Cardioprotective effect of fenugreek on isoproterenol-induced myocardial infarction in rats.

OBJECTIVES: This study is designed to evaluate the cardioprotective effect of fenugreek on isoproterenol- induced myocardial infarction and is investigated by an in vivo method in rats.
MATERIALS AND METHODS: Male Wistar albino rats were divided into four groups (n=10). Group I received 0.5% CMC treated as normal control group. Group II received isoproterenol (85 mg/kg body weight) intraperitoneal (i.p.) for two consecutive days (14(th) and 15(th) days). Group III received fenugreek (250 mg/kg body weight) intragastric intubation for 15 days. Group IV rats received fenugreek as in Group III and additionally isoproterenol was given for two consecutive days (14(th) and 15(th) days).
RESULTS: The results described the cardioprotective effect that observed in Group IV showed significantly (P< 0.05) decreased levels of TBARS and enhanced the activities of both enzymatic and non-enzymatic antioxidants (SOD, CAT, GPx and GSH) in myocardial infarcted rats when compared to Groups II and III. Histopathological studies were also co-relating with the above biochemical parameters.
CONCLUSION: These findings concluded the cardioprotective effect of fenugreek on lipid peroxidation and antioxidant defense system during isoproterenol-induced myocardial infarction in rats.

Introduction

Myocardial infarction is a major public health problem and the leading cause of mortality in both developed and developing countries. According to the world health organization report, heart disease and stroke will become the leading causes of death and disability worldwide by the year 2020.[1] The rat model of isoproterenol induced myocardial necrosis, out of many well-known models, has been used to evaluate several cardiac dysfunctions.[2]

Isoproterenol [1-(3, 4-dihydroxyphenyl)-2-isopropylamino ethanol hydrochloride] (ISO), a synthetic catecholamine and b-adrenergic agonist, causes severe oxidative stress in the myocardium, resulting in infarct-like necrosis of the heart muscle. It is also known to generate free radicals and to stimulate lipid peroxidation, which may be a causative factor for irreversible damage to the myocardial membrane.[3] New therapies are needed to treat myocardial ischemia because current treatment has only a limited impact on survival and annual cost.

Fenugreek (Trigonella foenum graecum) belongs to the leguminosae family and cultivated predominantly in India, the Mediterranean and North African regions. Fenugreek is known to have several pharmacological effects such as hypoglycemia,[4] hypocholesterolemia,[5] chemopreventive,[6] anti-oxidant,[7] antipyretic and anti-inflammatory effects.[8] The seeds of fenugreek contain lysine and L-tryptophan rich proteins, mucilaginous fiber and other rare chemical constituents such as saponins, coumarin, fenugreekine, nicotinic acid, sapogenins, phytic acid, scopoletin and trigonelline, which are thought to account for many of its presumed therapeutic effects.

Though there are several studies on the medicinal properties of fenugreek seeds, the antioxidant activity of the seeds in traditional medicinal uses has prompted us to investigate the in vivo cardioprotective effects of the seeds in detail. Therefore the current study is designed to evaluate the cardioprotective effect of fenugreek on isoproterenol-induced biochemical and histopathological changes in rats.

Materials and Methods

Chemicals

Isoproterenol was obtained from the Sigma Chemical Company, St. Louis, MO, USA. All the other chemicals and reagents used were of analytical grade.

Formulation and administration of fenugreek

Fenugreek powder was suspended in 0.5% carboxy methyl cellulose (CMC) and each animal belonging to two different groups received 1.0 ml of fenugreek suspension at a dose of 250 mg/kg body weight everyday respectively by intragastric intubation.

Induction of myocardial infarction

The myocardial infarction was induced by intraperitoneal (i.p.) injection of isoproterenol hydrochloride (85 mg/kg body weight, dissolved in physiological saline, for two consecutive days (14th and 15th days).[9]

Experimental animals

Forty male albino Wistar rats weighing 110-150 g were selected for the study. They were housed in plastic cages with filter tops under controlled conditions of 12h light and 12h dark cycles, 50% humidity at 28˚C. Standard pellet diet was fed to rats throughout the experimental period and water was given ad libitum. The study was conducted after obtaining a clearance from the institutional animal ethical committee (IAEC) (Reg .no P.Col/52/2010/IAEC/VMCP) of Vinayaka Mission College of Pharmacy, Salem, Tamil Nadu.

Treatment schedule

The rats were randomly assigned in-to four groups of ten animals each. The rats in Group I received 1.0 ml of 0.5% CMC every day via intragastric intubation and served as the untreated control. Group II rats received isoproterenol (85 mg/kg body weight) intraperitoneally twice at an interval of 24 h on the 14th and 15th days. The rats in Group III received fenugreek via intragastric intubation at a daily dose of (250 mg/kg body weight) respectively for a period of 15 days. Group IV rats received fenugreek as in Group III for 15 days and at the last of the experimental period on 14th and 15th days rats received isoproterenol (85 mg/kg body weight) injections intraperitoneally twice at an intravel of 24 h. The experiment was terminated after 15 days and all the animals were killed by cervical decapitation after an overnight fast.

Biochemical estimation

Hemoglobin was determined by the method of- Drabkin[10] Hemoglobin was oxidized to methemoglobin that combines with cyanide to form cyanmethemoglobin that was measured at 540nm. The amount of hemoglobin was expressed as mg/dl blood. Lipid peroxidation was estimated by measuring the level of thiobarbituric acid reactive substances (TBARS) in tissues via the method of Ohkawa.[11] and plasma via the method of Yagi.[12] The pink chromogen produced by the reaction of thiobarbituric acid with malondialdehyde, a secondary product of lipid peroxidation, was measured at 532nm. The values are expressed as nmoles/100 g tissue or nmoles/mg Hb. Reduced glutathione (GSH) content was determined via the method of Ellman.[13] GSH determination is based on the development of yellow colur when 5, 5’ dithio 2-nitro benzoic acid (DTNB) is added to compounds containing sulfhydryl groups. The values are expressed as nmoles g-1wet tissue. Glutathione peroxidase (GPx EC.1.11.1.9) activity was assayed via the method of Rotruck[14] with a modification: a known amount of enzyme preparation was incubated with H2O2 in the presence of GSH for a specified time period. The amount of H2O2 utilized was determined via the method of Ellman.[13] The values are expressed as μmoles of GSH utilized/min/mg Hb or protein. Superoxide dismutase (SOD EC.1.15.1.1) was assayed using the method of Kakkar and Viswanathan[15] based on the 50% inhibition of the formation of NADH- phenazine methosulfate-nitroblue tetrazolium formazan at 520nm. One unit of the enzymes is taken as the amount of enzyme for 50% inhibition of NBT reduction/min/mg protein. The activity of catalase (CAT EC. 1.11.16) was determined via the method of Sinha:[16] dichromate in acetic acid was reduced to chromic acetate when heated in the presence of hydrogen peroxide (H2O2), with the formation of perchromic acid as an unstable CAT intermediate. The chromic acetate formed was measured at 590 nm. Catalase was allowed to split H2O2 for different periods of time. The reaction was stopped at different time intervals via the addition of a dichromate acetic acid mixture, and heating the reaction mixture and measuring chromic acetate colorimetrically and determined the remaining H2O2. The values are expressed as μmoles of H2O2 utilized/min/mg protein.

Preparation of hemolysate

Blood was collected in heparinized tubes and plasma was separated by centrifugation at 2000×g for 10 min. After the separation of plasma, the buffy coat was removed and packed cells (RBCs) were washed thrice with cold physiological saline. To determine the activity of RBC antioxidant enzymes, RBC lysate was prepared by lysing a known volume of RBCs with hypotonic phosphate buffer, pH 7.4 and centrifuged at 3000×g for 10min at 2°C to separate the hemolysate.

Preparation of tissue homogenate

Heart tissue were removed immediately and washed with ice-cold saline and homogenized in the appropriate buffer in a tissue homogenizer.

Histopathological studies

At the end of the study, all the rats were sacrificed by cervical decapitation and the hearts were dissected out, washed in ice cold saline. Then myocardial tissue was immediately fixed in 10% buffered neutral formalin solution. After fixation, tissues were embedded in paraffin and serial sections were cut and each section was stained with hematoxylin and eosin. The slides were examined under light microscope and photographs were taken.

Statistical analysis

The results presented here are the means ± SD of 10 rats in each group. The results were analyzed using one-way analysis of variance [ANOVA] and the group means were compared using Duncan's multiple range test [DMRT] using SPSS version 12 for Windows. The findings were considered as statistically significant if P<0.05.[17]

Results

Normal architecture of the cardiac cells was observed with no evidence of microscopic changes in the control and fenugreek treated groups [Figure 1]. In isoproterenol-treated rats’ heart, histological changes such as perivascular cuffing of vasa vasorum with intimal fibrosis, disruption of medial elastic fibers with diffuse interstitial fibrosis and myocytolysis were seen. In fenugreek (250 mg/kg body weight) and isoproterenol (85 mg/kg body weight) treated rats there was no appreciable change in the heart.

Figure 1

Histopathology changes in the myocardial infarction of control and experimental rats. Effect of fenugreek on the histopathology of the myocardium. (a) Normal rats (group I) showing normal cardiac cells. (b) Isoproterenol (85 mg/kg bodyweight)- induced rats (group II) showing perivascular cuffi ng of vasa vasorum with intimal fi brosis, disruption of medial elastic fi bers with diffuse interstitial fi brosis and myocytolysis. (c) Fenugreek (250 mg/kg bodyweight) treated rats (group III) showing normal architecture of cardiac cells. (d) Fenugreek (250 mg/kg body weight) + isoproterenol (85 mg/kg bodyweight)- induced rats showing normal architecture of cardiac cells

The circulatory and tissue levels of TBARS in normal and experimental rats are shown in Table 1. Rats treated with isoproterenol showed a significant (P<0.05) increase in these levels when compared with normal control rats. Rats pre-treated with fenugreek (250 mg/kg body weight) and isoproterenol-induced rats significantly (P<0.05) decrease the levels of TBARS when compared with the isoproterenol induced rats.

Table 1

Effect of fenugreek on circulating and tissue lipid peroxidation (TBARS) of control and experimental rats

Table 2 represents the activities of antioxidants in the normal, control and the isoproterenol-induced rats. The isoproterenol-induced rats showed a significant (P<0.05) decrease in the antioxidants (SOD, CAT, GPx and GSH) when compared with normal control rats. On treatment with fenugreek (250 mg/kg body weight) daily for a period of 15 days, a significant (P<0.05) increase in the activities of antioxidant was observed in the isoproterenol-treated rats when compared with the isoproterenol induced rats.

Table 2

Effect of fenugreek on circulatory and tissue antioxidants of control and experimental rats

Discussion

Our result shows that fenugreek maintained the levels of lipid peroxides and antioxidants of heart in myocardial infarcted rats.

Histopathological findings also support the findings of this study. The isoproterenol induced myocardium showed perivascular cuffing of vasa vasorum with intimal fibrosis and disruption of medial elastic fibers with diffuse interstitial fibrosis. Normal rats treated with fenugreek (250 mg/kg body weight) showed normal cardiac fibers without any pathological changes. Myocardial section of Group IV rats showed normal architecture of cardiac cells. This action produced by the fenugreek reflects the protection offered during myocardial infarction.

In our study we observed an increase in the concentration of plasma, erythrocyte and tissue TBARS which has been suggested to be due to an enhanced oxidative stress in experimentally induced myocardial injury. Oral treatment with fenugreek significantly decreased the concentration of TBARS in the isoproterenol-induced rats. Fenugreek protected the heart against lipid peroxidative damage by removal of excess free radicals generated by isoproterenol. The presence of phenolic groups in phytochemicals of fenugreek probably protected the heart from myocardial damage by scavenging free radicals and thereby suppressing the peroxidation of lipids. Thus, fenugreek being a phenolic compound might have inhibited lipid peroxidation in our study.

SOD and CAT are considered as primary enzymes since they are involved in the direct elimination of ROS. We have observed significant decrease in the activities of SOD and CAT in the isoproterenol-induced rats (Group II): this might be due to excessive generation of free radicals by isoproterenol. Administration of fenugreek restored the levels of SOD and CAT to near normal. This shows the antioxidant effect of fenugreek against myocardial injury caused by free radicals.

Significant decrease in the level of GSH and Gpx may be due to its increased utilization during the burst of reactive oxygen species production, in protecting ‘SH’ group containing proteins from lipid peroxidation.[18] The decreased activities of glutathione peroxidase in the heart of isoproterenol-induced myocardial infarction might be due to decreased availability of their substrate, reduced glutathione. Inactivation of the enzyme Gpx in the heart tissue leads to the accumulation of oxidized glutathione which in turn inactivates many enzymes containing SH groups.

Administration of fenugreek significantly prevented the alterations in the levels of GSH, GPx and restored the levels to near normal. This effect may be due to the free radical scavenging properties of fenugreek.[19] The scavenging activities of the phenolic substances are attributed to the active hydrogen-donating ability of the hydroxyl substitutions.[20] The presence of the phenolic groups in the phytochemicals especially naringenin and quercetin in fenugreek seeds could be responsible for .OH radical scavenging activity.

Thus, it is quite clear that changes observed in SOD, CAT, GPx and GSH could be attributed to the enhancement in antioxidant status in blood and tissues of normal rats. Previously it has been reported that fenugreek extracts have been shown to produce beneficial effects, such as neutralizing the free radicals and enhancing the antioxidant status. Phenolic compounds of fenugreek act by scavenging free radicals[21] and quenching the lipid peroxidative chain. Thus, fenugreek being a phenolic compound might have inhibited lipid peroxidation in our study.

In conclusion, our study reveals that administration of fenugreek proved to be more effective in reducing the extent of myocardial damage and significantly counteracted the oxidative stress during isoproterenol-induced myocardial infarction in rats.