Green synthesis of Ag–Se nanoparticles using pomegranate peel: A multifunctional approach for antimicrobial, antioxidant, and anticancer applications

Almutairi, H. H., Mohammad, M. H., Alahmari, A. S., and Alwutayd, K. M. (2026). "Green synthesis of Ag–Se nanoparticles using pomegranate peel: A multifunctional approach for antimicrobial, antioxidant, and anticancer applications," BioResources 21(4), 10326–10338.

Abstract

Silver–selenium nanoparticles (Ag–Se NPs) were successfully prepared using Punica granatum peel extract via a green, eco-friendly, and cost-effective approach. The formation of nanoparticles was confirmed by a characteristic UV–Vis absorbance peak at 436 nm, while FTIR analysis indicated the involvement of functional groups in reduction and stabilization. TEM analysis showed predominantly spherical nanoparticles with an average size of 44.9 ± 19 nm. The synthesized Ag–Se NPs exhibited effective antibacterial activity against Staphylococcus aureus, Escherichia coli, and Bacillus subtilis, with inhibition zones of 17.8, 20.3, and 16.2 mm, respectively. The MIC values ranged from 62.5 to 500 µg/mL, confirming concentration-dependent antimicrobial activity. The nanoparticles showed significant antibiofilm activity, reaching 62.3% inhibition against E. coli at higher concentrations. Cytotoxicity evaluation demonstrated selective activity, with IC₅₀ values of 209 µg/mL for Vero normal cells and 115 µg/mL for MCF-7 cancer cells. Furthermore, antioxidant activity assessed by DPPH and ABTS assays showed IC₅₀ values of 458 and 312 µg/mL, respectively. Overall, the results indicate that green-synthesized Ag–Se NPs possess multifunctional biological activities, making them promising candidates for antimicrobial, anticancer, and antioxidant applications.

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Green Synthesis of Ag–Se Nanoparticles Using Pomegranate Peel: A Multifunctional Approach for Antimicrobial, Antioxidant, and Anticancer Applications

Hayfa Habes Almutairi ,^a,* Maryam Hadi Mohammad ,^b Amirah S. Alahmari ,^c and Khairiah M. Alwutayd ^c

DOI: 10.15376/biores.21.4.10326-10338

Keywords: Punica granatum peel; Silver–selenium nanoparticles; Green synthesis; Antimicrobial activity; Cytotoxicity; Antioxidants

Contact information: a: Chemistry Department, College of Science, King Faisal University, Al-Ahsa, Saudi Arabia; b: College of Medicine, Mustansiriyah University, Department of Microbiology, Baghdad, Iraq 10052; c: Department of Biology, College of Science, Princess Nourah bint Abdulrahman University, Riyadh 11671, Saudi Arabia; *Corresponding author: [email protected]

INTRODUCTION

Nanotechnology is widely recognized as a key driver of modern technological advancements, offering substantial opportunities for breakthroughs in biological and medical sciences throughout the twenty-first century (Soliman et al. 2026). In recent years, the large-scale production and application of nanomaterials including metal oxide nanoparticles, have increased significantly worldwide. Among these, metal-based nanoparticles have attracted considerable interest due to their unique physicochemical properties, such as enhanced optical, electrical, mechanical, and catalytic behaviors, which arise primarily from their nanoscale dimensions. Typically ranging between 1 and 100 nm, nanoparticles exhibit a high surface-to-volume ratio, resulting in increased surface reactivity and improved functional performance (Alfattah et al. 2025).

Among various nanomaterials, silver (Ag), selenium (Se), and their bimetallic composites have gained particular attention due to their versatile applications in biomedical and environmental fields. These nanoparticles have been reported to exhibit antimicrobial, antioxidant, and anticancer activities. Moreover, it has been demonstrated that reducing nanoparticle size can enhance therapeutic efficiency while minimizing adverse effects, highlighting the importance of nanoscale engineering (Aldayel et al. 2026). Selenium-based nanomaterials, in particular, have shown promising anticancer properties, although the exact mechanisms remain under investigation (Soliman and Salem 2025b). Silver nanoparticles have demonstrated significant potential in modulating reactive oxygen species (ROS), thereby reducing oxidative stress and contributing to their therapeutic effects. Additionally, their strong antibacterial activity against a wide range of pathogens highlights their importance in combating antimicrobial resistance (Bouqellah et al. 2026). On the other hand, selenium nanoparticles are considered highly attractive due to their lower toxicity compared to other selenium forms and their potent biological activities, including antioxidant and antimicrobial effects (Soliman et al. 2024).

Various physical, chemical, and biological methods have been employed for nanoparticle synthesis; however, green synthesis approaches have gained increasing attention due to their simplicity, sustainability, cost-effectiveness, and elimination of hazardous chemicals (Aljarba et al. 2026a). In contrast, plant-mediated synthesis offers a safer and more efficient alternative, as plant extracts are rich in bioactive compounds such as flavonoids, phenolics, alkaloids, terpenoids, and proteins, which act as natural reducing and capping agents (Soliman and Salem 2025a). Furthermore, this approach enhances nanoparticle stability, reduces aggregation, and improves biocompatibility, while also being economically advantageous (Saied et al. 2025).

Punica granatum L. (pomegranate) is a widely cultivated medicinal plant belonging to the family Lythraceae. It is known for its rich phytochemical composition and diverse therapeutic properties. Various parts of the plant, particularly the peel, are abundant in bioactive compounds such as polyphenols, flavonoids, tannins, ellagic acid, and anthocyanins, which exhibit strong antioxidant, antimicrobial, anti-inflammatory, and anticancer activities (Maphetu et al. 2022). Due to this high phytochemical content, pomegranate peel has gained significant attention as an effective natural reducing and stabilizing agent in the green synthesis of nanoparticles. Moreover, the utilization of Punica granatum peel, an agricultural waste material, adds value by promoting sustainability and eco-friendly nanomaterial production. Consequently, pomegranate peel-mediated synthesis has emerged as a promising approach for developing biocompatible nanoparticles with potential applications in biomedical and pharmaceutical fields.

This study biosynthesized and characterized bimetallic silver–selenium nanoparticles (Ag–Se NPs) using Punica granatum peel extract as a green reducing and stabilizing agent. In addition, the study investigated the biological activities of the synthesized nanoparticles, including antioxidant, antibacterial, antibiofilm, and anticancer properties, to explore their potential applications in biomedical and pharmaceutical fields.

EXPERIMENTAL

Materials

All chemicals used in this study were of analytical grade and used without further purification. AgNO₃, Na₂SeO₃, ascorbic acid, DPPH, ABTS, and potassium persulfate were obtained from Sigma-Aldrich (USA). Cell culture media and related reagents were purchased from Gibco (USA). Fresh peels of Punica granatum were collected locally and used for extract preparation, while all solutions were prepared using double-distilled water.

**Production of Ag–Se NPs via Green Method**

Ag–Se nanoparticles were synthesized via a green method using the aqueous peel extract of Punica granatum as a natural reducing and capping agent. Briefly, fresh pomegranate peels were washed, air-dried, and finely powdered. About 10 g of the powder was extracted in 100 mL distilled water at 60 to 70 °C for 30 min, followed by filtration to obtain the plant extract. For nanoparticle preparation, equal volumes of 1 mM AgNO₃ and Na₂SeO₃ solutions were mixed under continuous stirring, and 10 mL of the extract was added dropwise. The reaction mixture was maintained at 60 °C for 2 h, during which a visible color change confirmed nanoparticle formation. The synthesized Ag–Se NPs were separated by centrifugation at 10,000 rpm for 15 min, washed repeatedly with distilled water and ethanol, and dried at 50 °C. The phytochemicals present in the extract, particularly polyphenols and flavonoids, facilitated the reduction of metal ions and stabilized the formed nanoparticles, making this approach simple, eco-friendly, and cost-effective (Khormi et al. 2025).

Characterization of Ag–Se NPs

The optical properties and surface plasmon resonance were analyzed by UV–Visible spectroscopy (JASCO 730) over a wavelength range of 200 to 800 nm. Morphological features and particle size distribution were examined using transmission electron microscopy (TEM, JEOL-2100), where a drop of the nanoparticle suspension was placed on carbon-coated copper grids and dried overnight under vacuum. Surface morphology and elemental composition were further evaluated by SEM coupled with energy-dispersive X-ray spectroscopy (EDS) (JEOL JSM-6510 LV). The functional groups were identified by FTIR spectroscopy (JASCO 3600) in the 400 to 4000 cm^-1 range (Soliman et al. 2024).

In vitro Antimicrobial Assay

Staphylococcus aureus, Escherichia coli, and Bacillus subtilis were the tested pathogens used to evaluate the efficacy using the agar well diffusion method. Briefly, microbial suspensions were spread onto TSA plates, and wells (6 mm) were filled with nanoparticle suspensions, followed by incubation at 37 °C for 24 to 48 h to measure inhibition zones. The MIC was determined using the broth microdilution method in 96-well plates with serial nanoparticle concentrations. After incubation, MIC was defined as the lowest concentration inhibiting visible growth. The MBC was assessed by subculturing samples from non-growth wells onto agar plates and identifying the lowest concentration showing no colony formation (Aljarba et al. 2026b).

Assay for Biofilm Inhibition

The antibiofilm activity of Ag–Se NPs was assessed against Escherichia coli and Staphylococcus aureus using the microtiter plate assay. Bacterial cultures were diluted in glucose-supplemented TSB and treated with different concentrations of nanoparticles in 96-well plates, followed by incubation at 37 °C for 24 h. After removing planktonic cells, wells were washed with PBS, and the adhered biofilms were fixed with methanol, stained with crystal violet, and rinsed. The retained dye was solubilized with acetic acid, and biofilm biomass was quantified by measuring absorbance at 540 nm. The percentage of inhibition was calculated relative to untreated controls (Soliman et al. 2026).

Anticancer Activity

The cytotoxic effect of Ag–Se NPs was evaluated using the MTT assay on MCF-7 cancer cells and Vero normal cells. Cells were seeded in 96-well plates and incubated for 24 h at 37 °C with 5% CO₂ to allow attachment. After washing with PBS, cells were treated with different concentrations of Ag–Se NPs and incubated for 48 h, while untreated cells served as controls. Subsequently, MTT solution was added and incubated to allow formazan formation. The crystals were dissolved in DMSO, and absorbance was measured at 570 nm using a microplate reader. Cell viability was calculated relative to control cells to determine cytotoxicity (Soliman et al. 2023).

Cell viability and cytotoxicity percentages were calculated using the following equations, where OD stands for optical density:

Viability % = OD of test/ OD of control X 100 (1)

Inhibition % = 100 − Viability % (2)

Antioxidant Assays

The antioxidant activity of Ag–Se nanoparticles was evaluated using DPPH and ABTS assays. For the DPPH assay, a freshly prepared DPPH solution in methanol was mixed with different concentrations of nanoparticles and incubated in the dark at room temperature. The reduction in absorbance was measured at 517 nm, with ascorbic acid used as a positive control.

For the ABTS assay, the ABTS•⁺ radical was generated by reacting ABTS with potassium persulfate and incubating in the dark. The solution was diluted and mixed with Ag–Se NPs at various concentrations, followed by incubation at room temperature. Absorbance was then measured spectrophotometrically, and radical scavenging activity was calculated relative to the control, using ascorbic acid as a standard reference, where A is the absorbance.

Antioxidant activity(%) = (Control A − Ag–Se nanoparticles A)/ Control A*100 (3)

RESULTS AND DISCUSSION

Characterization

The UV–Visible spectrum (Fig. 1a) revealed a prominent absorbance peak at 436 nm, indicating the successful green synthesis of Ag–Se nanoparticles. The appearance of a deep reddish-brown coloration further confirmed the reducing potential of the plant extract in facilitating the formation of bimetallic nanoparticles. Comparable color changes and characteristic UV–Vis absorption profiles have been documented in previous studies during nanoparticle synthesis. For example, ZnSe nanoparticles synthesized using seaweed extract exhibited distinct absorption bands at 250 and 360 nm (Mirzaei et al. 2021). Likewise, Ag–Se nanoparticles produced using Ocimum tenuiflorum inflorescence extract demonstrated a surface plasmon resonance (SPR) peak at 433 nm (Olawale et al. 2022).

The functional groups involved in the biosynthesis of Ag–Se NPs were further investigated using FTIR spectroscopy. The FTIR spectra of the synthesized Ag–Se NPs and the Punica granatum peel extract were recorded over the range of 400 to 4000 cm⁻¹ (Fig. 1b). The spectrum of Ag–Se NPs exhibited characteristic absorbance bands at 3465, 2179, 1596, 1334, 1023, 620, and 455 cm⁻¹. In contrast, the peel extract showed distinct peaks corresponding to various phytochemical constituents responsible for nanoparticle formation. The broad band observed around 3465 cm⁻¹ is attributed to O–H stretching vibrations of hydroxyl groups, indicating the presence of polyphenols and alcohols. The peak at 2179 cm⁻¹ may be assigned to C≡C or C≡N stretching vibrations. The absorption band at 1596 cm⁻¹ corresponds to C=O stretching, suggesting the presence of carbonyl groups such as aldehydes, ketones, or carboxylic acids. The peak at 1334 cm⁻¹ is associated with C–H bending or –CH₃ deformation, while the band at 1023 cm⁻¹ indicates C–O stretching, which may arise from glycosidic linkages or alcohol groups (Méndez et al. 2021). The low-frequency bands at 620 and 455 cm⁻¹ are attributed to the vibrational modes of Se–O and Ag–O bonds, confirming the formation of Ag–Se nanoparticles (Ragab et al. 2022). Similar observations have been reported in previous studies, where plant-derived biomolecules played a crucial role in the reduction and stabilization of nanoparticles (Ettadili et al. 2022). The observed shifts and changes in peak intensity suggest the involvement of bioactive compounds present in Punica granatum peel extract in the reduction of metal ions and capping of the formed nanoparticles, thereby preventing aggregation and enhancing their stability (Ranaware et al. 2025).

Figure 2 illustrates the morphological and structural characteristics of the biosynthesized Ag–Se NPs. TEM images (Fig. 2a) revealed that the nanoparticles were predominantly spherical with occasional oval-shaped structures, indicating slight morphological variation. This diversity in shape and size may be attributed to the complex interactions occurring during the green synthesis process. The particle size distribution histogram (Fig. 2b) showed that the synthesized nanoparticles ranged between approximately 20 and 100 nm, with an average particle size of 44.9 ± 19 nm, suggesting relatively good dispersion with moderate polydispersity. Similar findings have been reported in previous studies, where Ag–Se NPs exhibited variable morphologies depending on the synthesis conditions and biological reducing agents (El-Behery et al. 2023). Comparable results were documented for Ag₂Se nanoparticles synthesized using microbial systems, which exhibited particle sizes in the range of 30 to 40 nm (Ozdal 2024). In addition, near-spherical and polymorphic Ag–Se nanoparticles with an average size of 33.1 nm were reported using Ocimum tenuiflorum extract (Olawale et al. 2021). SEM analysis (Fig. 2c) demonstrated that the Ag–Se NPs possessed a semi-spherical to irregular morphology with noticeable aggregation, which is a common feature in green-synthesized nanoparticles. The relatively larger apparent size observed in SEM compared to TEM can be attributed to surface aggregation and the presence of organic capping agents derived from Punica granatum peel extract, which may enhance particle clustering and influence the observed morphology (Martínez-Vieyra et al. 2020). The presence of phytochemicals on the nanoparticle surface confirms their role as reducing and capping agents during synthesis. EDX analysis (Fig. 2d) verified the elemental composition, showing silver (Ag) and selenium (Se) as the main components, along with carbon and oxygen signals attributed to plant-derived biomolecules. These results confirm the successful formation of Ag–Se nanoparticles and highlight the role of natural compounds in their stabilization, in agreement with previous studies (Numan et al. 2022).

Fig. 1. (a) UV–Vis and (b) FTIR of Ag–Se NPs

Fig. 2. (a) TEM image, (b) Particle size distribution (PSA-TEM), (c) SEM image and (d) EDX spectrum confirming the elemental composition of the Ag–Se NPs

Antimicrobial and Antibiofilm Activity

The increasing emergence of multidrug-resistant bacteria has directed attention toward bimetallic nanomaterials because of their enhanced antimicrobial performance (Hao et al. 2024). Both monometallic SeNPs and bimetallic Ag–Se NPs have demonstrated antibacterial activity against Gram-positive and Gram-negative bacteria through membrane damage and disruption of essential cellular processes (El-Behery et al. 2023; Soliman et al. 2024). Ag–Se NPs exhibited broad-spectrum antibacterial activity against Staphylococcus aureus, Escherichia coli, and Bacillus subtilis (Fig. 3a). E. coli was the most sensitive, with MIC and MBC values of 62.5 and 125 µg/mL, respectively. The corresponding values were 250 and 250 µg/mL for S. aureus and 500 and 1000 µg/mL for B. subtilis. Pomegranate-peel polyphenols, flavonoids, and tannins retained on the nanoparticle surface may contribute to this activity by increasing membrane permeability and interfering with microbial proteins (Maphetu et al. 2022). However, their independent contribution cannot be confirmed because the extract alone was not evaluated. These findings agree with previous reports on the antibacterial activity of bimetallic nanoparticles (Padilla-Cruz et al. 2021; Shumbula et al. 2024). The observed activity may result from complementary interactions between silver and selenium, together with the small particle size and high surface area of the nanoparticles (Singh et al. 2022; Jena et al. 2020).

Ag–Se NPs also produced concentration-dependent biofilm inhibition against S. aureus and E. coli, with greater activity against E. coli (Fig. 3b). This effect may involve interference with bacterial adhesion, EPS production, and quorum-sensing pathways (Rasheed et al. 2024). Previous studies have similarly reported antibiofilm activity for monometallic AgNPs and SeNPs and bimetallic Ag–Se NPs (El-Behery et al. 2023; Khormi et al. 2025; Fahmy et al. 2025). Metal-based nanoparticles have also been proposed for coating biomedical surfaces to reduce microbial colonization (Uthra et al. 2024). In addition, pomegranate-peel phytochemicals present on the nanoparticle surface may contribute to biofilm inhibition by reducing bacterial adhesion and EPS formation; however, this effect requires confirmation using an extract-only control.

Fig. 3. Evaluation of antibacterial (a) and antibiofilm (b) activities of biosynthesized Ag–Se nanoparticles against S. aureus, E. coli, and B. subtilis

Cytotoxicity of Ag–Se NPs on Normal and Cancer Cell Lines

Evaluating the cytotoxic profile of newly synthesized nanomaterials against normal cells represents a critical initial step in determining their biomedical applicability. In the present study, the cytotoxic effects of biosynthesized Ag–Se NPs were assessed on Vero normal cells and MCF-7 human breast cancer cells at various concentrations (Fig. 4). The results demonstrated a clear concentration-dependent response. At lower concentrations (15.6 to 31.25 µg/mL), Ag–Se NPs exhibited minimal cytotoxic effects on Vero cells, maintaining high cell viability (93.35 to 98.56%), indicating good biocompatibility. However, increasing concentrations (500 to 1000 µg/mL) resulted in a significant reduction in cell viability, confirming dose-dependent toxicity. The IC₅₀ value for Vero cells was calculated to be 209.3 µg/mL, suggesting relatively low toxicity toward normal cells. In contrast, Ag–Se NPs exhibited pronounced anticancer activity against MCF-7 cells, where cell viability decreased sharply with increasing nanoparticle concentration. The IC₅₀ value for MCF-7 cells was determined to be 114.7 µg/mL, indicating higher sensitivity of cancer cells compared to normal cells. These findings highlight the selective cytotoxic potential of Ag–Se NPs toward tumor cells. Such selective behavior may be attributed to differences in cellular metabolism, membrane composition, and oxidative stress responses between normal and cancer cells. Similar observations have been reported for various bimetallic nanoparticles, which exhibit enhanced anticancer properties compared to monometallic systems (Nieto-Argüello et al. 2022). For instance, pomegranate peel-mediated Ag–ZnO nanoparticles showed significant anticancer activity against MCF-7 cells with IC₅₀ values comparable to the present findings (Ehsan et al. 2022).

Fig. 4. Dose-dependent cytotoxicity and cell viability of Ag–Se NPs against Vero and MCF-7 cell lines

Antioxidant Activity

The antioxidant activity of the biosynthesized Ag–Se nanoparticles was assessed using DPPH and ABTS assays (Fig. 5). The results showed a clear concentration-dependent scavenging effect across the tested range. In both assays, Ag–Se NPs demonstrated moderate antioxidant activity compared to ascorbic acid, which exhibited superior efficiency. These findings are consistent with previous reports highlighting the dose-dependent antioxidant behavior of bimetallic nanoparticles (Hassanin et al. 2024). The observed antioxidant activity of Ag–Se NPs can be attributed to their ability to donate electrons or hydrogen atoms, thereby neutralizing free radicals such as DPPH and ABTS (Li et al. 2011). Additionally, the enhanced surface area-to-volume ratio of nanoparticles facilitates greater interaction with reactive species, improving their scavenging efficiency. Plant-mediated synthesis further contributes to antioxidant activity due to the presence of bioactive phytochemicals acting as capping agents (ŞEVK et al. 2025).

Fig. 5. DPPH and ABTS radical scavenging activity of Ag–Se NPs compared with ascorbic acid

CONCLUSIONS

Silver–selenium nanoparticles were successfully synthesized using Punica granatum peel extract via a green, eco-friendly approach, and their formation was confirmed using multiple characterization techniques.
The synthesized nanoparticles exhibited effective antibacterial activity against both Gram-positive and Gram-negative bacteria, with clear concentration-dependent effects.
Ag–Se NPs showed notable antibiofilm activity, indicating their potential in controlling biofilm-associated infections.
The nanoparticles demonstrated selective cytotoxicity, showing lower toxicity toward normal cells while exerting stronger anticancer effects against cancer cells.
Moderate antioxidant activity was observed in both DPPH and ABTS assays, supporting their potential biomedical applications.

ACKNOWLEDGMENTS

This research was funded by Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2026R461), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia. This work was supported by the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia (Grant No. KFU264499).

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Article submitted: May 3, 2026; Peer review completed: August 8, 2026; Revised version received and accepted: August 13, 2026; Published: September 4, 2026.

DOI: 10.15376/biores.21.4.10326-10338