Characterization of zinc oxide and iron oxide nanoparticles stabilized with morin hydrate, and investigation and comparison of their antioxidant, antimicrobial, apoptotic, and anticarcinogenic properties.

Introduction
Nanoparticles are utilized in a wide variety of industries, including materials science, drug delivery, electronics, optics, environmental rehabilitation, and cosmetics, among others. Nanoparticles play a crucial role in advancing technology and are used across various fields. Their unique characteristics make them extremely useful as building blocks for the development of novel materials, technologies, and systems [1, 2]. Nanoparticles are particles ranging in size from 1 to 100 nm that have unique properties and behaviors that differ from larger particles; these differences are primarily due to their small size and high surface-to-volume ratio. Nanoparticles, particularly metal oxides, are attractive for applications such as drug delivery, gene therapy, and biosensors due to their high surface-to-volume ratios and the ease with which their size, shape, morphology, and related electronic properties can be tuned [3–5]. Inorganic nanoparticles include nanoparticles devoid of carbon or organic components. Representative examples of this category include metallic, ceramic, and semiconductor nanoparticles. Metal nanoparticles consist solely of metal precursors and can be classified as monometallic, bimetallic, or polymetallic [6, 7]. Nanoparticles are synthesized using several techniques, including chemical, physical, and biological approaches. Among these technologies, “green synthesis” is an eco-friendly approach that employs biological resources, including plants, bacteria, and fungi. This sustainable approach seeks to reduce the utilization of detrimental chemicals and ecological consequences [8, 9]. i/) Nanotechnology extensively utilizes zinc oxide (ZnO) and iron oxide (Fe₃O₄/γ-Fe₂O₃) nanoparticles. Their unique structural properties make them useful in a wide range of fields. Zinc oxide nanoparticles are essential in electronic devices, functional coatings, and cosmetic product formulations because they are very effective at killing bacteria and acting as photocatalysts. They are also promising for anticancer treatments and cleaning up the environment because they can cause apoptosis by making reactive oxygen species, stop cell growth, and control oxidative stress. Iron oxide nanoparticles that show superparamagnetic behavior have also changed the biomedical field. They are important as contrast agents in magnetic resonance imaging, as vectors for the targeted delivery of therapeutic agents, and in magnetic hyperthermia applications, which make chemotherapy and radiotherapy more effective by causing localized thermal damage in tumor tissue [10–14]. The green production of ZnO/Fe3O4 nanocomposites enhances their biomedical applications while promoting sustainable development by eliminating hazardous chemicals. Phytochemicals derived from natural sources serve as reducing, capping, and stabilizing agents, enhancing synthesis efficiency and environmental safety [15].
UV–visible spectrophotometry (UV–Vis), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), and Fourier transform infrared spectroscopy (FTIR) are extensively employed for the characterization of manufactured nanoparticles [5, 16–20]. Morin hydrate is a yellow dietary flavonoid typically extracted from the fruits, leaves, and stems of plants in the Moraceae family. This natural chemical is characterized by its 3,5,7,2′,4′-pentahydroxyflavone structure and exhibits diverse pharmacological effects that significantly benefit health. Morin’s primary and notable characteristics are its powerful antioxidant and anti-inflammatory qualities. These two primary pathways also account for their various additional therapeutic potentials. Research has demonstrated that morin possesses free radical scavenging, cardioprotective, neuroprotective, antidiabetic, and antibacterial effects. Moreover, morin is believed to be efficacious against multiple cancer types, especially breast, ovarian, and colorectal malignancies, through the modulation of diverse cellular signaling pathways. It has been documented to provide protective and restorative effects in various chronic and degenerative disease models, including arsenic-induced toxicity, pulmonary and hepatic damage, neuroinflammatory diseases, diabetes, and myocardial infarction [21–30]. Using natural flavonoids like morin hydrate to stabilize zinc oxide and iron oxide nanoparticles is a big step forward in nanoparticle technology. It demonstrates the medical value of green synthesis and is essential for remedying the deficiencies of existing therapies [31]. ZnO nanoparticles are prominent among biocompatible metal oxides that exhibit broad-spectrum, potent antimicrobial activity due to their high surface area, photocatalytic properties, and ROS-generating capabilities [32–35]. In contrast, iron oxide nanoparticles generally show weaker antibacterial effects but are considered as functional components in biomedical imaging, targeting, and composite materials because of their magnetic properties and redox-active Fe centres [36, 37]. The objective of this study was to synthesize and characterize zinc oxide and iron oxide nanoparticles stabilized with morin hydrate and to perform comparative analyses of their antioxidant, antimicrobial, apoptotic, and anticarcinogenic properties in in vitro models. This study will demonstrate how the green synthesis method enhances the biological activity of the nanoparticles and identify the most promising candidates for biomedical applications such as targeted drug delivery, antimicrobial control, and cancer treatment. In addition, the findings of this study reveal the potential of environmentally friendly nanoparticles in environmental, biomedical, and many other fields. Therefore, the biological activities of morin-stabilized zinc oxide and iron oxide nanoparticles were compared to evaluate the effectiveness of metallic chemicals, which are commonly used as precursors in nanoparticle synthesis, on biological activity.

Material and methods

Materials
In this study, cell culture medium (DMEM-F12/RPMI 1640; Sigma-Aldrich Catalog No: D0697/R8758, USA), fetal bovine serum (FBS; Sigma-Aldrich Catalog No: F7524, USA), 1% penicillin/streptomycin (Sigma-Aldrich Catalog No: P4333, USA), Morin hydrate.
(Acros Organics, Catalog number 354000500) L-glutamine (Sigma-Aldrich Catalog No: 59202 C, USA), trypsin–EDTA solution (Sigma-Aldrich Catalog No: 59417 C, USA), dimethyl sulfoxide (DMSO) (Sigma-Aldrich Catalog No: PHR1309, USA), MTT (3-(4,5-Dimethylthiazol-2-yl)−2,5-Diphenyltetrazolium Bromide; Sigma-Aldrich), Culture plates (96-well) were used from Nunc (Brand products, Denmark). Catalog No: M2128, USA).

Synthesis and characterization of zinc and iron nanoparticles
In the first step, 300 mL of morin hydrate solutions were prepared at a concentration of 0.05 M. Additionally, 100 mL of zinc acetate dihydrate (Zn(CH3COO)2·2H2O) and iron(III) chloride hexahydrate (FeCl3·6H2O) solutions at a concentration of 0.1 M were prepared, which were ready to be used in the synthesis reactions. Two different metal salts were used to carry out the synthesis processes separately: 100 mL of 0.1 M Zn(CH3COO)2·2H2O solution was mixed with 300 mL of 0.05 M morin hydrate solution under a magnetic stirrer. The mixture was stirred with a magnet at 70 °C for 60 min. As a result of the reaction, an orange or orange-red (coral) solution was formed, indicating the formation of nanoparticles (Zinc-morin Complex, (For 1:1 stoichiometry [Zn(C15H9O7)2(H2O)2]). 100 mL of 0.1 M FeCl3·6H2O solution was added to 300 mL of 0.05 M morin hydrate solution while stirring with a magnet. This mixture was stirred with a magnet for 60 min at 70 °C. This reaction resulted in a black nanoparticle solution (Iron(III)-morin complex, (for 1:1 stoichiometry [Fe(C15H9O7)(H2O)4]Cl3 intensely colored complex cation) (Fig. 1). Both synthesized nanoparticle solutions were centrifuged at ~ 3300 g for 20 min.The supernatant formed after centrifugation was filtered. The resulting precipitates were washed with distilled water to remove residue and centrifuged again. This washing and centrifugation process was repeated until the particles were purified. The nanoparticle precipitates obtained after the purification process were dried in a 95 °C oven for 3 h to obtain stable products in powder form. SEM was used to image the synthesized nanoparticles, EDX was used to determine their elemental composition, and FTIR was used to determine the molecular groups they contained.

Methodology for determination of antimicrobial activity
The study utilized reference strains Pseudomonas aeruginosa ATCC 9027, Escherichia coli ATCC 11229, Klebsiella pneumoniae ATCC 13883, Staphylococcus aureus ATCC 25923, and Candida albicans ATCC 10231, provided by Microbiologics. The antimicrobial susceptibility of bacteria was evaluated using the disk diffusion method in line with clinical and laboratory standards. Fresh cultures of each test microorganism were cultured in Nutrient Broth (NB) at 37 °C until a turbidity equivalent to a 0.5 McFarland standard (about 1.5 × 108 CFU/mL) was achieved. The inoculum’s standard density was verified spectrophotometrically by calibrating the absorbance at 625 nm to a range of 0.08–0.10. Aseptic technique was employed to transfer 100 µL of the generated bacterial suspensions, which were then evenly distributed on the surface of Nutrient Agar solid medium. Subsequently, sterile disks were positioned on the surface of the medium, and these disks were infused with the essential oils under examination. Bacterial strains (E. coli, P. aeruginosa, K. pneumoniae, S. aureus) were incubated at 37 °C for 24 h, while the yeast strain (C. albicans) was incubated at 30 °C for 48 h. Following incubation, the diameters (mm) of the inhibition zones surrounding the disks were measured with a digital caliper. All experiments were conducted in triplicate and separately. The Sulbactam/Ampicillin (SAM, 20 µg) antibiotic disk served as a positive control to confirm the method’s validity and the bacteria’s sensitivity [38–40].

Analyses of antioxidant capacity (DPPH, FRAP, and CUPRAC)
The radical scavenging activity was subsequently determined by measuring the reduction in absorbance relative to the control sample (DPPH solution alone). The reduction capabilities of the samples were assessed using the FRAP method. An equal proportion of FRAP reagent (an acidic buffer containing 2,4,6-Tripyridyl-s-triazine (TPTZ) and a 20 mM FeCl3 solution was included in various concentration solutions of each sample. Following the incubation of the reaction mixtures, the quantity of the blue TPTZ-Fe2+ complex generated from the reduction of ferric ions (Fe3+) to ferrous ions (Fe2+) by antioxidants was determined by measuring the absorbance at 593 nm [41]. The CUPRAC approach, established by Apak et al. (2007), was utilized with modifications to ascertain the reducing capabilities of copper ions. For the analysis, 500 µL of 0.01 M copper(II) chloride (CuCl2), 500 µL of 7.5 × 10−3 M neocuproine (ethanolic solution), and 500 µL of 1 M ammonium acetate buffer solution (pH 7.0) were included into solutions of varying concentrations for each sample, respectively. Following the incubation of the combinations for a specified duration, the intensity of the yellow chelate complex generated between neocuproine and Cu+ ions was assessed at a wavelength of 450 nm [42, 43].

Cytotoxicity test

Cell culture
The following cancer lines (HCT-116, Colon Carcinoma), obtained from ATCC and stored in liquid nitrogen, were used in the study. HCT-116 cells were cultured in DMEM-F12/RPMI-1640 medium supplemented with 10% Fetal Bovine Serum (FBS), 100 μg/mL streptomycin/100 IU/mL penicillin, in incubators at 37 °C under humidified conditions with 5% CO2.

Cell viability assay
The cytotoxic effects of Zn/Fe oxide nanoparticles on the HCT-116 cell line were assessed using the MTT ([3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide]) assay. Cells were plated in sterile 96-well plates at a density of 1 × 104 cells per well and incubated at 37 °C in a 5% CO2 environment for 24 h. At the conclusion of this period, following the successful adherence of the cells, the culture medium was discarded, and NP extracts were introduced at concentrations of 0, 2.5, 5, 10, 25, 50, 100, and 200 µg/mL. The cells underwent exposure to these extracts and were incubated for a duration of 48 h. Following the 48-h incubation period, 10 µL of MTT reagent (0.5 mg/mL stock solution) was added to each well, and the plates were incubated for an additional 4 h. During this period, metabolically active mitochondrial dehydrogenase enzymes in living cells converted the yellow MTT tetrazolium salt into purple, water-insoluble formazan crystals. The MTT-containing medium was subsequently removed, and 100 µL of DMSO was added to each well to dissolve the formazan crystals. The intensity of the purple color generated by the formazan solution was quantified at 570 nm utilizing a microplate reader (Thermo Multiskan GO, Thermo, USA), with scattering noise eliminated through reference measurements at 690 nm. Optical density (OD) values were utilized as indicators of cell viability. Experiments were conducted in triplicate, and cell viability percentages were derived from the collected data. Concentration-dependent viability curves were generated, and IC50 values were determined through statistical analysis.

Apoptotic effect of MrFeO and MrZnO nanoparticles on HCT-116 cancer cells by acridine Acridine orange/Ethidium bromide (AO/EB) staining
It was used to morphologically detect apoptosis of MrFeO and MrZnO (277.14/210.45 μg/mL) agents applied to HCT-116 cells. The extract-treated cell was washed with PBS after incubation and fixed with 70% ethanol. At the end of fixation, the cells were washed with distilled water, stained with Acridine orange/Ethidium bromide (Cat No./ID: A6014-E1510) (Sigma Aldrich, Germany) working solution, and images were taken under a fluorescence microscope. It was used to morphologically detect apoptosis of MrFeO and MrZnO (277.14/210.45 μg/mL) nanoparticles applied to HCT-116 cells. After incubation, the treated cells were washed with PBS and fixed with 70% ethanol. Following fixation, the cells were washed with distilled water, stained with Acridine Orange/Ethidium Bromide (Cat No./ID: A6014-E1510) (Sigma Aldrich, Germany) working solution, and images were obtained under a fluorescence microscope.

Evaluation of the apoptotic impact of MrFeO and MrZnO nanoparticles on the HCT-116 cell line by Annexin V staining
The induction of apoptosis by MrZnO and MrFeO nanoparticles in HCT-116 colorectal cancer cells was evaluated using an Annexin V-based apoptosis detection assay. The assay was conducted utilizing a commercial FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, New Jersey, USA) in accordance with the manufacturer’s instructions. HCT-116 cells were inoculated in 6-well plates at a density of 5 × 105 cells per well and permitted to adhere for 24 h. Subsequent to the adhesion period, cells were administered with specified half-maximal inhibitory concentration (IC50) dosages of nanoparticles, namely 277.14 μg/mL for MrZnO and 210.45 μg/mL for MrFeO, for a duration of 48 h. Subsequent to the treatment period, cells were collected via trypsinization, washed, and resuspended in 1X Binding Buffer at a concentration of 1 × 106 cells/mL. Subsequently, 100 μL of the cell suspension (about 1 × 105 cells) was aliquoted into a sterile tube and stained with 5 μL of fluorescein isothiocyanate (FITC)-conjugated Annexin V and 5 μL of Propidium Iodide (PI) solution. Samples were gently vortexed and incubated for 15 min at ambient temperature in the absence of light. After incubation, 400 µL of 1X Binding Buffer was introduced to each tube to terminate the reaction. The stained cells were subsequently examined using a flow cytometer (BD FACSVia, New Jersey, USA). Cell populations were categorized into viable cells (Annexin V−/PI−), early apoptotic cells (Annexin V+/PI−), late apoptotic cells (Annexin V+/PI+), and necrotic cells (Annexin V−/PI+) according to staining properties.

UV–Vis absorption spectrum
Ultraviolet–visible spectroscopy is a common analytical technique for measuring molecules, inorganic ions, nanoparticles, and complexes in solution, relying on the interaction of electromagnetic radiation with matter. The size, shape, density, aggregation state, and surface-adjacent refractive index are critical parameters affecting the optical properties of metal-based nanoparticles. Thus, UV–Vis spectroscopy is crucial for the identification, characterization, and study of nanoparticles. UV–visible spectra were obtained using a T80 + UV/Vis Spectrometer (PG Instruments Ltd.) to assess the optical properties of MrFeO and MrZnO. A clean water solution at room temperature was utilized to establish the baseline, with a wavelength (λ) selected between 200 and 800 nm [44, 45].

Fourier transform infrared spectroscopy (FTIR)
An FT-IR instrument (Perkin Elmer Spectroscopy 100) was used to identify the bonds and functional groups present in the structure of MrZnO and MrFeO nanoparticles. KBr pellets with wavelengths ranging from 400 to 4000 cm−1 were analyzed using FTIR. Descriptive analyses of the molecules or bonds found in the sample’s natural structure are determined by the color of the altered bonds. This aimed to obtain information about the molecules and functional groups (such as –NH2, –OH, and –COOH) present in the structure of the materials used in the production of these nanoparticles.

SEM–EDX
Surface morphology was determined using an SEM (HITACHI S-3000H). The elemental composition of the samples was analyzed using energy-dispersive X-ray (EDX). UV–Vis spectroscopy was used to demonstrate the absorption of visible light by chemical compounds, resulting in the production of distinct spectra.

Statistical analysis
The results were presented as mean ± standard deviation. Differences between groups were analyzed using one-way ANOVA (IBM SPSS). A p value of < 0.05 was considered statistically significant.

Results and discussion

UV–Vis and FTIR measurement results
Nanomedicine aims to address problems related to human disease at the nanoscale, where most biological molecules exist and function. The physicochemical characterization of nanometer-scale materials is crucial in biomedical research to better interpret results. Parameters such as shape, size, crystal structure, purity, hydrodynamic size, agglomeration, and aqueous stability have been proposed for NP characterization. For the nanoparticle spectra based on pure water solutions (pH = 10), MrZnO exhibited a sharp absorption peak at approximately 300 nm and a maximum absorbance of approximately 2.1 au, while MrFeO exhibited a broader peak at approximately 350 nm and a higher maximum absorbance of approximately 2.5 au. The MrZnO curve in the graph shows a strong absorption band between wavelengths of approximately 300 nm and 400 nm (Fig. 2). The beginning of this band is related to the characteristic band gap energy (Eg) of ZnO. ZnO is an n-type semiconductor with a wide band gap energy of 3.37 eV. The sharp decrease in the curve in this region suggests that the synthesized MrZnO nanoparticles have good crystallinity. It exhibited optical properties consistent with similar studies in the literature, with band-edge absorption typical of ZnO nanoparticles, typically reported around 360–380 nm in biosynthesized or functionalized forms. Furthermore, the MrFeO spectrum exhibited an absorption characteristic unique to Morin, in addition to the broad UV–visible absorption profile of pure iron oxide nanoparticles. The appearance of these bands in the MrFeO spectrum may be an indication that Morin was successfully immobilized on the iron oxide surface [46–53]. Morin is a flavonoid chemical found in plants. Morin can absorb ultraviolet light. Morin’s UV absorption wavelength is generally between 330 and 365 nm (UVA) [54, 55]. Both spectra confirm the synthesis of morin-functionalized nanoparticles, as the peaks differ from those of free morin. This may be due to charge transfer interactions between the iron-zinc and morin aromatic systems. The UV–Vis graph for the 0.05 M morin (Mr), 0.1 M Zn(CH3COO)2·2H2O, and 0.1 M FeCl3·6H2O solutions verifies that the synthesis has been completed (Fig. 2).
An FTIR spectrophotometer is a widely used phenotypic analysis method to study the functional groups and metal–ligand bonds of the synthesized compounds. The results of FTIR characterization of morin-ZnO and MrFeO complexes in the 4000–400 cm−1 range are presented in Fig. 3. Based on the results of FTIR characterization, the formation of metal-morin complexes can be observed in the spectral difference between morin ligands and the complex compounds. The dissociation of metal ions into NPs during the synthesis process and their further stabilization are indicated by the changes in the positions and intensities of the O–H, C=O, –COOH, C–O–C, N–H, and C–N bands. Between 2500 and 4000 cm−1, single bonds such as OH, CH, and NH can be observed. Between 2000 and 2500 cm−1, triple bonds (C≡N and C≡C) can be found, and between 1500 and 2000 cm−1, double bonds (carbonyl (C=O), nitro (N=O), and C=C) can be found [56–58]. Morin hydrate is a bioflavonoid belonging to the flavonol class and has a flavone (2-phenyl-1-benzopyran-4-one) backbone with a hydroxyl group at the 3-position. It is a 7-hydroxy flavonol with three additional hydroxy substituents at the 2′, 4′, and 5-positions [59, 60].
The intense and broad band observed at 3148 cm−1 can be attributed to the stretching of the hydroxyl group (–OH) of phenol or alcoholic water. The presence of multiple hydroxyl groups (–OH) in the morin structure may have caused the formation of this band. As in the MrZnO sample, a significant weakening and broadening of the –OH band at approximately 3527.29 cm−1 is observed. This reduces the number of OH groups and changes the vibration frequency. This proves that the –OH groups in morin interact strongly with the Fe ions (Fe2+/Fe3+) on the FeO surface, indicating successful binding to both ZnO and FeO nanoparticles. The values of 1618.94 cm−1 (MrZnO) and 1660.14 cm−1 (MrFeO) indicate C=O stretching (Fig. 3) [61]. The MrZnO peaks at 637.32, 585.17, 584.12, 457.05 cm−1, the MrFeO peaks at 637.32, 585.36, 564.61, 475.94 cm, and the metal-oxide vibration bands at around 400–600 cm−1 confirm the presence or structural composition of the nanoparticle core in both cases [62–64].
The FTIR spectra of the produced ZnO and FeO nanoparticles revealed the presence of both organic functional groups and characteristic metal–oxygen vibrations, confirming successful nanoparticle formation and surface stabilization. Broad absorption bands observed at 3527 and 3148 cm−1 correspond to hydroxyl and hydrogen-bonded functional groups, indicating the involvement of phenolic compounds and adsorbed water molecules derived from the stabilizing matrix. The bands at 1618 and 1508 cm−1 are attributed to aromatic C=C stretching, suggesting that morin acts as a reducing and capping agent during nanoparticle synthesis. Furthermore, peaks at 1177 and 1086 cm−1 are related to C–O and C–O–C stretching vibrations, further supporting the presence of morin on the nanoparticle surface. Particularly, the absorption bands below 700 cm−1, particularly at 637, 585, and 564 cm−1, are characteristic of Zn–O and Fe–O lattice vibrations, providing strong evidence for the formation of zinc oxide and iron oxide nanoparticles with preserved crystalline metal–oxygen frameworks (Table 1).

SEM, EDX Measurement Results
The morphology of nanoparticles is a defining factor in cellular uptake, circulation, biodistribution, and, in certain cases, toxicity. Nevertheless, these effects should always be assessed in conjunction with size, surface chemistry, flexibility, and biodegradability [73–75]. The elemental composition of MrZnO was analyzed, revealing a carbon content of 59.150 wt%, an oxygen content of 19.05 wt%, and a zinc concentration of 21.80 wt%. The SEM images of MrZnO NPs, examined at magnifications of 1000×, 2500×, 5000×, 7500×, 10,000×, 15,000×, 20,000×, and 30,000×, demonstrated a uniform size distribution, rectangular prism morphology, low aggregation, and smooth surface features. Both SEM and EDX tests confirm the successful fabrication of a morin-coated ZnO nanoparticle structure (Fig. 4). Examining the elemental composition of Mr-FeO reveals that the carbon content is 64.53 wt%, the oxygen content is 24.61 wt%, and the iron content is 21.80 wt%. (Fig. 5). The increased concentration of carbon atoms in both nanoparticles may be ascribed to the Morin molecule. This confirms the significant occurrence of organic–inorganic hybrid material in the sample (59% organic and 41% inorganic; 65% organic and 35% inorganic, respectively). SEM images of MrFeO at the same magnifications reveal a uniform size distribution and negligible aggregation, but the rectangular prism architecture of MrZnO exhibits greater consistency. The observation that these materials, despite varying chemical structures, exhibit identical geometric morphology (rectangular prism) indicates that our synthesis technique (chemicals, temperature, pH, etc.) was effectively controlled or that crystal growth was restricted in particular directions. The high organic content of Mr-FeO indicates enhanced morin coating/loading. This is likely due to the unique crystal structures and surface energies of the two metal oxides, along with the magnetic attraction of FeO. The colors and physicochemical characteristics of the two nanoparticles were significantly different. In biological activity studies, aqueous solutions of MrZnO nanoparticles were seen to desiccate upon air exposure, hindering their manipulation, while MrFeO nanoparticles yielded highly fluid solutions (Figs. 4 and 5). A study indicated that FexOyNPs displayed diverse shapes and morphologies, such as cubic, hexagonal, rhombohedral, monoclinic trigonal, and tetragonal structures with significant particle aggregations, whereas ZnO NPs primarily exhibited hexagonal nanorod structures comprised of densely packed, larger rod-shaped nanoparticles, in addition to spherical, face-centered rhombohedral, cubic, and hexagonal forms [64].
Mr–FeO nanoparticles possess an exceptionally effective morin coating; yet, agglomeration challenges may necessitate adjustment of dispersion before application. Mr-ZnO, however, seems to exhibit greater structural and morphological control and definition. Both materials exhibit successful synthesis characterized by their unique advantages: high morin loading and magnetism versus optimal shape and dispersion.

Antimicrobial activity results of nanoparticles
The increasing incidence of hospital- and community-acquired infections caused by pathogens has enabled the production of nanoparticles that significantly enhance their microbial fighting capabilities through nanotechnology. Furthermore, the development of resistance to currently used antibiotics increases the need to find new antimicrobial agents. Inorganic NPs, exemplified by materials such as ZnO, are known for their potent and broad-spectrum antimicrobial activity. ZnO can provide long-term antimicrobial activity due to its ability to support a variety of microorganisms and the slow release of antimicrobial ions. Nanometric ZnO is a significantly better antimicrobial agent than its micrometric counterparts because the size of the nanoparticles is directly proportional to their increased surface area and reactivity. While opinions are divided, these materials are said to be highly biocompatible, meaning they are not very harmful to human cells and are only harmful to bacteria. This unique combination of properties makes ZnO-NPs very useful for biological and materials science applications that require cell killing [32, 76–80].
In our study, the antimicrobial effects of MrZnO and MrFeO nanoparticles on five different microorganisms (E. coli, K. Pneumonia, P. Aeruginosa, S. Aureus, C. albicans) were compared in terms of inhibition zone diameter (mm) using a positive control, Ampicillin/sulbactam (SAM). While MrZnO nanoparticles showed inhibition against all tested microorganisms (E. coli: 11.0 mm; K. Pneumoniae: 9.0 mm; P. Aeruginosa: 12.0 mm; S. Aureus: 8.0; C. albicans: 9.0 mm), MrFeO nanoparticles showed inhibition only against E. coli (9 mm) and P. aeruginosa (11 mm) (Table 2). As expected, SAM showed much higher inhibition zones against all microorganisms. While MrZnO exhibited a broad spectrum of activity, MrFeO nanoparticles exhibited a narrower spectrum of activity and a more limited overall antimicrobial potential than MrZnO. Despite the high dose (20 mg/mL), the efficacy of both nanoparticles was lower than that of SAM. This aligns with prior research indicating that ZnO nanoparticles demonstrate enhanced antibacterial efficacy relative to their chemically produced equivalents (Fig. 6). A prior study indicated that green-synthesized ZnO nanoparticles had superior antibacterial efficacy against both gram-positive and gram-negative bacteria in comparison to pure ZnO nanoparticles [81–83]. Previous studies have found that green-synthesized FeO nanoparticles are more effective against gram-negative bacteria than gram-positive bacteria. In contrast, our synthesized MrFeO nanoparticle was found to penetrate more easily into gram-negative bacteria. These data suggest that MrFeO nanoparticles may be a specific and alternative antimicrobial for the elimination of gram-negative bacteria [84]. The fact that MrZnO’s antibacterial properties are noteworthy suggests that ZnO-NPs may have the ability to disrupt bacterial cell membranes, easily enter cells, produce ROS, and break the chemical bonds of bacterial organic matter, thus creating a bactericidal effect [85, 86].

Cytotoxicity analysis results of nanoparticles
The IC50 values ​​were calculated by treating with MrZnO/MrFeO nanoparticles at doses of 0–200 µg/mL for 48 h in cancer cell lines (Table 3). MrZnO/MrFeO nanoparticles and cells with the lowest IC50 value for cancer cells were detected, and are presented in Table 3.
The cytotoxic effects of MrFeO nanoparticles on cancer cells were investigated. MTT results revealed that MrFeO nanoparticles exhibited a cytotoxic effect on HCT-116 (Colon Carcinoma) (IC50 : 210.46 µg/mL) cells (Fig. 7). The cytotoxic effect of MrZnO nanoparticles on cancer cells was investigated. MTT results revealed that MrZnO nanoparticles exhibited a cytotoxic effect on HCT-116 (Colon Carcinoma) cells (IC50: 277.15 µg/mL) (Fig. 7).
In the forthcoming decades, the aggregate incidence of cancer cases (excluding non-melanoma skin cancer) is anticipated to rise by around 50%, from 1,534,500 in 2015 to 2,286,300 by 2050 [87]. Colorectal carcinoma impacts both genders and ranks as the second foremost cause of cancer-induced mortality globally. Colon cancer mortality rates are escalating globally due to contemporary lifestyles characterized by excessive meat consumption, alcohol intake, and insufficient physical activity. Thus, there is an increased demand for novel, less detrimental pharmacological therapies for colon cancer. Colorectal cancer (CRC) represents a substantial segment of the global population. The magnitude of side effects and resistance in CRC patients is presently under investigation, notwithstanding recent advancements that have enhanced patient care and survival rates. Various chemicals present in medicinal plants have exhibited antitumor and antiapoptotic properties against multiple cancers, including colorectal cancer, in animal studies [88].
NP-based drug delivery systems demonstrate superior efficacy relative to conventional systems, attributed to the prolonged half-life of sensitive drugs and proteins, enhanced solubility of hydrophobic drugs, and the capacity for controlled and targeted drug release at pathological sites [89]. Metal nanoparticles are progressively being explored as alternative chemotherapy agents in cancer research. Metal nanoparticles possess the capability to be utilized across a diverse range of applications [90–92]. Green nanoparticles are characterized by their small size and distinctive physicochemical properties, which generally enhance intracellular bioavailability. Considering the known anticancer properties of ZnO and Fe2O3 nanoparticles and the demonstrated therapeutic efficacy of these nanoparticles in various cancer cells, we propose that these chemicals will yield effective, natural-based nanoparticles for targeted therapy [93–95]. Magnetic nanomaterials such as Fe3O4 (magnetite) and Fe2O3 (maghemite) are widely used in various fields, including biomarker and cell separation, gene therapy, targeted cancer therapy, stem cell separation and manipulation, DNA analysis, and non-invasive imaging of human internal organs. Magnetic drug delivery is another powerful application of magnetic NPs in the biomedical field, where they can be used to enhance traditional cancer treatment methods [96]. Iron oxide is one of the important transition metal oxide nanoparticles due to its variable oxidation state, magnetite properties, and low cost [97]. Zinc oxide nanoparticles, considered one of the most common metallic nanoparticles worldwide, are preferred against cancer cells due to their cytotoxic effects arising from the destabilization of protein function and ROS formation. The enhanced antibacterial and anticancer properties of modified zinc oxide nanoparticles are attributed to their Zn2+ release and diffusion ability in addition to their ROS production. One study investigated the potential anticancer properties of curcumin-loaded zinc oxide nanoparticles in the rhabdomyosarcoma RD cell line using the MTT assay and evaluated their cytotoxic effects in human embryonic kidney cells using the resazurin assay. The remarkable aspect ratio of the ZnO structures was identified as a contributing factor to the increased cytotoxicity of the nanoparticles. ROS (primarily superoxide, hydroxyl radicals, and hydrogen peroxide) are continuously produced and removed in biological systems. ROS are known for their important roles in various normal biochemical functions and abnormal disease processes. High ROS production exceeding the capacity of the cellular antioxidant defense system causes oxidative stress in cells, damaging cellular components including lipids, proteins and DNA [98–103].
Prolonged oxidative stress leads to protein, DNA, and lipid damage, as well as dysfunction of mitochondria and the endoplasmic reticulum, ultimately resulting in apoptosis or ferroptosis due to a relative imbalance or failure of the intracellular free radical scavenging mechanism’s defensive capabilities. Ferroptosis is an iron-dependent lipid ROS accumulation process. Small molecule ferroptosis inducers or inhibitors targeting iron metabolism or lipid peroxidation have been extensively studied [91, 104–106]. When IC50 values, which indicate the concentration required to kill 50% of cells or inhibit their growth, were examined, MrFeO IC50 = 210.46 µg/mL, while MrZnO IC50 = 277.15 µg/mL (Table 2 and Fig. 7). In our study, both MrZnO and MrFeO exhibited significant cytotoxic effects on HCT-116 colon cancer cell lines. The IC50 values obtained are relatively high compared to many chemotherapeutic drugs used in clinical practice. This indicates room for improvement in dosage and efficacy. A lower IC50 value means that the cytotoxic effect is stronger. In our study, MrFeO demonstrated more cytotoxic activity than MrZnO.
The anticarcinogenic efficacy of any test agent is contingent upon its capacity to impede cancer cell proliferation, migration, invasion, and angiogenesis, induce apoptosis and/or autophagy, and modulate cell proliferation and metabolism. The effects arise from the agent’s direct interaction with cellular components and biomolecules, which inhibit biomolecular function or disrupt essential survival and apoptotic pathways, induce epigenetic gene regulatory changes, generate free radicals that cause oxidative damage, or modify membrane fluidity [107, 108]. ZnONPs synthesized with plant extracts show stronger anticancer properties compared to plant extracts in A549 lung and MG-63 bone cancer cells [109–111]. One study demonstrated that Fe2O3 NPs suppressed the growth of colon cancer cells, resulting in 85.3% cell death at 100 ppm, while another study demonstrated effective growth suppression of human colon cancer cells via biosynthesized iron oxide nanoparticles. Iron oxide nanoparticles stimulate anticancer activity through both direct and indirect pathways via non-toxic wavelength radiation that is readily absorbed by toxic triggers of ROS generation. Furthermore, the nature of the iron oxide particles is thought to enable them to covalently adhere to tumor sites [112–114]. Considering the specific apoptotic response of cancer cells, as demonstrated in this literature-compatible study, both ZnO NPs and FeO NPs appear quite promising as novel anticancer agents. However, the precise difference that causes cancer-specific toxicity remains largely unclear.

Acridine orange/ethidium bromide (AO/EB) staining
Acridine Orange/Ethidium Bromide (AO/EB) staining is a qualitative test that uses fluorescence microscopy to detect cell viability and signs of apoptosis. Cells with intact cell membranes only take up AO, which makes them appear green and yield green nuclei. AO stains the nuclei of live cells green, while EB stains cells with disrupted cell membranes (apoptotic or necrotic) red/black. The nuclei of late apoptotic and necrotic cells appear orange-red because EB fluorescence is more prevalent. Apoptotic cells also have condensed and fragmented nuclei. Early apoptotic cells exhibit chromatin condensation, while late apoptotic cells exhibit nuclear fragmentation [115–118]. In our study, increased orange/red signals were observed in HCT-116 cells after MrZnO and MrFeO application, indicating the induction of apoptosis. This suggests that the nanoparticles trigger cell death by disrupting the cell membrane or generating ROS. This finding highlights the selective toxicity of the nanoparticles by targeting cancer cells (Fig. 8).

Apoptotic cell rates in flow cytometry
Cell death can occur as programmed cell death (apoptosis), which is an active and controlled process, or as necrosis, which is a passive and uncontrolled process. Caspases, which are a type of cysteine protease, are what cause apoptosis. It is characterized by changes in the shape of cells, such as shrinking, condensing chromatin, breaking up the nucleus, and blebbing the membrane. At the end of apoptosis, the dead cells break up into apoptotic bodies, which are then taken away by phagocytic cells from the surrounding area or the immune system without causing inflammation. Necrosis, on the other hand, doesn’t depend on caspase activation. Instead, it is characterized by cellular swelling, organelle dysfunction, mitochondrial impairment, and rupture of the plasma membrane. Necrotic cells release their contents, such as proteins and nucleic acids, which makes inflammation worse than apoptosis [119–123]. Annexin-V/Propidium Iodide (PI) staining quantitatively separates cell populations by flow cytometry. Annexin-V (FITC-labeled) binds phosphatidylserine in the outer membrane of apoptotic cells, while PI stains the DNA of necrotic cells. In HTC116 cells without MrZnO treatment, the total apoptotic cell rate (Early + Late) was 0.3%, while in MrFeO treatment, it was 4.6% (Early + Late). While in HTC116 cells treated with MrZnO, the necrotic cell rate was 37.5%, while in MrFeO treatment, it was 2.4%. In HCT-116 cells treated with MrZnO and MrFeO nanoparticles, necrotic cells were 37.5% and 3.5% (black), respectively. In these groups, the total apoptotic cell rate (Early + Late) was 4.6% in MrZnO treatment, and 27.6% (Early + Late). The number of necrotic cells was more limited in MrFeO, and death was more prevalent in the apoptotic cell population, which was 27.6% higher (Early + Late) (Fig. 9. MrZnO demonstrates that necroptosis was effective in HCT-116 colon cancer cells, while MrFeO nanoparticles effectively induced apoptosis in HCT-116 colon cancer cells. ZnO nanoparticles have been shown to enter cells and induce oxidative stress, leading to the downregulation of anti-apoptotic proteins and disrupting plasma membrane structure, thereby triggering apoptosis in cancer cells [124]. It has been reported that in cancer cells treated with ZnO nanoparticles, the expression of both mRNA and protein levels of the tumor suppressor gene p53 and proapoptotic genes is increased, while the expression of the antiapoptotic gene Bcl-2 is decreased [125]. ZnO nanoparticles exhibit increased cytotoxicity through ROS formation, which, when the cell’s antioxidant capacity is exceeded, leads to oxidative stress and ultimately cell death. This has also been demonstrated in in vitro studies [126, 127]. Morphological evidence was provided with AO/EB, and quantitative confirmation with Annexin-V/PI. (Fig. 10). This suggests that both Zinc and FeO nanoparticles cause cell death through cellular damage, oxidative stress, DNA damage, or mitochondrial pathways.

Antioxidant activity test results
When examining the CUPRAC and FRAP tests, antioxidant activity is measured at the concentration (C0.5A) at which the absorbance is 0.5. A lower C0.5A value indicates higher antioxidant activity, as it demonstrates that the same effect is achieved at a lower concentration. In the DPPH test, the IC50 value indicates the concentration that neutralizes 50% of radicals. A lower IC50 value indicates higher antioxidant capacity. BHA (14.94 ± 0.12 µg/mL) showed the highest antioxidant activity with the lowest C0.5A value, while MrFeO (18.33 ± 0.10 µg/mL) ranked second after BHA (14.94 ± 0.12) and outperformed BHT (32.65 ± 0.08). MrZnO (37.86 ± 0.08 µg/mL) had an antioxidant activity that was better than Trolox (62.38 ± 0.05 µg/mL) but lower than MrFeO. Trolox showed the lowest activity in the CUPRAC assay (Fig. 11A and Table 4). MrFeO performed better than the standard antioxidants BHT and Trolox in the CUPRAC assay, but lower than BHA. MrZnO, It was less effective than MrFeO but better than Trolox. In the FRAP test, MrFeO (8.85 ± 0.20 µg/mL) exhibited the highest antioxidant activity with the lowest C0.5A value, while BHA (9.87 ± 0.40 µg/mL) was in second place with a performance close to MrFeO. MrZnO (12.02 ± 0.50 µg/mL) had better activity than Trolox (13.60 ± 0.25 µg/mL) but lower than MrFeO and BHA. BHT (22.80 ± 0.20 µg/mL) showed the lowest performance in the FRAP test (Fig. 11B and Table 4). MrFeO had a better antioxidant capacity than all standards (BHA, BHT, Trolox) in the FRAP test. MrZnO, It was more effective than Trolox, but lagged behind MrFeO and BHA. MrFeO (11.60 ± 0.20 µg/mL) had the highest radical neutralization capacity in the DPPH test with the lowest IC50 value. MrZnO (12.70 ± 0.13 µg/mL) performed close to MrFeO and was better than Trolox, BHA, and BHT. MrFeO and MrZnO had higher radical neutralization capacity than standard antioxidants (BHT, BHA, Trolox) in the DPPH test. MrFeO performed slightly better than MrZnO (Tables 4, 5). MrFeO showed higher antioxidant activity compared to MrZnO in all tests (CUPRAC, FRAP, DPPH). Iron oxide nanoparticles generally exhibit superior antioxidant activity compared to zinc oxide nanoparticles due to their ability to participate in electron transfer reactions that effectively scavenge ROS and their redox-active iron centers. This is further enhanced by the Fe2+/Fe3+ redox pair present on the surface of iron oxide nanoparticles, facilitating radical quenching through reversible electron donation and acceptance, thereby increasing their capacity to neutralize free radicals and inhibit oxidative chain reactions. Furthermore, the presence of surface hydroxyl groups and oxygen vacancies on iron oxide nanoparticles promotes ROS adsorption and stabilization, contributing to their strong antioxidant performance [128–130]. In contrast, zinc oxide nanoparticles stand out for their superior antimicrobial properties, despite exhibiting considerable antioxidant activity, which has been reconfirmed in our study [32, 131].

Conclusion
This study reveals the diverse potentials of these novel nanoparticles, providing a promising direction for sustainable nanotechnology. The synthesis process produced nanoparticles exhibiting various crystalline structures, morphologies, and functional groups, as demonstrated by comprehensive characterization techniques such as SEM–EDX, FTIR, and UV–Vis spectroscopy. Due to their strong scavenging abilities, the synthesized ZnO and FeO nanoparticles significantly reduced cell viability in cancer cells through various cellular death mechanisms such as ROS production, apoptosis, or necrosis. While MrZnO exhibited a broad spectrum of antimicrobial activity, MrFeO nanoparticles exhibited a narrower spectrum of activity and a more limited overall antimicrobial potential. MrFeO showed a higher cytotoxic effect and a higher antioxidant effect (CUPRAC, FRAP, DPPH) compared to MrZnO in HCT116 colon cancer cells. In conclusion, MrZnO nanoparticles exhibited higher antimicrobial activity than MrFeO, while MrFeO exhibited superior antioxidant properties and greater cytotoxic and apoptotic effects on cancer cells than MrZnO. These properties make MrFeO a potential candidate for biosafe and cost-effective pharmaceuticals. These findings suggest the sustainability of the synthesized nanoparticles, which hold promise in various fields, including biomedicine, catalysis, sensing, and environmental remediation. Furthermore, future research should be supported by in vivo and in vitro studies to optimize the properties of the synthesized nanoparticles and ensure their safer use.