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This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution
and sharing with colleagues.
Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information
regarding Elsevier’s archiving and manuscript policies areencouraged to visit:
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 dispersivespectroscopy (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.cholerae. Alteration in membrane permeability and respiration of the silver nanoparticle treated bacterialcells were evident from the activity of silver nanoparticles.
The development of reliable green process for the synthesis ofsilver nanoparticles is an important aspect of current nanotech-nology research. Nanomaterials such as Ag, Au, Pt and Pd havebeen synthesized by different methods, including hard template[1], using bacteria [2], fungi [3] and plants [4]. Among these, sil-ver nanoparticles play a significant role in the field of biology andmedicine due to its attractive physiochemical properties. Klabundeet al. demonstrated that the highly reactive metal oxide nanopar-ticles exhibit excellent bactericidal action against Gram-positiveand Gram-negative bacteria [5]. The strong toxicity of silver againstwide range of microorganisms is well known and silver nanopar-ticles have been recently shown to be a promising antimicrobialmaterial. Sondi et al. studied the antimicrobial activity of silvernanoparticles against Escherichia coli as a model of Gram-negativebacteria [6].
Interdisciplinary research has widened the horizons of materialresearch, drawing new inspirations from biological systems. Thetowering environmental concerns had triggered the researchers todevice novel methods of synthesizing the nanomaterials in biolog-ical systems such as bacteria, fungi and plants, termed as “greenchemistry” approaches. Biosynthesis of silver nanoparticles using
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
Fig. 1. Aqueous solution of 10−3 M AgNO3 with A. indica leaf extracts (a) before adding the leaf extract and (b) After addition of leaf extract at 30 min.
oratories Pvt. Ltd., Mumbai, India. The bacterial cultures of E. coli(MTCC-443) and V. cholerae (MTCC-3904) were obtained fromMicrobial Type Culture Collection, Chandigarh, India.
2.2. Preparation of plant extract
Aqueous extract of A. indica was prepared using freshly col-lected leaves (10 g). They were surface cleaned with running tapwater, followed by distilled water and boiled with 100 ml of dis-tilled water at 60 ◦C for 5 min. This extract was filtered throughnylon mesh, followed by Millipore filter (0.45 �m) and used forfurther experiments.
2.3. Synthesis of silver nanoparticles
For synthesis of silver nanoparticles, the Erlenmeyer flask con-taining 100 ml of AgNO3 (1 mM) was reacted with 12 ml of theaqueous extract of A. indica. This setup was incubated in dark (tominimize the photoactivation of silver nitrate), at 37 ◦C under staticcondition. A control setup was also maintained without A. indicaextract.
2.4. Characterization of silver nanoparticles
Synthesized silver nanoparticles was confirmed by sampling thereaction mixture at regular intervals and the absorption maximawas scanned by UV–vis spectra, at the wavelength of 200–700 nm inBeckman-DU 20 spectrophotometer. Further, the reaction mixturewas subjected to centrifugation at 75,000 × g for 30 min, resultingpellet was dissolved in deionized water and filtered through Mil-lipore filter (0.45 �m). An aliquot of this filtrate containing silvernanoparticles was used for SEM, HRTEM, XRD and EDS studies. Forelectron microscopic studies, 25 �l of sample was sputter coatedon copper stub and the images of nanoparticles were studied usingSEM (JEOL, Model JFC-1600) and HRTEM (JEOL-3010). For XRD stud-ies, dried nanoparticles were coated on XRD grid and the spectrawas recorded by using Philips PW 1830 X-ray generator operated ata voltage of 40 kV and a current of 30 mA with Cu K�1 radiation. In
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-wellmicrotitre 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. 2. UV–vis spectra of aqueous silver nitrate with A. indica leaf extract at differenttime intervals.
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2.6. Changes in membrane permeability of bacterial cells
To study the membrane permeability of bacterial cells, the viablebacterial cultures of E. coli and V. cholerae in nutrient broth weretreated with synthesized silver nanoparticles. Ten milliliters of logphase cultures were centrifuged at 6000 rpm for 10 min and thepellet was suspended in sterile distilled water. Five milliliters of thissuspension was exposed to 100 ppb of silver nanoparticles and theconductance was recorded after incubation of 1, 3, 6 and 24 h usinga conductivity meter (NAINA Model-NDC 732). The same procedurewas adopted in the control experiments i.e. cultures treated withAgNO3.
2.7. Determination of respiration activity of bacterial cells
Changes in the respiration of log phase cultures of E. coli and V.cholerae in nutrient broth were studied using Biological OxygenMonitor (YSI-Model-5300, USA). It provides a measure of oxy-gen consumption by the bacterial cultures. The changes in oxygenuptake among the untreated and silver nanoparticles treated cul-tures were recorded.
Fig. 3. Image of scanning electron microscopic observation of (a) silver nitrate and(b) 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.
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Fig. 5. EDS analysis of silver nanoparticles showed characteristic peaks.
2.8. Statistical analyses
The data were subjected to One-way Analysis of Variance(ANOVA) to determine the significance of individual differences atp < 0.05 level. Significant means were compared by the Duncan’smultiple range test. All statistical analyses were carried out usingSPSS statistical software package (SPSS, Version 10.0, Chicago, USA).
3. Results and discussion
Several approaches have been employed to obtain a bettersynthesis of silver nanoparticles such as chemical and biologicalmethods. Recently, synthesis of silver nanoparticles using plantextracts getting more popular [22,23]. Chandran et al. synthesizedsilver nanoparticles by using the Aloe vera extract at 24 h of incuba-tion [19]. Similarly, in the present study silver nanoparticles weresynthesized using leaves extract of A. indica. Interestingly, silvernanoparticles were synthesized rapidly within 30 min of incuba-tion period. The aqueous silver nitrate solution was turned to browncolor within 30 min, with the addition of leaf extract (Fig. 1a and b).Intensity of brown color increased in direct proportion to the incu-bation period. It may be due to the excitation of surface plasmonresonance (SPR) effect and reduction of AgNO3 [24]. The controlAgNO3 solution (without leaf extract) showed no change of color.The characteristic absorption peak at 420 nm in UV–vis spectrum(Fig. 2) confirmed the formation of silver nanoparticles. SPR pat-terns, characteristics of metal nanoparticles strongly depend onparticle size, stabilizing molecules or the surface adsorbed parti-cles and the dielectric constant of the medium. The single SPR bandin the early stages of synthesis corresponds to the absorption spec-tra of spherical nanoparticles. Many SPR bands resulted later, withincrease in the incubation period and two such bands were promi-nent with 8 h incubation, it indicates the formation of anisotropicmolecules that later stabilized in the medium.
3.1. Electron microscopic study
SEM analysis of the AgNO3 and synthesized silver nanaoparticleswere clearly distinguishable owing to their size difference. From theSEM image the size of the control silver nitrate obtained was greaterthan 1000 nm size (Fig. 3a), where as synthesized silver nanopar-ticles measured 20–30 nm in size (Fig. 3b). HRTEM micrographalso confirmed the size of nanoparticles in the range of 20–30 nm
(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-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. 6. XRD pattern of the silver nanoparticles synthesized from aqueous leafextracts of A. indica.
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3.3. Minimal inhibitory concentration of silver nanoparticles
Synthesized silver nanoparticles showed effective antibacterialactivity against the test pathogens. MIC was recorded as the low-est concentration at which no visible growth of the test pathogenswas observed. Among the different concentration of silver nanopar-ticles 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 theMIC for E. coli and V. cholerae (Fig. 7a and b). The least MIC of silvernanoparticles than silver nitrate may be due to the smaller size ofthe nanoparticles [25].
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
Fig. 7. Minimal inhibitory concentration of silver nitrate and silver nanoparticles on (a) E. coli and (b) V. cholerae.
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Fig. 8. Changes in the membrane permeability of E. coli and V. cholerae.
smaller size of the particles which leads to increased membranepermeability and cell destruction. However, the mechanism of bac-tericidal actions of silver nanoparticles is still speculative and notwell understood. However, Sondi and Salopek-Sondi reported theantimicrobial activity of silver nanoparticles was closely associatedwith the formation of ‘pits’ in the cell wall of bacteria, leading toincreased membrane permeability and resulting in cell death [6].While Yamanaka et al. indicated that bactericidal actions of the sil-ver ion are caused primarily by its interaction with the cytoplasm inthe interior of the cell. The silver ion appears to penetrate throughion channels without causing damage to the cell membranes; itdenatures the ribosome and suppresses the expression of enzymesand proteins essential to ATP production, which renders the dis-ruption of the cell [26]. In our present study bacterial culturestreated with silver nanoparticles showed increased conductance.This could be well attributed to the dissolution of the cellular con-tents in the culture broth, by the disruption of the cell membrane
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)
Fig. 9. Changes in the respiration activity of E. coli and V. cholerae.
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or as a result of complete destruction of the bacteria. It hasalso been hypothesized that Ag+ primarily affects the function ofmembrane-bound enzymes, which played vital role in the res-piratory chain [27]. Silver has a greater affinity to react withsulfur- or phosphorus-containing biomolecules in the cell. Thus,sulfur-containing proteins in the membrane or inside the cells andphosphorus-containing elements like DNA are likely to be the pref-erential sites for silver nanoparticle binding [28,29].
The possible mechanism of biosynthesis of nanoparticles by bio-logical system was reductases and any other equivalent reductantsas reported earlier [18]. The nitrate reductase from Fusarium oxys-porum has been documented to catalyze the reduction of AgNO3 tosilver nanoparticles utilizing NADPH as reducing agent [30]. Sev-eral naphthoquinones and anthraquinones having very high redoxpotentials have been reported from F. oxysporum that could actas an electron shuttle in metal reduction [31]. Although such sys-tems were not repeated in plant mediated synthesis nanoparticles,the phytochemical constituents are attributed to the formation ofnanoparticles. Caffeine and theophylline present in tea extractswere also reported to catalyze the synthesis of nanoparticles [32].Phyllanthin from Phyllanthus amarus was also reported as the cap-ping ligands in the synthesis of silver nanoparticles [33]. Quercetinand polysaccharides have been used for silver nanoparticle syn-thesis [34]. Quercetin belongs to a group of plant pigments calledflavonoids, the active constituent of the phytochemicals in the A.indica [35], may be responsible for the nanoparticles synthesis. TheWHO approved the use of silver as a drinking water disinfectant[36], hence silver nanotechnology can be used in water purificationsystems.
4. Conclusions
The biosynthesized silver nanoparticles using A.indica leavesextract proved excellent antimicrobial activity. The antimicrobialactivity is well demonstrated with MIC, change in membrane per-meability and respiration activity of bacterial cells treated withsilver nanoparticles. Hence, the biological approach appears to becost efficient alternative to conventional physical and chemicalmethods of silver nanoparticles synthesis and would be suitablefor developing a biological process for large-scale production. Thesesilver nanoparticles may be used in effluent treatment process forreducing the microbial load.
Acknowledgments
The authors convey their thanks to Director, CAS in Botany, Uni-versity of Madras, for providing laboratory facilities. Thanks alsodue to Head of the Department of Geology, Nuclear Physics, Uni-
versity of Madras for providing SEM, XRD and EDS facilities. Wethank SAIF, IIT-Madras, Chennai for HRTEM analysis.
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