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

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces

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

Synthesis of silver nanoparticles using Acalypha indica leaf extracts and its
antibacterial activity against water borne pathogens
C. Krishnaraj ∗ , E.G. Jagan, S. Rajasekar, P. Selvakumar, P.T. Kalaichelvan, N. Mohan ∗∗
Centre for Advanced Studies in Botany, University of Madras, Guindy campus, Chennai-600025, India

a r t i c l e i n f o a b s t r a c t

Article history: In the present study, biosynthesis of silver nanoparticles and its activity on water borne bacterial
Received 29 August 2009 pathogens were investigated. Silver nanoparticles were rapidly synthesized using leaf extract of Aca-
Received in revised form 4 October 2009 lypha indica and the formation of nanoparticles was observed within 30 min. The results recorded from
Accepted 7 October 2009
UV–vis spectrum, scanning electron microscopy (SEM), X-ray diffraction (XRD) and energy dispersive
Available online 14 October 2009
spectroscopy (EDS) support the biosynthesis and characterization of silver nanoparticles. From high-
resolution transmission electron microscopy (HRTEM) analysis, the size of the silver nanoparticles was
Keywords:
measured 20–30 nm. Further, the antibacterial activity of synthesized silver nanoparticles showed effec-
Acalypha indica
Silver nanoparticles
tive inhibitory activity against water borne pathogens Viz., Escherichia coli and Vibrio cholerae. Silver
MIC nanoparticles 10 ␮g/ml were recorded as the minimal inhibitory concentration (MIC) against E. coli and V.
Biosynthesis cholerae. Alteration in membrane permeability and respiration of the silver nanoparticle treated bacterial
cells were evident from the activity of silver nanoparticles.
© 2009 Elsevier B.V. All rights reserved.

1. Introduction
The development of reliable green process for the synthesis of
silver nanoparticles is an important aspect of current nanotech-
nology research. Nanomaterials such as Ag, Au, Pt and Pd have
been 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 and
medicine due to its attractive physiochemical properties. Klabunde
et al. demonstrated that the highly reactive metal oxide nanopar-
ticles exhibit excellent bactericidal action against Gram-positive
and Gram-negative bacteria [5]. The strong toxicity of silver against
wide range of microorganisms is well known and silver nanopar-
ticles have been recently shown to be a promising antimicrobial
material. Sondi et al. studied the antimicrobial activity of silver
nanoparticles against Escherichia coli as a model of Gram-negative
bacteria [6].
Interdisciplinary research has widened the horizons of material
research, drawing new inspirations from biological systems. The
towering environmental concerns had triggered the researchers to
device novel methods of synthesizing the nanomaterials in biolog-
ical systems such as bacteria, fungi and plants, termed as “green
chemistry” approaches. Biosynthesis of silver nanoparticles using
bacteria [7–9], fungi [10–12], yeast [13] and plants [14–16] were
well documented. However, exploration of the plant systems as the
potential nanofactories, has heightened interest in the biological
synthesis of nanoparticles. Sastry et al. reported the biosynthe-
sis of nanoparticles using plant leaf extracts and their potential
application. They studied bioreduction of chloraurate ions and
silver ions by extracts of geranium [17] and neem leaf [18]. Fur-
ther, synthesis of gold nanotriangles and silver nanoparticles using
Aloe vera plant extracts was reported [19]. Most of the above
research on the synthesis of silver or gold nanoparticles utilizing
plant extracts employed broths resulting from boiling fresh plant
leaves. Whereas, Huang et al. exploited the synthesis of silver and
gold nanoparticles using the sundried Cinnamomum camphora leaf
extract [20]. Acalypha indica (Euphorbiaceae), a traditional medic-
inal plant of South India, has the source of bio-reductant and
stabilizers. The present study was aimed to rapid synthesis of silver
nanoparticles using aqueous leaves extract of A. indica and evalu-
ates its antibacterial activity against water borne pathogens such as
Escherichia 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

∗ 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),
mohannatarajan@hotmail.com (N. Mohan).
2.1. Materials
The healthy leaves of A. indica were collected from campus
of University of Madras, India. AgNO3 , MTT (methyl thiozolyl
diphenyl-tetrazolium bromide) were purchased from Himedia Lab-

0927-7765/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.colsurfb.2009.10.008
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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 addition presence of metals in the sample was analyzed by energy
(MTCC-443) and V. cholerae (MTCC-3904) were obtained from dispersive spectroscopy (EDS).
Microbial Type Culture Collection, Chandigarh, India.

2.5. Minimal inhibitory concentration of silver nanoparticles

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 tap
water, followed by distilled water and boiled with 100 ml of dis-
tilled water at 60 ◦ C for 5 min. This extract was filtered through
nylon mesh, followed by Millipore filter (0.45 ␮m) and used for
further experiments.
Minimal inhibitory concentrations (MICs) of AgNO3 and silver
nanoparticles were determined by MTT assay by using 96-well
microtitre plate [21]. The mean of live cells of E. coli and V. cholerae
was recorded using ELISA reader (Emax precision microplate
reader). The MIC was determined based on different concentra-
tions, where there was no increase in the OD595 and was zero.

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 the
aqueous extract of A. indica. This setup was incubated in dark (to
minimize the photoactivation of silver nitrate), at 37 ◦ C under static
condition. A control setup was also maintained without A. indica
extract.

2.4. Characterization of silver nanoparticles

Synthesized silver nanoparticles was confirmed by sampling the

reaction mixture at regular intervals and the absorption maxima
was scanned by UV–vis spectra, at the wavelength of 200–700 nm in
Beckman-DU 20 spectrophotometer. Further, the reaction mixture
was subjected to centrifugation at 75,000 × g for 30 min, resulting
pellet was dissolved in deionized water and filtered through Mil-
lipore filter (0.45 ␮m). An aliquot of this filtrate containing silver
nanoparticles was used for SEM, HRTEM, XRD and EDS studies. For
electron microscopic studies, 25 ␮l of sample was sputter coated
on copper stub and the images of nanoparticles were studied using
SEM (JEOL, Model JFC-1600) and HRTEM (JEOL-3010). For XRD stud-
ies, dried nanoparticles were coated on XRD grid and the spectra
was recorded by using Philips PW 1830 X-ray generator operated at Fig. 2. UV–vis spectra of aqueous silver nitrate with A. indica leaf extract at different
a voltage of 40 kV and a current of 30 mA with Cu K␣1 radiation. In time intervals.
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52 C. Krishnaraj et al. / Colloids and Surfaces B: Biointerfaces 76 (2010) 50–56

2.6. Changes in membrane permeability of bacterial cells

To study the membrane permeability of bacterial cells, the viable

bacterial cultures of E. coli and V. cholerae in nutrient broth were
treated with synthesized silver nanoparticles. Ten milliliters of log
phase cultures were centrifuged at 6000 rpm for 10 min and the
pellet was suspended in sterile distilled water. Five milliliters of this
suspension was exposed to 100 ppb of silver nanoparticles and the
conductance was recorded after incubation of 1, 3, 6 and 24 h using
a conductivity meter (NAINA Model-NDC 732). The same procedure
was adopted in the control experiments i.e. cultures treated with
AgNO3 .

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 Oxygen
Monitor (YSI-Model-5300, USA). It provides a measure of oxy-
gen consumption by the bacterial cultures. The changes in oxygen
uptake among the untreated and silver nanoparticles treated cul-
tures were recorded.

Fig. 4. High resolution transmission electron microscopic image of silver nanopar-

ticles. (a) Individual nanoparticles through high resolution transmission electron
microscope. (b) High resolution image of single particle with clear lattice fringes
Fig. 3. Image of scanning electron microscopic observation of (a) silver nitrate and and (c) SAED pattern.
(b) Synthesized silver nanoparticles.
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C. Krishnaraj et al. / Colloids and Surfaces B: Biointerfaces 76 (2010) 50–56 53

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 at
p < 0.05 level. Significant means were compared by the Duncan’s
multiple range test. All statistical analyses were carried out using
SPSS statistical software package (SPSS, Version 10.0, Chicago, USA).
(Fig. 4a). The nanoparticles obtained are highly crystalline as shown
by clear lattice fringes (Fig. 4b) and selected area electron diffrac-
tion (SAED) pattern (Fig. 4c). Similar to our study, the SAED pattern
of silver nanoparticles was reported by Song and Kim [23]. The EDS
spectrum (Fig. 5) recorded from silver nanoparticles showed strong
signal of silver.

3. Results and discussion 3.2. XRD analysis

Several approaches have been employed to obtain a better XRD analysis showed three distinct diffraction peaks at 38.1◦ ,
synthesis of silver nanoparticles such as chemical and biological 44.1◦ and 64.1◦ , which indexed the planes 1 1 1, 2 0 0 and 2 2 0 of
methods. Recently, synthesis of silver nanoparticles using plant the cubic face-centered silver. The lattice constant calculated from
extracts getting more popular [22,23]. Chandran et al. synthesized this pattern was a = 4.086 Å and the data obtained was matched
silver nanoparticles by using the Aloe vera extract at 24 h of incuba- with the database of Joint Committee on Powder Diffraction Stan-
tion [19]. Similarly, in the present study silver nanoparticles were dards (JCPDS) file No. 04-0783. The average grain size of the silver
synthesized using leaves extract of A. indica. Interestingly, silver nanoparticles formed in the bioreduction process was determined
nanoparticles were synthesized rapidly within 30 min of incuba- using Scherr’s formula, d = (0.9 × 180◦ )/ˇcos and was estimated
tion period. The aqueous silver nitrate solution was turned to brown as 35 nm (Fig. 6).
color 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 plasmon
resonance (SPR) effect and reduction of AgNO3 [24]. The control
AgNO3 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 on
particle size, stabilizing molecules or the surface adsorbed parti-
cles and the dielectric constant of the medium. The single SPR band
in the early stages of synthesis corresponds to the absorption spec-
tra of spherical nanoparticles. Many SPR bands resulted later, with
increase in the incubation period and two such bands were promi-
nent with 8 h incubation, it indicates the formation of anisotropic
molecules that later stabilized in the medium.

3.1. Electron microscopic study

SEM analysis of the AgNO3 and synthesized silver nanaoparticles

were clearly distinguishable owing to their size difference. From the
SEM image the size of the control silver nitrate obtained was greater
than 1000 nm size (Fig. 3a), where as synthesized silver nanopar-
ticles measured 20–30 nm in size (Fig. 3b). HRTEM micrograph Fig. 6. XRD pattern of the silver nanoparticles synthesized from aqueous leaf
also confirmed the size of nanoparticles in the range of 20–30 nm extracts of A. indica.
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3.3. Minimal inhibitory concentration of silver nanoparticles 3.4. Changes in membrane permeability of bacterial cells

Synthesized silver nanoparticles showed effective antibacterial
activity against the test pathogens. MIC was recorded as the low-
est concentration at which no visible growth of the test pathogens
was 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 the
MIC for E. coli and V. cholerae (Fig. 7a and b). The least MIC of silver
nanoparticles than silver nitrate may be due to the smaller size of
the nanoparticles [25].
Membrane permeability test was performed to study the inter-
action of silver nanoparticles on the bacterial cell surfaces. Ten
milliliters of log phase cultures of E. coli and V. cholerae exposed
to 100 ppb of silver nanoparticle showed high conductivity at
24 h (235,215 ␮S/cm) than AgNO3 treated broth (210,180 ␮S/cm)
(Fig. 8). The increase in the membrane permeability may be due
to the serious damage of cell membrane structure caused by sil-
ver nanoparticles. The maximum conductivity was observed in
silver 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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C. Krishnaraj et al. / Colloids and Surfaces B: Biointerfaces 76 (2010) 50–56 55

Fig. 8. Changes in the membrane permeability of E. coli and V. cholerae.

smaller size of the particles which leads to increased membrane
permeability and cell destruction. However, the mechanism of bac-
tericidal actions of silver nanoparticles is still speculative and not
well understood. However, Sondi and Salopek-Sondi reported the
antimicrobial activity of silver nanoparticles was closely associated
with the formation of ‘pits’ in the cell wall of bacteria, leading to
increased 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 in
the interior of the cell. The silver ion appears to penetrate through
ion channels without causing damage to the cell membranes; it
denatures the ribosome and suppresses the expression of enzymes
and proteins essential to ATP production, which renders the dis-
ruption of the cell [26]. In our present study bacterial cultures
treated 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 inability
to sustain with the ATP production, necessary for maintaining the
membrane 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. The
interactions of silver nanoparticles and thiol containing groups
resulted in the generation of reactive oxygen species and conse-
quently damaging the cell [27]. In our present study, the respiration
rate of silver nanoparticle treated bacterial cells of E. coli and V.
cholerae was decreased (1,1 oxygen in nanomole) when compared
with untreated bacterial cultures (16, 18 oxygen in nanomole) as
shown in Fig. 9. This can be attributed to the inhibitory activity
of silver nanoparticles on the respiratory enzymes (cytochrome
oxidases, malate dehydrogenase and succinate dehydrogenase)

Fig. 9. Changes in the respiration activity of E. coli and V. cholerae.

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56 C. Krishnaraj et al. / Colloids and Surfaces B: Biointerfaces 76 (2010) 50–56
or as a result of complete destruction of the bacteria. It has
also been hypothesized that Ag+ primarily affects the function of
membrane-bound enzymes, which played vital role in the res-
piratory chain [27]. Silver has a greater affinity to react with
sulfur- or phosphorus-containing biomolecules in the cell. Thus,
sulfur-containing proteins in the membrane or inside the cells and
phosphorus-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 reductants
as reported earlier [18]. The nitrate reductase from Fusarium oxys-
porum has been documented to catalyze the reduction of AgNO3 to
silver nanoparticles utilizing NADPH as reducing agent [30]. Sev-
eral naphthoquinones and anthraquinones having very high redox
potentials have been reported from F. oxysporum that could act
as 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 of
nanoparticles. Caffeine and theophylline present in tea extracts
were 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]. Quercetin
and polysaccharides have been used for silver nanoparticle syn-
thesis [34]. Quercetin belongs to a group of plant pigments called
flavonoids, the active constituent of the phytochemicals in the A.
indica [35], may be responsible for the nanoparticles synthesis. The
WHO approved the use of silver as a drinking water disinfectant
[36], hence silver nanotechnology can be used in water purification
systems.
4. Conclusions
The biosynthesized silver nanoparticles using A.indica leaves
extract proved excellent antimicrobial activity. The antimicrobial
activity is well demonstrated with MIC, change in membrane per-
meability and respiration activity of bacterial cells treated with
silver nanoparticles. Hence, the biological approach appears to be
cost efficient alternative to conventional physical and chemical
methods of silver nanoparticles synthesis and would be suitable
for developing a biological process for large-scale production. These
silver nanoparticles may be used in effluent treatment process for
reducing the microbial load.
Acknowledgments
The authors convey their thanks to Director, CAS in Botany, Uni-
versity of Madras, for providing laboratory facilities. Thanks also
due to Head of the Department of Geology, Nuclear Physics, Uni-
versity of Madras for providing SEM, XRD and EDS facilities. We
thank SAIF, IIT-Madras, Chennai for HRTEM analysis.
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