Bulletin of Trends in Chemical Sciences

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1 Bulletin of Trends in Chemical Sciences Paper Vol. 1 (1), 2016, Open access Synthesis of silver nano particles using albedo extract of Punica granatum and evaluation of its antibacterial activity Daniel Deva Sangeeth. S,Vijaya Kumar.V* Department of chemistry, Voorhees college, Vellore, Tamilnadu, India ABSTRACT Silver nano particles were synthesized using albedo extract of punica granatum and silver nitrate solution of different concentrations. The synthesized nano particles were characterized by UV,FT-IR, XRD, SEM, TEM, AFM, EDAX and AAS techniques. The silver nitrate and albedo fruit extract reaction mixture exhibits the strong peak at 435nm. XRD Spectrum shows the eight distinct diffraction peaks observed in the 2θrange (32.3 0, ,54.8 0, , , ). The TEM figures show size of the formed nano particles were found to be within the range nm. The synthesized silver nanoparticles shows excellent anti bacterial activity against clinically isolated multi drug human resistant pathogens. Introduction Nano-technology is a rapidly expanding multi disciplinary field which includes physics, material science, Bio-Technology, super molecular chemistry, chemical engineering, mechanical engineering & medicine. High surface areas of Nano particles are responsible for their antimicrobial, magnetic, electronic and catalytic properties. Nanomaterials can be synthesized by methods such as heat evaporation [1], Electro chemical techniques [2], Chemical Reduction [3], thermal Decomposition [4], microwave assisted methods [5], surface passivator reagents such as Thiophenol, Thiourea, which are used to prevent nano particles from aggregation pollute the environment [6,7]. Biological methods are eco friendly and are cost effective. Microbial synthesis of metal nano particles can takes place either intra cellularly or extra cellularly [8-10]. Intra cellular synthesis of nano particles requires additional steps such as ultra sound treatment or reactions with suitable detergents to release the synthesized nano particles [11]. At the same time extra cellular Bio synthesis is cheap and it requires simple downstream processing. This favors large scale production of silver nano particles to explore its potential applications because of this many studies were focused on extra cellular methods for the synthesis of metal nano particles [12-14]. The plant material based production of nano materials has wide range of application such as antimicrobial property. Various plant materials have been studied so far for the synthesis of silver, gold, platinum and titanium nano particles in different sizes and shapes are tabulated in Table -1. Review of literature states that synthesis of silver nano particles using albedo of Punica Granatum has not been reported so far. In the present study, synthesis of silver nano particles using the albedo extract of the fruit Punica Granantum belongs to the family Punicaceae. The fruit albedo is pale yellow in colour, the fruit and its albedo are used for treatment of

2 Table-I Plant materials used for synthesis of noble metal nanoprticles S.No Plant Materials Metal Nano Particle Size Particles 1 Acaly pha idica Ag 20-30nm 2 Citrus Sinensis Ag 31-12mm 3 Curcuma Longa Ag 21-30nm 4 Cassica Fistula Ag 50-60nm 5 Aloe Vera Ag and Au 15.2±4.2m, gastro intestinal diseases as a traditional medicine in India especially southern parts of India. In this study, We used Albedo of Punica Granatun as the reducing agent to reduce Ag + ions to Ag 0 in aqueous solution. Materials and methods Collection of materials The dried fruit bodies of the Punica Granatum were collected from the local market in Vellore, Tamil Nadu. The fruit bodies were rinsed with water thrice followed by deionized water to remove the fine and dust materials and then the fruit bodies were dried under the direct sun light for one week to completely remove the moisture Preparation of fruit extracts The dried fruit bodies were pulverized and grind well with a well cleaned mixie to make a powder. Five grams of powered sample was mixed into 100 ml of deionized water and the mixture was boiled for 20 minutes. After cooling the fruit body extract was filtered with whatman No.1 filter paper. The filtrate was stored at 4 0 C for further use. Synthesis of silver nano particles The 100 ml of aqueous filtrate albedo extract of PunicaGranatum was taken in to 250ml of Erlenmeyer flask. Then the extract was mixed into silver nitrate (AgNO 3) to make the final volume concentration of 0.2mM solution. The reaction mixture was kept into a dark room condition until the colour change was arisen. The reaction solution colour changes have observed for the characterization of silver nano particles. Characterization studies The Biosynthesized silver nanoparticles were characterized by different methods. A colour change from pale yellow to reddish brown upon incubation was observed indicating the formation of nanoparticles. The UV spectra of the biosynthesized nanopartclies were recorded using an Elico SL- 159 UV Spectro photometer by continuous scanning from 300 to 700 nm. The silver nitrate fruit albedo extract reaction mixture exhibits strong peak at 435nm. The major factor liable for the biological reduction of silver ions (Ag + ) in to silver nanoparticles (Ag 0 ) present in the fruit albedo extract of Punica Granatum was identified using FTIR spectroscopy. Results and discussion FT-IR spectrum of synthesized silver nano particles indicates the major peek at cm 1 and other peakes were obtained at cm 1, cm -1 the peak at cm -1 and cm -1 were assigned to the stretching of primary and secondary amine s respectively while the corresponding vibration at cm -1 can the assinied to the corboxyl group of C- C stretching Vibration present in the fruit albedo extract. The comparison of FTIR Spectrum between the fruit albedo extract and silver nanoparticles were observed only minor changes in the position as well as the absorption bands. Due to the silver nanoparticles the O-H stretching vibration the slightly shifted from to cm -1. The X-ray diffraction studies were performed to confirm the crystalline structure of synthesized silver nanoparticles XRD Spectrum of fruit albedo extract of reduced silver nanoparticles were shows the Eight distinct diffraction peaks observed in the 2θ range can be indexed to the , ,54.8 0, , , the lattice plane value was observed which may indexed at (111), (200), (220), (311), and (222) planes of fcc silver. The (200), (220), (311) and (222) Bragg reflections are weak and broadened relative to the intense (111), reflection. This feature indicate that the nanocrystals are (111) oriented as confirmed by high resolution TEM measurements. The TEM figures 28

3 and the size of the formed nano particles were found to be within the range nm. Table-2 Particles sizes determined by various technique Sl. No Analysis techniques /methods XRD AFM TEM SEM Particle size (nm) 50 <100nm 33.9nm 44nm sample was redispersed in ethanol, sodiumpellet and the sample was prepared in thin films on carbon coated copper grid. Electron dispersive Analysis X-Ray spectrometer (EDAX) was used for elemental analysis of the sample. Scanning Electron Microscopic studies were carried out to study the morphology of the silver nano particles. The SEM pictures indicated that the formed particles were from spherical to oval in shape with a smooth morphology. EDAX analysis confirmed the presence of elemental silver as the major constituent. The synthesized silver nanoparticles structure morphology and size of the nanoparticles were characterized by the AFM images. The resultant silver nanoparticle images were observed as spherical in shape is as shown. It has been reported that the topographical image of irregular silver nanoparticles have documented [12]. The size of the silver nanoparticles ranges <100nm and it cannot be controlled by varying the synthesis condition. The fabricated silver nano particles were imaged by AFM to understand the exact configuration of the fabricated silver nanoparticles and also used to verify that the silver nanoparticles were more or less homogenous in size and were spherical in shape. The particle size was measured using line profile determination of individual particles in the range of nm. The silver nanoparticle crystalline structure and size were further characterized by TEM. The TEM images were recorded at different magnification to find the individual particles. The synthesized silver nanoparticles were observed as spherical in shape and average size of the particles was nm. The variation in the particle sizes such as 41.94, 26.71, and 24.58nm in size is possibly due to the fact that the nanoparticles and being formed at different times. AFM, SEM & EDAX images of the synthesized silver nano particles shows spherical or mostly spherical shapes with <100nm in size. The graphical images to determine the morphology of the synthesized silver nanoparticles using albedo extract, the sample was analysed with Zeiss 700 Scanning electron microscope (SEM). The re-dispersed nanoparticles were dried in an oven to obtain a powdered form. Then 10 mg of the Fig.1 SEM image of synthesized silver nanoparticles Fig.2 AFM image of synthesized silver nanoparticles 29

4 Table-2 Shows the particle sizes that were obtained by various techniques. The differences in the particle sizes may have been due to the differences in sample preparation. Antibacterial activity of synthesized silver nanoparticles The biologically synthesized silver nanoparticles shows excellent antimicrobial activity against clinically isolated multi drug resistant human pathogens such as Gram positive Bactria S-A-Stapholococcus aureus and Gram nagtive bacteria E-C Escherichia coli. The mean inhibitory zone of the replicates of diameter was measured and tabulated (Table 3). The Gram negative bacterium E. coli showed minimum zone of inhibition at mm which may due to the cell wall of Gram positive bacteria composed of a thick peptidoglycan layer, which consisting of linear polysaccharide chains cross linked by short peptides thus forming more rigid structure leading to difficult penetration of the silver nanoparticle compared to the gram negative bacteria where the cell wall possesses thinner peptidoglycan layer [14]. The high bactericidal activity is certainly due to the silver cations released from Ag nanoparticles 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. In general, silver ions from silver nanoparticles are believed to become attached to the negatively charged bacterial cell wall and rupture it, which leads to the denaturation of protein and finally cell death. Silver nano particles demonstrated greater bactericidal efficiency compared to Gentamicin. Silver has a greater affinity to react with sulfur- or phosphorus-containing biomolecules in the cell. Therefore, sulfur-containing proteins in the membrane or inside the cells and phosphoruscontaining elements like DNA are likely to be the preferential sites for silver nanoparticle binding. Fig.3 Antimicrobial activity of silver Nanoparticles against Multidrug Resistant Pathogen Staphylococcus aureus and Escherichia Coli Table -3 Antibacterial activity of samples determined by well diffusion method Sample 2b(0.2mM) Conc (µl/well) Zone of inhibition (mm) S.a E.c 11.00± ± ± ± ± ± ± ±00.00 Gentamicin ± ±00.78 Conclusion In this study we have demonstrated that the albedo extract of Punica Grantum as a reducing agent can effectively produce the spherical shape silver nanoparticles followed by the green chemistry approach. The synthesized silver nanoparticles using fruit albedo extract of Punica Granatum proved excellent antimicrobial activity against clinically isolated multi drug resistant human pathogens. The antimicrobial activity of silver nanoparticle was well demonstrated by the clear zone of inhibition against s-a staphylococcus aureus, E-C 30

5 Escheri chia Coli. The powdered diffraction study shows the face centered cubic silver nanoparticles were stable after 6 months in room temperature, TEM, SEM, EDAX and AFM studies revealed that spherical shaped nanoparticles in the range of 40nm. Moreover biological synthesis of silver nanoparticles using plant material is the most conventional and eco-friendly method compare to the clinical and physical synthesis. References [1] Bae, CH, Nam SH, Park SM. Formation of silver nanoparticles by laser ablation of a silver target in NaCl solution. Appl. Surf sci- 2 (2002), [2] Rashid A. Khaydarov, Rednat R Khaydarov, Olga Gapurova, Yuriestrin, Thomas Scheper Electro chemical meth For the synthesis of silver nanopaticles J Nanopart Res 11 (2009), [3] Lakshmipathy R, Reddy BP, Sarada NC, Chidambaram K, Basha SK Watermelon rind-mediated green synthesis of noble palladium nanoparticles: Catalytic applications. App nanosci. 5 (2015) [4] Plante IJL, Zeid TW, Yangab P, Mokari T. Synthesis of metal sulfide nanomaterials via thermal decomposition of single source precursors. J. Mater chem. 20 (2010) [9] Jain N, Bhargava A, Majumdar S. Tarafdar Jc, Panwar J (Extracellurlar biosynthesis and characterization of silver nanoparticles using A spergillus NJP08 a mechanism perspective nanoscale 3 (2011) [10] Kalishwaralar K. Deepak V. Pandian SRK, Kottaisamy M, Barath Manikanth S, Karikeyan B, Gurunathan S Biosynthesis of silver and gold nanoparticles using Brevibacterium casei coll surf B 77 (2010) [11] Kalimuthu K, Babu RS, Venkataraman D, Bilal M, Gurunathan S Biosynthesis of silver nanocrystals by Bacillus licheniformis coll surf B 65 (2008) [12] Duran N, Priscyla D, Marcato PD, Alves O, De Souza G, Esposito E Mechanistic aspects of biosynthesis of silver nanoparticles by several Fusarium oxysporum strains. J nanobiotechnol 3 (2005) 1-7. [13] N. Saifuddin, C.W. Wong, and A.A.N. Yasumira, Rapid biosynthesis of silver nanoparticles using culture supernatant of bacteria with microwave irradiation, E- Journal of chemistry, 6 (2009) [14] Ahmad R, Minaeian S, Shanverdi HR, Jamalifar H, Nohi A Rapid synthesis of silver nanoparticles using culture supernatants of Enterobacteria. A novel biological approach process Bio-chem 42 (2007) [5] Nadagouda MN, Speth TF, Varma Rs Microwave assisted green synthesis of silver nano structures Accounts chem. Res. 44 (2011) [6] Pattabi M. J Uchil. Synthesis of cadmium sulphide nanoparticles. Solar Energ. Mater solar cell. 63 (2000) [7] Gericke M, A Pinches. Biological synthesis of metal nanoparticles Hydrometall 83 (2006) [8] Ahamd A, Mukherjee P, Senapati. S, Mandal. D, Khan, MI, Kumar. R, Sastry M extracellular biosynthesis of silver nanoparticles using the fungus Fusarium oxysparum coll surf B 28 (2003)