Indian J.Pharm.Biol.Res.

2013; 1(4):16-24

CODEN (USA): IJPB07 ISSN: 2320-9267

Indian Journal of Pharmaceutical and Biological Research (IJPBR)

Journal homepage: www.ijpbr.in

Original Research Article

Synthesis, characterization and antibacterial potential of silver nanoparticles

by Morus nigra leaf extract
Anu Kumar, Kuldeep Kaur, Sarika Sharma*
Department of Biotechnology, Arni University Indora , Kangra (H.P), India.
ARTICLE INFO: ABSTRACT
Article history: The present study reports the synthesis of silver nanoparticle using Morus nigra leaf extract
Received: 26 October 2013 were used as reducing agent for reduction of silver nitrate solution. The synthesis of silver
Received in revised form:
nanoparticles was analyzed by UV-Visible spectroscopy, Scanning Electron Microscopy. The
14 November 2013
Accepted: 21 November 2013 SEM analysis has shown that size of silver nanoparticles synthesized from leaves extract of
Available online: 7 December 2013 M.nigra was 200 nm and seems to be spherical in morphology. Morphology of chemically
Keywords: synthesized silver nanoparticles is nearly spherical and of size ranges from 300-500 nm. The
average particle size analyzed from SEM analysis was observed to be 350 nm. This article has
Biosynthesis,
discussed the synthesis of silver nanoparticles generated from plant extract, characterization
Silver nanoparticles,
and antibacterial analysis. In this study the antibacterial activity was examined against six
Antimicrobial activity,
MTCC cultures collected from IMTECH Chandigarh, Including both gram positive and gram
Leaf extract,
negative bacteria such as P.aeruginosa, S.aureus, B.subtilis, E.coli, P.flourescens and
M.nigra.
Streptococus mutans. Out of these strains the antimicrobial activity of the silver nanoparticles
showed maximum zone of inbhition against P.flourescens (22 mm), P.aeruginosa (19 mm),
S.aureus (18 mm) and least effective against E.coli (15mm). In contrast chemically
synthesized silver nanoparticles were found most effective against S.aureus (13 mm) and
B.subtilis (12mm) and almost ineffective against Streptococcus mutans (6 mm) and
P.flourescens (4 mm). In the concluding remarks, the silver nanoparticles synthesized using
M.nigra leaves extract would be a better antimicrobial effective against various bacterial
species.

1.Introduction delivery, gene delivery, and artificial implants. Nanoparticles are

being sighted as fundamental building blocks of nanotechnology.
In recent years, the research is mainly focused on the metal
Nanotechnology broadly refers to a field of applied science and
nanoparticles due to their unique optical, electronic, mechanical,
technology whose special and unique properties could be
magnetic, and chemical properties that are significantly different
attributed to their small sizes and large surface areas [1]. It is the
from those of bulk materials [2]. There is a method known as
application of science and technology to manipulate the matter at
Green synthesis, which is biosynthesis of nanoparticles using
atomic and molecular scale. It has the ability to build micro and
plant extracts, which was exploited to a vast extent because the
macro materials and products with atomic precision. An
plants are widely distributed, easily available, safe to handle and
important aspect of nanotechnology concerns the development of
with a range of metabolites. Human beings are often infected by
experimental processes for the synthesis of nanoparticles of
microorganisms such as bacteria, molds, yeasts, and viruses in
different sizes, shapes and controlled dispersity. Currently it is
the living environment. Research in antibacterial material
employed as a tool to explore the darkest avenues of medical
containing various natural and inorganic substances has been
sciences in several ways like imaging, sensing, targeted drug
intensive. The use of environmentally benign materials like plant

Corresponding Author: Sarika Sharma, Department of Biotechnology, Arni University Indora, Kangra (H.P), India. E-Mail:
sharmasarika19@gmail.com Mobile: +91-9888599261
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leaf extract [3], bacteria [4], fungi [5] and enzymes[6] for the
synthesis of silver nanoparticles offers numerous benefits of eco-
friendliness and compatibility for pharmaceutical and other
biomedical applications as they do not use toxic chemicals for
the synthesis protocol. Numbers of plants have been successfully
used for the extracellular synthesis of silver nanoparticles
including tamarind [7], Helianthus annus [8], Cinnamomum
camphora,[9] Coriander [10],Capsicum annuum [11] Avena
sativa [12], Azardirachta indica [13] and Pelargonium
graveolens [14]. In the present study, we have explored the green
synthesis ofsilver nanoparticles using M.nigra leaves extract.
Synthesized nanoparticles were characterized UV-Visble
spectroscopy, SEM. Further, the antimicrobial activity of
synthesized silver nanoparticles against six MTCC cultures
collected from IMTECH Chandigarh, Including both gram
positive and gram negative bacteria such as P.aeruginosa,
S.aureus, B. subtilis, E.coli, P. flourescens and Streptococus
mutans were explored.
Figure 1: M.nigra plant leaves aqueous extract
II. Storage
Plant extract (filtrate) was stored at the temperature of 40C till
use for further use.
III. Synthesis of silver nanoparticles
AgNO3 solution of (1mm) of silver nitrate was prepared and
used for the synthesis of silver nanoparticles.
i. By using plant
1.5 ml of M.nigra leaves extract was added drop wise to 30 ml of
10-3 M AgNO3 aqueous solution in a 100 ml beaker and heated
on water bath at 75oC for 20-30 minutes. Colour change from
colourless to brown confirmed the reduction of silver nitrate to
silver ions.
Original Research Article
2. Material and Methods
2.1 Collection of plant material
The plant material used was the leaves of Morus nigra which
was collected from region of Indora, Kathgarh District Kangra
(H.P), India.
I. Preparation of Plant Extract
Fresh and healthy leaves of M.nigra were collected, washed
thoroughly with distilled water, incised into small pieces and air-
dried. About 20 g of leaves of M.nigra were weighed and
transferred into 500 ml beaker containing 100 ml distilled water,
mixed well and boiled for 10 min. Filtered the extract through
Whatman No.1 filter paper and collect the filtrate in a 250 ml
Erlenmeyer flask.
Figure 1: Showed plant extract Filtered through whatman No.1
filter paper. i.e. prepared by washing fresh and healthy leaves
thoroughly with distilled water and air-dried.
ii. By chemical reduction
5ml of 10-3 M AgNO3 was added to 30ml NaBH4 in a 50ml
beaker placed on ice packets and stirred on a magnetic stirrer for
10 minutes. Color change from colorless to yellowish brown
indicates the synthesis of silver nanoparticles.
IV. Purification of silver nanoparticles
The fully reduced solution was centrifuged at 10000 rpm for 20
minutes. Supernatant was discarded and pellet was obtained and
redispersed in distilled water. The process of centrifugation and
redispersion was repeated three times to wash off any absorbed
substance on the surface of silver nanoparticles.
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2.2 Characterization of Silver Nanoparticles
I. UV-Vis Analysis
Ultraviolet-visible analysis was done by using SYSTRONICS
PC Based Double beam spectrophotometer 2202 with resolution
of 1nm between 300 to 600nm. Reduction of Ag+ ions was
monitored by measuring UV-Vis spectrum of mixture. Reduction
of AgNO3 to Ag+ was confirmed by Color change from colorless
to brown. Formation of silver nanoparticles is easily detected by
measuring the optical density of solutions/suspensions.
II. SEM analysis of silver nanoparticles
Scanning Electron Microscopic (SEM) analysis was done using
Thermo scientific SEM machine. Thin films of the sample were
prepared on a carbon coated copper grid by just dropping a very
small amount of the sample on the grid, extra solution was
removed using a blotting paper and then the film on the SEM
grid were allowed to dry by putting it under a mercury lamp for 5
minutes.
III. The Antimicrobial Activity Analysis of AgNPs
i. Bacterial strains
Test bacteria used were Escherichia coli (MTCC 443),
Staphyalococcus aureus (MTCC 3160), Pseudomonas
aeruginosa (MTCC 4673), Bacillus subtilis (MTCC 441),
Pseudomonas flourescens (MTCC 103), Streptococcus mutans
(MTCC 890). These organisms were chosen because they are
commonly isolated from the hospitalized patient with intestinal
ailments, blood and skin infections. All these microorganisms
were obtained from IMTECH Chandigarh.
ii. Antimicrobial activity
Innoculum was standardized at 1×106 cfu/ ml. The effect of
chemically and biologically synthesized silver nanoparticles on
several bacterial strains was assayed by using the agar well
diffusion assay method.
iii. Agar well diffusion method
50 ml Nutrient broth (NB) medium was prepared and poured in
six tubes for inoculating six different MTCC culture and
sterilized by autoclaving at 121°C for 15 minutes at 15 psi
pressure. Then these NB tubes were inoculated with six different
bacterial strain used. Then Incubate at 35¬37°C for 24 hours.
Mueller-Hinton Agar (MHA) was prepared and was sterilized by
Figure 2: Solution of silver nitrate (3mM) (left) and reduced silver nitrate solution after addition of plant extract (right)
Original Research Article
autoclaving at 1210C for 15 minutes at 15 psi pressure and was
used to determine the antibacterial activity of silver
nanoparticles. Then sterile Mueller-Hinton Agar (20ml) was
poured aseptically into each sterile petridish and allowed to
solidify at room temperature under sterile conditions. After
solidification, petriplates were spreaded with MTCC cultures.
Then three wells were bored in each plate by using a sterile
borer. Then (20µl) of sample (Silver nanoparticles + DMSO),
DMSO (control) and silver nitrate was loaded in respective
wells. Then plates were incubated at 370C for 24 hours. After 24
hrs plates were observed for zone of inhibition in mm and the
zone was measured by using scale [15].
3. Result and Discussion
Today nanomaterials are at the primary stage of fast developing
nanotechnology phase. Nanomaterials are facilitating modern
technology to deal with nano size objects. Currently
nanomaterial’s are already being used in medical application
such as drug carrier, strong antibacterial, detection for
pathogens/proteins and tissue engineering. In the recent times,
researchers paid more attention on the green synthesis and
applications of silver nanoparticles using various plant extracts.
In this study work is carried out on extracellular synthesis of
silver nanoparticles from leaves extract of plant M.nigra and
their antibacterial activity was investigated. Antibacterial activity
of chemically synthesized silver particles was compared with
biologically synthesized silver nanoparticles.
3.1 Synthesis of silver nanoparticles
To verify the synthesis of silver nanoparticles, flasks containing
plant extract (without silver nitrate solution) in biological method
and sodium borohydride (NaBH4) in chemical method was
positive control and pure silver nitrate as negative control was
monitored visually.
The M.nigra leaves extract and solution of NaBH4 (positive
control) and silver nitrate solution (negative control) was
observed to retain its original color. The silver nitrate treated
with leaves extract of M.nigra turned brown after 5 minutes due
to deposition of silver nitrate. The color was mainly observed
due to the excitation of surface plasmon resonance of deposited
silver nanoparticles[16]. In case of negative control no color
change was observed even after 48 hrs.
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Figure 3: Solution of sodium borohydride (0.2 M) (left) and (right) reduced silver nitrate solution after addition of chemical
3. Results and discussion
3.1 Characterization of silver nanoparticles
I. UV-Vis spectrum Analysis
The formation of silver nanoparticles by reduction of aqueous
metal ions during the exposure of M. nigra leaves extract and
NaBH4 solution may be easily followed by UV-visible
spectroscopy. UV-visible absorption spectrum of silver
nanoparticles in the presence of leaves extract of M.nigra and
NaBH4 solution is shown in Figure 4 (a and b).The surface
Plasmon band in silver nanoparticles solution in case of
biological method remain closer to 400 nm while it is close to
450nm in case of chemically synthesized silver nanoparticles.
Figure 4(a): UV-Vis absorption spectrum of obtained biologically
synthesized silver nanoparticles
II. SEM Analysis
Scanning electron microscopy has been employed to characterize
the size, shape and morphology of formed silver nanoparticles.
Original Research Article
Silver nanoparticles synthesized from the leaf aqueous extract of
M. Nigra was evaluated through spectrophotometry at a
wavelength range of 350-700 nm. Plasmon absorption band of
400 nm indicating the presence of spherical or roughly spherical
Ag nanoparticles. It is generally recognized that UV-Vis
spectroscopy could be used to examine size and shape of
nanoparticles in aqueoussolution. Figure 4(a) shown UV-Vis
spectra recorded from reaction medium of biological method
when M.nigra leaves extract is used as reducing agent. Figure
4(b) shown UV-Vis spectra recorded from reaction medium of
chemical method when NaBH4 is used as a reducing agent.
Figure 4(b): UV-Vis absorption spectrum of obtained chemically
synthesized silver nanoparticles
SEM image of both samples are shown in Figure 5 (a) and 5 (b)
respectively.
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 Kumar et al. / Indian J. Pharm. Biol. Res., 2013; 1(4):16-24

Figure 5 (a): SEM images of biologically synthesized silver
nanoparticles
From Figure 5(a) SEM analysis image has shown that size of
silver nanoparticles synthesized from leaves extract of M.nigra
was 200nm and seems to be spherical in morphology. From
Figure 5(b) it is evidenced that morphology of chemically
synthesized silver nanoparticles is nearly spherical and of size
ranges from 300-500nm. The average particle size analyzed from
SEM image is observed to be 350nm. In our study size of silver
nanoparticles which were synthesized by plant extract found
smaller as compared to chemically synthesized silver
nanoparticles, so that biologically synthesized silver
nanoparticles are of great importance as compared to chemically
synthesized silver nanoparticles.
III. Evaluation of Antibacterial activity of Silver Nanoparticles
(a) Antibacterial activity
Recently, silver nanoparticles are found to be a best biocide, for
example in wound dressings and as an antimicrobial coating on
consumer products, little is known about its mode of toxicity. A
Figure 5 (b): SEM images of the chemically synthesized silver
nanoparticles
wide research reports were available for using silver
nanoparticles as a antimicrobial agent against S. aureus and E.
Coli. [17]. Silver nanoparticles have been used as antimicrobial
agent against a number of bacterial strains such as B. subtilis and
S. aureus and E. coli [18], Staphylococcus aureus, E.coli,
Corynebacterium diphtheria and Micrococcus sp., and Gram
positive strains (S. aureus, S.epidermidis and Gram negative
strains (E. coli, S. typhi, Pseudomonas aueroginosa, Klebsiella
pneumonia, Proteus vulgaris) [19]. In this study Antibacterial
activity of silver nanoparticles was investigated against six
MTCC cultures collected from IMTECH Chandigarh including
both gram positive and gram negative bacteria such as
P.aeruginosa, S.aureus, B. subtilis , E.coli, P.flourescens and
Streptococus mutans. The maximum activity i.e 22 mm zone of
inhibition was obtained againstP.flourescens,17mm zone of
inhibition against Streptococcus, 15 mm zone of inhibition
against B. subtilis then 14 mm zone of inhibition against E.coli
and 19 mm against P.aeruginosa.

Table no. 1: Evaluation of Antibacterial activity of silver nanoparticles synthesized by Plant (M. nigra) and chemical (sodium
borohydride)
Sr. No. Bacterial isolates Zone of Inhibition in mm
Biologically synthesized Chemically synthesized Control
silver nanoparticles silver nanoparticles
1. S. mutans 17mm 6mm 0mm
2. B. subtilis 15mm 12mm 2mm
3. E.coli 14mm 7mm 1mm
4. P. flourescens 22mm 4mm 0mm
5. S.aureus 18mm 13mm 1mm
6 P.aeruginosa 19mm 8mm 2mm

The antibacterial activity of chemically synthesized nanoparticles Streptococcus, 12 mm zone of inhibition against B.subtilis, 7 mm
was examined against same six MTCC cultures as discussed zone of inhibition for E.colithen 8 mm against P.aeruginosa and
above. The maximum activity i.e 13 mm zone of inhibition was 4 mm against P. flourescens.
obtained against S.aureus,6 mm zone of inhibition against

Original Research Article 20

 Kumar et al. / Indian J. Pharm. Biol. Res., 2013; 1(4):16-24

(A). S. mutans (B) B. subtilis

(C) E.coli (D) P. flourescens

(E) S.aureus (F) P.aeruginosa

Figure 6: (A-F) Antibacterial activity of biologically synthesized silver nanoparticles

Original Research Article 21

 Kumar et al. / Indian J. Pharm. Biol. Res., 2013; 1(4):16-24

(A). E.coli (B) P. flourescens

(C) S.aureus (D) B. subtilis

(E) P. aeruginosa (F) S.mutans

Figure 7: (A-F) Antibacterial activity of chemically synthesized silver nanoparticles

Original Research Article 22

 Kumar et al. / Indian J. Pharm. Biol. Res., 2013; 1(4):16-24
Table No.2: Comparative study of antibacterial activity of biologically synthesized AgNPs and chemically synthesized silver
nanoparticles
Zone of inhibition(mm)
S. mutans B.subtilis E.coli
Biologically
Synthesized Silver 16mm 15mm 14mm
Nanoparticles
Chemically
Synthesized Silver 6mm 12mm 7mm
Nanoparticles
From comparative study of antibacterial activity of plant and
chemically synthesized silver nanoparticles, it was concluded
that silver nanoparticles which were synthesized by using plant
aqueous extract has more antibacterial activity as compared to
chemically synthesized silver nanoparticles. Biologically
synthesized silver nanoparticles found most effective against P.
flourescens (22 mm), P.aeruginosa (19 mm) and S. aureus (18
mm) and least effective against E. coli (15mm). As these silver
nanoparticles were found to be very much effective against
S.aureus and P.Flourescens so these can be of great importance
in dealing with skin infections and can be used as antibacterial
drug. In contrast chemically synthesized silver nanoparticles
were found most effective against S. aureus (13 mm) and B.
subtilis (12 mm) and almost ineffective against Streptococcus
mutans (6mm) and P. flourescens (4 mm).These results clearly
indicate that silver nanoparticles synthesized by using plant
extract are more effective against bacterial strains as compared to
chemically synthesized silver nanoparticles and could provide a
safer alternative to conventional antimicrobial and antibacterial
agents. The major mechanism through which silver nanoparticles
manifest antibacterial properties was either by anchoring or
penetrating the bacterial cell wall, and modulating cellular
signaling [20]. The mechanism involved in the inhibition of
bacterial growth by silver nanoparticles is unclear. However, the
general mechanism of antibacterial activity of silver
nanoparticles was proposed by many researchers, but the detailed
mechanism remains to be understood. The bacterial growth was
inhibited by silver ions, which accumulated into the vacuole and
cell walls as granules. The silver nanoparticles attached the
surface of the cell membrane disturbing the permeability and
respiration functions followed by dysfunction of metabolic
pathways including, silver ions can interact with nucleic acids
they preferentially interact In the present study, plant based
AgNPs showed maximum inhibitory activity against the
P.flourescens and S.aureus isolated from hospitalized patient
with intestinal ailments, blood and skin infections. This study
suggests green synthesized nanoparticles have been effectively
used as an alternative to antibiotic of bactericides against skin
wounds by reducing antimicrobial activities. This biosynthesis of
Original Research Article
P. flourescens S.aureus P.aeruginosa
22mm 18mm 19mm
4mm 13mm 8mm
silver nanoparticles can prove potential candidates for medical
applications.
4. Conclusion
A critical need in the field of nanotechnology is the development
of reliable and eco-friendly processes for synthesis of metallic
nanoparticles. Here, we have reported a simple biological and
low-cost approach for preparation of stable silver nanoparticles
by reduction of silver nitrate solution with a bioreduction method
using Morus nigra leaf extract as a reducing agent. Morus nigra
leaf extract is found suitable for the green synthesis of silver
nanoparticles. Characterization of silver nanoparticles was done
by UV spectroscopy and SEM analysis. Silver exhibits the strong
toxicity in various chemical forms to a wide range of
microorganism is very well known and silver nanoparticles have
recently been shown to be a promising antimicrobial material. In
conclusion, the bio-reduction of aqueous Gab ions by the plant
extract of the M.nigra leaf extract has been demonstrated.
Conflict of interest statement
We declare that we have no conflict of interest.
Acknowledgement
The authors are thankful to the authorities of Arni University,
Kathgarh Indora, Kangra (H.P), India for providing support to
the study and other necessary facility to carry out research work.
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silver nanoparticles by Morus nigra leaf extract. Indian J. Pharm. Biol. Res.2013; 1(4):16-24.
All © 2013 are reserved by Indian Journal of Pharmaceutical and Biological Research.
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