Double hydrothermal synthesis of iron oxide/silver oxide nanocomposites with antibacterial activity**

: In this work, iron oxide "gamma phase" ( 𝛾 -Fe 2 O 3 )-silver oxide (Ag 2 O) nanocomposite is prepared by a double hydrothermal method combined with Punicaceae plant extract as reducing agents. X-ray diffraction (XRD) results confirmed the presence of 𝛾 -Fe 2 O 3 and Ag 2 O and delafossite silverferrite(AgFeO 2 )phases.FieldEmissionScanningElec-tron Microscopy-energy dispersive spectroscopy (FESEM-EDS) results revealed nanoparticles (NPs) with a shape like a cauliflower plant. Furthermore, the anti-bacterial activity results presented high inhibition rates against Klebsiella pneumoniae , Staphylococcus aureus , Staphylococcus epidermidis , Escherichia coli , and Candida albicans . The present study exhibits a new approach to the preparation of metal oxides using a facile and inexpensive method.


Introduction
Nanostructures have attracted unparalleled interest due to their unusual properties that differ greatly from their bulk state due to the increased surface area to volume ratio that increases the reactivity of surface electrons in materials [1]. Metallic oxides nanostructures have many applications, especially in optoelectronic systems, biomedicine, catalytic systems, and antimicrobial activity [2]. Ag 2 O have been extensively studied due to their great antibacterial activity and unique photoelectric properties. Its exhibit various modes of inhibiting microorganisms and are well-known antimicrobial agents due to their low production costs [3]. As a semiconductor matter, Ag 2 O is known to have a wide bandgap that may exceed 2.4 eV [4,5]. -Fe 2 O 3 nanostructures consist of iron and oxygen and have an energy gap of about 2.2 eV with a high absorption capacity for the visible light spectrum [6,7,8]. Due to its non-toxicity and low cost due to its superior magnetic properties, it is used in wide applications, especially in the fields of photocatalysts, drug delivery, durable dyes ultra, diagnostic MRI, waterproofing, heat therapy and microbial inhibition [9,10]. In the same context, the incorporation of metallic nano-oxides with each other is one of the important ways to improve the properties of these materials, especially their structural, optical and morphological properties, as well as their antibacterial properties [11,12,13]. The green plant extracts method is considered efficient and effective for the preparation of metal oxide nanostructures based on the active compounds in plants [14]. The primary mechanism in this process is plant-assisted reduction due to phytochemicals such as carboxylic acids, amides, quinones, terpenoids, ketones flavones, organic acids, and aldehydes. The phytochemicals are responsible for the immediate reduction of ions. It was suggested that the phytochemicals are affected directly in the reduction of ions and the creation of NPs. The active compound present in the plant extract reduces the monovalent metal ion to uncharged atoms that aggregate to reach nano-size [15,16]. The Punicaceae plant extract contains many active compounds maybe acts as a reducing agent to formation NPs, such as ascorbic acid (C 6

Preparation of Punicaceae plant extract
30 g of Punicaceae plants (pulp) was rinsed with DW to remove any impurities and dust. Then, the pulp was mixed with 250 mL of DW and boiled at a suitable temperature. After a while, the solution was filtered with filter paper (Whatman No.4 and pore size 20 µm) and centrifuge at (3000) rpm. Finally, the plant extracts are using for the fabrication of -Fe 2 O 3 -Ag 2 O nanocomposite.

Fabrication of -Fe 2 O 3 -Ag 2 O nanocomposite
In a typical fabrication, First; 1 g of (Fe (NO 3 ) 3 .9H 2 O) and 1 g of AgNO 3 were dissolved in 50 ml of DW under magnetic stirring for 30 min to obtain solation with a molar ratio of 13:9 (silver nitrate to iron nitrate). After the homogeneous of solution 6 ml of Punicaceae plant Extracts was added. Then 5 ml of 50 mM of NaOH (pH= 11) was added above the solution to obtain a reddish-black precipitate. After this, the precipitate was collected using centrifugation, regularly rinsed with DW and ethanol. Finally dried in an oven at 80 C.
Second; 1 g of reddish-black precipitate and 0.5 g (8.3 mM) of urea were suspended in 100 ml of DW under magnetic stirring for 30 min. After homogenate, the solution was put inside an autoclave and sealed tightly then placed in the oven at 120 ∘ C for 2 h. The precipitate was collected using centrifugation and rinsed with both DW and ethanol to get clean precipitate.
Third, the double hydrothermal method was used to decorate the precipitate via dissolved 70 mg (5 mM) of AgNO 3 and 0.7 g of reddish-black precipitate in 80 ml of DW under stirring for 30 min. Subsequently, 5 ml of NaOH was added to the as-prepared precursor solution. Then the solution was dissolved inside an autoclave and sealed tightly then placed in the oven at 120 ∘ C for 2 h. The resulting black precipitate was collected by centrifugation, repeat-edly washed with DW ten times and ethanol four times and dried at 75 ∘ C for 3 h.

Characterization
The crystal phase compositions the nanocomposite were studied using XRD (Miniflex II Rigaku, Japan) with a Cu Kα radiation (λ = 1.5408Å). The data in this work matching with 2Θ and intensity of XRD patterns of ICDD database (International Centre for Diffraction Data). The morphology of the sample was studied by FESEM-DES (Zeiss Sigma VP-Germany). The optical properties were analyzed using a xenon lamp spectrophotometer (Split-beam Optics, Dual detectors, made in Japan).

Antimicrobial activity
The antimicrobial activity of -Fe 2 O 3 -Ag 2 O nanocomposite was examined against bacterial cultures Gram-positive (Staphylococcus aureus and Staphylococcus Epidermidis), Gram-negative (Escherichia coli and Klebsiella pneumonia), and against fungi cultures gram-positive (Candida albicans). We have opted for these five different types of pathogenic microbes because of the following reasons: -Escherichia coli: naturally living in the gut and does not cause a disease, but virulent strains can cause diarrhea, neonatal meningitis, urinary tract infections, hemorrhagic colitis, neonatal meningitis, gastroenteritis, food poisoning, and sometimes fever [20]. -Klebsiella pneumoniae: causes many diseases such as very pneumonia, meningitis, diarrhea, urinary tract infections, sepsis, and soft tissue infections [21]. -Staphylococcus epidermidis: causes the growth of biofilms on "plastic devices" that are fixed inside the human body, usually covering intravenous medical catheters and prostheses [22]. -Staphylococcus aureus: causes food poisoning and skin infections including abscesses and respiratory infections. Pathogenic strains usually producing virulence agents such as potent protein toxins to increase infections [23]. -Candida albicans: caused by hospital-acquired infection. It considers the main source of fungal infection in immunocompromised patients or critically ill patients. Patients often develop thrush and oropharyngeal candidiasis, which affect drug absorption and cause malnutrition [24].
The agar well-diffusion method is using to evaluate the antimicrobial activity of sample. The nutrient agar medium was prepared by dissolving yeast extract that contributes vitamins, carbohydrates, nitrogen, and salts. Also, the peptone provides organic nitrogen. The ingredients are well mixed, then boiled, and poured into Petri-dishes until it becomes solidified. Then, the nanocomposite powder was dissolved in DMSO solvent at the concentration (1mg/mL). Each well (diameter of 8 mm) contains 50 µL of a sample and DMSO solvent as control. Finally, the sterile Petri dishes (diameter of 90 mm) were incubated at ±25 ∘ C for 24 h.

XRD investigation
The XRD pattern of the as-prepared sample is shown in  Debye-Scherrer equation using to calculate the average crystallite size of all phases, as shown in Table 1 2 . This discrepancy in crystal size may be attributed to the difference in ionic radius of iron ions (Fe +2 and Fe +3 ) and silver ion (Ag + ), where Fe ions have ionic radius (Fe +2 is 0.076 nm and Fe +3 is 0.064 nm) and Ag ion have an ionic radius (Ag + is 0.128 nm) [25,26]. The favorite direction trend for (222) and attributed to -Fe 2 O 3 with cubic system structure.

Optical measurements
The UV-Vis absorption spectrum of -Fe 2 O 3 -Ag 2 O nanocomposite is shown in Figure 4, and the results demonstrated a sharp absorbance peak at a wavelength of 310 nm. This peak corresponds to the formation of metal oxide nanostructures. In addition, a small absorption peak was observed at the wavelength of 375 nm and may be attributed to the surface plasmon of AgNPs.
The relation between photon energy and (αhν)² was used to calculate the energy gap (Eg) of -Fe 2 O 3 -Ag 2 O nanocomposite, as shown in Figure 5. The results confirm the nanocomposite has Eg of 3.05 eV. This blueshift of an absorption spectrum to an ultraviolet area (low wavelength) may be attributed to the inverse correlation between nano-  size and Eg when the material is reduced to the nanoscale [27,28]. When the particle size reduces, the molecules and atoms (electrons) in NPs will be restricted and the overlapping between the orbitals of an atom becomes limited. And this causes board Eg between the conduction band and valence band [28,29]. This increase in the energy gap after mixing metal oxides is consistent with the results obtained in the work [30].

Antibacterial activity
Antibacterial activity against microorganisms is intrinsically dependent on the type and components of the cell wall. Bacteria are usually exposed to unusual environmental conditions. Accordingly, bacteria can withstand difficult conditions and modify themselves depending on their high ability to withstand the effects of extremely challenging environments. In other words, bacteria naturally have a very  Figure 6 The results of obtained inhibition zones are shown in Table 2 and indicate the highest antibacterial performance of the as-prepared specimen. Before discussing the antibacterial activity, it is very important to regard that nanostructures are small particles and have a very high surface area within a very small size, which gives them properties that differ from their properties in the bulk case. According to the aforementioned, nanostructures work directly to inhibit the growth of bacteria by contacting their cell wall, and this, in turn, depends mainly on the surface area, the shape of the nanostructures, and their concentration.
The anti-bacterial activity of -Fe 2 O 3 -Ag 2 O nanocomposite is mainly associated with the release of silver ions (Ag + ) and Iron oxide ions (Fe +2 and Fe +3 ). Because these ions can bind to the cell wall of bacteria through electrostatic attraction and reacted with the thiol group of bacteria cell-wall and blocked the transport of nutrients through the cell wall and finally the bacteria death. Also, those ions can interact with the base of DNA and affect its replication, then causing cellular death (31). And in the case of metal oxides, the reactive oxygen species (ROS) can play an important role in inhibiting bacterial growth through oxidative stress and reduction processes that greatly affect the bacterial or fungal cell components(32).