Green biosynthesized silver nanoparticles using aqueous extract of Salix alba: antimicrobial and cytogenetic effects on mitosis

2.1 Plant collection and pretreatment

In September 2024, leaves bark of S. alba plant were gathered locally from Baghdad marketplaces. The plant was identified by Dr. Ibrahim S. Al–Jubouri/College of Pharmacy /Al–Mustansiriyah University/Iraq.

2.2 Preparation of the aqueous extract

The aqueous plant extract of S. alba was prepared using the traditional method (Ghazali et al., 2022) by washing the plant parts with D.W. well to remove contaminants from the surface and drying them well with dry air for three days. 50 g of willow bark was ground well and placed in a glass beaker with a capacity of 500 mL that contains 300 mL of D.W. The mixture was heated at 60°C for 30 minutes. Then the extract was filtered using Whatman No. 1 filter paper and stored at 4°C for later use.

2.3 Reagents and chemicals used

All chemicals used in this study were of analytical grade. Silver nitrate (AgNO₃, Sigma-Aldrich, Cat. No. S7653), potassium chloride (KCl, Sigma-Aldrich, Cat. No. P9541), and phosphate-buffered saline (PBS, Gibco, Cat. No. 10010023) were employed during nanoparticle synthesis and experimental procedures. Giemsa stain used for cytogenetic evaluation was obtained from Merck (Cat. No. 1.09204). All reagents were prepared according to the manufacturers’ instructions, and the amounts used in each experiment are detailed within the methodological subsections.

2.4 Biosynthesis of silver nanoparticles

Silver nanoparticles (AgNPs) were synthesized according to Subhani et al., (2024) using bark of S. alba aqueous extract as a plant-based reducing agent and silver nitrate (AgNO₃) as the silver source. The reaction mixtures were prepared by combining 9 mL of AgNO₃ at varying concentrations (1.0, 1.5, 2.0, and 2.5 mM) with 1 mL of the previously prepared willow bark extract. It should be noted that concentrations expressed in this study (mM) refer to molarity of AgNO₃ used during nanoparticles synthesis while all biological assays were performed using AgNPs synthesized from these precursor molarities. The mixtures were then incubated in a dark chamber at 30°C for 24 hours without agitation to prevent photoreduction of silver nitrate. The selected conditions were adopted from previously reported studies demonstrating efficient green synthesis of stable silver nanoparticles. The selected AgNO₃ concentrations were chosen based on preliminary optimization experiments and in accordance with commonly reported ranges (0.5-5 Mm) to ensure controlled nanoparticles formation and stability. These concentrations were specifically selected as they are known to influence nanoparticles formation, including particle size, morphology and yield. The nanoparticles were purified by centrifugation at 10000 rpm for 15 minutes, pellet was washed 3 times with distilled water. Each experiment was performed in triplicate (n = 3) to ensure reproducibility. pH was maintained at 7.0, and visual color change was monitored at regular intervals (every 2 hours) until stabilization. The molarities (1-2.5 mM) refer to the concentration of AgNO₃ used during nanoparticles synthesis. The synthesized AgNPs were used in antimicrobial assays.

All experiments were performed under identical conditions to ensure batch-to-batch reproducibility and each synthesis was conducted in triplicate (n = 3).

2.5 Characterization of the plant-based green synthesized of silver nanoparticles

The subsequent methods were used to characterize the plant-based green synthesized nanoparticles. Every test administered in the laboratory of Ministry of Science and Technology, Baghdad, Iraq.

2.5.1 Visual observation

Silver nanoparticles are characterized by a noticeable color change by different period, which serves as an important indicator for the early detection of green-synthesized NPs.

2.5.2 Ultraviolet visible is spectroscopy

One practical approach for identifying the formation of green-synthesized nanoparticles by (Masood et al., 2025) through UV–Vis spectrophotometry. The absorbance spectra were recorded using a UV-Vis spectrophotometer (SmartSpec 3000, Bio-Rad Laboratories, USA) over a wavelength range of 200-800 nm.

2.5.3 Atomic force microscopy (AFM) and scanning electron microscopy (SEM)

Atomic force microscopy (AFM) and scanning electron microscopy (SEM) analyses were conducted using an NT-MDT scanning probe microscope. The nanoparticle samples were first diluted with distilled water, then a drop of the diluted solution was placed on a clean glass slide (1×1 cm). After allowing the sample to air-dry completely, the slide was mounted on the AFM sample stage, and imaging was performed following standard operating procedures (Tran et al., 2025).

2.5.4 Antimicrobial activity

Agar well diffusion method was used to evaluate the antimicrobial activity of the synthesized silver nanoparticles. The bacterial sample that used in this study was provided by the Molecular Biology Laboratory for postgraduate research/Department of Biology/College of Science/University of Baghdad.

2.5.5 Antimicrobial activity of silver nanoparticles

Mueller Hinton agar was prepared by dissolving 38 g of the medium in 1 L of distilled water. The solution was heated until completely dissolved and sterilized by autoclaving at 121°C for 15 min. After cooling to 45–50 °C, the medium was poured into sterile Petri dishes (20–25 mm in depth) under aseptic conditions and allowed to solidify. For antimicrobial testing, two bacterial strains (Staphylococcus aureus and Escherichia coli) and one fungal strain (Candida albicans) were used. A single colony from each strain was inoculated into brain–heart infusion broth and incubated for 18 h at 37°C. After incubation, 1 mL of each culture was transferred into 5 mL of sterile saline and vortexed to achieve homogeneity. The turbidity of the suspensions was adjusted to match 0.5 McFarland standard (~1.5 × 10⁸ CFU/mL). By using sterile swabs, the standardized microbial suspensions were evenly spread across the entire surface of the agar plates to ensure uniform distribution. Then, 100 μL of green-synthesized AgNPs from S. alba extract were carefully applied to the surface using a sterile micropipette. Four concentrations of AgNPs (100, 75, 50, and 25 μg/mL) were tested. The inculated plates were incubated at 37°C for 18 h, after which antimicrobial activity was evaluated based on the diameter of inhibition zones formed around each application site.

2.5.6 Animal acclimatization and experimental design

Adult male Swiss Albino mice (Mus musculus), aged between 8–10 weeks and weighing 23–27 g, were obtained from the Biotechnology Research Centre at Al-Nahrain University. The animals were housed under standard laboratory conditions for acclimatization. The animals were randomly assigned into three groups, each consisting of Six mice: (1) Control group: Received normal saline. (2) AgNO₃ NP group: received a single dose of 150 mg/kg. (3) Salix alba L extract group: a single dose of 400 mg/kg. Following treatment, all animals were sacrificed and femoral bone marrow was extracted to calculate the mitotic index.

2.5.7 Euthanasia and anaesthesia procedures

All procedures involving animals, including anesthesia and euthanasia, were performed in accordance with the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2020). Prior to euthanasia, mice were briefly anesthetized using isoflurane inhalation (2–3% in oxygen) to minimize distress. Euthanasia was performed by cervical dislocation while under anesthesia, as recommended for small laboratory rodents. The dose of colchicine (0.25 mL of 1 mg/mL, intraperitoneal) used for metaphase arrest was applied following standard cytogenetic protocols and did not cause pain or distress at the time of euthanasia.

2.5.8 Chromosome preparation of the mouse bone marrow

The experiment was done according to (Allen et al., 1977) as follow: For cytogenetic preparation, each animal was intraperitoneally injected with 0.25 ml of colchicine (1 mg/ml) to arrest cells in metaphase. Following euthanasia by cervical dislocation, the femur was aseptically extracted, and the bone marrow was flushed using 5 ml of phosphate-buffered saline (PBS, pH 7.2) into a sterile test tube. The suspension was centrifuged at 2000 rpm for 10 min., and the supernatant was discarded. Cells were then treated with 5 ml of 0.075 M potassium chloride (KCl) as a hypotonic solution and incubated at 37°C with intermittent shaking. After a second centrifugation at the same speed and duration, a chilled fixative was added dropwise with gentle agitation to a final volume of 5 ml. The mixture was kept at 4°C for 30 min. and centrifuged multiple times to ensure proper fixation, with the final suspension adjusted to 2 ml of fixative. A few drops (4–5) were dropped vertically from a height of approximately 90 cm onto pre-chilled glass slides to ensure proper chromosome spreading. After air drying, the slides were stained with Giemsa solution for 15 min. and rinsed with distilled water. Three slides were prepared per animal for cytogenetic examination under the microscope. This section must include enough detail on the data sources and processes so that others can reproduce your research. The selected 150 mg/kg body weight chosen based on previously published studies (Kim et al., 2008; Asharani et al., 2009). The mitotic index was determined by counting metaphase –arrested cells following colchicine treatment. A total of 1000 cells were examined using standard formula: MI% = (number of metaphase cells/total number of counted cells)*100.

2.6 Ethics approval

All animal procedures in this study were reviewed and approved by the Animal Ethics Committee of Al-Nahrain University, College of Biotechnology. The study was conducted in full compliance with institutional guidelines for laboratory animal care and the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2020). No additional permits or animal licenses were required for this study; if this information is not available, this is because the institution does not issue separate license numbers for routine academic research involving mice.

2.7 Statistical analysis

The results were statistically analysed using one-way ANOVA followed by Tukey’s post-hoc test to compare differences between groups. A significance level of p < 0.05 was considered statistically significant. All analyses were carried out using SPSS version 25.0 (IBM Corp., Armonk, NY, USA). Data were tested for normality using the Shapiro-Wilk test and for homogeneity of variance using levene’s test prior to performing one-way ANOVA followed by Tukey’s post hoc test.

3.1 Biogenic silver nanoparticles

The formation of green silver nanoparticles was confirmed through noticeable changes in the color of the reaction mixture as well as spectrophotometric analysis (Hussain et al., 2025). To better illustrate the nanoparticle formation process,image of the reaction mixture at time zero (immediately after adding the extract to AgNO₃) and after 2h of incubation have been included in ( Figure 1), The solution initially appeared pale yellow and gradually intensified to light brown,eventually reaching a dark brown color after 24 hours. The darker color observed at 2 h compared to 24 h may be due to aggregation or change in nanoparticles size and distribution over time. This shift in color indicates the successful reduction of silver ions (Ag⁺) into elemental silver nanoparticles (Ag⁰), likely driven by the active phytochemicals present in the willow bark extract. The brown coloration is commonly linked to the excitation of surface plasmon resonance (SPR), which is characteristic of silver nanoparticles (Muhammad et al., 2025). Spectral analysis showed a distinct absorbance peak around 433 nm, confirming the presence of AgNPs. The intensity of this SPR peak increased with higher concentrations of AgNO₃, reaching its maximum at 2 mM, suggesting more efficient nanoparticle formation at this concentration. The synthesis process may have been influenced by hydrophilic and hydrophobic interactions among the components, which can promote nanoparticle stability and shape (Hindryawati et al., 2025).

Figure 1. Visual observation of green-synthesized silver nanoparticles at different time (a) zero time, (b) after 2 hours and (c) after 24 hours.

(A–D) represent willow bark extract loaded with silver nanoparticles at concentrations of 1, 1.5, 2, and 2.5 mM AgNO₃, respectively. (E) shows the aqueous willow bark extract without the addition of AgNO₃.

These findings are in line with previous reports showing that silver nanoparticles in aqueous solution typically exhibit brown coloration due to SPR excitation (Khalid et al., 2025). Additionally, factors such as the composition of the biological extract and the concentration of metal salts play a key role in determining nanoparticle yield and characteristics. The unique optical properties of noble metals like silver stem from their ability to support surface SPR, making them ideal for nanoparticle-based applications (Saini et al., 2025) ( Figure 2).

Figure 2. UV–visible absorption spectra of green-synthesized silver nanoparticles, showing a characteristic surface plasmon resonance peak at 433 nm.

Atomic force microscopy (AFM) imaging provided valuable insight into the surface morphology and topography of the green-synthesized silver nanoparticles at varying silver nitrate concentrations. As seen in ( Figure 3 A–D), the nanoparticles exhibited distinguishable differences in size distribution and surface roughness depending on the precursor concentration.

Figure 3. Atomic Force Microscopy (AFM) 3D surface morphology of green-synthesized silver nanoparticles at different AgNO₃ concentrations: (A) 1 mM, (B) 1.5 mM, (C) 2 mM, and (D) 2.5 mM.

At 1 mM ( Figure 3-A), the nanoparticles were densely distributed with an average particle size of approximately 79.57 nm and an average height of 75 nm, indicating a relatively uniform structure that similarity to Jilani et al., (2025).

As the concentration increased to 1.5 mM ( Figure 3-B), the average size decreased to 69.19 nm with a reduced height of 65 nm. This suggests that a moderate increase in AgNO₃ concentration may enhance the nucleation rate, yielding smaller and more compact nanoparticles due to limited aggregation (Koçer and Özçimen 2025).

Interestingly, at 2 mM ( Figure 3-C), the average particle size rose to 85.66 nm and the height reached 80 nm. This might be attributed to particle growth dominating over nucleation, likely caused by the saturation of reducing agents in the S. alba extract, resulting in fewer but larger particles (Benaissa et al., 2025).

However, when the concentration increased to 2.5 mM ( Figure 3-D), the particle size again decreased to around 70 nm with a height of 60 nm. This fluctuation indicates that beyond a certain concentration threshold, the stabilizing capacity of the phytochemicals becomes insufficient to control the growth, leading to partial aggregation and size variation (Elumalai et al., 2025).

The grain size distribution charts ( Figure 4 A-D), further support these observations, showing a shift in peak particle populations depending on the concentration. Most particles fell within the 50–100 nm range, consistent with effective nanoscale synthesis. The narrow size distribution observed, especially at 1.5 and 2.5 mM, suggests enhanced control over particle growth when silver ion concentration and phytochemical reducing agents are well balanced (Zhu et al., 2025).

Figure 4. AFM particle size distribution analysis of green-synthesized silver nanoparticles at different AgNO₃ concentrations: (A) average height of 75 nm and 79.57 nm for average size, (B) average height of 65 nm and 69.16 nm for average size, (C) average height of 80 nm and 85.60 nm for average size, and (D) average height of 60 nm and 70 nm for average size.

Overall, the AFM results confirm that silver nitrate concentration plays a crucial role in determining nanoparticle size, height, and distribution. Optimal synthesis appears to occur around 1.5 to 2 mM, where particle uniformity and stability are most favorable. These findings highlight the sensitivity of green synthesis to reactant concentrations and the importance of fine-tuning reaction parameters to achieve desirable nanoparticle characteristics (Wilson et al., 2025).

As the previous figure which presents AFM-based measurements of the surface heights of green-synthesized silver nanoparticles prepared at varying silver nitrate concentrations. The recorded average heights were 75 nm (1 mM), 65 nm (1.5 mM), 80 nm (2 mM), and 60 nm (2.5 mM), indicating noticeable morphological variation. These differences suggest that the concentration of silver precursor plays a key role in controlling nanoparticle growth and surface topology.

The SEM images as Figure 5 A–D reveal clear differences in surface morphology and particle aggregation of green-synthesized silver nanoparticles at various magnifications. Spherical to semi-spherical particles are visible with varying levels of distribution, indicating successful formation and surface interaction influenced by silver nitrate concentration (Ahmed and Rahmah, 2025). The SEM images show micrometer-scale structures, which are attributed to nanoparticle aggregation during sample preparation and drying. The features represent agglomerated clusters rather than individual nanoparticles. In contrast, AFM analysis and the size distribution results confirm that the nanoparticles are within the nanoscale range, indicating that SEM observation reflect aggregation rather than actual particle size. The aggregation behaviour is commonly observed in green-synthesized nanoparticles due to the presence of plant-derived biomolecules. It may influence biological activity by reducing surface area and cellular interaction while enhancing stability.

Figure 5. Scanning electron microscopy (SEM) images of green-synthesized silver nanoparticles at different silver nitrate concentrations: (A) 1 mM, (B) 1.5 mM, (C) 2 mM, and (D) 2.5 mM. All images were captured at a magnification of 5.00 kx with a uniform scale bar of 10 μm.

3.2 Antimicrobial activity

The experimental part of the study included several biological assays to evaluate the antimicrobial activity of the prepared extract against selected bacterial (E. coli and S. aureus) and fungal strains (C. albicans) under laboratory conditions, as shown in Figure 6 A-C.

Figure 6. Antibacterial activity of silver nanoparticles synthesized using aqueous extract of Salix alba L using AgNPs synthesized from different precursor of AgNO₃ (1, 1.5, 2, and 2.5 mM) against (A) Escherichia coli, (B) Staphylococcus aureus, and (C) Candida albicans.

In this study, the aqueous extract of S. alba was used to synthesize silver nanoparticles and tested for antimicrobial activity against E. coli in vitro ( Figure 6-A). Petri dishes containing nutrient medium were treated with different precursor molarity of (AgNO₃) (1.0, 1.5, 2.0, and 2.5 mM). The inhibition zones measured 8, 10, 14, and 15 mm, respectively. The gradual increase in inhibition zone diameter with higher concentrations confirms a dependent on the molarity of AgNO₃ used during synthesis antibacterial effect of the synthesized nanoparticles (Agrawal et al., 2025).

Where, the antibacterial activity of the S. alba based silver nanoparticle extract was evaluated against S. aureus using a nutrient-rich medium inoculated with the bacterial strain. Different precursor molarity of AgNO₃ (1, 1.5, 2, and 2.5 mM) produced inhibition zones measuring 13, 15, 17, and 18 mm, respectively. Previous studies which Turay (2025) who estimated that the gradual increase in zone diameter with higher concentrations confirms a positive correlation between extract dose and antibacterial effectiveness. Although, Shalaby et al., (2025). The gradual increase in zone diameter with higher concentrations confirms a positive correlation between extract dose and antibacterial effectiveness. This enhanced activity at elevated concentrations may be attributed to the increased availability of silver nanoparticles and phytochemicals, which together intensify cell wall disruption, protein denaturation, and oxidative stress within the bacterial cells (Wang et al., 2025), as shown in Figure 6-B.

Furthermore, the antifungal activity of the green-synthesized AgNO₃ NPs was evaluated against C. albicans. As shown in the Figure 6-C, four different concentrations of the S. alba NPs extract that have different molarity concentration (1, 1.5, 2, and 2.5 mM) were tested. Clear inhibition zones were observed around each well (13, 15, 17 and 18 mm), respectively, with their diameters increasing proportionally with concentration (Ibrahim et al., 2025). This suggests a dependent on the molarity of AgNO₃ used during synthesis antifungal effect, likely due to enhanced NPs interaction with the fungal cell membrane. The increased presence of bioactive compounds and silver ions may contribute to membrane disruption, oxidative stress, and enzyme inhibition in C. albicans (Beyatli, 2025).

When compared with other plant-mediated silver nanoparticles, the antimicrobial performance of S. alba appears to be equally strong or superior. For example, Cassia siamea-derived AgNPs produced inhibition zones of 10–14 mm against S. aureus (Turay, 2025), while Acacia mangium-based AgNPs showed zones of 11–15 mm against E. coli (Aji et al., 2025). Similarly, Xanthium strumarium-mediated nanoparticles demonstrated inhibition zones around 12–15 mm against C. albicans (Beyatli, 2025). In our study, S. alba-derived AgNPs exhibited larger inhibition zones (up to 18 mm against S. aureus and C. albicans), suggesting that the phytochemical composition of willow bark rich in salicin, flavonoids, and phenolic compounds may enhance both nanoparticle stability and biological activity. These comparative results highlight the distinctive contribution of S. alba among plant-based nanoparticle systems.

The antimicrobial and cytogenetic effects of AgNPs generally belong to a combination of physical, chemical, and biochemical mechanisms rather than a single pathway. At the microbial level, AgNPs can adhere to the negatively charged bacterial cell wall, damaging membrane integrity and increasing permeability, which cause leakage of essential ions and metabolites (Ibrahim et al., 2025). Once internalized, AgNPs release silver ions (Ag⁺), which interact with sulfur- and phosphorus-containing biomolecules such as proteins and DNA, thereby impairing enzymatic activity and replication processes (Beyatli, 2025). Another well-documented mechanism is the generation of reactive oxygen species (ROS), including superoxide anions, hydroxyl radicals, and hydrogen peroxide, which induce oxidative stress, damage lipids, oxidize proteins, and fragment nucleic acids (Wang et al., 2025). These results are support by previous studies (Darwich et al., 2025; Aljohani et al., 2026) which indicated that biosynthesized silver nanoparticles have antibacterial activity because of different mechanisms like, damage of cell membrane, induction of oxidative stress and bacterial DNA and proteins interaction.

3.3 Chromosome from somatic cells of the mouse bone marrow

The results clearly indicated a decrease in mitotic index in silver nanoparticles treated group as compared with negative control. While it increased in plant extract group to 7.8% as compared to negative group 5%. As shown in Table 1 and Figure 7 (A-B).

Figure 7. Cytogenetic effects of aqueous extract from Salix alba L in mice bone marrow cells. (A) Representative microscopic image showing a general field view of bone marrow cells. (B) Magnified view highlighting mitotic cells (400X).

At the cytogenetic level, AgNPs may directly interact with chromosomes and mitotic machinery. Several studies have demonstrated that AgNPs can cause chromosomal aberrations, spindle disruption and disruption of cell-cycle progression, which is consistent with the observed reduction in mitotic index (Tang, 2025; Osman et al., 2025). For cancer therapy for the recent decades natural products play an important role in the development of a drug. In addition to the possible relationship between natural products and regulation of telomerase working (Shalash et al., 2021). ROS-mediated DNA strand breaks, coupled with impaired repair pathways, further contribute to chromosomal instability (Zhang et al., 2025). Additionally, AgNPs have been reported to interfere with mitochondrial respiration, leading to ATP depletion and triggering apoptosis, which indirectly reduces the pool of actively dividing cells (Bentrad, 2025).

It is also worth noting that the biological response to AgNPs is influenced by multiple factors, including nanoparticle size, shape, surface charge, and the phytochemical composition of the reducing agent used in synthesis (Kirubakaran et al., 2025). The phytochemicals in S. alba extract such as flavonoids, tannins, and salicin are likely to play a important role: (i) reducing and stabilizing silver nanoparticles, and (ii) exerting protective antioxidant effects that mitigate genotoxicity. This may explain why the group treated with S. alba extract alone showed normal or elevated mitotic index, in contrast to the significant suppression observed in the AgNP-treated groups. Such duality highlights the importance of considering both nanoparticle characteristics and plant-derived biomolecules when evaluating biosafety as explained in (Chaudhary et al., 2025b).

Mitotic index in AgNP-treated groups decreased which can be mechanistically explained by nanoparticle-induced oxidative stress and genotoxic effects. Silver nanoparticles (AgNPs) have the ability to penetrate cellular membranes and generate reactive oxygen species (ROS), which cause damage of DNA, proteins, and lipids, led to disruption of cell-cycle progression and inhibition of mitosis. Several recent studies have demonstrated that AgNP exposure induces DNA strand breaks, chromosomal abnormalities, and cell-cycle arrest, which collectively affect cellular proliferation (Huang et al., 2025; Sánchez-Alarcón et al., 2025). A significant decrease in mitotic index have observed in vivo studies on bone marrow cells following AgNP exposure due to genotoxic stress and chromosomal damage (Mahmood & Ahmed, 2024).

In contrast, the relatively higher mitotic index observed in plant extract-treated groups may be attributed to the antioxidant activity of phytochemicals present in Salix alba L. This plant is rich in phenolic compounds, flavonoids, and related bioactive molecules that possess strong free radical scavenging properties. These compounds can reduce oxidative stress by neutralizing ROS and protecting cellular components from nanoparticle-induced damage, thereby preserving normal cell division. Recent studies have highlighted the role of plant-derived polyphenols in mitigating oxidative damage, enhancing cellular defense mechanisms, and supporting cell survival and proliferation under stress conditions (Sharma et al., 2024; Muscolo et al., 2024).

The cytogenetic effects by MI which reported in the present study are comparable to those reported for other plant mediated AgNPs in recent literature. In this study, AgNP-treated groups showed a marked reduction in MI (1.2 ± 0.14%) as compared to negative control (5 ± 0.30%). Similar reduction in previous studies, where plant mediated AgNPs decreased MI to 1-3% in bone marrow cells, depending on nanoparticles size, dose, and synthesis method (Mahmood & Ahmed, 2024; El-Naggar et al., 2022).

Overall, the results demonstrate that S. alba mediated AgNPs exhibit strong antimicrobial and measurable cytogenetic effects in a dose-dependent manner. To better interpret these outcomes, that compare our findings with previous studies and discuss possible mechanisms that may explain the observed biological activities.