Silver Nanoparticles Synthesized from Abies alba and Pinus sylvestris Bark Extracts: Characterization, Antioxidant, Cytotoxic, and Antibacterial Effects

In recent years, phytofunctionalized AgNPs have attracted great interest due to their remarkable biological activities. In the present study, AgNPs were synthesized using Abies alba and Pinus sylvestris bark extracts. The chemical profile of these bark extracts was analyzed by LC-HRMS/MS. As a first step, the synthesis parameters (pH, AgNO3 concentration, ratio of bark extract and AgNO3, temperature, and reaction time) were optimized. The synthesized AgNPs were characterized by ATR-FTIR spectroscopy, DLS, SEM, EDX, and TEM. Their antioxidant, cytotoxic, and antibacterial properties were evaluated by the DPPH, ABTS, MTT, and broth microdilution assays, respectively. Abies alba and Pinus sylvestris bark extract-derived AgNPs were well-dispersed, spherical, small (average particle size of 9.92 and 24.49 nm, respectively), stable (zeta potential values of −10.9 and −10.8 mV, respectively), and cytotoxic to A-375 human malignant melanoma cells (IC50 = 2.40 ± 0.21 and 6.02 ± 0.61 μg/mL, respectively). The phytosynthesized AgNPs also showed antioxidant and antibacterial effects.


Introduction
Among nanomaterials, silver nanoparticles (AgNPs) have gained increasing attention over the last years due to their strong antimicrobial activity and unique electrical, optical, and catalytic properties. Nowadays, AgNPs have been imposed as a desirable nanomaterial in different fields such as medicine (impregnation of catheters, meshes for wounds and tissue therapy, bone and cardiovascular implants, bio-imaging, bio-sensing), cosmetics, food preservation, environmental protection (water disinfection), paint coating industry, and catalysis of various processes [1]. Many of the biomedical applications of AgNPs are based on their broad-spectrum antimicrobial activity against bacteria, fungi, and viruses [2]. AgNPs exert higher toxicity against Gram-negative bacteria which is strongly related to the thinner peptidoglycan layer in the bacterial cell wall [3,4]. AgNPs are also active against drug-resistant bacteria, enhance the antibacterial activity of antibiotics [4], have

Preparation of Bark Extracts
Extraction was performed with ultra-pure water at a ratio of 1:10 as previously described. Briefly, bark powder (10 g) was mixed with ultra-pure water (100 mL) at 60 • C followed by 3 h stirring at room temperature, filtration, and addition of ultra-pure water to 100 mL final volume [28,29].
The total phenolic content was also determined in the reaction mixtures (bark aqueous extract and 2 mM AgNO 3 , 1:9, v/v) before and after the synthesis and separation of AgNPs.

Characterization of AgNPs
AgNPs were characterized by attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy, dynamic light scattering (DLS), scanning electron microscopy (SEM), energy dispersive X-ray analysis (EDX), and transmission electron microscopy (TEM). The procedures were carried out as previously described [29], with some changes. The functional groups on the surface of AgNPs, belonging to the compounds in bark extracts acting as capping/stabilizing agents, were detected by ATR-FTIR spectroscopy (Bruker Alpha-P ATR FTIR spectrometer, Bruker, Ettlingen, Germany). In brief, the aqueous bark extracts (30 mL of each) were concentrated under reduced pressure at 40 • C (Büchi R-210 rotary evaporator system, Büchi Labortechnik AG, Flawil, Switzerland) and freezedried (Unicryo TFD 5505 freeze dryer, UniEquip GmbH, Munich, Germany). Freeze-dried extracts and AgNPs pellets were subjected to ATR-FTIR spectroscopy in the spectral region of 4000-400 cm −1 with a resolution of 4 cm −1 . The size and morphology of AgNPs were investigated by SEM coupled with EDX and TEM. For SEM-EDX measurements, a Quanta 200 scanning electron microscope equipped with an energy dispersive spectrometer (FEI Company, Hillsboro, OR, USA) was used. AgNPs pellets were analyzed in low vacuum mode at an operating voltage of 10 kV. TEM analyses, operated at 100 keV, were performed using a Hitachi High-Tech HT7700 transmission electron microscope (Hitachi High-Technologies Corporation, Tokyo, Japan) equipped with a Bruker EDX detector. Prior to analysis, colloidal AgNPs solutions (a drop of each) were placed on carbon-coated copper grids (300-mesh) and subjected to 24 h vacuum-drying (room temperature). DLS measurements were made on a Malvern Zetasizer Nano-ZS (Malvern Instruments, Malvern, UK). The hydrodynamic diameters and polydispersity index (PDI) values of the colloidal AgNPs solutions were determined using He/Ne laser (λ = 633 nm); electrophoretic light scattering was used to measure zeta potential. The colloidal AgNPs solutions refer to the reaction mixtures in which the synthesis of AgNPs was completed (before separation of AgNPs by centrifugation).
2.9. Assessment of Antioxidant Activity 2.9.1. DPPH Radical Scavenging Assay The antioxidant potential of synthesized AgNPs was initially evaluated by the DPPH radical scavenging assay [37]. A volume of 1800 µL of 0.002% DPPH solution in methanol was mixed with 200 µL of various dilutions of AgNPs in ultra-pure water (50-500 µg/mL). After 30 min incubation at room temperature in dark, the absorbance was determined at 515 nm (Specord 210 Plus spectrophotometer, Analytik Jena, Jena, Thuringia, Germany). Trolox (50-500 µg/mL) was used as positive control. The DPPH radical scavenging activity (%) was calculated as follows: 100 × (A C − A S )/A C , where A C and A S are the absorbances of the control (DPPH solution) and sample (AgNPs, Trolox), respectively. The experiments were carried out in triplicate.

ABTS Radical Cation Scavenging Assay
The ABTS radical cation was generated by incubating ABTS (7 mM) with potassium persulfate (2.54 mM) for 16 h in dark at room temperature; the solution was further diluted with ethanol to an absorbance of 0.70 ± 0.02 at 734 nm (Specord 210 Plus spectrophotometer, Analytik Jena, Jena, Thuringia, Germany). In brief, the ABTS radical cation solution (1500 µL) was mixed with different concentrations (50-500 µg/mL) of AgNPs/Trolox (positive control). The absorbance was recorded at 734 nm after 6 min incubation at room temperature [31,38]. ABTS radical cation scavenging activity was calculated as described in Section 2.9.1. The assay was performed in triplicate.

Assessment of Cell Viability
A. alba and P. sylvestris bark extract-derived AgNPs were applied at different concentrations ranging from 3.125 to 25 µg/mL, for 24 and 48 h. Cell viability was evaluated using the MTT assay as previously reported [40,41], with minor changes. Briefly, the cells were detached with trypsin/EDTA, counted, resuspended in 96-well microplates (7 × 10 3 cells/well), and maintained in the conditions mentioned above (Section 2.10.1). Once the monolayer formed (24 h), the cells were treated with A. alba or P. sylvestris bark extract-derived AgNPs. After 24/48 h treatment, the medium was removed, and the cells were washed with phosphate-buffered saline, followed by the addition of fresh growth medium (100 µL/well) and MTT (5 mg/mL, 10 µL/well) and incubation for 3 h at 37 • C (5% CO 2 , 95% air). DMSO (100 µL/well) was further added to solve the insoluble formazan crystals generated after MTT reduction [42][43][44]. The absorbance of the formazan dye was quantified at 570 nm using a microplate reader (Biochrom, Berlin, Germany). The cell viability (%) was calculated as follows: A S /A C × 100, where A S and A C are the absorbances of the formazan dye produced in samples (treated cells) and control (untreated cells), respectively. The experiments were performed in triplicate.

Assessment of Cell Morphology
Morphological changes in Vero and A-375 cells after 24 and 48 h treatment were examined using a Nikon Eclipse TS100 inverted microscope (Nikon, Tokyo, Japan) equipped with a digital camera (MSHOT MS60).

Assessment of Antibacterial Activity
The antibacterial activity of AgNPs was evaluated using the broth microdilution method as previously described [45,46], with slight modifications. Prior to the experiment, stock solutions of AgNPs and ciprofloxacin were prepared (A. alba and P. sylvestris bark extract-derived AgNPs: 250 µg/mL, ciprofloxacin: 2560 µg/mL) and further subjected to two-fold serial dilutions. AgNPs and ciprofloxacin stock solutions and dilutions were prepared in sterile ultra-pure water. In brief, 30 µL of 0.5 McFarland standard bacterial inoculum was mixed with two-fold serial dilutions of AgNPs (30 µL) or antibiotic (10 µL) and Mueller-Hinton broth up to 200 µL; the mixtures were incubated for 24 h at 37 • C followed by an evaluation of bacterial growth. The minimum inhibitory concentration (MIC) value was the lowest concentration of an antibacterial agent that completely inhibited the growth of bacteria [46,47].

Data Analysis
The total phenolic content was expressed as mean ± standard deviation (SD). The antioxidant activity was expressed as EC 50 values (half maximal effective concentration) calculated by linear interpolation between the values above and below 50% activity; the results were expressed as mean ± SD. The cytotoxic activity was expressed as IC 50 values (concentration of sample producing 50% inhibition of cell viability) using the polynomial graphic plots by modeling the percentage of cytotoxicity vs. concentration of AgNPs; the results were expressed as mean ± standard error (SE). The paired t-test was used to assess the differences between samples and control.

Chemical Characterization of Bark Extracts
The results are presented in the Supplementary File (Supplementary Figure S1, Supplementary Table S1).

Optimization of Synthesis Parameters
For the synthesis of AgNPs, A. alba/P. sylvestris bark aqueous extract was mixed with AgNO 3 under continuous stirring. Different parameters (pH, AgNO 3 concentration, ratio between the volume of bark extract and AgNO 3 , temperature, and reaction time) were optimized for the synthesis of AgNPs which was monitored by UV-Vis spectroscopy ( Figure 1). The generation of AgNPs was indicated by a color change and a characteristic absorption band (Surface Plasmon Resonance (SPR) band) in the visible domain (400-500 nm), caused by the collective oscillations of the free electrons at the surface of metallic Ag when excited by light [48].
To evaluate the effect of pH on AgNPs synthesis, the process was conducted at various pH values (2, 4, 6, 8, and 10). For both bark extracts, the synthesis of AgNPs could not be achieved at acidic pH (pH 2). At pH 4 and 6, slightly prominent bands between 400 and 500 nm indicated that the synthesis of AgNPs was initiated. At basic pH (8 and 10), characteristic SPR peaks were observed in the region of 410-430 nm, supporting the formation of AgNPs. In the case of both bark extracts, at pH 10, SPR peaks were sharper and their absorption maxima slightly shifted to shorter wavelengths ( Figure 1A,B) suggesting the formation of smaller-sized AgNPs [49]. A single sharp SPR band at short wavelengths indicates the synthesis of small and spherical AgNPs while broader SPR bands (two or three) at longer wavelengths are characteristic of anisotropic and large AgNPs [50,51]. So, pH 10 was chosen for the synthesis of A. alba and P. sylvestris bark extract-derived AgNPs.
to 10 with 0.1 N NaOH) and stirred at 40 °C/30 °C for 120 min. AgNPs synthesis was checked at different time intervals (0-120 min) during stirring and at 24 h after the end of stirring. As shown in Figure 1I,J, for both bark extracts, the reduction of Ag + started immediately after the addition of the reducing agent (bark extract) to the AgNO3 solution (0 min). At each time interval, an absorbance peak at around 420 nm indicated the synthesis of AgNPs. The yield of AgNPs increased as the reaction time increased. Therefore, a period of 120 min stirring followed by 24 h at rest was considered optimal for the synthesis of AgNPs.
The optimized experimental parameters for the synthesis of AgNPs were pH 10, the temperature of 30 and 40 °C (for P. sylvestris and A. alba bark extract-derived AgNPs, respectively), 2 mM AgNO3, bark extract to AgNO3 (2 mM) ratio of 1:9 (v/v), and 120 min reaction time (followed by 24 h at rest). The concentration of AgNO 3 solution is another parameter that significantly affected the synthesis of AgNPs. As shown in Figure 1C,D, for both A. alba and P. sylvestris bark extract-derived AgNPs, the absorption increased up to 2 mM AgNO 3 . At 3 mM AgNO 3 , the absorbance started to decrease and the SPR bands became broader and slightly shifted to longer wavelengths. It seems that higher AgNO 3 concentrations impair the synthesis and moreover, lead to larger AgNPs. The best SPR peak (as intensity, shape, and position) was obtained at 2 mM AgNO 3 , and therefore this concentration was chosen as the optimal one for the synthesis of AgNPs.
The effect of the volume ratio of bark extract to 2 mM AgNO 3 on AgNPs synthesis was investigated in several proportions (1:9, 2:8, 3:7, 5:5, 7:3, and 9:1). In the case of A. alba bark extract, AgNPs were synthesized only when the bark extract and AgNO 3 were used at a ratio of 1:9 (v/v); no AgNPs synthesis was observed at other ratios used in the experiment ( Figure 1E). For P. sylvestris bark extract-derived AgNPs, the highest SPR band was achieved at a similar ratio of bark extract to 2 mM AgNO 3 (1:9, v/v); the intensity of the SPR band decreased as the volume of bark extract increased ( Figure 1F). Hence, a bark extract to AgNO 3 (2 mM) ratio of 1:9 (v/v) was chosen as optimal for the synthesis of AgNPs.
The effect of temperature on AgNPs synthesis was evaluated using temperature values ranging from 30 to 80 • C and previously optimized parameters. In the case of A. alba bark extract-derived AgNPs, sharp peaks appeared when the synthesis was conducted at 40, 60, and 80 • C (absorption maximum at 426, 428, and 427 nm, respectively). Smaller and broader peaks were generated at 30, 50, and 70 • C ( Figure 1G). For P. sylvestris bark extract-derived AgNPs, the best yields were obtained at 30 and 70 • C. At 30 • C, the absorption maximum was observed at 423 nm; at 70 • C, it shifted to 425 nm ( Figure 1H). The temperature values of 60, 80, and 70 • C shifted the absorption maximum of A. alba and P. sylvestris bark extractderived AgNPs, respectively, to longer wavelengths, indicating that AgNPs size increased. Therefore, 40 and 30 • C were chosen as optimal temperatures for the synthesis of AgNPs from A. alba and P. sylvestris bark extract, respectively.
To evaluate the effect of reaction time on AgNPs synthesis, A. alba/P. sylvestris bark aqueous extract was mixed with 2 mM AgNO 3 (1:9, v/v, pH of reaction mixture adjusted to 10 with 0.1 N NaOH) and stirred at 40 • C/30 • C for 120 min. AgNPs synthesis was checked at different time intervals (0-120 min) during stirring and at 24 h after the end of stirring. As shown in Figure 1I,J, for both bark extracts, the reduction of Ag + started immediately after the addition of the reducing agent (bark extract) to the AgNO 3 solution (0 min). At each time interval, an absorbance peak at around 420 nm indicated the synthesis of AgNPs. The yield of AgNPs increased as the reaction time increased. Therefore, a period of 120 min stirring followed by 24 h at rest was considered optimal for the synthesis of AgNPs.
The optimized experimental parameters for the synthesis of AgNPs were pH 10, the temperature of 30 and 40 • C (for P. sylvestris and A. alba bark extract-derived AgNPs, respectively), 2 mM AgNO 3 , bark extract to AgNO 3 (2 mM) ratio of 1:9 (v/v), and 120 min reaction time (followed by 24 h at rest).

Characterization of AgNPs
A. alba and P. sylvestris bark extract-derived AgNPs, synthesized under optimized conditions, were characterized by ATR-FTIR spectroscopy, DLS, TEM, and SEM with EDX analyses.
ATR-FTIR spectroscopy was used to identify the functional groups of the compounds in bark extracts involved in the synthesis and stabilization of AgNPs. As shown in Figure 2, A. alba and P. sylvestris bark extracts exhibited broad bands at 3259 and 3269 cm −1 , respectively, corresponding to the stretching vibrations of the hydroxyl groups of polyphenols and polysaccharides [52]. Bands at 2925 and 2929 cm −1 (A. alba and P. sylvestris bark extract, respectively) belong to the stretching vibrations of the methylene groups [53]. Bands around 1600 and 1500 cm −1 (both bark extracts), 1443 cm −1 (A. alba bark extract), and 1262 cm −1 (P. sylvestris bark extract) could be ascribed to the stretching vibrations of the aromatic rings and =C-O-C groups of flavonoids [29,54]. The bands around 1600 and 1500 cm −1 could also belong to the amide region in proteins [29,53,55]. The bands at 1027 and 1025 cm −1 (A. alba and P. sylvestris bark extract, respectively) are characteristic of the C-O-C stretching vibrations in carbohydrates [29,56,57]. The aromatic C-H out-of-plane bending vibrations are indicated by the bands at 816 cm −1 (both bark extracts) and 775 cm −1 (P. sylvestris bark extract), the latter suggesting the presence of flavonoids (B ring) [29,58]. ATR-FTIR spectra of AgNPs illustrate shifts in the bark extract bands confirming the involvement of some phytochemicals (polyphenols, carbohydrates, proteins) in the formation of AgNPs (reduction of Ag + to metallic Ag), but also in their stabilization. The bands at 3259 and 3269 cm −1 in A. alba and P. sylvestris bark extracts, respectively, not only shifted, but they attenuated and broadened, indicating the participation of polyphenols/polysaccharides in the synthesis of AgNPs.
aromatic rings and =C-O-C groups of flavonoids [29,54]. The bands around 1600 and 1500 cm −1 could also belong to the amide region in proteins [29,53,55]. The bands at 1027 and 1025 cm −1 (A. alba and P. sylvestris bark extract, respectively) are characteristic of the C-O-C stretching vibrations in carbohydrates [29,56,57]. The aromatic C-H out-of-plane bending vibrations are indicated by the bands at 816 cm −1 (both bark extracts) and 775 cm −1 (P. sylvestris bark extract), the latter suggesting the presence of flavonoids (B ring) [29,58]. ATR-FTIR spectra of AgNPs illustrate shifts in the bark extract bands confirming the involvement of some phytochemicals (polyphenols, carbohydrates, proteins) in the formation of AgNPs (reduction of Ag + to metallic Ag), but also in their stabilization. The bands at 3259 and 3269 cm −1 in A. alba and P. sylvestris bark extracts, respectively, not only shifted, but they attenuated and broadened, indicating the participation of polyphenols/polysaccharides in the synthesis of AgNPs.
Polyphenols majorly contributed to the synthesis and stabilization of AgNPs. Their contribution is also supported by a significant decrease in the phenolic contents of the reaction mixtures after the synthesis and separation of AgNPs (supernatants) (A. alba: 152.01 ± 11.16 µg/mL in the reaction mixture vs. 18.88 ± 3.09 µg/mL in supernatant; P. sylvestris: 608.15 ± 9.33 µg/mL in the reaction mixture vs. 432.71 ± 3.89 µg/mL in the supernatant).  Polyphenols majorly contributed to the synthesis and stabilization of AgNPs. Their contribution is also supported by a significant decrease in the phenolic contents of the reaction mixtures after the synthesis and separation of AgNPs (supernatants) (A. alba: 152.01 ± 11.16 µg/mL in the reaction mixture vs. 18.88 ± 3.09 µg/mL in supernatant; P. sylvestris: 608.15 ± 9.33 µg/mL in the reaction mixture vs. 432.71 ± 3.89 µg/mL in the supernatant).
DLS analysis was performed to determine the mean hydrodynamic diameter (Zaverage), PDI, and zeta potential values for estimating the size of AgNPs in colloidal suspension, their dispersion, and stability, respectively [59]. A. alba bark extract-derived AgNPs showed a Z-average value of 62.84 nm and 2 peak values in the hydrodynamic diameter distribution (25.84 and 190.3 nm) ( Figure 3A). For P. sylvestris bark extract-derived AgNPs, a single peak (106.5 nm) with a narrow base was obtained, indicating better homogeneity; the average particle size (Z-average) was 84.35 nm ( Figure 3B). PDI value is an indicator of the homogeneity of the size distribution of AgNPs in colloidal dispersion. Low PDI values (below 0.7) reflect a narrow range of particle sizes and high uniformity [60]. In this study, PDI values indicated that P. sylvestris bark extract-derived AgNPs had a narrower distribution and better homogeneity in comparison with those derived from A. alba bark extract (0.221 vs. 0.334) ( Figure 3A,B). The zeta potential indicates the charge on the surface of AgNPs and predicts their stability in colloidal dispersion, with values of ±10-20 mV reflecting a relatively stable colloid system [55,61]. Both A. alba and P. sylvestris bark extract-derived AgNPs showed similar negative values for zeta potential (−10.9 and −10.8 mV, respectively) ( Figure 3A,B). a narrower distribution and better homogeneity in comparison with those derived from A. alba bark extract (0.221 vs. 0.334) ( Figure 3A,B). The zeta potential indicates the charge on the surface of AgNPs and predicts their stability in colloidal dispersion, with values of ±10-20 mV reflecting a relatively stable colloid system [55,61]. Both A. alba and P. sylvestris bark extract-derived AgNPs showed similar negative values for zeta potential (−10.9 and −10.8 mV, respectively) ( Figure 3A,B). The morphology of AgNPsʹ surface and their elemental composition were investigated by SEM with EDX analyses. SEM micrograph images showed aggregations of spherical nanoparticles ( Figure 4C,D), most probably caused by the procedures used to isolate AgNPs from the reaction mixtures (centrifugation followed by freeze-frying). EDX analysis provides qualitative and quantitative information on the chemical composition of AgNPs [34]. The EDX spectra of both A. alba and P. sylvestris bark extract-derived AgNPs ( Figure 4A and 4B, respectively) revealed a pronounced signal for metallic Ag at 3 keV which confirmed the formation of AgNPs. Several other signals were registered for C, N, O, P, and Cl belonging to the compounds in bark extracts acting as capping agents [33,62]. The morphology of AgNPs' surface and their elemental composition were investigated by SEM with EDX analyses. SEM micrograph images showed aggregations of spherical nanoparticles ( Figure 4C,D), most probably caused by the procedures used to isolate AgNPs from the reaction mixtures (centrifugation followed by freeze-frying). EDX analysis provides qualitative and quantitative information on the chemical composition of AgNPs [34]. The EDX spectra of both A. alba and P. sylvestris bark extract-derived AgNPs ( Figure 4A,B, respectively) revealed a pronounced signal for metallic Ag at 3 keV which confirmed the formation of AgNPs. Several other signals were registered for C, N, O, P, and Cl belonging to the compounds in bark extracts acting as capping agents [33,62].
The spherical shape of A. alba and P. sylvestris bark extract-derived AgNPs was confirmed by TEM ( Figure 5A,B,D,E). In contrast to SEM micrographs, TEM images showed that the synthesized AgNPs were well-dispersed. This disparity might have its origin in sample preparation for analysis. As already mentioned, AgNPs as pellets, obtained by centrifugation and freeze-drying, were analyzed by SEM, whereas AgNPs as colloidal dispersion were subjected to TEM. Centrifugation followed by freeze-frying promoted AgNPs agglomeration, which was visible in SEM micrographs. Colloidal AgNPs formed in situ had a good dispersion rate, as illustrated by TEM analysis. Both A. alba and P. sylvestris bark extract-derived AgNPs showed lighter margins attributed to the compounds in bark extracts which adhered to the surface of AgNPs, thus capping and stabilizing them [63,64]. The presence of metallic Ag was revealed by TEM-EDX mapping ( Figure 5C,F) confirming the reduction of Ag + to Ag 0 by the compounds present in bark extracts. A. alba bark extract-derived AgNPs ranged from 6 to 13 nm, whereas P. sylvestris bark extract-derived AgNPs were larger (12-31 nm). By fitting the histogram data with a Gaussian distribution, the average particle size was found to be 9.92 and 24.49 nm for A. alba and P. sylvestris bark extract-derived AgNPs, respectively ( Figure 5G,H).
AgNPs agglomeration, which was visible in SEM micrographs. Colloidal AgNPs formed in situ had a good dispersion rate, as illustrated by TEM analysis. Both A. alba and P. sylvestris bark extract-derived AgNPs showed lighter margins attributed to the compounds in bark extracts which adhered to the surface of AgNPs, thus capping and stabilizing them [63,64]. The presence of metallic Ag was revealed by TEM-EDX mapping ( Figure 5C,F) confirming the reduction of Ag + to Ag 0 by the compounds present in bark extracts. A. alba bark extract-derived AgNPs ranged from 6 to 13 nm, whereas P. sylvestris bark extractderived AgNPs were larger (12-31 nm). By fitting the histogram data with a Gaussian distribution, the average particle size was found to be 9.92 and 24.49 nm for A. alba and P. sylvestris bark extract-derived AgNPs, respectively ( Figure 5G,H).

Cytotoxic Activity
A. alba and P. sylvestris bark extract-derived AgNPs negatively impacted cell viability in a time-and dose-dependent manner. After 48 h treatment, the viability of A-375 cells exposed to A. alba bark extract-derived AgNPs (12.5 µg/mL) significantly decreased to 22.32 ± 3.48%. At higher concentrations (25 µg/mL), a slight increase in cell viability (26.12 ± 0.57%) was noticed ( Figure 7A). A similar tendency was observed in Vero cells, namely 37.11 ± 1.84% and 40.90 ± 2.65% viability in cells treated with 12.5 and 25 µg/mL A. alba bark extract-derived AgNPs, respectively ( Figure 7B). When A-375 and Vero cells were exposed to P. sylvestris bark extract-derived AgNPs (12.5 and 25 µg/mL) for 48 h, the following cell viability percentages were detected: 27.02 ± 0.36%, 24.10 ± 0.72%, and 42.46 ± 1.20%, 38.95 ± 3.15%, respectively ( Figure 7C,D). According to the IC 50 values (after 48 h treatment), A. alba bark extract-derived AgNPs were more cytotoxic to A-375 cells; both bark extract-derived AgNPs were less cytotoxic to Vero cells (Table 1).  The microscopic investigation revealed a decrease in cell density and substrate adhesion capacity together with membrane shrinkage after exposure to A. alba and P. sylvestris bark extract-derived AgNPs, the processes being more intense after 48 h exposure. In contrast, the control cells showed a high confluence of the monolayer, intact cell membrane, and smooth morphology (without roughness) (Figure 8).

Figure 7. Viability of A-375 human malignant melanoma cells exposed to Abies alba (A) and
Pinus sylvestris bark extract-derived AgNPs (C); viability of African green monkey kidney (Vero) cells exposed to Abies alba (B) and Pinus sylvestris bark extract-derived AgNPs (D); significant differences (p < 0.001) between Abies alba/Pinus sylvestris bark extract-derived AgNPs (at all tested concentrations) and control.
The microscopic investigation revealed a decrease in cell density and substrate adhesion capacity together with membrane shrinkage after exposure to A. alba and P. sylvestris bark extract-derived AgNPs, the processes being more intense after 48 h exposure. In contrast, the control cells showed a high confluence of the monolayer, intact cell membrane, and smooth morphology (without roughness) (Figure 8).

Antibacterial Activity
The antibacterial activity of AgNPs was investigated against five pathogenic reference strains, including two resistant ones: methicillin-resistant S. aureus (MRSA) ATCC 43300 and S. epidermidis ATCC 12228. The former also showed resistance to oxacillin and gentamicin, whereas the latter proved to be resistant to tetracycline [65]. According to the MIC values (Table 2), A. alba bark extract-derived AgNPs possessed good antibacterial activity, being more active than P. sylvestris bark extract-derived AgNPs against all tested bacteria except P. aeruginosa ATCC 9027. In addition, A. alba bark extract-derived AgNPs inhibited the growth of resistant bacteria (MRSA ATCC 43300, S. epidermidis ATCC 12228)

Antibacterial Activity
The antibacterial activity of AgNPs was investigated against five pathogenic reference strains, including two resistant ones: methicillin-resistant S. aureus (MRSA) ATCC 43300 and S. epidermidis ATCC 12228. The former also showed resistance to oxacillin and gentamicin, whereas the latter proved to be resistant to tetracycline [65]. According to the MIC values (Table 2), A. alba bark extract-derived AgNPs possessed good antibacterial activity, being more active than P. sylvestris bark extract-derived AgNPs against all tested bacteria except P. aeruginosa ATCC 9027. In addition, A. alba bark extract-derived AgNPs inhibited the growth of resistant bacteria (MRSA ATCC 43300, S. epidermidis ATCC 12228) while P. sylvestris bark extract-derived AgNPs showed no activity against these two strains in the tested concentration range (0.24-250 µg/mL).

Discussion
The use of plant extracts for the preparation of AgNPs has important advantages over other methods (physical, chemical, mechanical, and microbial). Plant extract-mediated AgNPs synthesis is simple, fast, and cost-effective. Phytochemicals in plant extracts play a dual role, serving as both reducing agents and stabilizers of AgNPs. In addition, the method is eco-friendly as it produces less environmental contamination [8]. To date, different parts of coniferous trees (bark, stem, needles, cones, and even exudates such as gum) have been used for the synthesis of AgNPs with various biological effects (antimicrobial, anticancerous, antioxidant, and antipyretic) [19].
The biological effects of plant-derived AgNPs depend not only on the components of plant extracts but also on their physicochemical properties which are influenced by the synthesis parameters (pH, AgNO 3 concentration, ratio between the volumes of plant extract, AgNO 3 , temperature, and reaction time) [3,12,33,35]. Therefore, in our study, we optimized the reaction conditions, taking into consideration the aforementioned parameters and monitoring the synthesis of AgNPs by UV-Vis spectroscopy. pH significantly affects the synthesis and stability of AgNPs. An alkaline pH facilitates the deprotonation of phenolic hydroxyl groups, thus enhancing their reduction abilities and consequently the synthesis of AgNPs [35]. At the same time, the amount of negative charge on the surface of AgNPs increases, the electrostatic repulsion between AgNPs becomes stronger, and their aggregation is lowered, resulting in small-sized, well-dispersed, and stable AgNPs. In addition, AgNPs synthesized in alkaline conditions have more regular shapes, usually spherical and hexagonal [87]. Basic pH values (8)(9)(10)(11)(12) were found to be optimal for the synthesis of AgNPs using extracts prepared from Picea abies bark [88,89], Pinus eldarica bark [90], Pinus thunbergii cones [91], Bryophyllum pinnatum leaves [33], Oryza sativa (Thai pigmented rice, riceberry) [35], Syzygium jambos leaves and bark [92], and Givotia moluccana leaves [53]. On the other hand, there are also studies reporting an optimal synthesis of AgNPs at neutral pH [51]. In our study, the synthesis of AgNPs was supported by an alkaline medium (pH 10). Another factor which dramatically affects the synthesis and stability of AgNPs is AgNO 3 concentration. Its increase results in higher yields of AgNPs but also a higher agglomeration rate. Moreover, an increase in AgNO 3 concentration above 10 mM causes the deposition of AgNO 3 on the surface of AgNPs, which may enhance their toxicity. Most studies showed that 1 mM AgNO 3 is the optimal concentration for AgNPs synthesis [51]. However, in some cases, small-sized AgNPs were synthesized using higher AgNO 3 concentrations. AgNPs of 15 and 20-50 nm were synthesized using 2 mM AgNO 3 and extracts obtained from Crocus sativus wastages [93] and Chrysanthemum morifolium flowers [94], respectively. AgNPs of 30-50 nm were obtained from Chenopodium murale leaf extract and 5 mM AgNO 3 [95]. The synthesis of monodispersed, spherical, and 9 nm sized AgNPs was achieved using Hibiscus cannabinus leaf extract and 5 mM AgNO 3 [96]. In our study, 9.92 and 24.49 nm sized AgNPs (according to TEM data) were produced from A. alba and P. sylvestris bark aqueous extracts, respectively, and 2 mM AgNO 3 in a ratio of 1:9 (v/v); an increase in the bark extract volume (ratios of 2:8, 3:7, 5:5, 7:3, and 9:1) impaired the synthesis of AgNPs. Other studies reported an optimal synthesis of AgNPs using Picea abies bark extract [89], Aloe vera leaf extract [97], Rubus glaucus fruit extract [98], Thymus kotschyanus plant extract [99], Juglans regia seed extract [100] or Prunus serotina cherry extract [101] and AgNO 3 (1 mM) in a ratio of 1:10 (v/v). An increase in AgNO 3 volume (P. abies bark extract and AgNO 3 in ratios of 1:50 and 1:100) significantly reduced the accumulation of AgNPs [89]. Temperature plays an important role in the synthesis of AgNPs. An increase in temperature enhances the kinetic energy of molecules and accelerates the reaction rate resulting in smaller-sized AgNPs and less AgNPs agglomeration [102]. On the other hand, high kinetic energy might enable multiple collisions and consequently, the aggregation and growth of AgNPs [103]. In our study, the optimal temperatures for the synthesis of P. sylvestris and A. alba bark extract-derived AgNPs were 30 and 40 • C, respectively. According to the literature data, a high-yield synthesis of uniformly sized, small, and spherical AgNPs was achieved at temperatures of 30-40 • C using leaf extracts of Hibiscus cannabinus [96], aloe [103], Cynodon dactylon [104], Ocimum sanctum [105], Tephrosia purpurea [106], O. tenuiflorum, Solanum tricobatum, Syzygium cumini, and Centella asiatica and peel extract of Citrus sinensis [107]. Reaction time affects both the yield and stability of AgNPs. Prolonged reaction times increase the yield of AgNPs but promote agglomeration of AgNPs, decreasing their stability [108]. There have been reported various reaction times in plant-mediated synthesis of AgNPs, e.g., 20 [107], and 72 h [34]. In our study, a reaction time of 120 min (followed by 24 h at rest) was found to be optimal for the synthesis of AgNPs using A. alba and P. sylvestris bark extracts.
The optimized parameters (pH 10, 30, and 40 • C for P. sylvestris and A. alba bark extractderived AgNPs, respectively, 2 mM AgNO 3 , bark extract to 2 mM AgNO 3 ratio of 1:9, v/v, and 120 min reaction time, followed by 24 h at rest) led to the synthesis of well-dispersed, spherical, small, and relatively stable AgNPs. Both DLS and TEM determined smaller average particle sizes for A. alba bark extract-derived AgNPs (62.84 and 9.92 nm, respectively, vs. 84.35 and 24.49 nm, respectively, for P. sylvestris bark extract-derived AgNPs). The difference in the average size between A. alba and P. sylvestris bark extract-derived AgNPs could be attributed to the different temperature values used in AgNPs synthesis (40 • C for A. alba bark extract-derived AgNPs, 30 • C for P. sylvestris bark extract-derived AgNPs) and AgNPs outer shell consisting of capping/stabilizing compounds. According to Verma and Mehata [102], as the temperature increases, the reaction rate quickens, resulting in smaller-sized AgNPs. In contrast to TEM, DLS analysis determined larger sizes (62.84 and 84.35 nm for A. alba and P. sylvestris bark extract-derived AgNPs, respectively). The discrepancy might be ascribed to the fact that DLS estimates the hydrodynamic diameter of AgNPs (the metallic core of AgNPs with the compounds adsorbed on their surface and the solvation shell), whereas TEM measures the geometric size of AgNPs [109]. The synthesized AgNPs were negatively charged (zeta potential values of −10.9 and −10.8 mV for A. alba and P. sylvestris bark extract-derived AgNPs, respectively). As already mentioned, polyphenols were involved in the synthesis and stabilization of AgNPs. According to Yang and Li (2013), an alkaline pH enhances the deprotonation of the phenolic hydroxyl groups of capping AgNPs, resulting in negatively charged hydroxylate ions on the surface of AgNPs [110]. The electrostatic repulsive forces between negatively charged AgNPs prevent their agglomeration, resulting in high dispersity and stability [55,111].
The anticancer potential of AgNPs has recently gained much attention. Various mechanisms are involved in the anticancer activity of AgNPs such as alteration of cell/lysosomal membrane integrity, increase in oxidative stress, induction of apoptosis, cell cycle arrest, and inhibition of angiogenesis [48,118]. The anticancer activity of AgNPs is considerably influenced by their size, shape, and zeta potential. A smaller size, an irregular shape, and a negative zeta potential are essential for good anticancer activity [119]. The use of plant extracts for the synthesis of AgNPs has produced phytofunctionalized AgNPs with remarkable anticancer properties. Plant extract-derived AgNPs proved to be active against different human cancer cell lines (lung, breast, liver, cervical, prostate, colon, ovarian, etc.); some phytofunctionalized AgNPs displayed low IC 50 values (<100 µg/mL) and/or showed less cytotoxicity against normal cells [8,120,121]. To the best of our knowledge, the cytotoxic potential of phytofunctionalized AgNPs against human melanoma cells has been scarcely investigated [122]. González-Pedroza et al. [123] have recently studied the cytotoxic effects of AgNPs synthesized using Annona muricata leaf and fruit peel aqueous extracts on A-375 human malignant melanoma cells and found a remarkable activity (IC 50 = 2.004 and 1.285 µg/mL, respectively), with less toxicity to normal macrophages (IC 50 = 10.7 and 13.7 µg/mL, respectively). A. muricata aqueous extracts contain polyphenols, acetogenins, and citric acid, which are responsible not only for the synthesis and stabilization of AgNPs but also for their anticancer activity. Acetogenins, for example, are well known for their pro-apoptotic effects [123]. Another study reported a significant reduction in the viability of A-375 cells after exposure to AgNPs obtained from the homeopathic mother tinctures (ethanolic extracts, 70% ethanol, v/v) of Gelsemium sempervirens, Thuja occidentalis, Phytolacca decandra, and Hydrastis canadensis (IC 50 = 80, 120, 78, and 100 µg/mL, respectively). All AgNPs showed pro-apoptotic effects associated with DNA fragmentation, cell cycle arrest at the G2/M phase, and caspase-3 activation [119]. AgNPs synthesized using boswellic acid (isolated from the gum resin of Boswellia serrata) exerted cytotoxic activity on G361 human malignant melanoma cells, and the effect is comparable to that of doxorubicin [124]. In our study, A. alba and P. sylvestris bark extract-derived AgNPs showed significant cytotoxicity toward A-375 human malignant melanoma cells (IC 50 = 2.40 and 6.02 µg/mL, respectively), being less cytotoxic against normal Vero cells (IC 50 = 2.76 and 9.71 µg/mL, respectively) (after 48 h treatment). The smaller-sized A. alba bark extract-derived AgNPs exhibited higher cytotoxicity against both cell lines. Smaller AgNPs are more cytotoxic due to their more efficient interaction with cell membranes, resulting in a higher penetration rate and cellular uptake and consequently higher antitumor efficacy [3,13,121,125]. In addition, phytochemical capping AgNPs undoubtedly contribute to the anticancer activity of phytofunctionalized AgNPs. Our study showed that polyphenols in A. alba and P. sylvestris bark extracts are involved in the synthesis and stabilization of AgNPs. Among these polyphenols, catechins (catechin, epicatechin, gallocatechin, procyanidin oligomers) have been reported to inhibit melanoma cell growth and invasion by multiple mechanisms (cell cycle arrest, decrease in cyclooxygenase-2 expression, prostaglandin E 2 production, ERK1/2 phosphorylation, and NFκBp65 activation) [126]. Moreover, a pine bark extract-Pinus maritima (syn. Pinus pinaster), having 61.26 mg catechin/g, induced apoptosis in A-375 cells by increasing caspase-3 expression and activity. In addition, the extract reduced oxidative stress and down-regulated matrix metallopeptidase-9, an enzyme responsible for cell invasion and metastasis [127]. A. alba and P. sylvestris bark extract-derived AgNPs also altered cell morphology, most probably due to cytoskeleton damage [123].
AgNPs are well-known antimicrobial agents being active against Gram-positive and Gram-negative bacteria, fungi, and viruses, including drug-resistant strains. In brief, Ag-NPs adhere to the microbial cell surface through electrostatic forces disrupting the cell membrane integrity, penetrating inside the cell, and interacting with proteins, lipids, ribosomes, and DNA, causing severe cellular dysfunctions. AgNPs increase cellular oxidative stress and alter important signal transduction pathways (tyrosine phosphorylation) causing cell damage and death [4]. The antibacterial potential of AgNPs can be significantly improved by using plant extracts for their synthesis. Extracts from different parts of plants (leaves, flowers, fruits, seeds, peels, roots, rhizomes, bark, stem, and even gum, latex, and callus) and whole plants have been used for the synthesis of AgNPs with antibacterial effects. The capping phytochemicals but also the size of AgNPs significantly impact the antibacterial activity. A small size (<50 nm) provides a greater surface of interaction with the bacterial membrane, which enhances the antibacterial potency [12,128]. In our study, the stronger antibacterial activity of A. alba bark extract-derived AgNPs can be attributed, in large part, to their smaller size (9.92 vs. 24.49 nm for P. sylvestris bark extract-derived AgNPs). In addition, A. alba bark extract-derived AgNPs displayed the lowest MIC values (18.75 µg/mL) against two Gram-positive bacteria (S. aureus ATCC 25923 and S. epidermidis ATCC 12228) which supports the contribution of capping phytochemicals to the antibacterial activity of AgNPs. As already mentioned, "naked" AgNPs are more effective against Gram-negative bacteria, whereas the phytofunctionalized ones have a broader antibacterial spectrum, including Gram-positive bacteria. Polyphenols, which cap and stabilize A. alba and P. sylvestris bark extract-derived AgNPs, have antimicrobial properties [31]. Catechins, for example, are more effective against Gram-positive bacteria. The antibacterial activity of catechins is associated with cytoplasmic membrane damage, alteration of membrane integrity, and cellular leakage. Catechins easily bind to the peptidoglycan layer of Gram-positive bacteria but hardly penetrate the lipopolysaccharide layers of Gramnegative bacteria [129]. AgNPs synthesized using plant extracts demonstrated efficacy against various bacteria, including those tested in the present study. Eucalyptus globulus leaf extract-derived AgNPs displayed antibacterial activity against MSSA, MRSA, and extended-spectrum β-lactamase E. coli and P. aeruginosa clinical isolates with MIC values of 30, 27, 36, and 27 µg/mL, respectively [130]. The MIC values of AgNPs prepared using Musa paradisiaca peel extract against MSSA (S. aureus ATCC 6538), E. coli ATCC 8739, and P. aeruginosa ATCC 9027 were 5.1, 3.4, and 1.7 µg/mL, respectively [34]. AgNPs obtained from Alpinia katsumadai seed extract inhibited the growth of E. coli (MIC = 20 µg/mL) and P. aeruginosa (MIC = 40 µg/mL) [131].

Conclusions
The results of the present study clearly demonstrate that Abies alba and Pinus sylvestris bark can be successfully used for the synthesis of bioactive AgNPs. Abies alba and Pinus sylvestris bark extract-derived AgNPs were well-dispersed, spherical, and small-sized (9.92 and 24.49 nm, respectively). The synthesized AgNPs had a pronounced cytotoxic activity against A-375 human malignant melanoma cells, being less cytotoxic to normal cells. They were also found to have antioxidant activity and antibacterial effects against Grampositive and Gram-negative bacteria. Abies alba bark extract-derived AgNPs demonstrated stronger toxicity against A-375 human malignant melanoma cells as well as stronger antibacterial activity against Staphylococcus aureus (methicillin-sensitive and methicillinresistant), Staphylococcus epidermidis, and Escherichia coli. The bioactivity of synthesized AgNPs was influenced by their size and capping/stabilizing phytochemicals on their surface. In conclusion, Abies alba and Pinus sylvestris bark extract-derived AgNPs exhibited antioxidant, antibacterial, and cytotoxic effects against A-375 human malignant melanoma cells. Further studies are needed for an in-depth assessment of their antioxidant, anticancer, and antimicrobial potential and also biocompatibility by using more normal tumor cell lines and bacterial strains.

Data Availability Statement:
The data presented in this study are available upon request from the corresponding author (Anca Miron, anca.miron@umfiasi.ro).