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Ellagic acid

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Electron Physician. 2015 Aug; 7(4): 1153–1162. 

Published online 2015 Aug 10. doi: 10.14661/2015.1153-1162

PMCID: PMC4578534

PMID: 26396728

Ellagic acid improves electrocardiogram waves and blood pressure against global cerebral ischemia rat experimental models

Khojasteh Hoseiny Nejad,1 Mahin Dianat,2 Alireza Sarkaki,2 Mohammad Kazem Gharib Naseri,2 Mohammad Badavi,2and  Yaghoub Farbood2

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Global cerebral ischemia (GCIR) arises in patients that are shown a variety of clinical difficulty including cardiac arrest, asphyxia, and shock. In spite of advances in understanding of the brain, ischemia and protective effects to improve ischemic injury still remain unknown. The aim of our study was to investigate the effect of ellagic acid (EA) pretreatment in the rat models of global cerebral ischemia reperfusion.


This experimental study was conducted in 2014 at the Physiology Research Center of the Ahvaz Jundishapur University of Medical Sciences in Ahvaz, Iran. Adult male Wistar rats (250–300 g) were used in this study. GCIR was induced by bilateral vertebral and common carotid arteries occlusion (4-VO). 32 rats were divided randomly to four groups: 1) So (Sham) received normal saline as vehicle of EA, 2) EA, 3) normal saline + GCIR, and 4) EA + GCIR. After anesthesia (a mix of xylazine and ketamine), animal subjected to 20 minutes of ischemia followed by 30 minutes of reperfusion in related groups. EA (100 mg/kg, dissolved in normal saline) or 1.5 ml/kg normal saline was administered (gavage, 10 days) to the related groups. EEG was recorded from NTS in GCIR treated groups.


Present data showed that: 1) EEG in GCIR treated groups was flattened; 2) Blood pressure, voltage of QRS and P-R interval were reduced significantly in the ischemic groups compared to before ischemia, and pretreatment with EA prevented this reduction; and 3) MDA level and heart rate was increased by GCIR and pretreatment with EA reduced MDA level and restored the HR to normal level.


Results indicate that global cerebral ischemia-reperfusion impairs certain heart functions and ellagic acid as an antioxidant can restore these parameters. The results of this study suggest the possible utility of ellagic acid in patients with brain stroke.

Keywords: Global cerebral ischemia, Ellagic acid, Rat, ECG, Blood pressure, MDA

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1. Introduction

According to the report of World Health Organization (WHO) in developed countries, stroke is ranked as the second leading cause of death (1). Ischemic stroke is the most wrecked kind of stroke. Among several types of models for ischemia, high level mortality rates are shown for global cerebral ischemia (2), which occurs in patients who have a variety of clinical conditions, such as: shock, cardiac arrest, and in patients undergoing complex cardiac surgery (3). The severity of brain injury is dependent on the magnitude and duration of the interruption in the blood supply and subsequent damage induced by reperfusion (4). The solitary tract conveys afferent information from chemoreceptors and stretch receptors in the walls of the cardiovascular (5).

The role of solitary nucleus (or solitary tract) in cerebral ischemia was recognized in clinical and experimental animal models (6). The entry point for integration center in the brain that control autonomic nervous visceral circulatory, digestive, immune, and the larynx function is the nucleus tractus solitarii (NTS) and also has an important role in relay for vagal reflex (7). A variety of visceral mechanical and chemical signals and nociceptive stimuli travel to the nucleus tractus solitarii via vagus nerve fibers. For that reason, the peripheral nerve is in close contact with the central nucleus tractus solitarius. Hence, acupuncture stimulation on the body and the harmful stimulation to inner organs may converge at the nucleus tractus solitarii. Therefore, inner organ functions may be ultimately influenced with a “comprehensive contribution” (8). Some of the research on this matter has indicated that solitary nucleus is more susceptible to destruction on middle cerebral arteries occlusion (9), but some reports have shown the role of solitary nucleus on global cerebral ischemia patients (10).

Heart rate, blood pressure (BP) and electrocardiograph (EKG) were changed in globally ischemic brain. Previous studies indicated the regulatory role of heart related physiological parameters in cerebral ischemia (1112). Altogether, there is no exact clinical drug for stroke, so studies have historically focused on attenuating the destructive role of ischemia in the brain. The level of reactive oxygen species (ROS) increases during ischemia and then leads to oxidative stress (13). Therefore, using antioxidant scavengers such as polyphenol compounds is more useful in reducing the ROS. As the previous study has shown, the effects of these compounds are known in some diseases, such as cardiovascular disease (14). Ellagic acid is a polyphenol compound. Ellagic acid (EA) is one of the natural phenol antioxidant found in numerous fruits such as blackberry, pomegranate, strawberry, grape, apple, kiwi, various vegetables, and nuts (15). Polyphenol administration partially (for malondialdehyde levels) or completely (for superoxide dismutase and total antioxidant capacity) reduced the oxidative stress induced by ischemia/reperfusion injury (16). It was reported that EA enhanced antioxidant related enzymes such as superoxide dismutase, catalase and gluthatione level in ischemic brain (17). Therefore, EA is one of the best candidates for ischemia related researches.

Taken together in the current study, we examined the role of EA in global cerebral ischemia rats. First, severe ischemia group (4-vessel occlusion (4-VO)) underwent near-complete ischemia as showed by electroencephalography acknowledged ischemic neuronal damages. Second, we used ellagic acid to remedy ischemia, and then we measured the heart rate, BP and EKG parameters.

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2. Material and Methods

2.1. Study design and setting

This experimental study was conducted in 2014 at the Physiology Research Center of the Ahvaz Jundishapur University of Medical Sciences in Ahvaz, Iran. The present study included 32 male Wistar rats that were selected according to previous studies (18).

2.2. Chemicals

Ketamine and xylazine (Alfasan Co., Holland) were used for anesthesia. Ellagic acid (EA) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich Co., USA). EA was dissolved in DMSO/normal saline (10%), buffered to a pH of 7, and gavaged in such way that the rats received 100 mg/kg/day. The solution of DMSO/normal saline (10%) as the solvent was also gavaged at 1.5 ml/kg/day to the related group. The dissolved EA was prepared daily.

2.3. Animals

In the present study, 32 adult male Wistar rats (250–300 g), purchased from Animal House of Ahvaz Jundishapur University of Medical Sciences, were housed in cages under the following conditions: controlled temperature (22±2 °C), 50% humidity, and 12/12- hr light/dark cycle (light on 07:00–19:00 h). The rats had free access to tap water and standard rat pellet diet (Pars Co. IR). Before the commencement of testing; all animals were slightly handled for 5 days (10 minutes daily). The experiments were approved by the Animal Ethics Committee of Ahvaz Jundishapur University of Medical Sciences (No. ajums. B-9348). All efforts were made to minimize animal suffering, and to reduce the number of animals used (mortality rate is 30% in ischemic groups).

2.4. Experimental protocols

Rats were divided randomly into four groups of eight rats (in accordance with previous studies) (18), as follows: 1) sham-operated (SO) were only given 1.5 ml/kg/day of solvent orally by gavage for 10 consecutive days; 2) ellagic acid group (EA); 3) global cerebral ischemic-reperfusion (GCIR) group where animals received 1.5 ml/kg of solvent by gavage for 10 consecutive days and then were exposed to global cerebral ischemia for 20 minute and 30 minute reperfusion (19); and 4) global cerebral ischemic-reperfusion + ellagic acid (EA+GCIR) group where animals received ellagic acid (100 mg/kg/day) in 1.5 ml/kg of solvent orally by gavage for 10 consecutive days and then were exposed to global cerebral ischemia (20 minutes) followed by 30 minutes of reperfusion; animals received only ellagic acid (100 mg/kg/day) dissolved in 1.5 ml/kg solvent by gavage for 10 consecutive days. The dose of EA (100 mg/kg) was selected based on previous reports (2021) and our pilot experimental study (22). Induction of ischemic-reperfusion was carried out 24 hours after last gavage.

2.5. Surgery

Rats were anesthetized by intraperitoneal injection of ketamine/xylazine (50/5 mg/kg). The animal’s body temperatures were maintained at 36.5±0.5 °C using heating pads with their heads mounted in a stereotaxic device for electrode implantation surgery. A coated stainless steel Teflon bipolar metal wire electrode (0.005″ bare, 0.008″ coated, A-M systems, Inc. WA) was implanted in the NTS (SolM) with stereotaxic coordination of AP=−14.04 mm; ML= 0.4 mm to bregma, and DV=8 mm from the dura, correspondingly. Implantation of electrodes at the right location was determined by histological verification at the end of experiments. All implants were fixed to the skull by dental acrylic cement and two glass anchor small bolts (2324).

2.6. Local EEG recording

After induction of anesthesia, electrical field potentials (local EEG) from the NTS of rats were fed to a ML135 bio-amplifier (AD Instruments, 4-Channels Power Lab, Lab Chart software version 7, Australia) with 1 mV amplification, sample recording 400 Hz, and 0.3–70 Hz band pass filtration for 5 minute. The basic EEG variations period of 5 seconds were compared in all groups. Electrical power of frequency bands were measured as µV2/Hz (24). The local EEG recording was done 1st and 11th days before and after I/R initiation correspondingly. The animals were allowed to recover for ten days before the commencement of experiments.

2.7. Induction of global cerebral ischemic-reperfusion

On the first day, after anesthesia, animals underwent transient forebrain global ischemia, as described by Pulsinelli et al. (25). A neck ventral midline incision was made and the common carotid arteries were exposed and separated from the vagus nerves. Then, a sterile field around each common carotid artery was slowly placed, without interrupting the carotid blood flow; the incision was then sutured. A second incision, 1 cm in length, covering direct occipital bone at the back of the first two cervical vertebrae was then made. The muscles around the spine from the midline, separating the left and right alar hole of the first cervical vertebrae were (26) thus exposed. Vertebral artery spinal canal passed under the alar foramen before the posterior fossa. 0.5 mm diameter electrocautery needles (Bowie Monopolar Electrocautery, Cincinnati, Ohio) were placed through each vertebral artery foramen electrocauterized alar, and were permanently blocked.

On the second day, under anesthesia, both CCA were occluded by microclamps for 20 minutes (19) to produce 4-vessel occlusion (25). This demonstrated that, in this study, the method of 4-vessel occlusion (4VO) was indeed carried out. Reperfusion (30 minute duration) was started by opening the carotid clamps after 20 minutes of ischemia. Those rats with 4VO were only involved in tests if their EEG was flattened during the ischemia period (27). Similar procedures were carried out in SO and EA groups without vertebral arteries electro-coagulation and carotid arteries occlusion. (28). Blood samples were collected from the heart, and after, coagulation at room temperature was centrifuged at 4000 rpm (10 minutes at ambient temperature). The collected serum samples were stored at − 80 °C until analysis.

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