International Journal of Current Biotechnology

Anil Ramdas Shet, Pritam Ghose, Laxmikant Patil, and Veeranna Hombalimath, A preliminary study on green synthesis and antibacterial activity of silver nanoparticles, Int.J.Curr.Biotechnol., 2015, 3(2):1-6. International Journal of Current Biotechnology ISSN: 2321-8371 Journal Homepage : http://ijcb.mainspringer.com A preliminary study on green synthesis and antibacterial activity of silver nanoparticles Anil Ramdas Shet*, Pritam Ghose, Laxmikant Patil and Veeranna Hombalimath Department of Biotechnology, B. V. Bhoomaraddi College of Engineering and Technology, Hubli-580031, Karnataka, India. A R T I C L E I N F O Article History: Received 10 February 2015 Received in revised form 15 February 2015 Accepted 22 February 2015 Available online 28 February 2015 Key words: Antimicrobial effect, Fruit extract, Green synthesis, Silver nanoparticles. A B S T R A C T Silver nanoparticles have attracted increasing interest due to their unique physical, chemical and biological properties compared to their macro-scaled counterparts. A comparative study of chemical and biological method for Silver nanoparticle synthesis is done. Few locally available fruits including Lemon (Citrus aurantifolia), Orange (Citrus sinensis) and Tomato (Solanum lycopersicum), which are known to contain relatively high amount of ascorbic acid were selected for silver nanoparticle synthesis. Silver nanoparticles were prepared from aqueous solution of silver nitrate using different fruit extracts. The synthesized silver nanoparticles have been characterized by UV-visible spectroscopy. The UV-Vis spectrophotometer analysis showed a peak for silver nanoparticles between 2-6 hours for the samples kept at room temperature (37ºC). It was also observed that orange extract synthesized nanoparticles showed highest peak, indicating synthesis of maximum nanoparticles and Trisodium citrate synthesized nanoparticles showed the minimum. Further, the antibacterial activity of silver nanoparticles was evaluated by well diffusion method. Antibacterial activity of the synthesized silver nanoparticles was tested with one Gram-positive (Staphylococcus aureus) and one Gram- negative (Escherichia coli) bacterium. Antibacterial effect was found to be maximum for lemon extract synthesized nanoparticles, and it was found that the silver nanoparticles have antibacterial activity against Escherichia coli and Staphylococcus aureus. Introduction In recent days nanotechnology has induced great scientific advancement in the field of research and technology. Nanotechnology is the study and application of small object which can be used across all fields such as chemistry, biology, physics, material science and engineering. Nanoparticle is a core particle,which performs as a whole unit in terms of transport and property. As the name indicates nano means a billionth or 10-9 unit. Its size range usually from 1-100nm due to small size it occupies a position in various fields of nano science and nanotechnology (Asta et al., 2006; Pham et al., 2012). Nano size particles are quite unique in nature because nano size increase surface to volume ratio and also its physical, chemical and biological properties are different from bulk material. So the main aim to study its minute size is to trigger chemical activity with distinct crystallography that increases the surface area. Thus in recent years much research is going on metallic nanoparticle and its properties like catalyst, sensing to optics, antibacterial activity, data storage capacity (Quang et al., 2013). Among them silver nano particles (Ag-NPs or nanosilver) *Corresponding author. Email address: anilrshet@gmail.com Mobile no: +91-9449986649. have attracted increasing interest due to their unique physical, chemical and biological properties compared to their macro-scaled counterparts. Ag-NPs have distinctive physico-chemical properties, including a high electrical and thermal conductivity, surface-enhanced Raman scattering, chemical stability, catalytic activity and nonlinear optical behavior. These properties make them of potential value in inks, microelectronics, and medical imaging. Besides, Ag-NPs exhibit broad spectrum bactericidal and fungicidal activity that has made them extremely popular in a diverse range of consumer products, including plastics, soaps, pastes, food and textiles, increasing their market value (Quang et al., 2013). There are two methods of synthesis of metallic nanoparticles, which are chemical method and physical method. In chemical approach it includes chemical reduction, electrochemical technique and photochemical reduction. The chemical process is again subdivided into classical chemical method (Johnson et al., 2014) where some chemical reducing agent (such as hydrazine, sodium borohydride, hydrogen) are used, radiation chemical method generated by ionization radiation. In the physical approach it includes condensation, evaporation and laser ablation for metal nanoparticle synthesis. The biological synthesis of nanoparticle is a challenging concept, which is very well known as green synthesis (Velavam et al., 1 Int.J.Curr.Biotechnol. Volume 3; Issue 2; Feb, 2015

2012). The biological synthesis of nanomaterial can solve the environmental challenges like solar energy conservation, agricultural production, catalysis, electronics, optics and biotechnological area. Green synthesis of nanoparticle are cost effective, easily available, ecofriendly, nontoxic, large scale production and act as reducing and capping agent (Quang et al., 2013) compared to the chemical method, which is a very costly as well as it emits hazardous by-product, which can have some deleterious effect on the environment. Biological synthesis (Kaushik et al., 2013) utilizes naturally occupying reducing agent such as plant extract, microorganism, enzyme, polysaccharide which are simple and viable, which is the alternative to the complex and toxic chemical processes. Plants and fruit extracts can be described as nano factories which provide potential pathway to bioaccumulation into food chain and environment. Among the different biological agents fruit extracts/plant extracts provide safe and beneficial way to the synthesis of metallic nano particle as it is easily available so there is possibilities for large scale production apart from this the synthesis route is ecofriendly, the rate of production is faster in comparison to other biological models such as bacteria, algae and fungi. The most important and distinct property of nanoparticles is that they exhibit larger surface area to volume ratio. Specific surface area is relevant for catalytic reactivity and other related properties such as antimicrobial activity in AgNPs. As specific area of nanoparticles increased, their biological effectiveness can increase due to the increase in surface energy. Several studies proposed that AgNPs may attach to the surface of the cell membrane disturbing permeability and respiratory functions of the cell. It is also possible that AgNPs not only interact with the surface of membrane but also penetrate inside the microorganism. Thus, it is of interest to study the use of different fruit extracts for the synthesis of silver nanoparticles. In addition, antibacterial activity of the synthesized silver nanoparticles was also determined and reported. Materials and Methods Chemicals All analytical reagents and media components were purchased from HiMedia and Sigma Chemicals. Preparation of fruit Extract Fresh green Lemon (Citrus aurantifolia), Orange (Citrus sinensis) and Tomato (Solanum lycopersicum) were washed separately and later smashed inside a grinder. The smashed fruits were then filtered to remove the debris. The filtered juice was centrifuged at 10000 rpm for 10 minutes to obtain the liquid fruit extract (Kaushik et al., 2013). At last the clear solution was collected and stored at 4 o C for further experiment. Synthesis of Silver Nanoparticles by tri-sodium citrate Silver Nanoparticles were prepared by chemical reduction method. 50 ml of 0.001 M Silver nitrate was heated. To this solution 5 ml of 1% tri-sodium citrate was added drop by drop. During this process solution was mixed vigorously. Solution was heated until color change was evident (yellowish brown). Then it was removed from the heating element and stirred until cool to room temperature (Johnson et al., 2014). Synthesis of Silver Nanoparticles using fruit extracts Silver nitrate solution (50 ml) and fruit extract (12.5 ml) were mixed in the ratio 4:1 (V/V) and the mixture was constantly stirred. The mixture was kept in microwave oven at 60 o C for 3 minutes, and cooled to room temperature. The change in color was seen after a certain period of time indicating formation of silver nanoparticles. UV-Visible Spectroscopy The silver nanoparticles show the Plasmon resonance at 300 to 600 nm in the UV-Visible spectrum. The UV-Visible spectrum of synthesized silver nanoparticles was analyzed by spectrophotometer (LAB INDIA). Antibacterial activity of silver nanoparticles The agar well diffusion method was employed for determination of the antimicrobial activities. A suspension of the microorganism was spread on nutrient agar by a glass spreader. 20 l of aqueous solution of silver nanoparticles was added to each of the well. After that, the plates were incubated at 37 C for 24 h, and the zone of inhibition of bacteria was measured. The experiment was done in triplicates. Results and Discussion Green synthesis of AgNP s Tri-sodium Citrate, Lemon, Orange and Tomato extract were selected to react with 0.001M of AgNO 3 at different incubation time. At room temperature there was a color change, which indicated the formation of metal nanoparticles. Figure 1 showed that there was a gradual change in the color from yellow to reddish yellow and from reddish yellow to dark brown for mixtures containing aqueous solution of 0.001M silver nitrate, Lemon, Orange, Tomato and Tri-sodium citrate respectively indicating the formation of silver nanoparticles. These characteristic color variations are due to the metal nanoparticles. The color intensity also increased with the duration of incubation which indicated formation of more amount of nanoparticles. Orange extract changed the colorless AgNO 3 solution to black color in minimum time. It is the fastest reaction, which may be due to its high Vitamin C content. The high Vitamin C fruit has antioxidant activity, acting as reducing agent by reduction of Ag + from silver nitrate to Ag o. For tri-sodium citrate and the orange extract solution, color change was instantly while Lemon extract and Tomato extract took more time for color change indicating slow formation of silver nanoparticles. Tomato synthesized nanoparticles showed least degree of color change, which showed lesser degree of silver nano particles formation. The progress of reaction as function of time was followed by UV-visible spectroscopy. UV-visible spectroscopy analysis UV-VIS absorption results confirmed formation of silver nanoparticles prepared in liquid by chemical reduction method (silver nitrate reduced by Tri-sodium citrate) as well as biological reduction (green synthesis) method using fruit extracts (orange, tomato, lemon). UV-VIS measurements have shown that size of the nano particles in the mixtures between the experimentally mentioned ranges that is between 300-600 nm (Natthawan et al., 2012). The SP band in silver nano particles solution was found to be specifically between 300-350nm throughout the observation period which indicated formation of small sized particles (<10nm). Volume 3; Issue 2; Feb, 2015 Int.J.Curr.Biotechnol. 2

Figure - 1: Synthesis of silver nanoparticles using AgNO 3 and different fruit extract at room temperature for 10 hrs. Figure - 2: UV-visible spectra of AgNP prepared using 0.001M AgNO 3 and lemon extract for 24h at room temperature 3 Int.J.Curr.Biotechnol. Volume 3; Issue 2; Feb, 2015

Figure 3: UV-visible spectra of AgNP prepared using 0.001M AgNO 3 and orange extract for 24h at room temperature Figure 4: UV-visible spectra of AgNP prepared using 0.001M AgNO 3 and tomato extract for 24h at room temperature Figure 5: UV-visible spectra of AgNP prepared using 0.001M AgNO 3 and tri-sodium citrate for 24h at room temperature Volume 3; Issue 2; Feb, 2015 Int.J.Curr.Biotechnol. 4

Figure 6:Zone of inhibition: a) Orange synthesized AgNP s, b)lemon synthesized AgNP s c)tomato synthesized AgNP s d) Tri-sodium synthesized AgNP s. i) & ii) for Escherichia coli, iii) & iv) for Staphylococcus Aureus Escherichia coli Sample A Sample B Staphylococcus Aureus (i) (ii) (iii) (iv) The Wavelength did not change much with time, but there was a variation in the absorption. The Increase in absorption indicates formation of more nanoparticles (Basavaraj et al., 2012). Tri-sodium citrate mixture showed a dip in its absorbance indicating reduced formation of nanoparticles. From all the graphs it was observed that the maximum absorbance was at 4 hrs and at room temperature. Maximum absorbance was shown by the orange extract solution which confirmed formation of maximum number of nanoparticles. Antibacterial activity of AgNPs Antibacterial test by zone of inhibition was carried out to qualitatively determine the level of inhibition by using differently prepared colloidal silver nanoparticles. Silver nanoparticles inhibited bacterial growth by the clear bacterial inhibition zone (a certain concentration of 20ìl/ well) which is correlated with the Antibacterial activity of Ag nanoparticles. From figure 6 it was observed that for both sample A and sample B, lemon synthesized silver nanoparticles have greater antibacterial property and tri-sodium synthesized AgNPs have the least antibacterial property against the bacteria. It was also observed that the silver nanoparticles seem to have more anti-bacterial effect against the gram negative bacteria. The antibacterial effect against E.coli for sample B was observed to be more when compared with sample A. It may be due to the decrease in the AgNP s size which can lead to an increase in the specific surface of a bacterial specimen, inducing an increase in their ability to penetrate cell membrane and thus improving antibacterial activity (Padma et al., 2012; Manal et al., 2014). The bacterial activity is presumably due to certain changes in the membrane structure of bacteria cell as a result of the interaction with the embedded AgNPs which leads to the increased membrane permeability of the bacteria and consequently, leading to their death. Conclusions A critical need in the field of nanotechnology is the development of reliable and eco-friendly processes for synthesis of metallic nanoparticles. The comparision of fruit extracts with tri-sodium citrate as reducing agent for the synthesis of silver nanoparticles was studied and found that orange extract is the best reducing agent. It was also found that biosynthesis is a low-cost approach when compared with chemical synthesis for the preparation of silver nanoparticles. The preliminary characteristics of the obtained silver nanoparticles were studied using UV-Visible spectrophotometer. The results confirmed the reduction of silver nitrate to silver nanoparticles with high stability. At room temperature, orange extract showed maximum absorbance which indicated the formation of more nanoparticles. Biosynthesized silver nanoparticles showed greater antibacterial activity than chemically synthesized on both gram-positive and gram- negative bacteria. Lemon extract solution showed maximum Antibacterial effect. Conflict of Interests The authors declare that they have no conflict of interests. Acknowledgement The authors are thankful to the Department of Biotechnology, B V Bhoomaraddi College of Engg and Tech, Hubli for their support. References Quang H. T., Van Q. N. and Anh-Tuan L., 2013. Silver nanoparticles: synthesis, properties, toxicology, applications and perspectives. Adv. Nat. Sci. Nanosci. Nanotechnol. 4:1-20. Kaushik R., Supratim B. and Pataki C. B., 2013. Green synthesis of silver nanoparticles by using grape (vitis vinifera) fruit extract: characterization of the particles and study of antibacterial activity. Research Journal of Pharmaceutical, Biological and Chemical Sciences. 4:1271-5 Int.J.Curr.Biotechnol. Volume 3; Issue 2; Feb, 2015

1278. Pham V. D., Chu H. H., Le T. B. and Jörn K., 2012. Chemical synthesis and antibacterial activity of novel-shaped Silver nanoparticles. International Nano Letter. 2:1-9. Basavaraj U., Praveenkumar N., Sabiha T. S., Rupali S. and Samprita B.,2012. Synthesis and characterization of silver nanoparticles. International Journal of Pharmacy and Biological Sciences. 2:10-14. Johnson A. S., Obot I.B. and Ukpong U. S., 2014. Green synthesis of silver nanoparticles using Artemisia annua and Sida acuta leaves extract and their antimicrobial, antioxidant and corrosion inhibition potentials. J. Mater. Environ. Sci. 5: 899-906. Velavan S., Arivoli P and Mahadevan K., 2012. Biological reduction of silver nanoparticles using Cassia auriculata flower extract and evaluation of their in vitro antioxidant activities. Nanoscience and Nanotechnology: An International Journal. 2: 30-35. Asta S., Igoris P., Judita P., Algimantas J. and Asta g., 2006. Analysis of Silver Nanoparticles Produced by Chemical Reduction of Silver Salt Solution. Materials science. 12: 287-291. Padma S. V. and Dhara S., 2012. Biosynthesis of silver nanoparticles using lemon leaves extract and its application for antimicrobial finish on fabric. Appl Nanosci. 2: 163-168. Manal A. A., Awatif A. H., Khalid M. O., Dalia F. A. E. and Abdelelah A. G. A., 2014. Silver nanoparticles biogenic synthesized using an orange peel extract and their use as an anti-bacterial agent. International journal of physical sciences. 9: 34-40. Natthawan P., Ampa J. and Sirirat M., 2012. Green synthesis and antibacterial activites of silver nanoparticles. 1 st Mae Fah Luang university international conference. 1-13. Volume 3; Issue 2; Feb, 2015 Int.J.Curr.Biotechnol. 6