Asian Pacific Journal of Tropical Medicine (2013)95-101 95

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Asian Pacific Journal of Tropical Medicine

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Document heading

Larvicidal activity of green synthesized silver nanoparticles using bark aqueous

extract of Ficus racemosa against Culex quinquefasciatus and Culex gelidus
Kanayairam Velayutham1, Abdul Abdul Rahuman1*, Govindasamy Rajakumar1, Selvaraj Mohana
Roopan2, Gandhi Elango1, Chinnaperumal Kamaraj1, Sampath Marimuthu1, Thirunavukkarasu
Santhoshkumar1, Moorthy Iyappan1, Chinnadurai Siva1
1
Unit of Nanotechnology and Bioactive Natural Products, Post Graduate and Research Department of Zoology, C.Abdul Hakeem College, Melvisharam
- 632 509, Vellore District, Tamil Nadu, India
2
Organic & Medicinal Chemistry Research Laboratory, Organic Chemistry Division, School of Advanced Sciences, VIT University, Vellore - 632 014,
Tamil Nadu, India

ARTICLE INFO ABSTRACT

Article history: Objective: To investigate the larvicidal activity of synthesized silver nanoparticles (Ag NPs)
Received 19 August 2012 utilizing aqueous bark extract of Ficus racemosa (F. racemosa) was tested against fourth
Received in revised form 30 October 2012
instar larvae of filariasis vector, Culex quinquefasciatus (Cx. quinquefasciatus) and japanese
Accepted 7 December 2012
Available online 20 February 2013
encephalitis vectors, Culex gelidus (Cx. gelidus). Methods: The synthesized Ag NPs was
characterized by UV-vis spectrum, X-ray diffraction (XRD), Scanning electron microscopy (SEM)
and Fourier transform infrared (FTIR). The larvicidal activities were assessed for 24 h against
Keywords: the larvae of Cx. quinquefasciatus and Cx. gelidus with varying concentrations of aqueous bark
Ficus racemosa extract of F. racemosa and synthesized Ag NPs. LC50 and r2 values were calculated. Results: The
Culex quinquefasciatus maximum efficacy was observed in crude aqueous extract of F. racemosa against the larvae of
Culex gelidus Cx. quinquefasciatus and Cx. gelidus (LC50=67.72 and 63.70 mg/L; r2=0.995 and 0.985) and the
Electron microscopic study synthesized Ag NPs (LC50=12.00 and 11.21 mg/L; r2=0.997 and 0.990), respectively. Synthesized Ag
NPs showed the XRD peaks at 2毴 values of 27.61, 29.60, 35.48, 43.48 and 79.68 were identified as
(210), (121), (220), (200) and (311) reflections, respectively. The FTIR spectra of Ag NPs exhibited
prominent peaks at 3 425, 2 878, 1 627 and 1 382 in the region 500-3 000 cm-1. The peaks
correspond to the presence of a stretching vibration of (NH) C=O group. SEM analysis showed
shape in cylindrical, uniform and rod with the average size of 250.60 nm. Conclusions: The
biosynthesis of silver nanoparticles using bark aqueous extract of F. racemosa and its larvicidal
activity against the larvae of disease spreading vectors. The maximum larvicidal efficacy was
observed in the synthesized Ag NPs.

O wing to the problems associated with resistance and

1. Introduction effects on non-target species by chemicals[1]. Filariasis is
endemic in 17 States and six Union Territories, with about
Mosquitoes are important vectors of diseases, especially 553 million people at risk of infection[2]. However, chronic
in the tropics. Regulation of mosquito populations to reduce manifestations, such as lymphedema (elephantiasis) and
the incidence of disease like malaria, filariasis and several hydrocele are debilitating and estimated by the World
arboviruses are importance from public health viewpoint. H ealth O rganization to account for nearly five million
disability adjusted life years[3]. Japanese encephalitis is
*Corresponding author: Dr. A. Abdul Rahuman, Unit of Nanotechnology and
the most important cause of viral encephalitis in Eastern
Bioactive Natural Products, Post Graduate and Research Department of Zoology, C. and Southeast Asia. Up to 50 000 cases and 15 000 deaths
Abdul Hakeem College, Melvisharam - 632 509, Vellore District, Tamil Nadu, India.
Tel.: +91 94423 10155; +91 04172 269009 annually are due to JE especially in the rural areas[4,5].
Fax: +91 04172 269487
The target species vector control is facing a threat due
E-mail: [email protected]
96 Kanayairam Velayutham et al./Asian Pacific Journal of Tropical Medicine (2013)95-101

to the development of resistance to chemical insecticides failure) and reproduction. A comparative assessment of
resulting in rebounding vectorial capacity[6]. Insecticides the 48 h acute toxicity of synthesized Au, Ag, and Ag-Au
have provoked undesirable effects, including toxicity to bimetallic nanoparticles was conducted to determine their
non-target organisms and fostered environmental and ecological effect in freshwater environments through the use
human health concerns[7]. The Ag NPs which are less likely of Daphnia magna (D. magna)[22]. The current study aimed
to cause ecological damage have been identified as potential to explore the larvicidal activity of green synthesized Ag
replacement of synthetic chemical insecticides, hence the NPs using aqueous bark extract of F. racemosa to control C.
need to use green synthesized Ag NPs for the control of quinquefasciatus and C. gelidus.
disease vectors.
The Ag NPs may be released into the environment from
discharges at the point of production, from erosion of 2. Materials and methods
engineered materials in household products (antibacterial
coatings and silver-impregnated water filters) and from 2.1. Preparation of aqueous bark extract of F. racemosa
washing or disposal of silver containing products[8]. Silver
has been known to exhibit strong toxicity to a wide range F. racemosa bark was collected from Melvisharam, Tamil
of microorganisms and has been used extensively in many Nadu, India. The bark was washed thoroughly to remove
antibacterial applications[9]. The green synthesis of Ag NPs impurities and under shade dried for about three weeks to
by various plants has been reported, the potential of plants remove the moisture. The bark was cut into small pieces,
as biological materials for the synthesis of nanoparticles powdered in a mixer and then sieved using 20 mesh size
are yet to be fully explored[10]. Recent reports include the sieves to get uniform size range. A queous extract was
biosynthesis of Ag NPs using leaf extracts of Manilkara prepared by mixing 50 g of dried leaf powder with 500 mL
zapota (M. zapota)[11], Mimosa pudica (M. pudica)[12] and of water (boiled and cooled distilled water) with constant
fruit peel extract of Musa paradisiaca (M. paradisiaca)[13] stirring on a magnetic stirrer[23]. The suspension of dried
against Rhipicephalus microplus (R. microplus), the fourth- bark powder in water was left for 3 h, filtered through
instar larvae of Anopheles subpictus (An. subpictus), C. Whatman no. 1 filter paper, and the filtrate was stored in
quinquefasciatus, Anopheles stephensi (An. stephensi), and amber colored air tight bottle at 10 曟 and used within a
Culex tritaeniorhynchus (Cx. tritaeniorhynchus). week.
Ficus racemosa (F. racemosa) L.(Moraceae) has been
used in Indian folk medicine for the treatment of various 2.2. Synthesis of Ag NPs by F. racemosa bark extract
diseases/disorders including jaundice, dysentery, diabetes,
diarrhea and inflammatory conditions [14]. The compound For the production of aqueous extract, 2.5 g of F. racemosa
of racemosic acid, gluanol acetate, caoutchouc, tannins, bark powder was added to a 100 mL Erlenmeyer flask with
毬-sitosterol, stigmasterol, friedelin and hentriacontane 250 mL sterile distilled water and then boiled for 5 min. The
from the bark of F. racemosa[15]. The F. racemosa bark extract was filtered with Whatman filter paper No. 1. The
showed hepatoprotective, chemopreventive, anti-diabetic, filtrate was treated with aqueous 1 mM silver nitrate (AgNO3)
anti-inflammatory, anti-pyretic, anti-tussive, and anti- solution in an Erlenmeyer flask and incubated at room
diuretic effects [16] . T he crude aqueous extract of the temperature. 80 mL aqueous solution of 1 mM of AgNO3 was
latex of Ficus benghalensis (F. benghalensis) was tested reduced using 20 mL of bark extract at room temperature
against the fourth instar larvae of C. quinquefasciatus[17]. for 10 min, resulting in a brown solution indicating the
T he insecticidal efficacy of different concentrations of formation of Ag NPs[24].
fruit pericarp methanol extract of Artocarpus lakoocha (A.
lakoocha)(Moraceae) was evaluated against second and third 2.3. Insect rearing
instar larvae of Aedes aegypti (Ae. aegypti)[18].
The use of plants for synthesize of nanoparticles are rapid Cx. quinquefasciatus and Cx. gelidus larvae were collected
low cost, eco-friendly and safe for human therapeutic use[19]. from stagnant water area of Melvisharam (12°56 23″N, 79°
Evaluation of synthesized Ag NPs using leaf aqueous extract 14′23″ E) and identified in Zonal Entomological Research
of Lawsonia inermis (L. inermis) used to control Pediculus Centre, Vellore (12°55′48″ N, 79°7′48″ E), Tamil Nadu. To
humanus capitis (P. h. capitis) and Bovicola ovis (B. ovis) start the colony, the larvae were kept in plastic and enamel
[20]. Nair et al[21] reported that the Ag NPs did not have acute trays containing tap water. They were maintained and reared in the
toxicity against the fourth instar larvae of the aquatic midge laboratory as per the method[25]. The larvae of Cx. quinquefasciatus
Chironomus riparius (C. riparius), but exhibited chronic and Cx. gelidus were collected from the insect rearing cage
toxicity on the development (pupation and emergence and identified in Zonal Entomological Research Centre,
 Kanayairam Velayutham et al./Asian Pacific Journal of Tropical Medicine (2013)95-101
97

Vellore. One gram of aqueous leaf extract was first dissolved the reaction mixture was subjected to centrifugation at 5 000
in 100 mL of distilled water for bioassay test of plant extract rpm for 30 min; resulting pellet was dissolved in deionized
(stock solution). The larvicidal activity was assessed by the water and filtered through Millipore filter (0.45 毺m). Fourier
procedure of WHO[26] with some modification and as per the transform infrared (FTIR ) spectra of the samples were
method of Rahuman et al[27]. For the bioassay test, larvae measured using a Perkin Elmer Spectrum One instrument in
were taken in five batches of 20 in 249 mL of water and 1.0 mL the diffuse reflectance mode at a resolution of 4 cm-1 in KBr
of the desired plant extract concentration. Control was set pellets. Powder samples for the FTIR was prepared similarly
up with dechlorinated tap water. The numbers of dead as for powder diffraction measurements. The FTIR spectra
larvae were counted after 24 h of exposure, and the percent of bark extracts taken before and after synthesis of Ag NPs
mortality was reported from the average of five replicates. were analyzed which discussed for the possible functional
The experimental media, in which 100% mortality of larvae groups for the formation of Ag NPs. An aliquot of this filtrate
occurs alone, were selected for dose response bioassay. containing Ag NPs was used for X-ray diffraction (XRD) and
Synthesized Ag NPs toxicity test was performed by placing FTIR analysis. For XRD studies, dried nanoparticles were
20 mosquito larvae into 200 mL of sterilized double distilled coated on the XRD grid, and the spectra were recorded using
water with Ag NPs in a 250 mL beaker (Borosil). 100 mg of Phillips PW 1830 instrument operating at a voltage of 40 kV
synthesized Ag NPs was first dissolved in 1 L of Milli Q water and a current of 30 mA with CuK毩1 radiation. For scanning
(stock solution). From the stock solution, the nanoparticle electron microscopy studies, 25 毺L of sample was sputter-
solutions were diluted using Milli Q water as a solvent coated on copper stub, and the images of nanoparticles
according to the desired concentrations (5, 10, 15, 20 and (SEM; JEOL, Model JFC-1600).
25 mg/L). Each test included a set control group (distilled
2.6. Statistical analysis
water) with five replicates for each individual concentration.
Mortality was assessed after 24 h to determine the acute
T he average larval mortality data were subjected to
toxicities on fourth instar larvae of Cx. quinquefasciatus
probit analysis for calculating LC50 and other statistics at
and Cx. gelidus. To avoid settling of particles especially at
95% fiducial limits of upper confidence limit and lower
higher doses, all treatment solutions were sonicated for an
confidence limit were calculated by using the software
additional of 5 min prior to addition of the mosquito larvae.
developed by Reddy et al[28]. Results with P<0.05 were
considered to be statistically significant.
2.4. Dose-response bioassay

During the laboratory trial, the crude bark extract of

3. Results
F. racemosa and synthesized Ag NPs were subjected to
a dose-response bioassay for larvicidal activity against In the present study, the larvicidal aqueous crude bark
Cx. quinquefasciatus and Cx. gelidus. D ifferent extracts and synthesized Ag NPs of F. racemosa were noted;
concentrations ranging from 20, 40, 60, 80 and 100 mg/L (for however, the highest mortality was found in synthesized
aqueous plant extracts) and 5, 10, 15, 20, and 25 mg/L (for Ag NPs against the larvae of Cx. quinquefasciatus and Cx.
synthesized Ag NPs) were prepared for larvicidal activity. gelidus at the concentration of 25 mg/L. The larvicidal activity
The numbers of dead larvae were counted after 24 h of of aqueous crude bark extracts and synthesized Ag NPs of
exposure, and the percent mortality was reported from the F. racemosa showed the LC50 (UCL-LCL) values of 67.72
average of five replicates. However, at the end of 24 h, the (61.5-74.74) and 12.00 (9.3-13.01) mg/L; r2 values of 0.995
selected test samples turned out to be equal in their toxic and 0.997 against Cx. quinquefasciatus and 63.70 (57.3-
potential. 70.88) and 11.21(10.0-14.09) mg/L; r =0.985 and 0.990 against
2

Cx. gelidus, respectively (Figure 1). The larvicidal activity

2.5. Characterization of the synthesized nanoparticles results showed the highest mortality in synthesized Ag
NPs than the aqueous bark extract of F. racemosa. All the
Synthesis of Ag NPs solution with bark extract was observed tested components that showed lethal effect and mortality
by UV-vis spectroscopy. The bioreduction of the Ag+ ions were positively dose-dependent. The results showed that
in solutions was monitored by periodic sampling of aliquots the optimal hours for measuring the percent mortality
(1 mL) of the aqueous component after 20 times dilution in aqueous bark extract and synthesized Ag NPs against
and measuring the UV-vis spectra of the solution. UV-vis were 9, 26, 39, 57, 77 and 24, 42, 58, 79 and 100 against
spectra of these aliquots were monitored as a function of Cx. quinquefasciatus and 13, 27, 48, 60, 72 and 28, 39, 64,
time of reaction on a Schimadzu 1601 spectrophotometer in 82 and 100 against Cx. gelidus at 1, 6, 12, 18 and 24 h,
300-700 nm range operated at a resolution of 1 nm. Further, respectively (Figure 2).
 98 Kanayairam Velayutham et al./Asian Pacific Journal of Tropical Medicine (2013)95-101

T he presence of the sharp peak at 2 922 to 2 878 cm

-1
80
was assigned to C - H and C - H (methoxy compounds)
70 LC50=SE stretching vibration, respectively (Figure 5 A and B). The
60 SEM micrograph shows the synthesized nanoparticles were
cylindrical, uniform, rod shaped and with an average size of
Doses (mg/L)

50
250.60 nm (Figure 6A, B and C).
40

30

20 1.2

10 1.0
0
Synthesized 0.8
Aqueous Synthesized Aqueous
extract AgNPs extract AgNPs
0.6
Cx. quinquefasciatus Cx. gelidus
0.4
Figure 1. Graph showing the LC50 values of Cx. quinquefasciatus and
Cx. gelidus larvae. 0.2

0
120 0 200 400 600 800
Cx. quinquefasciatus
100 Cx. gelidus
Figure 3. UV-vis spectra of silver nanoparticles synthesized using
80 aqueous bark extracts of F. racemosa.
Mortality (%)

60
(211)
40 450 29.60
400
20
350
(220)
0 300 (210) 35.48
100 80 60 40 20 25 20 15 10 5 (200)
Intensity

250 27.61 43.48

Aqueous extract Synthesized Ag NPs (311)
200
79.68
Concentrations (mg/L) 150
100
Figure 2. Graph showing the larvicidal activity of aqueous extract of 50
F. racemosa and synthesized Ag NPs against fourth instar larvae of 10 20 30 40 50 60 70 80
Cx. quinquefasciatus and Cx. gelidus. 2 Theta scale

The pure AgNO3 without aqueous bark extract of F. racemosa Figure 4. XRD pattern of silver nanoparticles synthesized using
aqueous bark extracts of F. racemosa.
didn’t show any colour change and there was no proof for 100
the formation of Ag NPs. Ag NPs were synthesized rapidly 90 A
within 30 minutes of incubation period. The aqueous silver 80
nitrate solution was turned to brown color within 30mins,
1 627

70
with the addition of bark extract. Intensity of brown color
1 373

60

increased in direct proportion to the incubation period. 50

%T

40
A bsorption spectrum of synthesized A g NP s with bark 30
2 922
B
3 422

aqueous extract of F. racemosa at different wave lengths

2 878

20
ranging from 300 to 600 nm revealed a peak at 425 nm 10
1 382

( F igure 3 ). T he XRD patterns of vacuum dried A g NP s 0

3 425

1 620

synthesized using bark extract of F. racemosa. A number -10

4 000 3 500 3 000 2 500 2 000 1 500 1 000
of Bragg reflections with 2毴 values of 27.61, 29.60, 35.48,
43.48 and 79.68 sets of lattice planes were observed and Figure 5. FTIR spectrum of (A) bark powder of F. racemosa (B)
indexed to (210), (121), (220), (200) and (311) facts of silver, synthesized silver nanoparticles using aqueous bark extracts of
respectively (Figure 4). The XRD results also suggest that F. racemosa.
crystallization of the bioorganic phase occurs on the surface
of the Ag NPs. The FTIR band intensities in different regions
of the spectrum for the F. racemosa bark powder and
synthesized Ag NPs test samples were analyzed. There was a
shift in the following peak and the spectra showed sharp and
strong absorption band at 1 620 to 1 627 cm-1 assigned to the
stretching vibration of (NH) C=O group. The band 1 373 to
1 382 cm developed for C - C and C - N stretching,
-1
Figure 6. SEM micrograph A) ×1 500 10 毺m; B) ×5 000 5 毺m; C)
respectively and was commonly found in the proteins. ×10 000 1 毺m showing the silver nanoparticles synthesized using
bark aqueous extract of F. racemosa.
 Kanayairam Velayutham et al./Asian Pacific Journal of Tropical Medicine (2013)95-101
99

4. Discussion al[42] reported the maximum efficacy was observed in crude

methanol, aqueous, and synthesized Ag NPs using aqueous
In the present study, the larvicidal activity of aqueous leaf extract of Nelumbo nucifera (N.nucifera) against the
bark extracts and synthesized Ag NPs of F. racemosa was larvae of An. subpictus (LC50=8.89, 11.82, and 0.69 ppm)
noted. H owever, the activity was observed in aqueous and against the larvae of Cx. quinquefasciatus (LC50=9.51,
bark extract of F. racemosa and the synthesized Ag NPs 13.65, and 1.10 ppm), respectively. The larvicidal activity of
against Cx. quinquefasciatus and Cx. gelidus. Rahuman et synthesized Ag NPs utilizing aqueous extract from Eclipta
al[29] have reported that the bioassay-guided fractionation prostrata (E. prostrata) was investigated and the maximum
of acetone extract of F. racemosa led to the separation efficacy was observed in crude aqueous, and synthesized
and identification of a tetracyclic triterpenes derivative; Ag NPs against fourth instar larvae of Cx. quinquefasciatus
gluanol acetate was isolated and identified as a new mosquito (LC50=27.49 and 4.56 mg/L; LC90=70.38 and 13.14 mg/L), and
larvicidal compound and it was quite potent against fourth against An. subpictus (LC50=27.85 and 5.14 mg/L; LC90=71.45
instar larvae of Ae. aegypti (LC50 =14.55 and LC90 = 64.99 ppm), and 25.68 mg/L), respectively[43]. The LC50 values for second
An. stephensi (LC50 =28.50 and LC90 =106.50 ppm) and Cx. and fourth larval instars after 24 h of synthesized Ag NPs
quinquefasciatus (LC50=41.42 and LC90=192.77 ppm). The using Plumeria rubra (P. rubrum) latex exposure were 1.49,
maximum efficacy was observed in methanol extract with the 1.82 ppm against Ae. aegypti and 1.10, 1.74 ppm against
lethal concentration (LC50) values of F. benghalensis against An. stephensi, respectively and the crude aqueous latex of
early second, third and fourth larvae of Cx. quinquefasciatus P. rubrum were 181.67, 287.49 ppm against Ae. aegypti and
were 41.43, 58.21 and 74.32 ppm, respectively[30]. The milky 143.69, 170.58 ppm against An. stephensi, respectively[44].
sap of Ficus carica have a significant toxic effect against The median lethal concentrations (LC50) of synthesized stable
early fourth stage larvae of Ae. aegypti with an LC50 value of silver nanoparticles using A. squamosa leaf broth that killed
10.2 毺g/mL and an LC90 value of 42.3 毺g/mL[31]. fourth instar larvae of Ae. aegypti, Cx. quinquefasciatus and
Madhumitha et al[32] have reported the larval parasitic An. stephensi were 0.30, 0.41, and 2.12 ppm, respectively[45].
mortality observed in fruit peel aqueous extract of Annona Fungus mediated synthesis of Ag NPs using Chrysosporium
squamosa (A. squamosa) were 36%, 55%, 72%, 92%, 100% tropicum (C. tropicum) showed efficacy (LC50=3.47, 4, and
and 14%, 34%, 68%, 89%, and 100% at 200, 400, 600, 800, 2 ; LC 90= 12 . 30 , 8 . 91 , and 4 ; LC 99= 13 . 18 , 13 . 18 , and 7 . 58 ,
and 1 000 ppm, respectively, against An. subpictus and respectively) after 1 h against the second instar larvae of Ae.
Cx. quinquefasciatus and the highest parasite mortality aegypti[46]. The Ag NPs synthesized by filamentous fungus
was found after 24 h of exposure against fourth instar Cochliobolus lunatus (C. lunatus) and the efficacy tested
larvae of An. subpictus (LC50 = 327.27 ppm, r2=0.970), Cx. concentrations ( 10, 5, 2.5, 1.25, 0.625, and 0.3125 ppm)
quinquefasciatus (LC50=456.29 ppm, r2=0.974), respectively. against second, third, and fourth instar larvae of Ae. aegypti
The larvicidal effects of aqueous extracts from leaves of (LC50=1.29, 1.48, and 1.58; LC90=3.08, 3.33, and 3.41 ppm) and
Ricinus communis (R. communis) showed the LC50 values of against An. stephensi (LC50=1.17, 1.30, and 1.41; LC90 =2.99,
1 091.44, 1 364.58 and 1 445.44 ppm against 2nd, 3rd and 4th 3.13, and 3.29 ppm) were observed, respectively[47].
larval instars of Cx. quinquefasciatus[33]. 55% mortality was T he XRD pattern of pure silver ions was known
observed in 2.5% concentration of aqueous extract of dried to display peaks at 2 毴 = 7 . 9 °, 11 . 4 °, 17 . 8 °, 30 . 38 °
leaves of Caesalpinia bonduc (C. bonduc) tested against the and 44 ° [48] . J ayaseelan and R ahuman [49] reported
fourth instar larvae of Cx. quinquefasciatus[34]. that the XRD patterns of vacuum dried Ag NPs
A queous extracts of Azadirachta indica (A.indica), synthesized using the leaf extract of Ocimum canum
Gymnema sylvestre (G. sylvestre), Nerium indicum (O. canum) and the number of B ragg reflections with
(N. indicum) and Datura metel (D. metel) were 2毴 values of 27.74° (210), 32.15° (122) and 36.19° (128). XRD
tested in a laboratory for larvicidal properties against pattern of Ag NPs after reaction showed the diffraction peaks
Cx. quinquefasciatus and the results of A. indica at 2毴=38.28°, 46.40°, 64.21° and 77.78° assigned to the
seeds showed high toxicity with LC 50 value of 0 . 53 (111), (200), (220) and (311) planes of a faced center cubic
ppm and LC 90 value of 3 . 42 ppm; G. sylvestre and lattice of silver[50]. Therefore XRD results also suggest that
N. indicum also showed the LC50 values less than 2.00 ppm, crystallization of the bioorganic phase occurs on the surface
while D. metel showed 3.97 ppm value[35]. The effects of of the Ag NPs.
the aqueous extracts of whole plants of Striga hermonthica FTIR spectrum the most intense band at 1 620-1 636 cm
-1

(S. hermonthica) and Mitracarpus scaber (M. scaber) against represent carbonyl groups from polyphenols such as
the larvae of Cx. quinquefasciatus were investigated and catechin gallate, epicatechin gallate, epigallocatechin,
showed 100% mortality was observed at 1% and 0.5% of epigallocatechin gallate, gallocatechin gallate and the
S. hermonthica and M. scaber, respectively[36-40]. aflavin; the results suggest that molecules attached with
The maximum larvicidal activity was observed in the Ag NPs have free and bound amide groups. These amide
synthesized Ag NPs using leaf aqueous extract of Tinospora groups may also be in the aromatic rings. This concludes
cordifolia (T. cordifolia) against fourth instar larvae of An. that the compounds attached with the Ag NPs could be
subpictus and Cx. quinquefasciatus (LC50=6.43 and 6.96 mg/ polyphenols with an aromatic ring and bound amide
L; r =0.773 and 0.828), respectively[41]. Santhoshkumar et
2
region[51]. The peak at 1 381 cm-1 corresponds to the C-N
100 Kanayairam Velayutham et al./Asian Pacific Journal of Tropical Medicine (2013)95-101

stretching of the aromatic amine group[52]. This suggests the piperidine amide extracted from Piper longum L. fruit shows
attachment of some polyphenolic components onto Ag NPs. activity against Aedes aegypti mosquito larvae. J Agric Food Chem
This means that polyphenols attached to Ag NPs may have 2002; 50: 3765-3767.
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synthesized Ag NPs measured 25-150 nm in size[53]. 42: 4133-4139.
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it seems clear that in some cases, nanoscale specific Ramirez JT, et al. The bactericidal effect of silver nanoparticles.
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products or reduced cofactors play an important role in [11]Rajakumar G, Rahuman AA. Acaricidal activity of aqueous
the reduction of respective salts to nanoparticles. It seems extract and synthesized silver nanoparticles from Manilkara
quite probable that the phenols play an important part in zapota against R. microplus. Res Vet Sci 2011; 93(1): 303-309.
the reduction of ions to Ag NPs as the concept of antioxidant [12]Marimuthu S, Rahuman AA, Rajakumar G, Santhoshkumar T,
action of phenol compounds is not new. Kirthi AV, Jayaseelan C, et al. Evaluation of green synthesized
The present green synthesis shows that the environmentally silver nanoparticles against parasites. Parasitol Res 2011; 108(6):
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