Eco-friendly synthesis of silver nanoparticles using Anemone coronaria bulb extract and their potent anticancer and antibacterial activities

Abstract

Silver nanoparticles (AgNPs) have emerged as promising multifunctional agents in biomedical applications due to their notable antimicrobial and anticancer properties. In this study, we present a green, sustainable, and cost-effective method for synthesizing AgNPs using the bulb extract of Anemone coronaria, an underutilized plant part that allows year-round resource-efficient production. The synthesized AgNPs were characterized by UV–Visible spectroscopy, scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), X-ray diffraction (XRD), and Fourier-transform infrared spectroscopy (FTIR). The nanoparticles exhibited a characteristic UV–Vis absorption peak at 425 nm, spherical morphology with an average size of 29 nm, and a face-centered cubic crystalline structure. FTIR analysis confirmed the presence of phytochemicals involved in the reduction and stabilization of AgNPs. The biosynthesized AgNPs showed potent antibacterial activity against both Gram-positive and Gram-negative bacteria, and demonstrated significant cytotoxic effects on cancer cells while sparing normal cells. Apoptosis induction, cell cycle arrest, and gene expression analyses further validated their anticancer efficacy. These findings highlight the therapeutic potential and biocompatibility of Anemone coronaria-derived AgNPs, offering a green nanotechnological approach for future biomedical applications.

Introduction

Despite significant advances in the field, cancer remains one of the leading causes of death worldwide. It is thought that the number of deaths, currently estimated at approximately 10 million, will reach 30 million in the next 5–10 years. Chemotherapy, surgery, and radiotherapy are the first-line treatment strategies in cancer therapy. The toxicity and serious side effects of chemotherapy, which is frequently used before or after surgery, on healthy cells put healthy cells at a disadvantage¹. The development of nanotechnology based on nano-sized materials has enabled nanoparticles (NPs) to offer new opportunities as an alternative to conventional treatments in cancer². Rapid advances in this field over the last two decades and the regulatory approval of many nanomedicines promise the development of new nanoparticle-based drugs in the future³.

Many physical and chemical synthesis approaches are used in the production of nanoparticles, differing in various aspects such as price, efficiency, and energy consumption^4–6. Traditional methods used in nanoparticle production, such as vapor deposition, flame pyrolysis, the sol–gel process, thermal plasma synthesis, inert gas condensation, and laser pyrolysis, present significant disadvantages for biological applications due to the presence of toxic chemicals⁷. In recent years, green synthesis methods that do not contain toxic substances have come to the fore in the synthesis of biocompatible nanoparticles in nanotechnology, as they are environmentally friendly, cost-effective, safe, and sustainable⁸.

Biological synthesis of NPs using various plants is attracting more attention, as plant extracts serve as reducing and stabilizing agents, removing chemical substances. Nowadays, therapeutic plant parts with various bioactive components, such as polyphenols, flavonoids, and alkaloids, are frequently preferred in NP synthesis^9,10.

Green-synthesized silver nanoparticles (AgNPs) were produced using Anemone coronaria bulb aqueous extracts. Anemone, a genus of Ranunculaceae, has long been used in ethnomedicine worldwide. Anemone plant components have immunomodulatory, antiinflammatory, antioxidant, and antimicrobial activity. It contains various medicinal compounds, especially triterpenoid saponins, some of which have anticancer activity¹¹. A. coronaria contains anthocyanins (in flowers), triterpene glycosides (in bulbs), and alkaloids (in plant parts), and due to this rich bioactive content, it has been used in different regions for different purposes in traditional medicine^12,13. A. coronaria bulbs have been found to contain 17 different triterpene glycosides. These compounds are known to exhibit a wide range of biological and pharmacological properties, including antimicrobial, antiinflammatory, and antitumor properties^14,15.

In this study, the green synthesis method was used for the synthesis of AgNPs exhibiting antimicrobial activity against infectious microorganisms such as bacteria and fungi^16,17. A. coronaria bulbs with various biological activities were used. The synthesized AgNPs were characterized, and their antibacterial and anticancer activities were investigated. The use of the plant’s bulb extract is quite advantageous and may be interesting in nanodrug development, as it allows the production of anticancer AgNPs without waiting for the plant to be grown. AgNPs synthesized from A. coronaria bulb extract were characterized by UV–Vis and FT-IR spectroscopic methods as well as XRD, DLS, SEM–EDX, and TEM methods. Antibacterial activity was tested against Gram-positive Streptococcus aureus and Gram-negative Escherichia coli. In addition, the cytotoxic activities of the synthesized AgNPs were determined by MTT assay in various cancer cells (human lung carcinoma, A549; human pancreatic cancer, MIA PaCa-2; human prostate cancer, PC-3) and healthy cells (human embryonic kidney cells, HEK293). The antitumoral effect observed on cancer cells was demonstrated by analysis of apoptosis and cell cycle. The effect on cell apoptosis was supported by the gene expression level.

Materials and methods

Plant extract preparation and green synthesis of silver nanoparticles

The bulbs of A. coronaria were purchased from a commercial supplier (n11.com, Türkiye). As the plant material was obtained from a commercial source, no specific permits were required. The plant species was identified by the supplier, and a representative specimen has been retained by the authors for reference. All procedures involving plant material complied with relevant institutional, national, and international guidelines and regulations, including the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES).

To prepare the extracts of the bulb of the A. coronaria flower, the bulb (7 g) was first divided into 4 parts using a sharp knife. After the chopped bulb was placed in 250 mL of distilled water, it was boiled at 90 °C for 20 min. After the boiled bulb extract was cooled, it was kept in the refrigerator for one night and then filtered to prepare it for the green synthesis process. After the bulb extract was prepared, green synthesis of nanosilver was carried out. For the ratios and parameters used for synthesis, previous studies in the literature were taken into consideration^18–24. Silver nitrate (AgNO₃) and bulb extract were used for the generation of silver nanoparticles. Sigma Aldrich supplied the silver nitrate salt (99%). The first stage in the fabrication of silver nanoparticles was the preparation of a 0.1 mM concentration of AgNO₃ (0.6 g of AgNO₃ salt was added to 36 mL deionized (DI) water), and subsequent magnetic stirring at room temperature in order to create a homogeneous solution. After adding 4 mL (10%) of bulb extract, the reaction mixture’s color changed from translucent to nearly black in 4 min, indicating the creation of AgNPs, and stirring was then ended. Bulb extract preparation and green synthesis mechanism are shown in Fig. 1. The extract has a light yellow color. The synthesized black nano-silver particles are clearly visible in the beaker.

Fig. 1.

Green synthesized silver nanoparticles using Anemone coronaria bulb extract and its synthesis mechanism.

Characterizations

The microstructure and EDS chemical mapping of green-synthesized silver nanoparticles have been studied utilizing an 80 mm² X-MAX detector and an EDS attachment on a JEOL 7001F Field Emission (FE) Scanning Electron Microscope (SEM). The surface morphologies, distribution, and components of silver nanoparticles from bulb extracts were examined. After drying in an oven at 40 °C for 20 min, 1 mL of the colloidal solution’s nanoparticles was placed in an ultrasonic bath for 10 min before being deposited into the SEM sample holder. The noticeable aggregation in the SEM pictures occurred during the drying stage. The Mastersizer 3000 laser technology (ISO-13320) method was used for particle size analysis to calculate the average size of nanosilver. The average particle size for silver nanoparticles was estimated by fitting the particle size distribution histogram to the log-normal distribution function, as shown in Eq. (1);²⁵

1

where D corresponds to average particle size and σ_D is the standard deviation. The wavelength range for Ag-NPs is 300 to 700 nm, as measured by a UV–Vis spectrophotometer 48 h after biosynthesis (Perkin Elmer, Lambda 25). To analyze the effects of bulb extracts and chemical interactions with silver nanoparticles, FT-IR analysis (Bruker Tensor 27) was performed in the wavenumber range of 650–4000 cm-1. X-ray Diffraction (XRD) was done at a rate of 1°/min in the range of 20° ≤ 2θ ≤ 80° at room temperature using a Rigaku Smart Lab CuK radiation monochromatic filter. The crystallite size of silver nanoparticles was calculated using Debye–Scherrer’s Eq. (2). The Debye-Scherer equation is used to determine the size of nanocrystals in a material.

2

where D is the Crystallites size (nm), λ the X-ray wavelength (k = 0.1541 nm), K the so-called shape or geometry factor which usually takes a value of about 0.9 (Scherer constant), β the full width at half maximum (FWHM) of diffraction peak and ‘‘θ’’ the diffraction angle.

Antibacterial activities of green synthesized silver nanoparticles

The agar well diffusion method was used to determine antimicrobial activity²⁶. Mueller–Hinton Agar was used as the medium in our study. Bacterial strains from stock cultures of a Gram-negative bacteria Escherichia coli (ATCC 25,922) and a Gram-positive bacteria Staphylococcus aureus (NCTC-13552) were separately suspended in 5 mL broth. After adjusting the density in the petri dish, the bacterial suspension was 108 CFU/mL. Each container was seeded with 100 µL. The inoculation was performed with the spread plate method by gently applying a sterile swab across the petri dish at frequent intervals. All Petri plates were then left to dry at room temperature for 5–15 min. Each of the 10, 50, 100, 150 µL AgNPs solutions was transferred to wells with a diameter of 5 mm on agar. In addition, 10 µg of ampicillin was used as a positive control. After 24 h of incubation, the diameters of the inhibition zones formed around the wells were measured. Antimicrobial activity assays against all test microorganisms were performed in duplicate.

Cell culture

The anticancer potential of green synthesized AgNPs was investigated on human lung carcinoma cell line A549, human pancreatic cancer cell line MIA PaCa-2, and human prostate cancer cell line PC-3. HEK293 cells, a human embryonic kidney cell line, were used as healthy cells. A549, MIA PaCa-2, and HEK293 cells were cultured in high glucose (4.5 g/L) Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS and 100 U/mL penicillin, 100 μg/mL streptomycin, and 25 μg/mL amphotericin B; whereas PC-3 cells were cultured in RPMI 1640 medium. The cell lines were incubated under controlled conditions at 37 °C with 5% CO₂. When the cells reached 80–90% density, they were subcultured with 0.25% trypsin containing 0.02% EDTA. The cell lines used in this study were kindly provided by Prof. Dr. Fatih Kocabaş (Department of Bioengineering, Yeditepe University, Istanbul, Turkey).

Cytotoxic effect of green synthesized silver nanoparticles

The cytotoxic effect of AgNPs on human cancer and healthy cells was evaluated by a colorimetric assay, MTT (3-(4.5-Dimethylthiazol-2-yl)-2.5-Diphenyltetrazolium Bromide). Cells were seeded in 100 µL culture volume, in 96-well culture dishes (Costar), at 7.5 × 10³ cells per well, and incubated for 24 h in a 5% CO₂ incubator at 37 °C to allow cells to adhere to the surface. After incubation, cells were treated with varying concentrations of AgNPs (6.3 µg/mL, 6.8 µg/mL, 7.5 µg/mL, 8.3 µg/mL, 9.4 µg/mL, 10.7 µg/mL, 12.5 µg/mL). The control group consisted of cells cultured in their own medium. The vehicle control (vhc) group included cells cultured in medium containing sterile DI water used as a solvent for AgNPs. After 24 h of treatment with AgNPs, 5 mg/mL MTT solution prepared in Phosphate Buffered Saline (PBS, Sigma-Aldrich) was added in an amount equal to 10% of the culture volume. After adding 100 µL of MTT solvent solution to dissolve formazan crystals, absorbance values were measured 16 h later, at a 570 nm wavelength. Absorbance data were analyzed using Prism 7.00 (GraphPad Software, Inc) program, for cell viability and proliferation, and the dose–response curve was created to determine the nanoparticle concentration that caused at least 50 percent inhibition (IC50)²⁷.

Analysis of cell apoptosis

A flow cytometry-based Annexin V-Fluorescein isothiocyanate (FITC) apoptosis detection kit (Invitrogen, eBioscience™), was used to determine apoptosis induced by AgNPs in cells. After the cells were seeded in 6-well culture dishes as 5 × 10⁵ cells/well, AgNPs were applied at the determined IC50 concentration, and the cells were collected at the end of 24 h. Before fluorescent labeling, cell pellets were washed with 1X PBS and then suspended with 195 μL of 1 × Binding Buffer. Then, 5 μL of Annexin V-FITC was added to the cell suspension and incubated for 10 min at room temperature. After centrifugation, the cells were resuspended with 190 μL 1 × Binding Buffer; 10 μL PI (Propidium Iodide, 20 μg/mL) was added, and the cell suspensions were analyzed on a BD FACSCalibur™ flow cytometer using FITC signal detectors and phycoerythrin (PE) emission signal detectors. Ten thousand events were read for each sample, and positive FITC and/or PI cells were measured by quadrant analysis. Apoptotic cell rates were calculated for all samples.

Cell cycle analysis

The changes induced by AgNPs in the cell cycle of cancer cells were assessed under the guidance of the cell cycle analysis kit (Invitrogen, TaliTM Cell Cycle Solution) protocol. Briefly, cells were seeded in 6-well culture dishes at 5 × 10⁵ cells/well density and then exposed to AgNPs at IC50 concentrations for 24 h. After the treatment, cells were fixed in ice-cold 70% ethanol for 1 h. The cells were then washed twice by centrifugation, and incubated in 200 µL Tali® Cell Cycle Solution for 30 min at room temperature, in the dark. The DNA content of the stained cells was analyzed by flow cytometry and the cell cycle profiles were determined.

The mRNA expression levels of apoptosis related genes

The mRNA expression levels of apoptosis-related Bax and Bcl2 genes were determined using real-time polymerase chain reaction (RT-qPCR) in cancer cells after 24 h of treatment. Total RNAs were extracted using the GeneJET RNA Purification Kit (Thermo Scientific) and then converted into cDNA using the Protoscript First Strand cDNA Synthesis Kit (New England Biolabs). RT-qPCR reaction mixtures were prepared using Maxima SYBR Green qPCR Master Mix (Thermo Fisher Scientific), and reactions were performed on an Applied Biosystems 7500 Fast Real-Time PCR System (Thermo Fisher Scientific). The primer sequences used in RT-qPCR amplification are given in Table 1. The Comparative Ct method (2-ΔΔCt) was used to measure the mRNA expression levels of the amplified genes. RT-qPCR data were standardized using the expression of the housekeeping gene GAPDH.

Table 1.

Human primer sequences used in RT-PCR amplification.

Gene	Strand	Primer’s sequences (5′–3′)
BAX	Forward	5′-TCAGGATGCGTCCACCAAGAAG-3′
	Reverse	5′-TGTGTCCACGGCGGCAATCATC-3′
BCL-2	Forward	5′-ATCGCCCTGTGGATGACTGAGT-3′
	Reverse	5′-GCCAGGAGAAATCAAACAGAGGC-3′
GAPDH	Forward	5′-GTCTCCTCTGACTTCAACAGCG-3′
	Reverse	5′-ACCACCCTGTTGCTGTAGCCAA-3′

Statistical analysis

Statistical analyses were performed using GraphPad Prism version 7.0.0 (GraphPad Software, San Diego, California, USA) to determine MTT and IC50 values. Differences between experimental groups for MTT were statistically analyzed using one-way ANOVA. Dose–response data were analyzed using the dr4pl R package²⁸, which fits a four-parameter logistic (4PL) model. The model was specified as Response ~ Dose, with a “logistic” initialization, and a “Tukey” robust loss function to account for potential outliers. The IC50 value was estimated from the fitted model parameters. Apoptosis, cell cycle and gene expression analyses were performed using the 2-tailed Student t test to determine the level of significance. Results were expressed as mean ± SEM. If the values had p < 0.05 was considered significant (*), p < 0.01 was considered very significant (**) and p < 0.001 was considered extremely significant (***).

Results and discussion

Green synthesis mechanism

Anemone has been used for detoxification, treatment of dysentery, malaria, tinea, ulcers, traumatic injury, pharyngolaryngitis, parasitic infections, and hepatitis. Apart from that, it has a wide range of pharmacological activities, including analgesic, antimicrobial, antiinflammatory, and either antitumor or anticancer. Studies have shown that Anemone contains abundant photochemical compounds such as triterpenoids, saponins, steroids, lactones, fats and oils, saccharides, alkaloids, oleanolic acid, triterpenes^11,12,29. These components act as reducing agents and stabilizers for the green synthesis of silver nanoparticles. These phytochemicals stabilize and reduce metal ions (M⁺) to (M⁰) during synthesis. The reduction of metal nanoparticles occurs when (OH) groups from plant extracts are linked with carbon atoms in aromatic rings^30–33. The fundamental structure of hydroxyl groups is made up of two lone pairs that hold an oxygen atom and a hydrogen atom together. They easily combine to generate hydrogen bonds, which give an ion a net positive or negative charge. Members of this group can also participate in chemical reactions that create chains of fatty acids or carbohydrates by joining molecules together. A hydroxyl group increases the structure’s solubility in water and transforms many organic molecules into alcohols. In the manufacture of green syntheses, secondary metabolites such as terpenoids, phenolic acids, amino acids, polyphenols, enzymes, alkaloids, flavonoids, tannins, carbohydrates, phenols, saponins, and others that are obtained from plant extracts typically serve as both reductants and stabilizers. Consequently, they prevent the generated metallic NPs from clustering. The three phases of the bioreduction process for plant extract-derived metal nanoparticles (NPs). In the first stage, metal ions undergo reduction and form a nucleus. In the second stage, tiny nuclei are brought together by increasing the thermodynamic stability of NPs. NPs eventually take on their own shape following the development period^34,35. This mechanism ensures that nanoparticles are synthesized naturally from silver nitrate without the use of chemicals. The steps and the mechanism of green synthesis of silver nanoparticles using A. coronaria bulb extract are shown with the images given in Fig. 1, respectively.

Microstructural characterizations

The morphological and chemical properties of the green synthesized nanoparticles are shown in Fig. 2. As can be seen from the SEM image, the nanoparticles agglomerated during the sample preparation stage. The general image revealed that the synthesized nanoparticles were circular in geometry and successfully synthesized with A. coronaria bulb extracts. The P, O, and S elements appearing in the EDS mapping and spectrum results are thought to come from the bulb extract. Particle size distribution analysis results are also presented in Fig. 2. By applying the log-normal distribution function to fit the particle distribution histogram, the estimated average particle size was determined to be 29 nm with a standard deviation of 2.22 nm. The average particle size was found to be lower than in many studies in the literature, with plant roots and seeds^36–40. This result clearly showed that small-scale silver nanoparticles can be synthesized with A. coronaria bulb extracts.

Fig. 2.

SEM images, EDS mapping and spectrum analyzes and particle size distribution plot of silver nanoparticles.

UV–Vis spectrophotometer

UV–Vis spectroscopy was performed to describe the optical properties and to prove the formation of AgNPs after green synthesis. The size, shape, and morphology of the synthesized AgNPs have a significant influence on the peak appearing in the UV–Visible spectrum. Previous studies have revealed that the visible region of spherical AgNPs under UV–Visible spectroscopy varies in wavelength with amplitude from 400 to 460 nm. The wide variety of absorption bands that occur is associated with the various sizes and shapes of AgNPs^41,42. Figure 3 shows that the maximum wavelength is about 425 nm, which is consistent with the results of other researchers^43–46. The absorption spectra of UV–Vis light indicate that silver nanoparticles were synthesized.

Fig. 3.

UV–Visible absorption spectra of Anemone coronaria bulb extract capped AgNPs after 48 h.

X-ray diffraction analysis

XRD analysis was performed to confirm the crystallinity of Ag-NPs after their synthesis with A. coronaria bulb extract. All of the sharp peaks (111), (200), (220), and (311) in Fig. 4 were identified as Bragg’s reflections of the face-centered cubic (fcc) structure of silver at 2θ values of around 38˚, 44.2˚, 64.4˚, and 77.6˚, and this result was similar in other research^47–51. This pattern is based on JCPDS No. 04-0873, the standard metallic silver XRD pattern⁵². XRD patterns, show that silver nanoparticles were successfully synthesized by A. coronaria bulb extract. Other peaks in the spectrum are thought to originate from metabolites that carry out the synthesis ofsynthesize AgNPs and cap the particle surfaces. By combining the diffraction peaks obtained from the XRD spectra using the Scherrer Eq. (2), the crystallite sizes of the AgNPs were calculated. The average crystallite dimensions of the green synthesized silver nanoparticles were measured to be 10 nm.

Fig. 4.

XRD pattern of green synthesized silver nanoparticles.

Fouirer transform ınfrared spektrofotometre (FTIR)

FTIR analysis was used to obtain data on the interaction between the functional groups and organic contents of Ag and bulb extract, which results in the closure and reduction of the intermediate, leading to well-dispersed Ag-NPs in their colloidal solutions. In the current study, the FTIR analysis spectra presented in Fig. 5 revealed the presence of several functional groups in the synthesized Ag-NPs. FTIR analysis of green synthesized AgNPs and A. coronaria bulb extract revealed multiple absorption peaks between 650 cm^-1 and 4000 cm^-1. The FTIR spectrum showed the absorption bands at 696 cm^-1, 878 cm^-1, 1045 cm^-1, 1086 cm^-1, 1275 cm^-1, 1384 cm^-1, 1455 cm^-1, 1637 cm^-1, 2983 cm^-1, and 3324 cm^-1. The difference in the two curves is a result of the change in the chemical bonds of the nanoparticles after the biosynthesis process⁵³. The strong and broad peaks at 3324 cm⁻¹ are associated with phenolic compounds and the OH-group of alcohols, while the small band at 2983 cm⁻¹ indicates the stretching vibration of C-H alkane groups^54,55. The strong sharp bands at 1637 cm⁻¹ specify the carbonyl groups (C = O) stretching vibration; peaks in the range of 1384 cm⁻¹ represent the O–H bending of carboxylic acid; and peaks between 1045 and 1086 cm⁻¹ represent the CO–O-CO stretch^56–58. Small bands at 1384 and 1455 cm⁻¹ specified the stretching of C-N and bending of NH, respectively, which confirms the existence of aliphatic groups in amide II^59,60. The sharp peak observed at 878 cm⁻¹ in the spectrum of AgNPs is assigned to C-H bending of alkenes⁶¹. The broad bands from 640 to 686 cm⁻¹ denote metal oxide; this refers to the interaction of AgNPs with the (OH) group^62,63. These spectra indicate that the functional groups associated with these bands are mainly responsible for the biological reduction and stabilization of Ag⁺ ions to AgNPs.

Fig. 5.

FT-IR spectra of AgNPs and Anemone coronaria bulb extract.

Antibacterial activity of silver nanoparticles

The antibacterial activity of biosynthesized AgNPs against S. aureus and E. coli bacteria was investigated. Figure 6a,b show the inhibition zones of 10 µg ampicillin and 10, 50, 100, and 150 μg/mL AgNPs against bacteria on agar for two repeated tests. As can be seen in Fig. 6c, as the amount of AgNPs increases, the bacterial inhibition area enlarged, that is, the antibacterial activity rate increases. The highest antibacterial effect was measured at 15 mm for E. coli and 10 mm for S. aureus in the inhibition areas treated with 150 μg/mL AgNPs. Antibacterial test results revealed that biosynthesized silver nanoparticles can be as effective as Ampicillin when used at rates higher than 150 μg/mL. In addition, the results showed that silver nanoparticles biosynthesized with A. coronaria bulb extracts were effective against both Gram-positive and Gram-negative bacteria. The bacterial killing mechanism of nano silver is explained by the electrostatic interaction between bacterial cells (-) and silver ions ( +), leading to the rupture of the cell wall, leakage of the inner cell contents, and eventual death of the bacteria. The gradual release of free silver ions from the AgNPs solution inhibits the proliferation of bacterial cells^64–66. However, it was also found to be more effective against E. coli, Gram-negative bacteria. This may be due to the component differences in the cell wall structures of Gram-positive and Gram-negative bacteria. Phosphate, carboxyl, and amino groups in the cell membranes of bacteria create a negative charge⁶⁷. Electrostatic attraction occurs rapidly between positively charged AgNPs and negatively charged bacterial cells, and this is one of the factors that enhances the antibacterial activity of AgNPs against Gram-negative bacteria^68–71. Apart from this, the sizes and shapes of nanoparticles are among the factors affecting their antibacterial activities⁷². Antibacterial activities of nanoparticles synthesized using plant extracts differ according to their sizes, geometries, phenolic compounds, and synthesis conditions.

Fig. 6.

(a) First trial of bacterial growth zones of S. aureus. and E. coli, (b) Second trial of bacterial growth zones of S. aureus. and E. coli and (c) Graph of bacterial growth areas for 10, 50, 100, and 150 µg silver solutions against 10 μg ampicillin.

Cytotoxic effects of silver nanoparticles

The cytotoxic effect of the synthesized nanoparticles on cancer and healthy cells was determined by the MTT assay, a colorimetric method for determining metabolic activity using tetrazolium salt. The tetrazolium salt is reduced by mitochondrial dehydrogenases in metabolically active cells to the water-insoluble purple formazan crystal, which ultimately measures mitochondrial metabolic rate and reveals cell viability⁷³. In this study, all cells were treated with AgNPs at concentrations of 6.3, 6.8, 7.5, 8.3, 9.4, 10.7 and 12.5 µg/mL for 24 h. After treatment, a significant decrease in cell viability was observed in different cancer cell types, depending on the concentration (Fig. 7a–c). The toxic effect was weaker in healthy cells than in cancer cells (Fig. 7d).

Fig. 7.

The dose-dependent cell viability and IC50 concentration of AgNPs in different cancer and healthy cell lines were measured by MTT assay after 24 h of treatment in (a) A549 lung cancer cell lines, (b) MIA PaCa-2 pancreatic cancer cell lines, (c) PC-3 prostatic cancer cell lines, and (d) HEK293 healthy human embryonic kidney cells. Half maximal inhibitory concentrations (IC50) calculated for each cell are shown on the right. Data are shown as the mean ± standard deviation of three replicates. Statistical significance in treatment groups compared to vhc group was determined using Student’s t-test and significance levels are shown as *p < 0.05, **p < 0.01 and ***p < 0.001.

The half maximal inhibitory concentration value (IC50) of AgNPs after 24 h of treatment for each cell was calculated as 9.15 µg/mL (A549), 9.87 µg/mL (MIA PaCa-2), 12.1 µg/mL (PC-3) and 12.9 µg/mL (HEK293) (Fig. 7a–d right). While the most significant growth inhibition was observed in A549 cells with the lowest concentration, AgNPs showed weaker cytotoxic effects on healthy cells than on especially lung cancer cells; they had an IC50 value of 12.9 µg/mL. The anticancer effects of Ag nanoparticles synthesized via green methods have been demonstrated in many studies. Ahn et al. demonstrated that silver nanoparticles prepared using Carpesium cernuum extract were more cytotoxic in A549 cells compared to murine melanoma cells⁷⁴. In our study, AgNPs were determined to be more toxic in lung cancer A549 cells than in pancreatic and prostate cancer cells. The 50% inhibitory concentration of AgNPs synthesized using Dicoma anomala root extract, one of the medicinal plants widely used in cancer treatment, was determined to be low in A549 cells, consistent with our study⁷⁵. AgNPs prepared with butanol fraction of another medicinal plant, Pinus roxburghii, exhibited lower toxicity in lung cancer A549 cells and prostate cancer PC-3 compared to our results, and their IC50 concentrations were 11.28 μg/mL and 56.27 μg/mL, respectively⁷⁶. Green synthesized AgNPs with Lantana camara leaf extract also exhibited a lower cytotoxic effect on A549 cells and had an IC50 concentration of 49.52 μg/mL compared to the green synthesized AgNPs with A. coronaria bulb extracts⁷⁷. The IC50 values ​​of AgNPs synthesized using rosmarinic acid extract obtained from Perilla frutescens were reported to be 31.25 µg/mL for A549 cells and 51.8 µg/mL for PC-3 cells⁷⁸. In the study determining the anticancer activity of AgNPs containing Berberis thunbergii leaves against pancreatic cancer cells, the IC50 value determined at different concentrations in different cell lines was found to be 141 μg/mL for MIA PaCa-2 cell lines⁷⁹. Medicinal plants, which are prominent in the treatment of many diseases, especially cancer, are significant in phytotherapy due to the various bioactive components they contain and are also favored for the green synthesis of nanoparticles. The importance of these green synthesized NPs as cancer therapeutics is increasingly recognized. Although cytotoxic effects are supported in many studies; a crucial aspect is that the effective concentration is at low doses. The data obtained from our study showed that AgNPs synthesized from A. coronaria bulb extracts were effective at low doses in cancer cells. In addition, it was determined that the concentration of AgNPs synthesized in our study required to inhibit 50% of healthy cells was higher than that required, especially for lung cancer cells, indicating weaker toxicity against healthy cells. These results reveal that they have significant anticancer potential.

Anemone coronaria silver nanoparticles induce apoptosis in cancer cells

Apoptosis, defined as programmed cell death, is one of the important mechanisms targeted in cancer treatment, and activation of the apoptotic pathway is desired⁸⁰. Therefore, induction of apoptosis is important in anticancer therapeutic approaches. Annexin V-FITC apoptosis assay was performed to determine the apoptotic effect of AgNPs on cancer cells. The method is based on the principle that phosphatidylserine, expressed on the surface facing the inside of the cell in healthy cells, comes out of the cell during apoptosis and is detected by Annexin V. Propidium iodide (PI) used in the assay cannot pass through the cell membrane in healthy cells, but it enters cells whose viability is compromised and acts as an indicator of necrotic cells by binding to DNA^81,82. In our study, we analyzed the effects of AgNPs on cell apoptosis after 24 h of treatment with half-maximal inhibitory concentration of AgNPs on cancer cells. As shown in Fig. 8a–c, a significant increase (p < 0.01; p < 0.001) in the rate of early and late apoptotic cells was observed in A549, MIA PaCa-2 and PC-3 cells. Especially in the PC-3 prostate cancer cell line early and late apoptotic cells increased 12-fold and 14-fold, respectively (p < 0.001). AgNPs also induced a significant increase in necrosis in A549 lung and MIA PaCa-2 pancreatic cells, while the increase in necrosis was lower in PC-3 prostate cancer cells.

Fig. 8.

Flow cytometric analysis of apoptosis induction in A549 (a), MIA PaCa-2 (b) and PC-3 (c) cancer cells after 24 h of treatment with IC50 doses of AgNPs synthesized from Anemone coronaria bulb extracts. Flow plots are shown on the right. Annexin V-negative/PI-negative staining profile identifies viable cells, Annexin V-positive/PI-negative staining profile identifies early apoptotic cells, Annexin V-positive/PI-positive staining profile identifies late apoptotic cells, and Annexin V-negative/PI-positive staining profile identifies necrotic cells. Data are indicated as the mean ± standard deviation of three replicates, and differences between treatment groups compared to the vhc group were determined using Student’s t-test. Statistical significance levels are shown as *p < 0.05, **p < 0.01 and ***p < 0.001.

Green-synthesized AgNPs have been shown to induce apoptosis in cancer cells. In the study reported by Ullah et al., it was shown that treatment of green synthesized AgNPs with the aqueous extract of Fagonia indica at a concentration of 12.35 μg/mL for 24 h induced apoptosis in cancer cells⁸³. A study reported in recent years demonstrated that green synthesized AgNPs with Phoenix dactylifera seed extract induced dose-dependent apoptosis in A549 human lung adenocarcinoma cells and had an anticancer effect⁸⁴. Similarly, it has been reported that AgNPs induce apoptosis by causing mitochondrial damage in various cancer types and exhibit anticancer effects^85–87. In our study, in accordance with the literature data, green synthesized AgNPs with A. coronaria bulb extracts caused an approximately threefold increase in apoptotic cells in lung and pancreatic cancer cell lines and a more than tenfold increase in prostate cancer cells. These results suggest that AgNPs cause antiproliferative effects by inducing apoptosis in cancer cells.

Effects of silver nanoparticles on cell cycle regulation

It is important to examine cell cycle dynamics in determining the response of cancer cells to a treatment approach. In this context, we performed cell cycle analysis in A549, MIA PaCa-2, and PC-3 cell lines exposed to AgNPs at IC50 concentrations for 24 h. After application, DNA content in sub-G0/G1, G0/G1, S, and G2/M phases was determined by flow cytometry.

In A549 cells, treatment at IC50 concentration (9.15 µg/mL) lead to a significant decrease in the proportion of cells in sub-G0/G1 and G0/G1 phases, accompanied by arrest in the S phase and, particularly, the G2/M phase. While the rate of arrested cells in G2/M phase was 16.14% in the vhc group, this rate was 23.1% after treatment with AgNPs. These results were consistent with those reported by Kanipandian et al.⁸⁸. Similarly to our results (Fig. 9a), Lee et al. reported that AgNPs caused a decrease in the proportion of cells in the G1 and S phases in A549 lung cancer cells, accompanied by cell death in the G2/M phase⁸⁹. In addition, AgNPs synthesized from Bergenia ligulate plant caused a similar arrest in the G2/M phase in breast cancer cells⁹⁰. However, it is reported that AgNPs with different bioactive component contents cause different effects on cancer cells. One of the findings was that when the anticancer activity of Pinus roxburghii AgNPs was evaluated, it induced cell arrest in the sub-G0/G1 phase in lung (A549) and prostate cancer (PC-3) cells⁷⁶. In our study, the anticancer effects of AgNPs on different cancer cells were investigated. Cell cycle analysis showed similar results in MIA PaCa-2 pancreatic cancer cell lines as in lung cancer cells, exhibiting a statistically significant decrease in G0/G1 cell population along with a significant increase in both S and G2/M phases, particularly G2/M. After the applied IC50 concentration of 9.15 µg/mL, the cell percentages in the G0/G1, S, and G2/M phases were determined as 48.62%, 5.41%, and 33.05% in the vehicle control, respectively. In the AgNPs treatment groups, cell cycle arrest was indicated by the percentages of 30.15%, 7.34%, and 39.62%, respectively, indicating cell cycle arrest especially in the G2/M phase (Fig. 9b). In the studies conducted for different pharmaceutical and biomedical applications, NPs are observed to exhibit different effects on cancer cells. Consistent with our results, NPs caused cell cycle arrest in the sub-G0/G1 and G2/M phases in PANC-1 pancreatic cancer cells⁹¹ and in the G2/M phase in liver cancer cells, and were associated with an increase in apoptosis rate⁹². AgNPs produced with Bergenia ligulate caused significant accumulation in the G2/M phase in the MCF-7 breast cancer cell population compared to the vehicle control⁹⁰. Depending on cancer cell heterogeneity, different cellular responses occur in different cancer types. In our study AgNPs caused arrest in the sub-G0/G1 phase which is an indicator of, early apoptotic cell population, with significantly decreased cell populations in the G0/G1 and G2/M phases on prostate cancer cells. The cell cycle distributions in vehicle control cells were 5.35%, 52.26%, 7.93%, and 27.98% for sub-G0/G1, G0/G1, S, and G2/M phases, respectively, whereas in cells treated with IC50 concentrations, these values were 18.26%, 35.37%, 7.27%, and 13.55%, respectively. Significant cell arrest was observed in the sub-G0/G1 phase (Fig. 9c). Consistent with our study data, Pinus roxburghii AgNPs induced cell arrest at sub-G0/G1 phase in prostate cancer (PC-3) cells⁷⁶. Similarly, magnetic iron oxide nanoparticles prepared with seaweed aqueous extracts caused arrest in Jurkat leukemia cells⁸², and silver nanoparticles synthesized using soybean extracts caused arrest in the sub-G0 phase in breast cancer cells⁹³. However, it is reported that various therapeutic applications investigated for the treatment of prostate cancer induce G0/G1 phase arrest in cancer cells^94,95. The basis for this is thought to be the variability of cancer-causing mechanisms in different cancers.

Fig. 9.

Cell cycle analysis in A549 (a), MIA PaCa-2 (b), and PC-3 (c) cancer cells treated with AgNPs at IC50 concentration for 24 h. Representative flow cytometry images showing changes in cell cycle progression are given on the right. Bar graphs on the left represent the percentage of cells arrested at different phases of the cell cycle. Data are presented as the mean ± standard deviation of three replicates and differences between treatment groups compared to the vhc group were determined using Student’s t-test. Statistical significance levels are shown as *p < 0.05, **p < 0.01 and ***p < 0.001.

Expression of apoptosis-related genes with real time-PCR

To further support the effects of AgNPs on apoptosis induction, the expression levels of apoptosis-related gense were examined 24 h after the IC50 dose was applied to A549, MIA PaCa-2, and PC-3 cells. The results are presented in Fig. 10.

Fig. 10.

Demonstration of apoptosis-related gene expression levels in A549 (a), MIA PaCa-2 (b) and PC-3 (c) cancer cells after treatment with AgNP at the IC50 dose for 24 h, determined by real-time quantitative polymerase chain reaction. The expression level of the genes was normalized to the reference gene GAPDH. Data are presented as the mean ± standard deviation of three replicates and differences between treatment groups compared to the vhc group were determined using Student’s t-test. Statistical significance levels are shown as *p < 0.05, **p < 0.01 and ***p < 0.001.

In our study, the expression levels of pro-apoptotic Bax and anti-apoptotic Bcl-2 genes were examined to determine the effect of AgNPs on apoptosis-related gene expression. The expression of the Bax gene was significantly increased in A549, MIA PaCa-2 and PC-3 cells (p < 0.05), while the expression of the Bcl-2 gene was downregulated to 0.84 and 0.76-fold in A549 and MIA PaCa-2 cells, respectively. However, this decrease was not statistically significant. In PC-3 cells, the Bcl-2 gene was downregulated to 0.32-fold, and this downregulation was statistically significant (p < 0.01).

Disruptions in the apoptotic pathway, one of the cellular death mechanisms, play an important role in cancer development. Apoptosis induction is a key cellular response in anticancer treatment approaches. The B cell lymphoma 2 (BCL-2) protein family has different members exhibiting pro- or anti-apoptotic activities. Among these protein family members, Bax, which exhibits pro-apoptotic effects, and Bcl-2, which has anti-apoptotic properties, are important in the evaluation of apoptosis⁹⁶. Various studies support an increase in Bax gene expression and a decrease in Bcl-2 gene expression with apoptosis induction by silver nanoparticles investigated as cancer therapeutics^97,98 and our results of the present study are consistent with these findings, confirming the modulation of Bax and Bcl-2 expression in response to AgNPs.

Conclusions

In this study, AgNPs were successfully synthesized using A. coronaria bulb extract via a green synthesis approach. Structural characterization confirmed the spherical morphology, average size of 29 nm, and crystalline nature of the synthesized AgNPs. The nanoparticles demonstrated significant antibacterial activity, with inhibition zones reaching 15 mm against E. coli and 10 mm against S. aureus at 150 µg/mL, demonstrating their efficacy against both Gram-negative and Gram-positive bacteria. From a cytotoxic perspective, AgNPs exhibited effective anticancer activity. The anticancer activity of AgNPs was confirmed through the induction of apoptosis and cell cycle arrest in cancer cells. The apoptotic effect was further supported by the upregulation of pro-apoptotic (Bax) and downregulation of anti-apoptotic (Bcl-2) gene expression in treated cancer cells.

The advantages of this study lie in its use of **a novel plant part—the bulb—** which enables year-round and resource-efficient production without requiring full plant maturity. Additionally, the synthesis method is non-toxic, cost-effective, and environmentally sustainable, aligning with modern biomedical material design principles. However, several limitations should be acknowledged. The biological testing was confined to in vitro models, and in vivo validation remains necessary to confirm therapeutic efficacy. Also, long-term toxicity and pharmacokinetics of the AgNPs must be evaluated, which is essential for clinical translation. These findings contribute valuable insights into the utility of A. coronaria bulb-derived AgNPs as a promising agent for anticancer and antibacterial applications. Future studies should explore mechanistic pathways, target-specific delivery, and surface functionalization strategies to improve therapeutic indices and expand biomedical applications.

Author contributions

Melek Yüce: Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Visualization, Writing – original draft, Writing – review & editing Esra Albayrak: Investigation, Methodology, Visualization, Validation, Writing – original draft, Writing – review & editing Arife Kübra Yontar: Investigation, Methodology, Visualization, Validation, Writing – original draft, Writing – review & editing. Sinem Çevik: Conceptualization, Supervision, Visualization, Validation, Writing – review & editing Cagri Gümüskaptan: Visualization, Writing – review & editing.

Funding

The work was supported by Ondokuz Mayıs University Scientific Research Project Office (Grant Number PYO.KÖK.1908.23.001).

Data availability

All data generated or analyzed during this study are included in this published article.

Declarations

Competing interests

The authors declare no competing interests.