Ecofriendly production of CuO/ZnO nanocomposites from guava leaf biomass and their antimicrobial and selective anticancer activities

Alsalamah, S., Alawlaqi, M. M., Sumayli, M., Areshi, S. M., Hazzazi, Y., Alghonaim, M. I., and Almotayri, A. M. (2026). "Ecofriendly production of CuO/ZnO nanocomposites from guava leaf biomass and their antimicrobial and selective anticancer activities," BioResources 21(2), 2906–2924.

Abstract

Psidium guajava leaf extract was utilized to produce CuO NPs and CuO/ZnO nanocomposites in this work. X-ray diffraction patterns showed the proper phase of the synthesized sample, and using Scherrer’s equation, the mean crystallite sizes were calculated to be 13.05 nm and 13.07 nm for CuO nanoparticles (NPs) and CuO/ZnO nanocomposite, respectively. From transmission electron microscopy images and particle size distribution from ImageJ, the samples had larger sizes of 84.6 nm and 96.6 nm, respectively. Fourier transform infrared (FTIR) spectroscopy also indicated the successful fabrication of the CuO NPs and CuO/ZnO nanocomposite, as the IR spectrum showed peaks at 611 and 521 cm⁻¹ for Cu–O and Zn–O, respectively. The biological activities were found to be higher for the CuO/ZnO nanocomposite than the CuO NPs alone. The inhibition zones recorded were 28 mm and 25 mm for Bacillus. subtilis and Staphylococcus aureus, respectively, followed by 24 mm and 21 mm for Salmonella typhi and Klebsiella pneumoniae, respectively. The nanocomposite exhibited selective cytotoxicity properties because the IC₅₀ values for Wi38 (normal human fibroblast cells) and SKOV3 (human ovarian carcinoma cells) were 295.48 and 81.87 µg/mL, respectively. Antioxidant analysis of (2,2-diphenyl-1-picrylhydrazyl) (DPPH) radical scavenging activity was found to be 6.93 µg/mL for the nanocomposite.

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Ecofriendly Production of CuO/ZnO Nanocomposites from Guava Leaf Biomass and their Antimicrobial and Selective Anticancer Activities

Sulaiman A. Alsalamah ,^a,* Mohamed M. Alawlaqi ,^bMari Sumayli ,^b Sultan Mohammed Areshi ,^b Yehia Hazzazi ,^bMohammed Ibrahim Alghonaim ,^aand Abdullah M. Almotayri ^c

DOI: 10.15376/biores.21.2.2906-2924

Keywords: CuO/ZnO nanocomposites; Antimicrobial activity; Antioxidant; Cytotoxicity

Contact information: a: Department of Biology, College of Science, Imam Mohammad Ibn Saud Islamic University (IMSIU), Riyadh 11623, Saudi Arabia; b: Department of Biology, College of Science, Jazan University, P.O. Box 114, Jazan 45142, Saudi Arabia; c: Department of Biology, Faculty of Science, Al-Baha University, 65779 Al-Baha, Saudi Arabia;

* Corresponding author: SAAlsalamah@imamu.edu.sa

INTRODUCTION

Nanotechnological approaches have increasingly been applied in agricultural and medical fields (Abdelghany et al. 2023a; El-Batal et al. 2023; Amin et al. 2025). The concept of “green” production of nanoparticles (NPs) by means of plant mediators has become a preferred strategy in an attempt to avoid the ecological and economical toxicity involved in the chemical synthesis (Alghonaim et al. 2025; Selim et al. 2025a). The physical property of NP size with high surface-area-to-volume ratios results in an unmatched catalytic, optical, and reactive potential. Polymer, lipid, or metal- or metal oxide-based NPs have therefore been applied for energy storage, remediation, and medicinal uses, particularly for imaging or drug delivery (Abdelghany et al. 2018). Copper (II) oxide (CuO) NPs, for instance, exhibit strong antioxidant, antimicrobial, and anticancer potential (Abdelghany et al. 2020; Alsalamah et al. 2025). The subcellular size of NPs allows for targeted cellular uptake, which can offer improved results with minimal systemic damage to the host organism. Additionally, scientists have developed NP nanocomposites to offer even more selectivity for regenerative and cancerous therapies.

Similarly to CuO, Zn oxide-containing NPs have shown great potential due to the unique way that they behave biologically. Zn oxide-containing NPs function as a selective cytotoxic agent against MCF7 that produce reactive oxygen species (ROS) for a cascade of effects that eradicate cancer cells and microbes while sparing healthy cells of the Vero cell line CCL-81 (Qanash et al. 2024). The potential of Zn oxide-containing NPs also extends to agricultural applications as a nano-fertilizer to improve crop growth (Abdelghany et al. 2023b, 2024), to the food industry as a shelf-life preservative through antimicrobial packaging, and finally to cosmetic applications as well due to its photocatalytic and UV-shielding properties.

Composites of two metals or metal oxides combine the unique properties of each component to achieve enhanced functionality compared to single-phase nanoparticles (Al Abboud et al. 2024). In such biphasic metal or metal oxide nanocomposites, interactions between the two phases can improve its stability, reduce agglomeration, and induce biological activity (Selim et al. 2025b). For example, combining CuO and ZnO into a single nanocomposite can merge the potent biological properties of CuO with the biocompatibility and ROS-mediated activity of ZnO (Al-Rajhi et al. 2022). Additionally, the structural and electronic interactions in biphasic composites can enhance surface reactivity, facilitating improved cellular uptake, ROS generation, and overall therapeutic performance. Here, the term “biphasic” means that methods such as X-ray diffraction (XRD) will reveal two distinct phases. Such nanocomposites have broad applications in biomedicine, catalysis, and environmental remediation, making them a promising platform for multifunctional nanoscale materials (Das and Srivastava 2018).

Scientists think that green-synthesised NPs are safer to work with than chemically produced ones (Nguyen et al. 2018). In this study, the authors used guava (Psidium guajava) leaves extract as capping agent to create CuO and CuO/ZnO NPs. This plant is found in tropical and subtropical areas. Numerous physiological compounds with antioxidative and anti-inflammatory properties, including flavonoids, triterpenoids, alkaloids, and tannins, are found in high concentrations in its leaves (Park et al. 2024).

Plant extracts appear to serve as a mediator in the green synthesis of ZnO NPs. In contrast to noble metal NPs, where reduction of metal ions is essential, ZnO formation does not involve a change in oxidation state, as zinc exists in the +2 oxidation state both in zinc acetate precursors (for example) and in the final ZnO lattice (López-López et al. 2021). In this context, the role of extracts is not a redox-driven reduction but rather the promotion of Zn²⁺ hydrolysis, condensation, and controlled nucleation, leading to formation of ZnO (Hamed et al. 2023). Phytoconstituents act as complexing and chelating agents that regulate Zn²⁺ speciation, enable the formation of Zn(OH)₂ intermediates, and subsequently direct their dehydration into ZnO. Additionally, these biomolecules serve as stabilizing and capping agents, preventing NPs agglomeration and controlling morphology and particle size. Similar non-redox mechanisms have been extensively reported for the biosynthesis of ZnO NPs. While in some other cases, plant extracts have been shown to act as reducing agents, they appear to have a broader role in NPs formation rather than causing a change in zinc’s valence state (Matinise et al. 2017). Little is known about the multifunctional use of CuO/ZnO NPs compared with monophasic CuO NPs. Thus, this study established a comparison of monophasic and biphasic oxide NPs as biological agents in different pharmacological and medical uses.

EXPERIMENTAL

Extracting Plant Extract

Psidium guajava leaves were collected from the Botanical Garden of Al-Qanater El-Khyria (El-Qulyubia Governorate, Egypt). The collected plant biomass was rinsed with distilled deionized water and air-dried under room temperature in the shade for five days. Five grams of the plant powder were suspended in 100 mL double-distilled deionized water and heated at 70 °C for 20 min. The decoction was filtered through Whatman No. 1 filter paper, and the filtrate was kept at 4 °C for further use.

Preparation of CuO/ZnO Nanocomposites and CuO NPs

For combined-phase CuO/ZnO nanoparticles, 50 mL each of 0.5 M copper sulfate and 0.5 M of zinc acetate was mixed to form the precursor solution. The mixture was heated at 80 °C with magnetic stirring. 50 ml of leaves extract was added dropwise after reaching the desired temperature. The synthesis reaction was allowed to proceed for 50 min. The resultant precipitate was separated through centrifugation (4800 rpm, 15 min) and calcined at 450 °C for 3 h. The CuO nanoparticles were synthesized using the identical thermal and mechanical protocol using copper sulfate alone.

Characterization of Synthesized CuO/ZnO Nanocomposites and CuO NPs

The synthesized particles were characterized structurally and chemically. Fourier transform infrared by FTIR (Thermo Scientific Nicolet iS50 FT-IR spectrometer) spectroscopy was used to recognize the functional groups present during the synthesis. The Shimadzu XRD-6000 lists, SSI, (Japan) was used to determine the crystallinity of the CuO/ZnO Nanocomposites and CuO NPs. The Scherrer equations were used to measure the diameters of the nanoparticles. The average crystallite size of the biosynthesized NPs was determined using the Scherer equation (D = 0.9 λ/β cosθ) to estimate the particle size of NPs. Next, d-spacing (dhkl = λ/(2sin θ), micro-strain (ε = β/4tan θ), and dislocation density (δ = 1/D^2) were computed (Table 1).

Transmission electron microscopy (TEM) images were obtained using a JEM-2100 PLUS electron microscope (JEOL, Japan) running at 200 kV with a LaB6 source. The energy dispersive X-ray spectroscopy (EDX) investigation of these samples was performed using a JEM-2100 F (URP) device equipped with a Dry SD30GV detector and set at 200 kV.

Antimicrobial Assay (Agar Well Diffusion Method) of CuO/ZnO Nanocomposites and CuO NPs

The agar well diffusion method (Qanash et al. 2023) was employed to determine the antimicrobial activities of the as-synthesized CuO and CuO/ZnO nanocomposites. The test panel included four bacterial isolates: Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633), Klebsiella pneumoniae (ATCC 13883), and Salmonella typhi (ATCC 6539), as well as two fungal isolates: Candida albicans (ATCC 10221) and Aspergillus terreus (ATCC 20542). The inoculum was prepared by standardizing the turbidity of bacterial and fungal cultures to 0.5 McFarland turbidity standard. The medium was swabbed with the standardized inoculum to form confluent growth on sterile Mueller–Hinton agar (MH agar) plates (bacteria) or Sabouraud dextrose agar plates (fungi). After punching 6-mm wells in the media, 100 µL of each nanoparticle dispersion was added to the respective wells. Dimethyl sulfoxide (DMSO) was used as a control and Gentamicin (10 µg/mL) or Fluconazole (25 µg/mL) was used as the positive standard in the antibacterial or antifungal assay, respectively. A 30-minute pre-incubation step was added to allow the test compounds to diffuse, followed by separate incubation conditions for bacterial plates (37 °C for 24 h) and fungal plates (28 to 30 °C for 48 to 72 h), after which antimicrobial activity was measured using a digital caliper to measure the diameter of the zone of inhibition (ZOI) in millimeters.

Cytotoxicity Assay (MTT Assay) of CuO/ZnO Nanocomposites and CuO NPs

The cytotoxicity studies were performed on two cell lines: SKOV3 human ovarian carcinoma (ATCC® HTB-77™) and Wi38 normal human fibroblasts (ATCC® CCL-75™). Cell cultures were maintained at 37 °C with 5% CO₂ in a humidified incubator and in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. After seeding the cells (1 × 10⁴ cells/well in 96-well plates) and incubating for 24 h for cell attachment, the samples were treated with the CuO or CuO/ZnO nanoparticles in a concentration range of 31.25 to 1000 µg/mL for 24 h. The metabolic activity of the cells was measured by MTT assay. To each well, 20 µL of MTT reagent (5 mg/mL) was added and incubated for 4 h. After solubilization of the formazan crystals with 150 µL DMSO, the absorbance was measured at 570 nm. The changes in the cell morphology were also observed with an inverted phase-contrast microscope and the cell viability was calculated using the standard formula:

DPPH Radical Scavenging Assay of CuO/ZnO Nanocomposites and CuO NPs

Radical scavenging activity was determined using the DPPH method. Aqueous suspensions of equal volumes (1 mL) of nanoparticle solution and 0.1 mM methanolic DPPH were mixed and kept at room temperature in the dark for 30 min. The absorbance of the mixture was measured at 517 nm (Alawlaqi et al. 2023). The scavenging efficiency was calculated using Eq. 1,

where A_control is the absorbance of the DPPH solution without sample, and A_sampleis the absorbance of the tested sample. The concentration of each tested compound required to scavenge 50% of DPPH radicals (IC₅₀) was determined by plotting the percentage scavenging activity against the compound concentration and fitting a suitable regression curve.

Statistical Analysis

Data processing was performed using SPSS software (version 18, IBM Corp., Armonk, NY, USA). The statistical framework included One-Way analysis of variance (ANOVA), Shapiro-Wilk normality tests, and Homogeneity of Variance checks, supplemented by appropriate post-hoc comparisons and evaluated honest significant differences (HSD).

RESULTS AND DISCUSSION

Characterization of CuO and CuO/ZnO NPs

A limitation in the use of vegetal matrices in the biosynthesis of nanoparticles is the question of its mechanistic predictability, due to the complexity of the plant extract. Although the exact mechanism of various phytochemical interactions in this process is not fully understood yet, the reduction and stabilization of NPs is mainly due to the reductant action of some specific bioactive moieties, such as carbonyl, hydroxyl, and methoxide groups, all of which are richly available in plant metabolites, such as phenolic compounds, flavonoids, alkaloids, and proteins (Devi et al. 2016; Abd-ElGawad et al. 2025). To make a correlation between this chemical scenario and the physico-structural one, the synthesized monophasic (CuO) and biphasic (CuO/ZnO) species were characterized using TEM and EDX (Amin et al. 2024, 2025). The morphometric analysis with ImageJ software recorded a wide range of particle sizes (67.9 to 122.07 nm) (Fig. 1a, b). The calculated average particle size was also different for pure CuO and CuO/ZnO, i.e., 84.63 and 96.6 nm, respectively (Fig. 1c, d). The particles exhibited semi-spherical and irregular shapes (Fig. 1a,b). The morphology was not entirely consistent with the results from Cao et al. (2021) (semi-spherical CuO/ZnO-doped nanoparticles with size in the range of 20 to 130 nm). They observed spherical shapes only. In contrast, some simultaneous reports point that the one-phasic composites (either CuO or ZnO) usually have similar and small diameters.

After the preparation of the biphasic CuO/ZnO nanoparticles, their propensity to agglomerate was observed. This issue is one of the common reasons for the biphasic NPs’ aggregation as the van der Waals attractive forces are stronger than their repulsive ones. The most significant feature is that, according to Turabik et al. (2023), although the morphological characteristics of mono- and biphasic particles are essential, their functionality and versatility are primarily defined by the surface-related physicochemical parameters such as zeta potential, pore width, or specific surface area. Energy dispersive X-ray spectroscopy was also applied to ensure the presence of targeted elements (Fig. 2a,b).

The spectrum corresponding to monophasic CuO nanoparticles shows the most intense peaks corresponding to the Copper (Cu) and Oxygen (O). As for the carbon (C) signals, they are probably related to the plant extract-based biomolecules acting as capping agents to stabilize the NPs. The EDX spectrum of the prepared biphasic CuO/ZnO nanocomposites resolved the peaks of all target elements – copper, zinc, and oxygen. Carbon signals were also detected, which could also be assigned to the organic compounds that stabilized the nanostructures.

Fig. 1. TEM images (a, b) of CuO and CuO/ZnO NPs, respectively

To investigate the vibration properties of the biosynthesized samples and to confirm the identity of the Psidium guajava metabolites involved in capping and stabilization, FTIR spectroscopy was used (Fig. 3). The broad absorption bands present at 3473 cm⁻¹ and 3454 cm⁻¹ in the higher frequency region are assigned to the stretching vibrations of hydroxyl (-OH) groups and are possibly due to surface-adsorbed water molecules. The adsorption of atmospheric CO₂ is confirmed by the presence of a distinct peak at 2,355 cm⁻¹ (Senthilkumaar et al. 2008). The peak centered at around 1630 cm⁻¹ due to the C=O stretching mode of carboxylic acid moieties is a clear indicator of the role of these functional groups in the reduction process (Atiek et al. 2024). The bands present at 1400, 1413, and 1220 cm⁻¹ are assigned to the vibrations of aromatic rings and C–C/C–OH stretching of polyol compounds. These observations further validate that the bioactive organic ligands present in the leaf extract are adsorbed on the nanoparticle surface and contribute to the steric stability.

The formation of the inorganic core is evident from vibrations in the low frequency fingerprint region. The formation of copper oxide is confirmed by the Cu–O stretching vibrations centered at 610 cm⁻¹, 611, and 680 cm⁻¹ (Vishveshvar et al. 2018). The Zn–O stretching vibration is associated with the peak found at 423 cm^-1, confirming the ZnO production (Music et al. 2002). As confirmed by the associated EDX profile (Fig. 2), these spectral analyses reconfirmed that Cu, Zn, and O were the major elements present in the samples.

Fig. 2. EDX analysis (a, b) of CuO and CuO/ZnO NPs, respectively

Fig. 3. FTIR analysis of a) CuO and b) CuO/ZnO NPs

The XRD analysis was used to determine whether the produced nanocomposite was crystalline or amorphous. The successful doping and development of crystallographic CuO NPs and CuO/ZnO nanocomposite is indicated by the appearance of sharp and distinct peaks for Cu and Zn in the XRD analysis (Fig. 4). The XRD pattern of the CuO NPs is displayed in Fig. 3. Monoclinic CuO’s (111), (200), (220), and (311) planes are responsible for the peaks that were seen at 43.40°, 50.55°, 74. 30°, and 90.16° (COD Reference: 9012954 and 1509146). Additionally, the highest points found at 36.29°, 39, 54.33, and 70.04 were in accordance with the (002), (101), (110), and (103) planes of hexagonal ZnO, respectively (COD Reference: 9008781). The crystalline structure of copper oxide nanoparticles is demonstrated by their strong reflection and well-defined appearance in XRD patterns (Bashiri Rezaie et al. 2018). Their crystalline structure is revealed by the XRD spectrum’s strong peaks (Yallappa et al. 2013).

The Scherrer formula was used to determine the average crystallite sizes of CuO and CuO/ZnO nanoparticles and were found to have average crystallite diameters of 13.05 and 13.07 nm, respectively. Table 1 shows the different properties of XRD spectrum, which appeared in CuO and CuO/ZnO NPs. Increased dislocation density causes lattice distortions, leading to increased micro-strain, which in turn subtly alters the ideal d-spacing (interplanar distance) observed in XRD, often showing a slight decrease or shift from standard values, with composites showing complex interplay between CuO and ZnO properties. Higher strain/dislocations typically correlate with smaller crystallite sizes and vice versa, with CuO/ZnO composites often exhibiting modulated properties compared to pure phases due to heterojunctions and doping effects.

Table 1. XRD Parameters for CuO and CuO/ZnO NPs

Fig. 4. XRD analysis CuO and CuO/ZnO NPs

Biological Potential

Table 2 indicates that the antibacterial activity of the CuO/ZnO nanocomposite was improved when compared with CuO nanoparticles and the negative controls. Statistically, the CuO/ZnO nanocomposite showed highly significant increases of inhibition zones of pathogenic microorganisms. Of the six different tested microorganisms (two fungal strains and four bacterial strains), B. subtilis (Gram-positive) was the most sensitive. Zone of inhibition was 28 ± 0.1 mm for B. subtilis and 25 ± 0.1 mm for S. aureus, which may indicate that the nanocomposite penetrated the relatively thicker peptidoglycan layer in Gram-positive bacterial strains. For the Gram-negative strains, S. typhi and K. pneumoniae showed 24 ± 0.2 mm and 21 ± 0.2 mm zones of inhibition, respectively, even though they have stronger outer membrane layers compared to Gram-positive strains. The improved action against Gram-negative bacteria suggests that the biphasic CuO/ZnO composition was effective to disrupts membrane integrity and interferes with essential metabolic processes. The plates (Fig. 5) visually confirm the quantitative data, showing larger, clearer inhibition halos around the CuO/ZnO wells for all bacteria tested. Across all bacterial plates, the negative control produced no inhibition, validating the experiment, while the positive control showed moderate, consistent activity. The superior inhibition halos around the CuO/ZnO wells visually document the enhanced antibacterial potency and the combination effect of the composite nanoparticles. The enhanced bactericidal effect was attributed to the combined antibacterial effects from the copper and zinc oxide phases. This is mainly due to the catalytic generation of ROS as a possible mechanism based on previously reported literature (Fani et al. 2025; Ma et al. 2025), such as hydrogen peroxide, superoxide ions, and hydroxyl radicals, that cause oxidative damage to DNA, proteins, and lipids. At the same time, the dissolution of Cu²⁺ and Zn²⁺ enabled intracellular entry, which interfered with enzyme activity and metabolic balance. The physicochemical characteristics with a high surface area and strong electrostatic attraction enabled the nanohybrids to attach firmly to the bacterial surface. It destabilizes the dense peptidoglycan layer of Gram-positive bacteria and disrupts the recalcitrant outer membrane of Gram-negative bacteria, causing substantial changes in permeability and cytoplasmic leakage. Overall, the dual-phase system creates multiple simultaneous attacks, leading to enhanced broad-spectrum bactericidal activity. The multidrug-resistant S. aureus was inhibited by the ZnO–CuO nanocomposite, which produced a 24 mm zone of inhibition, whereas ZnO nanoparticles alone produced an 8 mm inhibition zone (Jan et al. 2019). Table 3 shows how our research compares to other studies on a number of biological and other applications.

Table 2. Comparative Antibacterial and Antifungal Activities of CuO/ZnO Nanocomposites, CuO Nanoparticles, and Control Treatments

Fig. 5. Agar well diffusion assay showing inhibition zones produced by CuO/ZnO nanocomposites, CuO nanoparticles, and controls against bacterial and fungal pathogens

Table 3. Comparison of the Present Work with Other Studies Concerning Several Biological and Other Applications of CuO/ZnO NPs

Table 4 summarizes the cytotoxic effects of CuO and CuO/ZnO nanoparticles on Wi38 normal fibroblast cells and SKOV3 ovarian cancer cells across a concentration range of 1000 to 31.25 µg/mL. The data demonstrates a concentration-dependent cytotoxic response, where cell viability increased at higher concentrations but declined sharply at lower concentrations. For Wi38 normal cells, CuO showed cytotoxicity at low concentrations, reducing viability to nearly zero at 250 to 31.25 µg/mL. In contrast, CuO/ZnO NPs preserved much higher viability at equivalent doses 49.3% at 250 µg/mL and 0.18% at 62.5 µg/mL, indicating better biocompatibility of the nanocomposite compared to CuO alone. For SKOV3 cancer cells, both nanoparticle types displayed clear antiproliferative activity, but CuO/ZnO NPs exhibited notably stronger anticancer effects. At low concentrations (250, 125, and 62.5 µg/mL), SKOV3 cell viability decreased substantially (87.17%, 79.08%, and 47.42%, respectively), while CuO NPs showed comparatively weaker inhibition. Statistically, CuO/ZnO NPs appeared the highest significant efficiency against SKOV3 cancer cells at low concentration compared to CuO NPs alone. The calculated IC₅₀ values further confirm selective toxicity, where it was 397.35 ± 2.76 µg/mL and 295.48 ± 1.93 µg/mL employing CuO NPs and CuO/ZnO NPs, respectively, against Wi38 cell line. Meanwhile, it was 122.25 ± 2.38 µg/mL and 81.87 ± 0.82 µg/mL employing CuO NPs and CuO/ZnO NPs, respectively, against SKOV3 cell line. These values clearly indicate that CuO/ZnO NPs are more cytotoxic to cancer cells than normal cells, highlighting their potential as selective anticancer agents.

This difference may be attributed to differences in cellular metabolism, membrane properties, and redox balance between cancerous and normal cells. Cancer cells typically exhibit higher metabolic activity and a more vulnerable oxidative status, which may enhance their sensitivity to nanoparticle-induced stress.

Figure 6 depicts the morphological changes in Wi38 normal fibroblast cells following exposure to increasing concentrations of CuO (a) and CuO/ZnO NPs (b). Untreated control cells maintained normal spindle-shaped morphology and strong confluence. At low nanoparticle concentrations (31.25 to 125 µg/mL), CuO induced milder cellular shrinkage, membrane damage, and loss of confluence, consistent with the viability drop shown in Table 4. However, Wi38 cells exposed to the nanocomposite (CuO/ZnO) displayed severe alterations, retaining adherence and structural integrity, which supports the lower IC₅₀.

Additionally, Fig. 7 illustrates the dose-dependent morphological responses of SKOV3 cancer cells to CuO (a) and CuO/ZnO NPs (b). Control cells exhibited typical epithelial-like morphology with dense, well-defined colonies. Upon treatment with low to moderate nanoparticle concentrations (31.25 to 250 µg/mL), SKOV3 cells underwent marked morphological deterioration, including rounding, detachment, cytoplasmic condensation, and reduced cell density. These changes were more pronounced in the CuO/ZnO nanoparticle group, consistent with their stronger cytotoxic effect. The observed cytotoxic effects of CuO and CuO/ZnO NPs can be explained via induction of ROS generation as a probable mechanism, membrane disruption, and/or nanoparticle–cell interactions. In addition, several studies have highlighted the enhanced anticancer potential of CuO-ZnO nanoparticles. For instance, CuO–ZnO NPs demonstrated significant cytotoxicity against the MCF7 human breast cancer cell line, with an IC50 of 239.99 μg/mL (Daimari and Deka 2024). Similarly, hexagonal ZnO-CuO biphasic nanoparticles synthesized using Sambucus nigra L. extract exhibited notable anticancer effects against lung and melanoma cancer cells (Cao et al. 2021). Moreover, spherical ZnO-CuO biphasic nanoparticles produced via green synthesis were reported to possess both strong antibacterial properties and potent anticancer activity against MCF-7 cells (Madeshwaran and Venkatachalam 2024). These results suggest that combining ZnO and CuO in nanoparticle form can improve anticancer properties, making them promising candidates for biomedical applications.

Table 4. Cytotoxicity % of CuO and CuO/ZnO NPs against Wi38 and SKOV3 Cell Lines

Fig. 6. Morphological changes of Wi38 cell lines in response to CuO (a) and CuO/ZnO NPs (b)

The antioxidant capacity was determined using the DPPH free-radical scavenging assay for ascorbic acid, CuO NPs, and CuO/ZnO nanocomposites, in the concentration range of 0 to 1000 µg/mL. All three samples showed dose-dependent results. At low doses of 1.95 to 7.81 µg/mL, standard ascorbic acid had an inhibitory range of 41.0 to 57.0%, CuO/ZnO nanocomposite (33.6 to 50.8%) showed a highly significant scavenging ability than CuO NPs (27.5 to 45.3%) (Table 5). At 125 µg/mL, the nanocomposite had 84.2% scavenging ability, which was better than pure CuO (79.8%), while CuO was less active than ascorbic acid (86.4%) (Table 5). The nanocomposite (93.7 to 95.7%) still had a better scavenging capacity than CuO NPs (90.2 to 93.7%) at high concentrations of 500 to 1000 µg/mL. IC₅₀ values of ascorbic acid, CuO/ZnO, and CuO were calculated as 3.61 ± 0.06, 6.93 ± 0.25, and 11.61 ± 0.33 µg/mL, respectively. Among all the samples, ascorbic acid showed the highest activity, which was followed by the CuO/ZnO composite and CuO NPs (Table 5). The observed greater activity of the nanocomposite is attributed to the combination interfacial effect. The reactivity of the nanocomposite was increased due to the introduction of ZnO, which enhanced the number of active sites and improved the transfer of electrons (Afifi et al. 2015).

Fig. 7. Morphological changes of SKOV3 cancer cell lines in response to CuO (a) and CuO/ZnO NPs (b)

The present results are also in line with recent studies (Daimari and Deka 2024; Azabi et al. 2025). The radicals are more stabilized in the heterojunction than in individual oxides. Thus, CuO/ZnO nanocomposites are expected to be applied in various fields for the scavenging of free radicals and oxidative stress, which include pharmaceutical, cosmetic, and food-preservation industries.

Table 5. Antioxidant Effect via DPPH Scavenging at Different Concentration of CuO and CuO/ZnO NPs Compared with Standard Compounds

CONCLUSIONS

Monophasic nanoparticles of Cu and biphasic nanoparticles of Cu/Zn oxide were prepared via green synthesis and characterized by X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, and transmission electron microscopy (TEM), which verified that ZnO and CuO formation.
The CuO/ZnO nanocomposite displayed potent antioxidant activity, indicating a strong combination effect between CuO and ZnO.
P. guajava-derived CuO/ZnO nanocomposite presents a promising multifunctional material with potential applications in antimicrobial therapies, antioxidant formulations, and anticancer strategies.

FUNDING

This work was supported and funded by the Deanship of Scientific Research at Imam Mohammad Ibn Saud Islamic University (IMSIU) (grant number IMSIU-DDRSP2601)

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Article submitted: November 26, 2025; Peer review completed: January 17, 2026; Revised version received: January 19, 2026; Further revised version received and accepted: January 21, 2026; Published: February 5, 2026.

DOI: 10.15376/biores.21.2.2906-2924