Volume 9, Issue 3 (Aug 2024)                   JNFS 2024, 9(3): 449-459 | Back to browse issues page

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Moazami M, Sedighi M, Rashidipour M, Haydari S, Ali Mahmoudi G, Ghaderpour S, et al . The Protective Effect of Ethanolic Extract of Olive Leaf on Aluminum Phosphide-Induced Cardiac Toxicity in Rats. JNFS 2024; 9 (3) :449-459
URL: http://jnfs.ssu.ac.ir/article-1-647-en.html
The Protective Effect of Ethanolic Extract of Olive Leaf on Aluminum Phosphide-Induced Cardiac Toxicity in Rats
Maryam Moazami , Mehrnoosh Sedighi , Marzieh Rashidipour , Sara Haydari , Ghafar Ali Mahmoudi , Saber Ghaderpour , Morteza Bakhshesh , Afshin Nazari , Maryam Moghimian *
Department of Physiology, School of Medicine, Gonabad University of Medical Sciences, Gonabad, Iran.
Keywords: Aluminum Compounds, Cardiotoxicity, Electrocardiography, Gastric lavage, Oxidative stress, Pesticides
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The Protective Effect of Ethanolic Extract of Olive Leaf on Aluminum Phosphide-Induced Cardiac Toxicity in Rats
Maryam Moazami; PhD1, Mehrnoosh Sedighi; PhD2, Marzieh Rashidipour; PhD3, Sara Haydari; PhD1, Ghafar Ali Mahmoudi; PhD4, Saber Ghaderpour; MSc5, Morteza Bakhshesh; PhD 6, Afshin Nazari; PhD2 & Maryam Moghimian; PhD*7
1 Razi Herbal Medicines Research Center, Lorestan University of Medical Sciences, Khorramabad, Iran; 2 Cardiovascular Research Center, Shahid Rahimi Hospital, Lorestan University of Medical Sciences, Khoramabad, Iran; 3 Nutritional Health Research Center, Lorestan University of Medical Sciences, Khoramabad, Iran; 4 Department of Internal Medicine, School of Medicine, Lorestan University of Medical Sciences, Khorramabad, Iran; 5 Department of Physiology, Tabriz Faculty of Medical Science, Tabriz University of Medical Sciences, Tabriz, Iran; 6 Molecular and Medicine Research Center, Khomein University of Medical Sciences, Khomein, Iran; 7 Department of Physiology, School of Medicine, Gonabad University of Medical Sciences, Gonabad, Iran.
ARTICLE INFO ABSTRACT
ORIGINAL ARTICLE Background: The purpose of this study is to evaluate a treatment for aluminum phosphide (ALP) poisoning, which is known as “rice tablet”. Methods: In the present study, the impact of different doses of ethanolic extract of olive leaves (100, 200, and 400 mg/kg) on ALP (120 mg/kg)-induced cardiotoxicity was evaluated in anesthetized-gastrotomized adult male Wistar rats. Thirty-five rats were randomly assigned into five groups (n=7) as follows: control (AC) and treatment groups [ALP+Olea100 (AO100), ALP+Olea 200 (AO200), and ALP+Olea 400 (AO400)]. Physiological data including blood pressure, heart rate, electrocardiogram (ECG), as well as oxidative stress markers were measured in heart tissues. Results: ALP-intoxication led to perturbed normal ECG and increased oxidative stress. Administration of olive leaf extract at various concentrations, however, mitigated bradycardia after 90 minutes, following ALP-intoxication (in AO200), hypotension (in AO100), and cardiac conduction disturbances (decreased QTC in the AO200 30 (P<0.05), 60 (P<0.001), and 90 (P<0.05) minutes after intoxication)   and decreased PR 60 (P<0.05) and 90 minutes (P<0.01) after intoxication. This was compared with baseline as well as detrimental changes in cardiac electrophysiology [mitigated ST-segment elevation  in AO200  and depressed T-wave in the AO200 (P<0.05) and  AO400 (P<0.01)  groups 90 minutes after intoxication] Conclusion: Based on these authentic results, it seems that olive leaf extracts can be useful in reducing the severity of symptoms in ALP-poisoned individuals and could be utilized in a poisoning emergency.
Keywords: Aluminum Compounds; Cardiotoxicity; Electrocardiography; Gastric lavage; Oxidative stress; Pesticides.
Article history:
Received: 31 May 2022
Revised: 7 Dec 2022
Accepted: 7 Jan 2023
*Corresponding author:
moghimian.m@gmu.ac.ir
Department of Physiology, School of Medicine, Gonabad University of Medical Sciences, Gonabad, Iran.

Postal code: 9691793718
Tel: +98 915 177 8595

Introduction
Avast majority of scientific texts have addressed a wide range of  beneficial properties of olive leaves and their active ingredient called oleuropein.  Based on traditional information, the most known medicinal feature of olive leaf is cardioprotection. Similarly, anti-inflammatory, antioxidant, antiarrhythmic, hypotensive, and antiatherosclerotic effects of olive leaf extract have been reported by different experimental  and clinical studies. Also, recent advancecs demonstrated some potent and protective effect of this leaf extract aginst lead poisoning and carbendazim, diazinon, doxorubicin, busulfan, thioacetamide intoxications, and so on.
 The toxicity of aluminum phosphide (ALP) also called "rice tablet”, which is broadly used for fumigation process of grains, is mainly due to the presence of the phosphorous compound that could be released as phosphine gas, which is categorized as protoplasmic toxin (Azad et al., 2001). The ultimate biological complications of phosphine are numerous cellular enzymatic and organelle dysfunctions and cell death (Singh et al., 1996). The intensity of poisoning is very high when ALP is absorbed by either gastrointestinal tract or respiratory tract, whereas contamination of skin with ALP does not cause a significant problem for human beings (Chaudhry, 1997, Moffat et al., 2011). Following the digestion of ALP in gastric acid in the stomach, it releases phosphine gas by which a variety of symptoms can occur within a few minutes (Anger et al., 2000, Christophers et al., 2002). In case of toxicity, a high chemical oxidation process may induce vomiting, abdominal pain, restlessness, tachycardia, tachypnea, acidosis, and hypotension. Finally, cardiovascular and renal failure and severe acidosis are the most lethal complications of aluminum phosphide poisoning (Singh et al., 1996).
Various observations indicated that medicinal plants can be useful in the emergency room due to their effectiveness against drug poisoning (Gurjar et al., 2011). Because of lower side effects of herbs in comparison with synthetic drugs, the use of medicinal herbs has increased in recent years (Nasri and Shirzad, 2013, Rafieia-Kopaei, 2011, Sewell and Rafieian-Kopaei, 2014). Myriad studies showed that olive has many medicinal properties. Olive oil is useful for treatment of gallstones, dry cough, and pyorrhea, and it acts as an aphrodisiac substance to increase libido (Somova et al., 2003, Trovato et al., 1993).
Materials and Methods
Study design and animal procedures
In this study, 35 adult male Wistar rats weighing 290-310 g were randomly divided into four groups (n=7) ,including a control group (AC), and ALP+Olea100 (AO100), ALP+Olea200 (AO200), and ALP+Olea400 (AO400) groups (Figure 1). The animals were housed in cages containing four animals each in an air-conditioned environment (24±4 °C) with a humidity of 65±5% and an artificial lighting with a light-dark cycle of 12:12 h. All the animals had free access to standard rat chow and tap water. To perform the experiment, animals were anesthetized with sodium thiopental. Afterwards, neck and abdomen were completely shaved and placed on a surgical bed. A small bulb was placed on the floor of the surgical bed to maintain the temperature of the animal's body at 37 °C. After preparation of the animals, their organs were fixed on the surgical bed. To perform tracheostomy, a longitudinal incision was made in the midline of neck about 1 cm from sternal manubrium to the top. After removing the muscles of cervical region, the trachea was detached and intubated. By placing a small cushion (for example, an insulin syringe barrel) under the neck of the animal, the head of the rat was placed in the right position, and after the removal of the cervical muscles, the right carotid artery was exposed, and the vagus nerve was isolated inside the carotid sheet. A ligature was passed beneath the vessel at the site of a carotid bifurcation, and then, the ends of ligature were clamped. After that, the vessel was closed by bulldog clamps at the proximity of the sternal manubrium, and the second knot (a loose knot) was made after the clamps on the first knot. A small incision was obliquely made about 1 cm above the site of clamps. A heparinized catheter (PE-50) was retrogradely inserted towards the midline to the heart. Next, the loose knot located on the top of the vessel and catheter was tightened. The catheter was then pushed down (should not enter the heart) after the removal of the clamps. Therefore, the carotid artery was cannulated to record blood pressure and was connected to the Power Lab® apparatus to record arterial pressure fluctuations. To perform gastrostomy, an incision was made in the abdominal region of the rats where the stomach was readily accessible (the left side of the abdomen beneath the ribs). Afterward, the animals’ stomach was exposed by blunt disection, and immediately, a small incision was made in the stomach to insert a special tube in it. The tube was connected and fixed to the stomach by a suture, and ALP was administered at a dose of 120 mg/kg in the stomach of each rat via gastrostomy tube. In treatment groups, the ethanolic extract of olive leaves was also administered via gastrostomy tube for 20 minutes at concentrations of 100, 200, and 400 mg/kg per body weight. Three sub-dermal needle electrodes from ECG recorder were connected to both forelimbs and left hind limb and monitored lead II of ECG during the experiment.
The olive leaves extraction procedure
At first, one kilogram of the aerial parts of olive leaves was collected from Razi Herbal Medicine Research Center affiliated with the University of Lorestan, in Lorestan, Iran. In order to obtain the extract of olive leaves, one liter of 80% ethanol was added to dried leaves and then was vigorously vortexed for 12 minutes. This procedure was carried out twice. The resulting extract was concentrated by utilization of a rotary vacuum evaporator. The final extract was completely freeze-dried and was kept at 4 °C until later use. The extraction efficiency was 11.4%, and the amount of oleuropein concentration was reported as 18.45%.
Measuring oxidative stress
In order to study oxidative stress, reactivity of catalase (CAT), glutathione peroxidase (GPx), and malondialdehyde (MDA) was measured in heart tissues of the rats. So, at the end of each protocol, the heart was excised under deep anesthesia, was immediately frozen in liquid nitrogen, and was stored at −70 °C; this was done to estimate MDA (Sigma, USA), CAT (ZelBio), and GPx (Pars Azmoon, Iran) using spectrophotometric methods of Ohkawa et al. and Khaper and Singal  using diagnostic kits (Khaper and Singal, 1997, Ohkawa et al., 1979).
Measuring hemodynamic parameters: After cannulation of carotid artery, the data of arterial blood pressure and heart rate were transmitted to power lab apparatus by a pressure transducer. In this protocol, different doses of olive extract were examined. The measured indices included systolic blood pressure (SBP), mean arterial pressure (MAP), heart rate and  Electrocardiografic (ECG) parameters (RR, PR, QRS, QTC, ST Height, T-amplitude). Hemodynamic information was continuously recorded from the baseline and after 30, 60, and 90 minutes following different treatments.
Ethical conciderations
Rats were obtained from Lorestan University of Medical Sciences, and all the experimental procedures were performed according to the protocols authorized by the Animal Care Committee of Lorestan University of Medical Sciences (IR. LUMS.REC.1396.323).
Data analysis
The Kolmogorov–Smirnov test was used to determine whether the data were distributed normally. Mean ± SEM was calculated for all groups. Hemodynamic parameters within the groups were measured using analysis of variance (ANOVA) and Tukey's post hoc test and  Bonferroni comparison between all groups. A value of P-value < 0.05 was considered significant.
Results
Hemodynamic parameters and cardiac function
Alterations in heart rate in AC, AO100, AO200, and AO400 groups during the various experiment periods were presented in Table 1. Comparing the heart rates at baseline, 30, 60, and 90 minutes after intoxication with ALP showed that in the AC group, after 60 (P<0.001) and 90 minutes (P<0.05), the heart rate was significantly decreased compared with the baseline. Moreover, in the AO100 group, the heart rate was significantly reduced following poisoning with ALP after 60 (P<0.001) and 90 minutes (P<0.05), compared with the baseline. On the other hand, in AO200 group, the heart rate of rats was significantly reduced (P<0.05) in the 30th and 60th minutes; however, the decrease in the heart rate of the animals was decreased in the 90th minuteof intoxication, compared with the baseline. In the AO400 group, the heart rate did not show any significant increase after poisoning with ALP and in comparison with the baseline; but, leaf extract could not halt the unbound decrease in the animals’ heart rate at this dosage.
Comparing the MAP changes
Changes in MAP values in the AC, AO100, AO200, and AO400 groups in the baseline and 30, 60, 90 minutes after intoxication with ALP were presented in Table 1. The results showed that MAP was significantly (P<0.01) diminished at 60 and 90 minutes following the poisoning, compared with the baseline. In AO100 group, the reduction of MAP was not statistically significant, compared with the baseline values and at any period of time. However, in the AO200 group, MAP was significantly diminished, compared with the baseline when the comparison was made at the 60th (P<0.01) and 90th minute (P<0.001) following the administration of ALP. Finally, in AO400 group, MAP level also showed a significant decrease after 60 and 90 minutes of intoxication with ALP (P <0.001), when compared with the baseline values.
Comparing systolic blood pressure
As shown in Table 1, changes in systolic arterial pressure during various periods were evaluated in all the experimental groups. Comparison of changes in systolic blood pressure (SBP) at baseline, 30, 60 and 90 minutes after intoxication with ALP showed that SBP was significantly reduced in the 90th minute (P<0.01)  in the AC group when compared with the baseline (before ALP intoxication). Nonetheless, in AO100 group, SBP was not significantly reduced at any period of time when compared with the baseline value. Furthermore, SBP values were significantly reduced in AO200 (P<0.01) and AO400 (P<0.01) groups in the 60th and 90th minutes in comparison with baseline values.
ECG parameters
Changes in QRS intervals in experimental groups during the various periods were presented in Table 1. Comparison of this variable at baseline and 30th, 60th, and 90th minutes following ALP intoxication did not show any significant difference in any of the groups (Figure 2).
Comparing changes in QTC intervals: The values regarding QTC intervals at various periods of time were demonstrated in Table 1. Comparison of changes in QTC intervals at baseline and in 30, 60, and 90 minutes after ALP intoxication showed a significant reduction of QTC in the control group 60 and 90 minutes after ALP intoxication compared to the baseline (P<0.05). Compared with the baseline, QTC interval was significantly reduced in AO200 group after 30 (P<0.05), 60 (P<0.001), and 90 (P<0.05) minutes . QTC was also considerably lowered in AO400 group compared with baseline value when the comparison was made in the 30th (P<0.01), 60th (P<0.01), and 90th (P<0.05) minutes following the intoxication.
Comparing changes in RR intervals: Variations in RR intervals of all the experimental groups at the different periods were presented in Table 1. Comparing the changes in RR intervals at baseline and 30th, 60th, and 90th minutes of intoxication showed a significant reduction of RR in the control group (P<0.05). RR interval was significantly reduced in AO200 group after 30 (P<0.05), 60 (P<0.001), and 90 (P<0.05) minutes compared with the baseline values. RR was also significantly lowered in AO400 group, compared with the baseline when the comparison was made in the 30th (P<0.01), 60th (P<0.01), and 90th (P<0.05) minutes of ALP intoxication. The findings of this study demonstrated that RR interval was significantly increased in the AO100 group in comparison to the AC group when compared with the 60th (P<0.001) and 90th (P<0.05) minutes of intoxication. Accordingly, RR interval was further increased in the 30th minute when compared with 60th  minute (P<0.01). In AO200 group, with respect to the baseline value, the increase in RR interval was significant only in the 60th minute, while AO200 group inhibited the increase in RR interval at this dose. In AO400 group, in comparison with baseline value, no significant difference was observed in RR interval at different periods. So, olive leaf extract suppressed ALP-induced increase of RR interval at this concentration.
Comparison changes in PR intervals: Alterations of PR intervals in all the experimental groups were presented in Table 1 in different periods of time. Comparing changes in PR interval at baseline and after 30, 60, and 90 minutes following ALP intoxication showed that in AO100 group, PR interval was significantly decreased in the 60th (P<0.05) and 90th minutes (P<0.01) compared with the baseline value. Such a decrease in PR interval was more (P<0.01) in the 60th and 90th minutes when compared with the 30th minute of intoxication. No significant changes were observed in PR intervals in other groups.
Comparing changes in T wave height: Differences in T wave height regarding all the experimental groups are depicted in Table 1 at the different periods of time. Comparison of changes in T wave height at baseline, and after 30, 60 and 90 minutes of ALP intoxication demonstrated that in the control group, the height of T wave was significantly (P<0.05) lower in the 90th minute compared with the 60th minute. Olive leaf extracts inhibited ALP-induced decrease of T wave height at different periods in AO100 group, compared with the baseline value. In the AO200 group, the height of T wave was significantly (P<0.05) lower in the 90th minute in comparison with baseline and 30th minute. In AO400 group, the height of  T wave was significantly decreased in the 90th (P<0.01) and 60th minutes (P<0.05) when compared with the baseline value.
Comparison of changes in the height of ST segment: Differences in ST segment height regarding all the studied groups at various time intervals were shown in Table 1. Comparison of alterations in ST height at  baseline, and 30, 60, and 90 minutes after ALP intoxication showed that in AO200, the height of ST wave was significantly increased inthe 30th minutes compared with the baseline value. On the other hand, the height of deviation was significantly (P<0.05) reduced in 90th minute in comparison with  30th minute. The height of  ST segment did not change substantially at  different periods of time in other groups.
Comparison of CAT activity in  90th minute after ALP intoxication
Changes in CAT enzyme activity after 90th minutes of  intoxication were illustrated in Figure 3. The level of myocardial enzyme activity in  AO200 group was significantly lower than the control group. In this dose, olive extract prevented the increase in catalase activity in the state of ALP-induced toxicity (P<0.05).
Comparing GPx activity in the 90th minute after ALP intoxication
Alterations in the level of GPx enzyme activity 90 minutes after ALP poisoning was shown in Figure 4. The results indicated that the activity did not significantly change among the studied groups when compared in the different time intervals.
Comparing MDA concentration in the 90th minute after  ALP intoxication
According to Figure 5, variations in the levels of MDA were compared regarding all the groups 90 minutes after poisoning with ALP. The results showed that the concentration of MDA was significantly elevated in all the groups treated with different doses of olive leaf extract.
Table 1. Average changes in the mean of studied variables by terms of groups.
Minutes after intoxication Base line Number Groups
90 60 30
Arterial pressure (mmHg).
52.12±19.55c 55.72±25.63c 85.40±40.79 108.00±44.46a 10   Control
57.61±19.31 73.07±36.41 84.34±44.64 90.29±48.35 8   ALP+Olea100
71.81±21.28c 76.88±20.39c 120.21±30.82 135.00±26.80 8   ALP+Olea200
197.00±47.06c 77.33±20.66c 118.53±73.33 148.94±33.72 8   ALP+Olea400
Heart rate (beat/min)
196.37±128.68b 134.70±77.32 227.54±103.02 329.80±48.06 10   Control
195.43±77.39 130.74±61.57c 241.00±78.54 292.00±53.86 8   ALP+Olea100
243.48±77.05 171.28±87.42 b 183.21±81.06 b 306.00±52.56 8   ALP+Olea200
227.14±137.19 147.25±90.94 125.90±85.53 253.54±114.39 8   ALP+Olea400
Systolic blood pressure (mmHg).
56.77±22.51b 65.27±32.99 98.05±48.82 114.81±45.48 10 Control       
59.27±19.74 76.37±38.31 88.10±47.45 69.11±51.62 8    ALP+Olea100
83.26±22.25c 88.44±11.21c 129.80±35.33 145.63±32.44 8    ALP+Olea200
67.53±17.23c 94.51±16.70c 127.86±32.54 160.30±33.28 8    ALP+Olea400
The QRS duration (ms).
22.33±4.38 21.13±2.93 18.7±3.93 18.06±3.15 10   Control       
22.83±2.65 21.10±3.8 19.91±2.71 20.3±3.21 8   ALP+Olea100
22.03±2.62 19.98±2.07 18.95±3.14 20.47±6.98 8   ALP+Olea200
34.69±29.50 33.51±25.30 27.63±15.68 30.56±21.76 8   ALP+Olea400
The QTC interval (ms).
91.60±23.47b 98.43±26.61b 125.80±48.53 149.00±44.89 10   Control       
110.58±19.27 110.98±33.39 131.61±40.90 148.96±35.76 8   ALP+Olea100
114.47±20.47b 107.08±17.05b 105.19±37.21c 163.58±35.56 8   ALP+Olea200
137.21±102.47 119.36±78.67 191.98±124.46c 183.67±37.99c 8   ALP+Olea400
The RR interval (ms).
574.12±386.22b 654.94±297.94c 336.12±186.60 185.65±27.66 10   Control       
491.57±186.23b 672.46±282.77c,d 307.32±216.73 211.08±38.85 8   ALP+Olea100
383.88±167.35 540.78±246.97b 459.67±294.35 200.91±37.14 8   ALP+Olea200
576.90±475.6 723.96±345.07 681.06±299.52 264.37±177.83 8   ALP+Olea400
The PR interval (ms).
41.92±4.20 45.08±5.85 48.90±5.21 47.29±5.27 10   Control       
42.58±2.74c 43.79±7.35b,d 53.98±6.89 52.73±5.39 8   ALP+Olea100
43.68±2.72 45.87±4.36 47.06±6.71 51.43±7.95 8   ALP+Olea200
70.56±30.56 78.5±21.03 58.69±22.76 76.42±20.99 8   ALP+Olea400
The height of T waves (μv).
101.72±92.84b,d 212.24±119.45 157.35±67.85 207.89±73.95 10   Control       
108.53±84.31 194.96±75.00 176.13±78.10 167.90±74.07 8   ALP+Olea100
55.05±39.92b,e 138.69±99.24 170.66±87.78 142.9±077.72 8   ALP+Olea200
40.78±36.71c 89.16±76.99b 198.49±144.84 263.69±154.73 8   ALP+Olea400
The ST segment deviations (μv).
42.08±32.69 65.46±63.04 92.23±57.94 69.65±60.98 10   Control       
51.88±41.31 75.62±39.15 72.67±60.59 62.89±58.79 8   ALP+Olea100
30.94±23.45d 47.03±30.91 117.07±88.76 87.87±50.88b 8   ALP+Olea200
52.77±40.73 89.76±67.66 41.37±29.06 105.00±84.41 8   ALP+Olea400
Parameters within the groups were measured using ANOVA, Tukey's post-hoc, and Bonferroni test. a: Mean±SEM; b: P<0.05 versus   its baseline; c: P<0.001 versus   its baseline; d: P<0.01 versus 30 minutes; e: P<0.05 versus 60 minutes

.




Discussion
The present study showed the beneficial effects of olive leaf extract on ALP-induced cardiotoxicity. The findings indicated that intoxication with ALP led to a progressive reduction in heart rate and blood pressure. Severe hypotension and shock are serious complications of ALP toxicity which usually results in death (Bayazit et al., 2000). In the rats intoxicated with ALP, electrocardiographic findings showed that pathological injuries were similar to the damages occurred in myocardial ischemia, causing perturbations in electrical conduction system of the heart. Apparently,  a reduction in cardiac output is one of the leading cause of ALP-induced hypotension. In the current study, in comparison with the control group, olive leaf extract, at a dose of 100 mg/kg prevented the reduction of MAP and SBP significantly after 60 and 90 minutes of intoxication. As shown in the results, in AO400 group, olive leaf extract significantly prevented the decrese in heart rate after 90 minutes in comparison with the control group. In similar studies conducted on murine models, at concentrations of 10, 20, 40 mg/kg, ALP induced detrimental changes in ECG, similar to what happens in myocardial ischemia. Such alterations usually lead to changes in biochemical factors (Lall et al., 1997). Based on the studies, olive leaf compounds have protective impacts against cardiovascular diseases (Baharvand-Ahmadi et al., 2015, Khosravi-Boroujeni et al., 2013, Madihi et al., 2013b, Shayganni et al., 2016). In 2004, Manna et al. for the first timefound that olive extract had protective effects against acute reperfusion ischemic injuries in an isolated heart model (Manna et al., 2004). In line with this study, in 2006, Andreadou et al. demonstrated protective effects of oleuropein on cardiovascular ischemic injuries through the reduction in oxidative stress and the size of infarction (Andreadou et al., 2006). As an inferation, several studies revealed that the administration of 100-200 mg/kg oleuropein attenuated doxorubicin-induced cardiotoxicity in a rat model (Andreadou et al., 2007). Therefore, olive leaf extract or its active components can be useful in reducing blood pressure and preserving the heart contractility. The changes in ECG which were mentioned  can be reversed within 10-14 days in patients who survived from ALP toxicity (Singh et al., 1985). However, the mortality rate is very high in poisoned patients, and their survival rates vary, depending on the severity of intoxication and the half life of ALP (Chugh et al., 1991). Following intoxication with ALP, the concentration of MDA, as a biomarker of lipid peroxidation, is increased in cardiac tissue and the levels of CAT and GPx are decreased in parallel fashion. In the present study, CAT levels were significantly lower in AO200 compared with the control group, indicating that olive leaf extract prevented the increased turn-over of antioxidant enzymes which might be due to the decrease in the rate of oxidative stress (Lall et al., 1997). Animal and human studies have revealed that intoxication with ALP leads to the increase in the rate of oxidative stress as monitored by measuring MDA reactivity (Baradaran et al., 2013, Dua and Gill, 2004, Rafieian-Kopaei et al., 2014). Several papers also indicated that ALP-induced oxidative stress could impair electron transport chain, leading to excessive generation of oxygen free radicals and the changes in antioxidant defense system, including the decrease in CAT levels (Madihi et al., 2013a, Rafieian-Kopaei, 2014, Shirzad et al., 2011). Regarding this study, it was shown that increase in ST segment and decrease in T wave height were not statistically significant in the control group. Also, administration of olive leaf extract at a dose of 200 mg/kg resulted in a decrease in ST segment deviations after 90 minutes, and administration of 100 mg/kg inhibited the decrease in T wave height, compared with the baseline values. Based on pharmacological research, olive extract has antiarrhythmic properties against arrhythmogenic potentials of calcium chloride and aconitine (Esmailidehaj et al., 2016, Mipando, 2004). In 1978, Petkow and Manolov showed that a single intravenous injection of oleuropein within 10-40 mg/kg possesses antiarrhythmic properties against barium chloride in rabbits. In ECG, ST segment implies ventricular depolarization and the initiation of repolarization (Petkov and Manolov, 1978). Previous studies indicated that ALP could paradoxically elevate/depress ST segment, which shows pericardial/myocardial injuries (Baghaei et al., 2014). The findings, however, demonstrated that intoxication of rats with ALP could elevate ST segment in ECG while the administration of olive leaf extract prevented such elevation. A growing body of evidence shows that there is a positive correlation between increase in the ST segment and  mortality rate. This parameter is capable of predicting the intensity and the effectiveness of therapeutic strategies in acute toxicity with ALP. PR and QTC intervals are indices of electrical conductance of heart which are both increased in response to intoxication with ALP. QTC is calculated based on  Bazett formula in which QT is divided by the square root of RR interval (Ahnve, 1985). This perturbation in electrical conductance mimics cardiac arrest, or ischemic injuries occurred following ALP toxicity (Baghaei et al., 2014, Soltaninejad et al., 2012). In the present research, the administration of olive leaf extract at a dose of 100 mg/kg diminished PR interval in 60th and 90th minutes after intoxication of rats with ALP, which suggested that olive extract can neutralize deleterious impact of ALP on electrocardiograph. In ECG, QT interval pertains to depolarization and repolarization of ventricles. QTC is normally decreased when heart rate is increased. In clinics, QTC is usually used (instead of QT) for demonstrating perturbations in electrical conductance in ventricles (Nazemi et al., 2016). In the control group of this research, which was treated only with ALP, QTC was significantly decreased after 60 and 90 minutes in comparison with the baseline. Furthermore, olive leaf extract prevented ALP-induced decrease of QTC at a dose of 100 mg/kg after 60 and 90 minutes. Considering that QTC has an inverse association with  square root of RR interval, an increase in RR interval implies the decrease in heart rate following intoxication with ALP.  It should be noted that olive leaf extract prevented the decrease in heart rate caused by ALP intoxication through reducing RR interval. Hence, it is concluded that olive leaf extract at a concentration of 100 mg/kg improved heart function and increased QTC; this showed the protective effects against ALP-induced oxidative stress. The levels of MDA and ROS were rapidly increased following intoxication with ALP. Conversely, the activity of CAT, GSH, and thiol-containing compounds were decreased in response to the ALP intoxication of rats. Reports indicated that poisoning with ALP resulted in elevation of the rate of oxidative stress as measured by the concentration of MDA, which was considered a biomarker of lipid peroxidation. However, the authors could not show any significant difference between control and treatment groups concerning the levels of MDA in heart tissue of rats. The main limitation of this study was the risk of using rice tablets for the researcher.
Based on the results obtained in this study, the authors recommendthe following points for future studies: 1-Evaluating olive leaf extract in mitochondrial oxidative stress; 2-Investigating other formulations of olive extract in ALP-induced cardiovascular diseases and 3-increasing the periods of time for  assessing   the effect of olive leaf extract on ALP-induced cardiotoxicity.
Conclusion
According to the findings, intoxication with rice tablets is associated with electrocardiographic abnormalities and oxidative stress. In addition, administration of olive leaf extract at different doses primarily improves bradycardia, hypotension, and conduction disturbances of the heart (QTC increased and PR decreased) caused by poisoning with ALP ,prevents increase of ST segment, and decreases T- wave height.
Acknowledgement
The authors would like to thank Student Research Committee of Razi Herbal Medicine Research Center, Lorestan University of Medical Sciences, Khorramabad, Iran for their financialsupport.
Authors’ contributions
Moazami M, Sedighi M, Haydari S, Rashidipour M, and Mahmoudi GA were involved to mesurements ad data collection. Moghimian M, Nazari A, Ghaderpour S, and Bakhshesh M were parcitipted to processed data, performed analysis and designed figures. Moghimian M and Nazari A drafted the manuscript and supervised the work. All authors read the final manuscript and verified for publication.
Conflict of interest
Authors declared no conflict of interest.
Funding
This research has been supported by Student Research Committee of Razi Herbal Medicine Research Center, Lorestan University of Medical Sciences, Khorramabad, Iran for their financial support.
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Type of article: orginal article | Subject: public specific
Received: 2022/05/31 | Published: 2024/08/20 | ePublished: 2024/08/20
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