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.

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

Klebsilla (gram negative); Bacillus cereus and Staph aureus (gram positive); Candida (yeast) and Aspirigullus (mold). The strains were kept at 4˚C on agar slant and sub cultured at 37˚C for 24 h on nutrient agar before any susceptibility test.

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 AgNO₃ was purchased from Sigma-Aldrich, Cairo, Egypt. Table 1 shows the different conditions of green synthesis of AgNPs. A 20 mM solution of AgNO₃ was prepared for production of AgNPs. Green reduction of Ag⁺ was monitored by visual observation and UV-visible spectroscopy.

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.

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

samples were prepared by placing drops of aqueous dispersion of AgNPs in distilled water on 200 mesh carbon coated copper grids and dried at ambient conditions for 10 to 12 h. The FT-IR spectra of the products recorded on a JASCO Asia Portal - FT/IR-6300 Spectrometer using the KBr pellet method.

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(c), increasing the silver nanoparticles concentration and changing the particle morphology. It was observed that the colour started to change after 0.5 min. and reached a maximum absorption capacity of UV after 7 min.

This period decreased with increasing the C. rotundifolia extract content. In addition, the UV-Vis spectroscopy investigation of the prepared AgNPs showed that the size and shape of AgNPs influence the shape and position of the UV-Vis absorption peaks from plasmon resonance. The UV-Vis absorption spectra of the synthesized AgNPs at ambient temperatures with time intervals up to 3 h are shown in Figures 2(a)-(d). The appearance of a strong peak at 437 nm indicated the formation of AgNPs that related to its surface plasmon resonance phenomena [30] . As shown in Figures 2(a)-(d), the absorbance value for AgNPs increased with an increase in C. rotundifolia extract ratio and indicated a higher concentration of AgNPs produced, which was due to the availability of more reducing bio-molecules in C. rotundifolia extract. The SPR peaks at a lower substitution of C. rotundifolia extract 20:80 (v/v) were broad band whereas with a higher substitution ratio the SPR peaks became sharper and narrower. The intensity of this absorption band increased with time with a slight shift to 441 nm. The organic compounds in the C. rotundifolia extract contain numerous functional groups that can interact with silver to form a complex. Furthermore, the cleavage of C-C bonds of organic molecules released electrons required for the reduction of Ag. When the complex compound Ag⁺⁻ C. rotundifolia was reduced to Ag⁰⁻ cissus, the organic molecules endow AgNPs with excellent dispersibility.

Figure 3 shows XRD patterns of the green synthesized AgNPs. The distinct diffraction bands at approximately 37.97, 44.29, 64.38 and 77.39 2 θ correspond to (111), (200), (220) and (311), respectively, were matched to planes of a face-centred cubic lattice of silver (ICSD No. 01-071-4613), which confirm the crystalline structure of prepared AgNPs. The extra peaks near 22.5, 26.2, 29.2 and 32.0 2 θ are due to the presence of organic compounds on the surface of AgNPs. The intensity of the peak at the (111) plane was greater than the other peaks, suggesting that this plane was the predominant one. According to Scherrer’s formula, the average sizes of AgNPs synthesized by C. rotundifolia leaf extracts at room temperature is 28 nm [31] [32] .

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 -CH₂ 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.

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, the inhibition zone area increased with a higher dose of AgNPs from 25 to 200 µml. The highest antibacterial activity was observed with increasing ratio of plant extract (d sample, 40%) against E. coli, K. Pneumoniae and B. Cereus. This is clearly attributed to antimicrobial efficiency of AgNPs, and not to the organic compounds

*Values are mean inhibition zone (mm) ± S.D of three replicates.

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] .