Comparative Antiarrhythmic and Anti-Ischaemic Activity of Some Flavones in the Guinea-Pig and Rat
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Comparative Antiarrhythmic and Anti-ischaemic Activity of some Flavones in the Guinea-pig and Rat F. Occhiuto,* G. Busa, S. Ragusat and A. De Pasquale Pharmaco Biological Department, School of Pharmacy, University of Messina, Messina, Italy t Institute of Pharmacology and Experimental Toxicology, University of Cagliari, Cagliari, Italy A comparative study of 16 flavones was carried out using 4 experimental models: 1, hyperkinetic ventricular arrhythmias induced by reperfusion in rat isolated heart; 2, arrhythmias following coronary artery ligation-reperfusion in anaesthetized guinea-pig; 3, pytressine arrhythmii by coronary spasm in anaes- thetized guinea-pig; and 4, anti-ischaemic activity using an ex uiuo perfusion heart technique after coronary occlusion in guinea-pig. The structure effect relationships of flavone, Ihydroxytlavone, &hydroxytlavone, 7-hydroxyflavone, 6,7-dihydroxytlavone, 7,8-dihydroxytlavone, apigenin, apiin, diosmin, 5-hydroxyfhvone, luteolin, luteolin glycoside, orientin, pratol, rhoifolin, viterdn, tested as antiarrhythmic and anti-ischaemic agents were compared to lidocaine and verapamil at equimolar doses. The results obtained demonstrated that most of these compounds exerted a protective effect against coronary-spasm and coronary-ligation induced ischaemic crisis, and against the ischaemic and the post-iscbaemic arrhythmias. The potencies were found to be structurally dependent for the flavones tested. Keywoh: flavones; isolated rat heart; coronary ligation; coronary spasm; reperfusion; ventricular ar- rhythmia; myocardial ischaemic area. INTRODUCTION In uiuo experiments Male guinea-pigs weighing 350- 500g, were anaesthetized with uretane (1 g/kg i.p.) and artificially ventilated with room air via a tracheal Flavonoids form a large class of plant-derived cannula attached to a Harvard Ventilator, Harvard 2-phenylchromone substances present in the diet and Apparatus, Chicago, IL, USA (50 strokedmin, which have low mammalian toxicity. They also exert 10 mL/stroke); the external jugular vein was cannu- many potentially beneficial actions at both organ and lated for the administration of drugs. Verapamil, biochemical levels. In recent years, an increasing lidocaine and flavones were injected at equimolar number of pharmacological effects have been ascribed doses of 5 mM/kg in saline 3 min before the coronary to the flavonoids. For example their anti- ligation and the infusion of pytressine into the inflammatory, anti-ulcer, antianginal, spasmolytic, external jugular vein. Control guinea-pigs were anti-allergic and antiviral activities (Gabor 1986) as treated in a similar manner but received vehicle only well as their significant hypotensive and cardiac effects (saline). We used 10 animals in each group, control (Occhiuto et al., 1986). and drugs-treated. We have studied the protective effect of some flavones on myocardial ischaemia and hyperkinetic Coronary spaslll--reperfusion arrhythmia. Myocardial ventricular arrhythmias by ischaemia (HVAI) and ischaemia by coronary spasm was induced with reperfusion (HVAR) in vivo and in vitro. These pytressine (Parke Davis) 2UI/kg i.v. by a short effects, following flavone administration, were com- infusion (10 s). The electrocardiogram (Battaglia pared to those induced by verapamil and lidocaine, Rangoni app., Mod. Simplex a-r) of lead I1 and injected at equimolar doses as standard drugs. augmented voltage left (AVL) was recorded con- tinuously for 60 min before the infusion of pytressine. MATERIALS AND METHODS The following parameters were monitored during the period of ischaemia and after resolution of the Drug. The pure flavones, flavone, 3-hydroxyflavone, coronary spasm: ST segment elevation (height (mm) 5-hydroxytlavone, 6-hydroxyflavone, 6,7-dihydroxy- duration (s)); voltage increase or inversion of T wave; flavone, 7,8-dihydroxyflavone, apigenin, apiin, rhoifo- and arrhythmias (incidence, importance and dura- lin, vitexin, pratol, luteolin, luteolin glycoside, tion). The importance of arrhythmias was quantified, orientin and diosmin used in this study were obtained according to Lown et al. (1975). from the laboratoires SarsyntMse, Merignac, France. Coronary ligatiowreperfusion arrhythmia. Myocardial Verapamil hydrochloride was from Knoll AG (Liestal, ischaemia by coronary ligation was induced as Switzerland) and lidocaine hydrochloride was from K. described by Clark et al. (1980). After anaesthesia and Gulden S.p.A. (Italy). tracheotomy the thorax was opened at the fourth * Author to whom correspondence should be sent. intercostal space and a 5-0 atraumatic suture was 0951-418X/91/oooS-0014 $05.00 0 1991 by John Wiley & Sons, Ltd. PHMOTHERAPY RESEARCH, VOL. 5, NO. 1, 1991 9 F. OCCHIUTO, G. BUSA, S. RAGUSA AND A. DE PASQUALE placed around the left coronary artery. After 15min means and their controls were statistically significant. of ligation, the coronary artery was reperfused by All data are expressed as a mean of 10 animals. cutting the ligature. The incidence and the importance Means were considered significantly different if of hyperkinetic ventricular arrhythmias by ischemia p < 0.05. (HVAI) and by reperfusion (HVAR) was monitored by ECG recording in lead I or 111. Measurement of the ischaemic area. The measurement RESULTS of the ischaemic area was carried out using the method of Lepran et al. (1983) with some modifications. In separate experiments, hearts were In uiuo experiments excised 15 min after coronary ligation and perfused retrogradely with 5 mL of 4% formaldehyde in The incidence and the importance of the arrhythmias isotonic saline. The blood could be washed out of the are shown in Tables 1 and 2. In 7 out of 10 control normal myocardium, whereas the ischaemic area guinea-pigs, severe ischaemic dysrhythmias occurred remained dark red. The colour difference could be after the coronary-spasm and coronary-ligation. The further intensified by additional perfusion with 1mL most frequent arrhythmias was ventricular tachycardia of 70% ethanol. (VT), being aggravated to fibrillation (VF) in some A computerized graphic system (IBM Personal cases. After cutting of the ligature (reperfusion), VT Computer Model AT IBM S.p.A. (Rome, Italy), and VF were observed in 8 out of 10 guinea-pigs, but equipped with Professional Graphic Adapter, 15 x 11 no serious arrhythmias were observed after the end of cherry graphic tablet, Hewlett Packard 7470A plotter the coronary spasm. and Hewlett Packard laser jet 500 + printer) were Almost all flavones tested decreased the incidence used to quantify the cardiac ischaemic area. After and the importance of hyperkinetic arrhythmias fixation, the heart were sectioned in 10 transverse during both ischaemia and reperfusion. In particular, sections of equal thickness (2mm) with an LKB in the coronary spasm model (Table l), the highest ultratome 111 LKB (Bromma, Sweden). The seriated antiarrhythmic effect was obtained for vitexin sections obtained were photographed and stamped all (comparable to that observed after verapamil in equal magnification. Finally, the images of heart treatment) and rhoifolin, but were lower for diosmin sections were evaluated by computer-assisted plani- and pratol. The more active flavones had an hydroxyl metry analysis and slice thickness measurements to group at carbons 4’ and 5. The 7-0-glycosides (apiin; convert the area measurements to volumes, using the rhoifolin) and the 8-C-glycosides (vitexin; orientin) Autocad program from Autodesk (1986), version 2.64 were more active than the corresponding aglycones (Harrigton, 1985). The ischaemic area was expressed (apigenin; luteolin). as a percentage of the total cross-sectional area. As can be seen from Table 1, no significant changes were observed in the duration of the ST segment In uitro experiments elevation after pretreatment of the animals with the flavones, but the height of the ST segment was Hyperkinetic ventricular arrhythmias by reperfusion significantly reduced by apigenin, apiin, rhoifolin, (HVAR) in rat isolated heart. Male wistar rats, luteolin, luteolin-glycoside and diosmin. weighing 250-300 g were used. After anaesthesia with In the coronary ligation-reperfusion arrhythmia pentobarbital sodium (50 mg/kg i.p.), 500 U of model, vitexin, 5-hydroxyflavone, rhoifolin and heparin were injected intraperitoneally and the rat orientin showed a marked protective effect against the was killed by exsanguination. The heart was excized HVA ischaemia, but, against the reperfusion ar- immediately, mounted on a Langendorff’s apparatus, rhythmias, the order of antiarrhythmic potency was as and perfused with oxygenated Ringer-Locke solution follows: orientin > luteolin glycoside > apiin > at 37°C. Perfusion pressure was kept at 50mmHg rhoifolin > 5-hydroxyflavone > 3-hydroxyflavone > with a coronary flow of 8mL/min (basal perfusion). apigenin (Table 2). After stabilization, the heart was underperfused at a pressure of 8mmHg with a coronary flow of Determination of the ischaemic area. Because of the 0.5 mL/min (ischaemic perfusion); after 30 min of coronary ligation, the myocardial ischaemic area could ischaemic perfusion, the perfusion pressure was not be perfused with 4% formaldehyde but the normal brought back to basal condition (reperfusion). myocardium could. Therefore, in contrast to the light The incidence and the importance of the HVAR pink colour of the normal area, the ischaemic area occurring after reperfusion were recorded by an remained dark red with a sharp borderline. Additional electrocardiogram for 30 min, from the leads attached