Research Journal of Recent Sciences ______ ______________________________ ______ ____ ___ ISSN 2277 - 2502 Vol. 4 ( IVC - 201 5 ), 47 - 5 2 (201 5 ) Res. J. Recent . Sci. International Science Congress Association 47 Biosynthesis of Silver Nanoparticles from Plant (Fenugreek Seeds) Reducing Method and their Optical Properties Rajesh Kumar Meena and Neelu Chouhan * Department of Pure and Applied Chemistry, University of Kota, Kota - 324005 Rajasthan, INDIA Available online at: www.isca.in , www.isca.me Received th 201 5 , revised th 20 1 5 , accepted th 20 1 5 Abstract In this paper, we synthesied the spherical silver nanoparticles (AgNPs) of 50 - 90 nm size , using AgNO 3 solution and the aqueous extract of Fenugreek plant seeds, which can act as a reducing, stablising and capping agent, at ambient condition. The formation of silver nanoparticles was confirmed by the XRD pattern and first excitonic peak of UV Vis. spectra that was supported by the change in colour of the solution (from colourless to dark pink). As synthesised Ag nanoparticles were characterized with the help of UV - Vis absorption spectroscopy analysis, Fourier Transform Infrared (FTIR) analysis, X - ray diffraction analysis (XRD), Scanning Electron Microscopy (SEM) analysis. Keywords , Fenugreek seed, bioreduction, AgNPs . Introduction In recent research, nanotechnology plays a vital role our day to day life becuase it can not only engineer shape and size of metal but the basic properties (chemical, physical, mechanical, optical and catalytic, etc) may also be changed in the useful manner 1 . Nanotechnology has achieve the importance in different fields such as health care, food and feed, cosmetics, energy science, electronics, mechanics, space industries, environmental health, biomedical science, chemical industries, drug and gene delivery 2 . It also have expansively been achieved for the treatments of cancer 3 , diabetes 4 , allergy 5 , infection 6 and inf lammation 7 . Green chemistry is an enhancement and development in such a fields, design, implementation of chemical products and processes to reduce the use and generation of substances that are harmful to human health and environment 8 . There are many ways to synthesize nanoparticles such as solid reaction, co - precipitation, chemical reaction, and sol gel method etc. In recent years green synthesis of NPs has a number of advantages over chemical synthesis, such as cost effectiveness and simplicity,. Moreover it is compatible for food applications and biomedical, and this method eliminates the use of toxic chemicals, temperature, energy and high pressure 9,10 . The growing need of environment friendly nanoparticles has attracted lots of researchers to use green synthesis methods of a variety of metal nanoparticles 11 due to their interesting, motivating, attractive and remarkable properties with a variety of applications over their bulk material 12 . Considering the chemical reduction methods, photochemical reductio n, electrochemical reduction and heat evaporation etc., the biological method is more beneficial and advantageous 13 . In this biosynthesis method, the plant extract has been used as reducing agent and capping agent for the synthesis of nanoparticles 14 due t o their reducing properties 15 . Some properties such as size, morphology and distribution of the particles are clearly obtained from the nanoparticles 16 . The synthesis of silver nanoparticles has been synthesized using green methods which are less usage of chemicals, non - toxic and low cost and Environmental friendly. Plants used for green Synthesis of silver nanoparticles like using plant extracts have been reported in Argemone maxicana 17 , Ocimum 18 , Cleome Viscosa 19 , Trigonella foenum - graecum 20 , Cycas 21 , Eucal yptus hybrida 22 , Iresine herbstii 23 , Avena sativa 24 , Lantana camara 25 , Citrus limon 26 , Calotropis gigantean 27 Achillea wilhemsii 28 , Tagetes erecta 29 Trachyspermum ammi 30 . Zea mays 31 , Azadirachta indica (Neem) 32 , Medicago sativa (Alfa alfa) 33,34 , Aloevera 35 , Emblica officinalis (Amla) 36 , Capsicum annuum 37 , Geranium sp. 38,39 , Diopyros kaki 40 , Magnolia kobus 41 and Coriandrum sp. 42 , etc.,. All the parts of the plant like leaf, stem, flower, seed and skin of the fruits were used earlier for the synthesis of AgNP s. Plants have been used for the synthesis of nanoparticles were coated by the plant extract which has medical benefits and can be used as drug and cosmetic applications 43 . The various nanoparticles like Ag 44 , Au 45 , Fe 46 , Pd 47 , ZnO/Au and ZnO/Ag 48 nanopart icles as well as quantum dots CdS 49 , among these, Silver nanoparticles places a major role because it has a number of important properties such as optical, electronic, chemical, photo electro chemical, catalytic, magnetic, antibacterial, and biological lab elling, antimicrobial, catalytic. Silver nanoparticle acts as antimicrobial agent which finds applications in medical field such as AgNPs coated blood collecting vessels, coated capsules, band aids etc 50 . The silver is non - toxic to animal cells and highly toxic to bacteria, and other microorganisms (E - coli, Pseudomonas aeruginosa, Staphylococcus aureus). Due to these phenomena it is considered to be safe and effective bactericidal metal 51 - 53 . In this report, Fenugreek is a self pollinating annual leguminou s bean which belongs to Fabaceae family 54 commonly known as Indian methi, It is one of the most ancient medicinal herbs 55 . Fenugreek seeds are the most important and useful part of Research Journal of Recent Sciences ______ _ _ _______________________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 4 ( IVC - 201 5 ), 47 - 5 2 (201 5 ) Res. J. Recent. Sci. International Science Congress Association 48 fenugreek plant. The fenugreek, plant mainly shows the presence of saponin and alkaloids are anti - nutritional factors 56 . The current investigation focuses on the aqueous seeds extract of Fenugreek used to synthesize AgNPs using different experimental conditions and thereby enhancing the importance of plant sources and implementin g green chemistry for the future research. Material and Methods Preparation of Dried Biomass, The seeds of Fenugreek Seeds were collected from herbal garden of University of Rajasthan Campus, Rajasthan, India. The seeds were thoroughly washed with deionis ed distilled water and crushed. The powder was further used for preparation of 10 g/L aqueous seeds extract. This extract was filtered and stored at 4 o C until further use for present investigation. Chemicals : Silver nitrate (AgNO 3 ) was purchased from Sigm a - Aldrich. Deionised distilled water was used throughout the experiment. All other chemicals were of analytical grade. Synthesis of nanoparticles : For biosynthesis of nanoparticles, 2.0 ml plant seeds extract was mixed with 25 ml of freshly prepared silve r nitrate 10 - 3 M AgNO 3 solution was prepared in 250 mL of deionised water in a sterile conical flask and kept in dark condition at room temperature. The reaction mixture was incubated for 30 min or till colour change to dark pink was observed. The nanopart icles were then synthesized by drying at 90 o C. Ch aracterization of nanoparticles: The syntheised nanoparticles were characterized using UV - Vis Spectroscopy ( LABINDIA UV - Visible 3000 + ) over a range of 200 - 800 nm. The topography of the nanoparticles was st udied by SEM (Scanning Electron Microscope) analysis. FTIR (Fourier Transform Infrared Spectroscopy) was performed to obtain wide spectrum of nanoparticles over a narrow range. This method gives us information about plant peptides that have covered the par ticles during synthesis procedure. The XRD (X - Ray Diffraction Analysis) was performed to note the size of the obtained nanoparticle. Results and Discussion Synthesis and characterization of silver nanoparticles, Aqueous seed extract of Fenugreek acts as a reducing agent 57 which reduces metallic silver to nanosilver and hence the colour change was obtained figure - 1 . It is well known that silver nanoparticles exhibit reddish pink colour in aqueous solution due to excitation of surface plasmon vibrations in s ilver nanoparticles. Ag 2+ ions of silver nitrate are found to be reduced to Ag atoms. It is generally recognized that UV - Vis spectroscopy could be used to examine size and shape controlled nanoparticles in aqueous suspensions. Figure 1 show the UV - Vis spec trum recorded from the reaction medium after 24 hours and gives rise to an absorption band at 420 nm. (A) (B) Figure - 1 (A) Aqueous seed extract (B) Aqueous extract and silver nitrate after incubation The synthesized nanoparticles were characterized using FTIR, SEM, XRD and UV Vis spectroscopy analysis. The reduction of silver ions to nano silver was monitored and confirmed using UV spectra. After the colour change was obtained a small aliquot of sample was diluted with distilled water and subjected to UV analysis. The characteristic peak value for silver nanoparticles is between 400 - 580nm. Figure - 2 . (A) (B) Figure - 2 UV Vis spectra of (A) Pure Seed Extracts (B) Silver nanoparticles produced by extract of Fenugreek Seeds 150 300 450 600 750 0.00 0.05 0.10 0.15 0.20 0.25 Absorbance/au Wavelength /nm Silver Nanoparticles 400 600 800 0 3 6 Intensity/ au Wavelength/ nm Pure Seed Extract Research Journal of Recent Sciences ______ _ _ _______________________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 4 ( IVC - 201 5 ), 47 - 5 2 (201 5 ) Res. J. Recent. Sci. International Science Congress Association 49 The spectrum of the sample was obtained for wavelength range in - between 400nm to 580nm. The λ max of the nanoparticles was observed at 470nm. This is because of a phenomenon called Surface Plasmon Resonance (SPR) exhibited by silver nanoparticles. The sil ver nanoparticles oscillate when exposed to electromagnetic radiation and this oscillation gives a typical peak value 58 . The SEM (FESEM, JEOL JSM - 6700F ) image of the nanoparticles represents the topography of t he particles is shown in image figure - 3 . Fi gure - 3 SEM image of silver nanoparticles produced by Fenugreek seeds extract The SEM image suggests the presence of roughly spherical silver nanoparticles. The incidence of Xrays on the powdered nanoparticles gives a particular pattern which helps to char acterize the nanoparti cles as shown in the XRD graph figure - 4 . 40 50 60 70 2 Theta (degree) (220) (200) (111) Intensity (counts) 2 Theta (degree) Ag Nanoparticles Figure - 4 XRD pattern of the silver nanoparticles produced by extract of Fenugreek seeds XRD (manual mode) was used to characterize the AgNp. The 2d angle is converted to the diameter using t he Scherrer formula (Dp = Kλ /β1/2 cosθ). The size of silver nanoparticles synthesised by green synthesis was estimated to be around 20 - 50 nm. FTIR (Bruker - Tensor Model) analysis also gives a set of peak values unique for the sample along with information of the plant peptides that are present in the sample as the plant ex tract acts as a reducing agent figure - 5. FTIR analysis is used to confirm the presence of plant peptides visible due to the bending produced by amide bonds 59 . 3726.21 3471.78 3307.14 2881.43 2825.73 1756.01 1647.69 1572.95 1491.94 1389.77 1148.79 1023.10 809.43 664.39 597.63 575.32 1000 1500 2000 2500 3000 3500 Wavenumber cm-1 86 88 90 92 94 96 98 100 Transmittance [%] Figure - 5 FTIR spectra of nanoparticles synthesized by extract of Fenugreek seeds Biological synthesis of silver nanoparticles is an alternative to chemical synthesis and it used the reducing properties of biological products for synthesis of silver nitrate to nanosilver. Biologic al synthesis of nanoparticles has been previously reported using plant seed extracts. The phytochemical in the seed reduce the silver salts and not only produce silver nanoparticles but also stabilize it by capping the nanoparticles with the plant peptides . The antimicrobial activity of the nanoparticles is thus enhanced due to the presence of plant proteins and phytochemical. Conclusion In this present study the synthesis of silver nanoparticles was synthesized by biological method using Fenugreek seed ex tract which acts as a reducing agent to reduce silver metal to nanosize particles. The synthesized silver nanoparticles were subjected to analysis such as SEM, UV Vis Spectroscopy, XRD, and FTIR in order to characterize them. To the best of our knowledge t his is the best information of the observations of the unique structures of fenugreek seeds extract mediated Ag nanoparticles. This opens a way to understand the synthesis mechanism of Ag nanoparticles formed from other plant seeds extracts . Reference 1. Alq udami A and Annapoorni S., Fluorescence from metallic silver and iron nanoparticles prepa red by exploding wire technique, Plasmonics ., 2(1) 5 - 13 (2007) 2. Korbekandi H and I ravani S., Silver Nanoparticles, Nanotechnology and Nanomaterials , 3 , 5 - 16 (2012) 3. Brig ger I, Dubernet C, Couvreur P., Nanoparticles in cancer therapy an d diagnosis, Adv Drug Deliv Rev ., 64 , 24 – 36 (2012) Research Journal of Recent Sciences ______ _ _ _______________________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 4 ( IVC - 201 5 ), 47 - 5 2 (201 5 ) Res. J. Recent. Sci. International Science Congress Association 50 4. Basarkar A, Singh J. Poly (lactide - co - glycolide) - polymethacrylate nanoparticles for intramuscular delivery of plasmid encoding interleukin - 10 to prevent autoimmune diabetes in mice , Pharm Res ., 26 , 72 – 81 (2009) 5. Roy K, Mao HQ, Huang SK and Leong KW , Oral gene delivery with chitosan - DNA nanoparticles generates immunologic protection in a murine model of peanut allergy , Nat. Med ., 5 , 387 – 391 (19 99) 6. Furno F, Morley KS, Wong B, Sharp BL, Arnold PL, Howdle SM et al. Silver nanoparticles and polymeric medical devices , a new appr oach to prevention of infection, J Antimicrob Chemother ., 54 , 1019 – 1024 (2004) 7. Wilson DS, Dalmasso G, WangL, Sitaraman SV, M erlin D and Murthy N. , Orally delivered thioketal nanoparticles loaded with TNF - α – siRNA target inflammation and inhibit ge ne expression in the intestines, Nat. Mater ., 9 , 923 – 928 (2010) 8. Mano Priya M, Karunai Selvia B, John Paul JA. Green Synthesis of Silve r Nanoparticles from the Leaf Extracts of Euphorbia Hirta and Nerium Indicum , Digest.J. Nanomat. Biostruct ., 6(2) , 869 – 877 (2011) 9. Chandrakant K, Tagad, Sreekantha Reddy Dugasanic, Rohini Aiyer, Sungha Parkc, Atul Kulkarni and Sushma Sabharwal., Green synth esis of silver nanoparticles and their application for the development of optical fiber based hydrogen peroxide sensor, Sensors and Actuators B ., 183 , 144 – 149 (2013) 10. Yamini Sudha Lakshmi G, Fouzia Banu, Ezhilarasan, Arumugam, Sahadevan., Green Synthesis of Silver Nanoparticles from Cleome Viscosa , Synthesis and Antimicrobial Activity., 5 (2011) 11. Christopher L, Kitchens Douglas E, Hirt Scott M, Husson Alexey A and Vertegel., Synthesis, Stabilization, and Characterization of Metal Nanoparticles , The Graduate S chool of Clemson University, (2010) 12. Hasna Abdul Salam, Rajiv P, Kamaraj M, Jagadeeswaran P, Sangeetha Gunalan and Rajeshwari Sivaraj., Plants , Green Route for Nanoparticle Synthesis , Inter.Res.J.Bio. Sci ., 1(5) , 85 - 90 (2012) 13. Geoprincy G, Vidhyasrr BN, Poon guzhali U, Nagendra Gandhi N, Renganathan S., A review on green synthesis of silver nano particles , Asian.J.Pharma.Clini.res ., 6(1) , 8 – 12 (2013) 14. Akl M Awwad1, Nidà M., Green Synthesis of Silver Nanoparticles by Mulberry Leaves Extract , Nanoscience and Nanot echnology ., 2(4) , 125 - 128 (2012) 15. Umesh B. Jagtap, Vishwas A. Bapat., Green synthesis of silver nanoparticles using Artocarpus heterophyllus Lam. Seed extract and its antibacterial activity , Industrial Crops and Products ., 46 , 132 – 137 (2013) 16. Nethra Devi C, Sivakumar P and Renganathan S., Green synthesis of silver nanoparticles using Datura metel flower extract and evaluation of their antimicrobial activity , Inter.J. Nanomat.Biostruct ., 2(2) , 16 – 21 (2012) 17. Singh D. Jain, M. K. Upad hyay, N. Khandelwal and H. N. Verma., Green Synthesis Of Silver Nanoparticles Using Argemone Mexicana Leaf Extract And Evaluation Of Their Antimicrobial Activities., Digest Journal of Nanomaterials and Biostructures , 5(2) , 483 - 489 (2010) 18. Mallikarjun K, Narsimha G, Dillip GR, Praveen B, S hreedhar B, Lakshmi S, Reddy VS and Raju DP., Green synthesis of silver nanoparticles using Ocimum leaf extract and their characterization. Digest J Nanomat Biostruct ., 6(1) , 181 - 186 (2011) 19. Yamini Sudha Lakshmi.G Singapore., Green Synthesis of Silver Nan oparticles from Cleome Viscosa , Synthesis and Antimicrobial Activity , International Conference on Bioscience, Biochemistry and Bioinformatics IPCBEE ., 5 (2011) 20. R.P. Singh, S. Magesh, C., Rakkiyappan formation of fenugreek (Trigonella foenum - graecum) extrac t mediated Ag Nanoparticles , mechanism and applications , International Journal of Bio - Engineering Sciences & Technology - IJBEST., 2(3) , 283 - 294 (2011) 21. Anal K. Jha K. Prasad., Green Synthesis of Silver Nanoparticles Using Cycas Leaf , International Journal of Green Nanotechnology , Physics and Chemistry ., 1 , 110 – 117 (2010) 22. Manish dubey, Seema bhadauria, B.S. Kushwah., Green synthesis of nanosilver particles from extract of Eucalyptus hybrida (safeda) leaf , Digest Journal of Nanomaterials and Biostructures ., 4(3 ) , 537 – 543 (2009) 23. Dipankar C, Murugan S., The green synthesis, characterization and evaluation of the biological activities of silver nanoparticles synthesized from Iresine herbstii leaf aqueous extracts , Colloids ., 98 , 112 - 119 (2012) 24. V. Armendariz, I. Her rer a, J. R. Peralta - Videa et al., Size controlled gold nanoparticle formation by Avena sativa biomass , use of plants in nanobiotechnology , Journal of Nanoparticle Research ., 6(4) , 377 – 382 (2004) 25. Thirumurugan A et al., Biological synthesis of silver nanopart icles by Lantana camara leaf extracts , International Journal of Nanomaterials and Biostructures ., 2(4) 65 - 69 (2012) 26. Prathna TC and Chandrasekaran N and Raichur, Ashok M and Mukherjee, Amitava., Biomimetic synthesis of silver nanoparticles by Citrus limon ( lemon) aqueous extract and theoretical prediction of particle size , In , Research Journal of Recent Sciences ______ _ _ _______________________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 4 ( IVC - 201 5 ), 47 - 5 2 (201 5 ) Res. J. Recent. Sci. International Science Congress Association 51 Colloids and Surfaces B , Biointerfaces ., 82(1) , 152 - 159 (2011) 27. J. Sivakumar, C.Premkumar, P. Santhanam and N. Saraswathi., Biosynthesis of Silver Nanoparticles Using Calotropis gigante an Leaf , African Journal of Basic & Applied Sciences ., 3(6) , 265 - 270 (2011) 28. Javad karimi andeani, Hojjatollah kazemi, Sasan mohsenzadeh, Afsanehs afavi., Biosynthesis of gold nanoparticles using dried flowers extra ct of achillea wilhelmsii plant, Digest Jo urnal of Nanomaterials and Biostructures. , 6(3) 1011 - 1017 (2011) 29. krishnamurthy NB, Nagaraj , Barasa malakar, liny P, and Dinesh R., Green synthesis of gold nanoparticles using tagetes erecta l.(mari gold) flower extract & evaluation of their antimicrobial activities , International Journal of Pharma and BioSciences ., 3(1) , 212 - 221 (2012) 30. Chouhan Neelu and Meena Rajesh Kumar., Biosynthesis of silver nanoparticles using Trachyspermum ammi and evaluation of their antibacterial activities , Int J Pharm Bio Sci ., 6(2) , 1077 – 1086 (2015) 31. Choi S. M. , Seo M.H., Kim H. J. and Kim W. B., Synthesis and characterization of graphene - supported metal nanoparticles by impregnation method with heat treatment in H 2 atmosphere , Synthetic Metals ., 161 , 2405 – 2411 (2011) 32. Shankar S., R ai S., Ahmad A. and 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 , 496 – 502 (2004) 33. Gardea - Torresdey J. L., Gomez E., Peralta - Vide a J., Parsons J. G., Troiani H. E. and Santiago P. , Formation and growth of Au nanoparticles inside live alfalfa plants , Nano Lett ., 2 , 397 – 401 (2002) 34. Gardea - Torresdey J. L., Gomez E., Peralta - Videa J ., Parsons J.G., Troiani H.E. and Santiago P., Alfalfa sprouts , a natural s ource for the sy nthesis of silver nanoparticles, Langmuir., 19 , 1357 – 1361 (2003) 35. Chandran S.P., Chaudhary M., Pasricha R., Ahmad A. and Sastry M., Synthesis of gold nanotriangles and silver nanoparticles using Aloe vera plant extract, Biotechnol. Prog ., 22 , 577 – 583 (2006) 36. Amkamwar B., Damle C., Ahmad A., Sastry M., Biosynthesis of gold and Ag nanoparticles using Emblica officinalis fruits extract, their phase transfer and transmetallation in an organic solution , J Nanosci. Nanotechnol ., 5 , 1665 - 1671 (2005) 37. Li S., Shen Y., Xie A., Yu X., Qiu L., Zhang L. and Zhang Q., Synthesis of silver nanoparticles u sing. Capsicum annum L. Extract, Green Chemistry ., 9 , 852 – 858 (2007) 38. Shankar S. S., Ahmad A. and Sastry M., Geranium Leaf Assisted Biosy nthesis of Silver Nanop articles, Biotechnol. Progress ., 19 , 1627 – 1631 (2003) 39. Vijayakumara M., Priya K., Nancy F.T., Noorlidah A. and Ahmed A.B. A., Biosynthesis, characterisation and antibacterial effect of plant - mediated silver nanoparticles using Artemisia nilagirica , Industria l Crops and Products ., 41 , 235 – 240 (2013) 40. Gan P. P., Ng S. H., Huang Y., Fong S. and Li Y., Green synthesis of gold nanoparticles using palm oil mill effluent (POME) , A low - cost a nd eco - friendly viable approach, Bioresource Technology., 113 , 132 – 135 (2012) 41. Song J.Y., Hyeon - Kyeong H.K. and Kim B.S., Biological synthesis of gold nanoparticles using Magnolia kobus and Dipyros kaki leaf extracts , Process Biochem ., 44 , 1133 - 1138 (2009) 42. Narayanan K. B. and Sakthivel N., Coriander leaf mediated bio synthesis of gold nanoparticles, Materials Letters. , 62 , 4588 – 4590 (2008) 43. Mallikarjunaa K, Narasimhab G, Dilli pa GR, Praveenb B, Shreedharc B and Sree Lakshmic C et al., Green Synthesis of Silver Nanoparticles Using Ocimum Leaf Extract and Their Characterization , Digest.J. Nanomat.Biostruct ., 6(1) , 181 – 186 (2011) 44. Song JY and Kim BS., Rapid biological synthesis of silver nanoparticles using plant leaf extracts , Bioprocess Biosyst Eng. , 32 , 79 - 84 (2009) 45. Huang J, Li Q, Sun D, Lu Y, Su Y, Yang X, Wang H, Wang Y, Shao W, He N, Ho ng J, Chen C., Biosynthesis of silver and gold nanoparticles by using novel sun - dried Cinnamomum camphora leaves , Nanotechnol ., 18 , 105 - 104 (2007) 46. Pattanayak M and Nayak PL., Green Synthesis and Characterization of Zero Valent Iron Nanoparticles from the L eaf Extract of Azadirachta indica ( Neem ), World Journal of Nano Science & Technology ., 2(1) , 06 - 09 (2013) 47. Nadagouda M.N. Varma R.S., Green synthesis of silver and palladium nanoparticles at room temperature using coffee and tea extract , Green Chem ., 10 , 85 9 – 862 (2008) 48. Fage ria P., Gangopadhyay S. and Pande S., Synthesis of ZnO/Au and ZnO/Ag nanoparticles and their photocatalytic application using UV and visible light. RSC Adv ., 4 , 24962 – 24972 (2014) 49. Ahmad, P. Mukherjee, D. Mandal, S. Senapati, M.I. Khan, R. Kumar and M. Sastry., Enzyme Mediated Extracellular Synthesis of CdS Nanoparticles by the Fungus, Fusariuoxysporum , J.Am. Chem. Soc ., 124 , 12108 – 12109 (2002) 50. Geoprincy G, Saravanan P, NagendraGandhi N and Renganathan S., A novel approach for studying the Research Journal of Recent Sciences ______ _ _ _______________________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 4 ( IVC - 201 5 ), 47 - 5 2 (201 5 ) Res. J. Recent. Sci. International Science Congress Association 52 c ombined antimicrobial effects of silver nano particles and antibiotics through agar over layer method and disk diffusion method , Digest.J.Nanomat.Biostruct , 6(4) , 1557 - 1565 (2011) 51. Kalimuthu K, Babu RS, Venkataraman D, Bilal M and Gurunathan S., Biosynthesi s of silver nanocrystals by Bacillus licheniformis , Colloids Surf B ., 65(1) , 150 - 153 (2008) 52. Wijnhoven SWP, Peijnenburg WJGM, Herberts CA, Hagens WI, Oomen AG and Heugens EHW et al., Nano – silver , a review of available data and knowledge gaps in human and en vironmental risk assessment , Nano toxicology. , 3 , 109 - 138 (2009) 53. Klueh U, Wagner V, Kelly S, Johnson A and Bryers JD., Efficacy of silver - coated fabric to prevent bacterial colonization and subsequent device - based biofilm formation , J. Biomed. Mater. Res ., 53(6) , 621 – 631 (2000) 54. Balch PA Prescription for dietary wellness (2 nd edn). Penguin group, New York 15 - 17 (2003) 55. Thomas JE, Bandara M, Lee EL, Driedger D and Acharya S., Biochemical monitoring in fenugreek to develop functional food and medicinal plant v ariants , N Biotechnol ., 28 , 110 - 117 (2011) 56. Jani R, Udipi SA. and Ghugre PS Mineral content of complementary foods , Indian J Pediatr 76 , 37 - 44 (2009) 57. Thombre RS, Mehta S, Mohite J and Jaisinghani P., Synthesis of silver nanoparticles and its cytotoxic effec t on THP - 1 cancer cell line. International Journal of Pharma and Biosciences . 4(1) , 184 - 192 (2013) 58. Smitha SL, Nissamudeen KM, Philip D and Gopchandran KG., Studies on surface Plasmon resonance and photoluminescence silver nanoparticles , Spectrochim. Acta A ., 71(1) , 186 – 190 (2008) 59. Jing A, Wang D, Luo Q and Yuan X., Antimicrobial active silver nanoparticles and Silver/polystyrene core - shell nanoparticles prepared in room - temperature ionic liquid, Materials Science and Engineering C , 29(6) ,1984 – 1989 (2009)