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Controllable synthesis of silver nanoparticles using
Neem leaves and their antimicrobial activity
Aparajita Verma, Mohan Singh Mehata
*
Laser-Spectroscopy Laboratory, Department of Applied Physics, Delhi Technological University, Bawana Road, Delhi
110042, India
article info
Article history:
Received 15 September 2015
Received in revised form
8 October 2015
Accepted 4 November 2015
Available online 13 December 2015
Keywords:
Silver nanoparticles
Azadirachta indica
Absorption
Photoluminescence
Green synthesis
abstract
Silver nanoparticles (AgNPs) were synthesized using aqueous extract of Neem (Azadirachta
indica) leaves and silver salt. XRD, SEM, FTIR, optical absorption and photoluminescence
(PL) were measured and analysed. The synthesized AgNPs exhibits lowest energy absorp-
tion band at 400 nm. The effects of various parameters i.e., extract concentration, reaction
pH, reactants ratio, temperature and interaction time on the synthesis of AgNPs were
studied. It was found that the formation of AgNPs enhanced with time at higher temper-
ature and alkaline pH. The AgNPs formed were found to have enhanced antimicrobial
properties and showed zone of inhibition against isolated bacteria (Escherichia coli) from
garden soil sample. Based on the results obtained, it can be concluded that the resources
obtained from plants can be efficiently used in the production of AgNPs and could be
utilized in various fields such as biomedical, nanotechnology etc.
Copyright ©2015, The Egyptian Society of Radiation Sciences and Applications. Production
and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
The field of nanotechnology has proved to be one of the most
active areas of research (Moore, 2006; Sergeev &Shabatina,
2008). Synthesis of nanoparticles is increasing exponentially
because of its wide range of applications in the field of opto-
electronics, biosensors, bio-nanotechnology, biomedicine etc.
(Bharali, Sahoo, Mozumdar, &Maitra, 2003; Mehata,
Majumder, Mallik, &Ohta, 2010; Mehata, 2012, 2015; Ratnesh
&Mehata, 2015; Saxena, Mozumdar, &Johri, 2006; Subbiah,
Veerapandian, &Yun, 2010).
Various physical and chemical methods have been formu-
lated for the synthesis of nanopartilces of desired shape and
size. However these methods are not economically feasible
and environment friendly. Therefore, green synthesis has been
considered as one of the promising method for synthesis of
nanopartilces because of their biocompatibility, low toxicity
and eco-friendly nature (Malik, Shankar, Malik, Sharma, &
Mukherjee, 2014). Various microorganism and plants have
proved to be a source of inspiration for nanomaterial synthesis.
Some well-known examples of nanoparticles synthesized by
microorganisms either intracellularly (Weiner &Dove, 2003)or
extracellularly (Bansal, Bharde, Ramanathan, &Bhargava,
2012) are: synthesis of magnetite by magnetotactic bacteria
(Dickson, 1999; Lovley, Stolz, Nord, &Phillips, 1987; Philipse &
Maas, 2002) and synthesis of siliceous material by radiolar-
ians and diatoms (Kr€
oger, Deutzmann, &Sumper, 1999; Mann,
1993; Oliver, Kuperman, Coombs, Lough, &Ozin, 1995). A
mixture of curiosity and unshakable belief that mother earth
*Corresponding author.
E-mail addresses: msmehata@gmail.com,mohan.phy@dce.edu (M.S. Mehata).
Peer review under responsibility of The Egyptian Society of Radiation Sciences and Applications.
HOSTED BY Available online at www.sciencedirect.com
ScienceDirect
Journal of Radiation Research and Applied
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journal homepage: http://www.elsevier.com/locate/jrras
Journal of Radiation Research and Applied Sciences 9 (2016) 109e115
http://dx.doi.org/10.1016/j.jrras.2015.11.001
1687-8507/Copyright ©2015, The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an
open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
has developed the best method for the synthesis of nano range
of materials have led to a new and exciting field of research
which involves microorganism and plants for the synthesis of
nanomaterials. The green synthesis methods include synthe-
sis of nanoparticles using microorganisms like bacteria, fun-
gus, yeasts (Narayanan &Sakthivel, 2010), plants (Jha, Prasad,
Prasad, &Kulkarni, 2009; Makarov et al., 2014; Mittal, Chisti, &
Banerjee, 2013) and DNA (Sohn, Kwon, Jin, &Jo, 2011). Multi-
ple species of bacteria and fungi have been investigated for the
growth of nanoparticles of different composition and size, for
example, synthesis of gold by Verticilliumsp (Narayanan &
Sakthivel, 2010), synthesis of CdS quantum dots using fungi
etc. (Ahmad et al., 2002).
Besides microbes, use of part of plants like stem, leaves,
roots etc. (Jha et al., 2009) for the synthesis of nanoparticles is
yet another exciting possibility that is relatively unexplored.
Advantage of using plants over microorganism is the elimi-
nation of the elaborate process of cell culture. Moreover,
nanoparticles synthesized using biological methods are more
compatible for medical use as compared to chemical and
physical methods where toxic material may adsorb on the
surface of the nanoparticles that may have adverse effect
when used for medicinal purpose. The biosynthesis method
employing plant extracts of Pelargonium graveolens,Medi-
cagosativa,Azadirachta indica,Lemongrass,Aploevera,Cinnamo-
mum Camphor (Jha et al., 2009; Makarov et al., 2014; Mittal et al.,
2013; Shankar, Rai, Ahmad, &Sastry, 2004) have drawn great
attention as an alternative to conventional methods, because
plants are found in abundance in nature.
In recent years, increasing antibiotic resistance by mi-
crobes is imposing serious threat to the health sector. Nano-
particles have proved to be a likely candidate for antimicrobial
agent since their large surface to volume ratio ensures a broad
range of attack on bacterial surface. One of the most prom-
ising nanoparticle which acts as a highly effective antimi-
crobial agent is silver. Various investigations on silver
nanoparticles have been done to study its antimicrobial ac-
tivity. AgNPs exhibited significant antibacterial activity
against Escherichia coli,Staphylococcus aureus and antifungal
activity against Trichophyton,Trichosporon beigelii and Candida
albicans (Gajbhiye, Kesharwani, Ingle, Gade, &Rai, 2009).
Considering the advantages of green synthesis over other
methods, this study aims at the synthesis of AgNPs using
aqueous Neem (Azadirachta indica) leaves extract. It focuses on
the study of the effects of various physicoechemical param-
eters on AgNps. We also attempt to investigate about the
antimicrobial effect of the synthesized nanoparticles. Azadir-
achta indica, which is a common plant known as Neem is found
abundantly in India and in nearby Indian subcontinents. It
belongs to Meliaceae family and is known for its various ap-
plications especially its medicinal property (Subapriya &
Nagini, 2005). Azadirachta indica leaf extract is used in the
synthesis of various nanoparticles like gold, zinc oxide, silver
etc. The phytochemicals present in Neem are namely terpe-
noids and flavanones, which act as reducing as well as
capping agent and helping in stabilizing the nanoparticles.
When silver salt is treated with Neem leaf extract, the silver
salt is reduced to AgNPs. The synthesized nanoparticles,
which are capped with neem extract also exhibit enhanced
antibacterial activity.
2. Materials and methods
Silver nitrate was obtained from Sigma-Aldrich chemical Co.
All the glassware were washed with distilled water and dried
in oven. The petri-plates and agar were autoclaved before use.
20 g of finely cut Neem leaves were boiled in 100 ml water for
10 min and filtered to obtain Neem leaves extract. The extract
of Neem leaves (5 ml) were mixed with 45 ml of 1 mM silver
nitrate (AgNO
3
) and colour change was observed indicating
the formation of AgNPs (Shankar et al., 2004). The effects of
various physicoechemical parameters were examined by
varying the reactant concentration, pH, temperature and re-
action time. Reduction of Ag
þ
ions was monitored after
diluting a small amount of sample 20 times. Absorption
spectra were recorded with UV/VIS/NIR spectrometer (Perkin
Elmer Lambda 750) and photoluminescence (PL) spectra were
recorded with Fluorolog-3 spectrofluorometer (Horiba Jobin
Yyon) equipped with double-grating at excitation and emis-
sion monochromators (1200 grooves/mm) and an R928P pho-
tomultiplier tube (PMT). The excitation source was a 450 Watt
CW xenon lamp. Effect of time was studied by measuring the
absorption spectra of the solution at the time interval of 5, 15,
25, 35 and 45 min.
Effect of pH was studied by varying the pH of both Neem
broth and silver salt solution. 0.1 N KOH and 0.1 N HCL was
added to adjust the pH of the solution. The pH variation was
observed from pH 8e12 with an accuracy of ±0.2. Effect of
temperature was measured by varying the temperature be-
tween 10 and 50 C with an accuracy of ±3C. Neem broth
containing AgNPs were centrifuged at 10,000 rpm for 15 min
and the precipitate was thoroughly washed with sterile
distilled water to get rid of any unwanted impurities. The
purified pellet was then dried at 60 C and the sample was
characterized using scanning electron microscope (SEM,
Hitachi S7000N) and X-ray diffractometry (XRD, Bruker D8
advanced). Biomolecules responsible for the reduction of sil-
ver salt were studied using Fourier transform infrared (FTIR)
spectrometer (Thermoscientific Nicolet 380). The synthesized
AgNPs were then tested for their antibacterial property
against bacteria obtained from garden soil samples. The bac-
teria were grown on 1.8% agar plates then a small amount of
AgNPs were added for the study of antibacterial property.
3. Results and discussion
3.1. Effect of reaction time and concentration on the
formation of AgNPs
When silver salt (AgNO
3
) is added to aqueous Neem leaf
extract it results into a colour change from pale yellow to
yellowish brown and finally to dark brown colour, as shown in
Fig. 1. The change in colour of the solution is due to the
presence of silver nanoparticles formed by the reduction of
silver salt. The reduction of silver salt to silver ions is due to
the presence of reducing agents. It was suggested that com-
pounds like caffeine and theophylline act as reducing agent
when Acalypha indica leaf extract was used (Krishnaraj et al.,
2010). However, in Neem leaves extract natural reducing
Journal of Radiation Research and Applied Sciences 9 (2016) 109e115110
agent like terpenoids and flavanones are present, which are
responsible for reduction of silver salt to AgNPs. AgNPs show
yellowish brown colour in aqueous solution due to the exci-
tation of surface plasmon vibrations (Shankar et al., 2004). It is
noticed that the complete colour change took after about
30 min, thereafter no further colour of the reaction mixture
changed. This indicates that silver salt present in the reaction
mixture has been reduced completely. Then the formation of
silver nanoparticles was examined and confirmed by obtain-
ing the respective absorption spectra. The absorption spec-
trum originated due to the strong surface plasmon resonance
(SPR), i.e., due to resonant absorption of photons by AgNPs.
The observed absorption band is size dependent, since SPR
band depends on size and the refractive index of the solution
(Amendola, Bakr, &Stellacci, 2010).
Fig. 2 shows the absorption spectra of AgNPs obtained from
the reaction of Neem leave extract and AgNO
3
recorded in the
range of 250e700 nm at different reaction time. Absorption
maximum is observed at 400 nm, which is at higher energy as
that obtained by Shankar et al. (2004) and with olive leaf
(Khalil, Ismail, El-Baghdady, &Mohamed, 2013), indicating
that the prepared AgNPs are smaller and has uniform size
distribution. It is observed that there is an increase in the
absorbance with the passage of time (inset Fig. 2), indicating
an enhancement in the formation of AgNPs. Change in colour
was observed initially after 5 min of adding the salt solution to
the Neem leaf broth. After 30 min, the colour of the solution
becomes nearly constant, indicating that no silver salt was left
for further reaction. The results are in complete correlation
with that of reported by Shankar et al. (2004). With the passage
of time, the intensity of SPR band increased without any shift
in peak wavelength. According to Mie theory (Rout, Lakkakula,
Kolekar, Mendhulkar, &Kashid, 2009), absorption spectra of
spherical AgNPs exhibit single SPR band, and as the anisot-
ropy increases the number of peaks increases. In the present
study, SPR band suggests that the synthesized nanoparticles
are spherical in shape which is further confirmed by SEM
study. The size of the Ag Nanoparticles is calculated using
modified Mie theory as given:
gðRÞ¼g0þðAyFÞ=R(1)
where g(R) is resonance broadening, Ais scattering process (3/
4 in case of Ag), g
o
is the velocity of bulk scattering (5 10
12
s
1
for Ag) and v
f
is the Fermi velocity. From equation (1), the size
of the AgNPs (obtained when silver salt was mixed with Neem
leaf broth and left for 45 min) comes out to be approximately
4.0 nm.
Fig. 3 shows the absorption spectra of AgNPs obtained with
changing the concentration of Neem broth and silver salt.
When the concentration of Neem leaf broth to silver salt is in
the ratio 1:16, a relatively weak absorption band is observed.
With increase in concentration of reaction mixture (1:02), the
peak intensity increases abruptly, indicating an enhancement
in the production of AgNPs. The absorption intensity in-
creases monotonically with increasing concentration of Neem
Fig. 1 eChange in colour of the solution with time when silver salt was added to Neem leaf broth.
Fig. 2 eAbsorption spectra of AgNPs observed at five
different reaction times of 10
¡3
M aqueous solution of
silver nitrate with Neem leaf broth. Inset figure shows an
increase of absorption intensity as a function of reaction
time.
Journal of Radiation Research and Applied Sciences 9 (2016) 109e115 111
broth (as shown in inset Fig. 3). Moreover, on increasing the
concentration of Neem leaf broth the absorption spectrum
shifted towards red (from 408 to 421 nm), indicating an in-
crease in the size of AgNPs.
3.2. Effect of pH on the formation of AgNPs
Another important parameter which affects the formation of
nanoparticles is the pH of the solution. Change in pH affects the
shape and size of the particles, as pH has the ability to alter the
charge of biomolecules, which might affect their capping as
well as stabilizing abilities. Fig. 4 shows change in peak ab-
sorption wavelength and intensity on varying the pH of the
solution. As the pH increases from 9 to 13, the absorption
maximum shifts from 383 to 415 nm. In addition to the spectral
shift, the absorption intensity increases with increasing pH.
This indicates that pH 13 is the most favourable pH for the
synthesis of AgNPs using Neem leaf extract. Further, it was
observed that the pH enhances the rate of reduction reaction,
the colour change was observed very fast when AgNO
3
mixed
with aqueous Neem leaf extract, i.e. within few minutes the
colour of the sample changed to dark brown. The shift in the
peak wavelength indicates that the size of the particles in-
creases with increasing pH of the solution. As the diameter of
the particles get larger, the energy required for excitation of
surface plasmon electrons decreases, as a result the absorption
maximum shifted towards the longer wavelength region.
Moreover, it was observed that at acidic pH i.e. pH <7, the
formation of nanoparticles is suppressed. At high pH, the
bioavailability of functional groups in Neem leaf extract pro-
moted the synthesis of nanoparticles. However, at very high pH
i.e. pH ~ 13, the particles became unstable and agglomerated,
when kept for overnight. A linear relation is observed between
absorption maximum and pH (inset Fig. 4). It has been
mentioned (Khalil et al., 2013) that the absorbance of the AgNPs
obtained from olive leave extract increases with increasing pH
of the solution from 2 to 8. Upon further increasing the pH of
the solution, the absorbance decreases. However, in the pre-
sent study upon increasing the pH from 9 to 13 the absorbance
increases monotonically, indicating that the alkaline pH is
more favourable for the synthesis of AgNPs (Vanaja et al., 2013).
3.3. Effect of temperature on the formation of AgNPs
Temperature is yet another important factor that affects the
synthesis of nanoparticles significantly. Fig. 5 shows the ab-
sorption spectra of AgNPs at different temperatures in the
range of 10e50 C. With the increase in temperature, the
reduction of silver salt is enhanced, as indicated by rapid
change in the colour of the solution. The peak absorption
wavelength shifted toward blue from 433 to 397 nm, as tem-
perature variesfrom 10 to 50 C. The shift in the band maximum
is due to localization of surface plasmon resonance of the
AgNPs. This indicates that the size of the synthesized nano-
particles decreaseswith increasing temperature, which may be
due to the faster reaction rate at higher temperature. At high
temperature, the kinetic energy of the molecules increases and
silver ions getsconsumed faster, thus leaving less possibility for
particle size growth. Thus, smaller particles of uniform size
distribution are formed at higher temperature. The present
study is in complete correlation with the work reported based
Fig. 3 eAbsorption spectra of silver nanoparticles at
various concentrations of Neem leaf broth and silver salt
(1:02, 1:04, 1:08, 1:12, 1:16). Inset figure shows an increase
of absorption intensity as a function of concentration.
Fig. 4 eAbsorption spectra of silver nanoparticles at
different pH values (pH ¼9, 10, 11, 12, 13) of the reaction
mixture. Inset figure shows a linear relation between the
absorption maximum and pH.
Fig. 5 eAbsorption spectra of silver nanoparticles obtained
at different reaction temperature of 10,20
,30
,40
and
50 C. Inset figure shows nearly a linear relation between
the absorption maximum and temperature.
Journal of Radiation Research and Applied Sciences 9 (2016) 109e115112
on the banana peel extract (Ibrahim, 2015), where the intensity
of peak maximum increases withincreasing temperature along
with the blue shift in peak wavelength.
3.4. Surface morphology of AgNPs
XRD pattern of AgNps was recorded in the range of 30e70at
2qangles and analysed (as shown in supporting information
Fig. S
1
). The XRD pattern shows that the AgNps obtained by
the reduction of silver salt using Neem leaf extract are crys-
talline in nature. The high intensity peaks at around 37,44
and 64corresponding to three diffraction faces of silver. The
XRD peak at around 37represent to the Bragg reflection cor-
responding to the (111) plane.
SEM images of the synthesized AgNPs obtained from Aza-
dirachta indica leaf extract is given in supporting information as
Figs. S
2
and Fig. S
3
. The obtained nanoparticles are spherical in
shape and crystalline in nature. The samples used to examine
the morphology of AgNPs were kept for longer time, which may
have been agglomerated, therefore showing larger particle size
than that obtained from optical absorption spectra.
Furthermore, to identify the possible biomolecules present
in Neem leaf broth which are responsible for capping and sta-
bilizing the metal nanoparticles FTIR spectra were measured.
The FTIR spectra were measured for pure Neem leaf broth and
purified AgNPs obtained after centrifugation of sample at
10,000 rpm (Fig. S
4
). The FTIR spectrum of Neem leaf broth
shows peaks at 3389, 1635 and 1390 cm
1
. These peaks corre-
spond to the groups present in the sample and are indicating to
the OeH stretching (around 3389 cm
1
), C]C group (around
1635 cm
1
) and geminal methyl group (around 1380 cm
1
),
which is in good correlation with that of the other reports
(Tripathy, Raichur, Chandrasekaran, Prathna, &Mukherjee,
2010). These bands suggest the presence of terpenoids in
Neem leaf. It can be inferred that terpenoids present in Neem
leaf extract acts as stabilizingas well as capping agents. Besides
terpenoids, presence of flavanones are also possible. Terpe-
noids and flavanones interact through carbonyl groups and are
adsorbed on the surface of the metal ions. Terpenoids reduces
metal ions by oxidation of aldehydic groups in the molecules to
carboxylic acid (Smitha, Philip, &Gopchandran, 2009).
3.5. Antimicrobial activity of AgNPs
Garden soil samples are taken and serially diluted on 1.8% agar
plates, and a pour plating technique is used to culture the mi-
croorganisms present in different dilutions. The plates are
marked for each dilution. Bacterial colony is observed after
incubating the agar plates overnight at 37 C. Bacterial colonies
are obtained from the plate which is serially diluted to
10
6
(dilution factor). Agar plates are four quadrants streaked to
isolate different colonies and to obtain pure culture. Biochem-
ical analysis is further done to determine the strain of bacteria.
Colonies of E.coli and S.aureus were obtained. In the present
study E.coli strain is used to study the antibacterial property of
silver nanoparticles.
The antibacterial activity of the synthesized AgNPs which
is tested against bacterial colony obtained from soil sample is
shown in supporting information (seeFig. S
5
). Different
quantities of AgNPs (0, 2, 4, 8, 10 and 12 mgml
1
) are added to
the agar plates containing bacterial colony. Zone of clearance
is observed maximum at 12 mgml
1
of AgNPs. The antibac-
terial activity of AgNPs can be explained due to the change in
the cell membrane permeability or degradation of enzymes in
bacteria. The zone of clearance observed at 12 mgml
1
of
AgNPs is 6 mm (see Fig. S
5
).
3.6. Photoluminescence of synthesized AgNPs at
different reaction conditions
Fig. 6 shows photoluminescence (PL) spectra of AgNPs obtained
with 380 nm excitation at various reaction time, the similar one
used for the absorption spectra(cf. Fig. 2). The PL spectra show a
strong and well defined peak at around 450 nm. With the pas-
sage of time, the peak intensity increases gradually, indicating
an enhancement in nanoparticles synthesis. Such an
enhancement in the PL intensity is also observed at higher
concentration of silver salt with respect to Neem broth (Fig. 7),
at alkaline pH and at higher temperature (see supporting
Fig. 6 ePhotoluminescence spectra of AgNPs recorded at
five different reaction times of 10
¡3
M aqueous solution of
silver nitrate with Neem leaf broth. Excitation wavelength
was 380 nm. Inset figure shows an increase of PL intensity
as a function of reaction time.
Fig. 7 ePhotoluminescence spectra of AgNPs at various
concentrations of Neem leaf broth and silver salt (1:02,
1:04, 1:08, 1:12, 1:16). Excitation wavelength was 350 nm.
Inset figure shows an increase of PL intensity as a function
of concentration.
Journal of Radiation Research and Applied Sciences 9 (2016) 109e115 113
information Figs. S
6
and S
7
). The increase in intensity with
increasingconcentration of Neem broth can be explained by the
presence of large number of functional groups that react with
silver salt and increases the production of AgNPs. With the
change in pH from acidic to alkaline, the intensity of PL band
increases, indicating that the rate of reduction increases at
higher pH. Higher temperature also support the rate of forma-
tion of AgNPs. It is known that the AgNPs emits light between
400 and 700 nm (Vigneshwaranet al., 2007), which occurs due to
relaxation from the electronic motion of surface plasmon,
when surface plasmon electrons absorbs light at resonant fre-
quency a part of the absorption energy transferred into heat
energy and part of it radiates as PL, and a recombination of sp
electrons with holes in the dband (Parang et al., 2012).
4. Conclusions
AgNPs have been successfully synthesized using a well-known
medicinal plant Neem leaf extract (Scheme 1). The synthesized
AgNps are crystalline in nature, polydispersed and exhibit high
energy SPR band at around 400 nm and a strong PL at around
450 nm (Scheme 1), depending on controllable parameters. The
synthesis is found to be efficient in terms of reaction time as
well as stability of the AgNPs. The rate of synthesize is faster in
case of Neem as compared to the other biological methods
microbes, DNA etc. Thus, the rate of reaction of biological
synthesis is comparable to that of the chemical methods. The
synthesis process, i.e., formation of AgNPs critically depends
on the pH, temperature, reactant concentration and reaction
time. By changing these environmental parameters, the size
and shape of the synthesized nanoparticles can be altered.
Synthesis of AgNPs is enhanced with time at higher tempera-
ture and alkaline pH. Green synthesized AgNPs are found to
have enhanced antibacterial activity against bacterial colony
isolated from soil sample. Due to the enhanced antimicrobial
activity of AgNPs, it is effectively used in the field of medicine as
well as in food and cosmetic industries.
Acknowledgement
A.V. thanks to all the lab members especially to Mr. R.K. Rat-
nesh for his constant help in the measurements. This work is
financially supported by the DAE-BRNS, Govt. of India (Grant
No. 2012/37P/20/BRNS/765).
Appendix A. Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.jrras.2015.11.001.
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