Colloids and Surfaces B: Biointerfaces 108 (2013) 80–84

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Colloids and Surfaces B: Biointerfaces

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

Origanum vulgare mediated biosynthesis of silver nanoparticles for its

antibacterial and anticancer activity
Renu Sankar a , Arunachalam Karthik a , Annamalai Prabu a , Selvaraju Karthik a ,
Kanchi Subramanian Shivashangari b,∗∗ , Vilwanathan Ravikumar a,∗
a
Department of Biochemistry, School of Life Science, Bharathidasan University, Tiruchirappalli 620024, Tamilnadu, India
b
Regional Forensic Science Laboratory, Tiruchirapalli, Tamilnadu, 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, we achieved silver nanoparticles using the aqueous extract of Origanum vulgare
Received 28 December 2012 (Oregano) by reducing 1 mM silver nitrate (AgNO3 ) solution. The green synthesized silver nanopar-
Accepted 23 February 2013 ticles were characterized by high throughput techniques like UV–vis spectroscopy, Fourier infrared
Available online 4 March 2013
spectroscopy (FT-IR), field emission-scanning electron microscopy (FE-SEM), X-ray diffraction (XRD)
and dynamic light scattering measurements. Morphologically, the nanoparticles were found to be
Keywords:
spherical with an average particle size distribution of 136 ± 10.09 nm. FT-IR spectral analysis illus-
Silver nanoparticles
trates the occurrence of possible biomolecules required for the reduction of silver ions. The obtained
Origanum vulgare
FT-IR
nanoparticles were stable (-26 ± 0.77 mV) at ambient temperature. The biosynthesized nanoparticles
Human pathogens were found to be impressive in inhibiting human pathogens. The green synthesized silver nanopar-
Dose–response activity ticles showed dose dependent response against human lung cancer A549 cell line (LD50 – 100
␮g/ml).
© 2013 Elsevier B.V. All rights reserved.

1. Introduction Significant attempts have been made to synthesize nanoparticles

Recent advancement in nanotechnology has engineered nano-
materials that are potentially safe toward human welfare. The
application of nanotechnology is endless with a multidisciplinary
facet including molecular diagnostics, catalysis, electronics, drug
delivery, sensing and surfaces-enhanced Raman scattering [1]. The
early phase of this technology blends both physical and chemical
methods for the synthesis of nanomaterials using toxic chemicals
and harsh reaction conditions [2,3]. These nanostructures have the
tendency to release harmful byproducts into the environment lead-
ing to toxicological issues. To overcome this, there is a need for
clean, non-toxic, bio-compatible and environment friendly mate-
rials synthesized via ‘green’ approach [4]. The ability plants to
produce nanoparticles have featured exciting loom toward the
escalation of natural nano-factories. This phenomenon has brought
an insight among researchers for development of molecules which
are precise and efficient by using an elegant and ingenious methods.
∗ Corresponding author at: Department of Biochemistry, School of Life Science,
Bharathidasan University, Tiruchirappalli 620024, Tamilnadu, India.
Tel.: +91 431 2407071.
∗∗ Corresponding author at: No-75, Second Street, Ashok Nagar, Kanchipuram 631
502, Tamil Nadu, India. Tel.: +91 44 2726292.
E-mail addresses: shivashangari@gmail.com (K.S. Shivashangari),
ravikumarbdu@gmail.com (V. Ravikumar).
such as silver, gold, platinum, palladium, etc. using plants of natural
origin [5]. These prepared nanomaterials compete with chemical
methodologies in terms of size, shape and exhibit advanced prop-
erties. Based on the available literatures, silver nanoparticles have
drawn the curiosity of researchers ever since their application in
the field of antimicrobials, integrated circuits, sensors, biolabelling,
filters and biomedical devices [6]. Moreover, silver compounds are
non-toxic to the human body at low concentration compared to
other noble metals [7].
This paper focus on exploiting Origanum vulgare which is
untapped for green synthesis of nanoparticles. O. vulgare is a tem-
perate culinary herb traditionally used worldwide for its medicinal
property respiratory disorders, indigestion, dental caries, rheuma-
toid arthritis and urinary tract disorders [8]. Earlier studies have
demonstrated that, the plant possesses anti-hyperglycemic, anti-
oxidant, anti-mutagenic, anti-fungal, anti-inflammatory, anti-viral
and potent anti-bacterial properties [9]. Herein, we report the
green synthesis of silver nanoparticles by reducing the silver
nitrate solution using an aqueous leaf extract of O. vulgare. The
green synthesized nanoparticles possess improved antibacterial
activity against human pathogenic bacterial strains compared to
standard reference drug (Chloramphenicol). Cytocompatability of
the nanoparticles was also evaluated against human lung cancer
A549 cell line. Indeed, our results provide conclusive evidence of
the cytotoxic effect against cancer cell line.

0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.colsurfb.2013.02.033
 R. Sankar et al. / Colloids and Surfaces B: Biointerfaces 108 (2013) 80–84 81

2. Materials and methods

2.1. Green synthesis of silver nanoparticles

The collected O. vulgare leaves were washed thoroughly with

distilled water and shade dried for 2–3 weeks. The dried leaves were
powdered to fine particles using a mixer grinder. For the prepara-
tion of aqueous leaf extract (ALE), 10 g of leaf powder was dissolved
in 100 ml of deionized water followed by boiling at 60 ◦ C for 10 min.
The ALE was allowed to cool, filtered (Whatman No.1 filter paper –
Fig. 1. Color change.
0.44 ␮m) and refrigerated until further use. For the green synthesis
of AgNPs to occur, 90 ml of 1 mM silver nitrate (AgNO3 ) solution
was mixed with 10 ml of O. vulgare leaf extract. The amalgamate further incubated for 4 h and then 200 ␮l of DMSO was added. The
was heated gradually at 60–90 ◦ C for 10 min using a water bath. formed crystals were dissolved gently by pipetting two to three
The transformation of pale to reddish brown color indicates the times slowly. The absorbance at 570–620 nm was measured using
formation of AgNPs. plate reader.

2.2. Characterization of silver nanoparticles 2.6. Statistical analysis

UV spectrum was recorded using a UV–vis double beam spec-
trophotometer (UV-1601, Shimadzu, Japan). Fourier transform
infrared spectroscopy (FT-IR) was performed by spectrum RX
1 instrument in diffuse reflectance mode operated at a resolu-
tion of 4 cm−1 of wavelength of about 4000–400 cm−1 . FE-SEM
images were acquired by Carl Zeiss, SIGMA instrument (UK). X-
ray diffraction analysis (Philps X’Pert Pro X-ray diffractometer) was
performed by preparing a thin film of powdered AgNPs. To study
the average particle size distribution and stability of nanoparticles
a Malvern Zetazier (Nano ZS90, UK) instrument was used.
2.3. Agar well diffusion assay
The antibacterial activity of green synthesized AgNPs was per-
formed by Agar well diffusion method [10]. A total of nine bacterial
strains namely Aeromonas hydrophilla, Bacillus sps., Escherichia coli,
E. coli (Enteropathogenic–EP), Klebsiella sps., Salmonella sps.,
Salmonella paratyphi, Shigella dysenteriae, Shigella sonnei were pro-
cured from the Department of Biomedical Science, Bharathidasan
University, Tiruchirappalli, India. Stock cultures were maintained at
4 ◦ C on agar slants of nutrient media. Prior to the experiment, pure
cultures were sub cultured onto Muller Hinton broth and incubated
overnight at 37 ◦ C. Later Muller Hinton agar plates were prepared
and punctured for wells and loaded with 30 ␮l (0.3 mg/ml) of test
sample (AgNPs). After 24 h of incubation the plates measured for
Zone of Inhibition (ZoI) using a vernier caliper in triplicate. Chlor-
amphenicol was used as a positive control.
2.4. Cell culture
The statistical analysis was done using SPSS software Version 16
(SPSS Inc., Chicago, IL, USA). The one-way ANOVA was done for the
expressing significance of the present study. Statistical significance
was accepted at a level of p < 0.05.
3. Results and discussion
3.1. Green synthesis of silver nanoparticles
Current research on green synthesis of nanomaterials using
plant extracts has opened a new era. Many researchers have
reported, the biosynthesis of metal nanoparticles by means of aque-
ous extracts of plants for several applications [12]. The present
study deals with the synthesis of silver nanoparticles using aque-
ous leaf extracts of O. vulgare. Temperature dependent approach
was exercised to fabricate nanoparticles by reducing silver nitrate
(AgNO3 ) solution. It has been observed that, the increase in temper-
ature results in the rapid biosynthesis of nanoparticles [13]. At this
junction, the conversion rate of silver ions to silver nanoparticles
at 60 ◦ C is 45% while 100% at 90 ◦ C respectively (data not shown).
This is further confirmed by the formation dark brown color (Fig. 1)
in the colloidal solution due to excitation of surface plasmon vibra-
tions [14]. No color change was observed 1 mM AgNO3 (control)
solution (Fig. 1).
3.2. Characterization of silver nanoparticles
The UV–vis spectrum in Fig. 2 showed surface plasmon reso-
nance (SPR) peak bands centered at 440 nm. The occurrence of the

Human lung cancer A549 cell line were obtained from the
National Center for Cell Science, Pune, India. It was cultured in Dul-
becco’s modified Eagle’s medium (DMEM: Hi Media Laboratories
Mumbai, India), supplemented with 10% fetal bovine serum and 1%
penicillin/streptomycin (Hi Media Laboratories Mumbai, India).

2.5. MTT assay

MTT (dimethyl thiazolyltetrazolium bromide) assay was per-

formed by using the method of Yuan et al. [11]. Briefly, cells in
suspension containing approximately 1 × 104 were added to each
well of a 96-well culture plate and incubated for 24 h at 37 ◦ C in a
humidified atmosphere of 95% air and 5% CO2 . Human lung A549
cancer cells were treated with a series of 10–500 ␮g/ml concentra-
tion of green synthesized AgNPs. Control cultures were treated with
DMSO. The cultures were again incubated as above. After 36 h, 20 ␮l Fig. 2. UV spectral peak of temperature dependent silver nanoparticles synthesized
of MTT solutions were added to each well and the cultures were at 90 ◦ C.
82 R. Sankar et al. / Colloids and Surfaces B: Biointerfaces 108 (2013) 80–84
Fig. 3. FT-IR spectrum.
absorption peak reflects the shape and size of the nanoparticles. We
believe that, formation of the absorption peak with a decrease in
band width and increased band intensity is a sign of smaller, spher-
ical shaped particle with some agglomeration [15]. The slow but
sure increase in temperature is a key feature suggesting an increase
in the concentration of silver nanoparticles. Besides, the frequency
and width of the surface plasmon absorption the dielectric function
of silver almost disappeared [16]. It is noteworthy that the presence
of proteins in the aqueous extract of O. vulgare leaf extract be able
to hydrolyze metal precursors to form metal oxides extracellularlly
and were further confirmed by FTIR analysis.
FT-IR has become an important tool in understanding the
involvement of functional groups in relation between metal par-
ticles and biomolecules. In the present study, FT-IR measurements
were performed to identify the possible biomolecules respon-
sible for capping and stabilizing the silver nanoparticles. Fig. 3
shows spectral peaks suggesting, the occurrence of bands rele-
vant to amide N-H stretching (3410 cm−1 ), nitrile C N stretching
(2250 cm−1 ) and aromatic C C bending (1604 cm−1 ). The exist-
ence of the band at 1393 cm−1 and 1048 cm−1 suggested a strong
acquaintance between C–O [17]. These functional groups may have
an effective role in the green synthesis of silver nanoparticles. But,
the possible mechanism is unclear and needs further investigation.
Field emission-scanning electron microscope (FE-SEM) was used
to understand the surface morphology of silver nanoclusters [12].
FE-SEM images of synthesized nanoparticles using O. vulgare plant
leaf extract were found to be spherical in shape with agglomeration
(Fig. 4). The size of silver nanoparticles was found to be 63–85 nm
lacking monodispersity. We accept as true that, higher concentra-
tion of bioactive compounds in the colloidal solution might cause
Fig. 4. Field emission-scanning electron microscopic studies.
Fig. 5. X-ray diffraction analysis.
the formation of nanoclusters. It has been reported that the shape
of the metal nanoparticles has considerably changed their optical
and electronic properties [18]. Moreover, there is a point of view
for nanoparticle to congregate or assemble each other at higher
intensification by microscope.
XRD analysis showed three distinct diffraction peaks at 38.080◦ ,
64.560◦ and 77.640◦ that indexed the planes 1 1 1, 1 1 3 and 0 8 0
of face-centered-cubic structure of silver [19]. The XRD patterns
clearly reveal the biosynthesized silver nanoparticles are crys-
talline in nature (Fig. 5). Scherrer’s formula disclosed the size of the
nanoparticles is about 65 nm substantiating electron microscopic
studies. The average size distribution of silver nanoparticles in col-
loidal solution was found to be 136 ± 10.09 nm (Fig. 6a). A negative
zeta potential of about −26.0 ± 0.77 mV was observed in the present
study that pose ideal surface charge (Fig. 6b). High absolute value
of zeta potential designate high electrical charge on the surface of
the nanoparticles, which can cause strong repulsive force among
particles to withhold agglomeration [20]. It is also demonstrated
that a high zeta potential value of about −25 mV ensures a high
energy barrier for the stabilization of nanosuspension. [21].
3.3. Antibacterial activity
Silver nanoparticles pretense to have strong bactericidal activ-
ity against gram-negative and gram-positive bacteria including
multidrug resistant strains [22]. In the present study, the silver
nanoparticles showed more than 10 mm zone of inhibition against
E. coli, E. coli (EP), A. hydrophila, Salmonella sps., S. dysenteriae, S.
paratyphi and Sh. Sonnei (Supplementary Fig. S1). A zone of less
 R. Sankar et al. / Colloids and Surfaces B: Biointerfaces 108 (2013) 80–84
Fig. 6. Dynamic light scattering measurements (a) particle size distribution analysis; (b) zeta potential measurement.
Fig. 7. In vitro cytotoxicity of the silver nanoparticles.
than 10 mm was inferred in Bacillus sps. and Klebsiella sps. Com-
paratively, chlormaphenicol was used as a positive control. It is
clear from the study, silver nanoparticles by green synthesis can
compete commercial antimicrobial agents (antibiotics) used for the
treatment of bacterial infections. Till date, quite a few method-
ologies have been put forth to explain the possible mechanism
involved in the antibacterial action of nanosilver [7]. In some case,
smaller nanoparticles induces more cell permeability by crafting
intracellular lose and thereby leading to cell death [23]. Altogether,
nanosilver may impinge the cell viability in response with sulfur-
containing proteins that lined with bacterial cell wall. It is also
believed that, DNA loses its replication ability and the cellular pro-
teins become inactive on silver ion treatment [24].
Supplementary Fig. S1 associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.
2013.02.033.
3.4. Cytotoxicity studies
In vitro cytotoxicity of the silver nanoparticles was evaluated
against human lung cancer A549 cells at different concentra-
tions (10–500 ␮g/ml). Our results unveils, that there is direct
dose-response relationship with tested cells at higher concentra-
tions. In relation to cell death, a minimum of 100 ␮g/ml of silver
nanoparticles is well enough to induce 50% of cell mortality. In
the present study, the silver nanoparticles were able to inhibit the
growth of cell line by 6% at low concentration. In contrast, the pres-
ence of 500 ␮g/ml of silver nanoparticles significantly inhibits the
cell growth by more than 85% (Fig. 7). A few in vitro studies ana-
lyzed the translocation of silver nanoparticles in cancer cell line
83
with a LD50 value of 300 ␮g/ml [25]. In fact, silver nanoparticles
may induce reactive oxygen species and cause damage to cellular
components leading to cell death [26]. In recent, Patel et al. [27]
have reported the anti-proliferative effects of carvacrol isolated
from O. vulgare against human prostrate cancer cell line, LNCap.
In the same way, the extracts of O. vulgare exhibited pronounced
anticancer effect on HeLA cell line [28]. Nevertheless, this is the first
report on cytotoxic effects of green synthesized silver nanoparti-
cles using O. vulgare plant extract against human lung cancer A549
cells. The improved cytotoxic effects of O. vulgare may be due to
the presence of bioactive compounds such as carvacrol, terpinen,
thymol, sabinine, linolool, terpinolene, quercetin, apigenin as cap-
ping agents in green synthesis of silver nanoparticles [29]. Further,
investigations are underway to identify the possible mechanism
involved in the anticancer activity.
4. Conclusion
To conclude, we report a simple, speedy and efficient green syn-
thesis of silver nanoparticles from the O. vulgare plant leaf extract.
The characterization with UV–vis spectroscopy, Fourier transmis-
sion infrared (FT-IR), field emission-scanning electron microscopic
(FE-SEM), X-ray diffraction (XRD) and dynamic light scattering
(DLS) analysis evidence the formation of nanoparticles. The synthe-
sized silver nanoparticles showed promising antimicrobial activity
against human pathogenic bacterial strains. To this end, in vitro
cytotoxic activity of green synthesized silver nanoparticles against
human lung cancer A549 cell line was remarkable with 50% of mor-
tality at 100 ␮g/ml.
84 R. Sankar et al. / Colloids and Surfaces B: Biointerfaces 108 (2013) 80–84
Acknowledgement
We are grateful to Department of Science and Technology (DST)
for providing financial assistance to Mr. Renu Sankar through
INSPIRE Fellowship scheme. We also extend our acknowledge-
ment to University Grant Commission (UGC), Science & Engineering
Research Board (SERB) and Department of Science and Technol-
ogy - Fund for Improvement of S & T Infrastructure in Universities
and Higher Educational Institutions (DST-FIST)for their financial
support.
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