respectively.
Determination of lipid peroxidation in serum and myocardium
Malondialdehyde (MDA), a thiobarbiturate reactive substance, was measured as a marker for oxidative stress in serum and myocardial homogenates using a method prescribed by Satoh . The lipid peroxide expressed as nanomole MDA production per gram heart tissue and nanomole per milliliter serum, were measured spectrophotometrically.
Data were presented as mean ± sem. One-way-ANOVA was used to make comparisons between the groups. If the ANOVA analysis indicated significant differences, a Student–Newman–Keuls post test was performed to compare the mean values between the treatment groups and the control. Any differences between groups were considered significant at P < 0.05.