Scholars Research Library. Synthesis of silver nanoparticles from leaf extracts of Wedelia chinensis (Osbeck) Merrill and their antimicrobial activity

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1 Journal of Microbiology and Biotechnology Research Scholars Research Library J. Microbiol. Biotech. Res., 2013, 3 (5):48-53 ( ISSN : CODEN (USA) : JMBRB4 Synthesis of silver nanoparticles from leaf extracts of Wedelia chinensis (Osbeck) Merrill and their antimicrobial activity Priyanka Kamath C., Rama Bhat P.*, Jenitta Emima Packiyam Post Graduate Department of Biotechnology, Alva s College, Moodbidri, Karnataka, India ABSTRACT Wedelia chinensis is a reputed herbal medicine in Ayurvedic, Siddha and Unani system of medicine. The pharmacological studies reported about the leaves of this herb that can be used for treatment of various disease ailments. In the present investigation aqueous plant extracts was used to prepare nanoparticles. After characterization of nanoparticles, were used to study antibacterial and antifungal activities against Escherichia coli, Bacillus subtilis, Proteus mirabilis, Staphylococcus aureus, Penicillium notatum and Aspergillus niger. The silver nanoparticles were synthesized from leaf extract of W. chinensis using dried leaf powder mixed with the aqueous solution of 1mM silver nitrate solution. Ultraviolet-visible spectroscopy, Fourier Transform Infrared Spectroscopy (FTIR) and Scanning electron microscopy (SEM) were used to characterize the Ag nanoparticles. The nanoparticle showed a peak at 358 nm. The FTIR studies also showed five peaks for different groups. The SEM study showed that the nanoparticles are hexagonal shape with a diameter of nm. All the tested microbial strains showed antimicrobial activity. Keywords: Silver nanoparticles, Wedelia chinensis, FTIR, SEM. INTRODUCTION Nanotechnology is emerging as a cutting edge technology interdisciplinary with biology, chemistry and material science. It involves the creation of useful and functional materials, devices and systems by the control of matter on atomic and molecular size [1]. Nanoparticles are a special group of materials with unique features and extensive applications in diverse fields. It provides the ability to engineer the properties of materials by controlling their size and this made for the optical properties, catalytic activity, magnetic properties and antibacterial properties [2]. Silver nanoparticles are non-toxic to humans and most effective against bacteria, virus and other eukaryotic microorganisms at low concentrations and without any side effects [3]. Increasing common application is the use of silver nanoparticles for antimicrobial coatings and many textiles, keyboards, wound dressings and biomedical devices now contain silver nanoparticles that provide protection against bacteria. Large amount of nanoparticles can be easily synthesized from plant and the majority of these are non-toxic. These have been used for inhibiting the bacteria and fungi, and preventing burns and wound infection. In the recent years, plant-mediated biological synthesis of nanoparticles is gaining importance due to its simplicity and eco-friendliness. There are some reports on synthesis of nanoparticles from stem and leaf extracts [4-7] and studies on the biological activities like antimicrobial activity of these silver nanoparticles [8-11]. But still there are some plants whose properties are yet to be explored. One among such plant was Wedelia chinensis belonging to Asteraceae family. The plant is used in the treatment of kidney dysfunction and cold. The methanol extract can be used for antioxidant activity, antiulcerogenic and mucosal protective agent and also used as antimicrobial agent. The main objective of the present study was to synthesis of silver nanoparticles from Wedelia chinensis. The synthesized nanoparticles were characterized using Ultravioletvisible spectroscopy, Fourier transform infrared spectroscopy (FTIR) and Scanning electron microscopy (SEM). Biologically synthesized nanoparticles are tested against selected bacterial and fungal strains to study its antimicrobial activity. MATERIALS AND METHODS Plant material Wedelia chinensis was collected from local village near Karkala taluk of Dakshina Kannada, Karnataka and maintained in pots. It is a scrabous procumbent perennial soft herb, m long, stem procumbent at the base and rooting at the lower nodes, more or less appressed hairy with camphor like odour. Leaves opposite, subsessile, oblong, entire or irregularly crenate-serrate, scabrous with short white hairs. The flowers are yellow in colour. Isolation of Bacterial culture The bacterial test organisms used for the present study were Escherichia coli, Bacillus subtilis, Proteus mirabilis and Staphylococcus aureus. The bacterial cultures were collected from the Alva s College of Medical Laboratory Technology, Moodbidri. The isolates were subcultured in slants on nutrient agar medium and maintained in the PG Dept. of Biotechnology. 48

2 Isolation of Fungal Culture The fungal test organisms used in this study were Aspergillus niger and Penicillium notatum. The fungal cultures were isolated and identified in the laboratory of Department of Biotechnology using relevant manuals, were subcultured on potato dextrose agar medium and maintained in PDA slants. Microscopic observation and Colony characterization The bacteria and fungi were microscopically characterized and bacterial and fungal isolates were observed using hand lens and the colony morphology was recorded with respect to color, shape, size and nature of colony. Preparation of the plant extracts Fresh leaves of Wedelia chinensis were collected washed with distilled water to remove dirt and it is shade-dried for 15 days. These are kept in hot air oven at 60 C for 48 hours. The dried leaves were ground into fine powder and passed through 40-mesh sieve. The powder was stored in an air tight container for further use. Synthesis of silver nanoparticles For the synthesis of silver nanoparticles, 1mM aqueous extract of silver nitrate was prepared. Exactly 3g of leaf powder was mixed with 350ml of silver nitrate solution and centrifuged at 2000rpm for 25 minutes. The supernatant were collected and heated at 95 C. A change in the colour of the solution was observed after heating the mixture for 2 hours. The extracts were stored at 4 C for further use. Characterization The characterizations of synthesized silver nanoparticles were done to understand the characteristic wavelength, to recognize the functional groups bound to the silver and size of the nanoparticles. It was done by using Ultravioletvisible spectroscopy, Fourier Transform Infrared Spectroscopy (FTIR) and Scanning electron microscopic (SEM). Ultraviolet-Visible Spectroscopic analysis The bioreduction of silver ions in solution was monitored by measuring the sample in UV- visible spectrophotometer. The optical absorbance was recorded in nm wave length range. Fourier Transform Infrared Spectroscopy (FTIR) The lyophilized powder sample was used and examined by Infra red (IR) spectrum at the spectral range of cm -1 by Fourier Transform Infrared Spectroscopy to recognize the functional groups bound to the silver surface. Scanning electron microscopic (SEM) Scanning electron microscopic (SEM) analysis was done by preparing a thin film of the sample on a carbon coated copper grid by just dropping a very small amount of sample on the grid and allowed it to dry by putting it under mercury lamp for 5 minutes. Determination of antimicrobial activity Well diffusion assay Nutrient Agar medium and Potato dextrose agar (PDA) medium was prepared and autoclaved. After solidification, 24 hour old culture of each of the test organism` grown in Agar medium and PDA medium was swabbed. Five wells of diameter of 10mm diameter were punched into each swabbed plates. Different concentrations of 50, 100 and 150µl of synthesized silver nanoparticles were added along with reference drug (Kanamycin) for bacterial plates and (Fluconazole) for fungal plates and silver nitrate solution as positive and negative control. Plates were incubated at 37 C (bacterial plates) and at room temperature 28 C (fungal plates) for 24 hours. The plates were observed for inhibition zones and the diameters of the inhibition zone were measured. RESULTS AND DISCUSSION A B Fig. 1A: Wedelia chinensis, B: Colour change of leaf extracts containing silver before and after synthesis of silver nanoparticlas Synthesis of silver nanoparticles The green synthesis of silver nanoparticles through plant extract was carried out. It is well known that silver nanaoparticles exhibit yellowish brown colour in aqueous solution due to excitation of surface Plasmon vibration in silver nanoparticles [12]. In Wedelia, leaf extracts can be easily monitored from the change in the colour of the 49

3 reaction mixture from yellowish brown to dark brown (Fig. 1B). The time duration of change in colour varies from plant to plant. Wedelia synthesized silver nanoparticles after 1 hour of incubation which indicated the formation of silver nanoparticles (Fig. 2). This formation indicates that the silver ions in reaction mixture medium have been converted to elemental silver having the size of nanometric range. Ultraviolet-Visible (UV-VIS) Spectroscopic analysis It is generally recognized that UV visible spectroscopy could be used to examine size- and shape-controlled nanoparticles in aqueous suspensions [13]. SNP s have free electrons, which give rise to an SPR absorption band [14], due to the combined vibration of electrons of metal nanoparticles in resonance with the light wave [15,16]. Silver nanoparticles are known to exhibit a UV-visible absorption maximum in the range of nm. In the present study absorption peak was observed at 358nm which is a characteristic of silver nanoparticles (Fig. 2). Fig. 2: UV-VIS absorption spectra of silver nanoparticlee synthesized from Wedelia chinensis leaves at 1Mm silver nitrate Fourier Transform Infrared Spectroscopy (FTIR) Saifuddin et al. [17] explained that the FTIR measurement to identify the possible biomolecules responsible for capping and efficient stabilization of the metal nanoparticles synthesized in leaf broth. In the present study the lyophilized nanoparticle samples were analyzed in FTIR to identify the possible bio-molecules responsible for the reduction of the silver ions by cell filtrate. The representative spectra of nanoparticles obtained manifests absorption peaks using the spectral range between cm -1 (Fig. 3). The absorption peaks were observed at 3708 cm -1, cm -1, cm -1, cm -1 and cm -1 can be assigned as absorption bands of NH group OH group of phenols, C-H aromatic stretch of groups, -NHCO group of amides and C-Cl functional groups. The FTIR analysis supported the reducing property of silver nanoparticles synthesized by Wedelia chinensis leaf extract which in turn imparted the high stability of the synthesized silver nanoparticles. Fig. 3: FTIR analysis of silver nanoparticles biosynthesis using W. chinensis leaf extract Scanning electron microscopic (SEM) study The SEM image has been employed to characterize the size, shape and morphology of synthesized silver nanoparticles. From the SEM image of synthesized silver nanoparticles, it is evident that the morphology of the synthesized silver nanoparticles are hexagonal in shape with the diameter range of nm (Fig. 4). Ankamwar [18] observed relatively spherical shaped nanoparticles. The SEM image showed the high density silver nanoparticles synthesized by the A. paniculata development of silver nano structures [19]. The diameter size in our study confirms the presence of nanoparticles have been synthesized. 50

4 Fig. 4: Scanning Electron Microscopy (SEM) study silver nanoparticles are hexagonal in shape with the diameter range of nm Determination of antibacterial activity Antibacterial activity of biogenic silver nanoparticles was examined against bacterial strains by using standard zone of inhibition. The synthesized nanoparticles showed inhibition zone against Staphylococcus aureus and Bacillus subtilis, Gram negative bacteria Escherichia coli and Proteus mirabilis. Different concentrations of 50µl, 100µl and 150µl of synthesized silver nanoparticles were used for the study of antibacterial activity (Figs. 5 i (a), 5(b), 5(c), 5(d), 5 (ii) are indicative of the diameters of zones of inhibition due to microbial susceptibility. 150µl concentration showed highest maximum zone of inhibition against test organisms (Escherichia coli, Bacillus subtilis, Proteus mirabilis and Staphylococcus aureus). Maximum zone of inhibition of 25mm showed against Escherichia coli. In 100 µl concentration of silver nanoparticles showed maximum zone of inhibition of 11mm against Proteus mirabilis and 50µl concentration of synthesized silver nanoparticles showed maximum zone of inhibition of 12mm against Staphylococcus aureus. Antibacterial potential of silver is known since many years [20]. The use of plant extracts is effective against various microorganism [21]. Ahmad et al. [22] mentioned that the pathogenic effect of nanoparticles can be attributed to their stability in the medium as a colloid, which modulates the phosphotyrosine profile of the pathogen proteins and arrests its growth. The SNPs synthesized from plant species are toxic to multidrug resistant microorganisms. From this study it was clear that they have great potential in biomedical applications. 5(a) Escherichia coli 5(b) Bacillus subtilis 5(c) Proteus mirabilis 5 (d) Staphylococcus aureus Fig. 5 (a-d): Antibacterial activity of silver nanoparticles synthesized from Wedelia chinensis leaf extracts 51

5 Rama Bhat P et al J. Microbiol. Biotech. Res., 2013, 3 (5): µl Determination of antibacterial activity 100µl 150µl Reference drug Silver nitrate solution Escherichia coli Bacillus subtilis proteus mirabilis Staphylococcus aureus Fig. 5 (i): Antibacterial activity of silver nanoparticles synthesized from Wedelia chinensis leaf extracts Determination of antifungal activity Antifungal activity of biogenic silver nanoparticles was done against Aspergillus niger and Penicillium notatum.. Different concentrations of 50µl, 100µl and 150 µl of synthesized silver nanoparticles were used for the study of antifungal activity, are indicative of the diameters of zones of inhibition due to microbial susceptibility (Figs. 6 i (a- b), 6(ii). 100µl concentration tion of synthesized silver nanoparticles showed maximum zone of inhibition of 19mm against Penicilllium notatum, whereas for Asperigillus niger maximum zone of inhibition of 17mm showed for 150µl concentration of silver nanoparticles. 6(a) Penicillium notatum 6(b) Aspergillus niger Fig. 6(ii): Antifungal activity of silver nanoparticles synthesized from W. chinensis leaf extracts CONCLUSION Synthesis of silver nanoparticles from the leaf of Wedelia chinensis was confirmed by colour change from yellow to dark brown. Which indicate the formation of silver nanoparticles. Therefore, the growing need of developing a ecofriendly nanoparticle synthesis is possible and it can be used for various medical applications. Acknowledgement The authors are grateful to Dr. R. Ragunathan, Principal, Maharaja Co-Education College, Erode, Tamil Nadu, India for providing SEM and FTIR facilities. REFERENCES [1] Probin Phanjom; Azmin Sultana; Himakshi Sarma; Jahnabi; Ramchiary; Kongkana Goswami, Baishya. Digest Journal of Nanomaterials and Biostructures, 2012, 7, [2] Debarat Baishya, Nakul Sharma. Archeives of Applied Science Research, 2012, 4, Pitambar 52

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