Oxidative stress induced apoptosis of human lung carcinoma (A549) cells by a novel copper nanorod formulation

Food and Chemical Toxicology 49 (2011) 2990–2996

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Food and Chemical Toxicology

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

Oxidative stress induced apoptosis of human lung carcinoma (A549) cells by anovel copper nanorod formulation

Menaka C. Thounaojam a, Ravirajsinh N. Jadeja a, Mayur Valodkar b, Padamanabhi S. Nagar c,Ranjitsinh V. Devkar a,⇑, Sonal Thakore b

a Department of Zoology, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara 390002, Gujarat, Indiab Department of Chemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara 390002, Gujarat, Indiac Department of Botany, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara 390002, Gujarat, India

a r t i c l e i n f o

Article history:Received 8 June 2011Accepted 19 July 2011Available online 26 July 2011

Keywords:Copper nanorodsCytotoxicityAlmond skin extractOxidative stressAnticancer

0278-6915/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.fct.2011.07.055

⇑ Corresponding author. Tel.: +91 9825935445.E-mail address: [email protected] (R.V. Devk

a b s t r a c t

This study elucidates the process of synthesis of copper (Cu) nanorods using almond skin extract as sta-bilizing cum capping agent. These nanorods were (about 200 nm long and 40 nm wide) characterized bytransmission electron microscopy (TEM). Further, cytotoxicity potential of these nanorods was evaluatedin A549 cells (Human lung carcinoma cell line) via cell viability assay and extracellular lactate dehydro-genase (LDH) release. Also, reduced glutathione (GSH), lipid peroxidation (LPO), cellular oxidative stress(Rhodamine 123 florescence) and apoptosis (Annexin V FITC/Propidium iodide staining) were also inves-tigated in control and treated cells. Results indicated that Cu nanorods induced apoptotic death of cancercells by induction of oxidative stress, depletion of cellular antioxidants and mitochondrial dysfunction.This study reports a novel process of synthesis of almond skin extract capped Cu nanorods and its poten-tial as an anticancer agent against A549 lung carcinoma cells.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Nanomaterials, such as nanoparticles (NPs), nanotubes andnanowires have gained popularity in biomedical applications(Bai et al., 2007; Julien et al., 2010) due to their functional plural-ity acquired after they are subjected to changes in morphology,surface charge, chemical composition, solubility or modificationof functional groups. These physical attributes also alter their ex-tent of affinity and potency of interaction with live cells or tissues(Lanone and Boczkowski, 2006). Use of biological processes(involving micro-organism such as bacteria, fungus and plant ex-tracts) for NP synthesis have emerged as simple and viable alter-native to more complex chemical procedures that involves use oftoxic synthetic chemicals (Vijayaraghavan and Nalini, 2010; Pan-da et al., 2011). Seeding approaches (Nadagouda and Varma,2006) and solution-phase synthesis (Wiley et al., 2007) are fewof the commonly employed approaches for large scale productionof metal nanowires, nanobelts and nanorods. But, these methodsare executed at very high temperatures and result in poor yield ofNP of desired structure. Hence, formulation of new procedure fornon-toxic synthesis of nanoparticles by an environmentally be-nign ‘green chemical process’ is a major challenge for nanotech-

ll rights reserved.

ar).

nologists worldwide. Almonds (Prunus dulcis (Mill.) D.A. Webb;Rosaceae) are rich in polyphenols and other antioxidants. About70% of total polyphenols present in an almond nut is containedin its skin (Milbury et al., 2006). But, almond skin is generallyconsidered as an industrial byproduct (low economic value) thatis removed from its kernel by blanching (Vargas, 2005). However,recent research is focused on exploring possible uses of suchbyproducts in the food industry or as nutritional supplements(Wijeratne et al., 2006).

Recent studies from our research group had reported synthesisof plant latex or starch capped silver and copper NP that exhibitedexcellent antimicrobial and cytotoxic potentials towards cancercells (Valodkar et al., 2010, 2011a,b). Present study is the firstreport on easy and rapid synthesis of high concentration of Cunanorods using aqueous almond skin extract as stabilizing agentand ascorbic acid as a reducing agent. This nanorod formulationhas a novel morphology (rod-like shape) and the same wassubjected to scrutiny for its cytotoxic potential and anticancerproperties against A549 cells (human lung carcinoma cells).

2. Materials and methods

2.1. Chemicals

Copper nitrate (Cu (NO3)2�3H2O), ammonia and ascorbic acid were purchasedfrom Merck Mumbai, India and solutions were prepared in deionized water.

M.C. Thounaojam et al. / Food and Chemical Toxicology 49 (2011) 2990–2996 2991

2.2. Synthesis of nanoparticles

Almonds (50) were purchased from the local market. Almond skin was peeledmanually and collected in a glass beaker and soaked in 100 ml of deionized water.Contents were boiled at 100 �C for 5 min and then left overnight at room tempera-ture. Later, the solution was centrifuged at 1000g to obtain a clear yellow coloredsupernatant that was stored at 4 �C. Ten milliliters of the supernatant was mixedwith 0.6 ml of 0.1 M Cu (NO3)2 solutions with constant stirring in 25 ml Erlenmeyerflask and at 0 �C. The pH was adjusted to 10 by adding ammonia solution. Later,0.6 ml of 25% ascorbic acid solution was added so as to re-adjust the pH to 3. Theformation of copper nanoparticles was marked by development of a characteristicwine red colored solution. Finally this nanoparticle solution was made basic(pH � 9) with ammonia. The solution was kept in a refrigerator at 4 �C for 48 h priorto its characterization.

2.3. Characterization of nanoparticles

Optical property of the Cu nanorods was recorded on PerkinElmer Lambda 35UV–vis spectrophotometer whereas their size and shape was determined usingTEM on a Philips, Holland Technai 20 model operating at 200 kV. The samples forTEM were prepared by putting one drop of the suspension on standard carbon-coated copper grids and then dried under an IR lamp for 30 min. X-ray diffraction(XRD) of the Cu nanorods obtained by ultracentrifugation was determined usingPANalytical ‘X’PERT-PRO XRPD of Cu Ka radiation (k = 0.15406 nm) with scanningrate of 2�/min and 2h ranging from 10� to 80�. Cyclic voltammograms were carriedout on CH 600 C INSTRUMENT, using a Pt electrode as the working electrode, Pt rodas the counter electrode and Ag/AgCl as the reference electrode.

2.4. Maintenance of A549 cells

Human lung carcinoma cells (A549) obtained from National Centre for Cell Sci-ences, Pune, India, were seeded (1 � 105 cells/25 mm T Flask) and cultured in Dul-becco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS)and 1% antibiotic/anti-mycotic solution at 37 �C with 5% CO2

� (Thermo scientific,forma II water jacketed CO2 incubator). Cells were subsequently sub-cultured everythird day by trypsinization with trypsin phosphate versus glucose solution (TPVG).All the reagents were sterile filtered through 0.22 l filter (Laxbro Bio-Medical AidsPvt. Ltd.) prior to use for the experiment.

2.5. Cytotoxicity and Anti-proliferative assay

Mitochondrial MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide) reduction assay, as a index of cytotoxicity and anti-proliferative indexwas performed as mentioned earlier (Valodkar et al., 2011b). For cell viability assay,A549 cells (5.0 � 103 cells/well) were maintained in 96 well plates (Tarson IndiaPvt. Ltd.) for a period of 24 h in presence of Cu nanorods (10–100 lg/ml) or vehicle(Almond skin extract) while, for anti-proliferative assay, the treatment schedulewas 72 h (Allow the cells to complete cell cycle). At the end of incubation period,10 ll of MTT (5 mg/ml) was added to wells and the plates were incubated for 4 hat 37 �C. At the end of 4 h, culture media was discarded and all the wells werewashed with phosphate buffer saline (Himedia Pvt. Ltd., Mumbai, India). This wasfollowed by addition of 150 ll of dimethyl sulphoxide (DMSO) and incubated for30 min with constant shaking. Absorbance was read at 540 nm in ELX800 UniversalMicroplate Reader (Bio-Tek instruments, Inc., Winooski, VT).

2.6. Lipid peroxidation assay

A549 cells (1 � 105 cells/well) were maintained in six well plates as describedearlier for 24 h. Subsequently, cells were collected from the plate with a cell scraper(Tarson India Pvt. Ltd.) into 2 ml centrifuge tube. Lipid peroxidation was assayed inthe cell suspension using TBA–TCA–HCl reagent (Buege and Aust, 1978).

2.7. Measurement of intracellular reduced glutathione

Reduced glutathione estimation in the cell lysate was performed by the methodof Moron et al. 1979. A549 cells (1 � 105 cells/well) were maintained in six wellplates as described earlier for 24 h. At the end of the experimental period, cell lysatewas mixed with 25% of tri chloro acetic acid, and centrifuged at 3000 rpm for15 min. Supernatants were collected in micro centrifuge tubes and diluted up to1 ml with 0.2 M sodium phosphate buffer (pH 8.0) and 2.0 ml of 0.6 mM dithioni-trobenzoic acid was added and incubated at room temperature for 10 min. andabsorbance was read at 405 nm.

2.8. Measurement of intracellular ROS generation

A549 cells (0.5 � 105 cells/well) were grown on cover slips using 12 well cellculture plates for 16 h. At the end of incubation period, cells in cover slips wereincubated with 7.5 lM CM-H2DCFDA at 37 �C for 30 min in dark (Jadeja et al.,

2011). Cells were observed in a Leica DMRB florescence microscope. The intracellu-lar mean fluorescence intensity was quantified using the ImageJ 1.40 g software(National Institutes of Health, USA).

2.9. Morphological analysis of A549 cells treated with Cu nanorods

A549 cells (1.0 � 105 cells/well) were maintained in six well plates (Tarson In-dia Pvt. Ltd.) for a period of 24 h in presence of Cu nanorods (10–100 lg/ml) or vehi-cle. At the end of experimental period, cells were fixed in 4% paraformaldehyde for10 min, mounted in glycerin and examined under Leica DMIL inverted microscope(40X) and photographed (Valodkar et al., 2011b).

2.10. Measurement of mitochondrial membrane potential

The changes in mitochondrial membrane potential were measured using thefluorescent cationic dye Rhodamine 123 (rho123) as per Jadeja et al. 2011. A549cells (1.0 � 105 cells /well) were maintained in six well plates (Tarson India Pvt.Ltd.) for a period of 24 h as described earlier. The cells were then incubated with1 lM rho123 for 10 min at 37 �C. The fluorescence was determined at excitationand emission wavelengths of 485 and 530 nm, respectively using spectroflurometer(Jasco FP-6350) and expressed as florescence intensity units (FIU).

2.11. Annexin V-FITC/Propidium iodide staining for apoptosis

Annexin-V FITC/Propidium iodide double-staining assay was used to quantifyapoptosis, according to the manufacturer’s protocol (Sigma–Aldrich Ltd., USA).A549 cells (1.0 � 105 cells /well) were maintained in six well plates (Tarson IndiaPvt. Ltd.) for a period of 24 h in presence of Cu nanorods (10–100 lg/ml) or vehicle.Later cells from each well were centrifuged, washed with PBS and suspended in100 lL binding buffer, then 5 ll of Annexin V FITC Conjugate and 10 ll of Propidi-um Iodide solution was added to each cell suspension and incubated for 10 min atroom temperature in the dark. The samples were analyzed on flow cytometer(MoFlo™ Cytomation, Modular Flow Cytometer) and Cell Quest software.

2.12. Statistical analysis

Data was analyzed for statistical significance using one way analysis of variance(ANOVA) followed by Bonferroni’s multiple comparison test and results were ex-pressed as mean ± SEM using Graph Pad Prism version 3.0 for Windows, GraphPad Software, San Diego, California, USA.

3. Result and discussion

3.1. Formation and stability of Cu nanorods

Digital photographs of the almond extract and freshly preparedCu nanorods were taken at precursor concentration of 0.05 and0.1 M (Fig. 1A). The formation of Cu nanorods in the solution wasalso confirmed by change in color (pale yellow to wine red) andoptical spectroscopy wherein, the plasmon absorption maxima ap-peared at 569 nm (Supplementary data). The absorption peaks oflocalized surface plasmon resonance (SPR) were characteristic ofCu NPs, which could be predicted by the Mie resonance condition(Valodkar et al., 2010, 2011a).

3.2. Shape, size and purity of Cu nanorods

TEM images of the nanoparticle formulation showed self-assembled and highly mono dispersed rod-like nanostructures(Fig. 1B). A higher magnification revealed formation of Cu nanorods(200 nm length and 40 nm width). The XRD pattern further justi-fied that Cu nanorod formulation consisted of pure elemental cop-per (Supplementary data) and was free of oxide contamination.Bragg’s reflections representing b111N, b200N and b311N planesof fcc crystal structures due to metallic copper were observed at43.57 �, 50.71 �, and 74.49�. The pattern obtained matched withthe cubic phase of Cu (JCPDS 04-0836).

3.3. Electrochemical analysis of extract stabilized Cu nanorods

Cyclic voltammogram (CV) of the almond extract and Cunanorods was recorded at a scan rate of 0.03 V s�1 in the potential

(A)

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Fig. 1. (A) Digital image and (B) TEM of the Cu nanorods stabilized by aqueous almond extract, (C) Cyclic voltammogram of (a) almond extract and (b) extract stabilized Cunanorods.

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window between �1.0 and 1.0 V (Fig. 1C). An oxidation peak ofalmond extract appeared at the potential of about 0.123 V possiblydue to the redox activity of the extract. On the other hand, Cunanorod formulation recorded reduced peak of Cu ions. This isbecause almond skin extract is a mild reducing agent and henceis unable to induce reduction of Cu ions. The reduction peaks at�0.07 and 0.123 V correspond to the reduction of CuO ? Cu2Oand Cu2O ? Cu, respectively. Semi-empirical AM1 (Monagaset al., 2007) method showed the optimized structure of dimer ofa prodelphinidin present in the almond skin Fig. 2. The formationof a rod-like cavity in the dimer is assumed to be responsible forthe formation of nanorods.

3.4. Morphological evaluation of Cu nanorods treated A549 cells

The cytotoxicity effect following exposure of A549 cell line toCu nanorods was observed under phase contrast microscope. Adistinct alteration in cell shape and morphology was detectedfollowing exposure to Cu nanorods in a dose dependent manner(Fig. 3). Observations revealed that majority of the cells appearedto be rounded and shrunken when exposed to higher concentra-tions of Cu nanorods as compared to the control cells. Thesealterations in cellular morphology were further substantiated withmaximal population of floating cells (non-viable) observed inhigher doses of Cu nanorods.

Fig. 2. Optimized structure of prodelphinidin present in almond skins using semi-empirical AM1 method.

-10 0 10 20 30 40 50 60 70 80 90 100 1100

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nsns

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μg/ml Cu nanorods

% C

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ity % Viability

Fig. 4. Effect of Cu nanorod exposure to A549 cells on cell viability and anti-proliferative potential. Results are expressed as Mean ± SEM for n = 3 (replicates).Where, nsp > 0.05, ⁄p < 0.05, ⁄⁄p < 0.01 and ⁄⁄⁄p < 0.001 compared to 0 lg/ml Cunanorod.

M.C. Thounaojam et al. / Food and Chemical Toxicology 49 (2011) 2990–2996 2993

3.5. Cytotoxicity and anti-proliferative potential of Cu nanorods

MTT is a tetrazolium dye that undergoes reduction by the mito-chondrial enzymes to form a blue colored formazan. Only cellswith viable mitochondria retain the ability to carry out this reac-tion; therefore the color intensity is directly proportional to cellviability. Hence, it is a useful tool to detect cytotoxicity and anti-proliferative potential of various compounds/nano formulations.Observations recorded in MTT assay revealed that there was a dosedependent cytotoxicity in A549 cells exposed to Cu nanorods for24 h (Fig. 4). We have also evaluated cytotoxicity of Cu nanorods

Fig. 3. Phase contrast photomicrographs of A549 cells exposed to (a) untreated (almondnanorod, (e) 75 lg/ml Cu nanorod and (f) 100 lg/ml.

in 3T3L1 fibroblast cells and the same showed a dose dependenttoxicity. However, the extent of toxicity was significantly less ascompared to the observations recorded in A549 cells (Supplemen-tary data). Elevated lactate dehydrogenase (LDH) activity in the cellsupernatant is a popular method to assess the in vitro cytotoxicitypotential of a compound, extract or formulation. In the presentstudy, dose dependent (10–100 lg/ml) increase in activity levelsof LDH was recorded (Data not shown).

In another set of experiment, anti-proliferative potential ofA549 cells was assessed in presence of various doses of Cu nano-rods. Since, the average doubling time for A549 cells in culture is48 h it was thought pertinent to treat the cells with Cu nanorods

skin extract), (b) 10 lg/ml Cu nanorod, (c) 20 lg/ml Cu nanorod, (d) 50 lg/ml Cu

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Fig. 5. Effect of Cu nanorod exposure to A549 cells on reduced glutathione (GSH)and lipid peroxidation (LPO). Results are expressed as Mean ± SEM for n = 3(replicates). Where, nsp > 0.05, ⁄p < 0.05, ⁄⁄p < 0.01 and ⁄⁄⁄p < 0.001 compared to0 lg/ml Cu nanorod.

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Fig. 7. Effect of Cu nanorod exposure to A549 cells on mitochondrial membranepotential and ROS generation. Results are expressed as Mean ± SEM for n = 3(replicates). Where, nsp > 0.05, ⁄p < 0.05, ⁄⁄p < 0.01 and ⁄⁄⁄p < 0.001 compared to0 lg/ml Cu nanorod.

2994 M.C. Thounaojam et al. / Food and Chemical Toxicology 49 (2011) 2990–2996

for 72 h. Significant decrement in the cell viability was recordedafter 72 h exposure to Cu nanorods (Fig. 4) indicating at theiranti-proliferative potential.

3.6. Cellular lipid peroxidation and antioxidant status

Various studies have reported the role of oxidative stress innanoparticle toxicity (Li et al., 2008; Heng et al., 2010; Pandaet al., 2011). Interaction of nanoparticles with mammalian cellscan induce oxidative stress by favoring cellular ROS productionin large quantities (Sanpui et al., 2011). These free radicals mayattack polyunsaturated fatty acids (PUFAs) of plasma membraneforming peroxyl radicals, which subsequently attack adjacent fattyacids causing a chain reaction of lipid peroxidation. Also, increasein the levels of lipid peroxides such as thiobarbituric acid reactivesubstances (TBARS) is the initial phase of oxidative damage. This iscoupled with depletion of free radical scavenging non-enzymatic

Fig. 6. Florescence photomicrographs of DCF-DA stained A549 cells exposed to (a) vehicleml Cu nanorod, (e) 75 lg/ml Cu nanorod and (f) 100 lg/ml.

antioxidant such as reduced glutathione (GSH) (Shukla et al.,2011). In the present study, the depletion in GSH levels along withincreased LPO (measured as TBARS) has been observed in Cu nano-rods treated cells compare to that of untreated cells (Fig. 5). It isinferable from these observations that Cu nanorods manifests adose dependent cytotoxicity by gross damages to the cell mem-brane, depletion of antioxidant reserves and by elevating produc-tion of intracellular free radicals.

3.7. Cellular oxidative stress

Owing to the results obtained in the biochemical investigation,a status check of intracellular oxidative stress was done using DCF-DA (20, 70-dichlorodihydrofluorescein diacetate) fluorescence dye.It was evident that, there was prominent green florescence ob-served in the high doses (10–100 lg/ml) of Cu nanorods than thecontrol cells (Fig. 6). This is due to the elevated production of

(almond skin extract), (b) 10 lg/ml Cu nanorod, (c) 20 lg/ml Cu nanorod, (d) 50 lg/

Fig. 8. Effect of Cu nanorod exposure to A549 cells on apoptosis evaluated by Annexine V-FITC/propodium iodide staining evaluation by flow cytometry. Where, live cells(FITC�PI), early apoptotic (FITC + PI�) and late apoptotic or necrotic cells (FITC + PI+).

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hydrogen peroxide and peroxyl radicals that bring about intracel-lular oxidation of DCF-DA dye. This assay provided conclusiveevidence of elevated levels of intracellular oxidative stress follow-ing Cu nanorods treatment. Results obtained herein are similar toplant latex capped Cu nanoparticles, wherein a strong DCF-DApositive response was elicited in A549 cells (Valodkar et al.,2011b).

3.8. Mitochondrial function

Mitochondria are the potential sites of ROS production in a celland subsequent oxidative stress following toxic insults (Hiuraet al., 1999, 2000). During oxidative phosphorylation, some elec-trons occasionally escape from respiratory chain and form an ex-tremely reactive superoxide anion radical (O2), which getsconverted to hydrogen peroxide (H2O2) and further undergoes par-tial reduction to a more deleterious hydroxyl radical (OH.) (Boon-stra and Post, 2004). Recently, different research groups havereported that nanoparticles of various dimensions and chemicalcompositions get localized in mitochondria and induce majorstructural damages by causing ROS production. This process causesdisruption of the electron transport chain, low ATP yield and finallyinduces apoptosis (Yacobi et al., 2007; Costa et al., 2010). In such acase, cell death due to apoptosis has been attributed to decrementin mitochondrial membrane potential. In the present study, a fluo-rescence dye rhodamine 123 (RH-123) was used to assess mito-chondrial membrane potential (Jadeja et al., 2011). Resultsindicated that treatment of A549 cells with Cu nanorods causedsignificant decrement in mitochondrial membrane potential caus-ing production of ROS and membrane damage (Fig. 7). Disruptionof mitochondrial membrane potential by Cu nanorods treated cellsis attributable to ROS induced increased cellular oxidative stressand lowered antioxidant levels.

3.9. Induction of apoptosis

Apoptosis is a process of cell death characterized by variousmorphological and biochemical alteration leading to cell disrup-tion and formation of apoptotic bodies (AshaRani et al., 2009). Ina critical stage of apoptosis, the phospholipid bilayer of plasmamembrane breaks up and exposes phosphatidylserine (PS) on theirouter side. Exposure of PS on cell surface is a hallmark of anapoptotic cell and is useful in quantifying apoptotis (AshaRani

et al., 2009). Cu nanorods mediated apoptosis can be measuredusing annexin-V stain because of the ability of this stain to bindto negatively charged PS on cell membrane. Also, a conjugate ofFITC and Annexin V is used to quantify apoptotic cells on a sin-gle-cell basis by flow cytometry. Double staining of cells withFITC–Annexin V and propidium iodide permits discrimination oflive cells (FITC�PI�), early apoptotic (FITC + PI�) and late apopto-tic or necrotic cells (FITC + PI+). Results revealed 20% of early apop-totic and 8% of late apoptotic cells at 100 lg/ml dose of Cunanorods (Fig. 8). It is evident from these results that Cu nanorodscaused death of A549 cells by inducing apoptosis. But a detailedstudy is required to decipher the mechanism involved in Cu nano-rods mediated induction and progression of apoptotic pathway inA549 cells.

4. Conclusions

This study is the first report on synthesis of almond extractcapped Cu nanorods, its toxicity assessment and possible antican-cer property. It can be concluded from the present study that Cunanorods have the ability to induce apoptotic cell death by elevat-ing oxidative stress, depleting cellular antioxidants and inducingmitochondrial dysfunction. The study reports potential applicationof almond skin extract capped Cu nanorod formulation as a possi-ble anticancer agent that warrants further study.

Conflict of Interest

The authors declare that there are no conflicts of interest.

Acknowledgement

The authors are grateful to the University Grants Commission,New Delhi for financial assistance. Special thanks to Dr. Geeta. S.Padate, Head, Department of Zoology for her encouragement andnecessary permission.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.fct.2011.07.055.

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