For the seeding of ZnO, zinc acetate and sodium hydroxide (NaOH) were supplied from lobacheme. For the production of CeO2, cerium nitrate Ce (NO3)3.6H2O was bought from Sigma Aldrich. Merck provided the Dimethylsulfoxide (DMSO) and nutrient agar media that were utilized to determine Zone of Inhibition (ZOI). All compounds were analytical grade and require no further purification.
Microwave digestion device
Microwave digestion Speed Wave XPERT device from Berghof, Germany, was used for synthesis of ZnO and CeO2 nanorod structures. The Magnetron power 1000 W with maximum temp. up to 260 °C and pressure 100 bar. The device was created by the Berghof Fluoroplastic Technology GmbH using eight 100 mL Teflon vessels with the highest quality standards. They are made of premium TFM-PTFE and impressed through dependability and an outstanding lifespan.
Preparation of Olea europaea leaf plant extract.
Fresh and healthy leaves of Olea europaea (O. e.) were washed multiple times with deionized water to get rid of the dust particles on their surface then they were dried in shade.
A healthy and undamaged leaf was carefully chopped, and stirred with deionized water at 85 °C for 2 h. The extract was filtered through Whatman filter paper after it was allowed to cool. The filtered leaf extract was used for further experiments as, capping agent, stabilizer and reducing agent for synthesis of the metal oxides nanomaterials, being usable within 2 weeks. The storage done in Argon atmosphere. (Chatterjee et al. 2016; Rosi et al. 2018).
Synthesis of ZnO nanomaterials
ZnO nanorods (NRs) are prepared via environmentally friendly and fast microwave-assisted green hydrothermal method. As in a normal procedure, 12 gm of Zinc acetate hydrated was dissolved in 100 mL of distilled water to form a transparent solution. 15 mL Sodium hydroxide (NaOH) (5Molar) was added to the above solution gradually and under vigorous stirring. After formation of Zn (OH)4 precipitate, it was filtered, cleaned with distilled water, and allowed to dry at room temperature. Then suitable amount from the dried powder was added to 50 mL of (O. e.) extract and/or 50 mL of bi-distilled water. The suspension was put under continuous stirring and sonication for 30 min at room temperature to form a homogeneous suspension. The suspension was then transferred either into four Berghof TFM-PTFE microwave Teflon vessels at 160 °C for 30 min, or for comparison, into 100 mL Teflon-lined stainless-steel autoclave at 160 °C for 48 h, as a traditional hydrothermal synthesis method. After that, the Teflon vessels were allowed to cool down naturally and the resultant nanoproduct was harvested by centrifugation and washed with distilled water, and finally dried at 60 °C in the oven and then milled in a porcelain mill (Abdelmordy 2017).
Synthesis of CeO2 nanomaterials
4 gm of cerium nitrate hexahydrate was dissolved in 100 mL of distilled water to form a transparent solution. Sodium hydroxide (NaOH) was added to the above solution gradually and under vigorous stirring. After formation of Ce (OH)4 precipitate, it was filtered, cleaned with distilled water, and allowed to dry at room temperature.
Then suitable amount of the dried powder was added to 50 mL of (O. e. L.) extract and/or 50 mL of bi-distilled water. The suspension was put under continuous stirring and sonication for 30 min at room temperature to form a homogeneous suspension. The suspension was then transferred either into four Berghof TFM-PTFE microwave Teflon vessels at 200 °C for 30 min, or for comparison, into 100 mL Teflon-lined stainless-steel autoclave at 200 °C for 12 h, as a traditional hydrothermal synthesis method. The Teflon vessels were then allowed to cool naturally, and the last product was obtained by centrifugation, washed with distilled water, and then dried at 60 °C in the oven before being milled in a porcelain mortar (Maqbool 2017).
X'pert Philips X-ray diffraction (XRD) Using Cu K radiation, 40 kV, 30 mA, and a scan rate of 50/min, was used to examine the surface characteristics of ZnO and CeO2 nanomaterials. Utilizing Scherer's formula, the crystallite size of the produced materials was calculated from the broadening of the relevant X-ray peaks (Wang et al. 2020). The morphology of the produced nanomaterials was determined by using a transmission electron microscope (TEM), JEM-2000 EX (JEOL, Tokyo, Japan). Brunauer–Emmett–Teller (BET) analysis (Quantachrome Instruments, NOVA series, USA) was used to calculate the specific surface area (m2 g−1) and pore volume of the powders at 77.35 K. A multipoint BET technique was used to calculate the specific surface area (SBET) using adsorption data at relative pressures (P/P0) between 0.05 and 0.25. The Barrett-Joyner-Halenda (BJH) method was utilized to determine the pore size distribution using the adsorption–desorption isotherm. The pore volume and average pore size were calculated using the nitrogen adsorption volume at the relative pressure (P/P0) of 0.976.
The Zeta Sizer Nano (ZS), a high performance two angles particle size, zeta potential, and molecular weight analyzer ideal for measurement of small volume samples at very low or high concentration and the detection of aggregates, was used to calculate the average particle size of synthesised nanomaterials using the Dynamic Light Scattering (DLS) technique. Non-Invasive Backscatter Optics (NIBS) performs significantly better than 90 scattering optics systems (Lopez et al. 2016).
The chemical composition and quality of the synthesized ZnO and CeO2 nanomaterials were evaluated using FTIR 430 spectrometer (Jasco-Japan) equipped with a diffuse reflectance accessory (Harrick, USA) in the range of 400–4000 cm−1.
Anti-bacterial activity assay
The antibacterial activity assay of the green synthesized ZnO and CeO2 nanomaterials compared with that prepared via traditional hydrothermal methods, has been studied against six carefully chosen bacterial strains; two-gram negative bacteria, i.e. E. coli (ATCC 8739) and Serratia marcescens brought from faculty of Pharmacy, Tanta University, Egypt and four-gram positive bacteria, i.e. Staphyllococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633), Streptococcus mutant (ATCC 25175), and Methicillin-Resistant Staphylococcus Aureus (MRSA) obtained from faculty of Aquatic and fisheries sciences-Kafr elsheikh university. The experiment is carried out in DMSO solvent using the agar diffusion method (Nabi et al. 2020). The Zone of Inhibition (ZOI) was determined by using Kirby–Bauer disc diffusion susceptibility technique (Ningappa et al. 2008). The experiment was carried out in accordance with the guidelines established by the National Committee for Clinical Laboratory Standards (NCCLS, 1999).The pathogens under investigation were grown in 10 cm test plates on nutrient agar. The test plates were incubated for 48 h at 37 °C. The zone of inhibition (in mm diameter) was observed and recorded after the incubation period. Three different types of assays were carried out to study the antimicorbial activity of the green-synthesized NPs in the concentration range of 50–200 µg mL1. This is in order to determine the most active conc. towards the bacterial strains under investigation. 50 µg mL−1 was chosen as the MIC that gives a remarkable antimicrobial effect.
The experiments were carried out in triplicate, and the results are given as means with standard deviations (SD) for three parallel measurements. Antibiotics amoxicillin and flucloxacillin were used as controls. DMSO was used as negative control.
In-vitro antitumor cytotoxicity assay
The antitumor activity of the microwave-assisted green synthesized ZnO and CeO2 nanorods has been examined on hepatocellular carcinoma cell lines, according to van de Loosdrecht et al. (1994). The cell viability was determined by using MTT assay method. In a typical method, hepatocellular carcinoma cell line was seeded in a plate of 96 wells plate (1 × 105 cells/well) and incubated for 1–5 h at 37 °C in a 5% CO2 incubator to allow cell MTT to be metabolized. After incubation, the media were replaced with new fresh media treated with serial concentrations of ZnO and/or CeO2 nanomaterials (31.25 to 1000 µg mL−1). After incubation, Formazan (MTT metabolic product) was resuspended in 200ul DMSO and was shaken at 150 rpm for 5 min to thoroughly mix the formazan into the solvent. The optical density was measured at 560 nm and background was subtracted at 620 nm. Optical density should be proportional to cell quantity. The IC50 values were determined using nonlinear regression analysis with log (inhibitor) vs. variable slope (four parameters) (Van de Loosdrecht 1994).
In-vivo antitumor toxicity assay
The studied sample was administered orally to clusters of mice, every consisting of six animals, in gradually increasing dosages ranging from 0.1 to 3 g kg−1, in accordance with the procedure outlined by Finny (1964). The identical conditions were used to receive and care for the control animals. For 1 h, animals were continuously checked for abnormal characteristics. After administration, each group's mortality rate was tracked for 24 h (Finny 1964; El-Shafey et al. 2020).