Green-synthesized silver nanoparticles as a novel control tool against dengue virus (DEN-2) and its primary vector Aedes aegypti

Abstract

Dengue is an arthropod-borne viral infection mainly vectored through the bite of Aedes mosquitoes. Recently, its transmission has strongly increased in urban and semi-urban areas of tropical and sub-tropical regions worldwide, becoming a major international public health concern. There is no specific treatment for dengue. Its prevention and control solely depends on effective vector control measures. In this study, we proposed the green-synthesis of silver nanoparticles (AgNP) as a novel and effective tool against the dengue serotype DEN-2 and its major vector Aedes aegypti. AgNP were synthesized using the Moringa oleifera seed extract as reducing and stabilizing agent. AgNP were characterized using a variety of biophysical methods including UV-vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), and sorted for size categories. AgNP showed in vitro antiviral activity against DEN-2 infecting vero cells. Viral titer was 7 log10 TCID50/ml in control (AgNP-free), while it dropped to 3.2 log10 TCID50/ml after a single treatment with 20 μl/ml of AgNP. After 6 h, DEN-2 yield was 5.8 log10 PFU/ml in the control, while it was 1.4 log10 PFU/ml post-treatment with AgNP (20 μl/ml). AgNP were highly effective against the dengue vector A. aegypti, with LC50 values ranging from 10.24 ppm (I instar larvae) to 21.17 ppm (pupae). Overall, this research highlighted the concrete potential of green-synthesized AgNP in the fight against dengue and its primary vector A. aegypti. Further research on structure-activity relationships of AgNP against other dengue serotypes is urgently required.

Full text

ORIGINAL PAPER
Green-synthesized silver nanoparticles as a novel control
tool against dengue virus (DEN-2) and its primary vector
Aedes aegypti
Vasu Sujitha
1
&Kadarkarai Murugan
1
&Manickam Paulpandi
1
&
Chellasamy Panneerselvam
1
&Udaiyan Suresh
1
&Mathath Roni
1
&Marcello Nicoletti
2
&
Akon Higuchi
3
&Pari Madhiyazhagan
1
&Jayapal Subramaniam
1
&Devakumar Dinesh
1
&
Chithravel Vadivalagan
1
&Balamurugan Chandramohan
1
&Abdullah A. Alarfaj
4
&
Murugan A. Munusamy
4
&Donald R. Barnard
5
&Giovanni Benelli
6
Received: 20 May 2015 /Accepted: 25 May 2015
#Springer-Verlag Berlin Heidelberg 2015
Abstract Dengue is an arthropod-borne viral infection main-
ly vectored through the bite of Aedes mosquitoes. Recently, its
transmission has strongly increased in urban and semi-urban
areas of tropical and sub-tropical regions worldwide, becom-
ing a major international public health concern. There is no
specific treatment for dengue. Its prevention and control solely
depends on effective vector control measures. In this study, we
proposed the green-synthesis of silver nanoparticles (AgNP)
as a novel and effective tool against the dengue serotype
DEN-2 and its major vector Aedes aegypti.AgNPweresyn-
thesized using the Moringa oleifera seed extract as reducing
and stabilizing agent. AgNP were characterized using a vari-
ety of biophysical methods including UV–vis spectroscopy,
Fourier transform infrared spectroscopy (FTIR), scanning
electron microscopy (SEM), energy-dispersive X-ray
spectroscopy (EDX), X-ray diffraction (XRD), and sorted
for size categories. AgNP showed in vitro antiviral activity
against DEN-2 infecting vero cells. Viral titer was 7 log
10
TCID
50
/ml in control (AgNP-free), while it dropped to 3.2
log
10
TCID
50
/ml after a single treatment with 20 μl/ml of
AgNP. After 6 h, DEN-2 yield was 5.8 log
10
PFU/ml in the
control, while it was 1.4 log
10
PFU/ml post-treatment with
AgNP (20 μl/ml). AgNP were highly effective against the
dengue vector A. aegypti, with LC
50
values ranging from
10.24 ppm (I instar larvae) to 21.17 ppm (pupae). Overall, this
research highlighted the concrete potential of green-
synthesized AgNP in the fight against dengue and its primary
vector A. aegypti. Further research on structure–activity rela-
tionships of AgNP against other dengue serotypes is urgently
required.
Keywords Botanical insecticides .Mosquito-borne diseases .
Moringa oleifera .silver nanoparticles .Aedes aegypti .
cytotoxicity
Introduction
Dengue is a mosquito-borne viral disease transmitted by fe-
male mosquitoes, mainly Aedes aegypti and, to a lesser extent,
Aedes albopictus. Recently, dengue transmission has strongly
increased in urban and semi-urban tropical areas worldwide,
becoming a major international public health concern. Over
2.5 billion people are now at risk from dengue. The World
Health Organization estimates that there may be 50–100 mil-
lions of dengue infections worldwide every year. There are
four distinct, but closely related, serotypes of the virus that
*Giovanni Benelli
g.benelli@sssup.it; benelli.giovanni@gmail.com
1
Division of Entomology, Department of Zoology, School of Life
sciences, Bharathiar University, Coimbatore 641 046, Tamil Nadu,
India
2
Department of Environmental Biology, Sapienza University of
Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy
3
Department of Reproduction, National Research Institute for Child
Health and Development, Tokyo 157-8535, Japan
4
Department of Botany and Microbiology, College of Science, King
Saud University, Riyadh 11451, Saudi Arabia
5
Center for Medical, Agricultural, and Veterinary Entomology,
USDA-ARS,1600SW23rdDrive,Gainesville,FL32608,USA
6
Department of Agriculture, Food and Environment, University of
Pisa, via del Borghetto 80, 56124 Pisa, Italy
Parasitol Res
DOI 10.1007/s00436-015-4556-2

cause dengue (DEN-1, DEN-2, DEN-3, and DEN-4).
Recovery from infection by one provides lifelong immunity
against that particular serotype. However, cross-immunity to
the other serotypes after recovery is only partial and temporary
(WHO 2012). Currently, there is no specific treatment for
dengue, even if the development of a vaccine is in progress
(Murrell et al. 2011;WHO2015). Its prevention and control
solely depends on effective vector control measures (Suresh
et al. 2015;WHO2015).
Aedes larvae and pupae are usually targeted using organo-
phosphates and insect growth regulators. Indoors residual
spraying and insecticide-treated bed nets are also employed
to reduce transmission of malaria in tropical countries.
However, synthetic chemicals have strong negative effects
on human health and the environment and induce resistance
in a number of mosquito species (Hemingway and Ranson
2000). In this scenario, eco-friendly control tools are urgently
needed. In the latest years, huge efforts have been carried out to
investigate the efficacy of botanical products against mosquito
vectors; many plant-borne compounds have been reported as
effective against Culicidae, acting as adulticidal, larvicidal, ovi-
cidal, oviposition deterrent, growth and/or reproduction inhibi-
tors, and/or adult repellents (e.g., Amer and Mehlhorn 2006a,b;
Panneerselvam et al. 2012; Benelli et al. 2015a,b).
Nanobiotechnologies have the potential to revolutionize a
wide array of applications, including drug delivery, diagnos-
tics, imaging, sensing, gene delivery, artificial implants, tissue
engineering, and pest management (Elechiguerra et al. 2005).
The plant-mediated biosynthesis (i.e., Bgreen synthesis^)of
metal nanoparticles is advantageous over chemical and phys-
ical methods, since it is cheap, single-step, does not require
high pressure, energy, temperature, and the use of highly toxic
chemicals (Goodsell 2004). A growing number of plants and
fungi have proposed for efficient and rapid extracellular syn-
thesis of silver and gold nanoparticles (see Rajan et al. 2015
for a recent review), which showed excellent mosquitocidal
properties, also in field conditions (e.g., Rajakumar and
Rahuman 2011;Dineshetal.2015;Sureshetal.2015;
Murugan et al. 2015a,b).
In this study, we proposed the green-synthesis of silver
nanoparticles (AgNP) as a novel and effective tool against
the serotype DEN-2 and its major vector A. aegypti.AgNP
were synthesized using the Moringa oleifera (Moringaceae)
seed extract as reducing and stabilizing agent. AgNP were
characterized using a variety of biophysical methods includ-
ing UV–vis spectroscopy, Fourier transform infrared spectros-
copy (FTIR), scanning electron microscopy (SEM), energy-
dispersive X-ray spectroscopy (EDX), X-ray diffraction
(XRD), and sorted for size categories. Green-synthesized
AgNP were tested for antiviral activity against the dengue
serotype DEN-2 using vero cell culture assays and growth
inhibition tests. Furthermore, M. oleifera seed extract and
green-synthesized AgNP were tested for larvicidal and
pupicidal toxicity against the primary dengue vector
A. aegypti.
Materials and methods
Study species and collection of plant material
M. oleifera is a small, fast-growing tree with an ancient tradi-
tion in ethno-pharmacology. Its plant parts are rich in flavo-
noids, tannins, saponins, terpenoids, proanthocyanadins, and
cardiac glycosides (Vinoth et al. 2012). M. oleifera seeds have
been studied for their anti-microbial, anti-tumor, anti-inflam-
matory, antispasmodic, and diuretic properties (Cárceres et al.
1992; Guevara et al. 1999; Bharali et al. 2003; Ali et al.
2004;Chuangetal.2007).
In this study, the seeds of M. oleifera were collected from
the campus of the Bharathiar University (Coimbatore, India).
Seeds were identified by an expert taxonomist at the
Department of Botany (Bharathiar University, Coimbatore).
Voucher specimens were stored in our laboratories and are
available under request.
M. oleifera-mediated biosynthesis of silver nanoparticles
M. oleifera seeds were washed carefully with distilled water
and shade-dried at room temperature (28±2 °C) for 2 days;
20 g of seeds werepowdered using anelectrical blender; 5 g of
seed powder were added in a 300-mL Erlenmeyer flask filled
with 100 mL of sterilized distilled water, then the mixture was
boiled for 5 min, before finally decanting it. The seed extract
was filtered using Whatman filter paper no. 1, stored at −4°C
and tested within 5 days. The filtrate was treated with aqueous
1mMAgNO
3
solution in an Erlenmeyer flask, and incubated
at room temperature. Silver nitrate was purchased from the
Precision Scientific Co. (Coimbatore, India). A dark brown
solution indicated the formation of AgNP, since aqueous silver
ions were reduced by the M. oleifera seed extract generating
stable Ag
0
nanoparticles.
Characterization of green-synthesized silver nanoparticles
Following the methods reported by Murugan et al. (2015a,b),
the biosynthesis of AgNP was confirmed by sampling the
reaction mixture at regular intervals (2 ml per sampling).
The maxima absorption was scanned by UV–vis spectroscopy
at wavelength of 200–600 nm, using a UV-3600 Shimadzu
spectrophotometer with 1-nm resolution. Furthermore, the re-
action mixture was subjected to centrifugation at 15,000 rpm
for 20 min. The resulting pellet was dissolved in de-ionized
water and filtered through Millipore filter (0.45 μm). An ali-
quot of this filtrate containing AgNP was used for SEM, EDX,
FTIR, and XRD. SEM studies were carried out using a FEI
Parasitol Res

QUANTA-200 SEM. EDX analyses were conducted using a
JEOL-MODEL 6390. XRD analysis of AgNP-coated glass
substrates was carried out on a Phillips PW1830 instrument
operating at 40 kV and current of 30 mA with Cu Kαradia-
tion. FTIR measurements were carried out using a Perkin-
Elmer Spectrum 2000 FTIR spectrophotometer. The AgNP
size distribution was determined using the particle analyzer
Malvern Nanosizer (Malvern Instrument, UK), where the size
was analyzed measuring size-dependent fluctuation of a scat-
tering laser light on AgNP.
Cells and virus
C6/36 and vero cells (i.e., originally propagated from African
green monkey kidney cells) were purchased from the National
Centre for Cell Sciences (NCCS, Pune, India). Both cell lines
were maintained and propagated in Eagle’s minimum essen-
tial medium (EMEM) containing 10 % fetal bovine serum.
Cultured C6/36 and vero cells were incubated at 28 and
37 °C, respectively, in 5 % CO
2
humidified chamber. For virus
propagation, the serum concentration of the medium was re-
duced to 2 %. Dengue virus type-2 (DEN-2) New Guinea C
strain was propagated using C6/36 cell line, and harvested
after cytopathic effect (CPE) presentation 7 days post-infec-
tion. After titration, viral stock was maintained at −70 °C.
Cytotoxicity assays
In citotoxicity assays, quadruplicate wells of confluent mono-
layers of vero cells were grown in 96-well tissue culture
plates. Cells were incubated with different concentrations of
AgNP. Then, we examined cell viability, as the ability of the
cells to cleave the tetrazolium salt MTT [3-(4,5-dimethyl-
thiazol-2ol)-2,5diphenyltetrazoliumbromide), Sigma Chem.
Co. St. Louis, USA], by the mitochondrial enzyme succinate
dehydrogenase which develops a formazan crystal. Each con-
centration was replicated three times.
Treatment of viral cells
AgNP were diluted in DMEM and sonicated for 5 min.
Monolayers from the vero cell culture and the AgNP/
DMEM mixture at 1:40 dilution (5×10
4
TCID/ml) were incu-
bated at room temperature with rotation for 1 h. Then, the
dengue virus, serotype DEN-2, was added to vero cells, seed-
ed to 90 % confluency. The viral suspension was allowed to
absorb for 1 h at 37 °C in 5 % CO
2
. Following absorption,
non-adherent DEN-2 was washed off using phosphate buff-
ered saline (PBS); DMEM supplemented with 2 % FBS was
added to the cells. Then, they were incubated at 37 °C in 5 %
CO
2
for 5 days, which is the time at which the cytopathic
effect was observed in about 80 % of the cells infected with
the untreated DEN-2. Each concentration was replicated three
times.
Virus growth inhibition assay
Confluent monolayers of vero cells in 12-well plates were
washed with PBS, then infected with DEN-2 at 0.1 multiplic-
ity of infection. The plates were continuously shaken for
45 min at room temperature in AgNP-free conditions, for vi-
rus adsorption. The solution was removed and replaced with
DMEM medium containing AgNP at 20 μg/ml. Viruses were
harvested at 8, 24, 36 h post-infection; the viral yield was
estimated by plaque assay on vero cells. Each concentration
was replicated five times. As control, DEN-2 infected cells
were incubated in AgNP-free medium over different time
intervals.
A. aegypti rearing
Eggs of A. aegypti were provided by the National Centre for
Disease Control (NCDC) field station of Mettupalayam
(Tamil Nadu, India). Eggs were transferred to laboratory con-
ditions [27±2 °C, 75–85 % R.H., 14:10 (L:D) photoperiod]
and placed in 18×13× 4-cm plastic containers filled 500 ml of
tap water, waiting for hatching. Larvae were fed daily with a
mixture of dog biscuits (Pedigree, USA) and hydrolyzed yeast
(Sigma-Aldrich, Germany) (3:1, w/w). Larvae and pupae were
collected, transferred to glass beakers filled with 500 ml of de-
chlorinated water, and tested in subsequent experiments
(Suresh et al. 2015).
Larvicidal and pupicidal toxicity in laboratory conditions
Following the method reported by Suresh et al. (2015), 25
A. aegypti larvae (I, II, III, and IVinstar) or pupae were placed
in a glass beaker filled with 250 ml of de-chlorinated water
plus the M. oleifera seed extract (50, 100, 150, 200, 250 ppm)
or green-synthesized AgNP (5, 10, 15, 20, 25 ppm). Larval
food (0.5 mg) was provided for each tested concentration.
Each concentration was replicated five times against all in-
stars. In control treatments, 25 larvae or pupae were trans-
ferred in 250 ml of de-chlorinated water. Percentage mortality
was calculated as follows:
Percentage mortality
¼Number of dead individuals=Number of treated individualsðÞ100:
Data analysis
SPSS software package 16.0 version was used for all analyses.
In mosquito experiments, LC
50
and LC
90
were calculated by
probit analysis, following the method by Finney (1971). All
Parasitol Res

toxicity data were analyzed using a two-way ANOVA with
two factors (i.e., the tested dose and the elapsed time for
anti-dengue experiments; the tested dose and the instar for
mosquitocidal experiments). Means were separated using
Tukey’s HSD test. P<0.05 was used for the significance of
differences between means.
Results and discussion
Synthesis and characterization of silver nanoparticles
When the AgNO
3
aqueous solution was added to the
M. oleifera seed extract, the color changed from pale yellow
to dark brown, indicating the reduction from Ag
+
to Ag
0
,and
the formation of AgNP (Fig. 1a–b). The absorption spectra of
AgNP at different time intervals showed highly symmetric
single band absorption peaks. A maximum absorption peak
was observed at 450 nm (Fig. 1c); broadening of the peak
indicated that the particles are polydispersed (Prasad and
Elumalai 2011). The peak steadily increased with reaction
time, and saturated after 240 min indicating complete reduc-
tion of the silver nitrate. The color variation may be linked to
the fact that AgNP have free electrons generating a surface
plasmon resonance absorption band, due to the combined vi-
bration of electrons of metal nanoparticles in resonance with
the light wave (Noginov et al. 2006).
SEM and size analysis showed that M. oleifera-synthesized
AgNP were predominantly spherical in shape (Fig. 2), with a
mean size of 100 nm (Fig. 3). In agreement with our results,
Fig. 1 The seed extract of Moringa oleifera before (a) and after (b)
treatment with an aqueous solution of AgNO
3
(1 mM); (c)UV–vis
spectra of the Moringa oleifera aqueous seed extract plus AgNO
3
at
different time intervals
Fig. 2 SEM micrograph showing the morphological characteristics of
silver nanoparticles bio-synthesized using the Moringa oleifera aqueous
seed extract
Fig. 3 Zeta potential and sizedistribution of AgNP biosynthesized using
the Moringa oleifera aqueous seed extract
Parasitol Res

spherical AgNP of similar size have been fabricated using
different plant-borne products. Good examples include the
leaf extracts of M. oleifera (Sathyavathi et al. 2011),
Cissus quadrangularis (Sivakama Valli and Vaseeharan
2012), and Calotropis gigantea (Vaseeharan et al. 2012).
The EDX pattern showed the chemical composition of
M. oleifera-synthesized AgNP (Fig. 4). It showed a strong
silver signal, confirming the presence of metallic silver, in
agreement with the UV–vis results. The sharp optical ab-
sorption band peak at 3 Kev is a typical absorption range
of metallic silver nanocrystallites (Magudapathy et al.
2001; Fayaz et al. 2010). Furthermore, the weak signals
linked to oxygen are due to the occurrence of secondary
metabolites present in the aqueous seed extract of
M. oleifera. These metabolites are extremely important
for nano-biosynthesis. Indeed, they often surround AgNP
with a thin layer of Bcapping^organic material from the
plant leaf broth, and this enhance the AgNP stability in
the solution for several weeks after synthesis (Dinesh
et al. 2015;Sureshetal.2015).
The XRD pattern showed intense peaks corresponding
to (111), (200), (220), and (311) Bragg's reflection based
on the face-centered cubic structure of AgNP (Fig. 5).
Thus, XRD analysis highlighted that AgNP formed by
the reduction of AgNO
3
with M. oleifera leaf extract
were crystalline in nature (Shameli et al. 2012).
Comparable results have been reported for AgNP fabricated
using Cochlospermum gossypium and Acalypha indica
(Kora et al. 2010;Krishnarajetal.2010).
FTIR spectroscopy was carried out to identify the
Bcapping^biomolecules that may help to stabilize
M. oleifera-synthesized AgNP in aqueous solution. FTIR
spectrum showed prominent peaks at 3,336.8, 1,637.6,
and 597.9 cm
−1
(Fig. 6). The absorption peak at 3,
336.8 cm
−1
may be due to stretching of –O–H bonds,
while the peak at 1,637.6 cm
−1
maybelinkedtothe
stretching of aldehyde C-H bonds; the peak at around
597.9 cm
−1
has been reported as characteristic of C–Br
bonds. Notably, we found only a broad band above 3,
000 cm
−1
, with no further evidences corresponding to
thepresenceofO–H (3,400 cm
−1
) and NH (3,166 cm
−1
)
stretching frequencies by –COOH and –NH
2
groups
(Kalwar et al. 2013). The peaks at 1,620-1,636 cm
−1
rep-
resent carbonyl groups from polyphenols such as catechin
gallate, epicatechin gallate, epigallocatechin, epigallocate-
chin gallate, gallocatechin gallate, and theaflavin. Our re-
sults suggest that the molecules capping AgNP may have
both free and bound amide groups. Amide groups may be
attached to aromatic rings. Overall, we hypothesize that
Fig. 4 EDX spectrum of silver nanoparticles synthesized using Moringa
oleifera aqueous seed extract
Fig. 5 XRD pattern of silver
nanoparticles synthesized using
Moringa oleifera seed extract
Parasitol Res

part of the compounds capping AgNP could be polyphenols
with an aromatic ring and bound amide region (Kumar et al.
2012).
Cytotoxicity against vero cells
In our experiments, the viability of vero cells exposed for 48 h
to different doses of green-synthesized AgNP was comparable.
Similarly, no substantial differences were evoked by a 72-h
exposure of the cells to AgNP (Fig. 7). No morphological
differences were observed among control vero cells (not treated
with AgNP), and cells exposed for 24 and 48 h to 40 μg/ml of
green-synthesized AgNP, while some cells appeared damaged
after 72 h of exposure to the same dosage (Fig. 8a–d). Our
findings corroborate a number of previous in vitro evidences,
indicating that biofabricated AgNP show reduced or no toxicity
on mammalian cells (Speshock et al. 2010; Vivek et al. 2012;
see also Miura and Shinohara 2009). Hu et al. (2014) showed
that 100 μg/ml of AgNP were non-toxic against vero cells.
Only slight cytotoxicity rates have been reported for
Penicillium spp.-synthesized AgNP against vero cells
(Shreshtha Verma et al. 2013). Sukirtha et al. (2012) reported
in vitro cytotoxicity of biosynthesized AgNP against human
epithelial carcinoma cell line, with dose-dependent activity.
DEN-2 growth inhibition assays
M. oleifera-synthesized AgNP showed in vitro antiviral activ-
ity against dengue virus DEN-2 infecting vero cells. Viral titer
was 7 log
10
TCID
50
/ml in control vero cells (infected with
DEN-2 virus, not treated with AgNP), while it dropped to
3.2 log
10
TCID
50
/ml after a single treatment with green-
synthesized AgNP tested at 20 microl/ml (Fig. 9). Figure 10
showed the size differences between vero cells infected by
DEN-2 and vero cells infected by DEN-2, then treated with
AgNP (20 μl/ml). Furthermore, a single treatment with low
doses of M. oleifera-synthesized AgNP strongly reduced the
dengue viral yield estimated by plaque assay. After 6 h, den-
gue viral yield was 5.8 log
10
PFU/ml in the control (AgNP-
free), while it was 1.4 log
10
PFU/ml post-treatment with
Fig. 6 FTIR spectrum of vacuum-dried powder of silver nanoparticles
synthesized using Moringa oleifera seed extract
Fig. 7 Viab ility o f vero cell s
treated with green-synthesized
AgNP over different exposure
times
Parasitol Res

AgNP (20 μl/ml) (Fig. 11). To the best of our knowledge, this
is the first report of anti-dengue activity of green-synthesized
AgNP. This is of particular interest, since no specific treat-
ments are currently available against this virus (Murrell et al.
2011;WHO2015).
However, nanosilver materials have already been pro-
posed as valuable tools in the fight against other viruses
(Rogers et al. 2008; Speshock et al. 2010). For instance,
Hu et al. (2014) investigated the interaction between
nanosilver and HSV-2, pointing out that 100 μg/ml of
AgNP shows a significant inhibition against HSV-2 prog-
eny. Park et al. (2014) demonstrated that magnetic hybrid
colloid decorated with AgNP (AgNP@MHCs) is able to
inactivate bacteriophage ΦX174, murine norovirus
(MNV), and adenovirus serotype 2 (AdV2). Mori et al.
(2013) proved antiviral activity of AgNP/chitosan com-
posites against H1N1 influenza A virus. Lara et al.
(2010) reported that AgNP exert anti-viral activity against
HIV-1 at an early stage of viral replication, since AgNP
may act as inhibitors of viral entry.
Nanoparticle toxicity against A. aegypti
In laboratory assays, the seed extract of M. oleifera was
toxic against larval instars (I–IV) and pupae of the dengue
vector A. aegypti.LC
50
values were 151.97 ppm (I instar),
185.74 ppm (II), 215.04 ppm (III), 241.92 ppm (IV), and
294.92 ppm (pupae) (Table 1). A dose-dependent effect
was found as previously described for other plant-borne
compounds (see Benelli et al. 2015b for a recent review).
In particular, the methanolic extract of M. oleifera has
been already reported for its larvicidal activity against
the malaria mosquito Anopheles stephensi (Prabhu et al.
2011). The toxic action of M. oleifera-borne products
Fig. 8 Vero cell viability after the
treatment with green-synthesized
AgNP (40 μl/ml): acontrol; b
after 24 h; cafter 48 h; dafter 72 h
Fig. 9 Antiviral activity of green-synthesized AgNP in vero cells
infected with DEN-2; the asterisk indicate a significant difference over
the control (P<0.01)
Parasitol Res

against mosquito vectors may be linked to the lectin con-
tent, which is able to affect digestive (amylase, trypsin,
and protease) and detoxifying (superoxide dismutase
(SOD), α-andβ-esterases) enzymes, also in
organophosphate-resistant mosquito populations (Agra-
Neto et al. 2014).
AgNP synthesized from the seed extract of M. oleifera
were highly effective against A. aegypti young instars,
with LC
50
of 10.24 ppm (I), 11.81 ppm (II), 13.84 ppm
(III), 16.73 ppm (IV) and 21.17 ppm (pupae) (Table 2).
Similarly, a growing number of biofabricated nanoparti-
cles showed comparable toxicity against a number of
mosquito vectors, including A. aegypti (Suresh et al.
2015), A. stephensi (Dinesh et al. 2015), and Culex
quinquefasciatus (Murugan et al. 2015a; Muthukumaran
et al. 2015). This novel control tool against mosquito
seems promising due to rapid and cheap production.
However, further efforts are required to understand the
possible non-target effects of green-synthesized
mosquitocidal nanoparticles in the aquatic environment
(Murugan et al. 2015b). Moreover, the importance of
M. oleifera-synthesized AgNP is growing in a number
of different fields. Indeed, they have been studied also
for their activity against pathogenic bacteria and fungi
(e.g., Staphylococcus aureus,Candida tropicalis,
Candida krusei,andKlebsiella pneumoniae)(Prasad
and Elumalai 2011) and human cervical carcinoma cells
(Vasanth et al. 2014).
Fig. 10 Morphological changes
induced in vero cells by a
treatment with green-synthesized
AgNP. Control= vero cells
infected with DEN-2. Treated=
vero cells infected with DEN-2,
after a single treatment with
green-synthesized AgNP
(20 μl/ml)
Fig. 11 Reduction in DEN-2
viral yield post-treatment with
green-synthesized AgNP at
different time intervals
Parasitol Res

Tab l e 1 Larvicidal and pupicidal toxicity of Moringa oleifera seed extract against the dengue vector A. aegypti
Target Mortality (%) ±SD LC
50
(LC
90
)
95 % confidence limit LC
50
(LC
90
) Regression equation χ
2
50 ppm 100 ppm 150 ppm 200 ppm 250 ppm LCL UCL
I larvae 25.8± 1.16
a
36.8± 1.72
b
48.6± 1.62
c
61.6± 1.01
d
74.8± 2.03
e
151.97 (348.24) 134.02 (305.14) 170.18 (418.72) y=0.140+0.001x0.11 n.s
II larvae 20.4± 1.49
a
29.2± 2.31
b
40.6± 1.62
c
53.0± 1.44
d
66.4± 2.05
e
185.74 (390.39) 167.13 (338.25) 209.065 (478.43) y=1.163+ 0.006x0.08 n.s
III larvae 17.4 ± 1.46
a
24.8± 1.72
b
33.6± 1.85
c
46.4± 2.15
d
59.0± 1.67
e
215.04 (433.07) 192.88 (369.32) 247.44 (546.07) y=1.264+0.006x1.17 n.s
IV larvae 13.6± 1.91
a
19.4± 1.82
b
29.6± 2.15
c
37.4± 1.01
d
54.0± 1.50
e
241.92 (457.91) 241.92 (388.45) 282.75 (582.97) y=1.435+0.001x0.58 n.s
Pupae 8.2± 1.16
a
12.2± 1.72
b
21.8± 1.32
c
27.8± 1.32
d
40.6± 1.62
e
294.92 (519.06) 257.83 (430.17) 394.58 692.28) y=1.686+ 0.006x0.38 n.s
Mortality rates are means± SD of three replicates
Values followed by the same letter(s) are not significantly different (DMRT, α=0.05)
LC
50
lethal concentration that kills 50 % of the exposed organisms, LC
90
lethal concentration that kills 90 % of the exposed organisms, LCL lower confidence limit, UCL upper confidence limit, χ
2
chi-
square value, n.s. not significant (α=0.05)
Tab l e 2 Larvicidal and pupicidal toxicity of Moringa oleifera-synthesized silver nanoparticles against the dengue vector A. aegypti
Target Mortality (%)± SD LC
50
(LC
90
)
95 % Confidence Limit LC
50
(LC
90
) Regression equation χ
2
5 ppm 10 ppm 15 ppm 20 ppm 25 ppm LCL UCL
I larvae 35.0 ±1.89
a
48.0± 1.74
b
62.2± 1.32
c
79.0± 1.20
d
98.6± 1.95
e
10.24 (23.05) 4.06 (18.52) 13.76 (36.07) y=1.024+ 0.100x9.25 n.s
II larvae 31.6 ± 1.85
a
42.6± 1.35
b
56.2± 2.24
c
73.8± 2.03
d
90.6± 2.05
e
11.81 (26.80) 10.20 (24.28) 13.21 (30.46) y= 1.010+ 0.085x2.91 n.s
III larvae 28.2± 1.56
a
37.8± 1.83
b
50.6± 2.15
c
66.0± 1.41
d
82.0± 1.78
e
13.84 (31.25) 12.16 (27.85) 15.41 (36.49) y= 1.019+ 0.074x1.18 n.s
IV larvae 23.0 ± 1.89
a
31.0± 1.51
b
43.0± 1.87
c
60.8± 1.32
d
71.2± 1.72
e
16.73 (35.67) 15.03 (31.35) 18.61 (42.64) y= 1.132+ 0.068x0.64 n.s
Pupae 18.4± 1.34
a
25.0± 1.58
b
30.6± 2.07
c
49.2± 1.64
d
59.4± 1.51
e
21.17 (42.59) 19.02 (36.46) 24.24 (53.34) y= 1.266+ 0.060x1.67 n.s
Mortality rates are means± SD of three replicates
Values followed by the same letter(s) are not significantly different (DMRT, α=0.05)
LC
50
lethal concentration that kills 50 % of the exposed organisms, LC
90
lethal concentration that kills 90 % of the exposed organisms, LCL lower confidence limit, UCL upper confidence limit, χ
2
chi-
square value, n.s. not significant (α=0.05)
Parasitol Res

Conclusions
Overall, we biosynthesized AgNP using a cheap aqueous ex-
tract of M. oleifera seeds as reducing and stabilizing agent.
These AgNP are easy to produce and stable over time, with
homogeneous size range. Our research highlighted the con-
crete potential of green-synthesized AgNP in the fight against
dengue (serotype DEN-2) and its primary vector A. aegypti.
Further research on structure–activity relationships of AgNP
against other dengue serotypes is urgently required.
Acknowledgments We are grateful to Prof. Heinz Mehlhorn and the
anonymous reviewers for improving an earlier version of the
manuscript. We would like to thank the Deanship of Scientific Research
at King Saud University for its financial support (project no. RGP-1435-
057). Dr. Jayapal Subramaniam is supported by the University Grant
Commission of New Delhi (UGC-BSR-RFSMS-Research Fellowship
in Science for Meritorious Students). We also thank the Research and
Development Centre of Bharathiar University for providing instrumenta-
tion facilities.
Conflicts of interest The authors declare no conflicts of interest. Dr.
Giovanni Benelli is currently an Editorial Board Member of Parasitology
Research, but this does not alter the authors’adherence to all the
Parasitology Research policies on sharing data and materials.
Compliance with ethical standards All applicable international and
national guidelines for the care and use of animals were followed. All
procedures performed in studies involving animals were in accordance
with the ethical standards of the institution or practice at which the studies
were conducted.
Informed consent Informed consent was obtained from all individual
participants included in the study.
References
Agra-Neto AC, Napoleão TH, Pontual EV, de Lima Santos ND, de
Andrade Luz L, de Oliveira CMF, de Melo-Santos MAV, Barroso
Coelho LCB, do Amaral Ferraz Navarra DM, Guedes Paiva PM
(2014) Effect of Moringa oleifera lectins on survival and enzyme
activities of Aedes aegypti larvae susceptible and resistant to organ-
ophosphate. Parasitol Res 113:175–184
Ali GH, El-Taweel GE, Ali MA (2004) The cytotoxicity and antimicro-
bial efficiency of Moringa oleifera seeds extracts. Int J Env Studies
61:699–708
Amer A, Mehlhorn H (2006a) Larvicidal effects of various essential oils
against Aedes,Anopheles,andCulex larvae (Diptera: Culicidae).
Parasitol Res 99:466–472
Amer A, Mehlhorn H (2006b) Repellency effect of forty-one essential
oils against Aedes,Anopheles,andCulex mosquitoes. Parasitol Res
99:478–490
Benelli G, Bedini S, Cosci F, Toniolo C, Conti B, Nicoletti M (2015a)
Larvicidal and ovideterrent properties of neem oil and fractions
against the filariasis vector Aedes albopictus (Diptera: Culicidae): a
bioactivity survey across production sites. Parasitol Res 114:227–36
Benelli G, Murugan K, Panneerselvam C, Madhiyazhagan P, Conti B,
Nicoletti M (2015b) Old ingredients for a new recipe? Neem cake, a
low-cost botanical by-product in the fight against mosquito-borne
diseases. Parasitol Res 114:391–397
Bharali R, Tabassum J, Azad MR (2003) Chemomodulatory effect of
Moringa oleifera Lam. on hepatic carcinogen metabolising en-
zymes, antioxidant parameters and skin papillomagenesis in mice.
Asian Pac J Cancer Prev 4:131–139
Cárceres A, Saraiva A, Rizzio S, Zabala L, De Leon E, Navy F (1992)
Pharmacological properties of Moringa oleifera. 2: screening for
antispasmodic, anti-inflammatory and diuretic activity. J
Ethnopharmacol 36:233–237
Chuang PH, Lee CW, Chou JY, Murugan M, Shieh BJ, Chen HM (2007)
Anti-fungal activity of crude extracts and essential oil of Moringa
oleifera Lam. Bioresour Technol 98:232–236
Dinesh D, Murugan K, Madhiyazhagan P, Panneerselvam C, Nicoletti M,
Jiang W, Benelli G, Chandramohan B, Suresh U (2015)
Mosquitocidal and antibacterial activity of green-synthesized silver
nanoparticles from Aloe vera extracts: towards an effective tool
against the malaria vector Anopheles stephensi? Parasitol Res 114:
1519–1529
Elchiguerra JL, Burt JL, Morones JR, Camacho-Bragado A, Gao X, Lara
HH, Yacaman MJ (2005) Interaction of silver nanoparticles with
HIV-1. J Nanobiotechnol 3:1477–3155
Fayaz AM, Balaji K, Girilal MYR, Kalaichelvan PT, Venketesan R
(2010) Biogenic synthesis of silver nanoparticles and their synergis-
tic effect with antibiotics: a study against gram-positive and gram-
negative bacteria. Nanomed Nanotechnol Biol Med 6:103–109
Finney DJ (1971) Probit analysis. Cambridge University, London, pp 68–
78
Goodsell DS (2004) Bionanotechnology: lessons from nature. Wiley,
Hoboken
Guevara AP, Vargas C, Sakurai H, Fujiwara Y, Hashimoto K, Maoka T,
Kozuka M, Ito Y, Tokuda H, Nishino H (1999) An antitumor pro-
moter from Moringa oleifera Lam. Mutation Res 440:181–188
Hemingway J, Ranson H (2000) Insecticide resistance in insect vectors of
human disease. Annu Rev Entomol 45:371–391
Hu RL, Li SR, Kong FJ, Hou RJ, Guan XL, Guo F (2014) Inhibition
effect of silver nanoparticles on herpes simplex virus 2. Gen Mol
Res 13:7022–7028
Kalwar NH, Sirajuddin, Sherazi STH, Khaskheli AR, Hallam KR, Scott
TB, Tagar ZA, Hassan SS, Soomro RA (2013) Fabrication of small
l-threonine capped nickel nanoparticles and their catalytic applica-
tion. Appl Catal A Gen 453:54–59
Kora AJ, Sasidhar RB, Arunachalam J (2010) Gum kondagogu
(Cochlospermum gossypium) a template for the green synthesis
and stabilization of silver nanoparticles with antibacterial applica-
tion. Carbohydr Polymer 82:670–679
Krishnaraj C, Jagan EG, Rajasekar S, Selvakumar P, Kalaichelvan PT,
Mohan N (2010) Synthesis of silver nanoparticles using Acalypha
indica leaf extracts and its antibacterial activity against water borne
pathogens. Coll Surf B Biointerf 76:50–56
Kumar P, Senthamil Selvi S, Lakshmi Prabha A, Prem Kumar K,
Ganeshkumar RS, Govindaraju M (2012) Synthesis of silver nano-
particles from Sargassum tenerrimum and screening phytochemi-
cals for its antibacterial activity. Nano Biomed Eng 4:12–16
Lara HH, Ayala-Nuñez VN, Ixtepan-Turrent L, Rodriguez-Padilla C
(2010) Mode of antiviral action of silver nanoparticles against
HIV-1. J Nanobiotechnol 8:1–10
Magudapathy P, Gangopadhyay P, Panigrahi BK, Nair KGM, Dhara S
(2001) Electrical transport studies of Ag nanoclusters embedded in
glass matrix. Physica 299:142–146
Miura N, Shinohara Y (2009) Cytotoxic effect and apoptosis induction by
silver nanoparticles in HeLa cells. Biochem Biophys Res Commun
390:733–7
Mori Y, Ono Y, Miyahira Y, Nguyen VQ, Matsui T, Ishihara M (2013)
Antiviral activity of silver nanoparticle/chitosan composites against
H1N1 influenza A virus. Nanoscale Res Lett 8:1–6
Murrell S, Wu SC, Butler M (2011) Review of dengue virus and the
development of a vaccine. Biotechnol Adv 29:239–247
Parasitol Res

Murugan K, Benelli G, Ayyappan S, Dinesh D, Panneerselvam C,
Nicoletti M, Hwang JS, Mahesh Kumar P, Subramaniam J, Suresh
U (2015a) Toxicity of seaweed-synthesized silver nanoparticles
against the filariasis vector Culex quinquefasciatus and its impact
on predation efficiency of the cyclopoid crustacean Mesocyclops
longisetus. Parasitol Res 114:2243–2253
Murugan K, Benelli G, Panneerselvam C, Subramaniam J, Jeyalalitha T,
Dinesh D, Nicoletti M, Hwang JS, Suresh U, Madhiyazhagan P
(2015b) Cymbopogon citratus-synthesized gold nanoparticles boost
the predation efficiency of copepod Mesocyclops aspericornis
against malaria and dengue mosquitoes. Exp Parasitol 153:129–138
Muthukumaran U, Govindarajan M, Rajeswary M, Hoti SL (2015)
Synthesis and characterization of silver nanoparticles using
Gmelina asiatica leaf extract against filariasis, dengue, and malaria
vector mosquitoes. Parasitol Res 14:1817–27
Noginov MA, Zhu G, Bahoura M, Adegoke J, Small C, Ritzo BA, Drachev
VP, Shalaev VM (2006) The effect of gain and absorption on surface
plasmon in metal nanoparticles. Appl Phys B 86:458–460
Panneerselvam C, Murugan K, Kovendan K, Mahesh Kumar P (2012)
Mosquito larvicidal, pupicidal, adulticidal, and repellent activity of
Artemisia nilagirica (family: Compositae) against Anopheles
stephensi and Aedes aegypti. Parasitol Res 111:2241–51
Park SJ, Park HH, Kim SY, Kim SJ, Woo K, Ko GP (2014) Antiviral
properties of silver nanoparticles on a magnetic hybrid colloid. Appl
Environ Microbiol. doi:10.1128/AEM.03427-13
Prabhu K, Murugan K, Nareshkumar A, Ramasubramanian N,
Bragadeeswaran S (2011) Larvicidal and repellent potential of
Moringa oleifera against malarial vector, Anopheles stephensi Liston
(Insecta: Diptera: Culicidae). Asian Pac J Trop Biomed 1:124–129
Prasad TNVKV, Elumalai EK (2011) Biofabrication of Ag nanoparticles
using Moringa oleifera leaf extract and their antimicrobial activity.
Asian Pac J Trop Biomed 1:439–442
Rajakumar G, Rahuman A (2011) Larvicidalactivity of synthesized silver
nanoparticles using Eclipta prostrata leaf extract against filariasis
and malaria vectors. Acta Trop 118:196–203
Rajan R, Chandran K, Harper SL, Yun SI, Kalaichelvan PT (2015) Plant
extract synthesized nanoparticles: an ongoing source of novel bio-
compatible materials. Ind Crop Prod 70:356–373
Rogers JV, Parkinson CV, Choi YW, Speshock JL, Hussain SM (2008) A
preliminary assessment of silver nanoparticle inhibition of monkey-
pox virus plaque formation. Nanoscale Res Lett 3:129–133
Sathyavathi R, Krishna MB, Rao DN (2011) Biosynthesis of silver nano-
particles using Moringa oleifera leaf extract and its application to
optical limiting. J Nanosci Nanotechnol 11:2031–2035
Shameli K, Ahmad M, Al-Mulla EAJ, Ibrahim NA, Shabanzadeh P,
Rustaiyan A, Abdollahi Y (2012) Green biosynthesis of silver nano-
particles using Callicarpa maingayistem bark extraction. Molecules
17:8506–8517
Shreshtha Verma S, Abirami V, Mahalakshmi (2013) Anticancer and
antibacterial activity of silver nanoparticles biosynthesized by
Penicillium spp. and its synergistic effect with antibiotics. J
Microbiol Biotech Res 3:54–71
Sivakama Valli J, Vaseeharan B (2012) Biosynthesis of silver nanoparti-
cles by Cissus quadrangularis extracts. Mater Lett 82:171–173
Speshock JL, Murdock RC, Braydich-Stolle LK, Schrand AM, Hussain
SM (2010) Interaction of silver nanoparticles with Tacaribe virus. J
Nanobiotechnol 8:19–27
Sukirtha R, Priyanka KM, Antony JJ, Kamalakkannan S, Thangam R,
Gunasekaran P, Krishnan M, Achiraman S (2012) Cytotoxic effect
of green synthesized silver nanoparticles using Melia azedarach
against in vitro HeLa cell lines and lymphoma mice model.
Process Biochem 47:273–279
Suresh U, Murugan K, Benelli G, Nicoletti M, Barnard DR,
Panneerselvam C, Mahesh Kumar P, Subramaniam J, Dinesh D,
Chandramohan B (2015) Tackling the growing threat of dengue:
Phyllanthus niruri-mediated synthesis of silver nanoparticles and
their mosquitocidal properties against the dengue vector Aedes
aegypti (Diptera: Culicidae). Parasitol Res 114:1551–1562
Vasanth K, Ilango K, Mohan Kumar K, Agrawal A, Dubey GP (2014)
Anticancer activity of Moringa oleifera mediated silver nanoparti-
cles human cervical carcinoma cells by apoptosis induction. Coll
Surf B: Biointerf 117:354–359
Vaseeharan B, Clara Gunapoorani S, Lin YC, Chen JC (2012) Green
synthesis of silver nanoparticles through Calotropus gigantea leaf
extracts and evaluation of antibacterial activity against Vib r io
alginolytics. Nanotechnol Dev 2:12–16
Vinoth B, Manivasagaperumal R, Balamurugan S (2012) Phytochemical
analysis and antibacterial activity of Moringa oleifera lam. Int J Res
Biol Sci 2:98–102
Vivek R, Thangam, Muthuchelian K, Gunasekaran P, Kaveri K, Kannan
S (2012) Green biosynthesis of silver nanoparticles from Annona
squamosa leaf extract and its in vitro cytotoxic effect on MCF-7
cells. Proc Biochem 47:2405–2410
WHO (2012) Handbook for integrated vector management. World Health
Organization, Geneva
WHO (2015) Dengue and severe dengue. Fact sheet N°117, Geneva,
World Health Organization
Parasitol Res