Bioactivity and Toxicity of Biogenic Nickel Oxide Nanoparticles Using Punica granatum Seed Extract

doi:10.21203/rs.3.rs-3549223/v1

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Bioactivity and Toxicity of Biogenic Nickel Oxide Nanoparticles Using Punica granatum Seed Extract

https://doi.org/10.21203/rs.3.rs-3549223/v1

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The biosynthesis of metal oxide nanoparticles has attracted attention because of their bioactivity, low cost, rapid reactivity, and simplicity. This study aims to synthesize and characterize the nickel oxide nanoparticles (NiO-NPs) by Punica granatum seed extract and evaluate their bioactivities and toxicity. The biosynthesized NiO-NPs physicochemical characteristics were explored using NTA, TGA, XRD, UV–Vis, and FT-IR, showing the formation of high-purity crystalline NPs with an average size of 10–60 nm. Furthermore, the antibacterial, antioxidant, and hemolytic activity of the biosynthesized NiO-NPs were explored. The results of this study showed that NiO-NPs possess a high antibacterial activity against Gram-negative (Pseudomonas aeruginosa and Escherichia coli) and Gram-positive bacteria (methicillin-resistant Staphylococcus aureus and Aerococcus viridans). However, the NiO-NPs inhibitory effect on Gram-positive bacteria was higher than on Gram-negative bacteria. Moreover, NiO-NPs also showed a DPPH, ABTS, and H₂O₂ radical scavenging activity with non-hemolytic activity at low concentrations (< 250 mg/L) in a dose-dependent manner. Regarding the toxicity of NiO-NPs, an enhanced effect on seed development of Vigna radiate at low concentrations (5 mg/L) was observed, which can be utilized as an alternative to conventional agrochemicals. Also, the cytotoxicity analysis was performed in the L-929 normal fibroblast cell line using MTT assay and IC₅₀ (125.5 mg/L), indicating that the biosynthesized NiO-NPs are biocompatible at different concentrations. The obtained data showed that the bioactivity of NiO-NPs might be due to their small size, which generates various active sites because of their high surface area. Generally, P. granatum seed extract showed ability as an efficient reducing and capping agent for NiO-NPs biosynthesis and played a vital role in enhancing their bioactivity.

Nanoparticles

Antibacterial activity

Antioxidant activity

Bioactivity

Biogenic nanoparticles

green synthesis

Nanoparticles toxicity

Nanomaterials are commercially accessible bulk materials in terms of electrical, magnetic, optical, catalytic, and medicinal properties (Khan et al., 2019). These characteristics may be related to the large surface area and quantum confinement effects per unit volume (Dubadi et al., 2023). Because of the simplicity of their production and promising physicochemical features, Nickel oxide nanoparticles (NiO-NPs) have several advantages (Barzinjy et al., 2020; Zhang et al., 2018). Several techniques have been employed to synthesize NiO-NPs, such as chemical precipitation (Shuihab and Khalf, 2018), green synthesis techniques (Barzinjy et al., 2020; Ezhilarasi et al., 2016), hydrothermal methods (Refat et al., 2022), pulsed laser deposition (Alheshibri et al., 2022), sol-gel process (Patel et al., 2022), spin coating, and thermal evaporation (Zheng et al., 2019). These complicated methods have drawbacks, including expensive chemicals, toxic organic solvents, formation of byproducts, severe reaction limits, difficulty of NPs separation, prolonged time, etc. (Sharma et al., 2022). Therefore, it is becoming increasingly essential to demonstrate green techniques for NPs synthesis.

NiO-NPs formed chemically and naturally have similar physical and chemical characteristics. In addition to being useful for treating wounds, gas sensors, and solar cells, NiO-NPs have antibacterial, catalytic, and biocidal properties (Barzinjy et al., 2020). Various techniques can be applied to generate NPs, including chemical, physical, biological, and hybrid processes. However, the ecologically friendly, non-toxic, and safe chemicals used in the green synthesis approach for NPs generation are safe and effective (Ezhilarasi et al., 2016; Sharma et al., 2022). Furthermore, Kganyago et al. (2018) reported the utilization of the local remedy Monsonia burkenea to synthesize NiO-NPs with an average size of 20 nm using a novel technique.

The development of NPs from plants is one of the acceptable, straightforward, and green ways to generate NPs (Dubadi et al., 2023; Khan et al., 2021; Srihasam et al., 2020). The Punicaceae family includes pomegranate (Punica granatum L.), a crucial source of polyphenols, flavonoids, and rich natural antioxidants (El-Beltagi et al., 2022). These components showed therapeutic applications as a traditional treatment since ancient times. Phytochemicals such as flavonoids, which seem to be the most bioactive components of P. granatum extracts, are linked to several biomedical applications (Moga et al., 2021).

To this end, this study aims to comprehensively evaluate the bioactivity of biogenic NiO-NPs synthesized using pomegranate seed extract. The study explores the antibacterial activity of the NiO-NPs against Gram-negative and Gram-positive bacterial pathogens, as well as their potential as antioxidative agents, by assessing their scavenging activity against DPPH, ABTS, and H₂O₂ radicals. Furthermore, the hemocompatibility of NiO-NPs will be evaluated to determine their safety for biological applications. Additionally, the study will investigate the phytotoxicity of NiO-NPs on Mung Bean seed growth, their potential ecotoxicity using Artemia salina larvae, and their biocompatibility for human use. The ultimate goal is to comprehensively understand the bioactivity and potential toxicity of NiO-NPs in human, aquatic, and terrestrial ecosystems, thereby providing valuable insights for their sustainable and responsible applications.

2.1. Pomegranate seed extract preparation

The pomegranate seed extract was prepared following Barzinjy et al. (2020) with minor modifications. Briefly, 10 g of fresh P. granatum L. seeds obtained from Kafr Elsheikh City, Egypt, were soaked in 200 mL double distilled water (DW), heated for 30 min at 60°C, and cooled to 25°C. The prepared solution was filtered to eliminate the residual impurities, and the filtrate was used as a reducing agent and capping material during the biosynthesis process.

2.2. Synthesis and capping of NiO-NPs

NiO-NPs synthesized and capping procedures are shown in Fig. 1. In brief, A weight of 0.2 mg of nickel nitrite [Ni(NO₃)₂·6H₂O] was dissolved in deionized water (40 mL) for 10 min. Then, a volume of 10 mL of sodium hydroxide [1 M; NaOH] was mixed with P. granatum seed extract mixed with nickel nitrate solution dropwise to attain the mixture pH value of 12. After that, the formed solution was heated for 30 min at 60°C and stirred until a dark brown color appeared to indicate the formation of NiO-NPs. The formed precipitate was removed by centrifugation for 15 min at 11,000 g. The formed NiOH-NPs were converted to NiO-NPs through thermal decomposition for 40 min at 600°C. Also, this process eliminates all impurities in the synthesized NiO-NPs, which will be subjected to further bioactivity applications.

2.3. NiO-NPs characterization

The physicochemical characteristics of NiO-NPs were detected using a UV-visible spectrophotometer (UV-vis, Shimadzu UV-1800). Nanoparticle Tracking and Analysis (NTA) was performed via the Nanosight UK-LM20 instrument to track and record prepared NPs sizes. Functional groups were detected using the FT-IR spectrophotometer (Perkin Elmer spectrophotometer-RXI). The compounds were subjected to TGA analysis by SDT (Q600) thermal analyzer (TAInstruments, USA) under a nitrogen atmosphere at the heating rate of 20°C/min. XRD analysis was performed by X-ray diffractometer (RikaguMiniflex) using Cu-α radiation operated at 40 kV and 20 mA with a scan speed of 2θ = 5°/min. Compositional phase distribution and crystal quality of the formed particles were calculated by detecting the value of full width at half maximum (FWHM) of the XRD spectrum following the Debye–Scherrer equation (Eq. 1):

$\text{C}\text{r}\text{y}\text{s}\text{t}\text{a}\text{l}\text{l}\text{i}\text{t}\text{e} \text{s}\text{i}\text{z}\text{e}=\frac{\text{K}.{\lambda }}{\text{F}\text{W}\text{H}\text{M}.\text{c}\text{o}\text{s}{\theta }}$ Eq. 1

where K = 0.95, λ is the X-ray wavelength (1.54 Å), θ is Bragg’s angle, and FWHM is full width at half maximum.

2.4. Bioactivity of the synthesized NiO-NPs

2.4.1. Antibacterial Activity

NiO-NPs antibacterial activity was explored against Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 9027, methicillin-resistant S. aureus ATCC 33592, and Aerococcus viridans ATCC 11563. The selected strains were taken from glycerol stock, streaked on nutrient agar (NA), and incubated at 35°C. The individual colonies of each strain were inoculated in the preferred medium and incubated overnight at optimal temperature. The bacterial pellets were obtained by centrifuge overnight bacterial cultures and dissolved in normal saline solution to obtain bacterial suspension (0.5 McFarland standard solution) to standardize the total plate count of each strain. Antibacterial activity of NiO-NPs was performed using the disk diffusion method to investigate the bacterial sensitivity on Muller Hinton agar (MHA). The bacterial strain culture individually was swabbed on agar media. The discs, 6 mm in diameter, were sterilized and placed into the solution of synthesized NiO-NPs of different concentrations (0, 5, 10, 25, and 50 mg/L) and controls. The standard antibiotics, amoxicillin (30 µg/disk), amikacin (10 µg/disk), penicillin (10 µg/disk), and piperacillin (100 µg/disk), were used as a positive control for E. coli, S. aureus, A. viridans, and P. aeruginosa, respectively. Further, the discs that contained compounds were placed onto the medium plates, which were inoculated with tested bacterial strains. After loading the synthesized compounds and controls on media plates, the media plates were placed in an incubator at 35°C for 24 h. NiO-NPs antibacterial activity was assessed by assessing the inhibition zone (IZ) diameters in millimeters after incubation.

2.4.2. Antioxidant activity

2.4.2.1. DPPH radical scavenging activity

The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging potential was detected following Xu et al. (2019). NiO-NPs samples were dissolved in ethanol to evaluate their free radical scavenging potential at final concentrations of 0, 5, 10, 25, and 50 mg/L. A volume of 1 mL of the prepared concentrations of NiO-NPs solution was mixed with 1 mL DPPH in ethanol solution (0.1 mmol/L). The mixture was incubated at 25°C in the dark for 30 minutes and centrifuged for 10 minutes at 5500 g. The supernatant absorbance was detected at 517 nm. Ascorbic acid (Vitamin C) was used as a positive control. The DPPH radical scavenging potential (%) was estimated by Eq. (2).

$$\text{S}\text{c}\text{a}\text{v}\text{e}\text{n}\text{g}\text{i}\text{n}\text{g} \text{a}\text{c}\text{t}\text{i}\text{v}\text{i}\text{t}\text{y} \text{o}\text{f} \text{D}\text{P}\text{P}\text{H} \text{r}\text{a}\text{d}\text{i}\text{c}\text{a}\text{l} \left(\text{%}\right)=\frac{\text{C}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l} \text{a}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e}-\text{S}\text{a}\text{m}\text{p}\text{l}\text{e} \text{a}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e}}{\text{C}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l} \text{a}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e}}\text{X} 100 \text{E}\text{q}. 2$$

2.4.2.2. ABTS^•+ scavenging activity

NiO-NPs antioxidant potential was detected by evaluating the change in the decolorization that occurs due to 2,2'-azinobis 3-ethylbenzothiazoline-6-Sulfonate (ABTS^•+) reduction to ABTS. Briefly, 2 mM of ABTS.⁺ was mixed with 2.5 mM of potassium persulfate and kept for 6 h at 25°C in the dark. ABTS.⁺ was mixed with 0.1 mM of sodium phosphate buffer (KH₂PO₄). After mixing 3 mL of different concentrations of NiO-NPs (0–50 mg/L) with 1 mL of ABTS.⁺ solution, the ABTS activity was measured by Eq. 3. Vitamin C was used as a positive control.

${\text{A}\text{B}\text{T}\text{S}}^{{\bullet }+} \text{s}\text{c}\text{a}\text{v}\text{e}\text{n}\text{g}\text{i}\text{n}\text{g} \text{a}\text{c}\text{t}\text{i}\text{v}\text{i}\text{t}\text{y} \left(\text{%}\right)=\frac{\text{C}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l} \text{a}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e}-\text{S}\text{a}\text{m}\text{p}\text{l}\text{e} \text{a}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e}}{\text{C}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l} \text{a}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e}}\text{X} 100$ Eq. 3

2.4.2.3. Hydrogen peroxide (H₂O₂) scavenging activity

NiO-NPs effect as a scavenging agent for the H₂O₂ is explored following the method described by Ibrahim et al. (2020). H₂O₂ (40 mM) was prepared using a phosphate buffer solution (PBS, pH 7.4). Different concentrations of NiO-NPs (5, 10, 25, and 50 mg/L) were treated with 0.6 mL of the prepared H₂O₂ and incubated for 10 min prior to optical density detection at 230 nm. Vitamin C was applied as a positive control.

2.4.3. Hemolysis analysis

To explore the hemocompatible nature of NiO-NPs, the hemocompatible assay was performed as described by Shnawa et al. (2022). A volume of 5 mL of fresh human blood was obtained from the healthy human in tubes with EDTA-anticoagulation. One mL of fresh human blood was centrifuged for 10 min at 525 xg. A volume of 200 µL of erythrocytes was mixed with 9 mL PBS (pH 7.2) in order to prepare erythrocyte suspension. A volume of 100 µL of erythrocyte suspension was added to different concentrations of NiO-NPs (5, 10, 25, 50, 100, 250, 500, 1000 mg/L) and incubated at 35°C for 1 h in a 96-well plate. The supernatant's optical density at 570 nm was measured to evaluate the released amount of hemoglobin. Sterilized PBS and deionized water were the negative and positive controls, respectively (Shnawa et al., 2022). Percent hemolysis was investigated following Eq. 4:

$$\text{H}\text{e}\text{m}\text{o}\text{l}\text{y}\text{s}\text{i}\text{s} \left(\text{%}\right)= \frac{{\text{A}}_{S}-{A}_{NC}}{{\text{A}}_{PS}-{\text{A}}_{NC}} X 100 \text{E}\text{q}.4$$

where A_S is the sample absorbance, A_NC is the negative control absorbance, and A_PC is the positive control absorbance.

2.5. Toxicity assays

2.5.1. Toxicity of brine shrimp

To evaluate the toxic effect of the synthesized NiO-NPs, brine shrimp mortality was performed in 96-well plates using Artemia salina larvae (Ali et al., 2017). A. salina (Ocean star, USA) eggs were incubated under light for 48 h at 28°C in simulated seawater (38 g of sea salt and 6 mg of dried yeast) and were dissolved in 1 L DW. Ten mature phototrophic nauplii were collected and transferred to plate wells. The effect of different NiO-NPs concentrations (5, 10, 25, and 50 mg/L) was explored. However, doxorubicin (DOX) and dimethyl sulphoxide (DMSO) were applied as negative and positive controls, respectively. Dead shrimps were detected, and the inhibition was estimated. The GraphPad Prism program detected half of the maximal inhibitory effect (IC₅₀).

2.5.2. Effect on seed growth and development

NiO-NPs bioactivities on seed growth and development were assessed by Mung Bean (Vigna radiata) seeds. Seeds were sterilized by soaking in sodium hypochlorite [NaClO; 5%] for 20min and then soaked in DW. Different concentrations (5, 10, 25, and 50 mg/L) of the synthesized NiO-NPs were prepared using sterilized DW. The prepared concentrations of NiO-NPs were added separately in five layers of filter paper and 25 V. radiata seeds in sterilized Petri plates. DW was used as a control. The prepared plates were incubated at 30°C in the dark to detect NiO-NPs' effect on the rate of seed development and the lengths of the shoot and root on the 5th and 10th day.

2.5.3. Biocompatibility and cell viability

The biocompatibility assessment of NiO-NPs was conducted using the L-929 normal fibroblast cell line through cell viability analysis. L-929 cells were cultured in flasks containing Dulbecco's Modified Eagle's Medium and M-199 medium supplemented with 10% fetal bovine serum and incubated at 37°C with 5% CO₂ for 24 h. After the incubation period, the adherent cells were trypsinized for 5 min to obtain individual cells, followed by centrifugation at 800 rpm for 10 min. The cells were then counted and distributed in a 96-well Enzyme-linked immunosorbent assay plate with 5000 cells per well and incubated for 24 h to achieve approximately 70–80% confluence as a monolayer (Nayak et al., 2015). The toxicity of NiO-NPs was determined at various concentrations (5, 10, 25, and 50 mg/L) in triplicates. To assess cell viability, a 200 µL solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to each well and incubated for 5 h. Subsequently, the MTT solution was removed, and 200 µL of DMSO solvent was added to each well in the dark, followed by an additional 20 min of incubation. The optical density (OD₅₉₅ nm) of the formazan product was then measured using a microplate reader (Bio-Rad, USA). Media and other reagents utilized in these experiments were procured from Sigma-Aldrich (St. Louis, MO).

2.6. Statistical analysis

The obtained results were statistically analyzed and visualized by GraphPad prism (V. 8.0.2.) software and SigmaPlot software version 12.5 (SigmaPlot, Systat Inc., 2011, UK). t-test was used for multiple comparisons between investigated groups. p-value < 0.05 is considered significant.

3.1. NiO-NPs synthesis

NiO-NPs biosynthesis and capping were performed, as shown in Fig. 1. The reduction phase is 1st stage where metal ions and atoms are reduced and subjected to nucleation. In the 2nd phase, small NPs comprise larger NPs because of the binding energy between metal atoms (growth phase). NPs agglomeration and different types of unevenly shaped NPs are produced after the growth phase. NPs agglomeration is inhibited by plants' metabolites, which act as a stabilizing and capping agent (Barzinjy et al., 2020). The 2nd stage induces NiO-NPs thermodynamic stability. In the 3rd stage (stabilization stage), NPs shapes are formed (Barzinjy et al., 2020; Singh et al., 2022). The stabilization can be performed by the calcination to form NiO-NPs (Barzinjy et al., 2020).

3.2. Characterization of NiO-NPs

For NiO-NPs determination of size and concentration, NTA was carried out to explore the synthesized NiO-NPs size and concentration. Figure 2A showed that NiO-NPs were in the range of 20 to 40 nm with an average size of 20 nm. Moreover, the total concentration of NiO-NPs was found to be 2.6 x 10⁶ particles/mL. UV-vis was detected to reflect the properties of the synthesized NPs, as presented in Fig. 2B; UV-vis analysis indicates an absorption peak at 330 nm showed the absorption edge of NiO, confirming the synthesis of NiO-NPs (Barzinjy et al., 2020). The quantum confinement effect is responsible for the blue-shifted phenomena (Anandha Babu et al., 2014; Barzinjy et al., 2020). As demonstrated in Fig. 2B, the synthesized NiO-NPs exhibited no additional peaks associated with impurities or structural flaws, indicating that the produced NiO-NPs are crystalline.

Thermogravimetric analysis (TGA) was carried out at 600°C in the atmosphere to look into the alterations that occurred during the heat treatment of NiO-NPs. As illustrated in Fig. 2C, it is clear that the TGA analysis showed three stages of weight decrease. The evaporation of the water molecule during the crystallization process was shown to be the first step (7 wt.%) at temperatures between 164 and 268°C. The second weight loss, which was determined to be 32%, is associated with the thermal degradation of nickel hydroxide into NiO-NPs and is caused by removing one water molecule between 272 and 361°C. Due to organic-groups disintegration and removal in the sample during the green synthesis process, the last phase began at 361°C and concluded at 428°C, around 56% of the actual weight (Sharma and Ghose, 2015). There was no substantial breakdown or reactivity over 428°C.

The XRD analysis between 20 to 90 °2θ indicates the NiO-NPs crystallinity with diffraction pattern at two theta positions 37.28°, 43.33°, 62.91°, 75.40° and 79.35° corresponding to miller indices of plane (111), (200), (220), (311), and (222) planes, as shown in Fig. 2D, respectively. The obtained data was similar to previous studies (Barakat et al., 2013; Barzinjy et al., 2020; Wardani et al., 2019). The XRD results indicate the NiO crystal quality, with the intense diffraction bands and narrow FWHM indicating the synthesis of highly crystalline material. Furthermore, because all of the bands observed are attributable to NiO, the absence of any other peak in the diffractogram supports the formation of highly pure material. Table 1 shows the high-quality NiO-NPs with a FWHM average of 0.5492 and an average NPs size of ~ 20 nm according to the Debye–Scherrer equation, which was previously indicated by NTA analysis.

Table 1

Calculation of NiO-NPs particle size by Debye–Scherrer’s equation from XRD analysis.
Position (°2 θ)	Planes	FWHM left (°2 θ)	Lattice Strain (ε)	Size (nm)
37.28	111	0.57725	0.00747	15.16
43.33	200	0.57273	0.00483	20.28
62.91	220	0.76299	0.00544	12.74
75.40	311	0.4175	0.00236	25.11
79.35	222	0.41531	0.00218	25.95
Average FWHM		0.549156	Average size	19.85

The FT-IR analysis is important to explore the formed bonds between the atomic components of NiO-NPs to understand the dominant functional groups (Fig. 3). From the FT-IR spectra peak, 450 cm^− 1 is assigned to Ni-O bond stretching (Khairnar and Shrivastava, 2019), while the band at 1035 cm^− 1 showed the vibration of C = O and C = C bond. The absorption peak at 1110 cm^− 1 showed Ni-O-Ni stretching (Saravanakkumar et al., 2018). The width of a peak shows that NiO-NPs are crystalline. Also, the peak at 1388 cm^− 1 may be because of C–O stretching vibrations in alcohols or ethers (Wongsaprom and Maensiri, 2010). The absorption peak at 1462 cm^− 1 showed CH₂ bending vibration. The absorption peak at 1629 cm^− 1 showed the formation of water molecules' bending mode (H-O-H) (Khodair et al., 2022). 2851 cm^− 1 showed C–H stretching vibration, which implies the presence of secondary metabolites and protein chains in the structure of modified NiO-NPs. The peak at 2925 cm^− 1 indicates the aliphatic CH stretching. However, 3410 cm^− 1 showed the stretching and bending vibrations of the hydroxyl (–OH) group. Formed peaks at 1035, 1388, 1462, 2851, and 2925 cm^− 1 indicated the formation of proteins and secondary metabolites, which act as a stabilizing agent for NiO-NPs during the preparation process.

3.3. Bioactivity of NiO-NPs

3.3.1. Antibacterial activity of NiO-NPs

Broad-spectrum antibiotic abuse and overuse recently have been the main reason for the growth of antimicrobial resistance (e.g., methicillin-resistant S. aureus) (Nanayakkara et al., 2021). Therefore, in the case of persistent drug exposure, assessment of the sensitivity of pathogens to novel antibacterial agents is essential to treat multidrug-resistant (MDR) bacteria. Hence, a novel bio-safe antibacterial agent is necessary to treat bacterial infections.

The antibacterial activity of NiO-NPs was performed In this study to indicate the inhibitory effect of Gram-positive (S. aureus and A.viridans) and Gram-negative bacteria (E. coli and P. aeruginosa) by assessing the formed IZ. NiO-NPs showed robust antibacterial activity against the selected bacteria with various sensitivities as a concentration-dependent activity. The IZ for E. coli, P. aeruginosa, S. aureus, and A.viridans reached 16.3 ± 1.5, 22.3 ± 2.3, 25.6 ± 2.5 and 28.6 ± 2.2 mm, respectively, at the maximum NiO-NPs concentration (50 mg/L). In comparison with the lowest concentration (5 mg/L), IZ after NiO-NPs treatment was 0.0 ± 0.0, 0.0 ± 0.0, 2.5 ± 0.7, and 3.4 ± 0.5 mm, respectively (Fig. 4). However, compared to control antibiotics, the statistical analysis using t-test showed that there was fluctuation in significant variation (p < 0.05) between the effect of standard positive control antibiotics (amoxicillin and piperacillin) and NiO-NPs on Gram-negative bacteria (E. coli and P. aeruginosa, respectively) based on the investigated concentration, as shown in Fig. 4A and B. The impact of NiO-NPs was significantly higher than the standard antibiotic at a concentration of more than 25 mg/L. However, similar findings were detected for Gram-positive bacteria (S. aureus and A.viridans) against standard control (amikacin and penicillin, respectively) (Fig. 4C and D). The statistical analysis showed that NiO-NPs were significantly higher than the standard antibiotic at a more than 10 mg/L concentration. Moreover, these results indicate that NiO-NPs potential towards Gram-positive bacteria was higher than Gram-negative bacteria.

In this regard, our obtained data agreed with Yusof et al. (2019), who stated that this could be because of differences in cell physiology, cell wall structure, degree of contact, or bacterial metabolic activities. Also, Nisar et al. (2019) stated that the potential for membrane damage is caused by electrostatic interaction between NPs and cell surfaces. The particular processes behind the bacteriocidal effects of heavy metals are still poorly understood. The potential mechanisms by which NiO-NPs exert their bactericidal effects were revealed in previous studies. Mohamad et al. (2023) stated that metal oxide NPs (MO-NPs) have a positive charge, and the microbial cell has a negative charge; there is an electromagnetic attraction between the two that causes oxidation and death of the organisms. Ahmad et al. (2022) found that the electrostatic contact negatively impacts the bacterial cell wall function, stability, cytoplasm, and organelles because of NiO-NPs positive charges.

Additionally, Köktürk et al. (2021) stated that NiO-NPs create reactive oxygen species (ROS) that cause DNA and membrane damage, as well as protein oxidation, which has a lethal impact on the bacterial cell. Several studies also demonstrated the antibacterial effect of NiO-NPs against a broad platform of bacterial pathogens (Alam et al., 2023; Haroon et al., 2019; Karagecili et al., 2023; Prabhu et al., 2022). Vijaya Kumar et al. (2019) stated the antibacterial efficacy of the biogenic NiO-NPs utilizing leaves of the crown flower (Calotropis gigantean) against Gram-negative and positive bacterial strains due to the formation of ROS because of the large surface area of NPs. The authors also stated that NiO-NPs generate (superoxide radical, singlet oxygen, hydroxyl, and alpha-oxygen), which causes DNA oxidation, lipid peroxidation, and oxidation of protein that can inhibit microbial growth. Also, Prabhu et al. (2022) stated that the synthesized NiO-NPs capped with butterfly pea flower extract demonstrated robust antibacterial efficiency. They explained it by the phytochemical components of the flower of butterfly pea extract coated on NiO-NPs surface and combined with NPs to enhance their bioactivity as bactericidal.

ROS are produced due to the electrostatic interaction of bacteria and NPs, which is thought to cause bacterial cell mortality (Haroon et al., 2019). The strong interaction of the nickel ions (Ni²⁺) with the negatively charged bacterial cells resulting in collapse is a likely bactericidal mechanism for NP interactions with bacteria. When light interacts with NiO, the second reaction causes electron induction from the valance to the conduction band. O₂ and further electrical response produce O₂ radicals, producing H₂O₂. The interaction of hydrogen ions with water led to the formation of OH^•. ROS, such as O₂ and OH species, are crucial for the protein and lipid molecules breakdown of the bacteria's outer cell membrane (Ren et al., 2020).

Additionally, Ni²⁺ enters the cell wall, harming the DNA, proteins, and mitochondria while interfering with electron transport, which leads to cell death. Protein leakage results from NiO-NPs accumulation on the bacterial plasma membrane, causing the dysfunction and alteration of the plasma membrane's permeability (Behera et al., 2019). Other investigations showed that NiO-NPs had more activity as antibacterial agents against Gram-positive strains than Gram-negative strains, especially against multidrug-resistant S. aureus (MRSA) (Behera et al., 2019; Haider et al., 2020). Additionally, the antibacterial activity of NPs is influenced by their concentration and particle size, where the activity improved with decreasing NPs size and increased concentration (Álvarez-Chimal et al., 2022). Due to its participation in several cellular processes, nickel is an essential nutrient for many bacterial species' development and metabolic activities. Numerous bacteria have nickel-specific permeases or can recognize cellular nickel ion concentrations and take up this nutrient. These metal ions are also included in nickel-dependent enzymes (Alfano and Cavazza, 2020). NiO-NPs' bactericidal activity may be caused by electrostatic interaction, the production of Ni²⁺ ions, or ROS generation. Therefore, comprehending the antibacterial activity of NiO-NPs may aid in developing a foresighted substitute to combat MDR bacterial strains.

3.3.2. Antioxidant activity

The biosynthesized NiO-NPs exhibited high DPPH scavenging ability, as shown in Fig. 5A. The findings in this study proved that all the explored samples showed dose-dependent antioxidative potential ranging from 19.1–82.6% for concentrations ranging from 5 to 50 mg/L, respectively. Furthermore, the ABTS^•+ and H₂O₂ radicals scavenging potential of the synthesized NiO-NPs was found to range from 15.3 to 68.2% (Fig. 5B) and from 18.1 5 to 73.2% (Fig. 5C) for the concentration ranged from 5 to 50 mg/L of NiO-NPs, respectively.

These observations showed that P. granatumas' phytochemicals are a rich source of natural antioxidants. Hence, P. granatumas, as a capping agent for NiO-NPs present on the surface of the NPs, could be the main reason for the high antioxidant potential of NiO-NPs. The obtained data of the antioxidant potential assays in this study are consistent with previous studies on biosynthesized NiO-NPs (Hussein and Mohammed, 2021; Khalil et al., 2017; Khan et al., 2021). NiO-NPs activity as an antioxidant agent may be because of the varied phenolics in P. granatum, including the isomers of tannin, anthocyanins, and punicalagin. These compounds can scavenge radicals and inhibit lipids' oxidation (Benchagra et al., 2021). The main bioactive constituent of P. granatum is the polyphenols (e.g., punicalagin) and is widely reported for its bioactivity (Benchagra et al., 2021). Also, Balli et al. (2020) stated that P. granatum had the highest ellagitannin content and showed the maximum antioxidant activity. Farag et al. (2020) indicated that pomegranate has the highest amounts of flavonoids, polyphenols, anthocyanins, and tannins compared to fig, guava, and olive. Therefore, the authors stated that pomegranates are an excellent source of compounds that promote health while exhibiting promising antioxidant activity that can be practically used as food supplements to delay lipid oxidation and increase healing from specific ailments through their capacity to scavenge free radicals.

Compared with other fruits, Zhang et al. (2008) found that pomegranate showed high antioxidant activity compared to orange and apple after exploring DPPH and ABTS radical scavenging bioactivities. Moreover, the authors found that the antioxidant activity of pomegranate peel, leaves, seed extract, and juice was evaluated by the DPPH and ABTS methods and found that leaves and peel showed robust antioxidant bioactivity and high polyphenolic content in comparison with juice and seeds. They had the same IC₅₀ (0.14 mg/mL) in the DPPH assay. Also, Yisimayili et al. (2019) explored the antioxidative capacities of pomegranate peel, seed, leaf, and flower ethanolic extracts using a DPPH assay. The author found that the peel extract showed the most potent antioxidant activity. The polyphenols content was considerably related to the antioxidant and antimicrobial effect to prove that polyphenols could be pomegranate's functional and active compounds. However, Zhao and Yuan (2021) stated that anthocyanin from pomegranate might play a role as a reducing agent, radical scavenger, metal chelator, and hydrogen donor.

Moreover, flavonoids from pomegranate can be considered potent antioxidants and may show their antioxidant activity in three routes: metal chelation activity, free radical scavenger, and enhanced other antioxidant activity. However, the authors stated that pomegranate antioxidative activity and mechanism are still unclear. Thus, this fluctuation in the results could be because of the importance of pomegranate harvesting season on the phenolic content and antioxidant bioactivities (Borochov-Neori et al., 2009). Several studies reported the variation in the pomegranate chemical content and its antioxidant activity, independent of the performed antioxidant assay. Various samples of pomegranates harvested in different fields showed significant differences in anthocyanin and phenolic content (Karagecili et al., 2023; Zhang et al., 2008). Prakash and Prakash (2011) reported that the antioxidative bioactivity detected by two assays (BPPH and ABTS) was related to the total phenolic compounds than with anthocyanins content. The antioxidant activity of pomegranate components is an important aspect to be explored and utilized as an alternative to synthetic antioxidants, which are increasingly restricted because of their side effects.

3.3.3. Hemocompatibility assay

Red blood cells (RBCs) hemolysis value represents the degree of the interaction between NPs and RBCs, quantifying the impact of NPs on the erythrocyte membrane, resulting in hemoglobin release (Neun et al., 2017). For the assessment of NiO-NPs' bio-safe properties, the hemocompatibility evaluation was performed against human RBCs. Thus, NiO-NPs hemocompatibility using various concentrations was explored, as illustrated in Fig. 6, and compared with negative (PBS) and positive (DW) controls. The hemolysis percentage of NiO-NPs showed that hemolysis is proportionally related to NiO-NPs concentration.

At low concentrations (5-100 mg/L), NiO-NPs showed a non-hemolytic effect, while at high concentrations (250, 500, and 1000 mg/L), NiO-NPs caused 3.66, 7.72, and 13.62% hemolysis. The lowest concentration of NiO-NPs (5, 10, 25, 50, and 100 mg/L) showed 0.15, 0.28, 0.49, 0.88, and 1.92% of RBCs lysis. The hemolysis percentage at various NiO-NPs concentrations was significantly (P ≤ 0.05) lower than distilled water (positive control). The result revealed that the biogenic NiO-NPs were biocompatible at all concentrations below 100 mg/L. Lingaraju et al. (2020) found that the biosynthesized NiO-NPs using Mexican fireplant exhibited non-hemolytic effects within the concentration from 40 to 100 µg and were biocompatible for bio-applications. Additionally, Binu et al. (2021) performed a NiO-NPs hemocompatibility assay and found that NiO-NPs showed non-hemolytic effects. The data showed hemolysis percentages at 50, 25, 10, and 5 mg/L were 1.85, 1.1, 0.56, and 0.31%, respectively. Thus, NiO-NP concentrations ≤ 50 mg/L are safe to be utilized. Based on the ASTM (American Society for Testing and Material) standards, hemolytic substances show more than 5% of hemolysis percentage, 2–5% are slightly hemolytic, and less than 2% are non-hemolytic (Aula et al., 2014). Therefore, NiO-NPs showed an advantage in contrast to several NPs. Numerous nanoparticles (NPs) are hemolytic agents, such as Ag-NPs, tricalcium phosphate (TCP), amorphous silica, and hydroxyapatite (HAP), and were inappropriate for medical applications (de la Harpe et al., 2019).

3.4. NiO-NPs ecotoxicity evaluation

3.4.1. Brine shrimp cytotoxicity

The ecotoxicity of the synthesized NiO-NPs against A. salina larvae was investigated. The larvae were subjected to NiO-NPs at various doses (5–50 mg/L) for 24 h. A significant dose-dependent cytotoxic reaction with an IC₅₀ value of 34.5 mg/L was determined, as shown in Fig. 7. The motile mobility of A. salina larvae was inhibited by increasing the dosages of NiO-NPs and reached 63 ± 6% at 50 mg/L. The attachment of NPs may have slowed larval motility as the NiO-NPs penetrated the larval body, inhibiting their metabolic activity and resulting in the death of larvae (Uddin et al., 2021). NiO-NPs' effective antioxidant, antibacterial, and cytotoxic activities might be attributed to their nano size, exact surface area, and adhesion capabilities.

3.4.2. NiO-NPs effect on seed growth and development

With multiple uses in agriculture sectors to improve nutrient absorption, seed growth, and development and lower the usage of toxic conventional fertilizers, NP-based bio-fertilizers have recently become a superior alternative. As a result, NPs produced via a green technique have more potential and provide an environmentally benign substitute for chemicals utilized in agricultural applications. Currently, several metallic NPs, such as ZnO-NPs, TiO-NPs., and other MO-NPs, are in use for agricultural applications (Paramo et al., 2020; Uddin et al., 2021; Younes et al., 2020).

In this study, NiO-NPs' impact on the germination of V. radiata seed and the length of shoot and roots was explored on the 5th and 10th day. As depicted in Fig. 8, the low concentration of NiO-NPs (5 mg/L) exhibited an induction influence on V. radiata seed development and germination compared to the control. However, at low dose (5 mg/L), seed germination was induced by 4.5%. However, at higher concentrations (10, 25, and 50 mg/L), the germination was inhibited by 12.5, 36.8 and 73.6%, respectively (Fig. 8A). A considerable enhancement in the shoot length by 35.7 and 39.3% was detected on the 5th and 10th day, respectively at a concentration of 5 mg/L in comparison with the control (Fig. 8B). The same pattern was detected regarding root length at 5 mg/L with a significant increase of 36.6 and 29.3% on 5th and 10th day, respectively compared with control (Fig. 8C). On the other hand, higher concentrations (25 and 50 mg/L) can caused a significant inhibition in shoot and root length of V. radiate. The obtained data was in agreement with the results of other studies that indicated the stimulatory and inhibitory properties of NiO-NPs on seed growth of numerous plants (Chaudhary et al., 2018; Khalil et al., 2017; Saleh et al., 2019). Moreover, Faisal et al. (2013) found that High NiO-NP concentrations may translocate into the cell's cytoplasm with apparent alterations in organelle structure, leading to oxidative stress and mitochondrial malfunction, which may induce the apoptotic process. NiO-NPs' ability to release Ni ions and initiate the intrinsic apoptotic pathway in plant cells has been confirmed by increased caspase-3-like protease (e.g., caspase-3 and caspase-7) activity. The root cells displaying NPs most likely caused cell death through a mitochondrial-dependent apoptotic route. Therefore, further investigation of the effect and mechanism of NiO-NPs in plant and animal cells should be performed.

3.4.3. NiO-NPs biocompatibility

Demonstrating NiO-NPs biocompatibility is essential to their effective biomedicine and human use applications. In order to verify the biocompatibility of NiO-NPs, their cytotoxicity was further assessed against the normal fibroblast cell lines L-929. Along with toxicity against normal cell lines, which may influence biological applications, the safety of using NiO-NPs is a serious problem. NiO-NPs have not been shown to inhibit the L-929 cell line at lower doses in the current investigation. As the concentration of NiO-NPs increases, the proportion of viable cells in normal fibroblast cells decreases (Fig. 9). NiO-NPs' IC50 value against L-929 cell lines was determined to be 125.45 ± 1.6 mg/L. The IC₅₀ value indicates the high biocompatibility and safe use of NiO-NPs in the human body. The cytotoxicity of the synthesized NiO-NPs was explored by Bala et al. (2022) over the rat skeleton cell line L-6. The MTT test indicated that the cell viability of the L-6 cell line decreased with the concentration of NiO-NPs. However, the NiO-NPs should be thoroughly studied for their safety and biocompatibility before their practical applications.

The green synthesis of NiO-NPs using P. granatum seed extract was performed in this study to assess their bioactivities. The bioactive compounds in P. granatum seed extract act as a capping agent in improving the bioactive properties of the synthesis of NiO-NPs and limiting their aggregation. The physicochemical characteristics showed that NiO-NPs were crystalline with high quality and low impurities with an average size of 20 nm. The obtained data showed that NiO-NPs, even at low concentrations, possess antibacterial activity against both Gram-positive and negative bacteria, with more activity against Gram-positive bacteria. It is assumed that NiO-NPs can change the bacterial membrane permeability and show strong oxidative stress, causing the formation of ROS with a negative impact on microbial metabolic activity and inhibiting bacterial growth. Moreover, according to DPPH, ABTS, and H₂O₂ radical scavenging activity, NiO-NPs exhibited strong antioxidant activity. The obtained data showed that NiO-NPs could be hemolytic at high concentrations of 250–1000 mg/L. However, at lower concentrations, the hemolysis percentage was less than 1%, which indicated the non-hemolytic nature of low concentrations of the biosynthesized NiO-NPs. In addition, NiO-NPs revealed low toxicity against brine shrimps and an enhancement effect on seed development and germination at low concentrations, which showed that the biosynthesized NiO-NPs are eco-friendly and could be used as antibacterial and eco-friendly agrochemicals as an alternative to synthetic agrochemicals at an industrial scale. Also, the cell viability and biocompatibility assay showed that the IC₅₀ value for the NiO-NPs was 125.5 mg/L, which suggests that they are biocompatible at different concentrations. Our subsequent studies will investigate microalgal and plant species to compare the biosynthesized NiO-NP and affecting factors and explore the characterization, bioactivity, and toxicity.

Compliance with ethical standards

Ethical approval: This article does not contain any studies with human participants or animals performed by any of the authors.

Funding

The authors declare they have no financial interests.

Credit author statement

Mohamed A. Hussein: Conceptualization, Investigation, Writing-Reviewing and Editing, Project administration, Funding acquisition, Esraa A. Abdelkarim: Writing-original draft preparation, Visualization, Software, Tamer Elsamahy: Software, Investigation, Writing-Reviewing and Editing, Software, Mabrouk Sobhy: Writing-original draft preparation, Software, Rasha M. El Bayomi: Writing-Reviewing and Editing, and Abd-Elsalam E. Hafez: Writing-Reviewing and Editing, Visualization, Supervision

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

Faculty of Veterinary Medicine, Zagazig University is highly acknowledged for providing research facility.

Availability of data and materials

All data are fully available without restriction.

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Bioactivity and Toxicity of Biogenic Nickel Oxide Nanoparticles Using Punica granatum Seed Extract

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Abstract

Figures

1. Introduction

2. Material and methods

2.1. Pomegranate seed extract preparation

2.2. Synthesis and capping of NiO-NPs

2.3. NiO-NPs characterization

2.4. Bioactivity of the synthesized NiO-NPs

2.4.1. Antibacterial Activity

2.4.2. Antioxidant activity

2.4.2.1. DPPH radical scavenging activity

2.4.2.2. ABTS^•+ scavenging activity

2.4.2.3. Hydrogen peroxide (H₂O₂) scavenging activity

2.4.3. Hemolysis analysis

2.5. Toxicity assays

2.5.1. Toxicity of brine shrimp

2.5.2. Effect on seed growth and development

2.5.3. Biocompatibility and cell viability

2.6. Statistical analysis

3. Results and discussion

3.1. NiO-NPs synthesis

3.2. Characterization of NiO-NPs

3.3. Bioactivity of NiO-NPs

3.3.1. Antibacterial activity of NiO-NPs

3.3.2. Antioxidant activity

3.3.3. Hemocompatibility assay

3.4. NiO-NPs ecotoxicity evaluation

3.4.1. Brine shrimp cytotoxicity

3.4.2. NiO-NPs effect on seed growth and development

3.4.3. NiO-NPs biocompatibility

4. Conclusion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Bioactivity and Toxicity of Biogenic Nickel Oxide Nanoparticles Using Punica granatum Seed Extract

Status:

Version 1

Abstract

Figures

1. Introduction

2. Material and methods

2.1. Pomegranate seed extract preparation

2.2. Synthesis and capping of NiO-NPs

2.3. NiO-NPs characterization

2.4. Bioactivity of the synthesized NiO-NPs

2.4.1. Antibacterial Activity

2.4.2. Antioxidant activity

2.4.2.1. DPPH radical scavenging activity

2.4.2.2. ABTS•+ scavenging activity

2.4.2.3. Hydrogen peroxide (H2O2) scavenging activity

2.4.3. Hemolysis analysis

2.5. Toxicity assays

2.5.1. Toxicity of brine shrimp

2.5.2. Effect on seed growth and development

2.5.3. Biocompatibility and cell viability

2.6. Statistical analysis

3. Results and discussion

3.1. NiO-NPs synthesis

3.2. Characterization of NiO-NPs

3.3. Bioactivity of NiO-NPs

3.3.1. Antibacterial activity of NiO-NPs

3.3.2. Antioxidant activity

3.3.3. Hemocompatibility assay

3.4. NiO-NPs ecotoxicity evaluation

3.4.1. Brine shrimp cytotoxicity

3.4.2. NiO-NPs effect on seed growth and development

3.4.3. NiO-NPs biocompatibility

4. Conclusion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

2.4.2.2. ABTS^•+ scavenging activity

2.4.2.3. Hydrogen peroxide (H₂O₂) scavenging activity