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Vernonia cinerea (L.) Less.

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

; 45 kV, 30 mA). The XRD pattern was analyzed to


determine peak intensity, position, and width. The particle size was calculated using
the following formula:
d 0:9k=b cos h;
where d is the mean diameter of the nanoparticles, k is the wavelength of the X-ray
radiation source, and b is the angular full-width at half-maximum (FWHM) of the
XRD peak at diffraction angle h [37]. Powder samples for analysis were prepared by
centrifugation at 13,000 rpm for 15 min followed by redispersion in sterile distilled
water to remove any uncoordinated biological molecules. Centrifugation and redi-
spersion were repeated thrice for better separation.
K. Sahayaraj et al.
1 3
Fourier-transform infrared (FTIR) spectroscopy analysis
After complete reduction of Ag
?
ions by V. cinerea extract, 10 mL solution of
silver nanoparticles was centrifuged at 4,000 rpm for 10 min, and the resulting
suspension was redispersed in 20 mL Millipore water; the process of centrifugation
and redispersion was repeated three times so that the nanoparticles were free from
proteins or other bioorganic compounds present in the solution. Thereafter, the
puried suspension was completely dried and analyzed by FTIR spectrometer in the
range of 4,000 to 400 cm
-1
at resolution of 4 cm
-1
. The FTIR spectra of leaf
extract taken before and after synthesis of silver nanoparticles were analyzed and
investigated for possible functional groups involved in the formation of silver
nanoparticles.
Antimicrobial assays
Diffusion method
Xanthomonas campestris pv. malvacearum was isolated from infected cotton plants
and used for the experiment. The pathogen was isolated, subcultured on nutrient
agar (HiMedia, India), and identied using standard protocol [38]. Antimicrobial
activity was assayed using the agar well diffusion method [39]. Petri plates (9 cm)
were prepared with 20 mL sterile MullerHinton agar (HiMedia, India). Wells were
made using a sterile cork borer under aseptic conditions. V. cinerea leaf extract
and synthesized nanoparticles with various concentrations (10, 20, 40, 80, and
160 lg/mL) was added to the respective wells. Chloramphenicol (0.1 %; HiMedia,
Mumbai, India) and deionized water were used as positive and negative control,
respectively. After incubation for 24 h at 37 C, a clear zone around the well was
evidence of antibacterial activity. The diameter of the inhibition zone was measured
in millimeters using a HiMedia ruler. Each test was performed in triplicate.
Determination of minimum inhibitory concentration (MIC)
The broth microdilution assay was used to determine the MIC [40]. Test samples
(75 lL) of various concentrations (10320 lg/mL) were added to sterile microtiter
plates. Bacterial cell suspension (75 lL) corresponding to 1 9 10
8
colony-forming
units (CFU)/mL was added in all wells except control. Control well contained sterile
distilled water and broth to check sterility, while the negative control wells were
lled with nutrient broth and bacterial suspension to check for adequacy of the broth
to support bacterial growth. The plates were placed in a sterile Petri plate and
incubated at 37 C for 24 h. To indicate bacterial growth, 40 lL 0.2 mg/mL p-
iodonitrotetrazolium chloride (INT, HiMedia) was added to each well, followed by
incubation for another 30 min [40]. The colorless tetrazolium salt acts as an electron
acceptor and is reduced to a red-colored formazan product by biologically active
organisms. Inhibition of bacterial growth was indicated by a clear colorless well,
and the presence of growth was detected by the presence of pinkred color. The
lowest concentration showing no color change was considered as the MIC.
Vernonia cinerea (L.) Less. silver nanocomposite
1 3
Results and discussion
We present a detailed study on extracellular synthesis of silver nanoparticles by
V. cinerea aqueous extract and the antibacterial effect on a cotton-infecting
phytopathogen. It is generally recognized that UVVisible spectroscopy can be used
to examine size- and shape-controlled nanoparticles in aqueous suspension [41].
Figure 1 shows the ltrate of V. cinerea biomass with Ag
?
ions at the initial time
point and at the end point after 1 h of reaction. After addition of the biomass, the
silver nitrate solution changed from colorless to pale yellow in about 30 min, the
nal color deepening to cream or yellowish-brown within 1 h. Silver nanoparticles
(AgNPs) have free electrons, which give rise to a surface plasmon resonance (SPR)
absorption band [42], due to the combined vibration of electrons of the silver
nanoparticles in resonance with the light wave [43, 44].
The synthesized silver nanoparticles exhibited an absorption maximum at
430 nm in the visible region (Fig. 2) with yellowish-brown or cream color [4548].
Due to the excitation of the plasmon resonances of interband transitions, some
metallic nanoparticle dispersions exhibit unique bands/peaks [49]. The broadness of
the peak is a good indicator of the nanoparticle size. As the particle size increases,
the peak becomes narrower with decreased bandwidth and increased band intensity
[45, 47]. Moreover, the possible explanation for the difference in the reduction time
could be due to the difference in the reduction potential for both metal ions. Silver
nanoparticles synthesized using V. cinerea were stable for more than 6 months
when stored at room temperature (2930 C).
SEM analysis of the biologically synthesized silver nanoparticles clearly showed
a particle size ranging from 5 to 50 nm (Fig. 3) and that the silver nanoparticles
were deposited on the surface. High-resolution transmission electron microscopy
(HR-TEM) provided further insight into the morphology and size details of the
silver nanoparticles. HR-TEM micrographs were recorded from drop-coated lms
of silver nanoparticles synthesized by treating silver nitrate solution with plant
extract for 1 h. Though shape variation was noticed, the majority of the
nanoparticles were found to be spherical and polydisperse under HR-TEM. HR-
TEM also conrmed the size of the nanoparticles to be in the range of 550 nm
(Fig. 4). Similarly, silver nanoparticles synthesized using neem [6] and black tea
leaf extract [50] were spherical and polydisperse, as observed for V. cinerea.
The XRD spectrum of the silver nanoparticles is shown in Fig. 5 and conrms
the formation of metallic silver. The AgNPs showed diffraction peaks characteristic
of metallic face-centered cubic silver phase at 2h values of 38.08 (111), 44.17
(200), 64.41 (220), and 77.34 (311). The average crystal size of the AgNPs was
calculated by applying the mentioned equation [37] and was found to be 33 nm.
Hence, from the XRD pattern it was clear that the silver nanoparticles formed using
V. cinerea broth were essentially crystalline in nature.
FTIR measurements were carried out to identify the possible biomolecules
responsible for the reduction of the Ag
?
ions and capping of the bioreduced AgNPs
synthesized by V. cinerea. The FTIR spectra of untreated and treated plant extract
samples containing AgNPs are depicted in Fig. 6a and b, respectively. The observed
peaks are more characteristic of tannins, which are abundant in V. cinerea extracts
K. Sahayaraj et al.
1 3
[33]. Generally, at alkaline pH, tannins are hydrolyzed to glucose and gallic acid
units [51]. At alkaline pH, gallic acid rapidly reduces silver nitrate to silver
nanoparticles at room temperature, but the particles form aggregates in solution, as
gallic acid is a poor stabilizing agent. The FTIR characterization after the reaction
of V. cinerea with Ag
?
indicated that glucose might be responsible for the
stabilization of the silver nanoparticles. Glucose is a weak reducing agent at room
temperature, but a good stabilizing agent at alkaline pH [51]. In the FTIR spectrum,
several absorption peaks were found centered at 666, 1,352, 1,633, 2,078, 2,432, and
3,431 cm
-1
, lying in the range 6003,500 cm
-1
(Fig. 6b). The widest spectral
Fig. 1 Synthesis of silver nanoparticles using aqueous extract of Vernonia cinerea : a 10
-3
M AgNO
3
,
b initial point of time, c nal point of time
Fig. 2 UVvisible absorption spectra recorded as a function of time for silver nanoparticles obtained
using aqueous extract of Vernonia cinerea aqueous extract: a 30 min, b 1 h, c 24 h
Vernonia cinerea (L.) Less. silver nanocomposite
1 3
absorption was observed at 1,633 (amide I, C=O groups) and 3,431 cm
-1
(OH
stretching), whereas the absorption peaks centered at 1,515 and 1,540 cm
-1
can be
attributed to stretching vibration of C=C (aromatic ring). A weak OH in-plane
bend of phenol (1,380 cm
-1
) was lost due to capping of the plant extracts. The
signal at 1,380 cm
-1
is characteristic of phenol functional group, as reported by
Poljansek and Matjaz Krajnc [52]. The various functional groups referred to above
are mainly derived from heterocyclic compounds, which are water-soluble
components of V. cinerea. So, it can be assumed that different water-soluble
heterocyclic compounds such as tannins worked as the capping ligand during the
synthesis of silver nanoparticles and the presence of oxygen atoms helped in the
stabilization of nanoparticles by facilitating absorption of heterocyclic compounds
on the nanoparticles.
The antibacterial activity results for V. cinerea (VC) and V. cinerea biosynthesized
silver nanoparticles (VCBSNs) against X. campestris are presented in Table 1. The
Fig. 3 Representative scanning
electron micrography image
illustrating silver nanoparticles
biologically synthesized by
reduction of Ag
?
ions using
aqueous extract of Vernonia
cinerea aqueous extract
Fig. 4 Representative TEM images illustrating silver nanoparticles biologically synthesized by reduction
of Ag
?
ions using aqueous extract of Vernonia cinerea aqueous extract: a 10 nm, b 20 nm
K. Sahayaraj et al.
1 3
crude extract showed no activity against the tested pathogen, whereas the VCBSNs
signicantly (P\0.05) inhibited growth of the pathogen (13.00 0.58 mm). The
minimum inhibitory concentration (MIC) was found to be 80 lg/mL. The mechanism
of the bactericidal action of silver nanoparticles remains under debate and is not well
understood, but is closely associated with the formation of pits in the bacterial cell
wall, leading to increased membrane permeability and resulting in cell death [53]. The
bactericidal action of silver ions results primarily from their interaction with the
Fig. 5 X-ray diffraction (XRD) spectrum of silver nanoparticles synthesized by reduction of Ag
?
ions
using aqueous extract of Vernonia cinerea aqueous extract
Fig. 6 FTIR spectra of a Vernonia cinerea aqueous extract and b silver nanoparticles synthesized by
using V. cinerea extract
Vernonia cinerea (L.) Less. silver nanocomposite
1 3
cytoplasm in the interior of the cell. Silver ions appear to penetrate through ion
channels without causing damage to the cell membrane; they then denature the
ribosome and suppress the expression of enzymes and proteins essential to production
of adenosine triphosphate (ATP), resulting in cell disruption [18, 54]. In the cell,
silver ions may deactivate cellular enzymes and deoxyribonucleic acid (DNA) by
reacting with electron-donating groups such as thiol (SH) groups and generate
reactive oxygen species (ROS) [55, 56]. It has also been hypothesized that Ag
?
ions
primarily affect the function of membrane-bound enzymes, which play a vital role in
the respiratory chain [57]. Silver has a greater afnity to react with sulfur- or
phosphorus-containing biomolecules in the cell. Thus, sulphur-containing proteins in
the membrane or inside the cells and phosphorus-containing elements such as DNA
are likely to be preferential sites for silver nanoparticle binding [58, 59]. When silver
nanoparticles enter the bacterial cell, they form a low-molecular-weight region in the
center of the bacteria to which the bacteria thus conglomerates, protecting the DNA
from the silver ions. The nanoparticles preferably attack the respiratory chain and cell
division, nally leading to cell death [53, 6062]. Thus, it is reasonable to infer that
biologically synthesized AgNPs could be used to manage the disease caused by
X. campestris in cotton plant; there is a high possibility of generating a new
antimicrobial agent.
The mean size of the silver nanoparticles interacting with bacteria was 5 2 nm.
Particles were found inside the bacterial cell membrane. Furthermore, the particle
penetration was size dependent. Particles with size between 1 and 10 nm only
interact preferentially with bacteria. Such particle penetration ability was veried
earlier [53]. The inuence of size on antimicrobial activity was investigated by
Baker et al. [63]. They recorded that the antibacterial properties were related to the
total surface area of the nanoparticles. Smaller particles with larger surface-to-
volume ratios have greater antibacterial activity [64]. The size of VCBSNs ranged
from 5 nm to 50 nm, thus this smaller size enabled higher activity against
X. campestris. The surface plasmon resonance plays a major role in the optical
absorption spectra of silver nanoparticles, shifting to longer wavelength with
increasing particle size. The small size of nanoparticles implies that they have a
Table 1 Antibacterial activity of aqueous silver nanoparticles biologically synthesized by V. cinerea
against Xanthomonas campestris pv. malvacearum
Concentration (lg/mL) VC VCBSNs
10 0.00 0.00
20 0.00 0.00
40 0.00 7.67 0.34
a
80 0.00 9.67 0.34
a
160 0.00 13.00 0.58
a
Positive control 22.34 0.89
Positive control 0.1 % chloramphenicol, VC V. cinerea aqueous extract, VCBSNs V. cinerea biosyn-
thesized silver nanoparticles
a
Signicant at 5 % level using Tukeys test
K. Sahayaraj et al.
1 3
large surface area to come into contact with the bacterial cells and hence a higher
percentage of interaction than for larger particles [61, 6569]. The VCBSNs were
spherical and polydisperse. It required 80 lg/mL of nanoparticles to inhibit
X. campestris. Thus, silver nanoparticles with different shapes have different effects
on bacterial cells.
Conclusions
Nanotechnology is emerging as a eld of applied science and technology. Synthesis
of nanoparticles can be achieved by various physical and chemical methods, but
biological systems are gaining attention as an ecofriendly technique. The
biosynthetic method employing plant parts has proved to be a simple and cost-
effective method for synthesis of nanoparticles. We demonstrated that use of a
natural, low-cost biological reducing agent, namely V. cinerea aqueous extract, can
produce silver nanostructures through an efcient green nanochemistry methodol-
ogy, avoiding the use of hazardous and toxic solvents and other synthetic chemicals.
The nanoparticles were characterized using UVVisible spectrophotometry, XRD,
FTIR, SEM, and TEM analyses. The biologically synthesized silver nanoparticles
showed promising antimicrobial activity against X. campestris, an economically
important pathogen of cotton plant causing severe yield loss worldwide. The
aforementioned advantages of the aqueous plant-based silver NPs make them ideal
for use in green industrial, medicinal, microbiological, agricultural, and other
applications. The present biological synthesis is a simple, green, rapid, and low-cost
approach for producing silver nanoparticles in the laboratory.
Acknowledgments Senior author K.S. acknowledges nancial support from the Ministry of Earth
Science, New Delhi (ref. no. MRDF/01/33/P/07) provided for this work. We would like to thank the DST
unit of Nanoscience IIT, Madras for TEM analysis and Karunya University, Coimbatore for XRD and
SEM analyses.
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