J Physiol Biochem DOI 10.1007/s13105-015-0411-2
ORIGINAL PAPER
Cardioprotective effect of linseed oil against isoproterenol-induced myocardial infarction in Wistar rats: a biochemical and electrocardiographic study Amal Derbali & Kais Mnafgui & Marwa Affes & Fatma Derbali & Raouf Hajji & Neji Gharsallah & Noureddine Allouche & Abdelfattah El Feki Received: 3 November 2014 / Accepted: 8 April 2015 # University of Navarra 2015
Abstract The present study was designed to evaluate the cardioprotective effect of Tunisian flaxseed oil (Linum usitatissimum) against isoproterenol-induced myocardial infarction in rats by studying hypertensive and cardiac damage markers especially electrocardiographic changes and troponin T serum level. In vitro, the extracted oil showed an important inhibition of angiotensin converting enzyme (ACE) with an IC 50 = 85.96 μg/ml. According to chemical analysis, this extract is composed essentially of alpha linolenic acid (ALA), an n-3 polyunsaturated fatty acid (58.59 %). Male rats were randomly divided into three groups, namely control (C), isoproterenol (ISO), and isoproterenol-treated group with flaxseed oil (FO+ ISO). Isoproterenol injection showed changes in ECG pattern, including ST-segment elevation (diagnostic of myocardial infarction), increase in the serum levels of Amal Derbali and Kais Mnafgui contributed equally to this work. A. Derbali : K. Mnafgui (*) : A. El Feki Laboratory of Animal Ecophysiology, Faculty of Sciences of Sfax, University of Sfax, P.O. Box 95, 3052 Sfax, Tunisia e-mail: [email protected] M. Affes : N. Allouche Laboratory of Chemistry of Natural Products, Faculty of Sciences of Sfax, B.P. 1171, 3000 Sfax, Tunisia F. Derbali : R. Hajji Internal Medicine Department, Hospital of Sidi Bouzid, Sidi Bouzid 9100, Tunisia N. Gharsallah Laboratory of Plant Biotechnology, Faculty of Sciences of Sfax, B.P. 1171, 3000 Sfax, Tunisia
Troponin T and cardiac injury markers (creatine kinaseMB (CK-MB), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), aspartate transaminase (AST), and alanine transaminase (ALT)). However, Linum oil preco-treatment prevented almost all the parameters isoproterenol-induced myocardial infarction in rats. Results of the present study proved that flaxseed oil has a significant effect by heart protection against isoproterenol-induced myocardial infarction through beneficial effect of the important fraction of ALA. Keywords Linum usitatissimum . Isoproterenol . Troponin . ECG . Myocardial infarction
Introduction Cardiovascular disease and their thrombotic complications are the leading cause of mortality in the world today. Coronary artery disease, also known as atherosclerotic or ischemic heart disease, is the most common type of heart disease and cause of heart attacks. In 2010, coronary heart disease was the leading cause of death worldwide (more than 7 million deaths compared to 5.2 million deaths in 1990) [12]. The prevalence of cardiac ischemic infarction was predicted to reach 20 million patients by 2015 [24]. It may affect individuals at any age but becomes dramatically more common at progressively older ages, with approximately a tripling with each decade of life [5]. Myocardial infarction is the common presentation of the ischemic heart disease, and it causes irreversible
A. Derbali et al.
damage. It is the acute condition of myocardial necrosis that occurs as a result of imbalance between coronary blood supply and myocardial demand [35]. In general, the incidence of myocardial infarction increases additively as a function of the number of conventional risk factors, including hypertension, diabetes mellitus, and hypercholesterolemia [18]. Cardiovascular dysfunction is a devastating disease which badly needs a medical breakthrough. Although modern drugs are effective in treating this condition, they are mostly accompanied with adverse effects. Therefore, many researchers have focused on screening natural sources that have been intended to treat cardiovascular disease with minimal side effects. Linum usitatissimum, commonly known as flaxseed or linseed, belongs to the family Linaceae. The flax plant is not a new crop and is native to West Asia and the Mediterranean. Flaxseed has been playing a major role in the field of diet and disease research due to its potential health benefits associated with high content of linolenic acid (ALA), which is an essential omega-3fatty acid and also because of a major lignan that is secoisolariciresinol diglucoside [34]. Flaxseed has gained much importance in recent times as ethnomedicine due to its wide pharmacological actions. In addition, its therapeutic potentials as antioxidant, anticancer, antidiabetic, antiviral, bactericide, antiinflammatory, and anti-atherosclerotic agent are identified [3, 4, 11, 22, 23, 27]. The model of isoproterenol-induced acute myocardial ischemia is considered as one of the most commonly used experimental model to study the beneficial effects of many drugs and cardiac function [7]. In fact, isoproterenol [1-(3,4-dihydroxyphenyl)-2isopropylaminoethanol hydrochloride], a synthetic catecholamine and β-adrenergic agonist, increases heart rate and exhaust energy reservoir of cardiac myocytes leading to cell death [19]. It induces myocardial necrosis via multiple modes of action in experimental animals. It is essentially manifested by its stimulation of sarcolemmal adenylate cyclase and Na+ and Ca2+ channels resulting in exaggerated influx of Ca2+ and energy consumption and consequent cell death [15]. The objective of the present investigation was to study the plausible cardioprotective effect of flaxseed oil which is the hexane extract concentrate extracted from seeds of L. usitatissimum on isoproterenolinduced myocardial infarction in Wistar rats.
Material and methods Chemicals Angiotensin converting enzyme (ACE) kit was purchased from Trinity Biotech, USA. All the other chemicals used were of analytical grade.
Preparation of Linum extracted oil Flaxseeds were collected from Sidi Bouzid (Tunisia). Air-dried and powdered seeds of L. usitatissimum (100 g) were macerated with hexane for 24 h three times at room temperature using a mechanical stirrer. The extract was filtered through filter paper and concentrated with a vacuum evaporator. The remaining solution was fractionated successively with hexane, ethyl acetate, ethanol, and butanol to obtain the corresponding fractions: hexane (31.07 g), ethyl acetate (14.6 g), butanol (8.84 g), and ethanol (9.37 g). Flaxseed oil, obtained by evaporating the hexane extract under reduced pressure, was used for the in vivo treatment.
Determination of fatty acid composition Fatty acid methyl esters (FAMEs) analysis was carried out after performing alkaline treatment obtained by dissolving the oil (0.05 g) in n-hexane (1 ml) and adding a solution of potassium hydroxide (1 ml; 2 N) in methanol. FAMEs were analyzed by gas chromatography by means of a Shimadzu 17A gas chromatograph equipped with a flame ionization detector (FID) and a capillary column. The operation conditions were as following: the column temperature was set at 180 °C, the injector temperature was set at 230 °C, the detector temperature was set at 250 °C, the carrier gas was nitrogen with a head pressure of 0.6 bar for the FID, the pressure of air was at 1.5 bar, and the hydrogen pressure was 0.8 bar. Separation was accomplished by injecting 1 μl of solution in a capillary column of 30 m length, 0.32 mm of diameter, with a film thickness of 0.25 μm. The polar stationary phase was cyanopropylmethyl/ phenylmethyl-polysiloxane 1/1. Peaks were identified by comparing their retention times with those of authentic reference compounds. The fatty acid composition was expressed as relative percentages of each fatty acid.
Cardioprotective effect of linseed oil
Determination of angiotensin converting enzyme in vitro The ACE inhibitory activity was assayed by the method reported by Wu et al. [33], with captopril as positive control. The total reaction volume of 100 ml was made up of 25 ml of 2.17 mM Hippuryl-L-histidyl-L-leucine (HHL), 25 ml of 2 mU of ACE, 25 ml tested samples in different concentrations (all prepared with 100 mM borate buffer, containing 300 mM NaCl, pH 8.3), or 25 ml borate buffer for control. The mixture of HHL and sample of flaxseed or ACE alone was incubated at 37 °C in shaking water bath type (HETO, SBD50, Denmark) with continuous agitation at 450 rpm for 10 min to achieve thermal equilibrium. The reaction was started by adding ACE to tube containing HHL and samples or borate buffer. The reaction was terminated after 30 min incubation by addition of 300 ml of 1 MHCl and the solution filtered through a 0.45 nm nylon syringe filter for reversed-phase (RP)-HPLC analysis. HPLC analysis was performed on a LC-10ATvp system equipped with a diode array detector (Shimadzu, Japan). ZORBAX SB-C18 column (4.6 mm id_150 mm, 5 mm particle size) was used, and HA (hippuric acid) and HHL were detected at 228 nm. External standard of HA sample was prepared freshly on a daily basis and used for the qualification of HA. Captopril is used as positive control. The peak area of HA was recorded, and the percentage inhibition of ACE activity was calculated using equation: absorbancecontrol −absorbancetest PI ¼ 100 absorbancecontrol Animals and treatment The assays of the present study were conducted on total 24 adult male Wistar rats, weighting 250±10 g, which were obtained from the local Central Pharmacy, Tunisia. All rats were housed in an environmentally controlled breeding room (temperature 20±2 °C, humidity 60± 5 %, 12 h dark/light cycle) where they had standard diets and free access to tap water. The experimental protocols were conducted in accordance with the guide for the care and use of laboratory animals issued by the University of Sfax, Tunisia, and approved by the Committee of Animal Ethics.
Induction of experimental myocardial infarction Isoproterenol was dissolved in normal saline and injected to rats (85 mg/kg body weight) subcutaneously at an interval of 24 h for 2 days to induce experimental myocardial infarction. Animals were sacrificed 48 h after the first dose of isoproterenol. Experimental protocols After acclimatization, the animals were randomly divided into three groups consisting of eight rats each: 1. Group (1): (Control) rats received standard laboratory diet and drinking saline water ad libitum and served as a control. 2. Group (2): Isoproterenol (ISO): rats received standard diet+saline water for 7 days and at the 6th day rats received saline water for 7 days and at the 6th day subcutaneously injected with isoproterenol (85 mg/kg, subcutaneously injected, once at an interval of 24 h for 2 consecutive days) to induce myocardial infarction [19, 25]; 3. Group (3): Flaxseed oil (FO+ISO): rats received standard diet+flaxseed oil given orally (50 mg /kg bw) for 7 days and were injected subcutaneously with isoproterenol (85 mg /kg bw) on day 6th and 7th. All rats are fasted overnight but had free access to water at the last administration of the drug.
Electrocardiography At the end of the experimental period, needle electrodes were inserted under the skin of the animals under light Ether anesthesia. ECG recordings were made using Single Channel Digital ECG System (BIOPAC, Santa Barbara, California), and changes in ECG pattern were considered. This particular dosage was fixed after trying out different dosages for different days prior to isoproterenol treatment. At the end of experimental period, the rats were sacrificed by cervical decapitation and blood was collected. The serum was separated by centrifugation (1500×g, 15 min, 4 °C), frozen and stored at −20 °C until biochemical investigations. The heart and the artery were removed and cleaned by NaCl solution. All samples were stored at −80 °C until used.
A. Derbali et al.
Biochemical determinations The collected serum was used for the estimation of ACE, cardiac marker enzymes such as creatine phosphokinase-MB (CK-MB), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and troponin T rates were measured in frozen aliquots of serum by standardized enzymatic procedures using commercial kits from (Biolabo, France) on an automatic biochemistry analyzer (Vitalab Flexor E, USA) at the clinic pathological laboratory of Sidi Bouzid Hospital using commercially available standard enzymatic kits (Biolabo, France). Statistical analysis Data are presented as means±standard deviation (SD). Determinations were performed from eight animals per group, and differences were examined by a one-way analysis of variance (ANOVA) followed by the Fisher test (Stat View). *P
ORIGINAL PAPER
Cardioprotective effect of linseed oil against isoproterenol-induced myocardial infarction in Wistar rats: a biochemical and electrocardiographic study Amal Derbali & Kais Mnafgui & Marwa Affes & Fatma Derbali & Raouf Hajji & Neji Gharsallah & Noureddine Allouche & Abdelfattah El Feki Received: 3 November 2014 / Accepted: 8 April 2015 # University of Navarra 2015
Abstract The present study was designed to evaluate the cardioprotective effect of Tunisian flaxseed oil (Linum usitatissimum) against isoproterenol-induced myocardial infarction in rats by studying hypertensive and cardiac damage markers especially electrocardiographic changes and troponin T serum level. In vitro, the extracted oil showed an important inhibition of angiotensin converting enzyme (ACE) with an IC 50 = 85.96 μg/ml. According to chemical analysis, this extract is composed essentially of alpha linolenic acid (ALA), an n-3 polyunsaturated fatty acid (58.59 %). Male rats were randomly divided into three groups, namely control (C), isoproterenol (ISO), and isoproterenol-treated group with flaxseed oil (FO+ ISO). Isoproterenol injection showed changes in ECG pattern, including ST-segment elevation (diagnostic of myocardial infarction), increase in the serum levels of Amal Derbali and Kais Mnafgui contributed equally to this work. A. Derbali : K. Mnafgui (*) : A. El Feki Laboratory of Animal Ecophysiology, Faculty of Sciences of Sfax, University of Sfax, P.O. Box 95, 3052 Sfax, Tunisia e-mail: [email protected] M. Affes : N. Allouche Laboratory of Chemistry of Natural Products, Faculty of Sciences of Sfax, B.P. 1171, 3000 Sfax, Tunisia F. Derbali : R. Hajji Internal Medicine Department, Hospital of Sidi Bouzid, Sidi Bouzid 9100, Tunisia N. Gharsallah Laboratory of Plant Biotechnology, Faculty of Sciences of Sfax, B.P. 1171, 3000 Sfax, Tunisia
Troponin T and cardiac injury markers (creatine kinaseMB (CK-MB), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), aspartate transaminase (AST), and alanine transaminase (ALT)). However, Linum oil preco-treatment prevented almost all the parameters isoproterenol-induced myocardial infarction in rats. Results of the present study proved that flaxseed oil has a significant effect by heart protection against isoproterenol-induced myocardial infarction through beneficial effect of the important fraction of ALA. Keywords Linum usitatissimum . Isoproterenol . Troponin . ECG . Myocardial infarction
Introduction Cardiovascular disease and their thrombotic complications are the leading cause of mortality in the world today. Coronary artery disease, also known as atherosclerotic or ischemic heart disease, is the most common type of heart disease and cause of heart attacks. In 2010, coronary heart disease was the leading cause of death worldwide (more than 7 million deaths compared to 5.2 million deaths in 1990) [12]. The prevalence of cardiac ischemic infarction was predicted to reach 20 million patients by 2015 [24]. It may affect individuals at any age but becomes dramatically more common at progressively older ages, with approximately a tripling with each decade of life [5]. Myocardial infarction is the common presentation of the ischemic heart disease, and it causes irreversible
A. Derbali et al.
damage. It is the acute condition of myocardial necrosis that occurs as a result of imbalance between coronary blood supply and myocardial demand [35]. In general, the incidence of myocardial infarction increases additively as a function of the number of conventional risk factors, including hypertension, diabetes mellitus, and hypercholesterolemia [18]. Cardiovascular dysfunction is a devastating disease which badly needs a medical breakthrough. Although modern drugs are effective in treating this condition, they are mostly accompanied with adverse effects. Therefore, many researchers have focused on screening natural sources that have been intended to treat cardiovascular disease with minimal side effects. Linum usitatissimum, commonly known as flaxseed or linseed, belongs to the family Linaceae. The flax plant is not a new crop and is native to West Asia and the Mediterranean. Flaxseed has been playing a major role in the field of diet and disease research due to its potential health benefits associated with high content of linolenic acid (ALA), which is an essential omega-3fatty acid and also because of a major lignan that is secoisolariciresinol diglucoside [34]. Flaxseed has gained much importance in recent times as ethnomedicine due to its wide pharmacological actions. In addition, its therapeutic potentials as antioxidant, anticancer, antidiabetic, antiviral, bactericide, antiinflammatory, and anti-atherosclerotic agent are identified [3, 4, 11, 22, 23, 27]. The model of isoproterenol-induced acute myocardial ischemia is considered as one of the most commonly used experimental model to study the beneficial effects of many drugs and cardiac function [7]. In fact, isoproterenol [1-(3,4-dihydroxyphenyl)-2isopropylaminoethanol hydrochloride], a synthetic catecholamine and β-adrenergic agonist, increases heart rate and exhaust energy reservoir of cardiac myocytes leading to cell death [19]. It induces myocardial necrosis via multiple modes of action in experimental animals. It is essentially manifested by its stimulation of sarcolemmal adenylate cyclase and Na+ and Ca2+ channels resulting in exaggerated influx of Ca2+ and energy consumption and consequent cell death [15]. The objective of the present investigation was to study the plausible cardioprotective effect of flaxseed oil which is the hexane extract concentrate extracted from seeds of L. usitatissimum on isoproterenolinduced myocardial infarction in Wistar rats.
Material and methods Chemicals Angiotensin converting enzyme (ACE) kit was purchased from Trinity Biotech, USA. All the other chemicals used were of analytical grade.
Preparation of Linum extracted oil Flaxseeds were collected from Sidi Bouzid (Tunisia). Air-dried and powdered seeds of L. usitatissimum (100 g) were macerated with hexane for 24 h three times at room temperature using a mechanical stirrer. The extract was filtered through filter paper and concentrated with a vacuum evaporator. The remaining solution was fractionated successively with hexane, ethyl acetate, ethanol, and butanol to obtain the corresponding fractions: hexane (31.07 g), ethyl acetate (14.6 g), butanol (8.84 g), and ethanol (9.37 g). Flaxseed oil, obtained by evaporating the hexane extract under reduced pressure, was used for the in vivo treatment.
Determination of fatty acid composition Fatty acid methyl esters (FAMEs) analysis was carried out after performing alkaline treatment obtained by dissolving the oil (0.05 g) in n-hexane (1 ml) and adding a solution of potassium hydroxide (1 ml; 2 N) in methanol. FAMEs were analyzed by gas chromatography by means of a Shimadzu 17A gas chromatograph equipped with a flame ionization detector (FID) and a capillary column. The operation conditions were as following: the column temperature was set at 180 °C, the injector temperature was set at 230 °C, the detector temperature was set at 250 °C, the carrier gas was nitrogen with a head pressure of 0.6 bar for the FID, the pressure of air was at 1.5 bar, and the hydrogen pressure was 0.8 bar. Separation was accomplished by injecting 1 μl of solution in a capillary column of 30 m length, 0.32 mm of diameter, with a film thickness of 0.25 μm. The polar stationary phase was cyanopropylmethyl/ phenylmethyl-polysiloxane 1/1. Peaks were identified by comparing their retention times with those of authentic reference compounds. The fatty acid composition was expressed as relative percentages of each fatty acid.
Cardioprotective effect of linseed oil
Determination of angiotensin converting enzyme in vitro The ACE inhibitory activity was assayed by the method reported by Wu et al. [33], with captopril as positive control. The total reaction volume of 100 ml was made up of 25 ml of 2.17 mM Hippuryl-L-histidyl-L-leucine (HHL), 25 ml of 2 mU of ACE, 25 ml tested samples in different concentrations (all prepared with 100 mM borate buffer, containing 300 mM NaCl, pH 8.3), or 25 ml borate buffer for control. The mixture of HHL and sample of flaxseed or ACE alone was incubated at 37 °C in shaking water bath type (HETO, SBD50, Denmark) with continuous agitation at 450 rpm for 10 min to achieve thermal equilibrium. The reaction was started by adding ACE to tube containing HHL and samples or borate buffer. The reaction was terminated after 30 min incubation by addition of 300 ml of 1 MHCl and the solution filtered through a 0.45 nm nylon syringe filter for reversed-phase (RP)-HPLC analysis. HPLC analysis was performed on a LC-10ATvp system equipped with a diode array detector (Shimadzu, Japan). ZORBAX SB-C18 column (4.6 mm id_150 mm, 5 mm particle size) was used, and HA (hippuric acid) and HHL were detected at 228 nm. External standard of HA sample was prepared freshly on a daily basis and used for the qualification of HA. Captopril is used as positive control. The peak area of HA was recorded, and the percentage inhibition of ACE activity was calculated using equation: absorbancecontrol −absorbancetest PI ¼ 100 absorbancecontrol Animals and treatment The assays of the present study were conducted on total 24 adult male Wistar rats, weighting 250±10 g, which were obtained from the local Central Pharmacy, Tunisia. All rats were housed in an environmentally controlled breeding room (temperature 20±2 °C, humidity 60± 5 %, 12 h dark/light cycle) where they had standard diets and free access to tap water. The experimental protocols were conducted in accordance with the guide for the care and use of laboratory animals issued by the University of Sfax, Tunisia, and approved by the Committee of Animal Ethics.
Induction of experimental myocardial infarction Isoproterenol was dissolved in normal saline and injected to rats (85 mg/kg body weight) subcutaneously at an interval of 24 h for 2 days to induce experimental myocardial infarction. Animals were sacrificed 48 h after the first dose of isoproterenol. Experimental protocols After acclimatization, the animals were randomly divided into three groups consisting of eight rats each: 1. Group (1): (Control) rats received standard laboratory diet and drinking saline water ad libitum and served as a control. 2. Group (2): Isoproterenol (ISO): rats received standard diet+saline water for 7 days and at the 6th day rats received saline water for 7 days and at the 6th day subcutaneously injected with isoproterenol (85 mg/kg, subcutaneously injected, once at an interval of 24 h for 2 consecutive days) to induce myocardial infarction [19, 25]; 3. Group (3): Flaxseed oil (FO+ISO): rats received standard diet+flaxseed oil given orally (50 mg /kg bw) for 7 days and were injected subcutaneously with isoproterenol (85 mg /kg bw) on day 6th and 7th. All rats are fasted overnight but had free access to water at the last administration of the drug.
Electrocardiography At the end of the experimental period, needle electrodes were inserted under the skin of the animals under light Ether anesthesia. ECG recordings were made using Single Channel Digital ECG System (BIOPAC, Santa Barbara, California), and changes in ECG pattern were considered. This particular dosage was fixed after trying out different dosages for different days prior to isoproterenol treatment. At the end of experimental period, the rats were sacrificed by cervical decapitation and blood was collected. The serum was separated by centrifugation (1500×g, 15 min, 4 °C), frozen and stored at −20 °C until biochemical investigations. The heart and the artery were removed and cleaned by NaCl solution. All samples were stored at −80 °C until used.
A. Derbali et al.
Biochemical determinations The collected serum was used for the estimation of ACE, cardiac marker enzymes such as creatine phosphokinase-MB (CK-MB), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and troponin T rates were measured in frozen aliquots of serum by standardized enzymatic procedures using commercial kits from (Biolabo, France) on an automatic biochemistry analyzer (Vitalab Flexor E, USA) at the clinic pathological laboratory of Sidi Bouzid Hospital using commercially available standard enzymatic kits (Biolabo, France). Statistical analysis Data are presented as means±standard deviation (SD). Determinations were performed from eight animals per group, and differences were examined by a one-way analysis of variance (ANOVA) followed by the Fisher test (Stat View). *P
