Green synthesis of antimicrobial silver nanoparticles with Brassicaceae seeds

Herein, we demonstrate a facile and green route for the synthesis of silver nanoparticles (AgNPs) from silver nitrate and seed extracts of different vegetable seeds of Brassicaceae family. All the nanocomposites were fully characterized in the solid-state via various techniques such UV–vis spectrophotometer (UV–Vis); x-ray diffraction (XRD), High-resolution transmission electron microscopy (HR-TEM), energy-dispersive x-ray spectroscopy (EDS), and Fourier transform infrared (FTIR) spectrometry. The experimental parameters such as variation in seeds extract concentration, temperature, stirring time and pH were noted and optimum condition of concentration (20 ml), temperature (80 °C) and pH 8.5 was selected for the synthesis of NPs. Optical absorbance of AgNPs at ≈425 nm indicated the formation of metallic silver through surface plasmon resonance. The successful capping of biological macromolecules was confirmed by FTIR spectroscopy. XRD pattern depicted the formation of face-centered cubic silver nano-composite with average crystal size ranges from ≈14–20 nm. Bio-synthesized Ag nanoparticles showed enhanced antibacterial potential against gram-positive (B. safensis, B. subtilis, B. pumilis and S. aureus) and negative gram (E. coli and S.typhi) strains by disc diffusion method. Highest antimicrobial activity was given by sample S3 (17 mm) against B. pumilis whereas, sample S2 and S5 also showed significant bactericidal potential against B. pumilis that is 15 mm. While highest zone of inhibition for sample S1 and S4 is 14 mm.

Generally, NPs are produced in numerous ways such as physical and chemical that are time-consuming, require toxic chemicals and disturb the natural environment. So, the development of a biologically-inspired procedure for the fabrication of NPs is highly recommended [2,14,24,25]. The physical method consists of laser ablation, chemical reduction and production of metal clusters. Radiation methods involve ultraviolet/ microwave radiation, photo-chemical and sono-electrochemical approaches [6,26]. Numerous other chemical procedures include chemical reduction of metal salt precursors in solution, spray pyrolysis, sono-chemical, microwave-assisted and micro-emulsions [22,27,28]. Silver nanoparticles have been synthesized by various phytoconstituents like alkaloids, flavonoids, and terpenoids for the Ag + reduction [27].
Green chemistry is a unique process because it requires only plants for NP-synthesis, which offers a good alternative to a chemical/physical method because they are cost-effective, environmentally sustainable and easily expanded for large-scale processing. Green method is free from high pressure, temperature and lethal chemicals. Recently plants, bacteria, fungi, hormones [29], proteins [30,31] and enzymes (urease) [32] are also employed for the synthesis of NPs. Bio-molecule functionalized NPs could be rapidly prepared at room temperature through a one-pot reaction, for example protein-functionalized NPs were successfully synthesized for a variety of proteins with a wide range of molecular weights and isoelectric points. The prepared NPs exhibited high quantum yield, high photostability, colloidal stability and high functionalization efficiency. Although green plant-based synthesis does not only reduce preparation costs but does not require specific techniques and plant preparations for the synthesis of nano-composites, they use locally available plant seeds [4,6]. We used plants for green synthesis in this study; however, the main disadvantage of this method is that it is difficult to determine the reactive components present in extract because plants contain a large number of organic compounds. The present study is conducted to synthesize and characterize silver NPs by vegetable seeds belonging to the family Brassicacea. Vegetables of Brassicaceae family widely used as a food spice and medicine. They are good source of antioxidants, vitamins, minerals, chlorophylls, glucosinolates and polyphenols. In addition, vegetable seeds possess several pharmacological activities for instance anti-inflammation, bacteriostatic and antiviral potential. They also combat various illnesses including obesity, depression, cancer, cataracts and diabetes [33][34][35]. The study aims to use green and cost benefit approach for the synthesis and characterization of environmentally benign NPs and elucidating their interaction with G (+ve) and (−ve) bacterial strains.

Materials
Silver nitrate was obtained from Sigma-Aldrich and Nutrient Agar was procured from Oxoid. Glass apparatus was cleaned with aqua regia and deionized water. Five different seeds of Brassicaceae family were collected Nursery. The seeds were Raphanus sativus-GC.Herb.Bot.3296, Raphanus sativus-GC.Herb.Bot.3297, Brassica rapa-GC.Herb. Bot.3298, Brassica campestris-GC.Herb.Bot.3299) and Brassica oleracea-GC.Herb.Bot.3300. These plants were authenticated by Dr Zaheer-ud-din Khan, Government College University Lahore, Pakistan. The analytical chemicals were used in current project. Bacterial strains were taken from PCSIR Laboratories Lahore, Pakistan.

Preparation of plant extract and synthesis of AgNPs
Seeds (10 g) of five different plants were soaked in 100 ml double distilled water for 12 h at 25°C. Next day, extracts were filtered and kept in refrigerator until further proceedings. The synthesis of AgNPs was performed by varying different factors like concentration of seed extract, pH, stirring time and temperature. Seed extracts of five different plants were used for the synthesis of AgNPs named S1, S2, S3, S4 and S5 (table 1). 20 ml of seeds extract was recorded as the best conditions for the synthesis of NPs. So, 20 ml of seeds extract were mixed with 100 ml aqueous solution of silver nitrate for reduction of Ag + into Ag°. The resultant solution was stirred for about 1 h at pH 8.5. Formation of Ag-NPs was confirmed by color change [36]. A schematic illustration of synthesis is illustrated in figure 1.

Characterizations samples
UV-vis spectrophotometer-UV-1700 Shimadzu was employed to record the absorption spectra at the wavelength of 200-800 nm for different AgNPs. Information about structure and mean crystal size of AgNPs were monitored by XRD (model: PAN alytical X'Pert PRO) with the 2θ range of 10°-80°, equipped by Cu-Ka radiation with λ=1.540 Å. The morphology of nanomaterial was characterized by Philips-CM30 along with microscope (JEOL JEM 2100F) to record HR-TEM micrographs, coupled with EDS detector. The active functional groups involve in biosynthesized NPs were investigated by BRUKER ALPHA Platinum-ATR spectrometer.

Antimicrobial activity assay
The synthesized AgNPs using plant extracts were examined for antibacterial potential by disc diffusion process against different bacterial strains using a reported method [24,37]. Standard culture media [CM145, CM271 and CM201] was used throughout the experiment and transferred to Petri plates aseptically. Sterile paper discs  (6 mm, Difco, USA) were impregnated with 20 μl freshly prepared silver nanoparticles. The discs were placed on freshly prepared Petri dishes with a control. Sterile water and streptomycin (1%, 20 μl) were employed as a negative control and positive control respectively. Antibacterial activity was performed in triplicate and microbes are grown in agar medium along with the preparation of nanoparticles impregnated disc. The Petri dishes were placed in incubator for 24 h, after incubation zones of inhibition were measured in millimeters.

Results and discussion
Various factors that affect the formation of AgNPs were analyzed by UV-Visible spectroscopy [2] such as concentration, pH, stirring time and temperature. The effect of concentration was studied by taking 10 ml, 20 ml and 30 ml seed extract in 100 ml AgNO 3 (1.0 mM). The values of λ max and absorbance depict that 20 ml concentration is most appropriate concentration for biosynthesis of NPs ( figure 1(a)) [38] pH of reaction mixture was varied as acidic (4.5), neutral and alkaline (8.5) and recorded their effect on biosynthesized silver nanoparticles. The best pH for the synthesis of AgNPs was recorded to be 8.5 being alkaline or basic ( figure 1(b)). Different stirring times (0, 2, 4, 6, 8 and 10 min) were used at room temperature to evaluate the stirring effect . These stirring times were not shown any significant effect on the formation of AgNPs ( figure 1(c)). The effect of temperature (20, 40, 60, and 80°C) was monitored on all five samples and it is confirmed from UV-vis absorption spectra that high temperature involves the formation of fine crystalline silver NPs (figure 1(d)) [39]. UV-Visible spectroscopic analysis of biosynthesized NPs has been depicted in figure 2. The absorption peaks that appeared at 410-450 nm reflected the surface plasmon resonance of silver NPs. There was no absorption in case of seed extracts while pristine samples showed a distinct peak at 425 nm as reported in literature [27]. However, higher and lower concentrations of seed extracts beyond the optimum value resulted in broader peaks with decreased absorption intensity and dark color reaction mixture indicates agglomeration between NPs. It was observed that biosynthesized AgNPs were stable in solution up to one month [40].
Green synthesis involves a variety of phytoconstituents that involve reduction of metal ions as well as stabilizing/ capping metal NPs. So, it is not easy to propose the exact mechanism for reduction of silver ions. The plausible mechanism is shown in figure 3. The phyto-constituents involve tannins, polyphenols, and gallic acid contain high density of -OH groups. Hydroxyl group of the polyphenols oxidizes and releases two electrons [41]. These electrons are responsible for the reduction of 2Ag + [42]. After the atomic silver-Ag 0 formation, many reactions need to occur for silver NPs formation, since atomic silver-Ag 0 is not considered a nanoparticle, its agglomeration forms NPs and these reactions are called nucleation. Complexation of polyphenol with metallic silver, and this bond with the biomolecules is responsible for the stabilization of the nanoparticles [43,44]. The electrochemical potential difference is the main reason behind the interaction of ionic silver and phyto-constituents [2].
The functional groups of biosynthesized NPs S1, S2, S3, S4 and S5 were investigated by FTIR studies conducted in the range of 500-4000cm −1 (figure 4(a)). The Significant absorbance peaks of Ag-NPs appeared at 3260, 2920, 1555, 1440, 1030 and 520 cm −1 correspond to mainly polyphenols and terpenoids, present in seed extract. Absorption spectra at 3260 cm −1 corresponds the stretching frequency of -OH and amine N-H [45]. While, peaks at 2920 cm −1 are associated with C-H stretch of an alkane. However, vibrational band at C=C 1555 cm −1 reveals the existence of C=C and 1440 cm −1 depicted the stretching vibrations of -COO-. Peaks transmitted in the region 1030 cm −1 reveals the presence of unsaturated ketone and ester. Variations in the FTIR spectra demonstrates that different biomolecules from plant extracts actively participate in the reduction of AgNO 3 and also contribute to the formation of specific size NPs either through cysteine residues or free amines  by the surface-binding proteins [46]. This suggests that various functional groups (methyl, hydroxyl, carboxylate, and carbonyl) involve in the synthesis of AgNPs [47][48][49]. Figure 4(b) represents the XRD patterns of biosynthesized silver nanoparticles. A number of Bragg diffraction peaks indexed as 38°(111), 44°(200), 64°(220) and 76°(311) were ascribed to the crystallinity and the face-centered cubic Ag [JCPDS 04-0783] [50]. Sharpness of diffraction peaks reflects that several biomolecules from seed extracts involve in the formation of AgNPs. Broad peak in sample S2 at 38°represents an incomplete reduction of silver ions might be indicating inadequate phytochemicals in seed while a small peak at 26°shows the bio-organic phase on the surface of particles [51]. The average crystal size calculated from Scherrer equation (d XRD ) was 14.7, 20.1,15, 14.3 and 18.9 nm for S1, S2, S3, S4 and S5 respectively.
HRTEM images demonstrate the morphology and crystal structure of biosynthesized NPs figures 5(a)-(e). The micrographs revealed agglomerated NPs with approximate size of less than 60 nm. HR-TEM presents Fast-Fourier Transform-FFT of particular area shown by bright Yellow Square in figures 5(a)-(b), describes high atomic resolution as well as structural information. Interlayer fringe spacing for sample S1 and S2 was measured to be 0.24 and 0.25 nm which corresponds to (111) facet of fcc silver crystal (JCPDS. No.01-087-0597) [52]. Which is well-matched with XRD results and reported data.
EDX examination was employed to investigate the elemental composition of phyto-synthesized AgNPs. Characteristic silver peaks were monitored at ∼3, 22 and 25 KeV ( figure 6). However, a strong spectral signal at 3 KeV is a typical energy value for metallic Ag Nano crystallites [53]. Whereas, additional spectral signals (carbon-C and sulphur-S) reveals the presence of extracellular bio-moieties that were adsorbed on the surface of NPs.
The bacteria are known to consist of cytoplasm, cell membrane and cell wall. The cell wall of gram-positive (G +ve) bacteria consists of one multilayer peptidoglycan polymer (20-80 nm) thickness while gram-negative (G -ve) bacteria consists of two cell membranes thickness 7-8 nm. The NPs stick to bacterial membrane due to opposite electrostatic charges on them causing cell shrinkage, perforation and ultimately cell death [54].
The five different samples of silver nanoparticles were screened for their antimicrobial potential. and the l diagrams of zone inhibition were presented in figure 7. Streptomycin (1%) was employed as control. The prepared samples were found to be active against most bacterial strains taken into account. B. safensis and B. pumilis were found to be inhibited by three samples out of five. S. typhi was inhibited actively by the four samples. The least active inhibition was recorded against E. coli. The AgNPs from Raphanussativus Eurpean (Red Radish, S1) were found to be potent against B. safensis, S. aureus and S. typhi. The AgNPs from Raphanussativus East Asia (White Radish, S2) inhibited three bacterial strains and the potential was in comparison to the reference and showing slight activity against S. aureus and E. coli. The nanoparticles from Brassica rapa (Turnip, S3) were active against B. pumilis. The AgNPs from Brassica campestris (Saag, S4) exhibited excellent potential against two bacterial strains (B. safensis and S.

Conclusion
This work presents, green method for the synthesis of Ag-NPs using seed extracts and reveals the Brassicaceae family as an efficient biological reducing agent that can be easily scaled up as its economic, facile and environmentally benign features. Furthermore, the optimum reaction conditions for the preparation of Ag-NPs were determined by investigating different experimental parameters. Specifically, concentration of seed extract, pH, stirring time and temperature. The optimum conditions were found to be 20 ml of seed extract, 80°C temperature, 30 min stirring time and 8.5 (alkaline) pH for nanoparticle synthesis. Active biological constituents of seed extract act as effective stabilizing and reducing agents that were investigated by FTIR studies. XRD and HRTEM provided information regarding the crystallite size and phase-purity, average crystalline size of the AgNPs was recorded in the range of 14-20 nm by Scherrer's formula. The evidence of chemical composition was collected by EDS spectral signals. The synthesized material act as an efficient bactericidal agent against gram-positive and negative bacterial strains. This rapid synthesis of NPs by Brassicaceae family provides a good alternate for chemical reduction methods owing to its non-toxic nature. This environment-friendly synthesis of nanomaterial opens up new horizons in biomedical field. Further research is needed to evaluate their cytotoxicity and field potential.