Antibacterial Activity of Green Synthesis Silver Nanoparticles Using Some Wild Edible Plants Commonly Used in Al Baha, Saudi Arabia

In the present study, aqueous extract of Cissus rotundifolia (Wild edible plants) was used as a reducing and capping agent in the formation of silver nanoparticles (AgNPs). UV-visible spectroscopy (Uv-Vis) was used to monitor the formation of AgNPs in the aqueous medium. The green-prepared AgNPs investigated using Fourier-transform infrared spectroscopy (FTIR) and X-ray powder diffraction (XRD). The morphology and size of the benign silver nanoparticles were carried out by the transmission electron microscope (TEM) and field emission scanning electron microscope (FE-SEM). The susceptibility of bacteria strains against the green synthesis AgNPs was determined using the disk diffusion method. The microorganisms employed were E. coli, K. pneumoniae, B. cereus, S. aureus, C. lbicans and Aspergillus. The results showed the characteristic surface plasmon resonance peak of the AgNPs appeared at approximately 418 446 nm. XRD revealed peaks at 38.2, 44.16, 64.24 and 77.22 θ, and the intensity of these peaks enhanced when using microwave curing compared to ambient temperature. SEM and TEM results showed that the silver nanoparticles have a spherical shape and the particle size for samples is less than 37 nm. FTIR spectroscopy measurements showed the binding of organic compounds on the surface of the silver nanoparticles. The highest antibacterial activity was enhanced with increasing of AgNPs dose and with increasing of extract ration against most of microorganisms.

glycosides, are present at very low concentrations. C. rotundifolia from Africa and Asia showed anti-diabetic [8] as well as anti-parasitic properties [9]. It has minor economic importance as a medicinal plant [10]. An animal study has already shown that these plant materials have hypocholesterolemic activity [11]. In Yemen, this plant is used in many traditional or popular medical applications, such as loss of appetite, anti-malarial, gastrointestinal troubles, skin diseases and burns [12] [13] [14] [15]. Siddiqui et al. [16] used the useless and discarded leaves of C. rotundifolia for preparing bio adsorbent to the removal of heavy metal pollutant of water. Al-Mamary [17] reported that C. rotundifolia has significant antioxidant efficiencies that could be attributed to the presence of phenolics.
Nanoparticles of metals have unique properties, such as surface area and particle size, which are mainly different from those of bulk materials [18] [19]. Solar energy systems, optics, catalytic and antibacterial capacities are main applications fields of AgNPs [20]. Recently, many studies conformed that silver nanoparticles (AgNPs) display effective antimicrobial capacity against both gram-positive and gram-negative bacterial strains. Whereas, toxicity and hazard of chemicals, in addition to high cost and power consumption, are the main drawbacks of chemical methods to preparing silver nanoparticles [21]. AgNPs were synthesized using a green, rapid, one-step, cost-effective and environmentally friendly method using Ziziphus Jujuba leaf extract [22]. A bio-synthesis of Ag-NPs using water extract of M. pendan was successfully carried out. Whereas, the water soluble flavonoids in the water extract were responsible in the reduction of Ag + to Ag 0 [23]. The synthesized silver nanoparticles using autoclave assisted gum extract of neem (Azadirachta indica) are exhibited antibacterial activity against clinical isolates of Salmonella enteritidis and Bacillus cereus [24]. Rajeshkumar [25] investigated the antibacterial activity of biosynthesized silver nanoparticles using the fresh bark extract of Pongamia pinnata against gram positive (Klebsiella planticola) and gram negative (Staphylococcus aureus) bacteria. Khan et al. [26] were attributed the antimicrobial activities of silver nanomaterials to the structural changes in the protein cell wall. Antibacterial tests using four bacteria  [28].
The current investigation was carried out to screen the antibacterial activity of green synthesis silver nanoparticles using wild edible plants extract against some pathogenic bacterial strains.

Collection of Plant Materials
Sample of fresh C. rotundifolia leaves were collected from a farm at Al-Mikhwah city, Al Baha region in as shown in Figure 1. About 1000 g of plant were washed and rinsed with distilled water. Sample was dried indoor and grinded to fine powder using kitchen blender (grinder of Moulinex blender, 400 W) for 2.5 min at high speed and then stored in plastic bags.

Microorganisms
Bacterial strains were isolated from food samples, which included E. coli and

Extraction of Material and Synthesis of Silver Nanoparticles
100 g of powdered sample with 750 ml of deionized water poured into pressure cooker and kept for 2 hr on the hot plate at 350˚C. The solid residues were removed by filtration and the extract kept in refrigerator to use time [29]. 99.9% pure AgNO 3 was purchased from Sigma-Aldrich, Cairo, Egypt. Table 1 shows the different conditions of green synthesis of AgNPs. A 20 mM solution of Ag-NO 3 was prepared for production of AgNPs. Green reduction of Ag + was monitored by visual observation and UV-visible spectroscopy.

Antimicrobial Assay
The antimicrobial activity of AgNPs was investigated by the disk diffusion method. The pure cultures of each strain were swabbed uniformly on the individual plates using sterile cotton at 35˚C on a rotary shaker at 200 RPM. Sterile filter paper discs (5 mm in diameter) impregnated with 25, 50, 100, 150 and 200 µL of the samples of silver nanoparticles solution then sited on the surface of these agar plates. The plates were then incubated at 37˚C for bacterial growth after which growth was determined by measuring the diameter of the inhibition zone (mm) using a digital caliper. Each extract was analyzed in triplicate, the mean values are presented. Tetracycline (30 mg/disc) was used as a gram-positive control.

Characterization Tools
The reduction of silver ions into silver nanoparticles monitored by UV-visible spectroscopy (SHIMADZU MODELUV 1800, Japan) at a wavelength of 350 -700 nm. A diffractometer XRD thin film PANalytical X pert PRO, Cu target, wave length 1.54 A, 45 kV, 40 mA made in Holland was used to determine the crystallinity of prepared AgNPs. Scanning electron microscopy (SEM) examination was performed using JEOL JSM 6360 DLA, Japan, at 30 kV, and the SEM-EDX analysis was performed by FEI Company, Quanta FEG250, Holland. Transmission electron microscopy image were taken using a Hitachi, H-800 TEM. TEM

Characterizations of AgNPs
Green reduction of Ag + by C. rotundifolia extract was monitored by observing the colour of silver solutions that changed from colourless, to yellow, brown and then reddish brown as evidence of silver ion reduction, Figure 1    The presence of organic compounds of C. rotundifolia extract on the surface of the nanoparticles was investigated using FTIR. The FTIR spectra of silver nanoparticles prepared with 40:60 (v/v) leaf extract: Ag + (d) at ambient is shown in Figure 4. The band at 3432 cm -1 is attributed to the hydroxyl group of organic compounds, the small bands appearing at 2927 and 2852 cm -1 may be due to C-H stretching of vibration of the -CH 2 group from the aliphatic chains, and a relatively strong band can be noted at 2065 cm −1 due to CN stretching vibration [33]. Additionally, the band at 1635 cm −1 is related to stretching of the carbonyl groups and bands at 1384 cm −1 and 1106 cm −1 related to the carboxylic groups. These absorption bands indicate the absorption of different organic. Figure 5 shows SEM images of the nanoparticles synthesized with 40:60 (v/v) BLE leaf extract: Ag + (d sample) at ambient. Mostly spherical and nearspherical shapes were observed for AgNPs in the 20 -40 nm size range. TEM analysis gives actual information about the morphology of the surface of the AgNPs.
TEM images of the prepared AgNPs with different magnifications are shown in Figures 6(a)-(c). They clearly show the formation of the best AgNPs with spherical and oval shapes in the size range of 22 -38 nm. These perfect particle sizes with various shapes of AgNPs may be related to different components of the plant extract [34]. ATEM image of AgNPs is shown in Figure 6(d). It clearly shows a lattice spacing of 0.21 nm related to the (111) plane of Ag that matches with the XRD pattern. The polycrystalline nature of prepared silver nanoparticles was confirmed by SAED, Figure 6(e), as the FCC structure of silver [35]. Furthermore, EDX analysis confirmed the presence of silver as a single element.

Antimicrobial Activity of Green Prepared AgNPs
Antibacterial activities of the prepared AgNPs using extract of C. rotundifolia against the tested organisms are shown in Table 2. All the AgNPs samples tested   showed antibacterial activity, however, the C. rotundifolia extract differ in its activities against the micro-organisms tested. As seen in Table 2  in the extract. The mechanism of AgNPs as an antimicrobial is not understood, but many studies suggest that it occurs through the interaction of silver nanoparticles with the DNA of microorganisms, forming free radicals and destructuring the cell walls [36] [37].

Conclusion
From the outcomes of this study, we can conclude that silver nanoparticles can be prepared using an aqueous extract of C. rotundifolia leaf. The stability of biosynthetic silver nanoparticles was monitored for up to six months. The prepared AgNPs showed antimicrobial activity against E. coli, K. pneumoniae, B. cereus, S. aureus, C. lbicans and Aspergillus and it increased with increasing the dose of plant extract.