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Green Synthesis of Silver Nanoparticles Using Ginkgo biloba and Their

Bactericidal and Larvicidal Effects

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DOI: 10.1166/nnl.2018.2630

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 Copyright © 2018 American Scientific Publishers Nanoscience and
All rights reserved Nanotechnology Letters
Printed in the United States of America Vol. 10, 422–428, 2018

Green Synthesis of Silver Nanoparticles Using Ginkgo

biloba and Their Bactericidal and Larvicidal Effects
Pannerselvam Balashanmugam1 2 , Hyung Joo Kim3 , Vijay Singh4 , and Rangarajulu Senthil Kumaran3 ∗
1
Centre for Advanced Studies in Botany, University of Madras, Chennai 600025, India
2
CSIR-Central Leather Research Institute, Chennai 600025, India
3
Department of Biological Engineering, Konkuk University, Seoul 143 701, South Korea
4
Department of Chemical Engineering, Konkuk University, Seoul 143 701, South Korea

The synthesis of metallic nanoparticles using plant extracts has attracted much attention. In this
study, silver nanoparticles (AgNPs) were synthesized using the Ginkgo biloba plant-leaf extract
and their antibacterial and larvicidal activities were investigated. AgNPs were characterized using
UV-visible, X-ray diffraction (XRD), Fourier transform infrared (FTIR), energy-dispersive X-ray (EDX)
spectroscopy and field emission scanning electron microscope (FESEM) analysis. The UV-visible
spectral analysis showed a surface plasmon resonance (SPR) peak at 430 nm, the FESEM anal-
ysis revealed size of AgNPs between 25–45 nm and the XRD data confirmed the formation of the
AgNPs using G. biloba-mediated green synthesis. AgNPs showed the highest antibacterial activities
against Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtills, Enterobacter aero-
gens, Salmonella paratyphi and Escherichia coli. Also, the AgNPs exerted a significant larvicidal
effect on the Culex mosquito larvae.
Keywords: Green Synthesis, Silver Nanoparticles, UV-Visible, Surface Plasmon Resonance,
Cytotoxicity.

1. INTRODUCTION reduction process is extracellular and rapid, leading to

Green nanotechnology is an emerging field of scientific
and global interest wherein the significant focus is the
facilitation of the manufacture of safer nanotechnology-
based ecofriendly products with a sustainable commercial
viability. The “green synthesis” of metallic nanoparticles
(NPs) receives greater attention due to the unusual opti-
cal, chemical, photochemical and electronic properties1
of these NPs. Further, it is noted that the advantages of
the green-synthesis process over the chemical and phys-
ical methods include cost-effectiveness, an environmental
amenability including the absence of toxic chemicals, an
ease regarding the scaling up of the process for large-scale
syntheses and high pressure, energy and temperature levels
are not involved.2 The plant-mediated synthesis methods
are actively being practiced by researchers for their posi-
tive advantages, such as the absence of the need to main-
tain the microbial culture, the reduced time consumption
and the cost-effectiveness.3 Further, it is shown that the
plant biomolecules induce the reduction of the silver ion
(Ag+  to the silver (Ag) NP. The biomolecule-mediated
∗
Author to whom correspondence should be addressed.
the formation of AgNPs. It is concluded that during gly-
colysis, the plant production of a large amount of the
hydrogen ion (H+  is influenced by the nicotinamide ade-
nine dinucleotide (NAD) cofactor; therefore, the NAD
acts as a strong reducing agent and is responsible for
the AgNP formation.4 In fact, the AgNP is one of
the most promising types of metallic NP. An interest-
ing alternative of the existing antibacterial methods is
the utilization of metallic NPs, and this is due to the
bactericidal properties they exhibit as advantageous for this
purpose.
In recent years, the increasing rates of the resistance
capabilities of pathogenic bacteria and fungi against com-
mercially available antimicrobial agents are alarming in
the scientific community, and have become seriously
problematic.5 The microorganisms of living environments
such as bacteria, molds, yeasts and viruses are often
pathogenic and cause severe infections in human beings.
On these grounds, it is shown that a growing demand for
the development of new types of cost-effective antimi-
crobial agent has led to a resurgence of the use of
Ag-based antiseptics that may be linked to broad-spectrum

422 Nanosci. Nanotechnol. Lett. 2018, Vol. 10, No. 3 1941-4900/2018/10/422/007 doi:10.1166/nnl.2018.2630
Balashanmugam et al. Green Synthesis of Silver Nanoparticles Using Ginkgo biloba and Their Bactericidal and Larvicidal Effects
activity and a far lower propensity regarding the induce-
ment of a microbial resistance compared to antibiotics.
It is noted that in higher concentrations, Ag is toxic to
human beings, but at lower concentrations it is nontoxic
with a remarkable antibacterial activity.6–8 Similarly, the
necessity global efforts for mosquito control is essential
since many mosquito species are vectors of malaria, filaria-
sis and numerous arboviral diseases besides the intolerable
biting nuisances they cause.9 10 Biotechnologists and ento-
mologists agree that, regarding the mosquito control effi-
ciency, a consideration of the selectivity for specific target
organisms needs to occur. Nonetheless, for the new control
methodologies that seek to reduce mosquito breeding sites
and mosquito biting activities, combinatory chemical–
biological control methods that are compatible with a num-
ber of the advocated mosquito bio-control methods have
been selected for the proposed reduction of the mosquito
population and the human–vector contact occasions.11
The percentage of disease spread mediated through the
contact with the mosquito is increasing rapidly without
proper control measures. Currently the importance has
been given for Culex mosquitos, which serves as vectors
for disseminating more dangerous life threatening viral
diseases in humans, birds and animals. It is a great chal-
lenge in controlling the larvae of Culex in an ecofriendly
approach through the application of biogenic nanopar-
ticles which is reviewed and investigated as currently
underway.
Recently, a major concern has arisen regarding the pro-
motion of botanicals as environmentally friendly pesti-
cides, microbial sprays, insect-growth regulators (IGRs)
and other control measures, such as the employment of
beneficial insects that necessitate an integration of the
supervised-control measures.12 Under the circumstances,
the development of IGRs has received considerable atten-
tion regarding the selective control of insects that is in
consideration of the medical and veterinary importance
and it has resulted in mortality due to the high neurotoxic
effects of IGRs.13 14 Therefore, the role of biological con-
trol in modern vector control programs remains important
due to the disadvantages that are associated with synthetic
pesticides, including the development of pesticide-resistant
strains, ecological imbalances and the harm noted to non-
target organisms. Also, the efforts to develop plant-origin
substances that are considered to be more environmentally
friendly due to their innate biodegradability and low tox-
icity regarding most organisms have been renewed in this
century.15
In the present investigation, an ecofriendly approach
has been adopted for the synthesis of AgNPs, for which
an extract of the medicinal plant Ginkgo biloba was
used. Consequently, the AgNPs that were synthesized were
investigated for their antibacterial activity against a selec-
tion of human pathogenic bacteria and the larvicidal activ-
ity of the Culex mosquito.
Nanosci. Nanotechnol. Lett. 10, 422–428, 2018
Fig. 1. (a) Ginkgo biloba leaves, (b)—(i) green-synthesized silver
nanoparticles and (ii) G. biloba leaf extract.
2. EXPERIMENTAL DETAILS
2.1. Materials
The Ag nitrate, with the chemical formula AgNO3 (Merck
Darmstadt, Germany), potassium dihydrogen phosphate
(KH2 PO4 , dipotassium hydrogen phosphate (K2 HPO4 ,
magnesium sulfate heptahydrate (H14 MgO11 S), ammonium
sulphate ([NH4 ]2 SO4  glucose (C6 H12 O6  and Muller–
Hinton agar were used.
2.2. Microorganism Sources
The Pseudomonas aeruginosa (BESK21), Escherichia coli
(BESK28), Salmonella paratyphi (BESK24), Enterobac-
ter aerogens (BESK25), Bacillus subtilis (BESK18) and
Staphylococcus aureus (BESK27) were obtained from the
department of biological engineering culture collection, as
received from Konkuk University.
2.3. Preparation of the Plantleaf Extract
The G. biloba plant leaves where then utilized, which are
shown in Figure 1(a). The collected plant leaves were
washed thoroughly in water, and then rinsed with distilled
water to remove any dust particles. The cleaned leaves
were next shade-dried for approximately 2–4 days. The
dried leaves were processed as powdered and then stored,
protecting them from light for further use. To that end the
plant powder (2 g) was dispersed in 50 mL of distilled
water and then boiled for 15 min. The extract was subse-
quently filtered through the Whatman Grade 1 qualitative
filtration paper (GE Healthcare Life Sciences, U.S.A.), fol-
lowed by its storage in a vial for further experiments.
2.4. Synthesis of the AgNPs
For the synthesis of the AgNPs, 1 mL of AgNO3 (1 mM)
was added to 9 mL of the aqueous extract of the G. biloba
leaves. Consequently, this solution was then incubated at
37  C under dark and static conditions. It is noted that the
solution became a brown color within 12 hr, as shown in
Figure 1(b), indicating the formation of the AgNPs. A con-
trol was also maintained without the addition of AgNO3 .16
423
Green Synthesis of Silver Nanoparticles Using Ginkgo biloba and Their Bactericidal and Larvicidal Effects
2.5. Characterization Techniques of the
Synthesized AgNPs
The ultraviolet-visible (UV-vis) absorption-spectra analy-
ses were carried out using the Hitachi U2900 spectropho-
tometer (Hitachi, Japan) within the wavelength region
of 300–700 nm. It is shown that the Fourier transform
infrared (FTIR) spectra were recorded using the Bruker
FTIR spectrometer (Bruker, Germany) in the transmit-
tance mode and within the range of 400–4000 cm−1 . The
X-ray powder diffraction (XRPD) data were thus acquired
using the Rich Seifert 3000 diffractometer (Seifert Compa-
nies, Germany), according to the Bragg–Brentano geome-
try and using the step-scan technique, while a Johansson
monochromator was used to produce pure CuK1 radia-
tion (1.5406 Å; 40 kV; 30 mA) from 20–90. The data
were matched with the International Centre for Diffraction
Data (JCPDS) Card No-087-0720. At this time, the crys-
talline size was calculated from the full width at half max-
imum (FWHM) values of the diffraction peaks using the
Debye–Scherrer formula, as follows: D = 089 / cos ,
where D is the mean grain size,  is the X-ray wave-
length for the Cu target,  is the FWHM of the diffrac-
tion peak and  is the diffraction angle. To demonstrate
for the transmission electron microscopy (TEM) analy-
sis, a drop of a sample containing the synthesized AgNPs
was placed on a carbon (C)-coated copper (Cu) grid and
then dried under a vacuum. In like manner the TEM
images were obtained using the FEI TECNAI G2 (T-30)
transmission electron microscope (ThermoFisher Scien-
tific, U.S.A.) with an accelerating voltage of 250 kV
and the energy-dispersive X-ray spectroscopy (EDX) anal-
ysis was carried out using the Hitachi S-3400N field-
emission scanning electron microscope (FESEM) from
Hitachi, Japan.
2.6. Antibacterial Activity of the
Green-Synthesized AgNPs
The antibacterial activity of the biologically synthesized
AgNPs was determined using an agar-well diffusion
method. The bacterial cultures from fresh colonies were
inoculated in a Mueller–Hinton broth. At that time the cul-
tures were then allowed to grow until the optical density
(OD) reached 0.2 at 600 nm, where the OD of 0.2 cor-
responds to the medium concentration of 108 CFU mL−1
and then 20 mL of a sterilized nutrient-agar medium was
poured into petri plates. At that time, the medium was
allowed to solidify and the culture broths in each petri
plate were cotton swabbed. Four wells were made in the
medium using a gel puncture. The antibacterial test was
performed by loading the biologically synthesized AgNPs
into the well “A”, the plant extract into “D”, the antibiotic
(streptomycin) into the well “C” and 30 L of a 1-mM
AgNO3 , which served as the control, was loaded into the
well “B”. Notably, the plates were then incubated at 37  C
for 24 hr. After the incubation period, the inhibition zone
424
Balashanmugam et al.
diameters (ISD) were measured. This procedure was sub-
sequently repeated for a minimum inhibitory concentration
(MIC) assay. Next, the different concentrations (10, 20, 30,
40 L) of the synthesized AgNP solution were added to
the four wells and the plates were incubated at 37  C for
24 hr. To this end, the lowest concentration that is below
the concentration where the plate did not show any growth
has been considered as the MIC. In this case, the standard
deviations were recorded using the mean values of 3 repli-
cates. The student’s t-test method was used to evaluate the
statistical significance between the test sample and control.
2.7. Larvicidal Activity
To evaluate the larvicidal activity of the green-synthesized
AgNPs, the Culex larvae were collected from the stagnant-
water area of a pond. Additionally, they were identi-
fied by using standard manuals and confirmed with the
expert in that area of specialization. To start a colony,
the larvae were kept in plastic and enamel trays con-
taining tap water. The larvae were then maintained and
reared in a laboratory. In particular, the larvicidal activity
was assessed using a modified World Health Organiza-
tion (WHO) procedure.17 For the bioassay, the test larvae
were stored in separate plates. At different concentrations
(5, 10, 15, 20, 25, 30 L), 2 mL of the aqueous plant-
extract AgNPs was added in triplicate to each plate. The
water that had been incubated at room temperature was
used as the control. In light of this, to avoid the settling
of the particles, especially at the higher doses, all of the
treatment solutions were ultrasonicated for 5 min prior to
the addition of the solutions to the Culex mosquito lar-
vae. Additionally, the mortality was assessed after 24 hr
to determine the acute toxicity. The number of dead lar-
vae was counted after 24 hr of exposure and the mortality
percentages were reported. The standard deviations were
then recorded using the mean values of 3 replicates. Statis-
tical significance of the variables was measured by using
t-test.
3. RESULTS AND DISCUSSION
3.1. Spectral Characterization
The change of the aqueous-solution color from pale yellow
to brown, as is evident in Figure 1(b), is due to the sur-
face plasmon resonance (SPR) of the AgNPs at 430 nm,
which appears in Figure 2(a). It is noted that the AgNPs
exhibited the SPR band due to the combined vibrations of
the AgNP electrons that was in resonance with the inci-
dent light wave (Priya et al., 2011). The aqueous AgNO3
solution was mixed with the G. biloba extract, and then
incubated at 37  C. The formation of the AgNPs was mon-
itored using the UV-vis absorption spectra. Therefore, the
UV-vis analysis revealed that the formation of the AgNPs
occurred after 6 hr; the SPR-band intensity was increased
up to 72 hr, as shown in Figure 2(b); and after 72 hr, the
intensity remained constant, indicating that the G. biloba
Nanosci. Nanotechnol. Lett. 10, 422–428, 2018
Balashanmugam et al. Green Synthesis of Silver Nanoparticles Using Ginkgo biloba and Their Bactericidal and Larvicidal Effects
Fig. 2. (a) Ultraviolet (UV)-visible (Vis) spectrum, (b) UV-Vis spec-
tra at different time intervals, and (c) Fourier transform infrared (FTIR)
spectrum of G. biloba-silver nanoparticles.
plant-extract-mediated synthesis of AgNPs involves only
the use of the room temperature (37  C) condition and
higher-temperature heating is not required.
The SPR band remained unchanged for up to 120 days,
indicating the higher stability of the green-synthesized
AgNPs. Consequently, this stability is the result of the
potential barrier that develops as a result of the compe-
tition between weak van-der-Waals attraction forces and
an electrostatic repulsion.18 The FTIR measurement of the
dried AgNPs was carried out to identify the possible con-
jugation of biomolecules with the AgNPs; the AgNPs were
actually stabilized in the solution by the biomolecules of
the G. biloba extract. At this time, the absorption bands
are observable at 3397 cm−1 , 2923 cm−1 , 1621 cm−1 and
1089 cm−1 and these correspond to the stretching vibra-
tions of the O–H, –C–H, –C–C and –C–O groups, respec-
tively, which can be seen in Figure 2(c). Likewise, the
band at 1621 cm−1 suggests the presence of the amide
group of proteins. Moreover, the results indicate that the
conjugation of the carbonyl group of proteins regarding
the AgNPs is strong, with the proteins forming a layer
along with the bio-organics, thereby securing the NPs.
This proves that the polyphenolics such as tannic acid con-
fer free energy to reduce the NP-conversion rate of Ag
ions and these results are supported by previously reported
results.19
3.2. XRPD Analysis
The crystalline nature of AgNPs has been confirmed by
the XRPD analysis. The Figure 3 shows the XRPD pattern
of the synthesized AgNPs. To that end the three diffrac-
tion peaks that are evident at 38.2, 44.4 and 64.6 in the
2 range correspond to the (111), (200) and (220) reflec-
tion planes of the face centered cubic (fcc) structure of
the AgNPs, respectively and these results are well matched
Nanosci. Nanotechnol. Lett. 10, 422–428, 2018
Fig. 3. X-ray diffraction (XRD) pattern of G. biloba-silver
nanoparticles.
Fig. 4. (a) Transmission electron microscopy (TEM) image and
(b) energy-dispersive X-ray spectroscopy (EDX) spectrum of G. biloba-
silver nanoparticles.
with the JCPDS Card No-010893722. Therefore, the deter-
mined mean size of the green-synthesized AgNPs is within
the region of 25–45 nm. The calculated lattice parameter
is 4.08 Å., thereby confirming the formation of metallic
Ag (Ag0  in terms of the literature information.16
3.3. TEM and EDX Analyses
The TEM images of the AgNPs clearly show that the sizes
of the AgNPs are in the region of 25–45 nm, while the
AgNP morphology has been identified as a spherical shape
and this can be seen in Figure 4(a). Namely, a careful
observation reveals a bio-moiety layer on the AgNP sur-
faces. We concur that this organic moiety plays an impor-
tant role in the reduction of the Ag+ ions to AgNPs and
it also protects the interparticle binding. In earlier stud-
ies, Balashanmugam et al.20 reported 14 nm as the average
size of biosynthesized AgNPs. Whereas the EDX analysis
of Balashanmugam et al. shows strong signals in the Ag
region, confirming the presence of AgNPs and the typical
EDX-spectrum peak at 3 keV is due to metallic AgNPs,
as shown in Figure 4(b).
3.4. Antibacterial Activity of
Green-Synthesized AgNPs
It is noted that the green-synthesized AgNPs exhibited
an excellent antibacterial activity against the bacterial
425
Green Synthesis of Silver Nanoparticles Using Ginkgo biloba and Their Bactericidal and Larvicidal Effects Balashanmugam et al.

Table I. Antibacterial activity of green-synthesized silver nanoparticles. Table II. Minimum inhibitory concentration (MIC) of silver nanopar-
Standard Deviation (±) showed the mean values of 3 replicates. The ticles against bacterial pathogens. Standard Deviation (±) showed the
results are statistically significant with p value <0.05. mean values of 3 replicates with statistical significance (p value <0.05).

Inhibition zone (mm) Inhibition zone (mm)

Plant Positive Bacteria name 10 L 20 L 30 L 40 L
Bacteria name AgNPs AgNO3 extract control
Staphylococcus aureus 10 (±1) 12 (±2) 13 (±2) 16 (±3)
Bacillus subtills 14 (±3) 12 (±2) – 28 (±4) Enterobacter aerogens 8 (±1) 10 (±1) 12 (±2) 13 (±2)
Enterobacter aerogens 13 (±2) 9 (±1) – 27 (±3) Bacillus subtillus 10 (±2) 10 (±1) 14 (±2) 15 (±3)
Pseudomonas aerginosa 17 (±4) 13 (±3) – 21 (±2) E. coli – 8 (±1) 9 (±2) 9 (±1)
Staphylococcus aureus 15 (±3) 11 (±2) – 23 (±3) Salmonella paratyphi 8 (±1) 9 (±1) 11 (±2) 12 (±2)
Salmonella paratyphi 12 (±2) 6 (±1) – 22 (±4) Pseudomonas aeroginosa 11 (±2) 13 (±2) 15 (±2) 19 (±3)
Escherichia coli 8 (±1) 6 (±1) – 18 (±3)

the E. coli, as shown in Table II. The binding of the par-

pathogens P. aeruginosa, S. aureus, B. subtilis, E. aero-
gens, S. paratyphi and E. coli. It has been reported that the
antibacterial effect is size and dose-dependent, and is more
pronounced against P. aeruginosa than the other bacteria.21
The antibacterial activities of the AgNPs against the Gram-
positive and Gram-negative pathogenic bacteria have been
investigated at the AgNP concentration of 40 L. The IDZ
values are represented in Table I. In the present study, the
AgNPs showed the highest inhibition against the Gram-
negative bacteria P. aeruginosa (17 mm), whereas their
inhibition is the lowest against the E. coli (8 mm). Like-
wise, the variables of the data results showed the statisti-
cal significance (p value <0.05) between control test and
treatments.
The Shrivastava et al.22 study reported that the interac-
tion of the NPs with the cell wall results in the entry of
the NPs inside bacterial cells and this is associated with
the rupture of the bacterial cellular components as the
tyrosine phosphorylation of the putative peptides is modu-
lated, thereby inhibiting the bacterial growth. As a conse-
quence, the extent of the differential sensitivity of each of
the Gram-positive and the Gram-negative bacteria toward
the AgNPs depends upon their cell-surface characteristics
and their interaction with the AgNPs. In that event, the
cell wall of the Gram-positive bacteria does not consist
of an outer membrane and it has been observed that the
negative charge on the cell surface of the Gram-negative
bacteria is higher than that on the surface of the Gram-
positive bacteria.21 Consequently, it is shown that the inter-
action between the Gram-negative bacteria and AgNPs is
certainly stronger than that regarding the Gram-positive
bacteria. Besides, in this case the cell wall of the Gram-
negative bacteria consists of an outer membrane that is
composed of a lipid, a protein and lipid polysaccharides
that act as a barrier and provide an effective protection
against antibacterial agents.23
The MIC was investigated using the pathogenic bac-
teria P. aeruginosa, S. aureus, B. subtilis, E. aerogens,
S. paratyphi and E. coli. The MIC was determined using
10, 20, 30 and 40 L of the AgNPs. Hence, the AgNP
MIC regarding the P. aeruginosa, S. aureus, E. aerogens
and B. subtillus bacteria is 10 L, while it is 20 L for
ticles to the bacteria depends on the surface area of the
available AgNPs. Additionally the smaller particles with
a larger surface area are available for interactions and
the corresponding bactericidal effect will be stronger than
that of the larger particles.22 The higher bactericidal activ-
ity is certainly due to the Ag cations that are released
from AgNPs and act as reservoirs for the Ag+ bacteri-
cidal agent. The attachment of either the Ag ions or the
AgNPs to the cell wall causes an accumulation of the
envelope-protein precursors, resulting in the dissipation of
the proton motive force.24 Under the circumstance, the
AgNPs also result in a destabilization of the outer mem-
brane and a plasma-membrane rupture, thereby causing
the depletion of the intracellular adenosine triphosphate,
or the ATP.25 The association of the Ag with oxygen (O)
and its reaction with the thiol (–S–H) groups on the cell
wall forms R–S–S–R bonds, thereby blocking respiration
and causing cell death. For this reason it is noted that the
Ag ions block the function of the electron-transport chain
most sensitively between the cytochrome reductase and
the cytochrome oxidase, whereas the least-sensitive block-
ing is between the reduced form of nicotinamide adenine
dinucleotide, or NADH (C21 H27 N7 O14 P2  and the succi-
nate dehydrogenase.26 The Santoro et al.27 and Cho et al.28
studies reported that AgNPs are effective at concentrations
that are significantly lower than those of the correspond-
ing ions.
3.5. Larvicidal Activity of the
Green-Synthesized AgNPs
The use of natural products that are coupled with the avail-
able nanotechnology reduces mosquito populations at the
larval stage and provides many associated vector control
benefits. In that event, since AgNPs are considered to be
potential agents for various biological applications includ-
ing antimicrobial ones, their application as a mosquito lar-
vicidal agent has been investigated. Accordingly, the data
that were obtained from the present study clearly indi-
cates that AgNPs provides excellent larval control with
respect to the Culex mosquito. Even so, a greater mortal-
ity was observed in the larvae that were treated with the
AgNPs as compared to the aqueous extract. The aqueous

426 Nanosci. Nanotechnol. Lett. 10, 422–428, 2018

Balashanmugam et al. Green Synthesis of Silver Nanoparticles Using Ginkgo biloba and Their Bactericidal and Larvicidal Effects
Table III. Larvicidal activity of silver nanoparticles on Culex mosquito
larvae.
No of dead larvae after
the incubation period
Concentration of AgNPs 4h 8h 12 h 16 h
Extract-control – – – –
5 L – – – 2
10 L – – 2 5
15 L – 1 3 7
20 L 1 4 5 8
25 L 3 4 6 8
30 L 4 5 7 10
Water-control – – – –
extract showed a slight effect, while the distilled water
showed no effect on the larval mortality. Although it is
noted that the higher concentrations of the aqueous plant
extract alone caused no larvae deaths until the 24 hr of
exposure had passed in that case (Table III). Addition-
ally, after 12 hr of exposure, 20 L of the AgNPs slightly
decreased the survival of the larvae to 50%, while a 100%
mortality of the larval population was observed with the
AgNP-solution concentration of 30 L after 16 hr. The
AgNPs (15 L) killed the larvae slowly and a mortality
of almost 90% was found after 24 hr of exposure. It is
noted that the maximum efficacy is evident at 30 L of
the AgNPs (Table III). It is possible that the mechanism
that causes the death of the larvae is the capability that
allows the NPs to penetrate through the larval membrane.
As a consequence, the AgNPs in the intracellular space
can bind to the available sulfur (S)-containing proteins or
phosphorus-containing compounds like deoxyribonucleic
acid (DNA), thereby leading to the denaturation of a num-
ber of organelles and enzymes. Subsequently, the decrease
of the membrane permeability and the disturbance of the
proton motive force, cause a loss of the cellular function
that is shown to eventually result in cell death.
Correspondingly, the Sundaravadivelan et al.29 study
suggested that the noted higher AgNP doses facilitate
greater uptakes of the NPs by the larval bodies. Con-
sequently, the AgNP penetration into the treated larval
membrane causes an interaction with the cell molecules
that result in larvae death. In addition, it is noted that
the AgNPs reach the midgut epithelial membrane, inacti-
vate the enzymes and generate peroxide, thereby leading
to cell death.30 31 In like manner, the penetration of the
AgNPs that is promoted by the attraction of a positive
Ag+ with a negatively charged cell membrane was demon-
strated by Kvitek et al.32 and Liang et al.33 The Raman
et al.34 study reported that the Pithecellobium dulce medi-
ated extracellular green-synthesized AgNPs, which showed
a significant larvicidal activity against Culex quinquefas-
ciatus at the concentrations of 10, 20, 30, 40 and 50 mg/L.
The Rajakumar et al.35 study synthesized the AgNPs by
utilizing the aqueous extract of Eclipta prostrate, which
Nanosci. Nanotechnol. Lett. 10, 422–428, 2018
showed a significant larvicidal activity against the instars
of C. quinquefasciatus at the concentrations of LC50 =
456 mg/L and LC90 = 1314 mg/L and for the larvicidal
activity against Anopheles subpictus, the concentrations of
24 h LC50 = 514 mg/L and LC90 = 2568 mg/L were sufficient;
moreover, it showed that the fourth instars are significantly
2 more susceptible.35 In addition, the NPs that were synthe-
5
7
sized from the fresh leaves, dried leaves and green berries
9 of Solanum nigrum were separately subjected to larvicidal
10 bioassays on the second and third instar larvae of C. quin-
10 quefasciatus and Anophele stephensi at the concentrations
10 of 2.5, 5 and 10 ppm. To this end, the 10-ppm concentra-
–
tion of the fresh- and dried-leaf synthesized NPs resulted
in a 100% mortality in both the second and third instars
of A. stephensi, while the 10-ppm concentration of synthe-
sized berry NPs resulted in the mortality rates of 96.65%
and 100% for the second and third instars of A. stephens,
respectively. Additionally, in the case of C. quinquefascia-
tus, the mortality rates of 100%, 96.65% and 85% were
observed when the combinations of AgNPs/dried leaves,
AgNPs/fresh leaves and AgNPs/berries were used, respec-
tively. The results of the present work are in sound agree-
ment with those that were reported by the Rawani et al.36
study, confirming that the mortality rate in all forms of
larvae decreases with the decreasing of the NP concentra-
tion. The values of the results showed the statistical sig-
nificance (p value <0.05) between the control test and the
treatments.
Comparatively, there are numerous reports are avail-
able on the use of plant extracts for larvicidal activity.
A very low mortality percentage was observed accordingly
in each of the following four solvents: petroleum ether,
water, acetone and ethyl acetate. As a consequence, the
aqueous extracts of G. biloba (0.125%) showed the least
mortality compared with the other solvents, but the same
extract with a 1% (w/v) concentration provided the highest
mortality percentage, while a 1% (w/v) concentration of
the ethanol extract of G. biloba showed the highest mor-
tality of 39%.37 Additionally, the mortality rate of 21%
was noted for the first instar larvae for which a treat-
ment of 6% (w/v) Leucas aspera extract was applied; how-
ever, it increased to 85% with a 14% treatment of the
L. aspera leaf extract.38 The Muthukrishnan et al.39 study
observed that the LC50 values of the ethyl acetate extract of
L. aspera regarding the first, second, third and fourth instar
larvae of C. quinquefasciatus are 75.40, 93.09, 132.20 and
138.60 ppm, respectively.39
In this instance, the selected plant extract of G. biloba,
contained two kind of bioactive compounds (flavonoids,
terpenes), which indicate several desirable medicinal prop-
erties including use as an antioxidant, anti-inflammatory
and cell regeneration therapy. The leaf extracts of the plant,
L. aspera exhibited higher mortality rates, especially dur-
ing the molding process and the subsequent melanization
and tanning processes.40 The Murugan and Jayabalan41
study reported the exhibition of a 90% larval mortality
427
 Green Synthesis of Silver Nanoparticles Using Ginkgo biloba and Their Bactericidal and Larvicidal Effects
with a 4% leaf-extract concentration against the fourth
instar larvae of A. stephensi. In this case, the hexane
extract of the plant showed the highest larvicidal activ-
ity against two of the mosquito vectors, followed respec-
tively by the results from the chloroform and ethanol. The
LC50 values of L. aspera against the first, second, third
and fourth instar larvae of C. quinquefasciatus are 122.50,
149.97, 193.43 and 230.71 ppm, respectively, while the
LC50 values against Aedes aegypti are 77.40, 144.00,
199.72 and 257.17 ppm, respectively.42
4. CONCLUSION
The AgNPs were synthesized using a simple, low-cost,
nontoxic and ecofriendly green approach for which the
G. biloba plant-leaf extract was utilized. The nanoconju-
gate of the G. biloba AgNPs showed an effective antibac-
terial activity as noted against the P. aeruginosa compared
with the results of the other bacteria. However, the MIC
of the AgNPs is 10 L for the inhibiting of the P. aerugi-
nosa, S. aureus, B. subtills, E. aerogens and S. paratyphi,
while it is 20 L for the E. coli. It has been proved that the
nanoconjugate of the G. biloba–AgNP extract is an effi-
cient alternative source of the chemical larvicidal agents
that are identified to presently cause environmental pollu-
tion. Equally important the present investigation facilitates
a consideration of the application of the AgNP-conjugated
biomolecules in integrated vector control programs.
Conflict of Interest
The authors have declared no conflict of interest in the
performance of this study.
Acknowledgments: This work was carried out with
the support of Cooperative Research Program for Agri-
culture Science and Technology Development (Project
No. PJ012550032018) Rural Development Administration,
Republic of Korea.
References and Notes
1. P. Mohanpuria, N. K. Rana, and S. K. Yadav, J. Nanopart. Res.
10, 507 (2008).
2. C. N. Lok, C. M. Ho, R. Chen, Q. Y. He, W. Y. Yu, H. Sun, P. K.
Tam, J. F. Chiu, and C. M. Che, J. Biol. Inorg. Chem. 12, 527 (2007).
3. M. A. Farooqui, P. S. Chauhan, P. Krishnamoorthy, and J. Shaik,
Dig. J. Nanomat. Biostruct. 5, 43 (2010).
4. N. Ahmad, S. Sharma, V. N. Singh, S. F. Shamsi, A. Fatma, and
B. R. Mehta, Biotechnol. Res. Int. 2011, 454090 (2011).
5. G. D. Wright, Adv. Drug Deliv. Rev. 57, 1451 (2005).
6. S. Pal, Y. K. Tak, and J. M. Song, Appl. Environ. Microbiol. 73, 1712
(2007).
7. J. S. Kim, E. Kuk, K. N. Yu, J. H. Kim, S. J. Park, H. J. Lee, S. H.
Kim, Y. K. Park, Y. H. Park, C. Y. Hwang, Y. K. Kim, Y. S. Lee,
D. H. Jeong, and Y. K. Kim, Nanomedicine: Nanotechnol., Biol.
Med. 3, 95 (2007).
428
View publication stats
Balashanmugam et al.
8. A. Ingle, A. Gade, S. Pierrat, C. Sonnichsen, and M. Rai, Curr.
Nanosci. 4, 141 (2008).
9. T. Youdeowei and M. W. Service, Pest Control and Management,
Longman, Singapore (1983).
10. C. F. Curtis, Med. Vet. Entomol. 8, 107 (1994).
11. F. H. Collins and S. M. Paskewitz, Ann. Rev. Entomol. 40, 195
(1995).
12. S. A. Radhika and K. Sahayara, J. Biopest. 7, 152 (2014).
13. C. B. Wandscheer, J. E. Duque, M. A. De Silva, Y. Fukuyama, J. L.
Wohlke, J. Adelmann, and J. D. Fontana, Toxicon 44, 829 (2004).
14. S. S. Nathan and K. Kalaivani, Biological Cont. 34, 93 (2005).
15. M. Frederich, J. M. Dogné, L. Angenot, and P. Mol, Curr. Med.
Chem. 9, 1435 (2002).
16. P. Balashanmugam and P. T. Kalaichelvan, Int. J. Nanomed. 10, 87
(2015).
17. A. A. Rahuman, G. Gopalakrishnan, B. S. Ghouse, S. Arumugam,
and B. Himalayan, Fitoterapia 71, 553 (2000).
18. T. C. Prathna, N. Chandrasekaran, A. M. Raichur, and A. Mukherjee,
Colloids Surf. B. Biointerfaces 82, 152 (2011).
19. S. P. Dubey, M. Lahtinen, and M. Sillanpaa, Process Biochem.
45, 1065 (2010).
20. P. Balashanmugam, S. Santhosh, D. J. Mukesh Kumar,
S. Anbazhakan, and P. T. Kalaichelvan, Indo American Journal of
Pharmaceutical Res. 4, 475 (2014).
21. J. M. Martinko and M. T. Madigan, Brock Biology of Microorgan-
isms, Prentice Hall, Illinois (2005).
22. S. Shrivastava, T. Bera, A. Roy, G. Singh, P. Ramachandrarao, and
D. Dash, Nanotechnology 18, 225103 (2007).
23. T. Maneerung, S. Tokura, and R. Rujiravanit, Carbohyd. Polym.
72, 43 (2008).
24. E. L. Yu-sen, R. D. Vidic, J. E. Stout, C. A. McCartney, and L. Y.
Victor, Water Res. 32, 1997 (1998).
25. C. N. Lok, C. M. Ho, R. Chen, Q. Y. He, W. Y. Yu, H. Sun, and
C. M. Che, J. Proteome Res. 5, 916 (2006).
26. V. S. Kumar, B. M. Nagaraja, V. Shashikala, A. H. Padmasri, S. S.
Madhavendra, B. D. Raju, and K. R. Rao, J. Mol. Catal. A. Chem.
223, 313 (2004).
27. C. M. Santoro, N. L. Duchsherer, and D. W. Grainger, Nanobiotech-
nology 3, 55 (2007).
28. K. H. Cho, J. E. Park, T. Osaka, and S. G. Park, Electrochim. Acta
51, 956 (2005).
29. C. Sundaravadivelan and M. Nalini, Asian Pac. J. Trop. Biomed.
2012, 1 (2012).
30. G. D. Shockman and J. F. Barren, Ann. Rev. Microbiol. 37, 501
(1983).
31. M. Raffi, F. Hussain, T. M. Bhatti, J. I. Akhter, A. Hameed, and
M. M. Hasan, J. Mat. Sci. Technol. 24, 192 (2008).
32. L. Kvitek, A. Panáček, J. Soukupova, M. Kolar, R. Vecerova,
R. Prucek, and R. Zboril, J. Phys. Chem. C 112, 5825 (2008).
33. Z. Liang, A. Das, and Z. Hu, Water Res. 44, 5432 (2010).
34. N. Raman, S. Sudharsan, V. Veerakumar, N. Pravin, and K. Vithiya,
Spectrochim. Acta A 96, 1031 (2012).
35. G. Rajakumar and A. A. Rahuman, Acta Trop. 118, 196 (2011).
36. A. Rawani, A. Ghosh, and G. Chandra, Acta Trop. 128, 613 (2013).
37. V. Karthikeyan, K. Sivakumar, A. Gokuldas, and
S. Mohanasundaram, Asian J. Pharm. Clin. Res. 5, 189 (2012).
38. K. Kovendan, K. Murugan, S. P. Shanthakumar, S. Vincent, and J. S.
Hwang, Parasitology Res. 111, 1481 (2012).
39. J. Muthukrishnan, E. Pushpalatha, and A. Kasthuribai, Insect Sci.
Appl. 17, 389 (1997).
40. R. W. Mwangi and H. Rembold, Entomol. Exp. Appl. 46, 103 (1998).
41. K. Murugan and D. Jayabalan, Curr. Sci. 769, 631 (1999).
42. Maheswaran, S. Sathis, and S. Ignacimuthu, Int. J. Integr. Biol.
2, 214 (2008).
Received: 7 January 2018. Accepted: 25 March 2018.
Nanosci. Nanotechnol. Lett. 10, 422–428, 2018