Rhizomes of C. dactylon were collected from the fields of Maragheh (Iran) in November and voucher samples were preserved for reference in the Herbarium of Department of Pharmacognosy, School of Pharmacy, Tabriz University of Medical Sciences. Powdered rhizomes (400 g) were extracted by maceration in MeOH/H2O (70:30, 3×1 liter) at room temperature for 3 days. The hydroalcoholic extracts were combined and concentrated in vacuo to yield 27 g dried extract. This hydroalcoholic extract was kept in refrigerator for all experiments.
The crude extract of C. dactylon rhizomes was screened for the presence of different classes of compounds using the standard methods by some modifications [17, 18]. Thin Layer Chromatography procedures were performed using precoated silicagel plates (Merck; GF254, 0.25 mm) to confirm the results of screenings . The following spray reagents were used for detection of respective classes of compounds: Neu's reagent for polyphenoles and flavonoids, Antimony trichloride in chloroform for steroidal saponins and sterols, Kedd reagent for cardiac glycosides, Dragendorrf's reagent for alkaloids, and 5% Ethanolic sodium hydroxide for anthraquinones [19, 20]. Since the dried extract contained mainly flavonoid glycosides, it was standardized in its flavonoid glycosides content (4.6%) by the reported method .
Characterization of fractions obtained from the total extract by HPLC
The dried total extract of C. dactylon rhizomes was fractionated by solid-phase extraction (SPE) method. The total extract (2 g) was dissolved in minimum possible volume of 20% of MeOH/H2O mixture and adsorbed on Sep-Pak (mega tube 10 g; Waters) cartridge and eluted by step gradient of MeOH/H2O mixtures (20%; 40%; 60%, 80; and 100%). The high content fractions (20% and 40%), based on weight, were characterized by HPLC finger print. The fractions dissolved in mobile phase and 20 μl of the samples were injected into the reverse phase column (C18). The mobile phase consisted of a gradient of 0–35% (over 35 min), 35–55% (over 15 mhn), and 55-0% (over 10 min) methanol in water (V/V) and was delivered at a flow rate of 1 ml/minute. The detection was performed at three wavelengths of 220, 280, and 350 nm.
Male Wistar rats (110–120 g; 4 weeks old) were used in this study. The animals were given food and water ad libitum. They were housed in the Animal House of Tabriz University of Medical Sciences at a controlled ambient temperature of 25 ± 2°C with 50 ± 10% relative humidity and with a 12-h light/12-h dark cycle. 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).
Animals were randomized into six groups of 10 rats in each. Group 1 (normal control) received an intraperitoneal (i.p) injection of physiological saline (0.5 ml) and untreated for the whole period of the experiment (4 weeks). Group 2 was injected (i.p) a single dose of monocrotaline (50 mg/kg; MCT group)  and two weeks later they were given 1 ml vehicle [0.5% carboxy methyl cellulose (CMC)] orally, twice daily. Groups 3 to 6 were injected the same dose of MCT and two weeks later they received digoxin (0.01 mg/kg) or 50, 100, and 200 mg/kg (in 1 ml) of hydroalcoholic extract of C. dactylon rhizomes orally, twice daily continued for fifteen days. The extracts were suspended in CMC (0.5%). At the end of the experiments (4 weeks), hemodynamic measurements were taken and then the animals were sacrificed under deep anesthesia, and their hearts and lungs were removed. To assess the direct cardiac effects of the extract, in the other sets of experiments, wistar rat hearts (n = 6) were removed and mounted on a Langendorf apparatus and were exposed to different concentrations of the extract. Left ventricular developed pressure (LVDP), Left ventricular end diastolic pressure (LVEDP), and heart rate (HR) were recorded continuously during the experiment.
Four weeks after MCT injection, the animals were anaesthetized with sodium pentobarbital (60 mg/kg; i.p). When the rats no longer responded to external stimuli, the trachea was cannulated for artificial respiration and systemic arterial blood pressure was recorded from a catheter inserted into the left carotid artery. A standard limb lead I ECG was monitored continuously throughout the experimental period. Mean Arterial Pressure (MAP) was calculated from the systolic and diastolic blood pressures trace. Heart rate (HR) was calculated from the ECG . After recording the above mentioned parameters, the thorax was opened and the right ventricle was punctured with a 23-gauge needle attached to a pressure transducer. After stabilization, right ventricular systolic (RVSP) and end diastolic (RVEDP) pressures were recorded over 5 to 10 min. RV dP/dtmax and RV dP/dt/P as two indices of myocardial contractility were also calculated from RVSP. All parameters were continuously recorded using Powerlab system (AD Instruments, Australia).
Following the hemodynamic measurements, animals were sacrificed by an overdose of pentobarbital. The hearts, lungs, livers, and kidneys were removed and weighed. Then, the tissues were cut into small pieces for drying at 55°C until a constant weight was reached. Wet to body weight ratios and wet to dry weight ratios of the tissues were calculated to assess the degree of the congestion.
Isolated heart perfusion
Rats were anaesthetized with sodium pentobarbital (60 mg/kg intraperitoneally) and given heparin sodium (300 IU). Hearts were rapidly excised and placed in ice-cold buffer and mounted on a constant pressure (100 mmHg) Langendorff-perfusion apparatus. They were perfused with modified Krebs-Henseleit bicarbonate buffer containing (in mM): NaCl 118.5, NaHCO3 25.0, KCl 4.8, MgSO4 1.2, KH2PO4 1.2, CaCl2 1.7 and D-glucose 12.0. All solutions were gassed with 95% O2/5% CO2 with pH maintained between 7.35 and 7.45 at 37°C. Temperature was continuously monitored by a thermo-probe inserted into the pulmonary artery and maintained between 36.5 and 37.5°C. A latex, fluidfilled, isovolumic balloon was introduced into the left ventricle through the left atrial appendage and connected to a pressure transducer (P-1000B; Narco Bio Instruments) and HR, LVDP, and LVEDP were recorded with a Narco Bio physiograph (MK-III-S, Narco Bio Systems, USA) . The hearts were allowed to stabilize for 30 minutes before any interventions. Krebs containing different concentrations of the extract was perfused for 1 min at 30 min intervals and maximum responses were recorded. The effects of the extract were expressed as percentage change from pre-perfusion control values.
Data were presented as mean ± SD. Comparisons between groups were made with Student's paired t-test or one way ANOVA as appropriate. If ANOVA analysis indicated significant differences, a Student-Newman-Keuls post test was performed to compare mean values between treatment groups and control. Differences between groups were considered significant at p < 0.05.