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Phytofabrication of silver nanoparticles using pomegranate fruit seeds

Article · January 2011

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International Journal of Nanomaterials and Biostructures

Universal Research Publications. All rights reserved

Original Article
Phytofabrication of silver nanoparticles using pomegranate fruit seeds
Shalini Chauhan1*, Mukesh Kumar Upadhyay1, Narayan Rishi2 and Sushma Rishi2
*E mail: shaluphd@gmail.com
1
School of Life Sciences, Jaipur National University, Jaipur, India
2
Department of Virology and Immunology,Amity University, Noida, India

Received 28 September 2011; accepted 10 October 2011

Abstract

The cost effective and ecofriendly technique of green synthesis of silver nanoparticles from the extract of seed of pomegranate
fruit was demonstrated. The reduction process was simple and convenient to handle and was monitored by UV-Vis
spectroscopy. The morphology and crystalline phase of synthesized nanoparticles were determined from transmission electron
microscopy (TEM) and X-ray diffraction (XRD) method. These biologically synthesized nanoparticles were found to highly
toxic against different multi-drug resistant human pathogens. Therefore, these particles can be exploited for curing of infection
and other nanotechnology based therapeutic industries.
© 2011 Universal Research Publications. All rights reserved
Key words: Green synthesis, TEM, XRD, Silver nanoparticles.

INTRODUCTION method as it is cost effective and environment friendly

Nanotechnology is the application of science to control
matter at nanoscale level. This technique increases the scope
of investigation and regulation of nanoparticles at cellular
stage (Du et al., 2007), drug delivery (Jong et al., 2008),
diagnostics imaging cancer detection (Kairemo et al (2008),
sensing (Hall et al., 2011), artificial implants (Adini et al.,
2011), tissue engineering (Banobre et al., 2011), HIV
inhibition (Elechiguera et al., 2005) and water filtration
(Morones et al., 2005). A number of approaches are available
via chemical and phototchemical reactions for syntheses of
silver nanoparticles such as reverse micelles, thermal
decomposition of silver compounds, radiation assisted,
electrochemical, sonochemical and microwave assisted
process (Parashar et al ., 2009). Chemical synthesis methods
lead to presence of some toxic chemical absorbed on the
surface that they may have adverse effect in the medical
applications (Jain et al., 2009). Now a day biosynthesis of
nanoparticles is used by most of researchers to overcome the
above problems (Vdayasoorian et al., 2011). The green
synthesis is more advantageous over chemical and physical
International Journal of Nanomaterials and Biostructures 2011; 1 (2) 17-21
17
(Kowshik et al., 2003; Nabhikhan et al., 2010). Silver has
long recognized as inhibitory effect on microbes present in
medical and industrial process ( Reda et al., 2011) and have
antimicrobial properties with low toxicity ( Jaya et al., 2009).
Therefore, it is incorporated into various medical
applications; plastic cathertes coated with AgNPs prevent bio
film formation (Virendra et al., 2008). In the present paper it
is reported first time of synthesis of silver nanoparticles by
cell free aqueous extract of pomegranate fruit seed and this
biologically synthesized nanoparticles was characterised by
TEM and XRD also investigated against different multi drug
resistant human pathogens.
Materials and methods
Preparation of the extract:
Ripe pomegranate seeds were used to make aqueous extract.
10 g of seed was added into 100 ml of distilled water and
boiled it for 15 min. The above cooled content was filtered
with the help of Whatmann filter paper No.1 and filtrate was
used as a reducing agent for the preparation of nanoparticles.
 0.5 silver
0.45
5 hour
0.4 3hour

0.35 1hour

0.3

absorbance
0.25

0.2

0.15

0.1

0.05

0
wavelength

35

37

39

41

43

45

47

49
Fig. 2: UV-Vis spectrum of synthesized silver nanoparticles
Fig. 1: Seed extract of pomegranate (A) and colour changed after 5 hrs of reaction.
after 4 hrs of incubation of extract with 1 mM solution of
AgNO3 (B).

Synthesis of Silver Nanoparticles:

10 ml of pomegranate seed extract (act as a reducing agent)
was added with the 90 ml of aqueous solution of 1 mM
silver nitrate for reduction of Ag+ ions and incubated at room
temperature for 5 hrs. The reduction of pure Ag+ ions was
monitored by measuring absorption of the reaction medium in
a range of 400 - 450 nm wavelength using UV-Vis
spectrophotometer.
XRD measurement:
The silver nanoparticles was purified by repeated
centrifugation of above synthesized brown suspension at
10,000 rpm for 20 min followed by redispersion of the pellet
of silver nanoparticles in deionised water and again
centrifuged in same way. The freeze dried of above pellet
nanoparticles was analysed by XRD, to determine the
crystalline domain size.
TEM analysis:
Sample for TEM was of prepared by placing drops of silver
nanoparticles suspension over carbon coated grid and
allowing the solvent to evaporate the solvent. TEM was
performed by JOEL Model 1200 FX instrument operating at
80 K voltage for the characterization of size and shape of
synthesized silver nanoparticles.
Antibacterial assay:
The antibacterial assays of silver nanoparticles were done on
human pathogens Escherichia coli by food poisoning and
disc diffusion method:(a) In disc diffusion method, 100 µl of
culture inoculum was spread on Luria bertani medium plates.
Sterile paper disc containing silver nanoparticles along with
two standard discs were placed in above Petri plate and
incubated at 37 °C for 48 hrs and observation was recorded.
(b) In food poisoning method, silver nanoparticles were
added in Luria bertani medium at the time of pouring,
solidified Petri plate streaked with culture of E. coli. Medium
devoid of silver nanoparticles was used as a control.
International Journal of Nanomaterials and Biostructures 2011; 1 (2) 17-21
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Fig. 3: (A) and (B) TEM images of nanoparticles formed
after addition of seed extract with AgNO3 solution
Antifungal assay:
The assay was carried out in Petri plates containing 10 ml
PDA supplemented with silver nanoparticles. Aspergillus
flavus MTCC 277 was point inoculated in above medium and
incubated at 28 ºC for 72 hrs. The diameter of mycelial
colony developing on the silver nanoparticles containing
PDA plates were compared with the diameter of colony
obtained on control plates. The inhibition of fungal growth
was calculated by following formula:
% inhibition = (C-E) X 100 / C
Where C = diameter of fungal mycelium on control plate and
E = diameter of fungal mycelium on experimental plate.
Results and discussion
The colour change was noted by virtual observation in
pomegranate fruit seed extract incubated with aqueous
solution of AgNO3. It started to change colour from watery to
yellowish brown due to the reduction of silver ions, this
exhibit the formation of silver nanoparticles (Fig. 1).
Absorption spectrum at different wavelengths ranging from
400-500 nm revealed a peak of λ max at 430 nm (Fig. 2). The
seed extract without silver nitrate solution did not show any
change in colour. The colour intensity increased with
duration of time and colour of extract change brown after 4
hrs of incubation and no significant change after words. The
 Fig. 4: XRD pattern of silver nanoparticles

Fig. 5: Effect of silver nanoparticles on the growth of E.coli : (A) Control (B) treated plates

A B
Fig. 6: Antibacterial activities of silver nanoparticles on E.coli along with antibiotics Kanamycin (A) and Penicillin (B).

International Journal of Nanomaterials and Biostructures 2011; 1 (2) 17-21

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Fig. 7: Antifungal activity of silver nanoparticles
against Aspergillus flavus MTTCC 277.
XRD pattern showed two intense peaks in the spectrum of 2θ
value ranging 10 to 50 (Fig. 4). The TEM images clearly
reveals that shape and size of silver nanoparticles was 30 nm
with spherical shaped. It was noticeable that edges of
particles were lighter than the centre (Fig. 3). It exhibit that
few biomolecules like proteins of extract capped the silver
particles. The shape diffraction spots clearly suggest that the
particle is of single crystal quality. The nanoparticles
synthesized by green route were tested against multi drug
resistant human pathogens E. coli by food poisoning and disc
diffusion method and was found that 50 ppm was minimum
inhibitory concentration (Fig . 5) In the disc diffusion method
5µl of 50 ppm nanoparticles was placed on disc and tested
against pathogen. The zone around the disc was compared
with inhibition zone of two standard antibiotic kanamycin
and penicillin (Fig. 6). The antifungal activity of silver
nanoparticles was also tested against Aspergillus flavus
MTCC 277 (Fig. 7)and gave significant result.
The present work demonstrated that pomegranate seed extract
are capable of producing Ag from aqueous solution of Ag +.
The size dispersity of silver nanoparticles as spherical in
shape could be controlled by simple variation of the amount
of extract with aqueous solution. The reduction of metal ions
through fruit extracts leading to formation of spherical silver
nanoparticles. Application of such ecofriendly nanoparticles
in bactericidal against highly pathogens other therapeutic use
and electronic applications. The green synthesis of
nanoparticles has more advantages over physical and
chemical synthesis therefore; it might be used for exploitation
of fruit extract for production of nanoparticles in
nanotechnology industries.
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Source of support: Nil; Conflict of interest: None declared

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