Comparative Antiarrhythmic and Anti-ischaemic Activity of some 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, , , , 5-hydroxyfhvone, , luteolin glycoside, , pratol, , 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 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, , 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 perfusion with alcohol fixed the perfusable myocar- to the base and the apex of the heart. Verapamil, dium and whitened its colour. The tridimensional lodocaine and flavones (0.25 mL of 10 mM solution in digitalized cardiac image obtained by reconstruction perfusion medium) were added to the perfusate within of the transverse sections showed that the nonper- 15 s, immediately before the reperfusion. 10 hearts fusable area extended subepicardially to the lateral were used in each of the control and drug-treated free wall of the left ventricle, while subendocardially it groups. included the whole left ventricle, even including the septum. Statistics. Student’s unpaired T-test was used to In the control animals, the percentage of the assess whether the differences between treatment ischaemic area was 32.34%. Pretreatment of the

10 PHYTOTHERAPY RESEARCH, VOL. 5, NO. 1, 1991 @ 1991 by John Wiley & Sons, Ltd. COMPARATIVE ACTIVITY OF SOME FLAVONES

____ Table 1. Effects of pretreatment with some flavones on pytressine-induced ECG alteration in guinea-pigs.

DRUG Hydroxylation Other mw Max. increase in Increase in ST time (s) Modifications HVAl substitution ST (mml Mean f SE Mean f SE T wave %b degreeC increased voltage Saline 6.4 f 1.69 26.6f 1.46 inversion 70 48 a a Verapamil 454 1.2 f 0.58 13.6 f 4.68 none 0 0 Lidocaine 234 6.0 f 0.31 27.0f 0.83 increased voltage 20 3 Flavone 222 4.0 f 0.90 26.2 f 2.00 increased voltage 60 4A 3-Hydroxy flavone 3 238 3.6 f 0.25 23.4f 1.80 increased voltage 60 3 5-Hydroxy a slightly increased flavone 5 238 2.6 f 0.10 24.6f 7.15 voltage 60 2 6-hydroxy flavone 6 238 3.0 f 0.30 26.4f 2.30 increased voltage 60 3 7-hydroxy flavone 7 238 4.0 f 0.60 25.0 f 1.90 increased voltage 60 4A 6,7-Di hydroxy flavone 6.7 254 3.0 f 0.80 22.6f 2.00 increased voltage 40 2 7.8-Dihydroxy flavone 7.8 254 4.0 f 0.80 25.6 f 1.20 increased voltage 60 48 a slightly increased Apigenin 5'7.4' 270 2.3 f 0.37 52.0 f 9.30 voltage 40 4A a slightly increased Apiin 5.4' 7-0-(apio-glu) 564 2.4 f 0.18 24.0f 8.02 voltage 20 4A a Rhoifolin 5.4' 7-0-(rha-glu) 578 1.7 f 0.48 24.2f 7.01 none 60 1 a Vitexin 5,7,4' 8-C-fi-O-gl u 432 5.6 f 1.07 55.0f 3.16 increased voltage 0 0 Pratol 7 4'-OMe 268 3.8 f 0.58 30.0f 6.32 increased voltage 60 48 a slightly increased Luteolin 5.7.3l.4' 286 2.4 f 0.24 29.0 f 11.22 voltage 40 48 a Luteolin-Gly 5,7,3' 4'-(glu) 448 2.0 f 0.30 20.2 f 0.90 increased voltage 20 3 Orientin 5.7,3'.4' 8-C-(glu) 448 4.5 f 2.00 46.4 f 12.38 increased voltage 20 4A 94'-OMe a increased voltage Diosmin 5,3' 7-04rha-g lu) 608 2.1 f 0.10 29.0f 7.73 negative 70 48 The drugs were administered at doses of 5 mM/kg i.v.; values are the means of 10 animals. a Significant differences from the control p < 0.05. Percentage of animals with arrhythmias. 'Arrhythmias classed according to Lown et a/. 1975. Degree, 0 = no ventricular ectopic beats; 1 = occasional, isolated ventricular premature beat (VPB); 2 = frequent VPB minor 30/h; 3 = multiform VPB; 4 = repetitive VPB (A, couplets, B, salvos); 5 = early VPB R/T.

guinea-pigs with the flavones significantly reduced the of VF and VT was 70%. Flavones in the perfusate ischaemic area. The decreasing order of inhibitory reduced the incidence and the severity of reperfusion potency was as follows: rhoifolin > apiin > vitexin > arrhythmias. In this experimental model, the antiar- orientin > 6-hydroxyflavone > 3-hydroxyflavone > rhythmic effect exhibited by apiin, vitexin, luteolin, 5-hydroxyflavone (Table 3, Fig. 1). From Table 3, it is luteolin-4'-glycoside, rhoifolin and 5-hydroxyflavone evident that the hydroxy group in the A ring is was at least that of pratol and of apigenin. No relevant to the effectiveness of the flavones in this antiarrhythmic effect were observed after diosmin test. treatment (Table 4).

In vitro experiments

During the 30min period of the ischaemic perfusion, DISCUSSION the severity of arrhythmias differed from experiment to experiment and ranged from sporadic VEBs to Some of the flavones studied showed a marked AVB. In one of the 10 control hearts VT was protective effect against experimental ischaemia. All observed. After reperfusion, however, the incidence flavones tested (including diosmin that showed the

0 1991 by John Wiley & Sons, Ltd. PHYTOTHERAPY RESEARCH, VOL. 5, NO. 1, 1991 11 F. OCCHIUTO, G. BUSA, S. RAGUSA AND A. DE PASQUALE

Table2. Effects of pretreatment with some Bavones on Table 4. Effects of some Bavones added to perfusate prior coronary-ligation reperfusion arrhythmia in anaes- the reperfusion in rat isolated heart. thetized guinea-pigs Treatment HVAR Treatment HVAR % degree' %a degreeb Saline 70 48 Saline 80 48 Verapamil 20 1 Verapamil 20 2 Lidocaine 40 2 Lidocaine 30 4A Flavone 60 4A Flavone 50 48 3-hydroxy flavone 40 4A 3-Hydroxy flavone 50 3 5-Hydroxy flavone 40 2 5-Hydroxy flavone 20 4A 6-Hydroxy flavone 60 3 6-Hydroxy flavone 50 3 7-Hydroxy flavone 60 4A 7-Hydroxy flavone 50 48 6.7-Dihydroxy flavone 30 4A 6,7-Dihydroxy flavone 50 3 7.8-Dihydroxy flavone 30 48 7.8-Dihydroxy flavone 60 3 Apigenin 70 3 Apigenin 60 3 Api in 20 1 Apiin 20 4A Rhoifolin 20 2 Rhoifolin '30 48 Vitexin 50 1 Vitexin 50 4A Pratol 70 3 Pratol 70 3 Luteolin 60 1 Luteolin 30 48 Luteolin-Gly 40 1 Luteolin-Gly 20 3 Orientin 50 3 Orientin 20 2 Diosmin 70 4B Diosmin 50 4A Values are the means of 10 hearts. The drugs were perfused at Values are means of 10 animals. Drugs were administered at doses Of 0.25 mL SOL. 10 mM. doses of 5 mM/kg. 'Arrythmias classified according to Table 1. 'Percentage of animals with arrhythmias. Arrythmias classified according to Table 1. and Crataegus oxyacantha L. (De Nunno, 1947; Circosta et al., 1984; Occhiuto et al., 1986; Costa et al., 1986). lowest activity) decreased the incidence and the Examination of structure activity relationships in each experimental model (Fig. importance of HVA indicated that the antiarrhythmic effect of flavones 2). The results obtained suggested that flavones are was related to the 2-phenylchromone structure. The responsible for the antiarrhythmic activity and number and position of the hydroxyl groups had a cardiovascular activity exhibited by herbal remedies marked effect on the ability of these compounds to used in both official and traditional medicine, e.g., inhibit HVA. The aglycones were less potent in their Harpagophytum procumbens D.C., Olea europaea L., antiarrhythmic and anti-ischaemic action than their corresponding glycosides. This suggests that the presence of a sugar moiety increased the antiar- Table 3. khaemic area (percentage of the total cross- rhythmic and anti-ischaemic activity of the flavones sectional area) after coronary ligation in guinea- studied here. The potency of these flavones with pigs. Effect of verapamil, lidocaine and flavones respect to inhibition of cardiac arrhythmias was (5 mM/kg i.V.) highest for the flavones that had a 5-hydroxy structure

Treatment a Area (mmzl in ring A and a 4'-hydroxy structure in ring B. Saline 32.34 f 3.20 Rhoifolin, vitexin, apiin, orientin and luteolin were Vera pam iI 7.93 f 2.50' among the most active compounds tested and some Lidocaine 31.12 f 3.60 are similar in potency to verapamil. Flavone 26.1 5 f 2.90 It is known that some flavonoids (such as ) 3-Hydroxy flavone 17.51 f 2.28' exert an inhibitory effect on Ca2+-uptake (by Ca2+ 5-Hydroxy flavone 24.27 f 4.30' channel blockade or by chelation), Ca2+-mobilization 6-Hydroxy flavone 11.85 f 2.80' (from extracellular, membrane or intracellular 7-Hydroxy flavone 32.43 f 3.10 6.7-Dihydroxy flavone 20.24 f 4.50 sources) or Ca*+-dependent functions (such as 7.8-Dihydroxy flavone 22.51 f 4.90 calmodulin or myosin) (Nishino et al., 1984). In Apigenin 22.91 f 3.70 addition it is known that during ischaemia and Apiin 10.29 f 2.18' reperfusion Ca2+-accumulation is responsible for Rhoifolin 9.35 f 2.40' ischaemic damage and for HVA (Bourdillon and Vitexin 15.41 f 2.95' Poole-Wilson, 1981). Consequently, the mechanism of Pratol 30.20 f 4.40 antiarrhythmic and anti-ischaemic action of the Luteolin 18.46 f 4.00' flavones studied might be mediated in part through Luteolin 4'-Gly 24.45 f 5.10 their ability to inhibit Ca2+ overload induced during Orientin 16.34 f 3.30' ischaemia, and particularly after reperfusion. Diosmin 26.52 3.80' f In conclusion, our study confirms earlier reports 'Statistically significant difference in the ischaemic area from the control group, calculated by Student's unpaired t-test; concerning flavone cardiovascular activity and might p < 0.05. Each value is the mean f SE of 10 animals. provide a stimulus for the investigation of the nature of their biochemical and electrophysiological mechan-

12 PHMOTHERAPY RESEARCH, VOL. 5, NO. 1, 1991 0 1991 by John Wiley & Sons, Ltd. COMPARATIVE ACTIVITY OF SOME FLAVONES

Figure 1. Tridimensional image obtained by computerized reconstruc- tion of the transverse sections of guinea-pig hearts perfused 15 min after coronary occlusion. The yellow area represents the nonperfusable ischaemic area. The effects of verapamil, rhoifolin and pratol at 5 mM/kg i.v. are shown.

Figure 2. Antiarrhythmic effect of some flavones on the three experimental models tested. A = HVAl (Model I): B = HVAR (model 111); C = HVAR (model 11).

@ 1991 by John Wiley & Sons, Ltd. PHYTOTHERAPY RESEARCH, VOL. 5, NO. 1, 1991 13 F. OCCHIUTO, G. BUSA, S. RAGUSA AND A. DE PASQUALE

Figure 2. (Continued.) ism of action. Particularly, our results suggest that and this could provide a basis for future drug flavone effects on the heart are structurally related development.

REFERENCES

Autodesk Inc. (1986). Autocad user reference manual. Activity Relationships pp. 471-480. Alan R. Liss, New York. Bourdillon, P. D. V., and Poole-Wilson, P. A. (1981). Effects of Harrigton, S. (1985). Computer Graphics-A programming ischemia and reperfusion on calcium exchange and Approach. McGraw-Hill. mechanical function in isolated rabbit myocardium. Lepran, I., Koltai M., Siegmund, W., and Szekeres, L. (1983). Cardiovasc. Res. 15, 121. Determination of the ischemic area in conscious Rats. J. Circosta, C., Occhiuto, F., Ragusa, S., Trovato, A., Tumino, G., Pharrnacol. Methods, 9,219-230. Briguglio, F., and De Pasquale, A. (1984). A drug used Lown, B., Calvert, A. F., Armington, R., Ryan M. (1975). in traditional medicine: Harpagophytum procumbens DC. Monitoring for serious arrhythmias and high risk of II. Cardiovascular activity. J. ethnopharmacol, 2, 359. sudden death. Circulation, 189, 51-52. Clark, C., Foreman, M. I., Kane, K. A., McDonald, F. M., and Nishino, H., Naito, E., Iwashima, A., Tonaka, K., Matsuura, T., Parratt, J. R. (1980). Coronary artery ligation in anes- Fujiki, H., Sugimura, T. (1984). Interaction between thetized rats as a method for the production of quercetin and Ca++ Calmodulin complex. Gann, 74, 311. experimental dysrhythmias and for the determination of Occhiuto, F., Circosta, C., Briguglio, F., Tommasini, A., and De infarct size. J. Pharmacol. Methods. 3, 357-368. Pasquale, A. (1986). Etude comparee de I'activit6 Costa, R., Occhiuto F., Circosta, C., Ragusa, S., Busa, G., cardiovasculaire de jeunes pousses de feuilles et de fleurs Briguglio, F., and Trovato, A. (1986). Etude comparee de de Crataegus oxyacantha L. 1. Activite electrique et tension I'activit6 cardiovasculaire de jeunes pousses, de feuilles et arterielle chez le rat. Plant. Med. Phytother. 20, 37. de fleurs de Crataegus oxyacantha L. 111. Action protectrice Occhiuto, F., Circosta, C., Costa, R., Briguglio, F., and sur la coeur isole de rat vis-a-vis des agents arythmogenes Tommasini, A (1986). Etude comparee de I'activite et dans les arythmies par reperfusions. Plant. Med. cardiovasculaire des pousses des feuilles et des fleurs de Phytother. 20, 115. Crataegus oxyacantha L. II. Action de preparations De Nunno, R. (1947). Rivista Clinica Medica, 47, 506. extractives et de pricipes actifs purs isoles sur le coeur Gabor, M. (1986). Anti-inflammatory and anti-allergic prop- isole de lapin. Plant. MBd. Phytother. 20, 52. erties of flavonoids. Plant Flavonoids in Biology and Medicine: Biochemical, Pharmacological, and Structure Received 16 January 1990; accepted (revised) 9 April 1990.

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