RESEARCH ARTICLE

Protective effect of Syzygium cuminiagainst pesticide-induced cardiotoxicity

Neha Atale & Khushboo Gupta & Vibha Rani

Received: 29 November 2013 /Accepted: 19 February 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract Pesticide-induced toxicity is a serious issue whichhas resulted in plethora of diseases all over the world. Theorganophosphate pesticide malathion has caused many inci-dents of poisoning such as cardiac manifestations. The presentstudy was designed to evaluate the effect of Syzygium cuminion malathion-induced cardiotoxicity. Dose optimization ofmalathion and polyphenols such as curcumin, (−)-epicatechin,gallic acid, butylated hydroxyl toluene, etc. was done byMTTcell proliferation assay. Nuclear deformities, ROS production,and integrity of extra cellular matrix components were ana-lyzed by different techniques. S. cumini methanolic pulpextract (MPE), a naturally derived gallic acid-enriched anti-oxidant was taken to study its effect on malathion-inducedtoxicity. Nuclear deformities, ROS production, and integrityof extra cellular matrix components were also analyzed.Twenty micrograms per milliliter LD50 dose of malathionwas found to cause stress-mediated responses in H9C2 cellline. Among all the polyphenols, gallic acid showed the mostsignificant protection against stress. Gallic acid-enrichedmethanolic S. cumini pulp extract (MPE) showed 59.76 %±0.05, 81.61 %±1.37, 73.33 %±1.33, 77.19 %±2.38 and64.19 %±1.43 maximum inhibition for DPPH, ABTS, NO,H2O2 and superoxide ion, respectively, as compared toethanolic pulp extract and aqueous pulp extract. Our studysuggests that S. cumini MPE has the ability to protect againstthe malathion-mediated oxidative stress in cardiac myocytes.

Keywords Gallic acid .Malathion . Organophosphate .

Reactive oxygen species (ROS) . Syzygium cumini .

Antioxidant

Introduction

The widespread use of pesticides along with its application inagriculture throughout the world has become a topic of envi-ronmental and global health concern (Bovet and Paccaud2012). Exposure to these harmful chemicals has been associat-ed with elevated risk of chronic diseases affecting non targetspecies (Pesticide residues in food, FAO and WHO2012; McCauley et al. 2006). The World Health Organizationhas reported that an estimated one to five million cases ofpesticide poisonings occur every year, resulting in several thou-sands of fatalities, including children (Bergman et al. 2012).“Although developing countries use 25 percent of the world’sproduction of pesticides, they experience 99 percent of thedeaths due to inadequate safeguards” (Childhood PesticidePoisoning Information for Advocacy and Action, UNEP,WHO and FAO 2004; Yáñez et al. 2002). Although thesecompounds play role in food conservation, their release arisingfrom non-approved use leads to potential health hazards likecardiovascular diseases, pulmonary diseases, neurotoxicity etc(Food and Agriculture Organization of the United Nations2003; Mostafalou and Abdollahi 2013; Shafiee et al. 2010).

Organophosphates (OP) constitute one of the mostwidely used classes of pesticides employed in both agri-cultural and landscape pest control. The number of humanpoisonings with OP pesticides is estimated at around3,000,000/year, and the number of deaths and casualtiesaround 200,000/year (Gargouri et al. 2011; Liu et al.2012). Malathion is an organophosphate pesticide used inagriculture, commercial extermination, fumigation, veteri-nary practices, etc (Brenner 1992). Its toxicity is caused

Responsible editor: Philippe Garrigues

Neha Atale and Khushboo Gupta contributed equally.

N. Atale :K. Gupta :V. Rani (*)Department of Biotechnology, Jaypee Institute of InformationTechnology, A-10, Sector-62, Noida 201307, Uttar Pradesh, Indiae-mail: vibhasandeep@rediff.com

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by enhanced production of reactive oxygen species (ROS),disrupting the balance of antioxidative enzymes leading tooxidative stress (Altuntas et al. 2003; Ahmed and Zaki2009). Occupational exposure to malathion is boundedwith increased lipid peroxidation, increased DNA damageand oxidative deterioration of biological macromoleculesand related health issues (Possamai et al. 2007; Gargouriet al. 2011; Alavanja et al. 2004).

Recently, there is a great deal of interest in identifyingnatural and safe source of antioxidants that hold health-promoting therapeutic potential. Plant polyphenols havedrawn increasing attention due to their potent antioxidantproperties, less toxicity, and their marked effects in theprevention of oxidative stress (Bailes 2002; Mates 2000).Recent studies have shown that the drugs with combinedantioxidative potential are more effective in treating dis-eases (Soman et al. 2010). Gallic acid is a polyphenol andis known to have a strong antioxidative potential(Javanmardia et al. 2003), and Syzygium cumini (family:Myrtaceae), a well-known antidiabetic plant is enrichedwith gallic acid, vitamin C, tannins, anthocyanins, etc(Rekha et al. 2008; Banerjee et al. 2005). Our grouprecently investigated the cardioprotective properties ofS. cumini extracts in stress conditions (Atale et al. 2011,2013); however, the antioxidative potential of fruit pulp ofS cumini in malathion-induced cardiotoxicity has not beenreported yet.

In the present study, we extended our research to furtherinvestigate the protective effect of most enriched methanolicextract of S. cumini pulp in pesticide (malathion) inducedcardiac stress in H9C2 cardiac myocytes.

Material and methods

Pulp collection

Fruits of S. cumini were collected from Noida, Uttar Pradesh,India, in the month of July and were identified and authenti-cated by Dr. Anshu Rani, Department of Botany, Govt. P.G.College, Abu Road, Rajasthan, India. Fruits were washedwithwater thoroughly and air dried (Martinez and Valle 1993). Thepulp was collected for the preparation of aqueous and organicsolvents extracts.

Preparation of aqueous and organic solvent extractof S. cumini fruit pulp

The pulp was processed with water (APE), ethanol (EPE), andmethanol (MPE) and then dried. The dried extract wasreconstituted in water at a concentration of 1 mg/ml.

Antioxidant activities of APE, EPE, and MPE of S. cumini

1,1-diphenyl-1-picrylhydrazyl assay

1,1-diphenyl-1-picrylhydrazyl (DPPH) free radical scaveng-ing activity of S. cumini APE, EPE, and MPE was estimatedby the method of Brand-Williams et al. (1995). Various con-centrations of extract were mixed with 0.135 mM DPPHprepared in methanol (1:1). The resulting mixture was incu-bated in the dark for 30 min and absorbance was measured at517 nm. The scavenging activity of all the extracts wascalculated using the following equation:

ODcontrol−ODsample

ODcontrol� 100

Where OD control is the absorbance of DPPH+methanol;OD sample is the absorbance of DPPH radical+sample (i.e.extract or standard). Ascorbic acid was used as standard.

2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)scavenging activity

The 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)(ABTS) scavenging activity of all three extracts was determinedby the method of Re et al (1999). ABTS (7 mM) solution andpotassium persulfate (2.4 mM) was added in equal ratio andincubated for 12 h at 37 °C in the dark. One milliliter of freshlyprepared ABTS·+ solution was added in the resulting mixture.The standard and extracts were mixed individually with theresultingmixture in equal proportion. After 15min of incubation,the absorbance was measured at 734 nm. The percent inhibitioncapacity of the extracts for ABTS·+ and standard butylatedhydroxyltoluene (BHT) was calculated as mentioned above.

Nitric oxide scavenging activity

The NO radical scavenging activity of all the extracts was deter-mined by the method of Garrat (1964). Two milliliters of 10 mMsodium nitroprusside was mixed with 0.5 ml of APE/EPE/MPEat various concentrations. Themixture was incubated at 25 °C for2.5 h. The Griess reagent wasmixed with the resulting solution inequal proportion and incubated at 37 °C for 40 min. The absor-bance was measured at 540 nm. The amount of nitric oxideradical inhibition was calculated by following equation:

%Inhibition ¼ Ao−A1

A1� 100

Where A0 is the absorbance before reaction (without sam-ple) and A1 is the absorbance after reaction has taken place.

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Hydrogen peroxide scavenging activity

Scavenging activity of H2O2 by S. cumini extracts was deter-mined by the method of Ruch et al (1989). Each extract atvarious concentrations were mixed with 4 mM H2O2 solution(prepared in 0.1 M phosphate buffer, pH 7.4) in equal propor-tion and incubated for 10 min at 37 °C. The absorbance of thesolution was taken at 230 nm. The percent inhibition capacityof H2O2 by all the three extracts was calculated using theequation mentioned above.

Scavenging activity of superoxide anion

The scavenging activity of superoxide anion was determined bythe method of Nishimiki et al (1972). The reaction mixturecontained 1ml of 156μMnitro blue tetrazolium (NBT) preparedin 100 mM phosphate buffer, 0.1 ml of extract at variousconcentrations and 1 ml of 468 μM NADH. One hundredmicroliters of 60 μM PMS was added with the mixture andincubated at 25 °C for 10 min. The absorbance was measured at560 nm. The percent of superoxide scavenging capacity wascalculated using the percent inhibition formulamentioned above.

Lipid peroxidation assays

The peroxide inhibition capacity of the extracts was deter-mined using ferric thiocyanate (FTC) and thiobarbituric acid(TBA) methods. The FTC method was used to evaluate theperoxides at the initiation of lipid peroxidation and TBAmethod was used to analyze them after the oxidation. Theinhibition of lipid peroxidation was estimated by the % inhi-bition formula.

Ferric thiocyanate method The inhibition of linoleic acidperoxides was evaluated using FTC method as describedKikuzaki et al 1991. The reaction mixture had 4 mg of sampleand standard BHT at various concentrations, 4.1 ml of 2.5 %linoleic acid in 99.5 % ethanol, 8.0 ml of 0.02 M phosphatebuffer 4 ml of 99.5 % ethanol, and 3.9 ml of distilled water.The mixture was incubated in dark for 30 min. To measure theperoxides, 9.7 ml of 75 % (v/v) ethanol was added in thereaction mixture, followed by 0.1 ml of 30 % ammoniumthiocyanate along with 0.1 ml of 0.02 M ferrous chloride in3.5 % hydrochloric acid. After 5 min, ferrous chloride wasadded and analyzed spectrophotometrically at 500 nm.

Thiobarbituric acid method The method of Ottolenghi wasused for the determination of free radicals present in all thethree extracts (Ottolenghi 1959). For TBA also, the samereaction mixture composition was taken which was used inFTC assay. Equal volume of 20 % trichloroacetic acid and0.67 % of thiobarbituric acid were added to the mixture. Theresulting mixture was incubated in water bath at 100°C for

15 min and centrifuged after cooling at 3,000 rpm for 20 min.The absorbance of the supernatant was measured at 552 nm.

Cell culture

Heart-derived H9C2 cardiomyoblast cells were obtained fromthe National Centre for Cell Science (NCCS), Pune, India.H9C2 cells were cultured with Dulbecco’s modified Eagle’sMedium (DMEM) supplemented with penicillin, streptomy-cin, glucose, L-glutamine, sodium bi-carbonate and 10 % fetalbovine serum (FBS) in humidified CO2 incubator (NewBrunswick) with 5 % CO2 at 37

oC (Sreejit et al. 2008).

Cell viability/cytotoxicity assay for dose optimizationof malathion and polyphenols

Cell viability and proliferation was measured by 3-(4,5-dimeth-yl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)(Ferrari et al. 1990). For MTT Assay, 8,000 cells were seededin each well of 96-well plates. Cells were exposed to variousconcentration of malathion using 1 % dimethylsulfoxide(DMSO) as solvent. MTT assay was also performed for doseoptimization of polyphenols which were freshly prepared bydissolving in DMSO. The control and treated cells were incu-bated for 48 h in 37 °C, 5 % CO2 incubator. After that, a 10-μlMTT solution (5 mg/ml) was added and incubated at 25oC for3 h. Supernatant was then aspirated and formazan salt crystalswere dissolved in 200 μl DMSO. Samples were analyzed in anELISA plate reader (Biorad) at 570 nm. Cell viability is definedrelative to untreated control cells as follows: cell viability=absorbance of treated sample/absorbance of control.

Treatment of cells with pesticide and polyphenols

H9C2 cells were cultured in serum-free DMEM supplementedwith ITS (insulin 50mg/ml, transferrin 27.5 mg/ml and selenium0.025 mg/ml) and induced with optimized dose of malathion. Inorder to see the effect of polyphenols, cells were treatedwith theiroptimal doses under similar culture conditions simultaneouslyalongwithmalathion in the culturemedia. Cells were seeded andtreatment was given for 48 h. Three different experimental setswere used during the entire study. (a) Cells were induced withincreasing concentrations of malathion to confirm the dose; (b)malathion-induced cells were treated with polyphenols; (c)malathion-induced cells were treated with methanolic extract ofS. cumini pulp. DMSO was taken as control in all the sets.

Morphological analysis of the treated cells

Cell morphology was observed microscopically for the con-firmation of stress induced by malathion and effect of poly-phenols on stressed cells. Cell size was also quantified byimage J software.

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Giemsa staining assay for determination of cell morphology

For the determination of changes in cell morphology, Giemsastaining was performed (Barcia 2007). H9C2 cells culturedovernight and after 48 h of incubation cells were washed with1× PBS and fixed with 100 % cold methanol, and 5%Giemsasolution was added to each well and incubated for 15 min at25oC to observe the morphological changes. Images werecaptured under the inverted microscope at 20× magnification.

Evaluation of oxidative stress by DCFH-DA assay

For the evaluation of oxidative stress and reactive oxygenspecies formation, 2′,7′-dichlorofluorescin diacetate (DCFH-DA) was added to the cultures, since DCFH-DA enters cellsand the acetate group on DCFH-DA is cleaved by cellularesterases, trapping the nonfluorescent DCFH inside the cells.Subsequent oxidation by ROS, particularly H2O2 and hydrox-yl radical, yields an increase in the fluorescent product DCF,which is suggestive of H2O2 or hydroxyl generation (Wangand Joseph 1999). To study the oxidative stress, cells wereseeded onto glass cover slips, grown overnight and treatmentwas given. After 48 h of incubation, the cells were PBSwashed and fixed with methanol. They were then incubatedwith DCFH-DA dye at a concentration of 3 μg/ml in PBSbuffer and observed under fluorescent microscope (Olympus)at ×40 magnification. The stained cells were then eluted andthe fluorescence intensity was measured by spectrofluorome-ter (PerkinElmer).

DAPI staining assay for assessment of nuclear morphology

A blue fluorescent dye, 4′,6-diamidino-2-phenylindole(DAPI) is that preferentially binds to A–T-rich regions andstains dsDNA. Cells were induced and fixed with methanol.Membrane-permeable fluorescent dye, DAPI (50 ng/ml), wasadded and incubated for 30 min at 25 °C. Cells were thenvisualized under fluorescent microscope using DAPI filter at×40 magnification. Bright field images were also taken.

Propidium iodide staining assay for evaluation of cell death

Propidium iodide, a red-fluorescence DNA binding dye isimpermeable to cells with an intact plasma membrane; hence,when the cell integrity becomes compromised, it gains accessto the nucleus where it complexes with DNA rendering thenucleus highly fluorescent. After 48 h of inductions, cellswere fixed and stained with propidium iodide (PI; 2.5 ng/μl)solution for 15 min in dark at 25° C (Brana et al. 2002). Slideswere observed under fluorescent microscope using the TRITCfilter. Images were captured at ×40 magnification and elutedstained cells were analyzed by spectrofluorometer.

Verhoeff van Gieson collagen staining assay

ECM turnover occurs during cardiac remodeling is a well-accepted paradigm. Oxidative stress is associated with break-ing down of ECM components such as collagen, integrin,fibronectin, etc. (Zamilpa and Lindsey 2010). Verhoeff VanGieson stain is a marker of collagen content in cells. A seriesof steps were followed for staining the cells with alcoholichematoxylin, FeCl3, Lugol’s iodide and counterstaining withvan Gieson’s stain. Images were taken at ×20 magnification.Collagen content was estimated at 540 nm by eluting thecollagen-bound stain (Marotta and Martino 1985).

Statistical analysis

Experimental results were expressed as mean±SD. Each ex-periment was conducted in triplicates. Statistical analysis wasdone by SPSS 16 software. A one-way ANOVA with theTukey’s test was used to evaluate the significance of theresults obtained. IC50 values were also calculated for MPE.All the results were significant at P<0.05 level.

Results

Malathion treatment leads to ROS-induced stress on H9C2cells

Cell viability assay was performed to identify LD50 dose ofmalathion on H9C2 cells (Fig. 1a). It was found to be20 μg/ml and cell viability was observed to decrease inconcentration dependent manner. Further, change in morphol-ogy and cell size was observed to analyze the alterationscaused by increasing concentrations of malathion. At20 μg/ml, a significant change in morphology was observedand approximately 25% decrease in the cell size was assessedas compared to control (Fig. 1b); therefore this dose was usedsubsequently for further experiments. To observe the effect ofmalathion-induced nuclear damage, PI assay was performed.Prominent nuclear deformities were found on increasing con-centrations of malathion. Spectroflourometer analysis showedabout twofold increase in fluorescence intensity at 20μg/ml ascompared to control (Fig. 1c). The formation of intracellularROS (as visualized by florescence micrographs) was alsofound to be elevated in dose-dependent manner (Fig. 1d).Fourfold increase in intensity was found in 20 μg/mlmalathion-induced cells as compared to control.

Gallic acid showed maximum protectionagainst malathion-induced stress

A number of natural and synthetic antioxidants were tested forcell proliferative studies as summarized in Table 1. BHT,

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trolox, quercetin, (−)-epicatechin, ascorbic acid,curcumin, and gallic acid treatments were given in

malathion-induced cells in different sets. Gallic acidpotentially increased cell size nearby control and

Fig. 1 Dose optimization and treatment of H9C2 cells with malathion. aLD50 dose optimization for malathion by MTT assay. A dosage of20 μg/ml was found to be LD50 dose of malathion. b Analysis of cellmorphology and size of malathion-induced cells with different experi-mental sets—Control, DMSO, 10, 15, 20, and 25 μg/ml malathion.Images taken at ×20 magnification (P≤0.05). c PI staining for alterationin nuclear morphology. Representative images (×40 magnification)showed the increase in nuclear deformities in dose-dependent manner.

Similar results were obtained from graphical data of fluorescence inten-sities (P≤0.05). d DCFH-DA assay showed ROS fluorescence in sameset of experiments. ROS production was also increased significantly byincreasing concentration of malathion (×40 magnification). Similar re-sults were observed after quantitation (P≤0.05). Bright field images alongwith the DAPI stained cells showed cellular and nuclear morphology inthe same set

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Fig. 1 (continued)

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significantly prevented the cells from malathion-inducedstress than other polyphenols (Fig. 2).

The optimal doses for natural polyphenols such ascurcumin, gallic acid, and (−)-epicatechin were found to be8, 20, and 50 μM, respectively at which the cell viability wasfound to be greater than 90% (Fig. 3a). In comparison to otherpolyphenols studied, treatment with gallic acid was found toefficiently combat the stress mediated by malathion inductionin cells (Fig. 3b). Gallic acid significantly reversed the effectof malathion on cell size. Cell morphology as observed byGiemsa staining was found to have undergone deteriorationon induction with malathion, while treatment with gallic acidshowed protection against these alterations in cell morphology(Fig. 4a). Gallic acid alone did not show any adverse effect onthe morphology of cells. Fluorescence micrographs of DCFH-DA assay showed that gallic acid has the ability to suppressthe free radical upregulation due to malathion-induced stress.Intensity of fluorescence decreased up to half significantly inmalathion-induced cells treated with gallic acid as comparedto the malathion-induced stressed cells (Fig. 4b).

The changes in nuclear morphology such as nuclear defor-mities or fragmentation were observed in malathion-inducedcells compared to induced cells treated with gallic acid by PI

(Fig. 4c). Treatment with gallic acid on malathion-inducedcells decreases the intensity of fluorescence nearby control,which was found to be increased up to twofold by malathioninduction. Cells stained with van Gieson stain indicated re-duced collagen content up to 30–35 % in malathion-inducedcells as compared to control cells. However, treatment withgallic acid increased the collagen content as shown in graph-ical representation (Fig. 4d). This can be due collagen imbal-ance and ECM remodeling in stressed conditions while gallicacid treatment showed the protective effect.

Methanolic pulp extract of S. cumini has maximumantioxidative activity

DPPH and ABTS assays were carried out to evaluate theantioxidative potential of APE, EPE, and MPE. MPE showedmaximum free radical scavenging activity in a dose-dependent manner. MPE has the highest potential to scavengefree radical as compared to EPE and APE as shown by DPPH(59.76 %±0.50) and ABTS (81.61 %±1.37) at the concentra-tion of 1 mg/ml. However DPPH scavenging was found to beless than that of ABTS with all the three extracts. The NO,H2O2, and superoxides scavenging activity was found to beincreased in a dose-dependent manner. MPE showed 73.33 %±1.33, 77.19 %±2.38, and 64.19 %±1.43 inhibition for NO,H2O2, and superoxides, respectively, at the concentration of1 mg/ml (Fig. 5a). MPE significantly inhibits the lipid perox-ides also with increasing concentrations followed by EPE andAPE. FTC and TBA assay showed 70.46 %±2.13 and76.72 %±1.29 lipid peroxide inhibition for MPE at 1 mg/mlconcentration compared to BHT standard (Fig. 5b).

The percentage inhibition of free radical scavenging activ-ities and the IC50 values of the MPE for DPPH, ABTS, NO,H2O2 superoxide and lipid peroxidation were summarized inTable 2.

Effect of MPE of S. cumini on malathion-induced stressin H9C2 cell line

Optimal dose for MPE of S. cumini on H9C2 cells wasidentified by MTT assay and was found to be 20 μg/ml(Fig. 6a). The effect of MPE was analyzed against themalathion-induced stress. Cell morphology clearly depictedthe reversal of stress in MPE-treated malathion-induced cells(Fig. 6b). A 10–15 % increase in cell size was seen on MPE-treated malathion-induced cells as compared to malathionalone. Further, Giemsa staining confirmed the effect of MPEon malathion-induced stress and cellular integrity was foundto be conserved on treatment with MPE (Fig. 6c). Stress-induced ROS elevation was significantly decreased by treat-ment with MPE. ROS scavenging activity of MPE-treatedmalathion-induced cells showed the fluorescence nearby two-fold lesser than malathion-induced cells (Fig. 6d). Reduced

Table 1 Optimum dosesfor natural and syntheticpolyphenols

Polyphenols Optimum dose (μM)

BHT 40

Trolox 50

Quercetin 30

Epicatechin 50

Ascorbic acid 12

Curcumin 8

Gallic acid 20

Fig. 2 Effect of various natural and synthetic polyphenols on malathion-induced cardiotoxicity. The treatment of BHT, trolox, quercetin, epicate-chin, ascorbic acid, curcumin and gallic acid was given in malathion-induced cells in different sets. The cell size was measured and gallic acidwas found to be a potent agent for preventing cells from malathion-induced stress among all the polyphenols (P≤0.05)

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nuclear deformities were observed with MPE in malathion-induced cells and only MPE did not cause any changes innuclear conformity (Fig. 6e). The quantitative measurementsof the eluted cells showed twofold increase in fluorescenceintensity in malathion-induced cells and MPE treatment to itsustained the level of fluorescence nearby control. MPE wasalso found to cause protection against ECM remodeling withrespect to gallic acid. The reduced collagen content by mala-thion induction was regained by 30 % on treatment with MPE(Fig. 6f).

Fig. 3 a Dose optimization ofgallic acid, curcumin, and (−)-epicatechin on H9C2 cells. Cellviability assay was done for doseoptimization. The dose with 90 %cell viability was taken as theoptimal dose for treatment ofcells. The optimal doses forcurcumin, gallic acid, and (−)-epicatechin were found to be 8,20, and 50 μM respectively. bMorphological analysis oftreatment with naturalpolyphenols. Gallic acidtreatment showed a significantprotective impact on themorphology of the malathion-induced cells than the other twopolyphenols

�Fig. 4 Treatment of malathion-induced cells with gallic acid. a Giemsastaining was done in five experimental sets; control, malathion-inducedmalathion+gallic acid, gallic acid alone, and DMSO alone. b DCFH-DAassay determined the ROS mediated stress in malathion stressed cells andsuppression of ROS level in malathion+gallic acid-treated cells. Similarresults were obtained by quantitative analysis (P≤0.05). Bright field andDAPI fluorescence images were also taken at ×40 magnification. c PIassay showed the intact nuclei in gallic acid-treated malathion-induced setas compared to control (quantitatively also). d Verhoeff van Giesonstaining for determination of collagen content. The eluted stained cellswere then quantitated (P≤0.05)

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Fig. 4 (continued)

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Discussion

The study was undertaken to determine the role of methanolicfruit pulp extract of S. cumini onmalathion-induced toxicity inH9C2 cardiac cell line. Antioxidants constitute the primary

defense system that limits the toxicity associated with freeradicals (Devasagayam et al. 2004). Therefore, there is agrowing interest in natural antioxidants as a dietary regimenand to explore natural sources of antioxidants especially frommedicinal plants. Natural antioxidants in foods may be from

Fig. 5 a Antioxidative and lipid peroxidation assays for S. cumini aque-ous (APE) ethanolic (EPE) and methanolic pulp extracts (MPE): DPPHscavenging activity; ABTS scavenging activity; NO scavenging activity;H2O2 scavenging activity; superoxide anion scavenging activity. All

assays were compared to their respective standards. b FTC assay andTBA assay of S. cumini aqueous, ethanolic, and methanolic pulp extracts.Values indicated as % inhibition of peroxides. ** showed the level ofsignificance (P≤0.05)

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phenol, flavonoid, terpenoid, and steroids products formed fromreactions during processing and additives from plant extracts(Gomathi et al. 2012). Natural polyphenols show a uniquecombination of chemical, biological, and physiological activitiesand play a significant role in pathology during toxicity.

Cellular levels may fluctuate according to balance betweenROS production and antioxidant regeneration due to malathi-on induction (Cabello et al. 2001). Morphological changes arethe marker of pesticide-induced stress. In the present study,morphology of cells seemed to be affected by malathiontreatment. DCFH assay confirmed the production of ROS inmalathion-induced cells. PI assay depicted increased stress onmalathion treatment that was accompanied by compromisedcellular integrity and nuclear deformities whichmay even leadto cell death. Thus, it was confirmed that malathion inductionshowed the deteriorating effects on the cells.

For the protection against the toxic effects, cells were treatedwith number of natural and synthetic antioxidants. Among them,gallic acid was found to have significant reversal and had theability to counteract cellular distortion on toxic exposure to

malathion. Further experiments were therefore performed withgallic acid as positive control. Morphological studies, cell size,ROS production, nuclear conformity, and collagen content indi-cated that treatment with gallic acid gave maximum protectionagainst malathion-induced stress in H9C2 cells.

Table 2 Free radical scavenging activities of APE, EPE and MPE of S. cumini at different concentration (standard values in parentheses)

Conc. (mg/ml) DPPHa

(%inhibition)ABTSa

(%inhibition)NOa

(%inhibition)H2O2

a

(%inhibition)Superoxidea

(%inhibition)FTCa

(%inhibition)TBAa

(%inhibition)(%inhibition) (%inhibition) (%inhibition) (%inhibition) (%inhibition) (%inhibition) (%inhibition)

0.2 (MPE) 29.72±0.99 35.95±5.61 39.65±2.43 46.29±3.27 41.74±1.12 43.36±1.36 39.40±0.79

(EPE) 23.61±1.58 33.53±2.12 31.39±3.16 26.00±5.14 22.40±2.28 44.13±3.24 27.72±1.73

(APE) 10.20±0.48 14.68±0.56 28.56±2.46 18.69±0.50 22.72±1.00 36.65±2.27 23.57±2.75

(49.09±1.28) (56.59±1.54) (43.53±5.02) (57.27±4.10) (42.92±1.35) (48.74±1.36) (47.72±0.66)

0.4 (MPE) 36.90±2.50 52.62±2.01 52.05±1.59 52.44±1.26 46.34±0.55 49.16±0.96 47.03±1.78

(EPE) 33.13±1.42 39.90±3.47 39.61±0.61 49.72±1.09 40.29±2.40 45.41±1.50 38.87±1.25

(APE) 16.21±0.57 35.57±2.81 38.57±5.19 37.90±1.78 30.54±2.03 41.36±1.91 27.60±1.04

(57.06±1.59) (58.94±4.09) (62.28±1.82) (68.21±1.0) (54.79±2.33) (56.25±3.21) (57.08±2.06)

0.6 (MPE) 43.73±4.48 65.07±5.53 65.06±2.85 59.63±0.54 51.50±1.30 56.52±2.45 47.94±0.34

(EPE) 40.95±0.45 61.24±1.90 58.76±4.01 51.71±1.33 42.04±1.90 57.09±0.28 44.04±1.67

(APE) 33.99±3.39 50.72±1.64 53.54±4.24 44.73±0.55 40.05±2.92 43.03±2.13 36.86±1.10

(58.98±1.63) (81.24±3.56) (71.08±0.93) (71.51±1.13) (68.17±2.79) (63.26±1.36) (65.88±2.32)

0.8 (MPE) 50.09±1.72 74.51±3.67 66.05±2.11 68.91±3.06 59.27±0.34 64.14±1.79 58.32±0.84

(EPE) 51.25±0.91 68.85±4.20 60.92±3.04 57.28±1.17 47.71±0.45 62.53±0.50 54.31±2.30

(APE) 39.62±0.76 60.28±1.99 62.17±0.72 55.59±2.93 40.75±1.28 44.95±1.89 39.39±0.73

(62.44±0.28) (84.83±1.59) (74.24±1.44) (81.60±1.17) (70.81±1.49) (70.89±1.61) (73.80±1.34)

1 (MPE) 59.76±0.50 81.61±1.37 73.33±1.33 77.19±2.38 64.19±1.43 70.46±2.13 76.72±1.29

(EPE) 53.71±1.58 73.06±1.94 72.83±1.65 69.46±1.16 52.40±2.37 63.25±0.63 55.50±2.35

(APE) 47.73±3.32 65.08±1.12 63.78±1.58 61.00±0.70 51.23±0.72 54.30±4.62 52.35±2.24

(69.17±1.06) (91.83±0.66) (78.87±1.25) (87.81±0.98) (74.35±5.35) (72.05±0.44) (84.87±1.39)

DPPH IC50 mg/ml ABTS IC50 mg/ml NO IC50 mg/ml H2O2 IC50 mg/ml SuperoxideIC50 mg/ml

FTC IC50 mg/ml TBA IC50 mg/ml

S. cumini MPE 0.80 0.22 0.57 0.75 0.59 0.55 0.56

Standard 0.60 0.15 0.28 0.15 0.35 0.32 0.20

The enteries given in italic are to differentiate and emphasize upon the results of Methanolic pulp extract which shows maximum antioxidative potentialand ability to scavenge free radicals than Aqueous pulp extract and Ethanoloc pulp extracta Values represent mean±SD

�Fig. 6 Dose optimization and treatment of H9C2 cells with MPE. a Theoptimized dose for MPE of S. cumini as identified by MTT assay wasfound to be 20 μg/ml. H9C2 cells were cultured in control, malathion-induced, malathion+MPE, and MPE-alone treated sets. bMorphologicalanalysis of H9C2 cells showed the protective effect of MPE onmalathion-induced stress as compared to control (×20 magnification). cGiemsa staining demonstrated the morphological changes in the experi-mental observations (×20 magnification) (P≤0.05). d Suppression ofROS level in malathion+MPE-treated cells by DCFH-DA Assay (P≤0.05). Bright field and DAPI fluorescence images showed the cellular andnuclear morphology on malathion-mediated stress and the protectiveeffect of MPE on it. e PI staining of cells showed decrease in nuclearanomalies in MPE-treated cells (×40 magnification) and it was validatedgraphically (P≤0.05) also. f MPE treatment of malathion-induced cellsprevents stress-mediated change in collagen content (×20 magnification)as shown by histogram also

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Fig. 6 (continued)

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As a number of synthetic polyphenols are not FDA-approved and can be harmful, therefore there is a thrust toidentify natural sources of antioxidants (Mennen et al. 2005).The crude extracts of plants have potent therapeutic efficaciesthan purified compounds due to the presence of various otherphytochemicals. A combination of various phytochemicals ismore effective and has better therapeutic potential. Therefore,we prepared various extracts of S. cumini as its fruit is reportedto be rich in gallic acid content and further studies wereconducted in this regard.

Free radicals such as NO, H2O2, and superoxide playimportant roles in the development of oxidative stress andhave the ability to penetrate biological membranes and dam-age biomolecules in living cells. Increasing evidence in bothexperimental and clinical studies suggests that free radicalsformed disproportionately during diseases lead to increasedlipid peroxidation. We therefore investigated the effect ofS. cumini APE, EPE, and MPE on peroxidation of lipids bymeasuring the synthesis of malondialdehyde (MDA), which isproduced based on the acid-catalyzed decomposition of lipidperoxides. The FTC method indicates the amount of peroxidein the initial stages of lipid peroxidation (Saha et al. 2004)whereas the TBAmethod shows the amount of peroxide in thesecondary stage of lipid peroxidation (Rahmat et al. 2003).Mechanisms other than lipid peroxidation include DNA dam-age, protein oxidation, and intracellular calcium increase dueto membrane permeability lesions (Kirkinezos and Moraes2001). Aggregation of these lipid peroxides in the cell increasethe production of MDA, which could be carcinogenic(Gultekin et al. 2000). In the present study, the increasedMDA levels in heart due to exposure of malathion indicateincreased generation of free radicals. The increased MDAlevel may be due to an increase in reactive oxygen speciesin heart tissues. Thus, oxidative stress can be suggested as oneof the mechanisms in malathion-induced cardiotoxicity.Experimental results showed that all extracts prepared frompulp of S. cumini are not equally pharmaceutically active. Thequantity and quality of the extracted antioxidant polypheno-lics varies owing due to the variation in the polarity of thesolvent used for the extraction of polyphenolics from naturalsources (Durling et al. 2007; Alothman et al. 2009). APE wasfound to be least enriched in its phytocontent and antioxida-tive potential as compared to EPE and MPE, respectively.MPE exhibits the greatest antioxidative activity through scav-enging the free radicals which participate in various patho-physiologies. Therefore, in our study, protective effects ofS. cumini MPE were observed in malathion-inducedcardiotoxicity.

Further, cells were treated with MPE of S. cumini to deter-mine its protective properties. The severity of malathion-induced stress-mediated morphological changes and cell sizewas reduced on treatment with MPE. ROS levels and nucleardeformities have also decreased on MPE treatment Increment

in collagen content could also be clearly observed. Therefore,MPE could effectively ameliorate the malathion-induced ox-idative stress to a large extent. This shows the antioxidativeproperty and protective role of S. cumini in malathion-inducedcardiotoxicity.

This study is important as poisoning with organophosphateis a critical clinical problem and if not managed rapidly andeffectively results in cardiovascular collapse and death(Shadania et al. 2007). Cardiovascular disease (CVD) is theleading cause of death with 17 million deaths worldwide for atotal of 57 million annually and 80 % of all CVD deaths nowoccur in low- and middle-income countries (WHO report.2011). As these countries have majority of their populationinvolved in agricultural practices, therefore individual-levelstrategies for the treatment of cardiovascular abnormalitiesdue to pesticide exposure often incur very high costs are notfeasible, which emphasizes the need to carefully select inter-ventions that are affordable and highly effective in curing thediseases caused by pesticide-induced toxicity. Highly cost-effective medications or supplements of naturally extractedand easily available crude plant polyphenols is a favorableapproach to combat the fatal consequences of toxicity causedby malathion and other organophosphate pesticides.

Conclusion

The present study demonstrates that malathion produced tox-icity in cardiac cells by changes in cell morphology, increasedROS production and ECM remodeling. S. cumini has thepotential to alienate and suppress the effects of oxidative stresscaused by toxic exposure of malathion. Novel therapeuticstrategies can be designed to enhance its pharmacologicalpotential as certain barriers like bioavailability and poor ab-sorption tend to limit its activity. In the future, further under-standing of the mechanisms of S. cumini-mediated protectionagainst pesticides under stress can help to combat the effect ofpesticide exposure on human health.

Conflict of Interest The authors declare no conflict of interest.

References

Ahmed MM, Zaki IN (2009) Assessment the ameliorative effect ofpomegranate and rutin on chlorpyrifos-ethyl-induced oxidativestress in rats. Nat Sci 7:49–61

Alavanja MC, Hoppin JA, Kamel F (2004) Health effects of chronicpesticide exposure: cancer and neurotoxicity. Annu Rev PublicHealth 25:155–197

Alothman M, Rajeev B, Karim AA (2009) Antioxidant capacity andphenolic content of selected tropical fruits from Malaysia, extractedwith different solvents. Food Chem 115:785–788

Environ Sci Pollut Res

Altuntas I, Delibas N, Doguc DK, Ozmen S, Gultekin F (2003) Role ofreactive oxygen species in organophosphate insecticide phosalonetoxicity in erythrocytes in vitro. Toxicol In Vitro 17:153–157

Atale N, Jaiswal A, Chhabra A,Malhotra U, Kohli S, Mohanty S, Rani V(2011) Phytochemical and antioxidant screening of Syzygium cuminiseed extracts: a comparative study. J Pharm Res 4:4530–4532

Atale N, Chakraborty M, Mohanty S, Bhattacharya S, Nigam D, SharmaM, Rani V (2013) Cardioprotective role of Syzygium cumini againstglucose-induced oxidative stress in H9C2 cardiac myocytes.Cardiovasc Toxicol. doi:10.1007/s12012-013-9207-1

Bailes BK (2002) Diabetes mellitus and its chronic complications. AORN76:265–274

Banerjee A, Dasgupta N, De B (2005) In vitro study of antioxidantactivity of Syzygium cumini fruit. Food Chem 90:727–733

Barcia JJ (2007) The giemsa stain: its history and applications. Int J SurgPathol 15:292–296

Bergman A, Heindel JJ, Jobling S, Kidd AK, Zoeller TR (2012) State ofthe science of Endocrine Disrupting chemicals. United NationsEnvironment Programme and the World Health Organization 978-92-807-3274-0 (UNEP) and 978 92 4 150503 1 (WHO)

Bovet P, Paccaud F (2012) Cardiovascular disease and the changing faceof global public health: a focus on low andmiddle income countries.Pub Health Rev 33:397–415

Brana C, Benham C, Sundstrom L (2002) A method for characterizingcell death in vitro by combining propidium iodide staining withimmunohistochemistry. Brain Res Protocol 10:109–114

Brand-WilliamsW, Cuvelier ME, Berset C (1995) Use of a radical methodto evaluate antioxidant. Lebensm-Wiss U-Technol 28:25–30

Brenner L (1992) Malathion fact sheet. J Pest Reform 12:1–17Cabello G, Valenzuela M, Vilaxa A, Duran V, Rudolf I, Hrepic N,

Calaf G (2001) A rat mammary tumor model induced by theorganophosphorous pesticides parathion and malathion, possiblythrough acetylcholinesterase inhibition. Environ Health Perspect109:471–479

Childhood Pesticide Poisoning Information for Advocacy and Action,UNEP, WHO & FAO (2004)

Devasagayam TP, Tilak JC, Boloor KK, Sane KS, Ghaskadbi SS,Lele RD (2004) Free radicals and antioxidants in human health:current status and future prospects. J Assoc Phys India 52:794–804

Durling NE, Catchpole OJ, Grey JB, Webby RF, Mitchell KA, Foo LY,Perry NB (2007) Extraction of phenolics and essential oil from driedsage (Salvia officinalis) using ethanol-water mixtures. Food Chem101:1417–1424

Ferrari M, Fornasiero MC, Isetta AM (1990) MTTcolorimetric assay fortesting macrophage cytotoxic activity in vitro. J Immunol Methods131:165–172

Food and Agriculture Organization of the United Nations (2003)International Code of Conduct on the Distribution and Use ofPesticides (Revised). FAO, Rome

Gargouri B,Mansour BR, Abdallah BF, Elfekih A, Lassoued S, Khaled H(2011) Protective effect of quercetin against oxidative stress causedby dimethoate in human peripheral blood lymphocytes. LipidsHealth Dis 10:149–152

Garrat DC (1964) The quantitative analysis of drugs, 3rd edn. Chapman& Hall, Japan

Gomathi D, Ravikumar G, Kalaiselvi M, Devaki K, Uma C (2012)Efficacy of Evolvulus alsinoides (L.) on insulin and antioxidantsactivity in pancreas of streptozotocin induced diabetic rats. JDiabetes Metab Disord. doi:10.1186/2251-6581-12:39

Gultekin F, Ozturk M, AkdoganM (2000) The effect of organophosphateinsecticide chlorpyrifosethylon lipid peroxidation and antioxidantenzymes (in vitro). Arch Toxicol 74:533–538

Javanmardia J, Stushnoffb C, Lockeb E, Vivancob JM (2003)Antioxidant activity and total phenolic content of Iranian Ocimumaccessions. Food Chem 83:547–550

Kikuzaki H, Usuguchi J, Nakatani N (1991) Constituents ofZingiberaceae I. Diarylheptanoid from the rhizomes of gin-ger (Zingiber officinale Roscoe). Chem Pharm Bull 39:120–122

Kirkinezos IG, Moraes CT (2001) Reactive oxygen species and mito-chondrial diseases. Semin Cell Dev Biol 12:449–457

Liu SH, Lin JL, Weng CH, Yang HY, Hsu CW, Chen KH (2012) Heartrate-corrected QT interval helps predict mortality after intentionalorganophosphate poisoning. PLoS One 7(5):e36576

Marotta M, Martino G (1985) Sensitive spectrophotometric methodfor the quantitative estimation of collagen. Anal Biochem 150:86–90

Martinez SB, Valle MJ (1993) Storage stability and sensory quality ofduhat (Syzygium cumini Linn.) anthocyanins as food colorant. In:Mazza G, Miniati E (eds) Anthocyanins in fruits, vegetables andgrains. CRC, London, pp 139–147

Mates JM (2000) Effects of antioxidant enzymes in the molecular controlof reactive oxygen species toxicology. Toxicol 16:83–104

McCauley AL, Anger KW, Keifer M, Langley R, Robson GM,Rohlman D (2006) Studying health outcomes in farmworkerpopulations exposed to pesticides. Environ Health Perspect 114:953–960

Mennen IL, Walker R, Bennetau-Pelissero C, Scalbert A (2005) Risksand safety of polyphenol consumption. Am J Clin Nutr 81:326S–329S

Mostafalou S, Abdollahi M (2013) Pesticides and human chronic dis-eases: evidences, mechanisms and perspectives. Toxicol ApplPharmacol 268:157–177

Nishimiki M, Rao NA, Yagi K (1972) The occurrence of super-oxideanion in the reaction of reduced phenazine methosulfateand molecular oxygen. Biochem Biophys Res Commun 46:849–853

Ottolenghi A (1959) Interaction of ascorbic acid and mitochondria lipids.Arch Biochem Biophys 79:355–363

Pesticide residues in food (2012) Report of the Joint Meeting of the Foodand Agriculture Organization and World Health Organization.Rome 2013. 0259-2517

Possamai FP, Fortunato JJ, Feier G, Agostinho FR, Quevedo J, FilhoDW,Dal-Pizzol F (2007) Oxidative stress after acute and sub-chronicmalathion intoxication in Wistar rats. Env Toxicol Pharmacol 23:198–204

Rahmat A, Kumar V, Fong LM, Endrini S, Sani HA (2003)Determination of total antioxidant activity in three types of localvegetables shoots and the cytotoxic effect of their ethanol ex-tracts against different cancer cell lines. Asia Pac J Clin Nutr12:292–295

Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C (1999) Antioxidant activity: applying an improvedABTS radical cation decolorization assay. Free Radic BiolMed 26:1231–1237

Rekha N, Balaji R, Deecaraman M (2008) Effect of aqueous extract ofSyzygium cumini pulp on antioxidant defense system instreptozotocin induced diabetic rats. Iran J Pharmacol Ther 7:137–145

Ruch RJ, Cheng SJ, Klaunig JE (1989) Prevention of cytotoxicityand inhibition of intercellular communication by antioxidantcatechins isolated from Chinese green tea. Carcinogens 10:1003–1008

Saha K, Lajis NH, Israf DA, Hamzah AS, Khozirah S, Khamis S, SyahidaA (2004) Evaluation of antioxidant and nitric oxide inhibitory activ-ities of selected Malaysian medicinal plants. J Ethnopharmacol 92:263–267

Shadania S, Darabi D, Pajoumand A, Salimi A, Abdollahi M (2007) Asimplified acute physiology score in the prediction of acute organ-ophosphate poisoning outcome in an intensive care unit. Him ExpToxicol 26:623–627

Environ Sci Pollut Res

Shafiee H, Mohammadi H, Rezayat MS, Hosseini A, Hassani S,Mohammadirad A, Bayrami Z, Abdollahi M (2010) Prevention ofmalathion-induced depletion of cardiac cells mitochondrial energyand free radical damage by a magnetic magnesium-carrying nano-particle. Toxicol Mech Methods 20:538–543

Soman S, Rauf AA, Indira M, Rajamanickam C (2010) Antioxidant andantiglycative potential of ethyl acetate fraction of Psidium guajavaleaf extract in streptozotocin-induced diabetic rats. Plant Foods HumNutr 65:386–391

Sreejit P, Kumar S, Verma RS (2008) An improved protocol for primaryculture of cardiomyocyte from neonatal mice. In Vitro Cell Dev BiolAnim 44:45–50

Wang H, Joseph JA (1999) Quantifying cellular oxidative stress bydichlorofluorescein assay using micro plate reader. Free Radic BiolMed 27:612–616

World Health Organization (2011) Global status report onnoncommunicable diseases 2010. WHO, Geneva

Yáñez L, Ortiz D, Calderon J, Batres L, Carrizales L, Mejia J, Neito EJ,Barriga FD (2002) Overview of human health and chemical mix-tures: problems facing developing countries. Environ HealthPerspect 110:901–909

Zamilpa R, Lindsey MI (2010) Extracellular matrix turnover and signal-ing during cardiac remodeling following MI: causes and conse-quences. J Mol Cell Cardiol 48:558–563

Environ Sci Pollut Res