Resource-Efficient Technologies 3 (2017) 303–308
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Resource-Efficient Technologies
journal homepage: www.elsevier.com/locate/reft
Research paper
Green synthesis of silver nanoparticles using Capsicum frutescence and
its intensified activity against E. coli
✩
Thangaraj Shankar
a
, Perumal Karthiga
a
, Kalaiyar Swarnalatha
a , ∗
, Kalaiyar Rajkumar
b
a
Manonmaniam Sundaranar University, India
b
Madras Veterinary College, India
a r t i c l e i n f o
Article history:
Received 25 July 2016
Revised 7 November 2016
Accepted 17 January 2017
Available online 6 March 2017
Keywords:
Silver nanoparticles
Sweet pepper fruit extract
Biosynthesis
Bactericidal
Capsicum frutescence
a b s t r a c t
The purpose of this study was to expand a trouble free biological method for the synthesis of silver
nanoparticles using the fruit extract of Capsicum frutescence (Sweet pepper) to act as reducing and stabi-
lizing agent. Water soluble organics played a vital role for the reduction silver ions into silver nanopar-
ticles. The fruit extract was exposed to silver ions and the resultant biosynthesize d silver nanoparti-
cles characterized by UV–Vis spectrophotometry indicated the surface plasmon resonance band at 385–
435 nm. X-ray diffraction spectrum showed crystalline structure while scanning electron microscope anal-
yses exposed the monodispersed distribution and particle size of 20–25 nm. The elemental analysis dis-
played strong signal at 3 keV that agrees to silver ions and confirms the presence of metallic silver.
The antibacterial activity of silver nanoparticles was determined by agar well diffusion method against
gram positive and gram negative bacteria. Maximum and minimum zones of inhibition were renowned
against Escherichia coli (11.5 mm) and Bacillus subtilis (10.5 mm), respectively. This study exposed that sil-
ver nanoparticles retained good bactericidal activity at 80 μg/ml concentration.
©2017 Tomsk Polytechnic University. Published by Elsevier B.V.
This is an open access article under the CC BY-NC-ND license.
(
http://creativecommons.org/licenses/by-nc-nd/4.0/ )
1. Introduction
The exploit of green chemistry for the synthesis of biocompati-
ble silver nanoparticles has gained substantial awareness in the lat-
est years for prospective appliance in biomedicine. Metal nanopar-
ticles are of interest in both research and technology, due to their
particular properties not offered in isolated molecules or bulk met-
als. Because of these properties nanoparticles have many impera-
tive applications in catalysis, sensing and imaging etc. Among the
gracious metals (e.g. Ag, Pt, Au and Pd), silver (Ag) is the metal
of abundance for prospective applications in the field of biological
systems, living organisms and medicine. Due to their elite proper-
ties, silver nanoparticles (AgNPs) may have quite a lot of applica-
tions, such as catalysts in chemical reactions
[1,2] electrical bat-
teries and in spectrally discriminative coatings for absorption of
solar energy [3,4] as optical elements, pharmaceutical works and
in chemical sensing and biosensing
[5–7] . The pace of synthesis
✩
Peer review under responsibility of Tomsk Polytechnic University.
∗
Corresponding author. Manonmaniam Sundaranar University, Tirunelveli, India.
Fax: 0462 2334363.
E-mail address:
swarnalatha@msuniv.ac.in (K. Swarnalatha).
of nanoparticles through plant extract is as good as to those of
chemical methods and more rapidly than green synthesis by mi-
croorganisms.
The plant used in this research, belongs to the genus Cap-
sicum frutescence (Sweet pepper) in the family Solanaceae
[8] . The
sharp taste of Capsicum peppers is due to a fusion of seven al-
lied alkaloids of which capsaicin is the most ubiquitous. The sub-
stances responsible for the pungency are the capsaicinoid alkaloids.
They are characterized by means of a high biological activity and
their pharmacological, neurological and dietetic activities are well
known. When used at minimum levels in the usual diet, they ex-
tensively decrease serum, myocardial and aortic entire cholesterol
levels [9] . The biological activity mainly predicts the bactericidal
activity. Capsicum frutescence fruit can be used for the synthesis
of silver nanoparticles, since this fruit extract contains many sec-
ondary metabolites which potentially act as a best reducing and
stabilizing agent of the silver ions. In this paper we report the
synthesis of silver nanoparticles by reduction of Ag
+
with C. frutes-
cence fruit aqueous extract. The formation of the nanoparticles was
recorded by UV-Vis spectroscopy, whereas the size and shape were
determined by scanning electron microscope (SEM). The relation
of nanoparticles with C. frutescence fruit extract was confirmed
https://doi.org/10.1016/j.reffit.2017.01.004
2405-6537/© 2017 Tomsk Polytechnic University. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license.
(
http://creativecommons.org/licenses/by-nc-nd/4.0/ )
304 T. Shankar et al. / Resource-Efficient Technologies 3 (2017) 303–308
using FT-IR spectroscopy. The crystallite size and elemental posi-
tion were authenticated via XRD and EDAX. The bactericidal effect
of fruit extract and silver nanoparticles were evaluated with the
help of suitable clinical pathogens like Escherichia coli and Bacillus
subtilis .
2. Experimental methods
2.1. Chemicals
All the chemicals used for synthesis [Silver nitrate (AgNO
3
),
Potassium Bromide (KBr)], were of analytical grade from Merck
Limited, Mumbai, India. Nutrient Agar, Nutrient Broth, Agar Agar,
Muller Hinton Agar (MHA) purchased from Himedia Laboratories,
Mumbai, India. The aqueous solutions were prepared using triple
distilled water.
2.2. Preparation of fruit extract
Fresh and healthy fruits of Capsicum frutesence ( Fig. 1 ) were
washed several times with deionized water to remove the filth par-
ticles and then air dried to remove the residual moisture and cut
in to small pieces. Twenty-five grams of fruit in a round bottomed
flask with 100 ml deionized water and refluxed for half an hour,
cooled at room temperature and filtered with Whatman No.1 filter
paper.
2.3. Biosynthesis of silver nanoparticles
The nanoparticles were synthesized by known concentration of
C. frutesences broth was interacted with 1 mM silver nitrate. For the
reduction of silver ions, 10 ml of fruit extract make up to 100 ml
volume in 250 ml Erlenmeyer flask. The flask was incubated in a
rotary shaker at 150 rpm speed for a desired time at room temper-
ature for the development of silver nanoparticles.
2.3.1. Characterization of synthesized silver nanoparticles
The nanoparticle solution thus obtained was purified by re-
peated centrifugation at 20,0 0 0 rpm for 30 min followed by re-
dispersion of the pellet in distilled water. UV-vis spectra were
recorded as a function of reaction time on a Perkin Elmer UV-Vis
spectrophotometer operated at resolution of 1 nm. After drying of
the purified silver particles, the structure was predicted by scan-
ning electron microscopy (SEM, Hitachi S-2500C), energy disper-
sive X-ray spectroscopy (EDS, Sigma). The crystalline character of
silver nanoparticles was confirmed with the help of XRD. The X-ray
patterns were obtained in 2 theta configuration and the range was
selected from 20 ° and 80 °. These were performed by using Pana-
Fig. 1. Fresh and healthy fruits of Capsicum frutescence.
lytical X’pert Powder X’ Celerator Diffractometer. The alterations in
the chemical group were confirmed by Fourier transform infra red
spectroscopy (FT-IR, Jasco) by employing KBr pellet technique. The
FT-IR spectra were taken at a resolution of 4 cm
1
in the transmis-
sion mode (40 0 0-40 0 cm
−1
).
2.3.2. Antibacterial activity of fruit extract and nanoparticles
The bacterial strains used were E. coli and B. subtilis. The strains
were obtained from the Department of Microbiology, Sri Para-
makalyani College, Alwarkurichi, Tamil Nadu. Stock cultures were
maintained at 4 °C on slopes of nutrient agar. Dynamic cultures for
experiments were prepared by transferring a loopful of cells from
the stock cultures to test tubes of Mueller Hinton Broth (MHB)
for bacteria. The cultures were incubated for 24 hours at room
temperature. Agar well diffusion method [10] for bactericidal sus-
ceptibility was carried out according to standard method to as-
sess the presence of antibacterial activity of the synthesized sil-
ver nanoparticle. The concentration of the nanoparticle used in
the experiment was 20, 40, 60 and 80 μL. Well of about 6 mm
diameter were made aseptically using gel puncture instrument.
The plates were swabbed with gram negative strains like E. coli
and a gram positive strain B. subtilis . Then the plates were incu-
bated at 37 °C for 24 hours. Antibacterial activity was evaluated
by measuring the diameter of the zone of inhibition around the
well.
3. Results and discussion
3.1. Visual observation and UV-vis spectral analysis
The optical properties of silver nanoparticles were studied by
absorption spectroscopy. The structural change of the particles can
be easily examined by the UV-Visible absorption spectrum, which
can help us to know the complex formation. It is the primary
method to indicate the bioreduction of silver from aqueous sil-
ver nitrate solution to silver nanoparticles. Surface Plasmon reso-
nance bands play a vital role in size, shape, morphology
[11] . The
synthesized Ag NPs exhibit reddish brown color due to the exci-
tation of surface Plasmon resonance in Ag NPs. The optical ab-
sorption spectra of metal nanoparticles is dominated by the SPR,
which shows a shift toward the red end or blue end depending
upon the particle size, shape, state of aggregation and the sur-
rounding dielectric medium [12] . After 24 hr the settling of syn-
thesized silver nanoparticles at the bottom of the Erlenmeyer flask
reveals the reduction of silver metal into silver nanoparticles was
completed.
The secondary metabolite capsaicin alkaloid and other antioxi-
dants present in the fruit extract acting both as reducing and stabi-
lizing agent to form the nanoparticles. The reduction of silver ions
and the development of nanoparticles occurred within hour due to
excitation of surface Plasmon vibrations in the nanoparticles [13] .
Different concentrations of silver nitrate were taken for the study
to synthesize silver nanoparticles were analyzed using UV spec-
tra of resonance band at around 380–450 nm but 1 mM shows the
band at around 385 nm may be due to the strong activity of cap-
saicin. 2 mM- 5 mM concentration Plasmon band were similar to
previously reported literature [14] . If we increase the silver nitrate
concentration simultaneously, there is increase in wavelength up to
450 nm shown in Fig. 2 . The slight variation leads to slight changes
in shape and particle size. The results were similar to the reported
literature with slight variations
[15] . The intensity of the SRP peaks
increases as reaction time increases, which designates the increase
in concentration of the silver nanoparticles. The result reflects that
the Ag nanoparticles prepared by Capsicum frutesence fruit extract
T. Shankar et al. / Resource-Efficient Technologies 3 (2017) 303–308 305
Fig. 2. UV-Vis spectra of biosynthesized silver nanoparticles at different concentra-
tions from 1 mM to 5 mM.
are constant without aggregation This similar report were matched
with Psidium guajava leaf extract
[16] .
3.2. Fourier transform infrared analysis
FTIR spectroscopic studies were carrie d out to find out the pos-
sible chemical changes present in the extract. The spectra were
recorded before and after addition of silver nitrate solution. The
broad and narrow peaks of the fruit extract and nanoparticles
shown in
Fig. 3 . The peak at 3271 cm
−1
belongs to N
–
H stretching
of amine and the weak band at 2913.07 cm
−1
indicates the H
–
C
–
H
symmetric stretching of alkanes. Capsaicin which is alkaloids hav-
ing N
–
H stretch, this specific compound involved in the synthesis
and act as a backbone for the nanoparticles formation. The band
at 1016.90 cm
−1
corresponds to C
–
O stretching in fruit extract. Si-
multaneously in nanoparticles the band at 3251 cm
−1
shows the
hydrogen bonded O
–
H stretch phenols and alcohols. The band at
2923.64 cm
−1
and 2853.07 cm
−1
represents the H
–
C
–
H symmetric
stretching of alkanes. The strong peak at 1020.03 cm
−1
denotes the
C
–
O stretching of ethers. Some of the secondary metabolites, pro-
teins may also bind with the silver ions to the formation of silver
nanoparticles.
3.3. XRD analysis
The crystalline character of silver nanoparticles confirmed from
the X-ray diffraction (XRD) pattern. The prominent peaks [38.48 °,
44.39 °, 64.92 °,77.67 °] are indexed as (111), (200), (220), (311)
shown in
Fig. 4 . These peaks indicates that the crystals are
anisotropic reported by Daizy Philip
[17] . The average size of
nanoparticles is calculated by Scherrer’s equation by determin-
ing the width of the prominent peak which was found to be
19 nm also which was agreed with the SEM measurement. Gen-
erally, the broad peak in this pattern will attribute the size of
the particles
[18] . The obtained XRD pattern was compared and
matched with the joint committee powder diffraction standards
JCPDS file No. 04–0783. It might be considered that the unas-
signed peaks are owing to the crystallization of bioorganic phases
that occur on the surface of the silver nanoparticles
[19] .Our
results were well agreed with Calliandra haematocephala leaf
extract [20] .
Fig. 3. FT-IR spectra of the aqueous fruit extract and biosynthesized silver nanopar-
ticles.
3.4. Energy dispersive X-ray spectroscopy
This technique was to verify the presence of specific element
and it showed some small peaks along with the specific element as
C and O. The peak observed around 3 KeV shown in
Fig. 5 predicts
the binding energy of AgL which proves the confirmation of pure
silver due to the surface plasmon resonance [21] . Some weaker
elements like C and O also appeared due to the minor impuri-
ties. The amount of energy released by transferring electrons de-
pends on which shell its transferring from as well as which shell it
is transferred to furthermore, the atom of every element releases
x-ray with its unique amount of energy during the transferring
process.
3.5. Scanning electron microscopy
SEM Analysis is used to visualize the size and shape of the
nanoparticles. SEM micrographs of silver nanoparticles are given
in
Fig. 6 with different magnifications. The absorption of Ag NPs
shows the broad peak which represents that the particles were
in monodispersed in nature. In this, the secondary metabolites
Fig. 4. XRD pattern of biosynthesized silver nanoparticles using fruit extract of Cap-
sicum frutescence.
306 T. Shankar et al. / Resource-Efficient Technologies 3 (2017) 303–308
Fig. 5. EDX profile of biosynthesized silver nanoparticles at 3 KeV.
present in the plant also plays a vital role in the morphological
changes. Among the secondary metabolites capsaicin, this was the
major compound present in the fruit extract of C. frutescence in-
volved in the synthesis part. The particles get aggregated with one
another and the image was taken after 24–48 hr, this similar re-
sult was discussed by Chandran et al.
[22] . However the particles
aggregate may be due to cross linking. The particle size obtained
from SEM images is well correlated with the particle size deter-
mined from XRD using according to the Scherrer formula and the
average of the synthesized nanoparticles was in the range of 15–
20 nm.
3.6. Bactericidal efficacy of fruit extract and silver nanoparticles
Silver nanoparticles interactions toward the clinical pathogens
are depends on the size and shape of the nanoparticles [23] . An-
tibacterial activity is investigated against E. coli and B. subtilis for
silver nanoparticles and fruit extract by well diffusion and disc
method. It is well known E. coli is the common clinical pathogen
causes intestinal infection includes diarrhea, abdominal pain, and
fever. More severe cases can lead to bloody diarrhea, dehydra-
tion, or even kidney failure. So the specific strain was selected.
Table 1
Antibacterial activity of fruit extract and silver nanoparticles with positive and neg-
ative controls against the human clinical pathogens ( E. coli and B. subtilis ).
S. no Antibacterial agents Escherichia coli Bacillus subtilis
1 Fruit extract 8.8 ± 1 7.4 ± 1
2 Silver nitrate 9.9 ± 1 8.5 ± 1
3 Silver nanoparticles 14.5 ± 1 10.5 ± 1
4 Antibiotic disc 11.1 ± 1 9.8 ± 1
The increased zone of inhibition was good at gram negative bac-
teria E. coli when compared to B. subtilis . The zone of inhibition
around each well with silver nanoparticles and fruit extract for
both strains represented in
Figs. 7 and 8 . The zone of inhibition is
also clearly reported in
Table 1 . Positive and negative control was
used against both strains. The mechanisms of antibacterial activ-
ity of silver nanoparticles are by binding on the membrane of mi-
croorganisms through electrostatic interactions, cell wall disruption
andaffecting the intracellular processes such as DNA, RNA and pro-
tein synthesis
[24–27] . It shows immense possibilities in biomedi-
cal applications. Similar observations were found with Allium cepa
[28] .
T. Shankar et al. / Resource-Efficient Technologies 3 (2017) 303–308 307
Fig. 6. SEM images of silver nanoparticles synthesized using Capsicum frutescence (a) 1 μm (b) 500 nm and (c) 400 nm.
Fig. 7. Antibacterial activity of fruit extract FE (a) and silver nanoparticle Ag NP (b)
against Escherichia coli.
Fig. 8. Antibacterial activity of fruit extract FE (a) and silver nanoparticle Ag NP (b)
against B. subtilis.
4. Conclusion
In the present study we found that fruits also act as a best
source for the formation of silver nanoparticles. This green chem-
istry approach toward the nanoparticles has immense merits
like economic viability. The green synthesized silver nanoparti-
cles show excellent bactericidal activity against the gram negative
bacteria and moderate activity against the gram positive bacteria.
Our findings indicating that biosynthesized silver nanoparticles us-
ing the plant source will afford unique opportunities toward the
growth of nanomedicine and thus has the budding for utilize in
biomedical applications.
Acknowledgement
Author, PK gratefully acknowledges the UGC-PDFWM, University
Grants Commission, NewDelhi, India for funding research devel-
opment (Ref No:
F. 15-1/2012-13/PDFWM-2012-13-SC-TAM-21783 )
(SA-II) – dated 18.04.2014. We would like to acknowledge the sup-
port of Dr. G.Annadurai , Centre for Environmental Sciences, Al-
warkurichi, for microbiological studies.
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