Evaluation of Allelopathic Potential of Adhatoda Vasica by ...
Green Synthesis of Silver Nanoparticles Using Adhatoda Vasica Methanolic Extract and Its Biological...
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Jamonline / 2(4); 2012 / 282-291 Shalini Bandi & K Vasundhara
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Research Article
Journal of Atoms and Molecules
An International Online Journal ISSN – 2277 – 1247
GREEN SYNTHESIS OF SILVER NANOPARTICLES USING Adhatoda vasica
METHANOLIC EXTRACT AND ITS BIOLOGICAL ACTIVITIES
Shalini Bandi(1)*
, Dr. K Vasundhara(2)
(1) MSc Biotechnology, Hindu college postgraduate courses, Guntur, A P, India
(2) Head of the department, Department of Biotechnology, Hindu college postgraduate courses,
Guntur, A P, India
Received on: 15-07-2012 Revised on: 03-08-2012 Accepted on: 25-08-2012
Abstract:
There is an increasing commercial demand for nanoparticles due to their wide applicability in
various areas such as electronics, catalysis, chemistry, energy, and medicine. Metallic nanoparticles
are traditionally synthesized by wet chemical techniques, where the chemicals used are quite often
toxic and flammable. In this work, we describe a cost effective and environment friendly technique
for green synthesis of silver nanoparticles from 1mM AgNO3 solution through the Methanolic
extract of Adhatoda vasica as reducing as well as capping agent. Nanoparticles were characterized
using UV–Vis absorption spectrophotometry, FTIR and SEM. SEM analysis showed the average
particle size of 15-20nm as well as spherical to oval in shape. The synthesized nanoparticles show
high DPPD free radical scavenging activity and reducing power activity. Further these biologically
synthesized nanoparticles were found to be an anti diabetic agent and highly toxic against different
human pathogens.
Keywords: Adhatoda vasica, Green synthesis, nanoparticles, SEM, Biological activities
Introduction:
Nanotechnology is an enabling
technology that deals with structures ranging
from approximately 1_100nm in at least one
dimension (British Standards Institute [BSI]
2007) (1)
. The nano size results in specific
physicochemical characteristics that may
* Corresponding author
Shalini Bandhi,
Email: [email protected]
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differ from those of the bulk substance or
particles of larger size. This effect is mainly
attributed to high surface area to volume ratio,
which potentially results in high reactivity.
Because of these specific characteristics the
use of substances in nano form may have
advantages over the use of bulk chemicals.
Among the noble metals (e.g., Ag, Pt,
Au and Pd), silver (Ag) is the metal of choice
for potential applications in the field of
biological systems, living organisms and
medicine (2)
. Due to their exclusive properties,
silver nanoparticles (Ag-NPs) may have
several applications, such as catalysts in
chemical reactions (3)
, electrical batteries and
in spectrally selective coatings for absorption
of solar energy (4,5)
, as optical elements (6)
,
pharmaceutical components and in chemical
sensing and biosensing (7,8)
.
Various strategies are employed for
synthesis of silver nanoparticles (9)
.
Nanoparticles are synthesized by reduction in
solutions (10)
, thermal decomposition of silver
compounds (11)
, microwave assisted synthesis
(12), Laser mediated synthesis
(13) and
biological reduction method. All these
methods of synthesis of nano particles
involves the usage of hazardous chemicals,
cost effective and high laboratory resources
are required and are polluting the atmosphere.
Biosynthesis of nanoparticles using plant
extracts is the favorite method of green, eco-
friendly. Production of nanoparticles and
exploited to a vast extent because the plants
are widely distributed, easily available, safe to
handle and with a range of metabolites. The
plant material used for biosynthesis of
nanoparticles includes the plants such as Ulva
fasciata(14)
, leaf extract of Diopyros kaki(15)
,
Carica papaya (16)
, Trianthema decandra (17)
ect and several activities has been studied for
the synthesized nanoparticles.
In this study, the synthesis and
characterization of silver nanoparticles(Ag-
NPs ) / Adhatoda vasica (Ag/ Adhatoda
vasica) by a green method reported. Ag-NPs
were prepared using silver nitrate as silver
precursor and methanol extract of Adhatoda
vasica leaf as reducing agent and stabilizer.
Materials and Methods:
All chemical and reagents used were
analytical grade and are purchased from Merk
chemicals pvt ltd, Mumbai.
Extract Preparation:
Areal parts of the plant Adhatoda
vasica were washed and dried in an oven
dryer at 40 °C for 48 h. The dried plant parts
were then ground into powder, stored in dark
glass bottles and kept at low temperatures
until further analyses. The finely ground
Adhatoda vasica powder (20 g) were
extracted with methanol using Saxlet
apparatus. After filtration with Whatman filter
paper No 1 using vacuum pump, the residue
was re-extracted. The solvent was completely
removed using a rotary vacuum evaporator at
40 °C. The concentrated extract was then kept
in dark bottles at 4 °C until used.
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Synthesis of Ag/ Adhatoda vasica Emulsion:
Different concentrations of extracts
(1ml, 3ml and 5ml) were taken separately and
to this 10ml of different concentrations of
Siver nitrate solution (1mM , 2mM, 4mM,
6mM, 8mM and 10mM was added with
constant stirring and exposed to different
conditions like sunlight radiation, UV
radiation, Room temperature and Direct
boiling (prevent overheating). The colour
change of the solution was checked
periodically. The color change of the leaf
extract from yellow to dark brown indicated
the silver nanoparticles were synthesized from
the plant extract. Bioreduction of silver ions
in the solution was monitored using UV-VIS
spectrophotometer. Then the volumetric
flasks were incubated at room temperature for
48 hours. The contents were centrifuged at
10,000rpm for 15 minutes. The supernatant
was used for the characterization of the silver
nanoparticles.
Production and Recovery of silver
nanoparticles by centrifugation
Among various concentrations and
methods used, room temperature at 48h
incubation method was very effective and 5ml
of homogenized extract and 1mM silver
nitrate concentration had shown more
synthesis of nanoparticles. Further it was
chosen for bulk production as 25ml leaf
extract in 25ml of 1mM Siver nitrate.After
bioreduction, the solution consisting of silver
nanoparticles.
Characterization of silver nanoparticles
The presence of basic silver particles
was identified by using Atomic Absorption
Spectrophotometer (AAS-model 210). AAS
confirm the presence of silver.
UV- Vis spectra analysis
The reduction of metallic Ag+ ions
was monitored by measuring the UV- Vis
spectrum after about 24 hours of reaction. A
small aliquot was drawn from the reaction
mixture and a spectrum was taken on a
wavelength from 250nm to 800nm on UV-Vis
spectrophotometer (Double beam
spectrophotometer uv-2301).
Fourier Transform Infrared spectroscopy
(FTIR):
FTIR measurements are carried out to
identify the possible biomolecules responsible
for the reduction of the Ag+ ions and capping
of the bio-reduced SNP’s synthesized by
A.vasica.
SEM analysis:
The size and shape of the
biosynthesized nanoparticles were observed
by Scanning Electron Microscope (SEM)
(JSM-6390 Scanning electron microscope).
Samples were prepared by drop coating the
Ag nanoparticles solutions on to carbon
copper grid. The films on the grids were
allowed to dry prior to measurement.
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Biological activities of the Ag-NPs of
Adhatoda vasica:
Total Antioxidant Activity
DPPH Radical Scavenging Assay:
The effect of nano particle on DPPH
radical was estimated using the method of
Liyana-Pathirana and Shahidi (18)
. A solution
of 0.135 mM DPPH (2,2-diphenyl-1-
picrylhydrazyl) in methanol was prepared and
1.0 ml of this solution was mixed with 1.0 ml
of synthesized Ag-NPs of Adhatoda vasica.
The reaction mixture was vortexed thoroughly
and left in the dark at room temperature for 30
min. The absorbance of the mixture was
measured spectrophotometrically at 517 nm.
Ascorbic acid and BHT were used as
references.
The ability to scavenge DPPH radical was
calculated by the following equation: DPPH
radical scavenging activity (%) = [(Abs
control – Abs sample)]/(Abs control)] x 100
where Abs control is the absorbance of DPPH
radical + methanol; Abs sample is the
absorbance of DPPH radical + synthesized
Ag-NPs solution/standard.
Measurement of Reducing Power:
The reducing power of synthesized
silver nanoparticles of Adhatoda vasica was
determined using the method described
previously (18)
. A serial dilution of the
nanoparticle solution was (performed 200,
100, 50, 25 and 12.5μL/mL) dissolved in 0.2
M phosphate buffer pH, 6.6 containing 1%
ferrocyanate. The mixture was incubated at 50
ºC for 20 minutes. 10% trichloroacetic acid
(TCA, 2.5 mL) was added to a portion of this
mixture (5 mL) and centrifuged at 3,000 g for
10 minutes. The supernatant was separated
and mixed with distilled water (2.5 mL)
containing 1% ferric chloride (0.5 mL). The
absorbance of this mixture was measured at
700 nm. The intensity in absorbance could be
the measurement of antioxidant activity of the
extract (18)
.
Anti diabetic activity:
The animals (Rabbits) were fasted for
16 hour prior to the induction of diabetes.
STZ freshly prepared in citrate buffer (pH
4.5) was administered i.p. at a single dose of
50 mg/kg. Development of diabetes was
confirmed by measuring blood glucose
concentrations 72 hour after injection of STZ.
Rabbits with blood glucose level of 250 mg/dl
or higher were considered to be diabetic and
selected for experiment. Diabetic animals
were randomly assigned to groups. Group I
contained normal animals and served as
normal control. Group II and III served as
diabetic. Groups II receive the synthesized
nano particle of the methanolic extract of the
Adhatoda vasica during the experiments,
while the Group III received the reference
standard drug glimeperide (0.1 mg/kg).
Estimation of Blood Glucose
Initial, 8th
14th
and 21st
day non fasting
blood glucose levels were determined just
before administering the drugs. On the last
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day of experiment, blood samples were
collected from each animal. The blood
glucose level was estimated with One Touch
Basic Glucometer (Accu Chek Active, Roche,
Germany).
Anti-microbial activities:
Antibacterial activity of biologically
synthesized Ag-NPs of A.vasica was
determined by cup diffusion method and disc
diffusion method on nutrient agar medium
(Anon, 1996). Cups were made in nutrient
agar plate using sterile cork borer (5 mm),
filter paper discs were made using whattmann
filter paper and inoculums of selected micro
organism were spread on the solid plates with
a sterile swab moistened with the bacterial
suspension. Then 50μl each of 20μl/mL
synthesized Ag-NPs solution were placed in
the cups and the solution was dipped in the
filter paper discs kept in inoculated plates.
The treatments also included 50 μl of extract
and 1mM silver nitrate solution separately
which served as control. The plates were
incubated for 24 h. at 37°C and zone of
inhibition if any around the wells were
measured in mm (millimeter). For each
treatment six replicates were maintained. The
data was subjected to statistical analysis;
results can be shown in table 1.
Results and Discussion:
The time of addition of extract into the
metal ion solution was considered as the start
of the reaction. It is well known that silver
nanoparticles exhibit dark brown color in
aqueous solution due to excitation of surface
plasmon vibrations in silver nanoparticles. As
the Adhatoda vasica extract was mixed in the
aqueous solution of the silver ion complex,
initially the color changed from pale
yellowish to dark brown due to the reduction
of silver ion. The formation of silver
nanoparticles was observed at a concentration
of 1mM silver nitrate effectively.
Figure 1: Synthesis of silver nano particles
of A. vasica
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UV-vis spectroscopy could be used to
examine uv absorption of the nanoparticles.
Figure 1 show the UV-vis spectra which are
recorded after the completion of the reaction.
For 1mM solution, the silver nanoparticles
have absorbance peak at 395nm. The
frequency and width of the surface plasmon
absorption depend on the size and shape of
the metal nanoparticles as well as on the
dielectric constant of the metal itself and the
surrounding medium.
Figure 1: UV-Visible absorption spectrum of
the silver nanoarticles. (1) spectra of
immediate addition of the 1mM silver nitrate
solution and the plant extract. (2) After 48h
incubation at room temperature
FT-IR spectrum shows that formation
of new bonds in the reacting solution. The
spectra is due to the active compounds in the
plant extract solution may react with the silver
nitrate solution and hence show specific bands
corresponding to the reacting molecules.
Figure 2: FT-IR spectrum of synthesized
Ag-NPs solution after blank correction
Scanning electron microscope results
shows the size and shapes of the formed
nanoparticles. The silver nanoparticles are in
oval to spherical in shape and are slightly
aggregated in solution. This is due to the
binding force between the AgNPs and the
capping molecules that may get decreased
with increasing temperature even though the
size of the nanoparticles is reduced. The size
of the formed nano particles is found to be 15-
20nm.
.
Figure 3: SEM image of the nanoparticles,
Reduction of silver ions present in the
aqueous solution of silver complex during the
reaction with the ingredients present in the
Adhatoda vasica plant extract as observed by
the atomic absorption spectroscopy revealed
the presence of basic silver and UV-Vis
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spectroscopy revealed the presence of
nanoparticles . SEM analyses showed the
particle size between 15-20nm as well oval to
spherical in shapes of the nanoparticles. FTIR
analysis confirmed that the bioreduction of
silver ions to silver nanoparticles was due to
the reduction by capping material of plant
extract. The present study thus showed a
simple green route for rapid and economical
synthesis of silver nanoparticles.
DPPH Radical Scavenging Assay:
The synthesized silver nanoparticle of
the extract show high DPPH radical
scavenging activity. The compound at a
concentration 0.2μL/mL show the high
activity when compared to the other
remaining concentrations tested. Results
obtained were compared with Ascorbic Acid
as standard solution (μg/mL). Figure 4 Show
the radical scavenging activity.
Figure 4: DPPH radical scavenging activity
of Ag/NPS of Adhatoda vasica.
Reducing Power:
The synthesized silver nanoparticle of
the extract show high reducing power. The
synthesized Ag-NPS solution at with different
concentrations (μL/mL) show good reducing
activity. Among the different concentrations
of the solution 6μL/mL sow the result similar
with the standard solution (Qurcetine in
μg/mL). Figure 5 Show the reducing activity.
Figure 5: Reducing power activity results
of Adhatoda vasica Ag-NPs.
Anti diabetic activity:
The data reveals that the
synthesized silver nanoparticles of
Adhatoda vasica decreses the blood glucose
levels statistically significant. The activity
of the solution was compared with the
standard drug Glimipride. The standard
drug show better results than the sample
solution. Whereas control doesn’t diabetic
positive response. Results shown in figure
6
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Figure 6: Diabetic activity of Ag-NPs of
Adhatoda vasica
Antimicrobial Assay:
The synthesized silver nanoparticles
inhibit the growth of microorganisms. Growth
inhibition zones values were obtained in
millimeter for the synthesized Ag/ Adhatoda
vasica nanoparticles against 4 different micro
organisms. The solution shows inhibition
activity in all the organisms under study.
Among those the solution shows more
activity on Bacillus subtilis and lest effet on
Klebsiella pneumonia. Antibacterial activity
of silver NPs of Adhatoda vasica was
presented in Table 1.
S.NO Micro Organism Zone of
Inhibition
in mm
1 Pseudomonas
aeruginosa
16.7
2 Bacillus subtilis 18.2
3 Klebsiella
pneumonia
14.7
4 Staphylococcus
aureus
15.2
Table 1: Antimicrobial activity results of
Adhatoda vasica Ag-NPs.
Figure 7: Antimicrobial activity zone inhibition.
0
100
200
300
0 50 100
Blo
od
glu
cose
leve
l in
mg/
dl
Time in Hours
Anti diabetic activity
Control
Glimipride
Ag NPS
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Conclusion:
In conclusion, we have demonstrated in this
study that the eco-friendly use of a plant
extract to synthesize silver nanoparticles.
Synthesis of Ag/NPs using green resources
like Adhatoda vasica is a better alternative to
chemical synthesis, since this green synthesis
is pollutant free and eco-friendly. The results
suggested that Adhatoda vasica plays an
important role in the reduction and
stabilization of silver to silver nanoparticles.
The synthesized Ag/NPs show high DPPH
radical scavenging activity, high reducing
power activity. The particles show high
antidiabetic activity and resistance against the
pathogenic microorganisms. Further studied
needs to carry out that the synthesized
Ag/NPs were better to use as a medicine to
cure diseases instead of using modern
medicine.
References:
1. British Standards Institution (BSI).
2007. PAS136 Terminology for
nanomaterials. Accessed from the
website: http://www.bsiglobal. com.
2. Jain, D.; Daima, H.K.; Kachhwaha, S.;
Kothari, S.L. Synthesis of plant-
mediated silver nanoparticles using
papaya fruit extract and evaluation of
their anti microbial activities. Dig.
J.Nanomater. Biostruct. 2009, 4, 723–
727.
3. Jiang, Z.J.; Liu, C.Y.; Sun, L.W.
Catalytic properties of silver
nanoparticles supported on silica
spheres. J. Phys. Chem. B. 2005, 109,
1730–1735.
4. Joerger, T.K.; Joerger, R.; Olsson, E.;
Granqvist, C.G. Bacteria as workers in
the living factory: Metal-accumulating
bacteria and their potential for
materials science. Trends Biotechnol.
2001,19, 15–20.
5. Moulin, E.; Sukmanowski, J.; Schulte,
M.; Royer, F.X.; Stiebig, H. Thin-film
silicon solar cells with integrated
silver nanoparticles. Thin Solid Films
2008, 516, 6813–6817
6. Nam, J.M.; Park, S.J.; Mirkin, C.A.
Bio-barcodes based on oligonucleotide
modified nanoparticles. J. Am. Chem.
Soc. 2002, 124, 3820–3821
7. Aymonier, C.; Schlotterbeck, U.;
Antonietti, L.; Zacharias, P.;
Thomann, R.; Tiller, J.C.; Mecking, S.
Hybrids of silver nanoparticles with
amphiphilic hyperbranched
macromolecules exhibiting
antimicrobial properties. Chem.
Commun. 2002, 24, 3018–3019.
8. Songping, W.; Shuyuan, M.
Preparation of ultrafine silver powder
using ascorbic acid as reducing agent
and its application in MLCI. Mater.
Chem. Phys. 2005, 89, 423–427.
Jamonline / 2(4); 2012 / 282-291 Shalini Bandi & K Vasundhara
All rights reserved© 2011 www.jamonline.in 291
9. Thabet M. Tolaymat, Amro M. El
Badawy, Ash Genaidy, Kirk G.
Scheckel, Todd P. Luxton, Makram
Suidan, Science of the Total
Environment, 2010, 408,999–1006
10. Maribel G. Guzmán, Jean Dille,
Stephan Godet, World Academy of
Science, Engineering and Technology,
2008, 43
11. S. Navaladian Æ B. Viswanathan Æ
R. P. Viswanath Æ, T. K. Varadarajan,
Nanoscale Res Lett, 2007, 2:44–48
12. K J Sreeram, M Nidhin and B U Nair,
Bull. Mater. Sci., 2008, Vol. 31, No. 7,
, 937– 94
13. Reza Zamiri, Azmi Zakaria, Hossein
Abbastabar, Majid Darroudi, Mohd
Shahril Husin, Mohd Adzir Mahdi,
International Journal of
Nanomedicine 2011:6 565–568.
14. S. Rajesh, D. Patric Raja, J.M. Rathi3
and K. Sahayaraj, Biosynthesis of
silver nanoparticles using Ulva
fasciata (Delile) ethyl acetate extract
and its activity against Xanthomonas
campestris pv. Malvacearum,
JBiopest, 5 (Supplementary) (2012):
119-128
15. Jae Yong Song Æ Eun-Yeong Kwon,
Biological synthesis of platinum
nanoparticles using Diopyros kaki leaf
extract, Bioprocess Biosyst Eng
(2010) 33:159–164.
16. D. Jain, h. Kumar daima, s.
Kachhwaha, s. L. Kothari, synthesis
of plant-mediated silver nanoparticles
using papaya fruit extract and
evaluation of their anti microbial
activities, Digest Journal of
Nanomaterials and
Biostructures(2009), Vol. 4, No. 3,
September , p. 557 – 563.
17. R.Geethalakshmi and D.V.L. Sarada,
Synthesis of plant-mediated silver
nanoparticles using Trianthema
decandra extract and evaluation of
their anti microbial activities,
International Journal of Engineering
Science and Technology (2010), Vol.
2(5), 970-975.
18. Yen, G.C.; Duh, P.D. Scavenging
Effect of Methanolic Extracts of
Peanut Hulls on Free Radical and
Active Oxygen Species. J. Agric.
Food Chem. 1994, 42, 629-632.