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Silver is known for its antimicrobial activity, silver nanoparticles are gaining great importance due to their antimicrobial activities. " Green technology " is the use of various plant materials for the biosynthesis of nanoparticles, as it
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   ISSN 2394-966X  International Journal of Novel Research in Life Sciences Vol. 5, Issue 2, pp: (25-31), Month: March - April 2018, Available at:  Page | 25 Novelty Journals BIOSYNTHESIS OF SILVER NANOPARTICLES USING  Azadirachta indica  LEAF EXTRACT AND ASSESSMENT OF ITS ANTIBACTERIAL ACTIVITY ON SOME PATHOGENIC ENTERIC BACTERIA Jamiu, A.T 1* , Bello, S.A 2 1,2 Department of Biological Sciences, Al-Hikmah University, P.M.B. 1601 Ilorin, Kwara State, Nigeria. *Corresponding author:    Abstract:  Silver is known for its antimicrobial activity, silver nanoparticles are gaining great importance due to their antimicrobial activities. “Green technology” is the use of various plant materials for the biosynthesis of nanoparticles, as it does not involve any harmful chemicals. Bioactive compounds such as flavonoids, terpenoids etc present in plant extracts have made them best material for the green synthesis of nanoparticles. In this study we have reported the synthesis of silver nanoparticles by reducing the silver ions present in the silver nitrate solution by the aqueous extract of  Azadirachta indica  leaf. Silver nanoparticles (AgNPs) were successfully synthesized using  A. indica  leaf extract and the formation and stability of the reduced silver nanoparticles in the colloidal solution were monitored using UV-Vis spectroscopy. The broad surface plasmon resonance (SPR) peak was at 437nm. The antibacterial effect of the synthesized AgNPs produced was studied using some enteric ptathogenic bacteria such as Salmonella Typhi,  Pseudomonas aeuruginosa and  Escherichia coli . From the disc diffusion results, the highest antibacterial activity of synthesized AgNPs was found against S . Typhi (14mm) and inhibitory zone of 12mm was recorded for  E. coli  and  P. aeruginosa  respectively. The synthesized AgNPs showed excellent antibacterial property compared to the AgNO 3  solution and  A. indica  leaf extract. It could be concluded that  A. indica leaf extract can be used effectively in the production of antimicrobial AgNPs for commercial applications.  Keywords:    Azadirachta indica , Antibacterial activity, Biosynthesis, Silver nanoparticles. 1.   INTRODUCTION Resistance of microorganisms to common antibiotics has become a great burden to general healthcare facilities, especially in developing nations with little or improper medical resources (Marasini et al ., 2015). The treatment of bacterial infection with antibiotics is a route which is rapidly becoming more and more difficult to sustain, due to increase in emergence and re-emergence of multidrug resistant pathogens (Taylor et al. , 2005). In pursuit of novel treatment, there is growing interest in the use of nanomaterials with antimicrobial potentials to combat the menace of pathogen cessation, since their large surface to volume ratio ensures a broad range of attack on bacterial surface. Microbes also find it difficult to acquire resistance toward nanoparticles as they target multiple bacterial components, contrary to the mechanistic action of conventional antibiotics (Dhanalekshmi et al ., 2013).   ISSN 2394-966X  International Journal of Novel Research in Life Sciences Vol. 5, Issue 2, pp: (25-31), Month: March - April 2018, Available at:  Page | 26 Novelty Journals Nanoparticles can be synthesized via different approaches namely chemical, physical, and biological (Shah et al., 2013). In comparison with chemical and physical methods, biological synthesis has many advantages: it is simple, cost effective, non-hazardous, environmentally friendly and easily scaled up for large scale synthesis (Veerasamy et al ., 2011). Also, processes employed for making nanoparticles using plant extracts are readily scalable and less expensive in comparison to the relatively expensive methods based on microbial processes and whole plants (Mittal et al ., 2013), nanoparticles produced from plant extract possesses medicinal properties and could be used in drugs, targeted drug delivery and cosmetic industry (Saranyaadevi et al ., 2014). One of the most promising nanoparticles which act as a highly effective antimicrobial agent is silver. Various investigations on silver nanoparticles have been done to study its antimicrobial activity, silver nanoparticle exhibited significant antibacterial activity against  Escherichia coli, Staphylococcus aureus  and antifungal activity against Trichophyton, Trichosporon beigelii  and Candida albicans  (Gajbhiye e t al ., 2009). Plant extracts have enzymes and phytochemicals such as terpenoids, flavonoids and phenolic compound (Rao et al ., 2013) which act as bioreductants as well as capping agent in the presence of metal salt for nanoparticles synthesis. This study deals with the synthesis of silver nanoparticles from neem leaf. Neem (  Azadirachta indica ) commonly called ‗India Lilac‘ or ‗Margosa‘, belongs to the family Meliac eae, subfamily Meloideae and tribe Melieae. Neem is the most versatile, multifarious trees of tropics, with immense potential. It possesses maximum useful non-wood products (leaves, bark, flowers, fruits, seed, gum, oil and neem cake) than any other tree species (Sreeram et al ., 2008). Various parts of the neem tree have been used as traditional Ayurvedic medicine in India, neem oil and the bark and leaf extracts have been therapeutically used as folk medicine to control leprosy, intestinal helminthiasis, respiratory disorders, and constipation and also as a general health promoter. Neem oil finds use to control various skin infections. Bark, leaf, root, flower and fruit together cure blood morbidity, biliary afflictions, itching, skin ulcers, burning sensations and phthisis (Sreeram et al ., 2008). Biosynthesis of silver nanoparticles has been performed using a number of plants, some of which include  Aloe vera extract (Chandran et al ., 2006),  Allium sativum (Garlic) extract (Von White, 2012) Svensonia hyderobadensis  (LingaRao and Savithramma, 2011),  Acalypha indica (Vasireddy et al ., 2012) and Shoreatum buggaia  (Venkateswarlu et al ., 2010). Considering the vast potentiality of plants as sources this work aims to investigate the use of  Azadirachta indica  leaf extract for biosynthesis of AgNPs and the synthesized AgNPs was evaluated for its antibacterial against on some selected pathogens. To the best of my knowledge, this research will represent the first reference to the use of  A. indica  leaf extract for green synthesis of silver nanoparticles in Nigeria. 2. MATERIALS AND METHODS   2.1 Collection of Plant materials Fresh and healthy  Azadirachta indica  leaf was collected from a garden near Federal University of Technology, Minna. The plant material was taken to the Department of Biological Sciences, Federal University of Technology, Minna, for identification by a taxonomist, Mr Muhammad. 2.2 Medicinal Plant Extraction Preparation The  Azadirachta indica  leaf was washed with distilled water, and then air dried for 7 days at ambient temperature. The plant material was ground to a fine powder using electrical blender. Five grams (5g) of the powdered leaf was soaked in 100ml distilled de-ionized water for 30 minutes under vigorous shaking in the water bath at 60˚C . The obtained leaf extract was filtered through Whatmann filter paper no.1 and was stored in refrigerator at 4ºC for further experiment (Devasenan et al ., 2016). 2.3 Preparation of bacterial isolate  Already isolated and identified clinical bacterial isolates was collected from the Microbiology Lab, Federal University of Technology, Minna. The bacterial isolates used include S. Typhi,  E. coli , and P. aeruginosa . The isolates was maintained on agar slants and refrigerated for further use. 2.4 Synthesis of Silver Nanoparticles One (1) mM AgNO 3  solution was prepared and stored in amber colour bottle. One (1) ml of  Azadirachta indica  Leaf Extract (ALE) was added to 9ml 1 mM aqueous silver nitrate. Nine (9) mL of 10 − 3  M volume of silver nitrate solution   ISSN 2394-966X  International Journal of Novel Research in Life Sciences Vol. 5, Issue 2, pp: (25-31), Month: March - April 2018, Available at:  Page | 27 Novelty Journals was considered as control. The resulting solution was kept under direct sunlight. Observation for gradual colour change was done and the result was recorded (Das et al ., 2016). 2.5 Characterization of the Nanoparticles  The bioreduction of pure Ag + to Ag o ions was confirmed by subjecting 2mL of the synthesized AgNPs to UV-Visible spectrophotometer (Model- Shimadzu UV-1800, Japan) in the range of 190-800nm (Das et al ., 2016). 2.6 Antibacterial Activity of the Synthesized TLE-AgNPs  The silver nanoparticles synthesized using  Azadirachta indica  was tested for antibacterial activity by standard agar well-diffusion method (Perez et al ., 1990) against pathogenic enteric bacteria which include S. Typhi,  E. coli , and P. aeruginosa . The pure culture of bacterial pathogens was subcultured on nutrient agar. 100μl of fresh overnight grown cultures of the respective bacteria was spread on Nutrient Agar containing Petri plates. Each strain was swabbed uniformly using sterile cotton swabs. Wells of 6mm diameter was made on nutrient agar using sterile cork borer and 100μL of the synthesized ALE -AgNPs, AgNO 3  solution and aqueous leaf extract (control) was loaded into the different wells. After incubation at 37°C for 24 hours, the zone of inhibition was measured (Kora et al. , 2009). 3. RESULTS & DISCUSSION Silver nanoparticles with their unique chemical and physical properties are proving to be an alternative for the development of new antibacterial agents. Silver nanoparticles (AgNPs) have also found diverse applications in the form of wound dressings, coatings for medical devices and silver nanoparticle impregnated textile fabrics etc (Rai et al., 2009). A detailed study on the biosynthesis of silver nanoparticles and its antibacterial activity against some pathogenic enteric bacteria was conducted. Plate 1: Biosynthesis of AgNPs using aqueous leaf extract of  Azadirachta indica   Key : A = 1 mM AgNO 3  solution B = Plant extract C = 1ml of the plant extract dispensed into 9ml AgNO 3  solution D = 1 mM AgNO 3  solution + Plant extract at zero minute E = Colloidal AgNPs after 20 minutes Previous studies reported that AgNPs can be synthesized by plants such as Capsicum annuum  (Bar et al ., 2009), Carica  papaya  (Jha and Prasad, 2010), Gliricidia sepium  (Raut et al ., 2010),  Eucalyptus hybrid   (Dubey et al ., 2009) and microorganisms such as  Aspergillus fumigatus   (Bhainsa and D‘Souza, 2006), Cladosporium cladosporioides  (Balaji et al ., 2009), Fusarium oxysporum  (Ahmad et al ., 2003), Pseudomonas aeruginosa  (Husseiny et al ., 2007) and   ISSN 2394-966X  International Journal of Novel Research in Life Sciences Vol. 5, Issue 2, pp: (25-31), Month: March - April 2018, Available at:  Page | 28 Novelty Journals  Rhodopseudomonas capsulate  (He et al ., 2007). In this study, aqueous silver ions were reduced to AgNPs after mixing with 5%  Azadirachta indica  leaf extract (1:9), a stable reddish brown colour was produced within 10 minutes under solar irradiation (Plate 1). This change in colour has been previously observed by several investigators (Saxena et al ., 2010, Khandelwal et al ., 2010). These authors suggested that the colour change appeared due to the surface Plasmon resonance of the deposited AgNPs (Noginov et al ., 2006). Figure 1: UV- vis spectra of silver nanoparticles synthesized by  Azadirachta indica  leaf extract. The presence of nanoparticles was confirmed by subjecting 2mL of the synthesized AgNPs to UV-visible spectroscopic analysis in the range of 190-800nm (Figure 1). From this analysis, the absorbance peak was found at around 437nm. It is earlier reported that absorbance around 430nm for silver is a characteristic of these noble metal particles (Nestor et al ., 2008). The antimicrobial activity of AgNPs was reported in a series of report (Rai et al ., 2009, Jha et al ., 2010, Khandelwal et al ., 2010). The antibacterial activity of AgNPs produced from  A. indica  leaf extract was studied against some selected pathogenic enteric bacteria, using the well diffusion method. The diameter of inhibition zones (mm) around each well with silver nanoparticles solution is represented in Table 1. The highest antimicrobial activity of the synthesized AgNPs was found against Salmonella  Tyhi (16mm), the diameters of the inhibition zones against  Escherichia   coli  and Pseudomonas   aeruginosa  were found to be 12mm respectively. The synthesized AgNPs showed higher antimicrobial activity against the three enteric bacteria when compared to AgNO 3  and  A. indica  leaf extract (Control). Table 1: Results of antibacterial activity test of the plant extract, AgNPs and AgNO 3 . Test organisms/ zone of inhibition(mm) Plant extract AgNO 3  AgNPs   S. Typhi   10 12 14  E. coli 09 10 12  P. aeruginosa 09 11 12 A number of theories for antimicrobial actions of colloidal silver solution have been proposed. For example, alteration of permeability of cell membrane (Sondi, and Sondi, 2004), release of lipopolysaccharides and membrane proteins (Amro et al ., 2000), generation of free radicals responsible for the damage of membrane (Kim et al ., 2007), and dissipation of the proton motive force resulting in the collapse of the membrane potential (Chun-Nam et al ., 2006), however; the exact mechanism has not been fully deciphered. Moreover, Tripathi et al. 2010 studied the effect of silver nano balls on  Escherichia coli , S. typhimurium ,  B. subtilis  and P. aeruginosa  by colony forming unit (cfu) and growth curve at a concentration of 40µg/ml and showed a significant reduction of bacterial population and their growth pattern at the studied concentration. Overall, the results of this study indicated that the nano-sized silver produced by  A. indica  showed excellent antibacterial property and high antimicrobial activity compared to the ionic silver.   ISSN 2394-966X  International Journal of Novel Research in Life Sciences Vol. 5, Issue 2, pp: (25-31), Month: March - April 2018, Available at:  Page | 29 Novelty Journals 4. CONCLUSION   In this study,  Azadirachta indica  conjugated silver nanoparticles were synthesized using their leaves extract. The biosynthesized silver nanoparticles were proved to have excellent antimicrobial performance against pathogenic enteric bacteria, S.  Tyhi, P. aeruginosa  and  E. coli , using  A. indica  leaves extract. Therefore, AgNPs producing  A. indica  may be potentially utilized for the economical production of AgNPs for many pharmaceutical applications. Conflict of Interest The author has declared no conflict of interest. Compliance with Ethics Requirements This article does not contain any studies with human or animal subjects. ACKNOWLEDGMENTS The authors acknowledge with thanks the Department of Microbiology and Centre for Genetic Engineering and Biotechnology, Federal University of Technology, Minna, Niger State, Nigeria providing the resources for the research work. REFERENCES [1]   Ahmad A Mukherjee P Senapati S Mandal D Khan MI Kumar R Sastry M (2003). Extracellular biosynthesis of silver nanoparticles using the fungus Fusarium Oxysporum , Colloid Surf. B 28:313  –  318. [2]   Amro NA Kotra LP Wadu-Mesthrige K Bulchevy A Mobashery, S and Liu, G.Y (2000) High resolution atomic force microscopy studies of the E. coli outer membrane: the structural basis for permeability, Langmuir 16:2789  –  2796. [3]   Balaji, D.S. Basavaraja, S. Deshpande, R. Bedre, Mahesh, D. Prabhakar, B.K and Venkataraman, A (2009). Extracellular biosynthesis of functionalized silver nanoparticles by strains of Cladosporium cladosporioides  fungus, Colloids Surf B 68 (1):88  –  92. [4]   Bhui, H., D.K. Sahoo, G.P. Sarkar, P. De, S.P and Misra, A (2009). Green synthesis of silver nanoparticles using latex of Jatropha curcas, Colloids Surf. A 339:134  –  139. [5]   Bhainsa, K.C. and D‘Souza, S.F (2006). Extracellular biosynthesis of silver nanoparticles using the fungus Aspergillus fumigatus, Colloids Surf B 47:160  –  164. [6]   Chandran, S.P., Chaudhary, M., Pasricha, R., Ahmad, A. and Sastry, M (2006). ―Synthesis of gold nanotriangles and silver nanoparticles using  Aloe vera  plant extract,‖  Biotechnology Progress , vol. 22,no. 2, pp. 577  –  583. [7]   Chun-Nam, L. Ho, C.M. Chen, R. He, Q.Y. Yu, W.Y. Sun, H.P. Tam, K.H. Chiu, J.F and Che, C.M (2006). Proteomic analysis of the mode of antibacterial action of silver nanoparticles, J. Proteome Res. 5:916  –  924. [8]   Das, A.J., Kumar, R., Goutam, S.P and Sagar, S.S (2016). Sunlight Irradiation Induced Synthesis of Silver Nanoparticles using Glycolipid Bio-surfactant and Exploring the Antibacterial Activity.  Journal Bioengineer & Biomedical Sciences, 6(208), 2-10. [9]   Devasenan, S., Beevi, N.H. and Jayanthi, S.S. (2016). Synthesis and characterization of Copper Nanoparticles using Leaf Extract of  Andrographis Paniculata  and their Antimicrobial Activities.  International Journal of ChemTech  Research,  9 (04), 725-730. [10]   Dhanalekshmi, K.I., Meena, K.S. and Ramesh, I. (2013). Synthesis and Characterization of Ag@TiO2 Core-shell Nanoparticles and Study of its antibacterial Activity.  International Journal of Nanotechnology and Application (IJNA),  3(5), 5-14. [11]   Dubey, M. Bhadauria, S and Kushwah, B.S (2009). Green synthesis of nanosilver particles from the extract of Eucalyptus hybrid (Safeda leaves), Digest J. Nanomater. Biostruct. 5:537  –   543
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