Scholars Research Library Green synthesis of silver nanoparticles ...

Journal of Microbiology and Biotechnology Research

Scholars Research Library J. Microbiol. Biotech. Res., 2013, 3 (5):27-32

(http://scholarsresearchlibrary.com/archive.html) ISSN : 2231 –3168 CODEN (USA) : JMBRB4

Green synthesis of silver nanoparticles using leaf extract of Mangifera indica and evaluation of their antimicrobial activity Vikas Sarsar, Krishan K. Selwal* and Manjit K. Selwal Department of Biotechnology, Deenbandhu Chhotu Ram University of Science and Technology, Murthal, Sonipat, India _____________________________________________________________________________________________ ABSTRACT The synthesis of bio inspired nanoparticles is an important branch in nanotechnology. Different biological methods are used for the production of silver nanoparticles (SNP) due to their several applications, such as role of catalysts in chemical reactions, electrical batteries and in spectrally selective coatings for absorption of solar energy, as optical objectives, pharmaceutical constituents and in chemical sensing and biosensing. One of the most significant applications of silver nanoparticles is their use as an antimicrobial agent. In this work, wedescribe a cost effective and environment friendly approach to explore the synthesis of silver nanoparticles (SNP) from leaf extract ofMangiferaindica. The aqueous extract of the leaves of Mangifera indica used as reducing and stabilizing agents.The rapid reduction of silver ions was monitored by using UV-visible spectrophotometer. UV-visible spectrum of the aqueous state containing silver ions demonstrated a peak value at 440 nm. The antibacterial property of silver nanoparticles has allowed its wide range of application from disinfecting devices. The synthesized silver nanoparticlesshowedantibacterial activity against various human pathogens such as Escherichia coli, Staphylococcus aureus, Pseudomonas fragi, Bacillus subtilis, Streptococcus agalactiaeandProteus vulgaris. Key words: Nanoparticle, Nanotechnology,Antimicrobial, and Disinfectant _____________________________________________________________________________________________ INTRODUCTION Nanotechnology is referred to as the term for fabrication, characterization, manipulativeand application of structures by controlling shape and size at nano scale. Nanotechnology is one of the most active research areas in modern materials science and the synthesis of nanoparticles is gaining importance all over the world. The nanoparticles are significantly novel, improved physical, chemical, and biological properties due to their nano-scaled size (1-100nm). Silver nanoparticles have a very large surface area which typically results in greater biochemical reactivity, catalytic activity and atomicbehaviour compared to larger particles of the same chemical composition [1]. The synthesis of silver nanoparticles have received considerable attention due to their potential applications in catalysis [2], plasmonics [3], optoelectronics [4], biological sensor [5,6] antimicrobial activities [7,8], DNA sequencing [9], and surface-enhanced Raman scattering (SERS) [10].NPs has delivered solution to various problems like climate change and pollution control [11], clean water technology [12], energy generation [13], information storage [14] and biomedical applications [15]. The synthesis of NPs has been remarkable developments in the field of nanotechnology from the last ten years and proves its potential [16]. The nanoparticles synthesized by many physical and chemical methods which aretime consuming, costly and toxic for environment.The physical methods such as lithography and laser ablation[17]and the chemical methods start

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Krishan K. Selwal et al J. Microbiol. Biotech. Res., 2013, 3 (5):27-32 ______________________________________________________________________________ with silver salt precursor (dissolved in solvent) that is reduced in a chemical reaction and the nanoparticles are formed through nucleation and growth [18].The development of environmental friendly and sustainable techniques for the production of silver nanoparticles (AgNPs) to be used in medical field is a big challenge. Reports showed that it can be synthesised by bacteria [19], fungi [20]and plants [21].Earlier this decade, the potential of various plants for the synthesis of nanoparticles were explored. The presence of a large of phytochemicals, enzymes, proteins and other reducing agents with electron-shuttling compounds is usually involved in the synthesis of silver nanoparticles by plant extracts. Since then, various plants have been employed for the synthesis of nanoparticles.However, to the best of our knowledge no studies were ever done on Mangiferaindica (Mango) plant and have great abundance. In this study,Mangiferaindica leaf extract for rapid biosynthesis of SNPs and its antibacterial activity have been studied. MATERIALS AND METHODS Preparation of Leaf Extract Mangifera indica plantleaveswerecollectedfrom campus of DCRUST, Murthal, India.The collected leaves were cleaned under running tap water followed by double distilled water. The leaves were dried under shadow at room temp and then weighed 10gm of the dried leaves and cut them in to small pieces using a sharp knife. The small pieces of leaves were boiled in 100ml of double deionized water at 100oCin Erlenmeyer flask for 20 min. The boiled mixtures filtered through Whatman No.1 filter paper (pore size 25 µm).Now the plant extractsareready to use and stored at 40C for further use. Preparation of silver nitrate and synthesis of Silver Nanoparticles Silver nitrate (AgNO3) used in the present investigation is of Analytical grade purchased from Hi Media Laboratories Pvt. Mumbai, India.The aqueous solution 10-3 M silver nitrate was prepared in double deionized water in sterile amber colour bottle and kept at room temperature for 24 hours.For the reduction of silver ions, 10 mL of leaf extract was mixed to 90 mL of 10-3 M aqueous solution of AgNO3 in 250 mL Erlenmeyer flask and incubated at room temperature in dark. A change in colour from light yellows to yellowishbrownappearedindicates the excitation of surface plasmon resonance due to reduction of silver nitrate[22]. UV -Vis spectroscopy analysis UV-visible spectroscopy analysis was carried out by using UV-Visible absorption spectrophotometer (Systronic115) with a resolution of 5.0 nm between 200 to 700 nm.Thereduction of silverions in to metallic silver nanoparticlewas monitored by UV–Visible spectra of silver nanoparticles in aqueous solution. The interactions of these particles with light occur as electrons on the metal surface undergo oscillations when excited by light at specific wavelengths. The silver nanoparticles exhibit a unique peak in the range of 400–460 nm[23]. Antimicrobial activity The antibacterial activity of synthesized silver nanoparticles wasdeterminedbyusing the method of Ales Panaceket al., 2008 with some modifications. Agar well diffusion method was employed against pathogenic bacteria i.e. Staphylococcus aureus, Escherichia coli, Pseudomonas fragi, Bacillus subtilis, Streptococcus agalactiaeandProteus vulgaris. All the test cultureswere procured from the Microbial Type Culture Collection Center (MTCC), Chandigarh, India.The cultures were maintained at 4°C on nutrientagar (Hi-Media, India). Nutrient agar medium plates were prepared, sterilized and solidified. After solidification bacterial cultures were swabbed on these plates.Then the nanoparticle solution was poured into each well on all the plates with the help of micropipette (10 µl/ml).The plates were incubated at 37°Cfor 24 h and zone of inhibition was measured. RESULTS AND DISCUSSION Biosynthesis of silver nanoparticles Several approaches have been employed to obtain a better synthesis of silver nanoparticles such as chemical and biological methods.Plant provide a better platform for SNPs synthesis as they are free from toxic chemical and use of plant extracts also reduces the cost of microorganisms isolation and culture media enhancing the cost competitive feasibility over NPs synthesis by microorganisms.The other advantage of using plants for the synthesis of nanoparticles is that they are easily available, safe to handle and possess a broad variability of metabolites that may aid in reduction. In recent years plant mediated biological synthesis of nanoparticles is gaining importance due to its simplicity and eco friendliness. In our study the leaf extract of Mangifera indica was used for rapid biosynthesis of


Krishan K. Selwal et al J. Microbiol. Biotech. Res., Res. 2013, 3 (5):27-32 ______________________________________________________________________________ SNPs.. The reduction of silver ions into SNPs during exposure to the leaf extract of Mangiferaindicawas Mangifera specified by colour change. The light yellow colour solution changed into brownish yellow colour which indicates the formation of silver nanoparticles (Fig-1). The formation of brownish yellowcolour was developed within 5 to 25 min in all samples.

Fig. 1: Photograph of (a) Mangifera indica leaf extract and (b) after addition of silver nitrate

The colour became darker with the contact time of silver nitrate solution with the leaf extract (Fig-2). This colour was primarily due to surface plasmon resonance of deposited silver nanoparticles [24].. The leaf extract exhibited no change in colour until silver nitrate solution was not added even left for several week duration.

Fig. 2: photograph of mixture of Mangifera indica leaf extract and silver nitrate solution over 25 minutes time duration

UV-VIS spectral analysis The formation of SNPs was monitored by measuring the UV UV-Vis spectrum.The UV-Vis Vis spectrum for synthesis of silver nanoparticles from Mangifera indica has been recorded as a function of time (Fig 3). 3 Absorption spectra of Ag nanoparticles formed in the reaction media at 25min has has absorbance peak at 440nm broadening of peak indicated that the particles are dispersed. The weak absorption peak at shorter wavelengths may be due to the presence of several organic compounds/enzyme /enzyme which are known to interact with silver ion. The Mangifera indica shows an optical absorption band peak at 440nm, 44 typical of absorption for metallic silvernanoclusters, nanoclusters, due to the Surface Plasmon Resonance (SPR), which increased with time till reaction period. The intensity of the peak at 440 nm was increased with the time[23, 24].


Absorbance

Krishan K. Selwal et al J. Microbiol. Biotech. Res., 2013, 3 (5):27-32 ______________________________________________________________________________ 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

25 MIN 20 MIN 15 MIN 10 MIN 5MIN

Wavelength

Fig 3: Uv-Vis spectroscopy of silver nanoparticles synthesized from leaves of Mangifera indica along with contact time (leaf extract and metal ion concentration)(contact time 5,10,15,20,25 minute)

Antimicrobial Activity The synthesized SNPs show an effective antibacterial activity against pathogens of gram positive and gram negative bacteria.The result suggests that silver nanoparticles undergo an interaction with bacterial cell and displayed the strong action against Staphylococcus aureus, Escherichia coli, Pseudomonas fragi, Bacillus subtilis, Streptococcus agalactiaeandProteus vulgaris[25, 26].The mechanism of the bactericidal effect of silver colloid particles against bacteria is not very well-known. Silver nanoparticles may attach to the surface of the cell membrane and disturb its power function such as permeability and respiration. It is reasonable to state that the binding of the particles to the bacteria depends on the surface area available for interaction. Smaller particles having the larger surface area available for interaction will give more bactericidal effect than the larger particles [27]. NPs show an excellent bactericidal effect. It has been known for a long time that silver ions and silver compounds are highly toxic to most bacterial strains. Recently it was shown that the highly concentrated and nonhazardous nano-sized silver nanoparticles can easily be prepared in a cost effective manner and tested as a new type of bactericidal agents. In this study concentration of silver nanoparticles 10µl/ml was tested on bacterial strains. The formation of clear zone (restricted bacterial growth) around the cavity is an indication of antibacterial activity. The zone of inhibition of diameters was determined respectively(Graph 1).Silver ions have been demonstrated to interact with the protein and possibly phospholipids associated with the proton pump of bacterial membranes. ANTIMICROBIAL ACTIVITY OF SILVER NANOPARTICLES

ZONE OF INHIBITION IN MM

20 15 10 5 0 S. aureus

P. fragi

E. coli

B. subtilis S. aglactiae P. vulgaris

MICROORGANISMS

GRAPH-1: Antimicrobial activity of silver nanoparticles synthesized from leaf extract of Mangifera indica

Among the pathogens tested for antibacterial effect, the silver nanoparticles of Mangifera indicashowedhighest zone of inhibition against S. aureus and B. Subtilis(Fig.4 and Fig.5)[28].The mechanism depends on both the concentration of silver ions present and the sensitivity of the microbial species to silver ions. The use ofsilver


Krishan K. Selwal et al J. Microbiol. Biotech. Res., 2013, 3 (5):27-32 ______________________________________________________________________________ nanoparticles is effective against various microorganisms including plant pathogens. Application of silver nanoparticles in these fields is dependent on the ability of synthesis particles with different chemical composition, shape, size and nondispersity. These particles are chemically stable without undergoing degradation[29]. There are several physical and chemical methods for synthesis of metallic nanoparticles. Howeverdevelopment of simple and eco-friendly synthetic route would help promoting further interest in synthesis and application of metallic nanoparticles. Thus shows plant extracts have shown ability to reduce metal ions to form metallic nanoparticles.In this regard extracellular synthesis of silver nanoparticles achieved in study using plant leaf extracts may prove to be an important step in the right direction.

Figure 4: Antimicrobial activity shown by silver nanoparticles on S. Aureus

Figure IV: Antimicrobial activity shown by silver nanoparticles on B. Subtilis

CONCLUSION The silver nanoparticles were synthesized from leaf extract of Mangifera indica. The synthesized silver nanoparticles was confirmed by the change of colour of leaf extracts and characterized by UV-Vis spectroscopy. The synthesized silver nanoparticles showed potential anti-bacterial activity against both pathogenic and nonpathogenic gram positive and gram negative bacterial strains. Acknowledgments The first author wishes to thank University Grant Commission, New Delhiforprovidingthe junior research fellowship for the completion of the project. REFERENCES [1] K Govindaraju; S Tamilselvan; V Kiruthiga; G Singaravelu. J. Biopest,2010, 3, 394-399. [2] PV Kamat. J. Phy. Chem. B,2002, 106, 7729-7744. [3] SA Maier; ML Brongersma; PG Kik; S Meltzer; AAG Requicha; HA Atwater, Adv. Mat, 2001, 19, 1501-1505. [4] DH Gracias; J Tien; T Breen; C Hsu; Whitesides, G. M, Science, 2002, 289, 1170–1172.


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