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

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Spectrochimica Acta Part A: Molecular andBiomolecular Spectroscopy

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Green synthesis of silver nanoparticles using Alternanthera dentata leafextract at room temperature and their antimicrobial activity

http://dx.doi.org/10.1016/j.saa.2014.02.0581386-1425/� 2014 Elsevier B.V. All rights reserved.

⇑ Corresponding authors. Tel.: +91 09940800166 (V. Palanichamy). Address:Organic Chemistry Division, School of Advanced Sciences, VIT University, Vellore632 014, Tamilnadu, India. Tel.: +91 09865610356 (S.M. Roopan).

E-mail addresses: vpalanichamy@vit.ac.in (V. Palanichamy), mohanaroopan.s@gmail.com, mohanaroopan.s@vit.ac.in (S.M. Roopan).

Deenadayalan Ashok Kumar a, V. Palanichamy a,⇑, Selvaraj Mohana Roopan b,⇑a School of Biosciences & Technology, VIT University, Vellore 632 014, Tamilnadu, Indiab Chemistry Research Laboratory, School of Advanced Sciences, VIT University, Vellore 632 014, Tamilnadu, India

h i g h l i g h t s

� Green synthesis of silvernanoparticles is using Alternantheradentata for the first time.� The synthesized nanoparticles

solution was stable for more than fourmonths.� Preparation of AgNPs at room

temperature.� The synthesized AgNPs showed

pronounced activity against gramnegative bacteria.

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

a r t i c l e i n f o

Article history:Received 21 October 2013Received in revised form 8 February 2014Accepted 13 February 2014Available online 26 February 2014

Keywords:Green chemistryAlternanthera dentataBio-inspired silver nanoparticlesAntibacterial activityTEMSurface 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 con-firmed by Surface Plasmon Resonance (SPR) at 430 nm using UV–visible spectrophotometer. The reduc-tion of silver ions to silver nanoparticles by A. dentata extract was completed within 10 min. Synthesizednanoparticles were characterized using UV–visible spectroscopy; Fourier transformed infrared spectros-copy (FT-IR), X-ray diffraction (XRD), scanning electron microscopy and transmission electron micros-copy (TEM). The extracellular silver nanoparticles synthesis by aqueous leaf extract demonstratesrapid, simple and inexpensive method comparable to chemical and microbial methods. The colloidalsolution of silver nanoparticles were found to exhibit antibacterial activity against Escherichia coli, Pseu-domonas aeruginosa, Klebsiella pneumonia and, Enterococcus faecalis.

� 2014 Elsevier B.V. All rights reserved.

Introduction

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

properties. So, synthetic methods of such noble NPs are of greatinterest. Among noble metal NPs, AgNPs in particular are knownfor their versatile applications in medical industries. In the 21stcentury, nanotechnology is emerging as cutting edge technologyand has incredible applications in physics, chemistry, biology,material science and medicine. The major thrust has beendeveloping new materials and examining their properties bytuning the particle size, shape and distribution. Green nanotech-nology is gaining importance due to the elimination of harmful re-agents and provides effective synthesis of expected products in an

D.A. Kumar et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 168–171 169

economically manner [1–4]. Noble metal nanoparticles (MNPs)such as silver, gold and platinum are widely applied in medicinalapplications. There is a growing need to develop an environmen-tally friendly process for the synthesis of nanoparticles that doesnot employ toxic chemicals [5–8]. Generally, nanoparticles are pre-pared by a variety of chemical and physical methods. Which is notenvironmentally friendly. Green synthesis of MNPs is an economic,eco-friendly and simple method in the synthesis route [9–12]. Anumber of bio-molecules acts as reducing and protecting agentsin the green synthesis of MNPs. Green/biosynthesis of MNPs wereperformed by using bacteria; fungi and plant extract [13–15].Green synthesis appears to be a cost efficient alternative to con-ventional physical and chemical method of AgNPs synthesis andwould be suitable for developing a biological process for large-scale production. Nowadays plant extracts act as reducing and cap-ping agents for the synthesis of nanoparticles, which is moreadvantageous than chemical, microbial synthesis [16–18].

Alternanthera dentata is a small to medium perennial gardenshrub with dark purple foliage, commonly used for beautificationof gardens. Keeping the green approach in mind the present studyfocused on the biosynthesis of antimicrobial AgNPs from leaf ex-tract is reported. It is well known that the fresh leaf have been usedfor various medicinal applications. Hence, the present study wasaimed to rapidly green synthesize AgNPs using aqueous leaves ex-tract of A. dentata, to investigate the biomolecules responsible forsynthesis of AgNPs and finally to evaluate antibacterial effect ofAgNPs against bacterial strains. To the best of our knowledge, theuse of leaf extract for the green synthesis of noble nanoparticles,such as AgNPs has not been reported so far. In the present studyaqueous leaf extracts of the A. dentata is used for extracellular syn-thesis of silver nanoparticles having potential antimicrobialactivity.

350 375 400 425 450 475 500 525 5500.0

0.1

0.2

0.3

0.4

0.5

0.624 h 16 h 8 h 4 h AgNO3

Abs

orba

nce

Wavelength (nm)

Fig. 1. UV–vis spectra recorded as a function of time of reaction of 1 mM aqueoussolution of AgNO3 with aqueous leaf extracts of the Alternanthera dentata.

Materials and methods

Chemicals and plant collection

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

Preparation of the extract

The leaves of A. dentata were obtained from botanical garden,VIT University, Vellore. The obtained leaves were washed thor-oughly several times with deionized water. 5 g of leaves wereweighed, boiled for 5 min in 100 ml deionized water and the ex-tracts were filtered through Whatman filter paper No. 1. The fil-tered extract was stored in refrigerator at 4 �C. This extracts wereused 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 inErlenmeyer flask and 2 ml of leaves extract was added to it at roomtemperature. After 10 min the solution turns yellow to yellow–redto dark brown indicating the formation of silver nanoparticles.

Characterization

UV–visible analysis was carried out with Schimadzu UV spec-trophotometer (model UV-1800). Distilled water was used asblank. The transmission electron diffraction (SAED) pattern was ta-ken for morphological analysis of nanoparticles with JEOL-3010

field emission electron microscope with accelerating voltage of300 kv. The samples were characterized by preparing dilute solu-tion made in distilled water, drop castled on a carbon coated cop-per grid, followed by drying the sample at ambient conditionbefore it was attached to the sample holder on microscope. The ob-tained purified silver nanoparticles were subjected to X-ray diffrac-tion 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 characterizedusing FT-IR spectral measurements. The measurements were car-ried out on an instrument in the diffuse reflectance mode at a res-olution of 4 cm�1 in KBr pellets. The morphological studies of thesynthesized AgNPs were viewed by SEM instrument (HITACHImodel). The size and morphology of the synthesized AgNPs wasdetermined by Transmission Electron Microscope (JEOL 2100TEM).

Anti-microbial studies

The antibacterial assay was evaluated on pathogenic Escherichiacoli, Pseudomonas aeruginosa, Klebsiella pneumonia and, Enterococ-cus faecalis by agar diffusion method. Briefly Luria bertani (LB)broth (pH 7.4) was used to cultivate bacteria. Fresh overnight cul-tures as inoculums of each culture were seeded on Mueller Hintonagar plates by using sterile cotton swabs. Agar medium surface wasbored 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) werepoured into separate wells. Plates were incubated at 37 �C andzone of inhibition was measured after 48 h.

Results and discussion

UV–visible spectrum of silver nanoparticles

The addition of extract to 1 mM solution of AgNO3 changedfrom colorless to yellowish brown in about 15 min. The final colordeepened with increase in time. Previous reports clarify the pres-ence of AgNPs exhibiting yellowish brown color in solution dueto excitation of surface plasmon vibrations [19]. The reaction takesup to 75 min for completion at room temperature. At 60 �C, thereaction completes within 45 min (Fig. 1) and the color forms at10 min. this may be due to the temperature increment. The reac-tants are consumed rapidly eventually leading to the formationof smaller nanoparticles. In this method, time period and complete

170 D.A. Kumar et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 168–171

reduction of silver is lesser compared to that of earlier reports. Thereactants are consumed rapidly eventually leading to the forma-tion of smaller nanoparticles. In this biosynthesis method, timeperiod and complete reduction of silver is lesser compared to thatof earlier reports. It may be due to the excitation of Surface Plas-mon Resonance (SPR) of the synthesized AgNPs [20]. The SurfacePlasmon Resonance band at 430 nm confirmed the green synthesisof AgNPs at leaves extract [21]. Formation of AgNPs using 1 mMsolution of AgNO3 was confirmed using UV–visible spectral analy-sis. The characteristic Surface Plasmon Resonance band of biogenicAgNPs occurs at 430 nm for reaction carried out at room tempera-ture. It is observed that, the blue shift in the peak from 430 nm to420 nm with raising temperature. This clearly indicates the reduc-tion in the particle size at room temperature [22].

XRD pattern of synthesized AgNPs

The crystalline nature of AgNPs was confirmed by the analysisof XRD pattern as shown in Fig. 2. The XRD spectrum showed fourdistinct diffraction peaks at 38.28�, 44.33�, 64.33� and 77.53� corre-sponding lattice plane value was indexed at (111), (200), (220)and (311) planes of face centered cubic (fcc) silver with a latticeparameter of a = 4.08 Å which were in good agreement with refer-ence of fcc structure from joint committee of powder diffractionstandard (JCPDS). The mean size of AgNPs was calculated using

Fig. 2. XRD analysis in silver nanoparticles.

Fig. 3. FTIR analysis of silver nanoparticle

the Debye–Scherrer’s equation by determining the width of theBragg’s reflection [23]. The size of the nanoparticles was thusdetermined to be about 10–80 nm for AgNPs synthesized at roomtemperature. The XRD patterns obtained are consistent with earlierreports [24].

Electron microscopy studies

The SEM image provided further insight into the morphologyand size of the synthesized silver nanoparticles [25]. Silver nano-particles film was deposited on a carbon coated copper grid andSEM images of AgNPs synthesized by reduction of Ag+ ions byplant extract showed that AgNPs were 50–100 nm in size. Synthe-sized nanoparticles were found to be highly scattered due to itsspherical nature. In the present research study, small AgNPs areseen attached to the surface of very large bio-molecules. TEM mea-surements are also conducted in order to estimate the particle sizeand size distribution for the as-prepared samples. Fig. S1 (Supple-mentary document) shows representative TEM images recordedfrom the drop coated TEM grid of the as prepared Ag nanoparticles[26–28]. These micrographs show individual silver particles as wellas a number of aggregates with spherical shapes. Under carefulinspection of such images, these assemblies were found to beaggregates of silver nanoparticles. The nanoparticles were not indirect contact even within the aggregates. The separation betweenthe silver nanoparticles seen in the TEM image could be due to cap-ping effect of plant extract [29].

FTIR analysis of silver nanoparticles

Fig. 3 shows the FTIR measurements were carried out to identifythe possible biomolecules in leaf extract responsible for cappingleading to efficient stabilization of the silver nanoparticles [30].Peak at 3470 cm�1 and 1640 cm�1 corresponds to NAH stretchingand bending vibrations, respectively in amines from proteins ofplants. While stretching vibration of OAH bonds at 3280 cm�1

(alcohols and phenols) and CAH bonds at 2920 cm�1 arise fromplant metabolites, stretching vibrations of OAH at 2400 cm�1 peakand C@O at 1680 cm�1 peak, arise from carboxylic acid. Two peaksat 1600 cm�1 and 1500 cm�1 correspond to C@C stretching vibra-tions from aromatic rings, all from plant metabolites [31–33].Three peaks, viz., 1010 cm�1, 1190 cm�1 and 1080 cm�1 corre-spond to CAO stretching from alcohol, carboxylic acid, ester andether; all owing to functional groups of proteins and metabolitescovering the silver nanoparticles. Peak at 800 cm�1 is attributed

s synthesis by Alternanthera dentate.

D.A. Kumar et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 168–171 171

to aromatic groups. After bioreduction, there is a shift of theabsorption band of 1601–1595 cm�1 indicating the formation ofAgNPs and is capped with the biomoites. It confirms, the water sol-uble fractions in the extract played complicated roles in the biore-duction of the precursors and shape evolution of the nanoparticles.

Antibacterial effect of silver nanoparticles

Antibacterial study is an important field which covers all areassuch as medicinal chemistry [34]. The silver nanoparticles exhib-ited antibacterial activity against E. coli, P. aeruginosa, K. pneumoniaand, E. faecalis [35]. The zone of inhibition caused by the silvernanoparticles at different concentrations is shown in Figs. S2 andS3. Further the AgNPs are found to be highly toxic against bacteriaat concentration of 50 ppm. Antibacterial effect of silver nanopar-ticles (AgNPs) was size and dose dependent and was more pro-nounced against gram negative bacteria than gram positivebacteria. These findings are in agreement with previous studiesthat examined the antimicrobial activity of AgNPs against E. coli[36–39].

Conclusion

In this article we presented a simple and reproducible way forthe synthesis of silver nanoparticles. The use of natural extracts,distilled water and practically nontoxic reagents allows the syn-thesis pathways presented to be considered as ‘green’ and so per-mitting the synthesized AgNPs to be used in sensitive areas such asbiomedicine. The amount of plant material was found to play acritical role in controlling the size and size disparity of silver nano-particles in such a way that smaller silver nanoparticles and nar-row size distribution are produced when more plant extract isadded in the reaction medium. The methodology employed hereis very simple, easy to perform, inexpensive, and eco-friendly.The colloidal solutions are stable, suggesting that extract can beused as both reducing and stabilizing agent for the preparation ofAg nanoparticles.

Acknowledgements

We thank the Management of VIT University for providing nec-essary facilities to carry out this research study. Dr. S.M. Roopanthank to DBT-RGYI (BT/PR6891/GBT/27/491/2012) scheme for pro-viding the research funding. Authors also wish to thank CIF, Pondi-cherry University for SEM analysis and TEM analysis for PunjabUniversity.

Appendix A. Supplementary material

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

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