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Extract of ligustrum leaves (Ligustrum delavayanum Hariot [Oleaceae]) is well known in traditional Chinese medicine. One of the active components, oleuropein, displays vasodilating and hypotensive effects.
To analyze the effect of 0.008% lyophilized extract of ligustrum dissolved in 0.5% ethanol on heart function.
Experiments were done on isolated rat hearts perfused by the Langendorff method in control conditions and during ischemic-reperfusion injury.
Application of ligustrum induced positive inotropic and vasodilating effects in spontaneously beating hearts. Pretreatment of the hearts with ligustrum reduced left ventricular diastolic pressure measured during reperfusion and improved left ventricular contraction compared with hearts without any pretreatment. Ligustrum significantly suppressed the incidence and duration of cardiac reperfusion arrhythmias, expressed as G-score, from 7.40±0.58 in nontreated rats to 1.97±0.50.
Application of ligustrum or ethanol alone induced changes in coordination between atria and ventricles during ischemia-reperfusion injury. The ‘g-score’, a new parameter summing the incidence and duration of atrioventricular blocks, atrioventricular dissociation and cardiac arrest, is introduced. The g-scores with ligustrum pretreatment were higher during ischemia than during reperfusion. Ethanol significantly depressed myocardial contractility and coronary flow, and nonsignificantly decreased heart rate of isolated rat hearts. Electrical changes observed during coronary reperfusion in the presence of ethanol were accompanied by deterioration of contractile function.
Ligustrum had a significant protective effect on rat myocardium against ischemic-reperfusion injury. Ethanol partially attenuated the protective effect of ligustrum.
Screening programs of medicinal plants with the potential for treatment of cardiovascular diseases are often guided by the literature about the use of medicinal plants in traditional Asian medicine (1). Empirical clinical data of a healing effect of olive leaves in the case of hypertensive disease stimulated the isolation of iridoid oleuropein. It was found that the oleuropein group of iridoid compounds is typical and selective for the Oleaceae (2). The genus Ligustrum belonging to this family is known in traditional Chinese medicine as an immunomodulator and anticancer drug. It has also been used as a tonic in geriatric patients (3). In 1960 Damirov (4) described the potentiation of frog heart contraction after application of ethanol extract of Ligustrum japonicum leaves. Later pharmacological analysis of oleuropein showed its hypotensive (5), coronarodilating (6) and antiarrhythmic (7) actions, and prolongation of myocardial action potential duration (6).
In the present study, the effect of the extract of Ligustrum delavayanum leaves on ischemia-reperfusion (I-R) injury was observed in isolated spontaneously beating rat hearts. Pretreatment with the ligustrum extract was found to have a significant protective effect that was partially attenuated by the ethanol vehicle alone.
All the experiments were performed in accordance with the Proclamation of the Ministry of Agriculture of the Slovak Republic (June 9, 1998) for the Care and Use of Laboratory Animals (Law No 231/1998 in the Collection of Laws of the Slovak Republic). Male Wistar rats (270 to 330 g) were anesthetized with diethyl-ether and heparinized (500 U intraperitoneally). A rapid thoracotomy was performed and the aorta was quickly cannulated in situ. After ligation of the aorta the heart was removed from the thorax and immediately mounted in the Langendorff apparatus. The hearts were perfused retrogradely with filtered Krebs-Henseleit solution containing, in mmol/L, NaCl 118.00, KCl 4.70, MgSO4 1.66, KH2PO4 1.18, CaCl2 2.00, NaHCO3 25.00 and glucose 11.00, saturated with 95% O2 plus 5% CO2 (pH 7.4±0.05, 37°C). All chemicals were pro analysi quality (Lachema, Czech Republic).
Perfusion flow rate (coronary flow) was set up to achieve a coronary perfusion pressure of 7.5 to 7.9 kPa (10 mmHg = 1.33 kPa) by a peristaltic pump (VD ČSAV, Czech Republic) and measured above the aortic cannula with a pressure transducer (LMP 160, Tesla, Czech Republic) and an electromanometer (LDP 186, Tesla). Coronary effluent was collected into the calibrating cylinder. Ventricular pressure was measured by a thin latex balloon connected to the transducer and the electromanometer. The balloon, inserted into the left ventricle, was inflated with water (0.05 to 0.10 mL) to generate a ventricular preload of 1.3 to 1.5 kPa. An injection port proximal to the aortic cannula was used for infusion of ligustrum extract by micropump (P-1, Pharmacia, Sweden) at a rate set to 10% of the coronary flow. The final concentration in the coronary perfusate was 0.008% lyophilized ligustrum leaf extract in 0.5% ethanol.
Heart rate was recorded through two stainless steel electrodes implanted in the left ventricular wall on an electrocardiograph (Chiracard 600T, Chirana, Slovak Republic).
Following cannulation, the hearts were stabilized for 20 min or until two consecutive measurements were within 0.5% of one another. The hearts were then perfused with either ligustrum or 0.5% ethanol alone for 20 min followed by 10 min stop-flow ischemia with superfusion of the sinoatrial node and 10 min reperfusion (8).
All functional data – heart rate, left ventricular pressure (LVP), coronary perfusion pressure and coronary flow – were monitored and recorded on an electrocardiograph at a paper speed of 25 mm/s. Left ventricular developed pressure (LVPS-D) was expressed as the difference between the maximal (S) and minimal (D) ventricular pressures. Coronary vascular resistance was calculated as coronary perfusion pressure divided by coronary flow. Baseline measurements were taken at the end of the stabilization period before pre-treatment with either ligustrum or ethanol alone. Other measurements were taken before ischemia, and at the end of the ischemia and reperfusion periods.
Reperfusion-induced ventricular arrhythmias were classified from the electrocardiogram according to the Lambeth Conventions (9). For quantifying cardiac rhythm disturbances, the arrhythmia G score according to Curtis and Walker (8) was used:
where VPBS is the number of episodes of ventricular premature beats (VPB), VFS is the total duration of ventricular fibrillation, VTn is the number of episodes of ventricular tachycardia and VFn is the number of episodes of ventricular fibrillation.
All data are presented as mean ± SEM and evaluated by Student’s t test. Statistical significance was determined at the P≤0.05 level.
The L delavayanum Hariot plants were collected in the Arboretum Mlyňany (Slovak Republic) and dried at the Faculty of Pharmacy in Bratislava. A 50 mL decoction was prepared with 5 g of dried plant leaves according to the Pharmacopoea Bohemoslovaca IV, 1987, Avicenum, Prague. After cooling and filtration, the water extract was lyophilized and subsequently sonnicated in 50% ethanol. This mixture was dissolved in a 1:10 dilution of Krebs-Henseleit solution and used as a stock solution for perfusion.
In the initial experiments, perfusion of rat hearts (n=5) with 0.008% lyophilized extract of ligustrum dissolved in 0.5% ethanol nonsignificantly reduced LVPS-D and coronary flow by 30% relative to the corresponding values for control hearts (n=6) (Table 1). Administration of 0.5% ethanol alone (n=5) significantly (P≤0.05) decreased the contractile performance of the myocardium and coronary flow, and increased coronary vascular resistance compared with control (Table 1).
During the pretreatment period ethanol alone slowed the heart rate nonsignificantly compared with control or ligustrum (Table 1), but the number of VPB increased from the one to three VPB/min observed for ligustrum-treated hearts to two to 12 VPB/min. To separate the action of the ligustrum extract, the corresponding data for 0.5% ethanol and for 0.008% lyophilized ligustrum extract in 0.5% ethanol were compared, shown in Table 1. It was found that lyophilized ligustrum extract compared with ethanol alone increased LVPS-D and coronary flow by nearly 14% and 35%, respectively, and decreased end diastolic LVP and coronary vascular resistance by nearly 36% and 42%, respectively.
Reperfusion of the ischemic myocardium is frequently accompanied by myocardial stunning and reperfusion-induced arrhythmias. Table 2 shows that I-R injury of myocardium results in increased coronary vascular resistance, significantly reduced LVPS-D and increased diastolic pressure, clearly indicating failure of cardiac function. I-R injury led to considerable ventricular ectopic activity commencing within 30 s of coronary reflow. The time course and distribution of arrhythmias are illustrated in Figure 1. All hearts exhibited periods of ventricular tachycardia or ventricular fibrillation, rarely interrupted by ectopic beats. In four of six hearts, ventricular fibrillation persisted till the end of reperfusion. Ventricular arrhythmias that occurred during reperfusion gave a G-score of 7.40±0.58.
Because of the cardiotonic effect of ligustrum found in the isolated heart, its effect was tested during I-R injury. Following ligustrum administration, left ventricular contractility during reperfusion was significantly improved (Table 2) over the control. The heart rate during the ischemic period was reduced from 215±29 beats/min in control ischemia to 185±24 beats/min. Ventricular ectopic activities alternated with cardiac arrest in two of five hearts. The severity of the reperfusion-induced arrhythmia was much less pronounced in hearts pretreated with ligustrum (Figure 2). Fewer episodes of ventricular tachycardia or fibrillation terminated spontaneously before the end of the reperfusion period. The G-score decreased substantially to 1.97±0.50 compared with I-R injury with no pretreatment (Figure 3).
In the rat hearts treated with ethanol alone, reperfusion resulted in simultaneous significant suppression of LVPS-D and increase of diastolic ventricular pressure (Table 2), closer to the changes observed during reperfusion with no premedication. In this group, reduced coronary flow and higher coronary flow resistance compared with ligustrum pretreated rats were observed (Table 2). Heart rate fell to 90±10 beats/min, and the incidence and duration of the conduction blockade and of cardiac arrest appeared to increase during both ischemia and reperfusion. The distribution and time course of reperfusion-induced ventricular tachycardia and fibrillation were clearly reduced (Figure 4), as was the G-score (Figure 3).
Application of either lyophilized ligustrum extract in 0.5% ethanol or 0.5% ethanol alone induced changes in coordination between the atria and ventricles during I-R injury. Tachyarrhythmias (summed as G-score) combined with depressed conduction of the electrical signal led to quantification of these ‘slowing actions’ on the cardiac rhythm. A new parameter was derived, termed the ‘g-score’, a summation of the incidence and duration of atrioventricular blocks, atrioventricular dissociation and cardiac arrest, similar to the G-score:
where B[s] is the total duration of conduction blockades (blockade defined as an eventual absence of QRS), CA[s] is the total duration of cardiac arrest (arrest defined as a straight line in the electrocardiogram), DIS[n] is the number of episodes of atrioventricular dissociations (dissociation defined as a complete dissociation between atrial and ventricular excitation) and CA[n] is the number of episodes of cardiac arrest.
Calculated data (summarized in Figure 3) allowed quantitative comparison of the data on arrhythmias. Pretreatment of hearts with ligustrum induced rhythm changes calculated as the G- and g-scores during both the ischemic and the reperfusion periods. However, during I-R injury with no pretreatment, reperfusion-induced severe arrhythmias were observed, which provided the highest G-score. After pre-treatment with ligustrum the g-score during ischemia and reperfusion was 1.21±0.76 and 0.09±0.90, respectively (Figure 3). Pretreatment with 0.5% ethanol gave g-scores during ischemia and reperfusion of 1.54±0.98 and 0.012±0.001, respectively (Figure 3).
The effect of ligustrum leaf extract alone on I-R injury was estimated from corresponding data (showed in Table 2) obtained in the presence of lyophilized ligustrum extract in 0.5% ethanol and in the presence of 0.5% ethanol alone. It was found that ligustrum leaf extract improved left ventricular contractility by about 300%, reduced end diastolic pressure by about 44%, increased coronary flow by about 23% and reduced fibrillation severity during I-R injury of rat myocardium.
There is little information about the biological activity of plants from ligustrum species belonging to the Oleaceae family, the same genus as Oleum europaeum.
In the present study we have analyzed the biological effect of lyophilized water extract of L delavayanum Hariot leaves in isolated rat myocardium. The extract contains caffeinic acid derivatives (1.53%), total phenols (0.87%) and flavonoids (0.90%) (10). The hemodynamic effects of 0.008% lyophilized ligustrum leaf extract in 0.5% ethanol include depression of inotropy, chronotropy and dromotropy of rat myocardium. Occhiuto et al (6) described similar effects of a glyceroethanolic macerate of O europaeum leaves on mongrel dog hearts. The glyceroethanolic macerate (containing 30% ethanol) of O europaeum leaves selectively depressed conduction in atria and in His-Purkinje fibres, and decreased intraventricular conduction. Inhibition of conduction in sinoatrial and atrioventricular nodes resulted from prolongation of monophasic action potential duration and ventricular refractory periods. Negative inotropism for a 50% methanol extract of Leonurus cardiaca (Lamiaceae) was reported by Reuter and Diehl (11). Surprisingly they did not present any data about the activity of vehicle alone on the heart. In our study we showed that 0.5% ethanol significantly suppressed the contractile ability of myocardium and slowed the heart rate. These findings resemble the typical effect of higher doses of ethanol on the human cardiovascular system (12). Considering this, we tried to separate the effect of lyophilized ligustrum extract in 0.5% ethanol from the effect of vehicle alone. Comparisons of corresponding parameters disclosed positive effect of ligustrum extract on myocardial contraction, chronotropy and coronary vessel dilation.
Itoigawa et al (13) tested 16 flavonoids for their inotropic effect on guinea pig papillary muscle. The relative potency of the tested flavonoids showed the strongest inotropic response for quercetin. Because this native flavonoid was also detected in ligustrum leaves by mass spectroscopy (10) it may be hypothesized that quercetin may have a role in the cardiotonic activity of ligustrum leaves.
One of the characteristics of the ligustrum extract was the increase in coronary flow. The coronary dilating action of oleuropein, one of the iridoids present in the group of polyphenols, was described by Petkov and Manolov (7) in the isolated perfused rabbit heart. The vasodilating activity of oleuropein is similar to the clinically proven effect of coronary dilating drugs (crataemon).
The major findings of our study, performed on the rat model of myocardial ischemia and reperfusion, are that reperfusion-induced severe arrhythmias, especially ventricular fibrillation, were significantly suppressed by the ligustrum extract. In this regard, the cardioprotective effect of ligustrum leaves during I-R injury is of particular interest. It is generally accepted that free radicals generated at the time of reperfusion contribute substantially to damage of the myocardium during the reperfusion period (14). It is noteworthy that free radical scavenging activity is a well established feature of flavonoids (15,16), caffeine acid derivatives (17–19) and tyrozol (20,21), all of which were found in the ligustrum leaf extract. Identification of these compounds in ligustrum leaves allows speculation about the relation between protection of the heart during I-R injury and the antioxidant ability of these compounds.
In conclusion, our experiments show that ligustrum leaf extract not only induces positive cardiac and hemodynamic effects but also may effectively protect the heart against I-R injury.
The authors are grateful to I Zahradnik for critical comments and discussion during preparation of the manuscript. This study was supported in part by grants VEGA SR 1/7369/20, 1/8214/01 and 1/8220/01.