Pharmaceutical properties for green fabricated ZnO and Ag nanoparticle-mediated Borago officinalis: In silico predications study

2.1 Materials

Silver nitrate (AgNO₃, 99.9%) was purchased from Daejung, Korea, and Zinc nitrate hexahydrate (Zn (NO₃)₂·6H₂O, 99.0%) was purchased from Himedia, India. Ethanol was purchased from Duksan, Korea. Mueller Hinton Agar was purchased from Accumix, Spain. Nutrient broth was purchased from Oxoid, United Kingdom. The dried Borago officinalis flowers were purchased from a local market in Baghdad. Deionised water was used throughout the experiment.

2.2 Preparation of Borago officinalis flower extract

The purchased dried Borago officinalis flowers were extracted with 80% aqueous ethanol at 5:1,000 (w/v) for 3 h. The extract was filtered, and the excess ethanol was dried at 70°C using the oven. The obtained extract was stored in the fridge for further investigation. The extraction process was based on the studies by Rahmah et al. and Aziz et al. [15,16].

2.3 Preparation of Ag NPs

The green synthesis approach of Ag NPs was attained by the method of [17]. A 6.5 g of AgNO₃ was dissolved in 250 mL of deionised water using a magnetic stirrer (800 rpm) at room temperature (provide temperature, 25°C). Then, 100 mL of plant extract (B. officinalis) was the precursor solution. After 24 h, the colour of the mixing solvent alters to dark brown. The suspension was separated using a centrifuge, and the residue was washed with water and ethanol five times. The residue was dried using the oven at 40°C for 12 h, and Ag NPs were then obtained as the final product. Figure 1 illustrates the steps of Ag NPs preparation.

2.4 Preparation of ZnONPs

The green synthesis approach of Zn NPs was achieved by dissolving 7 g of zinc nitrate hexahydrate (Zn(NO₃)₂·6H₂O) in 200 mL of deionised water at 50°C for 120 min using a magnetic stirrer. Then, 150 mL of B. officinalis plant extract (5.5 mg·mL⁻¹) was mixed with the above solution and stirred for 60 min. Then, 50 mL of 1 M sodium hydroxide (NaOH) was added dropwise to the mixture solution. Then, the precipitation was washed by centrifuge with deionised water and ethanol several times. The resulting rainfall was dried at 100°C for 3 h to form powder, and impurities were removed [18,19]. Then, the calcination process was carried out at 550°C for 3 h, and ZnO NPs were formed. Figure 2 illustrates the steps of ZnO NP preparation.

Figure 1

Green synthesis of Ag NPs.

2.5 Characterisation

The optical properties of Ag NPs and ZnO NPs synthesised using the green method are determined using UV–visible spectroscopy (Shimadzu, UV-1800 spectrophotometer) at room temperature at 200–800 nm. The Fourier-transform infrared spectroscopy (FTIR) spectrum (Frontier FTIR-88091) was employed to analyse the boundary construction of resulted samples with an absorption range of 500–4,000 cm⁻¹. The functional gropes contained in each nanoparticle were identified via FTIR analysis. Field emission scanning electron microscopy (FESEM, TESCAN, MIRA3) was employed to capture images of Ag NP and ZnO NP samples to study their morphology. Energy-dispersive X-ray spectroscopy (EDS) also provided a straightforward and effective method for analysing the samples’ elemental composition. The structural analysis of the Ag NP and ZnO NP samples was performed using an X-ray Diffraction (XRD) diffractometer (XRD diffraction 6000, Shimadzu) with CuKα radiation (λ = 1.542 Å), operating at a current of 30 mA and a voltage of 40 kV. Data were collected in the 2θ range of 10–80°. The average particle size (D) was determined using the Scherrer equation, D = kλ/(β cosθ), where k is a constant (0.89), β represents the full width at half maximum, and θ corresponds to the Bragg angle.

2.6 Preparation of Mueller Hinton agar

Muller–Hinton (M–H) was prepared by adding 20 mL of the powdered agar into 1 L of distilled water. The mixture was heated under shaking conditions. M–H agar was sterilised at 121°C for 15 min. Then, it was cooled to 50°C and poured into a Petri dish. After solidifying the agar, the Petri dish was flipped and stored at 4°C.

2.7 Antibacterial activity

The antibacterial activity of the Ag NPs and ZnO NPs was performed using an agar well diffusion assay [20,21] against two Gram-positive bacteria, including Staphylococcus aureus and Streptococcus mutants, and two Gram-negative bacteria, including Klebsiella pneumonia and Escherichia coli. The bacterial samples were inoculated in 6-mm-diameter wells on an M–H agar petri dish. The final concentration (12.5, 25, 50, and 100 μg·mL⁻¹) of Ag NPs and ZnO NPs are added into the wells. The plates were incubated overnight at 37°C before measuring the inhibition zone diameter.

2.8 Biofilm inhibition

The antibiofilm activity of the Ag NPs and ZnO NPs against the Gram-positive and Gram-negative bacteria was conducted using crystal violet (CV) staining obtained by Al Rugaie et al. [22]. The bacterial strains were cultured in 96-well plates at a concentration of 1 × 10⁶, treated with Ag NPs and ZnO NPs, and then inoculated for 24 h. After incubation, the samples were washed with Phosphate-buffered saline (PBS). Adhered bacteria were stained with 0.1% CV and rinsed twice with distilled water. To assess biofilm formation, 0.2 mL of 95% ethanol was added to the CV-stained wells, followed by 2 h of incubation with shaking. The optical density was measured at 959 nm.

2.9 Bacterial reactive oxygen species (ROS) formation using DCFH-DA assay

Bacterial strains were treated with prepared nanoparticles. Then, 25 µM DCFH-DA in 1× PBS for 45 min was added. Samples were washed 2 times with 1× PBS buffer. Using the ZEISS Axioscope microscope with the Filter Set 38 HE (Oberkochen, Germany), the fluorescence was observed at a wavelength of 535 nm following the excitation of the sample at 485 nm.

2.10 DPPH assay

NPs were tested as antioxidants using (DPPH) method; 12 mg of DPPH was dissolved in 50 mL of methanol to obtain the stock solution. In the test tube, 3 mL of DPPH was combined with 100 μL of prepared NPs. Besides, 3 mL of DPPH solution in 100 μL of methanol can be represented as standard. After that, the tubes were kept in a dark place for 30 min. The absorbance was therefore determined at λ = 517 nm. The formula that used to calculate the percentage of antioxidants (%) = [(A _c – A _s) ÷ A _c] × 100% where A _c is the control absorbance and A _s is the testing sample absorbance.

2.11 MTT assay

This technique was used to investigate the cytotoxic effect of prepared nanoparticles against colon cancer cell line (HT-29). HT-29 cells were seeded in a 96-well plate at density 1 × 10⁴; after 24-h incubation; cells were treated with different concentrations of NPs (0, 12.5, 25, 50, and 100 μg·mL⁻¹). MTT solution at a concentration (2 mg·mL⁻¹) was added and then incubated for 3 h at 37°C; 100 µL of (DMSO) was added, followed by shaking for 15 min at room temperature, and the absorbance at 492 nm was measured using a microplate reader.

2.12 In silico analyses

The molecular structure was designed by materials studio software, and a sphere of ZnNPs and AgNPs with a radius of 3 Å was generated. Four targeted proteins were selected to investigate the bioactive properties of the ZnO NP and AgNP compounds. The target proteins, Colon Cancer Antigen 10 (ID: 2HQ6), MltA from E. coli (ID: 2GAE), S. mutans (ID: 3Q6A), and Peroxiredoxin from Aeropyrum pernix K1 (ID: 6KRR) were obtained from the Protein Data Bank www.rcsb.org. Protein preparation, including removing water molecules and heteroatoms, was performed using Biovia Discovery Studio software. Energy minimisation and checks for missing regions in the selected receptors were carried out using SWISS-PDBVIEWER (version 4.1.0, SPDBV). The molecular docking process was executed using AutoDock 4.2 software. It started with the addition of polar hydrogens to the proteins, followed by the application of Kollman charges and the assignment of Gasteiger charges. The grid box was configured to encompass the entire protein structure. Docking parameters were optimised using genetic algorithms, generating 50 conformations/poses. The best conformation was selected based on the lowest binding free energy, which reflected ligand–receptor non-covalent interactions. Hydrophobic interactions between the docked drug-like molecules and the target proteins were identified using LigPlus (LigPlot + v.2.2.8), while Biovia Discovery Studio was used to visualise the best-posed conformations.

2.13 Statistical analysis

The data were analysed using GraphPad Prism software (version 6). The mean ± standard deviation (SD) is used to display the data. All experiment was carried out at least three times. Tukey’s test was used for post-hoc comparisons between groups after an independent-sample t-test or a one-way analysis of variance was suitably used for statistical analysis. Statistical significance was determined at p < 0.05 [23,24].

3.1 Structural, optical, and morphological studies

Figure 3a reveals the UV–visible absorption spectra of Ag NPs synthesised using the green method. The UV–visible spectrum of Ag NPs was studied in a range of 200–800 nm. Figure 1a displays a broad absorption band around (406.6–538.6 nm) with a strong red shift edge at 437 nm. The broad peak of the surfers’ Plasmon resonance band around 437 nm has revealed the formation of Ag NPs. The SPR band related to Ag NPs was formed as a result of the presence of the reducing agent of compounds existing in the B. officinalis flower extract, which was employed for the incorporation of Ag NPs and the energy band gap by using the Tauc plot of the (αhυ)2 versus photon energy (hυ) curve with n = 1\2 for direct transition is about 3.76 eV, as shown in the inset of Figure 4(a). Figure 4(b) reveals the UV–visible absorption spectra of ZnO NPs synthesised by the green method. The B. officinalis flower extract–mediated ZnO NPs were characterised by a UV–visible spectrum (200–800 nm) range where ZnO NPs show a strong absorption peak at 376.7 nm, as shown in Figure 1b. Besides, the UV–visible spectroscopic reveals a sharp absorption peak and improves the stabilising agent as ZnO NPs are synthesised by the aqueous leaf of extract B. officinalis. In addition, the band gap of green fabricated Zn ONPs was calculated using the Tauc plot as 2.27 eV, as shown in the inset in Figure 4(b).

Figure 2

The green synthesis of ZnONPs.

Figure 3

The UV–visible spectrum of (a) Ag NPs and (b) synthesised by green method.

The XRD diffraction spectra of Ag NPs and ZnO NPs were analysed as illustrated in Figure 4(a) and (b). In Figure 4(a), the XRD patterns spectrum of Ag NPs display four prominent peaks at 2Ɵ = 38.11°, 44.30°, 64.44° and 77.40°, and these peaks‘ reflections correspond to planes of (111), (200), (220), and (311), respectively of cubic structure and matched with (JCPDS card-96-900-8460) of Ag nanoparticles [24]. These diffraction peaks indicate the preparation of silver nanoparticles using the green method by extracting plant compounds. In addition, a few peaks at 2θ = 27.8° and 32.33° were related to the green method using plant extract, revealing its role as a reducing agent of Ag ions. The average particle crystallite of Ag NPs was obtained using the Scherrer formula and found to be 18.25 nm from the peak intensity of Ag NPs (111). In state of ZnONPs prepared by green procedure using B. officinalis plant extract, the XRD patterns displays peaks at 2θ = 31.77°, 34.42°, 36.26°, 47.54°, 56.61°, 62.86°, 66.39°,67.96°, and 69.10° were related to planes (100), (002), (101), (102), (110), (103), (200), (112), and (201), respectively, as shown in Figure 4(b). The XRD data are related to the hexagonal structure of synthesised ZnO NPs by the green method [23,24]. The obtained data are matched with (JCPDS card-96-230-0451), and it can be revealed that the resulting crystallite ZnO NPs show high purity as shown in the XRD spectrum [25]. Moreover, the average particle crystallite of ZnO were obtained using the Scherrer formula and found to be 27.4, 27.6, and 47.6 nm from the peaks intensity of ZnO (100), (002), and (101), respectively. Moreover, using optimal concentrations of B. officinalis extract improves the crystallinity of AgNPs and ZnO NPs, as evidenced by sharper XRD peaks related to nanoparticle nucleation and growth rate. The Scherrer formula was employed to estimate crystallite size and confirm the structural properties relevant to the applications. Additionally, FESEM and Image J were used to validate particle morphology and size distribution, ensuring consistency in the synthesised nanoparticles.

The FTIR spectrum of biosynthesised AgNPs and ZnONPs in the range (500–4,000 cm⁻¹) are shown in Figure 6(a) and (b) at room temperature. The FTIR spectra of Ag NPs in Figure 6(a) showed the major absorption bands at (3,425–3,351 cm⁻¹), (2,981–2,844 cm⁻¹), 1,697, 1,568, 1,379, 1,125 and 732 cm⁻¹. The bands at (3,425–3,351 cm⁻¹), (2,981–2,844 cm⁻¹), and 1,379 cm⁻¹ were associated with stretching vibrating of hydroxyl groups (O–H) and alkane (C–H). Besides, the peaks at 1,697, 1,568, and 732 cm⁻¹ are according to the bending vibrating of alkene (C═O) groups. The band at 1,125 cm⁻¹ was related to the amide band containing carbonyl groups (C═O). The peak at 877 cm⁻¹ represented aromatic groups (C═H) for alkene. Moreover, these peaks are due to reduced sugars, flavonoids, saccharides, and proteins in the extract plant compounds. These functional groups form as reducing agents, providing stability and preventing agglomeration of Ag NPs in ZnONPs. Phytochemicals are essential in NP synthesis as they are crucial to stabilising the NPs by forming chelate complexes with metal ions. The primary mechanism explaining the formation of NPs summarised that the extract phytochemicals reduced and oxidised the ions. These compounds act as reducing agents, stabilisers, and capping agents, replacing the requirement for additional chemical stabilisers. Once the reaction is complete, the resulting product undergoes annealing to obtain NPs [26]. The FTIR spectrum of biosynthesised ZnONPs in the range of (500–4,000) cm⁻¹ is shown in Figure 6(b) at room temperature. The FTIR shows the prominent peak positions related to synthesised ZnO NPs using plant extract at 3,377 and 3,695 cm⁻¹, indicating broad peaks of O–H stretching vibrating. The beaks at 2,978 and 2,886 cm⁻¹ corresponded to the CH₃ stretching vibration. The peak at 1,527 cm⁻¹ indicates C═C bond stretching vibrating. The peak at 2,162 cm⁻¹ is related to triple bond stretching, and the peak at 1,384 cm⁻¹ indicates C–O–H bending vibration. The peal at 1,255 cm⁻¹ relates to C–O stretching in aromatic alcohols, while the peak at 1,079 cm⁻¹ indicates the C–O an aliphatic C–N bond stretching. Meanwhile, the peak at 586 cm⁻¹ corresponds to Zn-O stretching and deformation vibration (Figure 5).

Figure 4

The XRD diffraction of (a) AgNPs and (b) ZnONPs synthesised by a green method.

The resulting peaks reveal the formation of ZnO NPs using the extract of B. officinalis. The resulting peaks indicate the presence of functional groups like ketones, aldehydes, hydroxyl, alcohols, phenolic and carbocyclic acid with hydrogen bonds, which attributed to flavonoids, terpenoids, glycosides, and phenols and confirmed the green synthesised ZnO NPs by suing plant extracted as capping and stabilising agent reducing the aggregation of ZnONPs. The FESEM was used to study the surface morphology of Ag NPs and ZnO NPs, as shown in Figure 6(a)–(d). An expanded view in FESEM revealed that Ag NPs were relativity spherical. The average size of Ag NPs synthesised from the extract of B. officinalis flowers was measured to be in the range of (33–41 nm) as shown in Figure 6(a) and (b). in the case of ZnONPs synthesised from the extract of B. officinalis flowers, the morphology showed the spherical ZnO NPs with a size range of 50–80 nm, as shown in Figure 6(c) and (d). The resulting nanoparticles were in indirect contact, even within the aggregation, demonstrating the effect of a capping agent to stabilise the nanoparticles. Moreover, the FESEM images confirmed that using optimal extract concentration provides more nucleation sites, leading to the formation of smaller nanoparticles due to the availability of reducing and stabilising agents in the extract (flavonoids, polyphenols, and proteins). Moreover, the nanoparticles were developed by a quantitative element determined by using energy dispersive X-ray analysis (EDX) of Ag NPs and ZnO NPs, as shown in Figure 7(a) and (b), respectively. The EDX spectrum of Ag NPs displays the peaks of Ag, carbon, and oxygen elements, confirming Ag NPs formation with the highest peak intensity by using plant extracted as a capping agent without any additional peaks in EDX analysis, as shown in Figure 7(a). Furthermore, the particle size distribution obtained using image J analysis for Ag NPs and ZnONPs provides size measurements close to those observed in FESEM images, confirming its reliability. The results show that the measurements from Image J effectively match the resolution and details. In the state of ZnO NPs, the green synthesised ZnO NPs were further analysed through the EDX spectrum, as shown in Figure 7(b). The EDX analysis reveals the Zn, O, and carbon peaks, and Zn shows high peak intensity, confirming ZnO NP formation utilising plant extract as a capping agent. Besides, the appearance of Carbon elements in the EDX spectrum corresponds to biological molecules like amino, flavonoids, vitamins, and proteins in the plant extract. The average particle size for AgNPs is 44 and 69.5 nm for ZnO NPs, respectively, as shown in Figure 8(a) and (b). Image J provides an accurate representation of size variability by analysing many particles.

Figure 5

The FTIR spectrum of biosynthesised (a) Ag NPs and (b) ZnO NPs.

Figure 6

The FESEM images of (a) and (b) AgNPs and (c) and (d) ZnONPs.

Figure 7

EDS analysis of biosynthesised (a) Ag NPs and (b) ZnO NPs.

3.2 Antibacterial activity of synthesised NPs

The initial screening of in vitro antibacterial activities of green synthesised Ag NPs and ZnO NPs was conducted using an agar well diffusion assay. Four various contents (12.5, 25, 50, and 100 µg·mL⁻¹) of both Ag NPs and ZnO NPs were tested against two Gram-positive bacteria, S. aureus and S. mutans, and two Gram-negative bacteria, including K. pneumoniae and E. coli as shown in Table 1 and Figure 9. Ag NPs and ZnO NPs demonstrated vigorous antibacterial activities on S. mutans at the lowest amount of 12.5 µg·mL⁻¹ with an inhibition zone of 22 and 24 mm, respectively; meanwhile, the largest inhibition zone was recorded at 100 µg·mL⁻¹ of 28 and 30 mm, respectively, as shown in first-panel Figure 9. On the other hand, the second panel displays that the various concentrations of Ag NPs and ZnO NPs exhibited moderate and comparable antibacterial activities against S. aureus, with inhibition zones ranging from 12 to 22 mm. Notably, there was a direct correlation between the inhibition zone diameter and the concentrations of Ag NPs and ZnO NPs, as the inhibition zone diameter increased by 2 mm when doubling the concentrations of both Ag NPs and ZnO NPs. Regarding the results in the first panel, S. mutans was the bacteria most susceptible to Ag NPs and ZnO NPs compared to S. aureus due to its simple cell wall structure and lacking an outer membrane, which allows easy penetration by the nanoparticles and disturbs vital cellular functions [27]. Moreover, as shown in the third panel, Ag NPs and ZnO NPs produced less inhibition zone at 25 and 50 µg·mL⁻¹ concentrations against K. pneumoniae than the control. Based on the results, 100 µg·mL⁻¹ was the most effective against the Gram-negative bacteria K. pneumoniae with a maximum inhibition zone of 12 mm in. On the contrary, Ag NPs and ZnO NPs demonstrated strong and comparable antibacterial activity at the lowest concentration of 12.5 µg·mL⁻¹ with inhibition zones of 25 and 24 mm, respectively. There was a gradual and slight increase in the inhibition zone, coinciding with increasing the green synthesised nanoparticle concentrations. Table 2 shows the antibacterial activity of positive control (Genatmicin and Ampicillin) against tested bacterial strains compared with prepared NPs. However, the possible explanation is Ag NPs’ ability to accumulate on the bits on the cell wall owing to the nanoscale size, causing cytoplasmic wall denaturation or changing the cell membrane structure, thereby resulting in cell lysis [28]. Another interpretation is the Ag NPs’ ability to release silver ions, which adhere to the cell wall and cytoplasmic membrane due to the electrostatic attraction and affinity to sulphur proteins, as a consequence, disrupt adenosine triphosphate and deactivation of respiratory enzymes by producing ROS, which considered the main factor in destroying DNA and eventual cell death [29]. The findings of this study documented neither Ag NPs nor ZnO NPs inhibited the growth of Gram-negative bacteria K. pneumoniae at a low amount of 12.5 µg·mL⁻¹. As a comparison between the two Gram-negative bacteria, E. coli, as shown in the last panel, displayed more sensitivity to the green synthesised nanoparticles because of various factors, including the interactions between the cell wall and nanoparticles, the size of nanoparticles, as well as the E. coli cell wall lacking the thick layer of peptidoglycan, which make it vulnerable to the nanoparticles damaging impacts [30]. The mechanism of NPs’ antibacterial activity may be attributed to releasing toxic zinc and Ag ions and generating ROS, causing damage to the cell membrane and diffusing in the cytoplasm where they interact and affect various structures, leading to growth inhibition and cell death [31]. The Gram-positive bacteria consist of several peptidoglycan layers and teichoic acid, while the gram-negative bacteria consist of a thin layer of peptidoglycan surrounded by a secondary lipid membrane reinforced with transmembrane lipopolysaccharides and lipoproteins; the cell wall nature of the bacteria change due to the presence of NPs [32]. A semiconductor structure and surface defects characterise ZnO NPs; therefore, ZnO NPs are distinguished with magnificent photocatalytic features in the presence of light irradiation, leading to the production of ROS, including hydroxyl radicals (˙OH), singlet oxygen (¹O₂), superoxide anions (O²˙⁻), and hydrogen peroxide (H₂O₂), these ROS considered the essential factors responsible for the antibacterial functions as they cause oxidative damage to DNA and the bacterial membrane. Furthermore, ZnO NPs released Zn ions (Zn²⁺), which are necessary for modulating cell functions and disturbing bacterial growth [33]. On the other hand, the antibacterial activities of Ag NPs are derived from different mechanisms, including releasing silver ions (Ag⁺), which stimulate the production of intracellular ROS, disturbing bacterial cell and DNA integrity. Ag ions can also bind with bacterial membrane proteins, thus inhibiting essential enzymes required for bacterial survival [34]. In addition, the direct interaction of Ag NPs with the bacteria’s cell membrane increases permeability and damages the structure, causing cell death. They also modify microbial signal transduction pathways by affecting phosphotyrosine profiles in proteins involved in growth regulation [35].

Figure 8

The particle size distribution by using Image J of (a) Ag NPs and (b) ZnO NPs.

3.3 Antibiofilm activity of prepared NPs

Crystal violet assay assessed the effects of green Ag NPs and ZnO NPs in Gram-positive and Gram-negative bacteria biofilm formation. Figure 9 shows the susceptibility of tested bacteria biofilm to form AgNPs and ZnONPs using green method at different concentrations (12.5, 25, 50, and 100 μg·mL⁻¹). Compared to the control, 12.5, 25, 50, and 100 µg·mL⁻¹ concentrations inhibited biofilm formation. Ag NPs and ZnO NPs displayed comparable biofilm inhibition on S. aureus and S. mutans, ranging from 0.6 to 0.2 a.u., as shown in Figure 10.

Figure 9

Antibacterial activities of Ag NPs and ZnO NPs against S. mutans (first panel), S. aureus, K. pneumoniae, and E. coli (last panel): (A) Control, (B) 12.5 µg·mL⁻¹, (C) 25 µg·mL⁻¹, (D) 50 µg·mL⁻¹, and (E) 100 µg·mL⁻¹.

Similarly, Ag NPs and ZnO NPs exhibited comparable inhibition activity on K. pneumoniae biofilms. The lowest effect on the biofilm was displayed by Ag NPs of 0.5 a.u., at a concentration of 12.5 µg·mL⁻¹ (well B); ZnO NPs of 0.1 a.u displayed the highest effect. at a concentration of 100 µg·mL⁻¹ (well E), as reveal in Figure 10. Despite gradually doubling the Ag NPs and ZnO NPs concentrations, there was a slight difference in the biofilm inhibition of Gram-negative bacteria. In contrast, E. coli biofilm showed less sensitivity to both (Ag NPs and ZnO NPs). ZnO NPs exhibited the highest biofilm inhibition of 0.35 a.u. At 100 µg·mL⁻¹ (E) concentration compared to its effect on K. pneumoniae, which exhibited biofilm inhibition of 0.1 a.u. At the same concentrations (Figure 11).

Figure 10

Reduced biofilm formation by Ag NPs and ZnO NPs in S. aureus and S. mutans. (A) Control, (B) 12.5 µg·mL⁻¹, (C) 25 µg·mL⁻¹, (D) 50 µg·mL⁻¹, and (E) 100 µg·mL⁻¹.

The possible explanation suggested that nanoparticles can interact with and diffuse through biofilms by diffusing along the water channels, which serve as pathways for nutrient transport and can attach to the bacterial membranes, causing the reduction of exopolysaccharides secretion, which represents the primary component that forms the biofilm, eventually penetrate and accumulate inside the cells and leading to bacterial death [36]. Moreover, The enhanced antibiofilm activities of Ag NPs and ZnO NPs are primarily due to the rapid production of ROS and improved physical interactions between the nanoparticles and bacterial cells. These interactions cause significant damage to the bacterial cell membrane, hindering biofilm formation and reducing the likelihood of infection initiation and persistence. These mechanisms highlight the potential of green synthesised Ag NPs and ZnO NPs as powerful antibiofilm agents, particularly in preventing biofilm-related infections [37,38]. However, the overall results of this study can be summarised as follows: Despite the slight effect differences between Ag NPs and ZnO NPs on the bacterial biofilm, ZnO NPs are more potent as anti-biofilm.

3.4 Prepared nanoparticles induce bacterial ROS generation

In this study, the influences of NPs on bacterial ROS generation are investigated as indicated in Figure 12. The bactericidal effect of prepared NPs in bacteria is due to an increase in the cellular levels of ROS generation. The ROS generation assay findings verify that bacteria treated with prepared Ag and ZnO NPs produce ROS compared with control bacteria. This implies that the generation of ROS indirectly affects bacterial growth and contributes to bacterial death.

Figure 11

Reduced biofilm formation by Ag NPs and ZnO NPs in K. pneumoniae and E. coli: (A) Control, (B) 12.5 µg·mL⁻¹, (C) 25 µg·mL⁻¹, (D) 50 µg·mL⁻¹, and (E) 100 µg·mL⁻¹.

3.5 Antioxidant activity of green fabricated NPs

Because of their inertness to chemicals and thermal stability, inorganic nanoparticles can be used to immobilise natural antioxidants. Furthermore, using naturally occurring antioxidants coupled with nanoparticles enhances these antioxidants’ chemical stability under physiological circumstances. This strategy enables the goods to be supplied in their whole molecular form at a broader range of concentrations. Additionally, it is notable that this strategy is consistent with the antioxidants’ constant release [39]. Figure 13 illustrates the DPPH radical scavenging activities of prepared NPs, which were positively correlated with increasing concentrations. Each concentration of NPs demonstrated significant DPPH radical scavenging ability. Certain oxides of nanoparticles possess inherent physicochemical characteristics that enable them to actively scavenge nitrogen and oxygen species and alleviate various conditions stemming from oxidative stress [40].

Figure 12

Prepared NPs induce bacterial ROS generation.

3.6 Cytotoxic effect of NPs against colon cancer cell line

Green synthesis has become the most popular way to make nanoparticles. The body’s capacity to metabolise organic compounds, the relatively lower toxicity of plant-derived components, the compatibility of plant phytochemicals with therapeutic metal salts, and the environmental benefits of synthetic and organic moieties over strictly synthetic alternatives are the main factors contributing to the observed efficacy [34]. MTT assay was done to measure the antiproliferative activity of NPs against HT-29 cells. HT-29 cells were exposed to different concentrations of prepared NPs for 72 h. The results revealed the prepared NPs’ impact on the cell viability of the HT-29 cell line. However, the results indicated that the prepared NPs had no cytotoxic against normal cell line (MCF-10). These results were contrasted with control cells, reflecting a concentration-dependent manner, as shown in Figure 14. An in vitro study was prepared, and biosynthesised AgNPs were characterised. According to their findings, AgNPs were highly harmful to A549 cells. To investigate how AgNPs stop cancer cell growth, morphological alterations in A549 cells were analysed using an inverted microscope. The cell cycle distribution was measured using a flow cytometer. Furthermore, a DNA fragment test was used to investigate DNA damage caused by AgNPs in cancer cells; death in cancer cells and DNA fragmentation were observed [41,42]. ZnO nanoparticles had an IC₅₀ value of 38.60 μg·mL⁻¹ in their in vitro anticancer activity against the HeLa cell line, compared to the reference standard cisplatin. This discovery validates the strong cytotoxic effect of ZnO nanoparticles derived from shilajit extract on human cervical cancer cell lines [43].

Figure 13

Antioxidant activity of Ag NPs and ZnO NPs.

Figure 14

Antiproliferative activity of Ag NPs and ZnO NPs against HT-29 cells (upper panel) and MCF-10 cells (lower panel).

3.7 Molecular docking simulation

Zn and Silver possess several bioactive properties, making them valuable for medical and industrial applications, including antimicrobial, anti-inflammatory, antioxidant, anticancer, wound healing, and anti-biofilm. In silico docking simulations of ZnO NPs and AgNPs revealed a repulsive interaction, resulting in positive binding free energy. This result was expected, as the molecule was composed entirely of Zn and Ag atoms and had a spherical structure. Spherical shapes tend to interact with the surface of proteins rather than being inserted into them, which is thermodynamically favourable. ZnNPs and AgNPs showed the lowest binding free energy with 6KRR, followed by 2GAE, 3Q6A, and 2HQ6, as detailed in Tables 3 and 4. The binding energies suggest a repulsive interaction between ZnNPs and AgNPs and the targeted receptors, leading to a lack of inhibition. Consequently, Ki values were not generated in the DLG file, the output of the docking process, which aligns with the observed non-inhibitory effect. The only interaction that may occur with Cupper atoms is metal acceptor attraction alongside metal donor repulsion, as detected. ZnO NPs interacted with chain A of 2HQ6 through SER73, GLY74, GLN112, and ILE62, while 3Q6A interacted with chain G through GLU14 and TYR48. With 6KRR, ZnO NPs interacted with chain B through THR47, ALA58, and ASP145; on the other hand, with 2GAE, it interacted with chain A through ASN55, GLY57, HIS65, and GLU61, and AgNPs interacted with chain A of 2HQ6 through ASP23, GLY6, ALA21, and PRO9, while with 3Q6A, it interacted with chain G through GLU24 and TYR48. With 6KRR, AgNPs interacted with chain B through THR122, ALA133, and ASP132; on the other hand, with 2GAE, it interacted with chain A through ASN79, GLY108, HIS65, and GLU240. Figures 15–18 detect the various poses of the best conformers from the docking complex and the 3D and 2D visualisations of the hydrophobic interactions identified through LigPlus analysis [44]. Tables 5 and 6 show that 50 conformations were detected from the blind docking simulation between the ZnO NP and AgNP compound and the four targeted proteins, 2HQ6, 2GAE, 3Q6A, and 6KRR.

Figure 15

The best pose and 3D and 2D images of the best conformers resulted from the complexation reaction between ZnO NPs (a: 2HQ6) and (b: 3Q6A).

Figure 16

The best conformers’ best pose, 3D, and 2D images resulted from the complexation reaction between ZnO NPs and (a: 6KRR) and (b: 2GAE).

Figure 17

The best poses, 3D, and hydrophobic 2D images of the best conformers of the complexation reaction between (a: 2HQ6), and (b: 3Q6A) targeted proteins with AgNPs.

Figure 18

The best poses, 3D, and hydrophobic 2D images of the best conformers of the complexation reaction between (a: 6KRR), and (b: 2GAE) targeted proteins with AgNPs.