Int. J. Life. Sci. Scienti. Res.

eISSN: 2455-1716
Sharon et al., 2018
DOI:10.21276/ijlssr.2018.4.4.4

Research Article

Biosynthesis of Copper Nanoparticles using Artocarpus

heterophyllus against Dengue Vector Aedes aegypti
1 1 1*
E. Agnita Sharon , K. Velayutham , R. Ramanibai
1
Student, Department of Zoology, University of Madras, Guindy Campus, Chennai, Tamil Nadu, India
1
Research scholar, Department of Zoology, University of Madras, Guindy Campus, Chennai, Tamil Nadu, India
1
Professor, Department of Zoology, University of Madras, Guindy Campus, Chennai, Tamil Nadu, India

*Address for Correspondence: Dr. R. Ramanibai, Professor, Unit of Aquatic Biodiversity, Department of Zoology,
University of Madras, Guindy Campus, Chennai- 600025, Tamil Nadu, India, Fax: 044-22300899
Received: 04 Feb 2018/ Revised: 02 April 2018/ Accepted: 14 June 2018

ABSTRACT
In our present study, we account for eco-friendly biosynthesis of copper nanoparticles using aqueous leaves extract of Artocarpus
heterophyllus against first to fourth instar larvae of Aedes aegypti. The synthesized CuNPs were characterized by UV, XRD, FTIR
and SEM analyses were clearly distinguishable. The four different immature mosquito larval stages of A. aegypti were exposed to
varying concentrations of aqueous leaf extract of A. heterophyllus, copper sulphate (CuSO4) and synthesized CuNPs for 24 h. The
mortality was observed at aqueous extract (LC50= 48.40, 60.55, 70.36, and 82.79 mg/ml), CuSO4 (LC 50=21.81, 26.92, 41.38, and
55.12 mg/ml) synthesized CuNPs against first to fourth instars of A. aegypti (LC50= 3.85, 4.24, 4.66, and 5.08 mg/ml), respectively.
The novel properties created not only improve the quality of human’s life; it also helps in saving energy and environment.

Key-words: Copper nanoparticles, Jackfruit, Aedes aegypti, aqueous leaf extract

INTRODUCTION
Dengue is a viral infection transmitted between humans high cost, harmful effect on human health, other
by Aedes mosquitoes as a vector [1] Dengue is prevalent non-target populations, their non-biodegradable nature,
in more than 100 countries as it threatens the health of higher rate of biological magnification through the food
approximately 2.5 billion people [2] and around eighty chain, and increasing insecticide resistance on a global
million people are infected annually at an attacking rate scale [3]. Fusion of the herbal extracts into novel systems
of 4% worldwide. In India, 7-16 thousand cases of have certain advantages, such as their bulk dosing and
dengue are reported annually. Thus, it led to less absorption can be reduced which is a major problem
development of new vector-control strategies, antivirals, being faced, enticing the attention of major
and vaccines that can positively impact dengue control pharmaceutical corporations.
and prevention [1]. Medicinal plant products are proposed to be more
In later years, synthetic insecticides in mosquito control efficient and rapid in extracellular biosynthesis of
programme has been reduced due to lack of novel nanoparticles. Recently, elsewhere reported plant
insecticides, concern for environmental sustainability, fabricated nanoparticles have been studied for their
highly effective mosquitocidal properties. The green
How to cite this article
biosynthesis of nanoparticles is advantageous over
Sharon EA, Velayutham K, Ramanibai R. Biosynthesis of Copper
Nanoparticles using Artocarpus heterophyllus against Dengue chemical and physical methods since it is cheap; it is a
Vector Aedes aegypti. Int. J. Life. Sci. Scienti. Res., 2018; 4(4): single-step process and does not require high pressure,
1872-1879.
energy, temperature, and the use of highly toxic
chemicals [4].
Access this article online
Jackfruit (A. heterophyllus) belongs to the family
www.ijlssr.com
Moraceae. They grow abundantly in India, Bangladesh
and in many parts of Southeast Asia. A. heterophyllus is
an important evergreen tree in tropical areas and widely
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Int. J. Life. Sci. Scienti. Res. eISSN: 2455-1716
Sharon et al., 2018
DOI:10.21276/ijlssr.2018.4.4.4
[5]
grown in Asia including India . It has also several 12-hour light and dark photoperiod cycle. The larvae
chemical constituents such as artocarpesin, artocarpin, were fed dog biscuit and a brewer’s yeast powder
cynomacurin, isoartocarpin and norartocarpetin [6]. The mixture 3:1 ratio is used in the laboratory. After five
medicinal properties of jackfruit include anti- days, adult male mosquitoes were fed with 10% sucrose
inflammatory, anti-asthmatic, antioxidant, antimicrobial, solution. The emerging female mosquitoes obtained
antiviral, anti-tubercular, anticancer and anti-malarial blood meal from white albino rat for 2-3 hrs for eggs
activities [7]. The present study deals with biosynthesis of production.
copper nanoparticles using jackfruit leaf aqueous
extracts against dengue vector. Larvicidal bioassay- Larvicidal activity was assessed by
the procedure of WHO [10] with slightly few modifications
MATERIALS AND METHODS as per the method of Rahuman et al. [11].

Preparation of Aqueous leaves extract of A. Statistical Analysis- Mean percent larval mortality data
heterophyllus- The jackfruit leaves were collected from were subjected to analysis of variance and compared
our university campus and an identity authenticity by with Duncan’s multiple range tests to determine any
plant taxonomists. The leaves were washed thoroughly differences between plant species and within species
to remove impurities and under shade dried for about and concentration (SPSS, 2007). LC50 and their associated
one week to remove the moisture. The leaves powdered confidence intervals were estimated from 24 h
in a mixer and then sieved using 20 mesh size sieves to concentration mortality data using probit analysis [12].
get uniform size range. The aqueous extract was There was a strong relationship between the doses and
prepared by mixing 10 g of dried leaf powder with 100ml death rate of parasites showed in the linear correlation
distilled water at 60°C for 10 min and filtered through (r2) and all differences were considered significant if
Whatman No. 1 filter paper. P≤0.05.

Synthesis of copper nanoparticles- To synthesize CuNPs,

RESULTS
an Erlenmeyer flask containing 100 ml of 5 mM copper
UV- Analysis- Absorption spectrum of synthesized Cu
sulphate (CuSO4) was magnetically stirred for 3 hrs.
NPs with leaf aqueous extract of A. heterophyllus at
Following this, 60 ml of the aqueous extract of A.
different wave lengths ranging from 450 to 900 nm
heterophyllus was added with 40 ml of 5 mM CuSO4 at revealed a peak at 640 nm.
room temperature and was subsequently stirred for 24
hrs [8].
0.7
Characterization of Copper nanoparticles- The
bioreduction was observed using UV spectra, which was 0.6

done at regular intervals at 1nm resolution. The solution

0.5
mixture was subjected to centrifugation at 5,000 rpm for
Absorbance(Abs)

15min; resulting pellet was dissolved in distilled water 0.4

and filtered through 0.22 µm millipore filter. The

0.3
synthesized Copper nanoparticles were used for X-ray
diffraction (XRD), Fourier transform infrared 0.2
spectroscopy (FTIR), Scanning electron microscope
(SEM). 0.1

Mosquito rearing- The rearing and collection of Aedes 0.0

500 600 700 800 900
Wavelength(nm)
aegypti larvae were done based on the method
performed by Kamaraj et al. [9]. Aedes aegypti larvae Fig. 1: UV–Vis spectra of synthesized Cu NPs using
were collected from water bottles, tanks, containers and aqueous leaf extract of A. heterophyllus
small water courses. They were kept at a temperature of
28±2°C and 80±10% RH (Relative Humidity) under the
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Int. J. Life. Sci. Scienti. Res. eISSN: 2455-1716
Sharon et al., 2018
DOI:10.21276/ijlssr.2018.4.4.4

X- ray diffraction- The sample demonstrated a high FTIR Analysis- FTIR spectra of copper nanoparticles
crystallinity level with diffraction angles of 26.35°, 35.02° exhibited prominent peaks at 3428, 2925, 1614, 1382,
and 40.24°, which correspond to the characteristic face 1103, 655.8 cm−1 (Fig. 3).
centered cubic of copper lines indexed at (111), (200)
and (222), respectively (Fig. 2).

Fig. 2: XRD analysis of synthesized CuNPs using aqueous leaf extract of A. heterophyllus

Fig. 3: FTIR spectrum of color variations (Black indicating Aqueous extract, Green indicating copper sulphate and
Red indicating Synthesized CuNPs)

Scanning electron microscope (SEM) Analysis- SEM was clearly distinguishable, which measured in size 132
determinations of the sample showed the formation of nm (Fig. 4). CuNPs are asymmetrical dispersed and
nanoparticles. SEM analysis of the synthesized CuNPs aggregated infrequently to form free crystals structures.

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Int. J. Life. Sci. Scienti. Res. eISSN: 2455-1716
Sharon et al., 2018
DOI:10.21276/ijlssr.2018.4.4.4

Fig. 4: Scanning electron micrographs of CuNPs synthesized with A. heterophyllus leaf extract and magnified (A)
8.7mm×3.00 and (B) 8.7mm×5.00 inset bar represents 10 μm

Toxicity studies
and 26 at 100, 80, 60, 40 and 20 mg/L after 24 h (Fig. 5,
Synthesized CuNPs tested against first to fourth instar
Table 1). The control (distilled water) showed nil
larvae of A. aegypti- In the present study, the mosquito
mortality in the concurrent assay. The lethal effect first
larvicidal activity of aqueous leaf extracts, CuSo4 solution
to fourth instars larvae A. aegypti showed the values
(5mM) and synthesized CuNPs of A. heterophyllus are
ofLC50 =21.81, 26.92, 41.38 and 55.12 mg/ml and r2=
noted; however, the synthesized CuNPs showed 100%
0.993, 0.992, 0.997 and 0.965.
mortality in first to fourth instars larvae of A. aegypti at
Values of the efficacy of 5mM CuSo4 solution against first
the concentration of 10 mg/L.
to fourth instar larvae of A. aegypti reported the LC50
The percentage mortality of aqueous leaf extracts of A.
values of 48.40, 60.55, 70.36, 82.79 mg/ml with
heterophyllus against first to fourth instar larvae of A.
aegypti showed the values of 98, 82, 76, 64 and 48; 86, regression of 0.986, 0.969, 0.959, 0.965, respectively
(Fig. 6, Table 1).
76, 66, 58 and 44; 78, 66, 58, 48 and 36; 65, 58, 51, 43

Fig. 5: Larvicidal activity of aqueous leaf extracts of A. heterophyllus against first to fourth instar larvae of A. aegypti

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 Int. J. Life. Sci. Scienti. Res. eISSN: 2455-1716
Sharon et al., 2018
DOI:10.21276/ijlssr.2018.4.4.4

Fig. 6: Larvicidal activity of 5mM CuSo4 against A. aegypti

The synthesized Cu NPs by A. heterophyllus extract concentration. All the tested components that showed
against the first to fourth instar larvae of A. aegypti lethal effect and mortality were positively dose
showed LC50 values of 3.85, 4.24, 4.66 and 5.08mg/L and dependent. The results showed that the optimal hours for
r2 values of 0.992, 0.996, 0.990 and 0.990 respectively measuring the percent mortality of first to fourth instar
(Fig. 7, Table 1). In the present study, the mosquito larvae of A. aegypti synthesized CuNPs were 36%, 48%,
larvicidal activity results showed the highest mortality in 65%, 86% and 100%; 30%, 45%, 61%, 80% and 100%;
synthesized CuNPs than the aqueous leaf extract of A. 28%,41%, 56%, 78% and 96%; 24%, 38%, 52%, 75%
heterophyllus. Each test included a control group and 92% at 2, 4, 6, 8 and 24 h, respectively.
(distilled water) with three replicates for each individual

Fig. 7: Larvicidal activity of synthesized CuNPs against A. aegypti

Table 1: Mosquito larvicidal activity of aqueous leaf extracts of A. heterophyllus, CuSO4 (5 mM) and Synthesized
CuNPs against A. aegypti

Extracts/ Products Instars LC 50 mg/ml LCL-UCL (mg/ml) r2

Aqueous leaf extracts 1st 21.81 14.88-31.97 0.993

2nd 26.92 18.96-38.21 0.992
3rd 41.38 31.98-53.65 0.997

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Int. J. Life. Sci. Scienti. Res. eISSN: 2455-1716
Sharon et al., 2018
DOI:10.21276/ijlssr.2018.4.4.4
th
4 55.12 44.36-68.47 0.965

CuSO4 (5mM) 48.40 39.51-59.28 0.986

1st
2nd 60.55 51.88-70.66 0.969
3rd 70.36 62.99-78.58 0.959
4th 55.12 82.79-108.97 0.965
Synthesized Cu NPs 3.85 3.03-4.87 0.992
1st
2nd 4.24 3.56-5.05 0.996
3rd 4.66 3.96-5.47 0.990
4th 5.08 4.43-5.82 0.990

DISCUSSION
Mosquitoes are quite commonly held responsible for aqueous extracts with salt ions is a clear indicator of
distribution of major diseases like malaria, filariasis, nanoparticle formation.
dengue, chikungunya and Japanese encephalitis etc.
Mosquito borne diseases are distributed widely across
the globe. When a mosquito bites a human it injects it
saliva containing arbovirus which is responsible for
infecting a normal healthy human being.

UV- Analysis- The visual observation of aqueous leaf

extract of A. heterophyllus was pale yellow color before
the addition of copper sulphate solution the colour of
aqueous leaf extract of A. heterophyllus was pale yellow
colour which in turn changed into light brownish colour
(Fig. 8). The colour of the extract further changed to light
brown and later it changed to dark brown after two
hours of incubation period after which there was no Fig. 8: Visual observation of color change after addition
significant colour change. Therefore, the plant extracts of CuSO4
can act both as reducing agents and stabilizing agents in
X-Ray Diffraction- The average size of copper
the biosynthesis of nanoparticles. Different plant
nanoparticles was found to be 65 nm using Debye-
materials contain different organic reducing agents at
Scherrer equation. X-ray diffraction is a technique mainly
various concentrations and combinations [13] pertaining
used to establish the metallic nature of particles gives
to the synthesis of nanoparticles. It has been reported
information on translational symmetry size and shape of
that plant- mediated bioreduction of aqueous extract is
the unit cell from peak positions and information on
combined with relevant metal salt [14]. The reaction
electron density inside the unit cell, namely where the
occurs at room temperature and is generally completed
atoms are located from peak intensities [15]. XRD analysis
within few minutes and the process is relatively complex.
of the present study reveals the crystallographic
The reducing agents such as alkaloids, flavonoids which
structure of CuNPs and it also demonstrated a high
are present in the leaves extracts of various plants are
crystallinity level with diffraction angles of 26.35°, 35.02°
responsible for the reduction of copper ions. The
and 40.24° which correspond to the characteristic face-
reduction of Cu2+ ions to copper nanoparticles was
centered cubic of copper lines indexed at 111, 200 and
observed using UV-Vis spectroscopy where the surface
222, respectively.
plasma resonance showed a distinct peak at 640 nm (Fig.
1). A gradual increase in the characteristic peak FTIR Analysis- The peak at 3428 cm−1 overlaps with O–H
with increase in reaction time and the concentration of stretching and alcohol group. The sharp absorption peak

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Int. J. Life. Sci. Scienti. Res. eISSN: 2455-1716
Sharon et al., 2018
DOI:10.21276/ijlssr.2018.4.4.4
−1
at 2925 cm is assigned to C–H stretching. The peak at CuSO4 solution against first to fourth instar larvae of A.
1614 cm−1 overlaps with N-H bending. The peak at 1382 aegypti reported the LC50 values of 48.40, 60.55, 70.36,
cm−1 overlaps with C-H stretching. The sharp absorption and 82.79 mg/ml with regression of 0.986, 0.969, 0.959,
peak at 1103 cm−1 overlaps is assigned to C–O stretching and 0.965, respectively (Fig. 7 and Table 1). Ramyadevi et
and alcohol group and the peak at 655.8 cm−1 overlaps al. [17] have been reported copper acetate solution
with C-Cl stretching strong of alkyl halide. The peaks of against the larvae of A. subpictus and C. quinquefasciatus
CuNPs and aqueous extract also exhibited at 655.8 and (LC50 values of 23.47 and 15.24 mg/L).
659.6 cm−1, which overlap with C-Cl stretching strong of The synthesized Copper nanoparticles by A.
alkyl halide. Therefore the phenolic compounds and heterophyllus extract against the first to fourth instar
flavonoids present in the extract have high affinity to larvae of A. aegypti showed LC50 values of 3.85, 4.24,
bind towards CuNPs and help in the stabilization of 4.66 and 5.08 mg/L and r2 values of 0.992, 0.996, 0.990
CuNPs. Thus, possibly forming copper nanoparticles and and 0.990 respectively (Fig. 8 and Table 1). All the tested
also prevents possible aggregation. components that showed lethal effect and mortality
were positively dosed dependent. The results showed
Scanning Electron Microscope (SEM) analysis- SEM that the optimal hours for measuring the percent
determinations of the sample showed the formation of mortality of first to fourth instar larvae of A. aegypti
nanoparticles. SEM analysis of the synthesized Copper synthesized Copper nanoparticles were 36%, 48%, 65%,
nanoparticles was clearly distinguishable, which 86% and 100%; 30%, 45%, 61%, 80% and 100%;
measured in size 132 nm. The SEM picture indicates the 28%,41%, 56%, 78% and 96%; 24%, 38%, 52%, 75% and
size of polycrystalline particles with more or less uniform 92% at 2, 4, 6, 8 and 24 h, respectively. Our results
in size and shape. Generally, on the nanometer scale, showed that biosynthesized CuNPs using A.
metals tend to nucleate and grow into twinned and heterophyllus against the first to fourth instar larvae of A.
multiply twinned particles with their surfaces bounded aegypti showed the LC50 values of 3.85, 4.24, 4.66 and
by the lowest-energy facets (Fig. 4). Alike, results were 5.08 mg/L. The control showed no mortality of larvae
observed from the SEM micrographs of nanoparticles Santhoshkumar et al. [18] studied the aqueous and
obtained in the filtrate, which showed that the CuNPs synthesized AgNPs, which used Nelumbo nucifera plant
produced by Penicillium citrinum were spherical shaped extract against third instar larvae of A. subpictus (LC50
[16]
. values of 11.82, and 0.69 ppm and third instar larvae of
Synthesized Copper nanoparticles tested against first to C. quinquefasciatus (LC50 values of 13.65 and 1.10 ppm).
This implies that biosynthesized CuNPs has good
fourth instar larvae of A. aegypti- In the present study,
penetration capacity due to its small size and high
the mosquito larvicidal activity of aqueous leaf extracts,
CuSO4 solution (5mM) and synthesized Copper surface to volume ratio which results in disruption of
nanoparticles of A. heterophyllus are noted; however, organelles and enzymes in young juvenile instars as they
are easily susceptible to the action of CuNPs when
the synthesized Copper nanoparticles showed 100%
mortality first to fourth instars larvae of A. aegypti at the compared to higher instars.
concentration of 10 mg/L. CONCLUSIONS
The percentage mortality of aqueous leaf extracts of A. In this study, the biosynthesis of copper nanoparticles
heterophyllus against first to fourth instar larvae of A. using A. heterophyllus was studied. The physical property
aegypti showed the values of 98 %, 82 %, 76%, 64% and of synthesized nanoparticle was characterized using
48%; 86%, 76%, 66%, 58% and 44%; 78%, 66%, 58%, 48 appropriate techniques. This is the first report on
% and 36 %; 65 %, 58 %, 51 %, 43 % and 26 % at 100 %, evaluating larvicidal activity against Aedes aegypti using
80 %, 60 %, 40 % and 20 % mg/L after 24 h (Fig. 6 and CuNPs. Further research on CuNPs could bring a very
Table 1). The control (distilled water) showed nil promising avenue for vector control.
mortality in the concurrent assay. The lethal effect first
to fourth instars larvae A. aegypti showed the values of ACKNOWLEDGMENTS
LC50 =21.81, 26.92, 41.38 and 55.12 mg/ml and r2 =0.993, The authors are grateful to the University of Madras,
0.992, 0.997 and 0.965. Values of the efficacy of 5mM Department of Zoology for their help support in carrying
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Int. J. Life. Sci. Scienti. Res. eISSN: 2455-1716
Sharon et al., 2018
DOI:10.21276/ijlssr.2018.4.4.4

out the present study. Technical assistance and advice [9] Kamaraj C, Rahuman AA, Bagavan A. Antifeedant and
was provided by Prof. R. Ramanibai so we would like to larvicidal effects of plant extracts against Spodoptera
thank her for her support and encouragement. litura (F.), Aedes aegypti L. and Culex
quinquefasciatus Say. Parasitol. Res, 2008; 103(2):
CONTRIBUTION OF AUTHORS 325-331.
Dr. R. Ramanibai- Research Concept, Research Design, [10]WHO. Report of the WHO informal consultation on
Supervision, Funding, and Materials; the evaluation on the testing of insecticides,
E. Agnita Sharon- Data collection and processing, Data CTD/WHO PES/IC/96.1, 1996; pp. 1-69.
Analysis, Literature search, and Writing Article; [11]Rahuman AA, Gopalakrishnan G, Venkatesan P,
K. Velayutham- Critical review, Article editing, and Final Geetha K. Larvicidal activity of some Euphorbiaceae
approval. plant extracts against Aedes aegypti and Culex
quinquefasciatus (Diptera: Culicidae). Parasitol. Res,
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