Journal of Atoms and Moleculesjamonline.in/wp-content/uploads/2012/09/Green-Synthesis-Of-Silver... · Journal of Atoms and Molecules An International Online Journal ISSN – 2277

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]



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.



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.



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



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



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



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



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



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.

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Journal of Atoms and Moleculesjamonline.in/wp-content/uploads/2012/09/Green-Synthesis-Of-Silver... · Journal of Atoms and Molecules An International Online Journal ISSN – 2277