Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 104 (2013) 265270

Contents lists available at SciVerse ScienceDirect

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

journal homepage: www.elsevier.com/locate/saa

Sesbania grandiora leaf extract mediated green synthesis of antibacterial silver nanoparticles against selected human pathogens
J. Das a, M. Paul Das b, P. Velusamy a,
a b

Department of Biotechnology, School of Bioengineering, SRM University, Kattankulathur, Chennai 603 203, Tamil Nadu, India Department of Industrial Biotechnology, Bharath University, Chennai 600 073, Tamil Nadu, India

h i g h l i g h t s
" Green synthesis of silver

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

nanoparticles is using Sesbania grandiora for the rst time. " Plant proteins are found to be active molecules for nanoparticles synthesis. " Spherical shaped nanoparticles were obtained that were similar to previous reports. " It shows effective antibacterial properties against MDR pathogens.

a r t i c l e

i n f o

a b s t r a c t
Simple, effective and rapid approach for the green synthesis of silver nanoparticles (AgNPs) using leaf extract of Sesbania grandiora and their in vitro antibacterial activity against selected human pathogens has been demonstrated in the study. Various instrumental techniques were adopted to characterize the synthesized AgNPs viz. UVVis, FTIR, XRD, TEM, EDX and AFM. Surface Plasmon spectra for AgNPs are centered at 422 nm with dark brown color. The synthesized AgNPs were found to be spherical in shape with size in the range of 1025 nm. The presence of water soluble proteins in the leaf extract was identied by FTIR which were found to be responsible for the reduction of silver ions (Ag+) to AgNPs. Moreover, the synthesized AgNPs showed potent antibacterial activity against multi-drug resistant (MDR) bacteria such as Salmonella enterica and Staphylococcus aureus. 2012 Elsevier B.V. All rights reserved.

Article history: Received 12 September 2012 Received in revised form 7 November 2012 Accepted 23 November 2012 Available online 5 December 2012 Keywords: Sesbania grandiora Green synthesis AgNPs TEM AFM

Introduction 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. Metal nanoparticles have been extensively studied due to their specic characteristics such as catalytic activity, optical properties, electronic properties, antimicrobial properties and magnetic properties [14].

Corresponding author. Mobile: +91 8939731191; fax: +91 442357824.

E-mail address: velusamy.p@ktr.srmuniv.ac.in (P. Velusamy). 1386-1425/$ - see front matter 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2012.11.075

There are number of methods used for the synthesis of silver nanoparticles (AgNPs) including physical and chemical methods [58], electrochemical reduction [9,10], photochemical reduction [11] and thermal evaporation [12,13]. However, rapid and green synthesis method using plant extract has developed enormous interest in AgNPs synthesis due to green chemistry approach. Moreover, it is simple, cost effective, eco-friendly, easily scaled up for large scale synthesis, without using toxic and redundant chemicals in solid, liquid and gaseous form [14]. Indeed, a number of bacteria [15], fungi [16] and yeast have been well-known for synthesis of non-toxic noble nanoparticles. However the microbial mediated synthesis of nanoparticles is not industrially feasible as it requires expensive medium and maintenance of highly aseptic conditions [17]. Hence, exploration of the plant systems as the

266

J. Das et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 104 (2013) 265270

potential bio-factories has gained heightened interest in the biological synthesis of nanoparticles. Sesbania grandiora L. (also known as agati, syn. Aeschynomene grandiora) belongs to the family Fabaceae. It is one of the most popular green vegetables and also traditional medicinal plants of India. S. grandiora has been known to have antibacterial, antifungal, antidiabetic, antioxidant and antitumorigenic activities [18,19]. Hence, the present study was aimed to rapidly green synthesize AgNPs using aqueous leaves extract of S. grandiora, to investigate the biomolecules responsible for synthesis of AgNPs and nally to evaluate antibacterial effect of AgNPs against clinical isolates of bacterial pathogens, Salmonella enterica and Staphylococcus aureus. To the best of our knowledge, the use of S. grandiora leaf extract for the green synthesis of noble nanoparticles, such as AgNPs has not been reported so far. Materials and methods Chemicals and plant collection

which was allowed to dry for an hour. The micrographic images of silver nanoparticles were observed and recorded in different range of magnications. The shape and size of the AgNPs was also determined using atomic force microscope (AFM). The AFM studies were carried out by drop coating the dispersion containing the particles onto a glass slide after required reaction time and scanning at a rate of 100 mV/s in the range 50 lm 50 lm using Nanosurf Easysurf 2 (Switzerland). Fourier transform infrared spectroscopy (FTIR) spectrum of the sample was recorded by Fourier transform infrared spectroscopy (Lambda 45, PerkinElmer, USA). The FTIR spectrum ranged from 4000 to 450 cm1 at a resolution of 4 cm1 by making a KBr pellet with AgNPs. Bacterial pathogens The clinical isolates of bacterial strains of S. enterica and S. aureus were obtained from SRM medical college hospital and research centre, Kattankulathur, Chennai. Antibacterial property of silver nanoparticles

Silver nitrate (AgNO3) from Merck (Darmstadt, Germany), Nutrient agar and Mueller-Hinton agar medium were purchased from Hi Media (Mumbai, India). Fresh leaves of S. grandiora have been harvested from crop eld of Potheri, Chennai, India. Preparation of S. grandiora aqueous leaf extract Aqueous extract of S. grandiora was prepared using freshly collected leaves. The leaves were surface cleaned with running tap water, followed by double sterilized distilled water and then shade dried for 5 days to completely remove the moisture. A ne powder was obtained from the dried leaves using a kitchen blender. The leaf powder (10 g) was taken and mixed with 100 ml of Milli Q water and kept in a water bath at 60 C for 10 min. The extracts were ltered through a nylon mesh (0.2 lm) and followed by Whatman No. 1 lter paper. The ltered extract was stored at 4 C for further studies. Synthesis of silver nanoparticles For synthesis of AgNPs, an Erlenmeyer ask containing 10 ml of the aqueous extract of S. grandiora was mixed with 90 ml of AgNO3 (1 mM) solution. This setup was incubated in dark room at 37 C to avoid the photoactivation of silver nitrate. A control setup was also maintained without S. grandiora leaf extract. Characterization of the synthesized silver nanoparticles Surface Plasmon resonance of AgNPs was characterized using PerkinElmer double beam spectrophotometer (Lambda 45, PerkinElmer, USA) at the resolution of 1 nm from 300 to 650 nm. X-ray diffraction pattern of dried nanoparticles was obtained from an XPert Pro A Analytical X-ray difractometer instrument with XPert high score plus software operated at a voltage 40 kV and a current 30 mA with Cu Ka radiation in a h2h conguration. The presence of elemental silver was determined using EDX (Zeiss Evo 50, Switzerland). For scanning electron microscope (SEM), the sample was prepared with a drop of colloidal solution of nanosilver on a carbon-coated copper grid and a setting completely dried by vacuum desiccator. The image of the sample was obtained using a scanning electron microscope (JEOL 6380A, Japan). The morphological analysis of the particle was done with a transmission electron microscopy (TEM) (JEOL 2100, Japan) instrument accelerating voltage of 80 keV equipped with EDX. A drop of aqueous AgNPs sample was loaded on the carbon coated copper grid

The antibacterial activity of AgNPs was investigated by the standard Kirby-Bauer disc diffusion assay method against multi-drug resistant (MDR) strain such as S. enterica and S. aureus [18]. The bacterial suspension of 24 h grown MDR strains was swabbed on Mueller Hinton agar (MHA) plates using sterile cotton swab. Double sterilized paper disc (6 mm diameter) was placed on MHA plates. Stock solutions of AgNPs (1 mg/ml) were prepared in sterile distilled water and loaded on to each disc required concentrations of 3 lg, 6 lg, 9 lg, 12 lg, 15 lg and 20 lg/ml. The plates were incubated at 37 C for 24 h. After the incubation period, the zone of inhibition was determined by measuring the diameter using Hi Media antibiotic zone scale. Statistical analysis All values of inhibition zone are expressed as means (standard deviation). The results were analyzed using one-way analysis of variance (ANOVA), and the differences in means were analyzed using the TukeyKramer multiple-comparison test. A P value of <0.001 was considered to be signicant. The software GraphPad InStat was employed for the statistical analysis. Results and discussion Recently, biosynthesis of nanoparticles has received considerable attention due to the urgent need to develop environmentally benign technologies in material sciences. For instance, a great deal of effort has been put into the biosynthesis of nanoparticles, especially metal nanoparticles using plants [20]. The use of plants and plant products as sustainable and renewable resources in the synthesis of nanoparticles are more advantageous over prokaryotic microbes, which need expensive methodologies for maintaining microbial cultures and downstream processing. UVVis spectra of silver nanoparticles There have been several methods for biosynthesis of AgNPs using leaf extracts different plants [21,22]. More recently, Krishnaraj and co-workers reported the synthesis of silver nanoparticles within 30 min of incubation period by using leaves extract of Acalypha indica [23]. Similarly, in the present study silver nanoparticles were synthesized using aqueous leaves extract of S. grandiora. Interestingly, AgNPs were synthesized within 1 hr incubation period after addition of leaf extract. The color of the

J. Das et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 104 (2013) 265270

267

reaction mixture changed from colorless to brown, which indicated the formation of AgNPs. It is well-known that AgNPs exhibit yellowish brown color in water due to excitation of surface Plasmon vibration in metal nanoparticles [23]. The excitation spectra of the AgNPs samples were characterized using UVVis spectroscopy, and this technique has proved to be very useful for the analysis of nanoparticles [24]. As shown in Fig. 1, it was observed that the appearance of peak at 422 nm corresponds to the absorption intensity and steadily increased as a function of time reaction without any shift in the peak position. The peak at 422 nm was due to strong surface Plasmon resonance, which indicates the formation of AgNPs. The optical properties of AgNPs are related to excitation of Plasmon resonance or inter band transition particularly on the size effect [24]. XRD analysis The crystalline nature of AgNPs was conrmed 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 which were in good agreement with reference of FCC structure from joint committee of powder diffraction standard (JCPDS) Card No-087-0720. TEM and EDX study The result of FESEM and TEM showed that the morphology of the AgNPs was observed as a spherical shape within 25 nm (Fig. 3a and c). The EDX spectra recorded from the silver nanoparticles are shown in (Fig. 3b). The EDX prole shows the presence of strong characteristic silver signal at approximately 3 keV, which is typical for the absorption of metallic silver nanocrystals due to surface Plasmon resonance. Along with this weak oxygen peaks, from the biomolecules bound to the surface of the silver nanoparticles and chloride peaks due to presence of chloride ions in the plant extract were also found. It has been reported that nanoparticles synthesized using plant extracts are surrounded by a thin layer of some capping organic material from the plant leaf broth and are, thus, stable in solution up to 6 months after synthesis [25]. This is another advantage of nanoparticles synthesized using plant extracts over those synthesized using chemical methods. The EDX spectrum shows the characteristic silver peaks on the surface of the nanoparticles, suggesting successful silver nanoparticles synthesis using plant leaf extract.

Counts

( 1 1 1)
300

200

( 2 0 0) (220)
100

( 3 1 1)

0 40 50 60 Position [2Theta] 70 80

Fig. 2. XRD patterns of the synthesized AgNPs from aqueous leaf extracts of S. grandiora.

AFM analysis The surface morphology of the AgNPs was recorded using AFM. The two- and three- dimensional topography of the AgNPs was shown in Fig. 4a. Direct observation of the image revealed that the size of many of the AgNPs was in the order of 1025 nm. The particles appeared to be spherical in shape. The size distribution of the AgNPs was shown in Fig. 4b, which clearly indicates that the majority of the AgNPs fell in the average range of 1020 nm. The data obtained from the AFM correlated well with that of FESEM and TEM results. FTIR analysis of silver nanoparticles The FTIR spectroscopy is used to identify a major compound which is responsible for the biological reduction of silver ions (Ag+) into silver nanoparticles (Ag) from the leaf extract of S. grandiora. The IR spectrum of leaf extract alone showed the distinct peak in the range of 1075, 1245, 1381, 1561, 1628, 2919 and 3419 cm1 (Fig. 5a). The absorption peak located at around 1075 cm1 can be assigned as the absorption peak of ACAOACA or ACAOA [26]. Few less intense peaks centered at 1245 cm1, 1561 cm1 and 2919 cm1 are probably due to presence of CAH deformation vibration, stretching vibration of AC@CA and secondary amines respectively [26,27]. Moreover, strong intense peaks at 1381 cm1 and 1628 cm1 was mainly attributed to the CAN stretching vibrations as well as amide I bands of proteins [27,28] present in the leaf. In addition, there is a broad peak located at 3419 cm1, which can be assigned to the OAH stretching vibrations, indicating the presence of hydroxyl groups [26]. Further the FTIR spectrum of silver nanoparticles showed the distinct peak in the range of 1072, 1248, 1383, 1556, 1632, 2923 and 3417 cm1 (Fig. 5b). The comparison of FTIR spectrum between the leaf extract and silver nanoparticles showed only minor changes in the position as well as the absorption bands. This indicates that silver nanoparticles synthesized using the S. grandiora leaf extract are capped by proteins having functional groups of amines, alcohols, ketones and carboxylic acid. Antibacterial effect of AgNPs

2.0 1.8 1.6

8 hr

Absorbance (a.u.)

1.4 1.2 1.0

4 hr

2 hr
0.8 0.6

1 hr
0.4 0.2 300 350 400 450 500 550 600

8hr 4hr 2hr 1hr

650

Wavelength (nm)
Fig. 1. UVVis spectrum of aqueous leaf extract of S. grandiora that showed the production of AgNPs at different time intervals.

Antibacterial activity was investigated against S. enterica (Gram negative) and S. aureus (Gram positive) by disc diffusion method. The diameter of the zone of inhibition in millimeter around each well in different concentration level of AgNPs was determined

268

J. Das et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 104 (2013) 265270

Fig. 3. The images showing the shape and size of AgNPs by FESEM (a), EDX spectrum (b) and TEM (c).

Fig. 4. AFM Characterization of AgNPs. (a) Surface morphology. (b) Histogram analysis.

J. Das et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 104 (2013) 265270 Table 1 Growth inhibition of various pathogenic bacteria by AgNPs. Concentration of AgNPs (lg/ml)a Zone of inhibition (mm) (mean of three replicates) Salmonella enterica 3 6 9 12 15 20
a

269

Staphylococcus aureus 6.33 0.03 6.93 0.58 7.40 0.23 8.32 0.58 9.32 0.33 10.54 0.23

10.01 0.31 10.84 0.05 11.95 0.33 12.67 0.25 14.87 0.14 15.67 0.09

A stock solution at 1 mg/ml was used.

Fig. 5. FTIR spectrum: (a) leaf extracts (alone) and (b) synthesized AgNPs from leaf extract of S. grandiora.

cellular ATP. Sarkar et al. [35] reported that for S. aureus, silver nanoparticles demonstrated greater bactericidal efciency compared to penicillin. Silver has a greater afnity to react with sulfur or phosphorus-containing biomolecules in the cell [36]. Thus sulfur containing proteins in the membrane or inside the cells and phosphorus-containing elements like DNA are likely to be the preferential sites for silver nanoparticles binding [37,38] which leads to cell death.

(Fig. 6). The mean inhibitory zone of the three replicates of diameter was measured and tabulated (Table 1). A previously reported article stated that inhibitory concentrations values of AgNPs against S. enterica and S. aureus showed excellent antimicrobial activity by disc diffusion method [29]. The Gram negative bacterium S. enterica showed maximum zone of inhibition whereas the Gram positive bacterium showed lower zone of inhibition. This could be due to the presence of thicker peptidoglycan layer in Gram positive than Gram negative bacteria preventing the entry of AgNPs and its antimicrobial activity [30]. The high bactericidal activity is certainly due to the silver cations released from AgNPs that act as reservoirs for the Ag+ bactericidal agent. Big changes in the membrane structure of bacteria as a result of the interaction with silver cations lead to the increased membrane permeability of the bacteria [31,32]. Lin et al. [33] explained that in general, silver ions from AgNPs are believed to become attached to the negatively charged bacterial cell wall and rupture it, which leads to denaturation of protein and nally cell death. The attachment of either silver ions or nanoparticles to the cell wall causes accumulation of envelope protein precursors, which results in dissipation of the proton motive force. On the other hand, Lok [34] elucidated that AgNPs exhibited destabilization of the outer membrane and rupture of the plasma membrane, thereby causing depletion of intra-

Conclusion In this study, we have demonstrated that use of leaf extract of S. grandiora as a reducing agent can effectively produce spherical shape silver nanoparticles. The synthesized silver nanoparticles using leaf extract of S. grandiora proved excellent antibacterial activity against clinically isolated multi-drug resistant human pathogens. The antibacterial activity of silver nanoparticles was well demonstrated by the clear zone of inhibition against S. enterica and S. aureus. The powder diffraction study showed that the face centered cubic silver nanoparticles were stable after 6 months in room temperature. TEM and AFM studies revealed spherical shaped nanoparticles with size in the range of 1025 nm. Moreover biological synthesis of silver nanoparticles using plant material could be a conventional and eco-friendly method compared to chemical and physical synthesis.

Acknowledgments The authors convey their thanks to SRM University, Chennai, for providing laboratory facilities. Thanks are also due to Dean, School

Fig. 6. Antibacterial activity of AgNPs against multi-drug resistant pathogens of S. enterica and S. aureus.

270

J. Das et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 104 (2013) 265270 [19] R. China, S. Mukherjee, S. Sen, S. Bose, S. Datta, H. Koley, S. Ghosh, P. Dhar, Microbiol. Res. 167 (2012) 500506. [20] J.R. Lloyd, J.M. Byrne, V.S. Coker, Curr. Opin. Biotechnol. 22 (2011) 509515. [21] D. Philip, Spectrochim Acta A 78 (2011) 327331. [22] G. Rajakumar, A.A. Rahuman, Acta Trop. 118 (2011) 196200. [23] K.B. Narayanan, N. Sakthivel, Adv. Colloid Interface Sci. 169 (2011) 5979. [24] C. Krishnaraj, E.G. Jagan, S. Rajasekar, P. Selvakumar, P.T. Kalaichelvan, N. Mohan, Colloids Surf. B 76 (2010) 5056. [25] A. Ahmad, S. Senapati, M. Islam Khan, R. Kumar, R. Ramani, V. Srinivas, M. Sastry, Nanotechnology 14 (2003) 824828. [26] L. Lin, W. Wang, J. Haung, Q. Li, D. sun, X. Yang, H. Wang, N. He, Y. Wang, J. Chem. Eng. 162 (2010) 852858. [27] J.Y. Song, H.-K. Jang, B.S. Kim, Process Biochem. 44 (2009) 11331138. [28] N. Jain, A. Bhargava, S. Majumdar, J.C. Tarafdar, J. Panwar, Nanoscale 3 (2011) 635641. [29] V. Gopinath, D. MubaralAli, S. Priyadarshini, N.M. Priyadharsshini, N. Thajuddin, P. Velusamy, Colloids Surf. B 96 (2012) 6974. [30] S. Shrivastava, T. Bera, A. Roy, G. Singh, P. Ramachandrarao, D. Dash, Nanotechnology 18 (2007) 225103225111. [31] P. Dibrov, J. Dzioba, K.K. Gosink, C.C. Hase, Antimicrob. Agents Chemother. 46 (2002) 26682670. [32] I. Sondi, B. Salopek-Sondi, J. Colloid Interface Sci. 275 (2004) 177182. [33] Y.E. Lin, R.D. Vidic, J.E. Stout, C.A. McCartney, V.L. Yu, Water Res. 32 (1998) 19972000. [34] C. Lok, J. Proteome Res. 5 (2006) 916924. [35] S. Sarkar, A.D. Jana, S.K. Samanta, G. Mostafa, Polyhedron 26 (2007) 4419 4426. [36] M.J. Hajipour, K.M. Fromm, A. Akbar Ashkarran, D. Jimenez de Aberasturi, I.R. Larramendi, T. Rojo, V. Serpooshan, W.J. Parak, M. Mahmoudi, Trends Biotechnol. 30 (2012) 499511. [37] G. McDonell, A.D. Russell, Clin. Microbiol. Rev. 12 (1999) 147178. [38] D. MubarakAli, N. Thajuddin, K. Jeganathan, M. Gunasekaran, Colloids Surf. B 85 (2011) 360365.

of Bioengineering, SRM University, Chennai for providing valuable discussion and facilities. References
[1] H. Bar, D.K. Bhui, G.P. Sahoo, P. Sarkar, S.P. De, A. Misra, Colloids Surf. A 339 (2009) 134139. [2] T. Tuutijrvi, J. Lu, M. Sillanp, G.J. Chen, Hazard. Mater. 166 (2009) 1415 1420. [3] V. Gopinath, S. Priyadarshini, N. Meera Priyadharsshini, K. Pandian, P. Velusamy, Mater. Lett. 91 (2013) 224227. [4] L. Rassaei, M. Sillanp, R.W. French, R.G. Compton, F. Marken, Electroanalysis 20 (2008) 12861292. [5] D.-G. Yu, Colloids Surf. B 59 (2007) 171178. [6] Y. Tan, Y. Wang, L. Jiang, J. Colloid Interface Sci. 249 (2002) 336345. [7] C. Petit, P. Lixon, M.P. Pileni, J. Phys. Chem. 97 (1993) 1297412983. [8] S.A. Vorobyova, A.I. Lesnikovich, N.S. Sobal, Colloids Surf. A 152 (1999) 375 379. [9] Y.C. Liu, L.H. Lin, Electrochem. Commun. 6 (2004) 11631168. [10] G. Sandmann, H. Dietz, W. Plieth, J. Electroanal. Chem. 491 (2000) 7886. [11] K. Mallick, M.J. Witcombb, M.S. Scurrella, Mater. Chem. Phys. 90 (2005) 221 224. [12] C.H. Bae, S.H. Nam, S.M. Park, Appl. Surf. Sci. 197 (2002) 628634. [13] A.B. Smetana, K.J. Klabunde, C.M. Sorensen, J. Colloid Interface Sci. 284 (2005) 521526. [14] A. Saxena, R.M. Tripathi, R.P. Singh, Dig. J. Nanomater. Bios. 5 (2010) 427432. [15] S. Priyadarshini, V. Gopinath, N. Meera Priyadharsshini, D. MubarakAli, P. Velusamy, Colloids Surf. B 102 (2013) 232237. [16] A.M. Fayaz, M. Girilal, M. Rahman, R. Venkatesan, P.T. Kalaichelvan, Process Biochem. 46 (2011) 19581962. [17] M. Sathishkumar, K. Sneha, I.S. Kwak, J. Mao, S.J. Tripathy, Y.S. Yun, J. Hazard. Mater. 171 (2009) 400404. [18] K.P. Laldhas, V.T. Cheriyan, V.T. Puliappadamba, S.V. Bava, R.G. Unnithan, P.L. Vijayammal, R.J. Anto, J. Cell Mol. Med. 14 (2010) 636646.