Synthesis of silver nanoparticles using seed exudates of Sinapis arvensis as a novel bioresource, and evaluation of their antifungal activity
© Khatami et al.; licensee Springer. 2015
Received: 18 November 2014
Accepted: 25 February 2015
Published: 25 April 2015
In general, silver nanoparticles (AgNPs) are particles of silver with a size less than 100 nm. In recent years, synthesis of nanoparticles using plant extract has gained much interest in nanobiotechnology. In this concern, this study investigates green synthesis of AgNPs from silver nitrate using Sinapis arvensis as a novel bioresource of cost-effective nonhazardous reducing and stabilizing compounds. A stock solution of silver nitrate (0.1 M) was prepared. Different concentrations of silver nitrate (1, 2.5, 4, and 5 mM) were prepared from the above solution, then added to 5 mL of S. arvensis seed exudates. The mixtures were kept in 25°C. The synthesis of AgNPs was confirmed by the change in mixtures color from light yellow to brown. The antifungal activity of synthesized AgNPs was investigated in vitro.
The resulting AgNPs were characterized by UV-vis spectroscopy, X-ray diffraction (XRD), transmission electron microscopy (TEM), and Fourier transform infrared spectroscopy (FTIR). Formation of the AgNPs was confirmed by the change in mixture color from light yellow to brown and maximum absorption at 412 nm due to surface plasmon resonance of AgNPs. The role of different functional groups in the formation of AgNPs was shown by FTIR. X-ray diffraction was shown that the AgNPs formed in our experiments were in the form of nanocrystal, and TEM analysis showed spherical particles with an average size of 14 nm. Our measurements indicated that S. arvensis seed exudates can mediate facile and eco-friendly biosynthesis of colloidal-spherical AgNPs with a size range of 1 to 35 nm. The synthesized AgNPs showed significance antifungal activity against Neofusicoccum parvum cultures.
The AgNPs were synthesized using a biological source. This synthesis method is nontoxic, eco-friendly, and a low-cost technology for the large-scale production. The AgNPs can be used as a new generation of antifungal agents.
KeywordsSynthesis AgNPs Spherical AgNPs Antifungal
Silver nanoparticles (AgNPs) are particles of silver which are in the range between 1 and 100 nm. Nanostructure materials indicate unique physicochemical and biological environmental properties, including optical, magnetic, electronic, catalytic activity, and biological properties , which have increased their applications in medicine, agriculture, environment, and industry . AgNPs have high potential as commercial nanomaterials  and an effective antimicrobial agent.
Several techniques for synthesizing AgNPs have been proposed. Generally, AgNPs are prepared by different kinds of chemical and physical methods, but majority of these techniques are both expensive and environmentally hazardous . Furthermore, the synthesized nanoparticles may be unstable and tend to agglomerate rapidly and become useless unless capping agents are applied for their stabilization . Diverse chemical and physical methods have been used to prepare AgNPs with various sizes and shapes, such as UV irradiation [6,7], microware irradiation , chemical reduction , electron irradiation , photochemical , and lithography methods . However, most of these methods involve more than one step, high energy requirement, low material conversions, difficulty in purification, and hazardous chemicals . The synthesis of nanoparticles by chemical methods may lead to the production of some toxic chemical compound that may have adverse effects on their applications .
The biological synthesis of nanoparticles can potentially eliminate these problems. Biological synthesis of nanoparticles is nontoxic, eco-friendly, and a low-cost technology for the large-scale production of well-characterized nanoparticles . Therefore, there is a need to develop biological processes for nanoparticle synthesis. Recently, many live organisms such as bacteria, fungi, algae, and plants have been used for synthesis of nanoparticles [15-17]. The reduction of Ag+ to Ag0 took place by combinations of biomolecules such as proteins, polysaccharides, and flavonoids . Green synthesis of AgNPs has become very important in the recent years. Green AgNPs have the potential for large scale applications in the formulation of dental resin composites, bone cement [19,20], water and air filters [21,22], clothing and textiles, medical devices and implants , cosmetics , and packaging . Besides their antimicrobial properties, AgNPs and silver nanocomposites have other interesting characteristics which will further enable them to be used in catalysts, biosensors, conductive inks, and electronic devices [25,26]. They can be produced economically and in large industrial scale .
In this paper, we report biosynthesis of stable colloidal AgNPs using Sinapis arvensis seed exudates. This plant is an important medicinal crop in the southern regions of Iran. Antifungal activities of synthesized AgNPs were also investigated. Details of biosynthesis, physical characterizations, and antifungal activity of AgNPs are described.
Silver nitrate and potato dextrose agar (PDA) medium were obtained from Merck, Darmstadt, Germany. Seeds of S. arvensis were obtained from Pakanbazr, Isfahan, Iran. Strain of the fungus N. parvum was prepared from the Department of Plant Protection, Shahid Bahonar University of Kerman, Iran.
Seed exudates preparation
The surface of S. arvensis seeds were disinfected using 30% sodium hypochlorite for 5 min and rinsed with sterile distilled water three times. In the next step, the seeds were placed in 70% alcohol for 2 min and then washed four times with sterile distilled water and then imbibed in deionized (DI) water. (1 g dry weight/10 mL DI water) After being incubated at 25°C for 48 h in the dark, seeds were removed from the soaking medium. The supernatant phase was collected and centrifuged at 4,500 rpm for 10 min to separate the liquid fraction from any large insoluble particles and filtered by Whatman filter paper no. 1 (Sigma Aldrich, St. Louis, MO, USA). During the experiment, pH was 4.5.
Biosynthesis of AgNPs
For this reason, a 50 mL stock solution containing 0.1 M silver nitrate was prepared. Different concentrations of silver nitrate (1, 2.5, 4, and 5 mM) were prepared from the above solution, then added to 5 mL of S. arvensis seed exudates and incubated at 25°C as described previously . After treatment, the pale yellow color of reaction mixture was changed to brown indicating synthesis of AgNPs.
Characterization of AgNPs
The biosynthesis of AgNPs was monitored periodically using a UV-vis spectrophotometer (Scan Drop-type product, Analytik Jena, Germany) at different concentrations at room temperature. These measurements operated at a resolution of 1 nm and wavelength range between 300 and 650 nm .
The formation and quality of compounds were investigated by X-ray diffraction (XRD) technique. For this purpose, AgNPs was centrifuged (at 13,000 rpm; 25°C) for 10 min, washed with DI water and re-centrifuged in three cycles. Then purified AgNPs were dried and subjected to XRD experiment. X-ray diffraction was performed by STOE Stadi P (STOE & Cie. GmbH, Darmstadt, Germany) (λ = 1.54178 Å). The scanning was done in the region of 2θ from 10° to 80° .
Transmission electron microscopy
Transmission electron microscopy (TEM) was performed by using of a Carl Zeiss (Jena, Germany, 80 kV) for determining the morphology and size of AgNPs.
Inductively coupled plasma spectrometry
Antifungal activity of AgNPs
To determine the antifungal activity of AgNPs, mycelium growth inhibition test was used. Four concentrations of AgNPs (2.5, 5, 10, and 40 μg/mL) were prepared in PDA medium after autoclaving. 6-mm agar plugs of fresh culture of N. parvum prepared and transferred to centers of media containing different concentrations of synthesized AgNPs. Control plates contain no AgNPs. All plates incubated at 28°C. When in control, the fungus completely covered the entire surface of the medium, the mean radius of fungal growth in all plates measured and recorded . All treatments were performed in triplicates.
R is the mean radius of control and r is the radius of samples treated with nanoparticle. Data processed with SAS statistical analysis using Duncan’s test.
Results and discussion
Ultraviolet visible scanning spectroscopy studies
Strong peaks were observed at 2θ values at 38.09°, 44.15°, 64.67°, and 77.54°, corresponding to (111), (200), (220), and (311) Bragg’s reflection based on the face-centered-cubic (fcc) crystal structure of AgNPs . The broadening of Bragg’s peaks indicates the formation of AgNPs. The XRD pattern thus shows that the AgNPs formed by the reduction of Ag+ ions by S. arvensis seed exudates are crystalline in nature.
Transmission electron microscopy analysis
Inductively coupled plasma spectrometry
C f (μg/mL)
C 0 (μg/mL)
Antifungal activity of synthesized AgNPs
Percentage of growth inhibition due to the effect of AgNPs was analyzed by statistical software SAS. Results confirmed a significant effect of AgNPs on fungal growth inhibition at 1% confidence interval.
Metallic nanoparticles are traditionally synthesized by wet chemical synthesis techniques where the chemicals used are quite often toxic and flammable. But in this study, the S. arvensis seed exudates were successfully used for the single-pot biosynthesis of spherical AgNPs in ambient conditions with the size range from 1 to 35 nm, as inferred from the TEM imaging. This was achieved without the use of external stabilizing or capping agents. We concluded that S. arvensis seed exudates are a bioreductant and capping agent as well as an easily available plant source playing an important role in the synthesis of highly stable AgNPs. X-ray diffraction pattern strongly indicated a high purity of biosynthesized AgNPs. This pristine method is facile, cost effective, clean and green, and therefore is applicable for a variety of purposes. Moreover, it is easy to scale up the production of AgNPs to industrial scale using this method.
This green chemistry approach towards the synthesis of AgNPs has many advantages such as ease with which the process can be scaled up, economic viability, etc. Application of such nanoparticles in medicine and other applications makes this method useful for the large scale synthesis of other inorganic nanomaterials. Toxicity studies of AgNPs on pathogen open a door for a new range of antifungal and antibacterial agents. So in the present study, we demonstrated that AgNPs have significant antifungal activity. It was determined that the growth inhibitory effect of AgNPs strongly depends on the concentration of AgNPs and with the increase concentration of AgNPs in the medium the inhibitory effect on fungal growth increased. So AgNPs can be used as excellent new antifungal agent.
The authors are thankful to all members of the Department of Biotechnology Science, University of Kerman. We also acknowledge Mr. Abazari, Laboratory of Science, University of Kerman, for helping us. Also, the authors are thankful to the Iran Nanotechnology Council. Dr. Jian He Xu and Dr. Zolala J have made substantive intellectual contributions to this study, substantial contributions to the conception and design of it as well as to the acquisition, analysis, and interpretation of data.
- Phanjom P, Ahmed G (2015) Biosynthesis of silver nanoparticles by Aspergillus oryzae (MTCC No. 1846) and its characterizations. Nanoscience and Nanotechnology 5:14-21Google Scholar
- Song JY, Kim BS (2009) Rapid biological synthesis of silver nanoparticles using plant leaf extract. Bioprocess Biosyst Eng 32:79–84View ArticleGoogle Scholar
- Chaloupka K, Malam Y, Seifalian AM (2010) Nanosilver as a new generation of nanoproduct in biomedical applications. Trends Biotechnol 28:580–588View ArticleGoogle Scholar
- Sintubin L, Verstraete W, Boon N (2012) Biologically produced nanosilver: current state and future perspectives. Bioeng 109:2422–2436View ArticleGoogle Scholar
- Kassaee M, Akhavan A, Sheikh N, Sodagar A (2008) Antibacterial effects of a new dental acrylic resin containing silver nanoparticles. J Appl Polym Sci 110:1699–1703View ArticleGoogle Scholar
- Alt V, Bechert T, Steinrücke P, Wagener M, Seidel P, Dingeldein E, Doman E, Schnettler R (2004) An in vitro assessment of the antibacterial properties and cytotoxicity of nanoparticulate silver bone cement. Biomaterials 25:4383–4391View ArticleGoogle Scholar
- Jain P, Pradeep T (2005) Potential of silver nanoparticle-coated polyurethane foam as an antibacterial water filter. Biotechnol Bioeng 90:59–63View ArticleGoogle Scholar
- Sharma VK, Yngard RA, Lin Y (2009) Silver nanoparticles: green synthesis and their antimicrobial activities. Adv Colloid Interfac 145:83–96View ArticleGoogle Scholar
- de Mel A, Chaloupka K, Malam Y, Darbyshire A, Cousins B, Seifalian AM (2012) A silver nanocomposite biomaterial for blood-contacting implants. J Biomed Mater Res A Part A 100:2348–2357Google Scholar
- Kokura S, Handa O, Takagi T, Ishikawa T, Naito Y, Yoshikawa, T (2010) AgNPs as a safe preservative for use in cosmetics. Nanotechnol 6:6570–574Google Scholar
- Azeredo H (2009) Nanocomposites for food packaging applications. Res Int 42:1240–1253Google Scholar
- Tsuji M, Gomi S, Maeda Y, Matsunaga M, Hikino S, Uto K, Tsuji T, Kawazumi H (2012) Rapid transformation from spherical nanoparticles, nanorods, cubes, or bipyramids to triangular prisms of silver with PVP, citrate, and H2O2. Langmuir 28:8845–8861View ArticleGoogle Scholar
- Ghaffari-Moghaddam M, Hadi-Dabanlou R, Khajeh M, Rakhshanipour M, Shameli K (2014) Green synthesis of AgNPs using plant extracts. Korean J Chem Engineering 31:548–557View ArticleGoogle Scholar
- Banerjee P, Satapathy M, Mukhopahayay A, Das P (2014) Leaf extract mediated green synthesis of silver nanoparticles from widely available Indian plants: synthesis, characterization, antimicrobial property and toxicity analysis. Bioresources and Bioprocessing 1:3View ArticleGoogle Scholar
- Mohammadinejad R, Pourseyedi S, Baghizadeh A, Ranjbar S, Mansoori G (2013) Synthesis of silver nanoparticles using Silybum marianum seed extract. Int J Nanosci Nanotechnol 9:221–226Google Scholar
- Swamy M.K, Sudipta K, Jayanta K, Balasubramanya S (2015) The green synthesis, characterization, and evaluation of the biological activities of silver nanoparticles synthesized from Leptadenia reticulata leaf extract. Appl. Nanosci 5:1–9View ArticleGoogle Scholar
- Lei B, Zhang X, Zhu M, Tan W (2014) Effect of fluid shear stress on catalytic activity of biopalladium nanoparticles produced by Klebsiella Pneumoniae ECU-15 on Cr(VI) reduction reaction. Bioresources and Bioprocessing 1:28View ArticleGoogle Scholar
- Speth TF, Varma RS (2011) Microwave-assisted green synthesis of silver nanostructures. Accou of Chem Rese Acc of Cheml Resea 44:469–478View ArticleGoogle Scholar
- Song K, Lee S, Park T, Lee B (2009) Preparation of colloidal silver nanoparticles by chemical reduction method. Kore J of Che Eng 26:153–155View ArticleGoogle Scholar
- Li K, Zhang FS (2010) A novel approach for preparing silver nanoparticles under electron beam irradiation. J of Nanopa Resea 12:1423–1428View ArticleGoogle Scholar
- Harada M, Kawasaki C, Saijo K, Demizu M, Kimura Y (2010) Photochemical synthesis of silver particles using water-in-ionic liquid microemulsions in high-pressure CO2. J of Coll Interf Sci 343:537–545View ArticleGoogle Scholar
- Jensen MD, Malinsky CL, Haynes RPV (2000) Duyne, Nanosphere lithography: tunable localized surface plasmon resonance spectra of silver nanoparticles. J Phys Chem. 104 1059Google Scholar
- Philip D (2011) Mangifera indica leaf-assisted biosynthesis of well-dispersed AgNPs. Spectrochimica Acta 78:327–331View ArticleGoogle Scholar
- Bankar A, Joshi B, Kumar AR, Zinjarde S (2009) Banana peel extract mediated novel route for synthesis of AgNPs. Colloid Surf A Physicochem Eng 368:58–63View ArticleGoogle Scholar
- Dwivedi AD, Gopal K (2010) Biosynthesis of silver and gold nanoparticles using Chenopodium album leaf extract. Colloid Surf A Physicochem Eng Aspect 369:27–33View ArticleGoogle Scholar
- Velmurugan P, Shim J, Kamala-Kannan S, Lee KJ, Oh BT, Balachandar V (2011) Crystallization of silver through reduction process using Elaeis guineensis biosolid extract. Biotechnol Prog 27:273–279View ArticleGoogle Scholar
- Singh K, Panghal M, Kadyan S, Chaudhary U, Parkash YJ (2014) Antibacterial activity of synthesized silver nanoparticles from Tinospora cordifolia against multi drug resistant strains of pseudomonas aeruginosa isolated from burn patients. J Nanomed Nanotechnol 5:2Google Scholar
- Dubey M, Bhadauria S, Kushwah BS (2009) Green synthesis of nanosilver particles from extract of Eucalyptus hybrida (safeda) leaf. Dig J Nanomater Bios 4(34):537–543Google Scholar
- Kim SW, Jung JH, Lamsal K, Kim YS, Min JS, Lee YS (2012) Antifungal effects of silver nanoparticles (AgNPs) against various plant pathogenic fungi. Koren j of Microbiol 40:53–58Google Scholar
- Mulvaney P (1996) Surface plasmon spectroscopy of nanosized metal particles. Longmuir 12:788–800View ArticleGoogle Scholar
- Khatami M, Pourseyedi S (2015) Phoenix dactylifera (date palm) pit aqueous extract mediated novel route for synthesis high stable AgNPs with high antifungal and antibacterial activity. IET Nanobiotechnol 9:1–7View ArticleGoogle Scholar
- Yilmaz M, Turkdemir H, Kilic MA, Bayram E, Cicek A, Mete A, Ulug B (2011) Biosynthesis of AgNPs using leaves of Stevia rebaudiana. Mater Chem Phys 130:1195–1202View ArticleGoogle Scholar
- Kaviya S, Santhanalakshmi J, Viswanathan B, Muthumary J, Srinivasan K (2011) Biosynthesis of silver nanoparticles using Citrus sinensis peel extract and its antibacterial activity. Spectrochimica Acta 79:594–598View ArticleGoogle Scholar
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.