The leaves of C. argentea were obtained from the South Valley University farm and rinsed with deionized water. The washed and dried leaves were cut into little pieces. 20 g of C. argentea were combined with 200 mL of deionized water and heated for 30 min at 80°C. After cooling to ambient temperature, the mixture was filtered, collected, and stored in a refrigerator at 4°C for future experiments.

For comparison, two samples of ZnO NPs were synthesized under the same conditions: one with Sodium hydroxide as a reducing agent and the other with C. argentea extract. Two solutions were prepared following the coprecipitation approach. The precursor and reduction solutions in a typical synthesis, 5 g of zinc acetate [Zn(CH₃CO₂)₂], are dissolved in 100 ml of deionized water and stirred (1500 RPM) for 30 min. The reduction solution was 0.1 M of sodium hydroxide in the case of chemical ZnO NPs and 10 ml of fresh C. argentea extract in the case of biogenic ZnO NPs. The reducing solution was added to the precursor and stirred for 1 h (1500 RPM). Sodium hydroxide and hydrochloric acid were used to adjust the pH of the mixture to 10.4, and the mixture was left under stirring for another hour. After precipitation overnight at room temperature, NPs were collected by centrifugation (5000 RPM, 5 min, at room temperature), washed ten times with distilled water and one more time with ethanol, and dried in an oven at 100°C for 6 h (Figure 1).

X-ray diffraction analysis was conducted using the X'Pert PRO-PAN instrument with a copper target (Cu-K = 1.54056 A) operating at 40 kV and 30 mA. Additionally, the structural features of biogenic ZnO nanoparticles were investigated using a transmission electron microscope (JEOL, JEM 2100, Japan). Optical properties were measured using infrared spectroscopy (FTIR Model 6100, Jasco, Japan) and UV-visible spectrophotometry (SPECORD 200 PLUS, Analytik Jena, Germany).

Using a standardized (DPPH) assay, the free radical scavenging activity of biogenic ZnO NPs, C. argentea extract, and ascorbic acid was determined ^20,21. The stock solution of three samples (green ZnO NPs, C. argentea extract, and ascorbic acid) was generated first by dissolving 4 g in 4 ml methanol, followed by eight serial dilutions (1,000, 500, 250, 125, 62.5, 31.25, 15.625, and 7.8125 mg/ml). Second, 3 ml of DPPH was added to each test tube and incubated in the dark for 30 min at room temperature. Finally, at 517 nm, the absorbance (A) of the control and samples were measured spectrophotometrically.

The assessment of scavenging ability against DPPH free radicals was conducted for each sample and expressed as a percentage concerning a reference standard from Reference. ²⁰: Free radical scavenging activity % = Abs control − Abs sample Abs control × 100 (1)

The IC₅₀ was plotted as a function of concentration from a graph of free radical scavenging activity.

The agar disc diffusion method described by Mounyr Balouiri et al. was used to test the antibacterial activity against different bacterial strains: gram-positive [Staphylococcus aureus (ATCC 29213), Bacillus subtilis (ATCC 6633)] and gram-negative [E. coli (ATCC 25922) and Salmonella typhimurium (ATCC14028)]. Four samples were prepared in normal saline (0.9% NaCl). Biogenic ZnO NPs samples were prepared by sonication to get a dispersed NPs solution and diluted into three concentrations (25, 50, and 100 μg/ml). For comparison, a chemical ZnO NPs sample was used with the high concentration (100 μg/ml), C. argentea extract with concentration (20 μg/ml), and Gentamycin with concentration (20 μg/ml) as a positive control. Bacterial strains were cluttered in (Luria–Bertani) LB broth Overnight to obtain tested bacterial suspensions. Sterile Whatman filter paper discs were placed on the surface of the agar media, and then a proper volume containing the tested concentration of each sample was added. After incubating the plates for 24 h at 37°C, the diameters of the inhibitory zones were measured in millimeters. Each experiment was carried out three times to ensure accuracy.

The data were processed using the Statistical Package for the Social Sciences (SPSS 25.0) software, and the results were expressed as the mean standard deviation. The data were analyzed using (ANOVA), and the p < 0.05 value indicated a statistically significant difference.

Green synthesis of NPs is considered the most cost-effective, biocompatible, and flexible method for NPs synthesis. Biomolecules in the living organism have a high efficacy in reducing the metal ions to produce functionalized NPs with superior biocompatibility. To investigate the role of C. argentea extract on the formation of ZnO NPs, XRD analysis was performed for dried C. argentea extract, chemically and biogenic ZnO NPs. Figure 2A shows the XRD pattern of dried C. argentea extract. Mostly, the diffraction peaks at 2θ 14.97° and 22.88° belong to the fibrous cellulose (Manimaran et al., 2019; Ilaiya Perumal and Sarala, 2020). The successful formation of ZnO NPs was confirmed in both chemically and biogenic samples by the presence of fingerprint peaks in the XRD pattern, Figure 2B. The results in contact with JCPDS card No. 36-1451 for ZnO NPs (Wahab et al., 2014) and with the reported results of green ZnO NPs synthesized from different plants (Manokari and Shekhawat, 2017; Yusof et al., 2020; Sadhasivam et al., 2021). No additional peaks were observed for biomolecules from plant extract, indicating the absence of impurities and reflecting the prepared sample’s purity. Also, the sharp and narrow peak at 2θ ∼ 36° indicates the crystalline nature of the synthesized green ZnO NPs.

For chemical ZnO NPs, the diffraction peaks at 2θ (31.79, 34.43, 36.25, 47.54, 56.58, 62.84, 67.93 and 69.08) corresponding to the plane (100, 002, 101, 012, 110, 013, 112 and 201) respectively. A small shift in the peak position and decrease in the peak intensity was observed in the XRD pattern of biogenic ZnO NPs to be (31.5, 32.23, 36, 47.32, 56.42, 62.65, 67,82 and 69) corresponding to the plane (100, 002, 101, 012, 110, 013, 112 and 201) respectively. This peak shift was accompanied by a decrease in the peak intensity, suggesting that green synthesis decreases the crystallinity of ZnO NPs.

In the case of ZnO wurtzite structure a = b = 3.249 A° and c = 5.206 A°. The lattice constant a was calculated for the plane (100) using the relation (Gatou et al., 2023): a = λ 3 sin ⁡ θ (3)

Lattice constant c calculated from for the plane (002) using the relation (Gatou et al., 2023): c = λ sin ⁡ θ (4)

The unit cell volume for the hexagonal structure was calculated using the following expression (Gatou et al., 2023): V = 3 2 a 2 c (5)

The calculated unit cell parameters for chemical ZnO NPs were almost identical to the reported standard values (a = 3.247 A°, c = 5.203 A°, and unit cell volume V = 47.53 A°). While biogenic ZnO NPs showed some expansion in all unit cell directions (a = 3.275 A°, c = 5.548 A° and unit cell volume V = 51.55 A°), Table 2.

XRD pattern analysis is a method that is frequently utilized to analyze the structure of materials, particularly nanomaterials. The Debye-Scherer and Williamson-Hall techniques are two of the more common methodologies used in the XRD investigation of metal oxide nanomaterials. The Debye-Scherer method is used to calculate crystal size ( D _L ). The approach presupposes that X-rays enter the material and interact with all its crystallites. Using the Debye-Scherer equation, the main diffraction pattern (101) is then utilized to calculate the crystal size and microstrain: D L = k λ / β ⁡ cos ⁡ θ (5a) ɛ = β cos θ / 4 (6) Where k is a constant, λ wavelength, β diffraction peak, and θ is the Bragg angle.

In contrast, the Williamson-Hall method considers the impact of crystal size and lattice strain on the diffraction pattern. The approach posits that the X-rays interact with a finite number of crystallites in the sample and that the broadening of the diffraction peaks is caused by both the crystallites’ finite size and the presence of lattice strain. According to this model, the average crystallite size (D_L) and average micro-strain (ε) were found by taking the reciprocal of the intercept of (β cos θ) versus (4 sin θ) (Figures 3A, B). The primary distinction between the two methodologies lies in their underlying assumptions. The Debye-Scherer technique operates under the premise that the observed diffraction pattern is exclusively attributable to the finite dimensions of the crystallites. Conversely, the Williamson-Hall method considers the impact of crystal size and the influence of lattice strain on the resulting diffraction pattern.

The synthesized biogenic ZnO NPs were imaged by a high-resolution transmission microscope (HRTEM), Figures 4A, B. Chemical ZnO NPs showed a non-uniform spherical shape. While an accumulation of the formed semispherical particles of biogenic ZnO NPs was observed. It is reported that using biomolecules as reducing agents increases particle aggregation due to the electrostatic interaction between metal ions and biomolecules. Similar results were obtained for the biosynthesis of green ZnO NPs from different plant extracts (Manokari and Shekhawat, 2017; Yusof et al., 2020; Sadhasivam et al., 2021). ImageJ software was used to estimate the particle size from HETEM images. The number of counted particles was 100 per image. The average particle size was 26.48 ± 4.56 and 21.55 ± 4.73 nm for chemical and biogenic ZnO NPs, respectively.

The following expression can be used to calculate the specific surface area (SSA), which is defined as the total surface area per unit mass and is applicable for adsorption and reactions that take place on the surface of the material: SSA = S f   / β .   Ρ (7)

S_f represents the form factor of ZnO NPs, β is the mean particle size, and ρ is the density of ZnO (5.61 g/cm⁻³).

The SSA for biogenic ZnO NPs is shown in Table 2 using the assumption that the particles are spherical (S_f = 6), where particle sizes were measured using different methodologies.

From the structural analysis, it can be concluded that the biogenic synthesis of ZnO NPs from C. argentea extract didn’t change the structure of ZnO NPs. However, the produced NPs possess smaller size, lower microstrain, and higher specific surface area than chemically synthesized ZnO NPs.

The antioxidant, antibacterial, and anticancer capabilities of ZnO NPs can be improved by increasing their specific surface area and decreasing their particle size value. The larger number of reactive sites on the surface of ZnO NPs enhances their ability to scavenge free radicals and their antioxidant activity (Ramesh et al., 2022). The increased surface area of the NPs prevents oxidative damage to cells and tissues by efficiently adsorbing free radicals. Accordingly, this promises to treat various medical issues, including cardiovascular illness, neurological disease, and cancer (Hamed et al., 2023). ZnO nanoparticles (NPs) have a greater surface area relative to their volume, increasing the interaction between the NPs and bacterial cells and leading to greater antibacterial activity. This improved connection can potentially increase the NPs’ antibacterial activity by facilitating their penetration of the bacterial cell wall and disrupting cellular processes. As a result, such materials are used in the development of antibacterial coatings for use on medical equipment and surfaces and in the management of bacterial infections.

To generate reactive oxygen species (ROS) and cause cellular damage in cancer cells, ZnO NPs with a high specific surface area induce apoptosis (programmed cell death). The enhanced surface area of the nanoparticles facilitates more effective production of reactive oxygen species (ROS), enhancing their anticancer efficacy (Snezhkina et al., 2019). The large surface area can penetrate the tumor microenvironment and home in cancer cells more easily while avoiding collateral damage to healthy tissue. The process may help in the development of more precise tumor-targeting cancer medicines.

Fourier transformation Infrared spectroscopy (FTIR) was employed to investigate the possibility of Zn ions reduction and the formation of ZnO NPs. Several absorption bands were observed due to the presence of ZnO NPs and the organic molecules of C. argentea Figure 5A. Vibrational bands were observed at 452 and 526 cm⁻¹, corresponding to Zn-O stretching vibration. The absorption bands appearing at 698, 1,410, and 2,384 cm⁻¹ are due to C-H bending vibrations, while the band at 890 cm^-1 can be attributed to N-H stretching. C=C stretching vibration appeared at 1,620 cm⁻¹, while C=O appeared at 2,508 cm⁻¹.

Finally, the broad band observed was approximately between 3,000 and 3,500 cm⁻¹ corresponding to the OH vibration of the hydroxyl group. The formation of ZnO NPs was confirmed by the presence of Zn-O vibrational bands at 452 and 526 cm⁻¹ in the FTIR spectra, which are the fingerprints of inorganic NPs (Yusof et al., 2020; Sadhasivam et al., 2021). Some functional groups were detected due to the interaction of Zn⁺² ions with the biomolecules of C. argentea leaves, suggesting the successful reduction of ZnO and the formation of ZnO NPs (Parvathy et al.). Figure 5B shows the absorption spectrum of C. argentea, chemical, and biogenic ZnO NPs with an absorption peak at 255, 248, and 342 nm, respectively (Press et al., 2017). The optical band gap was calculated using the Tauc equation as the following (Gatou et al., 2023): α h υ = A h υ − E g n (8) where α is the absorption coefficient, h is the blank constant, A is the proportional constant, and E_g is the band gap energy. Herein, the value of constant n depends on the type of the transition process; for direct transition, n = 2, while n 1/2 for indirect optical transitions. For ZnO NPs, the possible transition is indirect, so that n will be considered = 1/2 (Rahman et al., 2020). Plotting the relation between (αhυ)² and the incident photon energy Eg (hυ), the band gap energy can be calculated by extrapolating the linear region on the energy axis, Figure 5C. The calculated band gap energy was 3.31 and 3.24 e.V for chemical and biogenic ZnO NPs, respectively. These values are smaller than the reported value (3.37 e.V). The decrease can be explained as the biosynthesis with plant extract modifying the surface of ZnO NPs, resulting in increased electron densities and lower energy bands (Alharbi et al., 2023).

The antioxidant activity of biogenic ZnO NPs is attributed to their ability to donate hydrogen. The formation of electron-hole pairs on the surface of ZnO NPs has great potential for reducing H₂O molecules, which can work as scavengers of DPPH molecules (Bisht and Rayamajhi, 2016). However, the biogenic synthesis of ZnO NPs can contribute to modulating the scavenging ability due to phytochemicals such as phenolics and polyphenolic compounds in the plant extract (Madan et al., 2016). These phytochemicals have antioxidant abilities, which contribute to the inhibition of free radical scavenging.

Figure 6 shows the DPPH radical scavenging activity of biogenic ZnO NPs compared with chemical ZnO NPs C. argentea and ascorbic acid (ASC) as standards. These results reflect a dose-dependent behavior in the scavenging activity of biogenic ZnO NPs with Ic₅₀ = 91.24 μg/ml compared with Ic₅₀ = 131.08, 58.76, and 14.37 μg/ml for chemical ZnO NPs, C. argentea and ascorbic acid, respectively. Similar results were reported for green ZnO NPs synthesized using the DPPH assay in Table 3. The Ic₅₀ of the aqueous extract of C. argentea was 30 μg/ml, which was attributed to its phenolic content as reported by Rub et al. (2013). Hence, the biosynthesis of ZnO NPs using C. argentea extract enriched its surface with biomolecules and improved its scavenging activity.

In this study, the antibacterial activity of biogenic ZnO NPs from C. argentea extract was evaluated against four types of bacterial strains: two gram-positive [Staphylococcus aureus (ATCC 29213), Bacillus subtilis (ATCC 6633)] and two gram-negative [E. coli (ATCC 25922) and Salmonella typhimurium (ATCC14028)]. The diameter of the inhibition zone of green ZnO NPs, chemical ZnO NPs, C. argentea extract, and Gentamycin as a positive control was determined using an agar disc diffusion method and presented in Figure 7. The highest concentration (100 μg/ml) was chosen for chemical ZnO NPs in order to compare its antibacterial activity. Biogenic ZnO NPs showed significant antibacterial activity against the four bacterial strains compared with the positive control and chemical ZnO NPs. Similar activity had been reported for green synthesized ZnO NPs from different plants (Table 4). Moreover, the inhibition zone of C. argentea extract against the four bacterial strains was significantly larger than that of Gentamycin, which was due to the presence of bioactive compounds such as alkaloids, flavonoids, phenols, terpenoids, starch, and cellulose (Malomo et al., 2011; Santana et al., 2021).

Regarding the bacterial strain type, biogenic ZnO NPs showed a higher bactericidal effect in gram-positive strains (S. aureus and B. subtilis) than in gram-negative strains (E. coli and Salmonella typhimurium). The explanation of this result was investigated by Vijayakumar et al. and attributed to the differences in the structure and components of gram-positive bacteria, such as the peptidoglycan layer, which facilitates the attachment and internalization of ZnO NPs to the cell wall (Sharma and Kaushal, 2022). The same behavior was observed in the case of chemical ZnO NPs, except that the diameter of the inhibition zone was smaller.

Additionally, the inhibition zone of biogenic ZnO NPs increased dramatically with concentration increments from 25 to 100 μg/ml in the four bacterial strains. This behavior is due to the increased number of ZnO NPs diffused in the agar medium, which is in contact with the reported results (Singh et al., 2018; Sadhasivam et al., 2021; Publishers, 2023).

Several mechanisms were reported for the antibacterial activity of green ZnO NPs; the main three possible mechanisms were illustrated in Figure 8 (Ambika and Sundrarajan, 2015; Mendes et al., 2022; González et al., 2021). The proposed mechanisms include:

• The release of Zn⁺² is due to the interaction of ZnO NPs with the bacterial cell. The negatively charged bacterial membrane attracted the released Zn⁺² via Van der Waals forces, leading to defragmentation of the cell membrane, inhibition of cell growth, and finally cell death.

The physiochemical properties such as size, shape, surface chemistry, and high surface-to-volume ratio are responsible for the antibacterial action of green ZnO NPs on different bacterial strains (Table 4).

One of the main advantages of ZnO NPs as an anticancer agent is their selectivity for cancer cells. The ability of NPs to kill cancer cells varies depending on their intrinsic properties, such as their ability to produce ROS. These ROS interact with the cellular components and initiate apoptosis. From a genetic point of view, apoptotic induction can occur through intrinsic or extrinsic pathways due to the activation of caspases. Although many genes can induce apoptosis, p53 is the master guardian of the cell, maintaining genomic stability through activated cell-cycle checkpoints, DNA repair, and apoptosis (Bisht and Rayamajhi, 2016; Tabrez et al., 2022). Cell cycle arrest is initiated by DNA damage and the p53 gene, enabling the possibility of either self-mediated programmed cell death or repair of the damage (Król et al., 2017; Levy et al., 2017).

The Bcl-2 level reduces resistance to apoptotic stimuli, resulting in apoptosis (Tang et al., 2017). Intrinsic pathways of apoptosis are the source of the mitochondria-integrated signals. The cell death resistance is motivated by the activation of NF-κB, which leads to downstream target gene expression (Albensi and Braun, 2019). The NF-κB activation in the mitochondria leads to cytochrome c release, thus triggering caspase cascades and programmed cell death.