GREEN SYNTHESIS OF SILVER NANOPARTICLES USING LEAF EXTRACT OF
MENTHA ARVENSIS AND ITS ANTIBACTERIAL ACTIVITY
Ms. A. Anitha Mary, Ms. B. Rabaka Sweety Mrs. Sangeetha
Jayaraj Annapackiam college for women (Autonomous)
Periyakulam, Tamilnadu
ABSTRACT
In the present study bio synthesis of silver Nanoparticles using
aqueous extract of M. arvensis and its antibacterial activity
against different micro organisms were investigated. In this work,
we describe a cost effective and environment friendly technique for
green synthesis of silver nanoparticles from 10ml of aqueous
extract of M. arvensis added with 90ml of 0.2 AgNo3 (1mM) solution,
the resulting mixture was incubated at 37º C under static
condition. The development of dark brown color indicated the
formation of Ag-Np’s. The Ag-Np’s monitored with the help of
UV-visible spectrophotometer at the wavelength of 200-800 nm. The
observed absorbance peak at 400 nm indicated the formation of
Ag-Np’s. The particle size of was determined by using particle
analyzer and the results showed that average size range was found
to be 0. 516 nm. Nanoparticles were characterized using UV-Vis
absorption spectroscopy, Fourier Transform Infra-Red (FT-IR)
Spectroscopy, X- ray diffractometer (XRD) and antibacterial
activity. The antibacterial activity of M. arvensis Ag-Np’s was
evaluated against both Gram positive and Gram negative pathogenic
microorganism by disc diffusion method.
Keywords – Nano silver, Mentha arvensis, UV-Vis Absorption, XRD,
FT-IR, Anti-bacterial activity.
Introduction The plant – mediated synthesis is a rapid, flexible
and suitable process for large- scale prodiction of nanoparticles.
Nowadays, plant parts like seed [1], leaf [2,3], bark [4], steam [
3,5] and fruit[6] extracts have been effectively used for synthesis
of nanoparticles. Among nanoparticles, silver nanoparticles have
been used enormously due to their potent anti-bacterial
activity[7]. Silver nanoparticles have found tremendous
applications in the field of high sensitivity bio-molecular
detection, diagnostics, catalysis and micro-electronics. Green
silver nanoparticles (AgNPs) have been synthesized using various
natural products like Azadirachta indica [8], Glycine max [9],
Cinnamon zeylanicum [10], Camellia sinensi [11], Peel extract of
Pomegranate [12] and Callicarpa maingayi stem bark extract [13].
However, there is still a need for economic, commercially viable as
well environmentally clean route to synthesize AgNPs. The
morphology and crystalline phase of the NPs were determined from
UV-Vis spectroscopy, Fourier Transform Infra-Red (FT-IR)
Spectroscopy, X- ray diffractometer (XRD) and antibacterial
activity.
The development of new resistant strains of bacteria to current
antibiotics has become a serious problem in public health,
therefore, there is a strong incentive to develop new bactericides.
Silver has long been known to exhibit a strong toxicity to a wide
range of microorganisms for these reason silver-based compounds
have been used extensively in many bactericidal applications.
Silver compounds have also been used in the medical field to treat
burn and a variety of infections. Jiang et al., [14] reported that
the Ag has long been recognized as having an inhibitory effect
toward many bacterial strains and microorganisms commonly present
in medical and industrial processes. AgNPs are reported to possess
anti-fungal [15], anti-inflammatory [16], and anti-viral
activity[17].
Hence, the aim of the present study was develop a novel approach
for green synthesis of Ag-NPs using leaf extract of Mentha arvensis
and exploring its antibacterial activity against Escherichia coli,
Salmonella typhi, Shigella flexneri, Micrococcous luteus,
Pseudomonas fluoresens, and Vibrio cholerae and characterization of
silver nanoparticles by UV-visible spectrometer, FT-IR analysis,
and X-ray diffraction studies.
Collection of Leaf and Preparations of Leaf Extraction:
The fresh leaves (15gms) of Mentha arvensis sample was collected
from Periyakulam. The collected leaves were washed finely cut and
then 100ml boiling double distilled water for 15 minutes and was
filtered through whatman filter paper no.1
Preparation of Silver Nitrate Solution:
0.02mmol aqueous solution of silver nitrate was prepared by
adding 0.0337g of silver nitrate in the 100ml double distilled
water.
Synthesis of Silver Nanoparticles in Mentha arvensis Leaf
Extract:
A typical experiment, silver nanoparticals was synthesized by
talking 10 ml of leaf extract and then it added into 90 ml 0.2
AgNO3 solution and kept in dark for 2 hours. The reduction of
silver ions silver nanoparticles during exposure to the leaf
extract was followed by colour changes from light brown to dark
brown. The synthesized nanoparticles were screened for its
antimicrobial by disc diffusion method.
Characterization of Silver Nanoparticles:
UV-Visible Spectral Analysis:
Synthesis of silver nanoparticles by reducing, the respective
metal ion solution with leaves extract may be easily observed by
UV- Vis spectroscopy. The absorption spectra of leaves extract
quantities and metal concentration was measured using a
spectrophotometer in 300-1000 nm range. The formation and
completion of silver nanoparticles was characterized by UV-visible
spectroscopy using a double beam spectrophotometer
Fourier Transform Infra-Red Spectroscopy:
The chemical composition of the synthesized silver nanoparticles
was study by using FT-IR spectrometer (Perkin-Elmer LS
-55-Lumiescence spectrometer). The solution were dried in hot air
oven for 5 day in 750c. FTIR spectrum of the sample mixed with KBR
powder, in a morter and pressed in to a pellet for measurement.
X-Ray Diffraction Analysis:
X- ray diffraction (XRD) analysis of drop- coated films of
silver nanoparticles in sample was prepared for the determination
of the formation of Ag nanoparticles by an X’ Pert pro X- ray
diffract meter (X’pert High score plus program) operated at a
voltage of kV and a current of 30 mA with Cu Kα radiation.
Antibacterial Activity
The antibacterial assays against, E.coli, S.typhi, M. luteus,
P.flurescens, S.flexneri and V.cholerae. Were also performed by
standard disc diffusion method. Nutrient ager(1g beef extract , 1g
peptone ,0.5g Nacl dissolved in 100ml of double distilled water)
was used to cultivated bacteria. The media was autoclaved and
cooled. The media was poured in the petridiscs and kept for
30minutes for solidification. After 30 minutes the fresh overnight
cultures of inoculums (100µl) of different culture were spread on
the solidification nutrient ager plates. Sterile paper disc made of
whatman filter paper, in 5mm diameter (dipped in silver
nanoparticles) than the disc were placed in each plates. The
cultured ager plates were incubated at 370c for 24 hours. After 24
hours of incubation the zone of inhibition was investigated.
RESULTColor change:
Biosynthesis of silver nanoparticlesWhen the leaf extract
incubated with silver nitrate, it was turned light brown to dark
brown color (Fig. 1) because of reduction reaction appeared in the
biological synthesis process. The conformation of these
nanoparticles are as followed by different characterizations.
(B) (A)
Figure:1 Colour change after synthesis of nanoparticles
A-leaf extract
B-Synthesis nanoparticles
UV-visible spectroscopy:
The bioreduction of silver aqueous solution was monitored by
periodic sampling of aliquoits of the mixture and subsequently
measuring UV-Vis spectral analysis was done by using Shimadzu
UV-1800 double beam spectrophotometer. The absorption peaks are
measured in the range 300-1000nm.
UV-visible spectroscopy analysis showed that the wavelength of
silver nanoparticles synthesized using M. arvensis leaf extract
centered at 320 nm due to the excitation of surface Plasmon
vibrations in the silver nanoparticles.The figures (2) represent
the UV-visible spectrum of silver nanoparticles.
Figure:2 UV-Vis Absorption Spectra of silver nanoparticles
synthesized from M.arvensis
Wavelength nm.Abs.
320.000.34
FTIR Analysis :
The FTIR analysis indicates various functional groups present at
different positions. FTIR spectroscopy study has confirmed that the
carbonyl groups of amine acid residues and peptides of protein has
strong ability to bind metal, so that the proteins could most
possibly form coat covering the metal nanoparticles (i.e capping
agent AgNPs) to prevent the agglomeration of the particles, and
thus the nanoparticles are stabilized in the medium. In this result
peaks in the region 3423 to 2372 were assigned to O-H stretching of
Carboxylic Acid and another peaks in the region 1732 was assigned
Aromatic C=C Bending. Then peak in the region 1660 correspond to
Alkenyl C=C Stretch. The peaks between 829 to 650 correspond to
aromatic C-H bending (Table 1), (Fig. 3) FTIR analysis reveals the
dual function of biological molecule possibly responsible for the
reduction and stabilization of silver nanoparticles in aqueous
solution.
Figure.3 FTIR Spectra of nanoparticles synthesized from M.
arvensis
Peaks Obtained
(/cm)
Bond
Functional Group
3423,2924, 2372
O-H stretching
Carboxylic Acid
1732
C=C stretching
Aromatic Bending
1660
C=C stretching
Alkenyl
829,738,650
C-H stretching
Aromatic Bending
Table.1: FTIR Analysis Data showing various functional
groups
XRD Analysis
The silver nanoparticles solution obtained was purified by
repeated centrifugation at 5000rpm for 20 minutes followed by
redispersion of the pettet of silver nanoparticles, the structure
and deionized water. After freeze drying of the purified silver
nanoparticles, the structure and composition were analyzed by XRD.
The crystallite domain size was calculated from the width of the
XRD peaks, assuming that they are free from non-uniform strains,
using the Scherer formula (fig 4) show the XRD spectrum of silver
nanoparticles.
D=
Where;
D-is the average crystallite domain size perpendendicular to the
reflecting planes,
λ – is the X-ray wavelength,
β –is the full width at half maxium (FWHM),
θ – is the direction angle.
The XRD value confirm that the synthesized particles were
nanometric in size of the silver nanoparticles. Thus estimated was
found to be 6.117 nm.
Figure.4 XRD pattern of silver nanoparticles synthesized from M.
arvensis
Antibacterial activity:
In the present study, the antibacterial activity of green
synthesized silver nanoparticles was tested E.coli, S.typhi, M.
luteus, P.flurescens, S.flexneri and V.cholerae and the results are
shown in figures(5,6.1-6.6). The range of inhibition zone of
M.arvensis leaf extract varied from (1-7mm)
Maximum inhibition zone was obtained against M.luteus (7mm)
followed by S,typhi (5mm) and E.coli and P.fluroecens (3mm) by the
silver nanoparticles. Among the green synthesized silver
nanoparticles of M.arvensis against four human pathogens maximum
inhibition zone was obtained againest M.luteus (7mm) followed by
S.typhi (5mm) E.coli, P.flurescens, S.flexneri (3mm) respectively
(Table 2).
Table: 2 Antibacterial activity of against different pathogenic
strain
Human pathogens
Zone of inhibition (mm)
Experimental
Control
Escherichia coli
3
2
Salmonella typhi
5
3
Micrococcus luteus
7
6
Pseudomonas flurescens
3
2
Shigella flexneri
2
2
Vibrio cholerae
1
0
Figure: 5 Antibacterial activity of silver nanoparticles against
human pathogen
Figures .6 Antibacterial activity of silver nanoparticles
against human pathogens
Fig: 6.1
Control- Silver Nitrate
Sample- Silver nanoparticles
Pathogen -Escherichia coli (Gram negative bacteria)
Fig: 6.2
Control- Silver Nitrate
Sample - Silver nanoparticles
Pathogen - Salmonella typhi (Gram Negative Bacteria)
Fig: 6.3
Control -Silver Nitrate
Sample - Silver nanoparticles
Pathogen -Micrococcus luteus (Gram Positive Bacteria)
Fig: 6.4
Control -Silver Nitrate
Sample - Silver nanoparticles
Pathogen - Pseudomonas flurescens (Gram Negative Bacteria)
Fig: 6.5
Control- Silver Nitrate
Sample - Silver nanoparticles
Pathogen – Salmonella typhi (Gram Negative Bacteria)
Fig: 5.6
Control -Silver Nitrate
Sample - Silver nanoparticles
Pathogen – Vibrio cholera (Gram Negative Bacteria)
Result and DiscussionIn this project an attempt has been made to
develop a fast, ecofriendly and convenient method for the green
synthesis of AgNPs using M.arvensis. The observed colour change
from light brown to dark brown colour solution indicated the
formation of Mentha arvensis AgNPs. It is showed in the figure (1).
The aqueous silver ions when exposed to herbal extracts were
reduced in solution.
UV-visible spectra of silver nanoparticles were taken in water
medium. Reduction of Ag+ ions during exposure to the extract of
M.arvensis was earily followed UV-Vis spectroscopy absorbance peak
at 320 nm showed in the reaction mixture indicated Silver
nanoparticles were formed (Fig-2). As the plant extract was mixed
in the aqueous solution of the silver ions complex, it started to
change the colour from light brown to dark brown (Jain et al.,
2009). UV-Vis spectral analysis was done by using synthesized
nanoparticles showed the colour changes. UV-Vis spectrophotometer
at the range of 300-700nm and observed the absorption peaks at 320
nm regions which are identical to the characteristics UV-Visible
spectrum of metallic silver it was recorded. The reduction of
silver ions and the formation of stable nanoparticles occurred
rapidly within an hour of reaction, making it on the fastest
bioreducting methods to produce. Ag nanostructure reported till
data (Shiv Shankar et al., 2009).
FTIR spectrums indicate various functional groups present at
different positions. IR spectroscopy study has confirmed that the
carbonyl group of amino acid residues and peptides of protein has a
stronger ability to bind metal, so that the protein could most
possibly form coat covering the metal nanoparticles (i.e capping of
AgNPs) to prevent the agglomeration of the particles and thus the
nanoparticles are stabilized in medium.
The previous studies of silver nanoparticles by FTIR analysis
confirm that have been found to be responsible for the reduction of
metal ions when using the plant extract synthesis of silver
nanoparticles similar to the use of metal nanoparticles. In the
present study appearance of peaks in the region 3423 to 2372 were
assigned to O-H stretching of Carboxylic Acid and another peaks in
the region 1732 was assigned Aromatic C=C Bending. Then peak in the
region 1660 correspond to Alkenyl C=C Stretch. The peaks between
829 to650 correspond to aromatic C-H stretching, respectively; FTIR
analysis reveals the dual function of biological molecules possibly
responsible for the reduction and stabilization of silver
nanoparticles in the aqueous medium.
The XRD pattern showed 40.49562 ×m instant peak in the whole
spectrum 2θ value ranging from 10 to 100.X ray diffraction (XRD)
patterns for silver nanoparticles were shows in (Figure 4). The
diffraction that peak indicates that the dimension of the resultant
nanoparticles is (Nano size in word). The XRD values confirm that
the synthesized particles were nanometric in size. The size of the
silver nanoparticles thus estimated was found to be 6.7114nm.
Antibacterial potential of silver is known for many years (Raut
Rajesh et al., 2009). The anti-bacterial activity of the leaf
extract of M. arvensis was studied against Gram-positive & Gram
negative bacteria. The leaf extract of M. arvensis exhibited a
significant anti-bacterial activity. The antibacterial activity of
Micrococcous luteus was higher than the other bacteria. The
inhibition zone diameter of M.arvensis was 7mm.
The antibacterial activity of Vibrio cholerae was the lowest and
the inhibition zone rang 1mm.The inhibition zone of Escherichia
coli, Salmonella typhi, Pseudomonas flurescens, Shigella flexneri
was ranging from 1-5mm. This symbolizes that the antibacterial
potential of M. arvensis silver nanoparticles is higher than that
of silver ions at their respective concentration used in the study.
Bio reduced silver nanoparticles showed considerable growth.
Inhibition of two of the well-known pathogenic bacteria species
Coupling of in herent property of M.arvensis extract with that of
silver nanoparticles has really proved to be beneficial to minimize
the dose that needs to be administered for total microbial
reduction.
SummarySilver ion and silver compounds have been known to strong
antibacterial activity using nanoparticles lead to an increase in
number of particles per unit area thus a antibacterial effects can
be maximized. In this present work silver nanoparticles was
prepared using the leaf extract of Mentha arvensis and it was
carried out UV-visible spectrophotometer, FTIR analysis and X-ray
diffraction activity. The green synthesis of silver nanoparticles
confirmed by the colour change occurs in the silver nitrate. In
M.arvensis of silver nanoparticles was indicated by light brown in
colour. The reduction of silver nitrate to silver nanoparticles was
indicated by the colour change from light brown to dark brown
colour.
Characterization of M.arvensis silver nanoparticles was done by
the UV-visible spectrophotometer and FTIR. The surface Plasmon band
occurs in the visible region of the light spectrum with absorbance
peak was at 320nm in M.arvensis
The following chemical groups were identified from FT-IR
analysis such as O-H band, carboxylic acid, C=C stretch is aromatic
bending and also the same stretching C=C, Alkenyl functional group
is present. Finally the C-H stretching present in the function
group is aromatic bending. Thus these chemical groups may play an
important role in the reduction of silver nitrate into silver
nanoparticles formed from test plant which were responsible for the
inhibition of tested human pathogens.
X- ray diffraction technique is a powerful method for the
investigation of the fine structure of the compound. XRD has been
widely used for the determination of crystallinity, crystal
structure are lattice parameters of nanoparticles. The XRD values
confirm that the synthesized particles were nanometric in size. The
size of the silver nanoparticles thus estimated was found to be
6.117nm.
It has been concluded that the tested plant M. arvensis was
capable of producing silver nanoparticles quite stable in solution
due to capping likely by the proteins present in the extract and
able to inhibit the tested bacterial pathogens E. coli, S. typhi,
M. luteus, P. flurescens, S. flexneri and V. cholerae. Therefore
nanoparticles of silver in combination with commercially available
antibiotics could be used as an antibacterial agent after future
trials on experimental animal.
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ExprimentalEscherichia coliSalmonella typhiMicrococcus
luteusPseudomonas flurescens Shigella flexneri Vibrio
cholerae357321ControlEscherichia coliSalmonella typhiMicrococcus
luteusPseudomonas flurescens Shigella flexneri Vibrio
cholerae236220
Bacterial pathogens
Inhibition zone in mm
2-theta (deg)
Intensity (counts)
20 40 60 80
0
200
400
600
500750125017502250275032503750
1/cm
10
12.5
15
17.5
20
%T
3957.93
3909.71
3863.42
3753.48
3414.00
2924.09
2372.44
1732.08
1666.50
1598.99
1498.69
1436.97
1381.03
1300.02
1251.80
1168.86
1107.14
1037.70
894.97
829.39
738.74
650.01
570.93
538.14
478.35
