A fresh pumpkin (C. maxima) was procured from Jagatpura, Jaipur, India. As per the institutional ethical approval committee, no formal approval was required to conduct this study. The specimen was confirmed by Dr. A. Khan (Department of Botany, AMU, Aligarh, India) and was deposited in a departmental herbarium containing voucher number 84/21. 5.0 g pumpkin seeds were dried and grinded to a fine powder. The powdered seeds were refluxed for 1 h in deionized water in a round-bottom flask and cooled at room temperature (RT). The resultant solution was filtered through Whatman filter paper to obtain a purified crude extract. The filtrate was then stored in a cool environment for future requirements.

The biogenic synthesis of MgONPs required 20 ml of pumpkin seed extracts and 80 ml of an aqueous solution of Mg(NO₃)₂ (0.1 M) under continuous magnetic stirring for 4 h at 60–80°C. As a result, the solution developed a brownish colloidal appearance, indicating the formation of MgONPs. The solution was then centrifuged at 10,000 rpm for 10 min, dried in an oven at 50–60°C, and calcinated in a muffle furnace at 500°C to get the white powder of MgONPs. A tentative mechanism of MgONP biosynthesis is depicted in Figure 1.

The formation of MgONPs was confirmed by UV–vis absorption spectroscopy (UV-1800, Japan) with a resolution of 1 nm ranging from 200–800 nm. Moreover, Fourier-transform infrared spectroscopy (FTIR) analysis was applied to detect and measure the functional group of pumpkin seed extracts involved in synthesizing MgONPs in the range of 4,000–400 cm⁻¹ (Perkin Elmer Spectrum 2000 FTIR) by the KBr pellet technique. Cu Kα radiation was employed to determine the X-ray diffraction (XRD) spectrum of MgONPs at 40 kV. The obtained XRD pattern of synthesized nanoparticles was compared with the Joint Committee on Powder Diffraction Standards (JCPDS) file. The characteristic diffraction peak in the XRD pattern of MgONPs was indexed as the face-centered cubic phase. In addition, the particle distribution, elemental composition, and surface morphology of biosynthesized MgONPs were evaluated by scanning electron microscopy (Nova nano, FE-SEM 450 FEI). The particle size and shape were confirmed by transmission electron microscopy (TEM, TECNAI G-20).

The ovarian teratocarcinoma cell line (PA-1) was procured from the National Centre for Cell Science (NCCS), Pune, India. The cells were grown in DMEM high-glucose media with 100 units/ml of penicillin/streptomycin and 10% fetal bovine serum and maintained in a humidified atmosphere of 5% CO₂ at 37°C.

The cytotoxicity of MgONPs was evaluated by 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) dye reduction assay, which is dependent on the conversion to the water-insoluble blue formazan compound by mitochondrial dehydrogenases (Alharthy et al., 2022). In brief, PA-1 cells were harvested and seeded in a 96-well plate and exposed to different concentrations (2.5–17.5 μg/ml) of MgONPs for 24 h at 37°C in 5% CO₂. After the treatment, the medium was aspirated, and the treated cells were exposed to 10 µl of MTT for another 2 h in the dark at 37°C. At the end of the incubation period, the purple formazan crystals were solubilized with 50 µl DMSO and measured spectrophotometrically on a microplate reader at 595 nm. The following formula calculated the percentage of cytotoxicity: Cell   proliferation   Inhibition   ( % ) = Mean   absorbance   of   the   control   cells − Mean   absorbance   of   the   treated   cells   Mean   absorbance   of   the   control   cells   X   100 .

The concentrations of MgONPs showing 50% reduction in cell viability (IC₅₀) were evaluated by extrapolating the dose–response curve.

After treating with different concentrations of MgONPs, the PA-1 cells were examined under a light microscope (×10 magnification). The photomicrographs of the cells were thoroughly evaluated for morphological changes such as shrinkage, detachment, membrane blebbing, and distorted shape.

To investigate apoptosis or necrosis, acridine orange/ethidium bromide (AO/EB) double fluorescent labeling was used according to the method described by Tabrez et al. (2022b). Briefly, the cells were seeded in a 6-well plate (5 × 10⁴ cells/well) and treated with different concentrations of MgONPs (10, 12.5, and 15 μg/ml) for 24 h. After treatment, the cells were washed with cold PBS and stained with a mixture of AO/EB (1:1; 100 μg/ml) for 5 min. After staining, the cells were monitored by using a fluorescence microscope (×40 magnification). The number of cells showing the feature of apoptosis was counted as a function of the total number of cells.

The intracellular reactive oxygen species (ROS) level was measured by the dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay, as mentioned previously (Alharthy et al., 2022; Tabrez et al., 2022b). The PA-1 cells were seeded in 6-well plates (2 × 10⁶ cells/well) and treated with different concentrations of MgONPs (10, 12.5, and 15 μg/ml) for 24 h at 37°C. After treatment, the cells were washed with PBS, and 25 μM of DCFH-DA was added for 30 min at 37°C. After that, the cells were washed with DMEM, and the fluorescence was recorded every 5 min, over to 30 min (excitation 485 nm, emission 535 nm), using a spectrofluorometer (Shimadzu, Columbia, United States). The increase in ROS production was calculated by a mean slope/min and normalized to the unexposed control.

The cells were separated at 0, 15, 30, 45, and 60 min intervals in 6-well plates, and the wells were rinsed with PBS to remove the weakly attached/unattached cells. The seeded cells were fixed with paraformaldehyde (5%) and crystal violet dye (1%) and fostered for 15 min. After incubation, crystal violet dye was bound to cellular proteins, and excess crystal violet was washed with PBS. The amount of crystal violet destained to protein was proportional to the number of cells in the well.

The scratch assay was performed according to the protocol defined by Goel and Gude (2011). In brief, the PA-1 cells were plated to make an 80% confluent monolayer. The monolayer of cells was scratched by using a sterile 200-microliter pipette tip to make a straight line to create scratches. After cleansing with PBS, the cells were treated with different doses of MgONP (10, 12.5, and 15 μg/ml) and maintained at 5% CO₂ and 37°C for 36 h. Later, the wound closure level was assessed, and microphotographs were taken at 0 and 24 h.

Data are presented as the mean ± SD of three independent values (wherever applicable). The control and treated cells were compared using a one-way ANOVA. p < 0.05 was considered as statistically significant.

The UV–vis spectrum of the biosynthesized MgONPs showed a peak at 292 nm, confirming the formation of small-sized nanoparticles (Figure 2). Single-surface plasmon resonance (SPR) bands that form at short wavelengths below 300 nm in UV–visible spectroscopy indicate the presence of small-sized particles. In contrast, other absorption peaks indicated the existence of several bioactive compounds, which may be essential for Mg(NO₃)₂ reduction to MgONPs (Pugazhendhi et al., 2019). Various studies have reported the UV–vis absorption spectra of MgONPs in the range of 250–350 nm synthesized from different biological sources (Pugazhendhi et al., 2019; Amina et al., 2020; Fouda et al., 2022). In our study, MgONPs were extracellularly synthesized and could be easily purified (Fouda et al., 2022). The pumpkin seeds contained large amounts of β- and γ-tocopherol, which are beneficial as reducing and capping agents that help in the synthesis of NPs (Shankar and Rhim, 2016; Kulczyński and Gramza-Michałowska, 2019). We used nitric acid during NP synthesis, which helps to synthesize NPs of smaller size, lower magnetization, better thermal properties, and higher stability (Nurdin et al., 2014).

FTIR spectra of the pumpkin seed extracts (Figure 3A) and biosynthesized MgONPs (Figure 3B) were monitored at the wavelength range of 4,000–400 cm⁻¹. The major IR vibration functional bands in the MgONPs spectrum were recorded at 479 cm⁻¹ (Figure 3B). The different bioactive moieties in pumpkin seed extracts act as reducing agents in synthesizing stable nanoparticles. The IR bands that appeared previously could be of an ester, ethers, carbonyl (polyols), or aromatic compounds. On the other hand, the peaks observed at 3,696 and 3,447 cm⁻¹ can be assigned to the hydrogen-bonded hydroxyl and free NH₂ groups. Similarly, the peaks around 2,928, 2,852, 1,631, 1,343, and 1,112 cm⁻¹ are attributed to the stretching vibration of the aromatic C=C bond, while the peak at 479 cm⁻¹ indicates the formation of MgONPs (Sharma et al., 2020). The FTIR analysis confirmed the role of bioactive compounds as reducing, capping, and stabilizing agents in MgONP biosynthesis. Our FTIR spectra are similar and concomitant with previously reported studies (Pugazhendhi et al., 2019; Amina et al., 2020; Fouda et al., 2022).

The XRD pattern of biogenic MgONPs was verified by the characteristic peak in the XRD image (Figure 4). The diffraction peaks at 2 theta values of 36.95°, 42.94°, 62.25°, 74.73°, and 78.83° are identified in the (111), (220), (220), (311), and 222) planes, respectively, of a faced center cubic lattice of magnesium, indicating the crystalline nature of MgONPs confirming the earlier study (Safaei-Ghomi et al., 2015). Moreover, a standard diffraction pattern of MgONPs (JCPDS file number 89-4248) showed similar patterns in the XRD of nanoparticles.

The morphology of biosynthesized MgONPs was characterized by SEM analysis at different magnifications. It showed agglomeration, resulting in moderately dispersed and slightly coalesced MgONPs (Figure 5A). The MgONPs were found to be of different sizes (<100 nm) and distinguished shapes. The observation of biosynthesized nanoparticles was cluster and randomly distributed forms. Similar data have been reported in the previous studies related to MgONPs biosynthesized from different sources (Pugazhendhi et al., 2019; Kgosiemang et al., 2020; Pamidimukkala, 2021).

The potential of nanoparticles is usually correlated with different characters that include shape, size, and distribution (Fouda et al., 2022). The electron diffraction patterns by TEM analysis identified the phase of nanoparticles and determined their particle size. The TEM image showed that the particles are nearly spherical and eclipsed in morphology (Figure 5B). The nanoparticles were moderately dispersed, and the particles’ average size was ∼100 nm. Earlier reports have also observed similar sizes of biogenic MgONPs synthesized from various sources (Amina et al., 2020; Fouda et al., 2022). Given the earlier scientific reports, the current MgONPs sized <100 nm are expected to be used in various biomedical and biotechnological applications.

The EDX analysis is used to detect the elemental composition of the biosynthesized MgONPs (Fouda et al., 2022). The production and quality of the synthesized nanomaterials are related to the existence of significant peaks in the EDX spectrum. Figure 6A shows an EDX spectrum of biogenic MgONPs that include atoms and mass percentages with a distinct peak corresponding to Mg and O and peaks of Na, Cl, and K in small amounts from sample preparation. Magnesium showed a high peak at approximately 1.2–1.3 keV, while oxygen showed a peak at approximately 0.5 keV. The quantitative analysis of the sample revealed the mass% of Mg and O ions was 42.4 and 49.3%, respectively, whereas the atomic percentages were 34.3 and 60.6%, respectively (Figure 6B). The compositions of the other elements were negligible, indicating the highly pure nature of our biogenic MgONPs. The EDX profile of MgONPs synthesized from algal extract also showed the weight percentages of Mg and O ions as 54.1 and 20.6%, respectively (Fouda et al., 2022). Another study also showed the average elemental percentage of Mg and O as 38.2%, and 27.9% in MgONPs biosynthesized from S. costus (Amina et al., 2020).

The results showed a concentration-dependent decrease in cell viability in PA-1 cells compared to the untreated control (Figure 7). The viability of cells starts declining at the lowest tested concentration of 2.5 μg/ml, and at the highest concentration (17.5 μg/ml), it was found to be 37.7% only. The IC₅₀ concentrations of biosynthesized MgONPs were found to be 12.5 μg/ml. Several researchers have also biosynthesized MgONPs from different sources, but the recorded IC₅₀ dose was much higher, indicating better anticancer efficacy of our biosynthesized MgONPs (Pugazhendhi et al., 2019; Amina et al., 2020; Fouda et al., 2022). The cytotoxicity could also depend on the types of cancer cells having abnormal metabolism and morphology, size, and shape of NPs, which in turn varies with the preparation method (Perde-Schrepler et al., 2019). Based on the estimated IC₅₀ value, three different concentrations (10, 12.5, and 15 μg/ml) of MgONPs were selected for the subsequent experiments.

The photomicrographs of MgONPs-treated PA-1 cancer cells showed a loss/alteration in cell morphology in a concentration-dependent manner (Figure 8). Cell shrinkage, detachment, membrane blebbing, and distorted shape were observed at the highest concentration. However, the cell morphology of the untreated control cells was found to be normal and intact. The entry of MgONPs into mammalian cells occurs mainly through endocytosis or macropinocytosis (Fouda et al., 2022). A similar alteration in cell morphology was reported earlier by green-synthesized MgONPs in different cancer cell lines, implying that the cytotoxicity of synthesized MgONPs may be attributed to their antineoplastic characteristics and ability to trigger cell death via a variety of molecular mechanisms (Farah et al., 2016; Al-Sheddi et al., 2018). Pugazhendhi et al. (2019) observed a similar pattern of cell rounding and shrinkage, membrane blebbing, apoptotic body formation, chromatin condensation, and reduced cell population after the treatment with MgONPs in A549 cells.

Figure 9 illustrates the AO/EB fluorescence pattern of PA-1 cells treated with MgONPs and showed the presence of early and late apoptotic cells, suggesting the apoptotic potential of MgONPs. Most of the untreated control cells showed an intact morphology and normal nuclear membrane. At 10 μg/ml MgONPs treatment, the PA-1 cells showed the initiation of nuclear fragmentation. However, MgONP treatment at higher concentrations (12.5 and 15 μg/ml) showed more intense nuclear fragmentation and advanced apoptotic cells.

During double-dye (AO/EB) staining, the AO permeates all the cells and give green fluorescence. On the other hand, EB interacts with the DNA of those cells that have lost their membrane integrity and stains the nuclei red for apoptotic cells. Double staining is a convenient, economical, and reliable in vitro tool to distinguish normal cells, early apoptotic cells, late apoptotic cells, dead cells, and nuclear morphology (Liu et al., 2015). The AO/EB staining interprets live cells as the usual green nucleus, early apoptotic cells as yellow fluorescence, which indicates a condensed or fragmented nucleus containing chromatin, and late apoptotic cells as orange fluorescence, which indicates chromatin condensation or fragmentation and cell necrosis (Liu et al., 2015). Dose-dependent cell growth inhibitions could happen due to the microscopically visible cell permeability disruption, reactive oxygen species (ROS) production, activation of caspase cascade-mediated apoptosis, and cell cycle arrest (Martínez-Torres et al., 2018). Moreover, the apoptotic induction and associated DNA damage have also been linked to excessive production of ROS, oxidative stress, and Sub-G1 arrest of cancer cells (Salehi et al., 2018). The modulation of apoptotic pathways is challenging as several pathways are involved in this process. However, our biosynthesized MgONPs showed promising apoptosis-inducing potential against ovarian cancer cells. Our results agree with the earlier reports that have observed apoptosis induction via different mechanisms by MgONPs, green synthesized from various sources against different cancer cell lines (Pugazhendhi et al., 2019; Amina et al., 2020; Fouda et al., 2022).

We observed a dose-dependent rise in ROS production in MgONP-treated PA-1 cells (Figure 10). The untreated control cells showed a dull green fluorescence. However, the MgONPs-treated (10, 12.5, and 15 μg/ml) cells showed a strong DCF stained green fluorescence indicating the production of intracellular ROS. The intricate association of ROS levels and cancer progression depends on the precise control of ROS generation and their scavenging. Cancer cells have more evolved antioxidant systems than normal cells and survive at slightly higher ROS levels than their normal counterparts. This characteristic makes cancer cells more vulnerable to outside factors, upregulating the generation of ROS. Increasing the ROS levels explore a growing number of therapeutic approaches to exceed the redox adaption of the same cells (Perillo et al., 2020). Our result corroborated well with the earlier studies that report a rise in ROS production in response to MgONPs treatment (Behzadi et al., 2018; Pugazhendhi et al., 2019). Various nanoparticles have been reported to induce ROS formation, eventually stimulating various anticancer events, such as oxidative stress, lipid peroxidation, DNA damage, and destabilization of mitochondria, resulting in cell death by apoptosis and necrosis (Alharthy et al., 2022; Tabrez et al., 2022b; Zughaibi et al., 2022). A low level of ROS has been suggested to regulate cancer cell tumorigeneses, whereas a high-level causes severe cellular damage (Youhannayee et al., 2019; Silva et al., 2021). Some studies also indicated that treating cells with various stress factors, such as anticancer drugs, increases ROS levels and induces cellular apoptosis by triggering proapoptotic signaling molecules (Lampiasi et al., 2009; Azizi et al., 2017). It is also assumed that oxidative stress-causing factors can selectively lead to cancer cell death compared with normal cell due to the higher production of ROS (Azizi et al., 2017).

The cell adhesion assay evaluates the ability of cell attachment to the extracellular matrix. In this assay, the PA-1 cells were implanted with various doses of MgONPs (10, 12.5, and 15 μg/ml). The MgONP-treated cells showed an aberrated cell adhesion pattern (Figure 11). These abnormal cells could reduce cancer progression and metastasis (Lin et al., 2020; Pallavi et al., 2020).

The cell migration assay is an essential aspect of cancer cell progression, which utilizes the ability of cells to migrate. The wound scratch assay showed a significant wound closure in untreated control PA-1 cells. However, the MgONP-treated cells showed a delay in the closure of the scratched area, suggesting a decrease in the motility and migration of cancer cells. The remnant scar region was found to be the highest compared to the starting gap area at the highest tested concentration of MgONPs (15 μg/ml). Our results showed a similar trend in inhibiting PA-1 cell migration as observed by other anticancer agents (Das et al., 2020; Haque et al., 2021). For comparison purposes, we have enlisted a table comprising the efficacy of green-synthesized MgONPs from various sources (Table 1). As far as our knowledge is goes from the available scientific literature, we are reporting the anticancer potential of green-synthesized MgONPs from pumpkin seed extracts for the first time. Experimental evidence suggests fascinating nutraceutical qualities of pumpkin seeds because they possess many bioactive compounds (Dotto and Chacha, 2020). Pumpkin seeds are a rich source of health-promoting compounds such as fatty acids, tocopherols, carotenoids, phenolic compounds, phytosterols, squalene, and minerals (Gedi and Mariod, 2022). A recent report suggests an inverse association of the risk of several types of cancer, such as breast, rectal, and lung cancer, to pumpkin seed intake (Batool et al., 2022).

Overall, the cellular modifications we observed could be explained as the invaded MgONPs induced ROS production, damaging the mitochondrial membrane integrity and activating the apoptotic pathway, resulting in cell death (Fouda et al., 2022). Subsequently, MgONPs’ potential to cause cellular injury could also be size-dependent, where smaller MgONPs increased ROS generation, improved interactions with cellular components, and improved membrane penetration to release Mg⁺ ions (Fouda et al., 2022). Despite their therapeutic potential, the challenge is appropriate bioavailability and cancer targeting by the compounds present in pumpkin seeds. The nanoparticle-based formulations are expected to overcome these challenges, and could provide substantial benefits. Even though the exact mechanism of MgONPs action is still obscure, it has been reported to induce apoptosis and ROS formation. Our results are concurrent with earlier reports, highlighting the significant therapeutic potential of our biogenic MgONPs against the studied cell lines.