protective effect of momordica charantia fruit extract on

Research ArticleProtective Effect of Momordica charantia Fruit Extract onHyperglycaemia-Induced Cardiac Fibrosis

Razif Abas1 Faizah Othman2 and Zar Chi Thent2

1 Department of Human Anatomy Faculty of Medicine and Health Science Universiti Putra Malaysia 43400 Serdang Malaysia2 Department of Anatomy Faculty of Medicine Universiti Kebangsaan Malaysia Jalan Raja Muda Abd Aziz50300 Kuala Lumpur Malaysia

Correspondence should be addressed to Zar Chi Thent zarrchiigmailcom

Received 2 June 2014 Revised 12 September 2014 Accepted 14 September 2014 Published 13 October 2014

Academic Editor Vittorio Calabrese

Copyright copy 2014 Razif Abas et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

In diabetes mellitus cardiac fibrosis is characterized by increase in the deposition of collagen fibers The present study aimed toobserve the effect of Momordica charantia (MC) fruit extract on hyperglycaemia-induced cardiac fibrosis Diabetes was inducedin the male Sprague-Dawley rats with a single intravenous injection of streptozotocin (STZ) Following 4 weeks of STZ inductionthe rats were subdivided (n = 6) into control group (Ctrl) control group treated with MC (Ctrl-MC) diabetic untreated group(DM-Ctrl) diabetic group treated with MC (DM-MC) and diabetic group treated with 150mgkg of metformin (DM-Met)Administration ofMC fruit extract (15 gkg body weight) in diabetic rats for 28 days showed significant increase in the body weightand decrease in the fasting blood glucose level Significant increase in cardiac tissues superoxide dismutase (SOD) glutathionecontents (GSH) and catalase (CAT) was observed following MC treatment Hydroxyproline content was significantly reducedand associated morphological damages reverted to normal The decreased expression of type III and type IV collagens wasobserved under immunohistochemical staining It is concluded that MC fruit extract possesses antihyperglycemic antioxidativeand cardioprotective properties which may be beneficial in the treatment of diabetic cardiac fibrosis

1 Introduction

The incidence of diabetes mellitus (DM) has increased glob-ally Cardiovascular complications are the leading cause ofmorbidity and mortality among diabetic patient Interest-ingly in the absence of hypertension and coronary arterydisease there is an alteration in themorphology of the cardiactissue Chronic hyperglycaemic disorder in diabetic statecauses glycation of body proteins and stimulates increasein free radical formation which includes reactive oxygenspecies (ROS) [1 2] Thus there is a disturbance in theoxidant and antioxidant activities As a consequence compli-cations like diabetic cardiomyopathy may develop due to theincreasedmyocardial fibrosis andmitochondrial dysfunction[3]Themechanism related to the diabetic cardiac diseasewasreported in both in vitro and in vivo studies [4]

In DM there is the primary defect in the stimulationof glycolysis and glucose oxidation in the cardiac tissues A

major restriction to glucose utilization in the diabetic heartis the slow rate of glucose transport into the myocardiumIt enhances the peripheral insulin resistance and triggerscell death [5] DM influences the myocardium in severalaspects [6] Increased myocardial fibrosis with an increasein the deposition of connective tissues is a major findingAccumulation of collagen in the myocardium as a result ofglycosylation of lysine residues impairs the collagen degra-dation As a result fibrosis leads to injury and stiffness inthe myocardial tissue [7] It was assumed that type III andtype IV collagens were mainly responsible for the increase inthe deposition of connective tissue in the diabetic myocardialtissue [8] Myocardial fibrosis attenuates the cardiac atrophyand eventually it results in cardiac failure [9] There arereports of an increase in the connective tissue deposits in theleft ventricle following induction of diabetes [9]

Dietary foods were reported to possess significant antiox-idant and antidiabetic properties in diabetic state [10] Thus

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2014 Article ID 429060 8 pageshttpdxdoiorg1011552014429060

2 Oxidative Medicine and Cellular Longevity

the daily intake of the antioxidant rich dietary foods improvesthe glycaemic status and increases the antioxidant level Thisresults in protection of the cardiac tissue damage [10] Amongthe dietary foods Momordica charantia (MC) fruit is widelyconsumed in south-east Asian countries like Malaysia Itbelongs to the family of Cucurbitaceae commonly known asbitter gourd or bitter melon in English and ldquoPeriardquo in localMalaysia To date the effects of MC fruit extract on diabeticcardiac disease and myocardial fibrosis were not elucidated

In the present study we observed the effect of the MCfruit extract on cardiac fibrosis and morphological damagesin the diabetic stateThe antioxidant effect ofMC fruit extractand its role in hydroxyproline in the cardiac tissues were alsoinvestigated We also examined the antioxidant effect of MCfruit extract and its role in hydroxyproline in the cardiactissues

2 Methods

21 Animals A total of thirty male Sprague-Dawley rats(weighing 250 plusmn 50 g) were used in the present study Ratswere placed individually in plastic cages under specific-pathogen-free conditions with a 12 h lightdark cycle Ani-mals were given free access to water and standard rat chowAll experimental procedures were performed according tothe protocol approved by the animal ethic committee Uni-versiti Kebangsaan Malaysia

22 Induction of Diabetes Following acclimatization all therats were fasted 12 hrs prior to induction of diabetes Baselinefasting blood glucose (FBG) level was recorded Diabetes wasinduced in the experimental group (119899 = 18) by a single doseof streptozotocin (STZ) with a dose of 60mgKg IV (SigmaAldrich Germany) which was dissolved in 10mM of normalsaline Control rats (119899 = 12) received the same volume ofnormal saline Diabetes was confirmed at 72 hrs followingSTZ injection bymeasuring the FBG levelThe FBG level wasmeasured with Accu- Chek advantage II glucometer (Accu-Chek Instant Germany) The rats with FBG level more than8mmolL were labelled as diabetic [11]

23 Preparation of MC Fruit Extract by Aqueous ExtractionMethod MC fruits (5 Kg) were purchased from the localmarketThewhole plant was identified by a botanist (voucherspecimenmdashUKMB 40067) Dried fruit of MC underwent areflux extraction process using 15 L water (dH

2O) between 75

and 80∘C for 3 hours Subsequently the extract was filteredand solidified in the minus80∘C refrigerator for 24 hrs It wasthen freeze-dried for 3ndash5 days until the powder of aqueousextract was formed In the present study the oral dosage of15 gkg bodyweight was used for the treatment of control anddiabetic groups [1]

24 Experimental Design Following 4 weeks of STZ induc-tion the control group was equally subdivided (119899 = 6) intocontrol group without treatment (Ctrl) and control grouptreated with MC extract 15 gkg (Ctrl-MC) Similarly theexperimental group was further subdivided (119899 = 6) into

diabetic group without treatment (DM-Ctrl) diabetic grouptreated with MC extract 15 gkg (DM-MC) and diabeticgroup treated with 150mgkg metformin (DM-Met) Thedose of metformin was adapted from a previous study [12]Body weight and fasting blood glucose levels were measuredat baseline pretreatment and posttreatment periodThe totalperiod of treatment for bothMC extract and metformin was28 days At the end of the study the rats were sacrificed andcardiac tissues were taken out

25 Determination of Antioxidant Levels Cardiac sampleswere homogenised in HEPES Homogenates were cen-trifuged at 15000 rpm for 5min in a Centrikon H-401(Germany) centrifuge at 4∘C Following centrifugation thesupernatant was collected and frozen at minus30∘Cuntil analysedAll enzymatic assays were carried out at 25 plusmn 05∘C using aPower Wavex microplate scanning spectrophotometer (Bio-Tek Instruments USA) in duplicate in 96-well microplates(UV Star Greiner Bio-One Germany) The enzymatic reac-tions beganwith the addition of the tissue extractThe specificassay was measured

251 Measurement of Superoxide Dismutase (SOD) ActivityAn amount of 20120583L of the SOD standard was diluted with198mL of sample buffer (dilute) 200120583L of the dilutedradical detector and 10 120583L of standard were added per wellThe reactions were initiated by adding 20 120583L of dilutedxanthine oxidase to all the wells Then the 96-well plate wasincubated on a shaker for 20 minutes at room temperatureThe absorbance was read at 440ndash460 nm using plate readerWe followed an earlier protocol for the measurement of SODactivity [13]

252 Measurement of Glutathione (GSH) Activity Anamount of 50 120583L of standard and sample were added perwell in the plate Assay cocktail was prepared by mixing thefollowing reagents in a 20mL vial MES buffer (1125mL)reconstituted cofactor mixture (045mL) reconstitutedenzyme mixture (21mL) water (23mL) and reconstitutedDTNB (045mL) Then 150120583L of the freshly preparedassay cocktail was added to each of the wells containingstandards and samples using a multichannel pipette Theplate was incubated on an orbital shaker The absorbance inthe wells was measured at 405ndash414 nm using a plate reader atfive-minute intervals for a period of 30 minutes (a total of 6measurements) [14]

253 Measurement of Catalase (CAT) Activity The reactionmixture contained 100120583L of diluted assay buffer 30 120583L ofmethanol and 20120583L of the sample were added The sampleswere diluted with diluted sample buffer or concentrated withan Amicon centrifuge concentrator with a molecular weightcut-off of 100000 Reactions were initiated by adding 20120583Lof diluted hydrogen peroxide to all the wells The plate wasincubated on a shaker for 20 minutes at room temperatureDiluted potassium hydroxide (30 120583L) and catalase Purpald(Chromagen) (30 120583L) were added to each well The plate waslater incubated for 10 minutes at room temperature on the

Oxidative Medicine and Cellular Longevity 3

Table 1 Levels of SOD GSH CAT and hydroxyproline in cardiac tissues of control and experimental groups of rats

Groups Ctrl Ctrl-MC DM-Ctrl DM-MC DM-MetSOD (UmL) 282 plusmn 066 365 plusmn 047 230 plusmn 063 621 plusmn 057lowast 362 plusmn 048GSH (120583M) 51655 plusmn 4522 60583 plusmn 6616 46960 plusmn 7016 101526 plusmn 7020lowast 55056 plusmn 6680CAT (120583M) 4293 plusmn 073 4444 plusmn 049 4120 plusmn 055 4888 plusmn 043lowast 4312 plusmn 056Hydroxyproline (120583g120583L) 00081 plusmn 00015 00083 plusmn 00010 00179 plusmn 00015 00091 plusmn 00011lowast 00086 plusmn 00016Each value was expressed as mean plusmn SEM (119899 = 6) lowast119875 lt 005 versus DM-Ctrl group 119875 gt 005 versus Ctrl group SOD superoxide dismutase activity GSHglutathione activity CAT catalase activity

shaker Then 10120583L of the catalase potassium periodate wasadded and incubated for 5 minutes at room temperature ona shaker The absorbance was read at 540 nm using a platereader The entire procedure of determination of catalaseactivity was according to an earlier method described byJohansson and Borg [15]

26 Determination of Cardiac Tissue Hydroxyproline LevelCardiac tissue was homogenized in dH

2O using 100 120583L H

2O

for every 10mg of tissue To 100 120583L sample of homogenate100 120583L concentrated HCL was added in a pressure-tightTeflon capped vial and hydrolyzed at 120∘C for 3 hoursFor the reaction process hydrolyzed sample of 10 120583L wastransferred to a 96-well plate and evaporated to dryness undervacuum An amount of 100 120583L of chloramine T reagent wasadded to each sample and standard and incubated at roomtemperature for 5minutesThen 100120583Lof theDMAB reagentwas added to each well and incubated for 90 minutes at60∘C The absorbance at 560 nm in a microplate reader wasmeasured [16]

27 Histological Examination From the collected cardiactissues six tissues per each group were collected (2-3mmin length) and fixed in 10 buffered formalin Then thetissues underwent dehydration in graded alcohol series Thetissues were cleared with xylene embedded in paraffin andsectioned at 5 120583m thickness in order to perform histologicaland immunohistochemical examination Histological evalu-ation of ventricular cardiac tissues was performed by HampEstaining Massonrsquos trichrome (MT) and Azanrsquos trichrome(AT) staining Transverse sections of cardiac tissues werecaptured digitally using NIH ImageJ 137 V An average of100 cells from each section were measured and mean cross-sectional cardiomyocytes were calculated under HampE stainThe ratio of blue stained connective tissue area to red stainedtotalmyocardial areawas calculated underMasson trichromestain to obtain the fraction of fibrosis () Both measure-mentswere performedunder image analyzer software [16 17]

28 Immunohistochemical Examination Regarding collagentype III and type IV expression after perfusion and paraffinembedding the slides were deparaffinized and rehydrated byimmersing the slides through the wells xylene (twice for 5minutes each) 100 EtOH (twice for 5 minutes each) 95EtOH (5 minutes) 70 EtOH (5 minutes) 50 EtOH (5minutes) and dH

2O for rinsed until ready for staining [18]

3 Results

31 Measurement of Body Weight The body weight ofdiabetic rats and nondiabetic control rats was measured(Figure 1) The weight was measured during baseline pre-treatment and posttreatment period At the end of pretreat-ment body weight in all DM groups (DM-Ctrl DM-MC andDM-Met) was found to be reduced compared to the non-DMgroups (Ctrl and Ctrl-MC) In posttreatment period DM-MC group rats showed a significant increase (119875 lt 005) inthe body weight compared to the DM-Ctrl group HoweverDM-Met group showed an increase in the body weight butit was statistically insignificant MC treated control group(Ctrl-MC) showed no significant increase in the body weightcompared to the control group (Ctrl)

32 Measurement of Fasting Blood Glucose Level Following4 weeks of STZ injection diabetic groups showed significantelevation in the FBG level compared to the control groups(Figure 2) The blood glucose level did not differ betweenCtrl group and Ctrl-MC group Following 8 weeks of STZinjection DM-MC group and DM-Met group animals hadremarkably lower fasting blood glucose levels compared tothe DM-Ctrl group (119875 lt 005) Administration withMC fruitextract for 28 days significantly reduced the fasting bloodglucose level in diabetic rats

33 Antioxidant Levels At the end of the study the animalswere sacrificed with an overdose of diethyl ether The cardiactissue was taken out and processed for further biochemicalanalyses The cardiac tissue antioxidant levels (SOD GSHand CAT) were measured It was observed the antioxidantactivities in cardiac tissues of DM-MC group rats had signifi-cantly increased compared to the DM-Ctrl group (119875 lt 005)Statistically significant impairment in SOD GSH and CATactivities was observed in DM-Ctrl group rats SurprisinglyDM-Met group rats did not show any significant changes inthe antioxidant level (Table 1) Oral administration of MCfruit extraction diabetic rats showed a significant increase inthe antioxidant enzymes

34 Hydroxyproline Level Cardiac tissueswere homogenizedto determine the hydroxyproline level using ELISA methodIt was observed that DM-MC group rats showed significantdecrease in the hydroxyproline level (119875 lt 005) compared totheDM-Ctrl groupDM-Met group also revealed a significant


0

100

200

300

400

Baseline Pretreatment Posttreatment

Body

wei

ght (

g)CtrlCtrl-MCDM-Ctrl

DM-MCDM-Met

lowast

Figure 1 Effect of MC fruit extract on body weight (g) of Sprague-Dawley rats Data were presented as means plusmn SEM (119899 = 6) lowast119875 lt 005versus DM-Ctrl group 119875 gt 005 versus Ctrl group

0

5

10

15

20

25

30


Fasti

ng b

lood

glu

cose

le

vel (

mm

olL

)

CtrlCtrl-MCDM-Ctrl

DM-MCDM-Met

lowast

Figure 2 Effect ofMC fruit extract on fasting blood glucose level (mmolL) of Sprague-Dawley rats Data were presented as means plusmn SEM(119899 = 6) lowast119875 lt 005 versus DM-Ctrl group 119875 gt 005 versus Ctrl group

decrease in the hydroxyproline level An increase in thehydroxyproline level showed that there was an increase inthe collagen deposition in the diabetic myocardial tissueHowever Ctrl-MC group did not show any prominent changein the hydroxyproline level compared to the Ctrl group(Table 1)

35 Histological Findings Figure 3 showed histologicalchanges in the left ventricular tissue in control and diabeticrats following 8 weeks of STZ induction A transverse sectionof cardiomyocytes was observed under HampE staining Asignificant decrease in the size of cardiomyocytes (119875 lt 005)was observed in DM-MC group rats compared to the DM-Ctrl group (Figure 3(a)) Prominent myocardial fibrosis wasobserved in the cardiac tissues of DM-Ctrl group rats whenviewed under MT staining However the amount of fibrosiswas observed to be significantly reduced (119875 lt 005) in DM-MC group rats compared to theDM-Ctrl group (Figure 3(b))The increased perivascular fibrosis was observed in thecardiac tissues of DM-Ctrl group rats (Figure 3(c)) Themorphological deteriorations appeared to be less in cardiactissues of DM-MC group and DM-Met group of rats

36 Immunohistochemical Staining Ventricular tissues fromDM-MC group rats displayed patchy or scattered immunos-taining for collagen type III and type IV Increased expressionof immunostaining was observed in few cardiomyocytes ofthe DM-Ctrl group rats (Figures 4(a) and 4(b)) Increasein type III and type IV collagens expressions indicated thesevere adhesion of collagen between the myofibers Howeverdecreased expression of type III and type IV collagen wasfound in the DM-MC group following treatment with MCfruit extract Similar findings were observed in cardiac tissuesof the DM-Met group rats

4 Discussion

The protective effect of MC fruit extract on type 1 DM hasbeen observed earlier [19] However the therapeutic effectof the MC fruit extract of hyperglycaemia-induced cardiacfibrosis was not yet explored to date

Two animal studies demonstrated the potential effect ofMC leaves extract on the diabetic cardiac tissues [19 20] Bothstudies focused on the effect of MC leaf extract Howeverthere is a paucity of experimental study with regard to MC


Ctrl Ctrl-MC

DM-MCDM-Ctrl DM-Met

lowast

lowast

nn

n

n

n

lowast

Ctrl

0

50

100

150

200

250

300

Ctrl-MC

DM

-Ctr

l

DM

-MC

DM

-Met

(120583m

)

lowast

(a)

Ctrl Ctrl-MC

DM-MCDM-Ctrl DM-Met

0102030405060708090

100

Ctrl

Ctrl-MC

DM

-Ctr

l

()

DM

-MC

DM

-Met

lowast

(b)

Ctrl Ctrl-MC

DM-Ctrl DM-MC DM-Met

(c)

Figure 3 (a)Therapeutic effect ofMC fruit extract on transverse section of left ventricular myocardial tissues of experimental rats examinedby hematoxylin and eosin staining (times400 bar = 50 120583m) Cytoplasmic space (lowast) n = nucleus Quantitative analysis of transverse diameter ofcardiomyocyte (bar graph) lowast119875 lt 005 versus DM-MC group (b)Therapeutic effect ofMC fruit extract on transverse section of left ventricularmyocardial tissues of experimental rats examined by Masson trichrome staining (times400 bar = 50 120583m) Collagen deposition (yellow arrowsblue staining) The bar graph showing fibrosis fractions in the myocardial tissue lowast119875 lt 005 versus DM-MC group (c) Therapeutic effect ofMC fruit extract on transverse section of left ventricular myocardial tissues of experimental rats examined by Azan trichrome staining (times400bar = 50 120583m) Perivascular connective tissue depositions (stained dark blue red arrows)

fruit To date the definite role of MC fruit extract in thecardiac fibrosis in type 1 DM still remains unknown In orderto address such issues the effect of MC fruit extract wasstudied in the animal model

In the present study the basic physiological such as bodyweight and biochemical such as FBG level parameters were

measured DM-MC group showed a significant increase (119875 lt005) in the body weight compared to DM-Ctrl group Thisis in accordance with a previous study where body weight ofexperimental diabetic animals was found to be reduced [21]Theoretically the reduction in the body weight may be dueto the increased catabolic rate in the state of DMThe protein


Ctrl Ctrl-MC

DM-Ctrl DM-MC DM-Metlowastlowast

(a)

Ctrl Ctrl-MC


(b)

Figure 4 (a)Therapeutic effect ofMC fruit extract on transverse section of left ventricular myocardial tissues of experimental rats examinedby immunohistochemical staining for type III collagen expression (times400) Expression (stained light brown lowast) (b) Therapeutic effect ofMCfruit extract on transverse section of left ventricular myocardial tissues of experimental rats examined by immunohistochemical staining fortype IV collagen expression (times400) Expression (stained purple blue) Note in DM-Ctrl group the outline of cardiomyocytes is distinct fromthe rest of the groups

degradation in the diabetic state results from unavailabilityof utilizing the carbohydrate as an energy source [2] Thesupplementation with the extract served as a source of energyand nutrients for the body metabolic activity [22] MC hasbeen shown to enhance the number of pancreatic beta cellsthus promoting the insulin secretion Moreover an activecompound present in MC extract known as charantin isproven to increaseGLUT4 thus increasing glucose utilizationin the liver and muscle tissue [23] The present finding withregard to body weight was comparable to DM-Met group(Figure 1) However there was no significant difference in thebody weight of Ctrl-MC compared to the Ctrl groups

Besides body weight it was found out that there was asignificant decrease in the FBG in DM-MC group comparedto DM-Ctrl group The effect of STZ on glucose and insulinhomeostasis reflects the toxin-induced abnormalities in betacell function [24]MC fruit extract is enriched with flavonoidcompounds such as charantins insulin-like peptides andalkaloids [25] It was reported that the antihyperglycaemicactivity of MC fruit was comparable to the usage of gliben-clamide in diabetic rats [26] Based on the present findings itwas found out thatMC fruit extract possesses similar effect tometformin (DM-Met) in decreasing the fasting blood glucoselevel in diabetic rats All the above preliminary investigationswere important for the confirmation of the diabetic status

DM is associated with an increased production of freeradicals and the antioxidants defence system is impaired inchronic hyperglycaemic state [27] Decrease in the activityof SOD GSH and CAT in the homogenized cardiac tissueof DM group was observed in the present study This maybe due to the increase in production of ROS that can reducethe activity of these enzymes Following administration withthe MC fruit extract restores the activity of the antioxidantenzymes significantly (119875 lt 005) and may help to avoid thedeleterious effects of free radicals in the diabetic state [18]Increase in the total antioxidants properties may be due to

the presence of active compounds such as charantosides inthe MC fruit extract However in the present study therewas no significant increase in the antioxidant levels of Ctrl-MC compared to Ctrl group In contrast to DM-MC groupDM-Met group did not show any significant increase in theantioxidant level in the cardiac tissues

It was assumed that diabetic cardiac disease is probablydue to an increase in collagen deposits in the myocardialtissue In order to confirm this we measured the hydrox-yproline content in the cardiac tissues of the individual ratsin the present study DM-Ctrl group showed a significantincrease (119875 lt 005) in hydroxyproline content Previousstudy demonstrated that the formation of advanced glycationend products (AGEs) in chronic hyperglycaemia acceleratedthe deposition of collagen and the development ofmyocardialstiffness in type 1 DM [28] However the significant decreasein hydroxyproline level was observed in the DM-MC groupIt is in accordance with a previous study which reported thatMC protects against the accumulation of collagen in DManimal models [29] It was believed that an active compoundof MC luteolin is responsible for reducing the collagencontent in cardiac tissue [30 31] Similar results were noted inthe DM-Met group in which hydroxyproline level was foundto be reduced

To support our assumption for collagen depositionhistological investigation was carried out The architecturalalteration in the myocardium of DM-Ctrl group of rats andreduce in transverse diameter of cardiomyocytes under HampEstain were observed in myocardial tissue of DM-MC group(Figure 3(a)) This is in accordance with the previous studyin which the deterioration in the normal myocardium suchas disorganization of myofibers reduced cardiomyocytestransverse diameter and scattered nuclei with increase incytoplasmic space was detected [21] These morphologicaldisturbances in the cardiac tissues are associated with loss ofcontractile protein myocyte dropout with interstitial fibrosis


and caloric deprivation In our study increased depositions ofconnective tissue with increased percentage of fibrotic depo-sition and perivascular fibrosis were observed in the DM-Ctrlgroup compared to the DM-MC group under MT and ATstaining respectively (Figures 3(b) and 3(c)) Studies showedthat the increase expression of connective tissue growth factor(CTGF) transforming growth factor (TGF) and increase incollagen deposition lead to the development of fibrosis indiabetic cardiac tissue [32] The increased glycogen levelsin the diabetic state lead to the formation of perivascularfibrosis which produces the stiffness of the cardiac musclewall [28] These changes were found to be reduced with theadministration of MC extract and metformin following 4weeks of treatment The mechanism that explains the effectof MC towards the diabetic cardiac disease has not beenproven yet It can be explained that the reduction in bloodglucose level may increase the peripheral glucose utilizationin cardiac tissue [20] As a result DM-MC group graduallyrestored the integrity of the connective tissue by reducing thesusceptibility of the cardiovascular complications

Although we assumed that the development of diabeticcardiac disease is most probably due to the increase depo-sition of collagen it is important to ascertain the definitivecollagen which is themajor factors responsible for the cardiacfibrosis Type III and type IV collagens are the importantcomponents of the extracellular matrix in the heart Studieshave shown that increased expression of type III collagen inthe rat model leads to the increase risk of cardiac fibrosis andcardiac failure [33]

In the present study DM-Ctrl group showed increasedexpression of type III and type IV collagens in cardiactissue This indicated the increased cardiac remodeling ofthe extracellular matrix which leads to the impairment ofcardiac tissue function Collagen accumulation in diabeticmyocardium is due to the impaired collagen degradationresulting from glycosylation of the lysine residues on collagen[34 35]

In a study carried out by Bhoomi et al (2013) it wasdemonstrated that the MC fruit extract reduced the cardiacfibrosis in diabetic rats [36] It could be suggested thattreatment withMC extract reduced the expression of type IIIand type IV collagen by improving the free radical scavengingactivity and reducing the ROS which leads to a decrease inthe oxidative stress and collagen accumulation [37] This wasalso demonstrated in the present study in which DM-MCgroup showed reduced expression of type III and type IVcollagen compared to the DM-Ctrl group Metformin (DM-Met) group also proved to reduce the expression of type IIIand type IV collagen in myocardial tissues (Figures 4(a) and4(b))

5 Conclusion

To the best of our knowledge the present study is the firstof its kind to prove that oral administration with MC fruitextract could protect the cardiac remodeling in type 1 diabeticanimal model by increasing the antioxidant level reducingthe collagen deposition and specific collagen expressionsFurther detailed studies are needed to explore the underlying

mechanism The present study highlighted the protectiveeffect of theMC fruit extract on diabetic cardiac fibrosis andencourages future research to useMC fruit as a supplement totreat cardiac complications arising due to diabetes mellitus

Conflict of Interests

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

Authorsrsquo Contribution

Razif Abas researched the data Razif Abas and Zar ChiThentreviewed the paper Razif Abas Zar Chi Thent and FaizahOthman contributed to the initial discussion and overseeingof the project Razif Abas and Zar Chi Thent wrote andreviewed the paper

Acknowledgments

This research study was financially supported by the Uni-versiti Kebangsaan Malaysia (GGPM-060-2013) Young Re-searchers Grant by Centre of Research and InstrumentationManagement (CRIM) The authors would like to acknowl-edge Professor Dr Srijit Das for his expert guidance towardsthis study

References

[1] J Virdi S Sivakami S Shahani A C SutharMM Banavalikarand M K Biyani ldquoAntihyperglycemic effects of three extractsfromMomordica charantiardquo Journal of Ethnopharmacology vol88 no 1 pp 107ndash111 2003

[2] D Sathishsekar and S Subramanian ldquoAntioxidant propertiesofMomordica charantia (bitter gourd) seeds on Streptozotocininduced diabetic ratsrdquo Asia Pacific Journal of Clinical Nutritionvol 14 no 2 pp 153ndash158 2005

[3] IM Stratton A I AdlerHAWNeil et al ldquoAssociation of gly-caemia withmacrovascular andmicrovascular complications oftype 2 diabetes (UKPDS 35) prospective observational studyrdquoBritish Medical Journal vol 321 no 7258 pp 405ndash412 2000

[4] E Arikawa R C W Ma K Isshiki et al ldquoEffects of insulinreplacements inhibitors of angiotensin and PKC1205731015840s actionsto normalize cardiac gene expression and fuel metabolism indiabetic ratsrdquo Diabetes vol 56 no 5 pp 1410ndash1420 2007

[5] J D Malhotra and R J Kaufman ldquoEndoplasmic reticulumstress and oxidative stress a vicious cycle or a double-edgedswordrdquo Antioxidants and Redox Signaling vol 9 no 12 pp2277ndash2294 2007

[6] H P Codinach and P R Freixa ldquoDiabetic cardiomyopathyconcept heart function and pathogenesisrdquo Anales de MedicinaInterna vol 19 no 6 pp 313ndash320 2002

[7] E Nemoto R P Darveau B L Foster G R Nogueira-Filhoand M J Somerman ldquoRegulation of cementoblast function byP Gingivalis lipopolysaccharide via TLR2rdquo Journal of DentalResearch vol 85 no 8 pp 733ndash738 2006

[8] M Shimizu K Umeda N Sugihara et al ldquoCollagen remod-elling inmyocardia of patients with diabetesrdquo Journal of ClinicalPathology vol 46 no 1 pp 32ndash36 1993


[9] O Bilim Y Takeishi T Kitahara et al ldquoDiacylglycerol kinasezeta inhibits myocardial atrophy and restores cardiac dysfunc-tion in streptozotocin -induced diabetesmellitusrdquoCardiovascu-lar Diabetology vol 4 no 7 pp 1186ndash1475 2008

[10] P Scartezzini and E Speroni ldquoReview on some plants ofIndian traditional medicine with antioxidant activityrdquo Journalof Ethnopharmacology vol 71 no 1-2 pp 23ndash43 2000

[11] S L Teoh A A Latiff and S Das ldquoThe effect of topicalextract ofMomordica charantia (bitter gourd) onwoundhealingin nondiabetic rats and in rats with diabetes induced bystreptozotocinrdquoClinical and Experimental Dermatology vol 34no 7 pp 815ndash822 2009

[12] A J Scheen ldquoLimited add-on value of oral glucose-loweringagents in type 1 diabetesrdquo Revue Medicale de Liege vol 68 no1 pp 16ndash21 2013

[13] C M Maier and P H Chan ldquoRole of superoxide dismutasesin oxidative damage and neurodegenerative disordersrdquo TheNeuroscientist vol 8 no 4 pp 323ndash334 2002

[14] C H Foyer M Lelandais and K J Kunert ldquoPhotooxidativestress in plantsrdquo Physiologia Plantarum vol 92 no 4 pp 696ndash717 1994

[15] L H Johansson and L A H Borg ldquoA spectrophotometricmethod for determination of catalase activity in small tissuesamplesrdquo Analytical Biochemistry vol 174 no 1 pp 331ndash3361988

[16] O Bilim Y Takeishi T Kitahara et al ldquoDiacylglycerol kinase120577 inhibits myocardial atrophy and restores cardiac dysfunctionin streptozotocin-induced diabetes mellitusrdquo CardiovascularDiabetology vol 7 article 2 2008

[17] N Nozaki T Shishido Y Takeishi and I Kubota ldquoModula-tion of doxorubicin-induced cardiac dysfunction in toll-likereceptor-2-knockout micerdquo Circulation vol 110 no 18 pp2869ndash2874 2004

[18] C Heymes M Vanderheyden J G F Bronzwaer A M Shahand W J Paulus ldquoEndomyocardial nitric oxide synthase andleft ventricular preload reserve in dilated cardiomyopathyrdquoCirculation vol 99 no 23 pp 3009ndash3016 1999

[19] U N Tripathi and D Chandra ldquoThe plant extracts ofMomordica charantia and Trigonella foenum graecum haveantioxidant and anti-hyperglycemic properties for cardiac tis-sue during diabetes mellitusrdquo Oxidative Medicine and CellularLongevity vol 2 no 5 pp 290ndash296 2009

[20] O A Komolafe D A Ofusori O S Adewole A O Ayoka andA A Abiodun ldquoMomordica charantia protects against cardiacdamage in streptozotocin-induced diabetic wistar ratsrdquo Journalof Pharmaceutical and Scientific Innovation vol 1 no 3 pp 32ndash36 2012

[21] Z C Thent T S Lin S Das and Z Zakaria ldquoHistologicalchanges in the heart and the proximal aorta in experimentaldiabetic rats fed with Piper sarmentsoumrdquo African Journal ofTraditional Complementary and Alternative Medicines vol 9no 3 pp 396ndash404 2012

[22] R I Bakare O A Magbagbeola A I Akinwande and O WOkunowo ldquoNutritional and chemical evaluation ofMomordicacharantiardquo Journal of Medicinal Plants Research vol 4 no 21pp 2189ndash2193 2010

[23] J K Grover and S P Yadav ldquoPharmacological actions andpotential uses of Momordica charantia a reviewrdquo Journal ofEthnopharmacology vol 93 no 1 pp 123ndash132 2004

[24] T Szkudelski ldquoThe mechanism of alloxan and streptozotocinaction in B cells of the rat pancreasrdquo Physiological Research vol50 no 6 pp 537ndash546 2001

[25] A Raman and C Lau ldquoAnti-diabetic properties and phyto-chemistry of Momordica charantia L (Cucurbitaceae)rdquo Phy-tomedicine vol 2 no 4 pp 349ndash362 1996

[26] L Ali A K A Khan M I R Mamun et al ldquoStudies onhypoglycemic effects of fruit pulp seed and whole plant ofMomordica charantia on normal and diabetic model ratsrdquoPlanta Medica vol 59 no 5 pp 408ndash412 1993

[27] S Penckofer D Schwertz and K Florczak ldquoOxidative stressand cardiovascular disease in type 2 diabetes the role ofantioxidants and pro-oxidantsrdquo The Journal of CardiovascularNursing vol 16 no 2 pp 68ndash85 2002

[28] R Candido J M Forbes M C Thomas et al ldquoA breaker ofadvanced glycation end products attenuates diabetes-inducedmyocardial structural changesrdquo Circulation Research vol 92no 7 pp 785ndash792 2003

[29] G N Jyothirmayi B J Soni M Masurekar M Lyons and T JRegan ldquoEffects ofmetformin on collagen glycation and diastolicdysfunction in diabeticmyocardiumrdquo Journal of CardiovascularPharmacology andTherapeutics vol 3 no 4 pp 319ndash326 1998

[30] S Kumar andA K Pandey ldquoChemistry and biological activitiesof flavonoids an overviewrdquo The Scientific World Journal vol2013 Article ID 162750 16 pages 2013

[31] E Basch S Gabardi and C Ulbricht ldquoBitter melon(Momordica Charantia) a review of efficacy and safetyrdquoThe American Journal of Health-System Pharmacy vol 60 no4 pp 356ndash359 2003

[32] K J Way K Isshiki K Suzuma et al ldquoExpression of connectivetissue growth factor is increased in injured myocardium associ-ated with protein kinase C 1205732 activation and diabetesrdquoDiabetesvol 51 no 9 pp 2709ndash2718 2002

[33] Q-Y Huang X-F Li and S-P Liu ldquoEndothelin-1 and typeIII collagen alterations in the heart of rats following cisplatin-induced toxicityrdquo Molecular Medicine Reports vol 5 no 3 pp651ndash654 2012

[34] V Soetikno KWatanabe F R Sari et al ldquoCurcumin attenuatesdiabetic nephropathy by inhibiting PKC-120572 and PKC-1205731 activityin streptozotocin-induced type I diabetic ratsrdquoMolecular Nutri-tion and Food Research vol 55 no 11 pp 1655ndash1665 2011

[35] Y Pan G Zhu Y Wang et al ldquoAttenuation of high-glucose-induced inflammatory response by a novel curcumin derivativeB06 contributes to its protection from diabetic pathogenicchanges in rat kidney and heartrdquo The Journal of NutritionalBiochemistry vol 24 no 1 pp 146ndash155 2013

[36] K Bhoomi B Patel and K Parmar ldquoEffects of momordica cha-rantia fruit on cardiac abnormalities associated with metabolicsyndromerdquo Journal of Advance Research in Biological Sciencesvol 1 no 1 pp 20ndash27 2013

[37] K E Sullivan and L D Black III ldquoThe role of cardiac fibroblastsin extracellular matrix-mediated signaling during normal andpathological cardiac developmentrdquo Journal of BiomechanicalEngineering vol 135 no 7 Article ID 071001 pp 71ndash100 2013

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

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


the daily intake of the antioxidant rich dietary foods improvesthe glycaemic status and increases the antioxidant level Thisresults in protection of the cardiac tissue damage [10] Amongthe dietary foods Momordica charantia (MC) fruit is widelyconsumed in south-east Asian countries like Malaysia Itbelongs to the family of Cucurbitaceae commonly known asbitter gourd or bitter melon in English and ldquoPeriardquo in localMalaysia To date the effects of MC fruit extract on diabeticcardiac disease and myocardial fibrosis were not elucidated

In the present study we observed the effect of the MCfruit extract on cardiac fibrosis and morphological damagesin the diabetic stateThe antioxidant effect ofMC fruit extractand its role in hydroxyproline in the cardiac tissues were alsoinvestigated We also examined the antioxidant effect of MCfruit extract and its role in hydroxyproline in the cardiactissues

2 Methods

21 Animals A total of thirty male Sprague-Dawley rats(weighing 250 plusmn 50 g) were used in the present study Ratswere placed individually in plastic cages under specific-pathogen-free conditions with a 12 h lightdark cycle Ani-mals were given free access to water and standard rat chowAll experimental procedures were performed according tothe protocol approved by the animal ethic committee Uni-versiti Kebangsaan Malaysia

22 Induction of Diabetes Following acclimatization all therats were fasted 12 hrs prior to induction of diabetes Baselinefasting blood glucose (FBG) level was recorded Diabetes wasinduced in the experimental group (119899 = 18) by a single doseof streptozotocin (STZ) with a dose of 60mgKg IV (SigmaAldrich Germany) which was dissolved in 10mM of normalsaline Control rats (119899 = 12) received the same volume ofnormal saline Diabetes was confirmed at 72 hrs followingSTZ injection bymeasuring the FBG levelThe FBG level wasmeasured with Accu- Chek advantage II glucometer (Accu-Chek Instant Germany) The rats with FBG level more than8mmolL were labelled as diabetic [11]

23 Preparation of MC Fruit Extract by Aqueous ExtractionMethod MC fruits (5 Kg) were purchased from the localmarketThewhole plant was identified by a botanist (voucherspecimenmdashUKMB 40067) Dried fruit of MC underwent areflux extraction process using 15 L water (dH

2O) between 75

and 80∘C for 3 hours Subsequently the extract was filteredand solidified in the minus80∘C refrigerator for 24 hrs It wasthen freeze-dried for 3ndash5 days until the powder of aqueousextract was formed In the present study the oral dosage of15 gkg bodyweight was used for the treatment of control anddiabetic groups [1]

24 Experimental Design Following 4 weeks of STZ induc-tion the control group was equally subdivided (119899 = 6) intocontrol group without treatment (Ctrl) and control grouptreated with MC extract 15 gkg (Ctrl-MC) Similarly theexperimental group was further subdivided (119899 = 6) into

diabetic group without treatment (DM-Ctrl) diabetic grouptreated with MC extract 15 gkg (DM-MC) and diabeticgroup treated with 150mgkg metformin (DM-Met) Thedose of metformin was adapted from a previous study [12]Body weight and fasting blood glucose levels were measuredat baseline pretreatment and posttreatment periodThe totalperiod of treatment for bothMC extract and metformin was28 days At the end of the study the rats were sacrificed andcardiac tissues were taken out

25 Determination of Antioxidant Levels Cardiac sampleswere homogenised in HEPES Homogenates were cen-trifuged at 15000 rpm for 5min in a Centrikon H-401(Germany) centrifuge at 4∘C Following centrifugation thesupernatant was collected and frozen at minus30∘Cuntil analysedAll enzymatic assays were carried out at 25 plusmn 05∘C using aPower Wavex microplate scanning spectrophotometer (Bio-Tek Instruments USA) in duplicate in 96-well microplates(UV Star Greiner Bio-One Germany) The enzymatic reac-tions beganwith the addition of the tissue extractThe specificassay was measured

251 Measurement of Superoxide Dismutase (SOD) ActivityAn amount of 20120583L of the SOD standard was diluted with198mL of sample buffer (dilute) 200120583L of the dilutedradical detector and 10 120583L of standard were added per wellThe reactions were initiated by adding 20 120583L of dilutedxanthine oxidase to all the wells Then the 96-well plate wasincubated on a shaker for 20 minutes at room temperatureThe absorbance was read at 440ndash460 nm using plate readerWe followed an earlier protocol for the measurement of SODactivity [13]

252 Measurement of Glutathione (GSH) Activity Anamount of 50 120583L of standard and sample were added perwell in the plate Assay cocktail was prepared by mixing thefollowing reagents in a 20mL vial MES buffer (1125mL)reconstituted cofactor mixture (045mL) reconstitutedenzyme mixture (21mL) water (23mL) and reconstitutedDTNB (045mL) Then 150120583L of the freshly preparedassay cocktail was added to each of the wells containingstandards and samples using a multichannel pipette Theplate was incubated on an orbital shaker The absorbance inthe wells was measured at 405ndash414 nm using a plate reader atfive-minute intervals for a period of 30 minutes (a total of 6measurements) [14]

253 Measurement of Catalase (CAT) Activity The reactionmixture contained 100120583L of diluted assay buffer 30 120583L ofmethanol and 20120583L of the sample were added The sampleswere diluted with diluted sample buffer or concentrated withan Amicon centrifuge concentrator with a molecular weightcut-off of 100000 Reactions were initiated by adding 20120583Lof diluted hydrogen peroxide to all the wells The plate wasincubated on a shaker for 20 minutes at room temperatureDiluted potassium hydroxide (30 120583L) and catalase Purpald(Chromagen) (30 120583L) were added to each well The plate waslater incubated for 10 minutes at room temperature on the






2O using 100 120583L H

2O





3 Results






0

100

200

300

400


Body

wei

ght (


DM-MCDM-Met

lowast


0

5

10

15

20

25

30


Fasti

ng b

lood

glu

cose

le

vel (

mm

olL

)

CtrlCtrl-MCDM-Ctrl

DM-MCDM-Met

lowast





4 Discussion




Ctrl Ctrl-MC

DM-MCDM-Ctrl DM-Met

lowast

lowast

nn

n

n

n

lowast

Ctrl

0

50

100

150

200

250

300

Ctrl-MC

DM

-Ctr

l

DM

-MC

DM

-Met

(120583m

)

lowast

(a)

Ctrl Ctrl-MC

DM-MCDM-Ctrl DM-Met

0102030405060708090

100

Ctrl

Ctrl-MC

DM

-Ctr

l

()

DM

-MC

DM

-Met

lowast

(b)

Ctrl Ctrl-MC


(c)






Ctrl Ctrl-MC


(a)

Ctrl Ctrl-MC


(b)













5 Conclusion







Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















2O using 100 120583L H

2O





3 Results






0

100

200

300

400


Body

wei

ght (


DM-MCDM-Met

lowast


0

5

10

15

20

25

30


Fasti

ng b

lood

glu

cose

le

vel (

mm

olL

)

CtrlCtrl-MCDM-Ctrl

DM-MCDM-Met

lowast





4 Discussion




Ctrl Ctrl-MC

DM-MCDM-Ctrl DM-Met

lowast

lowast

nn

n

n

n

lowast

Ctrl

0

50

100

150

200

250

300

Ctrl-MC

DM

-Ctr

l

DM

-MC

DM

-Met

(120583m

)

lowast

(a)

Ctrl Ctrl-MC

DM-MCDM-Ctrl DM-Met

0102030405060708090

100

Ctrl

Ctrl-MC

DM

-Ctr

l

()

DM

-MC

DM

-Met

lowast

(b)

Ctrl Ctrl-MC


(c)






Ctrl Ctrl-MC


(a)

Ctrl Ctrl-MC


(b)













5 Conclusion







Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

100

200

300

400


Body

wei

ght (


DM-MCDM-Met

lowast


0

5

10

15

20

25

30


Fasti

ng b

lood

glu

cose

le

vel (

mm

olL

)

CtrlCtrl-MCDM-Ctrl

DM-MCDM-Met

lowast





4 Discussion




Ctrl Ctrl-MC

DM-MCDM-Ctrl DM-Met

lowast

lowast

nn

n

n

n

lowast

Ctrl

0

50

100

150

200

250

300

Ctrl-MC

DM

-Ctr

l

DM

-MC

DM

-Met

(120583m

)

lowast

(a)

Ctrl Ctrl-MC

DM-MCDM-Ctrl DM-Met

0102030405060708090

100

Ctrl

Ctrl-MC

DM

-Ctr

l

()

DM

-MC

DM

-Met

lowast

(b)

Ctrl Ctrl-MC


(c)






Ctrl Ctrl-MC


(a)

Ctrl Ctrl-MC


(b)













5 Conclusion







Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Ctrl Ctrl-MC

DM-MCDM-Ctrl DM-Met

lowast

lowast

nn

n

n

n

lowast

Ctrl

0

50

100

150

200

250

300

Ctrl-MC

DM

-Ctr

l

DM

-MC

DM

-Met

(120583m

)

lowast

(a)

Ctrl Ctrl-MC

DM-MCDM-Ctrl DM-Met

0102030405060708090

100

Ctrl

Ctrl-MC

DM

-Ctr

l

()

DM

-MC

DM

-Met

lowast

(b)

Ctrl Ctrl-MC


(c)






Ctrl Ctrl-MC


(a)

Ctrl Ctrl-MC


(b)













5 Conclusion







Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Ctrl Ctrl-MC


(a)

Ctrl Ctrl-MC


(b)













5 Conclusion







Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















5 Conclusion







Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















protective effect of momordica charantia fruit extract on

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