Synthesis of silver nanoparticles using Dioscorea bulbifera tuber extract and evaluation of its synergistic potential in combination with antimicrobial agents.

Abstract

Development of an environmentally benign process for the synthesis of silver nanomaterials is an important aspect of current nanotechnology research. Among the 600 species of the genus Dioscorea, Dioscorea bulbifera has profound therapeutic applications due to its unique phytochemistry. In this paper, we report on the rapid synthesis of silver nanoparticles by reduction of aqueous Ag(+) ions using D. bulbifera tuber extract.
Phytochemical analysis revealed that D. bulbifera tuber extract is rich in flavonoid, phenolics, reducing sugars, starch, diosgenin, ascorbic acid, and citric acid. The biosynthesis process was quite fast, and silver nanoparticles were formed within 5 hours. Ultraviolet-visible absorption spectroscopy, transmission electron microscopy, high-resolution transmission electron microscopy, energy dispersive spectroscopy, and x-ray diffraction confirmed reduction of the Ag(+) ions. Varied morphology of the bioreduced silver nanoparticles included spheres, triangles, and hexagons. Optimization studies revealed that the maximum rate of synthesis could be achieved with 0.7 mM AgNO(3) solution at 50°C in 5 hours. The resulting silver nanoparticles were found to possess potent antibacterial activity against both Gram-negative and Gram-positive bacteria. Beta-lactam (piperacillin) and macrolide (eryth-romycin) antibiotics showed a 3.6-fold and 3-fold increase, respectively, in combination with silver nanoparticles selectively against multidrug-resistant Acinetobacter baumannii. Notable synergy was seen between silver nanoparticles and chloramphenicol or vancomycin against Pseudomonas aeruginosa, and was supported by a 4.9-fold and 4.2-fold increase in zone diameter, respectively. Similarly, we found a maximum 11.8-fold increase in zone diameter of streptomycin when combined with silver nanoparticles against E. coli, providing strong evidence for the synergistic action of a combination of antibiotics and silver nanoparticles.
This is the first report on the synthesis of silver nanoparticles using D. bulbifera tuber extract followed by an estimation of its synergistic potential for enhancement of the antibacterial activity of broad spectrum antimicrobial agents.

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synthesis of silver nanoparticles using
Dioscorea bulbifera tuber extract and evaluation
of its synergistic potential in combination
with antimicrobial agents
http://dx.doi.org/10.2147/IJN.S24793
sougata Ghosh1
sumersing Patil1
Mehul Ahire1
rohini Kitture2
sangeeta Kale3
Karishma Pardesi4
swaranjit s cameotra5
Jayesh Bellare6
Dilip D Dhavale7
Amit Jabgunde7
Balu A chopade1
1Institute of Bioinformatics and
Biotechnology, University of Pune,
Pune, 2Department of electronic
science, Fergusson college, Pune,
3Department of Applied Physics,
Defense Institute of Advanced
Technology, Girinagar, Pune,
4Department of Microbiology,
University of Pune, Pune, 5Institute
of Microbial Technology, chandigarh,
6Department of chemical engineering,
Indian Institute of Technology,
Mumbai, 7Garware research centre,
Department of chemistry, University
of Pune, Pune, India
correspondence: Balu A chopade
Institute of Bioinformatics and
Biotechnology, University of Pune,
Pune 411007, India
Tel +91 20 2569 0442
Fax +91 20 2569 0087
email directoribb@.unipune.ac.in
Background: Development of an environmentally benign process for the synthesis of silver
nanomaterials is an important aspect of current nanotechnology research. Among the 600 species
of the genus Dioscorea, Dioscorea bulbifera has profound therapeutic applications due to its
unique phytochemistry. In this paper, we report on the rapid synthesis of silver nanoparticles
by reduction of aqueous Ag+ ions using D. bulbifera tuber extract.
Methods and results: Phytochemical analysis revealed that D. bulbifera tuber extract is
rich in flavonoid, phenolics, reducing sugars, starch, diosgenin, ascorbic acid, and citric acid.
The biosynthesis process was quite fast, and silver nanoparticles were formed within 5 hours.
Ultraviolet-visible absorption spectroscopy, transmission electron microscopy, high-resolution
transmission electron microscopy, energy dispersive spectroscopy, and x-ray diffraction con-
firmed reduction of the Ag+ ions. Varied morphology of the bioreduced silver nanoparticles
included spheres, triangles, and hexagons. Optimization studies revealed that the maximum
rate of synthesis could be achieved with 0.7 mM AgNO3 solution at 50°C in 5 hours. The
resulting silver nanoparticles were found to possess potent antibacterial activity against both
Gram-negative and Gram-positive bacteria. Beta-lactam (piperacillin) and macrolide (eryth-
romycin) antibiotics showed a 3.6-fold and 3-fold increase, respectively, in combination with
silver nanoparticles selectively against multidrug-resistant Acinetobacter baumannii. Notable
synergy was seen between silver nanoparticles and chloramphenicol or vancomycin against
Pseudomonas aeruginosa, and was supported by a 4.9-fold and 4.2-fold increase in zone
diameter, respectively. Similarly, we found a maximum 11.8-fold increase in zone diameter of
streptomycin when combined with silver nanoparticles against E. coli, providing strong evidence
for the synergistic action of a combination of antibiotics and silver nanoparticles.
Conclusion: This is the first report on the synthesis of silver nanoparticles using D. bulbifera
tuber extract followed by an estimation of its synergistic potential for enhancement of the anti-
bacterial activity of broad spectrum antimicrobial agents.
Keywords: Dioscorea bulbifera tuber extract, silver nanoparticles, antimicrobial synergy
Introduction
Nanoparticles have received considerable attention in recent years due to their wide
range of applications in the fields of catalysis, photonics, optoelectronics, biological
tagging, pharmaceutical applications environmental pollution control, drug delivery
systems, and material chemistry.1–3 Although there has been extensive research on the
chemical synthesis of nanoparticles, these particles have several potential hazards,
including carcinogenicity, genotoxicity, cytotoxicity, and general toxicity.4,5 There is a
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need to develop clean, nontoxic, and ecofriendly procedures
for synthesis and assembly of nanoparticles.4 Synthesis of
metal nanoparticles is solely dependent on knowledge of
micro-organisms and their behavior, which play a crucial
role. Against this background, researchers have been working
extensively on extracellular and intracellular synthesis of
metal nanoparticles using bacteria, fungi, yeasts, and many
other biological sources.6–8 One of the major drawbacks to
using microbes for bioreduction is the need to maintain asep-
tic conditions, which is not only labor-intensive but also very
expensive in terms of industrial production costs. There are
reports on the synthesis of nanoparticles using lemon grass
and neem leaves.9,10 Leaf extracts have been used for synthesis
of silver nanoparticles, which has highlighted the possibility
of rapid synthesis and may also reduce the steps involved in
downstream processing, thereby making the process more
economical and cost-efficient.11–13 Sastry et al, Shankar et al,
and Ankamwar et al have worked extensively on the biosyn-
thesis of metal nanoparticles from various plant sources.6,11,14
They have reported on synthesis of nanoparticles having
exotic shapes and morphologies.9 The spectacular success in
this field has opened up the prospect of developing “green
synthesis” methods for metal nanoparticles with tailor-made
structural properties using benign starting materials. Silver
has been used as a healing and antibacterial agent throughout
the world for thousands of years.15 Its benefit over the use
of antibiotics can be used as a powerful strategy to combat
the increasing spread of multidrug resistance resulting from
broad use of antibiotics. Therefore, the clinical efficacy of
antibiotics has been compromised. Antibiotic resistance has
become a serious problem threatening the health of human
beings.16–20 Of all the inorganic antimicrobial agents, silver
elements and nanoparticle compounds have been the most
extensively tested. Sondi and Salopek-Sondi recently dem-
onstrated that silver (0) nanoparticles are quite effective
antimicrobial agents against Escherichia coli.21 In addition,
silver nanoparticles have excellent properties in terms of
conformational entropy in polyvalent binding, which makes
it easy for them to attach to flexible polymeric chains of
antibiotics.22,23 Moreover, silver nanoparticles have well
developed surface chemistry and chemical stability, and
are of appropriate size. They are able to maintain a constant
shape and size in solution. Thus, silver nanoparticles are
a good choice as inorganic nanomaterials in combination
with various classes of antibiotics for use against pathogenic
micro-organisms.
Dioscorea bulbifera is unique among 600 species of the
genus Dioscorea due to its species-specific phytochemistry,
supporting its widespread application in therapeutics.24
D. bulbifera tuber extract is rich in polyphenolic compounds,
especially flavonoids and catechin, which have contributed to
its pronounced antioxidant and antidiabetic properties.25–27 In
an earlier report, we reported synthesis of gold nanoparticles
using D. bulbifera.28 However, to date, there are no reports on
the synthesis of silver nanoparticles using D. bulbifera tuber
extract. In view of this background, a detailed investigation
is required into its potential for use in the synthesis of silver
nanoparticles, along with characterization and application
in therapeutics. Similarly, there are no reports including
thorough evaluation of the role of silver nanoparticles in the
enhancement of efficacy of a diverse group of antibiotics,
ie, β-lactams, cephalosporins, aminoglycosides, rifamycins,
glycopeptides, quinolones, tetracyclines, trimethoprim, car-
bapenems, cyclic peptides, and macrolides, against a wide
group of Gram-positive and Gram-negative bacteria.
In this paper, we report for the first time the synthesis of
silver nanoparticles by reduction of aqueous Ag+ ions with the
assistance of D. bulbifera tuber extract. We have used tuber
broth for the biogenic reduction of Ag+ ions. Our objective
in this work was to reduce ionic silver to nanoparticles using
a biomaterial that addresses two major factors, ie, the need
for the biomaterial to be environmentally benign and produce
no toxic industrial waste and for it to be cost-effective and
easily produced. We also investigated the effects of reaction
conditions, such as temperature and AgNO3 concentration,
on the rate of synthesis of silver nanoparticles. We studied
their antimicrobial effects at various concentrations against
a wide range of Gram-negative and Gram-positive bacteria.
Furthermore, we evaluated the silver nanoparticles in com-
bination with various groups of antibiotics for synergistic
enhancement of antimicrobial activity against pathogenic
bacteria. Herein, we present the first detailed study of the
synergistic potential of biogenic silver nanoparticles in
combination with 22 different antibiotics against 14 potent
bacterial pathogens.
Materials and methods
Plant material and preparation of extract
D. bulbifera tubers were collected from the Western
Ghats region in Maharashtra, India, chopped into thin
slices, and dried for two days at room temperature. The
tuber extract was prepared by putting 5 g of thoroughly
washed and finely ground tuber powder into a 300 mL
Erlenmeyer flask with 100 mL of sterile distilled water,
boiling the mixture for 5 minutes, then decanting it. The
extract obtained was filtered through Whatman No 1 filter

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synthesis of silver nanoparticles by D. bulbifera
paper. The filtrate was collected and stored at 4°C for
further use.
Phytochemical analysis
Total flavonoid content
A 0.5 mL sample of D. bulbifera tuber extract was mixed with
0.5 mL of 2% AlCl3 in methanol and incubated for 10 minutes
at room temperature. After exactly 10 minutes, absorbance
was recorded at 368 nm. The total phenolic content was
quantified from a standard quercetin curve.29
Total phenolic content
A 0.125 mL sample of D. bulbifera tuber extract was mixed
with 0.5 mL of deionized water. Folin-Ciocalteau reagent
0.125 mL was added and incubated for 5 minutes at room
temperature followed by addition of 1.25 mL of 7% Na2CO3
solution. The volume was made up to 3 mL with distilled water
followed by incubation for 90 minutes at room temperature.
Absorbance was recorded at 760 nm. Total flavonoid content
was estimated from a standard gallic acid curve.30
starch content
Five grams of dry D. bulbifera tuber powder was repeatedly
washed using 70% ethanol to remove sugars until the wash-
ings did not show color with an anthrone reagent. The residue
was dried and boiled in 100 mL of distilled water to obtain
D. bulbifera tuber extract without any sugars. A 1 mL sample
of D. bulbifera tuber extract was evaporated to dryness and
reconstituted in 60% perchloric acid. Thereafter, 4 mL of
anthrone reagent was added, followed by boiling in a water
bath for 8 minutes. The intensity of the dark green color was
recorded at 630 nm, and the starch content was estimated by
comparison with a standard glucose curve.31
Total reducing sugar
A 100 mL quantity of D. bulbifera tuber extract was made
up to a volume of 1 mL using distilled water, and 1 mL of
dinitrosalicylic reagent was added. The mixture was kept
at 100°C for 5 minutes, and absorbance was recorded at
540 nm. Total reducing sugar was quantified from a standard
maltose curve.32
Diosgenin content
A 1 mL sample of D. bulbifera tuber extract was evaporated
to dryness, and 5 mL of 60% perchloric acid was added to the
dry residue and mixed thoroughly. Absorbance was recorded
at 410 nm after 10 minutes, and the diosgenin content was
determined from a standard diosgenin curve.33
Ascorbic acid content
A 2 mL sample of D. bulbifera tuber extract was evaporated
to dryness, followed by reconstitution in oxalic acid 4%.
The sample was then brominated and 1 mL of dinitrophe-
nyl hydrazine reagent was added, followed by two drops of
10% thiourea. After mixing thoroughly, the tube was kept at
37°C for 3 hours. The orange-red osazone crystals formed
were dissolved in 7 mL of 80% sulfuric acid. Absorbance
was measured at 540 nm. The content of ascorbic acid in
the D. bulbifera tuber extract was quantified by comparison
with a standard curve for ascorbic acid.34
citric acid content
A 1 mL quantity of D. bulbifera tuber extract was evaporated
to dryness and reconstituted in 1 mL of 5% trichloroacetic
acid. The tube was stoppered after dropwise addition of
8 mL of anhydrous acetic anhydride, followed by incubation
at 60°C for 10 minutes in a water bath. Thereafter, 1 mL of
pyridine was added to each tube and restoppered, after which
the tube was incubated at 60°C for 40 minutes. At the end
of this period, the tube was transferred to an iced water bath
for 5 minutes and absorbance was recorded at 420 nm. The
total citric acid content was determined from a standard citric
acid curve in the range of 10–400 µg/mL.35
synthesis of silver nanoparticles
Ag+ ions were reduced by addition of 5 mL of D. bulbifera
tuber extract to 95 mL of 10–3 M aqueous AgNO3 solution.
Reduction of the Ag+ ions was monitored by measuring the
ultraviolet-visible spectrum of the solution at regular intervals
on a spectrophotometer (SpectraMax M5, Molecular Devices
Corporation, Sunnyvale, CA) operating at a resolution
of 1 nm. The effects of temperature on the rate of synthesis
and size of the prepared silver nanoparticles were studied by
carrying out the reaction in a water bath at 4°C–50°C with
reflux. The concentration of AgNO3 solution was also varied
from 0.3 to 5 mM.
TeM, hrTeM, and dynamic light
scattering measurements
The surface morphology and size of the bioreduced silver
nanoparticles was determined using a transmission elec-
tron microscope (TEM, Tecnai 12 Cryo, FEI, Eindhoven,
The Netherlands). The morphology and size of the silver
nanoparticles were also characterized by a higher resolution
transmission electron microscope (HRTEM, JEOL-JEM-
2100, Peabody, MA). Energy-dispersive spectra for the silver
nanoparticles taken using an energy dispersive spectrometer

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Ghosh et al
equipped with a JEOL JSM 6360A analytical scanning
electron microscope at an energy range 0–20 keV confirmed
the synthesis of silver nanoparticles using D. bulbifera
tuber extract. The size of the particles in 3 mL of the reac-
tion mixture was analyzed using dynamic light scattering
equipment (Zetasizer Nano-2590, Malvern Instruments Ltd,
Worcestershire, UK) in a polystyrene cuvette.
X-ray diffraction measurements
Phase formation of the bioreduced nanoparticles was studied
using x-ray diffraction. Diffraction data for thin thoroughly
dried nanoparticle films on glass slides were recorded on an
x-ray diffractometer (D8 Advanced, Brucker, Germany) with
a Cu Kα (1.54 Å) source.
Fourier transform infrared spectroscopy
A dry nanoparticle powder was obtained in the following
manner. Silver nanoparticles synthesized after 5 hours of
reaction of 1 mM AgNO3 solution with D. bulbifera tuber
extract were centrifuged at 10,000 rpm for 15 minutes at
room temperature, after which the pellet was redispersed
in sterile distilled water. The process of centrifugation and
redispersion in sterile distilled water was repeated three
times to ensure better separation of free entities from the
nanoparticles. The purified pellet was then dried and sub-
jected to Fourier transform infrared (FTIR, IRAffinity-1,
Shimadzu Corporation, Tokyo, Japan) spectroscopy measure-
ment using the potassium bromide (KBr) pellet technique
in diffuse reflection mode at a resolution of 4 cm−1. The
nanoparticle powder was mixed with KBr and exposed to
an infrared source of 500–4000 cm−1. A similar process was
used for the FTIR study of D. bulbifera tuber extract before
and after bioreduction.
Bactericidal activity and in combination
The effects of the silver nanoparticles synthesized using
D. bulbifera tuber extract, standard antibiotics, and a com-
bination of both were tested against both Gram-positive
and Gram-negative bacterial strains using the disc diffusion
method on Muller-Hinton agar plates. A single colony of
each test strain was grown overnight in liquid Muller-Hinton
medium on a rotary shaker (200 rpm) at 37°C. The cells
grown overnight were washed twice in sterile phosphate-
buffered saline and the inocula were prepared by dilution
with phosphate-buffered saline to a 0.4 McFarland standard
and applied to plates along with the nanoparticles and stan-
dard antibiotics. For determination of the combined effect,
discs containing 500 µg of different antibiotics were further
impregnated with 5 µL of freshly prepared silver nanopar-
ticles at a final concentration of 30 µg/disc. After incubation
at 37°C for 18 hours, the zones of inhibition were measured.
The assays were performed in triplicate.
Results
Phytochemical analysis
D. bulbifera is known to be rich in diverse phytochemicals
like phenolics, flavonoids, carbohydrates, and vitamins that
might play a significant role in the bioreduction of silver
nanoparticles. Phytochemical analysis of D. bulbifera tuber
extract revealed a high level of flavonoid content up to
4 mg/mL. Similarly, D. bulbifera tuber extract was found
to be rich in phenolic content. Total reducing sugar was
also found to be comparatively high (up to 3.41 mg/mL),
followed by starch. Dioscorea is known to contain a saponin
known as diosgenin. Table 1 shows the presence of diosgenin
26 µg/mL in D. bulbifera tuber extract. In addition to other
phytochemicals, ascorbic acid and citric acid were detected
in a range of 0.1–0.3 mg/mL.
synthesis of silver nanoparticles
and optimization study
Bioreduction of Ag+ to Ag0 mediated by D. bulbifera tuber
extract was monitored by recording the absorption spectra as
a function of time. The well known yellowish-brown color of
silver nanoparticles arises due to excitation of surface plasmon
vibrations with an absorbance maxima at 450 nm (Figure 1).
This might play a key role in the bioreduction process. A col-
loidal solution with development of intense brown coloration
indicated the synthesis of silver nanoparticles, which showed
a steady increase with time on shaking at 150 rpm and 40°C.
Table 1 Phytochemical content per mL of Dioscorea bulbifera tuber extract
Sample Phytochemicals (mg/mL)
Total flavonoid
content
Total phenolic
content
StarchTotal reducing
sugar
DiosgeninAscorbic acidCitric acid
DBTe
4 ± 0.120.53 ± 0.041.08 ± 0.023.41 ± 0.15 0.026 ± 0.006 0.17 ± 0.005 0.28 ± 0.01
Note: Values are means ± standard deviation (n = 3).
Abbreviation: DBTe, D. bulbifera tuber extract.

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synthesis of silver nanoparticles by D. bulbifera
Ultraviolet-visible spectra of the reaction mixture of
D. bulbifera tuber extract and AgNO3 were recorded at regular
time intervals (Figure 2). Although there was no significant
synthesis at t = 0 minutes and t = 30 minutes, the rate of
synthesis increased at t = 60 minutes at a very rapid rate and
was completed in 5 hours. The silver nanoparticles formed
employing D. bulbifera tuber extract were found to be very
stable, possibly because of the starch present in the extract
that prevented agglomeration, even after 30 days.
Optimization of the AgNO3 concentration was carried
out by plotting absorbance at the peak wavelengths of silver
nanoparticles against time. Variation in the reaction kinetics
was observed at different concentrations (Figure 3). The rate
of synthesis was found to be maximum at a concentration
of 0.7 mM, while the higher concentrations had a com-
paratively low rate of bioreduction, with 0.3 mM showing
the slowest rate of synthesis. Thus, the optimization study
showed a significant effect of concentration on the synthesis
of silver nanoparticles.
We further investigated the role of temperature on the
reaction rate by varying the temperature from 4°C to 50°C.
Maximal synthesis of silver nanoparticles was achieved at
50°C (Figure 4). The reaction was slow at lower tempera-
tures, and no significant difference was found at 4°C, 20°C,
and 30°C. However, an increase in the reaction rate was
observed with an increase in temperature.
TeM, hrTeM, and energy dispersive
spectrometry
The shape and size of the synthesized nanoparticles
were elucidated with the help of TEM and HRTEM. The
TEM images confirmed formation of silver nanoparticles
(Figures 5 and 6). Formation of silver nanotriangles is
a very rare event. The majority were formed as silver
nanorods and triangles in the size range of 8–20 nm
(Figure 5A). HRTEM images clearly show that the nano-
particles were mostly spherical in shape. Nanoparticles
of larger dimensions (about 75 nm) were observed. TEM
and HRTEM analysis also occasionally demonstrated
hexagonal nanoparticles along with the triangles and rods
Figure 1 solution of 1 mM AgNO3 (A) before and (B) after bioreduction by
Dioscorea bulbifera tuber extract at 40°c.
300 min
240 min
180 min
120 min
60 min
30 min
0 min
0
300 400500
Wavelength (nm)
600
OD
700800
0.1
0.2
0.3
0.4
0.5
0.6
Figure 2 Ultraviolet-visible spectra recorded as a function of reaction time of 1 mM AgNO3 solution with Dioscorea bulbifera tuber extract at 40°c.

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(Figures 5C and 6D). The silver nanoparticles were aniso-
tropic and included triangular, spherical, and ellipsoidal
particles (Figure 5D, E, and F). The phytochemicals pres-
ent in the tuber were believed to be the agents responsible
for reducing the Ag+ to Ag0, but the shape is believed to be
controlled by chemical factors like ascorbic acid and citric
acid. Similarly, the starch content of D. bulbifera tuber
extract may play a vital role in capping. Many triangular-
shaped nanoparticles were observed in addition to the rods.
Energy dispersive x-ray spectroscopy results confirmed the
presence of significant amounts of silver with no contami-
nants (Figure 7). The optical absorption peak was observed
at approximately 3 keV, which is typical for the absorption
of metallic silver nanocrystallites due to surface plasmon
resonance. The particle size distribution of the silver nano-
particles determined by dynamic light scattering as shown
in Figure 8 was found to be about 13.54 nm, which is well
in agreement with the TEM analysis.
0
0123
Time (h)
Absorbance at 450 nm
456
5 mM
4 mM
3 mM
2 mM
1 mM
0.7 mM
0.5 mM
0.3 mM
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Figure 3 Time course of silver nanoparticles formation obtained with different concentrations of AgNO3 using Dioscorea bulbifera tuber extract at 40°c.
0
02
Time (h)
Absorbance at 450 nm
46
50ºC
40ºC
30ºC
20ºC
4ºC
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Figure 4 Time course of silver nanoparticles formation obtained with 1 mM AgNO3
using Dioscorea bulbifera tuber extract with different reaction temperatures.
AB
C
D
200 nm
200 nm
200 nm
200 nm
Figure 5 characterization of silver nanoparticles formed with 1 mM AgNO3 and
5% Dioscorea bulbifera tuber extract at 40°c by transmission electron microscopy.
(A) silver nanotriangles and nanorods, (B) anisotropic silver nanoparticles, (C), silver
nanohexagon, and (D) irregular silver nanoparticles.

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synthesis of silver nanoparticles by D. bulbifera
full width at half the maximum intensity in radians, and θ
is the Bragg angle. Using the above formula, the calculated
crystallite size was approximately 10 nm.
FTIr analysis
FTIR absorption spectra of D. bulbifera tuber extract
before and after reduction of silver are shown in Figure 10.
D. bulbifera tuber extract shows hydroxyl group in alcoholic
and phenolic compound which is supported by the presence
of a strong peak at approximately 3300 cm−1. This sharp
peak representing O–H bond is not seen in D. bulbifera
tuber extract after bioreduction. This indicates that the poly-
ols are mainly responsible for reduction of Ag+ into silver
nanoparticles. This may lead to the disappearance of the
O–H bond. We could observe free hydroxyl groups in the
FTIR of silver nanoparticles at 3600 cm−1. The absorbance
bands at 2931 cm−1, 1625 cm−1, 1404 cm−1, and 1143 cm−1
are associated with the stretch vibrations of alkyl C–C,
conjugated C–C with a benzene ring, bending of C–O–H
and C–O stretch in saturated tertiary or secondary highly
symmetric alcohol in D. bulbifera tuber extract, respectively.
The presence of peaks at 3749 cm−1 and 1523 cm−1 indicate
that the silver nanoparticles may be surrounded by amines,
because the peaks indicate –NH2 symmetric stretching and
N–O bonds in nitro compounds.
Antimicrobial effects of antibiotics alone
and combined with nanoparticles
Silver is well known as one of the most universal antimicrobial
substances. The individual and combined effects of the
bioreduced silver nanoparticles with 22 different antibiotics
were investigated against 14 bacterial strains using the disc
diffusion method. The diameter of the silver nanoparticle
inhibition zones against different pathogenic cultures were
observed (Table 2). Silver nanoparticles exhibited maximum
cytotoxicity against E. coli and Pseudomonas aeruginosa,
while moderate activity was found for Salmonella typhi
and Bacillus subtilis. Silver nanoparticles were found to be
comparatively less active in killing Staphylococcus aureus.
Combined with the silver nanoparticles, the antibacterial
activity of the antibiotics showed wide variation, not only
between groups but also between members of the same
group (Table 3). When combined with aminoglycosides,
ie, amikacin and gentamicin, the silver nanoparticles
showed comparable synergy (a 0.1–2.2-fold increase), and
kanamycin was found to be superior against P. aeruginosa,
showing a 4.2-fold enhancement in inhibition when used
in combination. However, the best synergy was found with
AB
CD
20 nm
10 nm10 nm
10 nm
Figure 6 characterization of silver nanoparticles formed with 1 mM AgNO3 and 5%
Dioscorea bulbifera tuber extract at 40°c by high-resolution transmission electron
microscopy. (A) silver nanoparticles at a resolution of 20 nm, (B and C) spherical
silver nanoparticles, and (D) hexagonal silver nanoparticles.
1086
keV
Counts
42
SiL2,3
SiKn
AgLasc
AgL1
AgLI
AgLb2
AgLb
AgLa
0
0
500
1000
1500
2000
2500
Figure 7 Representative spot energy-dispersive spectrometer profile confirming
the presence of silver nanoparticles.
X-ray diffraction analysis
The diffraction data for the dry powder were recorded on a
Brucker x-ray diffractometer using a Cu Kα (1.54 Å) source.
Phase formation was confirmed from characteristic peaks
such as (111), (200), (220), and (311). The data matched with
the standard Joint Committee for Powder Diffraction Set, Card
040783, confirming a face-centered cubic structure for the silver
nanoparticles (Figure 9). The lack of any peaks resembling
metal or metal oxide other than for pure silver in the diffraction
data confirmed the purity of the synthesized silver nanopar-
ticles. Broadening of the peak indicated a smaller particle size.
The crystallite size was calculated using Scherrer’s formula:
d = 0.9λ/βcos
where 0.9 is the shape factor, generally taken for a cubic
system, λ is the x-ray wavelength, typically 1.54 Å, β is the

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Ghosh et al
streptomycin against E. coli, where inhibition increased by
11.8-fold. Among the β-lactams, the activity of ampicillin
was enhanced against Klebsiella pneumoniae, Neisseria
mucosa, and P. aeruginosa, with an appreciable increase
in activity of 3–4.2-fold. On the other hand, the activity
of penicillin was found to be enhanced moderately
against Acinetobacter baumannii, Enterobacter cloacae,
P. aeruginosa, B. subtilis, and Pseudomonas koreensis, and
was highest against K. pneumoniae. It is important to note
that the efficacy of piperacillin in combination with silver
nanoparticles was selectively enhanced against A. baumannii
by up to 3.6-fold. Feropenem showed negligible or no synergy
in combination with silver nanoparticles against any of
the organisms tested, except for K. pneumoniae. Similarly,
in the cephalosporin group of antibiotics, synergy was
observed only for ceftazidime against E. coli. Polymyxin,
a cyclic peptide antibiotic, showed negligible variation in
activity whether used alone or in combination with silver
nanoparticles against all the test pathogens. Even Gram-
negative bacteria like K. pneumoniae, Proteus mirabilis, and
P. aeruginosa, were found to be inhibited in the presence
of a combination of vancomycin and silver nanoparticles,
350030002500
Wavenumber cm−1
% transmittance
20001500 10004000
B
A
Figure 10 Fourier transform infrared absorption spectra of dried Dioscorea bulbifera
tuber extract (A) before bioreduction and (B) after complete bioreduction of
Ag+ ions at 50°c.
0
8.721
Size (nm)
Volume (%)
11.7
15.6921.0428.2143.8258.7778.82105.7141.8190.1
5
10
15
20
25
Figure 8 histogram of size distribution of silver nanoparticles synthesized by Dioscorea bulbifera tuber extract.
0
4050
100
200
300
400
500
600
700
60
(220) (200)
(111)
(311)
2θ
Intensity
7080
Figure 9 Representative x-ray diffraction profile of thin film silver nanoparticles.

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491
synthesis of silver nanoparticles by D. bulbifera
which otherwise showed a resistant pattern in the presence
of the antibiotic alone. Erythromycin showed enhanced
activity of 1.5–3.0-fold against K. pneumoniae, P. mirabilis,
P. aeruginosa, S. typhi, and A. baumannii in combination
with silver nanoparticles. Silver nanoparticles increased
the efficacy of nalidixic acid, a member of the quinolone
group of antibiotics, against both Gram-positive and Gram-
negative microbes, in particular E. coli and P. koreensis.
Rifampicin and tetracyclines showed identical activity in the
presence and absence of silver nanoparticles against most
of the tested pathogens. The activity of chloramphenicol
against P. aeruginosa was remarkably enhanced by up to
4.9-fold in the presence of silver nanoparticles, but moderate
synergy was observed against the other Gram-positive
pathogens tested. Combination of silver nanoparticles with
nitrofurantoin and trimethoprim showed identical selective
synergistic efficacy against K. pneumoniae.
Discussion
We have successfully demonstrated a rapid and efficient
route for synthesis of silver nanoparticles using D. bulbifera
tuber extract. The development of intense yellowish brown
color owing to the surface plasmon resonance confirmed the
synthesis of the silver nanoparticles.10 D. bulbifera tuber is
a rich source of flavonoids and phenolic acid derivatives.36,26
Flavonoids play a vital role in the reduction process for
synthesis of nanoparticles.37 Thus, the high flavonoid and
phenolic content in D. bulbifera tuber extract revealed in the
phytochemical analysis strongly supports the potential of
D. bulbifera tuber extract to bioreduce Ag+ to Ag0. Similarly,
the reducing sugars that are known to play a vital role in
bioreduction were found to be predominant in D. bulbifera
tuber extract.38 It is important to note that D. bulbifera
contains ascorbic acid and citric acid in notable amounts.39,40
This is in agreement with the presence of both ascorbic
and citric acid in D. bulbifera tuber extract in our study
as well. Widespread use of ascorbic and citric acid in the
synthesis of silver nanoparticles provides a strong rationale
for use of D. bulbifera tuber extract in the synthesis and
shaping of silver nanoparticles.41–43 Diosgenin is reported
to be a predominant saponin in D. bulbifera39,44 which might
contribute to the surfactant properties of D. bulbifera tuber
extract. Plant surfactants are widely used in the synthesis
of silver nanoparticles.45 Likewise, the significant starch
content in D. bulbifera tuber extract reflects the capping
properties of the extract, and starch is widely used in vari-
ous synthetic processes for capping and stabilizing silver
nanoparticles.46 Thus, phytochemical studies support the use
of D. bulbifera tuber extract as a suitable agent to facilitate
the synthesis of silver nanoparticles. The complete reduc-
tion of Ag+ within 5 hours indicates that synthesis was
much faster as compared with other plants like Sesuvium
portulacastrum L that take 24 hours for complete synthe-
sis of silver nanoparticles to occur.47 Optimization studies
showed that the sharpness of the peak increased with an
increase in temperature. Most likely, this occurs due to an
increase in the reaction rate of conversion of metal ions to
nanoparticles at higher temperatures.48 Recently, different
plant systems have been used to synthesize spherical and
irregular silver nanoparticles.47 Although nanospheres dom-
inated the population of the synthesized silver nanoparticles
as evident from HRTEM and TEM micrographs, in some
cases anisotropy was observed similar to other reports.49
Anisotropy among the silver nanoparticles could be related
to the recent report by Huang et al mentioning that nascent
silver crystals formed using Cinnamomum camphora leaf
powder were gradually enclosed by protective molecules,
which as a result eliminated rapid sintering of smaller
nanoparticles, resulting in formation of nanotriangles.12
The spot energy-dispersive spectrometry data confirmed
the purity of the metallic nanosilver, while the particle size
was calculated to be approximately 10 nm from the x-ray
diffraction data.50,51 The FTIR absorption spectra offered
information regarding chemical changes in the functional
groups involved in bioreduction.52 D. bulbifera tubers are
known to contain various bioactive compounds, including
flavonoids, terpenoids, phenanthrenes, amino acids, protein,
and glycosides.53–59 It is speculated that the alcohol groups
are oxidized to carbonyl groups in the course of reduction,
which supports the suggestion that polyol groups are pri-
marily responsible for the bioreduction process. Thus, the
water-soluble fractions of D. bulbifera played key roles in
the bioreduction of the precursors and evolution of shape
in the nanoparticles.
Silver ion and silver-based compounds are highly toxic
to micro-organisms, showing a strong biocidal effect against
microbial species because these are highly reactive species
with a large surface area.60–63 Silver nanoparticles produced
using microbes and plant extracts are known to exhibit potent
antimicrobial activity.47 Antimicrobial activity determined
using the disc diffusion method confirmed that combining
antibiotics with silver nanoparticles resulted in a greater
bactericidal effect on the test pathogens than either of the
antibacterial agents used alone. This experiment provides
solid evidence of the antibacterial synergy between antibiot-
ics and silver nanoparticles. A very interesting observation

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492
Ghosh et al
Table 2 Zone of inhibition (mm) of different concentrations of silver nanoparticles on the test pathogens by disc diffusion method
AgNPs
μg/disc
Zone diameter (mm)
Acinetobacter
baumannii
Enterobacter
cloacae
Escherichia
coli
Haemophilus
influenzae
Klebsiella
pneumoniae
Neisseria
mucosa
Proteus
mirabilis
1000
500
300
100
50
30
20
10
13
12
11
10
8
NI
NI
NI
12
10
8
NI
NI
NI
NI
NI
18
15
14
13
12
10
9
NI
12
11
9
8
NI
NI
NI
NI
16
15
14
12
11
9
8
NI
21
20
18
16
15
14
12
10
NI
NI
NI
NI
NI
NI
NI
NI
Table 3 Zone of inhibition (mm) of different antibiotics against test strains in absence and in presence of silver nanoparticles
(30 µg/disc)
Antiboitics Acinetobacter
baumannii
Enterobacter
cloacae
Escherichia
coli
Haemophilus
influenzae
Klebsiella
pneumoniae
Neisseria
mucosa
Proteus
mirabilis
ABCABCABCABCABCABCABC
Aminoglycosides
Amikacin
Gentamycin
Kanamycin
streptomycin
β-Lactams
Amoxicillin
Ampicillin
Penicillin
Piperacillin
Carbapenem
Feropenem
Cephalosporin
ceftazidime
ceftriaxone
cefotaxime
Cyclic peptide
Polymyxin
Glycopeptides
Vancomycin
Macrolide
erythromycin
Quinolones
Nalidixic acid
Rifamycin
rifampicin
Tetracycline
Tetracycline
Doxycycline
Others
chloramphenicol
Nitrofurantoin
Trimethoprim
23.0
13.0
7.0
16.0
23.0
14.0
10.0
16.0
0.0
0.2
1.0
0.0
19.0
17.0
18.0
23.0
20.0
17.0
18.0
23.0
0.1
0.0
0.0
0.0
10.0
17.0
14.0
7.0
18.0
17.0
16.0
25.0
2.2
0.0
0.3
11.8
23.0
23.0
22.0
24.0
23.0
23.0
22.0
24.0
0.0
0.0
0.0
0.0
17.0
18.0
7.0
22.0
17.0
18.0
9.0
22.0
0.0
0.0
0.7
0.0
20.0
30.0
26.0
28.0
26.0
42.0
26.0
28.0
0.7
1.0
0.0
0.0
17.0
20.0
17.0
20.0
17.0 0.0
20.0 0.0
17.0 0.0
20.0 0.0
7.0
7.0
7.0
7.0
9.0
12.0
13.0
15.0
0.7
1.9
2.4
3.6
7.0
14.0
7.0
18.0
8.0
14.0
12.0
0.0
0.3
0.0
1.9
15.0
12.0
29.0
10.0
15.0
12.0
29.0
10.0
15.0
0.0
0.0
0.0
0.0
10.0
10.0
13.0
17.0
10.0
10.0
13.0
17.0
0.0
0.0
0.0
0.0
7.0
7.0
7.0
18.0
10.0
15.0
16.0
18.0
1.0
3.6
4.2
0.0
40.0
22.0
46.0
40.0
40.0
50.0
48.0
40.0
0.0
4.2
0.1
0.0
16.0
7.0
14.0
18.0
16.0 0.0
7.0
14.0 0.0
18.0 0.0
0.0
17.017.00.013.013.00.018.018.00.025.025.00.07.015.03.648.048.00.015.015.0 0.0
8.0
11.0
10.0
8.0
11.0
130
0.0
0.0
0.7
20.0
20.0
23.0
20.0
20.0
23.0
0.0
0.0
0.0
9.0
7.0
7.0
18.0
7.0
7.0
3.0
0.0
0.0
17.0
13.0
14.0
17.0
15.0
17.0
0.0
0.3
0.5
9.0
16.0
16.0
9.0
16.0
16.0
0.0
0.0
0.0
32.0
47.0
30.0
32.0
47.0
30.0
0.0
0.0
0.0
20.0
18.0
15.0
20.0 0.0
18.0 0.0
15.0 0.0
15.015.00.013.013.00.07.08.00.310.010.00.016.016.00.016.018.00.37.07.00.0
12.012.00.07.07.00.018.019.00.123.023.00.07.013.02.424.025.00.17.012.0 1.9
7.014.03.07.08.00.37.08.00.318.018.00.07.011.01.525.027.00.27.011.0 1.5
7.010.01.011.012.00.27.01403.08.08.00.07.07.00.014.014.00.07.09.00.7
20.020.00.013.013.00.015.015.0.0.0.16.0
16.00.010.015.31.344.044.00.0.15.015.0 0.0
17.0
21.0
17.0
21.0
0.0
0.0
21.0
18.0
21.0
18.0
0.0
0.0
27.0
12.0
27.0
12.0
0.0
0.0
25.0
24.0
25.0
24.0
0.0
0.0
18.0
13.0
18.0
13.0
0.0
0.0
34.0
34.0
38.0
34.0
0.2
0.0
18.0
10.0
18.0 0.0
10.0 0.0
10.0
7.0
7.0
10.0
11.0
11.0
0.0
1.5
1.5
22.0
9.0
16.0
22.0
9.0
16.0
0.0
0.0
0.0
26.0
8.0
16.0
26.0
8.0
170
0.0
0.0
0.1
17.0
10.0
7.0
17.0
12.0
13.0
0.0
0.4
2.4
7.0
7.0
7.0
12.0
16.0
16.0
1.9
4.2
4.2
32.0
8.0
25.0
32.0
31.0
26.0
0.0
14.0
0.1
16.0
9.0
7.0
16.0 0.0
9.0
7.0
0.0
0.0

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493
synthesis of silver nanoparticles by D. bulbifera
Pseudomonas
aeruginosa
Salmonella
typhi
Serratia
odorifera
Vibrio
parahaemolyticus
Bacillus
subtilis
Paenibacillus
koreensis
Staphylococcus
aureus
10
9
8
NI
NI
NI
NI
NI
12
11
10
9
NI
NI
NI
NI
10
9
8
NI
NI
NI
NI
NI
12
11
9
8
NI
NI
NI
NI
17
16
15
13
11
10
9
NI
12
11
9
8
NI
NI
NI
NI
8
7
NI
NI
NI
NI
NI
NI
Note: All experiments were performed in triplicate, and standard deviations were negligible.
Abbreviations: NI, no inhibition; AgNPs, silver nanoparticles.
Pseudomonas
aeruginosa
Salmonella
typhi
Serratia
odorifera
Vibrio
parahaemolyticus
Bacillus
subtilis
Paenibacillus
koreensis
Staphylococcus
aureus
ABCABCABCABCABCABCABC
14.0
27.0
7.0
24.0
22.0
27.0
16.0
32.0
1.5
0.0
4.2
0.8
7.0
7.0
7.0
7.0
8.0
9.0
10.0
10.0
0.3
0.7
1.0
1.0
20.0
26.0
26.0
23.0
20.0
26.0
26.0
23.0
0.0
0.0
0.0
0.0
24.0
30.0
22.0
30.0
24.0
30.0
23.0
30.0
0.0
0.0
0.1
0.0
20.0
20.0
23.0
26.0
20.0
20.0
23.0
37.0
0.0
0.0
0.0
0.1
15.0
20.0
20.0
23.0
20.0
22.0
22.0
23.0
0.8
0.2
0.2
0.0
11.0
17.0
16.0
16.0
15.0
17.0
17.0
18.0
0.9
0.0
0.1
0.3
7.0
8.0
7.0
26.0
12.0
16.0
13.0
26.0
1.9
3.0
2.4
0.0
7.0
20.0
18.0
15.0
12.0
27.0
24.0
18.0
1.9
0.8
0.8
0.4
20.0
27.0
20.0
24.0
20.0
27.0
20.0
24.0
0.0
0.0
0.0
0.0
11.0
13.0
15.0
20.0
11.0
14.0
15.0
20.0
0.0
0.2
0.0
0.0
10.0
20.0
7.0
10.0
10.0
34.0
11.0
14.0
0.0
1.9
1.5
1.0
7.0
11.0
7.0
7.0
10.0
13.0
11.0
15.0
1.0
0.4
1.5
1.3
7.0
18.0
15.0
15.0
10.0
19.0
15.0
15.0
1.0
0.1
0.0
0.0
12.013.00.014.014.00.025.025.00.030.030.00.034.034.00.022.023.00.118.018.00.0
20.0
18.5
15.0
20.0
18.5
15.0
0.0
0.0
0.0
16.0
15.0
18.0
16.0
18.0
20.0
0.0
0.4
0.2
24.0
24.0
25.0
25.0
24.0
25.0
0.1
0.0
0.0
20.0
15.0
16.0
20.0
18.0
18.0
0.0
0.4
0.3
21.0
14.0
15.0
21.0
14.0
16.0
0.0
0.0
0.1
16.0
15.0
18.0
17.0
15.0
18..0
0.1
0.0
0.0
12.0
17.0
20.0
150
17.0
20.0
0.6
0.0
0.0
14.014.00.07.08.00.314.014.00.011.014.00.67.09.00.711.012.00.27.08.00.3
7.016.04.218.022.00.57.08.00.323.023.00.024.025.00.018.018.00.015.015.00.0
7.013.02.47.013.02.47.08.00.320.020.00.019.019.00.016.020.00.67.08.00.3
7.013.02.48.0 10.00.69.0 14.01.4 11.0 12.00.29.0 12.00.87.0 13.02.47.0 11.01.5
15.0 15.00.0 21.0 21.00.0 13.0 13.00.0 18.0 18.00.0 16.016.00.017.017.00.07.07.00.0
19.0
11.0
20.0
12.0
0.1
0.2
17.0
21.0
20.0
21.0
0.4
0.0
21.0
20.0
22.0
20.0
0.1
0.0
25.0
28.0
25.0
28.0
0.0
0.0
30.0
19.0
39.0
19.0
0.7
0.0
22.0
19.0
24.0
20.0
0.2
0.1
25.0
11.0
25.0
11.0
0.0
0.0
7.0
7.0
7.0
17.0
11.0
11.0
4.9
1.5
1.5
22.0
7.0
7.0
24.0
9.0
7.0
0.2
0.7
0.0
22.0
7.0
7.0
22.0
8.0
13.0
0.0
0.3
2.4
21.0
13.0
7.0
21.0
19.0
15.0
0.0
1.1
3.6
24.0
11.0
7.0
32.0
20.0
20.0
0.8
2.3
7.2
23.0
7.0
7.0
25.0
13.0
13.0
0.2
2.4
2.4
7.0
7.0
7.0
10.0
7.0
10.0
1.0
0.0
1.0
Notes: All experiments were done in triplicate, and standard deviations were negligible. Fold increases (C) for different antibiotics against five bacterial pathogens were
calculated as (B2 − A2)/A2, where A and B are the inhibition zones in mm for antibiotic only and antibiotic in combination with silver nanoparticles, respectively. In the absence
of bacterial growth inhibition zones, the disk diameters (7 mm) were used to calculate the fold increase (c).
Abbreviation: AgNPs, silver nanoparticles.

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Ghosh et al
that A. baumannii, a nosocomial pathogen resistant to almost
all known antibiotics and metal salts commonly used in
medicine, could also be sensitized in the presence of silver
nanoparticles.64–67 Therefore, this combinational approach
of antibiotics and silver nanoparticles would provide a
strategy for effective control of A. baumannii. From the
results, we can conclude that the antimicrobial effect of
silver nanoparticles is the key contributor to the synergistic
effect observed with a combination of silver nanoparticles
and antibiotics.
Scientists have established the antibacterial mechanism
of β-lactams, chloramphenicol, aminoglycosides, tetracy-
clines, and glycopeptides.68,69 However, there has not been
a consistent explanation for the antimicrobial mechanism
of silver, although application of silver to burn wounds has
been done for more than a century.70 This can be attributed
to the fact that silver at low concentrations does not enter
cells, but is adsorbed onto the bacterial surface just as silver
tends to adsorb to other surfaces.67 Thus, silver ions resist
dehydrogenation because respiration occurs across the cell
membrane in bacteria rather than across the mitochondrial
membrane as in eukaryotic cells.71 Again, some hypotheses
indicate that catalytic oxidation of silver ions, with nascent
oxygen, reacts with bacterial cell membranes, leading to cell
death. A pronounced antimicrobial effect of antibiotics in
combination with silver nanoparticles was observed in this
study. This clearly indicates that enhancement of efficacy
was due to the synergistic antibacterial action between
antibiotics and silver nanoparticles. As discussed above,
silver nanoparticles and antibiotics can kill bacteria with
a different mechanism. Thus, the synergistic effect can act
as a very powerful tool against resistant micro-organisms.
A bonding reaction between antibiotics and silver nanopar-
ticles by chelation ultimately increases the concentration of
antimicrobial agents at specific points on the cell membrane.
This may be attributed due to the selective approach of
silver nanoparticles towards the cell membrane that con-
sists of phospholipids and glycoprotein. Therefore, silver
nanoparticles facilitate the transport of antibiotics to the cell
surface acting as a drug carrier. As the silver nanoparticles
bind to the sulfur-containing proteins of the bacterial cell
membrane, the permeability of the membrane increases,
facilitating enhanced infiltration of the antibiotics into the
cell. More recently, it was shown that silver (I) chelation
prevents unwinding of DNA. Silver nanoparticles are com-
posed of silver (0) atoms.72 Silver nanoparticles are larger in
size than silver (I) ions, which makes them react with more
molecules, leading to more antimicrobial activity.
Conclusion
In an attempt to find natural, environmentally benign, and
easily available plant-based agents for synthesis of metal
nanoparticles, we have demonstrated for the first time the
efficiency of D. bulbifera tuber extract in the rapid syn-
thesis of stable silver nanoparticles possessing a variety
of fascinating morphologies owing to its diverse groups of
phytochemicals like phenolics, flavonoids, reducing sugars,
ascorbic acid, citric acid, and diosgenin. Based on our kinetic
studies, together with evidence obtained from FTIR, we pro-
pose that the main biomolecules responsible for nanoparticle
synthesis were polyphenols or flavonoids. The results of our
study show that the combination of silver nanoparticles and
antibiotics had synergistic antibacterial efficiency against
the test microbes, particularly P. aeruginosa, E. coli, and
A. baumannii. To elucidate the mechanism of this synergistic
antibacterial effect, more elaborate experimental evidence
will be required. Overall, this combinational approach of
antibiotic and silver nanoparticles seems to be one of the best
strategies for control of antibiotic-resistant micro-organisms
and hence therapeutic management in infection control.
Acknowledgment
SG acknowledges financial support for this work from the
Institute of Bioinformatics and Biotechnology, University
of Pune, India.
Disclosure
The authors report no conflicts of interest in this work.
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