Biotechnol. Bioinf. Bioeng.

2012, 2(1):632-637

Studies on antibacterial and antifungal activity of silver nanoparticles

synthesized using Artocarpus heterophyllus leaf extract
Rebecca Thombre, Fenali Parekh, Parvathi Lekshminarayanan, Glory Francis

Department of Biotechnology, Modern College of Arts, Science and Commerce, Shivajinagar,

Pune, Maharashtra 411005, India; Email: rebecca.thombre@gmail.com

ABSTRACT
A simplistic method for synthesis of colloidal silver nanoparticles was developed using leaf extract of
Artocarpus heterophyllus. The biostabilized silver nanoparticles were characterized by UV-Visible
Spectroscopy, SEM, EDS, XRD and FTIR. The silver nanoparticles demonstrated potent antibacterial activity
against Escherichia coli, Staphylococcus aureus and Bacillus subtilis. The nanoparticles also demonstrated
antifungal activity against Aspergillus niger and the yeast, Pichia pastoris.

Keywords: silver nanoparticles, Artocarpus heterophyllus, antibacterial activity, green synthesis

INTRODUCTION

Nanotechnology has tremendous potential to change the society with its varied applications.
Metallic nanoparticles of ZnO, Fe, Au, Ag and Pb have applications in various fields like
pharmaceuticals, agriculture and diagnostics. Recent research has made it possible to incorporate
silver nanoparticles in food, packaging, automotive industry, environment, disinfectant, cosmetics,
paints, etc. Elemental silver and silver nanoparticles have varied applications due to their medicinal
properties. Silver nanoparticles can be synthesized using physical, chemical and biological
reduction methods [1-7]. Biological method for synthesis of nanoparticles uses plant based extracts,
products, enzymes, reducing factors, proteins, peptides, antioxidants, triglycerides, saponins,
glycoproteins, antioxidants, pigments, latex, gums, polysaccharides, phytochemical constituents like
terpenoids, flavonoids, tannins and vitamins and cofactors for reduction and/or stabilization of
nanoparticles. The synthesis of nanoparticles using plant extracts is often termed as green synthesis
method that reduces or eliminates use and/or generation of hazardous substances.
The plant Artocarpus heterophyllus Lam. (Jackfruit) belongs to family Moraceae and is a
common fruit in India and Asia. The plant extracts of jackfruit contain antioxidants, prenylflavones,
jacalin, saponins, polyphenolics and flavonoids [8]. Thus, the advantage of using green synthesis
method for synthesis of silver nanoparticles over chemical and physical method is that it is
economic, the by-products are non- toxic, the need of controlled physical parameters is reduced and
the silver nanoparticles coated with plant based proteins have greater antibacterial property [9]. In
the present investigation, the silver nanoparticles were synthesized by the leaf extract of Artocarpus
heterophyllus. This is a simple and sustainable synthesis method that prevents pollution by using
reducing properties and enzymes of plant extracts. The silver nanoparticles produced by this method
are stable due to plant peptides and proteins. These nanoparticles are known to lyse the bacterial and
fungal cell and thus have antagonistic effects. As the silver nanoparticles possess antibacterial and
antifungal property, they find wide applications in the development of new pharmaceutical drugs, in
reduction of burn infection and disinfection.

Research Article, Biotechnol. Bioinf. Bioeng. 2012, 2(1):632-637

© 2012 Society for Applied Biotechnology; pISSN 2249-9075, eISSN 2249-9938
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MATERIALS AND METHODS

All the reagents were of analytical grade. Silver nitrate was purchased from Fisher Scientific, USA.
Nutrient agar medium was purchased from Himedia, India. All aqueous extracts were prepared in
autoclaved double distilled water. UV Visible spectroscopy analysis was performed using UV
visible spectrophotometer (Shimadzu, UV Vis-1800). The morphology of the obtained nanoparticles
was characterized by using analytical scanning electron microscope (Jeol, JSM 6360). FTIR spectra
were measured on Jasco FT/IR-16100. The XRD analysis was performed on Bruker AXS
D8ADVANCE.
Fresh leaves of Artocarpus heterophyllus were collected from Nigdi, Pune and authenticated by
Dr. Neeta Patil (Department of Botany, Modern College, Pune). The leaves were washed with
sterile distilled water thrice. The leaves were crushed and boiled in distilled water at 75°C for
appropriate time and then filtered to prepare 10 % (w/v) aqueous extract by hot extraction method.
Silver nanoparticles were synthesized by addition of Artocarpus heterophyllus leaf extract to equal
volume of 10 mM silver nitrate. The reaction mixture was incubated for 5-7 min at 30°C or till
appearance of dark brown colour. The particles were isolated by centrifugation, repeated washing
and drying at 80°C.
The antibacterial activity was studied using the well diffusion method as described by Thombre
et al. [10]. The antibacterial and antifungal activity was assessed by seeding 0.1 ml of test culture
(optical density at 600 nm = 0.5) on nutrient agar plates of 2.5 mm thickness. 6 mm wells were
made on agar surface with a sterile cork borer. The silver nanoparticles (20 µl) were added in the
well at appropriate concentration (40 mg/ml). The plates were incubated at 37°C for 24 h and the
zone of inhibition was recorded and expressed as average of six readings.

RESULTS AND DISCUSSION

The plant extract of Artocarpus heterophyllus reduced the silver nitrate solution to the metallic
silver which was evident due to the color of the reaction mixture to dark brown after incubation
(Figure 1). The extracts of the plant leaves acts as a reducing agent and also as capping agent, thus
mediating synthesis as well as stabilization of the silver nanoparticles. Thus this is a simple one step
green synthesis process of silver nanoparticles. The synthesized silver nanoparticles were
characterized by SEM (scanning electron microscope), XRD (X-ray diffraction), EDS (energy
dispersive X-ray spectroscopy), UV-Vis Spectroscopy and FTIR (fourier transform spectroscopy).
The reduction of pure silver ions was estimated by measuring the UV-Vis spectrum of the reaction.
A small aliquot of the sample was diluted with distilled water. The spectrum of sample was taken
with the frequency range 250 nm to 700 nm at room temperature. A unique property of silver
nanoparticles is that the SPR peak wavelength lies in the range of 400 nm to 570 nm. The λmax of the
silver nanoparticles was observed at around 428 nm. This characteristic phenomenon of silver
nanoparticles is known as Surface Plasmon Resonance (SPR). Due to the strong interaction of light
with the silver nanoparticles, the conduction electrons undergo oscillation. A typical SEM image
which provides the details about the surface topography, composition and other properties like
electrical conductivity of silver nanoparticles as observed in figure 2.
The silver nanoparticles are roughly spherical in shape and are suitable for applications in
medicine and pharmaceutical preparations. The elemental analysis or chemical characterization of
the sample was performed using EDS (Figure 3). The EDS demonstrates characteristic set of peaks
on the X-Ray spectrum as each element has a unique atomic property. The spectroscopic analysis
reveals peaks for silver (Ag) confirming the presence of silver nanoparticles. The FTIR spectrum of
the silver nanoparticles was analyzed in the range of 4000-400 cm-1 as KBr pellets. The FTIR
spectra shows presence of various functional groups like stretching vibration of C-C aromatic ring,

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CN triple bond, N-H bending which suggest the presence of amide linkage in proteins. This also
confirms the presence of the plant peptides and phytochemicals as they serve as reducing and
capping agents for the synthesis of silver nanoparticles. The nanoparticles were further
characterized using X-Ray diffraction (XRD). Figure 4 depicts the XRD analysis of the
nanoparticles which is used for the study of crystalline substances and estimation of the size of
nanoparticles using the Scherrer formula [9]. The X-ray diffraction (XRD) of obtained nanoparticle
powder was performed using P W1840 diffractometer control, Philips with scan speed 0.1, scale 20
to 80°C. The XRD pattern shows intense peaks at 2 theta scale confirming the crystalline nature of
the nanoparticles. The average crystallite size (t) calculated using the Scherrer’s relation: t = 0.94/β
cosθ [9], where l is the wavelength of X-ray and β is the half maximum line width was found to be
30-40 nm.

Figure 1. Synthesis of silver nanoparticles using leaf extract of Jackfruit; A: Leaf extract, B: colour
change after addition of silver nitrate in leaf extract.

Figure 2. SEM photograph of silver nanoparticles.

Since ancient times silver nanoparticles have reported to possess antimicrobial activity. The
antibacterial activity of the silver nanoparticles synthesized from Artocarpus heterophyllus was
screened using gram positive and gram negative organisms namely Escherichia coli,
Staphylococcus aureus and Bacillus subtilis. The antifungal activity was screened using strains of
Aspergillus niger and the yeast Pichia pastoris. The human commensal, E. coli is a gram-negative
bacterium found in the lower part of the intestine. Most strains are harmless but may occasionally

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cause severe food poisoning. They acquire drug resistance by transfer of R plasmids. They play an
essential part in the metabolism in the human intestine. The antibacterial activity of the aqueous
solution of silver nanoparticles was assessed against E. coli and the zone of inhibition was observed
to be 20 mm (Figure 5) [11]. Staphylococcus aureus is a gram-positive cocci with yellow colored
pigment. This bacterium is found in the respiratory tract and is the normal flora of human skin. It
also causes skin infections like pimples, impetigo, boils, carbuncles and abscesses [12] and is
increasingly developing resistance to drugs. Many multidrug drug resistant strains (MDR) are
causing serious infections and hence we assessed the antibacterial activity of silver nanoparticles
against it.

Figure 3. EDS of silver nanoparticles synthesized using Artocarpus heterophyllus.

Figure 4. XRD spectra of silver nanoparticles.

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Figure 5. Zone of inhibition (mm) for silver nanoparticles against bacterial and fungal strains.

The silver nanoparticles synthesized from Artocarpus heterophyllus were screened for
antibacterial activity against Staphylococcus aureus and the zone of inhibition recorded was 17 mm
(Figure 5). Bacillus subtilisis is a gram-positive, rod-shaped bacilli which forms protective
endospore. This endospore allows the organism to tolerate extreme environmental conditions. This
species is commonly found in soil but is also reported to be present in the normal gut commensal in
humans. The antibacterial activity of aqueous silver nanoparticles was assessed over this organism
and the zone of inhibition was reported to be about 20 mm (Figure 5) [13]. One of the most common
species of the Aspergillus genus is Aspergillus niger. It is a filamentous fungi, that is a food
contaminant and causes a disease called black mold on grapes, onions and peanuts. It is largely
found in soil and indoors and forms black colonies [14]. The antifungal activity of silver
nanoparticles synthesized from Artocarpus heterophyllus leaf extract was done using the well
diffusion method and the observed zone of inhibition was 15 mm (Figure 5). Pichia pastoris is yeast
having high growth rate and is used for large scale production of proteins using recombinant DNA
technology [15]. The antagonistic activity of silver nanoparticles was tested against Pichia pastoris
and the observed zone of inhibition was 19 mm (Figure 5).
The antimicrobial activity of the silver nanoparticles was assessed. The biostabilised
nanoparticles were effective against gram positive and negative bacteria, fungi and yeast. There are
previous reports on synthesis of silver nanoparticles using seed extract of the Indian jackfruit plant
[16]. However we report the synthesis using mature leaf extract which is economically feasible and
has more advantages as the leaves are available in abundant quantity throughout the year.
Stabilization of silver in biopolymers is highly advantageous as they are biocompatible and have
potential for applications in biomedical and nanomedical fields [17].

CONCLUSION

Silver is known since ancient times for its potent antimicrobial properties. The applications of silver
nanoparticles are thus imperative due to the beneficial effects of the noble metal. The production of
the silver nanoparticles using chemical synthesis method poses a potential threat to the environment

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due to use of toxic chemicals and precipitating agents. We have described a simple and sustainable
nanoparticles synthesis method that prevents pollution by using reducing properties and enzymes of
plant extracts. The Indian jackfruit (Artocarpus heterophyllus) leaf extract was used effectively for
nanoparticles synthesis. The silver nanoparticles produced by this method were stable due to plant
peptides, proteins and phytochemicals. These nanoparticles are known to lyse the bacterial and
fungal cell and thus have potent antagonistic effects. The effect of these nanoparticles was observed
against test organisms namely Escherichia coli, Staphylococcus aureus, Bacillus subtilis,
Aspergillus niger and Pichia pastoris. The nanoparticles inhibited the bacteria and fungi and thus
they may find wide applications in the development of new drugs, in reduction of burn infection and
in control of diseases.
Acknowledgements: We thank Board of College and University Development, University of
Pune for financial support.

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