Dietary fenugreek(Trigonella foenum-graecum)seeds and garlic(Allium sativum)alleviates oxidative stress in experimental myocardial infarction

P.Mukthamba,K.Srinivasan

Department of Biochemistry and Nutrition,CSIR–Central Food Technological Research Institute,Mysore 570 020,India

Abstract Soluble fibe-rich fenugreek seeds(Trigonella foenum-graecum)and garlic(Allium sativum)are understood to exert cholesterol-lowering and antioxidant effects.The cardioprotective influenc of a combination of fenugreek seeds and garlic by their antioxidant influenc was evaluated in hypercholesterolemic rats administered isoproterenol.Wistar rats were maintained on high-cholesterol diet for 8 weeks along with dietary interventions of fenugreek(10%),garlic(2%)and their combination.Myocardial infarction was induced with isoproterenol injection.Increased circulatory troponin,disturbed activities of cardiac ATPases,increased serum iron and decreased ceruloplasmin confirme myocardial infarction.Elevated lipid peroxides accompanied with reduced antioxidant molecules caused by isoproterenol and altered activities of antioxidant enzymes in serum and heart in induced myocardial necrosis were countered by dietary fenugreek,garlic,and fenugreek+garlic.Dietary fenugreek seeds and garlic ameliorated isoproterenol-induced compromised antioxidant status,the cardioprotective effect being higher by the combination of fenugreek seeds and garlic.

Keywords: Fenugreek seeds;Garlic;High cholesterol diet;Isoproterenol;Myocardial infarction;Oxidative stress

1.Introduction

Despite several advancement in medical sciences,cardiovascular disease (CVD) remains one of the leading causes for deaths.Consumption of high-cholesterol diet,overweight,and less physical activity are understood to be risk factors for cardiovasculardiseases.Oxidativedamageatthecellularorsubcellular level is now considered to be a major event in disease processes such as CVD.Oxidative stress is involved in the pathogenesis of atherosclerosis and other vascular injuries.Oxidative stress increases production of free radicals such as superoxide radical,hydrogen peroxide,peroxyl,nitric oxide,and hydroxyl radical and decreased levels of antioxidants in the myocardium damaging the myocardial cells and causing CVD[1].

It is generally accepted that a diet high in fibe,particularly soluble fibe is useful in controlling high cholesterol levels thus contributing to cardiovascular health.The role of fibe-rich fenugreek (Trigonella foenum-graecum) seeds in this regard could be worth investigating.Dietary fenugreek seeds decrease lipid peroxidation and counter the alteration in circulatory antioxidant molecules in experimental diabetic rats[2]and help in restoration of tissue antioxidant molecules in diabetic situation[3].The mode of action of theAlliumspice garlic in exerting the hypolipidemic and antioxidant action is different from that of fenugreek seeds [4].Garlic extracts are reported to lower plasma and erythrocyte lipid peroxide levels in atherosclerotic conditions[5].Consumption of garlic extract increases blood antioxidant potential,oxidation resistance,and superoxide radical scavenger activity,and decrease lipid peroxides in hypercholesterolemic situations[6].Hence,there is a possibility of an additive effect or even synergistic effect when fibe-rich fenugreek seeds and theAlliumspice garlic are consumed in combination.

Isoproterenol induced myocardial necrosis has been generally studied to evaluate the beneficia effects of food ingredients on cardiac dysfunctions.Isoproterenol induces myocardial necrosis by a multiple step mechanism and has been reported to show metabolic and morphologic aberrations in the heart tissue of experimental animals similar to those observed in humans [7].Free radicals generated by oxidative stress in the myocardium due to isoproterenol[1-(3.4-dihydroxyphenyl)-2-isopropyl aminoethanol hydrochloride],a synthetic catecholamine and β-adrenergic agonist stimulates lipid peroxidation resulting in infarction-like necrosis of the heart muscle leading to irreversible damage to the myocardial membrane[8].

We have recently reported the cardio protective influenc of dietary fenugreek seeds and garlic mediated through beneficia modulation of tissue lipid concentrations in experimental myocardial infarction induced in rats,with a higher effect seen with their combination[9].We have also reported that the susceptibility oflow density lipoproteins(carriers of cholesterol in blood) for oxidation is significantl reduced by dietary fenugreek seeds and garlic[10].The present study has particularly examined the influenc of dietary fenugreek seeds and garlic on antioxidant molecules and antioxidant enzymes in blood and heart under conditions of myocardial infarction,since oxidant stress plays a major role in the etiology of CVD.The study has also evaluated ceruloplasmin,iron concentration,and total iron binding capacity in serum,as well as the activity of ATPases in heart muscle which are indices of myocardial damage in induced myocardial infarction.

2.Materials and methods

2.1.Materials

Reduced glutathione,oxidized glutathione,nicotinamide adenine dinucleotide phosphate-reduced form (NADPH),ter-butyl hydroxyperoxide,glutathione reductase,xanthine oxidase,cytochrome-C,α-tocopherol,isoproterenol hydrochloride,p-phenylenediammine,sodium azide,bathophenanthroline sulfonate,ferrous ammonium sulfate,Tris-ATP,and ouabain were procured from Sigma-Aldrich Chemical Co.,(St.Louis,USA).1-Chloro-2,4-dinitrobenzoic acid,ethylene diamine tetraacetic acid,trichoroacetic acid,m-phosphoric acid,and sodium citrate were obtained from SISCO research laboratory (Mumbai,India).All other chemicals and solvents were obtained from SISCO Research Laboratory(Mumbai,India).Fenugreek seeds were purchased from local market and powdered,and stored at 4°C.Garlic bulbs used for the study was purchased from local market.Peeled garlic cloves were freeze-dried,powdered and stored at 4°C.

2.2.Animals and treatment

Theanimalstudywascarriedouttakingappropriatemeasures to minimize pain or discomfort taking all precautions regarding the care and use of animals for experimental procedures and with due clearance from the Institute’s Animal Ethics Committee.Male Wistar rats weighing 70-75 g from our Institute’s Experimental Animal Production Facility were used.Ten groups of these animals (n=6 per group) were housed and were maintained on various experimental diets and waterad libitumfor 8 weeks duration as described by us previously[9].The compositions of the basal diet and various experimental diets were as described previously[9].The three intervention diets contained 10% fenugreek seed powder,2% freeze-dried garlic powder,and 10% fenugreek seed powder+2% garlic powder,respectively.Thus,the eight groups were:(1) basal control (C),(2)C+fenugreek,(3)C+garlic,(4)C+fenugreek+garlic,(5)high cholesterol diet(HCD),(6)HCD+fenugreek,(7)HCD+garlic,and (8) HCD+fenugreek+garlic.After 8 weeks of diet regimen,myocardial infarction was induced with isoproterenol as described earlier [9].Two additional animal groups:(9) basal control and (10) HCD which were not administered isoproterenol served as normal and HCD controls,respectively.The overnight fasted animals were sacrifice under ether anesthesia.Blood was collected by heart puncture and serum was separated by centrifugation.Liver and heart were quickly excised,weighed and stored frozen until further analyses.

2.3.Concentration of troponin

The concentration of troponin in serum and cardiac tissue was estimated by immunosorbant assay(ELISA)technique using a standard kit(Cusabio Biotech Co.,Wuhan,China).

2.4.Ceruloplasmin,free iron concentration and total iron binding capacity in serum

Serum ceruloplasmin was estimated spectrophotometrically according to Houchin [11]by following the oxidation ofpphenylene diamine.Serum iron concentration was estimated spectrophotometrically according to Lee and Stumm[12]using bathophenanthraline reagent.Total iron binding capacity was determined according to the dipyridyl method of Ramsay[13].

2.5.Activities of ATPase in heart tissue

Activity of Na+,K+-ATPase in the myocardial tissue was assayed according to Kaplay[14]after isolating the microsomal fraction.The released inorganic phosphate by using Tris-ATP as substrate both in the presence and absence of ouabain was estimated.The enzyme activities were expressed as mmol Pi formed per h per mg protein.Activities of Ca++and Mg++-ATPases in heart tissue were independently measured by the method of Ramanadham and Kaplay[15].

2.6.Activities of antioxidant enzymes

Activities of antioxidant enzymes were determined in serum,liver homogenate and heart homogenate using standard procedures.Catalase (CAT) activity was assayed according to the method of Aebi[16]by following the decomposition of hydrogen peroxide,measured by recording the decrease in absorbance at 240 nm for 3 min.The enzyme activity was expressed as the amount of enzyme that decomposes 1 μM hydrogen peroxide per mg of protein.Superoxide dismutase (SOD) activity was measured by quantitating the inhibition of cytochrome-C reduction in the xanthine-xanthine oxidase system as described by Flohe and Otting[17].Glutathione peroxidase(GPX)was estimated using NADPH oxidation in a coupled reduction system of hydrogen peroxide and oxidized glutathione as described by Flohe and Gunzler [18].Glutathione reductase (GR) activity was assayed by measuring the oxidation of NADPH at 340 nm by oxidized glutathione according to the method of Carlberg and Mannervik [19].Glutathione-S-transferase (GST) activity was assayed by measuring the chlorodinitrobenzene-glutathione conjugate formed using 1-chloro-2,4-dinitrobenzene as the substrate,as described by Warholm et al.[20].

2.7.Antioxidant molecules

Glutathione in serum,liver homogenate and heart homogenate was determined by using Ellman’s reagent according to Beutler et al.[21].Ascorbic acid was estimated spectrophoto-metrically by measuring 2,4-dinitrophenylhydrazine derivative of dehydroascorbic acid using the method of Omaye et al.[22].α-Tocopherol was determined by HPLC method described by Zaspel and Csallany [23]using ODS column (C-18) and an UV-visible detector (295 nm) and a solvent system acetonitrile-methanol(1:1,v/v).

2.8.Statistical analysis

Results are expressed as mean±SEM and comparisons between groups were made by means of one-way ANOVA for basal diet groups and another one-way ANOVA for high cholesterol diet groups.Comparisons among different groups were made applying Dunnette test.Differences were considered significan whenp＜0.05.

3.Results

3.1.Effect of dietary fenugreek and garlic on the circulatory level of troponin in isoproterenol induced infarcted rats

High cholesterol fed rats injected with isoproterenol showed a significan increase in the concentration of troponin in the serum confirmin myocardial infraction as a result ofisoproterenol administration (Fig.1A).Dietary interventions with fenugreek,garlic,and their combination significantl countered this increase in circulatory troponin level which would have leached from heart from injured myocardial tissue (p＜0.05).This countering of elevation in circulatory troponin as a result of the above dietary interventions was also accompanied by a concomitant retention of the same in myocardial tissue(Fig.1B).

Fig.1.Effect of dietary fenugreek and garlic on the level of troponin in (A)serum and (B) heart tissue in isoproterenol induced infarcted rats.Values are mean±SEM of 6 animals.*Significantl different from uninjected HCD group(p ＜0.05);**Significantl different from isoproterenoladministeredHCDgroup(p ＜0.05);HCD:high cholesterol diet;BC:basal control;F:fenugreek;G:garlic,and ISO:isoproterenol.

3.2.Effect of dietary fenugreek and garlic on serum ceruloplasmin activity and iron concentration and total iron binding capacity in infarcted rats

Serum ceruloplasmin activity was diminished by about 47%both in normal as well as in HCD-fed situation as a result ofisoproterenol administration(Table1).The decreased levels of serum ceruloplasmin in normal as well as in HCD-fed situation during induction of myocardial infarction by isoproterenol administration was significantl countered by dietary fenugreek and garlic,the beneficia effect being higher in the case of fenugreek+garlic.Free iron concentration was increased by isoproterenol administration by 20%in normal rats and by 58%in HCD-fed rats(Table1).The elevation in serum free iron upon isoproterenol administration in these rats was significantl prevented by dietary fenugreek,garlic,and fenugreek+garlic,the effect being higher in the case of combination.Concomitant with the increase in free iron concentration,total iron binding capacity was decreased by isoproterenol administration by 48%in normal rats and by 42%in HCD-fed rats(Table1).The extent of decrease in total iron binding capacity in serum upon isoproterenol administration in these rats was significantl countered by dietary fenugreek,garlic,and fenugreek+garlic,the effect being higher in the case of combination.The restoration in total iron binding capacity approached near normalcy in the case of dietary intervention with fenugreek+garlic.

Table1 Effect of dietary fenugreek and garlic on the level of serum free iron and ceruloplasmin activity in control and isoproterenol induced infarcted rats.

3.3.Effect of dietary fenugreek and garlic on heart ATPase activities in infarcted rats

Isoproterenol administration resulted in cardiac hypertrophy as indicated by increased heart to body weight ratio.There was a 16%increase in the heart weight(expressed as g/100 g body weight) in both normal and HCD-fed situation.The increased heart weight was effectively countered by fenugreek+garlic in HCD-fed situation.Myocardial Ca2+-ATPase activity was diminished by 48%and 56%,respectively,in normal and HCDfed rats as a result ofisoproterenol administration (Table2).Similarly,Mg2+-ATPase activity in heart tissue was diminished by 24%and 32%,respectively,in normal and HCD-fed rats as a result ofisoproterenol administration.The decreases observed in the activities of cardiac Ca2+-and Mg2+-ATPases upon isoproterenol administration in normal as well as HCD-fed situation was also countered by dietary fenugreek or fenugreek+garlic,the effect being higher in the case of combination.The recovery in heart Ca2+-ATPase activity was to an extent of 42%and 49%,while the same was by 16%and 24%in Mg2+-ATPase by dietary intervention with fenugreek+garlic(Groups 5 and 10).

Na+,K+-ATPase activities were generally decreased in the myocardial tissue ofinfarcted rats.Ouabain sensitive Na+,K+-ATPase activity in myocardial tissue was diminished by 39%and 49%,respectively,in normal and HCD-fed rats as a result ofisoproterenol administration(Table2).Total Na+,K+-ATPase activity in myocardial tissue was also similarly decreased by 36% and 44%,respectively,in normal and HCD-fed rats as a result ofisoproterenol administration.Dietary fenugreek and garlic had beneficia effect on Na+,K+-ATPase activities in heart tissue ofinfarcted rats in both normal as well as in HCD-fed situation.The disturbed activities of membrane bound ATPases as a result of myocardial infarction by isoproterenol administration was countered by dietary fenugreek,garlic,and fenugreek+garlic,the effect being higher in the case of combination.OuabainsensitiveNa+,K+-ATPaseactivityinmyocardial tissue was restored by 60%and 81%,respectively,in normal and HCD-fed rats by dietary intervention with fenugreek+garlic.Similarly total Na+,K+-ATPase activity in the myocardial tissue was restored by 50%and 64%,respectively,in normal and HCDfed rats as a result of dietary intervention with fenugreek+garlic(Table2).

3.4.Effect of dietary fenugreek and garlic on antioxidant status in serum ofinfarcted rats

Lipid peroxides were higher in the serum ofisoproterenol administered rats both in normal situation(Group 2)and HCDfed situation (Group 7) (42% and 46% increase as compared to corresponding uninjected control rats)(TableA1).This was associated with a significan decrease in the concentration of antioxidant molecules-ascorbic acid (46% decreases),glutathione (65% decrease) and α tocopherol (13% decrease) in normal situation (Group 2).The elevated lipid peroxides were accompanied by diminished α-tocopherol concentration (by 38%)in HCD-fed situation(Group 7).On the other hand concentrations of glutathione and ascorbic acid were higher as a result ofisoproterenol administration in HCD-fed situation (Group 7).The increase in the concentration of ascorbic acid and glutathione was significantl negated by dietary interventions with fenugreek,or fenugreek+garlic.

Activities of the entire fie antioxidant enzyme in serum were significantl elevated by isoproterenol administration in HCDfed situation (Table3).Dietary interventions with fenugreek,garlic,and fenugreek+garlic generally countered the increase in the activities of catalase,superoxide dismutase,glutathione reductase and glutathione-S-transferase in these rats.Activities of catalase,superoxide dismutase and glutathione-S-transferase were decreased by isoproterenol administration in normal rats(Group 2).The diminished activities of these enzymes were generally negated by dietary interventions with fenugreek,garlic,and fenugreek+garlic.

Table2 Effect of dietary fenugreek and garlic on heart weight and ATPase activity in the heart tissue ofisoproterenol induced infarcted rats.

3.5.Effect of dietary fenugreek and garlic on antioxidant status in heart ofinfarcted rats

The lipid peroxides of heart tissue were increased by isoproterenol administration in HCD-fed rats which was accompanied by a significan decrease in glutathione concentration(TableA2).Dietary intervention with fenugreek + garlic alleviated the isoproterenol induced increase in lipid peroxides with associated depletion in glutathione concentration.Dietary intervention with fenugreek enhanced the concentration of α-Tocopherol in the heart tissue ofisoproterenol administered rats in normal situation.Isoproterenol administration generally brought about a significan decrease in the activities of antioxidant enzymes in heart tissue of HCD-fed rats (Table4).The decrease in the activities of catalase,glutathione s transferase,glutathione reductase,and superoxide dismutase were significantly prevented by pretreatments with dietary fenugreek,garlic,and fenugreek+garlic.

3.6.Effect of dietary fenugreek and garlic on antioxidant status in liver ofinfarcted rats

Liver is an active organ involved with the entire body’s lipid homeostasis;the antioxidant status of the same is also susceptible for change during atherogenic situation such as in high cholesterol feeding.Hence,the concentrations of antioxidant molecules and activities of relevant antioxidant enzymes were also determined in this organ in the present study involving experimentally induced infarcted rats,besides blood and cardiac tissues.The elevated level oflipid peroxides in the liver tissue caused by isoproterenol administration in normal situation(Group 2)and HCD-fed situation(Group 7)was accompanied by reductions in the concentrations of hepatic ascorbic acid and glutathione(TableA3).Increase in hepatic lipid peroxides during induction of myocardial infarction in HCD-fed rats was countered by interventions with dietary fenugreek,garlic,and fenugreek+garlic and was accompanied by significan increases in hepatic glutathione and α-tocopherol concentration.

The compromise in the activities of superoxide dismutase,glutathione s transferase,and glutathione peroxidase by isoproterenol administration in normal situation (Group 2) were invariablycounteredbythedietaryinterventionswithfenugreek,garlic,and fenugreek+garlic (Table5).Compromised activity of glutathione-S-transferase as a result ofisoproterenol administration in HCD-fed situation was countered by dietary fenugreek or fenugreek+garlic.

4.Discussion

The cardio protective influenc of dietary fenugreek seeds and garlic during induced myocardial infarction was evaluated in rats especially by virtue of their antioxidant effect in terms oflipid peroxidation,concentration of antioxidant molecules and activities of antioxidant enzymes.The disturbed activities of cardiac membrane-bound ATPases,increased serum iron,decreased total iron binding capacity,and decreased ceruloplasmin confirme myocardial infarction in isoproterenoladministered rats.Cardiac troponins are important diagnostic markers for myocardial infarction or heart muscle cell death.Several evidences have suggested that measurement of cardiac troponin is specifiand sensitive [24].In our present study,dietary interventions with fenugreek,garlic,and their combination significantl countered the increase in circulatory troponin level which would have leached from the injured myocardial tissue as a result ofisoproterenol administration.

Table3 Effect of dietary fenugreek and garlic on antioxidant enzymes in serum ofisoproterenol induced infarcted rats.

Table4 Effect of dietary fenugreek and garlic on antioxidant enzymes in heart tissue ofisoproterenol induced infarcted rats.

ATPases play a significan role in the contraction and relaxation cycles of the cardiac muscle by maintaining normal ionlevels within the myocyte.Several factors are known to alter the activities of ATPases especially lipid peroxidation and membrane fluidit.It has been reported that isoproterenol treatment resulted in a decrease in the activities of membrane bound ATPases[25].The loss of ATPase activity in the ischemic stage may be responsible for causing not only functional damage but also reversible necrotic changes in the involved myocardial cells.Inactivation of membrane-bound ATPases has been correlated with peroxidation of membrane lipids [26].Peroxidation of membrane lipids is believed to inactivate ATPases due to oxidation of thiol groups present in their active site and consequent conformational changes in the enzyme structure[27].Decreased Ca2+-ATPase activity can increase intracellular free calcium and hence alter the signal transduction pathways and cellular fluid ity[28].The diminished activities of cardiac membrane-bound Na+,K+-ATPase and Ca2+-ATPase were generally countered by dietary interventions with fenugreek or fenugreek+garlic.

Table5 Effect of dietary fenugreek and garlic on antioxidant enzymes in liver ofisoproterenol induced infarcted rats.

Garlic has been shown to exhibit a cardio protective effect against ischemic injury by inhibiting lipid peroxidation and thus enhancing the recovery of cardiac function [29].The antioxidant effect of fenugreek and garlic is probably responsible for the decrease in the activities of cardiac marker enzymes in serum with concomitant increase of the same in the heart tissue observed previously[9].Antioxidant potential of fenugreek and garlic has been recently reviewed [4].Lipid peroxidationin vivohas been identifie as one of the basic deteriorative reaction during myocardial ischemia[30].Lipid peroxides were higher in serum and heart as a result ofisoproterenol administration.Peroxidation of endogenous lipids might be a major factor involved in the cytotoxic effect ofisoproterenol.Pretreatment of rats with mangiferin and squalene have been shown to significantly decrease lipid peroxides both in serum and heart tissue in isoproterenol infarcted rats[31,32].

Ceruloplasmin is an extracellular antioxidant that can scavenge superoxide radicals [33]and inhibit ferritin dependent lipid peroxidation by catalyzing the oxidative re-incorporation of released iron into ferritin.Since ceruloplasmin has both ferro oxidase and copper-binding capacity,it could have been used more to neutralize the excess amount of free radicals and hence isoproterenol induced myocardial infarcted rats showed a decreased level of ceruloplasmin.The decreased levels of serum ceruloplasmin in normal as well as in HCD-fed situation during induction of myocardial infarction by isoproterenol administration was significantl countered by dietary fenugreek and garlic,the beneficia effect being higher in the case of fenugreek+garlic.Dietary fenugreek and garlic by their antioxidant effect would have prevented the loss of ceruloplasmin,thereby reducing the iron and copper mediated myocardial damage.

Total iron binding capacity is inversely related to the risk of myocardial infarction [34,35].The restoration ofiron binding capacity by dietary interventions with fenugreek,garlic,fenugreek+garlic is consistent with proportionate decreases in free iron concentration in serum.Similar increases in iron binding capacity following mangiferin and garlic oil administration have been reported in experimentally infarcted rats [29,36].In isoproterenol induced myocardial necrosis,free iron is released from heme containing proteins-hemoglobin and myoglobin with concomitant decrease in iron-binding capacity.Increased mobilization ofiron from ferritin in the heart by the enzyme xanthine oxidase and over-production of free radicals results in myocardial damage [37].The elevated levels of serum iron upon isoproterenol administration in these rats was significantl countered by dietary fenugreek,garlic,and fenugreek+garlic,the effect being higher in the case of combination.The increased plasma iron binding could have prevented hemolysis and iron catalyzed lipid peroxidation.This could be the reason for the decreased level ofiron and increased plasma iron binding capacity in fenugreek/garlic pretreated rats.

The increased levels oflipid peroxides indicate the excessive formation of free radicals resulting in irreversible damage to the heart in rats subjected to isoproterenol stress.Lipid peroxide is an important pathogenic event in myocardial infarction and the accumulated lipid peroxides reflec the various stages of the diseaseanditscomplications[38].Increasedleveloflipidperoxides injures blood vessels,causing increasing adherence and aggregation of platelets to the injured sites.The significan increase in lipid peroxides in serum and heart ofisoproterenol infarcted rats observed in the current study is in accordance with earlier similar reports[39,40].The elevated lipid peroxides in serum and heart caused by isoproterenol administration in normal or in HCD-fed situation was accompanied by reductions in the concentrations of one or more antioxidant molecules among ascorbic acid,αtocopherol and glutathione,and particularly of glutathione.This was countered by the interventions with dietary fenugreek,garlic,andfenugreek+garlicwhichwereaccompaniedbyincreases in antioxidant molecules.Glutathione plays an important role in the regulation ofivarious cell functions and in cell protection from oxidative injury.Depletion of glutathione results in enhanced lipid peroxidation and excessive lipid peroxidation cause increased glutathione consumption [41]as observed in the present study.

Garlic pretreated isoproterenol administered rats are reported to have maintained the level oflipid peroxides to near normal when compared to un-pretreated controls.This has been attributed to the protective effect of garlic on the myocardium,reducing the myocardial damage thereby restricting the leakage of these marker enzymes.Isoproterenol administered rats showed a significan decrease in ascorbic acid and glutathione levels in serum and liver.Decreased glutathione levels in isoproterenol administration may be due to its increased utilization in protecting thiol-containing proteins from lipid peroxidation [29].GSH functions as a free radical scavenger in the repair of radical induced cellular damage.Decreased glutathione was observed during increased oxidative stress caused by isoproterenol administration [29].This observation supports our findin where we are reporting a decline in glutathione level in isoproterenol administered animals.Pretreatment with garlic oil helped maintain glutathione levels near normal during isoproterenol administration[42].

In this investigation,dietary interventions with fenugreek and garlic generally countered the alterations in the activities of antioxidant enzymes associated with myocardial necrosis.The decrease in the activities of antiperoxidative enzymes-catalase and superoxide dismutase,and of glutathione dependent antioxidant enzymes glutathione-S-transferase and glutathione reductase were significantl prevented by pretreatments with dietary fenugreek,garlic,and fenugreek+garlic in both normal and HCD-fed situations.The diminished activities of catalase,superoxide dismutase and glutathione-S-transferase in serum ofinormal as well as HCD-fed rats as a result ofisoproterenol administration were negated by dietary interventions with fenugreek,garlic,and fenugreek+garlic.Dietary interventions with fenugreek,garlic,fenugreek+garlic even countered the elevated activities of catalase,superoxide dismutase,and glutathione reductase and glutathione-S-transferase in heart caused by isoproterenol administration in HCD-fed situation.The reduction observed in the activity of GST in isoproterenol induced myocardial infarction might be due to decreased availability ofits substrate mainly reduced glutathione.The compromise in the activities ofivarious antioxidant enzymes by isoproterenol administration in normal situation were invariably countered by the dietary interventions with fenugreek,garlic,and fenugreek+garlic.Compromised activity of glutathione-S-transferase as a result ofisoproterenol administration in HCD-fed situation was countered by dietary fenugreek or fenugreek+garlic in the myocardial tissue.

It is intriguing to note that high cholesterol feeding improved antioxidants (when compared to controls) in a few instances.Such improvements in antioxidants are an adaptive mechanism by the organism and have been reported in the literature [43].Although there was no synergy among the dietary fenugreek and garlic in alleviating the oxidative stress,the beneficia effect was nevertheless higher in the case of majority of antioxidant parameters of blood,heart,and liver.

Murugesan et al.[44]and Panda et al.[45]have observed the cardioprotective effect ofintragastrically administered fenugreek(250 mg/kg body mass for 15 days)or trigonelline isolated from fenugreek seed,as evident from significantl decreased levels of TBARS and enhanced activities of antioxidant enzymes and antioxidant molecule glutathione in myocardial infarcted rats.Our present study which examined dietary fenugreek(10%)for 8 weeks also showed the cardio protective effect through lipid peroxidation and antioxidant defense system during isoproterenol induced myocardial infarction in rats.Our present study has elucidated the antioxidant role of whole garlic (2%freeze-dried powder).A marked reversal of decreased activities of antioxidant enzymes in the heart tissue of myocardial infarcted rats as a result of daily administration of commercial garlic oil (75 mg/kg body mass) has been reported [29].Similar to the results observed by Banerjee et al.[46]and Asdaq and Inamdar [47]with chronic administration of raw garlic(500 mg/kg body mass for 30 days),our dietary administration of the same also offered protection against isoproterenol induced myocardial necrosis and associated oxidative stress in myocardial tissue.Absence of any benefiof garlic on glutathione levels and the activity of glutathione peroxidase in the myocardium ofinfarcted rats agrees with the report of Banerjee et al.[46].Our present study additionally provides information on the cardio protective influenc of fenugreek and garlic not only individually consumed,but also when these two are consumed together.Evaluation of the cardio protective influenc through beneficia modulation antioxidant stress associated with myocardial infarction under condition of hypercholesterolemia is yet another novelty of our investigation.

In summary,the current study demonstrated that dietary fenugreek and garlic significantl alleviated isoproterenol induced myocardial necrosis.Decreased myocardial necrosis as evidenced by restored activities of cardiac membrane-bound ATPase enzymes and histoarchitectural changes and augmentation of endogenous antioxidants,all contribute to their cardio protective potential.Thus,pretreatment with dietary fenugreek seeds,garlic or fenugreek+garlic for 8 weeks was found to ameliorate the effect ofisoproterenol-induced pathological changes in heart,and compromised antioxidant status,the beneficia effect being higher in the case of dietary intervention with the combination of fenugreek seeds and garlic.Cardiac hypertrophy(heart to body mass ratio) observed in myocardial infarction of HCD-fed rats was effectively countered particularly by the combination of fenugreek+garlic It would be interesting to observe whether the improvements in cardiac antioxidant activities and ATPase pumps due to fenugreek,garlic,and fenugreek+garlic supplementation in the diseased hearts,actually translate into functional benefits Since inflammatio is also a major contributor in the development and progression of heart disease,it would be interesting to examine the effects of fenugreek and garlic supplementation both individually and in combination on inflammator markers such as TNF-α or IL-6.

Thus,dietary fenugreek seeds,garlic or fenugreek+garlic ameliorated the effect ofisoproterenol-induced pathological changes in heart,and compromised antioxidant status,the beneficia cardio protective effect being higher in the case of dietary intervention with the combination of fenugreek seeds and garlic.The antioxidant property of fenugreek and garlic as evidenced in this study has far-reaching nutraceutical value.The antioxidant properties of these spices are of particular interest in view of the impact of suppression of oxidative stress in the development of cardiovascular disease.Although the beneficia antioxidative influenc of these spices were seen with concentrations more than the normal levels found in Indian diets,such higher levels can be comfortably consumed to derive the health beneficia antioxidant effects.

Conflict ofinterest

The authors declare that there are no conflict ofinterest.

Acknowledgement

The firs author (PM) is grateful to the Indian Council of Medical Research,New Delhi for the award of Senior Research Fellowship.

Appendix A

Tables A1-A3.

TableA1 Effect of dietary fenugreek and garlic on antioxidant molecules in serum ofisoproterenol induced infarcted rats.

TableA2 Effect of dietary fenugreek and garlic on antioxidant molecules in heart tissue ofisoproterenol induced infarcted rats.

TableA3 Effect of dietary fenugreek and garlic on antioxidant molecules in liver ofisoproterenol induced infarcted rats.