Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 132 (2014) 743–750

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Spectrochimica Acta Part A: Molecular and

Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa

Catalytically and biologically active silver nanoparticles synthesized

using essential oil
Vidya Vilas a,b, Daizy Philip a,⇑, Joseph Mathew b
a
Department of Physics, Mar Ivanios College, Thiruvananthapuram 695015, India
b
Department of Chemistry, Mar Ivanios College, Thiruvananthapuram 695015, India

h i g h l i g h t s g r a p h i c a l a b s t r a c t

 Essential oil is used for the synthesis

of silver nanoparticles.
 Rapid, cost-effective, environmentally
benign method is suggested.
 Efficiency of the nanocatalyst is
portrayed in the degradation of a
cationic dye and an organic pollutant.
 The synthesized biogenic silver
nanoparticles act as potent free
radical scavengers and antibacterial
agent.

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

Article history: There are numerous reports on phytosynthesis of silver nanoparticles and various phytochemicals are
Received 21 January 2014 involved in the reduction and stabilization. Pure explicit phytosynthetic protocol for catalytically and bio-
Received in revised form 9 May 2014 logically active silver nanoparticles is of importance as it is an environmentally benign green method.
Accepted 14 May 2014
This paper reports the use of essential oil of Myristica fragrans enriched in terpenes and phenyl propenes
Available online 13 June 2014
in the reduction and stabilization. FTIR spectra of the essential oil and the synthesized biogenic silver
nanoparticles are in accordance with the GC–MS spectral analysis reports. Nanosilver is initially charac-
Keywords:
terized by an intense SPR band around 420 nm, followed by XRD and TEM analysis revealing the forma-
Silver nanoparticles
Myristica fragrans
tion of 12–26 nm sized, highly pure, crystalline silver nanoparticles. Excellent catalytic and bioactive
Catalysis potential of the silver nanoparticles is due to the surface modification. The chemocatalytic potential of
Antibacterial activity nanosilver is exhibited by the rapid reduction of the organic pollutant, para nitro phenol and by the deg-
Antioxidant activity radation of the thiazine dye, methylene blue. Significant antibacterial activity of the silver colloid against
Gram positive, Staphylococcus aureus (inhibition zone – 12 mm) and Gram negative, Escherichia coli (inhi-
bition zone – 14 mm) is demonstrated by Agar-well diffusion method. Strong antioxidant activity of the
biogenic silver nanoparticles is depicted through NO scavenging, hydrogen peroxide scavenging, reducing
power, DPPH and total antioxidant activity assays.
Ó 2014 Elsevier B.V. All rights reserved.

Introduction

Molecular manipulation at the nanoscale has undergone

⇑ Corresponding author. Tel.: +91 471 2530887; fax: +91 471 2530023. progressive changes ever since its dawn before 4th century AD.
E-mail addresses: daizyp@rediffmail.com, philipdaizy@yahoo.co.in (D. Philip). Though the methods of nanoparticle (NP) synthesis extends over

http://dx.doi.org/10.1016/j.saa.2014.05.046
1386-1425/Ó 2014 Elsevier B.V. All rights reserved.
744 V. Vilas et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 132 (2014) 743–750
a wide range, green synthesis methods has gained wider acclaim
owing to the increasing awareness on the undesirable changes of
the environment posed by several synthesis strategies. Phytosyn-
thesis of nanoparticles uses phytoconstituents of the plant parts
as reducing and capping agents. This ecofriendly method does
not demand the use of high temperature, pressure or energy [1].
Development of a facile, pure, specific phytosynthetic route leads
to the use of volatile aromatic oil. Kumar et al. [2] have synthesized
metal NPs embedded paints based on vegetable oil. The auto oxida-
tion of drying oils have been used for the reduction of metal salts to
synthesize metal NPs. Castor oil, palm oil and coconut oil have
been used for the synthesis of metal NPs [3–6]. In the first report
on essential oil mediated synthesis of metal NPs, Sheny et al. [7]
have used Anacardium occidentale as a reducing and capping agent
for the synthesis of hexagonal gold NPs.
The synthesis of silver (Ag) on a nanoscale is of importance, due
to its thermal conductivity, chemical stability, catalytic and anti-
bacterial activity [8,9]. The surface effects and quantum effects of
nanoAg affects their chemical reactivity [10] and gives them
unique mechanical, optical and electric properties [11]. Recent
reports [12–17] suggest biological method for synthesis of anti-
fungal, antibacterial, antioxidant, robust, biocompatible Ag NPs.
Size, shape, surface area to volume ratio and the nature of the sur-
face modifier act as limiting factors of the synthesized Ag NPs
imparting adequate therapeutic potency.
Exploitation of the antibacterial potential of nanoAg can be
traced back to ancient times. Oligodynamic action coupled with a
broad spectrum of targeted bacteria prompts the use of Ag based
nanostructured materials as efficient antibacterial agents [18]. The
rapid multiplication and proliferation of drug resistant bacteria is
curbed by Ag NPs fortifying its use in burn treatments, water filters
and as an antimicrobial finish on fabrics [15]. Antioxidant nanoAg
repairs reactive oxygen species (ROS) damage to cell components
and cell function disruption enhancing the immune system. The
conjoint application of nanoAg as an antibacterial and an antioxi-
dant agent ensures its use as a biomedical nanoproduct. NanoAg
extends its aptness as a catalyst in pollution treatment, due to its
small size, larger surface area to volume ratio and greater accessibil-
ity of the surface atoms. Ag NPs have attracted the interests of scien-
tists in its use as a competent catalyst in the reduction of major
pollutants of the dye industry that pose serious health risks.
Myristica fragrans (M. fragrans) is an evergreen tree, successfully
cultivated in South India. There are reports [19,20] on the
hepatoprotective and anti cancer activity of Myristicin and anti-
inflammatory property of t – Caryophyllene; the essential oil
constituents. The leaf oil exhibit significant antimicrobial and larvi-
cide activity and cytotoxic activity against MCF-7 breast cancer cell
line and A 357 epidermal skin cancer line [19].
In the present study, Ag NPs are synthesized using the essential
oil extracted from leaves of M. fragrans. The catalytic potential of
the synthesized Ag NPs is tested by studying the degradation of
para nitro phenol (4NP), an anthropogenic pollutant and methy-
lene blue (MB), a thiazine dye. The antibacterial activity of the bio-
genic Ag NPs against the pathogenic Escherichia coli (E. coli) and
Staphylococcus aureus (S. aureus) is studied using Agar well diffu-
sion method. The in vitro antioxidant activity of Ag NPs is investi-
gated through a series of assays.
Materials and methods
Fresh green leaves of M. fragrans were collected from Thiruva-
nanthapuram. Silver nitrate (AgNO3), 4NP and sodium borohydride
(NaBH4) were procured from Sigma–Aldrich; MB and acetone from
Merck were used. Demineralised water has been used throughout
the experiment.
300 g fresh leaves of M. fragrans are hydro distilled in a Cleveng-
er apparatus, yielding 3 mL essential oil (yield 1%). The aromatic
oil, diluted using acetone (1:170) is used for the synthesis of Ag
NPs. 1 mL of diluted oil is added, with vigorous stirring to 30 mL
of 2.14  104 M AgNO3 at 100 °C and pH 7 to get the colloid a1.
The experiment is repeated with 2, 3, 4, 5 mL diluted oil to get
colloids a2, a3, a4 and a5, respectively. Colour change from yellow
to golden yellow and to red, indicated the enhanced formation of
Ag NPs, with quantity of diluted oil. 2 mL diluted oil is added with
vigorous stirring to 30 mL boiling solution of AgNO3
(2.14  104 M) at varying pH conditions of 7, 8, 9 and 10 to obtain
colloids a2, a6, a7 and a8, respectively.
Catalytic activity of Ag NPs
The efficiency of Ag NPs as heterogeneous catalyst in the
reduction of 4NP and MB using NaBH4 is investigated. The stock
solutions, 7.1  103 M 4NP and 0.25 M NaBH4 are prepared. After
stirring 1 mL each of 4NP and NaBH4 in 23 mL aqueous medium,
1 mL of Ag nanocatalyst is added and vigorously stirred. UV–vis
spectra are recorded after regular intervals of time to study the
degradation of 4NP. The procedure is repeated with 9.9  102 M
NaBH4 and 103 M MB. The progress of the rapid degradation of
the cationic dye is studied through UV–vis spectra taken at regular
intervals.
Antibacterial and antioxidant potential of Ag NPs
Agar-well diffusion method is used for the antibacterial studies
of green synthesized Ag NPs. Mueller–Hinton plates are seeded
with S. aureus and E. coli. Wells of 10 mm are bored and samples
of 25, 50,100-lL concentration are added. After incubating at
37 °C for 24 h, the antibacterial activity is assayed by measuring
the diameter of the zone of inhibition formed around the well.
Experiments are done in triplicate and mean values are presented.
Gentamycin is used as the positive control [21,22].
Free radical scavenging activity, reducing power, total antioxi-
dant activity of the green synthesized Ag NPs is exemplified through
nitric oxide scavenging activity, hydrogen peroxide scavenging
activity, reducing power activity, 2,2-diphenyl -1-picryl hydrazyl
(DPPH)) assay and total antioxidant activity (description of the pro-
cedure of antioxidant assays is given in Supplemental information).
Absorbance of the Ag NP bound coloured complexes of radicals or
ions are determined (Atest) in addition to the absorbance of a similar
mixture without the test solution (Acontrol). Percentage inhibition
(%inhibition) is ascertained using the equation,
%inhibition ¼ ½ðAcontrol  Atest Þ=Acontrol 100 ð1Þ
Instrumentation
UV–vis spectra are recorded using Perkin–Elmer Lambda-35
spectrophotometer with a scanning speed of 480 nm/min and a slit
width of 1 nm. TEM samples are prepared by dropping the Ag col-
loid on carbon coated copper grids. The images are taken using
TecnaiG2 30 TEM after the evaporation of the solvents. XRD pattern
is obtained using XPERT-Pro diffractometer operating at 30 mA
current and 40 kV voltage for the scanning angle 2h from 20° to
100°. The diffraction pattern is obtained by irradiating with Cu
Ka radiation with k of 1.5406 Å. FTIR spectra are recorded using
IR Prestige-21 Shimadzu spectrophotometer. GC–MS analysis was
carried out on a HP Chem Gas Chromatogram fitted with a DB5 sil-
ica column coupled with a model 5973 mass detector. Essential oil
components were determined on comparison of the mass spectra
with Wiley 275.L database. (GC–MS spectra given in Fig. S1 of
Supplemental information).
 V. Vilas et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 132 (2014) 743–750 745

Results and discussion
The essential oil of M. fragrans is used for the reduction of Ag+ to
0
Ag . GC–MS spectral analysis reports reveal the relative percentage
composition of the leaf essential oil of M. fragrans. Amidst the
92.51% of identified components, the essential oil is primarily com-
prised of Myristicin (22.76%), (1S) b (-) Pinene (18.92%), Limonene
(10.94%), Sabinene (9.14%) and Terpinolene (6.64%). (% Composi-
tion given in Table S1 of Supplemental information).
The formation and growth of Ag NPs is assumed to be in accor-
dance with ‘LaMer Model’ [23]. The reduction of Ag+ ions is fol-
lowed by condensation and surface reduction to Ag NPs. Though,
the optimum conditions for the synthesis of nanoAg are found
out, factors that affect the formation like change in volume of
diluted oil and change in pH are studied with in experimental
limitations.
When metal NPs are subjected to light excitation, the conduc-
tion electrons on the metal surface, are set into oscillations. The
resonance attained between the frequencies of surface electron
oscillations and light photons is localised surface plasmon
resonance (LSPR) [24] depicted through UV–vis spectral studies.
The wavelength for maximum absorption (kmax) and band width
yield a unique spectral fingerprint for a plasmonic NP with a spe-
cific size and shape [25]. The increase in intensity of the SPR band
with quantity of diluted oil shows the enhanced formation of Ag
NPs. Fig. 1a shows symmetric curves with kmax varying from 420
to 424 nm, with change in oil quantity. SPR band shows an increase
in intensity with pH (Fig. 1b) indicating rapid formation of Ag NPs.
LSPR is sensitive to size, shape, material and dielectric environment
of NPs. Surface morphology and size of the green synthesized Ag
NPs is determined through TEM analysis. TEM images of the
colloids a2 and a5 are shown in Figs. 2(a and b) and 3(a and b)
respectively at different magnifications. TEM images reveal the
presence of 12–26 nm sized Ag NPs in the colloids. TEM images fur-
ther depict the enclosure of nanoAg aggregates by a thin organic
layer (Fig. S2 of Supplemental information). SAED pattern shows
bright circular diffraction rings which can be indexed from inner
to outer as (1 1 1), (2 0 0), (2 2 0), (3 1 1) and (2 2 2) confirming the
face centred cubic crystalline structure of nanoAg.
The peaks in the X-ray diffraction pattern (Fig. 4a) are due to
reflections from the (1 1 1), (2 0 0), (2 2 0), (3 1 1), and (2 2 2) planes
confirming the face centred cubic crystalline structure of the Ag

Fig. 1. UV–vis spectra of (a) colloids a1–a5 showing the enhanced formation of silver Ag NPs with volume of diluted oil, (b) colloids a2, a6, a7, a8 showing the enhanced
formation of Ag NPs with increase in pH of the medium.

Fig. 2. TEM images of colloid a2 at different magnification. Inset figure depicts the SAED pattern of Ag NPs showing bright diffraction rings corresponding to the indexed
planes.
746 V. Vilas et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 132 (2014) 743–750

Fig. 3. TEM images of colloid a5 at different magnifications.

Fig. 4. (a) XRD pattern exhibiting the fcc structure of Ag NPs. (b) FTIR spectra of Ag NPs and diluted essential oil.

Fig. 5. UV–vis spectra showing degradation of (a) para nitro phenol and (b) methylene blue.
 V. Vilas et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 132 (2014) 743–750
Fig. 6. Linear correlation between ln[A/A0] and time depicting (a) pseudo first order
degradation of 4NP and (b) pseudo first order degradation of MB.
NPs (JCPDS file no: 04-0783). Absence of any other peak in the
diffraction pattern indicates the high purity of the sample.
FTIR spectrum of diluted oil (Fig. 4b) shows prominent bands at
1636 cm1,1510 cm1,1432 cm1and 1130 cm1. Bands at 1636
cm1and 1500 cm1 are due to C@C stretching vibrations of aro-
matic rings. The band at 1432 cm1 shows the OH deformations
in the oil which shifts to 1382 cm1 upon the formation of nanoAg.
FTIR spectrum of oil, shows a number of bands between 1300 cm1
and 885 cm1 due to CAO stretching vibrations of alcohols, ethers
and esters. A strong broad band at 1055 cm1 is observed in the
FTIR spectrum of Ag NPs. FTIR measurements are in accordance
with GC–MS results of leaf essential oil of M. fragrans. Weak
tertiary alcohols are good reducing agents at 100 °C in the presence
of acetone. This is shown by the shift from 1432 cm1 to
1382 cm1. Carbonyl group of the carboxylic acid, with high
affinity, then coordinates to Ag, reducing and stabilising them.
Fig. 7. Mueller–Hinton plates showing inhibition zones by (a) diluted essential oil, (b) silver nitrate, (c) colloid a2 and (d) colloid a5 at different concentrations against S.
aureus.
747
Reduction catalysis using Ag NPs
The rapid reduction of 4NP is followed through UV–vis spectral
analysis. Fig. 5a shows an intense band at 400 nm due to the
formation of sodium nitrophenolate ion [26] which shows a
decrease in intensity with time and a concomitant band at
296 nm indicating para amino phenol (4AP) [27–29]. Fig. 5b shows
the degradation of the cationic, water soluble dye, MB. Reduction
in the intensity of the band at 664 nm is due to the formation of
colourless leucomethylene blue [30,31]. In the absence of the
nanocatalyst, the reduction reaction proceeds at a very slow rate.
The intermediate redox potential of Ag+/Ag system with respect
to the donor – acceptor system, E0(A/A) < E0(Ag+/Ag) < E0(D+/D)
enables an efficient reaction between the donor and acceptor,
owing to the double electron transfer through the metal cluster
relay; referred to as the electron relay effect [32]. Analysis of the
kinetic data of both the degradation reactions reveals pseudo-first
order reaction kinetics, due to the constant concentration of NaBH4
taken in excess. The linear plot of ln[A/A0] versus time (Fig. 6) in
both the cases supports the kinetic studies.
Antibacterial potential of Ag NPs
The antibacterial activity of Ag NPs against two bacterial strains,
S. aureus and E. coli has been investigated. Figs. 7(a–d) and 8(a–d)
clearly depicts the enhanced activity of biogenic Ag NPs (a2)
against the bacterial strains in comparison to the diluted oil.
The marked difference in the diameter of the zone of inhibition
of the two samples a2 and a5, is attributed to a thin oil coating
around the Ag NPs in a5. However, the zone of inhibition increased
on increasing the concentration of a2 (Table 1). The green synthes-
ised Ag NPs showed greater activity against E. coli than S. aureus.
The lower efficacy of Ag NPs against S. aureus is attributed to the
difference in the membrane structure, distinctively, thickness of
the peptidoglycan layer between the two bacteria [33]. During
antibacterial action, Ag+ ions, the soft acid interacts strongly with
the phosphorus and sulphur compounds (soft base) (Figs. 9 and
10) leading to breakdown of mitochondrial function, DNA damage
748 V. Vilas et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 132 (2014) 743–750

Fig. 8. Mueller–Hinton plates showing inhibition zones by (a) diluted essential oil, (b) silver nitrate, (c) colloid a2 and (d) colloid a5 at different concentrations against E. coli.

Table 1
Inhibition zone diameter (mm) of silver nitrate solution, silver nanoparticles and
dilute oil against E. coli and S. aureus using Gentamycin as positive control.

Sample Concentration (lg) Zone of inhibition (mm)

E. coli S. aureus
Gentamycin 23 23
Silver nitrate 25 NZ NZ
50 NZ NZ
100 11 12
Colloid a2 25 10 NZ Fig. 10. Strong interaction between Ag (soft acid) and sulphur (soft base) of
50 12 10 cysteine residues of proteins [HSAB Theory] result in protein denaturation.
100 14 12
Colloid a5 25 NZ NZ
50 NZ NZ
100 NZ NZ or denaturation of protein, ultimately resulting in cell death
Dilute oil 25 NZ NZ
[34–38]. Also, ESR studies [38] have revealed the role of free radi-
50 NZ NZ cals generated from Ag NPs as a cause of its bactericidal action.
100 9 NZ Further, strong association of silver nanoparticles with the cell wall
of the bacteria may result in the formation of pits, affecting the
NZ = no zone of inhibition.
permeability and ultimately in cell death [39]. Antibacterial

Fig. 9. Strong interaction between Ag (soft acid) and phosphorus (soft base) of phosphate molecule of DNA [HSAB Theory] result in DNA damage.
 V. Vilas et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 132 (2014) 743–750
Fig. 11. Antioxidant activity of Ag NPs exhibited through the assays (a) NO scavenging activity, (b) hydrogen peroxide scavenging activity, (c) reducing power activity, (d)
DPPH assay and (e) total antioxidant activity.
activity of the silver nanoparticles depends on the available
particle surface area. Smaller particles with a larger surface area
available for interaction would show a higher antibacterial action
than larger particles [1,40].
In vitro antioxidant activity of Ag NPs
Interaction of the human system with oxygen generates free
radicals, neutralised by glutathione/GSH, the master antioxidant.
Oxidative stress induced by the changing environment and life
style result in well known and wide spread diseases. Metal NPs
are potent antioxidants that can delay the onset or slow the rate
of oxidation of autooxidizable materials.
Despite its beneficial effects, NO produces nitrite ions on react-
ing with superoxide, which may lead to tissue damage. Scavengers
of NO compete with oxygen, leading to reduced production of
nitrite ions. The NO scavenging activity of a2 and a5 increases with
increase in concentration of the samples (Fig. 11a.). Hydrogen
peroxide can cross cell membranes, rapidly and inside the cell,
on reaction with Fe2+ or Cu2+, they may form hydroxy radical, the
origin of many toxic effects. Positive hydrogen peroxide tests dem-
onstrate that Ag NPs are competent free radical scavengers
(Fig. 11b). Silver colloid a2 readily reduces Fe3+, exhibiting its
enhanced reducing power than a5 (Fig. 11c). Silver colloid a2 acts
as a better scavenger of the stable DPPH free radical. The power
to scavenge DPPH is found to increase with concentration
(Fig. 11d). An increase in optical density at 695 nm due to the
formation of green phosphomolybdenum complex, exhibits the
dose dependent antioxidant activity of the synthesized Ag NPs
(Fig. 11e).
Conclusion
Increasing global concern over environmental protection with
the emergence of non-biodegradable pollutants and antibiotic drug
resistant bacteria, urge the need for potent chemocatalytic
bioactive green synthesized nanoparticles. The study points out
the pertinence of using essential oil of M. fragrans for the synthesis
of 12–26 nm sized, spherical, crystalline Ag NPs. Terpenes and
phenyl propenes in essential oil function as reducing and capping
749
agents, as revealed by FTIR and GC–MS analysis. Ag NPs of high
purity and crystallinity are obtained as depicted by the XRD and
SAED patterns. The study demonstrates the strong chemocatalytic
potential and antibacterial activity of Ag NPs. The phytosynthe-
sized biogenic Ag NPs exhibit strong electron donating, reducing
and free radical scavenging ability. Using a refined phytosynthetic
design the study supports the potential of green synthesized
nanoAg to be used in diverse fields.
Acknowledgements
The authors are grateful for the technical supports by NIST and
CESS, Thiruvananthapuram. Vidya Vilas (one of the authors)
acknowledges KSCSTE, Govt of Kerala for the research fellowship.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.saa.2014.05.046.
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