Protective effects of saffron (Crocus sativus) against lethal ventricular arrhythmias induced by heart reperfusion in rat: A potential anti-arrhythmic agent

Abstract

Context: Saffron (Crocus sativus L.) has been used as a cuisine spice in eastern and western societies for thousands of years. In traditional medicine, saffron is recommended for the treatment of various kinds of disorders including heart palpitations.

Objective: We investigated the hypothesis of the protective effect of saffron on lethal cardiac arrhythmias induced by heart ischemia-reperfusion in rat.

Materials and methods: Animals were divided into a control (CTL) group that received tap water, Saf50, Saf100 and Saf200 groups that were orally treated with aqueous extracts of saffron, at dosages of 50, 100 and 200 mg/kg/day, respectively, and amiodarone (Amio) group that orally received 30 mg/kg/day for seven days. On day 8, heart ischemia-reperfusion was induced by ligation and releasing of the left anterior descending coronary artery.

Results: During reperfusion, the numbers and durations of ventricular fibrillation (VF) decreased in all groups compared to the CTL group (p < 0.05). Ventricular tachycardia (VT)/VF numbers (3.2 ± 1.2), durations (4.9 ± 2.6) and also arrhythmia severity (1.9 ± 0.35) were decreased significantly in the Saf100 group versus CTL group values (18.4 ± 11.6, 52 ± 31 and 3.3 ± 0.3, respectively). The PR and QTcn intervals of ECG were significantly longer in the Saf200 group (p < 0.001 versus CTL). The other doses of saffron only significantly prolonged the QTcn interval.

Conclusion: The results suggest that pretreatment with saffron, especially at the dosage of 100 mg/kg/day, attenuates the susceptibility and incidence of fatal ventricular arrhythmia during the reperfusion period in the rat. This protective effect is apparently mediated through reduction of electrical conductivity and prolonging the action potential duration.

• Heart
• ischemia-reperfusion
• ventricular fibrillation
• ventricular tachycardia

Introduction

In many cases, restoration of coronary flow by spontaneous recanalization, primary percutaneous coronary intervention (PCI) or thrombolysis is accompanied by reperfusion-induced lethal cardiac arrhythmias. Previous studies reported that sustained ventricular tachycardia or fibrillation (VT/VF) occurred in more than 5% of patients with ST-elevation myocardial infarction (STEMI) undergoing primary PCI (Mehta et al., 2009, 2012) and the occurrence of VT/VF was associated with increased 90-day mortality (Mehta et al., 2009). Reperfusion-induced arrhythmias were also observed in 30% of patients with successful reperfusion by streptokinase thrombolysis (Buckingham et al., 1986). In addition, reperfusion arrhythmia can be a major cause of sudden cardiac death. Available anti-arrhythmic drugs have been associated with adverse effects that their safety has been questioned in many clinical cases. For example, the major side effects of class 2 (β-adrenergic receptor blockers) and class 4 (calcium channel blockers) in patients with coronary heart disease are hypotension and bradycardia. Increase of re-entrant circuits due to slowing conduction velocity can account for class 1 (Na⁺ channel blockers) of anti-arrhythmic drugs. Finally, torsades de pointes, a serious cardiac arrhythmia and other side effects may be associated with administration of class 3 anti-arrhythmic drugs (Chabner & Knollman, 2011). Hence, the need to discover new effective and safe anti-arrhythmic drugs is inevitable. The use of dried stigmas of Crocus sativus L. (saffron) as a seasoning for some foods in western and eastern societies and also for therapeutic purposes in folk medicine is well known (Rios et al., 1996). Saffron has been recommended in traditional medicine to treat hypertension, arteriosclerosis and heart attack (Lokhande et al., 2006). Avicenna, the great Iranian physician (980–1037 A.D.), advised the use of saffron for the treatment of heart palpitations (Chishti, 1991). The cardioprotective effect of saffron and one of its constituents, crocin, in a stressful condition following the injection of high dose of isoproterenol has been confirmed in recent studies (Goyal et al., 2010; Joukar et al., 2010; Sachdeva et al., 2010). In addition, the attenuation effect of saffron on ischemic-reperfusion injury of skeletal muscle (Hosseinzadeh et al., 2009), kidney (Hosseinzadeh et al., 2005) and ischemic brain tissue injury (Vakili et al., 2011) is reported. The effect of saffron on electrophysiological aspects such as slowing the electrical conductivity of the heart, long QT interval (Joukar, 2012) and increasing the Wenckebach block cycle length and nodal functional refractory period in vitro (Khori et al., 2011), was also documented.

Considering the popularity and therapeutic properties of saffron as a known herb in traditional healing and recently in modern medicine, particularly its cardiovascular effects, in the present study we have investigated the hypothesis of anti-arrhythmic effect of this spice and whether saffron is able to prevent or reduce the occurrence of ventricular arrhythmias induced by reperfusion in the rat heart.

Materials and methods

Saffron extract

Dried stigmas of saffron were prepared from the farms of Zarand (Kerman province, Iran), identified and confirmed by Dr A. A. Maghsoudi Moud, Department of Botany, Bahonar University of Kerman, Iran. A voucher specimen of the plant was deposited in the herbarium center, Faculty of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran. The stigmas were chopped and macerated in tap water for three days. Then the mixture was filtered and different concentrations of saffron solution were prepared separately (Joukar et al., 2010).

Animals

This study conformed with the national guidelines for conducting animal studies (Ethic committee permission No. 86/123KA-Kerman University of Medical Sciences) and was performed on male Wistar rats aged 3 months and weighing 250–350 g. Animals were randomized to five groups and housed in a temperature-controlled room and 12 h light–dark cycle. The control group (CTL) received 1 ml/kg of tap water by gavage for seven days. Saffron groups, Saf50, Saf100 and Saf200, were fed with the saffron extract at dosages of 50, 100 and 200 mg/ml/kg/day, respectively, for seven days. Finally, the Amio group, positive control group, orally was treated with 30 mg/ml/kg/day of amiodarone, a classic anti-arrhytmic drug, for seven days.

Heart ischemia-reperfusion (I/R) and parameter recording

On day 8, animals were anesthetized with sodium thiopental (50 mg/kg i.p.) (Joukar et al., 2012). The right common carotid artery was cannulated with a filled catheter (saline with 15 IU/ml heparin) and connected to a pressure transducer and a PowerLab analog to digital converter (AD Instruments, Australia) to record the heart rate (HR) and arterial blood pressure (BP). The trachea was cannulated and animals were artificially ventilated with room air at 50 strokes/min (stroke volume 0.8 ml/100 g of body weight) during chest surgery and ischemia-reperfusion protocol. The chest was opened by a left thoracotomy and the 5th and the 6th ribs were separated by a small retractor. The pericardium was opened and a 6.0 silk suture was placed under the left anterior descending coronary artery (LAD) at the level of the left atrial appendage. Time window for animal recovery from surgery was 15 min. Electrocardiogram (ECG) limb lead II and BP were continuously recorded. Animals with cardiac arrhythmia or with a sustained drop in mean arterial BP below 70 mmHg during the stabilization period were excluded from the study. Then, two ends of the suture were passed through a small plastic tube and myocardial ischemia was induced by pulling the tube and suture that were clamped and pressing together against the myocardium for 10 min. Ischemia was confirmed by myocardial color change and ST segment elevation of ECG. Reperfusion was initiated by releasing the clamp and removing the tube and tension on the suture and recording of BP and ECG were continued for 15 min (Krzemiński et al., 2010).

Measured and calculated parameters

The mean arterial pressure (MAP) was calculated by “MAP = Pd + (Ps − Pd)/3 formula”, where Pd is the diastolic arterial pressure and Ps is the systolic arterial pressure. Pressure-rate product (PRP), an indirect measure of myocardial oxygen demand, was determined as the product of the HR and MAP [(MAP × HR) × 1000⁻¹]. PR interval and QT interval of basal ECG in each group were determined by mean of 1 min ECG recorded strips. To obviate the dependence of QT interval on HR, corrected QT (QTc) intervals were measured using Bazett’s formula normalized as QTcn-B = QT/(RR/f)^1/2, where RR is R–R interval and f = 150 ms (Kmecova & Klimas, 2010). Ventricular arrhythmias during reperfusion were analyzed according to the Lambeth conventions and they were defined as premature ventricular beats (PVBs) or premature ventricular contraction (PVC); discrete and identifiable premature QRS complexes, salvo; two or three consecutive PVBs, VT; a run of four or more consecutive ventricular premature beats, VF; a signal where individual QRS deflections could not easily be distinguished from each other and where rate could no longer be measured (Walker et al., 1988). The episodes PVB, salvo, VT and VF were counted and the duration of each episode of VT and VF in seconds was measured. The number of episodes and durations of VT and VF were also summed. In addition, the severity of arrhythmias in different groups was presented quantitatively by a previous scoring system with some modifications (Thuc et al., 2011), which are defined as follows: 0, <10 PVCs; 1, ≥10 PVCs; 2, 1–5 episodes of VT; 3, >5 episodes of VT or 1 episode of VF; 4, 2–5 episodes of VF; 5, >5 episodes of VF.

Statistical analysis

The results were presented as mean ± S.E.M. Comparison of hemodynamic data (HR, BP and PRP) among different groups was performed using a one-way ANOVA and post hoc Tukey’s test. Hemodynamic differences within each group during the various stages of the experiment were determined using repeated measures of ANOVA and the Bonferroni test. Arrhythmia episodes, duration and scores and percentage of animals with VT/VF in each group were compared using nonparametric Kruskal–Wallis and Mann–Whitney U tests. p Value <0.05 was considered as statistically significant.

Results

Mortality and exclusion

One animal from the CTL group was excluded from the study due to MAP <70 mmHg during the stabilization period. Three of the remaining 12 rats of the CTL group (n = 9, surviving), 2 of 9 Saf50 group rats (n = 7, surviving), 1 of 10 Saf100 group rats (n = 9, surviving), 1 of 8 Saf200 group rats (n = 7, surviving) and 1 of 10 Amio group rats (n = 9, surviving) died during the experiment period from an episode of VF. Only data from surviving rats were further analyzed (n = 41).

Hemodynamic measurements

The basal systolic, diastolic and mean arterial pressure, just before the induction of ischemia, showed no significant difference among different animal groups. On the other hand, basal HR was lower in Amio groups (p < 0.01 compared with others groups) and in the Saf200 group (p < 0.01 versus Saf50, and p < 0.05 versus CTL and Saf100 groups) (Table 1). At the ischemia period, hemodynamic measurements showed a significant reduction in MAP, systolic and diastolic pressure (Figure 1) in all groups versus their related basal values. In this period, HR of the CTL group and PRP of all groups also reduced (p < 0.05 versus related basal values) (Table 1). During reperfusion, BP in all groups showed an ascending trend, but still, at the end of this period, MAP, systolic and diastolic pressure were significantly lower in CTL, Saf100 and Saf200 groups compared to their baseline values. In addition, at the end of the experiment, only the HR of the Amio group increased compared to its basal value (p < 0.01). In the reperfusion stage, high dosage of saffron consumption (Saf200) was associated with the lowest level of BP than other groups. Comparison between groups showed that at the end of the reperfusion, diastolic and mean blood pressures were significantly higher in the Saf50 group versus the Saf200 group (p < 0.01). In addition, systolic BP level was higher in the Saf50 group versus the Saf200 group (p < 0.05) and diastolic BP was significantly higher in the Saf50 group versus the Saf100 group (p < 0.05). HR was also significantly more in Saf50 versus all other groups except the Saf100 group. The highest PRP was observed in Saf50, which was significant compared to the other groups except the Amio group (Table 1).

Figure 1. The strips of arterial blood pressure and lead II of ECG that simultaneously were recorded from a rat of the CTL group during different periods of experiments. Normal blood pressure and normal sinus rhythm during the pre-ischemic stage (Basal, a), drop of blood pressure and normal sinus rhythm with ST elevation (↑) at the onset of ischemic stage (b) and severe drop of blood pressure and VT/VF rhythm at the beginning of the reperfusion stage (c).

Figure 2. Basal PR interval and QT interval as Bazett’s formula normalized (QTcn-B) in each animal groups. Saffron with the dose of 200 mg/kg and amiodarone significantly increased PR interval (a). All doses of saffron and amiodarone prolonged the QT interval but the effect of high dose of saffron (200 mg/kg) was more prominent and was similar to amiodarone. **p < 0.01, ***p < 0.01 compared to CTL group. †p < 0.05 compared to Saf50 and Saf100 groups. CTL: control group, Saf: saffron, Amio: amiodarone.

Table 1. Blood pressure, heart rate and PRP of all animal groups during periods of experiment.

	Basal					End ischemia					End reperfusion
Group	S (mmHg)	D (mmHg)	MAP (mmHg)	HR (beat/min)	PRP	S (mmHg)	D (mmHg)	MAP (mmHg)	HR (beat/min)	PRP	S (mmHg)	D (mmHg)	MAP (mmHg)	HR (beat/min)	PRP
CTL (n = 9)	117 ± 5	96 ± 5	103 ± 5	377 ± 12	39 ± 2	66 ± 10^aa	48 ± 8^aa	54 ± 8^aa	306 ± 25^a	16 ± 3^a	91 ± 8^aa	75 ± 7^a,b	80 ± 7^a	365 ± 7	26 ± 4
SAF50 (n = 7)	123 ± 7	104 ± 4	110 ± 5	420 ± 17	47 ± 3	80 ± 7^aa	61 ± 8^a	67 ± 8^a	327 ± 37	22 ± 4^ba	101 ± 5^g	88 ± 8^h,j	92 ± 6^j	443 ± 23^b,k,l	41 ± 4^b,m
SAF100 (n = 9)	114 ± 5	89 ± 7	97 ± 6	386 ± 17	38 ± 3	71 ± 9^a	48 ± 8^aa	56 ± 8^aa	337 ± 36	18 ± 2^a	84 ± 5^a	58 ± 5^a	67 ± 5^a	392 ± 12	26 ± 2
SAF200 (n = 7)	119 ± 10	98 ± 10	105 ± 10	323 ± 15^d,e	35 ± 4	55 ± 5^aa	35 ± 6^aa	42 ± 6^aa	325 ± 14	14 ± 2^a	68 ± 3^aa	52 ± 3^aa	57 ± 3^aa	358 ± 19	20 ± 1^e
AMIO (n = 9)	122 ± 5	99 ± 4	107 ± 5	302 ± 16^f	33 ± 3	65 ± 10^aa	48 ± 10^aa	54 ± 10^aa	365 ± 15	20 ± 5^a	88 ± 10	70 ± 9	76 ± 9^a	383 ± 11^aa	30 ± 4

Values are mean ± SEM. CTL: control, Saf: saffron, S: systolic pressure, D: diastolic pressure, MAP: mean arterial pressure, HR: heart rate, PRP: pressure-rate product/1000.

^aap < 0.01 versus its basal value.

^ap < 0.05 versus its basal value.

^bp < 0.05 versus its ischemic value.

^dp < 0.05 versus Saf100 and CTL groups.

^ep < 0.01 versus Saf50.

^fp < 0.01 versus all other groups except Saf200.

^gp < 0.05 versus Saf200.

^hp < 0.05 versus Saf100.

^jp < 0.01 versus Saf200.

^kp < 0.01 versus CTL and Saf200 groups.

^lp < 0.05 versus Amio group.

^mp < 0.05 versus Saf100 and CTL groups.

Basal ECG

Basal ECG of treated animal groups was associated with some alterations. PR interval significantly increased in the Amio group (p < 0.001versus CTL, p < 0.05 versus Saf50 and Saf100 groups) and the Saf200 group (p < 0.01versus CTL) (Figure 2a). Corrected QT (QTc), which is presented as Bazett’s formula normalized (QTcn-B), significantly increased in the Amio and Saf200 groups (p < 0.0001 versus CTL group) and also in Saf50 and Saf100 groups (p < 0.001 compared with CTL group) (Figure 2b).

Arrhythmias during reperfusion

Strip samples of ECG and arterial BP of an animal during pre-ischemia, at the onset of ischemia and at the early stage of reperfusion are shown in Figure 1. The results showed a nonsignificant reduction of the incidence of PVB (PVC), salvo and VT in Amio and all saffron groups (Figure 3a–c). However, the incidence and duration of VF were reduced significantly in all tested groups compared to the CTL group (p < 0.05) (Figure 3e and f). In addition, a significant decrease in the incidence of VT/VF was observed in the Saf100 group (p < 0.05) (Figure 3g). Duration of VT and VT/VF was lower significantly in the Saf100 group whenever compared to the CTL group (p < 0.05 and p < 0.01, respectively) (Figure 3d and h). Duration of VT/VF was also reduced significantly in the Amio group (p < 0.05) (Figure 3h). The score of arrhythmia severity was attenuated in Amio and Saf100 compared with the CTL group (p < 0.05) (Figure 4a). The percent of animals showing episodes of VT/VF also reduced in Amio and Saf100 groups (Figure 4b).

Figure 3. Effects of different doses of saffron on reperfusion-induced ventricular arrhythmia in animal groups. Data are mean ± SEM (n = 7–9 for each group). Saffron (100 mg/kg) administration for seven days had no significant effect on the number of PVB (a), Salvo (b) and VT episodes (c) but reduced the duration of VT (d) and the total number and total duration of VT/VF episodes (g, h). All doses of saffron decreased the number and duration of VF as well as amiodarone (e, f). *p < 0.05, **p < 0.01 compared with CTL group. ▾ p < 0.05 versus other groups. PVB: premature ventricular beat, VT: ventricular tachycardia, VF: ventricular fibrillation, CTL: control group, Saf: saffron, Amio: amiodarone.

Figure 4. Score of arrhythmia severity and percent of animals that showed VT/VF in each experimental group. Data are mean ± SEM. (n = 7–9 for each group). Saffron (100 mg/kg) and amiodarone significantly decreased the score of arrhythmia severity (a) and percent of animals with VT/VF (b) versus CTL group. Scores were defined as: 0, <10 VPCs; 1, ≥10 VPCs; 2, 1–5 episodes of VT; 3, >5 episodes of VT or 1 episode of VF; 4, 2–5 episodes of VF; 5, >5 episodes of VF. *p < 0.05, compared with CTL group. CTL: control group, Saf: saffron, Amio: amiodarone.

Discussion

The present study demonstrated the anti-arrhythmic properties of saffron in an in vivo experimental model. Administration of the saffron extract at the dose of 100 mg/kg conferred more pronounced anti-arrhythmic effects and was comparable with the anti-arrhythmic effect of amiodarone. This effect was revealed as a decrease in the incidence, duration and severity of lethal cardiac arrhythmia and reduction in animals with VT/VF and mortality rate during reperfusion. In the Saf50 group, despite the better improvement in BP during the reperfusion period, the value of PRP, an index of myocardial oxygen demand, was higher than other groups. Increase in myocardial oxygen demand indicates increase of heart work. In stressful conditions, because of high cardiac workload, increase of heart damage and increase of reperfusion arrhythmias is more probable (Opie, 2004; Simonis et al., 2012). This may explain, at least a part, the greater susceptibility to ventricular arrhythmia in the Saf50 group than the Saf100 group during the reperfusion period as seen in this study. Recovery of BP along with lower PRP, lower cardiac work and hence lower cardiac injury in the Saf100 group can account for the more prominent anti-arrhythmic effects of 100 mg/kg dose of saffron. In addition, this dose of saffron was associated with fewer changes on basal ECG parameters including PR and QTc intervals versus amiodarone, which in turn is the other advantage. Amiodarone is able to block potassium and calcium channels; as well as antagonize beta-adrenergic receptors (Miller & Zipes, 2001). Its electrophysiologic effects as to other class 3 anti-arrhythmic drugs, through inhibition of potassium channels, are to prolong the action potential duration and effective refractory periods (Miller & Zipes, 2001) and hence prolongation of QT interval (Chabner & Knollman, 2011). Moreover, similar to class 4 anti-arrhythmic drugs, through inhibition of calcium channels, amiodarone can decrease the slope of pacemaker potential, reduction of HR and increase the PR interval (Torres et al., 1986; Winslow et al., 1990). Consistent with previous reports in our study amiodarone induced the PR and QTc interval prolongation that explains its anti-arrhythmic effect.

Electrophysiological effects of saffron with high dose (200 mg/kg) similar to amiodarone were revealed as PR and QTc interval prolongation. Low and intermediate doses of saffron (50 and 100 mg/kg) were only associated with QTc prolongation. Saffron caused slowing the electrical conductivity of the heart, long QT interval (Joukar, 2012) and increasing the Wenckebach block cycle length and nodal functional refractory period in the in vitro model (Khori et al., 2011). In agreement with previous findings, our study confirmed the depressant effect of saffron on atrio-ventricular conductivity as PR prolongation and lengthening of the refractory period as QTc prolongation in the in vivo model. On the other hand, it is documented that the extract of saffron has a potent inhibitory effect on the calcium channel of guinea-pig isolated heart (Boskabady et al., 2008). Inhibition of calcium channel can cause the reduction of the slope of pacemaker potential, and hence slowing the electrical conductivity of the heart. During the early seconds of reperfusion, action potentials heterogeneity may occur at the border and within the ischemic area. Heterogeneity in amplitudes and duration of action potentials of different cells within the ischemic and border zone can lead to the occurrence of re-entry phenomena (Coronel et al., 1992; Janse & Downar, 1977). Other mechanisms such as trigger activity, due to excess calcium entry into cells during ischemia, are also involved in ventricular arrhythmia during reperfusion (Kimura et al., 1984; Marban et al., 1990).

Anti-arrhythmic effects of saffron, which was seen in this study, could be due to its amiodarone-like effects. Saffron through prolonging the action potential and effective refractory period, seen as prolonged QT interval on ECG, may prevent the occurrence of the re-entry currents and hence lethal ventricular arrhythmias. On the other hand, regarding the inhibitory effect of saffron on calcium channels (Boskabady et al., 2008), similar to anti-arrhythmic drugs of class 4, it is possible that a part of its anti-arrhythmic effect prevents calcium entry into cells during heart ischemia.

The redox status of heart cells can affect its excitability. The shift of the cellular redox potential to increase oxidation can enhance action potential heterogeneity by modulating ion channels (Brown & O’Rourke, 2010). In addition, increased oxidation directly activates SarcKATP (sarcolemmal-ATP-dependent potassium) channels (Tokube et al., 1996, 1998) and changes the inactivation kinetics of l-type calcium channels (Belevych et al., 2009). SarcKATP channels opening reduces the action potential duration and effective refractory period (Aidonidis et al., 2009; Ferrier & Howlett, 2005) which in turn increases the incidence of cardiac arrhythmia (Billman, 2008; Billman et al., 2004). There is credible evidence that during reperfusion, production of oxygen free radicals increase in ischemic tissue (Bolli et al., 1989; Manning et al., 1988) and the use of ROS (reactive oxygen species) scavengers is associated with successful decrease in the reperfusion-induced arrhythmias (Cho et al., 2007; Hicks et al., 2007; Konya et al., 1992). Existing documents show the antioxidant properties of saffron (Asdaq & Inamdar, 2010; Hosseinzadeh et al., 2005). In a previous study, we demonstrated that pretreatment with saffron through augmentation of antioxidant levels decreased the isoproterenol-induced myocardial damage (Joukar et al., 2010). Therefore, a part of the anti-arrhythmic effects of saffron may be induced by its antioxidant features and stabilizing the redox balances in stressful condition. A possible reason for the diminution of anti-arrhythmia effect of high doses of saffron may be excess prolongation of the QT interval, which provides conditions for the occurrence of early afterdepolarizations, and hence increases the risk of reentry and a polymorphic dangerous ventricular tachycardia known as torsade de pointes (TdP) (Gupta et al., 2007).

Conclusion

In summary, our findings suggest that pretreatment with saffron 100 mg/kg provides a prominent protective effect against reperfusion-induced lethal cardiac arrhythmias in the rat. This beneficial effect may be mediated through increasing the effective refractory period of cardiac cells and prevention of redox imbalance. The potent anti-arrhythmic effect of saffron makes it possible to be a prospective therapeutic agent in clinical management of cardiac arrhythmia.

Declaration of interest

The authors declare that there are no conflicts of interest. Financial support of this study by Kerman University of Medical Sciences and Health Services of Iran (KUMS) is acknowledged. The data presented in this article are from a Master thesis (Elham Ghasemipour-Afshar) performed in the Department of Physiology and Physiology Research Center of KUMS.