Materials Science and Engineering C 49 (2015) 373–381

Contents lists available at ScienceDirect

Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Green synthesis and characterization of silver nanoparticles using Lantana camara leaf extract B. Ajitha a,⁎, Y. Ashok Kumar Reddy b, P. Sreedhara Reddy a a b

Department of Physics, Sri Venkateswara University, Tirupati 517502, India Department of Electrical Engineering, Korea Advanced Institute of Science and Technology, 335 Gwahangno, Yuseong-gu, Daejeon 305-701, South Korea

a r t i c l e

i n f o

Article history: Received 19 September 2014 Received in revised form 13 November 2014 Accepted 7 January 2015 Available online 9 January 2015 Keywords: Green synthesis Leaf extract Particle size Absorption Antibacterial activity

a b s t r a c t In this work, we have investigated on Lantana camara mediated silver nanoparticles (AgNPs) with different leaf extract (LE) quantity for the evaluation of efficient bactericidal activity. The AgNPs were prepared by simple, capable, eco-friendly and biosynthesis method using L. camara LE. This method allowed the synthesis of crystalline nanoparticles, which was confirmed by X-ray diffraction (XRD) and selected area electron diffraction (SAED) patterns. The X-ray photoelectron spectroscopy (XPS) analysis confirmed the formation of metallic silver and elucidates the surface state composition of AgNPs. UV–vis spectra of AgNPs and visual perception of brownish yellow color from colorless reaction mixture confirmed the AgNP formation. Involvement of functional groups of L. camara leaf extract in the reduction and capping process of nanoparticles was well displayed in Fourier transform infrared spectroscopy (FTIR). Decrement of particle size with an increment of leaf extract volume was evident in AFM, TEM images and also through a blue shift in the UV–vis spectra. The rate of formation and size of AgNPs were dependent on LE quantity. Meanwhile, these AgNPs exhibited effective antibacterial activity with the decrement of particle size against all tested bacterial cultures. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Nanoparticles are being expressed as fundamental building blocks of nanotechnology. Nanoparticles exhibit unique properties due to their size, distribution and morphology. In the late 1970's itself R.O. Becker et al. discovered the treatment of orthopedic infections by silver ions ensuing the promotion of bone growth by affecting the surrounding bacteria leading to cell death [1]. Nanocrystalline silver particles have many applications, but still there is the need for economic and biocompatible AgNPs to enhance the current thinking on the future of health care, food and consumer opinion that most “natural ones” are better and safer [2]. The bioinspired green synthesis of AgNPs is evolving as an important unique branch of nanotechnology [3]. Green synthesis of nanoparticles using microorganisms [4], enzymes [5], and plant extracts [6,7] offers several benefits over conventional physical and chemical methods. Biological synthesis pathways are economic, eco-friendly, easily scaled up for large scale synthesis and don't need to use toxic chemicals, high pressure, temperature and energy which enhance medical applicability [7,8]. However, the usage of plant materials is more advantageous than other biological processes as it eliminates the risk as well as the

⁎ Corresponding author. E-mail address: [email protected] (B. Ajitha).

http://dx.doi.org/10.1016/j.msec.2015.01.035 0928-4931/© 2015 Elsevier B.V. All rights reserved.

elaborate process of maintaining cell cultures and the reaction time decreases from several days to several hours [3,9]. The conjugation of plant material constituents with nanoparticles not only affords stabilization of the system, but also injects biocompatible functionalities into these NPs for enhancing further biological interactions. Green synthesized AgNPs have tremendous applications such as spectrally selective coatings for solar energy absorption and intercalation material for solar energy batteries, as optical receptors, catalysts in chemical reactions, biolabelling, and as antimicrobial agents [10,11]. Earlier literature reveals that AgNPs synthesized using plant (herbal) extracts have promising medical applications. Recently, bio-constituents of different plant extracts were recognized as potential synthesizers of metal nanoparticles. Therefore, biomolecules of plant extracts are currently being researched globally for control of size, shape and stability of the nanoparticles. In recent times, biosynthesis of AgNPs has been explored using Eucalyptus chapmaniana [6], Panax ginseng [7], Arbutus unedo [8], Datura metel [12], Malva parviflora [13], Cinnamon zeylanicum [14], Acalypha indica [15], Tribulus terrestris [16], Gliricidia sepium [17], and Ixora coccinea [18]. The extracts of different plant parts such as leaves, roots, seeds, stems and fruits containing antioxidants have been studied for the biosynthesis of AgNPs. In the current study attempts were made for the green synthesis of AgNPs using Lantana camara LE. L. camara (verbenaceae), is commonly known as wild or red sage and also regarded as both notorious weed and ornamental plant.

374

B. Ajitha et al. / Materials Science and Engineering C 49 (2015) 373–381

L. camara is a gregarious, erect, half climbing, hairy aromatic shrub. Different parts of the L. camara mainly the leaves have been used in the treatment of scratching, stomachache, rheumatism, wound healing, biliary fever, toothache, bronquitis, antiseptic and other infections [19, 20]. Nowadays, there has been a growing interest in the study and usage of traditional plants for pharmaceutical and medical applications because of its low toxicity and economic viability. The quantitative phytochemical tests on L. camara have revealed the presence of proteins, carbohydrates, common secondary metabolites, and minor components of phytosterols, saponins, tannins, and phycobatannin in the leaves [21]. Very recently, Bhakta et al. have confirmed the existence of phenolics, anthocyanins and proanthocyanidins in the leaves of L. camara [22]. Ganjewala et al. reported the presence of threefold total sugars and maximum amounts of phospholipid content in the leaves of L. camara lavender variety [21]. Hence, the reduction of Ag+ to Ag0 nanoparticles using L. camara leaf extract was ascribed due to the presence of phenolics, flavonoids, terpenoids, alkaloids, lipids, proteins and carbohydrates in the leaf extract [12,23]. Hence, leaf extract plays a dual role as both reducing and capping agents simultaneously without any involvement of chemicals. In this present work, AgNPs were synthesized by using L. camara leaf extract as a source for nanoparticle formation. 2. Materials and methods 2.1. Chemicals and plant material collection All the reagents purchased were of analytical grade and used without any further purification. Silver nitrate (AgNO3) was purchased from Sigma-Aldrich with ≥ 99.5% purity from India. Fresh leaves of L. camara have been harvested in the month of September 2013 from in and around Chittoor district, A.P., India. Milli-Q water was used for preparing aqueous solutions all over the experiments. 2.2. Preparation of leaf extract Fresh leaves of L. camara were collected and washed with tap water at first, then surface washed with Milli-Q water until no impurities remained. The dirt free leaves were shade dried for 10 days at room temperature to remove residue moisture. The dried leaves were pulverized in a sterile electric blender to obtain a fine powder and stored in an airtight bottle avoiding sunlight for further use. 10 g of leaf powder was mixed thoroughly with 100 ml of Milli-Q water and then boiled for 10 min at 60 °C followed by cooling and filtration through Whatman No. 1 filter paper to obtain LE [24]. The filtered extract was collected and used fresh for further studies. 2.3. Synthesis of nanoparticles 100 ml of 0.001 M aqueous solution of silver nitrate was prepared in an Erlenmeyer flask; and 5, 10, 15 and 20 ml of L. camara leaf broth was added to it separately at room temperature, thus the colloids A1, A2, A3 and A4 are synthesized respectively and the schematic synthesis procedure is depicted in Fig. 1. All the reaction flasks were covered with aluminum foil under continuous stirring for 10 min. The bio-reduction of the silver ions was rapid as the solution turned to brownish yellow within 5 min confirming the formation of AgNPs and there was no color change further. The obtained AgNPs were purified by repeated centrifugation at 15,000 rpm for 10 min followed by redispersion of the pellet twice in Milli-Q water to remove water soluble biomolecules such as proteins and secondary metabolites. Then the AgNPs were obtained and transferred into clean bottle for further studies. 2.4. Antibacterial assay The antibacterial assessment of AgNPs was carried out by using four different test pathogens; gram-negative Escherichia coli, Pseudomonas

Fig. 1. Schematic procedure of AgNP synthesis in green condition.

spp., gram-positive Bacillus spp., and Staphylococcus spp. The microbial cultures were maintained by the department of Microbiology, S.V. University, Tirupati, India. Antibacterial activity of the synthesized AgNPs was determined using Kirby-Bauer disc diffusion method. The bacterial test organisms were grown in nutrient broth for 24 h and used for the study. Nearly 20 ml of molten and cooled nutrient agar media was poured into sterilized petri plates and were left for solidification. Bacterial lawns were prepared by 100 μl of overnight cultures of each organism with the help of a sterile bent glass rod by swabbing on the top surface of the nutrient agar media. Then, double sterilized paper discs (4 mm) were kept on the agar plates and the AgNPs were synthesized with different leaf extract quantities i.e. A1, A2, A3 and A4 colloids of 6 μl were loaded onto each disc separately in each plate. At last, the petri plates with AgNPs discs were incubated at 37 °C for 24 h in an incubator. Positive test results were scored when the zone of inhibition surrounding each disc was observed. Then, the diameter of the zone of inhibition was measured with meter ruler and the mean value of inhibition around each disc was calculated and expressed in millimeter.

2.5. Characterization of the synthesized AgNPs The as-synthesized AgNPs were characterized by various instrumental analyses. Crystalline metallic silver was examined by Seifert 3003TT X-ray diffractometer, using Cu Kα radiation (λ = 0.1546 nm). In order to identify elements of surface, X-ray photoelectron spectroscopy (XPS) studies were performed using multi-purpose X-Ray photoelectron spectroscopy with a Sigma probe model. The deconvolution, curve fitting, and background subtraction were done using XPS Thermo Scientific™ Avantage software. The elemental compositional study was investigated through energy dispersive spectroscopy (EDS) by using Oxford Inca Penta FET x3 EDS instrument coupled with SEM. The morphological analysis of AgNPs was carried out using a ZEISS, SUPRA 55 field emission scanning electron microscopy (FESEM) and atomic force microscopy (AFM) of model SPA 400 measurements. The transmission electron microscopy (TEM) images were obtained using FETEM with Tecnai TF30 ST at an accelerating voltage of 300 kV. UV–vis spectroscopy measurements were recorded on a Perkin Elmer Lambda 950 UV–vis-NIR spectrophotometer with a wavelength resolution better than ± 0.2 nm. Photoluminescence spectra were analyzed using Horiba Jobin-Yvon Fluorolog-3 Spectrofluorometer (Model FL3-22PTI) in the wavelength range of 400–700 nm. Fourier transform infrared spectroscopy (FTIR) of the freeze-dried samples was recorded with ATR-FTIR using Bruker Vertex-80 spectrometer. Zeta potential values were measured using Nanopartica (HORIBA).

B. Ajitha et al. / Materials Science and Engineering C 49 (2015) 373–381

375

3. Results and discussion

(111)

3.1. Crystallographic analysis of AgNPs XRD analysis was employed to determine the crystal phase and structure of as prepared AgNPs. The XRD profiles of dried AgNPs prepared by the green reduction method are shown in Fig. 2. Four Bragg's reflections corresponding to (111), (200), (220) and (311) planes of the fcc crystal structure of metallic silver (JCPDS No. 04-0783) are interpreted from XRD. The (111) orientation is more intense than other peaks suggesting (111) as a predominant orientation. Increment of a leaf extract quantity, resulted in broadening of the diffraction peaks emphasizing the crystallite size decrement. No impurity peaks were observed revealing the formation of pure crystalline silver. The crystallite size of the AgNPs is calculated by Debye-Scherrer's formula [25],

A1

(200) (220) (311)

50

2

60

70

80

degree

D ¼ Kλ=βcosθ

O 1s

Ag 3d 3/2 Ag 3d 5/2

where D denotes the average crystallite size, K Scherer's constant (K = 0.94), λ X-ray wavelength (0.1546 nm), β full-width at half-maximum

(b)

900

800

600

500

400

300

200

S 2p P 2p Ag 4s Ag 4p Ag 4d

Intensity (a.u.)

700

Cl 2p

C 1s

Intensity (a.u.)

Ag 3s

Ag 3p 1/2

(a)

Ag 3p 1/2

Fig. 2. X-ray diffraction profiles of silver nanoparticles at different quantities of L. camara leaf extract.

ð1Þ

100

0

378

376

374

372

370

368

366

Binding energy (eV)

Binding Energy (eV)

(c)

Ag3d 5/2

40

Ag3d 5/2

30

Ag3d 3/2

20

Ag3d 3/2

Intensity (a.u)

A4

C -O-C

(d)

-O-C-C- O-

Intensity (a.u.)

Intensity (a.u.)

Ag-O

Ref. C

-C=O-

O=C -O

291

290

289

288

287

286

285

284

Binding energy (eV)

283

282

281

536

535

534

533

532

531

530

Binding energy (eV)

Fig. 3. XPS spectra of the green synthesized silver nanoparticles of (a) Survey, (b) Ag 3d, (c) C 1s and (d) O 1s.

529

528

527

526

376

B. Ajitha et al. / Materials Science and Engineering C 49 (2015) 373–381

(a)

(b)

(c)

Fig. 4. Typical (a) EDS spectrum, (b) quantitative analysis and (c) selected area elemental mapping of synthesized AgNPs at 20 ml of L. camara leaf aqueous broth.

A1

A4

Fig. 5. SEM images of biosynthesized AgNPs at (A1) 5 ml and (A4) 20 ml of L. camara leaf extract quantity.

B. Ajitha et al. / Materials Science and Engineering C 49 (2015) 373–381

377

A4

A1

Fig. 6. AFM morphologies of AgNPs at (A1) 5 ml and (A4) 20 ml of L. camara leaf extract quantity.

of diffraction line in radians and θ half diffraction angle. Increment in LE quantity found to result in decreased crystallite size from 24 nm to 11 nm. This suggests the increased reduction rate of the leaf extract and is in accordance with the earlier reports [13,25].

3.2. Chemical composition analysis by XPS The composition and oxidation state of the product were studied by XPS. Fig. 3(a) shows the typical XPS survey spectrum for the typical

25

(b)

(c)

14 nm

No of particles (%)

20

15

10

5

0 0

5

10

15

20

25

30

Particle diameter (nm)

Fig. 7. (a) TEM images of A1 (5 ml) and A4 (20 ml) samples, (b) SAED pattern and (c) particle diameter analysis of A4 sample of silver nanoparticles.

B. Ajitha et al. / Materials Science and Engineering C 49 (2015) 373–381

3.3. Elemental compositional analysis by EDS The elemental compositional analysis of the green synthesized AgNPs has been carried out using EDS as shown in Fig. 4(a). The presence of major emission peak around 3 keV indicates a characteristic strong signal for nanosized particles of silver, and reveals the existence of silver as a major component [14]. Besides this high intense peak, low intense peaks of C, O, S, Cl and P are also observed due to the capping action of biomolecules of leaf extract in AgNP formation. Fig. 4(b) shows the quantitative analysis of the prepared AgNPs. Quantitative analysis gives the weight % of the individual elements present in the sample. Fig. 4(c) displays the elemental mapping of the bio-reduced AgNPs. It confirms the homogeneous distribution of all the elements throughout the sample. 3.4. Surface morphology of AgNPs The FESEM, AFM and TEM are the best analyzing tools for structural and morphological properties. Fig. 5 shows the FESEM images of prepared AgNPs with a variation of leaf extract quantity (A1, A4). FESEM images indicate the nearly spherical nanoparticle formation. As leaf extract quantity is increased from 5 ml to 20 ml, the particle size is observed to be decreased from 34 nm to 20 nm. AFM is provided to be a beneficial tool for the purpose of studying various morphological features and parameters. Since, it has the advantage of probing in deep insights of surface topography qualitatively due to its both lateral and vertical nanometer scale spatial resolution. Fig. 6 shows the typical atomic micrographs of synthesized AgNPs. AFM images reveal the appearance of spherical nanoparticles and their respective particle size histograms clearly evidence the decrement trend in particle size from 31 nm to 17 nm, when the leaf extract quantity increased from 5 ml to 20 ml. TEM images are pictured in Fig. 7(a), which clearly confirms the formation of nearly spherical nanoparticles with homogeneous size distribution. Leaf extract quantity influences the size of the particles abruptly. When leaf extract quantity is increased to 20 ml, an obvious change in the size distribution of nanospheres was observed. With variation of an extract quantity the samples showed a decrement in particle

Table 1 The variation of particle size of AgNPs and zone of inhibitions against different pathogenic bacteria for A1–A4 colloids. Ag NPs (6 μl)

Particle size (nm)

Zone of inhibition of tested pathogenic bacterium (mm) E. coli

Staphylococcus spp.

Pseudomonas spp.

Bacillus spp.

A1 A2 A3 A4

27 22 18 14

3 4 4 6

3 4 4 6

6 7 7 7

3 4 8 8

size from 27 nm to 14 nm (Table 1). Zeyed et al. and Aromal et al. also observed the similar trend with the variation of extract quantity [13, 32]. This result suggests that the leaf extract serves as a size controller and affects the particle size. Fig. 7(b) shows a SAED pattern recorded by directing the electron beam perpendicular to one of the individual nanosphere. The characteristic bright circular fringes can be indexed to (111), (200), (220) and (311) of the pure face centered cubic (fcc) lattice structure. The average particle size of A4 sample was also measured as ~ 14 nm (Fig. 7(c)) and is in agreement with the particle size calculated from XRD spectra.

3.5. UV–vis absorption studies UV–vis spectroscopy is commonly employed for confirming metallic nanoparticle formation by studying the optical properties which depend particularly on size effect [15]. At first, the nanoparticles are visualized through the color change from colorless to brownish yellow immediately after the addition of leaf extract due to collective oscillation of free conduction electrons induced by an interacting electromagnetic field results in surface plasmon resonance (SPR) [13,33]. Fig. 8 presents the UV–vis spectra of aqueous solution of AgNPs prepared for different leaf extract quantities (A1–A4). The inset of the figure shows the color change and variation of SPR with quantity of extract. UV–vis spectra evince the blue shift of absorption band with increasing leaf extract quantity. For A1, A2, A3, A4 samples the absorption peak is centered around 440–400 nm and the location of characteristic surface plasmon resonance absorption peak of biogenic AgNPs is at 436, 421, 413 and 400 nm, respectively. UV–vis spectra corroborated the decrement of particle size with increment in extract quantity. In earlier papers the similar result was reported on the size variation of nanoparticles with an increase of leaf extract quantity [16,26,32].

440

430

Wavelength (nm)

product, indicating the existence of Ag, C, O, Cl, P and S. The strong signal of Ag 3d (~370 eV) indicates the presence of Ag metal. The C 1s peak observed at a binding energy of ~ 285 eV serves as a reference to correct the binding energy shift and it also stems from biomolecules of leaf extract capped to AgNPs. The spectrum also consists of O (~ 531 eV), Cl (~198 eV), S (~163 eV) and P (~133 eV) elements in their respective binding energy positions due to the interaction of biomolecules of leaf extract with AgNPs. From the XPS survey spectrum, the atomic percentages of Ag, C, O, P, S and Cl elements were also found to be 57.64%, 11.12%, 13.08%, 5.92%, 2.01% and 10.23%, respectively. The high-resolution XPS spectrum of Ag 3d is shown in Fig. 3(b). The Ag 3d3/2 and Ag 3d5/2 peaks can be divided into two pairs of peaks at 374.08/376.06 eV and 368.01/370.10 eV, respectively. The peaks at 374.08 and 368.01 eV are attributed to metallic Ag and are consistent with the XRD result [27]. Loss features are observed at a higher binding energy side of each spin-orbit component of Ag metal. The peak in the C 1s spectrum (Fig. 3(c)) can be fitted to several symmetrical peaks with binding energies of 284.21, 286.13, and 288.64 eV, which are assigned to the C 1s of the adventitious reference hydrocarbon, that of carbon \C\O\, and that of carboxyl carbon \O\C_O, respectively [28,29]. The peak of O 1s has also been deconvoluted into two bands as shown in Fig. 3(d). The peak at lower energy side can be ascribed to the O 1s core peak of O2 − bound to Ag+, while the other peak located at 531.05 eV should possibly be attributed to the oxygen in the carboxyl group (\C_O\) which are bound to the surface of the silver nanoparticles and at 532.34 eV corresponds to the back bone of \CH2\O\CH2\ [30,31].

Absorbance (a.u)

378

200

420

410

400

5

10

15

20

Extract quantity (ml)

A1 A2 A3 A4 300

400

500

600

700

800

900

Wavelength (nm) Fig. 8. UV–vis absorbance spectrum of AgNPs as a function of L. camara quantity. The inset of the figure shows the color change and variation of SPR with quantity of extract.

B. Ajitha et al. / Materials Science and Engineering C 49 (2015) 373–381

A1

379

1.0

A2

2122

0.9

A3 0.8 0.7

1635

Intensity (a.u)

Transmittance %

A4

0.6 0.5

3314

0.4 0.3 0.2 4000

3500

3000

2500

2000

1500

1000

-1

Wavenumber (cm )

350

400

450

500

550

600

650

700

Wavelength (nm) Fig. 9. Photoluminescence spectra of green synthesized AgNPs at different quantities of L. camara leaf aqueous broth with fixed quantity of AgNO3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.6. Photoluminescence emission studies Photoluminescence (PL) spectroscopy is a selective and extremely sensitive probe of discrete electronic states. The synthesized bio-genic AgNPs were found to be photoluminescent when excited with 370 nm. Enhanced photoluminescence intensity was observed with an increase of extract quantity (Fig. 9). A previous report suggested that photoemission wavelength is independent of the particle size while the intensity increases sharply with the decrease of particle size [16]. Photoluminescence spectra elicit the shifting of the emission peak towards higher energy wavelength with the increase of extract quantity. For A1, A2, A3 and A4 samples the emission peaks displayed a blue shift and enhancement in intensity owing to the decreased particle size at 462, 458, 453 and 447 nm, respectively.

A1

Fig. 11. FTIR spectrum of AgNPs synthesized with 20 ml L. camara leaf extract quantity.

3.7. Zeta potential measurements Zeta potential provides the information about the stability of nanoparticles and surface charge. Zeta potential of the synthesized AgNPs is pictured in Fig. 10. Bio-genic AgNPs displayed lower zeta potential value at lower extract quantity and higher zeta potential values at a higher quantity. The zeta potential value specifically increased from − 25 mV to − 50 mV for A1 and A4 colloids which expounds the increased stability of the prepared AgNPs with the increase of extract quantity. According to Salopek et al., when the zeta potential value lies in between −40 and −50 mV it assesses the good stability of the particles [34]. The high negative values illustrate the repulsion between the particles and thereby attainment of better stability of AgNP formation avoiding agglomeration. 3.8. FTIR analysis FTIR spectroscopy is helpful to analyze the possible interactions of AgNPs with different functional groups. FTIR spectrum shows the presence of different functional groups at various positions (Fig. 11). The

A4

Fig. 10. Zeta potential analysis of AgNPs prepared with (A1) 5 ml and (A4) 20 ml of L. camara leaf extract quantity.

380

B. Ajitha et al. / Materials Science and Engineering C 49 (2015) 373–381

Fig. 12. The plausible mechanism of the formation of AgNPs using L. camara leaf extract.

strong band at 1635 cm− 1 is attributed to carbonyl groups involved in the nanoparticle formation [35]. Also a less intense broad peak at 2122 cm− 1 corresponds to the O\C stretching mode [36]. Another strong, intense band at 3314 cm−1 is assigned to both \NH2 in primary aromatic amines and \OH groups in alcohols [37]. The IR peaks for amide I and amide II arise owing to carbonyl stretch and \N\H stretch vibrations in the amide linkage of the proteins and thereby they have a greater affinity to bind with AgNPs. The phytochemical tests on L. camara have revealed the presence of lipids, proteins, carbohydrates, and common secondary metabolites: phenolics, flavonoids, terpenoids, alkaloids and trace amounts of phytosterols, saponins, tannins, and phycobatannin in the leaves [20–22]. Particularly in lavender variety, maximum levels of phospholipids, lanthanedes and carbohydrates have been observed by Ganjewala et al. [21]. From the FTIR analysis, it can be stated that the hydroxyl and carbonyl groups present in carbohydrates, flavonoids, terpenoids, and phenolic compounds are powerful reducing agents and they may be accountable for the bioreduction of Ag+ ions leading to Ag0 nanoparticle synthesis. FTIR study confirms that the carbonyl groups of amino acid residues and peptides of proteins have a strong ability to bind metal ions and they may be encapsulated around the nanoparticles forming a protective coat-like membrane to avoid the agglomeration and thus results in nanoparticle stabilization in the medium. Thus, the LE components act as a bioreductants and surfactants too. The plausible

mechanism of the formation of AgNPs is shown in Fig. 12. It is pertinent to note that the leaf extracts specifically affect the size of the nanoparticles preventing the AgNPs from being oxidized. In this pursuit, proteins and all secondary metabolites of extract play a critical role in both reducing and capping mechanism for nanoparticle formation. 3.9. Antibacterial study The antimicrobial effects of silver have been known since ancient times [17]. The clear mechanism of AgNPs interaction with bacteria is not well known. AgNPs may attach to the cell wall thus leading to cell disruption by varying membrane permeability and cell respiration. AgNPs can penetrate inside the cell also since, they have a greater affinity to react with sulfur containing proteins in the cell wall and inside the cell and also phosphorous containing elements such as DNA [38,39]. Thus, they can easily bind to these constituents of the bacterial cell and disturb the normal functioning of the cell. Another possible way is the release of Ag cations from AgNPs which act as reservoirs for Ag+ bactericidal agent [40]. The green synthesized AgNPs showed great response against all tested pathogens and antibacterial activity is shown in Fig. 13. The diameters of zone of inhibition were analyzed by different leaf extract quantities. The highest inhibition zone was displayed by A4 colloid compared with other A1, A2 and A3 colloids against all tested organisms due to small

Fig. 13. Disc diffusion assay of bactericidal activity against bacterial organisms of prepared AgNPs (A1–A4).

B. Ajitha et al. / Materials Science and Engineering C 49 (2015) 373–381

size and all the mean values of all samples are tabulated in Table 1. It is well known that smaller nanoparticles having a high surface area interact more with bacteria compared to larger size particles [39]. Overall, AgNPs synthesized using L. camara leaf extract revealed the effective antibacterial property and can be used as a better antibacterial agent. 4. Conclusions Green synthesis of stable spherical AgNPs was demonstrated using L. camara leaf extract, where it acts as the reductant, size controller and stabilizer at room temperature. The obtained nanoparticles were well dispersed and antioxidized. The leaf extract quantity affects the size of the product critically and allows better control over the nanostructures. The functional groups responsible for the biosorption and bioreduction processes during the formation of AgNPs were identified through XPS and FTIR analysis. Both UV–vis and PL spectra elicited the blue shift, intended to the decrement of particle size with extract quantity. It is confirmed that the obtained nanoparticles are capable of rendering significant antibacterial efficacy and hence have potential applications in biomedical field. Finally, this protocol is simple, economic and raw material is also cheap, so it can be extendable to large scale commercial production of AgNPs. Acknowledgments One of the authors Ms. B. Ajitha would like to express her gratitude to the University Grants Commission (UGC letter No. F-5-99/2007 (BSR)), for awarding UGC-BSR Research Fellowship in sciences for meritorious students. References [1] R.O. Becker, J.A. Spadaro, Treatment of orthopaedic infections with electrically generated silver ions, J. Bone Joint Surg. 60-A (1978) 871–881. [2] H. Greathead, Plants and plant extracts for improving animal productivity, Proc. Nutr. Soc. 62 (2003) 279–290. [3] S.S. Shankar, A. Rai, A. Ahmad, M.J. Sastry, Rapid synthesis of Au, Ag and bimetallic Au shell nanoparticles using Neem, J. Colloid Interface Sci. 275 (2004) 496–502. [4] K.B. Narayanan, N. Sakthivel, Biological synthesis of metal nanoparticles by microbes, Adv. Colloid Interf. Sci. 156 (2010) 1–13. [5] H. Schneidewind, T. Schuler, K.K. Strelau, K. Weber, D. Cialla, M. Diegel, The morphology of silver nanoparticles prepared by enzyme-induced reduction, Beilstein J. Nanotechnol. 3 (2012) 404–414. [6] G.M. Sulaiman, W.H. Mohammed, T.R. Marzoog, A.A. Al-Amiery, A.A. Kadhum, A.B. Mohamad, G. Bagnati, Green synthesis, antimicrobial and cytotoxic effects of silver nanoparticles using Eucalyptus chapmaniana leaves extract, Asian Pac. J. Trop Biomed. 3 (2013) 58–63. [7] A.B. Vimalanathan, V. Tyagi, A. Rajesh, P. Devanand, M.G. Tyagi, Biosynthesis of silver nanoparticles using Chinese white ginseng plant root Panax ginseng, World J. Pharm. Pharm. Sci. 2 (2013) 2716–2725. [8] P. Kouvaris, A. Delimitis, V. Zaspalis, D. Papadopoulos, S.A. Tsipas, N. Michailidis, Green synthesis and characterization of silver nanoparticles produced using Arbutus unedo leaf extract, Mater. Lett. 76 (2012) 18–20. [9] V. Kumar, S.K. Yadav, Plant-mediated synthesis of silver and gold nanoparticles and their applications, J. Chem. Technol. Biotechnol. 84 (2009) 151–157. [10] N. Duran, P.D. Marcato, O.L. Alves, G. Souza, G.I.H. De Souza, E. Esposito, Mechanistic aspects of biosynthesis of silver nanoparticles by several Fusarium oxysporum strains, J Nanobiotechnol. 3 (2005) 8–14. [11] M. Kowshik, S. Ashtaputre, S. Kharrazi, W. Vogel, J. Urban, S.K. Kulkarni, K.M. Paknikar, Extracellular synthesis of silver nanoparticles by a silver-tolerant yeast strain MKY3, Nanotechnology 14 (2003) 95–100. [12] J. Kesharwani, K.Y. Yoon, J. Hwang, M. Rai, Phytofabrication of silver nanoparticles by leaf extract of Datura metel: hypothetical mechanism involved in synthesis, J. Bionanosci. 3 (2009) 39–44. [13] M.F. Zayed, W.H. Eisa, A.A. Shabaka, Malva parviflora extract assisted green synthesis of silver nanoparticles, Spectrochim. Acta A 98 (2012) 423–428.

381

[14] M. Sathishkumar, K. Sneha, S.W. Won, C.W. Cho, S. Kim, Y.S. Yun, Cinnamon zeylanicum bark extract and powder mediated green synthesis of nano-crystalline silver particles and its bactericidal activity, Colloids Surf. B 73 (2009) 332–338. [15] C. Krishnaraj, E.G. Jagan, S. Rajasekar, P. Selvakumar, P.T. Kalaichelvan, N. Mohan, Synthesis of silver nanoparticles using Acalypha indica leaf extracts and its antibacterial activity against water borne pathogens, Colloids Surf. B 76 (2010) 50–56. [16] S. Ashokkumar, S. Ravi, V. Kathiravan, S. Velmurugan, Synthesis, characterization and catalytic activity of silver nanoparticles using Tribulus terrestris leaf extract, Spectrochim. Acta A 121 (2014) 88–93. [17] W.R. Rajesh, R.L. Jaya, S.K. Niranjan, B.M.D.K. Sahebrao, Phytosynthesis of silver nanoparticle using Gliricidia sepium (Jacq.), Curr. Nanosci. 5 (2009) 117–122. [18] M. Karuppiah, R. Rajmohan, Green synthesis of silver nanoparticles using Ixora coccinea leaves extract, Mater. Lett. 97 (2013) 141–143. [19] E.L. Ghisalberti, Lantana camara L. (Verbenaceae), Fitoterapia 71 (2000) 467–486. [20] M.J. Deena, J.E. Thoppil, Antimicrobial activity of the essential oil of Lantana camara, Fitoterapia 71 (2000) 453–455 (D). [21] D. Ganjewala, S. Sam, K.H. Khan, Kham, Biochemical compositions and antibacterial activities of Lantana camara plants with yellow, lavender, red and white flowers, Eur. Asia J. Bio. Sci. 3 (2009) 69–77. [22] D. Bhakta, D. Ganjewala, Effect of leaf positions on total phenolics, flavonoids and proanthocyanidins content and antioxidant activities in Lantana camara (L), J. Sci. Res. 1 (2009) 363–369. [23] A.K. Mittal, Y. Chisti, U.C. Banerjee, Synthesis of metallic nanoparticles using plant extracts, Biotechnol. Adv. 31 (2013) 346–356. [24] J. Das, M.P. Das, P. Velusamy, Sesbania grandiflora leaf extract mediated green synthesis of antibacterial silver nanoaprticles against selected human pathogens, Spectrochim. Acta A 104 (2014) 265–270. [25] B.D. Cullity, Elements of X-ray Diffraction, Second ed. Addison-Wesley, MA, 1978. [26] M.F. Zayed, W.H. Eisa, Phoenix dactylifera L. leaf extract phytosynthesized gold nanoparticles; controlled synthesis and catalytic activity, Spectrochim. Acta A 121 (2014) 238–244. [27] P. Hu, X. Hu, C. Chen, D. Hou, Y. Huang, Biomaterial-assisted synthesis of AgCl@Ag concave cubes with efficient visible-light-driven photocatalytic activity, CrystEngComm 16 (2014) 649–653. [28] Z.P. Zhang, S.S. Feng, In vitro investigation on poly (lactide)-Tween 80 copolymer nanoparticles fabricated by dialysis method for chemotherapy, Biomacromolecules 7 (2006) 1139–1146. [29] M.T. Martinez, M.A. Callejas, A.M. Benito, M. Cochet, T. Seeger, A. Anson, J. Schreiber, C. Gordon, C. Marhic, O. Chauvet, J.L.G. Fierro, W.K. Maser, Sensitivity of single wall carbon nanotubes to oxidative processing: structural modification, intercalation and functionalisation, Carbon 41 (2003) 2247–2256. [30] J.L. Elechiguerra, L. Larios-Lopez, C. Liu, D. Garcia-Gutierrez, A. Camacho-Bragado, M.J. Yacaman, Corrosion at the nanoscale: the case of silver nanowires and nanoparticles, Chem. Mater. 17 (2005) 6042–6052. [31] G. Beamson, B.T. Pickup, W. Li, S.M. Mai, XPS studies of chain conformation in PEG, PTrMO, and PTMG linear polyethers, J. Phys. Chem. B 104 (2000) 2656–2672. [32] S.A. Aromal, D. Philip, Green synthesis of gold nanoparticles using Trigonella foenumgraecum and its size-dependent catalytic activity, Spectrochim. Acta A 97 (2012) 1–5. [33] K.B. Narayanan, N. Sakthivel, Green synthesis of biogenic metal nanoparticles by terrestrial and aquatic phototrophic and heterotrophic eukaryotes and biocompatible agents, Adv. Colloid Interf. Sci. 169 (2011) 59–79. [34] B. Salopek, D. Krasic, S. Filipovic, Measurement and application of zeta-potential, Rudarsko-Geolosko-Naftni Zbornik 4 (1992) 147–151. [35] C. Jayaseelan, R. Ramkumar, A.A. Rahuman, P. Perumal, Green synthesis of gold nanoparticles using seed aqueous extract of Abelmoschus esculentus and its antifungal activity, Ind. Crop. Prod. 45 (2013) 423–429. [36] C.O. Arean, G.T. Palomino, A.A. Tsyganenko, E. Garrone, Quantum chemical and FTIR spectroscopic studies on the linkage isomerism of carbon monoxide in alkali-metalexchanged zeolites: a review of current research, Int. J. Mol. Sci. 3 (2002) 764–776. [37] M. Noruzia, D. Zare, K. Khoshnevisan, D. Davoodi, Rapid green synthesis of gold nanoparticles using Rosa hybrida petal extract at room temperature, Spectrochim. Acta A 79 (2011) 1461–1465. [38] M.J. Hajipour, K.M. Fromm, A.A. Ashkarran, D.J.D. Aberasturi, I.R. Larramendi, T. Rojo, V. Serpooshan, W.J. Parak, M. Mahmoudi, Antibacterial properties of nanoparticles, Trends Biotechnol. 30 (2012) 499–511. [39] D. MubarakAli, N. Thajuddin, K. Jeganathan, M. Gunasekaran, Plant extract mediated synthesis of silver and gold nanoparticles and its antibacterial activity against clinically isolated pathogens, Colloids Surf. B 85 (2011) 360–365. [40] A. Azam, F. Ahmed, N. Arshi, M. Chaman, A.H. Naqvi, One step synthesis and characterization of gold nanoparticles and their antibacterial activities against E. coli (ATCC 25922 strain), Int. J. Theor. Appl. Sci. 1 (2009) 1–4.