In the study, CA leaves obtained from Büyük Çelikli Village of Sur County of Diyarbakır were used. AgNO3 (99.8% purity), colistin, vancomycin, and fluconazole were commercially purchased from Sigma Aldrich. E. coli ATCC 25922, P. aeruginosa ATCC27853, B. subtilis ATCC 11774, S. aureus ATCC 29213, and C. albicans were used to test the antimicrobial activities of AgNPs. Cytotoxicity tests (MTT) related to cell lines (CaCo-2/human colon epidermal adenocarcinoma; U118 MG/human brain glioma cells; SK-OV-3/human ovarian cancer cell line; HDF/human dermal fibroblasts) were performed in the Dicle University Central Research Laboratory.

Green leaves of CA were washed with deionized distilled water to remove residues and dehydrated at 25 ± 2°C. A total of 250 g of ground plant material was mixed with deionized pure water (500 ml) and boiled in a flask. After boiling, the cooled extract was filtered with a membrane filter (0.45 μm).

Firstly, an aqueous solution of 5 mM AgNO₃ with solid AgNO₃ was prepared. The CA extracts (500 ml) and 100 ml of AgNO3 were allowed to react in a glass vessel (1:5 ratio) at room temperature. Maximum absorbance of biologically synthesized AgNPs was determined by wavelength scanning (UV-vis spectroscopy) at various time periods (15, 30, 45, 60, 120, and 180 min) depending on the color change. At the end of the synthesis, the solution, which became a dark color depending on time, was subjected to centrifugation (6000 rpm, 20 min). The purpose of this process is to separate the synthesized nanoparticles from plant residues. The solid fraction obtained at the end of centrifugation was washed several times with distilled water and the resulting residue (AgNPs) was dried in an oven at 60°C for 72 h.

The maximum absorbance of synthesized AgNPs was measured at the 300–800 nm wavelength range with a spectrophotometer (Agilent CARY 60). Size, morphology, crystal structure, surface distribution, and zeta potential (ZP) values of AgNPs were revealed by scanning electron microscopy (SEM) (EVO 40 LEQ), transmission electron microscopy (TEM) (Quanta), field emission scanning electron microscopy (FE-SEM) (Quanta FEG240), electron dispersive X-ray (EDX) (Quanta FEG 240), X-ray diffraction analysis (XRD) (Rad B-DMAX II), and Zetasizer (Malvern Ins. Ltd.). The crystal dimension of AgNPs was calculated according to the D = Kλ/(β cosθ) equation (Asadi et al., 2018; Baran et al., 2018). In addition, Fourier transform infrared spectroscopy attenuated total reflectance (FT-IR ATR) was used to identify the functional groups present in the CA extract, and the functional groups responsible for the reduction at the end of the reaction test analysis conditions of used instruments are given in Table 1.

Growth inhibition of plant-based AgNPs on Gram-positive (B. subtilis; S. aureus) and Gram-negative (E. coli; P. aeruginosa) strains and yeast (C. albicans) was determined using a 96-well microplate with minimum inhibition concentration (MIC) method. Mueller Hinton broth and cell culture growth medium (Roswell Park Memorial Institute medium/ RPMI) were added to the wells for the growth of bacteria and yeast. The AgNP solution was added to the wells with the culture medium and microorganisms to determine the MIC value. Firstly, 100 µL of mixed culture medium was taken from the wells each time and transferred to the next well. Then the microorganism solutions adjusted according to the 0.5 McFarland standard were added to the microplates and incubated at 37°C/24 h. The minimum concentration without growth after incubation was determined as the MIC value (Baran et al., 2020). Commercially purchased standard antibiotics (colistin, vancomycin, and fluconazole) and 1 mM AgNO₃ solution were used to compare the growth inhibitory activities of AgNPs on pathogen microorganisms.

The MTT method was performed to determine the plant-based AgNP ratio of cytotoxicity (viability suppressor) on cancerous and healthy cell cultures. T-75 T-flasks were used to prepare the culture medium. CaCo-2, HDF, and U118 cell lines were cultivated in DMEM solution. The human ovarian cancer cell line (SK-OV-3) was incubated in RPMI solution. Prepared cultures were incubated at 5% CO2, 37°C, and 95% air and humidity conditions. When the cells reached about 80% confluency in the hemocytometer measurement, cell cultures were suspended at different concentrations and transferred to microplates (96-well) for incubation (overnight). At the end of the period, the cultured cell lines were treated with nanoparticles at different concentrations (25, 50, 100, and 200 μg/ml) and incubated for 2 days. In the next step, the MTT solution was transferred to the microplate wells and incubated for 3 h, and then DMSO was added and kept at room temperature for 0.25 h. The absorbance (540 nm) of the microplates was measured with MultiScan Go (Thermo).

By using the below formula, the percentage viability of the cell lines was calculated.

% viability = U/C*100 (Vickers, 2017).

U: Absorbance of cells treated with AgNPs.

C: The absorbance values of control cells.

The color change was observed after the CA leaf extracts with AgNO3 solution were left to react in a container. The UV-vis spectrum of AgNPs appeared to change from light green to purple (Figure 2). Because of the surface plasmon resonance, AgNPs gave a peak at a specific absorbance value of about 417.47 nm. Similarly, some researchers reported that the absorption spectrum of AgNPs is between 425–461 nm (Udayasoorian et al., 2011).

SEM, FE-SEM, and TEM images of synthesized AgNPs are given in Figures 3, 4. According to these results, it was seen that the nanomaterial was mostly spherical, nano-sized, and in clusters that were not in direct contact with each other. This indicates the stabilization of the AgNPs. It was reported that AgNPs have a spherical morphology and nano-dimensions in similar studies (Ramkumar et al., 2017a; Pallela et al., 2018). The biosynthesized nanoparticles are expected to have stronger antimicrobial activity, on account of their relatively small size. In the particle measurement done with TEM, it was seen that the sizes of AgNPs were approximately between 6.11–9.66 nm and the average size was approximately 7.83 nm (Figure 4). In some studies, using different materials, the sizes of AgNPs were reported to be between 2–95 nm (Nguyen et al., 2018; Behravan et al., 2019).

The FT-IR spectroscopy analysis determined the functional groups involved in the plant-derived reduction. The frequency of all stretch in the range of 4500–500 cm⁻¹ was recorded with four scans at 1 cm⁻¹ resolution. Figure 4 shows a comparison of FT-IR spectra for the aqueous CA extract (Figure 5A) and synthesized AgNPs (Figure 5B). When the biomolecules involved in reduction during the formation of AgNPs were examined (Figures 5A,B), the absorption peak at 1635 cm⁻¹ corresponded to the C=O stretching vibration, indicating the presence of amide. Because of the phenolic compounds in the CA leaf extract, it can be concluded that the absorption peak at 2122 cm⁻¹ belongs to alkyne (C≡C) groups while the absorption peak at 3331 cm-1 belongs to O-H and N-H stretching (Atalar et al., 2021). Presumably, these determined functional groups are responsible for the reduction of metal ions (Mandal et al., 2015).

According to the EDX profile (Figure 6), it was confirmed that the biosynthesized nanoparticles had silver in their composition. It was also seen that the elemental composition of silver was high (Figure 6). AgNPs showed a typical optical absorption peak at about 3 KeV owing to the surface plasmon resonance (SPR). It can be said that the other emerging peaks are because of phytochemicals attached to the surface of AgNPs in the CA leaf pulp (Punuri et al., 2012). Khamhaengpol and Siri (2017) and Dada et al. (2019) also revealed the EDX silver peaks in their work.

The XRD spectrum model for the synthesized AgNPs is shown in Figure 7. In XRD analysis results, peaks of 111^o, 200^o, 220^o, and 311^o, which coincide with 37.96, 44.29, 64.32, and 77.33, respectively at 2θ, were sharp peaks representing the spherical crystal structure of silver. The peaks indicated that the AgNPs were cubic in structure. It has been reported in many studies that these peaks belong to silver (Huang et al., 2019; Keskin et al., 2021). The highest peak, 37.96, was taken as the peak angle. The size of the nanomaterials was calculated as approximately 12.17 nm according to the below equation ((Baran et al., 2021a). D = K λ / ( β ⁡ cos ⁡ θ )

In the equation, D = the size of the particle, K = the constant value (0.89), λ = the wavelength value of XRD (1.5418 Å), β = the FWHM value of the high peak, and cosθ = the Braggθ angle of the high peak.

The zeta potential analysis gives the electric charge on the surface of the surrounded material. The high negative value of the zeta potential prevents the particles from sticking together or clumping together. This indicates the stability of the AgNP colloid. On the other hand, nanoparticles with a significantly lower negative charge can enter the cell more easily (58–60). The zeta potential of the biosynthesized AgNPs was found to be −19.6 mV (Figure 8). This value indicated that the AgNPs were stable and uniformly distributed. The different zeta potential values of AgNPs synthesized from various materials have been reported previously (Ferreyra Maillard et al., 2018; Amer et al., 2020).

The antimicrobial effects of AgNPs have become more important due to microorganisms that cause disease in humans developing resistance to conventional antibiotics. S. aureus, B. subtillis, E. coli, and P. aeruginosa strains and C. albicans yeast are pathogenic microorganisms frequently encountered in food-borne diseases (Yang et al., 2017; Mostafa et al., 2018). It was determined that the biosynthesized AgNPs significantly inhibited the growth of these microorganisms even at low concentrations (Table 2). It was observed that AgNPs strongly inhibited the growth of S. aureus and B. subtilis when compared to other microorganisms. Since silver has a strong tendency to interact with phosphorus and sulfur atoms in the bacterial cell wall, it interacts with the thiol and phosphorus groups in the bacterial cell membrane, thereby disrupting the bacterial respiration process. This causes the death of bacteria (Hamouda and Baker Jr, 2000). On the other hand, since the cell wall of Gram-positive bacteria has a hard polysaccharide layer, the transition to the Gram-positive bacterial wall is more difficult when compared to Gram-negative bacteria. Therefore, the inhibitory activity of AgNPs in Gram-positive bacteria is stronger than in Gram-negative bacteria (Tamboli and Lee, 2013). Thuc et al. (2016) reported that Gram-positive S. aureus has approximately 2–3 times higher resistance to AgNPs than Gram-negative E. coli and P. aeruginosa (Thuc et al., 2016). These effective inhibitory activities of AgNPs on different bacteria strains and yeasts were also reported by many researchers (Niknejad et al., 2015; Aygün et al., 2020).

AgNPs obtained by biosynthesis of chickpea leaf extract were applied to healthy cells (HDF) and three different cancer cell lines (CaCo-2, U118, and Sk-ov 3), and the results obtained after 48 h are shown in Table 3 and Figure 9. According to these results, it was seen that there was no toxic effect in HDF with a survival rate of 79.70% at a 25 μg/ml concentration. It was determined that the most suppressed concentration of viability was on CaCo-2 cells at 200 mg/ml (Table 3). Despite the increase in the concentration of AgNPs in other cancer cell lines, the increase in the percentage of viability can be explained by the proliferative properties of AgNPs for these cells (Morais et al., 2020).

It is known that AgNPs show strong oxidative properties (Wongpreecha et al., 2018). AgNPs tend to settle in biomolecules such as cell membranes and nuclei. They exert a toxic effect by stimulating apoptosis with an increase in ROS after localization (Gliga et al., 2014; Morais et al., 2020). The concentration, exposure time, shape, size, charge, degree of deposition, and chemistry of the surface composition can have a significant impact on the toxicity of AgNPs (Swamy et al., 2015). In studies conducted to examine the toxic effects of AgNPs on CaCo-2 cells, it was reported that concentrations above 3.75–5.5 μg/ml showed toxic effects (Zein et al., 2020). Inhibitory concentrations in Sk-ov-3 cells were reported to be 9.4 μg/ml (Fahrenholtz et al., 2017) and 29.36 μg/ml (Noor et al., 2021). Zhang et al. (2010) reported that the 100 μg/ml concentration of silver nanoparticles was toxic on cell viability on HDF cell lines (Zhang et al., 2010).