Broccoli triggers the hemocompatibility of green nanoparticles

Abstract

Green synthesis provides an eco-friendly strategy for producing metal oxide nanoparticles (MONPs) with improved biological performance. Among their biomedical applications, hemocompatibility is particularly critical for drug delivery, anticoagulant therapies, and blood-contacting medical devices. In this work, the hemocompatibility of four types of metal oxides; Calcium oxide (CaO), magnesium oxide (MgO), zinc oxide (ZnO), and titanium dioxide (TiO₂) nanoparticles were evaluated. The selected nanoparticles were greenly synthesized using Brassica oleracea (broccoli) extract and compared with chemically precipitated counterparts. XRD confirmed phase-pure structures (hexagonal ZnO, anatase TiO₂, and cubic MgO and CaO) with lattice modifications induced by biomolecules, including expansion in GZnO (unit cell volume 48.239 ų vs. 47.532 ų for ZnO) and compressive strain in GTiO₂ (135.214 ų vs. 135.989 ų for TiO₂). TEM revealed particle size reduction in GZnO (18.42 ± 3.46 nm vs. 21.12 ± 4.93 nm) and GTiO (19.14 ± 4.0 nm vs. 19.6 ± 3.62 nm), while GCaO and GMgO exhibited modest increases. Optical studies showed slight red-shifts in absorption peaks with narrowed band gaps for GZnO (2.967 eV vs. 3.042 eV) and GTiO₂ (3.037 eV vs. ₂3.068 eV), whereas GCaO displayed a marginal increase (4.07 eV vs. 4.056 eV). Colloidal stability in PBS was markedly improved, with green MONPs retaining 80–90% absorbance after 720 min compared to 15–65% for chemical MONPs. FTIR confirmed the involvement of phenolics, flavonoids, and proteins in capping and stabilization. Hemocompatibility assays demonstrated minimal hemolysis (< 5%) and preserved red blood cell morphology for green-synthesized MONPs. Our findings suggest that broccoli can be used to synthesize biocompatible metal oxide NPs with heightened hemocompatibility sustainably and efficiently, opening the door for their application in anticoagulant treatments, medication transport, and other biological domains.

Introduction

Metal oxide nanoparticles (MONPs) represent a versatile class of nanoparticles (NPs) with tunable physicochemical properties, exceptional stability, and broad applicability across fields such as catalysis, pollutant degradation, water purification, biosensing, and biomedical applications [1–3]. In recent years, green nanotechnology has emerged as a sustainable approach for NPs synthesis, overcoming limitations of conventional physical and chemical methods [4, 5]. By utilizing biomolecules from microorganisms or plants, green synthesis reduces environmental impact, lowers costs, and provides NPs with enhanced surface functionality [6, 7]. Among biological systems, plant extracts have attracted particular interest because they are abundant in bioactive compounds such as polyphenols, alkaloids, flavonoids, and terpenoids that act as dual-function agents. During synthesis, these molecules reduce metal precursors to NPs form and stabilize them against aggregation, producing biocompatible and environmentally friendly NPs [8, 9].

In biomedical applications involving direct or prolonged contact with blood such as drug delivery, biosensing, imaging, and tissue engineering, hemocompatibility is a critical safety parameter. It is typically evaluated through coagulation assays, hemolysis studies, and platelet aggregation tests. Several reports have shown that green-synthesized MONPs display superior hemocompatibility compared to their chemically synthesized counterparts [10, 11]. Factors influencing NPs hemocompatibility include:

(i) Particle size and surface area: Smaller particles exhibit higher surface reactivity, which can increase interactions with red blood cell (RBC) membranes and promote reactive oxygen species (ROS) generation [12]. (ii) Antioxidant phytochemicals from green synthesis: Polyphenols from plant extracts reduce ROS levels and stabilize nanoparticle surfaces, thereby lowering cytotoxicity and hemolytic potential [13, 14]. (iii) Synthesis method and surface chemistry: Chemically synthesized NPs often lack stabilizing organic molecules, resulting in elevated ROS generation and greater disruption of cell membranes [15, 16]. (iv) NPs concentration: Hemolysis percentage rises with increasing concentration, indicating that higher NPs concentrations amplify ROS effects and cellular interactions with blood components [17–19]. Experimental evidence supports these principles. For example, conventionally synthesized TiO₂ and ZnO NPs induced hemolysis rates of 52.5% and 65.2% at 250 ppm, respectively [20], whereas their green-synthesized counterparts remained below the 5% hemolysis threshold even at higher concentrations [21]. Similarly, TiO₂ NPs significantly accelerated clotting times and triggered platelet aggregation [22], while plant-derived coatings minimized these adverse effects [23]. MgO NPs have shown relatively low effects on platelet aggregation and coagulation [24]; green-synthesized MgO NPs from Mentha arvensis extract demonstrated high safety and hemocompatibility [25]. The incorporation of MgO NPs into a silk-polycaprolactone (PCL) matrix was reported. The MgO-Silk-PCL composite demonstrated good cell viability, adhesion, and hemocompatibility. It showed increased cell proliferation and differentiation, indicating that the presence of MgO NPs positively influenced the biological performance of the composite. In vivo studies revealed moderate inflammation at two weeks post-implantation, which subsided by four weeks, with no toxic effects observed in vital organs or blood parameters [26]. Likewise, CaO NPs synthesized using Citrullus colocynthis fruit extract exhibited good stability and biocompatibility with low hemolytic activity [27].

Despite these promising findings, the hemocompatibility of MONPs remains highly dependent on controlled synthesis and surface functionalization. In this study, we investigate the hemocompatibility of four green-synthesized metal oxides (TiO₂, ZnO, MgO, and CaO) produced using Brassica oleracea L. var. italica (broccoli) extract. Broccoli, rich in polyphenols and other phytochemicals, has been recognized for its antioxidant, antibacterial, and anticancer potential [28, 29]. Its use as a green reducing and stabilizing agent provides an opportunity to enhance the hemocompatibility of MONPs for safe biomedical applications.

Materials and methods

Chemicals and reagents

The MONPs were synthesized and modified using the following materials. These materials were all used without further purification. Titanium tetraisopropoxide (TTIP), Zinc nitrate hexahydrate [Zn (NO₃)₂·6H₂O], Magnesium nitrate hexahydrate [Mg (NO₃)₂·6H₂O] and Calcium nitrate tetrahydrate [Ca (NO₃)₂·4H₂O]) were purchased from Merck KGaA, Darmstadt, Germany. 0.05% Trypsin-EDTA, Dulbecco’s modified Eagle’s medium (DMEM), Phosphate buffered saline (PBS), Dimethyl Sulfoxide (DMSO) and Fetal bovine serum (FBS) were purchased from Himedia-India. HCl Hydrochloric acid (2 N), Sodium hydroxide and ethanol were purchased from Samchun chemicals. Broccoli was purchased Local market. 96-well transparent bottom, Eppendorf (2 mL), Falcon tubes (15 mL) and cell culture Flasks were purchased from Sarstedt.

Synthesis of MONPs

Aqueous extract of broccoli

Fresh and healthy broccoli flowers were collected from local market and washed using distilled water to remove any soil, dust, or residues of pesticides [30]. Following, broccoli flowers were cut into small pieces and dried for 24 h. 100 g of the dried broccoli were dissolved in 200 mL of distilled water under stirring at 60–70 °C in for 30 min. The final extract was then filtered three times with Whatman paper number #1and stored at 4 °C for future use [31].

Synthesis of MONPs

A simple chemical precipitation method was employed to synthesize eight samples: four using sodium hydroxide as the precipitating agent (hereafter denoted as MONPs) and their green counterparts prepared using broccoli extract as the reducing and stabilizing agent (denoted as GMONPs). For each synthesis, two solutions were prepared: a precursor solution and a reducing agent solution, as summarized in Table 1. The reducing agent solution was added dropwise to the precursor solution under continuous stirring at 1100 rpm for 1 h. The reaction pH was adjusted to the desired value (Table 1) using 2 M sodium hydroxide (NaOH) to promote nanoparticle formation. The resulting precipitates were allowed to settle for 24 h, followed by sequential washing three times with distilled water and once with ethanol. The washed precipitates were then dried at 100 °C for 8 h. The dried powders were ground into fine particles using an agate mortar and pestle and subsequently calcined under optimized conditions: 450 °C for 2 h for TiO₂, ZnO, and MgO NPs, and 650 °C for 3 h for CaO NPs (Fig. 1).

Table 1.

Synthesis conditions used in the study including sample code, precursor solution, reducing agent solution and rection pH

Sample	Precursor solution	Reducing agent solution	Rection pH	Refrences
TiO2 NPs	20 mL of Ti [OCH(CH3)2]4 (TTIP) to 100 mL of distilled water	50 mL of NaOH	10	[32]
GTiO2 NPs		50 mL of broccoli extract
ZnO NPs	5 g of (Zn (NO3)2·6H2O) to 100 mL of distilled water	50 mL of NaOH	10	[33]
GZnO NPs		50 mL of broccoli extract
MgO NPs	5 g of (Mg (NO3)2·6H2O) to 100 mL of distilled water	50 mL of NaOH	12	[34]
GMgO NPs		50 mL of broccoli extract
CaO NPs	5 g of (Ca (NO3)2·4H2O) to 100 mL of distilled water	50 mL of NaOH	12	[35]
GCaO NPs		50 mL of broccoli extract

Fig. 1.

Schematic illustration of synthesis of MONPs

Characterization of MONPs

Structural analysis was studied using X-ray diffraction (XRD, X’Pert PRO-PAN analytical diffractometer with Cu-Kα radiation (λ = 1.54056 A˚) at 40 kV and 30 mA) and High-Resolution Transmission Electron Microscope (JEOL, model JEM 2100, Japan). The particle size distributions were measured from the TEM images using ImageJ software (version 1.53t) with a minimum of 200 particles counted per sample. Optical properties were conducted using infrared spectroscopy (FTIR Model 6100, Jasco, Japan) and UV-visible spectrophotometry (SPECORD 200 PLUS, Analytik Jena, Germany). Thermal stability was assessed using Shimadzu (TGA-50 H) instrument at heating rate 20 °C/min under a constant flowing nitrogen atmosphere. Colloidal stability of MONPs in PBS was evaluated using UV-visible spectroscopy. Nanoparticle suspensions (100 µg/mL) were prepared in PBS (pH 7.4) and monitored at their characteristic absorption wavelengths over 720 min at room temperature. Absorption intensity was recorded at 0, 10, 20, 30, 60, 120, 180, 240, and 720 min.

Quantification of total phenolic compounds

The phenolic content of both broccoli extract and the synthesized NPs was measured employing the Folin-Ciocalteu assay, following established protocols with adjustments [36]. 100 µL of each sample was combined with 500 µL of a diluted Folin-Ciocalteu reagent (1:10 with distilled water). After a 5 min incubation period, 1.5 mL of a 7.5% sodium carbonate solution was introduced to the mixture. The reaction was kept in darkness for 30 min before the absorbance was recorded at 765 nm using a UV-Vis spectrophotometer. A calibration curve was constructed with gallic acid standards (0–500 µg/mL) to quantify the results, which are presented as milligram gallic acid equivalents per gram of sample (mg GAE/g). Each analysis was conducted in three independent replicates to ensure reliability.

Evaluation of free radical scavenging capacity

The antioxidant potential was assessed via the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical inhibition assay, adapted from literature methods [37]. Sample solutions (broccoli extract and NPs suspensions) were prepared across a concentration gradient from 1.95–500 µg/mL. A volume of 1 mL from each dilution was reacted with 2 mL of a 0.1 mM DPPH solution in methanol. The mixtures were shaken and then left to stand in the dark for 30 min. The reduction in the purple color of DPPH was measured spectrophotometrically at 517 nm, with methanol serving as the blank. Ascorbic acid was utilized as a positive control. The radical scavenging activity was computed using the following equation:

Here, Abs_control represents the absorbance of the DPPH solution alone, and Abs_sample is the absorbance with the test sample. The IC₅₀ value was derived from the dose-response curve using regression analysis. Triplicate measurements were performed for all samples to confirm result consistency.

Hemocomptibilty of MONPs

Blood sampling

Procedures for blood collection were approved by the Ethics Committee of the Faculty of Science, South Valley University (Permit Number: 002/12/24). Informed consent was obtained from five healthy donors prior to participation. Venous blood was drawn into EDTA anticoagulated tubes. To minimize variability, strict donor exclusion criteria were applied: individuals were required to be non-smokers and not to have taken any medication during the two weeks prior to blood collection. Blood from each donor was processed independently, and red blood cells or plasma were freshly prepared for each experiment. All assays were performed in triplicate for each donor, and the final results were expressed as mean ± standard deviation across the five donors. T.

Hemolysis assay

Hemolysis percentage was determined spectrophotometrically by measuring hemoglobin released from ruptured red blood cells (RBCs). Fresh human blood was collected in EDTA tubes according to approved institutional ethics and centrifuged at 1500 rpm for 10 min to separate RBCs. The RBCs were washed three times with phosphate-buffered saline (PBS, pH 7.4) and resuspended to obtain a 2% hematocrit suspension. Stock suspensions of MONPs were prepared in PBS at concentrations of 5, 10, 20, 50, 100, 150, and 200 µg/mL. Each nanoparticle suspension (1 mL) was then mixed in a 1:1 ratio with 1 mL of the RBC suspension, resulting in final working NPs concentrations of 2.5, 5, 10, 25, 50, 75, and 100 µg/mL during the assay. PBS served as the negative control, while 1% Triton X-100 was used as the positive control. Samples were incubated at 37 °C for 2 h and subsequently centrifuged at 1500 rpm for 10 min. The supernatants were collected, and hemoglobin release was quantified by measuring absorbance at 540 nm. The percentage of hemolysis was calculated using the following formula [38]:

1

where A_s is the absorption of test sample, A_NC is the absorption of negative control and A_PC density is the absorption of positive control of control.

Erythrocyte sedimentation rate (ESR)

The sedimentation rate of RBCs is considered a valuable parameter in diagnosis of blood diseases. It can be an indicator for inflammation. When inflammation is present in the body, certain proteins (acute-phase reactants) cause RBCs to climb together and fall more quickly than normal. The ESR was measured using Westergren method [39]. In this method 2 mL of anti-coagulated blood is incubated with MONPs at final concentrations ranging from 2.5 to 100 µg/mL under constant shaking for 3 h at room temperature. Following incubation, the treated blood was transferred into vertical, graduated Westergren tubes (200 mm in length, 2.5 mm in internal diameter). The tubes were left undisturbed in an upright position for 1 h at room temperature. The ESR value was recorded as the distance (in mm) from the upper level of the plasma column to the top of the sedimented RBCs.

Coagulation time

Coagulation time was evaluated to assess the influence of MONPs on blood clotting pathways. Two parameters were measured: Prothrombin Time (PT), which monitors the extrinsic pathway, and Activated Partial Thromboplastin Time (APTT), which monitors the intrinsic pathway. Platelet-poor plasma was obtained by centrifuging citrated whole blood at 3000 rpm for 15 min. Plasma samples were then incubated with MONPs at 100 µg/mL for 30 min at 37 °C. Untreated plasma (without NPs) served as the control. For PT, 100 µl of plasma was mixed with 100 µl of thromboplastin reagent, and clotting time was recorded. For APTT, 100 µl of plasma was mixed with 100 µl of APTT reagent and 100 µl of CaCl₂ (0.025 M), and clotting time was recorded.

Cytotoxicity of MONPs

Cell viability was assessed using the Methyl Thiazolyl Tetrazolium (MTT) assay. Human RBCs were cultured in complete DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were seeded in 96-well plates at a density of 1 × 10⁵ cells/well and allowed to adhere for 24 h at 37 °C in a 5% CO₂ incubator. MONPs were tested at concentrations of 500, 250, 125, 62.5, 31.25, 15.62, 7.81, 3.9, and 1.95 µg/mL. Control wells included (i) untreated cells cultured in complete medium (negative control) and (ii) cells treated with 10% DMSO (positive control for cytotoxicity). After 24 h incubation, each well received MTT solution (0.5 mg/mL) and plates were incubated for 4 h to allow viable cells to reduce MTT into formazan crystals. The crystals were dissolved in DMSO, and absorbance was measured at 570 nm using a microplate reader. Each concentration was tested in triplicate. Cell viability (%) was calculated using the equation [40]:

2

where (A570)_sample is the absorption of the test sample at 570 nm and (A570)_control is the absorption of the control at 570 nm.

Hematological parameters

Blood samples were collected from healthy donors and drawn directly into an EDTA anticoagulated tube. 2 mL of anticoagulated blood incubated with NPs with a concentration of 2 mg/mL under shaking for 3 h. Afterward the incubation time the total number of red and white blood cells was manually counted using a hemocytometer. The blood’s hemoglobin (Hb) level was measured spectrophotometrically at a wavelength of 540 nm. The microcentrifuge method was used to measure the hematocrit (Hct) using heparinized micro hematocrit capillary tubes (75 mm at 7000 g for 10 min) as standards. A minimum of 100 leucocytes were counted under a light microscope to determine the percentages of leukocyte types. Using the total RBC count, Hb concentration, and Hct, the hematological indices of mean cell hemoglobin concentration (MCHC), mean cell hemoglobin (MCH), and mean cell volume (MCV) were calculated.

Statistical analysis

Data were expressed as mean ± standard deviation (SD), and comparisons were made using one-way ANOVA test with Tukey’s post-hoc test.

Results and discussion

Characterization of MONPs

X-ray diffraction (XRD)

Phase identification and confirmation of the successful reduction of MONPs was achieved by comparing the XRD diffraction patterns of the prepared MONPs samples with the standard. Mathematical formulations were used calculation of lattice constants and unit cell volume of the investigated samples. For hexagonal systems a = b ≠ c (ZnO and TiO₂ NPs) lattice constants were calculated using the equations [41]:

3

The unit cell volume was calculated using [42]:

4

For cubic FCC a = b = c (CaO and MgO NPs) lattice constants were calculated using the equations [43]:

5

The unit cell volume was calculated using [43]:

6

where d_hkl is the interplanar space for the hkl plane and a, b and c are the lattice constants.

The average crystal size of the prepared samples was calculated using Scherer’s formula [44]:

7

where D is the average mean diameter of NPs, λ is the wavelength of X-ray radiation source, K is a constant crystalline shape factor equal 0.90, θ is the Bragg’s diffraction angle, and FWHM is the angular full width at half maximum of XRD peaks recorded at diffraction angle 2θ.

Regarding ZnO NPs, the hexagonal wurtzite structure is present in both ZnO NPs and GZnO NPs, and their diffraction peaks closely resemble standard data (JCPDS No. 36-1451) [45]. In contrast to ZnO NPs, GZnO NPs exhibit minor shifts to lower 2θ values, which suggests lattice expansion, Fig. 2a. The calculated lattice parameters were a = b = 3.247 Å, c = 5.203 Å and unit cell volume of 47.532 ų for ZnO NPs, whereas for GZnO NPs it expanded to a = b = 3.262 Å, c = 5.2319 Å and 48.239 ų, respectively. The observed phenomenon can be explained by the interaction of biomolecules, particularly flavonoids and other polyphenols, with the ZnO crystal lattice during nanoparticle synthesis. In previous studies on green synthesized ZnO NPs, similar shifts in XRD peaks were reported, with plant extracts acting as both reducing and stabilizing agents [46, 47]. Furthermore, ZnO NPs had an estimated particle size of 21.474 ± 3.72 nm, whereas GZnO NPs were 11.55 ± 0.83 nm. This suggests that the plant extract is essential in reducing particle size by preventing agglomeration. This structural change, along with the decreased crystallite size, improves GZnO’ s surface characteristics and reactivity, making it appropriate for uses like catalysis and biomedicine that call for high surface-to-volume ratios [48].

Fig. 2.

XRD patterns of MONPs and their green counterparts GMONPs: ZnO NPs (a), TiO₂ NPs (b), CaO NPs (c) and MgO NPs (d)

The distinctive anatase phase of TiO₂ NPs is demonstrated by peaks that match the JCPDS 21-1272 standard in both TiO₂ and GTiO₂ samples [49]. Nonetheless, minor changes in the 2θ values for GTiO₂ imply that the interaction of the biomolecules in the broccoli extract has altered the lattice structure, Fig. 2b. Additional information on the structural variations between the samples can be found in the unit cell volume and lattice constants. The lattice parameters for TiO₂ NPs are a = b = 3.7899 Å and c = 9.4675 Å, and their unit cell volume is 135.989 ų. By contrast, GTiO₂ has a unit cell volume of 135.214 ų with lattice parameters of a = b = 3.781 Å and c = 9.4579 Å. Compressive strain within the crystal structure is suggested by GTiO₂ small decrease in unit cell volume and lattice properties. Due to interactions between biomolecules and the MONPs framework, which result in lattice modifications, such effects are frequently seen in green synthesis techniques [9]. In addition, the particle size of TiO₂ NPs (20.03 ± 2.53 nm) is substantially larger than that of GTiO₂ (16.43 ± 1.47 nm). Such effects are commonly observed in green synthesis methods, where bio-organic molecules interact with the MONPs framework, causing lattice adjustments [9]. This strain could potentially influence the mechanical and electronic properties of TiO₂, making it suitable for various applications where modified surface and electronic properties are desired [50].

The presence of a face-centered cubic (FCC) structure, which complies with JCPDS No. 77-2376, verified the successful synthesis of CaO NPs, Fig. 2c [51]. Although both CaO and GCaO exhibit strong crystallinity, the small shifts in GCaO NPs diffraction peaks suggest that interactions with the broccoli extract’s biomolecules have caused structural modifications. These shifts suggest a modification of the crystal structure, enhancing the material’s surface properties, similar to findings that were reported for green CaO NPs [52].

The average particle size of CaO NPs is 13.17 ± 2.4 nm, with the GCaO NPs showing slightly larger sizes 15.16 ± 3.2 nm. This suggests that while the broccoli extracts stabilize the NPs, they do not significantly hinder particle growth. The lattice parameters of GCaO NPs (a = 4.252 Å) are slightly larger than that of CaO NPs (a = 4.204 Å), which may be due to the incorporation of biomolecules into the CaO NPs lattice, leading to lattice expansion [35].

Similarly, XRD patterns for MgO NPs verify the face-centered cubic (FCC) structure, which is in line with the rock salt arrangement that has been extensively reported (JCPDS No. 87–0653), Fig. 2d [53]. The extremely crystalline peaks with a strong (200) plane are present in MgO NPs, demonstrating exceptional crystallinity [54]. Modest changes in the 2θ values in the GMgO NPs sample indicate that the broccoli extract’s biomolecule interactions have changed the lattice structure. The lattice constant a = 4.204 ± 0.0041 Å with unit cell volume 74.313 ± 0.21 ų for MgO NPs increased to 4.224 ± 0.006 Å with unit cell volume 75.414 ± 0.332 ų for GMgO NPs. This suggests that the biomolecules from the broccoli extract might slightly expand the lattice, possibly due to the interaction of biomolecules with the surface of the NPs [55]. It is noteworthy that the particle size of GMgO (11.3 ± 0.778 nm) is slightly bigger than that of MgO NPs (10.63 ± 0.75 nm). This is because plant extracts have the ability to promote nucleation. These findings align with previous research on green synthesis, which has shown that biomolecules can occasionally serve as stabilizing agents while simultaneously encouraging a small amount of aggregation [56, 57].

It is clear from comparing the structural and morphological alterations across the many MONPs types that the green synthesis approach continuously affects the unit cell volume, lattice properties, and crystallite size. Because the plant-derived biomolecules have strong stabilizing and capping properties, the green approach significantly reduces the particle size of ZnO NPs and TiO₂. Lattice strain or expansion occurs in tandem with this reduction, enhancing the NPs surface characteristics and reactivity. On the other hand, the green synthesis approach causes a minor increase in particle size for MgO and CaO, presumably because of the biomolecules ability to promote aggregation. Nonetheless, structural alterations such lattice expansion remain noticeable, indicating robust interactions between the broccoli extract and MONPs surface [13, 58–60].

Transmission electron microscopy (TEM)

The synthesis and characterization of metal oxide nanoparticles through both conventional chemical methods and a green-mediated approach using broccoli extract were successfully accomplished, as confirmed by detailed TEM analysis, Fig. 3. The morphological examination revealed significant differences between the two synthesis routes across all four MONPs systems studied—ZnO, TiO₂, CaO, and MgO. While all NPs exhibited predominantly quasi-spherical to polyhedral structures, their dispersion states and size uniformity showed substantial variation dependent on the synthesis methodology [61]. The chemically synthesized NPs demonstrated pronounced agglomeration with broad size heterogeneity, suggesting uncontrolled nucleation and growth processes without adequate surface passivation. In contrast, the green-synthesized counterparts displayed markedly improved dispersion with reduced particle clustering, indicating that the phytochemical constituents in the broccoli extract effectively modulated the nucleation-growth equilibrium by functioning as both reducing and steric stabilization agents [62].

Fig. 3.

HRTEM imges of MONPs and their Broccoli mediated green counterparts GMONPs. a ZnO NPs, b GZnO NPs, c TiO₂ NPs, d GTiO₂ NPs, e CaO NPs, f GCaO NPs, g MgO NPs and h GMgO NPs

Particle size distributions were quantitatively analyzed using ImageJ software (version 1.53t) with measurements obtained from multiple TEM images containing at least 200 particles per sample. The size distribution histograms (Fig. 4) confirmed the visual observations, revealing that green-synthesized NPs generally exhibited narrower size distributions despite variations in mean particle diameters. The measured sizes showed that green-synthesized ZnO nanoparticles averaged 23.0 ± 8.64 nm compared to 24.39 ± 8.37 nm for their conventional counterparts, while green-synthesized TiO₂ NPs measured 21.42 ± 7.77 nm versus 23.46 ± 7.85 nm for chemically synthesized TiO₂ NPs. Interestingly, the alkaline earth metal oxides exhibited a different trend, with green-synthesized CaO NPs measuring 23.7 ± 7.8 nm compared to 19.5 ± 5.7 nm for conventional CaO, and green-synthesized MgO at 23.96 ± 7.56 nm versus 22.07 ± 6.23 nm for conventional MgO. Despite these variations in mean particle sizes, the green-synthesized NPs consistently exhibited narrower size distributions across all systems, reflecting improved control over nucleation kinetics and growth processes.

Fig. 4.

Particle size distribution histograms of chemically synthesized and broccoli-mediated MONPs obtained by measuring over 200 particles using ImageJ software. Scale bars: 100 nm. a ZnO NPs, b GZnO NPs, c TiO₂ NPs, d GTiO₂ NPs, e CaO NPs, f GCaO NPs, g MgO NPs and h GMgO NPs

The observed morphological and size distribution patterns can be attributed to the complex biochemical composition of the broccoli extract, which contains various phytochemicals including flavonoids, glucosinolates, phenolics, and reducing sugars. These biomolecules function synergistically in multiple roles: as reducing agents by facilitating controlled formation of metal hydroxide nuclei, as stabilizers and capping agents preventing particle agglomeration through steric and electrostatic hindrance, and as growth modulators that selectively bind to specific crystal facets thereby altering surface energies and growth dynamics [63]. This multifaceted interaction creates a regulated microenvironment for NPs formation that stands in sharp contrast to conventional chemical precipitation methods, which typically lack temporal and spatial control over particle nucleation. The metal-specific effects observed, where zinc and titanium oxides showed size reduction while calcium and magnesium oxides exhibited moderated growth with improved dispersion, highlight the tailored interactions between different metal ions and the complex phytochemical profile of the broccoli extract [64, 65].

The enhanced morphological characteristics achieved through green synthesis have significant implications for the functional performance and potential applications of these NPs. The improved monodispersity and reduced aggregation directly enhance colloidal stability, which is crucial for applications requiring stable suspensions and predictable behavior in biological or environmental systems. The presence of phytochemical residues on NPs surfaces may further contribute to enhanced biocompatibility and reduced oxidative stress, while potentially enabling synergistic therapeutic effects [66, 67].

UV-visible spectroscopy (UV-vis)

Significant variations between chemically and green-synthesized MONPs were found in their UV-visible absorption spectra, which were impacted using plant extracts in the green synthesis process. A slightly red-shifted peak at 345 nm for GZnO was observed compared with the absorption peak at 341 nm for ZnO, Fig. 5a. Likewise, GTiO₂ displayed a red-shifted peak at 305 nm, whereas TiO₂ displayed an absorption peak at 301 nm, Fig. 5b. For CaO NPs, the absorption peak was found at 240 nm, while GCaO exhibited a slightly red-shifted peak at 242 nm, Fig. 5c. GMgO NPs displayed a peak at 280 nm, whereas MgO NPs displayed a blue -shifted peak at 277 nm, Fig. 5d.

Fig. 5.

UV-vis spectra of broccoli extract, MONPs and their green counterparts GMONPs: ZnO NPs (a), TiO₂ NPs (b), CaO NPs (c) and MgO NPs (d)

As can be seen from the shifts in the absorption peaks, green synthesis induces slight but consistent variations in the optical characteristics of the various MONPs. These changes are ascribed to interactions between the bioactive compounds in the broccoli extract and the MONPs’ surface, which improve stability and modify their electronic surroundings [60, 68]. The absorption peak of the broccoli extract at 253 nm further supports the role of plant compounds in stabilizing the MONPs and modifying their optical properties [28]. The distinctive interactions between the MONPs and the broccoli biomolecules are reflected in the modest variations in the magnitude of these shifts and their consequences amongst the MONPs types [8, 9, 69–72]. Overall, these findings support the viability of broccoli extract-based green synthesis as a technique for synthesis stable MONPs with altered and improved optical characteristics.

A key factor in defining the electrical, optical, and photocatalytic characteristics of MONPs is their optical band gap energy (Eg). Significant variations between the chemical and broccoli extract-synthesized MONPs were shown by the Tauc plots, Fig. 6(a–d).

Fig. 6.

Optical bandgap as calculated from Tauc’s equation for MONPs and their green counterparts GMONPs: ZnO NPs (a), TiO₂ NPs (b), CaO NPs (c) and MgO NPs (d)

GZnO showed a slightly smaller optical band gap of 2.967 eV than ZnO NPs, which had an optical band gap of 3.042 eV, Fig. 6a. Similar TiO₂ NPs were found to have an optical band gap of 3.068 eV, which is somewhat greater than the 3.037 eV that calculated for GTiO₂ NPs, Fig. 6b. Additionally, the same behavior was observed for MgO NPs, which displayed the highest band gap energies among the four types of MONPs. The observed band gap MgO NPs was 4.703 eV, while the GMgO NPs’ band gap decreased to 4.671 eV, Fig. 6d [73, 74]. These slight drops in Eg suggest that broccoli biomolecules have a comparable impact on the electronic structure of MONPs. One possible explanation for the uneven morphologies seen in TEM for the green MONPs is that broccoli extract may modulate the nucleation process, resulting in subtle differences in defect density and electronic band structure [3, 4].

Conversely, the optical band gap of GCaO (4.07 eV) was marginally higher than that of chemically synthesized CaO NPs, which showed an optical band gap of 4.056 eV, Fig. 6c. The upward trend in Eg for GCaO NPs indicates that the capping effect of the biomolecules generated from broccoli has improved surface stability and decreased defect states [35, 75].

In comparison to their chemically synthesized counterparts MONPs, green synthesis using broccoli extract consistently demonstrated a tendency to either preserve or slightly reduce the optical band gap energies of all the MONPs under study. This suggests that subtle changes in electrical characteristics may result from the surface passivation and defect stabilization that the plant extracts probably introduce [71, 76]. Together with the noted morphological and size variations, these band gap energy variations are significant because they demonstrate how green synthesis can be used to customize the optical characteristics of MONPs for particular uses.

Fourier transformation spectroscopy (FTIR)

Several ways can be used to explain how biomolecules from broccoli extract interact with MONPs. The reduction of metal ions (Ti⁴⁺, Zn²⁺, Mg²⁺, and Ca²⁺) to their NPs forms is mostly dependent on the hydroxyl groups from phenolic compounds and terpenoids in the plant extracts [45, 46]. The hydroxyl and carbonyl groups found in plant-based compounds stabilize NPs electrostatically by forming hydrogen bonds and interacting with other dipoles. The phenolic compounds can also promote further stabilization through surface passivation, which inhibits aggregation and preserves colloidal stability in solution [1, 56].

We begin by examining the broccoli extract’s FTIR spectra to evaluate its impact on the green synthesis of the investigated MONPs. Several significant peaks are visible in the broccoli extract FTIR analysis, Fig. 7(a–d). Several bioactive chemicals, such as phenolic polyphenols, flavonoids, and terpenoids, are present in broccoli extract and are essential for stabilizing and lowering NPs. Flavonoids and phenolic acids, which are known to be important in the reduction of NPs, are suggested to be present by the peaks at 1589 cm^− 1 (C=C stretch) and 1688.3 cm^− 1 (C=O stretch). These substances have hydroxyl groups that can donate electrons to metal ions, which causes NPs to form [28, 77]. Proteins and amino acids typically exhibit C-H stretch and C-H bending vibrations, which are represented by peaks such as 3037.33 cm^− 1 and 1461.7 cm^− 1, respectively [28, 77]. These bioactive molecules can stabilize NPs by inhibiting their aggregation and improving their colloidal stability. Terpene C-H stretching vibrations, which aid in the stability of the NPs surface and the decrease of metal ions, are consistent with the peak at 2981.4 cm^− 1 [28, 77].

Fig. 7.

FTIR spectra of broccoli extract, MONPs and their green counterparts GMONPs: ZnO NPs (a), TiO₂ NPs (b), CaO NPs (c) and MgO NPs (d)

All MONPs have a distinctive fingerprint region in their FTIR spectra. ZnO NPs exhibit distinctive Zn-O stretching vibrations at 791.6 cm^− 1 and hydroxyl groups at 3435.5 cm^− 1. These peaks represent the water or hydroxyl groups on the surface of the NPs and the ZnO lattice, Fig. 7a. Additional peaks are introduced by the green synthesis using broccoli extract [78, 79]. It is suggested that the phenolic compounds from broccoli interact with the ZnO surface to stabilize the NPs [80] by shifting the hydroxyl band (e.g., at 3782.7 cm^− 1) and exhibiting C = O stretching at 1496.5 cm^− 1.

Ti-O vibrations at 850.4 cm^− 1 and 493.6 cm^− 1 are characteristic of TiO₂ NPs tetragonal crystal structure. The surfaces of the GTiO₂ NPs exhibit notable changes and new peaks due to the green synthesis. The peaks at 1374 cm^− 1 and 1641.12 cm^− 1 indicate that the surface of GTiO₂ NPs is contacted by phenolic groups found in broccoli’s flavonoids, Fig. 7b. These interactions provide a bio-functional layer that aids in the NPs stability [81, 82].

Ca-O bond vibrations are detected at 873.6 cm^− 1 and 1194.7 cm^− 1 in the FTIR spectra of CaO NPs, Fig. 7c [83]. Significant changes in the hydroxyl and C = O peak were observed in the FTIR spectrum of GCaO, indicating that the phenolic groups from the broccoli interact with the CaO surface to stabilize the particles [59, 84].

Mg-O stretching and bending vibrations are represented by the distinctive peaks at 389.5 cm^− 1 and 1588 cm^− 1 for chemical MgO NPs, Fig. 7d [53, 85]. GMgO NPs spectra exhibit noticeable alterations. Other peaks, such as 2981.4 cm^− 1, occur, indicating the presence of lipids or hydrocarbons from broccoli extract. Additional evidence that broccoli’s bioactive molecules aid in the stability and surface modification of MgO NPs is provided by the hydroxyl stretching, which is approximately 3428.8 cm^− 1 [59, 84].

Thermogravimetric analysis (TGA)

Thermogravimetric analysis (TGA) was carried out to evaluate the organic content of the synthesized MONPs and to confirm the incorporation of broccoli extract components during the green synthesis process (Fig. 8). The TGA curves of all samples displayed three characteristic weight-loss stages, and the detailed values are summarized in Table 2.

Fig. 8.

Thermogravimetric analysis (TGA) curves of green- and chemically synthesized MONPs: ZnO NPs (a), TiO₂ NPs (b), CaO NPs (c) and MgO NPs (d)

Table 2.

TGA weight loss (%) of green- and chemically synthesized MONPs across three thermal stages

Sample	Stage I (30–200 °C)	Stage II (200–600 °C)	Stage III (600–800 °C)	Total loss (30–800 °C)
GTiO2 NPs	0.5953	4.0394	0.3353	4.9700
TiO2 NPs	0.1984	1.6388	0.1428	1.9800
GZnO NPs	0.4997	3.6200	0.3050	4.4247
ZnO NPs	0.2403	1.4298	0.0993	1.7693
GMgO NPs	0.6081	4.0992	0.9847	5.6920
MgO NPs	0.2952	2.0234	0.5041	2.8227
GCaO NPs	0.9135	5.5843	1.9129	8.4107
CaO NPs	0.5236	3.0672	1.0735	4.6643

The first stage, occurring between 30 and 200 °C is attributed to the decomposition of organic matter, primarily originating from broccoli extract in the green-synthesized samples. All samples exhibited less than 1% weight loss in this region, indicating that the drying procedure effectively removed most of the residual moisture and resulted in relatively dry samples. Pronounced differences were evident between the two synthesis routes. For instance, GTiO₂ showed a 4.04% weight loss compared to 1.64% for TiO₂–NaOH, while GZnO exhibited 3.62% versus 1.43% for ZnO–NaOH. Similarly, GMgO displayed 4.10% compared to 2.02% for MgO–NaOH, and GCaO showed 5.58% versus 3.07% for CaO–NaOH. These increases confirm the incorporation of broccoli extract phytochemicals within the green-synthesized NPs.

The second stage, observed between 200 and 600 °C, is primarily associated with the decomposition of organic matter originating from the broccoli extract in the green-synthesized NPs. This stage displayed the most pronounced differences between green- and chemically synthesized MONPs. For instance, GTiO₂ NPs exhibited a weight loss of 4.63% compared to only 0.14% for TiO₂–NaOH, representing an approximately 33-fold increase. Similarly, GZnO showed a weight loss of 3.62% versus 0.09% for ZnO–NaOH (40-fold increase). GCaO experienced a 1.91% weight loss compared to 1.07% for its chemically synthesized counterpart (78% increase), while GMgO demonstrated 0.98% compared to 0.50% for MgO–NaOH (96% increase). These substantial increases confirm the successful incorporation of broccoli extract phytoconstituents in the green-synthesized NPs.

The final stage, occurring between 600 and 800 °C, corresponds to structural changes, including the complete decomposition of any residual organic matter and possible phase transitions within the metal oxide lattice. The weight loss in this region was minimal (< 0.5%) for all samples.

The TGA results provide definitive quantitative proof that broccoli extract components are successfully incorporated onto the NPs surfaces during green synthesis. The dramatically higher weight loss in Stage II for all green-synthesized MONPs confirms that the synthesis method effectively deposits organic matter rather than simply providing a reducing environment.

The organic content values (1–5 wt%) are consistent with previous reports of plant extract-mediated nanoparticle synthesis. Hameed et al. reported similar organic loading (2–4 wt%) for green-synthesized ZnO NPs [86], while Buazar et al. found 1–3 wt% organic content in potato extract-mediated ZnO synthesis [87]. The variation in organic content among different metal oxide types reflects the different surface chemistries and binding affinities: GTiO₂ (4.63% organic content has multiple coordination sites and can exist in various oxidation states, providing numerous anchoring points for organic molecules. The high surface reactivity of TiO₂ enables strong coordination bonds with polyphenolic compounds through Ti-O-C linkages [88, 89]. While GZnO NPs (3.62% organic content) surfaces readily interact with organic molecules through coordination bonds between zinc ions and electron-rich functional groups (hydroxyl, carbonyl) present in broccoli extract [90]. The moderate organic loading provides optimal surface coverage without excessive multilayer formation. Moreover,

GCaO (1.91% organic content) shows moderate organic binding, likely through electrostatic interactions and weak coordination bonds. The alkaline earth metal shows lower affinity for organic ligands compared to transition metals, explaining the intermediate organic content [91]. Further, MgO (0.98% organic content) exhibits the lowest organic loading among the studied materials. The modest organic content suggests that even small amounts of surface functionalization can provide significant benefits for this highly reactive material [92].

Colloidal stability assessment

The colloidal stability of all synthesized MONPs was evaluated in PBS (pH 7.4) using UV–visible spectroscopy over 720 min. Stability was determined by monitoring absorption intensity at the characteristic wavelength of each NPs type over time, with a decrease in absorption indicating aggregation and sedimentation.

As shown in Fig. 9, green-synthesized MONPs consistently demonstrated superior colloidal stability compared to their chemically synthesized counterparts. GTiO₂ NPs retained ~ 85% of their initial absorption intensity after 720 min, while TiO₂ NPs declined to ~ 55%. Similarly, GZnO NPs preserved ~ 90% stability compared to ~ 80% for ZnO NPs. The largest stability difference was observed for MgO: GMgO maintained ~ 80% stability, whereas MgO-NaOH dropped drastically to ~ 15%, indicating severe aggregation. CaO and GCaO displayed intermediate stability, with GCaO retaining ~ 85% compared to ~ 65% for CaO.

Fig. 9.

Time-dependent UV–visible absorption spectra and absorption edge plots of chemically synthesized and green-synthesized MONPs in PBS (pH 7.4) over 720 min

Absorption edge analysis confirmed these observations, showing that green-synthesized NPs maintained more consistent optical properties over time, reflecting superior dispersion stability.

The enhanced colloidal stability of green-synthesized MONPs can be attributed to the adsorption of bioactive compounds from broccoli extract onto the NPs surfaces, as confirmed by TGA analysis (Sect. 3.1.7). The organic content ranging from 0.98% to 4.63% contributes to stabilization through a combination of electrostatic and steric effects. Polyphenolic compounds, flavonoids, and glucosinolates, which are abundant in broccoli and contain multiple hydroxyl and carboxyl groups, exhibit strong coordination with metal oxide surfaces [93]. This surface functionalization lowers the surface energy and minimizes direct particle–particle interactions, provides electrostatic stabilization via ionizable functional groups, creates steric barriers that hinder particle aggregation, and scavenges reactive oxygen species (ROS) that would otherwise accelerate aggregation. Together, these mechanisms explain the superior stability observed in green-synthesized MONPs compared to their chemically synthesized counterparts.

Previous studies on plant extract–mediated nanoparticle synthesis have consistently reported stabilization effects like those observed in the present work. Polyphenol-rich plant extracts not only facilitate the reduction of metal ions but also play a crucial role in stabilizing the resulting inorganic NPs . The functional groups most frequently implicated in these processes include ketones, amines, aldehydes, alcohols, and carboxylic acids, with polyphenols recognized as the principal contributors to stabilization through surface coordination and capping [94]. Green synthesis approaches are therefore increasingly regarded as effective strategies for minimizing the adverse environmental and biological impacts associated with conventional chemical methods [95].

Importantly, the present study extends these findings by providing the first systematic comparison of colloidal stability across multiple metal oxide nanoparticle types (TiO₂, ZnO, CaO, MgO) synthesized using the same plant extract. This comparative analysis demonstrates that while the fundamental stabilization mechanism mediated by bioactive compounds is broadly applicable, the degree of stabilization exhibits material-specific variations related to the intrinsic surface chemistry of each metal oxide.

Quantification of total phenolic compounds

The comprehensive phytochemical characterization of broccoli extract and green-synthesized NPs revealed significant findings regarding their total phenolic content (TPC), Table 3. The TPC results demonstrated that broccoli extract possesses substantial phenolic compounds, quantified at 152.34 ± 7.15 mg GAE/g. This high phenolic content established a strong foundation for the extract’s functionality as both a reducing and capping agent in the nanoparticle synthesis process. The successful incorporation of these phytochemicals onto nanoparticle surfaces was confirmed through TPC measurements of the synthesized NPs, which followed the hierarchy: GZnO NPs (42.15 ± 2.05 mg GAE/g) > G-CaO NPs (38.42 ± 1.85 mg GAE/g) > GTiO₂ NPs (35.28 ± 1.65 mg GAE/g) > GMgO NPs (31.56 ± 1.45 mg GAE/g). This metal-dependent variation in phenolic adsorption provides crucial evidence for the differential binding affinities between broccoli phytochemicals and various metal oxide surfaces, consistent with previous reports on plant-mediated NPs synthesis [66, 96].

Table 3.

Total phenolic content (TPC) broccoli extract and green-synthesized NPs

Sample	Total phenolic content (mg GAE/g)
Broccoli extract	152.34 ± 7.15
GZnO NPs	42.15 ± 2.05
GCaO NPs	38.42 ± 1.85
GTiO2 NPs	35.28 ± 1.65
GMgO NPs	31.56 ± 1.45

Evaluation of free radical scavenging capacity

The antioxidant activity assessment through DPPH radical scavenging assay yielded results that strongly correlated with the TPC findings, Fig. 10. Broccoli extract exhibited remarkable antioxidant capacity with an IC₅₀ value of 50.85 ± 3.45 µg/mL, approaching the potency of the reference standard ascorbic acid (IC₅₀ = 45.16 ± 2.3 µg/mL). The green-synthesized nanoparticles maintained significant antioxidant activity in the same hierarchical pattern as their phenolic content: GZnO NPs (IC₅₀ = 112.16 ± 3.76 µg/mL) > GCaO NPs (IC₅₀ = 130.49 ± 5.27 µg/mL) > GTiO₂ NPs (IC₅₀ = 143.13 ± 5.65 µg/mL) > GMgO NPs (IC₅₀ = 155.77 ± 4.22 µg/mL). This consistent pattern across both parameters (R² = 0.96) establishes a direct relationship between the surface-adsorbed phenolic compounds and the observed antioxidant activity, supporting similar findings in previous studies on biogenic NPs [97].

Fig. 10.

Dose-response curves showing the percentage of DPPH scavenging (% SCV) for Ascorbic Acid, Broccoli Extract, and green-synthesized nanoparticles (G-ZnO, G-TiO₂, G-MgO, G-CaO NPs) across a range of concentrations. The corresponding IC₅₀ values are displayed in the legend

The high phenolic content of broccoli extract directly facilitates the reduction of metal ions during nanoparticle formation while simultaneously providing effective surface passivation through the capping action of antioxidant compounds, as documented in green synthesis protocols [67]. The variation in phenolic incorporation among different metal oxides can be attributed to several factors, including the coordination chemistry between phenolic hydroxyl groups and metal cations, surface charge characteristics, and the stability of metal-phenolate complexes [98]. The superior performance of GZnO NPs in both phenolic content and antioxidant activity suggests particularly favorable interactions between zinc ions and the antioxidant phytochemicals in broccoli extract, possibly through the formation of stable chelation complexes, which has been observed in other plant-mediated zinc oxide syntheses [99].

The preserved antioxidant activity in green-synthesized NPs, though reduced compared to the pure extract, demonstrates the successful creation of a bioactive interface that extends beyond mere morphological control. This functionality has profound implications for biomedical applications, where antioxidant nanoparticles can potentially mitigate oxidative stress in biological environments, as demonstrated in recent studies on therapeutic NPs [9]. The differential antioxidant potency among various GMONPs provides a strategic framework for selecting appropriate NPs for specific applications, with GZnO and GCaO NPs being particularly suitable for scenarios requiring enhanced antioxidant protection [100]. Furthermore, the demonstrated relationship between phenolic content and antioxidant activity validates the green synthesis approach as a viable method for producing functionally active NPs with inherent biological properties that surpass those of conventional synthesis methods.

Hemocomptibilty of MONPs

Hemolysis

The interaction of NPs with RBCs can cause membrane rupture, oxidative stress, or other negative consequences that cause hemoglobin to be released, which in turn causes hemolysis. Depending on the degree of hemolysis, this condition not only compromises the integrity of RBCs but also has repercussions for human health, such as anemia, thrombosis, and inflammation [101]. Numerous variables, including the NPs physicochemical characteristics such as their size, shape, surface charge, and surface functionalization affect how much hemolysis they cause. Electrostatic interactions, for example, make positively charged NPs more likely to damage negatively charged RBC membranes. In a similar vein, smaller NPs may be more likely to cause hemolysis due to their higher surface area-to-volume ratios and increased reactivity [102, 103].

The hemolysis percentage of the four MONPs and their green counterparts highlight the comparative cytotoxicity of these MONPs in terms of red blood cell (RBC) damage, Fig. 11(a-d). Among the MONPs, ZnO NPs exhibit the highest hemolytic activity, followed by TiO₂ NPs, CaO NPs, and MgO NPs. In comparison to their chemically synthesized counterparts, green-synthesized MONPs consistently demonstrate decreased hemolytic activity; the most noticeable decreases are shown in GZnO NPs and GMgO NPs. This pattern emphasizes how green synthesis might improve biocompatibility and lessen cytotoxic effects, most likely by adding biomolecules that stabilize and lessen the reactivity of MONPs [8, 9, 69, 104].

Fig. 11.

Hemolysis percentage of MONPs and their green counterparts GMONPs: ZnO NPs (a), TiO₂ NPs (b), CaO NPs (c) and MgO NPs (d)

According to reports, NPs surface modification significantly affects their hemolytic activity. Flavonoids, phenolics, proteins, and other biomolecules found in natural extracts function as capping agents, altering the surface characteristics of NPs. This surface alteration improves their biocompatibility by lowering their reactivity and minimizing their interactions with RBC membranes [105, 106].

Erythrocyte sedimentation rate (ESR)

The erythrocyte sedimentation rate (ESR) is a critical parameter for evaluating the inflammatory response triggered by NPs (NPs). Across all MONPs types, green-synthesized MONPs exhibit significantly lower ESR values, underscoring their enhanced biocompatibility and reduced pro-inflammatory effects.

The erythrocyte sedimentation rate (ESR) is a crucial indicator for evaluating the inflammatory response triggered by NPs. Compared to all other MONPs types, green-synthesized MONPs have been found to have much lower ESR values, indicating their enhanced biocompatibility and reduced pro-inflammatory effects, Fig. 12 (a–d). ESR values of all types of MONPs clearly increase with concentration, reaching a high of 200 µg/mL. This sharp increase suggests that MONPs cause a greater inflammatory response as their concentration increases because of their unchanged surface chemistry, which can activate immune cells and produce reactive oxygen species (ROS), both of which contribute to inflammation [107, 108]. However, green MONPs consistently show lower ESR values, indicating a significant reduction in their ability to cause inflammation. This decrease in inflammation was most likely brought on by the surface modification of NPs during the green synthesis, in which biomolecules from broccoli, like antioxidants and polyphenols, are integrated into the NPs’ structure. By stabilizing the surface of the NPs, these biomolecules reduce reactivity and minimize interactions with inflammatory mediators such plasma proteins and immune cells .

Fig. 12.

ESR (mm/h) of MONPs and their green counterparts GMONPs: ZnO NPs (a), TiO₂ NPs (b), CaO NPs (c) and MgO NPs (d)

In terms of MONPs type, ZnO NPs exhibit the highest propensity for inflammation, followed by TiO₂ NPs, CaO NPs, and MgO NPs. The higher inflammatory response of ZnO NPs is mostly due to their high reactivity and propensity to produce ROS, whereas TiO₂, CaO, and MgO NPs elicit milder reactions, most likely because of their decreased reactivity [109–112].

Coagulation time

Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) were monitored to examine the impact of MONPs and their green counterparts on blood coagulation cascades, Fig. 13. Both kinds of MONPs were found to have coagulation times ranging from 14 to 16 s, indicating a regular interaction with coagulation pathways. The literature indicates that NPs often have no effect on coagulation when their surface properties are carefully controlled to avoid platelet activation or protein denaturation [113, 114]. There are no discernible changes between MONPs (ZnO, TiO₂ CaO, MgO, ) and their green counterparts (GZnO, GTiO₂ GCaO, GMgO, ), suggesting that the green synthesis does not alter the intrinsic ability of MONPs to interact with blood plasma.

Fig. 13.

Coagulation times: prothrombin time (PT) and activated partial thromboplastin time (APTT) of MONPs and their green counterparts GMONPs

In comparison to both MONPs, the broccoli extract shows somewhat longer coagulation periods. This is in line with other research showing that plant-based extracts high in bioactive substances, including glucosinolates or flavonoids, can have modest anticoagulant effects by lowering platelet aggregation or blocking coagulation factors [115, 116]. Nevertheless, this anticoagulant characteristic has little effect on the behavior of the NPs when the broccoli extract is utilized in green synthesis. According to Yang et al. in their work on bioactive NPs coatings, this is probably caused by the encapsulation of active chemicals during production or surface changes that lessen their direct contact with coagulation pathways [117].

Toxicity

The results of cell viability of RBCs after exposure to the four types of MONPs and their green-synthesized counterparts are shown in Fig. 14 (a–d). A concentration dependent decrease in the cell viability was observed for all MONPs, reaching 74.2, 70.9, 73.6 and 74.5% for ZnO, TiO₂, CaO and MgO NPs at concentration 500 µg/mL, respectively. In the contrary, greener MONPs didn’t affect the cell viability significantly. These findings demonstrate that green-synthesized MONPs continuously perform better in terms of biocompatibility than their chemically synthesized counterparts. A substantial body of research supports this conclusion, demonstrating that green-synthesized MONPs can reduce adverse effects by modifying their surface with biomolecules such as proteins, polyphenols, and flavonoids. Nehru et al. showed that green-synthesized ZnO NPs were more compatible with cells and produced fewer ROS than standard ZnO NPs [118]. Likewise, biomolecule-capped MgO NPs exhibited no toxicity to blood cells, according to Velsankar et al. [105].

Fig. 14.

Cell viability of RBCs after treatment with broccoli extract, MONPs and their green counterparts GMONPs for 24 h as evaluated from MTT assay. ZnO NPs (a), TiO₂ NPs (b), CaO NPs (c) and MgO NPs (d)

Furthermore, these findings are consistent with hemolysis and ESR. RBCs are more cytotoxically affected by MONPs, which also demonstrated increased hemolysis percentages and an elevated ESR (a sign of inflammatory reactions). This implies that the main causes of decreased RBC viability are increased oxidative stress and membrane instability brought on by MONPs. On the other hand, the green-synthesized MONPs exhibit better biocompatibility, as seen by increased RBC survivability at all tested concentrations, despite having lower hemolysis percentages and ESR values.

ZnO NPs exhibited the highest hemolytic activity, lowest cell viability, and the most pronounced effects on ESR and coagulation. GZnO NPs mitigated these effects, showcasing the largest improvement in biocompatibility.

TiO₂ and CaO NPs demonstrated moderate hemolysis and cytotoxicity, while their green-synthesized counterparts (GTiO₂ and GCaO) exhibited significant reductions in toxicity. MgO NPs showed the least hemolytic activity and cytotoxicity among the conventional MONPs, with GMgO further enhancing biocompatibility.

Hematological parameters

Various effects on blood parameters are revealed by the observed hematological parameters of the investigated samples, broccoli extract MONPs and GMONPs, which provide information on their biological or therapeutic potential, Table 4. Most hematological parameters were stabilized by broccoli extract, however mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and white blood cells (WBCs) were somewhat decreased. This stabilizing impact might be explained by broccoli’s inherent antioxidant effects, which support the preservation of physiological function. The minor drop in MCHC and WBCs, however, suggests that there is limited immunological modulation, and that broccoli extract would be more suited as a mild stabilizer than an enhancer of blood parameters. This is supported by the cell viability results, which showed high RBC viability at all broccoli extract concentrations, demonstrating the extract’s non-toxic nature and potential for blood parameter stabilization without negative side effects.

Table 4.

Biochemical parameters exerted from hemogram of complete blood image after exposure to the test samples

Sample	HB (%)	HCT (%)	RBCs (%)	MCV (%)	MCH (%)	MCHC (%)	PLT (%)	WBCs (%)
Broccoli-extract	99.23	101.0	93.22	99.78	95.80	97.058	101	92.592
ZnO NPs	103.8	101	93.22	100.4	98.95	100	97.81	101.85
GZnO NPs	106.1	101	98.3	101.1	97.55	108.82	100.3	94.444
TiO2 NPs	101.5	96.72	94.91	100.1	104.1	108.82	99.63	103.70
GTiO2 NPs	100.7	98.77	96.61	99.35	104.5	105.88	98.54	100
CaO NPs	99.23	100	96.61	100.4	104.4	100	100.2	101.85
GCaO NPs	102.3	99.38	91.52	99.03	101.0	100	100	94.444
MgO NPs	103	101.2	86.44	99.03	98.95	100	99.27	90.740
GMgO NPs	103.0	101.2	86.44	99.03	98.95	100	99.27	90.740

All hematological parameters showed improvements with ZnO NPs, although hemoglobin (HB) and white blood cell counts were particularly noteworthy. These findings demonstrate the function of zinc in immune regulation and enzymatic activity, which makes ZnO NPs attractive options for applications needing enhanced oxygen transport and immunological support. The effects of the GZnO NPs were considerably more noticeable, as seen by the notable increases in MCHC, RBC counts, and HB. This improvement may be explained by the green-synthesized MONPs that increased biocompatibility, which lessens their harmful effects [119]. This is further supported by the hemolysis data, which showed that at moderate doses, GZnO NPs had hemolysis percentages that were much lower than those of ZnO NPs at all tested concentrations. Moreover, both ZnO and GZnO NPs maintained excellent RBC vitality; however, GZnO NPs demonstrated somewhat higher viability rates, confirming their appropriateness for therapeutic uses.

TiO₂ NPs marginally decreased hematocrit (HCT) while dramatically raising WBC numbers and MCHC. TiO₂ NPs’ interactions with immune cells are probably the cause of the immunostimulatory impact indicated by the rise in WBCs. Although the modest decrease in HCT might be due to slight impacts on red cell formation, TiO₂ NPs are still promising for immune-stimulating applications like cancer immunotherapy and infection prevention. GTiO₂ NPs showed increases in MCH and MCHC without significantly altering WBC or platelet (PLT) levels. When compared to TiO₂ NPs, GTiO₂ NPs had significantly reduced hemolysis levels, remaining below the 5% threshold even at higher concentrations. These results are consistent with hemolysis data. This strengthens GTiO₂ NPs’ biocompatibility and suggests that they could be a safer substitute for biomedical applications. Additionally, these results are further supported by cell viability data, which show that GTiO₂ NPs exhibit lower cytotoxicity than TiO₂ NPs by consistently maintaining high RBC viability.

Hematological parameters were kept in balance by CaO NPs, with all values being near baseline. Because of their neutrality, which indicates little interaction with blood cells, CaO NPs can be used in applications where physiological stability is essential. However, the RBC and WBC counts were lower for the green-synthesized GCaO NPs, which could indicate minor cytotoxic effects or disruption of blood cell formation. This is supported by hemolysis data, which shows that both CaO NPs and GCaO NPs must be further optimized to improve their safety profiles because they both surpass the 5% hemolysis threshold at greater doses. Further indicating possible cytotoxic issues at higher doses, cell viability data also revealed a modest decrease in RBC viability at higher concentrations of CaO and GCaO NPs.

Significant drops in RBC and WBC counts were seen by MgO NPs and its green-synthesized counterpart, GMgO NPs, which raised questions regarding their possible cytotoxicity. This reduction in hematological parameters raises the possibility that MgO and GMgO NPs are unsuitable for systemic or direct blood contact without additional toxicity-reducing processing [101]. The results of hemolysis support these conclusions, demonstrating that both MgO and GMgO NPs exhibit considerable hemolysis at concentrations over 100 µg/mL, with only slight improvements shown in the green-synthesized form. These results underline the necessity of thoroughly assessing their safety and modes of action. Higher concentrations of MgO and GMgO NPs consistently resulted in considerable decreases in RBC viability, according to cell viability data, highlighting their cytotoxic nature and the necessity of cautious dose management.

Conclusion

The present investigation establishes the successful synthsis of four metal oxide NPs, specifically (zinc oxide, titanium dioxide, calcium oxide, and magnesium oxide) through a sustainable approach utilizing broccoli extract. This green synthesis pathway offers an environmentally conscious alternative to traditional chemical methods, maintaining essential NPs characteristics while enhancing their biological relevance.

Characterization analyses confirmed that the botanical extract served dual functions as both a reducing agent and a stabilizer, yielding NPs with favorable morphological attributes including enhanced dispersion stability and minimized aggregation tendencies. Significant improvements were observed in the biological performance of these green-synthesized NPs, particularly their exceptional blood compatibility and controlled hemolytic behavior. Furthermore, these NPs demonstrated modulatory effects on coagulation parameters, indicating their potential utility in hematological applications.

The inherent antioxidant capacity transferred from the broccoli extract to the NPs presents an additional advantage for biomedical implementations, especially in scenarios involving oxidative stress. Variation in biological activity across different metal oxides provides a foundation for selective application development, with zinc- and calcium-based nanoparticles exhibiting particularly favorable properties.

While this research validates the viability of plant-mediated synthesis for producing biocompatible NPs, several aspects require further exploration. Subsequent studies should emphasize standardized phytochemical documentation, implementation of orthogonal characterization methodologies, and rigorous in vivo assessment to completely establish the therapeutic potential of these eco-friendly nanoparticles for targeted drug delivery, advanced wound management, and other medical applications.

Author contributions

Mary Samir did the experimental part and Alaa Hassan Said analyzed the data and wrote the original manuscript. G.A. Gamal, A. A. Ebnalwaled revised the original manuscript. All authors read and approved of the final manuscript.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability

All data supporting the findings of this study are available within the paper and its Supplementary Information.

Declarations

Ethics approval and consent to participate

This study was approved by the Ethics Committee of the Faculty of Science, South Valley University (Permit Number: 002/12/24). All procedures involving human participants and the collection of blood samples were conducted in full accordance with the ethical standards of the institutional research committee and with the 1964 Declaration of Helsinki and its later amendments, or comparable national ethical standards. Written informed consent was obtained from all participants prior to sample collection.

Consent for publication

All authors are aware of this submission.

Clinical trial number

Not applicable.

Competing interests

The authors declare no competing interests.