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EnhancedantibacterialactivityofIronOxideMagneticNanoparticlestreatedwithArgemoneMexicana,L.leafextract:anInvitrostudyARTICLEinMATERIALSRESEARCHBULLETINSEPTEMBER2013ImpactFactor:1.97DOI:10.1016/j.materresbull.2013.05.059

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ArokiyarajSelvarajSeoulNationalUniversity26PUBLICATIONS88CITATIONS

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SaravananMuthupandianMekelleUniversity31PUBLICATIONS127CITATIONS

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UdayaPrakashNyayiruKannaianMARINALABS84PUBLICATIONS204CITATIONS

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VijayakumarBadathalaVeltechHighTechDr.RangarajanDr.Sakunt22PUBLICATIONS38CITATIONS

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Availablefrom:SaravananMuthupandianRetrievedon:18June2015

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Materials Research Bulletin 48 (2013) 33233327

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Contents lists available at SciVerse ScienceDirect

Materials Rese

l s e1. Introduction

Antibiotic resistance, a well-known phenomenon in natureassumes signicant public health importance when it getsamplied many folds due to human misuse and neglect [1]. Ithas become a serious public health concern with economic andsocial implications throughout the world [2]. To overcome this, anewer area of research, i.e. nanoparticles in controlling bacterialgrowth is carried out by many authors. The antimicrobial activitiesof aluminum oxide, silver nanoparticles, gold nanoparticles andiron oxide nanoparticles have been previously reported [35]. Dueto their antibacterial activities, metallic nanoparticles represent aneffective solution for overcoming bacterial resistance [6].

Magnetic nanoparticles have applications in biomedicine due totheir controllable size of less than 100 nm which give them theability to attach with microbial cells. Magnetic biomaterialsprovide the ability to be directed and concentrated within thetarget tissue by means of external magnetic eld and be removedonce therapy was completed [7]. The drug loaded magneticnanoparticles helps in the controlled release of drug which reduceside effects due to their lower dosage and minimize or prevent

drug degradation by using pathways other than gastrointestinaland have several advantages such as, small particle size, largesurface area, magnetic response, bio-compatibility and non-toxicity [8].

The synergistic impact of nanoparticles as antimicrobials isalready studied and showed enhanced result. The synergisticimpact of silver nanoparticles with b-lactam antibiotics as anantibacterial agent [9], silver nanoparticles and antibiotics [10] asan effective antibacterial agent, antibacterial effect of iron oxideand titania [11], magnetic nanoparticle and microwave exposure[12], magnetic nanoparticle with glycol chitosan [13] and magneticnanoparticles and cephalosporins [14] were reported.

Similarly, the antiseptic qualities of medicinal plants have beenlong recognized. A major part of total population in developingcountries still uses folklore medicines obtained from plantresources [15]. Biologically active compounds which are presentin plants have always been of great interest. Recently there hasbeen a revival of interest in herbal medications due to a perceptionthat there are lower incidences of adverse reactions to plantpreparations compared to synthetic pharmaceuticals [16].

Argemone mexicana L. (Papaveraceae), commonly known asMexican prickly poppy, a native of Tropical America is introducedand naturalized in India and generally occurs as wasteland weed inalmost every part of the country. It is a prickly, glabrous, branchingherb with yellow juice and showy yellow owers. The plant

Available online 24 May 2013

Keywords:

A. Magnetic materials

B. Chemical synthesis

C. Electron microscopy

C. X-ray diffraction

particles of uniform size ranged between 10 and 30 nm and the crystallites were determined through

XRD. The peaks in XRD pattern are in good agreement with that of face-centered cubic form of iron oxide

nanoparticles. FT-IR spectroscopy conrmed the attachment of bioactive molecules of plant on the IO-

NPs surfaces. Furthermore, the antibacterial efcacy of IO-NPs, plant extract and IO-NPs treated with

plant extract was screened against Escherichia coli MTCC 443, Proteus mirabilis MTCC 425 and Bacillus

subtilis MTCC 441. The results showed a noteworthy inhibition on P. mirabilis and E. coli with IO-NPs

treated plant extract. This outcome may pave a way for using the magnetic nanoparticles as a drug

carrier system to cure bacterial diseases.

2013 Elsevier Ltd. All rights reserved.

* Corresponding author. Tel.: +91 9443077097; fax: +91 044 27453903.

E-mail address: bioinfosaran@gmail.com (M. Saravanan).

0025-5408/$ see front matter 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.materresbull.2013.05.059Enhanced antibacterial activity of iron oArgemone mexicana L. leaf extract: An i

S. Arokiyaraj a, M. Saravanan b,*, N.K. Udaya PrakasaR&D, Vel Tech High Tech Dr.Rangarajan Dr.Sakunthala Engineering College, Avadi, ChbDepartment of Biotechnology, SRM University, Kattankulathur, Chennai 603203, IndicDepartment of Bio-Environmental Chemistry, Chungnam National University, DaejeondDepartment of Advanced Zoology and Biotechnology, Loyola College, Chennai 600034

A R T I C L E I N F O

Article history:

Received 8 November 2012

Received in revised form 30 April 2013

Accepted 7 May 2013

A B S T R A C T

The present study intende

characterization and eval

extract. The formation of

spectroscopy. The morph

jo u rn al h om ep age: ww w.ede magnetic nanoparticles treated withvitro study

, M. Valan Arasu c, B. Vijayakumar a, S. Vincent d

ai 600062, India

764, South Korea

ia

r the chemical synthesis of iron oxide nanoparticles (IO-NPs) followed by

on of antibacterial activity after treating with Argemone mexicana L. leaf

Ps was conrmed by the colour change and further examined by UVvis

y was characterized by using SEM and TEM, which showed spherical

arch Bulletin

vier .c o m/lo c ate /mat res b u

S. Arokiyaraj et al. / Materials Research Bulletin 48 (2013) 332333273324contains alkaloids as berberine, protopine, sarguinarine, optisine,chelerytherine and the seed oil contains myristic, palmitic, oleic,and linoleic acids. In Ayurveda, the plant is used as a diuretic andpurgative. Roots, leaves, seeds and yellow juice are used to cureleprosy, skin-diseases, inammations and bilious fevers [17].

The objective of this study is to synthesize iron oxide magneticnanoparticles (magnetite) by chemical co-precipitation methodand to characterize using UVvisible spectrophotometer, XRD, FT-IR, SEM, TEM and evaluation of antibacterial activity after treatingwith A. mexicana L. leaf extract. In the present study a newapproach has been taken to study the antibacterial property of A.mexicana ethanolic extracts treated with IO-NPs by disc diffusionmethod against Escherichia coli, Proteus mirabilis and Bacillussubtilis. To the best of our knowledge, the synergistic impact ofmagnetic nanoparticles of iron oxide treated with any of the plantextract as an antibacterial agent is not yet reported so far in theearlier literature and hence this study is conducted to ndthe antibacterial activity of magnetic nanoparticles.

2. Materials and methods

2.1. Plant material collection and preparation of the extract

A. mexicana was collected from Chengelpet district belonging tothe state of Tamil Nadu, India and it was acknowledged by one ofthe authors (N.K. Udaya Prakash, Botanist) and deposited in theherbarium collections, Department of Biotechnology, Veltech HighTech Dr.RR Dr.SR Engineering College, Chennai, India. The leavesthat are collected were investigated for pathogenic infections andhealthy leaves were selected after the examination. Thus theselected leaves were washed in running tap water for removingthe surface pollutants. The washed leaves were dried at roomtemperature for 23 days under dark condition. After drying theleaves were powdered using electric blender. The extract wasprepared using Soxhlet apparatus with ethanol as a solvent. 15 g ofpowder was made into three bags containing 5 g each, were used inSoxhlet apparatus to form extract. Approximately 150 mL ofethanol was used for making extract. The plant extract (PE), thusformed was ltered and concentrated using rotary evaporator. Theconcentrated extract was stored in refrigerator for further use.The stored product was reconstituted again by using the samesolvent for required concentration.

2.2. Synthesis of iron oxide nanoparticles

The iron oxide nanoparticles (magnetite) were synthesized byusing co-precipitation method in alkaline media. Solutions of Fe2+

and Fe3+ with the molar ratio of 0.0125 M and 0.025 M in deionizedwater were stirred and 29.5% of ammonium hydroxide was addedat a rate of 0.2 mL/min to adjust the pH at 10 in a non-oxidizingenvironment. The black precipitate, thus obtained was washedthrice with deionized water and twice with ethanol. The blackcolored nanoparticles were separated by magnetic decantation toform magnetite. The magnetites formed were washed 3 times withdeionized water and 2 times with ethanol, and dried at roomtemperature.

2.3. Characterization methods

The synthesized nanoparticles were analyzed using ultravioletvisible spectroscopy (UVvis) (Perkin-Elmer Lambda 2Spectrophotometer) in 300800 nm wave length range. The XRDpatterns of the synthesized magnetite nanoparticles were recordedby X-ray diffractometer (Bruker D8 Advance, Bruker AXS,Germany) equipped with Ni lter and CuKa (l = 1.54056 A)radiation with scan range of 1070 A at 40 kV and 40 mA. Theparticle size of IO-NPs was calculated using full width at halfmaximum of face-centered cubic (3 1 1) using DebyeScherrerequation (D = Kl/b cos u) from the highest intensity of XRD pattern[18]. Fourier transform infrared (FT-IR) spectrometer was used totest the physicochemical interaction between leaf extract of A.mexicana and IO-NPS. FT-IR spectra were recorded in the range4000400 cm1 with Thermo Nicolet Avatar 370 FT-IR spectro-photometer, using KBr pellet method. The morphology of thesynthesized IO-NPs was determined by scanning electron micro-scope (JSM-6390LV, JEOL, Japan) resolution at 300 nm at anaccelerating voltage of 15 kV. A thin lm was prepared by dropcoating synthesized IO-NPs on to carbon coated copper SEM gridsand then the extra sample solution was removed using a blottingpaper. The thin lm on the SEM grid was allowed to stand for 5 minto dry prior to measurement. The ne powder of IO-NPs has beendispersed in ethanol on carbon coated copper grid and the size andshapes of the IO-NPs were obtained with TEM (TEM-JEOL 1200EX,Japan) at an accelerating voltage of 120 kV.

2.4. Microorganisms

Three bacterial strains chosen for studies of antibacterialassay were two Gram positive bacteria such as E. coli MTCC 443,P. mirabilis MTCC 425 and one Gram negative bacteria B. subtilisMTCC 441 consigned from Institute of Microbial Technology(IMTECH), Chandigarh, India. Bacterial cultures were prepared inMueller Hinton Broth (Hi-Media, Pvt. Ltd., Mumbai, India) andmaintained for 24 h at 37 8C. The cell suspensions were dilutedwith sterile MHB to provide an initial cell count of about106 CFU/ml.

2.5. Treatment of IO-NPs with ethanolic extract of A. mexicana

Plant extract of A. mexicana and synthesized particles (IO-NPs)were taken in the ratio of 1:1 and dissolved in 1 mL of ethanol. Thetreated mixture was incubated in BOD incubator shaker at 45 8C for12 h. After incubation this mixture was used for antibacterial assayto nd the bactericidal activity.

2.6. Antibacterial assay of IO-NPs with ethanolic extract of A.mexicana

The antibacterial efcacy was assayed by the standard KirbyBauer disc diffusion method against the bacterial pathogen [19].The bacterial suspension (106 CFU/ml) swabbed on the MullerHinton Agar (MHA) plates using sterile cotton swab. The sterileWhatman No. 1 paper disc of 6 mm dimension was impregnatedwith the three solution components such as IO-NPs alone,ethanolic extract of A. mexicana and IO-NPs treated with A.mexicana in three different concentrations i.e. 12.5 mg/disc, 25 mg/disc and 50 mg/disc respectively. The discs with streptomycin(Himedia, 30 mg/disc) was placed on the MHA plates maintained ascontrol. The disc were gently pressed and incubated in invertedposition for 24 h at 37 8C. After the incubation period, thesusceptibility of the test organisms was determined by measuringthe diameter of the zone of inhibition using Himedia zone scale andthe obtained results were tabulated for evaluation. Theseexperiments were performed in triplicates for achieving theoptimum results.

3. Results and discussion

3.1. Synthesis and characterization of IO-NPs

UVvis absorption spectrum measures the wavelength of thelight that the nanoparticles absorb. The absorption spectrum of

Fig. 1. UVvisible spectral analysis of synthesized IO-NPs and the peak notedaround 315 nm.

Fig. 2. (a) FT-IR spectroscopy analysis of IO-NPs and Argemone mexicana leaf extractand (b) FT-IR spectroscopy analysis of IO-NPs treated with Argemone mexicana.

Fig. 3. XRD pattern of iron oxide magnetic nanoparticles.

S. Arokiyaraj et al. / Materials Research Bulletin 48 (2013) 33233327 3325the synthesized IO-NPs recorded the peak at 315 nm (Fig. 1).Parallel results were observed by Cornell and Schwertmann, 2003[20]. The FT-IR spectrum of the magnetite showed three distinctbands at 566 cm1, 1635 cm1 and 3426 cm1 (Fig. 2a). The FeOstretching was established by vibration at 566 cm1, OHstretching vibration at 3426 cm1 and OH distorted vibrationat 1635 cm1 are due to surface adsorbed water. Similar resultswere reported by Yang et al. [21]. FT-IR spectrum of plant extractshows HCH stretching and bending vibrations at 29172849 cm1 and 14661400 cm1 respectively (Fig. 2a) due to n-alkane [22]. The FT-IR spectrum of Fe3O4 nanoparticles aftertreatment with the plant extract also showed HCH stretchingvibrations at 2972 and 2882 cm1 and HCH bending vibrationsat 1450 cm1 (Fig. 2b). From the FT-IR data, it is clear that thebioactive molecules present in the leaves extract of A. mexicanainteracted with the synthesized Fe3O4 nanoparticles.

The XRD pattern of the synthesized magnetite displayed sixcharacteristic peaks (2u = 30.308, 35.718, 43.318, 53.318, 57.618 and62.818), marked by their indices [(2 2 0), (3 1 1), (4 0 0), (4 2 2),(5 1 1), and (4 4 0)] are quite identical to the peaks of Fe3O4 crystalwith the cubic spinal structure (Fig. 3). The results are inagreement with the standard XRD pattern of Fe3O4 (JCPDS 75-0033). From the Scherrers formula the average crystallite size ofthe nanoparticles was found to be 11 nm. Similar results wereobtained by Gerko [23].

Fig. 4 shows the micrographs of scanning electron microscope

of the synthesized magnetite, the morphology of the particles was

Fig. 4. SEM image of synthesized IO-NPs.

observed to be spherical. The size and morphology of thesynthesized magnetite were also characterized by transmissionelectron microscopy (Fig. 5). The magnetites were found to havesmooth surface with well dispersed particles and the size rangedbetween 10 and 30 nm. The results are in agreement withobservation from SEM images [24].

3.2. Antibacterial activity

The antibacterial assay of IO-NPs, ethanolic extract of A.mexicana and combination effect of IO-NPs with A. mexicanaagainst the bacterial pathogens was identied (Table 1 and Fig. 6).

Fig. 5. TEM image of iron oxide nanoparticles.

Table 1Antibacterial activity of IO-NPS, plant extract and IO-NPs treated with Argemone mexic

Bacteria IO-NPs (mg/disc) Plant extract (mg/disc)

12.5 25 50 12.5 25

E. coli 7 7 8 8

P. mirabilis 7 7 8 9 10

B. subtilis 7 7 8 8

Values are expressed in mean of three independent experiments, () no zone of inhibi

S. Arokiyaraj et al. / Materials Research Bulletin 48 (2013) 332333273326Fig. 6. The antibacterial assay of (a) IO-NPs alone, (b) ethanolic extract of Argemonemexicana and (c) combination effect of IO-NPs with Argemone mexicana against the

bacterial pathogens.The mean zone diameter of different concentration level of IO-NPswas also determined. Based on the observations, it can beperceived that very lesser amount of activity against the testedbacterial strains was reported. Similar antibacterial effect of ironoxide nanoparticles was reported by Tran et al. [25]. Thebactericidal effect of IO-NPs may be due to their smaller size.Lee et al. [26] reported that the inactivation of E. coli by zero-valentiron nanoparticles could be because of the penetration of the smallparticles (sizes ranging from 10 to 80 nm) into E. coli membranes,leading to oxidative stress and causes disruption of the cellmembrane.

The ethanolic extract of A. mexicana showed moderate activity,and this inhibitory activity is may be due to the presence ofphytochemical alkaloids. This phytochemical is known to be aDNA intercalator and an inhibitor of DNA synthesis throughtopoisomerase inhibition [27,28]. Whereas the IO-NPs treatedwith ethnaolic extract of A. mexicana showed higher activityagainst the E. coli and P. mirablis which is reected in the meanzone values of 13 mm and 18 mm respectively. The recordedactivity is higher than that of the standard streptomycin (14 mm).In the case of B. subtilis the activity was found to be less andmaximum inhibition was observed at 50 mg/disc. Taylor et al. [29]have also reported similar concentration dependent bacteriainhibition. Mechanism: the antibacterial drugs and antibioticsdevelop oxidative stress by generating reactive oxygen species[30]. Reactive oxygen species (ROS) can cause damage to proteinsand DNA in bacteria [31]. This bactericidal property has beenevidenced by Park et al. [32] from silver metals. So, the inhibitoryproperty of IO-NPs treated with ethanolic extract of A. mexicanacould be due to the synergistic effect of the phytochemicals andgeneration of ROS by IO-NPs.

4. Conclusion

The synthesized IO-NPs treated with leaf extract of A. mexicanaL. proved to have outstanding antimicrobial efcacy against thebacterial pathogens. The biological approach on IO-NPs treatedwith A. mexicana L. leaf extract material is the most rapid and eco-friendly method and it has a wide scope in opting as an excellentdrug delivery system. The present study proved that theimmobilized nanomaterials of magnetite can effectively improvethe drug loading and the antibacterial efciency against themicrobial pathogens. It is conclude that further studies on this eldare of enormous importance in developing eco-friendly bio-

ana.

IO-NPs + plant extract (mg/disc) Streptomycin (mg/disc)

50 12.5 25 50 30

10 11 12 13 14

12 16 17 18 14

9 7 8 10 9

tion.nanomaterial and is highly recommended for biomedicines.

Conict of interest statement

The authors have declared no conict of interest.

Acknowledgement

The authors express their gratitude to Sophisticated AnalyticalInstrumentation facility, Indian Institute of Technology, Chennai600 036, India for the characterization study.

References

[1] H.C. Neu, Science 257 (1992) 10641073.[2] A. Kapil, Indian J. Med. Res. 121 (2005) 8391.[3] J.S. Kim, E. Kuk, Y.K. Nam, K. Jong-Ho, S.J. Park, L. Hu, Nanomed. Nanotechnol. Biol.

Med. 3 (2007) 95101.[4] W.Y. Chen, L. Ju-Yu, C. Wei-Jen, L. Liyang, D. Eric Wei-Guang, C. Yu-Chie, Nano-

medicine 5 (2010) 755764.[5] M. Valodkar, S. Modi, A. Pal, S. Thakore, Mater. Res. Bull. 46 (2011) 384389.[6] M. Saravanan, A. Nanda, Colloids Surf. B: Biointerfaces 77 (2010) 214218.[7] T.K. Indira, P.K. Lakshmi, Int. J. Pharm. Sci. Nanotechnol. 3 (2010) 10351042.[8] Y. Zheng, Y. Cheng, F. Bao, Y.S. Wang, Mater. Res. Bull. 41 (2006) 525529.[9] P. Li, L. Juan, W. Changzhu, W. Qing sheng, L. Jian, Nanotechnology 16 (2005)

19121917.[10] A.R. Shahverdi, A. Fakhimi, H.R. Shahverdi, S. Minaian, Nanomed. Nanotechnol.

Biol. Med. 3 (2007) 168171.[11] W.Y. Chen, L. Ju-Yu, C. Wei-Jen, L. Liyang, E.W. Guang Diau, Y.C. Chen, Nanome-

dicine 5 (2010) 755764.[12] P. Antoniea, C. Dorina, N. Claudia, T.F. Mihai, Afr. J. Microbiol. Res. 6 (2012) 1054

1060.[13] B.S. Inbaraj, T.Y. Tsai, B.H. Chen, Sci. Technol. Adv. Mater. 13 (2012).[14] D.E. Mihaiescu, A.M. Grumezescu, A.S. Rumezescu, D.E. Buteica, P.C. Mogosanu,

O.M. Balaure, Dig. J. Nanomater. Biostruct. 7 (2012) 253260.[15] D.S. Fabricant, N.R. Farnsworth, Environ. Health Prospect. 109 (2001) 6975.[16] C.M. Chiariandy, C.E. Seaforth, R.H. Phelps, G.V. Pollard, B.P. Khambay, J. Ethno-

pharmacol. 64 (1999) 265270.

[17] R.N. Chopra, S.L. Nayar, I.C. Chopra, Glossary of Indian Medicinal Plants, Council ofScientic and Industrial Research, New Delhi, 1986.

[18] Y.S. Kim, Y.H. Kim, J. Magn. Magn. Mater. 267 (2003) 105110.[19] A.W. Bauer, W.M.M. Kirby, M. Sherries, Am. J. Clin. Pathol. 45 (1966) 493496.[20] R.M. Cornell, U. Schwertmann, The Iron Oxides: Structure, Properties, Reactions,

Occurrences and Uses, 2nd ed., Wiley-VCH, Weinheim, 2003.[21] J. Yang, S.B. Park, H.G. Yoon, Y.M. Huhd, S. Haam, Int. J. Pharm. 324 (2006)

185190.[22] I. Bhattacharjee, A. Ghosh, N. Chowdhury, S.K. Chatterjee, G. Chandra, S. Laskar,

Z. Naturforsch. C 65 (2010) 533536.[23] O. Gerko, J. SolGel Sci. Technol. 37 (2006) 161164.[24] L. Stanciu, Y.H. Won, M. Ganesana, S. Andreescu, Sensors 9 (2009) 29762999.[25] N. Tran, A. Mir, D. Mallik, A. Sinha, S. Nayar, T.J. Webster, Int. J. Nanomed. 5 (2010)

277283.[26] C. Lee, J.Y. Kim, W.I. Lee, K.L. Nelson, J. Yoon, D.L. Sedlak, Environ. Technol. 42

(2008) 49274933.[27] L. Dassonneville, K. Bonjean, M.C. Pauw-Gillet, P. Colson, C. Houssier, J. Quetin-

Leclercq, L. Angenot, C. Bailly, Biochemistry 38 (1999) 77197726.[28] L. Guittat, P. Alberti, F. Rosu, S. Van Miert, E. Thetiot, L. Pieters, V. Gabelica, E. De

Pauw, A. Ottaviani, J.F. Roiu, J.L. Mergny, Biochemistry 85 (2003) 535541.[29] J.L.S. Taylor, T. Rabe, L.J. McGaw, A.K. Jager, J. Van Staden, Plant Growth Regul.

(2001) 2337.[30] M.A. Kohanski, M.A. DePristo, J.J. Collins, Mol. Cell 37 (2010) 311320.[31] H. Sies, Exp. Physiol. 82 (1997) 291295.[32] H. Park, J. Kim, J. Lee, J. Hahn, M. Gu, J. Yoon, Water Res. 43 (2009) 1027

1032.

S. Arokiyaraj et al. / Materials Research Bulletin 48 (2013) 33233327 3327

Enhanced antibacterial activity of iron oxide magnetic nanoparticles treated with Argemone mexicana L. leaf extract: An in vitro studyIntroductionMaterials and methodsPlant material collection and preparation of the extractSynthesis of iron oxide nanoparticlesCharacterization methodsMicroorganismsTreatment of IO-NPs with ethanolic extract of A. mexicanaAntibacterial assay of IO-NPs with ethanolic extract of A. mexicana

Results and discussionSynthesis and characterization of IO-NPsAntibacterial activity

ConclusionConflict of interest statementAcknowledgementReferences