Green-synthesis of Ag 2 O nanoparticles for antimicrobial assays**

: Silver oxide nanoparticles (Ag 2 O NPs) in the aqueous colloidal state were synthesized using the green method. Aqueous silver nitrate was prepared and mixed jointly with an aqueous extract of Lawsonia inermis (henna) leaf and heated with stirring at 75 ∘ C for 1h. Then, an aqueous colloidal solution of Ag 2 O NPs with a dark brown colour is forming. The physicochemical characterization of Ag 2 O NPs was studied using different techniques. A polycrystalline structure of (Ag 2 O/Ag) in face-centred cubic and cubic phases was revealing via grazing incident X-ray diffraction (GIXRD) patterns. Energy-dispersive X-ray analysis (EDX) spectra confirmed GIXRD results through peaks corresponding to the silver and oxygen elements making up the accurate composition of the silver oxide. UV-Vis absorbance peak of the localized surface plasmon resonance (SPR) appeared at the visible region and exhibited a blueshift at ∼ 425 nm with an energy bandgap ∼ 2.8 eV. The surface morphology and the size of the silver nanoparticles were analyzed using high resolution (FE-SEM) microscopy. FTIR spectra of Ag 2 O NPs has showed a shift in the bands compared to those produced by aqueous extract of the henna leaf (only). (0.4 molars) Ag 2 O NPs has showed excellent antimicrobial activity assays against all the pathogens microbe’s strains. Henna plant extract (only) has showed poor activity compared to Ag 2 O NPs. In comparison, the inhibition zone diameter of the gram-negative Bacteria is more considerable than the gram-positive bacteria. Moreover, Ag 2 O NPs activity against Bacteria is more prominent than fungi.


Introduction
In recent years, nanotechnology development was flourished, and green chemicals were used to synthesize various metallic nanoparticles without any external chemicals that might pollute the atmosphere [1,2,3]. As a comparison to other methods, the green approach is more advantageous due to its simplicity, cleanliness and results in the costeffective development of nanoparticles with defined properties [4,5]. Starch, proteins, phenolic acids, terpenoids, carbohydrates, alkaloids, and polyphenols, are bioactive compounds found in plant extracts. These compounds can help the form nanoparticles by reducing and capping agents [6,7]. Among the numerous metallic nanoparticles investigated, the noble metal silver occupies a prominent position in nanomaterial science due to its specific properties applied to various fields. The antimicrobial properties of Ag 2 O NPs, commonly used in antibacterial and antifungal applications, are due to electrical variations when interacting with Bacterial membrane. These modifications improve further the reactivity of Ag nanoparticle surfaces [8]. Furthermore, metal nanoparticles' in vitro bactericidal efficacy enhances by their stability in conditions of culture and their ability to remain effective for long periods without decomposition [9]. The antibacterial activity of the nanoparticles is probably due to electrostatic interaction with the cell membrane of the Bacteria and internalization of the Ag 2 O NPs in the microbial cell, which leads to the production of reactive oxygen species (ROS) and membrane damage [10]. ROS are solid oxidizing agents that oxidize lipids and proteins present in the cell and cause DNA damage. It causes oxidative stress and disrupts normal cellular functions due to the inactivity of essential proteins, and disarray in replication and protein synthesis leads to DNA damage. It also alters or inhibits the metabolism or respiratory cycles of the Bacteria. These mechanisms finally lead to cell death and, therefore, suppression of Bacterial growth **Paper included in the Special Issue entitled: Proceedings of Mustansiriyah International Conference on Applied Physics -2021 (MICAP-2021) [11,12]. Xiang et al. found that AgNPs can significantly inhibit the growth and development of fungi hyphae, destroy the cell membrane permeability of fungi hyphae, inhibit the synthesis of soluble proteins, destroy DNA structure, and inhibit DNA replication [13]. Sondi and Salopek-Sondi investigated the antimicrobial activity of silver nanoparticles against E. coli as a model for gram-negative Bacteria. Also, they report another antimicrobial activity mechanism that depends on the electrostatic attraction between negatively charged Bacterial cells and positively charged Ag NPs [9]. Panacek et al. discovered that Ag 2 O NPs have excellent antimicrobial activity against gram-negative and gram-positive Bacteria, and the mechanism depends on the size of silver particles [14]. Flores-Lopez et al. synthesized Ag 2 O/Ag nanoparticles using Aloe vera plant extract via a green synthesis method. It shows excellent antibacterial activity against gram-negative and gram-positive Bacteria E. coli and S. aureus. Moreover, high antifungal activity against some various species from Candida [15]. Recently, Ghojavand et al. successfully synthesized AgNPs from an aqueous extract of Felty germander using a green approach, and the resulting AgNPs has an excellent antifungal activity [16]. This paper aims to provide an alternative eco-friendly method to obtain silver oxide nanoparticles species and assess their bactericidal activity. Also, reducing the cost of synthesizing Ag 2 O NPs with a green eco-friendly method, using a low-cost and commercial Lawsonia inermis (henna) extract. (Ag 2 O NPs) shows excellent antimicrobial activity against some microbial pathogen strains (e.g., S-aureus, P-aeruginosa, E-coli, Penicillium spp., Aspergillus spp., and Candida albicans).

Preparation of extract
Collection fresh Lawsonia inermis (henna) leaves from nurseries of the plant in Wasit /Iraq. It is clean with tap water, gently brushed to remove soil and other dust particles, and washed with distilled water. Then, the henna leaves were sliced into small pieces and distributed evenly to facilitate homogenous drying. Henna leaves were dehydrated via the shade air-dry method during the summer in dry conditions and shaded areas to prevent microbial fermentation and subsequent degradation of metabolites of plant material for ten days. The dry leaves parts grinding into smaller particles using a mechanical grinder to shred the plant tissues to powder. The quantity of 2g of henna powder was dissolved in 100 ml distilled water at pH4.2 by heated and stirred at (50 ∘ C) for 1h. using a hot plate stirrer. The henna solution was filtered via vacuum filtration using a Buchner funnel, a side-arm flask, and filter paper. Finally, keeping the final aqueous solution at room temperature for additional usage.

Green-synthesis of Ag 2 O NPs
Silver nitrate (AgNO 3 ) provided by (Glentham life sciences LTD, U.K.) and plant extract of Lawsonia inermis (henna) were employed to synthesize silver oxide nanoparticles via the green-synthesis approach. One molar of aqueous silver nitrate solution was synthesized by dissolving AgNO 3 in 100 ml distilled water. The dissolution was performed at (75 ∘ C) under enthusiastic mixing at (700 rpm) for 1h. Then, 100 ml of the henna plant extract was slowly added to 100 ml of the silver nitrate aqueous solution, continuously stirred and heated at (75 ∘ C) for 1h. After that, a dark brown coloured aqueous colloidal mixture of the green synthesized Ag 2 O NPs is forming. To study the crystal structure, surface morphology, and identify the elemental composition of Ag 2 O NPs by GIXRD, FE-SEM / EDX techniques, the final colloidal solution of Ag 2 O NPs was deposited on a glass substrate to form a layer film via the drop-casting method. The dropcasting process was carried under a temperature below (60 ∘ C) using a micropipette and hot plate stirrer.

Preparation of bacteria and fungi for sensitivity test
The antimicrobial activity of the colloidal Ag 2 O NPs and henna plant extract was studied using an agar well diffusion method [17,18]. The Mueller Hinton agar (MHA) medium was prepared for the antimicrobial test since it is the best medium for developing the most pathogenic microbes. Three types of pathogenic Bacterial strains: gram-positive Staphylococcus aureus, gramnegative Pseudomonas aeruginosa, and Escherichia coli, were used to study the antibacterial assays. Also, three types of pathogenic fungal strains: Penicillium spp., Candida albicans, and Aspergillus spp, were used to study the antifungal assays. To stimulate the microbes, they were grown in a rich medium culture such as tryptic soy agar and incubated at (37 ∘ C) overnight for the sensitivity test. After that, The microbes at 25 ∘ C dissolved in physiological saline solution (0.85%). Then, the comparing between the turbidity of the suspension and (1/2) McFarland turbidity standard tube equal to (10 8 CFU /ml) were made; McFarland was synthesized according to MacFaddin (2x10 3 to 9.950) ml of (1%) sulphuric acid (H 2 SO 4 ) with (0.050 ml) of (1.176%) barium chloride dehydrate (BaCl 2 -2H 2 O). The norm liquidates into (5ml) screw-capped tubes of the same size used in this process, packed in the dark at room temperature, and shaken before use. After that, the microbe's suspension was swabbed on the superficies of the MHA under sterile conditions using the swabbing method. After the microbes dry up, make a well in the MHA with a diameter of 6 mm. The wells were puncturing with the backside of a sterile blue micropipette tip. 100 ml of the antimicrobial agent (i.e., Ag 2 O NPs or henna plant extract) is introduced into the well using a micropipette. All the Bacteria strains were incubating in dishes for 18h. (not more than 24h) at (37 ∘ C) overnight. While the fungi strains, Penicillium spp and Aspergillus spp at (25±2 ∘ C) for seven days, and Candida albicans at (25±2 ∘ C) for four days old culture. Finally, the positive growth inhibition zones diameter around each well was read in mm.

Characterization of green synthesized Ag 2 O NPs
Different techniques were used to study the physicochemical characterization of the green synthesized Ag 2 O NPs. The crystalline structure of Ag 2 O NPs layer film was analyzed using GIXRD model PHILIPS X-Ray Diffractometer, PW 1730, which measures intensity as a function of Bragg's angle, subject to the following conditions: Copper (Cu) is the target, with a wavelength of 1.54060 ∘ A, a current of 30 mA, and a voltage of 40 kV. Scanning angle 2Θ (change) in the range of (10-80) degrees at a speed of (5 degrees per minute). The surface morphology, particle size, and identify the elemental composition of the Ag 2 O NPs layer film was analyzed using field emission-scanning electron microscopy (FE-SEM / EDX) model TESCAN Mira3. While, the FTIR spectra of the Ag 2 O NPs colloidal solution in the wavenumber range of 400-4000 cm -1 were confirmed by Fouriertransform infrared spectroscopy (FTIR) model (IRAffinity-1, SHIMADZU). Moreover, evaluate the optical properties by UV-Vis spectrophotometer 1900i, type (SHIMADZU).

GIXRD analysis
Silver oxide nanoparticles (Ag 2 O NPs) layer film crystal structure was analyzed using the GIXRD technique. This technique used to obtain small incident Bragg diffraction angles at surface layers and near regions is the explicit form of X-ray diffraction of grazing incidence. So that, it is used to study the surface of the Ag 2 O NPs layer film, not the glass, because wave penetration is limited. Figure 1 shows GIXRD patterns sharp peaks of the (Ag 2 O NPs) layer film in a polycrystalline structure.  [19]. Besides, GIXRD patterns exhibited diffraction peaks at 2Θ equal 44.27 ∘ , 64.45 ∘ , and 77.42 ∘ can be wellmatched with the cubic phase structure and correspond to (200), (220), and (311) crystal planes, respectively of cubic Ag NPs which is well-matched with the JCPDS card number (00-004-0783) [20]. Table 1 illustrates the structural properties of the green synthesized Ag 2 O NPs. The average crystallite size of the maximum three peaks was determined utilizing the Debye-Scherer Eq. (1) and found to be ∼37.4 nm.
Here, D is the crystallite size, λ =1.5406 ∘ A is the wavelength of X-ray Cu-Kα radiation, β is the full width at half maximum (FWHM), Θ is the diffraction angle (Bragg's angle), and K is the crystallite form constant (0.94 for spherical shapes) [21,22]. The distance between the crystalline levels (d) was measure using Eq. (2). nλ = 2d sin θ Here, d is the distance between atomic layers in a crystal, λ is the wavelength of the incident X-ray beam, 2Θ is the diffraction angle (Bragg's angle), and n is the order of the diffraction peak [23,24,25].

FE-SEM / EDX analysis
Surface morphology of the green-synthesized (Ag 2 O NPs) layer film validated using a high-resolution microscope (FE-SEM). Figure 2 shows the FE-SEM images of the Ag 2 O NPs with different magnifications. The Ag and Ag 2 O NPs were shaped in oval and spherical with aggregation and lacked monodispersity. Figure 2d gives the average particle size distribution of Ag/Ag 2 O NPs around ∼39.1 nm. It was reported a similar result in a previous study [19]. Since each element in Ag 2 O NPs has a distinct atomic structure and it emits a spectrum with a specific collection of peaks. Therefore, the X-ray energy dispersive spectroscopy (EDX) technique is employed to confirm GIXRD results. Figure 3 gives peaks corresponding to silver and oxygen elements of silver oxide nanoparticles. Due to surface plasmon resonance, a strong absorption peak in the silver (Ag Lα1) was observed at 3 KeV, confirming the existence of silver nanocrystals. Also, a weak absorption peak in the oxygen (O Kα1), sodium (Na Kα1), and chlorine (CL Lα1) regions were observed at 0.5, 1.06, and 2.15 KeV, respectively. These elements are inherently present in the henna plant tissues. This result agrees with [26,27,28]. Figures 4a, b, and c show the dot mapping corresponds to silver and oxygen distribution. While. Figure 4 d shows the dot mapping corresponding to silver and oxygen distribution combine with (FE-SEM) surface morphology of Ag 2 O NPs, as shown in Figure 2c. Besides, silver and oxygen elements with a normalized concentration in weight percentage are equal to 78.48% and 21.52%, respectively. Also, the atomic weight percentage of silver and oxygen elements is 36.66% and 63.34%, respectively. A weak oxygen signal is due to X-ray emission from carbohydrates, proteins, and enzymes in the henna leaves [29]. Furthermore, the formation of silver oxide nanoparticles after synthesizing Ag 2 O NPs reacts with water in the solution is due to the nanoparticles' high surface-to-volume ratio, making them highly reactive [30].

FTIR analysis
The FTIR spectroscopy in the wavenumber range 400-4000 cm -1 was used to analyze the chemical bonds and functional groups of both henna plant extract (only) and colloidal Ag 2 O NPs, as shown in Figure 5. FTIR spectra of Ag 2 O NPs showed a shift in the bands compared to those produced by henna plant extract (only  [31,32], O-H bending vibration of an adsorbed water molecule on the surface of Ag 2 O NPs, which may be essential for antimicrobial assays [31,33], and weak stretching vibrations C=C [34], respectively. Furthermore, Ag 2 O NPs show an Ag-O bending mode of vibration at (715.31) cm -1 , confirming metal-oxygen bonding formation [9,31,35,36]. Figure 6a shows the UV-Vis absorption spectrum of colloidal Ag 2 O NPs. The presence of a narrow absorption peak agrees with the nanocrystalline nature of the Ag 2 O NPs sample. The intensity absorbance band of the localized surface plasmon resonance (SPR) appeared at the visible region and exhibited a blueshift at ∼425 nm due to Ag nanoparticles' small particle size [37]. The energy bandgap (Eg) of colloidal Ag 2 O NPs were accessed using Tauc's Eqs. (3) and (4), and the direct bandgap was estimated by extrapolating a straight line on the energy axis for Ag 2 O NPs.

UV-Vis analysis
Where Eg is optical band gap energy, hν is photon energy and equal to 1240 ev/λ, α is absorption coefficient, λ  is the wavelength (nm), k is absorbance, α 0 = band tailing parameter (constant), h, is plank's constant, ν is radiation frequency, n is the power factor for transition mode, it has values 1/2, 2, 3/2, and 3 for various types of transition, i.e., not allowed, allowed, and forbidden, respectively [32,33]. In this paper, n = 1/2 gave the best fit of our experimental data. Therefore, the obtained value of Eg by plotting (αhν) 2 vs hν as shown in Figure 6b, and 2.85 eV, was the Eg value of Ag 2 O NPs. Based on the above findings, Ag 2 O NPs colloidal solution energy bandgap is higher when compared with the reported values; it may be due to the particle size and quantum confinement effect [38].    cellent antimicrobial activity. The Ag 2 O NPs react with the microbe's cell wall and inhibit the respiratory process by interacting Ag NPs with the respiratory enzymes which are presented in the microbial cell walls. As shown in Figure 7, gram-negative Bacteria E.coli and P. Aeruginosa shows large inhibition zone diameters (38mm) and (37mm), respectively. In contrast, gram-positive S. aureus Bacteria shows a small inhibition zone diameter (32mm). The difference in the inhibition diameter is attributing to the thickness of the Bacteria cell walls. Gram-negative Bacteria have a thin layer of flexible lipopolysaccharides at the exterior. In contrast, the cell wall in gram-positive Bacteria is principally composed of a thick layer from zwitterionic and rigid peptidoglycan [39].

Antimicrobial assays
In the antifungal assay test, four different molar concentrations (0.1,0.2,0.3, and 0.4 M) of Ag 2 O NPs used against Penicillium spp. and Aspergillus spp. In addition, (0.4 M) of Ag 2 O NPs were used individually in Candida albicans. As shown in Figure 8, a large inhibition zone diameter is seen at (0.4M) Ag 2 O NPs against Candida albicans more than the other two types of fungus. Also, notice an increase in the diameter of the inhibition zone as the molar concentration of silver oxide nanoparticles increases (see Table 2). Furthermore, this result shows excellent antifungal assays of (0.4 M) Ag 2 O NPs against the three pathogens funges strains better than henna plant extract (only). Moreover, it offers an inhibition zone diameter against Bacteria larger than fungi. The X-ray and FE-SEM tests found that the silver oxide synthesized by the green approach is within the nanoscale range. Thus, the mechanism of antimicrobial activity in the current study is attributed to the high surface area to volume ratio between Ag NPs and microbes. Also, free radicals such as hydroxyl radicals as confirmed by FTIR, the release of Ag+ ions, and reactive oxygen species (ROS) production.

Conclusion
The green-synthesis approach successfully prepared silver oxide nanoparticles. GIXRD pattern of the Ag 2 O NPs layer film reveals two phases; face-centred cubic represents Ag 2 O NPs, and cubic represent Ag NPs with an average crystallite size of ∼37 nm. FE-SEM analysis illustrates that most particles were spherical with aggregation and lacked monodispersity with an average particle size of ∼39 nm. EDX analysis confirms the GIXRD result; the energy-dispersive Xray spectrum shows a strong absorption peak in the silver region (Lα1) was observed at 3 KeV, confirming the existence of silver nanocrystals. Also, a weak absorption peak in the oxygen region (Kα1) was observed at 0.5 KeV, confirming the presence of oxygen element. FTIR analysis confirmed fatty acids and carbohydrates related to Lawsonia inermis plant extract and other hydroxyl radicals in Ag 2 O NPs. UV-Vis absorbance peak of the localized surface plasmon resonance (SPR) appeared at the visible region and exhibited a blueshift at (∼425 nm) due to Ag nanoparticles' small particle size. Based on the above findings, Ag 2 O NPs colloidal solution energy bandgap is significant (∼2.8 eV) compared with the reported values due to the particle size and quantum confinement effect. Ag 2 O NPs shows an excellent antibacterial activity assay against E. coli, S. aureus, and P. aeruginosa better than antifungal activity assay against Penicillium spp., Aspergillus spp., and Candida albicans. Also, the antimicrobial activity assay of Lawsonia inermis plant extracts is less than green-synthesis Ag 2 O NPs.