Cardiovascular pharmacology

Diosmin exhibits anti-hyperlipidemic effects in isoproterenol inducedmyocardial infarcted rats

S.Sharmila Queenthy a,b, Babu John c,n

a Department of Chemistry, Manonmanium Sundarnar University, Tirunelveli, Tamil Nadu, Indiab Department of Biotechnology, Waljat College of Applied Sciences, Muscut, Omanc P.G and Research Department of Chemistry, Government Arts College, C.Mutlur, Chidambaram, Tamilnadu, India

a r t i c l e i n f o

Article history:Received 16 February 2013Received in revised form15 August 2013Accepted 30 August 2013Available online 12 September 2013

Keywords:DiosminElectrocardiogramIsoproterenolMyocardial infarctionLipidsLipo proteins

a b s t r a c t

The aim of the present study was to evaluate the protective effects of diosmin on experimentally inducedmyocardial infarcted rats. Diosmin (5 and 10 mg/kg body weight) was administered orally as pretreat-ment daily for a period of 10 days. Then isoproterenol (100 mg/kg) was injected subcutaneously into ratsat an interval of 24 h for 2 days (on 11th and 12th day). Isoproterenol-induced myocardial infarcted ratsshowed significant changes in electrocardiogram and an increase in the levels of cardiac markers,compared with normal rats. Additionally, increased plasma lipid peroxidation products and altered lipidmetabolism in the plasma were observed in the isoproterenol-induced myocardial infarcted rats.Pretreatment with diosmin (5 and 10 mg/kg body weight) minimized the electrocardiographic changes,decreased the levels of serum cardiac marker enzymes reduced plasma lipid peroxidation and minimizedthe alterations in the lipid metabolism of isoproterenol-induced myocardial infarcted rats. Also, diosmininhibited the enhanced activity of liver HMG CoA reductase. The in vitro study revealed the free radicalscavenging activity of diosmin. The free radical scavenging and anti-hyperlipidaemic effects are thereasons for the cardioprotective effects of diosmin.

& 2013 Elsevier B.V. All rights reserved.

1. Introduction

Myocardial infarction is a disease that occurs when the cor-onary arteries reduce the blood supply to a part of the myocar-dium that leads to hypoxia (Sangeetha and Darlin Quine, 2008).Cardiovascular risk factors have long been studied in order to gaininsight into the pathophysiology, epidemiology and therapy ofacute myocardial infarction (López Messa et al., 2004). As datafrom the original Framingham cohort were analyzed, newercohorts were added over the years, such as life style and diet,smoking, high blood pressure, altered lipid profile and obesity asimportant risk factors for myocardial infarction (O’Donnell andElosua, 2008).

Therapeutic doses of isoproterenol (L-β-(3, 4-dihydroxyphenyl)-α-isopropyl aminoethanol hydrochloride), a β-adrenergic receptor ago-nist are regulating heart function. But in excessive dose, isoproterenoldepletes the energy reserve of cardiomyocytes and thus results inbiochemical and structural changes, which are responsible for thedevelopment of irreversible damage (Aman Upaganlawar et al., 2011).Isoproterenol also increases the intracellular levels of calcium, there-by causing myocardial infarction. It serves as a well accepted and

standardized model to study the cardio protective effects of new drugsin preclinical trials.

The existing pharmacotherapy is effective in managing themyocardial infarction but with multiple adverse effects and drug–drug interactions (Wiffen et al., 2002). These disadvantages haveenhanced the need for alternative phytoconstituents, which maybe used to prevent the cardiovascular diseases. Flavonoids arepresent in fruits and various plants, which are pharmacologicallyactive and can be used for the treatment of degenerative diseases.They have multiple biological activities including vasodilator(Duarte et al., 1993), anticarcinogenic, anti-inflammatory, antibac-terial, immune-stimulating, antiallergic, and antiviral effects(Brown, 1980; Middleton and Kandaswami, 1992). In general,antioxidant activity of flavonoids is closely related to preventiveeffects on various diseases including arteriosclerosis, liver injury,and carcinogenesis (Ho et al., 1994).

Diosmin (3′, 5,7-Trihydroxy-4′-methoxyflavone 7-rutinoside) isan unsaturated flavone that can be found mainly in Hyssopand Rosemary (Del Baño et al., 2004; Camarda et al., 2007). Itexhibits anti- inflammatory, antioxidant and anti-mutagenic prop-erties (Kuntz et al., 1999) Literature survey revealed that there areno scientific reports available on the effects of diosmin in myo-cardial infarction. The aim of the study is to understand theprotective effect of diosmin in isoproterenol induced myocardialinfarcted rats.

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journal homepage: www.elsevier.com/locate/ejphar

European Journal of Pharmacology

0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.ejphar.2013.08.031

n Corresponding author. Tel.: þ91 4144 292159; fax: þ91 4144 292159.E-mail address: babu.bj@gmail.com (B. John).

European Journal of Pharmacology 718 (2013) 213–218

2. Materials and methods

2.1. Experimental animals

All the experiments were carried out with healthy male albinoWistar rats (Rattus norvegicus) weighing 180–200 g, purchasedfrom Mahaveer Enterprises, Hyderabad, India. They were housedin polypropylene cages (47�34�20 cm3) (3 rats per cage) linedwith husk, renewed every 24 h under a 12 h light/dark cycle ataround 22 1C with 50% humidity. The rats had free access to waterand food. The rats were fed on a standard pellet diet (Pranav AgroIndustries Ltd., Pune, and Maharashtra, India). The experimentswere carried out according to the guidelines of the Committee forthe Purpose of Control and Supervision of Experiments on Ani-mals, New Delhi, India and approved by the Institutional AnimalEthical Committee of Jayamukhi College of Pharmacy (Approvalno. 02; 10/06/2011).

2.2. Chemicals

Diosmin, a rhamnoglucoside (rutinoside), extracted from citrusspecies at 95% purity, and isoproterenol were purchased from SigmaChemical Co., St. Louis, MO, USA. Sodium sulfite, dimethyl sulfoxide,potassium tetra borate, nitroblue tetrazolium and hydroxylaminehydrochloride were purchased from Himedia, Mumbai, India. Allother chemicals and reagents used in the study were of analyticalgrade.

2.3. Induction of experimental myocardial infarction

Isoproterenol (100 mg/kg body weight) was dissolved in salineand injected subcutaneously into rats at an interval of 24 h for2 days to induce myocardial infarction (Punithavathi and StanelyMainzen Prince, 2010; Stanely Mainzen Prince, 2011).

2.4. Experimental design

The rats were divided into six groups of six rats each. Group I:normal rats were given 2 ml of saline orally by gastric intubationdaily for a period of 10 days; Group II: rats were treated withdiosmin (5 mg/kg) dissolved in 2 ml of saline orally by gastricintubation daily for a period of 10 days; Group III: rats weretreated with diosmin (10 mg/kg) dissolved in 2 ml of saline orallyby gastric intubation daily for a period of 10 days; Group IV: ratswere given 2 ml of saline orally by gastric intubation daily for aperiod of 10 days and then injected subcutaneously with isopro-terenol (100 mg/kg) in 2 ml of saline at an interval of 24 h for2 days (on 11th and 12th day); Group V: rats were pretreated withdiosmin (5 mg/kg) dissolved in 2 ml of saline orally by gastricintubation daily for a period of 10 days and then injectedsubcutaneously with isoproterenol (100 mg/kg) at an interval of24 h for 2 days (on 11th and 12th day); Group VI: rats werepretreated with diosmin (10 mg/kg) dissolved in 2 ml of salineorally by gastric intubation daily for a period of 10 days and theninjected subcutaneously with isoproterenol (100 mg/kg) at aninterval of 24 h for 2 days (on 11th and 12th day).

2.5. Electrocardiogram

Electrocardiographic patterns in the normal and experimental ratswere recorded according to the method of Mari Kannan and DarlinQuine (2011). Twenty four hours after the second dose of isoproter-enol, rats of all the groups were anesthetized with ketaminehydrochloride (100 mg/kg body weight) intraperitoneally and theelectrocardiographic patterns were recorded by a 16 channel poly-graph (Biopac systems Inc., USA). The types of alterations (P wave,

QRS complex, ST-segment elevation, RR interval) in the normal andexperimental rats were recorded.

2.6. Analysis of cardiac markers.

The level of cardiac troponin-I in the serum was estimated byVITROS immunodiagnostic kit purchased from the Ortho-ClinicalDiagnostics, Inc. New York, USA. Creatine kinase-MB in the serumwas estimated by a standard diagnostic kit obtained from AccurexPrivate Ltd, Mumbai, India.

2.7. Estimation of lipid peroxidation products

The levels of plasma thiobarbituric acid reactive substances wereestimated by the method of Yagi et al. (1998). Four ml of 0.083 Nsulfuric acid were added to 0.5 ml of plasma. To this mixture, 0.5 mlof 10% phosphotungstic acid was added and mixed thoroughly. Afterallowing the tubes to stand at room temperature for 5 min, themixture was centrifuged at 3000� g for 10 min. The supernatant wasdiscarded and the sediment was mixed with 2 ml of sulfuric acid and0.3 ml of 10% phosphotungstic acid. The mixture was shaken welland centrifuged at 3000� g for 10 min. The sediment was suspendedin 4 ml of double distilled water and 1 ml of thiobarbituric acidreagent was added. The reaction mixture was heated at 95 1C for60 min. After cooling, 5 ml of n-butanol was added and the mixturewas shaken vigorously and centrifuged at 3000� g for 15 min. Thecolor extracted in the n-butanol layer was measured at 530 nm in aUV-Spectrophotometer.

Lipid hydroperoxides in the plasma were estimated by themethod of Jiang et al. (1992). 1.8 ml of Fox reagent was added to0.2 ml of plasma and incubated for 30 min at room temperature andthe absorbance was measured at 560 nm in a UV-Spectrophotometer.

2.8. Estimation of lipid profile

Lipid profile was estimated in the plasma and heart tissuehomogenates. Lipids were extracted from the heart tissue by themethod of Folch et al. (1957). This lipid extract was used forfurther analysis. The total plasma cholesterol and triglyceridecontents were estimated by reagent kits (Qualigens Diagnostics,Mumbai, India). Plasma high density lipoprotein (HDL) level wasestimated by a reagent kit (Auto Zyme: HDL-Cholesterol) fromAccurex Diagnostics Private Limited, Mumbai, India. Plasma lowdensity lipoprotein (LDL) cholesterol and very low density lipo-protein (VLDL) cholesterol were calculated as follows: LDL–cho-lesterol¼Total cholesterol– (HDL-cholesterol þVLDL-cholesterol);VLDL-cholesterol¼Triglycerides/5 (Rajadurai and Stanely MainzenPrince, 2006).

2.9. Assay of 3-hydroxy-3 methyl glutaryl CoA reductase(HMG CoA reductase)

The activity of HMG-CoA reductase was assayed in the liver bythe method of Rao and Ramakrishnan (1975). The ratio of HMGCoA to mevalonate was taken as an index of enzyme activity whichcatalyzes the conversion of β-hydroxy-β-methyl glutaryl CoA tomevalonate. Lower ratio of β-hydroxy-β-methyl glutaryl CoA tomevalonate indicates higher enzyme activity and higher ratio of β-hydroxy-β-methyl glutaryl CoA to mevalonate indicates lowerenzyme activity.

2.10. Assessment of cardiac indices and left ventricular hypertrophy

The heart was then removed, cleaned from fat and fibroustissue, and dried with filter paper before determining the weightof the whole heart and the left ventricle. Cardiac indices were

S.Sharmila Queenthy, B. John / European Journal of Pharmacology 718 (2013) 213–218214

calculated as the ratio of whole heart weight to body weight, andthe ratio of left ventricle weight to body weight was calculated toassess left ventricular hypertrophy (El-Bahai et al., 2009).

2.11. Determination of superoxide radical scavenging activity in vitro

The in vitro superoxide radical scavenging activity of diosminwas determined by the method of Sabu and Kuttan (2002). One mlof NBT (100 mM of NBT in 100 mM phosphate buffer, pH 7.4), 1 mlof NADH solution (14.68 mM of NADH in 100 mM phosphatebuffer) and varying volumes of diosmin (10–50 μg/ml)were mixedwell. The reaction was started by the addition of 100 mM of PMS in(60 mM / 100 mM phosphate buffer, pH 7.4). The reaction mixturewas incubated at 30 1C for 15 min. The absorbance was measuredat 560 nm in a spectrophotometer. Incubation without diosminwas used as blank. Butylated hydroxytoluene was used as apositive control for comparison. Decreased absorbance of thereaction mixture was recorded 517 nm in a Systronics UV–visiblespectrophotometer. Decreased absorbance of the reaction mixtureindicates increased superoxide anion scavenging activity. All thesamples were treated in a similar manner. Absorbance wasrecorded and the percentage of inhibition was calculated.

2.12. Determination of hydroxyl radical scavenging activity in vitro

The in vitro hydroxyl radical scavenging capacity of diosmin wasmeasured using modified method as described by Halliwell et al.(1987). The incubation mixture in a total volume of 1 ml contained0.1 ml of 100 mM of potassium dihydrogen phosphate-KOH buffer,varying volumes of diosmin (10–50 mM), 0.2 ml of 500 mM of ferricchloride, 0.1 ml of 1 mM of ascorbic acid, 0.1 ml of 1 mM of EDTA,0.1 ml of 10 mM of H2O2 and 0.2 ml of deoxyribose. The contentswere mixed thoroughly and incubated at room temperature for60 min. Then 1 ml of 1% TBA (1 g in 100 ml of 0.05 N NaOH) and 1 ml

of 28% TCA were added. All the tubes were kept in a boiling waterbath for 30 min. The absorbance was read in a spectrophotometer at532 nm with reagent blank containing distilled water in place ofdiosmin. The percentage scavenging activity was determined.Decreased absorbance of the reaction mixture indicated increasedhydroxyl radical scavenging activity. The hydroxyl radical scavengingactivity of diosmin was reported as the percentage of inhibition ofdeoxyribose degradation.

2.13. Statistical analysis

Results are expressed as mean7standard deviation. One wayanalysis of variance (ANOVA) was carried out, and the statisticalcomparisons among the groups were performed with Duncan'smultiple range test (DMRT) using a software, statistical package forthe social science (SPSS) version 16.00. Throughout the report,wherever we have used the word “significant” to describe results,we mean “statistically significant at an alpha level of 0.05(Po0.05) for the two-sided alternative hypothesis.

3. Results

We analyzed the function of cardiac conduction system byrecording electrocardiogram patterns of normal and experimentalrats (Fig. 1). We observed the changes in the various phases of eachcardiac cycle. Normal (Group-I) and diosmin treated rats (Group-II) showed no change in the cardiac conduction activity with nochanges in depolarization waves and repolarization wave. The ratsinduced with MI by isoproterenol (Group-III) showed significantchanges in ST-segment, QT interval, P wave, QRS complex and RRinterval compared to normal rats. These changes are almostsimilar to the indications of MI. Oral pretreatment with diosminin isoproterenol-induced rats (5 and 10 mg/kg) (Group-IV and V)

Fig. 1. (A–F): Effects of diosmin on electrocardiographic patterns in normal and experimental rats. (A). Electrocardiogram pattern of normal rats showing normalcardiograph. (B). Electrocardiogram pattern of diosmin (5 mg/kg) treated rats showing normal cardiograph. (C). Electrocardiogram pattern of diosmin (10 mg/kg) treated ratsshowing normal cardiograph. (D). Electrocardiogram pattern of isoproterenol (100 mg/kg)-induced rats showing pathological changes such as ST-segment elevation. (E).Electrocardiogram pattern of diosmin (5 mg/kg) treated isoproterenol-induced rats showing minimized ST-segment elevation. (F). Electrocardiogram pattern of diosmin(10 mg/kg) pretreated isoproterenol-induced rats showing almost normal cardiograph without any elevation in ST-segment.

S.Sharmila Queenthy, B. John / European Journal of Pharmacology 718 (2013) 213–218 215

showed a significant improvement in the ECG parameters, com-pared to isoproterenol-induced rats (Group-III).

In the isoproterenol-induced myocardial infarcted rats, signifi-cant (Po0.05) increases in the levels of cardiac markers such astroponin- I and creatine kinase—MB fraction in the serum wereobserved. Pretreatment with diosmin significantly (Po0.05)decreased the levels of these cardiac markers in the serum,compared to non-treated isoproterenol-induced myocardialinfarcted rats (Table 1).

The isoproterenol-induced myocardial infarcted rats showed asignificant (Po0.05) increase in the levels of plasma thiobarbituricacid reactive substances and lipid hydroperoxides compared withthe normal rats. Pretreatment with diosmin significantly (Po0.05)reduced the levels of plasma thiobarbituric acid reactive sub-stances and lipid hydroperoxides, compared to rats induced withisoproterenol alone that reflecting the anti lipid-peroxidativeeffect of diosmin (Fig. 2).

Isoproterenol-induced myocardial infarcted rats showed a sig-nificant (Po0.05) increase in the levels of plasma total cholesterol,LDL-cholesterol, VLDL-cholesterol, triglycerides and a significantdecrease in the levels of plasma HDL-cholesterol compared withnormal group. Pretreatment with diosmin significantly (Po0.05)decreased the levels of plasma total cholesterol, LDL-cholesterol,VLDL-cholesterol and significantly (Po0.05) increased the levelsof plasma HDL-cholesterol (Table 2).

The significant (Po0.05) increase in the activity of HMG-CoAreductase in the liver of isoproterenol-induced myocardial infarctedrats was indicated by the lower ratio of HMG-CoA/mevalonate.Pretreatment with diosmin significantly (Po0.05) decreased theactivity of HMG-CoA reductase, compared to isoproterenol-inducedmyocardial infarcted rats and it is indicated by higher ratio of HMGCoA/mevalonate (Fig. 3).

The mean body weight of rats in all experimental groups hadno significant change (data not shown in the results). The ratio ofleft ventricle weight to body weight was significantly (Po0.05)increased in isoproterenol-induced rats as compared to the normalrats (Fig. 4). Pretreatment with diosmin significantly (Po0.05)reduced the ratio of left ventricle weight to body weight indicatingthe antihypertrophic effects of diosmin.

For all the biochemical parameters studied, treatment withdiosmin (5 mg/kg and 10 mg/kg) (Groups III and IV) daily for aperiod of 10 days to the normal rats did not show any significanteffect. Also, pretreatment with diosmin (10 mg/kg body weight)(Group VI) showed a higher effect than the lower dose (5 mg/kgbody weight) (Group V).

Diosmin showed positive response with the scavenging ofsuperoxide and hydroxyl free radicals in vitro. The percentagescavenging effects of diosmin on superoxide free radical atdifferent concentrations (10, 20, 30, 40 and 50 mM) were found

Table 1Effects of diosmin on serum cardiac markers.

Groups CK MB (IU/l) Troponin–I (ng/ml)

Normal 7.7170.59a 0.2370.01a

Diosmin (5 mg/kg) 7.9370.48a 0.2470.01a

Diosmin (10 mg/kg) 7.7370.57a 0.2370.01a

ISO (100 mg/kg) 17.9271.07b 0.9870.06b

Diosmin (5 mg/kg)þ ISO 13.2270.84c 0.5770.05c

Diosmin (10 mg/kg)þ ISO 9.3770.59d 0.3670.02d

ISO—isoproterenol, each value is mean7standard deviation for six rats in eachgroup, values not sharing a common superscript (a,b,c,d) differ significantly witheach other (Po0.05), Duncan's multiple range test).

* * * §

* * * §

0 2 4 6 8

10 12 14 16

I II III IV V VI

TBA

RS:

nm

ol/m

L;

LOO

H: v

alue

s x1

0-5nm

ol/m

l/dl.

Groups

Thiobarbituric acid substances Lipid hydroperoxides

Fig. 2. Effects of diosmin on plasma lipid peroxidation products. Group I: Normal;Group II: Diosmin (5 mg/kg); Group III: Diosmin (10 mg/kg); Group IV: Isoproter-enol -induced rats (100 mg/kg); Group V: Diosmin (5 mg/kg)þ Isoproterenol;Group VI: diosmin (10 mg/kg)þ Isoproterenol. Each column is mean7standarddeviation for six rats in each group. Columns not sharing a symbol (n, †, ‡, §)differ significantly with each other. Unitsn: TBARS: nmol/ml; LOOH: values�10–5 mmol/Dl.

Table 2Effects of diosmin on lipid profile in the plasma.

Normal Diosmin (5 mg/kg) Diosmin (10 mg/kg) ISO (100 mg/kg) Diosmin (5 mg/kg)þISO (100 mg/kg)

Diosmin (10 mg/kg)þISO (100 mg/kg)

Total cholesterol (mg/dl) 83.7273.42a 82.3775.90a 81.9975.63a 131.5779.28b 114.7976.06c 91.9775.51d

HDL-Cholesterol (mg/dl) 37.7072.21a 36.2972.24a 35.6072.44a 20.0671.83b 25.8071.97c 32.0273.31d

LDL-Cholesterol (mg/dl) 39.4472.82a 39.5475.83a 39.9376.60a 100.4177.68b 80.3375.86c 52.3276.82d

VLDL-Cholesterol (mg/dl) 6.5870.36a 6.5570.38a 6.4670.36a 11.1070.65b 8.6670.45c 7.6470.51d

Triglycerides (mg/dl) 32.5771.34a 33.7571.57a 32.3071.84a 55.9572.48b 42.5771.62c 37.0171.66d

ISO—isoproterenol, each value is mean7standard deviation for six rats in each group, values not sharing a common superscript (a,b,c,d) differ significantly with each other(Po0.05, Duncan's multiple range test).

* * * §

0

1

2

3

I II III IV V VI

Rat

io o

f 3-h

ydro

xy-3

-met

hyl-

glut

aryl

-CoA

to m

eval

onat

e

Groups

Fig. 3. Effects of diosmin on liver HMGCo-A reductase activity. Group I: Normal;Group II: Diosmin (5 mg/kg); Group III: Diosmin (10 mg/kg); Group IV: Isoproter-enol -induced rats (100 mg/kg); Group V: Diosmin (5 mg/kg)þ Isoproterenol;Group VI: Diosmin (10 mg/kg)þ Isoproterenol. Each column is mean7standarddeviation for six rats in each group. Columns not sharing a symbol (n, †, ‡, §) differsignificantly with each other.

S.Sharmila Queenthy, B. John / European Journal of Pharmacology 718 (2013) 213–218216

to be 20.24, 35.48, 52.37, 75.66 and 92.47% respectively. Thepercentage scavenging effects of diosmin on hydroxyl radical atdifferent concentrations (10, 20, 30, 40 and 50 mM) were found tobe 18.73, 33.89, 47.54, 72.56 and 87.65% respectively. These resultsclearly show the effects of diosmin to scavenge superoxide andhydroxyl free radicals even at lower doses.

4. Discussion

ECG is a good tool to analyze the function of heart. Theanatomical difference of the atria and the ventricles, their sequen-tial activation, depolarization and repolarization are the reasons toproduce clearly differentiable deflections in electrical conductivityand these deflections are recorded using ECG. The decreased QRScomplex may lead to inadequate contractile activity in theisoproterenol-induced myocardial infarcted rats.

Another strong finding was positive correlation between ST-segment elevation and a hyper acute T wave. Pharmacoepidemio-logical analysis clearly indicated that ST segment elevationremains one of the adverse predictors of myocardial infarctionand it is also considered as a reliable biomarker in the clinical trials(Cannon et al., 1997). We also observed pathological Q wave in theisoproterenol-induced myocardial rats. These changes clearly indi-cate that there is some abnormality with respect to the conduc-tivity and contractility of the heart. In this context, a previousstudy revealed that after 24 h of isoproterenol administration,decreased QRS complex, ST-segment elevation and a hyper acute Twave were observed in ECG of Wistar rats (Mari Kannan andDarlin Quine, 2013). Pretreatment with diosmin significantlyreduced the pathological changes in the ECG of isoproterenol-induced rats.

The levels of troponins are the ideal biochemical markers,which are present at high concentration in the myocardium,absent in non-cardiac tissue and released rapidly following myo-cardial damage. The release of cardiac troponins has been corre-lated with the severity of infarction in experimental animalmodels (Bertinchant et al., 2000). The higher levels of troponin-Iobserved in the serum indicate that there is a myocardial damagein isoproterenol-induced myocardial infarcted rats. The increasedactivity of serum creatine kinase MB (CK-MB) in the isoproterenol-induced myocardial infarcted rats confirmed that there is myo-cardial damage. Pretreatment with diosmin (5 and 10 mg/kg)blocked the toxic effect of excessive dose of isoproterenol andinhibited cardiac damage, which was indicated by the decreasedlevels of cardiac markers in the diosmin pretreated rats. The

results showed the cardioprotective effects of diosmin in themyocardium.

The excessive free radicals produced by isoproterenol, damageslipophilic cell membrane which is indicated by the elevated lipidperoxidation in terms of thiobarbituric acid reactive substancesand lipid hydroperoxides. The observed increase in the plasmalipid peroxidation products in isoproterenol-induced myocardialinfarcted rats indicating that there is an increased lipid peroxida-tion. Becker (2004) reported that Haber–Weiss reaction stimulatedby excessive amount of superoxide radicals formed by isoproter-enol, and initiating lipid peroxidation. Rats pretreated with dios-min significantly reduced lipid peroxidation in the plasma ofisoproterenol- induced myocardial infarcted rats by its antilipidperoxidation property.

The altered lipid profile is a matter of great concern given theirrole in MI. It has been suggested that hyperlipidaemia andhypertriglyceridaemia are directly related to coronary artery dis-eases. The obtained results of diosmin encouraged us to focus onits antihyperlipidaemic effect for better understanding of its modeof action. In the present study, an altered plasma lipid profile wasobserved in isoproterenol-induced myocardial infarcted rats. Car-diac cyclic adenosine monophosphate enhanced lipid biosynthesisduring the hypoxic condition is the reason for the increased levelsof lipids in isoproterenol-induced myocardial infarcted rats(Leroith, 2007). Pretreatment with diosmin significantly decreasedthe levels of plasma total cholesterol in isoproterenol-inducedmyocardial infarcted rats by its antihypercholesterolemic effects.

Paritha and Devi (1997) reported that decreased activity oflipoprotein lipase reduced the uptake of triglycerides from thecirculation. This is the reason for the observed increased levels ofplasma triglycerides in isoproterenol-induced myocardial infarctedrats. Pretreatment with diosmin significantly reduced the levels ofplasma triglycerides in isoproterenol-induced myocardial infarctedrats.

Decreased number of LDL receptors in isoproterenol-inducedmyocardial infarcted rats is the reason for high levels of LDL-cholesterol. In addition, the decreased levels of HDL make theheart vulnerable to oxidized LDL and cause myocardial infarction.The increased lipolysis induced by isoproterenol is the reason forincreased VLDL-cholesterol. Pretreatment with diosmin signifi-cantly minimized the alterations in lipoproteins and regulatedlipid profile. Diosmin was reported for its protective effect onisolated human lipoproteins from in vitro peroxidation (Dumonet al., 1994). The same action may be responsible for the protectiveeffects against lipoprotein changes in isoproterenol-induced myo-cardial infarcted rats.

Most of the drugs acting positively on lipid profile are actingthrough HMG-CoA reductase, a rate limiting enzyme in cholesterolsynthesis. The beneficial effects of diosmin on lipid profile stimulatedus to find out its effect on the activity of HMG-CoA reductase. Theobserved increased activity of HMG-CoA reductase in the liver ofisoproterenol-induced myocardial infarcted rats resulted in increasedlevels of cholesterol. Fascinatingly, we observed that diosmin sig-nificantly decreased the activity of this enzyme in the liver ofmyocardial infarcted rats. The observed effect of diosmin on theHMG-CoA reductase is the reason for the decreased levels of lipids.

Free radicals play a deleterious role in the biological systemsand act as a major cause for degenerative disorders. Generally,flavonoids inhibit oxygen free radical formation. As diosminproduced cardioprotective effects in this study, we are interestedto find out the superoxide and hydroxide radical scavengingeffects of diosmin in vitro. The in vitro studies on diosmin showedits free radical scavenging activity. The hydroxyl free radicalscavenging and superoxide free radical scavenging activities ofdiosmin were evidenced by the in vitro studies. The dose depen-dent activity of diosmin confirmed that the small quantity of the

§***

0

0.0005

0.001

0.0015

0.002

0.0025

I II II IV V VIGroups

Rat

io o

f lef

t ven

tric

le w

eigh

t (m

g)

/who

le b

ody

wei

ght (

g)

Fig. 4. Effects of Diosmin on left ventricular hypertrophy. Group I: Normal; GroupII: Diosmin (5 mg/kg); Group III: Diosmin (10 mg/kg); Group IV: Isoproterenol-induced rats (100 mg/kg); Group V: Diosmin (5 mg/kg)þ Isoproterenol; Group VI:Diosmin (10 mg/kg)þ Isoproterenol. Each column is mean7standard deviation forsix rats in each group. Columns not sharing a symbol (n, †, ‡, §) differ significantlywith each other.

S.Sharmila Queenthy, B. John / European Journal of Pharmacology 718 (2013) 213–218 217

drug can also produce positive impact. This free radical scavengingmechanism of diosmin may be the reason for its protective effectagainst isoproterenol-induced cardio toxicity.

In conclusion, pre-treatment with diosmin provides cardiopro-tection by preventing the accumulation of lipids by its free radicalscavenging and antihyperlipidaemic effects. Also, diosmin pre-treatment appears to be instrumental in correcting myocardialrhythm by regulating electrical conductivity within the myocar-dium. The improvement in status of lipid profile by HMG-CoAreductase inhibiting capacity of diosmin further contributes to itsoverall cardio protective property.

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