The free radical scavenging (FRSC) activity was evaluated using a standardized DPPH assay (Alghamdi et al., 2023). Test samples and standard material were dissolved in methanol to prepare a series of concentrations, as shown in Table 1. Subsequently, 3 mL of DPPH was added to each test tube and kept in the dark at room temperature for 30 min. Ultimately, the absorbance (A₅₁₇) of the test samples and the standard was detected spectrophotometrically at 517 nm, and FRSC activity against DPPH was evaluated using Equation 1 (Table 1).

The capacity of the test samples to induce bacterial cell death was evaluated using the agar diffusion disk method (Balouiri et al., 2016). Luria-Bertani (LB) broth medium was used to grow the four bacterial strains listed in Table 1 and acquire the bacterial suspensions. The next day, hygienic disks with a 6-mm diameter containing test samples and standard material with the concentrations listed in Table 1 were used to cover the cultured bacterial plates, which were then incubated at 37°C for 24 h. The zone of inhibition was then measured in millimeters.

The cytotoxicity of test samples and standard material against two cell lines, listed in Table 1, was screened using the MTT assay (Kamal et al., 2022). Cells were seeded with a density of 10,000 cells per well in a 96-well plate in complete DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin and incubated for 24 h at 37°C with 5% CO₂. The next day, after the cells reached a confluency of 80%, detached cells were removed by washing the plates with PBS, and 100 μL of the new culture medium containing test samples and standard with the concentrations listed in Table 1 was added to each well under incubation conditions (at 37°C with 5% CO₂). The exposure time was set to be 24 h; after this period, the cells were washed again with PBS, and 80 µL of FBS-free medium mixed with 20 µL of MTT reagent was added to each well under incubation conditions (at 37°C with 5% CO₂). After 3 h, 100 µL of stooping regent DMSO was added to each well to halt the reaction, and the mixture was left under shaking in the dark for 15 min. Finally, the absorbance at 590 nm was measured, and cell viability was evaluated using Equation 2 given in Table 1.

Oxidative stress (ROS) of the MgO NPs was evaluated using the DCFH-DA assay (Amin et al., 2023). Generally, the generated ROS oxidizes the non-fluorescent DCFH-DA into the brightly fluorescent compound dichlorofluorescein (DCF) (λ_EX/λ_EM = 485 nm/535 nm). This assay was performed in both HepG2 and HUVEC cell lines under the same culturing conditions as the MTT assay. Cells were exposed to test samples at the concentrations mentioned in Table 1 for 24 h. The cells were then washed with PBS and incubated with 100 µL of the fresh culture medium containing 80 µL of the serum-free medium and 20 µL of the DCFH-DA reagent under incubation conditions (at 37°C with 5% CO₂) for half an hour in the dark. After treatment, the cells were washed with PBS, and the microplate reader detected the fluorescence intensity at λ_EX/λ_EM = 485 nm/535 nm.

The anti-inflammatory activity of the sample was determined using a protein denaturation assay (Fahaduddin, 2024). Any material with anti-inflammatory properties could stabilize protein structures and prevent denaturation. The tested samples and standards were prepared in PBS at the concentrations mentioned in Table 1. A measure of 3 mL of 1% bovine serum albumin (BSA) was added to each tube and incubated in a water bath at 55°C for 20 min. The absorbance was detected at 660 nm, and the inhibitory percentage was evaluated using Equation 5 (Table 1).

The successful formation of both MgO NPs and MgO–neem NPs was confirmed by the presence of distinct fingerprint diffraction peaks characteristic of MgO NPs, as illustrated in Figure 2. These peaks indicate the crystalline nature and structural integrity of the synthesized NPs, confirming that the synthesis methods used effectively produced high-quality MgO NPs. Five diffraction peaks appeared in both samples at 2θ = 36.7, 42.76, 62.08, and 78.4 for MgO NPs, which shifted to lower 2θ = 36.46, 42.4, 61.84, 73.84, and 78.22 for MgO–neem NPs, respectively. These diffraction peaks belonged to (111), (200), (220), (311), and (222) diffraction plans, respectively. The detected diffraction peaks were assigned to cubic face-centered crystal (FCC) structure MgO NPs, which is in line with the reported card (JCPDS No. 87-0652) (Wang et al., 2024).

The cubic FCC structure is characterized by lattice parameters (a = b = c = 4.215787 A°). The lattice parameter for both samples was calculated using Equation 6 (Lee et al., 2016): d h k l = a h 2 + k 2 + l 2 . (6)

The average crystal size was calculated using Scherer’s Equation 7 (Kamal et al., 2022): D = 0.9 λ Γ Cos θ , (7) where D is the mean crystal size, λ is the X-ray wavelength source, 0.9 is constant for crystal shape, θ is the diffraction angle, and Γ is the full-width at half-maximum of the diffraction peak. The dislocation density (δ, nm^-2) was calculated as 1/D².

A slight increase was observed in the crystal size of bioinspired MgO–neem NPs compared with MgO NPs from 21.807 ± 2.053 to 23.09 ± 2.78 (nm) due to the coating action of biomolecules in the neem extract. This increase was combined with an enlargement in the lattice parameter a from 4.225 to 4.248 (A°) and an increase in the dislocation density (see Table 2). The results establish that neem extracts are reducing agents for synthesizing MgO NPs without distorting the crystal structure. Similar results were reported for the green synthesis of MgO NPs from different biological sources (Moorthy et al., 2015; Aravind Kumar et al., 2019).

The high-resolution transmission electron microscope (HRTEM) technique was used to study the structural future of NPs in terms of particle size and shape. The formed MgO NPs showed semispherical shapes (Figure 3). In HRTEM images, some aggregations of MgO NPs and MgO–neem NPs were observed. These aggregations were also observed in SEM images, mainly due to surface attractive interactions in the nanoscale. The green synthesis of NPs can contribute to this agglomeration by coating the MgO NP surface with biomolecules, which can interact with the surrounding molecules via many Coulombic interactions (Arabi et al., 2020; Shanavas et al., 2020). ImageJ software was used to calculate the particle size of MgO NPs. A total of 100 particles per image were selected, and then particle size distribution was plotted. The mean particle size of MgO NPs was 16.97 ± 3.4 nm, while the mean particle size of MgO–neem NPs was 17.78 ± 3.42 nm. These results were in line with the XRD data and reported results for bioinspired MgO NPs (Nijalingappa et al., 2019; Vijayakumar et al., 2021; Pachiyappan et al., 2020).

UV-vis spectrometry was used to characterize the photocatalytic activity of NPs (Devanand et al., 2013). The absorption edge was detected for the neem extract at 252 nm, while for MgO and MgO–neem NPs, it was 283 and 285 nm, respectively (Figure 4A). The literature indicates that the successful reduction of metal ions and the generation of metal oxide NPs were suggested by an apparent absorption edge in the 260–300-nm range when sodium hydroxide and plant extract were utilized in the NP manufacturing process.

To correlate the optical properties with the observed structural alterations of bioinspired MgO–neem NPs, Tauc’s Equation 8 was utilized to determine the optical band gap, E_g (Devanand et al., 2013): α hv = A hv − E g m 2 , (8) where α is the absorption factor, hν is the energy of the incident photon, and A and m are constants depending on the nature of the transition. Plotting the relationship between photon energy hν and (αhv)² yielded the predicted optical band gap energy E_g of bioinspired MgO–neem NPs (Figure 4B). The band gap of bioinspired MgO–neem NPs (E_g = 4.701 eV) was greater than that of MgO NPs (E_g = 4.534 eV). This discrepancy was in line with the published findings and connected to the observed shift in the particle size of bioinspired MgO–neem NPs (Sharmila et al., 2019; Fatiqin et al., 2021).

In the Fourier-transform infrared (FTIR) spectrum, three vibrational bands were identified as MgO NP fingerprints. The first band, which is related to the stretching vibration of magnesium oxide, is often observed between 600 and 880 cm⁻¹. The second band, caused by the stretching of magnesium carboxylate, emerged between 1,600 and 1,640 cm ⁻¹. Water is represented as moisture by the stretching hydroxyl (O-H) in the third broadband that appears between 3,350 and 3,550 cm⁻¹. These bands were observed in the MgO and bioinspired MgO–neem NP spectra (Figure 5).

To investigate the role of biomolecules in the neem extract in the reduction of MgO NPs, the FTIR spectrum of the neem extract was analyzed. The broadband that was visible between 3,350 and 3,550 cm⁻¹ was associated with the O-H vibration, which is indicative of amino acids and carbohydrates. Aromatic aldehyde stretching vibrations of C-H were observed at 2,935 cm⁻¹. Conversely, at 1,630 cm⁻¹, C=O stretching, which primarily originates from alkene compounds in proteins, was observed. The carbonyl group found in flavonoids was identified as the band observed at 1,590 cm⁻¹. At approximately 1,400 cm⁻¹, C-H bending vibration became apparent. Furthermore, the C-O stretching vibration originated in the aliphatic amine band at 1,029 cm⁻¹.

Examining the FTIR spectra more closely revealed that the bioinspired MgO–neem NPs contained the functional groups (O-H, C-H, C=O, and C-O) that are associated with terpenoids, flavonoids, and proteins of the neem extract. Thus, the reduction and production of MgO NPs are mostly caused by these biomolecules. Research indicates that proteins’ C-N and C=O functional groups serve as capping agents to aid in the production of NPs (Chandrasekaran et al., 2024). Additionally, proteins’ amine linkages have a strong attraction to metals, which causes them to form a stabilized layer on the surface of the metal NPs.

The stability of NPs is one of the main challenges that hinder their potential applications. Measurement in conditions similar to in vitro or in vivo environments is an important yet hard feature of NP characterization. One of the critical factors is the stability and amount of aggregation of NPs under physiological conditions (e.g., plasma) or in various media relevant to biotechnological applications (e.g., culture medium). Several studies have demonstrated that the stability of NPs in various culture media can be significantly reduced depending on ionic and protein content, influencing NP characteristics and their functions in both in vitro and in vivo applications (Kadir et al., 2023; Proniewicz et al., 2024).

For screening the stability of MgO NPs, UV spectroscopy was used. Both MgO NP samples were suspended in complete DMEM, and then UV spectra were recorded during different time intervals from 0 to 480 min (Figure 6). Both samples showed good stability during the measurement period (8 h), and there was no significant change in the absorption edge.

Thermogravimetric analysis (TGA) is a straightforward analytical method that calculates a material’s weight change (or gain) in relation to temperature. When materials are heated, they may lose weight by straightforward processes like drying or chemical reactions that release gases. The composition and structure of the material are closely linked to these thermal processes. To determine the material’s thermal behavior, TGA curves for any material may be divided into segments based on how the material’s weight loss changes with temperature.

For MgO NPs, the weight loss was observed in three segments (Figure 7). The first segment starts from ambient temperature to 260^°C with an approximate weight loss of 2.4% for MgO NPs and 3% for MgO–neem NPs. This weight loss was mainly due to the loss of absorbed water from moisture (Chandrasekaran et al., 2024; Hirphaye et al., 2023). The slight increase in the weight loss of bioinspired MgO–neem NPs due to the larger content of water was confirmed by the higher intensity of the stretching O-H band in the FTIR spectrum. The second segment, from 260 to 500^°C, reflects the decomposition of organic molecules and the transition of MgO NPs (Poonguzhali et al., 2022; Leung et al., 2014). The weight loss in this segment was 5% for both MgO NPs and MgO–neem NPs, while the third segment lies between 500 and 1,000°C, in which the residue of organic molecules decomposes with smaller weight loss at approximately 1.6% and 1.5% for MgO NPs and MgO–neem NPs, respectively. This smaller weight loss indicates that both samples were stabilized in the crystalline phase above 500°C, which is in line with the reported thermal stability of MgO NPs (Leung et al., 2014; Sharmila and Selvaraj, 2024a).

Creating free radicals is a cascade-like process; it starts with gaining or losing an electron, and this electron eventually hits another atom or molecule to create more free radicals. As a result, the reaction continues to produce an increasing number of these free radicals (Abdallah et al., 2019; Dobrucka, 2018). Antioxidant molecules are sufficiently stable to donate an electron to form a stable molecule and mitigate the damaging effects of free radicals. The literature suggests that the capacity to donate hydrogen is the reason behind the antioxidant activity of bioinspired MgO NPs. The production of an electron–hole pair on the surface of MgO NPs can significantly reduce H₂O molecules, which can act as DPPH molecules’ scavengers. Nevertheless, because the plant extract contains phytochemicals, including phenolics and polyphenolic compounds, the green synthesis of MgO NPs may help modify the scavenging activity (Khan et al., 2020; Akshaykranth et al., 2021).

The capability of the bioinspired MgO–neem NPs as a scavenger for DPPH radicals is compared to that of MgO NPs, neem extract, and ascorbic acid (ASC) as the standard and presented in Figure 8. A dose-dependent behavior was observed in the scavenging activity of all tested samples. The calculated IC₅₀ value for bioinspired MgO–neem NPs was 69.03 μg/mL. The IC₅₀ values for MgO NPs, neem extract, and ascorbic acid were 131.62 μg/mL, 84.7 μg/mL, and 15.35 μg/mL, respectively. The improved scavenging activity of the MgO–neem NPs can be mainly attributed to the presence of biomolecules from the neem extract, which enhance the NP efficacy. Previous studies on bioinspired MgO NPs from different biological sources have shown comparable DPPH scavenging activity (Table 3). These comparable scavenging values are bio-source-dependent, which indicates the importance of optimization of green MgO NP synthesis to achieve the desired bioactivity.

The MgO NPs’ antibacterial activity can be ascribed to two distinct mechanisms: the antimicrobial effect mediated by reactive oxygen species and the non-reactive species. MgO NPs create H₂O₂, which causes oxidative stress in the microbial system (Dobrucka, 2018; Nguyen et al., 2023). This leads to the production of reactive oxygen species (ROS), ultimately resulting in cell death. Furthermore, MgO NPs have been linked to cellular membrane disruption and contents leaking after physical contact. They may penetrate cells more quickly due to their smaller size, and they can interact with cells more extensively due to their larger surface area. Cell, protein, and DNA damage occur at higher MgO NP concentrations (Kumar et al., 2021; Breijyeh et al., 2020). For comparing the antibacterial activity of the investigated samples, a clearer zone of inhibition was measured in millimeters and is shown in Figure 9.

Bioinspired MgO–neem NPs recorded a significant activity for Gram-positive and Gram-negative bacterial strains in a concentration-dependent manner compared with MgO NPs and neem extract at all concentrations. This activity is close to the positive control Gentamicin, especially at the higher concentration (100 μg/mL). This behavior was attributed to the functionalization of MgO–neem NP surfaces with biomolecules from the neem extract. It is reported that the biological synthesis of NPs enhances the ROS generation ability, improving the antibacterial activity (Umaralikhan and Jamal, 2018; Suresh et al., 2018). The neem extract was also identified as a powerful antibacterial agent due to the high content of phenolic and flavonoid phytochemicals (Moorthy et al., 2015). The antibacterial action of bioinspired MgO NPs from different biological sources was reported against many pathogens (Table 3).

Moreover, when comparing the inhibition zones, Gram-negative bacterial strains (S. typhimurium and E. coli) exhibited smaller inhibition zones than the examined Gram-positive strains (S. aureus and B. subtilis) after treatment with all test samples. This behavior was reported for most Gram-negative bacterial strains, which was attributed to the stronger structure of Gram-negative bacteria than that of Gram-positive bacteria. Due to this structural variation, Gram-negative bacterial strains possess higher resistance to destruction than Gram-positive bacterial strains. However, bioinspired MgO–neem NPs showed a higher activity than MgO NPs at the three concentrations. These findings reflect the impact of the green synthesis of MgO NPs in treating different pathogens.

As a distinct property, MgO NPs were characterized by the ability to generate ROS due to their high surface-to-volume ratio. The physicochemical properties of MgO NPs, such as size, shape, and surface reactivity, control the number of generated ROS. Upon entering the cellular membrane, these ROS cause oxidative stress, which, in turn, causes DNA damage, protein oxidation, mitochondrial malfunction, and, eventually, cell death (Velsankar et al., 2023; Majeed et al., 2018). The percentage of cell viability was determined for the tested samples by the colorimetric MTT assay as a function of mitochondrial activity and normalized to its respective control (Figures 10A, B). A significant concentration-dependent reduction in the cell viability of HepG2 cancer cells was observed after treatment with all samples. Bioinspired MgO–neem NPs showed distinct cancer cell-killing activity with an IC₅₀ value of 94.58 μg/mL compared with doxorubicin (IC₅₀ = 26.62 μg/mL), MgO NPs (IC₅₀ = 241.77 μg/mL), and neem extract (IC₅₀ = 361.68 μg/mL) (Figure 10A). There was no significant effect on the cell viability of normal cells except for cells treated with doxorubicin (IC₅₀ = 123.57 μg/mL), (Figure 10B). These results demonstrate the high selectivity of MgO–neem NPs toward cancer cells, which is stimulated by their green synthesis and linked with antibacterial activity results (Fouda et al., 2021; Suba et al., 2022; Mangalampalli et al., 2019; Kessler et al., 2022; Mittag et al., 2019). Comparable findings were reported for bioinspired MgO NPs after exposure to different types of cancerous cells (Table 3).

MgO NPs cause ROS production under physiological conditions due to their large band gap, enabling them to donate hydrogen ions easily (Majeed et al., 2018; Mittag et al., 2019; Krishnamoorthy et al., 2012). This study assessed the generation of ROS following the exposure of two distinct cell lines to MgO NPs, MgO–neem NPs, and neem extract with variable concentrations for 24 h. Figure 11A shows an increase in ROS generation in HepG2 cancer cells that is dosage-dependent, which is consistent with the cytotoxicity and antibacterial findings, given that MgO–neem NPs exhibited increased toxicity against HepG2 cells compared with MgO NPs and neem extract. These findings show that exposure to MgO–neem NPs increases ROS levels, which, in turn, causes oxidative stress and cellular damage. In contrast, there was no notable increase in the level of ROS in either the untreated or normal HUVEC cells (Figure 11B). Many researchers have observed that MgO NPs have a stronger selectivity against cancer cells (Mittag et al., 2019; Poljsak et al., 2013; Joó et al., 2023). Since cancer cells have high rates of metabolism and proliferation, the presence of additional chemical and signaling components in MgO NPs increases their reactivity. These findings also aligned with the high scavenging activity of the obtained bioinspired MgO–neem NPs. In normal cell lines, the number of generated ROS is limited and can be eliminated by the antioxidant scavengers’ enzymes. Due to the high selectivity in cancer cells, the number of generated ROS is huge due to the high surface-to-volume ratio, which disturbs the balance between antioxidant activity and ROS and cannot be neutralized by the action of antioxidant scavengers’ enzymes (Gong et al., 2020; Alqahtani et al., 2020). These unique bioactivities of bioinspired MgO–neem NPs increase their potential as alternative agents in the biomedical field.

The antidiabetic activity of MgO NPs is evaluated by the estimation of α-amylase and α-glucosidase inhibition percentages. It is reported that these two digestive enzymes break down carbohydrates into glucose. These two enzymes are crucial factors influencing the conversion of disaccharides and oligosaccharides into monosaccharides (Evans et al., 2002; Forman and Zhang, 2021). Therefore, the inhibition of these two enzymes is critical for the treatment of type-2 diabetes. The percentage of α-glucosidase inhibition was calculated spectrophotometrically using yeast α-glucosidase and pNPG after treatment with MgO NPs, MgO–neem NPs, neem extract, and acarbose with variable concentrations, as shown in Figure 12A.

Compared with acarbose as the standard with an IC₅₀ value of 11.16 μg/mL, MgO–neem NPs showed concentration-dependent powerful inhibition with an IC₅₀ value of 50.6 μg/mL. This behavior was linked to the high antioxidant activity of MgO–neem NPs. Moreover, neem extract and MgO NPs demonstrated comparable inhibition with IC₅₀ values of 82.64 and 172.25 μg/mL, respectively. Similar behavior was observed for α-amylase inhibition percentage, and the test samples followed the same trend, starting with acarbose as the standard with an IC₅₀ value of 12.41 μg/mL, MgO–neem NPs with an IC₅₀ value of 61.53 μg/mL, neem extract with an IC₅₀ value of 133.96 μg/mL, and MgO NPs with an IC₅₀ value of 227.75 μg/mL (Figure 12B). It is reported that oxidative stress plays a critical role in the development of diabetes. An excess blood glucose level causes oxidative stress, which, in turn, causes glucose to auto-oxidize and free radicals to develop (Caturano et al., 2023; Chen et al., 2018; Dunkelberger and Song, 2010). Therefore, the treatment with MgO–neem NPs led to scavenging these free radicals, causing the inhibition of α-glucopyranoside and α-amylase enzymes. These results agreed with the report on bioinspired MgO NPs (Table 3).

As a defense mechanism, inflammation involves blood vessels, immune cells, and molecular mediators. Inflammation has three main functions: it dissolves injured cells and tissues, starts tissue healing, and removes the source of cell harm. White blood cells in the human body use the process of inflammation to defend the body against harm or infection from external intruders like bacteria and viruses. A low level of inflammation may risk the organism’s life by allowing hazardous stimuli, like germs, to gradually destroy tissue (Ammulu et al., 2021; Sharmila and Selvaraj, 2024b), 102. An anti-inflammatory medication works to lessen inflammation. Regrettably, organisms’ tissues and organs may suffer more harm from unchecked inflammation. Therefore, finding natural sources of anti-inflammatory drugs is critical.

A protein denaturation assay was used to assess the anti-inflammatory action of MgO NPs, and the degree of denaturation inhibition indicated high anti-inflammatory action. The percentage of inhibition of diclofenac sodium as standard, MgO NPs, bioinspired MgO–neem NPs, and neem extract was calculated and is shown in Figure 13. Significant inhibition was recorded for MgO–neem NPs with an IC₅₀ value of 6.66 μg/mL compared with that of diclofenac sodium with an IC₅₀ value of 20.56 μg/mL, while neem extract and MgO NPs showed less inhibition with IC₅₀ values of 48.56 and 102.44 μg/mL, respectively. Traditionally, neem extract is used to treat inflammation. The primary cause of the enhanced anti-inflammatory properties of MgO–neem NPs is their phytochemical content, mainly the phenolic and flavonoid components. These phytochemicals enhance ROS generation, as we mentioned before, leading to the activation of pro-inflammatory cytokines and interleukins (Gatou et al., 2024; Sharmila and Selvaraj, 2024a).