Indian J.Pharm.Biol.Res. 2013; 1(4):16-24 CODEN (USA): IJPB07 ISSN: 2320-9267 Indian Journal of Pharmaceutical and Biological Research (IJPBR) Journal homepage: www.ijpbr.in Original Research Article Synthesis, characterization and antibacterial potential of silver nanoparticles by Morus nigra leaf extract Anu Kumar, Kuldeep Kaur, Sarika Sharma * Department of Biotechnology, Arni University Indora, Kangra (H.P), India. ARTICLE INFO: Article history: Received: 26 October 2013 Received in revised form: 14 November 2013 Accepted: 21 November 2013 Available online: 7 December 2013 Keywords: Biosynthesis, Silver nanoparticles, Antimicrobial activity, Leaf extract, M.nigra. ABSTRACT The present study reports the synthesis of silver nanoparticle using Morus nigra leaf extract were used as reducing agent for reduction of silver nitrate solution. The synthesis of silver nanoparticles was analyzed by UV-Visible spectroscopy, Scanning Electron Microscopy. The SEM analysis has shown that size of silver nanoparticles synthesized from leaves extract of M.nigra was 200 nm and seems to be spherical in morphology. Morphology of chemically synthesized silver nanoparticles is nearly spherical and of size ranges from 300-500 nm. The average particle size analyzed from SEM analysis was observed to be 350 nm. This article has discussed the synthesis of silver nanoparticles generated from plant extract, characterization and antibacterial analysis. In this study the antibacterial activity was examined against six MTCC cultures collected from IMTECH Chandigarh, Including both gram positive and gram negative bacteria such as P.aeruginosa, S.aureus, B.subtilis, E.coli, P.flourescens and Streptococus mutans. Out of these strains the antimicrobial activity of the silver nanoparticles showed maximum zone of inbhition against P.flourescens (22 mm), P.aeruginosa (19 mm), S.aureus (18 mm) and least effective against E.coli (15mm). In contrast chemically synthesized silver nanoparticles were found most effective against S.aureus (13 mm) and B.subtilis (12mm) and almost ineffective against Streptococcus mutans (6 mm) and P.flourescens (4 mm). In the concluding remarks, the silver nanoparticles synthesized using M.nigra leaves extract would be a better antimicrobial effective against various bacterial species. 1.Introduction Nanotechnology broadly refers to a field of applied science and technology whose special and unique properties could be attributed to their small sizes and large surface areas [1]. It is the application of science and technology to manipulate the matter at atomic and molecular scale. It has the ability to build micro and macro materials and products with atomic precision. An important aspect of nanotechnology concerns the development of experimental processes for the synthesis of nanoparticles of different sizes, shapes and controlled dispersity. Currently it is employed as a tool to explore the darkest avenues of medical sciences in several ways like imaging, sensing, targeted drug delivery, gene delivery, and artificial implants. Nanoparticles are being sighted as fundamental building blocks of nanotechnology. In recent years, the research is mainly focused on the metal nanoparticles due to their unique optical, electronic, mechanical, magnetic, and chemical properties that are significantly different from those of bulk materials [2]. There is a method known as Green synthesis, which is biosynthesis of nanoparticles using plant extracts, which was exploited to a vast extent because the plants are widely distributed, easily available, safe to handle and with a range of metabolites. Human beings are often infected by microorganisms such as bacteria, molds, yeasts, and viruses in the living environment. Research in antibacterial material containing various natural and inorganic substances has been intensive. The use of environmentally benign materials like plant Corresponding Author: Sarika Sharma, Department of Biotechnology, Arni University Indora, Kangra (H.P), India. E-Mail: sharmasarika19@gmail.com Mobile: +91-9888599261 16
leaf extract [3], bacteria [4], fungi [5] and enzymes[6] for the synthesis of silver nanoparticles offers numerous benefits of ecofriendliness and compatibility for pharmaceutical and other biomedical applications as they do not use toxic chemicals for the synthesis protocol. Numbers of plants have been successfully used for the extracellular synthesis of silver nanoparticles including tamarind [7], Helianthus annus [8], Cinnamomum camphora,[9] Coriander [10],Capsicum annuum [11] Avena sativa [12], Azardirachta indica [13] and Pelargonium graveolens [14]. In the present study, we have explored the green synthesis ofsilver nanoparticles using M.nigra leaves extract. Synthesized nanoparticles were characterized UV-Visble spectroscopy, SEM. Further, the antimicrobial activity of synthesized silver nanoparticles against six MTCC cultures collected from IMTECH Chandigarh, Including both gram positive and gram negative bacteria such as P.aeruginosa, S.aureus, B. subtilis, E.coli, P. flourescens and Streptococus mutans were explored. 2. Material and Methods 2.1 Collection of plant material The plant material used was the leaves of Morus nigra which was collected from region of Indora, Kathgarh District Kangra (H.P), India. I. Preparation of Plant Extract Fresh and healthy leaves of M.nigra were collected, washed thoroughly with distilled water, incised into small pieces and airdried. About 20 g of leaves of M.nigra were weighed and transferred into 500 ml beaker containing 100 ml distilled water, mixed well and boiled for 10 min. Filtered the extract through Whatman No.1 filter paper and collect the filtrate in a 250 ml Erlenmeyer flask. Figure 1: Showed plant extract Filtered through whatman No.1 filter paper. i.e. prepared by washing fresh and healthy leaves thoroughly with distilled water and air-dried. II. Storage Plant extract (filtrate) was stored at the temperature of 40C till use for further use. III. Synthesis of silver nanoparticles AgNO3 solution of (1mm) of silver nitrate was prepared and used for the synthesis of silver nanoparticles. i. By using plant 1.5 ml of M.nigra leaves extract was added drop wise to 30 ml of 10-3 M AgNO 3 aqueous solution in a 100 ml beaker and heated on water bath at 75 o C for 20-30 minutes. Colour change from colourless to brown confirmed the reduction of silver nitrate to silver ions. Figure 1: M.nigra plant leaves aqueous extract ii. By chemical reduction 5ml of 10-3 M AgNO3 was added to 30ml NaBH4 in a 50ml beaker placed on ice packets and stirred on a magnetic stirrer for 10 minutes. Color change from colorless to yellowish brown indicates the synthesis of silver nanoparticles. IV. Purification of silver nanoparticles The fully reduced solution was centrifuged at 10000 rpm for 20 minutes. Supernatant was discarded and pellet was obtained and redispersed in distilled water. The process of centrifugation and redispersion was repeated three times to wash off any absorbed substance on the surface of silver nanoparticles. Original Research Article 17
2.2 Characterization of Silver Nanoparticles I. UV-Vis Analysis Ultraviolet-visible analysis was done by using SYSTRONICS PC Based Double beam spectrophotometer 2202 with resolution of 1nm between 300 to 600nm. Reduction of Ag + ions was monitored by measuring UV-Vis spectrum of mixture. Reduction of AgNO 3 to Ag + was confirmed by Color change from colorless to brown. Formation of silver nanoparticles is easily detected by measuring the optical density of solutions/suspensions. II. SEM analysis of silver nanoparticles Scanning Electron Microscopic (SEM) analysis was done using Thermo scientific SEM machine. Thin films of the sample were prepared on a carbon coated copper grid by just dropping a very small amount of the sample on the grid, extra solution was removed using a blotting paper and then the film on the SEM grid were allowed to dry by putting it under a mercury lamp for 5 minutes. III. The Antimicrobial Activity Analysis of AgNPs i. Bacterial strains Test bacteria used were Escherichia coli (MTCC 443), Staphyalococcus aureus (MTCC 3160), Pseudomonas aeruginosa (MTCC 4673), Bacillus subtilis (MTCC 441), Pseudomonas flourescens (MTCC 103), Streptococcus mutans (MTCC 890). These organisms were chosen because they are commonly isolated from the hospitalized patient with intestinal ailments, blood and skin infections. All these microorganisms were obtained from IMTECH Chandigarh. ii. Antimicrobial activity Innoculum was standardized at 1 106 cfu/ ml. The effect of chemically and biologically synthesized silver nanoparticles on several bacterial strains was assayed by using the agar well diffusion assay method. iii. Agar well diffusion method 50 ml Nutrient broth (NB) medium was prepared and poured in six tubes for inoculating six different MTCC culture and sterilized by autoclaving at 121 C for 15 minutes at 15 psi pressure. Then these NB tubes were inoculated with six different bacterial strain used. Then Incubate at 35 37 C for 24 hours. Mueller-Hinton Agar (MHA) was prepared and was sterilized by autoclaving at 121 0 C for 15 minutes at 15 psi pressure and was used to determine the antibacterial activity of silver nanoparticles. Then sterile Mueller-Hinton Agar (20ml) was poured aseptically into each sterile petridish and allowed to solidify at room temperature under sterile conditions. After solidification, petriplates were spreaded with MTCC cultures. Then three wells were bored in each plate by using a sterile borer. Then (20µl) of sample (Silver nanoparticles + DMSO), DMSO (control) and silver nitrate was loaded in respective wells. Then plates were incubated at 370C for 24 hours. After 24 hrs plates were observed for zone of inhibition in mm and the zone was measured by using scale [15]. 3. Result and Discussion Today nanomaterials are at the primary stage of fast developing nanotechnology phase. Nanomaterials are facilitating modern technology to deal with nano size objects. Currently nanomaterial s are already being used in medical application such as drug carrier, strong antibacterial, detection for pathogens/proteins and tissue engineering. In the recent times, researchers paid more attention on the green synthesis and applications of silver nanoparticles using various plant extracts. In this study work is carried out on extracellular synthesis of silver nanoparticles from leaves extract of plant M.nigra and their antibacterial activity was investigated. Antibacterial activity of chemically synthesized silver particles was compared with biologically synthesized silver nanoparticles. 3.1 Synthesis of silver nanoparticles To verify the synthesis of silver nanoparticles, flasks containing plant extract (without silver nitrate solution) in biological method and sodium borohydride (NaBH 4 ) in chemical method was positive control and pure silver nitrate as negative control was monitored visually. The M.nigra leaves extract and solution of NaBH 4 (positive control) and silver nitrate solution (negative control) was observed to retain its original color. The silver nitrate treated with leaves extract of M.nigra turned brown after 5 minutes due to deposition of silver nitrate. The color was mainly observed due to the excitation of surface plasmon resonance of deposited silver nanoparticles[16]. In case of negative control no color change was observed even after 48 hrs. Figure 2: Solution of silver nitrate (3mM) (left) and reduced silver nitrate solution after addition of plant extract (right) Original Research Article 18
Figure 3: Solution of sodium borohydride (0.2 M) (left) and (right) reduced silver nitrate solution after addition of chemical 3. Results and discussion 3.1 Characterization of silver nanoparticles I. UV-Vis spectrum Analysis The formation of silver nanoparticles by reduction of aqueous metal ions during the exposure of M. nigra leaves extract and NaBH 4 solution may be easily followed by UV-visible spectroscopy. UV-visible absorption spectrum of silver nanoparticles in the presence of leaves extract of M.nigra and NaBH 4 solution is shown in Figure 4 (a and b).the surface Plasmon band in silver nanoparticles solution in case of biological method remain closer to 400 nm while it is close to 450nm in case of chemically synthesized silver nanoparticles. Silver nanoparticles synthesized from the leaf aqueous extract of M. Nigra was evaluated through spectrophotometry at a wavelength range of 350-700 nm. Plasmon absorption band of 400 nm indicating the presence of spherical or roughly spherical Ag nanoparticles. It is generally recognized that UV-Vis spectroscopy could be used to examine size and shape of nanoparticles in aqueoussolution. Figure 4(a) shown UV-Vis spectra recorded from reaction medium of biological method when M.nigra leaves extract is used as reducing agent. Figure 4(b) shown UV-Vis spectra recorded from reaction medium of chemical method when NaBH 4 is used as a reducing agent. Figure 4(a): UV-Vis absorption spectrum of obtained biologically synthesized silver nanoparticles II. SEM Analysis Scanning electron microscopy has been employed to characterize the size, shape and morphology of formed silver nanoparticles. Figure 4(b): UV-Vis absorption spectrum of obtained chemically synthesized silver nanoparticles SEM image of both samples are shown in Figure 5 (a) and 5 (b) respectively. Original Research Article 19
Figure 5 (a): SEM images of biologically synthesized silver nanoparticles From Figure 5(a) SEM analysis image has shown that size of silver nanoparticles synthesized from leaves extract of M.nigra was 200nm and seems to be spherical in morphology. From Figure 5(b) it is evidenced that morphology of chemically synthesized silver nanoparticles is nearly spherical and of size ranges from 300-500nm. The average particle size analyzed from SEM image is observed to be 350nm. In our study size of silver nanoparticles which were synthesized by plant extract found smaller as compared to chemically synthesized silver nanoparticles, so that biologically synthesized silver nanoparticles are of great importance as compared to chemically synthesized silver nanoparticles. III. Evaluation of Antibacterial activity of Silver Nanoparticles (a) Antibacterial activity Recently, silver nanoparticles are found to be a best biocide, for example in wound dressings and as an antimicrobial coating on consumer products, little is known about its mode of toxicity. A Figure 5 (b): SEM images of the chemically synthesized silver nanoparticles wide research reports were available for using silver nanoparticles as a antimicrobial agent against S. aureus and E. Coli. [17]. Silver nanoparticles have been used as antimicrobial agent against a number of bacterial strains such as B. subtilis and S. aureus and E. coli [18], Staphylococcus aureus, E.coli, Corynebacterium diphtheria and Micrococcus sp., and Gram positive strains (S. aureus, S.epidermidis and Gram negative strains (E. coli, S. typhi, Pseudomonas aueroginosa, Klebsiella pneumonia, Proteus vulgaris) [19]. In this study Antibacterial activity of silver nanoparticles was investigated against six MTCC cultures collected from IMTECH Chandigarh including both gram positive and gram negative bacteria such as P.aeruginosa, S.aureus, B. subtilis, E.coli, P.flourescens and Streptococus mutans. The maximum activity i.e 22 mm zone of inhibition was obtained againstp.flourescens,17mm zone of inhibition against Streptococcus, 15 mm zone of inhibition against B. subtilis then 14 mm zone of inhibition against E.coli and 19 mm against P.aeruginosa. Table no. 1: Evaluation of Antibacterial activity of silver nanoparticles synthesized by Plant (M. nigra) and chemical (sodium borohydride) Sr. No. Bacterial isolates Zone of Inhibition in mm Biologically synthesized Chemically synthesized Control silver nanoparticles silver nanoparticles 1. S. mutans 17mm 6mm 0mm 2. B. subtilis 15mm 12mm 2mm 3. E.coli 14mm 7mm 1mm 4. P. flourescens 22mm 4mm 0mm 5. S.aureus 18mm 13mm 1mm 6 P.aeruginosa 19mm 8mm 2mm The antibacterial activity of chemically synthesized nanoparticles was examined against same six MTCC cultures as discussed above. The maximum activity i.e 13 mm zone of inhibition was obtained against S.aureus,6 mm zone of inhibition against Streptococcus, 12 mm zone of inhibition against B.subtilis, 7 mm zone of inhibition for E.colithen 8 mm against P.aeruginosa and 4 mm against P. flourescens. Original Research Article 20
(A). S. mutans (B) B. subtilis (C) E.coli (D) P. flourescens (E) S.aureus (F) P.aeruginosa Figure 6: (A-F) Antibacterial activity of biologically synthesized silver nanoparticles Original Research Article 21
(A). E.coli (B) P. flourescens (C) S.aureus (D) B. subtilis (E) P. aeruginosa (F) S.mutans Figure 7: (A-F) Antibacterial activity of chemically synthesized silver nanoparticles Original Research Article 22
Table No.2: Comparative study of antibacterial activity of biologically synthesized AgNPs and chemically synthesized silver nanoparticles Biologically Synthesized Silver Nanoparticles Chemically Synthesized Silver Nanoparticles Zone of inhibition(mm) S. mutans B.subtilis E.coli P. flourescens S.aureus P.aeruginosa 16mm 15mm 14mm 22mm 18mm 19mm 6mm 12mm 7mm 4mm 13mm 8mm From comparative study of antibacterial activity of plant and chemically synthesized silver nanoparticles, it was concluded that silver nanoparticles which were synthesized by using plant aqueous extract has more antibacterial activity as compared to chemically synthesized silver nanoparticles. Biologically synthesized silver nanoparticles found most effective against P. flourescens (22 mm), P.aeruginosa (19 mm) and S. aureus (18 mm) and least effective against E. coli (15mm). As these silver nanoparticles were found to be very much effective against S.aureus and P.Flourescens so these can be of great importance in dealing with skin infections and can be used as antibacterial drug. In contrast chemically synthesized silver nanoparticles were found most effective against S. aureus (13 mm) and B. subtilis (12 mm) and almost ineffective against Streptococcus mutans (6mm) and P. flourescens (4 mm).these results clearly indicate that silver nanoparticles synthesized by using plant extract are more effective against bacterial strains as compared to chemically synthesized silver nanoparticles and could provide a safer alternative to conventional antimicrobial and antibacterial agents. The major mechanism through which silver nanoparticles manifest antibacterial properties was either by anchoring or penetrating the bacterial cell wall, and modulating cellular signaling [20]. The mechanism involved in the inhibition of bacterial growth by silver nanoparticles is unclear. However, the general mechanism of antibacterial activity of silver nanoparticles was proposed by many researchers, but the detailed mechanism remains to be understood. The bacterial growth was inhibited by silver ions, which accumulated into the vacuole and cell walls as granules. The silver nanoparticles attached the surface of the cell membrane disturbing the permeability and respiration functions followed by dysfunction of metabolic pathways including, silver ions can interact with nucleic acids they preferentially interact In the present study, plant based AgNPs showed maximum inhibitory activity against the P.flourescens and S.aureus isolated from hospitalized patient with intestinal ailments, blood and skin infections. This study suggests green synthesized nanoparticles have been effectively used as an alternative to antibiotic of bactericides against skin wounds by reducing antimicrobial activities. This biosynthesis of silver nanoparticles can prove potential candidates for medical applications. 4. Conclusion A critical need in the field of nanotechnology is the development of reliable and eco-friendly processes for synthesis of metallic nanoparticles. Here, we have reported a simple biological and low-cost approach for preparation of stable silver nanoparticles by reduction of silver nitrate solution with a bioreduction method using Morus nigra leaf extract as a reducing agent. Morus nigra leaf extract is found suitable for the green synthesis of silver nanoparticles. Characterization of silver nanoparticles was done by UV spectroscopy and SEM analysis. Silver exhibits the strong toxicity in various chemical forms to a wide range of microorganism is very well known and silver nanoparticles have recently been shown to be a promising antimicrobial material. In conclusion, the bio-reduction of aqueous Gab ions by the plant extract of the M.nigra leaf extract has been demonstrated. Conflict of interest statement We declare that we have no conflict of interest. Acknowledgement The authors are thankful to the authorities of Arni University, Kathgarh Indora, Kangra (H.P), India for providing support to the study and other necessary facility to carry out research work. References 1. Nanda A and Saravanan M. Biosynthesis of silver nanoparticles from Staphylococcus aureus and its antimicrobial activity against MRSA and MRSE. Nanomed.: Nanotechnol., Biol. Med.,2009;5: 452 456. Original Research Article 23
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