research article cardioprotective activity of pongamia...

Research ArticleCardioprotective Activity of Pongamia pinnata inStreptozotocin-Nicotinamide Induced Diabetic Rats

Sachin L Badole1 Swapnil M Chaudhari1 Ganesh B Jangam1

Amit D Kandhare2 and Subhash L Bodhankar2

1Department of Pharmacology PESrsquos Modern College of Pharmacy Sector 21 Yamuna Nagar Nigadi Pune 411 044 India2Department of Pharmacology Poona College of Pharmacy Bharati Vidyapeeth Deemed University Paud RoadErandwane Pune 411 038 India

Correspondence should be addressed to Sachin L Badole sachinbadolerediffmailcom

Received 17 February 2015 Revised 29 March 2015 Accepted 30 March 2015

Academic Editor Kazim Husain

Copyright copy 2015 Sachin L Badole 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

Pongamia pinnata (L) Pierre has been used in traditional medicine for the treatment for diabetes and metabolic disorder Theaim of this study was to investigate the effect of petroleum ether extract of the stem bark of P pinnata (known as PPSB-PEE) oncardiomyopathy in diabetic rats Diabetes was induced in overnight fasted Sprague-Dawley rats by using injection of streptozotocin(55mgkg ip) Nicotinamide (100mgkg ip) was administered 20min before administration of streptozotocin Rats were dividedinto group I nondiabetic group II diabetic control (tween 80 2 10mLkg po) as vehicle and group III PPSB-PEE (100mgkgpo) The blood glucose level ECG hemodynamic parameters cardiotoxic and antioxidant biomarkers and histology of heartwere carried out after 4 months after STZ with nicotinamide injection PPSB-PEE treatment improved the electrocardiographichemodynamic changes LV contractile function biological markers oxidative stress parameters and histological changes in STZinduced diabetic rats PPSB-PEE (100mgkg po) decreased blood glucose level improved electrocardiographic parameters (QRSQT and QTc intervals) and hemodynamic parameters (SBP DBP EDP max 119889119875119889119905 contractility index and heart rate) controlledlevels of cardiac biomarkers (CK-MB LDH and AST) and improved oxidative stress (SOD MDA and GSH) in diabetic ratsPPSB-PEE is a promising remedy against cardiomyopathy in diabetic rats

1 Introduction

Diabetic cardiomyopathy is defined as the changes inducedby diabetes mellitus in cardiac structurefunction in theabsence of ischemic heart disease hypertension or othercardiac pathologies [1] Diabetes is multifactorial and hasbeen associatedwith various disorders including obesity dys-lipidemia thrombosis infarction hypertension endothelialdysfunction and coronary artery disease [2] However dias-tolic and systolic dysfunctions are mainly altered apart fromtraditional cardiac risks parameters such as hypertensionatherosclerosis and dyslipidemia [3] Diabetic patients havean increased risk of cardiovascular diseases and these arethe major cause of death in them [4 5] Cardiomyopathyis a prevalent cause of death in patients with diabetes [6]

The world prevalence of diabetes among adults (aged 20ndash79years) will be 64 affecting 285 million adults in 2010 andwill increase to 77 affecting 439 million adults by 2030[7] The global prevalence of diabetes mellitus is forecast toreach 300 million by 2025 and over three quarters of thedeaths amongst this population will be expected to resultfrom cardiovascular disease [8] Individuals with diabetesare at a significantly greater risk of developing both micro-and macrovascular disease and have a cardiac mortalityequivalent to that in nondiabetic patients with confirmedheart disease [9]

Pongamia pinnata (L) Pierre (Fabaceae) is popularlyknown as Indian beech in English [10] In previous study wereported that petroleum (PPSB-PEE) and alcoholic extract(PPSB-ALE) of the stem bark of P pinnata (L) showed

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 403291 8 pageshttpdxdoiorg1011552015403291

2 BioMed Research International

antihyperglycaemic activity in diabetic mice [11 12] Furtherwe observed that concomitant administration of PPSB-PEEwith synthetic oral hypoglycemic drugs produced synergisticeffect in diabetic mice [13] The preliminary phytochemicalanalysis of PPSB-PEE showed the presence of alkaloidsterpenoids triterpenes flavonoids steroids and volatile oils[12] Cycloart-23-ene-3120573 25-diol (B2) isolated from the stembark of P pinnata possesses antidiabetic activity in diabeticanimals [14 15] Cycloart-23-ene-3120573 25-diol improved theabnormalities of diabetic conditions in diabetic mice due toincreased glucagon-like peptide 1 (GLP-1) insulin secretion[16] and has a protective effect on vital organs like heart andkidney [17] The literature review indicated that P pinnatabark was used by traditional practitioners of medicine fortreatment of diabetes and its complications However morescientific work is necessary to validate the ethnobotanicalclaim relating to diabetic complications Hence our objectiveof this studywas to evaluate cardioprotective activity of PPSB-PEE in diabetic rats

2 Materials and Methods

21 Collection Identification and Authentication of PlantMaterials The fresh bark of Pongamia pinnata (L) Pierrewas collected in August-September 2011 from mature treenear Padali village Parner Taluka Ahmednagar districtMaharashtra state India The bark of Pongamia pinnatawas identified and authenticated at Department of BotanyAgharkar Research Institute Pune India and voucher spec-imen was deposited at that institute (Pongamia pinnatavoucher specimen sample number AHMA-23892)

22 Drugs and Chemicals Streptozotocin (STZ) nicoti-namide (NTM) epinephrine hydrochloride superoxide dis-mutase (SOD) andmalondialdehyde (MDA)were purchasedfrom Sigma Chemical Co USA Petroleum ether (60 80)diethyl ether (Merck India) tween 80 (Research-Lab India)reduced glutathione (GS H) 551015840-dithiobis-(2-nitrobenzoicacid) (DTNB) (Hi media India) thiobarbituric acid (TBA)thiopentone sodium injection (Thiosol Sodium) (NeonLabo-ratories Ltd India) creatine kinase-MB isoenzyme (CKMB)(Randox Laboratory UK) LDH (Ecoline Merck India) andGODPOD kit (Accurex India) were purchased from respec-tive vendors All chemicals used were of analytical grade

23 Preparation of PPSB-PEE PPSB-PEE was prepared byusing previously reported method [12] Briefly weighedquantity (500 g) of air-dried powder (Mesh size-16) of thestem bark of P pinnata was macerated with petroleum ether(3000mL) at room temperature for 7 days with occasionalshaking This process was repeated with same volume ofpetroleum etherMacerates were pooled transferred to previ-ously weighed Petri dish and evaporated to dryness at roomtemperature to obtain 16ww dried extract

24 Experimental Animals and Research Protocol ApprovalMale Sprague-Dawley (SD) rats (200ndash250 g) were procured atour institution animal house facility Pune India and housedin an air-conditioned room at a temperature of 25 plusmn 2∘C

and relative humidity of 45 to 55 under 12-h light 12-h dark cycle The animals had free access to food pellets(Chakan Oil Mills Pune India) Water was provided adlibitum The experimental protocol was approved by theInstitutional Animal Ethics Committee (IAEC) constituted inaccordance with the rules and guidelines of the Committeefor the Purpose of Control and Supervision on ExperimentalAnimals (CPCSEA) New Delhi India (protocol numberMCPIAEC202011)

25 Preparation of Drugs Solution The semisolid PPSB-PEEwas emulsified with 2 tween 80 in distilled water to preparethe drug solution Streptozotocin was dissolved in citratebuffer (pH 45) and nicotinamide was dissolved in normalphysiological saline

26 Induction of Diabetes Diabetes was induced in overnightfasted Sprague-Dawley rats by using streptozotocin injec-tion (55mgkg ip) Nicotinamide (100mgkg ip) wasadministered 20min before administration of streptozotocin[16] Hyperglycaemia was confirmed after 3 days using theglucose oxidase peroxidase (GODPOD) method Glycemiclevel control of diabetic rats was made with subcutaneousinjections of exogenous human NPH insulin (2 unitsday ofNPH insulin) Those rats that showed blood glucose levelsabove 300mgdL (diabetic) were selected for the study

27 Dose Selection of PPSB-PEE In our previous reportedefficacy study PPSB-PEE (100mgkg po) showed mostpromising antihyperglycemic effect in diabetic mice [12 13]Based on our previous results we selected dose (100mgkgpo) of PPSB-PEE for this study

28 Effect of Chronic Administration of PPSB-PEE in DiabeticRats Male Sprague-Dawley rats weighing 200ndash250 g bodyweight were divided into three groups and each groupcontained six rats (119899 = 6) as follows group I nondiabeticgroup II diabetic control (tween 80 2 10mLkg po) asvehicle and group III PPSB-PEE (100mgkg po) Vehicleand PPSB-PEE were administered for 4 months (once a day)at predetermined time (15 days after injection of streptozo-tocin and nicotinamide) The condition of the animals wasmonitored daily at the time of dosing as per experimentaldesign Blood was collected using the retroorbital plexus ofeach rat under mild ether anesthesia Blood glucose wasdetermined by using GODPOD method at end of the studyperiod

29 Serum Cardiac Markers At the end of experimentalperiod rats were anaesthetized with diethyl ether Bloodwas collected by retroorbital puncture and following serumwas separated Serum levels of aspartate aminotransferase(AST) creatine kinase-MB isoenzyme (CK-MB) and lactatedehydrogenase (LDH) enzymes were measured by auto-mated chemistry analyzer (Microlab 300 Merck USA) usingreagent kits

210 Electrocardiographic (ECG) Hemodynamic ParametersThe rats were anesthetized with urethane (125 gkg ip)ECG was recorded using 8-channel recording Power Lab

BioMed Research International 3

System (AD Instruments LABCHART 6 software Australia)and QRS interval QT interval heart rate and QTc intervalwere recorded Simultaneously blood pressure was recordedby polyethylene cannula (PE 50) filledwith heparinised saline(100 IUmL) and connected to pressure transducer The rightcarotid artery was cannulated transducer was filled withheparinised saline and connected to pressure transducer formeasurement of blood pressure and systolic blood pressure(SBP) diastolic blood pressure (DBP) and mean arterialblood pressure (MABP) were recorded Further left ventric-ular systolic pressure was measured by means of a Millarmikro-tip transducer catheter (Millar instrument TX USA)inserted into the left ventricle via the right carotid artery andconnected to a bioamplifier and end diastolic pressure (EDP)maximum 119889119875119889119905 contractility index and minimum 119889119875119889119905were recorded as standardized in our laboratory

211 Effect on Enzymatic Biomarkers of Oxidative Stress Therats were humanely euthanized after recoding all parametersby cervical dislocation method and the heart was removedanddivided into two portionsOne portionwas used formea-surement of myocardial endogenous antioxidant enzymesHeart tissues were isolated from all rats and were cut intosmall pieces placed in chilled 025M sucrose solution andblotted on a filter paper The tissues were homogenized in10 chilled Tris hydrochloride buffer (10mM pH 74) bytissue homogenizer (Remi Motors India) and centrifugedat 12000 rpm for 15min 0∘C using Eppendorf rsquos 5810-Rhigh speed cooling centrifuge The endogenous antioxi-dant enzymes estimated were malondialdehyde reducedglutathione and superoxide dismutase Malondialdehydecontent in supernatant of the rat heart was determined bymethod of Slater and Sawyer [18] The assay of reducedglutathione was carried out by method of Moron et al [19]The superoxide dismutase activitywas determined bymethodof Misra and Fridovich [20] Protein concentrations weredetermined using the method of Lowry et al [21]

212 Histopathology of Heart by Hematoxylin-Eosin StainingThe isolated tissue was trimmed into small pieces andpreserved in 10 formalin for 24 h Specimens were cut insections of 3ndash5 120583m in thickness by microtome and stainedby hematoxylin-eosin The specimens were mounted bydisterene phthalate xylene (DPX) The photomicrographsof each tissue section were observed using Cell Imagingsoftware for Life Science microscopy (Olympus Soft ImagingSolution GmbH Munster Germany)

213 Statistical Analysis The data was expressed as mean plusmnstandard error of mean (SEM) One-way analysis of variance(ANOVA) was applied to test the significance of differencebetween average biochemical and ECG parameters of differ-ent groups and multiple comparisons were determined bypost hocDunnettrsquos testThe statistical analysis was performedusing GraphPad Prism 50 software (GraphPad software SanDiego CA USA) 119901 lt 005 was considered statisticallysignificant

0

100

200

300

400

500

600

700

NondiabeticDiabetic control

Time (months)

Plas

ma g

luco

se le

vel (

mg

dL)

minus2 minus1 0 1 2 3 4 5

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

PPSB-PEE (100mgkg)

Figure 1 Effect of PPSB-PEE on blood glucose level in rats Valuesare mean plusmn SEM 119899 = 6 in each group and statistical analysis wascarried out by one-way ANOVA followed by post hoc Dunnettrsquostest performed using GraphPad Prism lowastlowast119901 lt 001 lowastlowastlowast119901 lt 0001ns nonsignificant compared with diabetic group 119901 lt 0001 nsnonsignificant compared with nondiabetic group

3 Results

31 Effect of PPSB-PEE on Blood Glucose Level in DiabeticCardiomyopathy in Rats In this study we observed thatdiabetic control groupwas significantly (119901 lt 0001) increasedin blood glucose level compared to normal control groupTreatment with PPSB-PEE (100mgkg po) once a day for 4months at predetermined time caused significant (119901 lt 0001)reduction in blood glucose level compared to diabetic controlgroup It is thus apparent that PPSB-PEE treatment resulted inbetter control of blood glucose level in diabetic rats (Figure 1)

32 Effect of PPSB-PEE on ECG in Rats ECG recording wasfound normal in nondiabetic group QRS interval of diabeticcontrol was significantly (119901 lt 001) decreased compared tonondiabetic group After treatment with PPSB-PEE the QRSinterval was significantly (119901 lt 005) increased comparedto diabetic control group QT and QTc interval of diabeticcontrol showed significant (119901 lt 0001) increase compared tonondiabetic group QT and QTc intervals showed significant(119901 lt 005) decrease in PPSB-PEE treatment group comparedto diabetic control group The heart rate of diabetic controlshowed significant (119901 lt 001) decrease compared to nondia-betic group After treatment with PPSB-PEE the heart rateshowed significant (119901 lt 005) increase (Table 1)

33 Effect of PPSB-PEE on Hemodynamic and LV Functionin Rats Diabetic control group showed significant (119901 lt0001) decrease in SBP DBP EDP and MABP compared tonondiabetic group A significant (119901 lt 0001) increase wasfound in SBP DBP and EBP in PPSB-PEE treated diabeticgroups However nonsignificant fluctuation was observed inMABP after PPSB-PEE treatment (Table 1) Diabetic group


Table 1 Effect of PPSB-PEE treatment on electrocardiographic hemodynamic changes and LV contractile function in rats

Parameters Nondiabetic Diabetic control PPSB-PEE (100mgkg)

ECG

Heart rate (BPM) 35348 plusmn 1512 25795 plusmn 3252 33888 plusmn 4108

QRS interval (ms) 2312 plusmn 523 1374 plusmn 203 1823 plusmn 345

QT interval (ms) 6726 plusmn 124 9100 plusmn 287 7363 plusmn 202

QTc interval (ms) 13108 plusmn 245 19491 plusmn 388 15244 plusmn 089

Hemodynamic

SBP (mmHg) 12431 plusmn 122 8751 plusmn 354 11584 plusmn 108

DBP (mmHg) 9141 plusmn 144 6772 plusmn 102 7865 plusmn 132

EDP (mmHg) 1491 plusmn 211 713 plusmn 102 1828 plusmn 427

MABP (mmHg) 12225 plusmn 432 7992 plusmn 012 9865 plusmn 078

LV functionMax 119889119875119889119905 (mmHgs) 392193 plusmn 32234 261929 plusmn 8041 369572 plusmn 28795

Min 119889119875119889119905 (mmHgs) minus251314 plusmn 45765 minus169452 plusmn 44721 minus214329 plusmn 39847Contractility index 5618 plusmn 565 3775 plusmn 318 4352 plusmn 298

Data are expressed as mean plusmn SEM (119899 = 6) and analyzed by one-way ANOVA followed by post hoc Dunnettrsquos test 119901 lt 005 119901 lt 0001 ns nonsignificant

compared with diabetic group 119901 lt 005 119901 lt 001 119901 lt 0001 ns nonsignificant compared with nondiabetic group

Table 2 Effect of PPSB-PEE treatment on cardiac serum biomarkers in rats

Parameters Nondiabetic Diabetic control PPSB-PEE (100mgkg)CK-MB (IUL) concentration 112132 plusmn 10154 203215 plusmn 16331 133014 plusmn 4562

LDH (IUL) concentration 129075 plusmn 9623 281642 plusmn 11357 176666 plusmn 9875

AST (IUL) concentration 16341 plusmn 2187 53192 plusmn 4741 33250 plusmn 2362

Data are expressed as mean plusmn SEM (119899 = 6) and analyzed by one-way ANOVA followed by post hoc Dunnettrsquos test 119901 lt 005 119901 lt 001 119901 lt 0001 nsnonsignificant compared with diabetic group 119901 lt 0001 ns nonsignificant compared with nondiabetic group

showed significant decrease in max 119889119875119889119905 (119901 lt 001)contractility index (119901 lt 0001) and min 119889119875119889119905 (119901 lt 005)compared to nondiabetic group while PPSB-PEE treatmentgroup showed significant (119901 lt 005) increase in max 119889119875119889119905and contractility index (119901 lt 0001) compared to diabeticcontrol group (Table 1)

34 Effect of PPSB-PEE on Serum Cardiac Markers in RatsSerum AST LDH and CK-MB level were significantlyincreased (119901 lt 0001) in diabetic group compared to non-diabetic group PPSB-PEE treatment significantly reducedserum CK-MB (119901 lt 0001) LDH (119901 lt 001) and AST(119901 lt 005) level compared to diabetic group (Table 2)

35 Effect of PPSB-PEE on Oxidative Stress Markers in RatsThe SOD and GSH levels were significantly reduced (119901 lt0001) in the diabetic compared to nondiabetic group PPSB-PEE treated group showed significantly higher levels of SOD(119901 lt 0001) and GSH (119901 lt 001) compared to diabeticgroup However MDA level in diabetic group significantly(119901 lt 0001) increased compared to nondiabetic groupTreatment of PPSB-PEE showed significant decrease (119901 lt0001) compared to diabetic group (Figure 2)

36 Effect of PPSB-PEE on Histopathology of Heart in RatsHematoxylin-eosin staining of nondiabetic rat heart showednormal architecture of heart (Figure 3(a)) The photomicro-graphs of diabetic control rat heart showed massive necrosisof heart muscle fibers along with focal mass and frag-mentation Disorganized arrangement with no well-definedboundaries vascular congestion cytoplasmic vacuoles and

cytoplasmic eosinophilia were observed severely in myocar-dial cells (Figure 3(b)) Treatment with PPSB-PEE showedlesser loss of myocardial fibers myocardial hypertrophy andcytoplasmic eosinophilia causing pink coloration of fibersindicating myocardial injury as compared to diabetic controlgroup indicating moderate protection with respect to theseparameters (Figure 3(c))

4 Discussion

Diabetic cardiomyopathy is characterized by an early dias-tolic and later systolic dysfunctionwith intracellular retentionof calcium and sodium and loss of potassium Cardiacdysfunction occurs at 6 to 12 weeks and even early at 2weeks from onset of diabetes [22] In this work PPSB-PEE (100mgkg po) treatment significantly reduced bloodglucose levels in streptozotocin-nicotinamide induced dia-betes in Sprague-Dawley rats PPSB-PEE reduced bloodglucose levels due to its ability to stimulate the secretion ofinsulin from the surviving pancreatic 120573 cellsThe preliminaryphytochemical analysis of PPSB-PEE showed the presenceof alkaloids terpenoids triterpenes flavonoids steroids andvolatile oils But flavonoids are responsible for stimulatinginsulin from the surviving pancreatic 120573 cells in diabetic mice[12]

Autonomic neuropathy is common complication of bothtype 1 and type 2 diabetes mellitus QT interval prolongationis important manifestation of diabetic neuropathy and hasbeen used to screen diabetic patients at risk for suddencardiac death [23] Experimental studies with animal modelsdemonstrated that the prolonged action potential resulted


0

2

4

6

8

10

Treatment

SOD

(Um

g pr

otei

n)

0

10

20

30

40

0

1

2

3

4

5

lowastlowastlowast

lowastlowastlowast

lowastlowast

Diabetic controlNondiabetic

GSH

(120583g

of G

SHm

g pr

otei

n)

MD

A (n

M o

f MD

Am

g pr

otei

n)

PPSB-PEE100mgkg)(

Treatment

Diabetic controlNondiabetic PPSB-PEE100mgkg)(

Treatment


Figure 2 Effect of PPSB-PEE on oxidative stress markers in rats Values are mean plusmn SEM 119899 = 6 in each group and statistical analysis wascarried out by one-way ANOVA followed by post hocDunnettrsquos test performed using GraphPad Prism lowastlowast119901 lt 001 lowastlowastlowast119901 lt 0001 comparedwith diabetic control group 119901 lt 0001 compared with nondiabetic group

from the decreased outward repolarizing potassium currentsespecially transient outward potassium currents [24] How-ever the exact mechanism of action is not clear yet Theelectrocardiogram showed that the QT period was extendedin diabetic cardiomyopathy QTc represents corrected QTinterval [22 23] In this study we observed that ECG ofdiabetic control shows prolonged QT interval and QTcinterval compared with nondiabetic group Moreover PPSB-PEE (100mgkg) protected the cardiac function by reducingthe QT interval and QTc interval compared with diabeticcontrol QRS complex reflects the rapid depolarization of theright and left ventricles [23] In this study QRS complex wasdecreased in diabetic control compared to nondiabetic andPPSB-PEE (100mgkg) significantly improved this condition119875 wave inversion was observed in diabetic group which isindicative of abnormality of SA node PPSB-PEE treatedgroup did not show inversion of 119875 wave

STZ induced diabetes animals produced systolic anddiastolic dysfunction [25] Diabetic control showed reducedsystolic blood pressure diastolic blood pressure end diastolicblood pressure and mean arterial blood pressure PPSB-PEE(100 mgkg) treated animals showed significant increase insystolic blood pressure diastolic blood pressure and end

diastolic blood pressure The max 119889119875119889119905 and min 119889119875119889119905are the maximum rates of pressure development duringcontraction and relaxation [26] As previously reported invivo LV cannulation showed decreased LV max 119889119875119889119905 andmin 119889119875119889119905 after 15 days of STZ injection [27] and after 12weeks of STZ injection in rats [28] In this study diabetic ratsshowdepression in the rate of relaxation (min119889119875119889119905) and rateof contraction (max119889119875119889119905) relative to PPSB-PEE (100mgkg)treated rats This result suggests that PPSB-PEE providessufficient contractile reserve to ameliorate the detrimentaleffects of diabetes on cardiac contractility Overall PPSB-PEEsignificantly improves contractility index

Diagnosis of cardiac enzymes is prerequisite in case ofdiabetic cardiomyopathy Aspartate aminotransferase (AST)creatine kinase-isoenzyme (CK-MB) and lactate dehydroge-nase (LDH) are commonly used as biomarkers formyocardialinfarction Serum creatine phosphokinase activity is a moresensitive indicator in early stage of myocardial ischemiawhile peak rise in LDH is roughly proportional to the extentof injury to the myocardial tissue Also the integrity of thecardiac apparatus in drug biotransformation andmetabolismcould be assessed by evaluating the levels of AST CK-MBand LDH in serum These enzymes are tightly bound to


(a) (b)

(c)

Figure 3 Effect of PPSB-PEE on histopathology of heart in rats A-group I nondiabetic B-group II diabetic control (tween 80 2 10mLkgpo) andC-group III PPSB-PEE (100mgkg po) (a) Nondiabetic rat showing normal architecture of heart (Grademinusminus) (b) Diabetic controlrat heart showing massive necrosis of heart muscle fibers along with focal mass and fragmentation (+++) (c) PPSB-PEE (100mgkg po)treated rat heart showing minimum pathological changes that is swelling of myocardial fibers and focal degeneration (++) Grade minus noinjury Grade ++++ severe injury Grade ++ moderate injury (magnification 10x)

the contractile apparatus of the cardiac muscle tissue andany serious insult to the heart muscle will evoke the releaseof these enzymes into the serum Diabetic animals showincreased levels of CK-MB and LDH in serum [29] In thisstudy PPSB-PEE (100mgkg) significantly reduced levels ofcardiac enzymes such as CK-MB LDH and AST comparedwith diabetic control indicating prevention of cardiac damageand offering cardioprotection

Hyperglycemia results in the production of reactive oxy-gen and nitrogen species which leads to oxidativemyocardialinjury Oxidative stress induced by reactive oxygen andnitrogen species derived from hyperglycemia causes abnor-mal gene expression altered signal transduction and theactivation of pathways leading to programmed myocardialcell deaths The resulting myocardial cell loss thus plays acritical role in the development of diabetic cardiomyopathy[30 31] In this respect superoxide dismutase is considereda primary enzyme since it is involved in the direct elim-ination of reactive oxygen species Superoxide dismutase

is an important defense enzyme which catalyzes the dis-mutation of superoxide radicals [32] Superoxide dismutaselevel decreased in diabetes [15 33] Reactive oxygen speciesdegrade polyunsaturated lipids forming malondialdehydeThis compound is a reactive aldehyde and is one of the manyreactive electrophile species that cause toxic stress in cellsand form covalent protein adducts referred to as advancedlipoxidation end-products (ALE) in analogy to advanced gly-cation end-products (AGE) The production of this aldehydeis used as a biomarker to measure the level of oxidative stressin an organism [34 35] Diabetes leads to increase in levelsof malondialdehyde [15] Glutathione a tripeptide presentin millimolar concentrations in all the cells is an importantantioxidant Reduced glutathione normally plays the roleof an intracellular radical scavenger and is the substrate ofmany xenobiotic elimination reactions [32] Diabetes leadsto decrease in levels of reduced glutathione [15 36] In thisstudy we observed increase in malondialdehyde decreasein reduced glutathione and superoxide dismutase in heart


tissue indicated an increase in oxidative stress in diabeticcontrol PPSB-PEE was found to possess antioxidant activityPPSB-PEE (100mgkg) significantly reduced oxidative stressby increasing superoxide dismutase reducing glutathionelevels and decreasingmalondialdehyde levels compared withdiabetic control

Histopathological studies reported that mild endomy-ocardial necrosis was present at 8 weeks of diabetes anda severe focal endomyocardial necrosis was found after 12weeks of diabetes [22 37] In this study histological changesin diabetic heart showed massive necrosis of heart musclefibres along with focal mass and fragmentation as com-paredwith nondiabetic heart whereas PPSB-PEE (100mgkg)treated diabetic group showedminimal pathological changesthat is swelling of myocardial fibers and focal degenerationmay be due to reduction in oxidative stress and hyper-glycemia PPSB-PEE offered protection to the heart at cellularlevel

In conclusion PPSB-PEE (100mgkg po) decreasedblood glucose level improved electrocardiographic param-eters (QRS QT and QTc intervals) and hemodynamicparameters (SBP DBP EDP max 119889119875119889119905 contractility indexand heart rate) controlled levels of cardiac biomarkers (CK-MB LDH and AST) and improved oxidative stress (SODMDA and GSH) in diabetic rats Therefore PPSB-PEE is apromising remedy against diabetic cardiomyopathy There isopen avenue for further cellular andmolecular research of thePPSB-PEE

Conflict of Interests

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

Acknowledgment

Theauthors would like to acknowledge PESrsquosModernCollegeof Pharmacy University of Pune India for providing neces-sary facilities to carry out the study

References

[1] L Maya and F J Villarreal ldquoDiagnostic approaches for diabeticcardiomyopathy and myocardial fibrosisrdquo Journal of Molecularand Cellular Cardiology vol 48 no 3 pp 524ndash529 2010

[2] J Wang Y Song Q Wang P M Kralik and P N EpsteinldquoCauses and characteristics of diabetic cardiomyopathyrdquo TheReview of Diabetic Studies vol 3 no 3 pp 108ndash117 2006

[3] Y F Zhi J B Prins and T H Marwick ldquoDiabetic cardiomy-opathy evidence mechanisms and therapeutic implicationsrdquoEndocrine Reviews vol 25 no 4 pp 543ndash567 2004

[4] W B Kannel M Hjortland and W P Castelli ldquoRole ofdiabetes in congestive heart failure the Framingham studyrdquoTheAmerican Journal of Cardiology vol 34 no 1 pp 29ndash34 1974

[5] S Boudina and E D Abel ldquoDiabetic cardiomyopathy revisitedrdquoCirculation vol 115 no 25 pp 3213ndash3223 2007

[6] M Sulaiman M J Matta N R Sunderesan M P Gupta andM Periasamy ldquoResveratrol an activator of SIRT1 upregulatessarcoplasmic calcium ATPase and improves cardiac function

in diabetic cardiomyopathyrdquo American Journal of PhysiologymdashHeart and Circulatory Physiology vol 298 no 3 pp H833ndashH843 2010

[7] J E Shaw R A Sicree and P Z Zimmet ldquoGlobal estimates ofthe prevalence of diabetes for 2010 and 2030rdquoDiabetes Researchand Clinical Practice vol 87 no 1 pp 4ndash14 2010

[8] H King R E Aubert and W H Herman ldquoGlobal burdenof diabetes 1995ndash2025 prevalence numerical estimates andprojectionsrdquo Diabetes Care vol 21 no 9 pp 1414ndash1431 1998

[9] S Murarka and M R Movahed ldquoDiabetic cardiomyopathyrdquoJournal of Cardiac Failure vol 16 no 12 pp 971ndash979 2010

[10] A Krishnamurthi The Wealth of IndiamdashRaw Materials Ph-Re vol 8 Publication and Information Directorate Council ofScientific and Industrial Research New Delhi India 1998

[11] S L Badole and L Bodhankar ldquoAntihyperglycemic activity ofPongamia pinnata stem bark in diabetic micerdquo PharmaceuticalBiology vol 46 no 12 pp 900ndash905 2008

[12] S L Badole and S L Bodhankar ldquoInvestigation of antihypergly-caemic activity of aqueous and petroleum ether extract of stembark of Pongamia pinnata on serum glucose level in diabeticmicerdquo Journal of Ethnopharmacology vol 123 no 1 pp 115ndash1202009

[13] S L Badole and S L Bodhankar ldquoConcomitant administrationof petroleum ether extract of the stem bark of Pongamiapinnata (L) Pierre with synthetic oral hypoglycaemic drugs inalloxan-induced diabetic micerdquo European Journal of IntegrativeMedicine vol 1 no 2 pp 73ndash79 2009

[14] S L Badole and S L Bodhankar ldquoAntihyperglycaemic activityof cycloart-23-ene-3120573 25-diol isolated from stem bark ofPongamia pinnata in alloxan induced diabetic micerdquo ResearchJournal of Phytochemistry vol 3 no 1 pp 18ndash24 2009

[15] S L Badole and S L Bodhankar ldquoAntidiabetic activity ofcycloart-23-ene-3120573 25-diol (B2) isolated from Pongamia pin-nata (L Pierre) in streptozotocin-nicotinamide induced dia-betic micerdquo European Journal of Pharmacology vol 632 no 1ndash3pp 103ndash109 2010

[16] S L Badole P P Bagul S PMahamuni et al ldquoOral L-glutamineincreases active GLP-1 (7-36) amide secretion and improvesglycemic control in stretpozotocin-nicotinamide induced dia-betic ratsrdquo Chemico-Biological Interactions vol 203 no 2 pp530ndash541 2013

[17] S L Badole S L Bodhankar and C G Raut ldquoProtec-tive effect of cycloart-23-ene-3120573 25-diol (B2) isolated fromPongamia pinnata L Pierre on vital organs in streptozotocin-nicotinamide induced diabetic micerdquo Asian Pacific Journal ofTropical Biomedicine vol 1 supplement no 2 pp S186ndashS1902011

[18] T F Slater and B C Sawyer ldquoThe stimulatory effects of car-bon tetrachloride and other halogenoalkanes on peroxidativereactions in rat liver fractions in vitro General features of thesystems usedrdquo Biochemical Journal vol 123 no 5 pp 805ndash8141971

[19] M S Moron J W Depierre and B Mannervik ldquoLevels of glu-tathione glutathione reductase and glutathione S-transferaseactivities in rat lung and liverrdquo Biochimica et Biophysica Actavol 582 no 1 pp 67ndash78 1979

[20] H PMisra and I Fridovich ldquoThe role of superoxide anion in theautoxidation of epinephrine and a simple assay for superoxidedismutaserdquoThe Journal of Biological Chemistry vol 247 no 10pp 3170ndash3175 1972


[21] O H Lowry N J Rosenbrough A L Farr and R J RandellldquoProtein measurement with the folin phenol reagentrdquo TheJournal of biological chemistry vol 193 no 1 pp 265ndash275 1951

[22] S Xi G Zhou X Zhang W Zhang L Cai and C ZhaoldquoProtective effect of total aralosides of Aralia elata (Miq)Seem (TASAES) against diabetic cardiomyopathy in rats duringthe early stage and possible mechanismsrdquo Experimental andMolecular Medicine vol 41 no 8 pp 538ndash547 2009

[23] Y Li Y Gu Y Song et al ldquoCardiac functional analysis by elec-trocardiography echocardiography and in situ hemodynamicsin streptozotocin-induced diabetic micerdquo Journal of HealthScience vol 50 no 4 pp 356ndash365 2004

[24] D Qin B Huang L Deng et al ldquoDownregulation of K+channel genes expression in type I diabetic cardiomyopathyrdquoBiochemical and Biophysical Research Communications vol 283no 3 pp 549ndash553 2001

[25] RWichi CMalfitano K Rosa et al ldquoNoninvasive and invasiveevaluation of cardiac dysfunction in experimental diabetes inrodentsrdquo Cardiovascular Diabetology vol 6 article 14 8 pages2007

[26] O Yamaguchi Y Higuchi S Hirotani et al ldquoTargeted deletionof apoptosis signal-regulating kinase 1 attenuates left ventricularremodelingrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 100 no 26 pp 15883ndash158882003

[27] G R Borges M de Oliveira H C Salgado and R Fazan JrldquoMyocardial performance in conscious streptozotocin diabeticratsrdquo Cardiovascular Diabetology vol 5 article 26 2006

[28] D Sun M Shen J Li et al ldquoCardioprotective effects oftanshinone IIA pretreatment via kinin B2 receptor-Akt-GSK-3120573dependent pathway in experimental diabetic cardiomyopathyrdquoCardiovascular Diabetology vol 10 article 4 2011

[29] E Edet M Akpanabiatu A Eno I Umoh and E Itam ldquoEffectof Gongronema latifolium crude leaf extract on some cardiacenzymes of alloxan- induced diabetic ratsrdquo African Journal ofBiochemistry Research vol 3 pp 366ndash369 2009

[30] L Cai and Y J Kang ldquoOxidative stress and diabetic cardiomy-opathy a brief reviewrdquo Cardiovascular Toxicology vol 1 no 3pp 181ndash193 2001

[31] A C Maritim R A Sanders and J B Watkins III ldquoDiabetesoxidative stress and antioxidants a reviewrdquo Journal of Biochem-ical and Molecular Toxicology vol 17 no 1 pp 24ndash38 2003

[32] S Moussa ldquoOxidative stress in diabetes mellitusrdquo RomanianJournal of Biophysics vol 18 pp 225ndash236 2008

[33] L Cai J Wang Y Li et al ldquoInhibition of superoxide generationand associated nitrosative damage is involved in metalloth-ionein prevention of diabetic cardiomyopathyrdquo Diabetes vol54 no 6 pp 1829ndash1837 2005

[34] E E Farmer and C Davoine ldquoReactive electrophile speciesrdquoCurrent Opinion in Plant Biology vol 10 no 4 pp 380ndash3862007

[35] D Del Rio A J Stewart and N Pellegrini ldquoA review ofrecent studies on malondialdehyde as toxic molecule andbiologicalmarker of oxidative stressrdquoNutritionMetabolism andCardiovascular Diseases vol 15 no 4 pp 316ndash328 2005

[36] M Hamblin D B Friedman S Hill R M Caprioli H MSmith and M F Hill ldquoAlterations in the diabetic myocardialproteome coupled with increased myocardial oxidative stressunderlies diabetic cardiomyopathyrdquo Journal of Molecular andCellular Cardiology vol 42 no 4 pp 884ndash895 2007

[37] A Akula M K Kota S G Gopisetty et al ldquoBiochemical his-tological and echocardiographic changes during experimentalcardiomyopathy in STZ-induced diabetic ratsrdquoPharmacologicalResearch vol 48 no 5 pp 429ndash435 2003

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

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OphthalmologyJournal of


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Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


antihyperglycaemic activity in diabetic mice [11 12] Furtherwe observed that concomitant administration of PPSB-PEEwith synthetic oral hypoglycemic drugs produced synergisticeffect in diabetic mice [13] The preliminary phytochemicalanalysis of PPSB-PEE showed the presence of alkaloidsterpenoids triterpenes flavonoids steroids and volatile oils[12] Cycloart-23-ene-3120573 25-diol (B2) isolated from the stembark of P pinnata possesses antidiabetic activity in diabeticanimals [14 15] Cycloart-23-ene-3120573 25-diol improved theabnormalities of diabetic conditions in diabetic mice due toincreased glucagon-like peptide 1 (GLP-1) insulin secretion[16] and has a protective effect on vital organs like heart andkidney [17] The literature review indicated that P pinnatabark was used by traditional practitioners of medicine fortreatment of diabetes and its complications However morescientific work is necessary to validate the ethnobotanicalclaim relating to diabetic complications Hence our objectiveof this studywas to evaluate cardioprotective activity of PPSB-PEE in diabetic rats

2 Materials and Methods

21 Collection Identification and Authentication of PlantMaterials The fresh bark of Pongamia pinnata (L) Pierrewas collected in August-September 2011 from mature treenear Padali village Parner Taluka Ahmednagar districtMaharashtra state India The bark of Pongamia pinnatawas identified and authenticated at Department of BotanyAgharkar Research Institute Pune India and voucher spec-imen was deposited at that institute (Pongamia pinnatavoucher specimen sample number AHMA-23892)

22 Drugs and Chemicals Streptozotocin (STZ) nicoti-namide (NTM) epinephrine hydrochloride superoxide dis-mutase (SOD) andmalondialdehyde (MDA)were purchasedfrom Sigma Chemical Co USA Petroleum ether (60 80)diethyl ether (Merck India) tween 80 (Research-Lab India)reduced glutathione (GS H) 551015840-dithiobis-(2-nitrobenzoicacid) (DTNB) (Hi media India) thiobarbituric acid (TBA)thiopentone sodium injection (Thiosol Sodium) (NeonLabo-ratories Ltd India) creatine kinase-MB isoenzyme (CKMB)(Randox Laboratory UK) LDH (Ecoline Merck India) andGODPOD kit (Accurex India) were purchased from respec-tive vendors All chemicals used were of analytical grade

23 Preparation of PPSB-PEE PPSB-PEE was prepared byusing previously reported method [12] Briefly weighedquantity (500 g) of air-dried powder (Mesh size-16) of thestem bark of P pinnata was macerated with petroleum ether(3000mL) at room temperature for 7 days with occasionalshaking This process was repeated with same volume ofpetroleum etherMacerates were pooled transferred to previ-ously weighed Petri dish and evaporated to dryness at roomtemperature to obtain 16ww dried extract

24 Experimental Animals and Research Protocol ApprovalMale Sprague-Dawley (SD) rats (200ndash250 g) were procured atour institution animal house facility Pune India and housedin an air-conditioned room at a temperature of 25 plusmn 2∘C

and relative humidity of 45 to 55 under 12-h light 12-h dark cycle The animals had free access to food pellets(Chakan Oil Mills Pune India) Water was provided adlibitum The experimental protocol was approved by theInstitutional Animal Ethics Committee (IAEC) constituted inaccordance with the rules and guidelines of the Committeefor the Purpose of Control and Supervision on ExperimentalAnimals (CPCSEA) New Delhi India (protocol numberMCPIAEC202011)

25 Preparation of Drugs Solution The semisolid PPSB-PEEwas emulsified with 2 tween 80 in distilled water to preparethe drug solution Streptozotocin was dissolved in citratebuffer (pH 45) and nicotinamide was dissolved in normalphysiological saline

26 Induction of Diabetes Diabetes was induced in overnightfasted Sprague-Dawley rats by using streptozotocin injec-tion (55mgkg ip) Nicotinamide (100mgkg ip) wasadministered 20min before administration of streptozotocin[16] Hyperglycaemia was confirmed after 3 days using theglucose oxidase peroxidase (GODPOD) method Glycemiclevel control of diabetic rats was made with subcutaneousinjections of exogenous human NPH insulin (2 unitsday ofNPH insulin) Those rats that showed blood glucose levelsabove 300mgdL (diabetic) were selected for the study

27 Dose Selection of PPSB-PEE In our previous reportedefficacy study PPSB-PEE (100mgkg po) showed mostpromising antihyperglycemic effect in diabetic mice [12 13]Based on our previous results we selected dose (100mgkgpo) of PPSB-PEE for this study

28 Effect of Chronic Administration of PPSB-PEE in DiabeticRats Male Sprague-Dawley rats weighing 200ndash250 g bodyweight were divided into three groups and each groupcontained six rats (119899 = 6) as follows group I nondiabeticgroup II diabetic control (tween 80 2 10mLkg po) asvehicle and group III PPSB-PEE (100mgkg po) Vehicleand PPSB-PEE were administered for 4 months (once a day)at predetermined time (15 days after injection of streptozo-tocin and nicotinamide) The condition of the animals wasmonitored daily at the time of dosing as per experimentaldesign Blood was collected using the retroorbital plexus ofeach rat under mild ether anesthesia Blood glucose wasdetermined by using GODPOD method at end of the studyperiod

29 Serum Cardiac Markers At the end of experimentalperiod rats were anaesthetized with diethyl ether Bloodwas collected by retroorbital puncture and following serumwas separated Serum levels of aspartate aminotransferase(AST) creatine kinase-MB isoenzyme (CK-MB) and lactatedehydrogenase (LDH) enzymes were measured by auto-mated chemistry analyzer (Microlab 300 Merck USA) usingreagent kits

210 Electrocardiographic (ECG) Hemodynamic ParametersThe rats were anesthetized with urethane (125 gkg ip)ECG was recorded using 8-channel recording Power Lab






0

100

200

300

400

500

600

700


Time (months)

Plas

ma g

luco

se le

vel (

mg

dL)


lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

PPSB-PEE (100mgkg)


3 Results







ECG





Hemodynamic



















4 Discussion




0

2

4

6

8

10

Treatment

SOD

(Um

g pr

otei

n)

0

10

20

30

40

0

1

2

3

4

5

lowastlowastlowast

lowastlowastlowast

lowastlowast


GSH

(120583g

of G

SHm

g pr

otei

n)

MD

A (n

M o

f MD

Am

g pr

otei

n)

PPSB-PEE100mgkg)(

Treatment


Treatment








(a) (b)

(c)











Acknowledgment


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















0

100

200

300

400

500

600

700


Time (months)

Plas

ma g

luco

se le

vel (

mg

dL)


lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

PPSB-PEE (100mgkg)


3 Results







ECG





Hemodynamic



















4 Discussion




0

2

4

6

8

10

Treatment

SOD

(Um

g pr

otei

n)

0

10

20

30

40

0

1

2

3

4

5

lowastlowastlowast

lowastlowastlowast

lowastlowast


GSH

(120583g

of G

SHm

g pr

otei

n)

MD

A (n

M o

f MD

Am

g pr

otei

n)

PPSB-PEE100mgkg)(

Treatment


Treatment








(a) (b)

(c)











Acknowledgment


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















ECG





Hemodynamic



















4 Discussion




0

2

4

6

8

10

Treatment

SOD

(Um

g pr

otei

n)

0

10

20

30

40

0

1

2

3

4

5

lowastlowastlowast

lowastlowastlowast

lowastlowast


GSH

(120583g

of G

SHm

g pr

otei

n)

MD

A (n

M o

f MD

Am

g pr

otei

n)

PPSB-PEE100mgkg)(

Treatment


Treatment








(a) (b)

(c)











Acknowledgment


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

2

4

6

8

10

Treatment

SOD

(Um

g pr

otei

n)

0

10

20

30

40

0

1

2

3

4

5

lowastlowastlowast

lowastlowastlowast

lowastlowast


GSH

(120583g

of G

SHm

g pr

otei

n)

MD

A (n

M o

f MD

Am

g pr

otei

n)

PPSB-PEE100mgkg)(

Treatment


Treatment








(a) (b)

(c)











Acknowledgment


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c)











Acknowledgment


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Acknowledgment


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article cardioprotective activity of pongamia...

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