Preparation and Characterization of Zinc Oxide Nanoparticles Using Leaf Extract of Sambucus ebulus

: Plants are one of the best sources to obtain a variety of natural surfactants in the ﬁeld of green synthesizing material. Sambucus ebulus , which has unique natural properties, has been considered a promising material in traditional Asian medicine. In this context, zinc oxide nanoparticles (ZnO NPs) were prepared using S. ebulus leaf extract, and their physicochemical properties were investigated. X-ray di ﬀ raction (XRD) results revealed that the prepared ZnO NPs are highly crystalline, having a wurtzite crystal structure. The average crystallite size of prepared NPs was around 17 nm. Green synthesized NPs showed excellent absorption in the UV region as well as strong yellow-orange emission at room temperature. Prepared nanoparticles exhibited good antibacterial activity against various organisms and a passable photocatalytic degradation of methylene blue dye pollutants. The obtained results demonstrated that the biosynthesized ZnO NPs reveal interesting characteristics for various potential applications in the future. (UV-visible spectrophotometer, PerkinElmer and Cary ﬂuorescence to survey the and An electron paramagnetic resonance (EPR) investigation on a spectrometer was conducted to study the paramagnetic defects in the structure. The GC-MS particles’ morphology and size, as well as concentration. The smaller the NP size, the higher their toxic effects on microorganisms, which have relatively large interfacial area and can easily penetrate bacterial membranes, thereby increasing their antibacterial effectiveness. The biosynthesized ZnO NPs used in the present work present an average diameter of 55–75 nm. It is reported that spherical shape nanoparticles (with average 21 nm size) were obtained using plant extract of Ocimum americanum , which showed significant antimicrobial activity against Gram-positive, Gram-negative, and fungal pathogens [24]. ZnO NPs prepared using aqueous Tabernaemontana divaricata green leaf extract showed a spherical shape with particle size ranging from 20 to 50 nm [25]. A higher antibacterial activity against S. aureus and E. coli and lesser antibacterial activity against Salmonella Paratyphi ( S. paratyphi ) compared to the standard pharmaceutical

ZnO nanoparticles (NPs) can be synthesized through many physiochemical routes, such as sol-gel processes, co-precipitation, laser vaporization, microemulsion, and ball milling [7][8][9][10] Commonly, these preparation methods face several limitations, such as the high cost of equipment, the large area required for equipment set up, and additional use of capping agents, stabilizers and toxic chemicals [11]. Most of these chemical methods are not environmentally friendly due to the use of harsh chemicals for stabilizing processes, which will bind to the ZnO NPs, and limit their biological applications [1,11]. To overcome these limitations, green chemistry procedures have attracted zinc acetate dihydrate was dissolved in 20 mL absolute ethanol and kept under continuous stirring for 30 min [37][38][39][40][41][42][43][44]. Approximately 2 mL of extract (as a natural surfactant) was added to the solution, followed by stirring for 2 h at 80 • C; the suspension was centrifuged at 7168 g for 15 min, and the precipitate was dried at 60 • C in a hot air oven until dried NPs were obtained. The prepared NPs were calcined at 450 • C for 2 h. A schematic representation of the experimental work and possible chemical bond formed between the extracted molecules and zinc oxide are shown in Figure 1.
Appl. Sci. 2020, 10, x FOR PEER REVIEW  3 of 19 for 15 min, and the precipitate was dried at 60 °C in a hot air oven until dried NPs were obtained. The prepared NPs were calcined at 450 °C for 2 h. A schematic representation of the experimental work and possible chemical bond formed between the extracted molecules and zinc oxide are shown in Figure 1.

Figure 1.
A schematic illustration of the synthetic route for production of ZnO nanoparticles (NPs).

Antibacterial Activity Test
The antibacterial activity of the biosynthesized ZnO NPs against pathogenic microorganisms, namely Gram-positive Bacillus cereus (KNIH28) and Staphylococcus aureus (ATCC6538 P), and Gramnegative Escherichia coli (E. coli)ATCC27853), was carried out on Muller-Hilton agar dishes using the disk diffusion method [45]. Nutrient agar broth (Himedia, Bombay) was used as bacterial culture medium in the bacterial assays. Pure cultures of the microorganisms were provided by the Alborz hospital, Karaj, Iran. The bacterial pathogens were freshly incubated for 4 h to standardize the culture to McFarland standards (10 6 CFU/mL).
All of the chemicals used for the cell culturing were of analytical grade. Cells were grown in agar media and humidified incubator at 37 °C under 5% CO2. The concentration of bacterial cells was adjusted to 10 6 CFU/mL, and ciprofloxacin (10 µg/disc) was used as a standard. Paper disks (6 mm diameter, Whatman No. 1 filter paper) infused with 100 µg/mL concentrations of prepared ZnO NPs and pure extract (10 µg/mL) suspended in deionized water were used to ascertain the antibacterial activity, followed by incubation at 37 °C for a period of 24 h. Inhibition zones, that formed around the disk, were investigated by measuring the corresponding diameter. The antibacterial experiments were repeated three times.

Antioxidant Activity
The antioxidant activity was determined using the hydrogen peroxide (H2O2) free radical scavenging assay [46]. A quantity of 1 mL (with various concentrations) of the biosynthesized ZnO NPs and ascorbic acid as a standard were mixed with 50 mM (0.6 mL) H2O2 solution and then incubated at room temperature for 5 min. Absorbance was detected with a UV-vis spectrophotometer at 230 nm. Phosphate-buffered saline without H2O2 was used as a blank test [46]. The inhibition percentage of hydrogen peroxide scavenging was obtained using the following equation: % H2O2 free radicals = {1 − ( )} × 100 (1) where A c is the absorbance of the control and A s is the absorbance of the sample.

Antibacterial Activity Test
The antibacterial activity of the biosynthesized ZnO NPs against pathogenic microorganisms, namely Gram-positive Bacillus cereus (KNIH28) and Staphylococcus aureus (ATCC6538 P), and Gramnegative Escherichia coli (E. coli)ATCC27853), was carried out on Muller-Hilton agar dishes using the disk diffusion method [45]. Nutrient agar broth (Himedia, Bombay) was used as bacterial culture medium in the bacterial assays. Pure cultures of the microorganisms were provided by the Alborz hospital, Karaj, Iran. The bacterial pathogens were freshly incubated for 4 h to standardize the culture to McFarland standards (10 6 CFU/mL).
All of the chemicals used for the cell culturing were of analytical grade. Cells were grown in agar media and humidified incubator at 37 • C under 5% CO 2 . The concentration of bacterial cells was adjusted to 10 6 CFU/mL, and ciprofloxacin (10 µg/disc) was used as a standard. Paper disks (6 mm diameter, Whatman No. 1 filter paper) infused with 100 µg/mL concentrations of prepared ZnO NPs and pure extract (10 µg/mL) suspended in deionized water were used to ascertain the antibacterial activity, followed by incubation at 37 • C for a period of 24 h. Inhibition zones, that formed around the disk, were investigated by measuring the corresponding diameter. The antibacterial experiments were repeated three times.

Antioxidant Activity
The antioxidant activity was determined using the hydrogen peroxide (H 2 O 2 ) free radical scavenging assay [46]. A quantity of 1 mL (with various concentrations) of the biosynthesized ZnO NPs and ascorbic acid as a standard were mixed with 50 mM (0.6 mL) H 2 O 2 solution and then incubated at room temperature for 5 min. Absorbance was detected with a UV-vis spectrophotometer at 230 nm.
Phosphate-buffered saline without H 2 O 2 was used as a blank test [46]. The inhibition percentage of hydrogen peroxide scavenging was obtained using the following equation: where A c is the absorbance of the control and A s is the absorbance of the sample.

Photocatalytic Activity
The photocatalytic activity of the biosynthesized ZnO NPs was investigated via the photocatalytic degradation of MB (a dye normally resistant to biodegradation). The photocatalytic experiments were performed under UV light (24 W UV lamp, 360 nm) irradiation. The photocatalytic decomposition of MB solution (50 ppm) by ZnO NPs as a photocatalyst (0.02 g) was performed in a 100 mL beaker at 35 • C under UV irradiation and continuous stirring to achieve the homogeneity of the solution. Absorbance was measured with a UV-Vis spectrophotometer.

Analytical Methods
The X-ray diffraction (XRD) pattern of ZnO NPs was recorded using a PANalytical PW3050/60 X-ray diffractometer equipped with Cu-Kα radiation source (λ = 1.5406 Å) operated at 40 kV and 30 mA. Transmission electron microscopy (TEM; Philips CM120), field emission scanning electron microscopy (FE-SEM; Mira3 Tescan), energy-dispersive X-ray spectroscopy (EDX; Mira3 Tescan), and X-ray photoelectron spectroscopy (XPS; Thermo Scientific K-Alpha) were employed to investigate the morphology and topography of the samples along with elemental chemical surface analysis.
The FE-SEM instrument was operated at an accelerating voltage at 10 kV. The synthesized NPs were subjected to UV, Fourier-transform infrared (FTIR; range 4000-400 cm −1 ), and photoluminescence (PL; range 200-800 nm) spectroscopy (UV-visible spectrophotometer, PerkinElmer and Cary fluorescence spectrophotometer) to survey the optical properties, functional groups, and structural defects. An electron paramagnetic resonance (EPR) investigation on a Bruker ELEXSYS E500 X-band (~9.45 GHz) spectrometer was conducted to study the paramagnetic defects in the structure. The GC-MS analysis of the plant extract was conducted using an Agilent 7890 A instrument under computer control at 70 eV that comprises an AOC-20i auto-sampler and a gas chromatograph interfaced to a mass spectrometer (GC-MS) equipped with an Agilent 5975 C fused capillary column (30 × 0.25 µm ID × 0.25 µm df). For GC-MS detection, an electron ionization system was operated in electron impact mode with an ionization energy of 70 eV. Helium gas (99.999%) was used as a carrier gas at a constant flow rate of 1 mL/min, and an injection volume of 2 µL was employed (a split ratio of 10:1). The injector temperature was maintained at 260 • C. The ion-source temperature was kept at 270 • C. The oven temperature was programmed from 40 • C with an increase rate of 3 • C/min to 250 • C. Surface potential was investigated using a Malvern Zetasizer Nano-series (Malvern Instruments Zen 3600, Malvern, UK)

Physicochemical Characterisation
The XRD pattern of the biosynthesized ZnO NPs presented in Figure 2a reveals well-defined relatively broad peaks with high intensity which indicates the formation of nanocrystalline phase. The observed peaks are indexed within the hexagonal ZnO wurtzite-type structure, in agreement with Joint Committee on Powder Diffraction Standards (JCPDS) card no. 36-1451 [38]. No additional peaks can be detected within the resolution limit of the X-ray diffractometer, thereby confirming the formation of single ZnO phase. Using the Debye-Scherrer formula (D = 0.9 λ/β cosθ, where θ is the diffraction angle, λ = 1.5406 Å, and β is the peak width at half maximum), crystallite size of prepared ZnO nanoparticles was calculated around 17 nm [38]; also, lattice parameters (a, c) were found to be about 3.306 and 5.199, respectively.

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The elemental information of the synthesized ZnO NPs was confirmed by EDX ( Figure 2b); the spectrum indicates the existence of Zn and O elements which confirms again the purity of the prepared ZnO NPs.
The analysis of EPR curve based on the calculated g-values from a spectrum formed of a symmetrical Lorentzian shape, indicated the presence of two narrow resonances: one at g-value~1.985 and the other at g-value~1.987, corresponding to shallow donors and singly ionized oxygen vacancies, respectively ( Figure 2c) [47,48]. The elemental information of the synthesized ZnO NPs was confirmed by EDX ( Figure 2b); the spectrum indicates the existence of Zn and O elements which confirms again the purity of the prepared ZnO NPs.
The analysis of EPR curve based on the calculated g-values from a spectrum formed of a symmetrical Lorentzian shape, indicated the presence of two narrow resonances: one at g-value ~1.985 and the other at g-value ~1.987, corresponding to shallow donors and singly ionized oxygen vacancies, respectively ( Figure 2c) [47,48].
FTIR analysis helped to identify functional groups existing in the plant extract that contribute to the mechanism of bonding with ZnO NPs. As we mentioned previously, S. ebulus extract contains high amounts of polyphenolic derivatives. such as flavonoids, anthocyanins, and cyanidin-3glucoside, which are considered potential bioactive compounds for therapeutic treatments and to act as reducing agents [8][9][10][11][12][13][14][15]. A comparison between the FTIR spectrum of the plant extract and that of the biosynthesized ZnO NPs is shown in Figure 2d [49][50][51][52], whereas the broad stretching band at 3429 cm −1 indicates the presence of hydrogen-bonded groups. These results signify the existence of flavonoid derivatives in the S. ebulus extract. Any shift or change in the position and intensity of peaks in the sample spectrum can be correlated with the interaction of the functional groups of the flavonoids and phenols with the ZnO NPs. It can be mentioned that functional groups in the extract donate electrons that could reduce zinc ions (Zn +2 to Zn +1 ) and finally zinc NPs (Zn 0 ). FTIR analysis helped to identify functional groups existing in the plant extract that contribute to the mechanism of bonding with ZnO NPs. As we mentioned previously, S. ebulus extract contains high amounts of polyphenolic derivatives. such as flavonoids, anthocyanins, and cyanidin-3-glucoside, which are considered potential bioactive compounds for therapeutic treatments and to act as reducing agents [8][9][10][11][12][13][14][15]. A comparison between the FTIR spectrum of the plant extract and that of the biosynthesized ZnO NPs is shown in Figure 2d [49][50][51][52], whereas the broad stretching band at 3429 cm −1 indicates the presence of hydrogen-bonded groups. These results signify the existence of flavonoid derivatives in the S. ebulus extract. Any shift or change in the position and intensity of peaks in the sample spectrum can be correlated with the interaction of the functional groups of the flavonoids and phenols with the ZnO NPs. It can be mentioned that functional groups in the extract donate electrons that could reduce zinc ions (Zn 2+ to Zn +1 ) and finally zinc NPs (Zn 0 ). Moreover, the negative functional groups present in the extract could have a stabilizing effect. FTIR spectra of the biosynthesized ZnO NPs showed a small shift with slight changes in some related peaks and in their intensities, suggesting that the major biomolecules from the extract were capped or bonded to the surface of ZnO NPs. The major peak for the plant extract shifting from 3429 to 3426 cm −1 in ZnO was assigned to the O-H of the phenol groups and -NH 2 stretching vibrations, while another peak shifted from 1668 to 1579 cm −1 was ascribed to N-H binding. The appearance of new peaks at 700 and 2212-2230 cm −1 proved that ZnO NPs underwent C-H and C=C out-of-plane bending, respectively. Furthermore, the FTIR spectrum of the biosynthesized ZnO NPs showed a sharp and intense band at 546 cm −1 , indicating the existence of Zn-O vibrations [52]. The FTIR results demonstrated that phenol and flavonoid compounds were abundant in the prepared extract and ZnO NPs. In addition, according to the GC-MS technique and Table 1, the most important compounds in the extract were carbonyl acids, ethers, and phenolic compounds. There are many advantages of applying natural plants in NP synthesis, including coating NPs with different pharmacological species (such as phenolic and polyphenolic compounds) on the metal oxide layer, which facilitates the bonding of NPs with the receivers on bacterial membranes. The S. ebulus extract is rich in such compounds, which are responsible for enhancing the antibacterial properties of ZnO NPs. The possible mechanism for the formation of ZnO NPs using the plant extract could be related to the flavonoid/phenolic molecules reacting with Zn 2+ ions through the donor-acceptor mechanism. Oxygen or hydroxyl compounds present in the plant extract tend to donate an electron to electrophile Zn complexes resulting in oxidation of OH groups and reduction of electron deficient zinc ions to zinc atoms ( Figure 1).
The synthesized NPs were investigated by XPS surface analysis; Figure 3 depicts a full survey scan in the range 0-1100 eV. Two intense peaks located at~1024 and 1044 eV were observed, which are attributed to the Zn 2p 3/2 and Zn 2p 1/2 , respectively, corresponding to Zn-O bonding within the hexagonal wurtzite-type structure [53,54]. The O1s peak was also present in the spectrum at 529−530 eV, corresponding to Zn-O bonds in the intrinsic sites of the ZnO matrix, which is in good agreement with the FTIR spectrum of the biosynthesized ZnO NPs (sharp and intense band at 546 cm −1 , which indicates the existence of Zn-O vibrations). Appl. Sci. 2020, 10, x FOR PEER REVIEW 8 of 19  Figure 4a shows the absorption spectrum of the biosynthesized ZnO NPs with an absorption peak at around 376 nm, which was assigned to the band-gap absorption of ZnO, while the very weak peak at 350 nm could be related to the presence of S. ebulus leaf extract. The energy band gap was found to be ~3.3 eV using the formula E = hc/λ, where λ is the wavelength, h = 6.626 × 10 −34 Js (Plank's constant), and c = 3 × 10 8 m/s, which is in good agreement with the reported values in the literature [42,44].  Figure 4a shows the absorption spectrum of the biosynthesized ZnO NPs with an absorption peak at around 376 nm, which was assigned to the band-gap absorption of ZnO, while the very weak peak at 350 nm could be related to the presence of S. ebulus leaf extract. The energy band gap was found to bẽ 3.3 eV using the formula E = hc/λ, where λ is the wavelength, h = 6.626 × 10 −34 Js (Plank's constant), and c = 3 × 10 8 m/s, which is in good agreement with the reported values in the literature [42,44]. Moreover, using UV-vis diffuse reflectance spectroscopy (Figure 4b), the band-gap energy of the biosynthesized ZnO NPs was calculated using the Kubelka-Munk equation [55]:

Optical Characterisation
where R is the reflectance. By plotting (F(R) × E) 1/2 as a function of energy, the value of the band gap was obtained to be~3.3 eV (Figure 4b). The strong absorption in the UV region demonstrated that the biosynthesized ZnO NPs has a good capacity for UV-protection applications [24]. For example, the skin is one of the important protective barriers of the body and various events, such as trauma, cuts, and burns, can damage this natural layer. In these cases, a special cover must be used in the process of wound healing to repair the skin, improve the wound, protect it from UV rays, and reduce the risk of wound infection. The PL spectrum of ZnO normally consists of two main emission peaks: (1) a UV peak near the band-edge emission and (2) a visible peak resulting from deep level emission (DLE) bands [38,56]. Some optically active intrinsic defects influence the optical properties of ZnO, and different kinds of defects are created during synthesis, growth, and/or upon annealing. The PL spectra of bare ZnO and biosynthesized ZnO using the plant extract are shown in Figure 4c,d, respectively. A broad blue emission peak centered at~478 nm can be observed for the bare ZnO, which is normally associated with Zn defects, such as transitions from Zn i levels (or conduction band) to valence band or V Zn states (Figure 4c), while in Figure 4d, the PL spectrum of the biosynthesized ZnO using the plant extract is composed of a weak UV band at around 402 nm and an intense orange band at around 598 nm. UV emission is related to near band-edge transition, exciton-exciton collision, and free exciton recombination. The UV emission is attributed to transitions between Zn i to the valence band [38], while yellow-orange emission is generally attributed to the DLE bands and excess oxygen, such as the transition between the vacancies and interstitials of neutral and singly ionized oxygen [38]. In n-type ZnO, acceptor-like defects (O i ) are easier to form, which enables the transition between different band-gap energies. Yellow-orange luminescent materials are widely used in optical and medical treatments, and luminescent nanoproducts with potential bio-applications have gained great interest during the past years [57]. It can be concluded that during the chemical interaction between the plant extract molecules and ZnO NPs, some defects were passivated, which influenced the defect density and emission properties. As we mentioned previously, S. ebulus extract is rich in antioxidants (such as phenols and flavonols) through their carboxyl groups with binding to ZnO NPs, which can produce new levels or defects in the band gap of the host structure and, finally, influence the emission of ZnO. It is worth mentioning that many hundred years ago, Sambucus ebulus extract was used as a natural dye for textile applications; it is reported that silk fabrics were dyed successfully sing natural dyes extracted from Sambucus ebulus fruit [55].  Figure 4a shows the absorption spectrum of the biosynthesized ZnO NPs with an absorption peak at around 376 nm, which was assigned to the band-gap absorption of ZnO, while the very weak peak at 350 nm could be related to the presence of S. ebulus leaf extract. The energy band gap was found to be ~3.3 eV using the formula E = hc/λ, where λ is the wavelength, h = 6.626 × 10 −34 Js (Plank's constant), and c = 3 × 10 8 m/s, which is in good agreement with the reported values in the literature [42,44].  Moreover, using UV-vis diffuse reflectance spectroscopy (Figure 4b), the band-gap energy of the biosynthesized ZnO NPs was calculated using the Kubelka-Munk equation [55]:

Optical Characterisation
where R is the reflectance. By plotting ( ( ) × ) 1/2 as a function of energy, the value of the band gap was obtained to be ~3.3 eV (Figure 4b). The strong absorption in the UV region demonstrated

Microstructural Characteristisation
The uniform distribution and almost spherically shaped ZnO NPs can be observed in the FE-SEM image in Figure 5a. Some NPs are present with a hexagonal shape and the average particle size is around 40-45 nm. The NPs were dispersed in ethanol and sonicated in an ultrasonic bath for 20 min for TEM analysis. The TEM image in Figure 5b clearly shows that the NPs are mostly spherical with a size ranging from 25 to 30 nm. The selected area electron diffraction pattern, depicted in Figure 5b, confirms the crystalline nature of the biosynthesized ZnO NPs.

Microstructural Characteristisation
The uniform distribution and almost spherically shaped ZnO NPs can be observed in the FE-SEM image in Figure 5a. Some NPs are present with a hexagonal shape and the average particle size is around 40-45 nm. The NPs were dispersed in ethanol and sonicated in an ultrasonic bath for 20 min for TEM analysis. The TEM image in Figure 5b clearly shows that the NPs are mostly spherical with a size ranging from 25 to 30 nm. The selected area electron diffraction pattern, depicted in Figure  5b, confirms the crystalline nature of the biosynthesized ZnO NPs. It is known that the physicochemical properties of nanomaterials, including size, shape, surface area, zeta potential, and composition greatly effect on their toxicological effects. In this regard, ZnO NPs induce a significant cytotoxicity in a size-dependent trend; particularly soluble and smaller NPs provide more useful toxicological information on nanomaterials. ZnO-NP antimicrobial activity is significantly affected by particles' morphology and size, as well as concentration. The smaller the NP size, the higher their toxic effects on microorganisms, which have relatively large interfacial area and can easily penetrate bacterial membranes, thereby increasing their antibacterial effectiveness. The biosynthesized ZnO NPs used in the present work present an average diameter of 55-75 nm. It is reported that spherical shape nanoparticles (with average 21 nm size) were obtained using plant extract of Ocimum americanum, which showed significant antimicrobial activity against Grampositive, Gram-negative, and fungal pathogens [24]. ZnO NPs prepared using aqueous Tabernaemontana divaricata green leaf extract showed a spherical shape with particle size ranging from 20 to 50 nm [25]. A higher antibacterial activity against S. aureus and E. coli and lesser antibacterial activity against Salmonella Paratyphi (S. paratyphi) compared to the standard pharmaceutical It is known that the physicochemical properties of nanomaterials, including size, shape, surface area, zeta potential, and composition greatly effect on their toxicological effects. In this regard, ZnO NPs induce a significant cytotoxicity in a size-dependent trend; particularly soluble and smaller NPs provide more useful toxicological information on nanomaterials. ZnO-NP antimicrobial activity is significantly affected by particles' morphology and size, as well as concentration. The smaller the NP size, the higher their toxic effects on microorganisms, which have relatively large interfacial area and can easily penetrate bacterial membranes, thereby increasing their antibacterial effectiveness. The biosynthesized ZnO NPs used in the present work present an average diameter of 55-75 nm. It is reported that spherical shape nanoparticles (with average 21 nm size) were obtained using plant extract of Ocimum americanum, which showed significant antimicrobial activity against Gram-positive, Gram-negative, and fungal pathogens [24]. ZnO NPs prepared using aqueous Tabernaemontana divaricata green leaf extract showed a spherical shape with particle size ranging from 20 to 50 nm [25]. A higher antibacterial activity against S. aureus and E. coli and lesser antibacterial activity against Salmonella Paratyphi (S. paratyphi) compared to the standard pharmaceutical formulation, was also observed. ZnO NPs prepared using pomelo fruit juice with an average particle size of about 10-20 nm showed significant antibacterial activity against pathogenic strains such as S. aureus and K. aerogenes and a less significant role in E. coli pathogenic bacterial strains [26]. ZnO NPs green synthesized from Aristolochia indica of size 22.5 nm exhibited remarkable bactericidal activity [27].
A zeta-potential study was carried out to detect the surface charge and stability of the prepared NPs and indicated the presence of a distinct peak at −38 mV, which suggests that the synthesized NPs have good stability. This means that the capping molecules present on the surface of biosynthesized ZnO NPs are mainly comprised of negatively charged groups and also responsible for moderate stability of the nanoparticles. The rich source of proteins and flavonoids in the extract may be responsible for the reduction of metal ions and efficient stabilization of ZnO NPs.

Photocatalytic Tests
Photocatalytic technology is an important technique for eliminating dyes in sewage water. The photocatalytic degradation characteristics of the biosynthesized NPs in MB dye solution was conducted under UV irradiation. Under irradiation, electrons through the valence band are excited and travel to the conduction band, which creates holes in the valence band. Generally, the electrons form O − 2 species through reaction with oxygen, while the holes form hydroxyl radicals through reaction with OH ions. Hence, oxidizing species can attack and oxidize organic pollutants. Usually, the proper photocatalytic activity of ZnO products can be related to the number of defects [58,59].
The degradation rate of MB was calculated as follow [60]: where A 0 is the absorbance at t = 0 min and A is the absorbance after treatment. According to Figure 6a, it is obvious that the absorbance peak was reduced significantly (after 200 min). Indeed, the biosynthesized ZnO NPs show a noticeable reduction in intensity, thus indicating the degradation of the dye molecules. Figure 6b depicts the degree of degradation of the MB solution obtained with ZnO NPs after UV radiation over time. It is obvious that the maximum degradation of around 80% was obtained after 200 min. In a similar study on photocatalytic degradation of MB dye with ZnO-Camellia sinensis NPs, the same result of approximately 80% degradation in 120 min was obtained [61].
Appl. Sci. 2020, 10, x FOR PEER REVIEW 11 of 19 formulation, was also observed. ZnO NPs prepared using pomelo fruit juice with an average particle size of about 10-20 nm showed significant antibacterial activity against pathogenic strains such as S. aureus and K. aerogenes and a less significant role in E.coli pathogenic bacterial strains [26]. ZnO NPs green synthesized from Aristolochia indica of size 22.5 nm exhibited remarkable bactericidal activity [27]. A zeta-potential study was carried out to detect the surface charge and stability of the prepared NPs and indicated the presence of a distinct peak at −38 mV, which suggests that the synthesized NPs have good stability. This means that the capping molecules present on the surface of biosynthesized ZnO NPs are mainly comprised of negatively charged groups and also responsible for moderate stability of the nanoparticles. The rich source of proteins and flavonoids in the extract may be responsible for the reduction of metal ions and efficient stabilization of ZnO NPs.

Photocatalytic Tests
Photocatalytic technology is an important technique for eliminating dyes in sewage water. The photocatalytic degradation characteristics of the biosynthesized NPs in MB dye solution was conducted under UV irradiation. Under irradiation, electrons through the valence band are excited and travel to the conduction band, which creates holes in the valence band. Generally, the electrons form 2 − species through reaction with oxygen, while the holes form hydroxyl radicals through reaction with OH ions. Hence, oxidizing species can attack and oxidize organic pollutants. Usually, the proper photocatalytic activity of ZnO products can be related to the number of defects [58,59].
The degradation rate of MB was calculated as follow [60]: where A0 is the absorbance at t = 0 min and A is the absorbance after treatment. According to Figure  6a, it is obvious that the absorbance peak was reduced significantly (after 200 min). Indeed, the biosynthesized ZnO NPs show a noticeable reduction in intensity, thus indicating the degradation of the dye molecules. Figure 6b depicts the degree of degradation of the MB solution obtained with ZnO NPs after UV radiation over time. It is obvious that the maximum degradation of around 80% was obtained after 200 min. In a similar study on photocatalytic degradation of MB dye with ZnO-Camellia sinensis NPs, the same result of approximately 80% degradation in 120 min was obtained [61].

Antibacterial and Antioxidant Properties
Since no previous work has been reported yet on S. ebulus extract-loaded ZnO NPs, some antibacterial properties in this study were compared with similar studies but using different natural extracts and commercial/chemical synthesized ZnO . The antibacterial activity of the biosynthesized ZnO NPs using leaf extract and the pure extract was investigated on B. cereus, S. aureus, and E. coli bacteria (Figure 7a). The biosynthesized ZnO NPs exhibited antibacterial activity over all three bacteria, but was higher for Gram-negative bacteria compared to Gram-positive bacteria. Moreover, the ZnO NPs exhibited a considerable zone of inhibition for E. coli and B. cereus compared with S. aureus. Similar observations were reported for the antibacterial properties of ZnO NPs/plant extracts against E. coli strains [62,63]. The results show more activity toward Gram-negative bacteria, which can be due to the existence of a thick rigid layer that surrounds their cells. This effect has already been reported [63].

Antibacterial and Antioxidant Properties
Since no previous work has been reported yet on S. ebulus extract-loaded ZnO NPs, some antibacterial properties in this study were compared with similar studies but using different natural extracts and commercial/chemical synthesized ZnO . The antibacterial activity of the biosynthesized ZnO NPs using leaf extract and the pure extract was investigated on B. cereus, S. aureus, and E. coli bacteria (Figure 7a). The biosynthesized ZnO NPs exhibited antibacterial activity over all three bacteria, but was higher for Gram-negative bacteria compared to Gram-positive bacteria. Moreover, the ZnO NPs exhibited a considerable zone of inhibition for E. coli and B. cereus compared with S. aureus. Similar observations were reported for the antibacterial properties of ZnO NPs/plant extracts against E. coli strains [62,63]. The results show more activity toward Gramnegative bacteria, which can be due to the existence of a thick rigid layer that surrounds their cells. This effect has already been reported [63].  The antioxidant properties of the biosynthesized NPs are presented in Figure 7b. The H2O2 activity of ascorbic acid (standard), the biosynthesized ZnO NPs, and leaf extract exhibited IC50 values of 36, 43, and 62 µg/mL, respectively. Enhancement in the antioxidant properties of the biosynthesized ZnO NPs in comparison with the leaf extract could be a consequence of metal or Zn ions existing in the structure. Generally, it has been reported that phenolic species existing in plant extracts have revealed high antioxidant properties, which is an important characteristic for bioapplications [76]. For comparison, strong antioxidant activity has been found in ZnO NPs isolated from extracts of Allium sativum, Rosmarinus officinalis, and Ocimum basilicum for comparison [77].
Microorganisms are protected by a cell membrane covering them and through this membrane sufficient nutrient are transported [64,65]. Figure 8 shows different mechanisms adopted by Gramnegative bacteria for attachment of ZnO NPs and their transport inside the bacteria cell. Gramnegative bacteria have a triple thin layer of peptidoglycan compared with Gram-positive bacteria and show less toleration to nanoparticle interaction with the cell membrane. According to Figure 8, a Gram-negative bacterium (such as E. coli) has a layer of peptidoglycan in its cell wall where porins are present in the outer layer. Porins are a family of proteins that exist in the outer peptidoglycan layer that helps passive diffusion of NPs inside the cell. ZnO NPs prefer to dissolve in the aqueous medium containing bacteria and release Zn +2 ion (because of free energy) [78]. The interaction between zinc ions and the cell wall results in a change in surface tension which leads to the membrane depolarization at the point of contact. As a result, the bacterial membrane shows abnormal textures such as membrane rupture or membrane blebs. ZnO NPs, as an antibacterial agent, act with a series of mechanisms; in this way, cell membrane integrity loss (induced by disruption of the phospholipid bilayer) and oxidative stress (induced by reactive oxygen species (ROS) generation which causes cell death by inhibiting) are two important events ( Figure 8) [64,79]. ZnO and its related defects can be easily activated by UV and visible light, which leads to electron-hole pair generation [35]; the formed excitons could increase ROS concentration. ROS molecules could be superoxide, hydroxyl, singlet oxygen, and peroxide ions which are produced on the surface of NPs and possess high electronegativity that can enhance the antimicrobial activity. For example, the generated H2O2 molecules can penetrate into the bacterial cell membrane inducing structural changes to membranes and hence disturbing nutrient/protein transport and causing bacteria death. OH radicals are negatively charged, and thus cannot penetrate the outer cell membrane of E. coli, which is also negatively charged, and remain in direct contact with the outer surface of the bacterium [66][67][68]. The entry of ZnO NPs into the cell leads to integrity loss of phospholipid layer and blebbing of intracellular elements resulting in cell death. Nanoparticles react with membrane proteins and inactivate them decreasing membrane permeability and causing cell death. The antibacterial The antioxidant properties of the biosynthesized NPs are presented in Figure 7b. The H 2 O 2 activity of ascorbic acid (standard), the biosynthesized ZnO NPs, and leaf extract exhibited IC 50 values of 36, 43, and 62 µg/mL, respectively. Enhancement in the antioxidant properties of the biosynthesized ZnO NPs in comparison with the leaf extract could be a consequence of metal or Zn ions existing in the structure. Generally, it has been reported that phenolic species existing in plant extracts have revealed high antioxidant properties, which is an important characteristic for bio-applications [76]. For comparison, strong antioxidant activity has been found in ZnO NPs isolated from extracts of Allium sativum, Rosmarinus officinalis, and Ocimum basilicum for comparison [77].
Microorganisms are protected by a cell membrane covering them and through this membrane sufficient nutrient are transported [64,65]. Figure 8 shows different mechanisms adopted by Gramnegative bacteria for attachment of ZnO NPs and their transport inside the bacteria cell. Gram-negative bacteria have a triple thin layer of peptidoglycan compared with Gram-positive bacteria and show less toleration to nanoparticle interaction with the cell membrane. According to Figure 8, a Gram-negative bacterium (such as E. coli) has a layer of peptidoglycan in its cell wall where porins are present in the outer layer. Porins are a family of proteins that exist in the outer peptidoglycan layer that helps passive diffusion of NPs inside the cell. ZnO NPs prefer to dissolve in the aqueous medium containing bacteria and release Zn 2+ ion (because of free energy) [78]. The interaction between zinc ions and the cell wall results in a change in surface tension which leads to the membrane depolarization at the point of contact. As a result, the bacterial membrane shows abnormal textures such as membrane rupture or membrane blebs. ZnO NPs, as an antibacterial agent, act with a series of mechanisms; in this way, cell membrane integrity loss (induced by disruption of the phospholipid bilayer) and oxidative stress (induced by reactive oxygen species (ROS) generation which causes cell death by inhibiting) are two important events (Figure 8) [64,79]. ZnO and its related defects can be easily activated by UV and visible light, which leads to electron-hole pair generation [35]; the formed excitons could increase ROS concentration. ROS molecules could be superoxide, hydroxyl, singlet oxygen, and peroxide ions which are produced on the surface of NPs and possess high electronegativity that can enhance the antimicrobial activity. For example, the generated H 2 O 2 molecules can penetrate into the bacterial cell membrane inducing structural changes to membranes and hence disturbing nutrient/protein transport and causing bacteria death. OH radicals are negatively charged, and thus cannot penetrate the outer cell membrane of E. coli, which is also negatively charged, and remain in direct contact with the outer surface of the bacterium [66][67][68]. The entry of ZnO NPs into the cell leads to integrity loss of phospholipid layer and blebbing of intracellular elements resulting in cell death. Nanoparticles react with membrane proteins and inactivate them decreasing membrane permeability and causing cell death. The antibacterial activities of ZnO NPs strongly depend on shape, size, powder concentration, specific surface area, zeta potential, etc. For example, it is reported that ZnO NPs with positive surface potential showed higher antimicrobial properties compared with ZnO NPs of the same size but with negative surface potential [80]. Furthermore, it is reported that small-sized nanoparticles can penetrate the bacterial membrane easily and spherically shaped ZnO NPs release Zn 2+ ions more effectively than rod-shaped ions [81,82].
Appl. Sci. 2020, 10, x FOR PEER REVIEW 14 of 19 activities of ZnO NPs strongly depend on shape, size, powder concentration, specific surface area, zeta potential, etc. For example, it is reported that ZnO NPs with positive surface potential showed higher antimicrobial properties compared with ZnO NPs of the same size but with negative surface potential [80]. Furthermore, it is reported that small-sized nanoparticles can penetrate the bacterial membrane easily and spherically shaped ZnO NPs release Zn +2 ions more effectively than rod-shaped ions [81,82].

Figure 8.
A schematic diagram of the antibacterial activity mechanism.
As observed above, biosynthesized ZnO NPs depict better antibacterial activity against various microorganisms when compared to pure extract.
Previous studies imply that green ZnO nanoparticles showed more enhanced biocidal activity against various pathogens when compared to chemical ZnO nanoparticles [83]; furthermore, Susheela et al. reported that green synthesized nanoparticles show more effective antimicrobial activity than those of plant extracts [84].
Surface modifying reagent molecules result in differences in the release of Zn 2+ ions and the production of reactive oxygen species (ROS). S. ebulus leaf extract contains enormous numbers of carbonyl, carboxyl, and hydroxyl groups in polyphenols and flavonoids [35,[69][70][71]. These compounds can have a positive effect on human health because they can act as antioxidants and antiinflammatory and anticancer agents. Indeed, these compounds present a wide range of biological activities, and the presence of abundant hydroxyl and carbonyl groups is responsible for antibacterial applications [69][70][71]. These compounds can affect the release of Zn +2 and the production of ROS. The existence of these compounds in the extract and the surface helps ZnO NPs to adhere to the bacterial cell membranes. The chemical interaction between the extract molecules and ZnO NPs enhance the reactivity of NPs and improves penetration via bonding to the surface of the bacteria. Plant extract compounds attach to the surface of the bacteria and create new pathways near the surface for better transportation of or penetration by ions. For example, hydrogen in these compounds acts as both an oxidizing and a reducing agent (due to its two distinct oxidation states) which can easily form many bonds with other molecules [72,73].
In a recent study, it was found that Albizia lebbeck stem bark increased the antibacterial effect of biosynthesized ZnO NPs [74]. In another investigation, ZnO NPs (100-190 nm) were synthesized using Trifolium pratens flower extract and the high zone of their growth inhibition (29-31 mm) against E. coli, and S. aureus at ~1200 µg/mL was obtained [20]. In the work performed by Gunalan et al. [75], the biosynthesized ZnO NPs using Aloe vera extract showed enhanced antibacterial properties As observed above, biosynthesized ZnO NPs depict better antibacterial activity against various microorganisms when compared to pure extract.
Previous studies imply that green ZnO nanoparticles showed more enhanced biocidal activity against various pathogens when compared to chemical ZnO nanoparticles [83]; furthermore, Susheela et al. reported that green synthesized nanoparticles show more effective antimicrobial activity than those of plant extracts [84].
Surface modifying reagent molecules result in differences in the release of Zn 2+ ions and the production of reactive oxygen species (ROS). S. ebulus leaf extract contains enormous numbers of carbonyl, carboxyl, and hydroxyl groups in polyphenols and flavonoids [35,[69][70][71]. These compounds can have a positive effect on human health because they can act as antioxidants and anti-inflammatory and anticancer agents. Indeed, these compounds present a wide range of biological activities, and the presence of abundant hydroxyl and carbonyl groups is responsible for antibacterial applications [69][70][71]. These compounds can affect the release of Zn 2+ and the production of ROS. The existence of these compounds in the extract and the surface helps ZnO NPs to adhere to the bacterial cell membranes. The chemical interaction between the extract molecules and ZnO NPs enhance the reactivity of NPs and improves penetration via bonding to the surface of the bacteria. Plant extract compounds attach to the surface of the bacteria and create new pathways near the surface for better transportation of or penetration by ions. For example, hydrogen in these compounds acts as both an oxidizing and a reducing agent (due to its two distinct oxidation states) which can easily form many bonds with other molecules [72,73].
In a recent study, it was found that Albizia lebbeck stem bark increased the antibacterial effect of biosynthesized ZnO NPs [74]. In another investigation, ZnO NPs (100-190 nm) were synthesized using Trifolium pratens flower extract and the high zone of their growth inhibition (29-31 mm) against E. coli, and S. aureus at~1200 µg/mL was obtained [20]. In the work performed by Gunalan et al. [75], the biosynthesized ZnO NPs using Aloe vera extract showed enhanced antibacterial properties compared with chemically prepared ZnO NPs [75]. On the other hand, Arvanag et al. [76] synthesized ZnO NPs with Silybum marianum L. seed extract and reported effective growth inhibition of Gram-negative E. coli. In another study, silver-loaded ZnO nanowires were synthesized under UV light and exhibited strong antibacterial effects against E. coli [73]; it was found that UV exposure caused more ROS species formation that increased antibacterial activity. In our study, we achieved lower used concentration values of S. ebulus leaf extract-loaded ZnO NPs that exhibited more effective antimicrobial activity against E. coli. It should be mentioned that despite the novel properties of Sambucus ebulus, similar to many kinds of plants, they may exhibit toxicity when large amounts of them are used [18]. Briefly, it can be concluded that combining ZnO NPs with S. ebulus extract is a simple, safe, and cost-effective approach for producing new materials with great potential for future biomedical and industrial applications.

Conclusions
In this study, ZnO NPs were successfully prepared through a green synthesis method using S. ebulus leaf extract, which showed interesting properties. X-ray diffraction analysis confirmed the formation of a ZnO hexagonal wurtzite structure with a crystallite size of 17 nm. The prepared ZnO NPs exhibited high UV absorbance, strong orange emission at room temperature, and acceptable photocatalytic degradation of MB dye pollutant. Moreover, the prepared ZnO NPs demonstrated efficient antibacterial and antioxidant activities. Surface modifying reagent molecules from the extract resulted in better antibacterial activity, which may be due to the differences in the release of Zn 2+ ions and the production of reactive oxygen species. The results of our investigation demonstrated that biosynthesized ZnO NPs possess promising potential in medical care, food packaging, and industrial applications as an alternative to chemical compounds. Further experiments should be performed, including in vivo measurements, and side effects of ingesting this compound should be thoroughly investigated.