Research ArticleCardioprotective Activity of11987310158401015840119873101584010158401015840-Bis[5-methyl-2-oxo-12-dihydro-3H-indol-3-ylidene]carbonohydrazide Derivativeagainst Doxorubicin Induced Cardiotoxicity in Rats

Salma Tabassum1 Kiran Gangarapu23 Gouthami Thumma4

Sarangapani Manda5 and Rama Narsimha Reddy Anreddy1

1 Department of Pharmacology Vaageswari College of Pharmacy Rama Krishna Colony Beside LMD Police StationKarimnagar Andhra Pradesh 505 481 India

2Department of Pharmaceutical Chemistry Kakatiya Institute of Pharmaceutical Sciences Pembarthy HasanparthyWarangal 506 371 India

3 Center for Pharmaceutical Sciences IST JNTU Kukatpally Hyderabad 500085 India4Deparment of Pharmaceutics SVS School of Pharmacy SVS group of Institutions Ramaram HanamkondaWarangal 506 015 India5 Department of Pharmaceutical Chemistry University College of Pharmaceutical Sciences Kakatiya UniversityWarangal 506 009 India

Correspondence should be addressed to Rama Narsimha Reddy Anreddy anreddyramgmailcom

Received 25 May 2013 Accepted 18 November 2013 Published 28 January 2014

Academic Editor Isabel Seiquer

Copyright copy 2014 Salma Tabassum et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The present study was aimed at evaluating the cardioprotective effect of novel synthetic11987310158401015840119873101584010158401015840-bis[5-methyl-2-oxo-12-dihydro-3H-indol-3-ylidene]carbonohydrazide derivative by estimating the various biomarkers like creatine kinase-myoglobin (CK-MB)lactate dehydrogenase (LDH) aspartate aminotransferase (AST) and triglycerides (TG) in plasma and antioxidants like catalasesuperoxide dismutase in heart tissue homogenate and histopathological examination of heart tissues The results showed thesignificant (119875 lt 005) dose dependent decrease in elevated cardiotoxic biomarkers CK-MB LDH AST and TG levels Thehistopathological studies of heart tissues showed mild degeneration of muscle bundles and less interstitial edematous changesThe results showed the significant (119875 lt 005) dose dependent increase in antioxidant enzymes catalase and superoxide dismutasein heart tissue homogenates These observations enable us to conclude that11987310158401015840119873101584010158401015840-bis[5-methyl-2-oxo-12-dihydro-3H-indol-3-ylidene]carbonohydrazide has cardioprotective activity against doxorubicin induced cardiotoxicity

1 Introduction

Cardiotoxicity is the occurrence of heart electrophysiologydysfunction orand muscle damage The heart becomesweaker and is not as efficient in pumping and therefore circu-lating blood Cardiotoxicity may be caused by chemotherapyheavy metal intake or in an incorrect administration of druglike bupivacaine [1] Ischaemic heart disease is a leading causeof morbidity and mortality worldwide The World HealthOrganization (WHO) estimates there will be about 20millionCVD deaths in 2015 accounting for 30 percent of all deaths

worldwide [2] Myocardial infarction is the acute conditionof myocardial necrosis that occurs as a result of imbalancebetween coronary blood supply andmyocardial demandThepatient may experience significant disability or die [3]

Isatin and its derivatives are used in organic synthesisand they are used in evaluating new product that possessesdifferent biological activities In the past few decades isatinand its derivatives have received much attention due to theirchemotherapeutic values [4] Isatin and its analogs showconsiderable pharmacological actions such as antimicrobialanticancer antiviral anticonvulsant anti-inflammatory and

Hindawi Publishing CorporationJournal of ChemistryVolume 2014 Article ID 260672 7 pageshttpdxdoiorg1011552014260672

2 Journal of Chemistry

analgesic [5] From these results ideas for future molecularmodifications leading to compounds with greater favourablepharmacological properties may be derived

Doxorubicin is broad spectrum antibiotic used as an anti-cancer drug which was limited by its dependent cardiotoxiceffects [6]

In the present study we have evaluated the cardioprotec-tive effect of novel synthetic isatin derivative 11987310158401015840119873101584010158401015840-bis[5-methyl-2-oxo-12-dihydro-3H-indol-3-ylidene]carbonohy-drazide against doxorubicin induced cardiotoxicity in ratsby estimating various cardiotoxic biomarkers like creatinekinase-myoglobin (CK-MB) lactate dehydrogenase (LDH)aspartate aminotransferase (AST) triglycerides (TG) inplasma and histopathological studies of rat heart tissuesand the antioxidant capacity of test compound was knownby estimating catalase superoxide dismutase in heart tissuehomogenates and glutathione levels in plasma

2 Materials and Methods

21 Animals All the experiments were carried out withmale wistar albino rats (200 plusmn 20 g) supplied by Mahaveerenterprises Hyderabad They were housed individually incages under hygienic conditions and placed in a controlledenvironment with a 12 12 h light dark cycle at 25∘C + 2∘Cand 45 + 10 humidity for 7 days before the experimentThe animals were allowed a commercial standard rat diet andwater ad libitumThe animal care and experimental protocolswere in accordance with CPCSEAIAEC

22 Test Compound We have selected the following newlysynthesized isatin derivative to evaluate cardio protectiveactivity This compound was gifted by University Collegeof Pharmaceutical Sciences Kakatiya University WarangalIndia The structure and physicochemical properties of thecompound are as follows

23 Chemistry

11987310158401015840119873101584010158401015840-Bis[5-methyl-2-oxo-12-dihydro-3H-indol-3-ylidene]

carbonohydrazide (3) To a solution of 5-methyl isatin(004mol) and carbohydrazide (002mol) in the 30mLof ethanol a few drops of glacial acetic acid is addedto the reaction mixture and was refluxed for 2-3 hoursThe reaction was monitored by TLC Then the reactionmixture was cooled overnight and the precipitate wascollected by filtration The solids were purified by columnchromatography and recrystallized with ethanol

11987310158401015840119873101584010158401015840-Bis[(3Z)-5-methyl-2-oxo-12-dihydro-3H-indol-3-

ylidene]carbonohydrazide (3) yield 75 MP (∘C) 250ndash252 IR (KBr) cmminus1 3320 (NndashH) 3198 (NndashH) 3010 (CndashHAr) 2980 (CndashH str) 1730 (C=O) 1680 (s C=O lactam)1608 1472 (C=C Ar) 1620 (C=N) 1H-NMR (DMSO-d6)ppm 120575 1028 (s 2H) 948 (s 2H) 683ndash872 (m 6H) 231(s 6H) Mass (ESI-MS) mz 377 (M+1 68) 399 (M+Na100) Elemental analysis for C

19H16N6O3

calculated6063 C 428 H 2233 N found 6069 C 423 H2238 N

24 Induction of Cardiotoxicity In the present study car-diotoxicity was induced by a single dose of doxorubicin(15mgkg ip) treatment in rats [7]

3 Experimental Design

A total of 20 rats were randomly divided into 4 groups of fiverats each

Group I Rats were orally administered with 01 CMC for 8days as the normal control

Group II Rats were orally administered with 01 CMC for8 days and on 7th day single dose of doxorubicin (15mgkgip)

Group III Rats were pretreated with test compound(50mgkg) orally for 8 days and on 7th day single dose ofdoxorubicin (15mgkg ip)

Group IV Rats were pretreated with test compound(100mgkg) orally for 8 days and on 7th day single dose ofdoxorubicin (15mgkg ip)

4 Collection of Blood Samples and Organs

After 24 h following doxorubicin treatment blood sampleswere collected into Eppendorf tubes containing sodiumcitrate through retroorbital puncture After centrifugationplasma was separated and stored at minus20∘C for further bio-chemical assay

Rats were killed by overanesthesia and hearts wereremoved rapidlyThe tissues of heartwere excised andwashedwith prechilled physical saline homogenized with prechilledphysical saline in tissue homogenizer and then centrifuged at10000 rpm for 10min at 4∘C The supernatants were assayedimmediately

5 Assessment of Cardiotoxicity

Determinations of creatine kinase-myoglobin (CK-MB) lac-tate dehydrogenase (LDH) triglycerides (TG) were assayedusing commercial available reagents

51 Estimation of Lactate Dehydrogenase (LDH) The estima-tion of LDH is carried out based on the method of Andries[8] The principle involved is that LDH specifically catalyzesthe oxidation of lactate to pyruvate and transphosphorylationof ADP to ATP with subsequent formation of NADHThe rate at which NADH was formed is proportional toenzyme activity The increase in absorbance per minute as afunction of NADH production is measured calorimetricallyat 340 nm

52 Estimation of Creatine Kinase-Myoglobin (CK-MB) Theimmunoinhibition method of CK-MB analysis was per-formed using an enzymatic kit manufactured by CoralClinical Systems Verna Goa India The test was carried outas previously described [9]

Journal of Chemistry 3

NH

O

O

+ NH

CO

NH

NHN

H

O

N NH

CO

NH

N

O

5-Methylisatin Carbohydrazide

H3CH2N NH2

C2H5OH

glCH3COOH

H3C CH3

1 2 3

Scheme 1 Synthetic route for the bis-isatin derivative 3

53 Estimation of Triglycerides (TG) The levels of triglyc-erides were estimated by GPO-POD method [10] Brieflytriglycerides are hydrolysed by lipase to glycerol and free fattyacids Glycerol is phosphorylated by ATP in the presence ofglycerolkinase (GK) to glycerol-3-phosphate (G-3-P) whichis oxidized by the enzyme glycerol-3-phosphate oxidase (G-P-O) producing hydrogen peroxide Hydrogen peroxide soformed reacts with 4-aminopyrine and ESPAS in the presenceof the enzyme peroxidase (POD) to produce a brown colorcomplexThe intensity of the color developed is proportionalto triglycerides concentration

54 Determination of Antioxidant Enzyme Activities Bio-chemical estimation of catalase superoxide dismutase andglutathione peroxidase in plasma andor heart tissues wasmeasured by respective diagnosing kits and assay procedures

Catalasewas assayed according to themethod ofAebi [11]The enzyme superoxide dismutase (SOD) was determinedin erythrocytes using photooxidation method as reportedearlier in literature [12] Glutathione was measured spec-trophotometrically according to the method of Beutler et al1963 [13]

6 Histopathological Examination

One heart from each group was stored in formalin solutionTissues were embedded in paraffin sectioned at 4 120583m andstained with hematoxylin and eosin (HampE) The sectionswere examined under light microscope and then photomi-crographs were taken

7 Statistical Analysis

All the values were expressed as mean plusmn standard deviation(SD) (119899 = 5) Statistical comparisons between differentgroups were done by using one way analysis of variance(ANOVA) followed by Dunnettrsquos test 119875 value less than 005was considered as statistically significant

8 Results

We report the simple and efficient method for synthesis ofsome new bis-5-methyl isatin carbohydrazone derivatives asplanned in Scheme 1 The desired derivative is prepared bythe reaction of 5-methyl isatin with carbohydrazide in thepresence of glacial acetic acid in ethanol under reflux con-dition The synthesized compounds were purified by columnchromatography All of the derivatives were supported by

spectral data The FT-IR 1H NMR and mass spectra are inagreement with the proposed structures

The IR spectra of the compounds 3 showed the absorptionbands at around 3320 1730 and 1620 cmminus1 regions result-ing from the ndashNH C=O and C=N functions respectivelyAbsence of carbonyl (C=O) peak around 1715 cmminus1 revealsthe formation of carbohydrazone derivatives The 1H NMRspectra of compound 3 which displayed broad singlets in therange of 120575 110-130 are due to ndashNH protons The aromaticprotons are shown in multiplets around 120575 75-85 ppm Allcompounds have shown an excellent agreement betweenthe calculated and experimental obtained data for elementalanalysis Mass spectra the base peak are shown as M+1 peakand also characteristic of all the remaining derivatives

In the present study administration of doxorubicin ata single dose of 15mgkg (ip) to rats produces significantchanges in cardiotoxic biomarkers that include elevation ofplasma CK-MB LDH TG and AST levels Elevation of allthese parameters might be due to cardiac tissue damagewhich was reported in previous studies [14] This confirmsthat single dose of Doxorubicin 15mgkg ip will producecardiotoxicity in rats

Cardioprotective activity of test compound was evalu-ated by measuring CK-MB LDH TG AST levels and thehistopathological examination of rat hearts treated with testcompound The antioxidant capacity in rats after treatmentwith test compound was evaluated by estimating catalase andsuperoxide dismutase levels in heart tissue homogenate

9 Estimation of Plasma Parameters

91 Creatine Kinase-Myoglobin (CK-MB) Creatine kinase-myoglobin (CK-MB) is an indicator of myocardial damageThe results were as shown in Figure 1

In this study we found that single dose of doxorubicin torats causes significant (119875 lt 0001) elevation of plasma CK-MB levels This elevation indicates that cardiotoxicity is pro-duced with doxorubicin Pretreatment with test compoundat the dose of 50mgkg and 100mgkg before a single dose ofdoxorubicin administration showed a significant (119875 lt 001and 119875 lt 0001) decrease in CK-MB levels compared todoxorubicin group in Figure 1

92 Lactate Dehydrogenase (LDH) Lactate dehydrogenaseis an enzyme that presents in heart tissues If there is anydamage to heart tissues it releases LDH into blood streamThe results were as shown in Figure 2

4 Journal of Chemistry

lowastlowast

lowastlowastlowast

020406080

100120140160180200

Normal control

Dox + test (100 mgkg)

Dox + test(50 mgkg)

Dox(15 mgkg)

CK-M

B (I

UL

)

Figure 1 Effect of test compound on plasma CK-MB levels in ratstreated with doxorubicin

119875 lt 0001 versus normal control lowast119875 lt001 lowastlowastlowast119875 lt 0001 versus dox

lowast

lowastlowast

020406080

100120140160

LDH

(IU

L)

Normal control

Dox + test (100 mgkg)

Dox + test(50 mgkg)

Dox(15 mgkg)

Figure 2 Effect of test compound on plasma LDH levels in ratstreated with doxorubicin

119875 lt 0001 versus normal control lowast119875 lt005 lowastlowast119875 lt 001 versus dox

In this study we found that single dose of doxorubicin torats causes significant (119875 lt 0001) elevation of plasma LDHlevelsThis elevation indicates that cardiotoxicity is producedwith doxorubicin Pretreatment with test compound at thedoses of 50mgkg and 100mgkg before a single dose ofdoxorubicin administration showed a significant (119875 lt 005and 119875 lt 001) dose dependent decrease in LDH levels andthese results were comparable to doxorubicin group

93 Triglycerides (TG) Triglyceride is fatty substance that iscomposed of three fatty acids Triglycerides levels in bloodeither come from the diet or the liver High TG levels in bloodindicate cardiac complications like atherosclerosis whichmaycause toxic events in cardiac tissuesThe resultswere as shownin Figure 3

In this study we found that single dose of doxorubicin torats causes significant (119875 lt 0001) elevation of plasma TGlevelsThis elevation indicates that cardiotoxicity is producedwith doxorubicin Pretreatment with test compound at thedoses of 50mgkg and 100mgkg before a single dose ofdoxorubicin administration showed a significant (119875 lt 005and 119875 lt 001) dose dependent decrease in TG levelscompared to doxorubicin group

94 Aspartate Aminotransferase (AST) The elevation of ASTlevels in plasma indicates tissue damage The results were asshown in Figure 4

lowast

lowastlowast

020406080

100120140160180200

TG (m

gdL

)

Normal control

Dox + test (100 mgkg)

Dox + test(50 mgkg)

Dox(15 mgkg)

Figure 3 Effect of test compoundonplasmaTG levels in rats treatedwith doxorubicin

119875 lt 0001 versus normal control lowast119875 lt 005lowastlowast

119875 lt 001 versus dox

lowast

lowastlowast

020406080

100120140

Abso

rban

ce (I

UL

)

Series1

Normal control

Dox + test (100 mgkg)

Dox + test(50 mgkg)

Dox(15 mgkg)

Figure 4 Effect of test compound on plasma AST levels in ratstreated with doxorubicin

119875 lt 0001 versus normal control lowast119875 lt005 versus dox lowastlowast119875 lt 001 versus dox

In this study we found that single dose of doxorubicin torats causes significant (119875 lt 0001) elevation of plasma ASTlevelsThis elevation indicates that cardiotoxicity is producedwith doxorubicin Pretreatment with test compound at thedoses of 50mgkg and 100mgkg before a single dose ofdoxorubicin administration showed significant (119875 lt 005 and119875 lt 001) dose dependent decrease in AST levels comparedto doxorubicin group

10 Estimation of Antioxidants

101 Catalase Catalase is a common enzyme found in nearlyall living organisms that are exposed to oxygen To preventdamage caused by hydrogen peroxide it must be quicklyconverted into other less dangerous substances To endthis catalase is frequently used by cells to rapidly catalysethe decomposition of hydrogen peroxide into less reactivegaseous oxygen and water molecules Catalase activity inheart tissue homogenate represents antioxidant status incardiac tissues

In this study we found that single dose of doxorubicinto rats causes significant (119875 lt 0001) decrease in catalaselevels This reduction indicates that toxicity is produced withdoxorubicin Pretreatmentwith test compound at the doses of50mgkg and 100mgkg before a single dose of doxorubicinadministration showed a significant (119875 lt 005 and 119875 lt 001)

Journal of Chemistry 5

002040608

112141618

2

Cata

lase

activ

ity (I

UL

)

lowast

lowastlowast

Series1

Normal control

Dox + test (100 mgkg)

Dox + test(50 mgkg)

Dox(15 mgkg)

Figure 5 Effect of test compound on catalase levels in heart tissuehomogenate in rats treated with doxorubicin

119875 lt 0001 versusnormal control lowast119875 lt 005 versus dox lowastlowast119875 lt 001 versus dox

lowastlowast

0

5

10

15

20

25

30

35

Supe

roxi

de d

ismut

ase (

mU

)

lowast

Normal control

Dox + test (100 mgkg)

Dox + test(50 mgkg)

Dox(15 mgkg)

Figure 6 Effect of test compound on superoxide dismutase levelsin heart tissue homogenate in rats treated with doxorubicin

119875 lt

0001 versus normal control lowastlowast119875 lt 001 versus dox lowastlowastlowast119875 lt 0001versus dox

dose dependent increase in levels compared to doxorubicingroup

102 Superoxide Dismutase Superoxide dismutases are aclass of enzymes that catalyze the dismutation of superoxideinto oxygen and hydrogen peroxide It is an important antiox-idant defense in nearly all cells exposed to oxygen Superoxidedismutase activity was estimated in tissue homogenate withthe help of pure bovine superoxide dismutase standard

In this study we found that single dose of doxorubicinto rats causes significant (119875 lt 0001) decrease in superoxidedismutase levelsThis reduction indicates that toxicity is pro-duced with doxorubicin Pretreatment with test compound atthe doses of 50mgkg and 100mgkg before a single dose ofdoxorubicin administration showed a significant (119875 lt 001and 119875 lt 0001) dose dependent increase in levels comparedto doxorubicin group

103 Glutathione Glutathione the dominant intracellularthiol plays an important protective role against oxidativestress caused by reactive oxygen species It is an endogenousantioxidant Glutathione was estimated in plasma with thehelp of pure glutathione standard graph The values wereshown in Figure 7

020406080

100120140160

Glu

tath

ione

(120583M

)

Series1

Normal control

Dox + test (100 mgkg)

Dox + test(50 mgkg)

Dox(15 mgkg)

lowastlowast

lowastlowastlowast

Figure 7 Effect of test compound on glutathione levels in plasmain rats treated with doxorubicin

119875 lt 0001 versus normal controllowastlowast

119875 lt 001 versus dox lowast119875 lt 0001 versus dox

In this study we found that single dose of doxorubicinto rats causes significant (119875 lt 0001) decrease in glutathionelevels This reduction indicates that toxicity is produced withdoxorubicin Pretreatmentwith test compound at the doses of50mgkg and 100mgkg before a single dose of doxorubicinadministration showed a significant (119875 lt 001 and119875 lt 0001)dose dependent increase in levels compared to doxorubicingroup

11 Histopathological Examination

The heart tissue of different groups of animals was histo-pathologically examined and the results were as shown inFigure 8

Histopathological observations in rat heart tissues

Group I (Normal control) showed no lesions of pathologicalsignificance

Group II (Doxorubicin 15mgkg ip) showed degenerationof muscle bundles

Group III (Doxorubicin + test compound 50mgkg) showeddegeneration of muscle bundles and interstitial edematouschange

Group IV (Doxorubicin + test compound 100mgkg) showedmild degeneration of muscle bundles and less interstitialedematous change

These above results indicate that test compound showedcardioprotection against doxorubicin induced cardiotoxicity

12 Discussion

The present study was aimed to evaluate the cardioprotec-tive activity of test compound by measuring various car-diotoxic biomarkers antioxidant enzymes and histopatho-logical examination in rats

In the present study cardiotoxicitywas induced by a singledose of doxorubicin (15mgkg ip) in rats Doxorubicintreatment causes significant elevation in plasma CK-MB

6 Journal of Chemistry

(a)

(b)

(c)

(d)

Figure 8 Light micrograph of heart tissue from rats treated with(a) normal control (b) doxorubicin treated (c) doxorubicin +test compound 50mgkg and (d) doxorubicin + test compound100mgkg

LDH TG and AST when compared with normal controlgroupThere is a significant reduction in catalase superoxidedismutase levels in heart tissue and significance depletionin glutathione levels in plasma when compared with normalgroup This confirms the induction of cardiotoxicity in ratswhich is supported by histopathological changes observed inheart tissues [7]

Doxorubicin is broad spectrum antibiotic used as ananticancer drug which was limited by its dose dependentcardiotoxic effects [15] The role of oxygen free radicals

and oxidative stress has been well established in case ofdoxorubicin induced cardiac damage in rats Doxorubicinis converted into its semiquinone form in cardiac myocytesby CYP450 and flavin monooxygenases Then it reactswith molecular oxygen initiates cascade of reactions andproduces ROS ROS reacts with lipids proteins and othercellular constituents to cause damage to mitochondria andcell membranes of the heart muscle [16]

Pretreatment with 50mgkg and 100mgkg of test com-pound for 8 days shows significant protection against car-diotoxicity in rats by decreasing the cardiac biomarkers likeCK-MB LDH TG and AST and elevation of antioxidantenzymes like catalase superoxide dismutase levels in hearttissue and glutathione levels in plasma

Histopathological observations of hearts of animalstreated with a single dose of doxorubicin (15mgkg ip)showed degeneration of muscle bundles Pretreatment with50mgkg and 100mgkg of test compound for 8 days showedless or mild degeneration of muscle bundles and mild inter-stitial oedematous change was seen (Figure 8) This showsthat test compound has cardioprotection against doxorubicininduced cardiotoxicity

Catalase is a free radical scavenging enzyme whichhas cellular defence against oxidative stress and scavengingreactive oxygen radicals [17] In the present study doxoru-bicin treatment produced notable decrease in heart tissuecatalase levels when compared with normal Pretreatmentwith 50mgkg and 100mgkg of test compound for 8 daysshowed marked elevation in catalase levels compared withdoxorubicin group (Figure 5)

Superoxide dismutases are a class of enzymes that catalyzethe dismutation of superoxide into oxygen and hydrogenperoxide As such they are an important antioxidant defensein nearly all cells exposed to oxygen [12] In the present studydoxorubicin treatment produced decrease in SOD levelswhen compared with normal Pretreatment with 50mgkgand 100mgkg of test compound for 8 days showed markedelevation in SOD levels compared with doxorubicin group(Figure 6)

Glutathione is a most important antioxidant enzymepreventing damage to important cellular components causedby reactive oxygen species such as free radicals and peroxidesGlutathione is found almost exclusively in its reduced formsince the enzyme that reverses it from its oxidized formglutathione reductase is constitutively active and inducibleupon oxidative stress [13] It exhibits antioxidant activity byreacting with superoxide radicals peroxy radicals and soforth [18] In the present study doxorubicin treatment pro-duced decrease in plasma glutathione levels when comparedwith normal Pretreatment with 50mgkg and 100mgkg oftest compound for 8 days showed marked elevation in glu-tathione levels compared with doxorubicin group (Figure 7)

All the results were supported with previous reportsthatN-(benzo[d]oxazol-2-yl)-2-(5-bromo-2-oxoindolin-3-ylidene)hydrazinecarboxamide has cardioprotective activityagainst doxorubicin induced cardiotoxicity in rats Pretreat-mentwith compound significantly reduced the elevated levelsof cardiotoxic biomarkers in plasma [14]N-substituted isatinderivatives are more potent small molecules with enhanced

Journal of Chemistry 7

free radical scavenger properties and the cytoprotectiveeffect on the apoptosis of PC12 cells induced by H

2O2

was screened [19] These above reports mainly support thatisatin derivatives have free radical scavenger properties andcardioprotective activity The present study results show thatnovel synthetic isatin derivative decreases the cardiotoxicbiomarkers like CK-MB LDH TG and AST and elevates theantioxidant enzymes like catalase and superoxide dismutaseglutathione which supports that novel synthetic isatin deriva-tive has cardioprotective activity

13 Conclusion

On the basis of our findings it may be worthy to suggest thefollowing

(i) Novel synthetic isatin derivative has cardioprotectiveeffect against doxorubicin induced cardiotoxicity inrats by decreasing the cardiotoxic biomarkers likeCK-MB LDH TG and AST in plasma

(ii) Novel synthetic isatin derivative has antioxidanteffect evaluated by measuring antioxidant enzymesThere is an increase in catalase superoxide dismutasein heart tissues and glutathione levels in plasma indoxorubicin induced cardiotoxicity in rats

(iii) Novel synthetic isatin derivative has cardioprotectiveeffect against doxorubicin induced cardiotoxicity byobserving the histopathological changes in rat hearttissues

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The authors are thankful to the Secretary Vaageswari Collegeof Pharmacy Thimmapur Karimnagar for providing thenecessary facilities for carrying out this research

References

[1] C Huang X Zhang J M Ramil et al ldquoJuvenile exposureto anthracyclines impairs cardiac progenitor cell function andvascularization resulting in greater susceptibility to stress-induced myocardial injury in adult micerdquo Circulation vol 121no 5 pp 675ndash683 2010

[2] V Fuster and B B Kelly Promoting Cardiovascular Health in theDevelopingWorld ACritical Challenge to Achieve Global HealthNational Academies Press 2010

[3] K A Reimer and R B Jennings ldquoMyocardial ischemia hypox-ia and infarctionrdquoTheHeart and Cardiovascular System vol 2pp 1133ndash1201 1992

[4] B Bhrigu D Pathak N Siddiqui M S Alam and W AhsanldquoSearch for biological active isatins a short reviewrdquo Interna-tional Journal of Pharmaceutical Sciences amp Drug Research vol2 no 4 pp 229ndash235 2010

[5] S N Pandeya S Smitha M Jyoti and S K Sridhar ldquoBiologicalactivities of isatin and its derivativesrdquo Acta Pharmaceutica vol55 no 1 pp 27ndash46 2005

[6] P K Singal and N Iliskovic ldquoDoxorubicin-induced cardiomy-opathyrdquo The New England Journal of Medicine vol 339 no 13pp 900ndash905 1998

[7] M N Nagi and M A Mansour ldquoProtective effect of thymo-quinone against doxorubicin-induced cardiotoxicity in rats apossible mechanism of protectionrdquo Pharmacological Researchvol 41 no 3 pp 283ndash289 2000

[8] A J Bakker BMirchi J T Dijkstra F Reitsma H Syperda andA Zijlstra ldquoIFCC method for lactate dehydrogenase measure-ment in heparin plasma is unreliablerdquo Clinical Chemistry vol49 no 4 pp 662ndash664 2003

[9] M Panteghini F Ceriotti G Schumann and L SiekmannldquoEstablishing a reference system in clinical enzymologyrdquo Clini-cal Chemistry and Laboratory Medicine vol 39 no 9 pp 795ndash800 2001

[10] G Bucolo and H David ldquoQuantitative determination of serumtriglycerides by the use of enzymesrdquo Clinical Chemistry vol 19no 5 pp 476ndash482 1973

[11] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

[12] H P Misra and I Fridovich ldquoSuperoxide dismutase a pho-tochemical augmentation assayrdquo Archives of Biochemistry andBiophysics vol 181 no 1 pp 308ndash312 1977

[13] E Beutler O Duron and BM Kelly ldquoImprovedmethod for thedetermination of blood glutathionerdquoThe Journal of Laboratoryand Clinical Medicine vol 61 pp 882ndash888 1963

[14] A R N Reddy J Nagarajua G Rajyalaksmib and M Saranga-panib ldquoCardioprotective effect of N-(benzo[d]oxazol-2-yl)-2-(5-bromo-2-oxoindolin-3-ylidene)hydrazinecarboxamideagainst doxorubicin induced cardiotoxicity in ratsrdquo Toxico-logical amp Environmental Chemistry vol 94 no 10 pp 2012ndash2018 2012

[15] F S Carvalho A Burgeiro R Garcia A J Moreno R A Car-valho and P J Oliveira ldquoDoxorubicin-induced cardiotoxicityfrom bioenergetic failure and cell death to cardiomyopathyrdquoMedicinal Research Reviews vol 34 no 1 pp 106ndash135 2014

[16] P K Singal N Iliskovic T Li and D Kumar ldquoAdriamycincardiomyopathy pathophysiology and preventionrdquoThe FASEBJournal vol 11 no 12 pp 931ndash936 1997

[17] A R Lehenbauer Ludke A A-R S Al-Shudiefat S DhingraD S Jassal and P K Singal ldquoA concise description of car-dioprotective strategies in doxorubicin-induced cardiotoxicityrdquoCanadian Journal of Physiology and Pharmacology vol 87 no10 pp 756ndash763 2009

[18] R D Olson J S MacDonald and C J VanBoxtel ldquoRegulatoryrole of glutathione and soluble sulfhydryl groups in the toxicityof adriamycinrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 215 no 2 pp 450ndash454 1980

[19] G Chen Y Wang X Hao S Mu and Q Sun ldquoSimple isatinderivatives as free radical scavengers synthesis biological eval-uation and structure-activity relationshiprdquo Chemistry CentralJournal vol 5 no 1 article 37 2011

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CatalystsJournal of

2 Journal of Chemistry

analgesic [5] From these results ideas for future molecularmodifications leading to compounds with greater favourablepharmacological properties may be derived

Doxorubicin is broad spectrum antibiotic used as an anti-cancer drug which was limited by its dependent cardiotoxiceffects [6]

In the present study we have evaluated the cardioprotec-tive effect of novel synthetic isatin derivative 11987310158401015840119873101584010158401015840-bis[5-methyl-2-oxo-12-dihydro-3H-indol-3-ylidene]carbonohy-drazide against doxorubicin induced cardiotoxicity in ratsby estimating various cardiotoxic biomarkers like creatinekinase-myoglobin (CK-MB) lactate dehydrogenase (LDH)aspartate aminotransferase (AST) triglycerides (TG) inplasma and histopathological studies of rat heart tissuesand the antioxidant capacity of test compound was knownby estimating catalase superoxide dismutase in heart tissuehomogenates and glutathione levels in plasma

2 Materials and Methods

21 Animals All the experiments were carried out withmale wistar albino rats (200 plusmn 20 g) supplied by Mahaveerenterprises Hyderabad They were housed individually incages under hygienic conditions and placed in a controlledenvironment with a 12 12 h light dark cycle at 25∘C + 2∘Cand 45 + 10 humidity for 7 days before the experimentThe animals were allowed a commercial standard rat diet andwater ad libitumThe animal care and experimental protocolswere in accordance with CPCSEAIAEC

22 Test Compound We have selected the following newlysynthesized isatin derivative to evaluate cardio protectiveactivity This compound was gifted by University Collegeof Pharmaceutical Sciences Kakatiya University WarangalIndia The structure and physicochemical properties of thecompound are as follows

23 Chemistry

11987310158401015840119873101584010158401015840-Bis[5-methyl-2-oxo-12-dihydro-3H-indol-3-ylidene]

carbonohydrazide (3) To a solution of 5-methyl isatin(004mol) and carbohydrazide (002mol) in the 30mLof ethanol a few drops of glacial acetic acid is addedto the reaction mixture and was refluxed for 2-3 hoursThe reaction was monitored by TLC Then the reactionmixture was cooled overnight and the precipitate wascollected by filtration The solids were purified by columnchromatography and recrystallized with ethanol

11987310158401015840119873101584010158401015840-Bis[(3Z)-5-methyl-2-oxo-12-dihydro-3H-indol-3-

ylidene]carbonohydrazide (3) yield 75 MP (∘C) 250ndash252 IR (KBr) cmminus1 3320 (NndashH) 3198 (NndashH) 3010 (CndashHAr) 2980 (CndashH str) 1730 (C=O) 1680 (s C=O lactam)1608 1472 (C=C Ar) 1620 (C=N) 1H-NMR (DMSO-d6)ppm 120575 1028 (s 2H) 948 (s 2H) 683ndash872 (m 6H) 231(s 6H) Mass (ESI-MS) mz 377 (M+1 68) 399 (M+Na100) Elemental analysis for C

19H16N6O3

calculated6063 C 428 H 2233 N found 6069 C 423 H2238 N

24 Induction of Cardiotoxicity In the present study car-diotoxicity was induced by a single dose of doxorubicin(15mgkg ip) treatment in rats [7]

3 Experimental Design

A total of 20 rats were randomly divided into 4 groups of fiverats each

Group I Rats were orally administered with 01 CMC for 8days as the normal control

Group II Rats were orally administered with 01 CMC for8 days and on 7th day single dose of doxorubicin (15mgkgip)

Group III Rats were pretreated with test compound(50mgkg) orally for 8 days and on 7th day single dose ofdoxorubicin (15mgkg ip)

Group IV Rats were pretreated with test compound(100mgkg) orally for 8 days and on 7th day single dose ofdoxorubicin (15mgkg ip)

4 Collection of Blood Samples and Organs

After 24 h following doxorubicin treatment blood sampleswere collected into Eppendorf tubes containing sodiumcitrate through retroorbital puncture After centrifugationplasma was separated and stored at minus20∘C for further bio-chemical assay

Rats were killed by overanesthesia and hearts wereremoved rapidlyThe tissues of heartwere excised andwashedwith prechilled physical saline homogenized with prechilledphysical saline in tissue homogenizer and then centrifuged at10000 rpm for 10min at 4∘C The supernatants were assayedimmediately

5 Assessment of Cardiotoxicity

Determinations of creatine kinase-myoglobin (CK-MB) lac-tate dehydrogenase (LDH) triglycerides (TG) were assayedusing commercial available reagents

51 Estimation of Lactate Dehydrogenase (LDH) The estima-tion of LDH is carried out based on the method of Andries[8] The principle involved is that LDH specifically catalyzesthe oxidation of lactate to pyruvate and transphosphorylationof ADP to ATP with subsequent formation of NADHThe rate at which NADH was formed is proportional toenzyme activity The increase in absorbance per minute as afunction of NADH production is measured calorimetricallyat 340 nm

52 Estimation of Creatine Kinase-Myoglobin (CK-MB) Theimmunoinhibition method of CK-MB analysis was per-formed using an enzymatic kit manufactured by CoralClinical Systems Verna Goa India The test was carried outas previously described [9]

Journal of Chemistry 3

NH

O

O

+ NH

CO

NH

NHN

H

O

N NH

CO

NH

N

O

5-Methylisatin Carbohydrazide

H3CH2N NH2

C2H5OH

glCH3COOH

H3C CH3

1 2 3

Scheme 1 Synthetic route for the bis-isatin derivative 3

53 Estimation of Triglycerides (TG) The levels of triglyc-erides were estimated by GPO-POD method [10] Brieflytriglycerides are hydrolysed by lipase to glycerol and free fattyacids Glycerol is phosphorylated by ATP in the presence ofglycerolkinase (GK) to glycerol-3-phosphate (G-3-P) whichis oxidized by the enzyme glycerol-3-phosphate oxidase (G-P-O) producing hydrogen peroxide Hydrogen peroxide soformed reacts with 4-aminopyrine and ESPAS in the presenceof the enzyme peroxidase (POD) to produce a brown colorcomplexThe intensity of the color developed is proportionalto triglycerides concentration

54 Determination of Antioxidant Enzyme Activities Bio-chemical estimation of catalase superoxide dismutase andglutathione peroxidase in plasma andor heart tissues wasmeasured by respective diagnosing kits and assay procedures

Catalasewas assayed according to themethod ofAebi [11]The enzyme superoxide dismutase (SOD) was determinedin erythrocytes using photooxidation method as reportedearlier in literature [12] Glutathione was measured spec-trophotometrically according to the method of Beutler et al1963 [13]

6 Histopathological Examination

One heart from each group was stored in formalin solutionTissues were embedded in paraffin sectioned at 4 120583m andstained with hematoxylin and eosin (HampE) The sectionswere examined under light microscope and then photomi-crographs were taken

7 Statistical Analysis

All the values were expressed as mean plusmn standard deviation(SD) (119899 = 5) Statistical comparisons between differentgroups were done by using one way analysis of variance(ANOVA) followed by Dunnettrsquos test 119875 value less than 005was considered as statistically significant

8 Results

We report the simple and efficient method for synthesis ofsome new bis-5-methyl isatin carbohydrazone derivatives asplanned in Scheme 1 The desired derivative is prepared bythe reaction of 5-methyl isatin with carbohydrazide in thepresence of glacial acetic acid in ethanol under reflux con-dition The synthesized compounds were purified by columnchromatography All of the derivatives were supported by

spectral data The FT-IR 1H NMR and mass spectra are inagreement with the proposed structures

The IR spectra of the compounds 3 showed the absorptionbands at around 3320 1730 and 1620 cmminus1 regions result-ing from the ndashNH C=O and C=N functions respectivelyAbsence of carbonyl (C=O) peak around 1715 cmminus1 revealsthe formation of carbohydrazone derivatives The 1H NMRspectra of compound 3 which displayed broad singlets in therange of 120575 110-130 are due to ndashNH protons The aromaticprotons are shown in multiplets around 120575 75-85 ppm Allcompounds have shown an excellent agreement betweenthe calculated and experimental obtained data for elementalanalysis Mass spectra the base peak are shown as M+1 peakand also characteristic of all the remaining derivatives

In the present study administration of doxorubicin ata single dose of 15mgkg (ip) to rats produces significantchanges in cardiotoxic biomarkers that include elevation ofplasma CK-MB LDH TG and AST levels Elevation of allthese parameters might be due to cardiac tissue damagewhich was reported in previous studies [14] This confirmsthat single dose of Doxorubicin 15mgkg ip will producecardiotoxicity in rats

Cardioprotective activity of test compound was evalu-ated by measuring CK-MB LDH TG AST levels and thehistopathological examination of rat hearts treated with testcompound The antioxidant capacity in rats after treatmentwith test compound was evaluated by estimating catalase andsuperoxide dismutase levels in heart tissue homogenate

9 Estimation of Plasma Parameters

91 Creatine Kinase-Myoglobin (CK-MB) Creatine kinase-myoglobin (CK-MB) is an indicator of myocardial damageThe results were as shown in Figure 1

In this study we found that single dose of doxorubicin torats causes significant (119875 lt 0001) elevation of plasma CK-MB levels This elevation indicates that cardiotoxicity is pro-duced with doxorubicin Pretreatment with test compoundat the dose of 50mgkg and 100mgkg before a single dose ofdoxorubicin administration showed a significant (119875 lt 001and 119875 lt 0001) decrease in CK-MB levels compared todoxorubicin group in Figure 1

92 Lactate Dehydrogenase (LDH) Lactate dehydrogenaseis an enzyme that presents in heart tissues If there is anydamage to heart tissues it releases LDH into blood streamThe results were as shown in Figure 2

4 Journal of Chemistry

lowastlowast

lowastlowastlowast

020406080

100120140160180200

Normal control

Dox + test (100 mgkg)

Dox + test(50 mgkg)

Dox(15 mgkg)

CK-M

B (I

UL

)

Figure 1 Effect of test compound on plasma CK-MB levels in ratstreated with doxorubicin

119875 lt 0001 versus normal control lowast119875 lt001 lowastlowastlowast119875 lt 0001 versus dox

lowast

lowastlowast

020406080

100120140160

LDH

(IU

L)

Normal control

Dox + test (100 mgkg)

Dox + test(50 mgkg)

Dox(15 mgkg)

Figure 2 Effect of test compound on plasma LDH levels in ratstreated with doxorubicin

119875 lt 0001 versus normal control lowast119875 lt005 lowastlowast119875 lt 001 versus dox

In this study we found that single dose of doxorubicin torats causes significant (119875 lt 0001) elevation of plasma LDHlevelsThis elevation indicates that cardiotoxicity is producedwith doxorubicin Pretreatment with test compound at thedoses of 50mgkg and 100mgkg before a single dose ofdoxorubicin administration showed a significant (119875 lt 005and 119875 lt 001) dose dependent decrease in LDH levels andthese results were comparable to doxorubicin group

93 Triglycerides (TG) Triglyceride is fatty substance that iscomposed of three fatty acids Triglycerides levels in bloodeither come from the diet or the liver High TG levels in bloodindicate cardiac complications like atherosclerosis whichmaycause toxic events in cardiac tissuesThe resultswere as shownin Figure 3

In this study we found that single dose of doxorubicin torats causes significant (119875 lt 0001) elevation of plasma TGlevelsThis elevation indicates that cardiotoxicity is producedwith doxorubicin Pretreatment with test compound at thedoses of 50mgkg and 100mgkg before a single dose ofdoxorubicin administration showed a significant (119875 lt 005and 119875 lt 001) dose dependent decrease in TG levelscompared to doxorubicin group

94 Aspartate Aminotransferase (AST) The elevation of ASTlevels in plasma indicates tissue damage The results were asshown in Figure 4

lowast

lowastlowast

020406080

100120140160180200

TG (m

gdL

)

Normal control

Dox + test (100 mgkg)

Dox + test(50 mgkg)

Dox(15 mgkg)

Figure 3 Effect of test compoundonplasmaTG levels in rats treatedwith doxorubicin

119875 lt 0001 versus normal control lowast119875 lt 005lowastlowast

119875 lt 001 versus dox

lowast

lowastlowast

020406080

100120140

Abso

rban

ce (I

UL

)

Series1

Normal control

Dox + test (100 mgkg)

Dox + test(50 mgkg)

Dox(15 mgkg)

Figure 4 Effect of test compound on plasma AST levels in ratstreated with doxorubicin

119875 lt 0001 versus normal control lowast119875 lt005 versus dox lowastlowast119875 lt 001 versus dox

In this study we found that single dose of doxorubicin torats causes significant (119875 lt 0001) elevation of plasma ASTlevelsThis elevation indicates that cardiotoxicity is producedwith doxorubicin Pretreatment with test compound at thedoses of 50mgkg and 100mgkg before a single dose ofdoxorubicin administration showed significant (119875 lt 005 and119875 lt 001) dose dependent decrease in AST levels comparedto doxorubicin group

10 Estimation of Antioxidants

101 Catalase Catalase is a common enzyme found in nearlyall living organisms that are exposed to oxygen To preventdamage caused by hydrogen peroxide it must be quicklyconverted into other less dangerous substances To endthis catalase is frequently used by cells to rapidly catalysethe decomposition of hydrogen peroxide into less reactivegaseous oxygen and water molecules Catalase activity inheart tissue homogenate represents antioxidant status incardiac tissues

In this study we found that single dose of doxorubicinto rats causes significant (119875 lt 0001) decrease in catalaselevels This reduction indicates that toxicity is produced withdoxorubicin Pretreatmentwith test compound at the doses of50mgkg and 100mgkg before a single dose of doxorubicinadministration showed a significant (119875 lt 005 and 119875 lt 001)

Journal of Chemistry 5

002040608

112141618

2

Cata

lase

activ

ity (I

UL

)

lowast

lowastlowast

Series1

Normal control

Dox + test (100 mgkg)

Dox + test(50 mgkg)

Dox(15 mgkg)

Figure 5 Effect of test compound on catalase levels in heart tissuehomogenate in rats treated with doxorubicin

119875 lt 0001 versusnormal control lowast119875 lt 005 versus dox lowastlowast119875 lt 001 versus dox

lowastlowast

0

5

10

15

20

25

30

35

Supe

roxi

de d

ismut

ase (

mU

)

lowast

Normal control

Dox + test (100 mgkg)

Dox + test(50 mgkg)

Dox(15 mgkg)

Figure 6 Effect of test compound on superoxide dismutase levelsin heart tissue homogenate in rats treated with doxorubicin

119875 lt

0001 versus normal control lowastlowast119875 lt 001 versus dox lowastlowastlowast119875 lt 0001versus dox

dose dependent increase in levels compared to doxorubicingroup

102 Superoxide Dismutase Superoxide dismutases are aclass of enzymes that catalyze the dismutation of superoxideinto oxygen and hydrogen peroxide It is an important antiox-idant defense in nearly all cells exposed to oxygen Superoxidedismutase activity was estimated in tissue homogenate withthe help of pure bovine superoxide dismutase standard

In this study we found that single dose of doxorubicinto rats causes significant (119875 lt 0001) decrease in superoxidedismutase levelsThis reduction indicates that toxicity is pro-duced with doxorubicin Pretreatment with test compound atthe doses of 50mgkg and 100mgkg before a single dose ofdoxorubicin administration showed a significant (119875 lt 001and 119875 lt 0001) dose dependent increase in levels comparedto doxorubicin group

103 Glutathione Glutathione the dominant intracellularthiol plays an important protective role against oxidativestress caused by reactive oxygen species It is an endogenousantioxidant Glutathione was estimated in plasma with thehelp of pure glutathione standard graph The values wereshown in Figure 7

020406080

100120140160

Glu

tath

ione

(120583M

)

Series1

Normal control

Dox + test (100 mgkg)

Dox + test(50 mgkg)

Dox(15 mgkg)

lowastlowast

lowastlowastlowast

Figure 7 Effect of test compound on glutathione levels in plasmain rats treated with doxorubicin

119875 lt 0001 versus normal controllowastlowast

119875 lt 001 versus dox lowast119875 lt 0001 versus dox

In this study we found that single dose of doxorubicinto rats causes significant (119875 lt 0001) decrease in glutathionelevels This reduction indicates that toxicity is produced withdoxorubicin Pretreatmentwith test compound at the doses of50mgkg and 100mgkg before a single dose of doxorubicinadministration showed a significant (119875 lt 001 and119875 lt 0001)dose dependent increase in levels compared to doxorubicingroup

11 Histopathological Examination

The heart tissue of different groups of animals was histo-pathologically examined and the results were as shown inFigure 8

Histopathological observations in rat heart tissues

Group I (Normal control) showed no lesions of pathologicalsignificance

Group II (Doxorubicin 15mgkg ip) showed degenerationof muscle bundles

Group III (Doxorubicin + test compound 50mgkg) showeddegeneration of muscle bundles and interstitial edematouschange

Group IV (Doxorubicin + test compound 100mgkg) showedmild degeneration of muscle bundles and less interstitialedematous change

These above results indicate that test compound showedcardioprotection against doxorubicin induced cardiotoxicity

12 Discussion

The present study was aimed to evaluate the cardioprotec-tive activity of test compound by measuring various car-diotoxic biomarkers antioxidant enzymes and histopatho-logical examination in rats

In the present study cardiotoxicitywas induced by a singledose of doxorubicin (15mgkg ip) in rats Doxorubicintreatment causes significant elevation in plasma CK-MB

6 Journal of Chemistry

(a)

(b)

(c)

(d)

Figure 8 Light micrograph of heart tissue from rats treated with(a) normal control (b) doxorubicin treated (c) doxorubicin +test compound 50mgkg and (d) doxorubicin + test compound100mgkg

LDH TG and AST when compared with normal controlgroupThere is a significant reduction in catalase superoxidedismutase levels in heart tissue and significance depletionin glutathione levels in plasma when compared with normalgroup This confirms the induction of cardiotoxicity in ratswhich is supported by histopathological changes observed inheart tissues [7]

Doxorubicin is broad spectrum antibiotic used as ananticancer drug which was limited by its dose dependentcardiotoxic effects [15] The role of oxygen free radicals

and oxidative stress has been well established in case ofdoxorubicin induced cardiac damage in rats Doxorubicinis converted into its semiquinone form in cardiac myocytesby CYP450 and flavin monooxygenases Then it reactswith molecular oxygen initiates cascade of reactions andproduces ROS ROS reacts with lipids proteins and othercellular constituents to cause damage to mitochondria andcell membranes of the heart muscle [16]

Pretreatment with 50mgkg and 100mgkg of test com-pound for 8 days shows significant protection against car-diotoxicity in rats by decreasing the cardiac biomarkers likeCK-MB LDH TG and AST and elevation of antioxidantenzymes like catalase superoxide dismutase levels in hearttissue and glutathione levels in plasma

Histopathological observations of hearts of animalstreated with a single dose of doxorubicin (15mgkg ip)showed degeneration of muscle bundles Pretreatment with50mgkg and 100mgkg of test compound for 8 days showedless or mild degeneration of muscle bundles and mild inter-stitial oedematous change was seen (Figure 8) This showsthat test compound has cardioprotection against doxorubicininduced cardiotoxicity

Catalase is a free radical scavenging enzyme whichhas cellular defence against oxidative stress and scavengingreactive oxygen radicals [17] In the present study doxoru-bicin treatment produced notable decrease in heart tissuecatalase levels when compared with normal Pretreatmentwith 50mgkg and 100mgkg of test compound for 8 daysshowed marked elevation in catalase levels compared withdoxorubicin group (Figure 5)

Superoxide dismutases are a class of enzymes that catalyzethe dismutation of superoxide into oxygen and hydrogenperoxide As such they are an important antioxidant defensein nearly all cells exposed to oxygen [12] In the present studydoxorubicin treatment produced decrease in SOD levelswhen compared with normal Pretreatment with 50mgkgand 100mgkg of test compound for 8 days showed markedelevation in SOD levels compared with doxorubicin group(Figure 6)

Glutathione is a most important antioxidant enzymepreventing damage to important cellular components causedby reactive oxygen species such as free radicals and peroxidesGlutathione is found almost exclusively in its reduced formsince the enzyme that reverses it from its oxidized formglutathione reductase is constitutively active and inducibleupon oxidative stress [13] It exhibits antioxidant activity byreacting with superoxide radicals peroxy radicals and soforth [18] In the present study doxorubicin treatment pro-duced decrease in plasma glutathione levels when comparedwith normal Pretreatment with 50mgkg and 100mgkg oftest compound for 8 days showed marked elevation in glu-tathione levels compared with doxorubicin group (Figure 7)

All the results were supported with previous reportsthatN-(benzo[d]oxazol-2-yl)-2-(5-bromo-2-oxoindolin-3-ylidene)hydrazinecarboxamide has cardioprotective activityagainst doxorubicin induced cardiotoxicity in rats Pretreat-mentwith compound significantly reduced the elevated levelsof cardiotoxic biomarkers in plasma [14]N-substituted isatinderivatives are more potent small molecules with enhanced

Journal of Chemistry 7

free radical scavenger properties and the cytoprotectiveeffect on the apoptosis of PC12 cells induced by H

2O2

was screened [19] These above reports mainly support thatisatin derivatives have free radical scavenger properties andcardioprotective activity The present study results show thatnovel synthetic isatin derivative decreases the cardiotoxicbiomarkers like CK-MB LDH TG and AST and elevates theantioxidant enzymes like catalase and superoxide dismutaseglutathione which supports that novel synthetic isatin deriva-tive has cardioprotective activity

13 Conclusion

On the basis of our findings it may be worthy to suggest thefollowing

(i) Novel synthetic isatin derivative has cardioprotectiveeffect against doxorubicin induced cardiotoxicity inrats by decreasing the cardiotoxic biomarkers likeCK-MB LDH TG and AST in plasma

(ii) Novel synthetic isatin derivative has antioxidanteffect evaluated by measuring antioxidant enzymesThere is an increase in catalase superoxide dismutasein heart tissues and glutathione levels in plasma indoxorubicin induced cardiotoxicity in rats

(iii) Novel synthetic isatin derivative has cardioprotectiveeffect against doxorubicin induced cardiotoxicity byobserving the histopathological changes in rat hearttissues

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The authors are thankful to the Secretary Vaageswari Collegeof Pharmacy Thimmapur Karimnagar for providing thenecessary facilities for carrying out this research

References

[1] C Huang X Zhang J M Ramil et al ldquoJuvenile exposureto anthracyclines impairs cardiac progenitor cell function andvascularization resulting in greater susceptibility to stress-induced myocardial injury in adult micerdquo Circulation vol 121no 5 pp 675ndash683 2010

[2] V Fuster and B B Kelly Promoting Cardiovascular Health in theDevelopingWorld ACritical Challenge to Achieve Global HealthNational Academies Press 2010

[3] K A Reimer and R B Jennings ldquoMyocardial ischemia hypox-ia and infarctionrdquoTheHeart and Cardiovascular System vol 2pp 1133ndash1201 1992

[4] B Bhrigu D Pathak N Siddiqui M S Alam and W AhsanldquoSearch for biological active isatins a short reviewrdquo Interna-tional Journal of Pharmaceutical Sciences amp Drug Research vol2 no 4 pp 229ndash235 2010

[5] S N Pandeya S Smitha M Jyoti and S K Sridhar ldquoBiologicalactivities of isatin and its derivativesrdquo Acta Pharmaceutica vol55 no 1 pp 27ndash46 2005

[6] P K Singal and N Iliskovic ldquoDoxorubicin-induced cardiomy-opathyrdquo The New England Journal of Medicine vol 339 no 13pp 900ndash905 1998

[7] M N Nagi and M A Mansour ldquoProtective effect of thymo-quinone against doxorubicin-induced cardiotoxicity in rats apossible mechanism of protectionrdquo Pharmacological Researchvol 41 no 3 pp 283ndash289 2000

[8] A J Bakker BMirchi J T Dijkstra F Reitsma H Syperda andA Zijlstra ldquoIFCC method for lactate dehydrogenase measure-ment in heparin plasma is unreliablerdquo Clinical Chemistry vol49 no 4 pp 662ndash664 2003

[9] M Panteghini F Ceriotti G Schumann and L SiekmannldquoEstablishing a reference system in clinical enzymologyrdquo Clini-cal Chemistry and Laboratory Medicine vol 39 no 9 pp 795ndash800 2001

[10] G Bucolo and H David ldquoQuantitative determination of serumtriglycerides by the use of enzymesrdquo Clinical Chemistry vol 19no 5 pp 476ndash482 1973

[11] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

[12] H P Misra and I Fridovich ldquoSuperoxide dismutase a pho-tochemical augmentation assayrdquo Archives of Biochemistry andBiophysics vol 181 no 1 pp 308ndash312 1977

[13] E Beutler O Duron and BM Kelly ldquoImprovedmethod for thedetermination of blood glutathionerdquoThe Journal of Laboratoryand Clinical Medicine vol 61 pp 882ndash888 1963

[14] A R N Reddy J Nagarajua G Rajyalaksmib and M Saranga-panib ldquoCardioprotective effect of N-(benzo[d]oxazol-2-yl)-2-(5-bromo-2-oxoindolin-3-ylidene)hydrazinecarboxamideagainst doxorubicin induced cardiotoxicity in ratsrdquo Toxico-logical amp Environmental Chemistry vol 94 no 10 pp 2012ndash2018 2012

[15] F S Carvalho A Burgeiro R Garcia A J Moreno R A Car-valho and P J Oliveira ldquoDoxorubicin-induced cardiotoxicityfrom bioenergetic failure and cell death to cardiomyopathyrdquoMedicinal Research Reviews vol 34 no 1 pp 106ndash135 2014

[16] P K Singal N Iliskovic T Li and D Kumar ldquoAdriamycincardiomyopathy pathophysiology and preventionrdquoThe FASEBJournal vol 11 no 12 pp 931ndash936 1997

[17] A R Lehenbauer Ludke A A-R S Al-Shudiefat S DhingraD S Jassal and P K Singal ldquoA concise description of car-dioprotective strategies in doxorubicin-induced cardiotoxicityrdquoCanadian Journal of Physiology and Pharmacology vol 87 no10 pp 756ndash763 2009

[18] R D Olson J S MacDonald and C J VanBoxtel ldquoRegulatoryrole of glutathione and soluble sulfhydryl groups in the toxicityof adriamycinrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 215 no 2 pp 450ndash454 1980

[19] G Chen Y Wang X Hao S Mu and Q Sun ldquoSimple isatinderivatives as free radical scavengers synthesis biological eval-uation and structure-activity relationshiprdquo Chemistry CentralJournal vol 5 no 1 article 37 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Carbohydrate Chemistry

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Advances in

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Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

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Medicinal ChemistryInternational Journal of

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Chromatography Research International

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Applied ChemistryJournal of

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Theoretical ChemistryJournal of

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Quantum Chemistry

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Organic Chemistry International

ElectrochemistryInternational Journal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Journal of Chemistry 3

NH

O

O

+ NH

CO

NH

NHN

H

O

N NH

CO

NH

N

O

5-Methylisatin Carbohydrazide

H3CH2N NH2

C2H5OH

glCH3COOH

H3C CH3

1 2 3

Scheme 1 Synthetic route for the bis-isatin derivative 3

53 Estimation of Triglycerides (TG) The levels of triglyc-erides were estimated by GPO-POD method [10] Brieflytriglycerides are hydrolysed by lipase to glycerol and free fattyacids Glycerol is phosphorylated by ATP in the presence ofglycerolkinase (GK) to glycerol-3-phosphate (G-3-P) whichis oxidized by the enzyme glycerol-3-phosphate oxidase (G-P-O) producing hydrogen peroxide Hydrogen peroxide soformed reacts with 4-aminopyrine and ESPAS in the presenceof the enzyme peroxidase (POD) to produce a brown colorcomplexThe intensity of the color developed is proportionalto triglycerides concentration

54 Determination of Antioxidant Enzyme Activities Bio-chemical estimation of catalase superoxide dismutase andglutathione peroxidase in plasma andor heart tissues wasmeasured by respective diagnosing kits and assay procedures

Catalasewas assayed according to themethod ofAebi [11]The enzyme superoxide dismutase (SOD) was determinedin erythrocytes using photooxidation method as reportedearlier in literature [12] Glutathione was measured spec-trophotometrically according to the method of Beutler et al1963 [13]

6 Histopathological Examination

One heart from each group was stored in formalin solutionTissues were embedded in paraffin sectioned at 4 120583m andstained with hematoxylin and eosin (HampE) The sectionswere examined under light microscope and then photomi-crographs were taken

7 Statistical Analysis

All the values were expressed as mean plusmn standard deviation(SD) (119899 = 5) Statistical comparisons between differentgroups were done by using one way analysis of variance(ANOVA) followed by Dunnettrsquos test 119875 value less than 005was considered as statistically significant

8 Results

We report the simple and efficient method for synthesis ofsome new bis-5-methyl isatin carbohydrazone derivatives asplanned in Scheme 1 The desired derivative is prepared bythe reaction of 5-methyl isatin with carbohydrazide in thepresence of glacial acetic acid in ethanol under reflux con-dition The synthesized compounds were purified by columnchromatography All of the derivatives were supported by

spectral data The FT-IR 1H NMR and mass spectra are inagreement with the proposed structures

The IR spectra of the compounds 3 showed the absorptionbands at around 3320 1730 and 1620 cmminus1 regions result-ing from the ndashNH C=O and C=N functions respectivelyAbsence of carbonyl (C=O) peak around 1715 cmminus1 revealsthe formation of carbohydrazone derivatives The 1H NMRspectra of compound 3 which displayed broad singlets in therange of 120575 110-130 are due to ndashNH protons The aromaticprotons are shown in multiplets around 120575 75-85 ppm Allcompounds have shown an excellent agreement betweenthe calculated and experimental obtained data for elementalanalysis Mass spectra the base peak are shown as M+1 peakand also characteristic of all the remaining derivatives

In the present study administration of doxorubicin ata single dose of 15mgkg (ip) to rats produces significantchanges in cardiotoxic biomarkers that include elevation ofplasma CK-MB LDH TG and AST levels Elevation of allthese parameters might be due to cardiac tissue damagewhich was reported in previous studies [14] This confirmsthat single dose of Doxorubicin 15mgkg ip will producecardiotoxicity in rats

Cardioprotective activity of test compound was evalu-ated by measuring CK-MB LDH TG AST levels and thehistopathological examination of rat hearts treated with testcompound The antioxidant capacity in rats after treatmentwith test compound was evaluated by estimating catalase andsuperoxide dismutase levels in heart tissue homogenate

9 Estimation of Plasma Parameters

91 Creatine Kinase-Myoglobin (CK-MB) Creatine kinase-myoglobin (CK-MB) is an indicator of myocardial damageThe results were as shown in Figure 1

In this study we found that single dose of doxorubicin torats causes significant (119875 lt 0001) elevation of plasma CK-MB levels This elevation indicates that cardiotoxicity is pro-duced with doxorubicin Pretreatment with test compoundat the dose of 50mgkg and 100mgkg before a single dose ofdoxorubicin administration showed a significant (119875 lt 001and 119875 lt 0001) decrease in CK-MB levels compared todoxorubicin group in Figure 1

92 Lactate Dehydrogenase (LDH) Lactate dehydrogenaseis an enzyme that presents in heart tissues If there is anydamage to heart tissues it releases LDH into blood streamThe results were as shown in Figure 2

4 Journal of Chemistry

lowastlowast

lowastlowastlowast

020406080

100120140160180200

Normal control

Dox + test (100 mgkg)

Dox + test(50 mgkg)

Dox(15 mgkg)

CK-M

B (I

UL

)

Figure 1 Effect of test compound on plasma CK-MB levels in ratstreated with doxorubicin

119875 lt 0001 versus normal control lowast119875 lt001 lowastlowastlowast119875 lt 0001 versus dox

lowast

lowastlowast

020406080

100120140160

LDH

(IU

L)

Normal control

Dox + test (100 mgkg)

Dox + test(50 mgkg)

Dox(15 mgkg)

Figure 2 Effect of test compound on plasma LDH levels in ratstreated with doxorubicin

119875 lt 0001 versus normal control lowast119875 lt005 lowastlowast119875 lt 001 versus dox

In this study we found that single dose of doxorubicin torats causes significant (119875 lt 0001) elevation of plasma LDHlevelsThis elevation indicates that cardiotoxicity is producedwith doxorubicin Pretreatment with test compound at thedoses of 50mgkg and 100mgkg before a single dose ofdoxorubicin administration showed a significant (119875 lt 005and 119875 lt 001) dose dependent decrease in LDH levels andthese results were comparable to doxorubicin group

93 Triglycerides (TG) Triglyceride is fatty substance that iscomposed of three fatty acids Triglycerides levels in bloodeither come from the diet or the liver High TG levels in bloodindicate cardiac complications like atherosclerosis whichmaycause toxic events in cardiac tissuesThe resultswere as shownin Figure 3

In this study we found that single dose of doxorubicin torats causes significant (119875 lt 0001) elevation of plasma TGlevelsThis elevation indicates that cardiotoxicity is producedwith doxorubicin Pretreatment with test compound at thedoses of 50mgkg and 100mgkg before a single dose ofdoxorubicin administration showed a significant (119875 lt 005and 119875 lt 001) dose dependent decrease in TG levelscompared to doxorubicin group

94 Aspartate Aminotransferase (AST) The elevation of ASTlevels in plasma indicates tissue damage The results were asshown in Figure 4

lowast

lowastlowast

020406080

100120140160180200

TG (m

gdL

)

Normal control

Dox + test (100 mgkg)

Dox + test(50 mgkg)

Dox(15 mgkg)

Figure 3 Effect of test compoundonplasmaTG levels in rats treatedwith doxorubicin

119875 lt 0001 versus normal control lowast119875 lt 005lowastlowast

119875 lt 001 versus dox

lowast

lowastlowast

020406080

100120140

Abso

rban

ce (I

UL

)

Series1

Normal control

Dox + test (100 mgkg)

Dox + test(50 mgkg)

Dox(15 mgkg)

Figure 4 Effect of test compound on plasma AST levels in ratstreated with doxorubicin

119875 lt 0001 versus normal control lowast119875 lt005 versus dox lowastlowast119875 lt 001 versus dox

In this study we found that single dose of doxorubicin torats causes significant (119875 lt 0001) elevation of plasma ASTlevelsThis elevation indicates that cardiotoxicity is producedwith doxorubicin Pretreatment with test compound at thedoses of 50mgkg and 100mgkg before a single dose ofdoxorubicin administration showed significant (119875 lt 005 and119875 lt 001) dose dependent decrease in AST levels comparedto doxorubicin group

10 Estimation of Antioxidants

101 Catalase Catalase is a common enzyme found in nearlyall living organisms that are exposed to oxygen To preventdamage caused by hydrogen peroxide it must be quicklyconverted into other less dangerous substances To endthis catalase is frequently used by cells to rapidly catalysethe decomposition of hydrogen peroxide into less reactivegaseous oxygen and water molecules Catalase activity inheart tissue homogenate represents antioxidant status incardiac tissues

In this study we found that single dose of doxorubicinto rats causes significant (119875 lt 0001) decrease in catalaselevels This reduction indicates that toxicity is produced withdoxorubicin Pretreatmentwith test compound at the doses of50mgkg and 100mgkg before a single dose of doxorubicinadministration showed a significant (119875 lt 005 and 119875 lt 001)

Journal of Chemistry 5

002040608

112141618

2

Cata

lase

activ

ity (I

UL

)

lowast

lowastlowast

Series1

Normal control

Dox + test (100 mgkg)

Dox + test(50 mgkg)

Dox(15 mgkg)

Figure 5 Effect of test compound on catalase levels in heart tissuehomogenate in rats treated with doxorubicin

119875 lt 0001 versusnormal control lowast119875 lt 005 versus dox lowastlowast119875 lt 001 versus dox

lowastlowast

0

5

10

15

20

25

30

35

Supe

roxi

de d

ismut

ase (

mU

)

lowast

Normal control

Dox + test (100 mgkg)

Dox + test(50 mgkg)

Dox(15 mgkg)

Figure 6 Effect of test compound on superoxide dismutase levelsin heart tissue homogenate in rats treated with doxorubicin

119875 lt

0001 versus normal control lowastlowast119875 lt 001 versus dox lowastlowastlowast119875 lt 0001versus dox

dose dependent increase in levels compared to doxorubicingroup

102 Superoxide Dismutase Superoxide dismutases are aclass of enzymes that catalyze the dismutation of superoxideinto oxygen and hydrogen peroxide It is an important antiox-idant defense in nearly all cells exposed to oxygen Superoxidedismutase activity was estimated in tissue homogenate withthe help of pure bovine superoxide dismutase standard

In this study we found that single dose of doxorubicinto rats causes significant (119875 lt 0001) decrease in superoxidedismutase levelsThis reduction indicates that toxicity is pro-duced with doxorubicin Pretreatment with test compound atthe doses of 50mgkg and 100mgkg before a single dose ofdoxorubicin administration showed a significant (119875 lt 001and 119875 lt 0001) dose dependent increase in levels comparedto doxorubicin group

103 Glutathione Glutathione the dominant intracellularthiol plays an important protective role against oxidativestress caused by reactive oxygen species It is an endogenousantioxidant Glutathione was estimated in plasma with thehelp of pure glutathione standard graph The values wereshown in Figure 7

020406080

100120140160

Glu

tath

ione

(120583M

)

Series1

Normal control

Dox + test (100 mgkg)

Dox + test(50 mgkg)

Dox(15 mgkg)

lowastlowast

lowastlowastlowast

Figure 7 Effect of test compound on glutathione levels in plasmain rats treated with doxorubicin

119875 lt 0001 versus normal controllowastlowast

119875 lt 001 versus dox lowast119875 lt 0001 versus dox

In this study we found that single dose of doxorubicinto rats causes significant (119875 lt 0001) decrease in glutathionelevels This reduction indicates that toxicity is produced withdoxorubicin Pretreatmentwith test compound at the doses of50mgkg and 100mgkg before a single dose of doxorubicinadministration showed a significant (119875 lt 001 and119875 lt 0001)dose dependent increase in levels compared to doxorubicingroup

11 Histopathological Examination

The heart tissue of different groups of animals was histo-pathologically examined and the results were as shown inFigure 8

Histopathological observations in rat heart tissues

Group I (Normal control) showed no lesions of pathologicalsignificance

Group II (Doxorubicin 15mgkg ip) showed degenerationof muscle bundles

Group III (Doxorubicin + test compound 50mgkg) showeddegeneration of muscle bundles and interstitial edematouschange

Group IV (Doxorubicin + test compound 100mgkg) showedmild degeneration of muscle bundles and less interstitialedematous change

These above results indicate that test compound showedcardioprotection against doxorubicin induced cardiotoxicity

12 Discussion

The present study was aimed to evaluate the cardioprotec-tive activity of test compound by measuring various car-diotoxic biomarkers antioxidant enzymes and histopatho-logical examination in rats

In the present study cardiotoxicitywas induced by a singledose of doxorubicin (15mgkg ip) in rats Doxorubicintreatment causes significant elevation in plasma CK-MB

6 Journal of Chemistry

(a)

(b)

(c)

(d)

Figure 8 Light micrograph of heart tissue from rats treated with(a) normal control (b) doxorubicin treated (c) doxorubicin +test compound 50mgkg and (d) doxorubicin + test compound100mgkg

LDH TG and AST when compared with normal controlgroupThere is a significant reduction in catalase superoxidedismutase levels in heart tissue and significance depletionin glutathione levels in plasma when compared with normalgroup This confirms the induction of cardiotoxicity in ratswhich is supported by histopathological changes observed inheart tissues [7]

Doxorubicin is broad spectrum antibiotic used as ananticancer drug which was limited by its dose dependentcardiotoxic effects [15] The role of oxygen free radicals

and oxidative stress has been well established in case ofdoxorubicin induced cardiac damage in rats Doxorubicinis converted into its semiquinone form in cardiac myocytesby CYP450 and flavin monooxygenases Then it reactswith molecular oxygen initiates cascade of reactions andproduces ROS ROS reacts with lipids proteins and othercellular constituents to cause damage to mitochondria andcell membranes of the heart muscle [16]

Pretreatment with 50mgkg and 100mgkg of test com-pound for 8 days shows significant protection against car-diotoxicity in rats by decreasing the cardiac biomarkers likeCK-MB LDH TG and AST and elevation of antioxidantenzymes like catalase superoxide dismutase levels in hearttissue and glutathione levels in plasma

Histopathological observations of hearts of animalstreated with a single dose of doxorubicin (15mgkg ip)showed degeneration of muscle bundles Pretreatment with50mgkg and 100mgkg of test compound for 8 days showedless or mild degeneration of muscle bundles and mild inter-stitial oedematous change was seen (Figure 8) This showsthat test compound has cardioprotection against doxorubicininduced cardiotoxicity

Catalase is a free radical scavenging enzyme whichhas cellular defence against oxidative stress and scavengingreactive oxygen radicals [17] In the present study doxoru-bicin treatment produced notable decrease in heart tissuecatalase levels when compared with normal Pretreatmentwith 50mgkg and 100mgkg of test compound for 8 daysshowed marked elevation in catalase levels compared withdoxorubicin group (Figure 5)

Superoxide dismutases are a class of enzymes that catalyzethe dismutation of superoxide into oxygen and hydrogenperoxide As such they are an important antioxidant defensein nearly all cells exposed to oxygen [12] In the present studydoxorubicin treatment produced decrease in SOD levelswhen compared with normal Pretreatment with 50mgkgand 100mgkg of test compound for 8 days showed markedelevation in SOD levels compared with doxorubicin group(Figure 6)

Glutathione is a most important antioxidant enzymepreventing damage to important cellular components causedby reactive oxygen species such as free radicals and peroxidesGlutathione is found almost exclusively in its reduced formsince the enzyme that reverses it from its oxidized formglutathione reductase is constitutively active and inducibleupon oxidative stress [13] It exhibits antioxidant activity byreacting with superoxide radicals peroxy radicals and soforth [18] In the present study doxorubicin treatment pro-duced decrease in plasma glutathione levels when comparedwith normal Pretreatment with 50mgkg and 100mgkg oftest compound for 8 days showed marked elevation in glu-tathione levels compared with doxorubicin group (Figure 7)

All the results were supported with previous reportsthatN-(benzo[d]oxazol-2-yl)-2-(5-bromo-2-oxoindolin-3-ylidene)hydrazinecarboxamide has cardioprotective activityagainst doxorubicin induced cardiotoxicity in rats Pretreat-mentwith compound significantly reduced the elevated levelsof cardiotoxic biomarkers in plasma [14]N-substituted isatinderivatives are more potent small molecules with enhanced

Journal of Chemistry 7

free radical scavenger properties and the cytoprotectiveeffect on the apoptosis of PC12 cells induced by H

2O2

was screened [19] These above reports mainly support thatisatin derivatives have free radical scavenger properties andcardioprotective activity The present study results show thatnovel synthetic isatin derivative decreases the cardiotoxicbiomarkers like CK-MB LDH TG and AST and elevates theantioxidant enzymes like catalase and superoxide dismutaseglutathione which supports that novel synthetic isatin deriva-tive has cardioprotective activity

13 Conclusion

On the basis of our findings it may be worthy to suggest thefollowing

(i) Novel synthetic isatin derivative has cardioprotectiveeffect against doxorubicin induced cardiotoxicity inrats by decreasing the cardiotoxic biomarkers likeCK-MB LDH TG and AST in plasma

(ii) Novel synthetic isatin derivative has antioxidanteffect evaluated by measuring antioxidant enzymesThere is an increase in catalase superoxide dismutasein heart tissues and glutathione levels in plasma indoxorubicin induced cardiotoxicity in rats

(iii) Novel synthetic isatin derivative has cardioprotectiveeffect against doxorubicin induced cardiotoxicity byobserving the histopathological changes in rat hearttissues

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The authors are thankful to the Secretary Vaageswari Collegeof Pharmacy Thimmapur Karimnagar for providing thenecessary facilities for carrying out this research

References

[1] C Huang X Zhang J M Ramil et al ldquoJuvenile exposureto anthracyclines impairs cardiac progenitor cell function andvascularization resulting in greater susceptibility to stress-induced myocardial injury in adult micerdquo Circulation vol 121no 5 pp 675ndash683 2010

[2] V Fuster and B B Kelly Promoting Cardiovascular Health in theDevelopingWorld ACritical Challenge to Achieve Global HealthNational Academies Press 2010

[3] K A Reimer and R B Jennings ldquoMyocardial ischemia hypox-ia and infarctionrdquoTheHeart and Cardiovascular System vol 2pp 1133ndash1201 1992

[4] B Bhrigu D Pathak N Siddiqui M S Alam and W AhsanldquoSearch for biological active isatins a short reviewrdquo Interna-tional Journal of Pharmaceutical Sciences amp Drug Research vol2 no 4 pp 229ndash235 2010

[5] S N Pandeya S Smitha M Jyoti and S K Sridhar ldquoBiologicalactivities of isatin and its derivativesrdquo Acta Pharmaceutica vol55 no 1 pp 27ndash46 2005

[6] P K Singal and N Iliskovic ldquoDoxorubicin-induced cardiomy-opathyrdquo The New England Journal of Medicine vol 339 no 13pp 900ndash905 1998

[7] M N Nagi and M A Mansour ldquoProtective effect of thymo-quinone against doxorubicin-induced cardiotoxicity in rats apossible mechanism of protectionrdquo Pharmacological Researchvol 41 no 3 pp 283ndash289 2000

[8] A J Bakker BMirchi J T Dijkstra F Reitsma H Syperda andA Zijlstra ldquoIFCC method for lactate dehydrogenase measure-ment in heparin plasma is unreliablerdquo Clinical Chemistry vol49 no 4 pp 662ndash664 2003

[9] M Panteghini F Ceriotti G Schumann and L SiekmannldquoEstablishing a reference system in clinical enzymologyrdquo Clini-cal Chemistry and Laboratory Medicine vol 39 no 9 pp 795ndash800 2001

[10] G Bucolo and H David ldquoQuantitative determination of serumtriglycerides by the use of enzymesrdquo Clinical Chemistry vol 19no 5 pp 476ndash482 1973

[11] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

[12] H P Misra and I Fridovich ldquoSuperoxide dismutase a pho-tochemical augmentation assayrdquo Archives of Biochemistry andBiophysics vol 181 no 1 pp 308ndash312 1977

[13] E Beutler O Duron and BM Kelly ldquoImprovedmethod for thedetermination of blood glutathionerdquoThe Journal of Laboratoryand Clinical Medicine vol 61 pp 882ndash888 1963

[14] A R N Reddy J Nagarajua G Rajyalaksmib and M Saranga-panib ldquoCardioprotective effect of N-(benzo[d]oxazol-2-yl)-2-(5-bromo-2-oxoindolin-3-ylidene)hydrazinecarboxamideagainst doxorubicin induced cardiotoxicity in ratsrdquo Toxico-logical amp Environmental Chemistry vol 94 no 10 pp 2012ndash2018 2012

[15] F S Carvalho A Burgeiro R Garcia A J Moreno R A Car-valho and P J Oliveira ldquoDoxorubicin-induced cardiotoxicityfrom bioenergetic failure and cell death to cardiomyopathyrdquoMedicinal Research Reviews vol 34 no 1 pp 106ndash135 2014

[16] P K Singal N Iliskovic T Li and D Kumar ldquoAdriamycincardiomyopathy pathophysiology and preventionrdquoThe FASEBJournal vol 11 no 12 pp 931ndash936 1997

[17] A R Lehenbauer Ludke A A-R S Al-Shudiefat S DhingraD S Jassal and P K Singal ldquoA concise description of car-dioprotective strategies in doxorubicin-induced cardiotoxicityrdquoCanadian Journal of Physiology and Pharmacology vol 87 no10 pp 756ndash763 2009

[18] R D Olson J S MacDonald and C J VanBoxtel ldquoRegulatoryrole of glutathione and soluble sulfhydryl groups in the toxicityof adriamycinrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 215 no 2 pp 450ndash454 1980

[19] G Chen Y Wang X Hao S Mu and Q Sun ldquoSimple isatinderivatives as free radical scavengers synthesis biological eval-uation and structure-activity relationshiprdquo Chemistry CentralJournal vol 5 no 1 article 37 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

4 Journal of Chemistry

lowastlowast

lowastlowastlowast

020406080

100120140160180200

Normal control

Dox + test (100 mgkg)

Dox + test(50 mgkg)

Dox(15 mgkg)

CK-M

B (I

UL

)

Figure 1 Effect of test compound on plasma CK-MB levels in ratstreated with doxorubicin

119875 lt 0001 versus normal control lowast119875 lt001 lowastlowastlowast119875 lt 0001 versus dox

lowast

lowastlowast

020406080

100120140160

LDH

(IU

L)

Normal control

Dox + test (100 mgkg)

Dox + test(50 mgkg)

Dox(15 mgkg)

Figure 2 Effect of test compound on plasma LDH levels in ratstreated with doxorubicin

119875 lt 0001 versus normal control lowast119875 lt005 lowastlowast119875 lt 001 versus dox

In this study we found that single dose of doxorubicin torats causes significant (119875 lt 0001) elevation of plasma LDHlevelsThis elevation indicates that cardiotoxicity is producedwith doxorubicin Pretreatment with test compound at thedoses of 50mgkg and 100mgkg before a single dose ofdoxorubicin administration showed a significant (119875 lt 005and 119875 lt 001) dose dependent decrease in LDH levels andthese results were comparable to doxorubicin group

93 Triglycerides (TG) Triglyceride is fatty substance that iscomposed of three fatty acids Triglycerides levels in bloodeither come from the diet or the liver High TG levels in bloodindicate cardiac complications like atherosclerosis whichmaycause toxic events in cardiac tissuesThe resultswere as shownin Figure 3

In this study we found that single dose of doxorubicin torats causes significant (119875 lt 0001) elevation of plasma TGlevelsThis elevation indicates that cardiotoxicity is producedwith doxorubicin Pretreatment with test compound at thedoses of 50mgkg and 100mgkg before a single dose ofdoxorubicin administration showed a significant (119875 lt 005and 119875 lt 001) dose dependent decrease in TG levelscompared to doxorubicin group

94 Aspartate Aminotransferase (AST) The elevation of ASTlevels in plasma indicates tissue damage The results were asshown in Figure 4

lowast

lowastlowast

020406080

100120140160180200

TG (m

gdL

)

Normal control

Dox + test (100 mgkg)

Dox + test(50 mgkg)

Dox(15 mgkg)

Figure 3 Effect of test compoundonplasmaTG levels in rats treatedwith doxorubicin

119875 lt 0001 versus normal control lowast119875 lt 005lowastlowast

119875 lt 001 versus dox

lowast

lowastlowast

020406080

100120140

Abso

rban

ce (I

UL

)

Series1

Normal control

Dox + test (100 mgkg)

Dox + test(50 mgkg)

Dox(15 mgkg)

Figure 4 Effect of test compound on plasma AST levels in ratstreated with doxorubicin

119875 lt 0001 versus normal control lowast119875 lt005 versus dox lowastlowast119875 lt 001 versus dox

In this study we found that single dose of doxorubicin torats causes significant (119875 lt 0001) elevation of plasma ASTlevelsThis elevation indicates that cardiotoxicity is producedwith doxorubicin Pretreatment with test compound at thedoses of 50mgkg and 100mgkg before a single dose ofdoxorubicin administration showed significant (119875 lt 005 and119875 lt 001) dose dependent decrease in AST levels comparedto doxorubicin group

10 Estimation of Antioxidants

101 Catalase Catalase is a common enzyme found in nearlyall living organisms that are exposed to oxygen To preventdamage caused by hydrogen peroxide it must be quicklyconverted into other less dangerous substances To endthis catalase is frequently used by cells to rapidly catalysethe decomposition of hydrogen peroxide into less reactivegaseous oxygen and water molecules Catalase activity inheart tissue homogenate represents antioxidant status incardiac tissues

In this study we found that single dose of doxorubicinto rats causes significant (119875 lt 0001) decrease in catalaselevels This reduction indicates that toxicity is produced withdoxorubicin Pretreatmentwith test compound at the doses of50mgkg and 100mgkg before a single dose of doxorubicinadministration showed a significant (119875 lt 005 and 119875 lt 001)

Journal of Chemistry 5

002040608

112141618

2

Cata

lase

activ

ity (I

UL

)

lowast

lowastlowast

Series1

Normal control

Dox + test (100 mgkg)

Dox + test(50 mgkg)

Dox(15 mgkg)

Figure 5 Effect of test compound on catalase levels in heart tissuehomogenate in rats treated with doxorubicin

119875 lt 0001 versusnormal control lowast119875 lt 005 versus dox lowastlowast119875 lt 001 versus dox

lowastlowast

0

5

10

15

20

25

30

35

Supe

roxi

de d

ismut

ase (

mU

)

lowast

Normal control

Dox + test (100 mgkg)

Dox + test(50 mgkg)

Dox(15 mgkg)

Figure 6 Effect of test compound on superoxide dismutase levelsin heart tissue homogenate in rats treated with doxorubicin

119875 lt

0001 versus normal control lowastlowast119875 lt 001 versus dox lowastlowastlowast119875 lt 0001versus dox

dose dependent increase in levels compared to doxorubicingroup

102 Superoxide Dismutase Superoxide dismutases are aclass of enzymes that catalyze the dismutation of superoxideinto oxygen and hydrogen peroxide It is an important antiox-idant defense in nearly all cells exposed to oxygen Superoxidedismutase activity was estimated in tissue homogenate withthe help of pure bovine superoxide dismutase standard

In this study we found that single dose of doxorubicinto rats causes significant (119875 lt 0001) decrease in superoxidedismutase levelsThis reduction indicates that toxicity is pro-duced with doxorubicin Pretreatment with test compound atthe doses of 50mgkg and 100mgkg before a single dose ofdoxorubicin administration showed a significant (119875 lt 001and 119875 lt 0001) dose dependent increase in levels comparedto doxorubicin group

103 Glutathione Glutathione the dominant intracellularthiol plays an important protective role against oxidativestress caused by reactive oxygen species It is an endogenousantioxidant Glutathione was estimated in plasma with thehelp of pure glutathione standard graph The values wereshown in Figure 7

020406080

100120140160

Glu

tath

ione

(120583M

)

Series1

Normal control

Dox + test (100 mgkg)

Dox + test(50 mgkg)

Dox(15 mgkg)

lowastlowast

lowastlowastlowast

Figure 7 Effect of test compound on glutathione levels in plasmain rats treated with doxorubicin

119875 lt 0001 versus normal controllowastlowast

119875 lt 001 versus dox lowast119875 lt 0001 versus dox

In this study we found that single dose of doxorubicinto rats causes significant (119875 lt 0001) decrease in glutathionelevels This reduction indicates that toxicity is produced withdoxorubicin Pretreatmentwith test compound at the doses of50mgkg and 100mgkg before a single dose of doxorubicinadministration showed a significant (119875 lt 001 and119875 lt 0001)dose dependent increase in levels compared to doxorubicingroup

11 Histopathological Examination

The heart tissue of different groups of animals was histo-pathologically examined and the results were as shown inFigure 8

Histopathological observations in rat heart tissues

Group I (Normal control) showed no lesions of pathologicalsignificance

Group II (Doxorubicin 15mgkg ip) showed degenerationof muscle bundles

Group III (Doxorubicin + test compound 50mgkg) showeddegeneration of muscle bundles and interstitial edematouschange

Group IV (Doxorubicin + test compound 100mgkg) showedmild degeneration of muscle bundles and less interstitialedematous change

These above results indicate that test compound showedcardioprotection against doxorubicin induced cardiotoxicity

12 Discussion

The present study was aimed to evaluate the cardioprotec-tive activity of test compound by measuring various car-diotoxic biomarkers antioxidant enzymes and histopatho-logical examination in rats

In the present study cardiotoxicitywas induced by a singledose of doxorubicin (15mgkg ip) in rats Doxorubicintreatment causes significant elevation in plasma CK-MB

6 Journal of Chemistry

(a)

(b)

(c)

(d)

Figure 8 Light micrograph of heart tissue from rats treated with(a) normal control (b) doxorubicin treated (c) doxorubicin +test compound 50mgkg and (d) doxorubicin + test compound100mgkg

LDH TG and AST when compared with normal controlgroupThere is a significant reduction in catalase superoxidedismutase levels in heart tissue and significance depletionin glutathione levels in plasma when compared with normalgroup This confirms the induction of cardiotoxicity in ratswhich is supported by histopathological changes observed inheart tissues [7]

Doxorubicin is broad spectrum antibiotic used as ananticancer drug which was limited by its dose dependentcardiotoxic effects [15] The role of oxygen free radicals

and oxidative stress has been well established in case ofdoxorubicin induced cardiac damage in rats Doxorubicinis converted into its semiquinone form in cardiac myocytesby CYP450 and flavin monooxygenases Then it reactswith molecular oxygen initiates cascade of reactions andproduces ROS ROS reacts with lipids proteins and othercellular constituents to cause damage to mitochondria andcell membranes of the heart muscle [16]

Pretreatment with 50mgkg and 100mgkg of test com-pound for 8 days shows significant protection against car-diotoxicity in rats by decreasing the cardiac biomarkers likeCK-MB LDH TG and AST and elevation of antioxidantenzymes like catalase superoxide dismutase levels in hearttissue and glutathione levels in plasma

Histopathological observations of hearts of animalstreated with a single dose of doxorubicin (15mgkg ip)showed degeneration of muscle bundles Pretreatment with50mgkg and 100mgkg of test compound for 8 days showedless or mild degeneration of muscle bundles and mild inter-stitial oedematous change was seen (Figure 8) This showsthat test compound has cardioprotection against doxorubicininduced cardiotoxicity

Catalase is a free radical scavenging enzyme whichhas cellular defence against oxidative stress and scavengingreactive oxygen radicals [17] In the present study doxoru-bicin treatment produced notable decrease in heart tissuecatalase levels when compared with normal Pretreatmentwith 50mgkg and 100mgkg of test compound for 8 daysshowed marked elevation in catalase levels compared withdoxorubicin group (Figure 5)

Superoxide dismutases are a class of enzymes that catalyzethe dismutation of superoxide into oxygen and hydrogenperoxide As such they are an important antioxidant defensein nearly all cells exposed to oxygen [12] In the present studydoxorubicin treatment produced decrease in SOD levelswhen compared with normal Pretreatment with 50mgkgand 100mgkg of test compound for 8 days showed markedelevation in SOD levels compared with doxorubicin group(Figure 6)

Glutathione is a most important antioxidant enzymepreventing damage to important cellular components causedby reactive oxygen species such as free radicals and peroxidesGlutathione is found almost exclusively in its reduced formsince the enzyme that reverses it from its oxidized formglutathione reductase is constitutively active and inducibleupon oxidative stress [13] It exhibits antioxidant activity byreacting with superoxide radicals peroxy radicals and soforth [18] In the present study doxorubicin treatment pro-duced decrease in plasma glutathione levels when comparedwith normal Pretreatment with 50mgkg and 100mgkg oftest compound for 8 days showed marked elevation in glu-tathione levels compared with doxorubicin group (Figure 7)

All the results were supported with previous reportsthatN-(benzo[d]oxazol-2-yl)-2-(5-bromo-2-oxoindolin-3-ylidene)hydrazinecarboxamide has cardioprotective activityagainst doxorubicin induced cardiotoxicity in rats Pretreat-mentwith compound significantly reduced the elevated levelsof cardiotoxic biomarkers in plasma [14]N-substituted isatinderivatives are more potent small molecules with enhanced

Journal of Chemistry 7

free radical scavenger properties and the cytoprotectiveeffect on the apoptosis of PC12 cells induced by H

2O2

was screened [19] These above reports mainly support thatisatin derivatives have free radical scavenger properties andcardioprotective activity The present study results show thatnovel synthetic isatin derivative decreases the cardiotoxicbiomarkers like CK-MB LDH TG and AST and elevates theantioxidant enzymes like catalase and superoxide dismutaseglutathione which supports that novel synthetic isatin deriva-tive has cardioprotective activity

13 Conclusion

On the basis of our findings it may be worthy to suggest thefollowing

(i) Novel synthetic isatin derivative has cardioprotectiveeffect against doxorubicin induced cardiotoxicity inrats by decreasing the cardiotoxic biomarkers likeCK-MB LDH TG and AST in plasma

(ii) Novel synthetic isatin derivative has antioxidanteffect evaluated by measuring antioxidant enzymesThere is an increase in catalase superoxide dismutasein heart tissues and glutathione levels in plasma indoxorubicin induced cardiotoxicity in rats

(iii) Novel synthetic isatin derivative has cardioprotectiveeffect against doxorubicin induced cardiotoxicity byobserving the histopathological changes in rat hearttissues

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The authors are thankful to the Secretary Vaageswari Collegeof Pharmacy Thimmapur Karimnagar for providing thenecessary facilities for carrying out this research

References

[1] C Huang X Zhang J M Ramil et al ldquoJuvenile exposureto anthracyclines impairs cardiac progenitor cell function andvascularization resulting in greater susceptibility to stress-induced myocardial injury in adult micerdquo Circulation vol 121no 5 pp 675ndash683 2010

[2] V Fuster and B B Kelly Promoting Cardiovascular Health in theDevelopingWorld ACritical Challenge to Achieve Global HealthNational Academies Press 2010

[3] K A Reimer and R B Jennings ldquoMyocardial ischemia hypox-ia and infarctionrdquoTheHeart and Cardiovascular System vol 2pp 1133ndash1201 1992

[4] B Bhrigu D Pathak N Siddiqui M S Alam and W AhsanldquoSearch for biological active isatins a short reviewrdquo Interna-tional Journal of Pharmaceutical Sciences amp Drug Research vol2 no 4 pp 229ndash235 2010

[5] S N Pandeya S Smitha M Jyoti and S K Sridhar ldquoBiologicalactivities of isatin and its derivativesrdquo Acta Pharmaceutica vol55 no 1 pp 27ndash46 2005

[6] P K Singal and N Iliskovic ldquoDoxorubicin-induced cardiomy-opathyrdquo The New England Journal of Medicine vol 339 no 13pp 900ndash905 1998

[7] M N Nagi and M A Mansour ldquoProtective effect of thymo-quinone against doxorubicin-induced cardiotoxicity in rats apossible mechanism of protectionrdquo Pharmacological Researchvol 41 no 3 pp 283ndash289 2000

[8] A J Bakker BMirchi J T Dijkstra F Reitsma H Syperda andA Zijlstra ldquoIFCC method for lactate dehydrogenase measure-ment in heparin plasma is unreliablerdquo Clinical Chemistry vol49 no 4 pp 662ndash664 2003

[9] M Panteghini F Ceriotti G Schumann and L SiekmannldquoEstablishing a reference system in clinical enzymologyrdquo Clini-cal Chemistry and Laboratory Medicine vol 39 no 9 pp 795ndash800 2001

[10] G Bucolo and H David ldquoQuantitative determination of serumtriglycerides by the use of enzymesrdquo Clinical Chemistry vol 19no 5 pp 476ndash482 1973

[11] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

[12] H P Misra and I Fridovich ldquoSuperoxide dismutase a pho-tochemical augmentation assayrdquo Archives of Biochemistry andBiophysics vol 181 no 1 pp 308ndash312 1977

[13] E Beutler O Duron and BM Kelly ldquoImprovedmethod for thedetermination of blood glutathionerdquoThe Journal of Laboratoryand Clinical Medicine vol 61 pp 882ndash888 1963

[14] A R N Reddy J Nagarajua G Rajyalaksmib and M Saranga-panib ldquoCardioprotective effect of N-(benzo[d]oxazol-2-yl)-2-(5-bromo-2-oxoindolin-3-ylidene)hydrazinecarboxamideagainst doxorubicin induced cardiotoxicity in ratsrdquo Toxico-logical amp Environmental Chemistry vol 94 no 10 pp 2012ndash2018 2012

[15] F S Carvalho A Burgeiro R Garcia A J Moreno R A Car-valho and P J Oliveira ldquoDoxorubicin-induced cardiotoxicityfrom bioenergetic failure and cell death to cardiomyopathyrdquoMedicinal Research Reviews vol 34 no 1 pp 106ndash135 2014

[16] P K Singal N Iliskovic T Li and D Kumar ldquoAdriamycincardiomyopathy pathophysiology and preventionrdquoThe FASEBJournal vol 11 no 12 pp 931ndash936 1997

[17] A R Lehenbauer Ludke A A-R S Al-Shudiefat S DhingraD S Jassal and P K Singal ldquoA concise description of car-dioprotective strategies in doxorubicin-induced cardiotoxicityrdquoCanadian Journal of Physiology and Pharmacology vol 87 no10 pp 756ndash763 2009

[18] R D Olson J S MacDonald and C J VanBoxtel ldquoRegulatoryrole of glutathione and soluble sulfhydryl groups in the toxicityof adriamycinrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 215 no 2 pp 450ndash454 1980

[19] G Chen Y Wang X Hao S Mu and Q Sun ldquoSimple isatinderivatives as free radical scavengers synthesis biological eval-uation and structure-activity relationshiprdquo Chemistry CentralJournal vol 5 no 1 article 37 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Journal of Chemistry 5

002040608

112141618

2

Cata

lase

activ

ity (I

UL

)

lowast

lowastlowast

Series1

Normal control

Dox + test (100 mgkg)

Dox + test(50 mgkg)

Dox(15 mgkg)

Figure 5 Effect of test compound on catalase levels in heart tissuehomogenate in rats treated with doxorubicin

119875 lt 0001 versusnormal control lowast119875 lt 005 versus dox lowastlowast119875 lt 001 versus dox

lowastlowast

0

5

10

15

20

25

30

35

Supe

roxi

de d

ismut

ase (

mU

)

lowast

Normal control

Dox + test (100 mgkg)

Dox + test(50 mgkg)

Dox(15 mgkg)

Figure 6 Effect of test compound on superoxide dismutase levelsin heart tissue homogenate in rats treated with doxorubicin

119875 lt

0001 versus normal control lowastlowast119875 lt 001 versus dox lowastlowastlowast119875 lt 0001versus dox

dose dependent increase in levels compared to doxorubicingroup

102 Superoxide Dismutase Superoxide dismutases are aclass of enzymes that catalyze the dismutation of superoxideinto oxygen and hydrogen peroxide It is an important antiox-idant defense in nearly all cells exposed to oxygen Superoxidedismutase activity was estimated in tissue homogenate withthe help of pure bovine superoxide dismutase standard

In this study we found that single dose of doxorubicinto rats causes significant (119875 lt 0001) decrease in superoxidedismutase levelsThis reduction indicates that toxicity is pro-duced with doxorubicin Pretreatment with test compound atthe doses of 50mgkg and 100mgkg before a single dose ofdoxorubicin administration showed a significant (119875 lt 001and 119875 lt 0001) dose dependent increase in levels comparedto doxorubicin group

103 Glutathione Glutathione the dominant intracellularthiol plays an important protective role against oxidativestress caused by reactive oxygen species It is an endogenousantioxidant Glutathione was estimated in plasma with thehelp of pure glutathione standard graph The values wereshown in Figure 7

020406080

100120140160

Glu

tath

ione

(120583M

)

Series1

Normal control

Dox + test (100 mgkg)

Dox + test(50 mgkg)

Dox(15 mgkg)

lowastlowast

lowastlowastlowast

Figure 7 Effect of test compound on glutathione levels in plasmain rats treated with doxorubicin

119875 lt 0001 versus normal controllowastlowast

119875 lt 001 versus dox lowast119875 lt 0001 versus dox

In this study we found that single dose of doxorubicinto rats causes significant (119875 lt 0001) decrease in glutathionelevels This reduction indicates that toxicity is produced withdoxorubicin Pretreatmentwith test compound at the doses of50mgkg and 100mgkg before a single dose of doxorubicinadministration showed a significant (119875 lt 001 and119875 lt 0001)dose dependent increase in levels compared to doxorubicingroup

11 Histopathological Examination

The heart tissue of different groups of animals was histo-pathologically examined and the results were as shown inFigure 8

Histopathological observations in rat heart tissues

Group I (Normal control) showed no lesions of pathologicalsignificance

Group II (Doxorubicin 15mgkg ip) showed degenerationof muscle bundles

Group III (Doxorubicin + test compound 50mgkg) showeddegeneration of muscle bundles and interstitial edematouschange

Group IV (Doxorubicin + test compound 100mgkg) showedmild degeneration of muscle bundles and less interstitialedematous change

These above results indicate that test compound showedcardioprotection against doxorubicin induced cardiotoxicity

12 Discussion

The present study was aimed to evaluate the cardioprotec-tive activity of test compound by measuring various car-diotoxic biomarkers antioxidant enzymes and histopatho-logical examination in rats

In the present study cardiotoxicitywas induced by a singledose of doxorubicin (15mgkg ip) in rats Doxorubicintreatment causes significant elevation in plasma CK-MB

6 Journal of Chemistry

(a)

(b)

(c)

(d)

Figure 8 Light micrograph of heart tissue from rats treated with(a) normal control (b) doxorubicin treated (c) doxorubicin +test compound 50mgkg and (d) doxorubicin + test compound100mgkg

LDH TG and AST when compared with normal controlgroupThere is a significant reduction in catalase superoxidedismutase levels in heart tissue and significance depletionin glutathione levels in plasma when compared with normalgroup This confirms the induction of cardiotoxicity in ratswhich is supported by histopathological changes observed inheart tissues [7]

Doxorubicin is broad spectrum antibiotic used as ananticancer drug which was limited by its dose dependentcardiotoxic effects [15] The role of oxygen free radicals

and oxidative stress has been well established in case ofdoxorubicin induced cardiac damage in rats Doxorubicinis converted into its semiquinone form in cardiac myocytesby CYP450 and flavin monooxygenases Then it reactswith molecular oxygen initiates cascade of reactions andproduces ROS ROS reacts with lipids proteins and othercellular constituents to cause damage to mitochondria andcell membranes of the heart muscle [16]

Pretreatment with 50mgkg and 100mgkg of test com-pound for 8 days shows significant protection against car-diotoxicity in rats by decreasing the cardiac biomarkers likeCK-MB LDH TG and AST and elevation of antioxidantenzymes like catalase superoxide dismutase levels in hearttissue and glutathione levels in plasma

Histopathological observations of hearts of animalstreated with a single dose of doxorubicin (15mgkg ip)showed degeneration of muscle bundles Pretreatment with50mgkg and 100mgkg of test compound for 8 days showedless or mild degeneration of muscle bundles and mild inter-stitial oedematous change was seen (Figure 8) This showsthat test compound has cardioprotection against doxorubicininduced cardiotoxicity

Catalase is a free radical scavenging enzyme whichhas cellular defence against oxidative stress and scavengingreactive oxygen radicals [17] In the present study doxoru-bicin treatment produced notable decrease in heart tissuecatalase levels when compared with normal Pretreatmentwith 50mgkg and 100mgkg of test compound for 8 daysshowed marked elevation in catalase levels compared withdoxorubicin group (Figure 5)

Superoxide dismutases are a class of enzymes that catalyzethe dismutation of superoxide into oxygen and hydrogenperoxide As such they are an important antioxidant defensein nearly all cells exposed to oxygen [12] In the present studydoxorubicin treatment produced decrease in SOD levelswhen compared with normal Pretreatment with 50mgkgand 100mgkg of test compound for 8 days showed markedelevation in SOD levels compared with doxorubicin group(Figure 6)

Glutathione is a most important antioxidant enzymepreventing damage to important cellular components causedby reactive oxygen species such as free radicals and peroxidesGlutathione is found almost exclusively in its reduced formsince the enzyme that reverses it from its oxidized formglutathione reductase is constitutively active and inducibleupon oxidative stress [13] It exhibits antioxidant activity byreacting with superoxide radicals peroxy radicals and soforth [18] In the present study doxorubicin treatment pro-duced decrease in plasma glutathione levels when comparedwith normal Pretreatment with 50mgkg and 100mgkg oftest compound for 8 days showed marked elevation in glu-tathione levels compared with doxorubicin group (Figure 7)

All the results were supported with previous reportsthatN-(benzo[d]oxazol-2-yl)-2-(5-bromo-2-oxoindolin-3-ylidene)hydrazinecarboxamide has cardioprotective activityagainst doxorubicin induced cardiotoxicity in rats Pretreat-mentwith compound significantly reduced the elevated levelsof cardiotoxic biomarkers in plasma [14]N-substituted isatinderivatives are more potent small molecules with enhanced

Journal of Chemistry 7

free radical scavenger properties and the cytoprotectiveeffect on the apoptosis of PC12 cells induced by H

2O2

was screened [19] These above reports mainly support thatisatin derivatives have free radical scavenger properties andcardioprotective activity The present study results show thatnovel synthetic isatin derivative decreases the cardiotoxicbiomarkers like CK-MB LDH TG and AST and elevates theantioxidant enzymes like catalase and superoxide dismutaseglutathione which supports that novel synthetic isatin deriva-tive has cardioprotective activity

13 Conclusion

On the basis of our findings it may be worthy to suggest thefollowing

(i) Novel synthetic isatin derivative has cardioprotectiveeffect against doxorubicin induced cardiotoxicity inrats by decreasing the cardiotoxic biomarkers likeCK-MB LDH TG and AST in plasma

(ii) Novel synthetic isatin derivative has antioxidanteffect evaluated by measuring antioxidant enzymesThere is an increase in catalase superoxide dismutasein heart tissues and glutathione levels in plasma indoxorubicin induced cardiotoxicity in rats

(iii) Novel synthetic isatin derivative has cardioprotectiveeffect against doxorubicin induced cardiotoxicity byobserving the histopathological changes in rat hearttissues

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The authors are thankful to the Secretary Vaageswari Collegeof Pharmacy Thimmapur Karimnagar for providing thenecessary facilities for carrying out this research

References

[1] C Huang X Zhang J M Ramil et al ldquoJuvenile exposureto anthracyclines impairs cardiac progenitor cell function andvascularization resulting in greater susceptibility to stress-induced myocardial injury in adult micerdquo Circulation vol 121no 5 pp 675ndash683 2010

[2] V Fuster and B B Kelly Promoting Cardiovascular Health in theDevelopingWorld ACritical Challenge to Achieve Global HealthNational Academies Press 2010

[3] K A Reimer and R B Jennings ldquoMyocardial ischemia hypox-ia and infarctionrdquoTheHeart and Cardiovascular System vol 2pp 1133ndash1201 1992

[4] B Bhrigu D Pathak N Siddiqui M S Alam and W AhsanldquoSearch for biological active isatins a short reviewrdquo Interna-tional Journal of Pharmaceutical Sciences amp Drug Research vol2 no 4 pp 229ndash235 2010

[5] S N Pandeya S Smitha M Jyoti and S K Sridhar ldquoBiologicalactivities of isatin and its derivativesrdquo Acta Pharmaceutica vol55 no 1 pp 27ndash46 2005

[6] P K Singal and N Iliskovic ldquoDoxorubicin-induced cardiomy-opathyrdquo The New England Journal of Medicine vol 339 no 13pp 900ndash905 1998

[7] M N Nagi and M A Mansour ldquoProtective effect of thymo-quinone against doxorubicin-induced cardiotoxicity in rats apossible mechanism of protectionrdquo Pharmacological Researchvol 41 no 3 pp 283ndash289 2000

[8] A J Bakker BMirchi J T Dijkstra F Reitsma H Syperda andA Zijlstra ldquoIFCC method for lactate dehydrogenase measure-ment in heparin plasma is unreliablerdquo Clinical Chemistry vol49 no 4 pp 662ndash664 2003

[9] M Panteghini F Ceriotti G Schumann and L SiekmannldquoEstablishing a reference system in clinical enzymologyrdquo Clini-cal Chemistry and Laboratory Medicine vol 39 no 9 pp 795ndash800 2001

[10] G Bucolo and H David ldquoQuantitative determination of serumtriglycerides by the use of enzymesrdquo Clinical Chemistry vol 19no 5 pp 476ndash482 1973

[11] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

[12] H P Misra and I Fridovich ldquoSuperoxide dismutase a pho-tochemical augmentation assayrdquo Archives of Biochemistry andBiophysics vol 181 no 1 pp 308ndash312 1977

[13] E Beutler O Duron and BM Kelly ldquoImprovedmethod for thedetermination of blood glutathionerdquoThe Journal of Laboratoryand Clinical Medicine vol 61 pp 882ndash888 1963

[14] A R N Reddy J Nagarajua G Rajyalaksmib and M Saranga-panib ldquoCardioprotective effect of N-(benzo[d]oxazol-2-yl)-2-(5-bromo-2-oxoindolin-3-ylidene)hydrazinecarboxamideagainst doxorubicin induced cardiotoxicity in ratsrdquo Toxico-logical amp Environmental Chemistry vol 94 no 10 pp 2012ndash2018 2012

[15] F S Carvalho A Burgeiro R Garcia A J Moreno R A Car-valho and P J Oliveira ldquoDoxorubicin-induced cardiotoxicityfrom bioenergetic failure and cell death to cardiomyopathyrdquoMedicinal Research Reviews vol 34 no 1 pp 106ndash135 2014

[16] P K Singal N Iliskovic T Li and D Kumar ldquoAdriamycincardiomyopathy pathophysiology and preventionrdquoThe FASEBJournal vol 11 no 12 pp 931ndash936 1997

[17] A R Lehenbauer Ludke A A-R S Al-Shudiefat S DhingraD S Jassal and P K Singal ldquoA concise description of car-dioprotective strategies in doxorubicin-induced cardiotoxicityrdquoCanadian Journal of Physiology and Pharmacology vol 87 no10 pp 756ndash763 2009

[18] R D Olson J S MacDonald and C J VanBoxtel ldquoRegulatoryrole of glutathione and soluble sulfhydryl groups in the toxicityof adriamycinrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 215 no 2 pp 450ndash454 1980

[19] G Chen Y Wang X Hao S Mu and Q Sun ldquoSimple isatinderivatives as free radical scavengers synthesis biological eval-uation and structure-activity relationshiprdquo Chemistry CentralJournal vol 5 no 1 article 37 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

6 Journal of Chemistry

(a)

(b)

(c)

(d)

Figure 8 Light micrograph of heart tissue from rats treated with(a) normal control (b) doxorubicin treated (c) doxorubicin +test compound 50mgkg and (d) doxorubicin + test compound100mgkg

LDH TG and AST when compared with normal controlgroupThere is a significant reduction in catalase superoxidedismutase levels in heart tissue and significance depletionin glutathione levels in plasma when compared with normalgroup This confirms the induction of cardiotoxicity in ratswhich is supported by histopathological changes observed inheart tissues [7]

Doxorubicin is broad spectrum antibiotic used as ananticancer drug which was limited by its dose dependentcardiotoxic effects [15] The role of oxygen free radicals

and oxidative stress has been well established in case ofdoxorubicin induced cardiac damage in rats Doxorubicinis converted into its semiquinone form in cardiac myocytesby CYP450 and flavin monooxygenases Then it reactswith molecular oxygen initiates cascade of reactions andproduces ROS ROS reacts with lipids proteins and othercellular constituents to cause damage to mitochondria andcell membranes of the heart muscle [16]

Pretreatment with 50mgkg and 100mgkg of test com-pound for 8 days shows significant protection against car-diotoxicity in rats by decreasing the cardiac biomarkers likeCK-MB LDH TG and AST and elevation of antioxidantenzymes like catalase superoxide dismutase levels in hearttissue and glutathione levels in plasma

Histopathological observations of hearts of animalstreated with a single dose of doxorubicin (15mgkg ip)showed degeneration of muscle bundles Pretreatment with50mgkg and 100mgkg of test compound for 8 days showedless or mild degeneration of muscle bundles and mild inter-stitial oedematous change was seen (Figure 8) This showsthat test compound has cardioprotection against doxorubicininduced cardiotoxicity

Catalase is a free radical scavenging enzyme whichhas cellular defence against oxidative stress and scavengingreactive oxygen radicals [17] In the present study doxoru-bicin treatment produced notable decrease in heart tissuecatalase levels when compared with normal Pretreatmentwith 50mgkg and 100mgkg of test compound for 8 daysshowed marked elevation in catalase levels compared withdoxorubicin group (Figure 5)

Superoxide dismutases are a class of enzymes that catalyzethe dismutation of superoxide into oxygen and hydrogenperoxide As such they are an important antioxidant defensein nearly all cells exposed to oxygen [12] In the present studydoxorubicin treatment produced decrease in SOD levelswhen compared with normal Pretreatment with 50mgkgand 100mgkg of test compound for 8 days showed markedelevation in SOD levels compared with doxorubicin group(Figure 6)

Glutathione is a most important antioxidant enzymepreventing damage to important cellular components causedby reactive oxygen species such as free radicals and peroxidesGlutathione is found almost exclusively in its reduced formsince the enzyme that reverses it from its oxidized formglutathione reductase is constitutively active and inducibleupon oxidative stress [13] It exhibits antioxidant activity byreacting with superoxide radicals peroxy radicals and soforth [18] In the present study doxorubicin treatment pro-duced decrease in plasma glutathione levels when comparedwith normal Pretreatment with 50mgkg and 100mgkg oftest compound for 8 days showed marked elevation in glu-tathione levels compared with doxorubicin group (Figure 7)

All the results were supported with previous reportsthatN-(benzo[d]oxazol-2-yl)-2-(5-bromo-2-oxoindolin-3-ylidene)hydrazinecarboxamide has cardioprotective activityagainst doxorubicin induced cardiotoxicity in rats Pretreat-mentwith compound significantly reduced the elevated levelsof cardiotoxic biomarkers in plasma [14]N-substituted isatinderivatives are more potent small molecules with enhanced

Journal of Chemistry 7

free radical scavenger properties and the cytoprotectiveeffect on the apoptosis of PC12 cells induced by H

2O2

was screened [19] These above reports mainly support thatisatin derivatives have free radical scavenger properties andcardioprotective activity The present study results show thatnovel synthetic isatin derivative decreases the cardiotoxicbiomarkers like CK-MB LDH TG and AST and elevates theantioxidant enzymes like catalase and superoxide dismutaseglutathione which supports that novel synthetic isatin deriva-tive has cardioprotective activity

13 Conclusion

On the basis of our findings it may be worthy to suggest thefollowing

(i) Novel synthetic isatin derivative has cardioprotectiveeffect against doxorubicin induced cardiotoxicity inrats by decreasing the cardiotoxic biomarkers likeCK-MB LDH TG and AST in plasma

(ii) Novel synthetic isatin derivative has antioxidanteffect evaluated by measuring antioxidant enzymesThere is an increase in catalase superoxide dismutasein heart tissues and glutathione levels in plasma indoxorubicin induced cardiotoxicity in rats

(iii) Novel synthetic isatin derivative has cardioprotectiveeffect against doxorubicin induced cardiotoxicity byobserving the histopathological changes in rat hearttissues

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The authors are thankful to the Secretary Vaageswari Collegeof Pharmacy Thimmapur Karimnagar for providing thenecessary facilities for carrying out this research

References

[1] C Huang X Zhang J M Ramil et al ldquoJuvenile exposureto anthracyclines impairs cardiac progenitor cell function andvascularization resulting in greater susceptibility to stress-induced myocardial injury in adult micerdquo Circulation vol 121no 5 pp 675ndash683 2010

[2] V Fuster and B B Kelly Promoting Cardiovascular Health in theDevelopingWorld ACritical Challenge to Achieve Global HealthNational Academies Press 2010

[3] K A Reimer and R B Jennings ldquoMyocardial ischemia hypox-ia and infarctionrdquoTheHeart and Cardiovascular System vol 2pp 1133ndash1201 1992

[4] B Bhrigu D Pathak N Siddiqui M S Alam and W AhsanldquoSearch for biological active isatins a short reviewrdquo Interna-tional Journal of Pharmaceutical Sciences amp Drug Research vol2 no 4 pp 229ndash235 2010

[5] S N Pandeya S Smitha M Jyoti and S K Sridhar ldquoBiologicalactivities of isatin and its derivativesrdquo Acta Pharmaceutica vol55 no 1 pp 27ndash46 2005

[6] P K Singal and N Iliskovic ldquoDoxorubicin-induced cardiomy-opathyrdquo The New England Journal of Medicine vol 339 no 13pp 900ndash905 1998

[7] M N Nagi and M A Mansour ldquoProtective effect of thymo-quinone against doxorubicin-induced cardiotoxicity in rats apossible mechanism of protectionrdquo Pharmacological Researchvol 41 no 3 pp 283ndash289 2000

[8] A J Bakker BMirchi J T Dijkstra F Reitsma H Syperda andA Zijlstra ldquoIFCC method for lactate dehydrogenase measure-ment in heparin plasma is unreliablerdquo Clinical Chemistry vol49 no 4 pp 662ndash664 2003

[9] M Panteghini F Ceriotti G Schumann and L SiekmannldquoEstablishing a reference system in clinical enzymologyrdquo Clini-cal Chemistry and Laboratory Medicine vol 39 no 9 pp 795ndash800 2001

[10] G Bucolo and H David ldquoQuantitative determination of serumtriglycerides by the use of enzymesrdquo Clinical Chemistry vol 19no 5 pp 476ndash482 1973

[11] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

[12] H P Misra and I Fridovich ldquoSuperoxide dismutase a pho-tochemical augmentation assayrdquo Archives of Biochemistry andBiophysics vol 181 no 1 pp 308ndash312 1977

[13] E Beutler O Duron and BM Kelly ldquoImprovedmethod for thedetermination of blood glutathionerdquoThe Journal of Laboratoryand Clinical Medicine vol 61 pp 882ndash888 1963

[14] A R N Reddy J Nagarajua G Rajyalaksmib and M Saranga-panib ldquoCardioprotective effect of N-(benzo[d]oxazol-2-yl)-2-(5-bromo-2-oxoindolin-3-ylidene)hydrazinecarboxamideagainst doxorubicin induced cardiotoxicity in ratsrdquo Toxico-logical amp Environmental Chemistry vol 94 no 10 pp 2012ndash2018 2012

[15] F S Carvalho A Burgeiro R Garcia A J Moreno R A Car-valho and P J Oliveira ldquoDoxorubicin-induced cardiotoxicityfrom bioenergetic failure and cell death to cardiomyopathyrdquoMedicinal Research Reviews vol 34 no 1 pp 106ndash135 2014

[16] P K Singal N Iliskovic T Li and D Kumar ldquoAdriamycincardiomyopathy pathophysiology and preventionrdquoThe FASEBJournal vol 11 no 12 pp 931ndash936 1997

[17] A R Lehenbauer Ludke A A-R S Al-Shudiefat S DhingraD S Jassal and P K Singal ldquoA concise description of car-dioprotective strategies in doxorubicin-induced cardiotoxicityrdquoCanadian Journal of Physiology and Pharmacology vol 87 no10 pp 756ndash763 2009

[18] R D Olson J S MacDonald and C J VanBoxtel ldquoRegulatoryrole of glutathione and soluble sulfhydryl groups in the toxicityof adriamycinrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 215 no 2 pp 450ndash454 1980

[19] G Chen Y Wang X Hao S Mu and Q Sun ldquoSimple isatinderivatives as free radical scavengers synthesis biological eval-uation and structure-activity relationshiprdquo Chemistry CentralJournal vol 5 no 1 article 37 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Journal of Chemistry 7

free radical scavenger properties and the cytoprotectiveeffect on the apoptosis of PC12 cells induced by H

2O2

was screened [19] These above reports mainly support thatisatin derivatives have free radical scavenger properties andcardioprotective activity The present study results show thatnovel synthetic isatin derivative decreases the cardiotoxicbiomarkers like CK-MB LDH TG and AST and elevates theantioxidant enzymes like catalase and superoxide dismutaseglutathione which supports that novel synthetic isatin deriva-tive has cardioprotective activity

13 Conclusion

On the basis of our findings it may be worthy to suggest thefollowing

(i) Novel synthetic isatin derivative has cardioprotectiveeffect against doxorubicin induced cardiotoxicity inrats by decreasing the cardiotoxic biomarkers likeCK-MB LDH TG and AST in plasma

(ii) Novel synthetic isatin derivative has antioxidanteffect evaluated by measuring antioxidant enzymesThere is an increase in catalase superoxide dismutasein heart tissues and glutathione levels in plasma indoxorubicin induced cardiotoxicity in rats

(iii) Novel synthetic isatin derivative has cardioprotectiveeffect against doxorubicin induced cardiotoxicity byobserving the histopathological changes in rat hearttissues

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The authors are thankful to the Secretary Vaageswari Collegeof Pharmacy Thimmapur Karimnagar for providing thenecessary facilities for carrying out this research

References

[1] C Huang X Zhang J M Ramil et al ldquoJuvenile exposureto anthracyclines impairs cardiac progenitor cell function andvascularization resulting in greater susceptibility to stress-induced myocardial injury in adult micerdquo Circulation vol 121no 5 pp 675ndash683 2010

[2] V Fuster and B B Kelly Promoting Cardiovascular Health in theDevelopingWorld ACritical Challenge to Achieve Global HealthNational Academies Press 2010

[3] K A Reimer and R B Jennings ldquoMyocardial ischemia hypox-ia and infarctionrdquoTheHeart and Cardiovascular System vol 2pp 1133ndash1201 1992

[4] B Bhrigu D Pathak N Siddiqui M S Alam and W AhsanldquoSearch for biological active isatins a short reviewrdquo Interna-tional Journal of Pharmaceutical Sciences amp Drug Research vol2 no 4 pp 229ndash235 2010

[5] S N Pandeya S Smitha M Jyoti and S K Sridhar ldquoBiologicalactivities of isatin and its derivativesrdquo Acta Pharmaceutica vol55 no 1 pp 27ndash46 2005

[6] P K Singal and N Iliskovic ldquoDoxorubicin-induced cardiomy-opathyrdquo The New England Journal of Medicine vol 339 no 13pp 900ndash905 1998

[7] M N Nagi and M A Mansour ldquoProtective effect of thymo-quinone against doxorubicin-induced cardiotoxicity in rats apossible mechanism of protectionrdquo Pharmacological Researchvol 41 no 3 pp 283ndash289 2000

[8] A J Bakker BMirchi J T Dijkstra F Reitsma H Syperda andA Zijlstra ldquoIFCC method for lactate dehydrogenase measure-ment in heparin plasma is unreliablerdquo Clinical Chemistry vol49 no 4 pp 662ndash664 2003

[9] M Panteghini F Ceriotti G Schumann and L SiekmannldquoEstablishing a reference system in clinical enzymologyrdquo Clini-cal Chemistry and Laboratory Medicine vol 39 no 9 pp 795ndash800 2001

[10] G Bucolo and H David ldquoQuantitative determination of serumtriglycerides by the use of enzymesrdquo Clinical Chemistry vol 19no 5 pp 476ndash482 1973

[11] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

[12] H P Misra and I Fridovich ldquoSuperoxide dismutase a pho-tochemical augmentation assayrdquo Archives of Biochemistry andBiophysics vol 181 no 1 pp 308ndash312 1977

[13] E Beutler O Duron and BM Kelly ldquoImprovedmethod for thedetermination of blood glutathionerdquoThe Journal of Laboratoryand Clinical Medicine vol 61 pp 882ndash888 1963

[14] A R N Reddy J Nagarajua G Rajyalaksmib and M Saranga-panib ldquoCardioprotective effect of N-(benzo[d]oxazol-2-yl)-2-(5-bromo-2-oxoindolin-3-ylidene)hydrazinecarboxamideagainst doxorubicin induced cardiotoxicity in ratsrdquo Toxico-logical amp Environmental Chemistry vol 94 no 10 pp 2012ndash2018 2012

[15] F S Carvalho A Burgeiro R Garcia A J Moreno R A Car-valho and P J Oliveira ldquoDoxorubicin-induced cardiotoxicityfrom bioenergetic failure and cell death to cardiomyopathyrdquoMedicinal Research Reviews vol 34 no 1 pp 106ndash135 2014

[16] P K Singal N Iliskovic T Li and D Kumar ldquoAdriamycincardiomyopathy pathophysiology and preventionrdquoThe FASEBJournal vol 11 no 12 pp 931ndash936 1997

[17] A R Lehenbauer Ludke A A-R S Al-Shudiefat S DhingraD S Jassal and P K Singal ldquoA concise description of car-dioprotective strategies in doxorubicin-induced cardiotoxicityrdquoCanadian Journal of Physiology and Pharmacology vol 87 no10 pp 756ndash763 2009

[18] R D Olson J S MacDonald and C J VanBoxtel ldquoRegulatoryrole of glutathione and soluble sulfhydryl groups in the toxicityof adriamycinrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 215 no 2 pp 450ndash454 1980

[19] G Chen Y Wang X Hao S Mu and Q Sun ldquoSimple isatinderivatives as free radical scavengers synthesis biological eval-uation and structure-activity relationshiprdquo Chemistry CentralJournal vol 5 no 1 article 37 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of