Jamonline / 2(4); 2012 / 282-291 Research Article

Shalini Bandi & K Vasundhara

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) (2)

MSc Biotechnology, Hindu college postgraduate courses, Guntur, A P, India

Head of the department, Department of Biotechnology, Hindu college postgraduate courses, Guntur, A P, India

Received on: 15-07-2012 Abstract:

Revised on: 03-08-2012

Accepted on: 25-08-2012

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 UVVis 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: * Corresponding author Shalini Bandhi, Email: Shalini.bandhi@gmail.com 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 may

physicochemical characteristics that All rights reserved 2011

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Jamonline / 2(4); 2012 / 282-291 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)

Shalini Bandi & K Vasundhara 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) (17)

, Trianthema decandra

ect and several activities has been studied for the synthesized nanoparticles. In this study, the synthesis and characterization of silver nanoparticles(AgNPs ) / 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 extracted vasica with powder methanol (20 g) were Saxlet
(9)

. Due to their exclusive properties,

silver nanoparticles (Ag-NPs) may have several applications, such as catalysts in chemical reactions
(3)

, electrical batteries and

(6)

in spectrally selective coatings for absorption of solar energy

(4,5)

, as optical elements

pharmaceutical components and in chemical sensing and biosensing (7,8). Various strategies are employed for synthesis of
(10)

silver

nanoparticles

Nanoparticles are synthesized by reduction in solutions

(12)

, thermal decomposition of silver , microwave assisted synthesis synthesis method.

(13)

compounds , Laser

(11)

mediated reduction

and these

biological

All

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, ecofriendly. Production of nanoparticles and exploited to a vast extent because the plants

using

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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Jamonline / 2(4); 2012 / 282-291 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

Shalini Bandi & K Vasundhara

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

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

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 SNPs synthesized by A.vasica.

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

SEM analysis: The size and shape of the

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

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.

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.

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Jamonline / 2(4); 2012 / 282-291 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 of 0.135 mM DPPH
(18)

Shalini Bandi & K Vasundhara C 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

. A solution

(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)

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, 8 14 and 21 day non fasting blood glucose levels were determined just before administering the drugs. On the last
th th st

. A serial dilution of the

nanoparticle solution was (performed 200, 100, 50, 25 and 12.5L/mL) dissolved in 0.2 M phosphate buffer pH, 6.6 containing 1% ferrocyanate. The mixture was incubated at 50

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Jamonline / 2(4); 2012 / 282-291 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

Shalini Bandi & K Vasundhara 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.

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 50l each of 20l/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 l of extract and 1mM silver nitrate solution separately which served as control. The plates were incubated for 24 h. at 37C 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.

Figure 1: Synthesis of silver nano particles of A. vasica

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 All rights reserved 2011 www.jamonline.in 286

Jamonline / 2(4); 2012 / 282-291 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

Shalini Bandi & K Vasundhara

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 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 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 size of the nanoparticles is reduced. The size of the formed nano particles is found to be 1520nm. .

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 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 All rights reserved 2011 silver and UV-Vis

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Jamonline / 2(4); 2012 / 282-291 spectroscopy revealed the presence of

Shalini Bandi & K Vasundhara

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.

Reducing Power: The synthesized silver nanoparticle of the extract show high reducing power. The synthesized Ag-NPS solution at with different concentrations (L/mL) show good reducing activity. Among the different concentrations of the solution 6L/mL sow the result similar with the standard solution (Qurcetine in g/mL). Figure 5 Show the reducing activity.

DPPH Radical Scavenging Assay:

The synthesized silver nanoparticle of the extract show high DPPH radical

scavenging activity. The compound at a concentration 0.2L/mL show the high activity remaining when compared to the other Results Figure 5: Reducing power activity results of Adhatoda vasica Ag-NPs.

concentrations

tested.

obtained were compared with Ascorbic Acid as standard solution (g/mL). Figure 4 Show the radical scavenging activity.

Anti diabetic activity: The synthesized data silver reveals that the of

nanoparticles

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 Figure 4: DPPH radical scavenging activity of Ag/NPS of Adhatoda vasica. solution. Whereas control doesnt diabetic positive response. Results shown in figure 6

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Jamonline / 2(4); 2012 / 282-291 Anti diabetic activity

Blood glucose level in mg/dl 300 200 100 0 0 50 100

Shalini Bandi & K Vasundhara activity on Bacillus subtilis and lest effet on Klebsiella pneumonia. Antibacterial activity of silver NPs of Adhatoda vasica was

Control
Glimipride Ag NPS Time in Hours

presented in Table 1.

S.NO

Micro Organism

Zone of Inhibition in mm

Figure 6: Diabetic activity of Ag-NPs of Adhatoda vasica 1 Pseudomonas aeruginosa 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 4 2 3 Bacillus subtilis Klebsiella pneumonia Staphylococcus aureus

16.7

18.2 14.7

15.2

Table 1: Antimicrobial activity results of Adhatoda vasica Ag-NPs.

Figure 7: Antimicrobial activity zone inhibition.

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Jamonline / 2(4); 2012 / 282-291 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

Shalini Bandi & K Vasundhara 3. Jiang, Z.J.; Liu, C.Y.; Sun, L.W. Catalytic properties supported of on silver silica

nanoparticles

spheres. J. Phys. Chem. B. 2005, 109, 17301735. 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, 1520. 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, 68136817 6. Nam, J.M.; Park, S.J.; Mirkin, C.A. Bio-barcodes based on oligonucleotide modified nanoparticles. J. Am. Chem. Soc. 2002, 124, 38203821 7. Aymonier, C.; Schlotterbeck, U.; Antonietti, L.; Zacharias, P.;

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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