GREEN SYNTHESIS OF SILVER NANOPARTICLES USING CALLUS EXTRACT OF CAPSICUM ANNUUM L. AND THEIR...

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GREEN SYNTHESIS OF SILVER NANOPARTICLES USING CALLU S EXTRACT OF

CAPSICUM ANNUUM L. AND THEIR ACTIVITY AGAINST MICRO ORGANISMS

PRITI AGARWAL, VINOD KUMAR BAIRWA, SUMITA KACHHWAHA & S. L. KOTHARI

Department of Botany, University of Rajasthan, Jaipur, India

ABSTRACT

Several biological methods for the production of silver nanoparticles (Ag-NPs) are available due to their multiple

applications. Biosynthesis of silver nanoparticles using callus extract of Capsicum annuum offers an extracellular synthesis

of nanoparticles. It is eco-friendly and cost effective approach so it can be an economic and efficient alternative way for

the synthesis of nanoparticles on large scale. In this study the biosynthesis of silver nanoparticles using callus extract of

Capsicum annuum and antimicrobial activities has been reported. Silver nanoparticles were synthesized by bioreduction of

silver nitrate (AgNO3) solution with callus extract of C. annuum and characterized using UV-Vis absorption

spectrophotometer, Photoluminescence, FTIR, XRD, SEM, and EDS. A mixed phase, cubic and hexagonal, with the

average particle size of 15 nm, SNPs thus synthesized, were evaluated for their antimicrobial activity.

KEYWORDS : Silver Nanoparticles, Anti-Tubercular Agent, Callus Extract, Capsicum annuum, Bio Reduction,

Antimicrobial Properties

INTRODUCTION

Capsicum annuum (Family-Solanaceae) is an economically important plant used as spice in a variety of cuisines

all over the world. It is most widespread and widely cultivated species in the subtropics and temperate regions of the world

(Belletti et al., 1998). It provides flavour, colour and adds to taste in different food preparations (Ravishankar et al., 2003).

Capsicum is an excellent source of vitamin A, B, C and E (Simonne et al., 1997). Capsicum has been part of human lives

as common spice and folk medicine in many part of the world.

The therapeutic properties in Capsicum is attributed to a group of alkaloids known as capsaicinoids, it is used as a

counter irritant in Lumbao, neuralgia, rheumatic disorders and non allergic rhinitis. The plants have also been used as folk

remedies for dropsy, colic, diarrhea, asthma, arthritis, muscle cramps and toothache (Ravishankar et al., 2003).

Capsicum is not only an important agricultural crop due to its economical importance but also for its nutritional

value. It is a good source of antioxidant compounds which is playing an important role for prevention of many human

disease and act as an anti-aging factor (Simonne et al., 1997; Howard et al., 2000; Lee et al., 1995). The extract of

Capsicum annuum contains a lot of biomolecules like protein/enzyme, polysaccharides and vitamins (Collera-Zuniga et al.,

2005; Jagadeesh et al., 2004)

Synthesis of metallic and oxide nanoparticles by the use of plant extracts has been reported in the past

(Jain at al., 2009; Jha et al., 2009a; Jha et al., 2009b; Jae and Kim, 2009; Kumar and Yadav, 2008; Arangasamy and

Munusamy, 2008; Huang et al., 2007; Sharma et al., 2007; Shen et al., 2007; Shankar et al., 2003). Synthesis of silver

nanoparticles by the use of Capsicum annum plant extract has been reported in the past (Shen et al., 2007). The earlier

International Journal of Nanotechnology and Application (IJNA) ISSN(P): 2277-4777; ISSN(E): 2278-9391 Vol. 4, Issue 5, Oct 2014, 1-8 © TJPRC Pvt. Ltd.

2 Priti Agarwal, Vinod Kumar Bairwa, Sumita Kachhwaha & S. L. Kothari

Impact Factor (JCC): 1.8003 Index Copernicus Value (ICV): 3.0

work suggests that the non-fleshy fruit like chilli more treasure of candidate metabolites than the vegetative part of the

chilli plant as reported earlier (Shen et al., 2007).

Nanoparticles are such particles which have unique physical, chemical and biological properties. Due to their

small size and high surface area to volume ratio of nanoparticles provides a tremendous driving force for diffusion,

especially at elevated temperatures. It is very well known that the metal (e.g., Ag, Pt, Au and Pd) silver (Ag) is playing an

important role in the field of biological systems, living organisms and medicine (Jain et al., 2009). Metals are choice for

potential applications in due to their exclusive properties, silver nanoparticles (AgNPs) have several applications, used in

chemical reactions as catalysts (Jiang et al., 2005), electrical batteries and in spectrally selective coatings for absorption of

solar energy (Joerger at al., 2001; Moulin et al., 2008), as optical elements (Nam et al., 2002), pharmaceutical components

and in chemical sensing and biosensing (Aymonier et al., 2002; Songping et al., 2005). According to literature we found

that no attempt has so far been made to employ callus extract of Capsicum annuum for synthesis of silver nanoparticles.

MATERIAL AND METHODS

Synthesis of Nanoparticle

Whitish green bright callus (Figure 1) (25g) taken from 15 day old raised explants of Capsicum annuum cv.

Super wonder. Callus was washed thoroughly in distilled water, dried, cut into small fine pieces and crushed in 100 ml

distilled water. The extract was filtered through Whatman No. 1 filter paper (pore size 25 µm) and later through 0.6 µm

size filters. 1mM aqueous solution of Silver nitrate (AgNO3) was prepared and used for the synthesis of SNPs. 10 ml of

C. annuum callus extract was added into 90 ml of aqueous solution of 1 mM silver nitrate on a shaker (120 rpm) at room

temperature. The change in colour (Figure 2) indicated formation of SNPs which was confirmed on the basis UV-Vis

spectrum of the solution (Figure 3). The SNPs solution, thus obtained, was purified by repeated centrifugation at 5000 rpm

for 20 min, followed by re-dispersion of the pellet of SNPs into deionized water. After freeze-drying, the purified SNPs

were characterized.

Figure 1: White Bright Callus of Capsicum annuum

Characterization

UV-Visible Spectroscopy

The reduction of pure Ag+ ions was monitored by measuring the UV-Vis spectrum of the reaction medium after

5 hours and by diluting a small aliquot of the sample into distilled water. UV-Vis spectral analysis was done by using

UV-Vis spectrophotometer UV-2450 (Shimadzu) (Figure3)

Green Synthesis of Silver Nanoparticles Using Callus Extract of Capsicum annuum L. and Their Activity against Microorganisms 3


Photoluminescence (PL) Spectroscopy

The PL emission spectrum of the SNPs was recorded on RF5301 PL Shimadzu Spectrofluorimeter using a four

side polished quartz cuvette of path length 10 mm. (Figure 4)

FTIR Analysis

The FTIR of the sample was analyzed on the IR Affinity-1 Shimadzu Spectrometer in the diffuse reflectance

mode operating at a resolution of 4cm-1. The sample was dried and grinded with KBr. (Figure 5)

X-Ray Diffraction (XRD) Measurements

The dried powder of silver nanoparticles was analyzed by XRD for the formation of Ag nanoparticles using an

X’Pert Pro x-ray diffractometer (PAN alytical BV, The Netherlands) operated at a voltage of 45kV and current of 40mA

with Cu k (α) radiation of 1.54059 Ǻ wavelength. The scanning was done in the region of 2θ from 30° to 80° at 0.03/min

and the time constant as 1s. (Figure 6)

Scanning Electron Microscopy

SEM analysis was done using scanning electron microscope (Carl ZEISS EVOR -18) operated at 20kV. For the

SEM analysis sample was first sonicate for 15 min. A drop of this solution was loaded on carbon coated copper grids and

solvent was allowed to evaporate to leave a thin film on the substrate. (Figure 7)

Energy Dispersive Spectroscopy (EDS) Measurements

EDS measurement of the SNPs drop coated onto Al substrate was performed on Oxford instrument nano analysis

X-act EDS. (Figure 8)

Antibacterial Assay

The AgNPs synthesized were tested for bactericidal activity against pathogenic E. coli. Bacteria. Luria Bertani

(LB) broth/ agar medium was used to cultivate bacteria. In another assay E. coli. bacteria were treated with different

concentration of Ag NPs (0, 5, 10, 25, 50, 100 ppm). (Figure 9)

RESULTS AND DISCUSSIONS

UV-Vis Absorbance Studies

The pale green C. annuum extract changing to brownish colour after addition of AgNO3, suggested bio reduction

of aqueous silver ions (Ag+) by the callus extract and the formation of silver nanoparticles (SNP) (Figure 2).

Figure 2: A Visible Observation of Change in Colour during Silver Nanoparticles Formation

(a) AgNo3 (b) callus extract (c) 15min (d) 30 min (e) 1h (f) 2 h



The reaction mixture was checked periodically for change in colour from green to different shades of brown and

was monitored by UV spectrophotometer. Absorption spectra of silver nanoparticles had absorbance peak at 428 nm,

broadening of peak indicated that the particles were polydispersed (Figure 3).

Figure 3: UV-Vis Absorption Spectrum of Silver Nanoparticles

Photoluminescence (PL) Spectroscopy Analysis

The luminescence of Ag and that of noble metal is generally attributed to excitation of electron from occupied d

bands into states above the Fermi level (Lara et al., 2010). Wilcoxon and co-workers speculated that similar basic

mechanism is responsible for the nanocluster photoluminescence (Birla et al., 2009; Inoue et al., 2010). The synthesized

SNPs were found to be Photoluminescent. Figure 4 shows the luminescence spectrum of freshly prepared SNPs, Which

exhibited a sharp and strong peak near 370 nm and a broadened band between 500-600nm. (Figure 4)

Figure 4: Photoluminescence Spectra

FTIR Analysis

FTIR measurement carried out to identify the possible interaction between bio molecule and SNPs. The FTIR

measurements of biosynthesized silver nanoparticles show bands at around 755, 1560, 1715, 3156 and 3678 cm-1.

Absorption peak at 1715 cm-1 assigned to carbonyl functional group in unsaturated/aromatic carboxylic acids. The band at

3156 cm-1 is characteristic to hydroxyl functional group in alcohol and phenols present in high concentration while the

band at 3678 cm-1 show hydroxyl functional groups present in alcohol and phenols in low concentration. The absorption

peak at 755 cm-1 is due to the aromatic C-H group (Figure 5).



Figure 5: FTIR Spectrum

XRD Analysis

The crystal structure of silver nanostructures were further characterized and confirmed by XRD. The XRD pattern

of silver nanoparticles showed 4 XRD peaks appear at 38 (111), 46 (103), 54.5 (006), 77 (201) (JCPDF card file no.

00-041-1402). This indicates that the sample contained a mixed phase, cubic and hexagonal structures of silver

nanoparticles (Figure 6)

Figure 6: XRD Spectrum

SEM Observation of Silver Nanoparticles

SEM micrographs of Ag NPs being formed using callus extract of C. annuum. It was observed that Ag

nanoparticles were spherical in shape and particles were found to be round 50-70 nm (Figure7).

Figure 7: The Spherical Shaped Nanoparticles in the Range of 50-70 Nm



EDS Analysis

Spot profile spectra recorded from the silver nanoparticles showed a strong silver signal along with an Al signal

which is due to the thin film made on the Al substrate taken for the EDS measurement. (Figure 8)

Figure 8: EDS Spectrum

Antibacterial Activity of Silver Nanoparticles

The results indicated that silver nanoparticles synthesized from callus extract of C.annuum showed antibacterial

activity against E. coli. bacteria. It was observed that as the concentration of biosynthesized Ag NPs increased, microbial

growth decreased. (Figure 9)

Figure 9: Effect of Different Concentration of Ag NPs on the Growth of E. coli

CONCLUSIONS

The bio-reduction of aqueous Ag+ ions by the callus extract of C.annuum has been demonstrated. The reduction

of the metal ions through plant extracts has played the pivotal role in synthesis of silver nanoparticles.

This approach of green chemistry toward the synthesis of silver nanoparticles as an antitubercular agent has

advantages such as, ease with which the process can be scaled up and affordability. Biologically synthesized silver

nanoparticles could be of immense use in medical textiles for their efficient antibacterial and antimicrobial properties

(Shahverdi et al., 2007). Use of these eco-friendly nanoparticles as a bactericidal against other microorganisms, for wound

healing and other medical and electronic applications, makes this method potentially exciting for the large-scale synthesis

of other inorganic materials.



ACKNOWLEDGEMENTS

The authors are thankful to CSIR (Council for scientific and industrial Research) for their economic support and

Department of Physics, University of Rajasthan for providing instrument facility.

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