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Colloids and Surfaces B: Biointerfaces 76 (2010) 50–56

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces

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ynthesis of silver nanoparticles using Acalypha indica leaf extracts and itsntibacterial activity against water borne pathogens

. Krishnaraj ∗, E.G. Jagan, S. Rajasekar, P. Selvakumar, P.T. Kalaichelvan, N. Mohan ∗∗

entre for Advanced Studies in Botany, University of Madras, Guindy campus, Chennai-600025, India

r t i c l e i n f o

rticle history:eceived 29 August 2009eceived in revised form 4 October 2009ccepted 7 October 2009vailable online 14 October 2009

a b s t r a c t

In the present study, biosynthesis of silver nanoparticles and its activity on water borne bacterialpathogens were investigated. Silver nanoparticles were rapidly synthesized using leaf extract of Aca-lypha indica and the formation of nanoparticles was observed within 30 min. The results recorded fromUV–vis spectrum, scanning electron microscopy (SEM), X-ray diffraction (XRD) and energy dispersive

eywords:calypha indicailver nanoparticlesIC

spectroscopy (EDS) support the biosynthesis and characterization of silver nanoparticles. From high-resolution transmission electron microscopy (HRTEM) analysis, the size of the silver nanoparticles wasmeasured 20–30 nm. Further, the antibacterial activity of synthesized silver nanoparticles showed effec-tive inhibitory activity against water borne pathogens Viz., Escherichia coli and Vibrio cholerae. Silvernanoparticles 10 �g/ml were recorded as the minimal inhibitory concentration (MIC) against E. coli and V.

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. Introduction

The development of reliable green process for the synthesis ofilver nanoparticles is an important aspect of current nanotech-ology research. Nanomaterials such as Ag, Au, Pt and Pd haveeen synthesized by different methods, including hard template1], using bacteria [2], fungi [3] and plants [4]. Among these, sil-er nanoparticles play a significant role in the field of biology andedicine due to its attractive physiochemical properties. Klabunde

t al. demonstrated that the highly reactive metal oxide nanopar-icles exhibit excellent bactericidal action against Gram-positivend Gram-negative bacteria [5]. The strong toxicity of silver againstide range of microorganisms is well known and silver nanopar-

icles have been recently shown to be a promising antimicrobialaterial. Sondi et al. studied the antimicrobial activity of silver

anoparticles against Escherichia coli as a model of Gram-negativeacteria [6].

Interdisciplinary research has widened the horizons of materialesearch, drawing new inspirations from biological systems. The

owering environmental concerns had triggered the researchers toevice novel methods of synthesizing the nanomaterials in biolog-

cal systems such as bacteria, fungi and plants, termed as “greenhemistry” approaches. Biosynthesis of silver nanoparticles using

∗ Corresponding author. Tel.: +91 9840528499; fax: +91 044 22352494.∗∗ Corresponding author. Tel.: +91 9840097658; fax: +91 044 22352494.

E-mail addresses: krishnarajbio@gmail.com (C. Krishnaraj),ohannatarajan@hotmail.com (N. Mohan).

927-7765/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfb.2009.10.008

ne permeability and respiration of the silver nanoparticle treated bacterialtivity of silver nanoparticles.

© 2009 Elsevier B.V. All rights reserved.

bacteria [7–9], fungi [10–12], yeast [13] and plants [14–16] werewell documented. However, exploration of the plant systems as thepotential nanofactories, has heightened interest in the biologicalsynthesis of nanoparticles. Sastry et al. reported the biosynthe-sis of nanoparticles using plant leaf extracts and their potentialapplication. They studied bioreduction of chloraurate ions andsilver ions by extracts of geranium [17] and neem leaf [18]. Fur-ther, synthesis of gold nanotriangles and silver nanoparticles usingAloe vera plant extracts was reported [19]. Most of the aboveresearch on the synthesis of silver or gold nanoparticles utilizingplant extracts employed broths resulting from boiling fresh plantleaves. Whereas, Huang et al. exploited the synthesis of silver andgold nanoparticles using the sundried Cinnamomum camphora leafextract [20]. Acalypha indica (Euphorbiaceae), a traditional medic-inal plant of South India, has the source of bio-reductant andstabilizers. The present study was aimed to rapid synthesis of silvernanoparticles using aqueous leaves extract of A. indica and evalu-ates its antibacterial activity against water borne pathogens such asEscherichia coli and Vibrio cholerae. In addition, respiratory charac-teristics and membrane dynamics of the cells were studied to vali-date the antimicrobial activity of synthesized silver nanoparticles.

2. Experimental

2.1. Materials

The healthy leaves of A. indica were collected from campusof University of Madras, India. AgNO3, MTT (methyl thiozolyldiphenyl-tetrazolium bromide) were purchased from Himedia Lab-

C. Krishnaraj et al. / Colloids and Surfaces B: Biointerfaces 76 (2010) 50–56 51

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microtitre plate [21]. The mean of live cells of E. coli and V. choleraewas recorded using ELISA reader (Emax precision microplatereader). The MIC was determined based on different concentra-tions, where there was no increase in the OD595 and was zero.

Fig. 1. Aqueous solution of 10−3 M AgNO3 with A. indica leaf extracts (a)

ratories Pvt. Ltd., Mumbai, India. The bacterial cultures of E. coliMTCC-443) and V. cholerae (MTCC-3904) were obtained from

icrobial Type Culture Collection, Chandigarh, India.

.2. Preparation of plant extract

Aqueous extract of A. indica was prepared using freshly col-ected leaves (10 g). They were surface cleaned with running tap

ater, followed by distilled water and boiled with 100 ml of dis-illed water at 60 ◦C for 5 min. This extract was filtered throughylon mesh, followed by Millipore filter (0.45 �m) and used for

urther experiments.

.3. Synthesis of silver nanoparticles

For synthesis of silver nanoparticles, the Erlenmeyer flask con-aining 100 ml of AgNO3 (1 mM) was reacted with 12 ml of thequeous extract of A. indica. This setup was incubated in dark (toinimize the photoactivation of silver nitrate), at 37 ◦C under static

ondition. A control setup was also maintained without A. indicaxtract.

.4. Characterization of silver nanoparticles

Synthesized silver nanoparticles was confirmed by sampling theeaction mixture at regular intervals and the absorption maximaas scanned by UV–vis spectra, at the wavelength of 200–700 nm ineckman-DU 20 spectrophotometer. Further, the reaction mixtureas subjected to centrifugation at 75,000 × g for 30 min, resultingellet was dissolved in deionized water and filtered through Mil-

ipore filter (0.45 �m). An aliquot of this filtrate containing silveranoparticles was used for SEM, HRTEM, XRD and EDS studies. Forlectron microscopic studies, 25 �l of sample was sputter coated

n copper stub and the images of nanoparticles were studied usingEM (JEOL, Model JFC-1600) and HRTEM (JEOL-3010). For XRD stud-es, dried nanoparticles were coated on XRD grid and the spectra

as recorded by using Philips PW 1830 X-ray generator operated atvoltage of 40 kV and a current of 30 mA with Cu K�1 radiation. In

e adding the leaf extract and (b) After addition of leaf extract at 30 min.

addition presence of metals in the sample was analyzed by energydispersive spectroscopy (EDS).

2.5. Minimal inhibitory concentration of silver nanoparticles

Minimal inhibitory concentrations (MICs) of AgNO3 and silvernanoparticles were determined by MTT assay by using 96-well

Fig. 2. UV–vis spectra of aqueous silver nitrate with A. indica leaf extract at differenttime intervals.

5 rfaces B: Biointerfaces 76 (2010) 50–56

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.6. Changes in membrane permeability of bacterial cells

To study the membrane permeability of bacterial cells, the viableacterial cultures of E. coli and V. cholerae in nutrient broth werereated with synthesized silver nanoparticles. Ten milliliters of loghase cultures were centrifuged at 6000 rpm for 10 min and theellet was suspended in sterile distilled water. Five milliliters of thisuspension was exposed to 100 ppb of silver nanoparticles and theonductance was recorded after incubation of 1, 3, 6 and 24 h usingconductivity meter (NAINA Model-NDC 732). The same procedureas adopted in the control experiments i.e. cultures treated withgNO3.

.7. Determination of respiration activity of bacterial cells

Changes in the respiration of log phase cultures of E. coli and V.holerae in nutrient broth were studied using Biological Oxygen

onitor (YSI-Model-5300, USA). It provides a measure of oxy-

en consumption by the bacterial cultures. The changes in oxygenptake among the untreated and silver nanoparticles treated cul-ures were recorded.

ig. 3. Image of scanning electron microscopic observation of (a) silver nitrate andb) Synthesized silver nanoparticles.

Fig. 4. High resolution transmission electron microscopic image of silver nanopar-ticles. (a) Individual nanoparticles through high resolution transmission electronmicroscope. (b) High resolution image of single particle with clear lattice fringesand (c) SAED pattern.

C. Krishnaraj et al. / Colloids and Surfaces B: Biointerfaces 76 (2010) 50–56 53

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dards (JCPDS) file No. 04-0783. The average grain size of the silvernanoparticles formed in the bioreduction process was determinedusing Scherr’s formula, d = (0.9� × 180◦)/ˇcos�� and was estimatedas 35 nm (Fig. 6).

Fig. 5. EDS analysis of silver nano

.8. Statistical analyses

The data were subjected to One-way Analysis of VarianceANOVA) to determine the significance of individual differences at< 0.05 level. Significant means were compared by the Duncan’sultiple range test. All statistical analyses were carried out using

PSS statistical software package (SPSS, Version 10.0, Chicago, USA).

. Results and discussion

Several approaches have been employed to obtain a betterynthesis of silver nanoparticles such as chemical and biologicalethods. Recently, synthesis of silver nanoparticles using plant

xtracts getting more popular [22,23]. Chandran et al. synthesizedilver nanoparticles by using the Aloe vera extract at 24 h of incuba-ion [19]. Similarly, in the present study silver nanoparticles wereynthesized using leaves extract of A. indica. Interestingly, silveranoparticles were synthesized rapidly within 30 min of incuba-ion period. The aqueous silver nitrate solution was turned to brownolor within 30 min, with the addition of leaf extract (Fig. 1a and b).ntensity of brown color increased in direct proportion to the incu-ation period. It may be due to the excitation of surface plasmonesonance (SPR) effect and reduction of AgNO3 [24]. The controlgNO3 solution (without leaf extract) showed no change of color.he characteristic absorption peak at 420 nm in UV–vis spectrumFig. 2) confirmed the formation of silver nanoparticles. SPR pat-erns, characteristics of metal nanoparticles strongly depend onarticle size, stabilizing molecules or the surface adsorbed parti-les and the dielectric constant of the medium. The single SPR bandn the early stages of synthesis corresponds to the absorption spec-ra of spherical nanoparticles. Many SPR bands resulted later, withncrease in the incubation period and two such bands were promi-ent with 8 h incubation, it indicates the formation of anisotropicolecules that later stabilized in the medium.

.1. Electron microscopic study

SEM analysis of the AgNO3 and synthesized silver nanaoparticles

ere clearly distinguishable owing to their size difference. From the

EM image the size of the control silver nitrate obtained was greaterhan 1000 nm size (Fig. 3a), where as synthesized silver nanopar-icles measured 20–30 nm in size (Fig. 3b). HRTEM micrographlso confirmed the size of nanoparticles in the range of 20–30 nm

les showed characteristic peaks.

(Fig. 4a). The nanoparticles obtained are highly crystalline as shownby clear lattice fringes (Fig. 4b) and selected area electron diffrac-tion (SAED) pattern (Fig. 4c). Similar to our study, the SAED patternof silver nanoparticles was reported by Song and Kim [23]. The EDSspectrum (Fig. 5) recorded from silver nanoparticles showed strongsignal of silver.

3.2. XRD analysis

XRD analysis showed three distinct diffraction peaks at 38.1◦,44.1◦ and 64.1◦, which indexed the planes 1 1 1, 2 0 0 and 2 2 0 ofthe cubic face-centered silver. The lattice constant calculated fromthis pattern was a = 4.086 Å and the data obtained was matchedwith the database of Joint Committee on Powder Diffraction Stan-

Fig. 6. XRD pattern of the silver nanoparticles synthesized from aqueous leafextracts of A. indica.

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.3. Minimal inhibitory concentration of silver nanoparticles

Synthesized silver nanoparticles showed effective antibacterialctivity against the test pathogens. MIC was recorded as the low-st concentration at which no visible growth of the test pathogensas observed. Among the different concentration of silver nanopar-

icles tested 10 �g/ml proved to be MIC for E. coli and V. cholerae

Fig. 7a and b). Whereas, in AgNO3 20 �g/ml was recorded as the

IC for E. coli and V. cholerae (Fig. 7a and b). The least MIC of silveranoparticles than silver nitrate may be due to the smaller size ofhe nanoparticles [25].

Fig. 7. Minimal inhibitory concentration of silver nitrate an

B: Biointerfaces 76 (2010) 50–56

3.4. Changes in membrane permeability of bacterial cells

Membrane permeability test was performed to study the inter-action of silver nanoparticles on the bacterial cell surfaces. Tenmilliliters of log phase cultures of E. coli and V. cholerae exposedto 100 ppb of silver nanoparticle showed high conductivity at24 h (235,215 �S/cm) than AgNO3 treated broth (210,180 �S/cm)

(Fig. 8). The increase in the membrane permeability may be dueto the serious damage of cell membrane structure caused by sil-ver nanoparticles. The maximum conductivity was observed insilver nanoparticles treated cells than AgNO3, it may be due to

d silver nanoparticles on (a) E. coli and (b) V. cholerae.

C. Krishnaraj et al. / Colloids and Surfaces B: Biointerfaces 76 (2010) 50–56 55

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Fig. 8. Changes in the membrane

maller size of the particles which leads to increased membraneermeability and cell destruction. However, the mechanism of bac-ericidal actions of silver nanoparticles is still speculative and notell understood. However, Sondi and Salopek-Sondi reported the

ntimicrobial activity of silver nanoparticles was closely associatedith the formation of ‘pits’ in the cell wall of bacteria, leading to

ncreased membrane permeability and resulting in cell death [6].hile Yamanaka et al. indicated that bactericidal actions of the sil-

er ion are caused primarily by its interaction with the cytoplasm inhe interior of the cell. The silver ion appears to penetrate throughon channels without causing damage to the cell membranes; itenatures the ribosome and suppresses the expression of enzymes

nd proteins essential to ATP production, which renders the dis-uption of the cell [26]. In our present study bacterial culturesreated with silver nanoparticles showed increased conductance.his could be well attributed to the dissolution of the cellular con-ents in the culture broth, by the disruption of the cell membrane

Fig. 9. Changes in the respiration ac

eability of E. coli and V. cholerae.

structures with the loss of membrane permeability or the inabilityto sustain with the ATP production, necessary for maintaining themembrane dynamics.

3.5. Respiration activity of the bacterial cells

Respiration activity of test pathogens was performed to elu-cidate the possible mode of action of silver nanoparticles. Theinteractions of silver nanoparticles and thiol containing groupsresulted in the generation of reactive oxygen species and conse-quently damaging the cell [27]. In our present study, the respirationrate of silver nanoparticle treated bacterial cells of E. coli and V.

cholerae was decreased (1,1 oxygen in nanomole) when comparedwith untreated bacterial cultures (16, 18 oxygen in nanomole) asshown in Fig. 9. This can be attributed to the inhibitory activityof silver nanoparticles on the respiratory enzymes (cytochromeoxidases, malate dehydrogenase and succinate dehydrogenase)

tivity of E. coli and V. cholerae.

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r as a result of complete destruction of the bacteria. It haslso been hypothesized that Ag+ primarily affects the function ofembrane-bound enzymes, which played vital role in the res-

iratory chain [27]. Silver has a greater affinity to react withulfur- or phosphorus-containing biomolecules in the cell. Thus,ulfur-containing proteins in the membrane or inside the cells andhosphorus-containing elements like DNA are likely to be the pref-rential sites for silver nanoparticle binding [28,29].

The possible mechanism of biosynthesis of nanoparticles by bio-ogical system was reductases and any other equivalent reductantss reported earlier [18]. The nitrate reductase from Fusarium oxys-orum has been documented to catalyze the reduction of AgNO3 toilver nanoparticles utilizing NADPH as reducing agent [30]. Sev-ral naphthoquinones and anthraquinones having very high redoxotentials have been reported from F. oxysporum that could acts an electron shuttle in metal reduction [31]. Although such sys-ems were not repeated in plant mediated synthesis nanoparticles,he phytochemical constituents are attributed to the formation ofanoparticles. Caffeine and theophylline present in tea extractsere also reported to catalyze the synthesis of nanoparticles [32].

hyllanthin from Phyllanthus amarus was also reported as the cap-ing ligands in the synthesis of silver nanoparticles [33]. Quercetinnd polysaccharides have been used for silver nanoparticle syn-hesis [34]. Quercetin belongs to a group of plant pigments calledavonoids, the active constituent of the phytochemicals in the A.

ndica [35], may be responsible for the nanoparticles synthesis. TheHO approved the use of silver as a drinking water disinfectant

36], hence silver nanotechnology can be used in water purificationystems.

. Conclusions

The biosynthesized silver nanoparticles using A.indica leavesxtract proved excellent antimicrobial activity. The antimicrobialctivity is well demonstrated with MIC, change in membrane per-eability and respiration activity of bacterial cells treated with

ilver nanoparticles. Hence, the biological approach appears to beost efficient alternative to conventional physical and chemicalethods of silver nanoparticles synthesis and would be suitable

or developing a biological process for large-scale production. Theseilver nanoparticles may be used in effluent treatment process foreducing the microbial load.

cknowledgments

The authors convey their thanks to Director, CAS in Botany, Uni-ersity of Madras, for providing laboratory facilities. Thanks alsoue to Head of the Department of Geology, Nuclear Physics, Uni-

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B: Biointerfaces 76 (2010) 50–56

versity of Madras for providing SEM, XRD and EDS facilities. Wethank SAIF, IIT-Madras, Chennai for HRTEM analysis.

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