Phyto-mediated synthesis of copper oxide nanoparticles using Artemisia abyssinica leaf extract and its antioxidant, antimicrobial and DNA binding activities

ABSTRACT Copper oxide nanoparticles (CONPs) are one of the most important metal oxide nanoparticles (MONPs) in the emerging field of nanomedicine due to their remarkable features. Novel CONPs were synthesized using ethanolic (50%, v/v) leaf extract of the indigenous plant Artemisia abyssinica for the first time. The precursor salt concentration, extract volume, pH of the solution, and synthesis temperature were optimized with the UV-Vis spectrophotometer. Using UV-visible spectroscopy, the SPR peaks were observed at 408 nm, and the band gap was found to be 3.039 eV, revealing its semiconductor nature. The presence of phytochemicals on the surface of CONPs was confirmed by FTIR, TGA/DTA, and EDX analysis. The spherical nature and average crystal sizes of the particle 18.4 and 24.6 nm were determined using TEM and XRD analysis. CONPs showed promising antimicrobial activity against selected drug-resistant pathogenic bacterial and fungal strains. The highest inhibition zone was exhibited on Staphylococcus aureus (32.5 ± 0.02 mm with MIC value 10 μg/mL) among bacterial strains and Aspergillus flavus (22 ± 0.34 mm with MIC value 25 μg/mL) in fungal strains. CONPs revealed the strong antioxidant potential (88.81 ± 0.02%, at 200 μg/mL) with an IC50 value of 5.75 μg/mL. Furthermore, CONPs remarkably exhibited DNA binding activity with CT-DNA. GRAPHICAL ABSTRACT


Introduction
Nanotechnology has recently sparked tremendous research, with applications in science, engineering, agriculture, biotechnology, food science, environment, energy, electronics, space industry, medicine, and biology (1)(2)(3)(4). Because of their diverse, physicochemical and structural properties include as small size, large surface area, optical and electrical activity, and mechanical and magnetic features (5,6). Metal oxide nanoparticles (MONPs) is a promising tool for biomedical applications because of their high stability, simple preparation processes, engineering to the desired size, shape, and porosity, easy incorporation into hydrophobic and hydrophilic systems, and easy crosslinking by various molecules due to the negative charge of the surface (7)(8)(9) Consequently, MONPs have been considered as a vital and widespread area of research in biomedicine, including antiprotozoal (10), antimalarial (11), antiinflammatory (12), antimicrobial, and antioxidant (13)(14)(15), antiviral (16), antidiabetic, and anticancer therapeutics (17,18).
Metal oxide nanoparticles have been synthesized using a variety of physicochemical techniques (19). However, these methods have some drawbacks, including high cost, high energy consumption and releasing of toxic chemicals into the ecosystem (20). Synthesis of metal oxide nanoparticles from biological entities has great advantages over chemical and physical techniques. Biological (green) synthesis techniques prevent the formation of unwanted or hazardous byproducts using efficient, accessible, and ecofriendly synthesis techniques as well as enhance the biocompatibility of nanoparticles and hence can be utilized in many medical applications (21,22).
Green synthesis method encompasses bacteria, fungi, and plant phytochemicals as precursor materials. Bacteria (23), fungi (24), arthropods (25) and animal entities such as enzymes (26), were used for the synthesis of ecofriendly and biocompatible metal oxide nanoparticles (MONPs). However, compared to bacteria, fungi, and other animal entities-mediated synthesis, phytomediated nanoparticles synthesis is more simple, safe, and efficient way to harvest nanoparticles on a large scale (27,28). The availability of useful phyto-constituents in a variety of plant extracts, such as alkaloids, flavonoids, saponins, terpenoids, phenols, carbohydrates, proteins, carboxylic acids, and ascorbic acids, has led to a broad consideration of plant components for the synthesis of metal oxide nanoparticles (29). These components are capable of reducing metal salts into nanoparticles as well as used as stabilizing and capping agents of MONPs (27,30). Recent studies have also shown that the synthesis of MONPs using plant extracts, i.e. phytomediated synthesis, has some benefits, such as low cost, biocompatible and enhanced bioavailability (4,31).
Several studies have been demonstrated on the applications of phyto-mediated synthesized MONPs in biomedicine (32)(33)(34). However several metal oxide nanoparticles have a variety of biological and biochemical activities, copper oxide nanoparticles have gained great interest recently. This is because of their structural properties, less toxicity, and high biocompatibility (35).
Copper is needed by our body for a variety of metabolic and physicochemical functioning including serving as a cofactor for numerous enzymes. Therefore, CONPs are highly reactive and easily combine with several molecules, resulting in a wide range of biological functions (36). Consequently, CONPs have a great impact in biomedical applications recently, such as antimicrobial (37,38), antioxidant (39), antifungal (40), anticancer (41), anti-inflammatory and antdiabetic (42), antimalarial (43), and antiviral activities (44).
The Ethiopian indigenous Artemisia abyssinica Sch. Bip. ex A. Rich (Family-Asteraceae; Chikugn-Amharic) is a short-lived annual, aromatic, gray, silky hairy plant frequently utilized in traditional medicine and rituals (45). It is a well-known stimulant and reliever, with ternate, grey-green leaves that grow up to 10 cm long. The stems are superiorly grooved and sparsely branching. Infectious diseases (bacterial, viral, protozoan), bronchitis and other inflammatory disorders, cold and fever, anorexia, colic, headache, amenorrhea, and dysmenorrhea are treated by Artemisia abyssinica traditionally (46). In addition, the plant has also been used as an antimalarial, antispasmodic, antirheumatic, antioxidant, and antitumor agent (47)(48)(49). The major phytochemicals screened in Artemisia abyssinica are alkaloids, flavonoids, saponins, phenolic compounds, tannins, and essential oils (50).
The main objective of this study was to carry out green synthesis of novel CONPs using leaf extract of indigenous medicinal plant and to evaluate its biological activities. Instead of toxic reducing and stabilizing agents, using versatile phytochemicals that make synthesis of ecofriendly and biocompatible nanotherapeutics. Moreover, integration of bioactive molecules of medicinal plant with the CONPs is supposed to be tremendously important for enhancement of bioavailability and bioactivity due to the synergistic effect in varieties of ailments. Therefore, this study attempts to synthesize CONPs by greener route using the leaf extract of Artemisia abyssinica through optimizations of different reaction parameters. The biosynthesized CONPs were characterized by using UV-visible, FTIR, TGA, SEM-EDX, and TEM/ HRTEM-SAED techniques. The comprehensive reports of antimicrobial activity of biosynthesized CONPs on drugresistant pathogenic bacterial and fungal strains, its antioxidant and DNA binding activities was also provided.

Collection and preparation of plant extract
The fresh leaves of Artemisia abyssinica were collected from Jimma University garden, Oromia region, Ethiopia, and authenticated at the Addis Ababa University Herbarium (Voucher No. AUGH004). The leaves were frequently cleansed and rinsed with tap water, followed by distilled water to eliminate dust particles, and then air-dried for 15 days in the shade to eliminate moisture contents. Dry samples were mashed in a mechanical blender before being packed into brown bottles. The extractions were prepared using 10 g of powdered plant material in 250 mL conical flasks containing 100 mL deionized water, 50% ethanol (water & ethanol, 1:1 v/v), and nhexane. The flasks were subsequently covered with aluminum foil to avoid the effect of light. The mixtures were then shaken for 90 min at 120 rpm and 50°C in a mechanical shaker, allowed to warm for 30 min on a magnetic stirrer at 60°C and then allowed to cool to room temperature overnight. To obtain a clear solution, the prepared solutions were filtered through Whatman No.1 filter paper, centrifuged, and then stored at −4°C for the next works.

Phytochemical analysis of the extracts
The aqueous, 50% Ethanol, and n-hexane leaf extracts of Artemisia abyssinica were subjected to phytochemical screening, and major secondary metabolites such as alkaloids, polyphenols, flavonoids, terpenoids, saponins, protein, and amino acids, anthraquinones, glycosides, and tannins were analyzed by following the reported procedures (46,51).
Test for alkaloids: To 1 mL of extract in test tube 3 mL distilled water and 1 mL 35% HCl were added. The mixture was warmed for 15 min in a water bath, then cooled and filtered. After that, 1 mL of the filtrate was tested with 0.5 mL each of Mayer's and Dragendorff's reagents. a) Mayer's Test: A few drops of Mayer's reagent (solution of Potassium Mercuric Iodide) were added to 2 mL of extracts. The presence of alkaloids is indicated by the appearance of a light yellow color precipitate. b) Dragendroff's Test: Two drops of Dragendroff's reagent (solution of Potassium Bismuth Iodide) were added to 2 mL of extract. The presence of alkaloids is indicated by the production of a crimson precipitate.
Test for flavonoids: Flavonoids were determined by two alternative tests: a) Alkaline reagent test: A few drops of sodium hydroxide solution were added to 2 mL extracts. The presence of flavonoids is revealed by producing a bright yellow color that fades to colorless when dilute acid is added. b) Lead acetate test: A few drops of lead acetate solution were added to the 2 mL extracts. The presence of flavonoids is shown by the production of a yellow color precipitate.

Test for proteins and free amino acids
Biuret test: Equal amounts of 5% sodium hydroxide and 1% copper sulfate solutions were added to the 2 mL extracts. The presence of proteins and free amino acids is observed by the formations of pink or purple color. Test for phenols Ferric Chloride Test: To 3 mL of warmed extract in a water bath 2 mL ferric chloride solution was added. The presence of phenols was determined by the appearance of blue color.
Anthraquinones test: A few drops of diluted sulfuric acid extracted with benzene were added to 5 mL of extract to hydrolyze the solution. Finally, it was treated with 1 mL of dilute ammonia. The mild rose pink coloring suggested the minor reactions for anthraquinones.
Tannins test: To 5 mL of pure extract in a test tube, a few drops of 0.1% ferric chloride (FeCl 3 ) were added and allowed to stand sometimes. The brownish-green color observed indicates the existence of tannins.
Test for Saponins Foam test: A drop of sodium bicarbonates solution was added to a test tube holding around 5 mL of extracts. The test tube was firmly shaken for 3 min. The presence of saponins is indicated by the appearance of honeycomb-like foam.
Test for Terpenoids Salkowski test: To 5 mL of the extracts in a test tube 2 mL chloroform and 3 mL of saturated H 2 SO 4 was added. A reddish-brown coloring confirmed the presence of terpenoids near the interface.
Test for Glycosides Keller-killing test: To 0.5 mL extract in a test tube 1 mL glacial acetic acid containing traces of ferric chloride and 1 mL concentrated sulfuric acid were mixed. The creation of a reddish-brown color observes the presence of glycosides at the junction of two layers and the upper layer turning bluish-green.  (10,20,30,40, and 50 mL) at different pH (3, 5, 7.5, 9, and 11) and temperature (25, 40, 50, 60, 70 and 80°C) were used to synthesize CONPs. Visual observation of blue to brown color was used to assess the formation of CONPs in the solution. The reduction of copper ions and formation of CONPs was assessed periodically by using UV-visible spectrophotometer. The precipitated particles were isolated by centrifuging at 6000 rpm for 20 min and washed with deionized water. The precipitated pellets were dried in a hot-air oven at 80°C for six to eight hours, and stored in proper containers. The purified CONPs were then subjected to characterization.

Characterizations
The Surface Plasmon Resonance (SPR) peak in the wavelength range of 200-800 nm was determined using an ultraviolet-visible (UV-Vis) JENWAY 6405 Spectrophotometer. The possible biomolecules responsible for the reduction of copper ions into CONPs were tested using Fourier transform infrared spectroscopy (FTIR Shimadzu, Japan 8400S) with the potassium bromide (KBr) disk method over the wavenumber regions between 400 and 4000 cm −1 . Moreover, the existing biomolecules and thermal stability of biosynthesized CONPs were analyzed by TGA/DTA (DTG 60H Shimadzu, Japan), heated 0-800°C. The size and crystalline nature of biosynthesized CONPs were analyzed by A BRUKER D8 Advance XRD, AXS GMBH, and Karisruhe, West Germany, equipped with a Cu target for generating a Cu Kα radiation (wavelength 1.5406 Å) at GSE. The measurements were conducted at room temperature with an accelerating voltage of 40 kV and an applied current of 30 mA, respectively. The instrument was used in a step scan mode with a step time of 1s and a degree (2θ) of 0.020 0 , respectively, spanning a temperature range of 10-80 o . External morphology and surface features of CONPs were analyzed using a scanning electron microscope (Tescan Mira 3 LMU), and their elemental composition was assessed using an energy-dispersive X-ray (EDX) spectroscope with a resolution of 1 nm and a voltage of 15 kV. Morphology, particle size, and crystalline nature of CONPs were also characterized using TEM instrument JEOL, JEM-2100 (accelerating voltage up to 200 kV, LaB6 filament), EDS-1.5 Å TEM resolution.

Antioxidant activity test
Antioxidant activities of the precursor salt, extract, and bio-synthesized CONPs were assessed by DPPH (1, 1diphenyl-2-picrylhydrazyl) radical scavenging assay (RSA) (52). The solution composed of DPPH radical was stored uninterrupted for 3 h in order to confirm its stability. Constant λ max of the solution at 517 nm was observed, which confirms the solutions stability throughout the experiment. Then, 1 mL methanolic DPPH solution (0.1 mM) was mixed with 1 mL methanolic CONPs solutions (200, 150, 100, 50, and 25 g/mL). The reaction solution was mixed and incubated for 30 min at 27 ± 2°C in the dark. The absorbance of the solutions was read at 517 nm using a UV-vis spectrophotometer. Ascorbic acid and methanol were used as a positive and blank, respectively. Free radical scavenging activity was calculated using the following formula: where AB absorbance of the blank, and AS absorbance of the sample.

Antifungal activity
The antifungal activity of extract and CONPs against Aspergillus flavus, Aspergillus niger, and Candida albicans was analyzed by the agar well diffusion method (54).
Sabouraud dextrose agar (SDA) culture media plates were made, inoculated with the fungal strains, and incubated at 25°C for 10 days to see if fungus growth occurred. After 10 days, a core-borer was used to punch wells in the agar plate, and different concentrations (25, 50, 100, and 200 g/mL) of samples dissolved in DMSO were injected into each well. Fluconazole and DMSO were used as positive and negative controls, respectively. The plates were left at 25°C for 1 h to allow the test sample to diffuse before being incubated at 25°C for three days. The zone of inhibition against the examined fungus was evaluated after incubation.

Statistical data analysis
Data obtained from the analysis of the Artemisia abyssinica leaf extract and copper oxide nanoparticles (CONPs) samples were analyzed using one-way analysis of variance (ANOVA) of statistical package of social science (SPSS) version 20 and denoted as mean ± SD for triplicate experiments. Also, data analysis was carried out using Origin software (Originpro 9.0 64bit), Gatan Microscopy Suite ® software (GMS 64bit) version 2.x and ImageJ (imajej153-win java8\imagej\imagej.exe).

Biosynthesis synthesis of CONPs
Biosynthesis of CONPs was performed by using ethanollic (50%, v/v) leaf extract of Artemisia abyssinica. Reaction parameters (precursor salt concentration, extract volume, pH, and temperature) were optimized as described in Figures 1-4. The change in color from blue (aqueous salt solution) and dark brown (pure extract color) to reddish-brown indicates the formation of CONPs. Aside from the color transition, developing a distinctive absorption band associated with CONPs at 406-410 nm also validates the nanoparticles' formation via the green route (as indicated in Figures 1-4 (37, 56,   57). The characteristic band in the prescribed reported range for CONPs was noticed in all of the synthetic reactions carried out to optimize the synthesis approach of the CONPs (marked in Figures 1-4). This was linked to the fact that the existing biomolecules from the extract could not eliminate a large number of precursor ions present in the reaction media. In order to achieve surface stability, the reduced nanostructures were agglomerated on the unreacted salt molecules present in the reaction media. As a result, very big clusters evolved in the reaction media, leaving this reaction situation inappropriate for the biosynthesis of CONPs. The UV-Vis spectrum of the CONPs was further utilized to calculate the Eg values (eV) of the synthesized nanostructures by utilizing the following empirical formula: (αhv) 2 = (hv−Eg); where hv represents the optical energy, while α represents the absorptivity coefficient of the material (59). The Tauc plots (in Figure 1    extract in the reaction media increased, the characteristic peak shifted to longer wavelengths. The surface plasmonic resonance peak of CONPs was not shown at higher (30:40 and 40:40) and lower (5:40) volumes of extract. According to the findings, a moderate amount of the extract is required to synthesize CONPs successfully. Because a sufficient amount of phytochemicals was available in the reaction medium to enable the reduction and stability of CONPs at moderate volume of extracts. At a lower volume of extract (5:40), the amount of phytochemicals that exist could not stabilized the particles. However, by raising the phytochemical concentrations; to higher volume (30:40 and 40:40) of extract the small-sized nanostructures owing to their higher instability aggregated to give large-sized CONPs.

Optimization of the reaction parameters
As provided in Tauc plots (in Figure 2(b,c)), the band gap energy (Eg) values for 10:40 and 20:40 were 3.032 and 3.024 eV, respectively. Hence at 10:40 larger band gap indicates the formation of more small nanoparticles compared to 20:40, which has a smaller band gap. However, by 10:40, small nanoparticles with a high yield have been expected. Consequently, using a 10:40 extract to salt volume ratio is optimum for the biosynthesis of CONPs using leaf extract of Artemisia abyssinica (60).

Optimization of pH
The reaction process was also optimized to determine what pH value was required for CONPs synthesis. The UV-Vis spectral investigation results at various pH values (3, 5, 7.5, 9, and 11) in Figure 3 revealed that at lower pH values (3 and 5) greater absorbance with sharp peaks was observed, indicating the formation of CONPs. Abandoned surface plasmonic resonance peaks were recorded at high pH (7.5, 9, and 11), indicating the inhibition of the synthesis of CONPs. This is because changes in pH affect the type of charge in the extract's secondary metabolites (56). This change in charge nature impacts phytoconstituents binding and reducing capacities.
Because of the constituent metabolites in the extract of Artemisia abyssinica, the ability to reduce the precursor salt concentration was observed at low pH levels. However, due to neutralization and hydrolytic reactions, the acidic nature of metabolites such as polyphenols, flavonoids, and tannins was suppressed in the basic medium. As a result, the increased hydroxyl ion concentration in the medium favored agglomeration rather than reduction processes, prohibiting metabolites from producing CONPs at higher pH levels (61). As revealed by SPR absorption peaks and Tauc plots Figure 3(b-d) among lower pH, at pH 5 observed more intense peak with moderate band gap than the others, which means the small size of CONPs with high yield have been produced (62).

Optimization of temperature
Temperature is also an important factor that can determine the medium and yield of the reaction. Consequently, the effect of temperature on the biosynthesis of CONPs is also optimized by varying its value from 25 to 70°C with keeping all other reaction variables constant. Moreover, the spectral analysis of the synthesis reaction at various temperatures is described in Figure  4. Temperature variation altered the reaction properties of the medium in the same way as other parameters affected (63). Therefore, the heat stability of the phytochemicals was critical for the biosynthesis of CONPs. If the appropriate temperature were not used during the synthesis, the phytochemicals would decompose, and the process of nanostructure reduction and stabilization would suppress (64).
Temperatures of 50, 60, and 70°C were effective in the biosynthesis of CuONPs, as evidenced by the formation of the typical absorption peaks at 409, 408, and 407 nm, respectively. However, at a temperature of 70°C maximum absorbance was observed as more yields with small particles were expected. Nevertheless, moderate and low absorbance was observed at lower (25°C) and higher (80°C) temperatures; hence they were not optimum for the effective synthesis of CONPs. At higher temperature conditions (80°C), the phytochemicals become destabilizing, and the collision frequency of the nanocrystals will be increased, which leads to the aggregation of the nanostructures and hence lower absorbance has been detected. The Eg values (Figure 4(b-g)) also demonstrated the optimum temperature range for the biosynthesis of CONPs. The temperature of 70°C was considered optimum due to having higher band gap energy which produces a small size with more yield (64).

FTIR analysis of synthesized CONPs
FT-IR spectroscopy is an effective technique for the detection of responsible functional groups of biomolecules that act as capping and stabilizing agents in the biosynthesis of CONPs. Absorption bands at 3436, 2072, 1638, and 588 cm −1 have been observed in the FT-IR spectrum of Artemisia abyssinica leaf extract ( Figure 5(a)). The stretching and bending vibrational frequencies of phenolic −OH are assumed to be involved in the broad band at 3436 cm −1 (65). The small peak observed at 2072 cm −1 also attributed due to the stretching and bending C-H of methylene groups. The intense peak at 1638 cm −1 could be responsible for the stretching vibration of C = O of phenolic compounds. Broad band at 588 cm −1 could be attributed due to stretching of C-O or C-O-C (66).
Biosynthesized CONPs were shown in absorption bands at 3334, 3224, 2907, 2072, 1110, and 588 cm −1 , as indicated in Figure 5(b). The appearance of prominent IR peaks of Artemisia abyssinica at 3443-3224, 2072 and 588 cm −1 indicates the contribution of phytochemicals to the biosynthesis of CONPs. The generation of CONPs was also verified by a minor shift in the FT-IR bands of Artemisia abyssinica extract from 1638 to 1385 cm −1 , as well as the presence of new peaks at 2907 and 1110 cm −1 . The peak at 2907 cm −1 was due to the stretching vibration of atmospheric CO 2 . The peak at 1385 cm −1 corresponds to the stretching vibrations of the Cu-O bond (37). The FT-IR spectra demonstrated that nanoparticles are coated with biomolecules, particularly with the OH residues of alcohols and polyphenols. Polyphynols' OH residues have a great ability to bond with metal by coating their surface and inhibiting aggregation, which are essential for stabilization (66).

TGA analysis of CONPs
Thermogravimetric analysis was also performed to confirm the presence of biomolecules on the surface of CONPs, which are used as capping and reducing agents, as illustrated in Figure 6. The DTA thermogram revealed two distinct peaks in the temperature range of 41°C to 800°C. The first endothermic peak at 41°C to 202°C, which corresponds to the first 5.46 % weight loss in the TGA curve. The exothermic peak at 202°C to 345°C observed is linked with the second 16.18 % weight loss in the TGA curve. The first weight loss (5.32 %) observed is associated with the desorption of physically as well as chemically adsorbed water molecules on the surface of CONPs. The second weight reductions on the thermogram were found to be 16.18 % which is due to the decompostion of biomolecules involved in stability and reduction of CONPs. As a result, biomolecules such as alkaloids, flavonoids, and polyphenols are expected to be present in the Artemisia abyssinica leaf extract, which is responsible for the reduction and stabilization of CONPs, as previously mention in (Table 1 and Figure 5). The overall weight loss detected in the thermogram was 21.64 percent, indicating that the manufactured CONPs contain 78.36 percent copper oxide at a given temperature range.  (fcc) crystals of copper oxide nanoparticles (CuONPs) was observed, as determined by the JCPDS (card No. 00-048-1548, Tenorite-C2/c) (61). The average crystallite size was calculated to be 24.6 nm by using the Debye-Scherrer equation:

XRD analysis of synthesized CONPs
where 'λ' is the wavelength of X-ray (0.1541 nm), 'β' is FWHM (full width at half maximum), 'θ' is the diffraction angle, and 'D' is crystallite size.

SEM-EDX analysis of synthesized CONPs
Scanning electron microscopy with energy-dispersive Xray spectroscopy (SEM-EDX) techniques was used to characterize the morphology, structures and composition of the synthesized nanoparticles. As shown in Figure 8(a,b), SEM images of CONPs at low and high resolution was found to be nearly spherical. Moreover, the particles are dispersed across the surface without agglomeration, which might be associated with the presence of bioactive molecules from plant extract. The formation of crystalline CONPs was confirmed by EDX analysis (Figure 8(c)) which revealed an intense optical absorption peaks around 0.5 and 1 and 8 keV energy, confirming the desertion of copper nitrate and the generation of crystalline copper oxide nanoparticles. The minor peaks with neglected weight percentages observed are carbon and nitrogen, which could come from biomolecules bonded to the CONPs' surface.     Figure 9 shows Transmission Electron Microscope (TEM) images of biosynthesized CONPs with nearly spherical shapes and sizes ranging from 11.6 to 26.7 nm, with an average particle size of 18.4 nm as analyzed by ImageJ software. The five spots on the SAED pattern ( Figure 9 (d)) were found to correspond to specific CONPs crystal planes observed in XRD measurements in Figure 6. Colored concentric circles were used to indicate the most prominent five planes, which correspond to the (002), (111), (200), (202) and (311) planes, as well as the XRD result. Figure 9(b,c) also demonstrates the monocrystalline character of CONPs with an interplaner spacing (IPS) of 0.271 nm in the CuO (002) plane.

Antioxidant assay
In-vitro antioxidant potential of Artemisia abyssinica extract, precursor salt (CuNO 3 .3H 2 O) and biosynthesized copper oxide nanoparticles were evaluated against DPPH free radical. The DPPH free radical scavenging potential of each sample was determined compared to ascorbic acid (AA). The acquired results in Table 2 and Figure 10

Antimicrobial activity
This study analyzed in vitro antimicrobial activity of Artemisia abyssinica extract and green synthesized CONPs against selected bacterial and fungal strains using the Mueller-Hinton agar disc-diffusion method (53). The antibacterial activity of extract and CONPs were evaluated against drug-resistant bacterial strains such as Staphylococcus aureus (+), Streptococcus pneumoniae (+), Pseudomonas aeruginosa (-) and Escherichia coli (-) by measuring the zones of inhibition and chloramphenicol used as a positive control. In addition, the antifungal activity of extract and CONPs were also evaluated using drug-resistant human and plant pathogenic fungal strains (Aspergillus flavus, Aspergillus niger and Candida albicans).

Antibacterial activity
As shown in Table 3 and Figure 11, both extract and green synthesized copper nanoparticles have shown viable in vitro antibacterial activity in all selected drugresistant bacterial strains. In most cases, based on their concentrations, the efficacy of extract and CONPs was shown to be substantial when compared to Chloramphenicol (standard drug) for all bacterial strains. In the case of S. aureus, the highest zone of inhibition was identified. Following that, antibacterial activity was shown against S. pneumoniae, P.aeruginosa, and E. coli, as provided in Table 3.   Artemisia abyssinica extract has revealed antibacterial activity (>10 mm to <21 mm zone of inhibition) against all bacterial strains with the lowest and highest concentrations used. It showed the highest zone of inhibition (21 ± 0.00) against S. aureus and lowest zone of inhibition (18.5 ± 0.22) against E.coli bacterial strain with MIC (25 and 50 μg/mL), respectively.
As most studies reported properties of nanoparticles such as size, shape, surface area, and charge distribution can influence the pass of biological barriers and interactions of nanoparticles with cellular environment (67)(68)(69). The biosynthesized CONPs was revealed auspicious antibacterial activities in selected standard bacterial cell lines. This may be due to distinctive physicochemical properties of the synthesized nanoparticles including small size, large surface area, and high charge distributions. In addition, controlling parameters, including precursor salt concentrations, plant extract, pH, temperature, and reaction time, could contribute for these aspects. The precise mechanism underlying CONPs antibacterial activity is unspecified; however, adhesion of these particles to bacteria's surfaces, penetration inside the cell and destruction of bacterial biomolecules and intracellular structures, production of reactive oxygen species (ROS) and free radicals that cause cellular toxicity and oxidative stress, and modulation of the bacterial signal transduction pathway are among the most proposed mechanisms for their action in general (70).

Antifungal activity
As provided in Figure 12 in vitro antifungal activity of Artemisia abyssinica extract and biosynthesized copper oxide nanoparticles against conventional human and plant pathogenic fungal strains. In comparison with Fluconazole (positive control), CONPs showed promising antifungal activity while the extract showed the lowest inhibition zones against all fungal strains. Plant extract Table 3. Antibacterial activity inhibition zone (mean ± SD, in mm) of extract, CONPs and Chloramphenicol against standard E. coli, S. pneumoniae, P. aeruginosa and S. aureus bacterial strains.  Figure 11. Antibacterial activity extract and CONPs against selected bacteria strains.
in Table 4  However, the biosynthesized COPNs showed inspiring ability in both fungal and bacterial strains, it is demonstrating less activity in fungi when compared to bacteria. This could be linked to fungi having chitin made up of polysaccharides with N-acetylglucosamine and a nitrogen group, and a firmer cell wall. As a result, samples cannot easily pass from the outside layer of the cell wall to the interior layer. Nevertheless, bacteria's cell wall is composed of peptidoglycan (a polymer containing sugars and amino acids), which is less rigid and allows for easier passage of samples.

DNA binding activity
One of the most fundamental approaches for testing a compound's DNA binding ability is UV-visible spectral titration. CONPs' DNA binding capacity was examined using CT-DNA, one of the most important features in the most therapeutic molecule. The stability of CT-DNA was tested at room temperature for 1 hr at 10-min intervals, and the absorption peak stayed consistent (1.78 at 280 nm). The binding efficiency was observed by spectral changes of the CONPs during titration with an increasing amount of CT-DNA. The SPR maxima of CONPs revealed the blue shift (408-435 nm) by increasing the concentrations of CT-DNA from 15 to 240 μL. As shown in Figure 13, the absorption wavelengths of CONPs were 408, 411, 416, 423, 429, and 441 for concentrations of CT-DNA 0, 15, 30, 60, 120, and 240 μL. The blue shift in absorption spectra and lowering the absorbance of the CONPs indicate the strong stacking interactions of nanoparticles with CT-DNA (71).

Conclusion
In this study, constituent phytochemicals from Artemisia abyssinica leaf extract were employed as natural reducing and capping agents for the biosynthesis of novel CONPs in a simple and eco-friendly way. The generation of viable CONPs in the reaction mixture was confirmed by the revealed Surface Plasmon Resonance (SPR) peak at 408 nm UV-visible spectroscopy. The synthesized nanoparticles have band gap energy of 3.039 eV with  proper size and optical property for biological activity. The constituent functional groups from the extract phytochemicals were responsible for reducing and capping biosynthesized CONPs, as confirmed by the FTIR spectrum and TGA/DTA. According to XRD, SEM-EDX and TEM/HRTEM-SAED analysis, spherical CONPs with average particle and crystal sizes of 18 and 24 nm, respectively, and face-centered cubic geometry were investigated. The biological activity of synthesized green copper nanoparticles had tested and shown inspiring ability in vitro by scavenging DPPH radicals, inhibiting the growth of different drug-resistant bacterial and fungal strains, and interacting with CT-DNA.
In conclusion, biosynthesized CONPs from Artemisia abyssinica leaf extract could be promising candidates for the new therapeutic agent for microbial infection, antioxidant therapy, and DNA targeting drugs.