# Pomegranate Peel Extract-Mediated Green Synthesis of ZnO-NPs: Extract Concentration-Dependent Structure, Optical, and Antibacterial Activity

Authors: Adnan Alnehia; Abdel-Basit Al-Odayni; Annas Al-Sharabi; A. H. Al-Hammadi; Waseem Sharaf Saeed
Journal: Journal of Chemistry
 (2022)

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## Abstract

Plant-based nanoparticles (NPs) have many advantages over physical and chemical methods and featured with several medicinal and biological applications. In this study, zinc oxide NPs (ZnO-NPs) were synthesized usingpomegranate peel aqueous extract, under mild and ecofriendly conditions. The ZnO-NPs structure, morphology, and optical properties were investigated using X-ray diffraction (XRD), scanning electron microscope (SEM), Fourier transform infrared (FTIR), and ultraviolet-visible (UV-Vis). Antibacterial activity against Gram-positive and Gram-negative strains were evaluated using the disk diffusion method. The effect of extract concentration (20, 30, and 40 mL) on the final properties of NPs, as well as the NPs concentration used for antibacterial test (50, 100, and 200 mg/mL), were also studied. The results indicate a hexagonal structure with particle size increases as extract concentration increase (D  = 18.53, 29.88, and 30.34 nm), while the optical bandgap was decreased (Eg = 2.87, 2.80, and 1.92 eV). The antibacterial activity of ZnO-NPs indicated high efficiency, similar or even higher than that of the control azithromycin, more against S. aureus, increased with NPs concentration, and preferred when NPs prepared from high extract concentration. Such promising physicochemical properties support the usefulness and efficacy of the reported bio-route for production of ZnO-NPs and may encourage its application for large-scale production.


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## Body

## 1. Introduction

Plant-mediated synthesis of NPs is an innovative industrial technique with plenty of profitable and ecofriendly features [1, 2]. Green synthesis of metal and metal-oxide NPs is one interesting issue of nanoscience, with plants seem to be the best candidates for the large-scale application [3]. The use of plant extract for making such NPs is, on the one hand, cost-effective, easy to be scaled up, and environmentally benign and, on the other hand, the resulting nanoproduct is more stable and tailored in shapes and sizes compared to those obtained by other organisms [3, 4].Among metal oxide NPs, ZnO nanostructures are the forefront of research due to their unique features and wide applications. ZnO-NPs can be synthesized through several ways such as chemical (sol-gen and solvothermal), physical (evaporation-condensation and laser ablation), and biological methods. Due to the use of organic solvents and the nature of chemical reactions that may produce harmful chemicals for environment and human being which possibly adsorbed on the NPs’ surface, the chemical method is not favored in production of NPs. Likewise, physical methods are associated with some difficulties like the high cost and requirement of harsh conditions such as high pressure and temperature [5, 6]. Thus, biosynthesis has increasingly become a focus of research interest in this field, providing attractive alternative to the conventional chemical and physical methods, due to its simplicity, eco-friendliness, low price, and considerable antimicrobial activity [3, 7]. Besides of its simplicity, biosynthesis commonly needs no expensive equipment or training, while it provides pure products.It is known that the nature of biological entities (extract, enzymes, and proteins) used to reduce, and stabilizeation of NPs influence their end-properties, of the synthesized NPs including their structure, shape, size, and morphology, and thus, bioactivity. Using plants as biogenic source for biosynthesis of NPs, the plant type, extraction protocol, solvent employed, and extract concentration play an important role in the properties of the NPs, with precursor concentration being the significant factor affecting the morphology of the synthesized ZnO-NPs. Besides bacteria strain, the bioactivity has been reported to also depend on concentration and morphology of ZnO-NPs [8].ZnO-NPs prepared using plant extracts have shown favored optical and biological properties compared with those from conventional methods of synthesis [3, 5, 9–11]. In addition, biosynthesis of such metal-oxide NPs is one advantageous method, due to the wide concern of pollution, principally because the concept of environmental protection is now deeply rooted in the expectations of the population [8, 12].Pomegranate is a familiar, sweet tasting fruit with hard pericarps. A fruit yields about half of its weight in juice, which leads to a lot of peel waste [13, 14]. The peel contains a variety of biologically active compounds those evidently responsible for their reported higher antibacterial properties [13–17] than leaves and flowers.At this point,pomegranate peel was targeted for synthesis of ZnO-NPs through a fabricated route. The obtained ZnO-NPs was fully characterized for its structural and optical properties using FTIR, XRD, SEM, and UV-Vis techniques. Then, the bioactivity against selected bacteria strains was evaluated in reference to azithromycin as a standard drug using the diffusion method.


## 2. Materials and Methods

### 2.1. Materials

All chemicals, including zinc nitrate hexahydrate (Zn (NO3)2·6H2O; ≥99%), sodium hydroxide (NaOH; 98%), and ethanol (EtOH; 99.5) were purchased from BDH Chemical Ltd. (Pool, England, UK) and used as received without further purification. Distilled water (DW) was used wherever required.


### 2.2.Pomegranate Peel Collection

Thepeels of the pomegranate fruit (PP) were collected from a local market at Thamar city (Thamar, Yemen) during the second half of summer season, 2021. The fruit originally comes from Saadah farms, Saadah governorate, Yemen, and freshly sales within two-to-three weeks of harvesting. To remove the dust particles, the peels were washed thoroughly four-to-five times with tap water, then by DW three times. The clean peels were left to dry at room temperature for three weeks. After that, the dried peels were ground to fine powder with the help of electrical grinder.


### 2.3. Preparation of Aqueous Extract

Typically, 15 g of PP dry powder were mixed with 200 mL DW to prepare the extract (termed PPE). The mixture was stirred at room temperature (24 ± 2°C) for 30 min during which the color of the media was changed from colorless to yellow. Subsequently, the solution temperature was increased and left at boiling for 5 min, then cooled to room temperature, filtered with Whatman No. 2 filter paper and used freshly as obtained for the synthesis of the target ZnO-NPs.


### 2.4. Biosynthesis of ZnO-NPs

To 25 mL aqueous solution of Zn (NO3)2·6H2O (5 g, 0.67 M), 10 mL of NaOH aqueous solution (1.5 g, 3.75 M) was added slowly over about 5 min, followed by dropwise addition of 20 mL of freshly prepared PPE. Upon completion of addition, the mixture was stirred at room temperature for 90 min, then filtered. The obtained precipitate was thoroughly and sequentially washed with ethanol and DW and left to dry at room temperature for 48 h. After that, the dry powder was annealed at 200°C for 3 h to obtain ZnO (termed Z20). The same protocol was applied to prepare Z30 and Z40 in which the volumes of the extract used (i.e., PPE) were 30 and 40 mL, respectively. The overall scheme for biosynthesis of ZnO-NPs and its bioactivity are summarized in Figure 1.Figure 1
biosynthesis route of ZnO-NPs usingpomegranate peel extract, characterization methods, and bioactivity.


### 2.5. Biological Studies

#### 2.5.1. Antibacterial Test

Thein vitro antibacterial activity of the synthesized ZnO-NPs (Z20, Z30, and Z40) was evaluated by screening against Gram-positive (Staphylococcus aureus (S. aureus)) and Gram-negative (Escherichia coli (E.coli)) bacteria using the disk-diffusion method as described in the literature [18] and in reference to Azithromycin (AzM) standard drug. The test bacteria were kind gifts from Al-Jarfi medical Lab (Thamar city, Yemen). The media, nutrient agar (Hi-media, Mumbai, India) was prepared in accordance with the manufacturer’s recommendation. Hence, freshly bacteria suspension was made to an inoculum density equivalent to 0.05 McFarland (1.5 × 108 CFU (colony-forming unit)/mL)). The agar plates were inoculated with the test bacteria with the aid of sterilized swabs. The ZnO-NPs were suspended by sonication in DW to obtain 50, 100, and 200 mg/mL concentrations and used for antibacterial tests [5]. Sterile filter paper disks with diameters of 6 mm were fabricated and immersed in the NPs suspensions. Similarly, disks of AzM and DW were also prepared and used as positive and negative controls, respectively. The paper disks were placed aseptically on the surface of bacterially seeded Petri dishes and incubated at 37°C for 21–22 h. The zone of inhibition (ZOI) was determined by measuring the diameter of the inhibition area in mm [19, 20].


#### 2.5.2. Hemolytic Assay

The biosafe nature of the synthesized biogenic ZnO-NPs was assessed using the hemolytic assay against human red blood cells (RBC), following a method described elsewhere [21] with slight modification. Briefly, fresh blood was collected from healthy individuals with a sterile needle (a 25-year-old male volunteer with an O-positive-blood group) after the provision of informed consent. The blood was then dispersed in EDTA-containing tubes to prevent clotting, and RBCs were isolated by centrifugation (1 mL blood) at 4000 rpm for 10 min, followed by careful removal of supernatant and washing the pellet with normal saline solution (NS; 0.9 w/v% sodium chloride, pH 4.5–7.0; Pharmaceutical Solutions Industry, Jeddah, Saudi Arabia). Erythrocyte suspension was then prepared in NS to obtain 2% cell suspension. Test samples of ZnO-NPs were prepared in NS to final concentrations of 3.12–200 μg/mL. To previously marked test tubes containing 0.5 mL of the cell’s suspension, 0.5 mL of each test samples, NS (negative control), and DW (positive control) were added, immediately transferred into 37°C incubator, and left for 60 min. After incubation, the solutions were centrifuged at 4000 rpm for 10 min and the separated supernatant was photometrically measured for the released free hemoglobin at 540 nm.The hemolytic activity was calculated using the following formula (Equation (1):(1)%hemolysis=AS−ANAP−AN×100,where AS, AN, and AP are the absorbance of the test sample (ZnO-NPs), negative control (NS), and positive control (DW), respectively.


### 2.6. Characterization

The diffraction patterns of ZnO-NPs were obtained using an XD-2 X-ray diffractometer (Beijing Purkinje General Instrument Co., Ltd., Beijing, China), with CuKα1 radiation of λ = 1.54 Å, in the 2θ range of 10 to 80 and scanning rate of 0.02 min−1. The electronic spectra were recorded on a U-3900 UV-Vis spectrophotometer (Hitachi, Tokyo, Japan), over wavelength range of 200–900 nm at room temperature. Electron micrographs were obtained for NP sputter coated with gold samples, using a JSM-6360 LV SEM (Jeol Ltd., Tokyo, Japan). FTIR spectra were measured on a Nicolet iS10 FTIR spectrometer from Thermo Scientific (Madison, WI, USA), equipped with an attenuated total reflection (ATR, diamond crystal) accessory, over the range of 650–4000 cm−1, with 32 scans per spectrum and 4 cm−1 scanning resolution at room temperature.


## 2.1. Materials

All chemicals, including zinc nitrate hexahydrate (Zn (NO3)2·6H2O; ≥99%), sodium hydroxide (NaOH; 98%), and ethanol (EtOH; 99.5) were purchased from BDH Chemical Ltd. (Pool, England, UK) and used as received without further purification. Distilled water (DW) was used wherever required.


## 2.2.Pomegranate Peel Collection

Thepeels of the pomegranate fruit (PP) were collected from a local market at Thamar city (Thamar, Yemen) during the second half of summer season, 2021. The fruit originally comes from Saadah farms, Saadah governorate, Yemen, and freshly sales within two-to-three weeks of harvesting. To remove the dust particles, the peels were washed thoroughly four-to-five times with tap water, then by DW three times. The clean peels were left to dry at room temperature for three weeks. After that, the dried peels were ground to fine powder with the help of electrical grinder.


## 2.3. Preparation of Aqueous Extract

Typically, 15 g of PP dry powder were mixed with 200 mL DW to prepare the extract (termed PPE). The mixture was stirred at room temperature (24 ± 2°C) for 30 min during which the color of the media was changed from colorless to yellow. Subsequently, the solution temperature was increased and left at boiling for 5 min, then cooled to room temperature, filtered with Whatman No. 2 filter paper and used freshly as obtained for the synthesis of the target ZnO-NPs.


## 2.4. Biosynthesis of ZnO-NPs

To 25 mL aqueous solution of Zn (NO3)2·6H2O (5 g, 0.67 M), 10 mL of NaOH aqueous solution (1.5 g, 3.75 M) was added slowly over about 5 min, followed by dropwise addition of 20 mL of freshly prepared PPE. Upon completion of addition, the mixture was stirred at room temperature for 90 min, then filtered. The obtained precipitate was thoroughly and sequentially washed with ethanol and DW and left to dry at room temperature for 48 h. After that, the dry powder was annealed at 200°C for 3 h to obtain ZnO (termed Z20). The same protocol was applied to prepare Z30 and Z40 in which the volumes of the extract used (i.e., PPE) were 30 and 40 mL, respectively. The overall scheme for biosynthesis of ZnO-NPs and its bioactivity are summarized in Figure 1.Figure 1
biosynthesis route of ZnO-NPs usingpomegranate peel extract, characterization methods, and bioactivity.


## 2.5. Biological Studies

### 2.5.1. Antibacterial Test

Thein vitro antibacterial activity of the synthesized ZnO-NPs (Z20, Z30, and Z40) was evaluated by screening against Gram-positive (Staphylococcus aureus (S. aureus)) and Gram-negative (Escherichia coli (E.coli)) bacteria using the disk-diffusion method as described in the literature [18] and in reference to Azithromycin (AzM) standard drug. The test bacteria were kind gifts from Al-Jarfi medical Lab (Thamar city, Yemen). The media, nutrient agar (Hi-media, Mumbai, India) was prepared in accordance with the manufacturer’s recommendation. Hence, freshly bacteria suspension was made to an inoculum density equivalent to 0.05 McFarland (1.5 × 108 CFU (colony-forming unit)/mL)). The agar plates were inoculated with the test bacteria with the aid of sterilized swabs. The ZnO-NPs were suspended by sonication in DW to obtain 50, 100, and 200 mg/mL concentrations and used for antibacterial tests [5]. Sterile filter paper disks with diameters of 6 mm were fabricated and immersed in the NPs suspensions. Similarly, disks of AzM and DW were also prepared and used as positive and negative controls, respectively. The paper disks were placed aseptically on the surface of bacterially seeded Petri dishes and incubated at 37°C for 21–22 h. The zone of inhibition (ZOI) was determined by measuring the diameter of the inhibition area in mm [19, 20].


### 2.5.2. Hemolytic Assay

The biosafe nature of the synthesized biogenic ZnO-NPs was assessed using the hemolytic assay against human red blood cells (RBC), following a method described elsewhere [21] with slight modification. Briefly, fresh blood was collected from healthy individuals with a sterile needle (a 25-year-old male volunteer with an O-positive-blood group) after the provision of informed consent. The blood was then dispersed in EDTA-containing tubes to prevent clotting, and RBCs were isolated by centrifugation (1 mL blood) at 4000 rpm for 10 min, followed by careful removal of supernatant and washing the pellet with normal saline solution (NS; 0.9 w/v% sodium chloride, pH 4.5–7.0; Pharmaceutical Solutions Industry, Jeddah, Saudi Arabia). Erythrocyte suspension was then prepared in NS to obtain 2% cell suspension. Test samples of ZnO-NPs were prepared in NS to final concentrations of 3.12–200 μg/mL. To previously marked test tubes containing 0.5 mL of the cell’s suspension, 0.5 mL of each test samples, NS (negative control), and DW (positive control) were added, immediately transferred into 37°C incubator, and left for 60 min. After incubation, the solutions were centrifuged at 4000 rpm for 10 min and the separated supernatant was photometrically measured for the released free hemoglobin at 540 nm.The hemolytic activity was calculated using the following formula (Equation (1):(1)%hemolysis=AS−ANAP−AN×100,where AS, AN, and AP are the absorbance of the test sample (ZnO-NPs), negative control (NS), and positive control (DW), respectively.


## 2.5.1. Antibacterial Test

Thein vitro antibacterial activity of the synthesized ZnO-NPs (Z20, Z30, and Z40) was evaluated by screening against Gram-positive (Staphylococcus aureus (S. aureus)) and Gram-negative (Escherichia coli (E.coli)) bacteria using the disk-diffusion method as described in the literature [18] and in reference to Azithromycin (AzM) standard drug. The test bacteria were kind gifts from Al-Jarfi medical Lab (Thamar city, Yemen). The media, nutrient agar (Hi-media, Mumbai, India) was prepared in accordance with the manufacturer’s recommendation. Hence, freshly bacteria suspension was made to an inoculum density equivalent to 0.05 McFarland (1.5 × 108 CFU (colony-forming unit)/mL)). The agar plates were inoculated with the test bacteria with the aid of sterilized swabs. The ZnO-NPs were suspended by sonication in DW to obtain 50, 100, and 200 mg/mL concentrations and used for antibacterial tests [5]. Sterile filter paper disks with diameters of 6 mm were fabricated and immersed in the NPs suspensions. Similarly, disks of AzM and DW were also prepared and used as positive and negative controls, respectively. The paper disks were placed aseptically on the surface of bacterially seeded Petri dishes and incubated at 37°C for 21–22 h. The zone of inhibition (ZOI) was determined by measuring the diameter of the inhibition area in mm [19, 20].


## 2.5.2. Hemolytic Assay

The biosafe nature of the synthesized biogenic ZnO-NPs was assessed using the hemolytic assay against human red blood cells (RBC), following a method described elsewhere [21] with slight modification. Briefly, fresh blood was collected from healthy individuals with a sterile needle (a 25-year-old male volunteer with an O-positive-blood group) after the provision of informed consent. The blood was then dispersed in EDTA-containing tubes to prevent clotting, and RBCs were isolated by centrifugation (1 mL blood) at 4000 rpm for 10 min, followed by careful removal of supernatant and washing the pellet with normal saline solution (NS; 0.9 w/v% sodium chloride, pH 4.5–7.0; Pharmaceutical Solutions Industry, Jeddah, Saudi Arabia). Erythrocyte suspension was then prepared in NS to obtain 2% cell suspension. Test samples of ZnO-NPs were prepared in NS to final concentrations of 3.12–200 μg/mL. To previously marked test tubes containing 0.5 mL of the cell’s suspension, 0.5 mL of each test samples, NS (negative control), and DW (positive control) were added, immediately transferred into 37°C incubator, and left for 60 min. After incubation, the solutions were centrifuged at 4000 rpm for 10 min and the separated supernatant was photometrically measured for the released free hemoglobin at 540 nm.The hemolytic activity was calculated using the following formula (Equation (1):(1)%hemolysis=AS−ANAP−AN×100,where AS, AN, and AP are the absorbance of the test sample (ZnO-NPs), negative control (NS), and positive control (DW), respectively.


## 2.6. Characterization

The diffraction patterns of ZnO-NPs were obtained using an XD-2 X-ray diffractometer (Beijing Purkinje General Instrument Co., Ltd., Beijing, China), with CuKα1 radiation of λ = 1.54 Å, in the 2θ range of 10 to 80 and scanning rate of 0.02 min−1. The electronic spectra were recorded on a U-3900 UV-Vis spectrophotometer (Hitachi, Tokyo, Japan), over wavelength range of 200–900 nm at room temperature. Electron micrographs were obtained for NP sputter coated with gold samples, using a JSM-6360 LV SEM (Jeol Ltd., Tokyo, Japan). FTIR spectra were measured on a Nicolet iS10 FTIR spectrometer from Thermo Scientific (Madison, WI, USA), equipped with an attenuated total reflection (ATR, diamond crystal) accessory, over the range of 650–4000 cm−1, with 32 scans per spectrum and 4 cm−1 scanning resolution at room temperature.


## 3. Results

### 3.1. Structural Characterization

#### 3.1.1. FTIR Analysis

Figure2 represents the FTIR spectra of the PPE-mediated ZnO-NPs (Z20, Z30, and Z40). The spectra are accompanied with indicative peaks of the obtained NPs and traced capping agents from pomegranate peels. The broadband on the range 3070–3550 cm−1, in which at least three peaks were identifiable at 3396, 3278, and 3169 cm−1 and more clearly in the spectrum of the Z40 sample was attributed to various v (OH) and v (NHn) groups, including Zn-OH, free and H-bonding water-OH [22, 23], alcohols, and amides that possibly are a part of NPs-stabilizing compounds, and the capping agents. The peaks at 1675 and 1627 cm−1 assigned to C = O and C = C stretching bands of flavonoids and amides in the extract [24] are weak, suggesting their contribution in ZnO-NPs stabilization. These bands were highly overlapped with C-C absorbance around 1590 cm−1 and being almost invisible in the spectra of Z20 and Z30, possibly due to their sourced low contents. Absorption at 1590, 1591, and 1551 cm−1 in Z20, Z30, and Z40, respectively, is associated with C-C stretching band and its high intensity may indicate its abundance due to reduction of alkenes involved in the production of ZnO-NPs [24, 25]. Besides, the bands at 1396 and 1336 cm−1 may characterize C-N stretching or CH and OH bending vibrations of aromatic structures. Hence, it is reported that the amide group, amino, carbonyl group, and polyphenolic compounds in the PPE are a part of redox reaction, dispersion, capping, and stabilizers involved in the production of nanoparticles during the process of synthesis [24, 26, 27]. Bands at 1019 and 929 cm−1 are ascribed to asymmetric and symmetric stretching vibrations of C-O-O bonds [27].Figure 2
FTIR pattern of the synthesized PPE-mediated ZnO-NPs.The characteristic peaks of ZnO-NPs usually seen on the fingerprint frequencies, i.e., below 900 cm−1. However, due to capacity limit of the ATR-FTIR instrument (4000–650 cm−1), peaks below 650 cm−1 were, unfortunately, not reported. Nevertheless, the spectra are incorporated with strong peaks at 670 cm−1 which proposed the formation of ZnO-NPs [1].


#### 3.1.2. XRD Analysis

The XRD patterns of the synthesized PPE-mediated ZnO-NPs are shown in Figure3(a). The spectra exhibit nine diffraction peaks on the range of 2θ° 25–75 and some of these peaks are detailed in Table 1. The diffractograms have ascertained material purity with no other external peaks observed, suggesting that the applied method of synthesis is effective to obtain ZnO-NPs of high purity. However, unidentifiable peak at 2θ° of 30.10 in Z20 profile may be due to some organics in the extract. Furthermore, the analysis revealed a hexagonal phase as compared with the database (JCPDS No. 36–1451). It is obvious that the diffraction peaks are moderately broad and, thus, indicating highly crystalline ZnO-NPs. According to the literature [28, 29], the broad peak is an indication of small and fine NPs (nanoscale crystalline particles) while the narrow or low intensity peak signifies low crystallinity of the NPs. As shown in Figure 3(b) and Table 1, the average particle diameter (D) of the biosynthesized ZnO-NPs, which calculated using the Debye–Scherrer equation (Equation (2), were between 18.53 and 30.38 nm [30–32]. The dislocation density (δ) of the fabricated samples is specified using the Williamson and Smallman’s relation (Equation (3)) [33, 34].(2)D=kλβcosθ,(3)δ=1D2,where D is the crystallite size, k is a constant denotes the shape factor (0.94), λ is the diffraction wavelength of CuKα (λ = 1.5406 Å), β is the full width at half maximum (FWHM), θ is the diffraction angle, and δ is the dislocation density. Figure 3(a) (insert) represents the shifting in the peak position toward higher angle with crystallite size increase which, in turn, reflect the effect of preparation extract volume [35]. Other structural parameters such as d-spacing (Å), a (Å), c (Å), c/a ratio, unit cell volume v (Å)3, the volume of particles V (nm)3, atomic packing factor (%), and the degree of crystal lattice distortion (R) were also computed [36, 37], and the corresponding values are gathered in Table 2.Figure 3
(a) X-ray diffraction (XRD) pattern of the synthesized ZnO NPs (Z20, Z30, and Z40); inset is a magnification of the prominent peak of (101) plane. (b) Crystallite sizes (D, nm) and dislocation (δ) vs. extract volume (20–40 mL).
(a)(b)Table 1
Structural parameters of ZnO-NPs.
Samples2-Thetad-spacingFWHM (deg)Planes (hkl)Crystallite sizes (D, nm)Average (D, nm)Average dislocation (lines/m2) (1015)Z2031.8012.8110.56610014.5918.5342.9134.5392.5940.38100221.8336.3392.4700.47510117.6047.4231.9150.70310212.3456.7001.6220.34311026.31Z3032.1412.8540.34410024.0329.8781.1234.3212.6100.15700252.9636.6802.4480.34010124.6247.9791.8940.40510221.4757.0391.6130.42611021.22Z4032.2792.7710.34010024.3330.3781.0834.9392.5650.29400228.3236.7802.4410.39010121.4647.5351.9110.13910262.4557.0811.6120.59011015.33Table 2
Geometric parameters of ZnO-NPs as computed from XRD.
SamplesLattice parameters (Å)(c/a) ratioVolumes of unit cell (Å3)Volumes of particles, V (nm)3Atomic packing factor (%)Degrees of crystal lattice distortion (R)abcZ203.2463.2465.1991.60247.443331.8575.45821.019559Z303.2653.2655.2041.60048.0413958.375.826961.024541Z403.2163.2165.2221.62046.7714670.974.431531.005687


#### 3.1.3. SEM Analysis

Figure4 shows the SEM micrograph of the synthesized ZnO-NPs (Z20, Z30, and Z40) in which the ZnO were mainly composed of nanoplatelets with an overall quite dense morphology. The nanosheets’ thickness could be estimated to a tenth of nm; however, the thicker sheets may consist of several sheets aggregated to form the nanoplate network [27], with irregular NPs having almost spherical shapes [5]. Such aggregation and flaky agglomeration could be due to a high surface energy of the NPs and also perhaps due to densification of the narrow space between NPs [38]. A similar morphology of ZnO-NPs was also reported by a number of researchers [11, 39]. It seems that the aggregation, as well as the flake thicknesses, become less for ZnO-NPs produced at lower extract concentrations and this may explain the decreased bioactivity on the same order, i.e., antibacterial activity order Z20 > Z30 > Z40.Figure 4
Scanning electron microscope (SEM) image of ZnO-NPs. (a) Z20; (b) Z30; (c) Z40.
(a)(b)(c)


#### 3.1.4. UV-Vis Analysis

Figure5 shows the UV-Vis spectra of the ZnO-NPs prepared with various PPE concentrations (Z20, Z30, and Z3). The spectra revealed characteristic maximum absorbances at 370, 375, and 378 nm, respectively, indicating quantum size effects [38]. The bandgap energy (Eg) as calculated from Tauc’s plotting for the direct allowed transition, described by Equation (3) [40–42], was found at 2.87, 2.80, and 1.92 eV, respectively for ZnO-NPs (Z20, Z30, and Z40) prepared using different extract volumes (20, 30, and 40 mL) (Figures 5(b)–5(d))).This denotes that the extract volume has an impact on the resulting bandgap of the synthesized ZnO-NPs, i.e., with PPE extract volume increase, the Eg decreased.(4)αhν=Chν−Egn.Figure 5
Absorption spectra of ZnO nanoparticles (a) and determination ofEg (b, c, and d).
(a)(b)(c)(d)Typically, theEg depends on the structure, size, and shape of the nanoparticles and, therefore, the employment of different extract volume can adjust such properties. By increasing PPE volume, a red shift in the optical bandgap is observed. Such an effect is obviously the result of the organic compounds amount differences present in the extract. According to the literature [13], the PPE is rich with phenolic compounds that may drive production of the ZnO-NPs which further affected by the extract volume used, i.e., the magnitude of phytocompounds.


### 3.2. Antibacterial Activity of ZnO-NPs

The antibacterial activity of the biosynthesized ZnO-NPs was tested against both Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria using the disk-diffusion method. For this, three different concentrations 50, 100, and 200 mg/mL of the biosynthesized ZnO-NPs (Z20, Z30, and Z40) were prepared and used for antibacterial activity studies in comparison to AzM as a standard drug. The resulting zones of inhibition (ZOI) are summarized in Table 3, and selected images of inhibition plates are given in Figure 6. It was observed that all the tested ZnO-NPs have an inhibitory effect against S. aureus and E. coli similar to or higher than the AzM standard drug. The ZOI was higher against the Gram-positive bacteria (S. aureus) compared to Gram-negative bacteria (E. coli). This might be due to the fact that Gram-positive bacteria are less susceptible to antibacterial potency than Gram-negative bacteria, perhaps this is a result of their different cell wall structures [43].Table 3
Antibacterial activity of ZnO-NPs using the zone of inhibition method compared to Azithromycin standard drug.
SamplesBacteriaZone of inhibition (ZOI; diameter in mm) ± standard deviation (SD) at various concentrationsZnO-NPs concentration (mg/mL)Azithromycin (control)50 mg/mL100 mg/mL200 mg/mLZ20S. aureus27 ± 1.2531 ± 1.7532 ± 1.2528 ± 0.25E. coli22 ± 1.0526 ± 0.2527 ± 1.2523 ± 0.25Z30S. aureus29 ± 2.0026 ± 1.7534 ± 1.5028 ± 0.50E. coli22 ± 0.2523 ± 0.2525 ± 2.2523 ± 0.25Z40S. aureus13 ± 0.5015 ± 0.2517 ± 0.0028 ± 0.25E. coli12 ± 0.2515 ± 0.5016 ± 0.7523 ± 0.50Figure 6
(a) Selected plate images of antibacterial activity tests of ZnO-NPs prepared using 20, 30, and 40 mL PPE (from the bottom to the top: Z20, Z30, and Z40) againstS. aureus (left images) and E. coli (right images). Disks: (1) ZnO-NPs 50 mg/mL per disc; (2) ZnO-NPs 100 mg/mL per disc; (3) ZnO-NPs 200 mg/mL per disc; (4) Azithromycin antibiotics (positive control); (5) distilled water (negative control). (b) Histogram illustration for the corresponding zone of inhibition data; bars represent the standard error of the mean (n = 2).
(a)(b)In the Gram-positive strain, peptidoglycan is thick while being thinner in the Gram-negative strain, but contains an outer membrane consisting of lipopolysaccharides that provides the bacteria resistance to prepared ZnO and makes them less susceptible [44]. The antibacterial potency of ZnO-NPs against microorganisms depends on cell wall integrity [43, 45]. The results indicate that the use of pomegranate peel extract-mediated synthesis of ZnO nanoparticles can be more efficient against Gram-positive bacteria and Gram-negative bacteria. This may be due to the existence of the higher number of phenolic compounds. Moreover, the results illustrated that the prepared samples have a strong antibacterial activity against both the strains compared to Azithromycin.It is obvious that the ZOI is low for ZnO-NPs prepared using Z40, the case that can be attributed to the higher particle sizes of Z40 (30.34 nm) compared to those of Z20 (18.53 nm) and Z30 (29.88 nm). The highest antibacterial activity for the lower particle sizes, e.g., Z20, is due to their smaller size (18.53 nm) which, in turn, means increased active surface area that further facilitates ease interaction with the bacterial wall [40]. With the increase in ZnO-NPs concentration from 50 to 200 mg/mL, a gradual increase in the bacterial inhibition was also observed, supporting the concentration-dependence activity of the NPs [46, 47].The bioactivity of NPs could be attributed to various factors such as chemical composition, particle size and shape, concentration, surface charge, and exposure time. The destructive action of ZnO-NPs on microorganisms could be due to one or simultaneous mechanisms: (i) attachment of NPs to a bacterial surface [46], hence the following stepwise events were proposed to be involved in adsorption of NPs on the bacterial surface facilitated by surface potential, distortion of cell morphology, NPs penetration into cells, membrane damage due to structural and functional interruption, and leakage of cellular components, thus functionality loss [9, 47, 48]; (ii) Zn2+ release from ZnO-NPs which, up on penetration into the cell, can inhibit several bacterial activities including transports, metabolisms, and enzyme functions, leading to cell death; (iii) ZnO activity as a result of the formation of reactive oxygen species (ROS) which leads to oxidative stress and subsequent cell damage [49]. An illustration of the proposed mechanism is given in Figure 7.Figure 7
The proposed antibacterial mechanism of ZnO-NPs.


### 3.3. Biocompatibility of ZnO-NPs with Human Erythrocytes

Biocompatibility of ZnO-NPs was assessed usingin vitro hemolysis assay at different concentrations 3.12–200 μg/mL, against RBCs and in reference to NS and DW as negative and positive controls [21], respectively. Hemolysis is generally based on measuring hemoglobin released from RBC after sample-induced cell lysis. Figure 8 illustrates the averaged data obtained from the two experiments. As can be seen, the toxicity of ZnO-NPs at the evaluated highest concentration (200 μg/mL) was 17.4%, which agreed with the previous reports [11, 50]. The hemolysis effect at 3.12 and 6.25 μg/mL were around 4.6 and 7.5%, increased to 16.8% for 25 μg/mL above which no significant differences could be observed up to 200 μg/mL. Basically, substances with hemolysis <2% is standardized as nonhemolytic, 2–5% slightly hemolytic while >5% hemolytic [21]. The obtained values at low concentrations revealed low toxicity and are in agreement with the literature [50] stated that ZnO-NPs concentration lower than 5% is nontoxic, being slightly hemolytic under 5–40 μg/mL while hemolytic at > 40 μg/mL. Muhammad et at. [11], have reported a 21.8% hemolytic for ZnO-NPs at a concentration of 200 μg/mL, however researchers have detected no hemolysis at concentrations below 5 μg/mL. Meanwhile, the hemolytic effect of ZnO-NPs has been reported to be concentration-dependent [11, 50], other factors including test conditions (medium, cells, and positive and negative controls), parameters (sample concentration and incubation time), and the substance nature (biogenic source, particle shape, and size) have to be analyzed as well to elucidate slight differences among studies [51].Figure 8
Hemolysis of erythrocytes caused by ZnO-NPs at different concentrations (3.12–200μg/mL), normal saline (NS, negative control), and distilled water (DW, positive control). Insert is an illustration of samples absorption (OD) including controls.


## 3.1. Structural Characterization

### 3.1.1. FTIR Analysis

Figure2 represents the FTIR spectra of the PPE-mediated ZnO-NPs (Z20, Z30, and Z40). The spectra are accompanied with indicative peaks of the obtained NPs and traced capping agents from pomegranate peels. The broadband on the range 3070–3550 cm−1, in which at least three peaks were identifiable at 3396, 3278, and 3169 cm−1 and more clearly in the spectrum of the Z40 sample was attributed to various v (OH) and v (NHn) groups, including Zn-OH, free and H-bonding water-OH [22, 23], alcohols, and amides that possibly are a part of NPs-stabilizing compounds, and the capping agents. The peaks at 1675 and 1627 cm−1 assigned to C = O and C = C stretching bands of flavonoids and amides in the extract [24] are weak, suggesting their contribution in ZnO-NPs stabilization. These bands were highly overlapped with C-C absorbance around 1590 cm−1 and being almost invisible in the spectra of Z20 and Z30, possibly due to their sourced low contents. Absorption at 1590, 1591, and 1551 cm−1 in Z20, Z30, and Z40, respectively, is associated with C-C stretching band and its high intensity may indicate its abundance due to reduction of alkenes involved in the production of ZnO-NPs [24, 25]. Besides, the bands at 1396 and 1336 cm−1 may characterize C-N stretching or CH and OH bending vibrations of aromatic structures. Hence, it is reported that the amide group, amino, carbonyl group, and polyphenolic compounds in the PPE are a part of redox reaction, dispersion, capping, and stabilizers involved in the production of nanoparticles during the process of synthesis [24, 26, 27]. Bands at 1019 and 929 cm−1 are ascribed to asymmetric and symmetric stretching vibrations of C-O-O bonds [27].Figure 2
FTIR pattern of the synthesized PPE-mediated ZnO-NPs.The characteristic peaks of ZnO-NPs usually seen on the fingerprint frequencies, i.e., below 900 cm−1. However, due to capacity limit of the ATR-FTIR instrument (4000–650 cm−1), peaks below 650 cm−1 were, unfortunately, not reported. Nevertheless, the spectra are incorporated with strong peaks at 670 cm−1 which proposed the formation of ZnO-NPs [1].


### 3.1.2. XRD Analysis

The XRD patterns of the synthesized PPE-mediated ZnO-NPs are shown in Figure3(a). The spectra exhibit nine diffraction peaks on the range of 2θ° 25–75 and some of these peaks are detailed in Table 1. The diffractograms have ascertained material purity with no other external peaks observed, suggesting that the applied method of synthesis is effective to obtain ZnO-NPs of high purity. However, unidentifiable peak at 2θ° of 30.10 in Z20 profile may be due to some organics in the extract. Furthermore, the analysis revealed a hexagonal phase as compared with the database (JCPDS No. 36–1451). It is obvious that the diffraction peaks are moderately broad and, thus, indicating highly crystalline ZnO-NPs. According to the literature [28, 29], the broad peak is an indication of small and fine NPs (nanoscale crystalline particles) while the narrow or low intensity peak signifies low crystallinity of the NPs. As shown in Figure 3(b) and Table 1, the average particle diameter (D) of the biosynthesized ZnO-NPs, which calculated using the Debye–Scherrer equation (Equation (2), were between 18.53 and 30.38 nm [30–32]. The dislocation density (δ) of the fabricated samples is specified using the Williamson and Smallman’s relation (Equation (3)) [33, 34].(2)D=kλβcosθ,(3)δ=1D2,where D is the crystallite size, k is a constant denotes the shape factor (0.94), λ is the diffraction wavelength of CuKα (λ = 1.5406 Å), β is the full width at half maximum (FWHM), θ is the diffraction angle, and δ is the dislocation density. Figure 3(a) (insert) represents the shifting in the peak position toward higher angle with crystallite size increase which, in turn, reflect the effect of preparation extract volume [35]. Other structural parameters such as d-spacing (Å), a (Å), c (Å), c/a ratio, unit cell volume v (Å)3, the volume of particles V (nm)3, atomic packing factor (%), and the degree of crystal lattice distortion (R) were also computed [36, 37], and the corresponding values are gathered in Table 2.Figure 3
(a) X-ray diffraction (XRD) pattern of the synthesized ZnO NPs (Z20, Z30, and Z40); inset is a magnification of the prominent peak of (101) plane. (b) Crystallite sizes (D, nm) and dislocation (δ) vs. extract volume (20–40 mL).
(a)(b)Table 1
Structural parameters of ZnO-NPs.
Samples2-Thetad-spacingFWHM (deg)Planes (hkl)Crystallite sizes (D, nm)Average (D, nm)Average dislocation (lines/m2) (1015)Z2031.8012.8110.56610014.5918.5342.9134.5392.5940.38100221.8336.3392.4700.47510117.6047.4231.9150.70310212.3456.7001.6220.34311026.31Z3032.1412.8540.34410024.0329.8781.1234.3212.6100.15700252.9636.6802.4480.34010124.6247.9791.8940.40510221.4757.0391.6130.42611021.22Z4032.2792.7710.34010024.3330.3781.0834.9392.5650.29400228.3236.7802.4410.39010121.4647.5351.9110.13910262.4557.0811.6120.59011015.33Table 2
Geometric parameters of ZnO-NPs as computed from XRD.
SamplesLattice parameters (Å)(c/a) ratioVolumes of unit cell (Å3)Volumes of particles, V (nm)3Atomic packing factor (%)Degrees of crystal lattice distortion (R)abcZ203.2463.2465.1991.60247.443331.8575.45821.019559Z303.2653.2655.2041.60048.0413958.375.826961.024541Z403.2163.2165.2221.62046.7714670.974.431531.005687


### 3.1.3. SEM Analysis

Figure4 shows the SEM micrograph of the synthesized ZnO-NPs (Z20, Z30, and Z40) in which the ZnO were mainly composed of nanoplatelets with an overall quite dense morphology. The nanosheets’ thickness could be estimated to a tenth of nm; however, the thicker sheets may consist of several sheets aggregated to form the nanoplate network [27], with irregular NPs having almost spherical shapes [5]. Such aggregation and flaky agglomeration could be due to a high surface energy of the NPs and also perhaps due to densification of the narrow space between NPs [38]. A similar morphology of ZnO-NPs was also reported by a number of researchers [11, 39]. It seems that the aggregation, as well as the flake thicknesses, become less for ZnO-NPs produced at lower extract concentrations and this may explain the decreased bioactivity on the same order, i.e., antibacterial activity order Z20 > Z30 > Z40.Figure 4
Scanning electron microscope (SEM) image of ZnO-NPs. (a) Z20; (b) Z30; (c) Z40.
(a)(b)(c)


### 3.1.4. UV-Vis Analysis

Figure5 shows the UV-Vis spectra of the ZnO-NPs prepared with various PPE concentrations (Z20, Z30, and Z3). The spectra revealed characteristic maximum absorbances at 370, 375, and 378 nm, respectively, indicating quantum size effects [38]. The bandgap energy (Eg) as calculated from Tauc’s plotting for the direct allowed transition, described by Equation (3) [40–42], was found at 2.87, 2.80, and 1.92 eV, respectively for ZnO-NPs (Z20, Z30, and Z40) prepared using different extract volumes (20, 30, and 40 mL) (Figures 5(b)–5(d))).This denotes that the extract volume has an impact on the resulting bandgap of the synthesized ZnO-NPs, i.e., with PPE extract volume increase, the Eg decreased.(4)αhν=Chν−Egn.Figure 5
Absorption spectra of ZnO nanoparticles (a) and determination ofEg (b, c, and d).
(a)(b)(c)(d)Typically, theEg depends on the structure, size, and shape of the nanoparticles and, therefore, the employment of different extract volume can adjust such properties. By increasing PPE volume, a red shift in the optical bandgap is observed. Such an effect is obviously the result of the organic compounds amount differences present in the extract. According to the literature [13], the PPE is rich with phenolic compounds that may drive production of the ZnO-NPs which further affected by the extract volume used, i.e., the magnitude of phytocompounds.


## 3.1.1. FTIR Analysis

Figure2 represents the FTIR spectra of the PPE-mediated ZnO-NPs (Z20, Z30, and Z40). The spectra are accompanied with indicative peaks of the obtained NPs and traced capping agents from pomegranate peels. The broadband on the range 3070–3550 cm−1, in which at least three peaks were identifiable at 3396, 3278, and 3169 cm−1 and more clearly in the spectrum of the Z40 sample was attributed to various v (OH) and v (NHn) groups, including Zn-OH, free and H-bonding water-OH [22, 23], alcohols, and amides that possibly are a part of NPs-stabilizing compounds, and the capping agents. The peaks at 1675 and 1627 cm−1 assigned to C = O and C = C stretching bands of flavonoids and amides in the extract [24] are weak, suggesting their contribution in ZnO-NPs stabilization. These bands were highly overlapped with C-C absorbance around 1590 cm−1 and being almost invisible in the spectra of Z20 and Z30, possibly due to their sourced low contents. Absorption at 1590, 1591, and 1551 cm−1 in Z20, Z30, and Z40, respectively, is associated with C-C stretching band and its high intensity may indicate its abundance due to reduction of alkenes involved in the production of ZnO-NPs [24, 25]. Besides, the bands at 1396 and 1336 cm−1 may characterize C-N stretching or CH and OH bending vibrations of aromatic structures. Hence, it is reported that the amide group, amino, carbonyl group, and polyphenolic compounds in the PPE are a part of redox reaction, dispersion, capping, and stabilizers involved in the production of nanoparticles during the process of synthesis [24, 26, 27]. Bands at 1019 and 929 cm−1 are ascribed to asymmetric and symmetric stretching vibrations of C-O-O bonds [27].Figure 2
FTIR pattern of the synthesized PPE-mediated ZnO-NPs.The characteristic peaks of ZnO-NPs usually seen on the fingerprint frequencies, i.e., below 900 cm−1. However, due to capacity limit of the ATR-FTIR instrument (4000–650 cm−1), peaks below 650 cm−1 were, unfortunately, not reported. Nevertheless, the spectra are incorporated with strong peaks at 670 cm−1 which proposed the formation of ZnO-NPs [1].


## 3.1.2. XRD Analysis

The XRD patterns of the synthesized PPE-mediated ZnO-NPs are shown in Figure3(a). The spectra exhibit nine diffraction peaks on the range of 2θ° 25–75 and some of these peaks are detailed in Table 1. The diffractograms have ascertained material purity with no other external peaks observed, suggesting that the applied method of synthesis is effective to obtain ZnO-NPs of high purity. However, unidentifiable peak at 2θ° of 30.10 in Z20 profile may be due to some organics in the extract. Furthermore, the analysis revealed a hexagonal phase as compared with the database (JCPDS No. 36–1451). It is obvious that the diffraction peaks are moderately broad and, thus, indicating highly crystalline ZnO-NPs. According to the literature [28, 29], the broad peak is an indication of small and fine NPs (nanoscale crystalline particles) while the narrow or low intensity peak signifies low crystallinity of the NPs. As shown in Figure 3(b) and Table 1, the average particle diameter (D) of the biosynthesized ZnO-NPs, which calculated using the Debye–Scherrer equation (Equation (2), were between 18.53 and 30.38 nm [30–32]. The dislocation density (δ) of the fabricated samples is specified using the Williamson and Smallman’s relation (Equation (3)) [33, 34].(2)D=kλβcosθ,(3)δ=1D2,where D is the crystallite size, k is a constant denotes the shape factor (0.94), λ is the diffraction wavelength of CuKα (λ = 1.5406 Å), β is the full width at half maximum (FWHM), θ is the diffraction angle, and δ is the dislocation density. Figure 3(a) (insert) represents the shifting in the peak position toward higher angle with crystallite size increase which, in turn, reflect the effect of preparation extract volume [35]. Other structural parameters such as d-spacing (Å), a (Å), c (Å), c/a ratio, unit cell volume v (Å)3, the volume of particles V (nm)3, atomic packing factor (%), and the degree of crystal lattice distortion (R) were also computed [36, 37], and the corresponding values are gathered in Table 2.Figure 3
(a) X-ray diffraction (XRD) pattern of the synthesized ZnO NPs (Z20, Z30, and Z40); inset is a magnification of the prominent peak of (101) plane. (b) Crystallite sizes (D, nm) and dislocation (δ) vs. extract volume (20–40 mL).
(a)(b)Table 1
Structural parameters of ZnO-NPs.
Samples2-Thetad-spacingFWHM (deg)Planes (hkl)Crystallite sizes (D, nm)Average (D, nm)Average dislocation (lines/m2) (1015)Z2031.8012.8110.56610014.5918.5342.9134.5392.5940.38100221.8336.3392.4700.47510117.6047.4231.9150.70310212.3456.7001.6220.34311026.31Z3032.1412.8540.34410024.0329.8781.1234.3212.6100.15700252.9636.6802.4480.34010124.6247.9791.8940.40510221.4757.0391.6130.42611021.22Z4032.2792.7710.34010024.3330.3781.0834.9392.5650.29400228.3236.7802.4410.39010121.4647.5351.9110.13910262.4557.0811.6120.59011015.33Table 2
Geometric parameters of ZnO-NPs as computed from XRD.
SamplesLattice parameters (Å)(c/a) ratioVolumes of unit cell (Å3)Volumes of particles, V (nm)3Atomic packing factor (%)Degrees of crystal lattice distortion (R)abcZ203.2463.2465.1991.60247.443331.8575.45821.019559Z303.2653.2655.2041.60048.0413958.375.826961.024541Z403.2163.2165.2221.62046.7714670.974.431531.005687


## 3.1.3. SEM Analysis

Figure4 shows the SEM micrograph of the synthesized ZnO-NPs (Z20, Z30, and Z40) in which the ZnO were mainly composed of nanoplatelets with an overall quite dense morphology. The nanosheets’ thickness could be estimated to a tenth of nm; however, the thicker sheets may consist of several sheets aggregated to form the nanoplate network [27], with irregular NPs having almost spherical shapes [5]. Such aggregation and flaky agglomeration could be due to a high surface energy of the NPs and also perhaps due to densification of the narrow space between NPs [38]. A similar morphology of ZnO-NPs was also reported by a number of researchers [11, 39]. It seems that the aggregation, as well as the flake thicknesses, become less for ZnO-NPs produced at lower extract concentrations and this may explain the decreased bioactivity on the same order, i.e., antibacterial activity order Z20 > Z30 > Z40.Figure 4
Scanning electron microscope (SEM) image of ZnO-NPs. (a) Z20; (b) Z30; (c) Z40.
(a)(b)(c)


## 3.1.4. UV-Vis Analysis

Figure5 shows the UV-Vis spectra of the ZnO-NPs prepared with various PPE concentrations (Z20, Z30, and Z3). The spectra revealed characteristic maximum absorbances at 370, 375, and 378 nm, respectively, indicating quantum size effects [38]. The bandgap energy (Eg) as calculated from Tauc’s plotting for the direct allowed transition, described by Equation (3) [40–42], was found at 2.87, 2.80, and 1.92 eV, respectively for ZnO-NPs (Z20, Z30, and Z40) prepared using different extract volumes (20, 30, and 40 mL) (Figures 5(b)–5(d))).This denotes that the extract volume has an impact on the resulting bandgap of the synthesized ZnO-NPs, i.e., with PPE extract volume increase, the Eg decreased.(4)αhν=Chν−Egn.Figure 5
Absorption spectra of ZnO nanoparticles (a) and determination ofEg (b, c, and d).
(a)(b)(c)(d)Typically, theEg depends on the structure, size, and shape of the nanoparticles and, therefore, the employment of different extract volume can adjust such properties. By increasing PPE volume, a red shift in the optical bandgap is observed. Such an effect is obviously the result of the organic compounds amount differences present in the extract. According to the literature [13], the PPE is rich with phenolic compounds that may drive production of the ZnO-NPs which further affected by the extract volume used, i.e., the magnitude of phytocompounds.


## 3.2. Antibacterial Activity of ZnO-NPs

The antibacterial activity of the biosynthesized ZnO-NPs was tested against both Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria using the disk-diffusion method. For this, three different concentrations 50, 100, and 200 mg/mL of the biosynthesized ZnO-NPs (Z20, Z30, and Z40) were prepared and used for antibacterial activity studies in comparison to AzM as a standard drug. The resulting zones of inhibition (ZOI) are summarized in Table 3, and selected images of inhibition plates are given in Figure 6. It was observed that all the tested ZnO-NPs have an inhibitory effect against S. aureus and E. coli similar to or higher than the AzM standard drug. The ZOI was higher against the Gram-positive bacteria (S. aureus) compared to Gram-negative bacteria (E. coli). This might be due to the fact that Gram-positive bacteria are less susceptible to antibacterial potency than Gram-negative bacteria, perhaps this is a result of their different cell wall structures [43].Table 3
Antibacterial activity of ZnO-NPs using the zone of inhibition method compared to Azithromycin standard drug.
SamplesBacteriaZone of inhibition (ZOI; diameter in mm) ± standard deviation (SD) at various concentrationsZnO-NPs concentration (mg/mL)Azithromycin (control)50 mg/mL100 mg/mL200 mg/mLZ20S. aureus27 ± 1.2531 ± 1.7532 ± 1.2528 ± 0.25E. coli22 ± 1.0526 ± 0.2527 ± 1.2523 ± 0.25Z30S. aureus29 ± 2.0026 ± 1.7534 ± 1.5028 ± 0.50E. coli22 ± 0.2523 ± 0.2525 ± 2.2523 ± 0.25Z40S. aureus13 ± 0.5015 ± 0.2517 ± 0.0028 ± 0.25E. coli12 ± 0.2515 ± 0.5016 ± 0.7523 ± 0.50Figure 6
(a) Selected plate images of antibacterial activity tests of ZnO-NPs prepared using 20, 30, and 40 mL PPE (from the bottom to the top: Z20, Z30, and Z40) againstS. aureus (left images) and E. coli (right images). Disks: (1) ZnO-NPs 50 mg/mL per disc; (2) ZnO-NPs 100 mg/mL per disc; (3) ZnO-NPs 200 mg/mL per disc; (4) Azithromycin antibiotics (positive control); (5) distilled water (negative control). (b) Histogram illustration for the corresponding zone of inhibition data; bars represent the standard error of the mean (n = 2).
(a)(b)In the Gram-positive strain, peptidoglycan is thick while being thinner in the Gram-negative strain, but contains an outer membrane consisting of lipopolysaccharides that provides the bacteria resistance to prepared ZnO and makes them less susceptible [44]. The antibacterial potency of ZnO-NPs against microorganisms depends on cell wall integrity [43, 45]. The results indicate that the use of pomegranate peel extract-mediated synthesis of ZnO nanoparticles can be more efficient against Gram-positive bacteria and Gram-negative bacteria. This may be due to the existence of the higher number of phenolic compounds. Moreover, the results illustrated that the prepared samples have a strong antibacterial activity against both the strains compared to Azithromycin.It is obvious that the ZOI is low for ZnO-NPs prepared using Z40, the case that can be attributed to the higher particle sizes of Z40 (30.34 nm) compared to those of Z20 (18.53 nm) and Z30 (29.88 nm). The highest antibacterial activity for the lower particle sizes, e.g., Z20, is due to their smaller size (18.53 nm) which, in turn, means increased active surface area that further facilitates ease interaction with the bacterial wall [40]. With the increase in ZnO-NPs concentration from 50 to 200 mg/mL, a gradual increase in the bacterial inhibition was also observed, supporting the concentration-dependence activity of the NPs [46, 47].The bioactivity of NPs could be attributed to various factors such as chemical composition, particle size and shape, concentration, surface charge, and exposure time. The destructive action of ZnO-NPs on microorganisms could be due to one or simultaneous mechanisms: (i) attachment of NPs to a bacterial surface [46], hence the following stepwise events were proposed to be involved in adsorption of NPs on the bacterial surface facilitated by surface potential, distortion of cell morphology, NPs penetration into cells, membrane damage due to structural and functional interruption, and leakage of cellular components, thus functionality loss [9, 47, 48]; (ii) Zn2+ release from ZnO-NPs which, up on penetration into the cell, can inhibit several bacterial activities including transports, metabolisms, and enzyme functions, leading to cell death; (iii) ZnO activity as a result of the formation of reactive oxygen species (ROS) which leads to oxidative stress and subsequent cell damage [49]. An illustration of the proposed mechanism is given in Figure 7.Figure 7
The proposed antibacterial mechanism of ZnO-NPs.


## 3.3. Biocompatibility of ZnO-NPs with Human Erythrocytes

Biocompatibility of ZnO-NPs was assessed usingin vitro hemolysis assay at different concentrations 3.12–200 μg/mL, against RBCs and in reference to NS and DW as negative and positive controls [21], respectively. Hemolysis is generally based on measuring hemoglobin released from RBC after sample-induced cell lysis. Figure 8 illustrates the averaged data obtained from the two experiments. As can be seen, the toxicity of ZnO-NPs at the evaluated highest concentration (200 μg/mL) was 17.4%, which agreed with the previous reports [11, 50]. The hemolysis effect at 3.12 and 6.25 μg/mL were around 4.6 and 7.5%, increased to 16.8% for 25 μg/mL above which no significant differences could be observed up to 200 μg/mL. Basically, substances with hemolysis <2% is standardized as nonhemolytic, 2–5% slightly hemolytic while >5% hemolytic [21]. The obtained values at low concentrations revealed low toxicity and are in agreement with the literature [50] stated that ZnO-NPs concentration lower than 5% is nontoxic, being slightly hemolytic under 5–40 μg/mL while hemolytic at > 40 μg/mL. Muhammad et at. [11], have reported a 21.8% hemolytic for ZnO-NPs at a concentration of 200 μg/mL, however researchers have detected no hemolysis at concentrations below 5 μg/mL. Meanwhile, the hemolytic effect of ZnO-NPs has been reported to be concentration-dependent [11, 50], other factors including test conditions (medium, cells, and positive and negative controls), parameters (sample concentration and incubation time), and the substance nature (biogenic source, particle shape, and size) have to be analyzed as well to elucidate slight differences among studies [51].Figure 8
Hemolysis of erythrocytes caused by ZnO-NPs at different concentrations (3.12–200μg/mL), normal saline (NS, negative control), and distilled water (DW, positive control). Insert is an illustration of samples absorption (OD) including controls.


## 4. Conclusion

In this study, ZnO-NPs were biosynthesized usingpomegranate peel aqueous extract. The resultant NPs, obtained from various extract concentrations (20, 30, and 40 mL), were characterized for their structural, optical, and morphological properties using FTIR, SEM, XRD, and UV-Vis. The method resulted in a hexagonal crystallite, with averaged diameters of 18.53, 29.88, and 30.34 nm and optical bandgaps of 2.87, 2.80 and 1.92 eV, respectively. The antibacterial activity of this as-obtained ZnO-NPs at concentrations of 50, 100, and 200 mg/mL, examined on S. aureus and E. coli and compared with AzM standard drug, revealed comparable activity to that of AzM, higher when particle size is low, and more efficient against Gram-positive (S. aureus) bacteria than Gram-negative (E. coli) and at higher ZnO-NPs concentration. Hence, it could be concluded that pomegranate peel extract is a good candidate for biosynthesis of ZnO-NPs and the utilized green method is effective in the production of ZnO-NPs with a tailorable particle size and morphology.



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*Source: 9647793-2022-10-06.xml*