Green Synthesis of Silver Nanoparticles Using Nigella sativa Seeds and Evaluation of Their Antibacterial Activity

Synthesis of silver nanoparticles using seeds of Nigella sativa as a capping agent was evaluated in this study. Different concentrations of the aqueous extract of N. sativa with silver nitrate solution were exposed to sunlight; as a force for acceleration of the formulation. Then the silver nanoparticles were characterized by UV-Vis, scanning electron microscope (SEM) and X-ray diffraction (XRD) techniques. Antibacterial activity of the nanoparticles was investigated against Staphylococcus aureus and Escherichia coli by the disc diffusion method. The characterization of nanoparticles was detected by the change in color to yellow-brown which indicated the formulation of silver nanoparticles. Irregular shapes within range of nanoscale were detected using SEM and XRD techniques. The finding suggests that silver nanoparticles may be effectively used as antibacterial agent.


Introduction
In today's world, nanotechnology is a relatively new field, but its structural nanometer dimensions are not new, and in fact, these materials have much significance. Nanotechnology has the potential to provide novel, paradigm-shifting solutions to medical problems. Nanotechnology, which has been defined as the engineering and manufacturing of materials at the atomic and molecular scale, offers exclusive tools for developing safer and more efficient medicines (nanomedicines), and provides several potential advantages in drug formulation and delivery [1]. In pharmaceutical trade, nanotechnology may revolutionize the rules and possibilities of drug discovery and change the landscape of pharmaceutical industries. In medicine, nanotechnology application may be referred to as nanomedicine that explains various intriguing possibilities in the healthcare sector [2] [3]. Different methods are currently used for the preparation of metallic nanoparticles (NPs) including physical, chemical, and biological methods. Mechanical milling and high-energy mechanical milling are effective physical methods for synthesizing NPs. The chemical method usually involves use of chemicals for synthesis of nanoparticles which makes them certainly unsuitable against any application as it contains toxic compounds [4]. There has usually been a demand for sustainable, reliable, green, and eco-friendly approaches to synthesize metallic NPs minimizing or even disposing of the use of poisonous and risky chemical substances. synthesis of nanoparticles by biological method is through microbes like Aspergillus flavus, Phoma exigua and plant sources such as Cynodon Dactylon, Glycyrrhiza glabra, Nigella sativa, etc. [5] [6] [7]. Of all the nanoparticles developed and characterized thus far, silver nanoparticles (AgNPs) assume a significant position owing to their inherent characteristic of acting as an antimicrobial agent even in the solid state. Although, its significance was recognized much earlier, silver was not well exploited except for its use in oriental medicine and in coins [8]. It is estimated that nearly 320 tons of AgNPs are manufactured every year and used in nanomedical imaging, biosensing and food products [9].
There is a continuous increase in the number of multidrug resistant bacterial and viral strains due to mutation, pollution and changing environmental conditions [10]. To circumvent this predicament, scientists are trying to develop drugs for the treatment of such microbial infections. Many metal salts and metal nanoparticles have been found to be effective in inhibiting the growth of many infectious bacteria [11]. A number of investigations had emphasized the antimicrobial effect of nanoparticles synthesized from plants and was found to be effective against UTI causing bacteria usually Klebsiella spp., Escherichia coli and Staphylococcus aureus [12]. The present study was aimed to make an effective approach for synthesizing silver nanoparticles using seeds of Nigella sativa as a reducing agent and to assessing the potential for their use in the treatment of urinary tract infection.

Preparation of Aqueous Silver Nitrate
Silver nitrate solution (1 M) was prepared and stored in an amber colored bottle.

Preparation of Extract from Seeds of Nigella sativa
The seeds of Nigella sativa were washed several times with deionized water.

Synthesis of Silver Nanoparticles
Five different aliquots of the seed extracts (1 -5 mL) were taken separately and 10 mL of 1 mM silver nitrate solution was added with constant stirring and exposed to sunlight radiation. The colour change of the solution was checked periodically; from yellow to dark brown indicated that the silver nanoparticles were synthesized [13].

UV-Vis Spectroscopy Analysis
The reduction of pure Ag 2+ ions was monitored by measuring the UV-Vis spectrum of the silver nanoparticles solution after diluting a small aliquot of the sample in distilled water. UV visible spectroscopy was carried out on UV-1800 (Shimadzu) [13].

SEM Analysis
This technique was done using Scanning electron Microscope (SEM) (TESCAN MIRA). Thin films of samples were prepared on a carbon coated copper grid by dropping a very small amount of sample on the grid [13].

XRD Analysis
The silver nanoparticle solution was centrifuged at 2500 rpm for 20 min. The pellet was washed three times with 20 mL of deionized water. The dried powder of silver nanoparticles was collected for the determination of formation of silver nanoparticles (XRD-7000s/7000L Shimedazu) [13].

Antimicrobial Activity by Disc Diffusion Method
The prepared Mueller Hinton agar was poured on to sterile Petri-dishes plates and cultures of E. coli and S. aureus were swabbed on to the agar plates. Meanwhile, the sterile discs were impregnated with the silver nanoparticle solution and a positive control drug (Ciprofloxacin). The plates were incubated overnight at room temperature then the zone of inhibition was measured [14].

Synthesis of Silver Nanoparticles
Different optimization studies were carried out for the synthesis of silver nanoparticles from Nigella sativa seeds extract, results showed in Figure 1  dark brown because of the surface Plasmon resonance. The surface plasmon resonance (SPR) band is influenced by size, shape, morphology, composition and dielectric environment of the prepared nanoparticles [15].

UV-Vis Spectral Analysis
UV-Vis spectra were recorded for the N. sativa silver nanoparticles at different concentrations of silver nitrate solution, the result was showed in Figure 2. As the level of the absorption was found to be increase which indicate more synthesis of silver nanoparticles as was found in previous studies [16]. The visible range of UV spectra for silver ranging 400 to 600 nm, while in is this study the absorption ranged between 450 to 480 nm [17].

SEM Analysis
Using the Scanning electron microscope technique, a further insight into the morphological details of Nigella sativa AgNPS has been obtained. The SEM micrographs of the synthesized silver nanoparticles showed in Figure 3. The Figure   gives information about the irregular morphology and size of the synthesized silver nanoparticles when a voltage of 25 kV was applied. The size was found to be within the actual size range of nanoparticles, which is 1 -100 nm [13].

XRD Analysis
The XRD analysis was carried out for size distribution and crystal structure determination. The following results were obtained by the XRD analysis ( Figure 4).

Antimicrobial Activity
The antimicrobial activity was tested against two bacterial strains; Gram positive Staphlococcus aureus and Gram negative Escherichia coli. The synthesized AgNPs showed activity against S.aureus with inhibition zone of 13 mm obtained from the highest concentration (20 mg/ml) while the activity of AgNPs were found to be 6 mm against E. coli. Ciprofloxacin was used in this study as a positive control antibiotic; it obtained higher activity against S.aureus compared to E. coli (32 mm and 18 mm) respectively, which is mentioned in Table 1. Thus, the silver nanoparticles of N. sativa seeds significantly inhibits the pathogens, however, further investigation is required for understanding the mechanism of action [18].

Conclusion
In conclusion, the bioreduction of silver ions using seeds of Nigella sativa as reducing agent has been illustrated. From the present study, it is clear that the silver nanoparticles synthesized through the green method using seeds of N. sativa can inhibit the organisms causing urinary tract infection providing a significant inhibition zone. Thus, the silver nanoparticles from seeds of N. sativa may be used in the management of bacterial urinary tract infections.