Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 168–171

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Green synthesis of silver nanoparticles using Alternanthera dentata leaf extract at room temperature and their antimicrobial activity Deenadayalan Ashok Kumar a, V. Palanichamy a,⇑, Selvaraj Mohana Roopan b,⇑ a b

School of Biosciences & Technology, VIT University, Vellore 632 014, Tamilnadu, India Chemistry Research Laboratory, School of Advanced Sciences, VIT University, Vellore 632 014, Tamilnadu, India

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 Green synthesis of silver

nanoparticles is using Alternanthera dentata for the first time.  The synthesized nanoparticles solution was stable for more than four months.  Preparation of AgNPs at room temperature.  The synthesized AgNPs showed pronounced activity against gram negative bacteria.

a r t i c l e

i n f o

Article history: Received 21 October 2013 Received in revised form 8 February 2014 Accepted 13 February 2014 Available online 26 February 2014 Keywords: Green chemistry Alternanthera dentata Bio-inspired silver nanoparticles Antibacterial activity TEM Surface Plasmon Resonance

a b s t r a c t A green rapid biogenic synthesis of silver nanoparticles AgNPs using Alternanthera dentata (A. dentata) aqueous extract was demonstrated in this present study. The formation of silver nanoparticles was confirmed by Surface Plasmon Resonance (SPR) at 430 nm using UV–visible spectrophotometer. The reduction of silver ions to silver nanoparticles by A. dentata extract was completed within 10 min. Synthesized nanoparticles were characterized using UV–visible spectroscopy; Fourier transformed infrared spectroscopy (FT-IR), X-ray diffraction (XRD), scanning electron microscopy and transmission electron microscopy (TEM). The extracellular silver nanoparticles synthesis by aqueous leaf extract demonstrates rapid, simple and inexpensive method comparable to chemical and microbial methods. The colloidal solution of silver nanoparticles were found to exhibit antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia and, Enterococcus faecalis. Ó 2014 Elsevier B.V. All rights reserved.

Introduction Many researchers have widely used noble nanoparticles (NPs) in various technological applications because of their unique

⇑ Corresponding authors. Tel.: +91 09940800166 (V. Palanichamy). Address: Organic Chemistry Division, School of Advanced Sciences, VIT University, Vellore 632 014, Tamilnadu, India. Tel.: +91 09865610356 (S.M. Roopan). E-mail addresses: [email protected] (V. Palanichamy), mohanaroopan.s@ gmail.com, [email protected] (S.M. Roopan). http://dx.doi.org/10.1016/j.saa.2014.02.058 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.

properties. So, synthetic methods of such noble NPs are of great interest. Among noble metal NPs, AgNPs in particular are known for their versatile applications in medical industries. In the 21st century, nanotechnology is emerging as cutting edge technology and has incredible applications in physics, chemistry, biology, material science and medicine. The major thrust has been developing new materials and examining their properties by tuning the particle size, shape and distribution. Green nanotechnology is gaining importance due to the elimination of harmful reagents and provides effective synthesis of expected products in an

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economically manner [1–4]. Noble metal nanoparticles (MNPs) such as silver, gold and platinum are widely applied in medicinal applications. There is a growing need to develop an environmentally friendly process for the synthesis of nanoparticles that does not employ toxic chemicals [5–8]. Generally, nanoparticles are prepared by a variety of chemical and physical methods. Which is not environmentally friendly. Green synthesis of MNPs is an economic, eco-friendly and simple method in the synthesis route [9–12]. A number of bio-molecules acts as reducing and protecting agents in the green synthesis of MNPs. Green/biosynthesis of MNPs were performed by using bacteria; fungi and plant extract [13–15]. Green synthesis appears to be a cost efficient alternative to conventional physical and chemical method of AgNPs synthesis and would be suitable for developing a biological process for largescale production. Nowadays plant extracts act as reducing and capping agents for the synthesis of nanoparticles, which is more advantageous than chemical, microbial synthesis [16–18]. Alternanthera dentata is a small to medium perennial garden shrub with dark purple foliage, commonly used for beautification of gardens. Keeping the green approach in mind the present study focused on the biosynthesis of antimicrobial AgNPs from leaf extract is reported. It is well known that the fresh leaf have been used for various medicinal applications. Hence, the present study was aimed to rapidly green synthesize AgNPs using aqueous leaves extract of A. dentata, to investigate the biomolecules responsible for synthesis of AgNPs and finally to evaluate antibacterial effect of AgNPs against bacterial strains. To the best of our knowledge, the use of leaf extract for the green synthesis of noble nanoparticles, such as AgNPs has not been reported so far. In the present study aqueous leaf extracts of the A. dentata is used for extracellular synthesis of silver nanoparticles having potential antimicrobial activity.

field emission electron microscope with accelerating voltage of 300 kv. The samples were characterized by preparing dilute solution made in distilled water, drop castled on a carbon coated copper grid, followed by drying the sample at ambient condition before it was attached to the sample holder on microscope. The obtained purified silver nanoparticles were subjected to X-ray diffraction analysis (advance powder X-ray diffractometer, Bruker, Germany model D8). The scanning range was done between 10° and 90°. Purified AgNPs in the form of powder were characterized using FT-IR spectral measurements. The measurements were carried out on an instrument in the diffuse reflectance mode at a resolution of 4 cm 1 in KBr pellets. The morphological studies of the synthesized AgNPs were viewed by SEM instrument (HITACHI model). The size and morphology of the synthesized AgNPs was determined by Transmission Electron Microscope (JEOL 2100 TEM). Anti-microbial studies The antibacterial assay was evaluated on pathogenic Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia and, Enterococcus faecalis by agar diffusion method. Briefly Luria bertani (LB) broth (pH 7.4) was used to cultivate bacteria. Fresh overnight cultures as inoculums of each culture were seeded on Mueller Hinton agar plates by using sterile cotton swabs. Agar medium surface was bored by using sterile gel borer to make wells (7 mm diameter). 100 ll of the different concentrations of silver nanoparticles (20 lg/ml, 40 lg/ml, 60 lg/ml, 80 lg/ml and 100 lg/ml) were poured into separate wells. Plates were incubated at 37 °C and zone of inhibition was measured after 48 h. Results and discussion

Materials and methods

UV–visible spectrum of silver nanoparticles

Chemicals and plant collection

The addition of extract to 1 mM solution of AgNO3 changed from colorless to yellowish brown in about 15 min. The final color deepened with increase in time. Previous reports clarify the presence of AgNPs exhibiting yellowish brown color in solution due to excitation of surface plasmon vibrations [19]. The reaction takes up to 75 min for completion at room temperature. At 60 °C, the reaction completes within 45 min (Fig. 1) and the color forms at 10 min. this may be due to the temperature increment. The reactants are consumed rapidly eventually leading to the formation of smaller nanoparticles. In this method, time period and complete

Preparation of the extract The leaves of A. dentata were obtained from botanical garden, VIT University, Vellore. The obtained leaves were washed thoroughly several times with deionized water. 5 g of leaves were weighed, boiled for 5 min in 100 ml deionized water and the extracts were filtered through Whatman filter paper No. 1. The filtered extract was stored in refrigerator at 4 °C. This extracts were used as reducing as well as stabilizing agent. Synthesis of bio-inspired silver nanoparticles 20 ml of 1 mM aqueous solution of silver nitrate were taken in Erlenmeyer flask and 2 ml of leaves extract was added to it at room temperature. After 10 min the solution turns yellow to yellow–red to dark brown indicating the formation of silver nanoparticles.

0.6

24 h 16 h 8h 4h AgNO3

0.5

Absorbance

Silver nitrate (AgNO3) from Merck (Germany), Nutrient agar and Mueller–Hinton agar medium were purchased from Hi Media (Mumbai, India). Fresh leaves of A. dentata were collected from the campus of VIT University, Vellore, and Tamilnadu, India.

0.4

0.3

0.2

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Characterization 0.0

UV–visible analysis was carried out with Schimadzu UV spectrophotometer (model UV-1800). Distilled water was used as blank. The transmission electron diffraction (SAED) pattern was taken for morphological analysis of nanoparticles with JEOL-3010

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Wavelength (nm) Fig. 1. UV–vis spectra recorded as a function of time of reaction of 1 mM aqueous solution of AgNO3 with aqueous leaf extracts of the Alternanthera dentata.

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reduction of silver is lesser compared to that of earlier reports. The reactants are consumed rapidly eventually leading to the formation of smaller nanoparticles. In this biosynthesis method, time period and complete reduction of silver is lesser compared to that of earlier reports. It may be due to the excitation of Surface Plasmon Resonance (SPR) of the synthesized AgNPs [20]. The Surface Plasmon Resonance band at 430 nm confirmed the green synthesis of AgNPs at leaves extract [21]. Formation of AgNPs using 1 mM solution of AgNO3 was confirmed using UV–visible spectral analysis. The characteristic Surface Plasmon Resonance band of biogenic AgNPs occurs at 430 nm for reaction carried out at room temperature. It is observed that, the blue shift in the peak from 430 nm to 420 nm with raising temperature. This clearly indicates the reduction in the particle size at room temperature [22].

XRD pattern of synthesized AgNPs The crystalline nature of AgNPs was confirmed by the analysis of XRD pattern as shown in Fig. 2. The XRD spectrum showed four distinct diffraction peaks at 38.28°, 44.33°, 64.33° and 77.53° corresponding lattice plane value was indexed at (1 1 1), (2 0 0), (2 2 0) and (3 1 1) planes of face centered cubic (fcc) silver with a lattice parameter of a = 4.08 Å which were in good agreement with reference of fcc structure from joint committee of powder diffraction standard (JCPDS). The mean size of AgNPs was calculated using

the Debye–Scherrer’s equation by determining the width of the Bragg’s reflection [23]. The size of the nanoparticles was thus determined to be about 10–80 nm for AgNPs synthesized at room temperature. The XRD patterns obtained are consistent with earlier reports [24]. Electron microscopy studies The SEM image provided further insight into the morphology and size of the synthesized silver nanoparticles [25]. Silver nanoparticles film was deposited on a carbon coated copper grid and SEM images of AgNPs synthesized by reduction of Ag+ ions by plant extract showed that AgNPs were 50–100 nm in size. Synthesized nanoparticles were found to be highly scattered due to its spherical nature. In the present research study, small AgNPs are seen attached to the surface of very large bio-molecules. TEM measurements are also conducted in order to estimate the particle size and size distribution for the as-prepared samples. Fig. S1 (Supplementary document) shows representative TEM images recorded from the drop coated TEM grid of the as prepared Ag nanoparticles [26–28]. These micrographs show individual silver particles as well as a number of aggregates with spherical shapes. Under careful inspection of such images, these assemblies were found to be aggregates of silver nanoparticles. The nanoparticles were not in direct contact even within the aggregates. The separation between the silver nanoparticles seen in the TEM image could be due to capping effect of plant extract [29]. FTIR analysis of silver nanoparticles

Fig. 2. XRD analysis in silver nanoparticles.

Fig. 3 shows the FTIR measurements were carried out to identify the possible biomolecules in leaf extract responsible for capping leading to efficient stabilization of the silver nanoparticles [30]. Peak at 3470 cm 1 and 1640 cm 1 corresponds to NAH stretching and bending vibrations, respectively in amines from proteins of plants. While stretching vibration of OAH bonds at 3280 cm 1 (alcohols and phenols) and CAH bonds at 2920 cm 1 arise from plant metabolites, stretching vibrations of OAH at 2400 cm 1 peak and C@O at 1680 cm 1 peak, arise from carboxylic acid. Two peaks at 1600 cm 1 and 1500 cm 1 correspond to C@C stretching vibrations from aromatic rings, all from plant metabolites [31–33]. Three peaks, viz., 1010 cm 1, 1190 cm 1 and 1080 cm 1 correspond to CAO stretching from alcohol, carboxylic acid, ester and ether; all owing to functional groups of proteins and metabolites covering the silver nanoparticles. Peak at 800 cm 1 is attributed

Fig. 3. FTIR analysis of silver nanoparticles synthesis by Alternanthera dentate.

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to aromatic groups. After bioreduction, there is a shift of the absorption band of 1601–1595 cm 1 indicating the formation of AgNPs and is capped with the biomoites. It confirms, the water soluble fractions in the extract played complicated roles in the bioreduction of the precursors and shape evolution of the nanoparticles.

Antibacterial effect of silver nanoparticles Antibacterial study is an important field which covers all areas such as medicinal chemistry [34]. The silver nanoparticles exhibited antibacterial activity against E. coli, P. aeruginosa, K. pneumonia and, E. faecalis [35]. The zone of inhibition caused by the silver nanoparticles at different concentrations is shown in Figs. S2 and S3. Further the AgNPs are found to be highly toxic against bacteria at concentration of 50 ppm. Antibacterial effect of silver nanoparticles (AgNPs) was size and dose dependent and was more pronounced against gram negative bacteria than gram positive bacteria. These findings are in agreement with previous studies that examined the antimicrobial activity of AgNPs against E. coli [36–39].

Conclusion In this article we presented a simple and reproducible way for the synthesis of silver nanoparticles. The use of natural extracts, distilled water and practically nontoxic reagents allows the synthesis pathways presented to be considered as ‘green’ and so permitting the synthesized AgNPs to be used in sensitive areas such as biomedicine. The amount of plant material was found to play a critical role in controlling the size and size disparity of silver nanoparticles in such a way that smaller silver nanoparticles and narrow size distribution are produced when more plant extract is added in the reaction medium. The methodology employed here is very simple, easy to perform, inexpensive, and eco-friendly. The colloidal solutions are stable, suggesting that extract can be used as both reducing and stabilizing agent for the preparation of Ag nanoparticles.

Acknowledgements We thank the Management of VIT University for providing necessary facilities to carry out this research study. Dr. S.M. Roopan thank to DBT-RGYI (BT/PR6891/GBT/27/491/2012) scheme for providing the research funding. Authors also wish to thank CIF, Pondicherry University for SEM analysis and TEM analysis for Punjab University.

Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.2014.02.058.

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