Facile Green Synthetic Route to the Zinc Oxide (ZnONPs) Nanoparticles: Effect on Green Peach Aphid and Antibacterial Activity

In this study, zinc oxide nanoparticles (ZnONPs) were synthesized using Punica granatum peel extract in one– step reaction at room temperature. Zinc oxide nanoparticles were characterized by Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), ultraviolet visible spectroscopy (UV-vis) and scanning electron microscopy (SEM). The UV-vis absorption spectrum shows an absorption band at 278 nm due to ZnO nanoparticles. XRD characterized the final product as highly crystalline ZnO with sizes in the range 10-40 nm. The SEM results reveal a presence of network of randomly oriented ZnO nanoplatelets with an average size of 40 nm and thicknesses of about 8 nm. This study determined the effect of zinc oxide nanoparticles on green peach aphid and antibacterial activity.

In this research work, we developed a green and fast route for synthesis zinc oxide nanoparticles using Punica granatum peel aqueous extract. Also this research work determined the effect of synthesized ZnONPs on green peach aphid (GPA) and antibacterial efficacy against standard strains of Gram positive Staphylococcus aureus and Gram negative Escherichia coli.

Materials
Zinc acetate dihydrate (ZnC 4 H 6 O 2 ·2H 2 O) and potassium hydroxide (KOH) are pure grade purchased from SIGMA-Aldrich and used without further purification. Distilled water was used in all experimental work.

Preparation of Punica granatum Peel Extract
Fresh fruits of healthy Punica granatum were collected from local market, Amman, Jordan. Peels were washed with water to remove dust particles and then dried in shade for two weeks to remove the residual moisture. Peels aqueous extract was prepared by placing 20 g of dried fine powder in 500 ml glass beaker along with 400 ml of sterile distilled water. The mixture was boiled for 10 min until the color of aqueous solution changed from watery to brown. Then the mixture was cooled to room temperature and filtered with Whatman No. 1 filter paper before centrifuging at 1,200 rpm for 5 minutes to remove biomaterials. The aqueous extract was stored at room temperature in order to be used for further experiments.

Green Synthesis of Zinc Oxide Nanoparticles (ZnONPs)
In a typical reaction mixture, 1.2 g of zinc acetate dihydrate was dissolved in 100 ml of the distilled water in 250 mL conical flask and stirred magnetically at room temperature for 5 min. Punica granatum peel aqueous extract (pH 5) was adjusted by potassium hydroxide to pH > 12. Afterwards, the alkaline peels aqueous extract was added drop wise under stirring, as soon as, the peels extract comes in contact zinc ions spontaneous change the colorless of zinc ions to yellow color. The obtained yellow mixture was left under stirring at room temperature. After one min, the yellow mixture started changing to a yellow-white suspended mixture, indicating the formation of water soluble monodispersed zinc oxide nanoparticles.

Characterization Techniques
Scanning electron microscopy (SEM) analysis of synthesized zinc oxide nanoparticles was done using a Hitachi S-4500 SEM machine. Powder X-ray diffraction (XRD) was performed using a X-ray diffractometer, Shimadzu, XRD-6000 with CuKα radiation λ = 1.5405 Å over a wide range of Bragg angles (3 o ≤ 2θ ≤ 80 o ). Fourier transform infrared (FT-IR) spectroscopic measurements were done using Shimadzu, IR-Prestige-21 spectrophotometer. UV-vis spectrum of zinc oxide nanoparticles was recorded, by taking 0.1 ml of the sample and diluting it with 2 ml deionized water, as a function of time of reaction using a Schimadzu 1601 spectrophotometer in the wave length region 200 to 700 nm operated at a resolution of 1 nm.

Green Peach Aphid
Zinc oxide nanoparticles synthesized using aqueous Punica granatum peels was tested against the green peach aphid. Five concentrations of ZnONPs were used to find their effect on this global pest. The testing method is similar to that adopted by Ghidan et al. (2016). Two ways analysis of variance was used to compare between mortalities caused by the ZnONPs concentrations.

Antibacterial Activity
Zinc oxide nanoparticles synthesized using aqueous Punica granatum peel extract was tested for its potential antimicrobial activity against some selected microbes. To analyze the antimicrobial activity of the sample, the samples were subjected to Agar well Diffusion method. Diameter of the zone of inhibition was measured in mm and expressed as Mean ± Standard Deviation.

XRD Analysis
The X-ray diffraction (XRD) pattern of synthesized ZnO nanoparticles is illustrated in Figure 1

UV-vis
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Conclusion
In the present work, we first report an eco-friendly and simple method for the synthesis of zinc oxide nanoparticles using Punica granatum peel extract. FTIR analysis of aqueous Punica granatum peel extract indicated the presence of phyto-constituents such as amines, aldehydes, phenols, and alcohols which were the surface active molecules stabilized the zinc oxide nanoparticles. XRD analysis reveals that the average size of the nanoparticles was found to be 20 nm which was calculated by Debye-Scherrer equation. FT-IR and XRD results corroborated the purity of the synthesized ZnONPs. Green synthesized ZnONPs was evaluated against green peach aphid, the results showed significant effect on this aphid compared with the control treatment. Gram positive Staphylococcus aureus and Gram negative Escherichia coli showing significant effective activity. The method of the present study offers several important advantageous features. First, the synthesis route is economical and environmentally friendly, because it involves inexpensive and non-toxic materials for second, large scale synthesis.