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silver nanocomposite
and its antibacterial activity against a cotton pathogen
K. Sahayaraj
M. Roobadevi
S. Rajesh
S. Azizi
Received: 4 February 2014 / Accepted: 15 April 2014
Springer Science+Business Media Dordrecht 2014
Abstract Noble-metal nanomaterials are of particular interest today because of
their applications in many areas, including agriculture. The latter topic is one of the
most active areas of research in metal nanomaterials. Metal nanoparticles are tra-
ditionally synthesized by wet chemical techniques, in which the chemicals used are
often toxic and ammable. We report here biosynthesis of silver nanoparticles using
leaf extract of Vernonia cinerea (L.) Less. (Asteraceae). Treatment of aqueous
solution of AgNO
3
with V. cinerea leaf extract resulted in rapid formation of stable
silver nanoparticles. The growth of nanoparticles was monitored by UVVisible
spectrophotometry complemented by characterization using transmission electron
microscopy (TEM), X-ray diffraction analysis, and Fourier-transform infrared
spectroscopy. A feasible mechanism for the formation of nanomaterial and the
difference in the reduction time for silver nanoparticle synthesis is discussed. TEM
analysis revealed the presence of polydisperse silver nanoparticles with average size
of 550 nm. X-ray diffraction studies corroborated that the biosynthesized nano-
particles were crystalline silver. Furthermore, this green biogenic approach is a
rapid and simple alternative to chemical synthesis. The biologically synthesized
silver nanoparticles were found to be highly effective against Xanthomonas
campestris pv. malvacearum (13.00 0.58 mm) with minimum inhibitory
K. Sahayaraj (&) S. Rajesh
Crop Protection Research Centre, Department of Advanced Zoology and Biotechnology,
St. Xaviers College (Autonomous), Palayamkottai 627 002, Tamil Nadu, India
e-mail: ksraj48@gmail.com
M. Roobadevi
Department of Biotechnology, Nandha Arts and Science College, Erode 638052, Tamil Nadu, India
S. Azizi
Department of Chemistry, Faculty of Science, University Putra Malaysia, 43400 Serdang, Selangor,
Malaysia
1 3
Res Chem Intermed
DOI 10.1007/s11164-014-1676-8
concentration of 80 lg/mL. Hence, such biosynthesized silver nanoparticles can be
used in control of cotton bacterial blight.
Keywords Vernonia cinerea Xanthomonas campestris pv. malvacearum
Silver nanoparticles Antibacterial MIC
Introduction
The future success of nanotechnology is akin to capturing a wild horse: powerful
and full of potential, but it must be tamed before it becomes useful. The taming of
any beast requires deep understanding of the basic fundamental traits that govern its
behavior, which for a nanomaterial is primarily a combination of its composition,
size, and shape. To advance nanotechnology for antimicrobial applications,
bioengineered devices, and high-speed electronics, development of methods to
understand and control the behavior of nanomaterials is needed. A nanomaterial
may be dened as any material (insulator, conductor or semiconductor), which has
been controllably synthesized in the size range of roughly 1100 nm. At this size
and dimensional range, essentially any material will exhibit properties that are
different from those that it would show as an atomic cluster or as a larger, bulk
material [1].
Production of nanoparticles can be achieved through different methods, among
which chemical approaches are the most popular. However, some chemical methods
cannot avoid the use of toxic chemicals in the synthesis protocol. Since
nanoparticles of noble metals such as gold, silver, and platinum are widely applied
in contact with humans, there is a growing need to develop environmentally friendly
processes for nanoparticle synthesis that do not use toxic chemicals. Biological
methods of nanoparticle synthesis using microorganisms [24], enzymes [5], and
plant or plant extracts [6] have been suggested as possible ecofriendly alternatives to
chemical and physical methods of nanoparticle synthesis. Such methods can also be
suitably scaled up for large-scale synthesis of nanoparticles [7]. However, the major
drawback of using microbes for bioreduction is the difculty in maintaining aseptic
conditions, which not only requires technical effort but also greatly increases
production costs to a high level at the industrial level.
Only in recent years have biosynthetic methods employing plant extracts
received some attention as a simple and viable alternative to chemical procedures or
physical methods for synthesizing metal nanoparticles. Gardea-Torresdey et al. [8]
rst reported the formation of gold and silver nanoparticles by living plants.
Extracellular nanoparticle synthesis using plant leaf extracts rather than whole
plants would be more economical owing to the easier downstream processing.
Shankar et al. [6] reported synthesis of pure metallic silver and gold nanoparticles
by reduction of Ag
?
and Au
?
ions using neem (Azadirachta indica) leaf broth.
There have been recent reports on photosynthesis of silver and gold nanoparticles by
employing lemon grass extract [6], Aloe vera plant extract [9], Murraya koenigii
[10], green tea (Camellia sinensis) [11], coriander leaves [12], sun-dried
K. Sahayaraj et al.
1 3
Cinnamomum camphora leaves [13], Cinnamon zeylanicum [14], phyllanthin
extract [15], bioactive principle of henna leaves (apiin) [16], Acalypha indica [17],
Curcuma longa [18], Hibiscus rosa-sinensis [19], Panicum virgatum [20], and Rosa
rugosa [21]. Green synthesis is very efcient for production of silver nanoparticles
because of its simplicity, accuracy, and cost-effectiveness, in addition to its green
chemistry perspective and agricultural and biomedical applications.
The antimicrobial activity of silver and its compounds has been exploited
worldwide [2224]. In addition, silver is known to exhibit an oligodynamic effect
because of its ability to exert bactericidal activity even at minimum concentrations
[25]. Development of resistance to silver in microbes is improbable due to its action
on a broad spectrum of targets in the cell [26]. The advantage of silver nanoparticles
over bulk metals or salts is the slow and regulated release of silver from
nanoparticles, thereby providing long-lasting protection against bacteria [27]. There
are various reasons for considering silver nanoparticles (AgNPs) as a universal
microbicidal agent [2830].
Vernonia cinerea (L.) Less. (Asteraceae) is a slender-stemmed plant with
variable leaf shape and pinkish-purple owers, distributed in grassy areas of
Southeast Asia and Hawaii. It has been documented and recommended in Thai
traditional medicine, as in other countries, for smoking cessation and relief of
asthma, cough, fever, malaria, urinary calculi, and arthritis [30]. Active compound
of V. cinerea showed anti-inammatory, analgesic, and antipyretic activities [31],
and anti-oxidant and anti-inammatory activity [32]. Furthermore, the methanol
extract [33] and benzene extract fraction [34] of V. cinerea exhibited anti-
inammatory and antibacterial activity, respectively.
The genus Xanthomonas (Proteobacteria) is a diverse and economically
important group of Gram-negative bacterial pathogens. Bacterial blight caused by
Xanthomonas campestris pv. malvacearum (Smith) Dye (synonym Xanthomonas
axonopodis pv. malvacearum) [35, 36] has become an increasing problem in cotton
production worldwide. The focus of the present study is the synthesis of silver
nanoparticles using aqueous extract of V. cinerea for the rst time, and their
characterization. In addition, we recorded the antibacterial activity of the prepared
nanoparticles against a Gram-negative bacterium, X. campestris, an important
pathogen of the cotton plant. To the best of our knowledge, this is the rst report on
the use of V. cinerea as a biological system for synthesis of silver nanoparticles for
cotton pathogen management.
Materials and methods
Materials
Silver nitrate was obtained from Hi-Media, Mumbai. All glassware was washed
with distilled water and oven-dried before use. Fresh aerial part of V. cinerea was
collected from St. Xaviers College campus (8.7166N, 77.7333E), Palayamkottai,
identied, and deposited in St. Xaviers College Herbarium (XCH no. 25483) for
future reference.
Vernonia cinerea (L.) Less. silver nanocomposite
1 3
Synthesis of silver nanoparticles
Plant leaf broth solution was prepared by taking 5 g thoroughly washed and nely
cut leaves in a 300-mL Erlenmeyer ask with 100 mL sterile distilled water, then
boiling the mixture for 20 min before nally decanting it. Typically, 0.1 mL ltered
leaf broth was added to 100 mL 10
-3
M aqueous AgNO
3
solution for reduction of
Ag
?
ions at room temperature. The color of the solution changed from light yellow
to cream, indicating the formation of silver nanoparticles (AgNPs).
Characterization
UVVisible spectral analysis
Bioreduction of AgNO
3
in aqueous solution was monitored using UVVisible
spectrophotometry at regular intervals. UVVisible spectra were recorded from
samples in quartz cuvettes at resolution of 1 nm as a function of reaction time using
a Shimadzu UV-1601 spectrophotometer.
Transmission electron microscopy (TEM) and scanning electron microscopy (SEM)
analyses
The size and shape of the biosynthesized nanoparticles were observed by TEM and
SEM measurements, respectively. Samples of silver nanoparticles synthesized using
V. cinerea leaf broth for TEM analysis were prepared by placing a drop of
nanoparticle solution on a carbon-coated copper grid and allowing water to
evaporate. TEM measurements were performed using a JEOL model 3010
instrument operated at accelerating voltage of 120 kV. The morphology of the
nanoparticles was observed using a JSM-6390 SEM. Samples for SEM analysis
were prepared by drop-coating the Ag nanoparticle solution onto a carbon-coated
copper grid. The lms on the grids were allowed to dry prior to SEM measurement.
Powder X-ray diffraction
The X-ray diffraction (XRD) pattern of dry nanoparticle powder was obtained using
Cu K
a
radiation (1.5406 A