All chemicals used are analytical grades obtained from Sigma Aldrich, Cairo, Egypt. Magnesium nitrate hexahydrate [Mg(NO₃)₂.6H₂O] was used as a precursor for MgO-NPs synthesis. The antimicrobial activity was conducted using Muller Hinton agar media (ready-prepared-Oxoid), whereas the cell lines used to investigate the In-vitro cytotoxicity were obtained from the Holding Company for Biological Products and Vaccines (VACSERA), Dokki, Giza, Egypt. All the reactions in the current study were conducted using distilled water (dis. H₂O).

Biomass of the brown algae C. crinita was collected from the Western Red Sea coast of Egypt in Hurghada City (27° 17′ 02.5″ N, 33° 46′ 21.0″ E).

The collected biomass of C. crinita was washed several times with tap water to remove seawater salts followed by washing with double-distilled water to remove any attached debris and sediments. The epiphytes were removed manually. The washed samples were oven-dried at 70°C for 24 h, followed by grinding into a fine powder using an electric blender. Approximately 10 g of C. crinita powder was mixed with 100 ml dis H₂O, shaken well, and heated at 60°C using a magnetic stirrer for 120 min. After that, the mixture was centrifuged for 10 min at 1500 rpm, and the supernatant was collected and used as reducing and stabilizing for MgO-NPs as follows: 51.3 mg of metal oxide precursor [Mg (NO₃)₂.6H₂O] was dissolved in 10 ml dis. H₂O and added to 90 ml of obtained algal aqueous extract to get a final concentration of 2 mM (Figure 1). After 24 h of incubation, the change of color from pale yellow to yellowish-brown indicates the formation of MgO-NPs (Pugazhendhi et al., 2019). Finally, the resultant NPs were calcinated at 400°C for 4 h.

The maximum surface plasmon resonance (SPR) of biosynthesized MgO-NPs was detected through monitoring the absorbance spectra using a UV-Vis spectrophotometer (Jenway 6305, Staffordshire, United Kingdom) in the range of 200–800 nm. The role of functional groups present in the aqueous extract of C. crinita in the reduction and stabilizing processes was investigated by Fourier transform infrared (FT-IR) spectroscopy (Agilent system Cary 660 FT-IR model). The NPs sample was mixed with KBr and pressured to form a disk that was scanned in the range of 400–4000 cm⁻¹ (Badawy et al., 2021). The shapes of biosynthesized MgO-NPs and their sizes are investigated using Transmission Electron Microscopy (TEM) (JEOL 1010, Japan, acceleration voltage of 120 kV, X40000). A drop of MgO-NPs colloidal solution was added to the TEM grid and the solution excess was removed by contacting the gride to blotting paper. The loaded gride was undergone vacuum desiccation for 24 h before being placed on the TEM holder (Soliman et al., 2021). The elemental contents in the MgO-NPs sample were analyzed using Scanning Electron Microscopy linked with energy-dispersive X-ray (SEM-EDX) (JEOL, JSM-6360LA, Japan) (Shaheen and Fouda, 2018).

The X-ray diffraction (XRD) analysis was conducted by X’Pert PRO at a 2θ degree of 0°–80° (Philips, Eindhoven, Netherlands). The operation conditions were voltage at 40 kV, current at 30 mA, and the x-ray source was Cu Ka radiation. Based on XRD analysis, the Debye–Scherrer equation was used to measure the particle size (Gu et al., 2018) as follows: D= 0 .9×0 .154 / β   Cosθ (1) where D is average particle size, 0.9 is the Scherrer’s constant, 0.154 is the wavelength (nm) of X-ray radiation, β is the half of maximum intensity, and θ is the Bragg’s angle.

The X-ray photoelectron spectroscopy (XPS) analysis was accomplished by ESCALAB 250XI⁺ instrument (Thermo Fischer Scientific, Inc., Waltham, MA, USA) connected with monochromatic X-ray Al Kα radiation (1,486.6 eV). The analysis was conducted under the following conditions: the size of the spot was 500 μm, the samples were prepared under a pressure adjusted at 10^–8 mbar, the energy was calibrated with Ag3d_5/2 signal (∆BE: 0.45 eV) and C 1s signal (∆BE: 0.82 eV), and the full and narrow-spectrum pass energies were 50 and 20 eV, respectively (Hamza et al., 2021a).

All the results presented are the means of three independent replicates. Data were subjected to statistical analysis by a statistical package SPSS v17. The mean difference comparison between the treatments was analyzed by t-test or the analysis of variance (ANOVA) and subsequently by Tukey HSD test at p < 0.05.

The first monitor for phyco-synthesis of MgO-NPs was the color change from pale yellow to yellowish-brown due to the mixing of aqueous extract of C. crinita with Mg(NO₃)₂.6H₂O. This change was investigated by UV-vis spectroscopy to detect the maximum surface plasmon resonance (SPR), which mainly depends on the size, distribution, and shape of phyco-synthesized MgO-NPs in the colloidal solution (Fedlheim and Foss, 2001). In this reaction, the UV radiations react with the metals in the tested solution that support the transition of electrons from low to a higher energy state, then the SPR is obtained which gives a prediction about the shape and size of NPs in the range of (2–100 nm) (Poinern, 2014). In this study, the maximum SPR was appeared at 320 nm, whereas algal aqueous extract has two maximum peaks at 490 and 570 nm (Figure 2A). The shifting in maximum peak confirmed the successful formation of MgO-NPs by algal metabolites present in the aqueous extract that act as reducing and capping/stabilizing agents. Compatible with the current study, the aqueous extract of brown algae Sargasssum wightii was changed to yellowish-brown after being mixed with Mg(NO₃)₂ as an indication of MgO-NPs formation, and the maximum SPR was appeared at 322 nm (Pugazhendhi et al., 2019). The presence of adsorption peaks at wavelength 200–250 nm refers to the existence of different bioactive compounds in algal aqueous extract such as alginates and polyphenols that play a critical role in the reduction of Mg(NO₃)₂.6H₂O to form MgO-NPs (Pugazhendhi et al., 2019).

The synthesis of NPs in our study is extracellular mode that is more convenient as the produced NPs are easily purified. Several compounds aid as reducing agents. As previously reported, the dominant biomolecules in brown algae are sterols such as cholesterols, fucosterols, sulfated polysaccharides, and various functional groups including glucuroronic acid, muramic acid, alginic acid, and vinyl derivatives that could act as reducing as well as capping agents for the synthesis of NPs (Chaudhary et al., 2020). For synthesis conditions, the pH of reaction medium is one of the important used experimental parameters in our synthetic method (Fouda et al., 2018). Here, the conducted alkaline condition used for NPs synthesis, as compared with neutral condition, enhances the reducing power of functional groups and prevents the NPs agglomeration (Parial et al., 2012). Besides that, it helps in the NPs capping and stabilization by interacting with the amine groups of surface-bound proteins and their residual amino acids (Namvar et al., 2014). Further experiments for the effect of different pH values, temperature, time, static conditions, substrate concentration, and stirring to find the optimal reaction conditions and the best physical characteristics of NPs should be investigated for the validation of all experimental conditions.

The bioactive compounds present in the aqueous extract of brown algae C. crinita and their role in the reduction, capping, and stabilizing of MgO-NPs were identified by FT-IR analysis (Figure 2B). FT-IR of the algal aqueous extract confirms the presence of C=O from polysaccharide moieties at 1627 cm⁻¹, whereas the broadband at 2075 cm⁻¹ is related to -NCS stretching of fucoidan from biomass that explains the sulfone stretching peak (Hamza et al., 2020a; Hamza et al., 2020b). The strong broadness of bands at 3340 related to OH and NH stretching vibration. The appearance of absorption peaks in the range of 450–650 cm⁻¹ refers to the successful formation of Mg―O (Ramanujam and Sundrarajan, 2014; Pugazhendhi et al., 2019; Fouda et al., 2021c); this peak overlapped with the C-S band from the biomasses (Hamza et al., 2021b). The weak absorption peak at 840 cm⁻¹ corresponded to bending C═C of alkene. The peak at 1027 cm⁻¹ related to stretching C―O or bending C―H or stretching CN of primary amines (Coates, 2006; Hamza et al., 2019), whereas the peak at 1166 cm⁻¹ corresponded to stretching C―O of the alcohol and overlapped with stretching C―N of tertiary amines (Coates, 2006). The absorption peak at 1395 cm⁻¹ is related to the vibration of C―H bending (Ogunyemi et al., 2019), whereas the peak at 1438 cm⁻¹ is corresponded to stretching C═O of carboxylic salts and adsorption of CO₂ and CO₃ ^2– on the MgO-NPs surface (Coates, 2006; Hamza et al., 2021b). The absorption peak at 1628 cm⁻¹ is related to water adsorption in the sample (Moorthy et al., 2015), whereas the broad peak at 2000 cm⁻¹ is signified to C―H bending of aromatic compounds overlapped with -NCS of biomass (Hamza et al., 2020a). The weak absorption peak at 2557 cm⁻¹ is referred to stretching of the S―H group of thiol-containing compounds, whereas the peak at 2723 corresponds to N―CH₃ methylamine and stretching C―H bond (these data are matched with XPS analysis) (Coates, 2006). The small absorption peak at 2850 cm⁻¹ signifies to stretching vibration bond of C―H in CH₂ groups’ existence in the phyco-chemical compounds (Essien et al., 2020). Finally, the broad and strong peaks that appeared in the range of 3400 cm⁻¹ to 3700 cm⁻¹ corresponded to the N―H and O―H groups of different amino acids present in the aqueous extract of brown algae (Moorthy et al., 2015; Pugazhendhi et al., 2019). Jena and coworkers reported that the most common functional groups present in the algal aqueous extract and responsible for NPs fabrications are –NH₂–, –C═O–, and –SH– groups (Jena et al., 2014). According to the FT-IR analysis, the role of bioactive compounds such as amino acids, polysaccharides, primary and tertiary amines, and others that are present in the aqueous extract of brown algae has been confirmed to act as reducing, capping, and stabilizing of MgO-NPs.

The activity of NPs is usually correlated with different characters including shape, size, and distribution (Salem and Fouda, 2021). Therefore, it is important to detect the size and shape of NPs. As shown from the TEM analysis in Figure 3A, the bioactive compounds present in the aqueous extract of macroalgae C. crinita have the efficiency to fabricate spherical and well-dispersed MgO-NPs with a size range of 3.0–18.0 nm with an average size diameter of 10.65 ± 3.3 nm (Figure 3B). As mentioned in the previous studies, the activity of NPs is increased by decreasing their size. For example, the activity of MgO-NPs to inhibit the growth of pathogenic Gram-positive Bacillus subtilis was varied according to the sizes used. The inhibition percentages were 75.7%, 94.5%, and 96.1% for size 2145.9, 47.3, and 35.9 nm, respectively (Huang et al., 2005). Moreover, spherical MgO-NPs synthesized through harnessing metabolites of Penicillium chrysogenum exhibited a size range of 7–40 nm and showed inhibition activity against pathogenic Gram-positive bacteria, Gram-negative bacteria, and unicellular fungi as well against larvae and pupa of malarial vector Anopheles stephensi (Fouda et al., 2021a). The obtained size (3.0–18.0 nm) of MgO-NPs in the current study is expected to have high activity for various biomedical and biotechnological applications.

The EDX analysis is used to detect the elemental composition of the biosynthesized MgO-NPs. The EDX chart showed that the phyco-synthesized MgO-NPs are highly pure; it contains Mg and O ions which indicates the successful formation of MgO through harnessing metabolites present in the aqueous extract of brown algae C. crinita (Figure 4A). Also, the existence peak of Mg and O at bending energy between 0.5 and 1.5 KeV confirms the successful formation of MgO (Fouda et al., 2021e). The quantitative analysis revealed that the weight percentages of Mg and O ion in the sample were 54.1 and 20.6%, respectively, whereas the atomic percentages were 50.3% and 17.1%, respectively (Figure 4A). Similarly, the EDX profile of MgO-NPs fabricated by the Pterocarpus marsupium aqueous extract showed two peaks at 0.5 and 1.5 KeV for O and Mg ions with weight percentages of 69.3 and 30.6%, respectively (Ammulu et al., 2021). The presence of C indicates the bounding of algal metabolites to the MgO-NPs surface. Compatible with the current study, the presence of carbon in the EDX chart of MgO-NPs was attributed to the attached biomolecules secreted by brown algae Sargasssum wightii to the surface of MgO (Pugazhendhi et al., 2019).

The crystallinity of phyco-synthesized MgO-NPs was investigated using the XRD pattern. The data represented in Figure 4B showed five intense peaks at 2θ values of 36.9°, 42.6°, 62.2°, 74.7°, and 78.8° which corresponded to (111), (200), (220), (311), and (222), respectively. The obtained peaks confirmed that the phyco-synthesized MgO-NPs were a crystallographic and face-centered cubic structure (FCC) as compared with JCPDS file no.39–7746 (Pugazhendhi et al., 2019). The size of biosynthesized MgO-NPs can be calculated according to the width of the sharp XRD peak (200) located at a 2θ value of 42.7° by Debye Scherrer’s equation. Data showed that the mean crystal size of phyco-synthesized MgO-NPs was 21 nm based on XRD analysis. Similar XRD spectra were recorded for green synthesized MgO-NPs by different biological entities (Sharma et al., 2020; Ammulu et al., 2021; Hassan et al., 2021).

The XPS analysis of MgO-NPs was analyzed and characterized by several identified peaks. Figure 5A depicts the survey analysis spectra of the sample, which has several peaks of the constituents; this appears in C 1s, N 1s, Cl (1s, 2p), O (1s, KL1, 2s, and KL2), whereas the Mg was detected at different bending energy (BE) as 1s, 2s, 2p, and KL1-5 verifying a majority product of this element over the other elements.

Different profiles of deconvolutions and signals were shown in comparing the chemical constituents. The deconvoluted and assignment peaks of the most abundant groups or species are recorded. The C 1s shows five splitting internal peaks, which are assigned as C (C, N, H) at 284.09 eV, C(=N, O) or C-O-C at 285.51 eV, N-C=O (amide) at 288.18 eV, and O-C=O and O-C-O at 289.53 eV, respectively (Figure 5B) (Jurado-López et al., 2017; Hamza et al., 2019; He et al., 2021). Two internal splitting peaks were shown for the O 1s peak (Figure 5C) which was assigned for O (N, C, H) at 530.52 eV, and the other peak for O-C=O at 531.8 eV (Jurado-López et al., 2017; Hamza et al., 2021b; Wei et al., 2021). This proves the chemical composition of the hydrocarbon (i.e., carbohydrate moiety) produced by the brown algae is compatible with the EDX chart.

The N 1s was splitting into five internal peaks assigned at 398.82 eV for N (C, H), and 401.04 eV for N_tert, this is for polysaccharides moieties (Lu et al., 2019; He et al., 2021), whereas the medium of the sorption and the dissolved salt source was appeared by three peaks 402.84, 404.15, and 406.54 eV for NO, NO₂, and NO₃, respectively (Figure 5D) (Bartis et al., 2015).

As shown in Figures 5E–G the Mg is predominately an oxide over the hydroxide species; this was shown in the Mg1s which split into two peaks at 1303.65 eV with (at%) 88.83, and the other peak at 1304.77 eV for Mg(OH)₂ with 11.7% (at%) (Yao et al., 2013). Mg2p shows the same deduction as in the Mg1s, the majority for the MgO at 49.14 eV, (at%) 88.77 over Mg-OH 50.34 eV (at% 11.23%) (Dang et al., 2017; Le Febvrier et al., 2017), whereas the Mg2s deconvoluted twice at 87.93 eV with 90.76% (at%) for MgO and at 89.6 eV with 9.24% (at%) for Mg(OH)₂ (Dang et al., 2017). This emphasizes the predominantly abundant MgO species over Mg(OH)₂ species and the majority of Mg in the yield.

The agar well diffusion assay was used to evaluate the microbicidal activities of the phyco-synthesized MgO-NPs. Measurement of inhibition zones developed around agar wells demonstrated the dose-dependent activity of MgO-NPs against selected clinical pathogens (Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and the unicellular fungi (Candida albicans) as recently reported (Hassan et al., 2021). The results proved the divergent activity of the nanostructured MgO against all tested pathogens. The MgO-NPs at 50 μg mL⁻¹ exhibited a wide spectrum activity against all tested microbes. Bacillus subtilis was the most sensitive strain recording 13.3 ± 0.5 mm zone of inhibition (ZOI). Doubling the concentration of MgO-NPs (100 μg mL⁻¹) improved the antimicrobial activity, and B. subtilis retained the biggest ZOI (15.6 ± 0.5 mm), whereas E. coli had the smallest ZOI (13.6 ± 0.5 mm). The MgO-NPs concentration at 200 μg mL⁻¹ showed the maximum effectiveness against all microbes, B. subtilis recorded the largest ZOI (18.6 ± 0.5 mm), whereas it was 15.3 ± 0.5 mm for E. coli. Moreover, S. aureus, P. aeuroginosa, and C. albicans recorded 17.3 ± 0.5, 17.6 ± 0.5, and 16.6 ± 0.5 mm ZOIs, respectively ( Figure 6).

MgO-NPs synthesized by brown algae Sargassum wighitii showed antifungal and broad-spectrum antibacterial potential against Proteus mirabilis, Staphylococcus aureus, Serratia marcescens, Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa (Pugazhendhi et al., 2019). Similarly, Deepak and coworkers demonstrated the facile fabrication of silver-NPs by the brown seaweed Sargassum wightii, and they reported bactericidal efficacy against B. subtilis, B. cereus, P. aeruginosa, Salmonella typhimurium, Enterococcus faecalis, and Shigella flexneri (Deepak et al., 2018).

MgO-NPs are metal-based, light nanoparticles with antimicrobial potential; it is totally resorbed and metabolized in the body. The minimal fungicidal and bactericidal inhibitory concentrations of MgO-NPs against prevailing infectious yeasts and bacteria must be determined to be applied clinically (Nguyen et al., 2018). Herein, MgO-NPs inhibited the growth of all the tested microbes. However, MIC was divergent for the two Gram-positive bacteria, specifically 12.5 μg mL⁻¹ was the MIC for B. subtilis and 50 μg mL⁻¹ for S. aureus. In contrast, the concentration of MgO-NPs increased to 25 and 50 μg mL⁻¹ for realizing the MIC for the Gram-negative bacteria, P. aeruginosa, and E. coli, respectively. The antifungal properties of MgO-NPs were confirmed against C. albicans, registering the MIC value 12.5 μg mL⁻¹.

The antibacterial properties of MgO-NPs are attributed to various mechanisms as proposed by several authors as shown in Figure 7. These mechanisms include the enhancing reactive oxygen species (ROS) production, reaction of MgO-NPs with the bacterial cell wall and interfering with electron transport chains, the interaction between Mg²⁺ and active macromolecules inside the bacterial cells, and the production of alkaline conditions due to the release of Mg²⁺ inside the bacterial cells (Wong et al., 2020). The interaction between the bacterial cell wall and MgO-NPs might lead to the deformation of a cell wall structure and disrupt the electron transport chains, ultimately blocking the selective permeability function (Karthik et al., 2019). Also, this interaction led to the formation of toxic substances such as ^•OH, H₂O₂, and^–O₂ that irreversibly destroy the cell wall and its important components such as phospholipids. It might also interfere with protein and nucleic acids, which necessitates cell death (He et al., 2016). In addition, once MgO-NPs enter the cell, it presents their alkaline effects with the release of Mg²⁺ (Karthik et al., 2019; Abinaya et al., 2021). Likewise, the efficient toxicity of MgO-NPs against diverse multidrug-resistant clinical pathogens makes it a good candidate for alternative medicine (He et al., 2016).

Various nanoparticles showed anticancer properties and can be used in cancer therapy or as targeted delivery systems for anticancer drugs (Chaudhary et al., 2020). The safe use of NPs as an antiproliferative agent against carcinoma requires evaluation of their side effects against normal cell lines and protein structure (Behzadi et al., 2018). Consequently, an MTT assay was used to evaluate the cytotoxicity of the phyco-synthesized MgO-NPs against the human colon cancer cell lines (Caco-2) and the monkey’s healthy cell lines (Vero). The MTT assay colorimetrically estimates the viability and proliferation of active cells regarding their metabolic reduction potency.

Seven concentrations of phyco-synthesized MgO-NPs (7.8, 15.6, 31.25, 62.5, 125, 250, and 500 μg mL⁻¹) were prepared to study their effect on the viability of Caco-2 and Vero cell lines within 48 h. MgO-NPs exhibited cytotoxic effects against normal and cancer cell lines in a dose-dependent manner. After the incubation with MgO-NPs for 48 h, the cloned cells suffer a partial or complete breakdown of the characteristic integral monolayer of epithelial cells along with shrinkage, buoyancy and the cells appear grainy and spherical. Besides the changes that occurred in the typical epithelial morphology of the cell lines, there is a decrease in the cells population. These cellular modifications can be explained by the entry of MgO-NPs into mammalian cells via endocytosis or macro-pinocytosis followed by enhanced synthesis of reactive oxygen species (ROS) that damage the membrane mitochondrial potential and lead to the activation of the apoptotic pathway, which ends with cell death (Verma et al., 2020). Furthermore, the cellular damage induced by MgO-NPs is size-dependent, smaller-sized MgO NPs boosted the production of ROS, enhanced the interaction with cellular components, and improved membrane permeation to liberate Mg⁺ ions (Ratan et al., 2020). Likewise, Pugazhendhi et al. have reported the cytotoxic potential of MgO-NPs against lung cancer cell lines (A549), and observed cell rounding and shrinkage, membrane blabbing, apoptotic body formation, chromatin condensation, and reduced cell population after cells were incubated with MgO-NPs (Pugazhendhi et al., 2019).

Our results for the In-Vitro cytotoxicity assay demonstrated the dramatic effect of MgO-NPs on the viability of treated cell lines and increasing the concentration of MgO-NPs has significantly reduced cell viability ( Figure 8). However, the biocolloidal MgO reduced the viability of cancer, as well as normal cell lines. The IC₅₀ concentration against the Caco-2 cancer cell line was 113.4, whereas it was 141.2 μg mL⁻¹ for the normal Vero cell line. This threshold can be exploited clinically for selective targeting of cancer treatment. Accordingly, we claimed that MgO-NPs are more toxic to the cancer cell line than to the healthy cell line. Comparable cytotoxicity analysis concluded the enhanced anticancer potential of green synthesized MgO-NPs against cancer cell line compared to the normal cell line (Behzadi et al., 2018; Pugazhendhi et al., 2019; Amina et al., 2020).

The efficacy of Cystoseria spp. to fabricate different metal and metal oxides nanoparticles with varied shapes, sizes, and applications compared with the current study is shown in Table 2. As shown, the polysaccharide fucoidan secreted by Cystoseira barbata have the efficacy to fabricate spherical Ag-NPs and Se-NPs with sizes of 12.86 and 16.18 nm, respectively, and showed anti-Candida activity (Alghuthaymi et al., 2021). Moreover, the spherical CuO-NPs with the size of 11–80 nm were successfully synthesized by the aqueous extract of Cystoseira myrica and exhibit high activity against two cancerous cell lines, namely, HepG2 and MCF-7 (Mohamed et al., 2021). In a recent study, the aqueous extract of C. crinita was used to form rectangular ZnO-NPs with sizes ranging between 23 and 200 nm and showed antimicrobial and antioxidant activity (Elrefaey et al., 2022). To the best of our knowledge, this is the first report for the green synthesis of MgO-NPs using an aqueous extract of C. crinita. In the current study, a small size (3–18 nm) of spherical MgO-NPs was successfully formed by brown algae, C. crinita, and exhibited varied activity including antimicrobial, in-vitro cytotoxicity, larvicidal, and repellence activity.