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International Journal of Nanotechnology and Application (IJNA) ISSN 2277-4777 Vol. 3, Issue 4, Oct 2013, 11-18 © TJPRC Pvt. Ltd.

SYNTHESIS OF PLANT-MEDIATED SILVER NANOPARTICLES USING PLANT EXTRACT OF SONCHUS ASPER ABHA VERMA1, PRAKASH JOSHI2 & ARVIND ARYA3 1,3

Department of Biotechnology, Meerut Institute of Engineering and Technology, Meerut, Uttar Pradesh, India

2

Homoeopathic Pharmacopoeia Laboratory, Department of AYUSH, Ministry of Health and Family Welfare, Kamala Nehru Nagar, Ghaziabad, Uttar Pradesh, India

ABSTRACT This paper reports a convenient and extracellular method for the synthesis of silver nanoparticles by reducing silver nitrate with the help of aqueous extract prepared using a weed namely Sonchusasper.Theeffect of various parameters like amount of plant extract, incubation time, incubation temperature, silver nitrate concentration and pH wereinvestigated. The rapid reduction of silver ions was monitoredby changing color of plant leaf extracts from green to brown after treatment with AgNO3 and UV-Vis spectrophotometer. Ourresults suggests that Sonchusaspermediated silver nanoparticles could act as an effectiveantimicrobial agent andprove as an alternative for the development of new antimicrobial agents to combat resistance problem.

KEYWORDS: Silver Nanoparticles, Antimicrobial Activity, Sonchusasper INTRODUCTION Due to their unique optical, electronic, mechanical, magnetic, and chemical properties, metal nanoparticles have been a subject of research now a day. Their small sizes, large surface to volume ratios and crystallographic surface structure (Parameshwariet al., 2011)are responsible for their unique properties.An emerging new science which makes use of nanoparticles is Nanomedicine that is an outcome of fusion of nanotechnology and medicine. Silver nanoparticles can be synthesized by various techniques, such as chemical reduction of silver ions in aqueous solutions with or without stabilizing agents (Liz-Marzán and Lado-Tourino, 1996), thermal decomposition in organic solvents(Esumiet al., 1990), chemical reduction and photoreduction in reverse micelles (Pileni and Duxin, 2000; Sun and Xia, 2002), and radiation chemical reduction (Henglein, 1993; Henglein and Giersig, 1999). Most of these methods are extremely expensive and also involve the use of toxic, hazardous chemicals, which may pose potential environmental and biological risks. Metallic nanoparticles are mostly prepared from Nobel metals such as Gold, Silver, Platinum and Lead using chemical methods. Among the Nobel metals, Silver (Ag) is the metal of choice in the field of biological systems, living organisms and medicine(Parasharet al., 2009). Since noble metal nanoparticles are widely applied to areas of human contact (Song and Kim, 2009), there is a growing need to develop environmentally friendly processes for nanoparticles synthesis.Biological methods of synthesis have paved way for the “greener synthesis” of nanoparticles and these have proven to be better methods due to slower kinetics, they offer better manipulation and control over crystal growth and their stabilization. This has motivated an upsurge in research on the synthesis routes that allow better control of shape and size for various nanotechnological applications. The use of environmentally benign materials like plant extract (Jain et al., 2009), bacteria (Saifuddinet al., 2009), fungi (Vermaet al., 2010)and enzymes (Willneret al., 2006)for the synthesis of silver nanoparticles offer numerous benefits of eco-friendliness and compatibility for pharmaceutical and other biomedical applications as they do not use toxic chemicals for the synthesis protocol. The plant material may include weeds which are


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thought to be the unwanted flora growing in the fields and thereby competing with normal cultivars for nutrients and space. The approach will provide an effective alternative for the utilization of this unwanted flora.

MATERIALS AND METHODS Microbial Cultures The microbial cultures used were obtained from MTCC IMTECH Chandigarh in lyophilized form. The cultures used were Escherichia coli (MTCC No.1722) andStaphylococcus aureus (MTCC No. 96). Plant Material and Synthesis of Silver Nanoparticles Leaves of Sonchusasperwere collected from the fields. The leaves were washed and the cleaned leaves were air dried for 7 days and then ground to a fine powder. 200 mg of powdered leaf was dispensed in 100ml of sterile distilled water and boiled for one hour at 80ยบC. Then the leaf extract was collected in separate conical flasks by standard filtration method and stored in dark coloured bottles till further use. The 1mM Silver nitrate solution was prepared and stored in brown bottles. 5ml of herbal extracts was taken in BOD bottle separately and to this 95 ml of AgNO3 solution was added. The time of addition of extract into the aqueous AgNO3 solution was considered as the start of the reaction.The reaction mixture was centrifuged at 10,000 rpm for 30 minutes in order to obtain the pellet which is used for further study. Detection and Characterization of Phyto Silver Nanoparticles Visual Observation: After treatment of leaf extract with AgNO3, the colour change of the reaction mixture was visually observed. The time taken for the reaction mixture to change colour was noted. UV-Vis Spectrophotometric Analysis: The aliquots of reaction mixture were subjected to the measurement of absorbance by UV-visible spectrophotometer from 300 to 800 nm for the detection of silver nanoparticles.

Optimization of Various Parameters for Nanoparticles Synthesis Amount of Plant Extract To 10 ml of 0.001 M silver nitrate solution, different concentrations (0.1 ml, 0.3 ml, 0.5 ml and 0.7 ml) plant extract was added with constant stirring and was observed for change in colour and then subjected to UV-Vis spectrophotometric analysis. Incubation Time To 10 ml of 0.001 M silver nitrate solution, 0.5 ml of plant extract was added with constant stirring and was observed for change in colour. The reaction mixture was subjected to UV-Vis spectrophotometric analysis at 0 hrs, 2 hrs, 6hrs and 24 hrs. Silver Nitrate Concentrations To three test tubes containing 10 ml silver nitrate of different concentrations (0.001 M, 0.01 M and 0.05 M), 0.5 ml of plant extract was added with constant stirring and was observed for change in colour.The reaction mixture was then subjected to UV-Vis spectrophotometric analysis. Incubation Temperature To 10ml of 0.001 M Silver nitrate solution, 0.5ml of plant extract was added with constant stirring and then was exposed to different temperatures like 4ยบC, 27ยบC, 40ยบC and 90ยบC. The colour change of the solution was checked


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periodicallyand reaction mixture was subjected to UV-Vis spectrophotometric analysis Preparation of Inoculum The bacterial cultures were grown in nutrient agar at 37ºC. The growth obtained was suspended in sterile nutrient broth and optical density was set up to McFarland No. 5 standard i.e. approximately 0.132 at 600 nm. At this absorbance a concentration of 108 cells /ml was obtained. This suspension was used as inoculum for antibacterial susceptibility testing. Antibacterial Activity of Phyto Silver Nanoparticles The silver nanoparticles synthesized were tested for antimicrobial activity by disk diffusion method against mentioned bacterial cultures. The pure cultures of bacteria were sub-cultured on nutrient agar. Each strain was spreaded uniformly onto the individual plates using L shaped glass spreader. Disks impregnated with 20 μl of nanoparticles were placed on each plate. After incubation at 37ºC for 24 hours, the different levels of zone of inhibition were measured. Analysis of Bacterial Growth Curve The antibacterial activity of silver nanoparticles against Staphylococcus aureus was also studied analyzing its growth curve. Fresh colonies from agar media were inoculated into 10ml of nutrient broth media. The media was supplemented with 100 μg/ml and 30 μg/ml silver nanoparticles and bacterial cultures were incubated at 37ºC with continuous shaking. The growth of Staphylococcus aureus in broth media was indexed by measuring the optical density (at λ = 600nm) at regular intervals using UV-Vis spectrometer. Whereas control does not contain any exposure of silver nanoparticles synthesized from any plant extracts.

RESULTS AND DISCUSSIONS The synthesis and application of nanomaterial is in the limelight in modern nanotechnology. Plants including herbs, lower plants, higher plants, weeds etc. contain an array of secondary metabolites such as phenolic compound, terpenoids, essential oils, and flavonoids, which helps inthe reduction of metal ion and formation of nanoparticles(Haverkamp and Marshall, 2009). The present investigation demonstrate the formation of silver nanoparticles by the reduction of aqueous silver metal ions by plant extracts prepared using Sonchusasper.The nanoparticles synthesizedwere characterized by X-ray diffraction (XRD). Visual Inspection and UV-Vis Spectrophotometric Analysis The reduction ofsilver ions into silver particles during exposure to the plant extract is followed by colour changefrom colorless or pale yellow to yellowish brown. It is well known that silver nanoparticles exhibit yellowish brown colour in aqueoussolution due to excitation of surface plasmon vibrations in silver nanoparticles (Thirumuruganet al., 2010). It was observed that as the plant extract was mixed in the colorless aqueous solutionof the silver ion complex, it started to change the colour from watery to yellowish brown due toreduction of silver ion, which may be the indication of formation of silver nanoparticles(Jain et al., 2009).The color was observed to be enhanced gradually with time and became stable with no further change in colour after 24hrs. The synthesis of silver nanoparticles was further confirmed by measuring the UV-Visspectrum of the reaction mixture. The change in colourof the plant extract is shown in Figure 1. Colloidal solution of silver nanoparticles when subjected to UV-Vis spectrophotometric analysis showed a peak at 404 nm. Silver nanoparticlesare extraordinarily efficient at absorbing and scattering light and have a colour that depends uponthe size and shape of the particles(Sondi and Salopek-Sondi, 2004). The strong interaction ofsilver nanoparticles with light occurs because the conduction electron of the metal surfaceundergoes a collecting oscillation when excited by light at


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specific wavelength. This oscillationis known as surface plasmon resonance (SPR), results in unusually strong scattering andabsorption property.A unique property of spherical Silver nanoparticles is that SPR peak wavelength occurs between400-500 nm depending upon the particle size and the local refractive index near the particlesurface. Due to this unique optical property, a great deal of information about the physical stateof these particles can be obtained by analyzing their spectral property in solution.

Optimization of Nanoparticles Synthesis Amount of Plant Extract Figure 1 shows the effect of varying amount of leaf extract in silver nitrate solution. The observed dark colour, which can be attributed to silver nanoparticle formation, in 7 ml extract sample at all the point of time indicates an increased rate of silver nanoparticle synthesis. By increasing the amount of extract in similar environmental conditions, the observed wavelength of maximum peak does not change much, however a slight decrease in peak wave length (Figure 2) has been observed i. e. from 404 nm to 402 nm, which suggest the reduction in size of nanoparticles synthesized. On increasing the amount of plant extract an increase in intensity of peak is observed which suggest increase in the concentration of silver nanoparticles.

Figure 2: UV-Visible Spectra of Silver Nitrate and Different Amount of Sonchusasper Leaf Broth Mixture Incubation Time On incubating the silver nitrate and plant extract mixture a gradual increase in the intensity of colour change was observed. The colour starts changing after 2 hrs and indicates the nanoparticles synthesis. After 24 hrs the colour was stabilized with no further change. On subjecting the mixture to UV-Vis spectrum analysis, a peak occurs at 404 nm. With due course of time the intensity of the peak increases showing an increase in silver nanoparticles concentration (Figure 3). From the Figure it isalso evident that there is a reduction in the rate of formation of nanoparticles after 6 hrs, suggesting that the silver ions are completely reacting to form silver nanoparticles(Christensen et al., 2011)

Figure 3: UV-Visible Spectra of Silver Nitrate and Sonchusasper Leaf Broth Mixture at Different Time Intervals


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Figure 1: Photographs of the Mixture of Different Amount of Sonchusasper Broth and Silver Nitrate Solution over a Twenty Hours’ Time Period Silver Nitrate Concentrations The color change observed on adding plant extract to containers containing silver nitrate of different concentration indicates the reaction of silver nitrate reduction. Only 5 hrsafter the completion of reaction, silver particles were seen to be settled and accumulated at bottom of tubes in case of 0.005 M and 0.01 M silver nitrate. The results are in accordance to the results obtained by (Ghorbani et al., 2011).This accumulation was fargreater in case of 0.01 M situation. For analytical study of prepared samples, the amount of absorbance was observed within the range of 300 – 600 nm by UV-Vis spectroscopy. A surface Plasmon resonance was observed to be centered at 404 nm in case of 0.001 M silver nitrate and at 386 nm in case of 0.005 M (Figure 4). Such observation of surface Plasmon has been well documented for various metal nanoparticles with size ranging from 2 to 100 nm. At the highest concentration i.e. 0.01 M no peak was observed thus indicating no nanoparticles synthesis at this concentration. Presence of large amount of silver in small volume of the solution creates high attraction between silver atoms and nanoparticles conglomeration and formation of particles with large size at micro scale. This presence of very large particles (larger than nanometer) was the reason of fast settlement of particles in the container(Ghorbani et al., 2011).

Figure 4: UV-Visible Spectra of Different Concentrations of Silver Nitrate and Sonchusasper Leaf Broth Mixture Incubation Temperature The reaction temperature plays a significant role to control the process of nucleation(Mittal et al., 2012).On incubating the silver nitrate and plant extract mixture at different temperatures there was a considerable difference in colour change at 40C and rest of the temperatures considered. The change in colour was observed to be delayed at 40C and


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occurs after 6 hrs, while at other temperatures the colour change was observed at 2 hrs of incubation. From the results it may be concluded that low temperature is not favoring the reaction. The spectrum analysis of the mixtures at different temperatures reveals a peak occurring at 402 nm in case of 4 0C, 270C and 370C whereas at 404 nm in case of 900C. From Figure 5, it is evident that the concentration of silver nanoparticles is comparably low in case of 4 0C while approximately similar in other case.

Figure 5: UV-Visible Spectra of Silver Nitrate and Sonchusasper Leaf Broth Mixture at Different Temperatures

Antibacterial Activity of Phyto Silver Nanoparticles Biologically synthesized Silver nanoparticles using Sonchusasper were found to be active against Staphylococcus aureus among all the microbes used in the study (Figure 6). It was observedthat with the increase in concentration of silver nanoparticles, the diameter of inhibition zone also increases (Table 1).The mechanism of bactericidal effect of the silver nanoparticles is not very well known(Moroneset al., 2005; Sondi and Salopek-Sondi, 2004). Possibly the silver nanoparticles mayattach to the surface of cell membrane and disturb its permeability.The binding of the particle tothe bacteria depends upon surface area available for interaction. The smaller particle like silvernanoparticles having large surface area available for interaction will give more bactericidal effectthan the large particle It has been demonstrated that silver nanoparticles can also penetrateinside the bacteria, causing damage by interacting with phosphorus and sulphur containingcompound. Silver tends to have high affinity to react with such compounds. One more possibilityfor the antibacterial activity would be release of silver ions from the nanoparticles.

Figure 1: Antimicrobial Activity of SNPs Synthesized using Sonchus asper against Staphylococcus aureus


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Table 1: Antimicrobial Activity of SNPs Synthesized Name of Plant

Concentration of Nanoparticles in µg/ml

Sonchusasper

0.020 0.010 0.005

Zone of Inhibition in mm at Various Concentration of Herbal Nanoparticles, in Case of E. coli S. aureus 20 16 10

Analysis of Growth Curve Silver nanoparticles exhibits strong antibacterial activity due to their well-developed surface which provides maximum contact with the environment. Here, antibacterial effect of silver nanoparticles were studied by using optical intensity as function of time for 10 hrs with varying concentration of silver nanoparticles. From Figure 7 we can conclude that in the absence of silver nanoparticles there is increase in optical density showing bacterial growth but as the concentration of silver nanoparticles increases, there is reduction in the bacterial growth of Staphylococcus aureus, thereby decreasing the slope of bacterial growth curve(Ansari et al., 2011).

Figure 2: Growth Curve Analysis of Staphylococcus aureus

CONCLUSIONS It is concluded that the leaf extract of Sonchusasperis capable of producing silver nanoparticles. The terpenoids, flavonoids and other phenolic compounds are responsible in the synthesis of nanoparticles. Following the addition of leaf extract to the silver nitrate solution, silver nanoparticles began to form within 2 hrs. It was found that the increasing the extract amount increases the rate of reduction process. At low temperature the reduction is low and at other temperatures there is not much considerable difference in nanoparticles synthesis. The reduction of silver ions to silver nanoparticles was found to be better at 0.001M concentration of Silver nitrate. The antibacterial activity of synthesized nanoparticles suggests their use as antibacterial agents.

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