South Indian Journal of Biological Sciences 2015; 1(2); 115-118 Online ISSN:2454-4787 Green synthesis of Silver nanoparticles using aqueous extract of Taraxacum oicinale and its antimicrobial activity Selvaraj Arokiyaraja, *,†, Muthupandian Saravananb,†, Badathala Vijayakumarc a Institute of Green Bio Science & Technology, Seoul National University, Pyeongchang,Republic of Korea Institutes of Biomedical Science, College of Health Sciences, Mekelle University, Mekelle-1871, Ethiopia. c Department of Chemistry, Vel Tech High Tech Dr.RR Dr.SR Engineering College, Avadi, Chennai-600 062, India b † Author contributed equally Corresponding Author: Selvaraj Arokiyaraj E-mail: arokiyaraj16@gmail.com Tel.: +821040067121 Manuscript details Article History: Received 16 July 2015 Revised 31 August 2015 Accepted 4 September 2015 Published 29 September 2015 Keywords: Taraxacum oicinale, SEM, UV- vis spectrophotometer Abstract Aim of the present study is to focus on the biosynthesis of silver nanoparticles using Taraxacum oicinale loral extract. he color of solution changes into dark brown indicating the formation of silver nanoparticles (AgNPs) upon addition of 1 mM silver nitrate. hese nanoparticles were characterized by UV- Vis spectrophotometry which showed the absorption peak at 465 nm speciic for AgNPs. Scanning electron microscopic image showed that the synthesized AgNPs were seemed to be spherical in morphology. Dynamic light Scattering (DLS)was used to determine the particles size of the nanoparticles and average diameter of AgNPs is found to be 545 nm ± 5 nm. Antibacterial activity of these green synthesized AgNPs was evaluated against selected pathogns such as Enterococcus faecalis and Pseudomonas aeruginosa by disc difusion assay.hese synthesized AgNPs showed a good antimicrobial activity against the selected bacterial pathogens. Introduction he advancement in nanoparticle systems has an impact in scientiic areas. Metal nanoparticles such as silver, gold and copper were found to exhibit antibacterial and other biological activities. Development of biocompatible, non-toxic and eco-friendly methods for the synthesis of nanoparticles is a topic of concern. Biological methods are considered as safe, cost-efective, sustainable and environment friendly processes. Plant mediated synthesis process was more advantages over the chemical and physical methods (Anastas and Warner 1998; Rawel et al., 2015). When compared to the conventional chemical methods, this method is eco-friendly and cost efective. Recently there are several reports on green synthesis of AgNPs using plant extracts such as Jatropha curcas seeds, Acalypha indica leaf, Trianthema decandra roots, Ocimum sanctum stems and roots, Chrysanthemum indicum lowers, Mimusops elengi leaf extract (Bar et al., 2009; Krishnaraj et al., 2010; Geethalakshmi and Sarada 2010; Ahmad et al., 2010; Prakash et al., 2013;Arokiyaraj et al., 2014). Taraxacum oicinale is an herbaceous perennial plant of the family Asteraceae, commonly called dandelion. he plant grows in the temperate regions of the world, on roadsides, in lawns, on distributed banks, etc. he plant T. oicinale has been used traditionally for poor digestion, water retention and for disease of liver including hepatitis (Dearing et al., 2001). Pharmacological proiling of T. oicinale has shown diuretic, cholerectic, anti-inlammatory, anti-oxidative, anti-carcinogenic, analgesic, anti-allergic, anti-hyperglycemic and anti-thrombotic activities (Schütz et al., 2005; Clare et al., 2009; Awortwe et al., 2013). In the present study, we report the synthesis of AgNPs using aqueous lower extract of T. oicinale and the nanoparticles were characterised using UV-vis, 115 XRD, DLS and SEM techniques. Further more,he antimicrobial activities of green synthesized AgNPs have been evaluated against the selected Gram-negative pathogenic bacteria such as Enterococcus faecalis and Pseudomonas aeruginosa. 2. Materials and methods 2.1. Preparation of lower extract and synthesis of AgNPs Flowers of T. Oicinale were collected and washed frequently with Milli-Q deionized water. 2 g of powder was mixed with 500 mL of Milli-Q deionized water and kept at 40oC for 24 h. Ater incubation the crude extract thus obtained, was iltered by Whatman No.1 ilter paper. 5 mL of extract was assorted with 500 mL of 1mM silver nitrate (Sigma-Aldrich) solution. he reaction mixture was kept undisturbed until the conversion of colourless solution turns into reddish brown color which indicated the formation of AgNPs (Fig. 1). his process was conveyed out at microwave condition for 5 minutes. he particles were then puriied by centrifugation and stored in screw capped vials under ambient conditions for further characterization and applications. source (λ=1.5412 Å) in the range of 5o to 90o, in 2θ angles with a scanning rate of 0.5sec/step. he average crystallite size of AgNPs was calculated using Debye-Scherrer formula (D=Kλ/βcosθ). he particle size of the aggregates was measured by dynamic light scattering (DLS) experiments on a Malvern Zetasizer Nano ZS instrument equipped with a 4.0 mW He–Ne laser operating at a wavelength of 633 nm. he size and morphology of the green synthesised AgNPs was investigated with the scanning electron microscope 2.2. Phytochemical analysis he aqueous extract obtained from the lower of T. Oicinale was tested for the presence of the phytochemicals tannins, saponins, lavonoids, terpenoids, steroids, alkaloids, and glycosides according to the method described by Trease and Evans (2002). 2.3. Characterization of Ag NPs he reduction of silver ions was supervised by assessing the UV-vis spectrum of reaction mixture ater 8 min by using UV-Vis spectrophotometer (Cyber lab UV100, USA) in the wavelength range 300 - 700 nm. he crystalline structure of the synthesized AgNPs Fig. 2. Characterization of green synthesised AgNPs using T. oicinale (lower extract) (a-UV-vis, b- XRD, c-DLS and dEDAX) (JSM 35 CF JEOL) in a resolution of 60Å at 15 KV, magniication of 5.0 K. he images were taken by drop coating AgNPs on an aluminium foil. Energy dispersive absorption (EDAX) spectroscopy photograph of AgNPs were carried out by the SEM equipment, as mentioned above. 2.4. Antibacterial activity he antibacterial eicacy was assayed by disc difusion method against Gram-negative pathogenic bacteria Enterococcus faecalis and Pseudomonas aeruginosa (Moshi et al., 2006). he bacterial suspension (108 colony-forming units/mL) was swabbed on the Mueller Hinton Agar plates using sterile cotton swabs. he sterile disc which was 6 mm in diameter was impregnated with the AgNO3, AgNPs and streptomycin at the concentrations of 25 µg/disc. he discs were gently pressed and incubated at 37°C for 24 h. Antibacterial activities were determined by measuring the diameters of zones of inhibition in mm. he experiments were performed in triplicate. 3. Result and Discussion Fig. 1. Colour change observed in the sample at diferent time intervals upon addition of AgNO3. was investigated by X-ray difraction pattern using a Bruker D8 advance difractometer, operated at 40 kV and a current of 40 mA with Cu/kα radiation In this proposed study T. Oicinale was used for the biosynthesis of AgNPs. he change in color of the reaction mixture was noted by visual observation. When the 1mM silver nitrate solution was added to the lower extracts of T. Oicinale at the beginning of the reaction showed yellowish color, and gradually increased in color intensity to dark brown, with the increasing period of incubation (Prakash et al., 2013). 116 3.1. Phytochemical analysis 3.3. Antibacterial assay An aqueous extract of T. oicinale, showed various phytochemicals such as lavonoids, terpenoids, alkaloids and glycosides (Table 1). hese components may act as a possible reducing agents during the conversion of silver nitrate into silver nanoparticles. Many studies have showed that biomolecules like phenols, proteins, lavonoids and terpenoids play a role in reducing and capping the nanoparticles (Vedpriya et al., 2010). AgNPs showed antibacterial efect against the tested bacterial pathogens (Table 2). Results indicate that Table2. Antibacterial activity of green synthesised silver nanoparticle using disc difusion method. Bacteria Table 1. Phytochemical analysis of lower extract Phyto constituents Tannin Saponin Flavonoid Terpenoid Steroid Alkaloid Glycosides E.faecalis P.aeruginosa Taraxacumoicinale + + + + Zone of inhibition in mm T.oicinale Streptomycin 10± 0.50 12±0.27 11±0.76 14±0.90 Values are the means and standard deviations of the diameters of zone of growth inhibitions of three independent experiments. 3.2. Characterization he UV-Vis spectrum of silver nanoparticles prepared from aqueous extract of T.oicinale is shown in Fig. 2a. It shows a broad absorption band at 465 nm which indicates that the particles are poly dispersed. AgNPs exhibit surface Plasmon resonance (SPR) absorption band almost in identical shape i.e. almost symmetrical suggesting the formation of spherical shaped nanoparticles without much aggregation (Lina et al., 2010). he crystalline nature of the AgNPs that were synthesized using T.oicinale was conirmed by XRD analysis (Fig. 2b). he XRD pattern shows ive characteristic peaks at 2θ values of 38°, 44°, 64°, 77° and 81° corresponding to 111, 200, 220, 311 and 222 planes respectively for silver, suggesting that these silver nanoparticles are crystalline in nature (Baker et al., 2005). he average particle diameter of the synthesized silver nanoparticles was calculated from the XRD pattern using Scherrer’s equation and the average crystallite size of the silver nanoparticles is found to be 545 nm. Fig. 2c shows the particle size distribution of AgNPs determined by DLS. he intensity-average diameter of AgNPs is found to be 545 nm ± 5 nm. he value is in agreement with value obtained from Scherrer’s equation used in XRD. Figure 2d shows the elemental composition of the synthesized AgNPs. he peaks suggest presence of silver (Ag), chlorine (Cl), carbon (C), silicon (Si) and oxygen (O). he peak at 3 keV corresponds to Ag is strong (46%) and it has optical absorption in this range due to the surface plasma resonance (Bindhu et al., 2013). Other peaks are due to carbon, chlorine and oxygen indicate the presence of plant extract, which correspond to the biomolecules that are reducing and capping agents over the AgNPs. Figure 3 shows SEM images of silver nanoparticles and the particles are found to be spherical in shape. the NPs showed signiicant inhibitory efects against E. Faecalis and P.aeruginosa. Nanoparticles have been known for their inhibitory and bactericidal effects in the past decades. here are diferent mechanisms by which the microbes are being killed that includes cell wall damage, DNA damage and protein synthesis inhibition. AgNPs have the ability to attach to the bacterial cell wall and makes structural changes in the cell membrane like the permeability of the cell membrane leading to death of the cell. Many studies demonstrated AgNPs exhibit wide range of antibacterial property with close attachment of the nanoparticles themselves with the microbial cell and their antibacterial eicacy depends upon the size of nanoparticles (Saravanan et al., 2011; Shrivastava et al., 2007). Similarly, Kim et al., 2007 reported antimicrobial activity of AgNPs against E. coli and S. aureus. 4. Conclusion T.oicinale acts as good source for the biosynthesis of AgNPs. AgNPs synthesized from loral extract of T.oicinale showed good antibacterial activity against the selected bacterial pathogens. he synthesized AgNPs will explore their competence in pharmaceutical ield for the controlthe infections caused by E. faecalis, and P. aeruginosa. With the recent ongoing eforts in improving particle synthesis eiciency and exploring their biomedical applications, it is hopeful that the execution of the approach of and their commercial applications health care sector will be very much useful in the upcoming years. Conlict of interest statement We declare that we have no conlict of interest. Acknowledgements he authors would like to acknowledge their sincere appreciation to Seoul National University for supporting our research. References 1. Ahmad N, Sharma S, Alam MK, Singh VN, Shamsi SF, Mehta BR, Fatma A. (2010). Rapid synthesis of silver nanoparticles 117 using dried medicinal plant of basil. Colloids Surfaces B Biointerfaces, 81, 81- 86. 2. Anastas PT, Warner JC. (1998). Oxford University Press, New York, USA. 3. Arokiyaraj S, ValanArasu M, Vincent S, Udaya Prakash NK, Choi SH, Oh YK, Choi CK, Kim KH. (2014). Rapid green synthesis of silver nanoparticles form Chrysanthemum indicum L and its antibacterial and cytotoxic efects: an in vitro study. International Journal of Nanomedicine, 9, 379 - 388. 4. Awortwe C, Safo DO, Gyekye IJA, Sackeyi AC. (2013). he anti-inlammatory activity of Taraxacum oicinale leaves in ovalbumin sensitized guinea-pigs. International Journal of Pharmacy and Pharmaceutical Sciences, 5, 628 - 633. 5. Baker C, Pradhan A, Pakstis L, Pochan DJ, Shah SI. (2005). Synthesis and Antibacterial Properties of Silver Nanoparticles. Journal of Nanoscience and Nanotechnology, 5, 224 - 249. 6. Bar H, Bhui DK, Gobinda SP, Sarkar PM, Pyne S, Misra A. (2009). Green synthesis of silver nanoparticles using seed extract of Jatropha curcas. Physicochemical Engineering Aspects, 348, 212 – 216. 7. Bindhu MR, Umadevi M. (2013). Synthesis of monodispersed silver nanoparticles using Hibiscus Cannabinus leaf extract and its antimicrobial activity. Spectrochimica Acta Part A, 101, 184 – 190. 8. ClareBA, Conroy RS, Spelman K. (2009). he diuretic efect in human subjects of an extract of Taraxacum oicinale folium over a single day. Journal of Alternative and Complementary Medicine, 15, 929 – 934. 9. Dearing MD, Mangione AM, Karasov WH. (2011). Plant secondary compounds as diuretics: An overlooked consequence. American Zoology, 41, 890 – 901. 10. Geethalakshmi E, Sarada DV. (2010). Synthesis of plant-mediated silver nanoparticles using Trianthema decandra extract and evaluation of their antimicrobial activities. International Journal of Engineering Science and Technology, 2, 970 - 975. 11. Kim J, Kang HS, Chu GJ, Hong SB. (2008). Antifungal efectiveness of nano silver colloidal against rose powdery mildew in green house. Journal of Solid State Phenomena, 135,15 -18. 12. Kim JS, Kuk E, Yu KN, Kim JH, Park SJ, Lee HJ, Jeong DH, Cho MH. (2007). Antimicrobial efects of silver nanoparticles. Nanomedicine Nanotechnology Biology and Medicine, 3, 95 –101. 13. Krishnaraj C, Jagan EG, Rajasekar S, Selvakumar P, Kalaichel- van PT, Mohan N. (2010). Synthesis of silver nanoparticles using Acalypha indica leaf extracts and its antimicrobial activity against water borne pathogens. Colloids Surfaces B Biointerfaces, 76, 50 14. Lina L, Wang W, Huang J, Li Q, Sun D, Yang X, Wang H, He N, Wang Y. (2010). Nature factory of silver nanowires: plant-mediated synthesis using broth of Cassia istula leaf. Chemistry Journal. 16, 852 - 858. 15. Moshi MJ, Mbwambo ZH, Nondo RSO, Masimba PJ, Kamuhabwa A, Kapingu MC, homas P, Richard M. (2006). Evaluation of ethnomedical claims and Brine shrimp toxicity of some plants used in Tanzania as traditional medicines. African Journal of Traditional Complementary and Alternative Medicine, 3, 48 – 58. 16. Prakash P, Gnanaprakasam P, Emmanuel P, Arokiyaraj S, Saravanan M. (2013). Green synthesis of silvernanoparticles from leaf extractof Mimusops elengi Linn. for enhanced antibacterial activity against multi drug resistant clinical isolates. Colloids Surfaces B, 108, 255-259. 17. Rauwel P, Küünal S, Ferdov S, Rauwel E. (2015). A Review on the green synthesis of silver nanoparticles and their morphologies studied via TEM. Advances in Materials Science and Engineering, 9, 682749. 18. Saravanan M, Vemu AK, Barik SK. (2011). Rapid biosynthesis of silver nanoparticles from Bacillus megaterium (NCIM 2326) and their antibacterial activity on multi drug resistant clinical pathogens. Colloids Surfaces B, 88, 325-331. 19. Schütz K, Kammerer DR, Carle R, Schieber A. (2005). Characterization of phenolic acids and lavonoids in dandelion (Taraxacum oicinale WEB. ex WIGG.) root and herb by high-performance liquid chromatography/ electrospray ionization mass spectrometry. Rapid Communications in Mass Spectrometry, 19, 179–186. 20. Shrivastava S, Bera T, Roy A, Singh G, Ramachandrarao Dash P. (2007). Characterization of enhanced antibacterial effects of novel silver nanoparticles. Journal of Nanotechnology, 18, 225103-225112. 21. Trease GE, Evans WC,. (2002). Pharmacognosy. 15th Ed. London: Saunders Publishers, 42– 44. 22. Vedpriya, A. (2010). Living Systems:eco-friendlynanofactories. Digest Journal of Nanomaterials and Biostructures, 5, 9 – 21. 118