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Chapter 6 Green synthesis and characterization of silver nanoparticles using Jatropha seedcake extract This part has been communicated as: Anjali Bose, Haresh Keharia and M. P. Deshpande (2012). Eco-friendly phyto-synthesis of silver nanoparticles using Jatropha seedcake extract. Industrial Crops and Products.

JSC for silver nanoparticle synthesis Chapter 6

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6.1. Introduction

Nanoparticles (structures with at least one dimension approximately within 1 to 100

nm) have received considerable attention in both scientific and technological areas due to

their unique and unusual physico-chemical properties compared with that of bulk materials

(Bhushan 2004). Further, metal nanoparticles are particularly interesting because they can

be easily synthesized, modified chemically and applied for device fabrication (Feldheim

and Foss 2002).

Amongst various metal nanoparticles, silver nanoparticles (AgNPs) are perhaps most

widely recognized owing to its broad range of applications such as, in photonics (Gould et

al 2000), micro-electronics (Maekawa et al 2010), photocatalysis (Chen et al 2010),

lithography (Hulteen et al 1999), biosensing (Ngece et al 2011), etc. The properties and

applications of nanoparticles particularly depend on their morphology i.e. crystal structure

and dimensions. Consequently, significant research has been concentrated on developing

techniques to control their size, shape and distribution (Ozin 1992, Feldheim and Foss

2002). Different surface passivator reagents such as thiophenol, thiourea, mercapto-acetate

etc. have been employed in order to prevent the agglomeration of particles during and upon

their synthesis as well as to subsequently control their size (Li et al 2003, Stewart et al

2012). Unfortunately, these passivators are toxic (HSDB, TOXNET, U.S. NLM, NIH),

would raise environmental issues, if they are to be used in processes for large scale

synthesis of nanoparticles.

In addition, AgNPs have also been recognized as novel therapeutic agents extending

its use as antibacterial, antifungal, antiviral, anti-inflammatory and anti-cancerous agents

(Vaidyanathan et al 2009). The broad-spectrum antimicrobial properties of AgNPs

encourage its use in a large number of biomedical and environmental applications as well

as in cosmetics, clothing and different consumer products (Rai et al 2009). The

conventional synthesis of AgNPs involve number of chemical and physical methods that

are energy and capital intensive, employ toxic chemicals, thus limiting its biomedical

applications. Consequently, there has been growing need for synthesis of bio-compatible

metal nano-particles, without employing toxic chemicals in the synthesis protocols. In this

respect, use of microorganisms in biosynthesis of nano-particles has been demonstrated in

last two decades, particularly owing to their facile assembly of the nano-dimensioned


117

particles. However, slow reaction rates, difficulty in maintaining culture and aseptic

conditions during synthesis, directed research towards the use of plant part and extract for

the synthesis of metal nanoparticles. Gardea-Torresdey and co-workers first reported the

formation of AgNPs by living plants (Gardea-Torresdey et al 2003). Later on, the synthesis

of AgNPs has been reported using plants (Iravani 2011) and plant products like green tea

(Camellia sinensis) (Vilchis-Nestor et al 2008), natural rubber (Abu Bakar et al 2007),

Aloe-vera plant extract (Chandran et al 2006), latex of Jatropha curcas (Bar et al 2009),

mesocarp of coconut (Roopan et al 2013) etc.

In the present chapter, we discussed the use of Jatropha seedcake as reducing and

capping agent for biogenesis of AgNPs. The AgNPs thus obtained were characterized by

Transmission Electron Microscopy (TEM), X-Ray Diffraction (XRD), and Fourier

Transform Infra-Red (FTIR) spectroscopy.

6.2. Materials and Methods

6.2.1. Materials

Jatropha seedcake (JSC) was obtained as gratis from the Food Processing and

Bioenergy division, Anand Agriculture University, Anand, Gujarat, India. The seedcake

was sun dried and powdered. 10 g powdered JSC was boiled in 100 mL distilled water for 1

h and then filtered to get the extract. This extract was used as reducing agent and stabilizer.

Silver nitrate (AgNO3) analytical grade was purchased from Hi-Media, India.

6.2.2. Synthesis of Silver nanoparticles

In a typical reaction procedure, a measured volume of JSC extract was added to

appropriate volume of aqueous AgNO3 solution to get a final volume of 10 mL, the mixture

was heated at 90°C and the resulting solution became reddish in color after 15 min of

heating. The reaction was stopped by plunging the tubes in ice-cold water.

Reactions were performed to determine the effect of varying concentrations of JSC

extract and AgNO3 on the AgNPs synthesis.


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6.2.3. Characterization of the nanoparticles

Formation and stability of AgNPs in aqueous colloidal solution was confirmed by

UV-Visible spectrophotometry (UV-Visible spectrophotometer, Shimadzu UV-1601) in the

wavelength range of 300-700 nm operated at resolution of 1 nm.

The size and morphology of the nanoparticles was determined by transmission

electron microscopy (Techai 20; Philips, Holland). AgNPs were mounted on TEM grids by

placing a drop of the particle solution on a carbon-coated copper grid and dried overnight.

The SAED was obtained by directing the electron beam perpendicular to one of the

nanoparticle. The crystalline nature of the metal nanoparticles was confirmed XRD using

Powder X-ray diffractometer (X’pert MPD; Philips, Holland) with Cu-Kα radiation and data

were obtained over the range of 0 to 100° (2θ) with a scanning rate of 0.005°s-1 and step

size of 0.02°. The XRD peaks were indexed and crystallographic lattice parameters were

determined by Powder X-diffraction analysis software (Dong 1999).

6.2.4. FTIR analysis

The functional groups responsible for reduction and stabilization of the bioreduced

AgNPs were analyzed using FTIR spectroscopy (Spectrum GX; Perkin-Elmer). Samples

were prepared by grinding 1 to 2 mg of nanoparticles along with 250 mg KBr and

pelletized using hydraulic press at 20,000 prf.

6.2.5. Evaluation of antibacterial activity of the bioreduced silver nanoparticles.

The antibacterial activity of AgNPs was measured by agar gel diffusion method using

following bacterial test cultures: Escherichia coli MTCC 40, Salmonella para-typhi MTCC

735, Bacillus subtilis ATCC 6051 and Staphylococcus aureus MTCC 87.

To evaluate the minimum inhibitory concentration (MIC), 50 µL of 108 cfu/mL test

culture was added to nutrient broth supplemented with varying concentrations (25 to 500

µg/mL) of silver nanoparticles. Control tubes contained only inoculated broth. The tubes

were incubated at 37°C for 24 h. The turbidity of the tubes was measured using visible

spectrophotometer (Systronics, Ahmedabad, India) at 600nm.


119

6.3. Results and Discussion

6.3.1. Synthesis of silver nanoparticles

The formation of AgNPs in aqueous colloidal solution was marked by the change in

the colour of the reaction solution from pale yellow to deep red (Mulvaney 1996). The

characteristic surface plasmon resonance (SPR) band of colloidal silver was observed

between 424 to 438 nm, which is in good agreement with the earlier reports of bioreduced

AgNPs (Bar et al 2009, Kumar and Mamidyala 2011, Roopana et al 2013). Furthermore, it

was observed that with increase in volume fraction of JSC extract upto 0.1, the intensity of

SPR band increased and further increase in JSC extract volume fraction seized the reaction

(Fig. 6.1). The increase in the intensity of characteristic SPR band may be attributed to

higher concentration of AgNPs (Bar et al 2009). However, at higher volume fraction the

reaction mixture became hazy, probably due to the presence of excess biomaterial (Bar et al

2009).

Figure 6.1 UV-Visible absorption spectra of silver nanoparticles at varying volume fractions of JSC aqueous extract.

Figure 6.2 shows the effect of varying AgNO3 concentrations on AgNPs synthesis

when the reaction was carried out using fixed volume fraction (0.1) of JSC extract. The


120

intensity of SPR band, which corresponds to concentration of AgNPs, increased with

increasing concentration of AgNO3 upto 1 mM. At concentration of AgNO3 above 1 mM,

no further increase in intensity of SPR band was observed, indicating the saturation of

biomolecules for the reduction of silver ions to silver. Such effect has been earlier reported

for synthesis of nanoparticles employing the extract of Cinnamon zeylanicum bark

(Satishkumar et al 2009) and Banana peel (Bankar et al 2010).

Figure 6.2 UV-Visible absorption spectra of silver nanoparticles at varying AgNO3 concentration.

6.3.2. Characterization of the bioreduced silver nanoparticles

The bioreduced AgNPs were found to be predominantly spherical in shape, well

dispersed with no agglomeration (Fig. 6.3). The average particle size of AgNPs observed in

TEM image was computed to be 10.48 ± 2.74 nm (Fig. 6.4). The similar size of AgNPs was

reported by Philip (2010) and Valodkar et al (2011) using leaf extract of Hibiscus rosa

sinensis and latex of Euphorbia nivulia, while AgNPs of larger size have been reported by

Mude et al (2009) and Santhoshkumar et al (2011) using callus extract of Carica papaya

and leaf extract of Nelumbo nucifera as reducing and stabilizing agents. Such size variation


121

of metal nanoparticles synthesized using plant parts and products could be attributed to

difference in reductive potential and capping biomolecules from different plant origin.

Figure 6.3 TEM micrograph of uniformly distributed silver nanoparticles.

Figure 6.4 Particle size distribution of silver nanoparticles synthesized using JSC aqueous extract

The SAED pattern (Fig. 6.5) indicated the polycrystalline nature of bioreduced

AgNPs. Inter-planar spacing (also known as d-spacing) was calculated using Bragg

equation (Bragg and Bragg 1931):

RLd hkl

…………………………………………………………………...…………….. (1)


122

where, R (in mm) is the radius of diffraction pattern, L (320 mm) is the distance

between specimen and photographic film and λ (0.02736 Å) is the wavelength of the

electron based on the accelerating voltage (200 kV). The calculated values for d-

spacings (dhkl) (2.068 and 1.245 Å) were found consistent with the values from standard

JCPDS File No. 04-0783 for silver nanocrystals (Table 6.1). Subsequently, two distinct

diffraction planes were indexed as (200) and (311) facets which correspond to the cubic

phase of silver.

Figure 6.5 Selected Area Electron Diffraction pattern of bioreduced silver nanoparticles

Table 6.1: Comparison of Inter-planer spacings (dhkl) from standard silver diffraction data (JCPDS file no. 04-0783) with the calculated and experimentally observed values from SAED and XRD diffractogram

JCPDS No:04-0783 SAED XRD dhkl (Å) Intensity h k l Calculated dhkl (Å) Observed dhkl (Å) 2.359 100 1 1 1 ND 2.35720 2.044 40 2 0 0 2.068 2.03944 1.445 25 2 2 0 ND 1.44308 1.231 26 3 1 1 1.245 1.22844 1.1796 12 2 2 2 ND 1.17473

*ND denotes ring pattern not detected


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Further, the crystalline nature of the newly synthesized AgNPs was confirmed using

XRD analysis (Fig. 6.6). The presence of five intense diffraction peaks at 2θ values

38.148°, 44.383°, 64.524°, 77.666 ° and 81.949° correspond to (111), (200), (220), (311),

and (222) diffraction facets, respectively, for face centered cubic structures of silver

(JCPDS, File No. 04-0783). The presence of other peaks at 2θ values of 27.752°, 32.358°

and 46.172° corresponds to (211), (122), and (231) diffraction facets for orthorhombic

structure of AgNO3 (JCPDS, File No. 43-0649) which indicated the presence of the

unreacted AgNO3 as impurity. The XRD pattern thus clearly illustrates that the AgNPs

synthesized by the present green method were crystalline in nature.

Figure 6.5 X-Ray Diffraction pattern of bioreduced silver nanoparticles (* denotes diffraction facets for AgNO3)

For FCC structure, the lattice parameter (a) can be calculated from measured values

for the d-spacing of the (111) diffraction facet using the following equation,

222 lkh

ad hkl

…………………………………………….……………………… (2)

where, a is the lattice parameter, h, k and l are the Miller indices of the Bragg plane. The

calculated value for lattice parameter a was found to be 4.083 Å, which is in good


124

agreement with the standard lattice parameter value (a=4.086 Å) from standard JCPDS File

No. 04-0783 for silver nanocrystals.

The crystallite size of the bioreduced AgNPs was calculated from broadening of the

diffraction peaks according to Scherrer equation (Scherrer 1981):

cos2

KT ……………………………………………………………………………. (3)

where, T is the crystallite size, K (~1) is the Scherrer’s constant related to the shape and

index (h.k.l) of the crystal, λ is the wavelength of the X-ray used (Cu-Kα, 1.54056 Å), θ is

the Bragg’s diffraction angle (in degree), and β2θ is broadening of diffraction lines

measured at half of its maximum intensity (in radian). The average crystallite size was

calculated to be 5.58 ± 1.29 nm (Table. 6.2).

Table 6.2: Calculation for crystallite size of bioreduced silver nanoparticles from XRD data using Scherrer equation

FWHM β2θ (°)

FWHM β2θ (radians)

2θ (°)

θ (radians)

cos θ (radians)

T * (Å) h k l

1.8 0.0314159 27.752 0.242181682 0.970817072 50.51166761 1.9 0.03316123 32.358 0.282376581 0.960395943 48.37240705 1.9 0.03316123 38.148 0.33290382 0.945097394 49.15542442 1 1 1 1.1 0.01919861 44.383 0.387314414 0.925926752 86.66271677 2 0 0 1.9 0.03316123 46.172 0.402926371 0.919917469 50.50090366 2.2 0.03839721 64.524 0.563077648 0.845616297 47.44665408 2 2 0 1.95 0.03403389 77.666 0.677763136 0.778977297 58.10883986 3 1 1 2.1 0.03665188 81.949 0.71513933 0.755001887 55.67167461 2 2 2 T = 55.80378601

* T = Kλ/β₂θ cosθ, where K = 1 and λ = 1.54056 Å

6.3.3. FTIR analysis

It was observed that the AgNPs in aqueous solution remained stable over one year

with little aggregation. It has been reported that the carbonyl groups from the amino acid

residues and peptides have a strong affinity to bind with metals and act as encapsulating

agent, thus preventing the nanoparticles from agglomeration (Mitra and Das 2008). In FTIR

spectrum of AgNP, the bands at 1650.42, 1452.36 and 1241.89 cm−1 (Fig. 6.7)

corresponded to characteristic amide I, II and III bands (Coates 2000). The amide band I

was assigned to the stretch mode of the carbonyl group coupled to the amide linkage (NH)-


125

C=O. The band at 1452.36cm−1 was assigned to the methylene scissoring vibrations from

the proteins. The occurrence of these three bands indicated the presence of peptide moiety

probably as capping agent in bioreduced AgNPs.

Figure 6.7 FTIR spectra of bioreduced silver nanoparticles and JSC extract

In addition, the decrease in intensity at 3426.22, 1452.36 and 1406.13 cm−1 after

reduction of AgNO3 may be attributed to the involvement of polyphenols and amines in the

reduction process. The strong absorption band at 3426.22 cm-1 may be due to presence of

polyphenolic hydroxyl group while the bands at 1452.36 and 1406.13 cm-1 are

characteristic of amide II and are assigned to N-H stretching modes of vibration in the

amide linkage (Coates 2000).

This suggests that the biological molecules could possibly perform dual functions of

reduction and stabilization of AgNPs in the aqueous medium.

6.3.4. Application as antimicrobial agent

Silver is well known as one of the most universal antimicrobial substances. Silver ion

and silver-based compounds are highly toxic to microorganisms, exhibiting strong biocidal

effect against them (Rai et al 2009). However, silver ions or salts have limited usefulness as

antimicrobial agents due to interfering effects of salts and/or discontinuous release of

inadequate concentration of silver ions from metals (Dipankar and Murugan 2012). In


126

contrast, these kinds of limitation can be overcome using AgNPs because they are highly

reactive species due to their extremely large surface area, which provides better contact

with micro-organisms. The AgNPs produced biologically are known to exhibit potent

antimicrobial activity (Sharma et al 2009).

Figure 6.8 Anti-bacterial activity of silver nanoparticles against (a) E. coli MTCC 40, (b) S. paratyphi MTCC 735, (c) B. subtilis ATCC 6051 and (d) S. aureus MTCC 87. (CON indicates reaction control and AgNP indicates bioreduced silver nanoparticles)

The AgNPs described in present study were found to exhibit antibacterial activity

against both Gram positive and Gram negative bacteria tested (Fig. 6.8). Similar

observations regarding antibacterial activity of AgNPs have been reported by Sondi and

Salopek-Sondi (2004), Satishkumar et al (2009), Kora et al (2010) and Soo-Hwan et al

(2011). In addition, the AgNPs exhibited lower MIC against the Gram-positive bacteria


127

than the Gram-negative (Table 6.3). This difference in sensitivity of Gram-positive and

Gram-negative bacteria towards AgNPs may be attributed to the difference in their cell

wall structure. The cell wall of Gram-negative bacteria consists of an outer membrane

composed of lipids, proteins and lipopolysaccharides (LPS) which may act as a barrier and

provide higher protection against silver nanoparticles. But the cell wall of Gram-positive

bacteria do not consist of an outer membrane hence are more susceptible to silver

nanoparticles in comparison to Gram negative bacteria (Maneerung et al 2008, Xu et al

2009). Table 6.3: Antibacterial acitivity of bioreduced silver nanoparticles against bacterial strains

Test organism Inhibition Zone (mm) MIC* (µg/mL) Escherichia coli MTCC 40 18.5 100 Salmonella paratyphi MTCC 735 17.5 100 Bacillus subtilis ATCC 6051 1.9 50 Staphylococcus aureus MTCC 87 1.9 50

*MIC, minimum inhibitory concentration

Although a number of studies have tried to fully elucidate the mechanism behind the

biocidal action of AgNPs, no universal conclusion has been drawn so far. There is no doubt

that the antibacterial activity of AgNPs is a complex process, and several possible modes of

action are proposed, involving the direct attachment to cell membrane, disruption of

membrane integrity (Sondi and Salopek-Sondi 2004), increased membrane permeability

(Soo-Hwan et al 2011), formation of free radicals (Soo-Hwan et al 2011) or interaction

with DNA and/or proteins thus interrupting cell replication and metabolism (AshaRani et al

2009).

6.4. Conclusion

The present work demonstrates the competence of using biomaterial toward the

synthesis of AgNPs, by adopting the principles of green chemistry. The experiments were

carried out using the aqueous extract of Jatropha seedcake as reducing and capping agents.

The bioreduced AgNPs thus produced were predominately spherical in shape with a narrow

particle size distribution ranging from 3 to 19 nm. The average particle size, as obtained

from TEM analysis was found to be 10.48 ± 2.74 nm. The X-ray diffraction pattern of


128

AgNPs indicated a face-centred cubic crystalline phase of silver nanoparticles with lattice

parameter estimated to be 4.083 Å. AgNPs prepared through this route were quiet stable

and remained intact for over one year at 4°C. The AgNPs synthesized in present study

exhibited potential antibacterial activity, suggesting its prospective applications in

antibacterial formulations.


129

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