Bio-capped multifunctional CuO nanoparticles via Knautia arvensis for dye removal, antibacterial and antifungal efficiency, and molecular docking

Copper oxide nanoparticles (CuONPs) synthesized using Knautia arvensis flower extract in an environmentally friendly and one-step procedure were characterized by UV–vis, FT-IR, SEM and DLS. The in vitro antibacterial and antifungal activities of CuONPs were determined using E. coli, S. aureus and A. niger. In silico antibacterial and antifungal evaluation of CuONPs were performed by molecular docking analysis using chitin deacetylase of A. niger, topoisomerase IV of E. coli and tyrosyl-tRNA synthetase of S. aureus. The best binding energy was determined using these microbial targets in molecular docking analyses and the antimicrobial mechanisms between the microorganism and the CuONP were elucidated. The degradation potential of Remazol brilliant blue R (RBBR) and Napthol blue black (NBB) dyes in the presence of CuONPs were investigated. The peak obtained at 289 nm as a result of UV–vis analysis revealed the presence of CuONPs. The spherical morphology of CuONPs and the particle size varying between 88–289 nm were visualized by SEM. DLS analysis pointed out the mean diameter of CuONPs was 189 nm along with the PDI value of 0.324. The 600 and 595 cm−1 vibrations attained in the FT-IR spectroscopy showed the presence of CuONPs. In addition, the presence of phenolic compounds found in the plant extract responsible for bio-capping of copper ions into CuONPs were enlightened by the FT-IR analysis. Dye degradation activity of CuONPs was found as 69% and 71% using NBB and RBBR at 50 °C in 90 min, respectively. Antifungal and antibacterial interactions of CuONPs with chitin deacetylase of A. niger, topoisomerase IV of E. coli and tyrosyl-tRNA synthetase of S. aureus were analyzed in order to reveal the antimicrobial mechanisms of CuONPs and it was found that CuONPs demonstrate significant interactions with those proteins with binding energies −7.25, −7.14 and −7.89 kcal mol−1, respectively.


Introduction
Metallic nanoparticles have a wide range of applications therefore; their synthesis is a rapidly advancing topic for nanotechnology and biotechnology.Metallic nanoparticles consist of various metals and their oxidized forms, and important physical properties such as shape and size can be changed by changing the ambient conditions during their synthesis.The obtained nanoparticles are highly reactive to environmental substances due to their strong surface energies [1].Perhaps because of these properties, its antibacterial, antifungal, anti-inflammatory, antioxidant and antitumor activities have been reported in many studies [2].The biosynthesis of nanoparticles is an environmentally friendly method, mostly in one-step process that is fast and low cost.This route, which makes it possible and includes green nanotechnological methods, uses microorganisms or plants as a source.More traditional methods include chemical and physical synthesis methods, which lead to the emergence of toxic chemicals during and after synthesis [3].Environmentally friendly plant-mediated synthesis, which is an alternative to traditional methods, overcomes the obstacles of chemical synthesis such as energy consumption, expensive product input and by-product output that are harmful to human health [4].Taking advantage of natural resources, especially for nanotechnological production, has gained great momentum in recent years, as it is beneficial for the environment and human health.Within this trend, many metal and metal oxide nanoparticles have been synthesized and sustainable processes have been developed.
In the current period, environmentally friendly synthesis protocols are preferred to obtain industrially needed products.The nanoparticle biosynthesis has recently alter to widespread because it is beneficial for simple scaling, non-toxicity, reproducibility, and definite morphology [5].Plants are the most used organisms in this biosynthesis.Since the metal nanoparticle synthesis potential and product of each plant are different, they have been investigated and their usage areas have been determined.Plant-derived nanoparticle synthesis provides more stable and high yield nanoparticles compared to microbial synthesis [6].Because of this feature, it is a more effective method.There are various metabolites, biochemicals and phytochemicals such as polyphenols in the plant extract obtained from some parts of the plants for example fruits and roots.These phytochemicals are used in biosynthesis as they play a dual role in nanoparticle synthesis by both reducing metal ions and stabilizing the metal core as nanoparticles [7].Because they are created in a single process, biologically or environmentally generated nanoparticles are significantly superior than those made by physical or chemical means in a number of areas, including increased stability and appropriate dimensions.The structural characteristics of synthesis products derived from biological precursors are contingent upon several reaction parameters, including temperature and pH levels.Chemicals found in plant extracts for instance flavonoids, terpenoids, aldehydes, ketones and phenolic compounds are used in the biosynthesis of metal or metal oxide nanoparticles [8].Since the plant part functions as a stabilizing agent, other stabilizators are no longer needed in the suggested biological synthesis [9].These components are capable of capping, reducing and stabilizing.Plantderived nanoparticle synthesis is simple, cost-effective and environmentally friendly as it avoids the use of toxic chemicals.Because of these features, plant-derived biosynthesis has an increasing importance as a good alternative method to traditional synthetic methods [10].
In this study, the CuONPs were biosynthesized using the flowers of Knautia arvensis.The plant, commonly known as field scabious, is a herbaceous representative of the Dipsacaceae family [11].It is widespread in grasslands across Europe, as well as in adjacent regions of Asia and Africa.This perennial oak species may be found growing beside roadsides, in open woodlands, dry slopes, meadows, and pastures.Its stem can be straight or branching [12].K. arvensis, which is present in the natural flora of Turkey, was collected from the Istanbul-Marmara region.The flora of Turkey includes 10 Knautia species, 2 of which are endemic [13].Biosynthesis, characterization, antibacterial and antifungal studies of the synthesized CuONPs were performed.In this study, the characterization, in silico and in vitro studies of CuONPs obtained from K. arvensis are reported for the first time in the literature.The dye removal efficiency of CuONPs was tested on RBBR and NBB at different temperatures.The antibacterial and antifungal activity of the synthesized CuONPs was demonstrated by in silico molecular docking analysis.

Synthesis of CuONPs
Knautia arvensis flowers collected from Selimpasha/Istanbul region were kept at room temperature to dry.The dried flowers were grinded and 5 g extract mixed with 50 ml of ultrapure water, then left for 10 min at 60 °C.The obtained solution was filtrated and cooled down and complemented with distilled water to 100 ml volume.40 ml of extract was mixed with 1 ml Cu(SO 4 ).5H 2 O (0.1 M).The mixture was left in a water bath for 2 h at 60 °C.The obtained solution was pictured in order to evaluate the color change and the aggregation.The CuONP solution was washed three times and centrifuged for 5 min.The final solution was oven dried overnight at 50 °C.
For the optimization of CuONP biosynthesis, the extract obtained from plant supply was mixed with Cu(SO 4 ).5H 2 O solution in selected ratios (1:5, 1:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, v/v) at pH: 5.6.UV-vis spectroscopy was applied and the absorbances of the CuONP samples were obtained in order to find the best ratio.Another optimization parameter is pH, and the CuONP synthesis was carried out by adjusting the pH of the plant extract according to pH 3.0, 7.0, and 9.0 values using the concentration rate 50:1 (K.arvensis extract : Cu(SO 4 ).5H 2 O solution, v/v).At the same time, the absorbance of CuONPs obtained by synthesis at pH 5.6, which is the inherent pH value of the plant extract, was measured.The optimum pH value was determined by the absorption spectrum obtained.Spectroscopic examination was performed at room temperature by a Shimadzu UV2400 the in a quvartz cuvette using route length of 10 mm.Experiments were applied as a function of reaction time at 200-800 nm.

Characterization of nanoparticles
The characteration of CuONPs were performed using UV-vis, DLS, FT-IR and SEM.The average particle size and polydispersity index (PDI) number of the produced CuONPs were certified by means of a DLS analysis performed using a Zetasier Malvern Nano ZS equipment.CuONPs were analyzed using FT-IR to provide information on their binding characteristics, with the use of a Perkin Elmer 1600.Potassium bromide (KBr) and K. arvensis extract were combined at concentration of 1:10 (v/v), and the pellet was studied from 400 to 4000 cm −1 of wavenumbers.The CuONP shape was ascertained by SEM studies using a Hitachi S5500.One drop of the CuONP solution was placed on carbon-coated grids and rinsed twice with ultrapure water in order to obtain SEM samples.

Antibacterial activity
The antibacterial activity of the K. arvensis mediated CuONPs was confirmed by the disk diffusion method.E. coli and S. aureus were incubated in nutrient broth for 24 h at 37 °C and the proliferating colonies were diluted to 10 8 CFU/mL considering the McFarland 0.5 turbidity standard.20 μl of the diluted bacterial suspensions were inoculated into solid media.20 μl CuONP solution (1 mg mL −1 ) was was added to the 5 mm diameter wells opened in the medium.Plates were incubated at 37 °C for 24 h.The inhibition zones formed at the end of incubation were measured in mm.The positive control in this experiment was streptomycin, whereas the negative control was distilled water.Further, optical examination was conducted by allowing the strains to grow in nutrient broth at 37 °C overnight.A mixture of 2 ml of broth including bacterial cells and CuONP solution (1 ml), and 1 ml of broth as negative control (not including bacterial cells) were used in the experiments.The samples were incubated for 24 h in quvartz cuvettes while OD 600 measurements were taken at 1st, 2nd, 3rd, 4th, 6th, and 24th h.Each experiment was performed in triplicate.

Antifungal activity of CuONPs
The antifungal susceptibility of the CuONPs were analyzed by agar diffusion test against Aspergillus niger stock culture.Fungal cells were grown on PDA prior to testing.20 μl of activated fungal strain, at 10 8 CFU/mL concentration considering McFarland 0.5 turbidity standard, was inoculated into PDA medium.Inhibition zones formed after incubated for 24 h at 37 °C, were measured at the end of the period.The negative and positive controls of this study were distilled water and Amphotericin B, respectively.The strains were quantified by incubating in potato dextrose broth at 37 °C overnight.2 ml of sample containing fungal cells were combined with 1 ml of CuONP solution.1 ml of broth devoid of fungi was used as the negative control.In UV cuvettes, this combination was incubated for 24 h while OD 600 measurements were taken at 1st, 2nd, 3rd, 4th, 6th, and 24th hours.Each experiment was performed in triplicate.

Dye removal performance of CuONPs
Dye elimination ability of the CuONPs was tested by mixing 2 mg ml −1 CuONPs with 10 mg/l NBB and RBBR at pH 8.0.The reaction depends on the color removal and the performance of CuONPs was monitored for 90 min to calculate the influence of time on color removal.Each experiment was conducted in triplicate.The degradation rate was quantified spectrophotometrically with Shimadzu Pharmaspec UV-1700 and calculated by decrease in absorbance at the specific λ max of dyes using the formula: Ci 100 DE = Dye removal efficiency Ci = Absorbance of the dye Cf = Absorbance of the dye upon contact with the CuONPs

In silico molecular docking of CuONPs
The antibacterial and antifungal efficiency of CuONPs were evaluated via in silico molecular docking.Crystal structures of chitin deacetylase (PDB ID 7BLY), topoisomerase IV (PDB ID 3FV5) and tyrosyl-tRNA synthetase (PDB ID 1JIJ) were downloaded for A. niger, E. coli and S. aureus, respectively.In molecular docking studies, predefined inhibitory binding sites for topoisomerase IV of E. coli and tyrosyl-tRNA synthetase of S. aureus were used as active binding sites of receptors.The binding site of chitin deacetylase was found using AutoSite (v1.1) of AutoGridFR (AGFR v1.2) tool [14].The monoclinic crystal structure of CuONPs was compiled using Avogadro molecular editor software (v1.2) as reported in our previous work [15,16].Molecular docking analysis and file prepocessing were performed in AutoDock Vina (v1.2.3) [17].Binding model with the best energy was analyzed with the UCSF Chimera (v1.16) [18].

UV-vis spectroscopy
The characterization studies of the obtained CuONPs were started up using UV-vis spectrophotometry.Spectrophotometric analyses of CuONPs obtained from Knautia arvensis flower extract were performed in the wavelength range of 200-800 nm.An absorbance peak was observed at 289 nm at the end of the analysis.In figure 1(A), it is possible to see the CuONPs obtained as a precipitate as a result of synthesis.This morphological examination includes both the color change distinguished by dark brown and the precipitate in which CuONPs are visibly aggregated.Figure 1(B) shows the spectral band analyzed by UV-vis.The spectrum of nanoparticles obtained from K. arvensis flowers clearly indicates particle formation, and the obtained spectral edge is in the distinctive range for copper oxide characterization.The band at 289 nm indicates that the stable nanoparticles are bionanofactured because the optical character of metal oxide nanoparticles depends on the nanoparticle size acting on the SPR band wavelength.Therefore, the results of the spectral analysis confirmed that the nanoparticles obtained were CuONPs when compared with other studies in the literature by keeping in mind that SPR absorbance is susceptible to the environment and the distance between nanoparticles [19,20].
The synthesis was optimized via two parameters: plant extract concentration and pH.Firstly, plant extract: Cu(SO 4 ).5H 2 O solution were obtained in proportions 1:5, 1:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1 (v/v).The optimum synthesis ratio was approved as the sample ratio of the highest absorbance.Figure 2(A) displays the UV-vis spectrum between 200-800 nm of CuONPs obtained with various ratios of K. arvensis flower extract.The greatest peak was obtained with the sample 40:1 (plant extract: Cu(SO 4 ).5H 2 O solution, v/v) therefore, this mixture was selected as the optimum synthesis ratio.The decrease in the band area seen in the figure is based on the narrowing of the particulate crystal lattice and the strengthening of the interatomic bonds due to the decrease in the plant extract concentration [21].Also, CuONPs were biosynthesized applying plant extracts at various pH and their absorbance was quantified by UV-vis.The ideal pH of the plant extract utilized in the study to obtain CuONPs was taken as the value of pH with the highest absorbance.Figure 2(B) displays the absorbance of CuONPs between 200-800 nm obtained with K. arvensis extract at pH values 3.0, 5.6, 7.0 and 9.0.The highest peak was monitored at pH 5.6 and this value was chosen as the ideal pH value of the synthesis.pH 5.6 is also the natural pH value of K. arvensis flower extract.Concentration is of great importance in synthesis because higher or lower than optimal concentrations exert a significant effect on CuONPs.High concentrations of the plant extract will slow down the formation of aggregates as the excess amount of phytochemicals means more reducing and stabilizing power [22].The decrease in the peak area seen with the pH change indicates the importance of the acid-base rate of the medium in metal oxide nanoparticle synthesis.The characteristic SPR band of CuONP, which almost disappears when moving away from the optimum pH, was also clearly obtained at pH of 5.6 the natural pH of the K. arvensis flower extract.

FT-IR spectroscopy
The functional groups of CuONPs and K. arvensis flower extract were examined by FT-IR analyses between 4000-500 cm −1 (figure 3).There are 3320, 2122, 1634, 591 and 567 cm −1 peaks in the FT-IR spectrum of the plant extract, respectively.When we consider nanoparticles, there is a shift in the absorbance peak of the decreasing band intensity monitored from 3320 cm −1 to 3270 cm −1 , indicating that copper ions bind with the hydroxyl group of the extract.The peaks at 2915, 1632, 1588, 1538, 1494, 1417, 1357, 1265, 1201, 1170, 1068, 1039, 833, 800, 600 and 595 cm −1 which are not found in the extract are observed in the CuONP IR.The peak at 2915 cm −1 is attributed to the alkane C-H stretch while the peak at 1632 cm -1 is mainly connected to the C=O stretch of polysaccharides.The 1588 and 1538 cm −1 peaks, which appears with the fabrication of CuONPs, corresponds to the presence of carboxylic and carbonyl groups of I and II amides and amide II region characteristic of proteins/enzymes, respectively.The peaks at 1494 cm −1 and around are attributed to the presence of the −COO group whereas 1417 cm −1 can be assigned to the bending of O-H groups of alcohol.The peak at 1265 cm −1 is attributed to C-0 stretching of ethers and the peak at 1170 cm −1 can be corralated to C−O and C−C stretching of carbohydrates.The broad band at 1068 cm −1 corresponds to C-O stretching vibrations of nucleic acids and that of 1039 cm −1 is the C-O stretching/bending of (C-O-C) carbohydrates.Vibrations of 833 cm −1 and around are attributed to M-O stretching vibrations of CuO nanoparticles.The peak at 800 cm −1 can be corresponded to the bending of aromatic C-H.The peaks at 600 and 595 cm −1 can be attributed to the pure and successful formation of CuO as reported previously [20,23].By these results, the presence of CuONPs, polysaccharides, amines, amides (groups I and II), alcohols, ethers, carbohydrates, nucleic acids were verified by the FT-IR analysis.

SEM analyses
The morphological properties and dimensions of CuONPs were investigated by Scanning electron microscopy (SEM) analyses.In the SEM images shown in figure 4, the nanoparticles were seen as spherical, well-dispersed, and the nanoparticle dimensions extended between 88 to 289 nm.The low tendency of agglomeration distinguished in the figures may be attributed to the phenolic content of the K. arvensis flower extract.The phenolic compounds acting as capping and stabilizing organic agent that causes dispersion may be adsorbed on the surface of the CuONPs or the CuONPs may be coated with a layer of phenolic compounds [16,24].The smooth surface morphology can be seen in SEM microstructure, which can be explained by biochemical constituents of the K. arvensis.These constituents determine the capping reactant content by the inclusion of phytochemicals naturally found in the plant extract.The phytochemicals such as flavonoids, phenols, proteins, terpenoids, and tannins, play role as reducing and stabilizing agents, reducing the metallic salts into the corresponding nanoparticles [25].The plant extract produces electrons that lead to copper salts being reduced therefore, CuONPs are synthesized by the reaction of phytochemicals with the copper ion subsequently to the reduction [26].The results of SEM imaging indicated the successful biosynthesis and enlightened the morphological characteristics of the K. arvensis mediated CuONPs.

DLS analyses
Dynamic Light Scattering (DLS) of CuONPs obtained via K. arvensis flower extract was performed with Zetasier Malvern Nano ZS device.Polydispersity index (PDI) value and average particle size of CuONPs were verified.Figure 5 shows the hydrodynamic size distribution of the fabricated CuONPs.This granulometric distribution graph was expressed against size of the particles and their intensity.The result of the DLS analysis indicated the CuONPs have an average diameter of 189 nm.The size of CuONPs found in in the DLS analysis is larger than found in the SEM results.Because with DLS, information about the hydrodynamic structure in the liquid environment is obtained, whereas electron microscopy makes a dry measurement and gives a different result compared to the hydrated medium.In DLS, when a monochromatic light beam strikes a solution containing spherical particles moving with Brownian motion, the light undergoes a doppler shift, which alters the wavelength of the incoming light, and this alteration provides the measurement of the size of the particle [22].addition, the liquid environment contains biochemical constituents transferred from the plant extract and found on the surfaces of CuONPs.Therefore, measuring together with all these metabolites may be one of the reasons for the high DLS result.Another reason is the hydrostatic interactions of nanoparticles in the aquatic environment.These weak interactions change the distribution of nanoparticles and affect the size reflected in the DLS result.The PDI value of CuONPs was measured as 0.324, which indicates that the nanoparticles were  monodispersed.In general, nanoparticles have PDI ranges of 0.01-0.7,and particles with a broad dimensional distribution possess PDI values bigger than 0.7 [27].

Antibacterial and antifungal activity
The antibacterial activity of CuONPs was determined by disk diffusion method using Gram negative E. coli and Gram positive S. aureus bacteria.The inhibition zone of CuONPs synthesized by K. arvensis flower extract against E. coli was quantified as 16 mm, and the inhibition zone against S. aureus was quantified as 20 mm (table 1).The zone of inhibition of Streptomycin (the positive control) against E. coli and S. aureus were measured as 20 mm and 23 mm, respectively.At the end of the study, it was determined that the biosynthesized CuONPs showed good antibacterial activity against both type of bacteria.It was also seen that they showed better antibacterial activity against gram positive bacteria.CuONP solution used to measure antibacterial activity is 20 μl (1 mg mL −1 ), and as stated in previous reports, inhibition of bacterial growth is directly proportional to nanoparticle concentration in the medium [28].The strong antibacterial performance of CuONPs obtained in this study can be attributed to the chemical structure of the nanoparticle.The weak bonds in the structure of the metal nanoparticles take electrons to the conduction band, thus making it easier for the nanoparticle to attach to the bacterial cell surface and enter the cell.Therefore, even low amounts (20 μl) of biosynthesized CuONPs have high reactive properties.As confirmed by the US-EPA, it is known that copper ions have antibacterial properties [29], but the antibacterial activity of the nanoparticle form of copper and copper oxide is higher than that of the ionic form [30]. Thus, it is undeniable that CuONPs are potent agents, especially if biosynthesized by green methods.As stated above, nanoparticle toxicity on bacteria can occur with the effect of electrostatic interactions caused by weak bonds in the structure (attachment to the bacterial membrane -breaking the membrane structure -penetration).It may also occur in the presence of free radicals by the oxidative stress.Reactive oxygen species that emerge when metal ions form free radicals cause fatal damage to vital compartments of bacteria such as membranes and mitochondria [31].In this study, the antibacterial activity of the CuONPs was also quantified optically by OD 600 measurements.Optic density of the CuONP containing samples and the control were measured for 24 h using E. coli and S. aureus strains (figure 6).The results indicated that CuONP containing samples had low absorbance while the control that was not including CuONP showed an increasing turbidity with increasing absorbance.The results of the optic measurements evaluated that presence of CuONPs have a strong inhibitory effect on the proliferation of both bacteria.Antifungal activity of the synthesized CuONPs against A. niger was investigated by agar diffusion test.The inhibition zone of nanoparticles was quantified as 21 mm and the inhibition zone of Amphotericin B (positive control) was measured as 28 mm.The negative control, dH 2 O, did not form any zone.As a consequence, antifungal efficiency of the synthesized CuONPs was quite good.In addition, the figure obtained by measuring the optical density evaluated that A. niger growth was negatively affected in the presence of CuONPs and the OD 600 value, which is an indicator of microbial growth, was very low (figure 7).In this study, the control group that did not contain any CuONP, was spectrophotometrically examined for 24 h, and the rising optical density reached 1.9 at the end of the period.This result indicated that CuONPs strongly inhibited the growth of A. niger.CuONPs complement traditional organic antimicrobial agents with better qualities because they have a high surface area to volume ratio, which supplies novel characteristics in means of optical, chemical and mechanical to emerge that are distinct from those of the ionic form [32].In this study, the CuONPs have been shown to be powerful in the context of microbial defense.Therefore, the antifungal activity of the obtained CuONPs can be used in agricultural control against fungal plant pathogens, as well as in pharmaceutical and medical applications.

Dye removal activity
The dye elimination performance of the CuONPs was studied against industrial azo dye Naphthol Blue Black (NBB) and anthraquinone dye Remazol Brilliant Blue R (RBBR).The dye degradation spectra of the nanoparticles against both industrial dyes was monitored for 90 min by employing 2 mg mL −1 CuONPs with 10 mg/l NBB and RBBR at pH 8.0.The maximum decolorization of NBB and RBBR was achieved as 42% and 45% at 25 °C after 90 min incubation, respectively (figure 8).However, decolorization efficiency improved to 69% (NBB) and 71% (RBBR) by the increase in temperature to 50 °C in same period of time.RBBR removal efficiency of the CuONPs was better than NBB at pH 8.0 at both temperatures and increased as the time increased.The significant increase of the decolorization of NBB from 42% to 69% by the rise of temperature from 25 to 50 °C is mainly due to the optimum activity temperature of the CuONPs.The situation is similar for RBBR, decolorization increased from 45% to 71% by increasing the temperature from 25 to 50 °C.According to these results, it is seen that the biosynthesized CuONPs are more reactive at 50 °C.The fast and powerful decolorization feature of CuONPs, which removes appraximately 70% of industrial dyes in the environment in  as short as 90 min, indicates their potential use in the field of polluted water treatment.RBBR and NBB dyes, which are used in the industry, especially in the field of textiles, constitute an important branch of organic pollutants.These dyes, which cause environmental problems, were chosen as a model in the control of water pollution in our study, were removed in a very short time.The results showed that CuONPs acted very quickly, decolorizing about 50% of both dyes at 50 °C in 60 min.This effective performance has proven that biosynthesized CuONPs will be useful in the treatment of pollution of aquatic environments.

Molecular docking analysis
In order to elucidate the antifungal and antibacterial mechanisms of CuONPs, molecular docking analysis of nanoparticles with various protein structures from different microorganisms were carried out.The best binding energies of CuONPs with chitin deacetylase (CDA) of A. niger, topoisomerase IV (TopoIV) of E. coli and tyrosyl-tRNA synthetase (TyrRS) of S. aureus were −7.25, −7.14 and −7.89 kcal mol −1 , respectively.CDA, which regulates the production of chitosan by removing the acetic group of the chitin substrate, has been suggested as a fungicide target due to its support for virulence and immunological invasion in the fungal cell wall, especially for agricultural approaches [33,34].In the analysis with AGFR, the binding site of CDA was found at 40.165, 44.564, and −7.430 for x-y-z, respectively.In the binding region (20x20x20 Å), it has been observed that CuONPs especially interact with CDA via Ser62, Arg67, Gln89, Glu90, and Gly91 residues in addition to hydrogen bond with Lys65 with interactions varying lengths ranging from 2.23-3.70Å (figure 9(A)).
One of the most favorable targets for the development of antibacterial agents is DNA topoisomerases, which modulate the division of bacteria [35].Simply this vital enzyme is responsible for the decatenation of DNA knots during DNA replication [36].Its modulatory role in the bacterial division is well defined and has therefore been subjected to molecular docking with CuONPs.In the TopoIV of E. coli interaction of CuONPs, 25 interactions with the length of 2.50-3.69Å were observed, resulting in −7.14 kcal mol −1 binding energy, mostly through Asn42, Glu46, Met74 and Pro75, without hydrogen bonding (figure 9(B)).
TyrRS, a member of the aminoacyl-tRNA synthetase family, is one of the promising bacterial targets involved in the production of bacterial proteins, especially in the elimination of drug resistant pathogens [37].When the antibacterial mechanisms of CuONPs on S. aureus were examined in the TyrRS, it was observed that the interactions with the best binding energy arose via the four residues of TyrRS, His47, His50, Lys84, and Asp195 (figure 9(C)).On the other hand, it is remarkable that there were 2 hydrogen bonds in the interactions that occur as −7.294 kcal mol −1 in the TyrRS CuONP molecular docking and were observed as the third best binding.The molecular docking analysis of this study showed that CuONPs can significantly interact with various vital proteins of microorganisms, thus providing antibacterial and antifungal activity.

Conclusion
In this study, biosynthesis of multifunctional CuONPs were obtained using Knautia arvensis flower extract as a green method.The CuONP solution gave an absorbance peak at 289 nm by UV-vis spectrophotometry which was confirmed the biosynthesis using the plant extract.In the FT-IR analysis, the functional groups of both CuONPs and the plant extract were elucidated.The 600 and 595 cm −1 vibrations seen in the spectrum of CuONPs and belong to stretching between metal and oxygen, showed that CuONPs were successfully synthesized.The size and morphological properties of CuONPs were characterized by SEM.The nanoparticles were found to be spherical and homogeneously distributed, ranging in size from 88-289 nm.The DLS analysis resulted in the average particle size was 189 nm and the PDI value was 0.324.The PDI values implied that CuONPs are monodisperse systems.It was determined that CuONPs showed good antifungal and antibacterial activity against A. niger, E. coli and S. aureus strains.Dye removal experiments of biosynthesized CuONPs against industrial dyes NBB and RBBR were successfully concluded, and a fast and high performance was achieved with an average of 70% decolorization in 90 min.The molecular docking analyses showed that the antibacterial efficiency of CuONPs can be achieved through disruption of cell division, or vital proteins, while supporting that its activity on fungus may be through inhibition of the synthesis of structural proteins.However, it is noteworthy that the energies at which the best bindings were observed in the analyses with the three target proteins were close (i.e., from −7.14 to −7.89 kcal mol −1 ).Although our in vitro and in silico analyses demonstrated the potential of CuONPs in antifungal and antibacterial approaches, it also points to the need for further studies that will comprehensively address the target selectivity of CuONPs.It was demonstrated by this study that one step biosynthesis is an practical and economical way of obtaining multifunctional CuONPs.

Figure 1 .
Figure 1.(A) Image of dark brown aggregate of CuONPs in the solution obtained via K. arvensis flower extract.(B) UV-vis spectrum of CuONPs synthesized using K. arvensis between 200-800 nm wavelengths.

Figure 6 .
Figure 6.Antibacterial efficiency of CuONPs obtained via K. arvensis flower extract based on OD 600 measurement versus time using (A) E. coli (B) S. aureus.

Figure 7 .
Figure 7. Antifungal activity of CuONPs obtained via K. arvensis flower extract based on OD 600 measurement versus time using A. niger.

Figure 8 .
Figure 8.The impact of temperature and time on CuONPs' dye removal activity.

Figure 9 .
Figure 9. Interactions of the best binding model of CuONPs with CDA of A. niger (A), TopoIV of E. coli (B), and TyrRS of S. aureus (C), (it was conducted via UCSF Chimera after molecular docking with AutoDock Vina).The interactions and energies of models have summarized in the chart at the left bottom (The green line, yellow line, red and gold spheres, and ball & stick stand for interactions, hydrogen bond, CuONPs, and residues in the binding site of the receptor, respectively).

Table 1 .
Zone of inhibition of the biosynthesized CuONPs.