BIOFABRICATION OF SILVER NANOPARTICLES USING AQUEOUS LEAF EXTRACT OF MELIA DUBIA, CHARACTERIZATION AND ANTIFUNGAL ACTIVITY

Abstract

ABSTRACT
Objective: To investigate the bio-fabrication of silver nanoparticles (AgNPs) using aqueous leaf extract of Melia dubia (ALM) and test the antifungal
activity of AgNPs against pathogenic fungi Aspergillus niger and Candida tropicalis.
Methods: 10 ml of aqueous leaf extract of Melia dubia was added to 90 ml of 1 mM silver nitrate and incubated for 8h at room temperature. After
incubation, the color change was observed from light yellow to dark brown. The synthesized AgNPs were characterized using UV-Vis spectroscopy,
Fourier Transform Infra red spectroscopy (FTIR), Energy Dispersive X-ray Spectroscopy (EDX), Scanning Electron microscopy (SEM), X-ray
diffraction analysis (XRD) and Atomic Force Microscopy (AFM). Antifungal activity against Aspergillus niger and Candida tropicalis was carried out
by employing the disc diffusion method.
Results: UV-Vis spectra confirmed the synthesis of AgNPs by showing characteristic peak between 380-450 nm*. FTIR spectra showed the
functional groups possibly involved in the synthesis of AgNPs. EDX confirms the presence of elemental silver at 3 Kev. SEM and AFM showed the
synthesized AgNPs were spherical in shape with size ranging between 20-40 nm*. XRD analysis revealed the crystalline nature of AgNPs with face
centred cubic (FCC) lattice. AgNPs was found to be very effective against the tested pathogenic fungi A. niger and C. tropicalis and formed the
inhibition zones 13.0 and 14.5 mm respectively.
Conclusion: It is concluded that the bio-fabrication of AgNPs using aqueous leaf extract of Melia dubia was robust and rapid. The AgNPs were stable
and proved to be excellent antifungal agents.
Keywords: Melia dubia, Silver nanoparticles, FTIR, SEM, Antifungal activity

Full text

BIOFABRICATION OF SILVER NANOPARTICLES USING AQUEOUS LEAF EXTRACT OF MELIA
DUBIA, CHARACTERIZATION AND ANTIFUNGAL ACTIVITY
Original Article
VASUDEVA REDDY NETALA1, VENKATA SUBBAIAH KOTAKADI2, SUKHENDU BIKASH GHOSH3,PUSHPALATHA
BOBBU1, VENKATESWARLU NAGAM4, SHARMA KK5, VIJAYA TARTTE4*
1Department of Biotechnology, Sri Venkateswara University, Tirupati, A. P. India, 2DST-PURSE Centre, Sri Venkateswara University,
Tirupati, A. P. India, 3NABTD, DAE-BRNS, BARC, Mumbai, M. H. India, 4Department of Botany, Sri Venkateswara University, Tirupati, A. P.
India, 5
Received: 22 Aug 2014 Revised and Accepted: 22 Sep 2014
Department of Microbiology, SVIMS University, Tirupati, A. P. India.
Email: tvijayasvu@yahoo.com
ABSTRACT
Objective: To investigate the bio-fabrication of silver nanoparticles (AgNPs) using aqueous leaf extract of Melia dubia (ALM) and test the antifungal
activity of AgNPs against pathogenic fungi Aspergillus niger and Candida tropicalis.
Methods: 10 ml of aqueous leaf extract of Melia dubia was added to 90 ml of 1 mM silver nitrate and incubated for 8h at room temperature. After
incubation, the color change was observed from light yellow to dark brown. The synthesized AgNPs were characterized using UV-Vis spectroscopy,
Fourier Transform Infra red spectroscopy (FTIR), Energy Dispersive X-ray Spectroscopy (EDX), Scanning Electron microscopy (SEM), X-ray
diffraction analysis (XRD) and Atomic Force Microscopy (AFM). Antifungal activity against Aspergillus niger and Candida tropicalis was carried out
by employing the disc diffusion method.
Results: UV-Vis spectra confirmed the synthesis of AgNPs by showing characteristic peak between 380-450 nm*. FTIR spectra showed the
functional groups possibly involved in the synthesis of AgNPs. EDX confirms the presence of elemental silver at 3 Kev. SEM and AFM showed the
synthesized AgNPs were spherical in shape with size ranging between 20-40 nm*. XRD analysis revealed the crystalline nature of AgNPs with face
centred cubic (FCC) lattice. AgNPs was found to be very effective against the tested pathogenic fungi A. niger and C. tropicalis and formed the
inhibition zones 13.0 and 14.5 mm respectively.
Conclusion: It is concluded that the bio-fabrication of AgNPs using aqueous leaf extract of Melia dubia was robust and rapid. The AgNPs were stable
and proved to be excellent antifungal agents.
Keywords: Melia dubia, Silver nanoparticles, FTIR, SEM, Antifungal activity.
INTRODUCTION
In the recent years, research on silver nanoparticles (AgNPs) has
attracted many scientists a considerable attention due to their
remarkable optical, electrical, magnetic and catalytic properties [1].
Due to these properties, AgNPs find several applications in different
fields. AgNPs was employed in electrical batteries [2], polarizing
filters, catalysts, sensors [3], drug delivery [4], DNA sequencing [5],
anticytotoxic agents [6] and antimicrobial agents [7,8]. Many recent
studies reported that AgNPs are proved to be excellent antifungal
agents [9]. Many physical and chemical methods which include
sonochemical assisted [10], thermal assisted [11], and polyol [12]
processes were reported for the synthesis of AgNPs. But all of these
methods involved the application of toxic, hazardous chemicals
leading to environmental and health risks. By considering all these
risks, researchers moved to biosynthetic approach. Biological
approaches are simple, eco-friendly and rapid. Biological approaches
includes plant extract mediated synthesis, microbial assisted and
enzyme assisted synthesis. Microbial and enzyme mediated
approaches are involved elaborate culturing processes, aseptic
environment, while plant extract mediated synthesis does not
involve all of these processes. AgNPs has been extensively
synthesized using various plant extracts including Acalypha indica
[13], Eucalyptus citriodora, Ficus bengalensis [14], Cissus
quadrangularis [15], Cassia alata [16], Catharanthus roseus [17] and
Andrographis paniculata [18]. In the present work, we have reported
the bio-fabrication of AgNPs from the aqueous leaf extract of Melia
dubia (ALM), an important medicinal plant belongs to the family
Meliaceae. The characterization of biofabricated AgNPs was done to
determine their formation, size, shape and crystalline nature by
employing different spectroscopic methods which include UV-Vis
spectroscopy, Fourier Transform Infra Red spectroscopy (FTIR),
Scanning Electron microscopy (SEM), Energy dispersive X-ray
spectroscopy (EDX), Atomic force microscopy (AFM) and X-ray
diffraction (XRD) analysis. Antifungal activity of the biofabricated
AgNPs was carried out to evaluate their biomedical importance. AgNPs
were very effective against Aspergillus niger and Candida tropicalis
MATERIALS AND METHODS
Collection of plant material
Leaves of Melia dubia were collected and authenticated by
Taxonomist, Department of Botany,
Sri Venkateswara University, Tirupati, A. P. India. The voucher
specimen was deposited in the herbarium (VJ-0912).
Preparation of aqueous leaf extracts
Fresh leaves were washed with tap water and then with distilled
water (DW) for 2-3 times. 10 g of leaves were weighed, cut into fine
fragments, boiled in 100 ml of DW for 15 m at 60 º C, cooled to room
temperature and then filtered. The filtrate named. Aqueous leaf
extract (ALM) was used for the synthesis of AgNPs.
Bio-fabrication of AgNPs
Silver nitrate (AgNO3) was purchased from Molychem, India. 10 ml
of ALM was added to 90 ml of 1 mM AgNO 3
Characterization of AgNPs
and incubated in a dark
place at room temperature for about 8h. After incubation, the
synthesis of AgNPs was initially observed visually and further
detected by UV-Vis spectral analysis.
UV-Vis analysis of AgNPs
1 ml of AgNPs solution was diluted with 10 ml of DW. An aliquot of
this diluted sample was used for UV-Vis spectroscopic (Analytical
International Journal of Pharmacy and Pharmaceutical Sciences
ISSN- 0975-1491 Vol 6, Issue 10, 2014
Innovare
Academic Sciences

Tartte et al.
Int J Pharm Pharm Sci, Vol 6, Issue 10, 298-300
299
technologies Ltd, India) analysis. UV-Vis spectrum was recorded
between 300-700 nm* with a resolution of 1 nm*.
FTIR analysis of AgNPs
100 ml of AgNPs solution was taken in a rotator evaporator,
evaporated, concentrated and dried to get fine powder of AgNPs.
This fine powder was used for FTIR analysis. FTIR analysis was
carried out using FTIR spectrometer (ALPHA interferometer; Make:
Bruker, Germany) between 500-4000 cm-1 range at 2 cm-
1
SEM and EDX analysis of AgNPs
resolution.
To confirm the morphology and size of the bio-fabricated AgNPs,
SEM analysis was performed. An aliquot of the suspension of AgNPs
was taken on a clean glass slide and evaporated to dryness. SEM
analysis was done at 15 kV with magnification X 2.5 k, followed by
EDX spectroscopy to confirm elemental silver (SEM Hitachi- S520).
AFM analysis of AgNPs
The AgNPs were tested for surface morphology by using AFM
(Solver NEXT). Surface topographical image was obtained in tapping
mode at a high resonance frequency by oscillating the cantilever
using piezoelectric materials. Characterization was done by
observing the patterns of surface topography and data analysis was
done using Nova PX software.
XRD characterization of AgNPs
The AgNPs powder was used for XRD characterization to know the
crystalline nature of synthesized AgNPs. X-ray diffractometer
(Rigaku, Tokyo) was operated at a voltage of 40 kV and a current of
50 mA with Cu Kα as a radiation source with step size of 2θ.
Antifungal activity of AgNPs
Antifungal activity of the AgNPs was evaluated for their biomedical
importance. Antifungal activity of the AgNPs was checked against
fungal species Aspergillus niger and Candida tropicalis. Disc diffusion
method was employed for antifungal activity [19]. 15 ml of
autoclaved PDA medium was taken into each plate and allowed to
solidify. After solidification, 200 µl of fungal inoculum were swabbed
with cotton. Each PDA plate comprises of 3 discs. One disc
impregnated with ALM. Second with antibiotic Voriconazole, third
one impregnated with 25 µl of AgNPs solution. PDA plates were
incubated for 260
RESULTS AND DISCUSSION
C for 24 h and tested for inhibition zone.
In the present investigation, bio-fabrication of AgNPs from the ALM
was carried out. The bio-fabricated AgNPs were well characterized
and then evaluated for their biomedical importance as antifungal
agents. The bio-fabrication of AgNPs initially detected by the color
change of the solution from light yellow to dark brown. This color
change was due to Surface Plasmon Resonance (SPR) vibrations
within the AgNPs [17,18].
Fig. 1: UV-Vis spectra of AgNPs showing specific SPR peak. (Inset
shows the dark brown colored solution of AgNPs).
UV-Vis spectra confirmation of AgNPs
To further strengthen the evidence that the biofabrication of AgNPs
was carried out using ALM, UV–Vis spectral analysis was carried in
the range of 300– 800 nm to see the appearance of specific SPR peak
of AgNPs. The spectrum showed SPR peak between 380-450 nm and
confirmed the synthesis of AgNPs from ALM (Fig 1)
FTIR Analysis
FTIR analysis of the AgNPs synthesized showed intensive peaks at
1025 cm-1, 1265 cm-1, 1610 cm-1 and 2273 cm-1. The peak at 1025
cm-1 corresponding to C-N stretching vibrations of aliphatic amines.
Peak at 1265 cm-1 corresponding to O-H group of the phenols. Peak
at 1610 cm-1 could be assigned to amide I band. The peak at 2273
cm-1
SEM and EDX characterization of AgNPs
corresponding to methylene stretch of the proteins. FTIR
results indicated that phenols play an important role in the
biosynthesis of AgNPs, while proteins involved both in synthesis and
stabilization of AgNPs.
The SEM characterization determined that AgNPs were 20-40 nm in
size with the roughly spherical shape. SEM micrograph (Fig 2)
showed that AgNPs were partially aggregated. EDX showed the
characteristic peak of elemental silver by sharp signals 3 Kev and thus
confirmed the presence of elemental silver. The absorption peak in the
range of 3 to 4 KeV is characteristic for the elemental silver [8].
Fig. 2: Representative SEM micrograph of synthesized AgNPs
from ALM.
Fig. 3: AFM micrograph showing the topography of the AgNPs
synthesized from ALM. (Arrows indicates roughly spherical
shaped AgNPs with 20-40 nm* in size)
AFM analysis of AgNPs
The surface topology micrograph of AgNPs was shown in Fig 3. The
nicrograph shows AgNPs were uneven distributed, aggregated with
roughly spherical shape and the size ranged between 20-40 nm*.
XRD characterization of AgNPs
XRD characterization of AgNPs revealed the crystalline nature. The
XRD pattern showed the two different diffraction peaks at 38.30 and

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Int J Pharm Pharm Sci, Vol 6, Issue 10, 298-300
300
46.40
Antifungal activity of the AgNPs was tested against fungal
pathogens Aspergillus niger and Candida tropicalis.
which were indexed to (111) and (200) planes respectively of
the lattice of face centred cubic (FCC) symmetry and thus confirmed
the crystallization of AgNPs [18,19].
Antifungal activity of the AgNPs
The results were shown in the Table 1. It is noted that ALM also
showed antifungal activity against Aspergillus niger and Candida
tropicalis. and formed of inhibiton zones of 6 mm and 8 mm
diameter respectively. AgNPs synthesized from ALM formed the
inhibiton zones against Aspergillus niger and Candida tropicalis of
13 mm and 14.5 mm respectively and thus proved their
biomedical importance.
Table 1: Antifungal activity of AgNPs synthesized, compared with ALM and Voriconazole, a standard fungicide
(Mean values ± SD of three replicates).
S. No. Tested Pathogen Diameter of inhibition zone (mm)
ALM
AgNPs
Voriconazole
1.
Aspergillus niger
6.0±0.1
13.0±0.3
16.5±0.2
2.
Candida tropicalis
8.0±0.4
14.5±0.2
16.2±0.3
CONCLUSION
In the present study, we have investigated the bio-fabrication of
Silver nanoparticles using aqueous leaf extract of Melia dubia. Color
change of the reaction solution and UV-Vis spectra confirmed the
formation of silver nanoparticles. The possible mechanism was
reported using FTIR spectra. Plant secondary metabolites which
include phenols, flavonoids and proteins were possibly involved the
reduction of Silver ions (Ag+)to silver nanoparticles (Ag0
1. Alivisatos AP. Perspectives on the physical chemistry of
semiconductor Nanocrystals J. Phys Chem 1996:100;13226–39.
). SEM and
AFM studies showed that the size ranging from 20-40 nm with the
spherical shape. XRD confirmed their crystalline nature. The bio-
fabricated AgNPs were proved as excellent antifungal agents. This
method is very simple, eco-friendly and very rapid approach.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interest
ACKNOWLEDGEMENT
We are very thankful to NABTD, DAE-BRNS for financial assistance.
We are also thankful to IICT-Hyderabad for technical assistance.
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