International Journal of Pharma and Bio Sciences
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1 Research Article Nanotechnology International Journal of Pharma and Bio Sciences ISSN BIOGENESIS OF SILVER NANOPARTICLES USING ALTERNANTHERA FICOIDEA LEAF EXTRACT AND ITS ANTIBACTERIAL POTENTIAL MRINAL KUMAR BAISHYA 1, DEBABRAT BAISHYA 2* AND MOHAN CHANDRA 3 KALITA 1 Department of Biotechnology, Gauhati University, Guwahati , Assam, India 2 Department of Bioengineering and Technology, Institute of Science and Technology, Gauhati University, Guwahati , Assam, India 3 Department of Biotechnology, Gauhati University, Guwahati , Assam, India ABSTRACT The synthesis of nanomaterials is a cornerstone of nanotechnology. Most of the synthesis methods encompassing chemical routes have led to environmental pollution, especially the bi-products associated with metal production. Here we report an environmentally benign and cost effective approach in synthesizing silver nanoparticles using leaf extracts of Alternanthera ficoidea. The surface plasmon resonance of silver nanoparticles produced a peak centered near 410nm was characterized by UV-Visible spectrophotometer. Silver nanoparticles were circular in shape with maximum particles in size range within 4 22 nm as observed under Transmission electron microscope (TEM). As-synthesized silver nanoparticles were also found to inhibit the growth of two gram negative clinical bacterial isolates Escherichia coli, and Serratia marcescens. KEYWORDS: Alternanthera ficoidea, silver nanoparticles, transmission electron microscope, antibacterial potential DEBABRAT BAISHYA Department of Bioengineering and Technology, Institute of Science and Technology, Gauhati University, Guwahati , Assam, India *Corresponding author P - 104
2 INTRODUCTION The field of nanotechnology is one of the most active research areas in modern material science. There have been impressive developments in the field of nanotechnology in the recent past years, with numerous methodologies developed to synthesize nanoparticles of particular shape and size depending on specific requirements. Moreover, new applications of nanomaterials are emerging rapidly. Silver nanoparticles are one of the promising products in the nanotechnology industry. The development of consistent processes for the synthesis of silver nanomaterials is an important aspect of current nanotechnology research. The methods currently being used encompass chemical routes. A big impediment to encouragement of these methods is that the byproducts associated with metal production have become a great concern with respect to environmental pollution, additionally; some of these processes are expensive. In addition, the synthetic procedures involve conditions such as high temperature, pressure and environmental inertness, which are cost intensive 1. Ever increasing pressure to develop environmentally benign technique for nanoparticle synthesis has lead to a renewed interest in biotransformation as a route to growth of nano-scale structures. Biological systems have a unique ability to control the structure, phase and nano-structural topography of the inorganic crystals 2. It is well known that microbes such as bacteria 3,4, Yeast 5, fungi 6,7 and algae 8,9 are able to adsorb and accumulate metals and can be used in the reduction of environmental pollution and also for the recovery of metals from waste. The biosynthetic method employing plant extracts have drawn attention as a simple and viable alternative to chemical procedures and physical methods. Bioreduction of gold and silver ions to yield metal nanoparticles using living plants 10,11, Geranium leaf broth 12, Neem leaf broth 13 lemongrass extract 14, Tamarind leaf extract 15 and Aloe vera plant extracts 16 have been reported. In the present study A. ficoidea leaf extract was used to synthesize silver nanoparticles. As-synthesized nanoparticles were then tested for its antibacterial activity. MATERIALS AND METHODS (i) Collection of chemicals and plant material: Silver nitrate was purchased from HiMedia Laboratories Pvt. Ltd., Mumbai. All other chemicals were purchased from Merck, Mumbai. The plant material i.e Alternanthera ficoidea was collected from the Botanical garden of Gauhati University. The bacterial strains were obtained from Guwahati Medical College Hospital, Guwahati, Assam. (ii) Preparation of Alternanthera leaf extract 20 g fresh leaves of Alternanthera ficoidea were collected and washed three times with distilled water to remove dust particles. Leaves were finely chopped and added to 100 ml of ethanol and filtered with Whatman paper No. 1. Filtrate was collected and was stored at 4 0 C for further analysis. (iii) Biosynthesis of Silver nanoparticles 550 µl of leaf extract was added to 2.45 ml of 3mM AgNO 3 solution at room temperature. The mixture was then kept in the dark and change in the colour was noted. (iv) Characterization of prepared nanoparticles To observe the optical property of biosynthesized silver nanoparticles, samples were analyzed for UV VIS spectroscopic studies (SHIMEDZU-1800) at room temperature operated at a resolution of 1 nm between 250 and 500 nm ranges.. Characterization of size and shape of biosynthesized silver nanoparticles was done by Transmission electron microscopy (TEM, Philips CM 200). The sample was first sonicated (Vibronics VS 80) for 15 min. A drop of this solution was loaded on carbon-coated copper grids, and solvent was allowed to evaporate under Infrared light for 30 min. TEM was operated at an accelerating voltage of 200 kv. P - 105
3 (v) Antimicrobial Activity The silver nanoparticles synthesized using Alternanthera ficoidea leaf extract was tested for antimicrobial activity by agar well diffusion method against two clinical isolates Escherichia coli, and Serratia marcescens. The pure cultures of bacteria were subcultured on nutrient agar medium. Each isolates was swabbed uniformly onto the individual plates using sterile cotton swabs. Wells of 5mm diameter were made on nutrient agar plates using gel puncture. Using a micropipette, 10 µl of nanoparticles solution was poured onto each well on all plates. After incubation at 37 0 C for 24 hours, the different levels of zone of inhibition of bacteria were measured. RESULTS AND DISCUSSION The addition of Alternanthera ficoidea leaf extract to silver nitrate solution resulted in change in colour from dark greenish to dark yellow (Fig 1). These color changes arise because of the excitation of surface plasmon vibrations with the silver nanoparticles 17. Figure 1 Synthesis of silver nanoparticles using A. ficoidea. a) A. ficoidea leaf extract; b) Silver nanoparticles synthesis as seen by dark yellow colour The surface plasmon resonance of silver nanoparticles produced a peak centered near 410nm, which indicates the reduction of AgNO 3 into silver nanoparticles (Fig 2). UV-VIS spectroscopy could be used to examine size and shape controlled nanoparticles in aqueous suspensions 18. The UV-VIS spectrum was recorded after the completion of the reaction. P - 106
4 Int J Pharm Bio Sci 2013 July; 4(3): (P) S1 Abs wavelength (nm) Figure 2 UV-VIS Spectroscopic analysis of Silver Nanoparticles synthesized from leaf eaxtract of Alternanthera ficoidea The TEM images of the prepared silver nanoparticles at 50, 20 and 5 nm scales are shown in the Fig. 4 a, b, and c, respectively. It was observed that Ag nanoparticles were circular in shape with maximum particles in size range within 4 22 nm It was also observed that silver nanoparticles were evenly distributed in the sample. a) b) P - 107
5 Int J Pharm Bio Sci 2013 July; 4(3): (P) c) Figure 4 TEM images of biosynthesized silver nanoparticles using Alternanthera ficoidea leaf extracts at a) 50nm, b) 20nm and c) 5nm scales Antibacterial activity was observed against E. coli and S. marsescens and the diameter of zone of inhibition was recorded to be 5mm and 2mm respectively. However, the leaf extract of Alternanthera ficoidea, taken as control, found to have no inhibitory effect on the tested bacterial strains (Fig.5). Although the exact mechanism of inhibitory action of nanoparticles against bacteria was not well known, it is thought that formations of free radicals from the surface of the silver nanoparticles were responsible for the antibacterial function.19 a) b) c) d) Figure 5 Antimicrobial activity of biosynthesized silver nanoparticles using Alternanthera ficoidea. a) Alternanthera ficoidea leaf extract against E. coli, b) Alternanthera ficoidea leaf extract P - 108
6 against S. marsescens, c) Biosynthesized silver nano particles against E. coli and d) Biosynthesized silver nano particles against S. marsescens CONCLUSION The present study of silver nanoparticles employing the alcoholic extract of leaf of Alternanthera ficoidea is advantageous in many aspects. It is devoid of any such synthetic reducing agents which are harsh and toxic. The ethanolic extract was found to be highly efficient in synthesizing the silver nanoparticles under the set laboratory conditions. The UV-VIS analysis of the surface plasmon resonance property suggested the formation of silver nanoparticles with an absorption peak at 410nm. TEM analysis shows the spherical shape of the silver nanoparticles with size ranging between 4-22 nm. The biosynthesized silver nanoparticles can be good candidate as antimicrobial agent as evidence from its activity against E. coli and S. marsescens. Thus, the silver nanoparticles synthesized using the leaf extract of Alternanthera ficoidea is believed to have immense potential for application in the field of pharmaceutical industries and in the other fields as well. REFERENCES 1. Rao C.N.R., Kulkarni G.U., John Thomas P., Agrawal V. V., Gautam U. K. and Ghosh M., Nanocrystals of metals, semiconductors and oxides: novel synthesis and applications. Curr. Sci., 85: 1041, (2003) 2. Cui D. and Gao H., Advance and prospects of bionanomaterials. Biotechnol. Prog., 19: 683, (2003) 3. Beveridge T. Y. and Murray R. G. E., Sites of metal deposition in the cell wall of Bacillius subtilis. J. Bacteriol., 141: 876, (1980) 4. Brierley J. A. (1990) In: Biosorption of Heavy Metals (Ed. Volesky, B) Boca Raton, USA. Pp Huang, C.P., Juang, C.P., Morehart, K. and Allen, L., The removal of copper(ii) from dilute aqueous solutions by Saccharromyces cerevisiae, Water Res., 24: 433, (1990) 6. Frilis N. and Myers-Keith P., Biosorption of uranium and lead by Streptomyces longwoodensis, Biotechnol. Bioengg., 28: 21, (1986) 7. Volesky B. and Holan Z. R., Biosorption of heavy metals. Biotechnol. Progr., 11: 235, (1995) 8. Sakaguchi T., Tsuji T., Nakajima A. and Horikoshi T., Accumulation of cadmium by green microalgae. Eur. J. Appl. Microbiol., 8: 207, (1997) 9. Darnall D. W., Greene B., Henzel M. J., Hosea M., McPherson R.A., Sneddon J. and Alexander M. D., Selective recovery of gold and other metal ions from an algal biomass, Environ. Sci. Technol., 20: 206, (1986) 10. Gardea-Torresdey, J. L.; Gomez, E.; Peralta-Videa, J. R.; Parsons, J. G.; Troiani, H.; Jose-Yacaman, M. Alfalfa sprouts: A natural source for the synthesis of silver nanoparticles. Langmuir, 19: , (2003) 11. Gardea-Torresdey, J. L.; Rodriguez, E.; Parsons-Jason, G.; Peralta-Videa, J. R.; Meitzner, E. G.; Cruz-Jimenez, G. Use of ICP and XAS to determine the enhancement of gold phytoextraction by chilopsis linearisusing thiocyanate as a complexing agent. Anal. Bioanal. Chem., 382: , (2005) 12. Shivshankar, S.; Ahmad, A.; Sastry, M. Geranium leaf assisted biosynthesis of silver nanoparticles. Biotechnol. Prog., 19: , (2003) 13. Shivshankar, S.; Rai, A.; Ahmad, A.; Sastry, M. Rapid synthesis of Au, Ag, and bimetallic Au core Ag shell nanoparticles using Neem (Azadirachta indica) leaf broth. J. Colloid Interface Sci., 275: , (2004) 14. Shivshankar, S.; Rai, A.; Ahmad, A.; Sastry, M. Controlling the optical properties of lemongrass extract P - 109
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