Ribes nigrum L. Extract-Mediated Green Synthesis and Antibacterial Action Mechanisms of Silver Nanoparticles

Silver nanoparticles (Ag NPs) represent one of the most widely employed metal-based engineered nanomaterials with a broad range of applications in different areas of science. Plant extracts (PEs) serve as green reducing and coating agents and can be exploited for the generation of Ag NPs. In this study, the phytochemical composition of ethanolic extract of black currant (Ribes nigrum) leaves was determined. The main components of extract include quercetin rutinoside, quercetin hexoside, quercetin glucuronide, quercetin malonylglucoside and quercitrin. The extract was subsequently employed for the green synthesis of Ag NPs. Consequently, R. nigrum leaf extract and Ag NPs were evaluated for potential antibacterial activities against Gram-negative bacteria (Escherichia coli ATCC 25922 and kanamycin-resistant E. coli pARG-25 strains). Intriguingly, the plant extract did not show any antibacterial effect, whilst Ag NPs demonstrated significant activity against tested bacteria. Biogenic Ag NPs affect the ATPase activity and energy-dependent H+-fluxes in both strains of E. coli, even in the presence of N,N’-dicyclohexylcarbodiimide (DCCD). Thus, the antibacterial activity of the investigated Ag NPs can be explained by their impact on the membrane-associated properties of bacteria.


Introduction
The emergence of antibiotic resistance is a serious challenge for both human and veterinary medicine. Several mechanisms of antibiotic-resistance development are known [1][2][3]. Although there are some approaches to overcome this threat, no final solution is found yet. The antibacterial properties of silver date back to ancient times [4]. Silver (positively charged silver ions (Ag + ), when dissolved in aqueous environment) provides strong antimicrobial activity against a wide spectrum of microorganisms. Silver ions serve as multifunctional agents which, for instance, can produce pores in bacterial cell walls through interaction with the peptidoglycan components [5]. The other mechanism of antimicrobial action of silver cations involves its ability to penetrate the bacterial cell membrane, which leads to the inhibition of cellular respiration and, consequently, the generation of the reactive oxygen species (ROS). Moreover, Ag + is highly toxic to microorganisms due to its ability to disrupt not only DNA but also their replication cycle [5]. The current study is mainly focused on the understanding of the underlying mechanisms of antibacterial activities of Ag NPs, produced by the leaf extracts of R. nigrum harvested from high-altitude Armenian landscape against Escherichia coli strains (including drug-resistant ones).

The Total Phenolic-Flavonoid Composition of R. nigrum Extracts
The total flavonoid and phenol contents in R. nigrum extracts were estimated from the calibration curve of quercetin and gallic acid, respectively. The total flavonoid and phenolic contents of 35.42 ± 1.52 mg quercetin equivalent (QE)/g DW) and 84.1 ± 1.6 mg gallic acid equivalent (GAE)/g DW) were quantified, respectively. The presence of high flavonoid and phenolic content in the R. nigrum extract indicates its potential as a reducing agent.

Identification of Major Polyphenols in R. nigrum Extract
The HPLC results have revealed the presence of about 30 major constituents comprising mainly of flavan-3-ols, flavonols, hydroxycinnamates, lignans, naphthols and furanocoumarins ( Figure 1). Each compound was assigned to one of the above-mentioned groups by the careful analysis of its UV-Vis spectrum. Finally, the compounds were identified based on the full scan MS, MS2 spectra, obtained for major m/z signals recorded in negative ion mode, retention time, and bibliography (Table 1).
lower than fruit extract [27]. There is no literature concerning the acute toxicity, genotoxicity, reproductive and developmental toxicity or carcinogenicity of this plant extract [28]. Literature reveals several reports on the green synthesis of Ag NPs using R. nigrum fruit or pomace extracts [29,30]; however, there is no literature about the utilization of leaf extracts.
The current study is mainly focused on the understanding of the underlying mechanisms of antibacterial activities of Ag NPs, produced by the leaf extracts of R. nigrum harvested from high-altitude Armenian landscape against Escherichia coli strains (including drug-resistant ones).

The Total Phenolic-Flavonoid Composition of R. nigrum Extracts
The total flavonoid and phenol contents in R. nigrum extracts were estimated from the calibration curve of quercetin and gallic acid, respectively. The total flavonoid and phenolic contents of 35.42 ± 1.52 mg quercetin equivalent (QE)/g DW) and 84.1 ± 1.6 mg gallic acid equivalent (GAE)/g DW) were quantified, respectively. The presence of high flavonoid and phenolic content in the R. nigrum extract indicates its potential as a reducing agent.

Identification of Major Polyphenols in R. nigrum Extract
The HPLC results have revealed the presence of about 30 major constituents comprising mainly of flavan-3-ols, flavonols, hydroxycinnamates, lignans, naphthols and furanocoumarins ( Figure 1). Each compound was assigned to one of the above-mentioned groups by the careful analysis of its UV-Vis spectrum. Finally, the compounds were identified based on the full scan MS, MS2 spectra, obtained for major m/z signals recorded in negative ion mode, retention time, and bibliography (Table 1).  Table 1.  O 9 ). The further MS2 fragmentation spectra revealed the identity of these two compounds as caffeoyl quinic acid isomers. In the case of compounds 14, 24 and 25, it was not possible to assign Compound 12 showed the base peak ion in the MS spectrum at m/z 521.20294 (C 26 H 33 O 11 ) followed by the cleavage of hexose moiety (−162 amu) in MS2 spectrum. Thus, this compound was classified as a lignan and tentatively assigned as lariciresinol glucoside.
A similar pattern of fragmentation has also been observed for compounds 28 and 30. The fragmentation of precursor ions at m/z values of 407.13468 (C 20 H 23 O 9 ) and 449.14526 (C 22 H 26 O 10 ) for compounds 28 and 30, respectively, have provided the same aglycon ion at m/z 245.08138 (C 14 H 13 O 4 ) due to the loss of hexose and acetyl-hexose moieties. Therefore, these compounds were tentatively identified as marmesin glucoside and marmesin acetyl glucoside, respectively.

Radical Scavenging Capacity of R. nigrum Extract
The potential antioxidant activity of R. nigrum extract was confirmed by DPPH assay. The radical scavenging activity of plant extract was compared with that of catechin (IC 50 = 12.62 ± 0.8 µg/mL), which is a well-known antioxidant and exhibits excellent Antibiotics 2022, 11, 1415 6 of 17 DPPH scavenging properties. In our study, R. nigrum extract provided an IC 50 value of 63.59 ± 1.63 µg/mL (R 2 = 0.9462) which clearly indicated that the extract was able to scavenge 50% of the radicals at the concentration of around 63.59 µg/mL.

Antioxidant Profiling by HPLC Coupled Post-Column Derivatization
Post-column derivatization of analytes with ABTS reagent was performed during HPLC analysis of R. nigrum extract. This investigation has allowed us to identify the specific compounds responsible for the antioxidant activity. As observed in the ABTS colorimetric tests, the reduction reaction has led to a significant shift in the UV-Visible spectrum, resulting in ABTS reagent absorption change (discoloration). The presence of antioxidants in the eluate has provided negative peaks in the chromatogram, recorded after derivatization at 734 nm ( Figure 1B). The profile obtained after derivatization indicates that almost all of the compounds identified in R. nigrum extract exhibit antioxidant activity. The highest antioxidant activity was observed for flavan-3-ols, followed by hydroxycinnamates and flavonols.

Metal Chelating Capability of R. nigrum Leaf Extract
R. nigrum leaf extract provided 56.53 ± 0.7% of metal chelating activity, whilst the same concentration of positive control (EDTA) provided 90 ± 0.8%.

Production of Silver Nanoparticles Using Plant Extract
The phytochemical profiling of R. nigrum leaf extract affirmed the presence of polyphenols whose reducing nature paved the way for the green synthesis of Ag NPs. Experimental data have shown that the synthesis of nanoparticles occurs in the presence of light. The formation of Ag NPs was confirmed by exploiting UV-Vis spectroscopy ( Figure 2). The results have indicated the emergence of an absorbance peak at around 445 nm, which is specific for Ag NPs in the samples ( Figure 2) [9].

Radical Scavenging Capacity of R. nigrum Extract
The potential antioxidant activity of R. nigrum extract was confirmed by DPPH assay. The radical scavenging activity of plant extract was compared with that of catechin (IC50 = 12.62 ± 0.8 µ g/mL), which is a well-known antioxidant and exhibits excellent DPPH scavenging properties. In our study, R. nigrum extract provided an IC50 value of 63.59 ± 1.63 µ g/mL (R² = 0.9462) which clearly indicated that the extract was able to scavenge 50% of the radicals at the concentration of around 63.59 µ g/mL.

Antioxidant Profiling by HPLC Coupled Post-Column Derivatization
Post-column derivatization of analytes with ABTS reagent was performed during HPLC analysis of R. nigrum extract. This investigation has allowed us to identify the specific compounds responsible for the antioxidant activity. As observed in the ABTS colorimetric tests, the reduction reaction has led to a significant shift in the UV-Visible spectrum, resulting in ABTS reagent absorption change (discoloration). The presence of antioxidants in the eluate has provided negative peaks in the chromatogram, recorded after derivatization at 734 nm ( Figure 1B). The profile obtained after derivatization indicates that almost all of the compounds identified in R. nigrum extract exhibit antioxidant activity. The highest antioxidant activity was observed for flavan-3-ols, followed by hydroxycinnamates and flavonols.

Metal Chelating Capability of R. nigrum Leaf Extract
R. nigrum leaf extract provided 56.53 ± 0.7% of metal chelating activity, whilst the same concentration of positive control (EDTA) provided 90 ± 0.8%.

Production of Silver Nanoparticles Using Plant Extract
The phytochemical profiling of R. nigrum leaf extract affirmed the presence of polyphenols whose reducing nature paved the way for the green synthesis of Ag NPs. Experimental data have shown that the synthesis of nanoparticles occurs in the presence of light. The formation of Ag NPs was confirmed by exploiting UV-Vis spectroscopy ( Figure 2). The results have indicated the emergence of an absorbance peak at around 445 nm, which is specific for Ag NPs in the samples (Figure2) [9].  In our examination, the color of the reaction mixture changed from light yellow to brown in the presence of the plant extract after 18 h of incubation ( Figure 3). This change in color affirmed the production of Ag NPs, whilst the change of color from brown to dark brown or black could indicate the possible oxidation of Ag NPs as a result of the interaction of Ag NPs with oxygen, leading to the formation of silver oxides as described in the literature [31] and will be investigated in the follow-up project using X-Ray Diffraction (XRD). This change in color from brown to dark brown or black may also be due to possible aggregation [32]. No color change was observed in the absence of plant extract. Since photon energy is necessary for the formation of Ag NPs from Ag + in the presence of plant extracts, the experiments were performed in the presence of light [33].
In our examination, the color of the reaction mixture changed from light yellow to brown in the presence of the plant extract after 18 h of incubation ( Figure 3). This change in color affirmed the production of Ag NPs, whilst the change of color from brown to dark brown or black could indicate the possible oxidation of Ag NPs as a result of the interaction of Ag NPs with oxygen, leading to the formation of silver oxides as described in the literature [31] and will be investigated in the follow-up project using X-Ray Diffraction (XRD). This change in color from brown to dark brown or black may also be due to possible aggregation [32]. No color change was observed in the absence of plant extract. Since photon energy is necessary for the formation of Ag NPs from Ag + in the presence of plant extracts, the experiments were performed in the presence of light [33].

Characterization of Ag NPs
The biosynthesized Ag NPs were characterized by analytical techniques such as dynamic light scattering (DLS), zeta potential (ZP), scanning electron microscopy (SEM), transmission electron microscopy (TEM) analysis and inductively coupled plasma-optical emission spectrometry (ICP-OES).
Dynamic light scattering (DLS) measures the average size of nanoparticles based on the method of laser beam diffraction [34]. The results of DLS analysis provide a Z-average size of nanoparticles as 61.14 ± 0.17 d.nm. Polydispersity index (PDI) indicates the heterogeneity of a sample based on size [35]. The value of PDI ranges from 0.0 for a very homogeneous sample to 1.0 for a very heterogeneous sample. In our experiments, this value ranged from 0.1 to 0.4 for tested samples, which mean that the investigated suspension was somewhat polydisperse [30]. This relatively high value of PDI was expected as the sample was only dispersed in aqueous medium and no surfactants were utilized to have a more practical values when applied in biological testing.
The Z-potential (ZP) values provide information about the nanoparticle surface charge and the probability of their aggregation [36,37] which in our sample was observed to be around −19.2 ± 1.02 mV. The obtained ZP value indicated that the investigated NPs were prone to agglomeration, which may be the reason for further aggregation.
The SEM and TEM were performed to determine the particle size and shapes as described above. SEM images revealed that Ag NPs range in size from 1 to 50 nm ( Figure  4a,b). TEM image has also confirmed the above-mentioned sizes of the investigated NPs (Figure 4c,d). Moreover, it affirmed that the particles were spherical in shape with low agglomeration. EDX revealed a spectral signal in silver region affirming the presence of Ag NPs [38]. The spectral signals of carbon, oxygen, chlorine, and sulfur were also observed, which are generally present in biological material (plant extracts of R. nigrum) [7].

Characterization of Ag NPs
The biosynthesized Ag NPs were characterized by analytical techniques such as dynamic light scattering (DLS), zeta potential (ZP), scanning electron microscopy (SEM), transmission electron microscopy (TEM) analysis and inductively coupled plasma-optical emission spectrometry (ICP-OES).
Dynamic light scattering (DLS) measures the average size of nanoparticles based on the method of laser beam diffraction [34]. The results of DLS analysis provide a Zaverage size of nanoparticles as 61.14 ± 0.17 d.nm. Polydispersity index (PDI) indicates the heterogeneity of a sample based on size [35]. The value of PDI ranges from 0.0 for a very homogeneous sample to 1.0 for a very heterogeneous sample. In our experiments, this value ranged from 0.1 to 0.4 for tested samples, which mean that the investigated suspension was somewhat polydisperse [30]. This relatively high value of PDI was expected as the sample was only dispersed in aqueous medium and no surfactants were utilized to have a more practical values when applied in biological testing.
The Z-potential (ZP) values provide information about the nanoparticle surface charge and the probability of their aggregation [36,37] which in our sample was observed to be around −19.2 ± 1.02 mV. The obtained ZP value indicated that the investigated NPs were prone to agglomeration, which may be the reason for further aggregation.
The SEM and TEM were performed to determine the particle size and shapes as described above. SEM images revealed that Ag NPs range in size from 1 to 50 nm (Figure 4a,b). TEM image has also confirmed the above-mentioned sizes of the investigated NPs (Figure 4c,d). Moreover, it affirmed that the particles were spherical in shape with low agglomeration. EDX revealed a spectral signal in silver region affirming the presence of Ag NPs [38]. The spectral signals of carbon, oxygen, chlorine, and sulfur were also observed, which are generally present in biological material (plant extracts of R. nigrum) [7].  The total silver content was determined by employing inductively coupled plasmaoptical emission spectrometry (ICP-OES) which provided a value of 83.50 ± 3.32 µ g/mL [39]. Considering the operating principles of ICP-OES, most likely, only small particles may have been detected, and the large particles may have been discarded by the instrument.

Effect of Biogenic Ag NPs on Bacterial Growth Rate, FOF1-ATPase Activity, and H + -Fluxes through the Membrane in Escherichia coli ATCC 25922 and Drug-Resistant E. coli pARG-25 Strains
Our previous investigations have shown antibacterial activity of biogenic Ag NPs against different Gram-positive and Gram-negative bacteria [7,9]. Intriguingly, the Gramnegative bacteria were more susceptible to this agent, perhaps due to the possible interaction of these particles with bacterial cell walls [7,9]. Therefore, in order to investigate the mechanism of antibacterial activities, two strains of Escherichia coli i.e., ATCC 25922 and drug-resistant E․ coli pARG-25 were employed in this study.
Since the disk-diffusion method revealed an MIC value of 10 µ g/mL for Ag NPs, a similar concentration was employed to investigate the antibacterial mechanisms of biogenic Ag NPs against E. coli ATCC 25922 and kanamycin resistant E. coli strains. It should be mentioned that R. nigrum extract did not show any antibacterial effect (Figure 5). At a concentration of 10 µ g/mL, the biogenic Ag NPs decreased the growth rate of E. coli wild The total silver content was determined by employing inductively coupled plasmaoptical emission spectrometry (ICP-OES) which provided a value of 83.50 ± 3.32 µg/mL [39]. Considering the operating principles of ICP-OES, most likely, only small particles may have been detected, and the large particles may have been discarded by the instrument.

Effect of Biogenic Ag NPs on Bacterial Growth Rate, F O F 1 -ATPase Activity, and H + -Fluxes through the Membrane in Escherichia coli ATCC 25922 and Drug-Resistant E. coli pARG-25 Strains
Our previous investigations have shown antibacterial activity of biogenic Ag NPs against different Gram-positive and Gram-negative bacteria [7,9]. Intriguingly, the Gramnegative bacteria were more susceptible to this agent, perhaps due to the possible interaction of these particles with bacterial cell walls [7,9]. Therefore, in order to investigate the mechanism of antibacterial activities, two strains of Escherichia coli i.e., ATCC 25922 and drug-resistant E. coli pARG-25 were employed in this study.
Since the disk-diffusion method revealed an MIC value of 10 µg/mL for Ag NPs, a similar concentration was employed to investigate the antibacterial mechanisms of biogenic Ag NPs against E. coli ATCC 25922 and kanamycin resistant E. coli strains. It should be mentioned that R. nigrum extract did not show any antibacterial effect (Figure 5). At a concentration of 10 µg/mL, the biogenic Ag NPs decreased the growth rate of E. coli wild type and drug-resistant strain by~2.5 and 2.3-fold, respectively, in comparison with control Antibiotics 2022, 11, 1415 9 of 17 cells (without Ag NPs addition) as presented in Figure 5. Thus, the biogenic NPs have demonstrated excellent antibacterial activity against both tested strains of bacteria.
Antibiotics 2022, 11, x FOR PEER REVIEW 9 of 17 type and drug-resistant strain by ~2.5 and 2.3-fold, respectively, in comparison with control cells (without Ag NPs addition) as presented in Figure 5. Thus, the biogenic NPs have demonstrated excellent antibacterial activity against both tested strains of bacteria. To understand the mechanisms underlying the antibacterial activity of the biogenic Ag NPs, H + -translocating FOF1-ATPase activity and H + -fluxes through the bacterial membrane were determined. In the case of E. coli ATCC 25922, no significant effect of Ag NPs on the total FOF1-ATPase activity in membrane vesicles was observed, whilst the addition of the Ag NPs decreased ATPase activity in drug-resistant strain by ~3.4 fold, in comparison with control (without Ag NPs addition), as shown in Figure 6. Moreover, DCCDsensitive ATPase activity in E. coli pARG-25 decreased in the presence of Ag NPs by ~6 fold (Figure 6). In the case of E. coli ATCC 25922, DCCD-sensitive ATPase activity was observed to be ~1.6-fold lower than control cells ( Figure 6). To understand the mechanisms underlying the antibacterial activity of the biogenic Ag NPs, H + -translocating F O F 1 -ATPase activity and H + -fluxes through the bacterial membrane were determined. In the case of E. coli ATCC 25922, no significant effect of Ag NPs on the total F O F 1 -ATPase activity in membrane vesicles was observed, whilst the addition of the Ag NPs decreased ATPase activity in drug-resistant strain by~3.4 fold, in comparison with control (without Ag NPs addition), as shown in Figure 6. Moreover, DCCD-sensitive ATPase activity in E. coli pARG-25 decreased in the presence of Ag NPs by~6 fold ( Figure 6). In the case of E. coli ATCC 25922, DCCD-sensitive ATPase activity was observed to bẽ 1.6-fold lower than control cells ( Figure 6).
Antibiotics 2022, 11, x FOR PEER REVIEW 9 of 17 type and drug-resistant strain by ~2.5 and 2.3-fold, respectively, in comparison with control cells (without Ag NPs addition) as presented in Figure 5. Thus, the biogenic NPs have demonstrated excellent antibacterial activity against both tested strains of bacteria. To understand the mechanisms underlying the antibacterial activity of the biogenic Ag NPs, H + -translocating FOF1-ATPase activity and H + -fluxes through the bacterial membrane were determined. In the case of E. coli ATCC 25922, no significant effect of Ag NPs on the total FOF1-ATPase activity in membrane vesicles was observed, whilst the addition of the Ag NPs decreased ATPase activity in drug-resistant strain by ~3.4 fold, in comparison with control (without Ag NPs addition), as shown in Figure 6. Moreover, DCCDsensitive ATPase activity in E. coli pARG-25 decreased in the presence of Ag NPs by ~6 fold (Figure 6). In the case of E. coli ATCC 25922, DCCD-sensitive ATPase activity was observed to be ~1.6-fold lower than control cells ( Figure 6).  Furthermore, biogenic Ag NPs were observed to affect the H + -translocating ATPase which is closely related to the decrease of ATPase activity as observed in the presence and absence of DCCD, an inhibitor of H + -translocation systems. Moreover, the FOF1-ATPase can be considered a target of Ag NPs responsible for the antimicrobial effect of biogenic Ag NPs.
The analysis of energy-dependent H + -fluxes through the bacterial membrane demonstrated that Ag NPs enhanced H + -fluxes in both strains of E. coli by~1.2-1.3 fold (Figure 7). A decrease in H + -fluxes was observed in the presence of DCCD. Moreover, Ag NPs increased DCCD-sensitive H + -fluxes by ~2 and 1.5-fold in E. coli ATCC 25922 and E. coli pARG-25, respectively (Figure 7).

Discussion
The leaves of R. nigrum are widely used in European traditional medicine due to the presence of high content of biologically active phytochemicals including flavonoids and phenolic compounds. Such active moieties are mostly associated with redox character responsible for antioxidant activity. The high content of such active metabolites in R. nigrum extract makes them suitable to serve as reducing agent not only in chemical-based tests, but also in the biogenesis of silver nanoparticles. Several other scientific publications affirm the notion that such extract could be employed for the synthesis of Ag NPs [7,[10][11][12]15,28,40].
Phenolic fraction of R. nigrum extract was investigated for the identification of compounds and the results have revealed the presence of around 30 major constituents comprising mainly of flavan-3-ols, flavonols, hydroxycinnamates, lignans, naphthols and furanocoumarins. The literature data also suggest quercetin derivatives as the main components of R. nigrum leaf extracts, which constitute around 80% of polyphenolic compounds on a dry mass basis [41]. The presence of such a variety of active ingredients explains the excellent antioxidant as well as reducing capacity of R. nigrum extract. Numerous studies have shown that extracts rich in phenolic content exhibit strong antioxidant activity [7,[42][43][44]. Moreover, the post-column derivatization of analytes with ABTS

Discussion
The leaves of R. nigrum are widely used in European traditional medicine due to the presence of high content of biologically active phytochemicals including flavonoids and phenolic compounds. Such active moieties are mostly associated with redox character responsible for antioxidant activity. The high content of such active metabolites in R. nigrum extract makes them suitable to serve as reducing agent not only in chemical-based tests, but also in the biogenesis of silver nanoparticles. Several other scientific publications affirm the notion that such extract could be employed for the synthesis of Ag NPs [7,[10][11][12]15,28,40].
Phenolic fraction of R. nigrum extract was investigated for the identification of compounds and the results have revealed the presence of around 30 major constituents comprising mainly of flavan-3-ols, flavonols, hydroxycinnamates, lignans, naphthols and furanocoumarins. The literature data also suggest quercetin derivatives as the main components of R. nigrum leaf extracts, which constitute around 80% of polyphenolic compounds on a dry mass basis [41]. The presence of such a variety of active ingredients explains the excellent antioxidant as well as reducing capacity of R. nigrum extract. Numerous studies have shown that extracts rich in phenolic content exhibit strong antioxidant activity [7,[42][43][44]. Moreover, the post-column derivatization of analytes with ABTS reagent indicated, that almost all of the compounds identified in R. nigrum extract exhibit antioxidant activity.
Again, due to the high flavonoid and phenolic content, the R. nigrum leaf extract expresses the metal chelating ability which is important to stabilize the Ag NPs by preventing their aggregation and agglomeration [45,46]. In our investigations, this capacity was not strong enough to provide the long-term stability of the nanoparticles and eventually, some aggregation was observed which also led to a decrease in the activity of biogenic nanoparticles. This tendency for agglomeration was also confirmed by the obtained ZP values of the investigated samples. The particles have provided excellent antibacterial activities against both strains of E. coli perhaps due to the relatively small size and round shapes of nanoparticles and by changing the H + -translocating ATPase activity and energy-dependent H + -fluxes (Figure 8).
not strong enough to provide the long-term stability of the nanoparticles and eventually, some aggregation was observed which also led to a decrease in the activity of biogenic nanoparticles. This tendency for agglomeration was also confirmed by the obtained ZP values of the investigated samples. The particles have provided excellent antibacterial activities against both strains of E. coli perhaps due to the relatively small size and round shapes of nanoparticles and by changing the H + -translocating ATPase activity and energydependent H + -fluxes (Figure 8). FOF1-ATPase plays a crucial role in bacterial energetics as it mediates several energydependent processes, including ion transport, and regulation of the enzymatic activity of the membrane. FOF1-ATPase is directly involved in secondary solute transport systems, such as K + uptake, Trk-like or KtrI system forming H + /K + -exchanging pump. The H + /K +exchanging pump, therefore, plays a crucial role to provide antibacterial activity [47].
Assuming all of the above-mentioned findings, it is possible to conclude, that the biogenic Ag NPs provide antibacterial activity via the changes in membrane permeability as affirmed by another study using chemically synthesized Ag NPs [48].

Chemicals and Reagents
Folin-Ciocalteu (FC) reagent, ethanol, gallic acid, 2,2-Diphenyl-1-picrylhydrazyl (DPPH), EDTA, AgNO3, kanamycin, ampicillin, Mueller-Hinton agar and catechin were F O F 1 -ATPase plays a crucial role in bacterial energetics as it mediates several energydependent processes, including ion transport, and regulation of the enzymatic activity of the membrane. F O F 1 -ATPase is directly involved in secondary solute transport systems, such as K + uptake, Trk-like or KtrI system forming H + /K + -exchanging pump. The H + /K +exchanging pump, therefore, plays a crucial role to provide antibacterial activity [47].
Assuming all of the above-mentioned findings, it is possible to conclude, that the biogenic Ag NPs provide antibacterial activity via the changes in membrane permeability as affirmed by another study using chemically synthesized Ag NPs [48].

Plant Material Collection, Identification and Extraction
The plant material (R. nigrum L.) was collected from Lori province (Armenia, 1600-1650 m a.s.l.) during the fruiting period (July 2019). The identification of the plant was carried out at the Department of Botany and Mycology, Yerevan State University (YSU), Armenia. Plant samples were deposited at the Herbarium of the Department of Botany and Mycology, YSU, Armenia. The collected leaves were washed, dried in the shadow at room temperature and subsequently crushed to obtain the powder, which was stored in a dry and dark place until use. Plant material was extracted using ethanol, as described in the European Pharmacopeia [49]. The obtained dried extracts were stored at 4 • C until further use.

LC-Q-Orbitrap HRMS Analysis
The phytochemical analysis of R. nigrum extract was performed using a Dionex Ultimate 3000 UHPLC system (Thermo Scientific TM , Dionex, San Jose, CA, USA) equipped with Synergi TM Hydro-RP A (150 × 4.5 mm, 4 µm, Phenomenex) column, held at a temperature of 30 • C as described in the literature [52].
Raw data from high-resolution mass spectrometry was elaborated with Compound Discoverer (v. 2.1, Thermo, Waltham, MA, USA), which facilitated the peak recognition, retention times arrangement, profile assignment, and isotope pattern. Major metabolite identification was based on accurate mass and mass fragmentation pattern spectra against MS-MS spectra of compounds available on a customized database of different classes of phytochemicals created on the basis of literature data and implemented in the software. Raw data from three experimental replicates and a blank sample were processed using a workflow presented by Kusznierewicz, Mróz, Koss-Mikołajczyk, and Namieśnik [52]. The chromatograms before derivatization were recorded at 270 nm in DAD detector. The eluate stream from the DAD detector was directed to the post-column derivatization instrument. The post-column derivatization with ABTS reagent was carried out according to methods described in the literature with slight modification [53,54]. A stream of methanolic ABTS solution (1 mM) was introduced to the stream of eluate at a rate of 0.1 mL/min and then directed to the reaction loop (1 mL, 130 • C). The antioxidant profiles were recorded in a UV-Vis detector at 734 nm.

Chelating Capability of R. nigrum Leaf Extract
Fe 2+ chelating capability (CC) of the R. nigrum leaf extract was determined according to methods described in the literature [15]. EDTA (1 mg/mL) was used, as a reference chelating agent.

Synthesis of Ag NPs Using R. nigrum Extracts
A stock solution of R. nigrum extract was prepared by dissolving 5 mg of plant extract in 10 mL of Milli-Q water (18.2 MΩ·cm at 25 • C) and Ag NPs were synthesized by mixing the solutions of AgNO 3 (10 mM) and plant extract in 1:9 ratio to achieve a final concentration of 1 mM for AgNO 3 [6,55]. A control sample excluding plant extract was also prepared similarly. The samples were agitated on a shaker (150 rpm) under dark and light (normal room light) conditions at a temperature of 23 ± 2 • C for 18 h.

Characterization of Biosynthesized Ag NPs
The optical properties of Ag NPs were characterized by exploiting a UV-Vis spectrophotometer (Lambda 35, Perkin Elmer, Waltham, MA, USA) [7]. The physical stability of the investigated samples was evaluated by the zeta potential (ξ-potential) measurement and dynamic light scattering (DLS), employing a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). The size of biosynthesized Ag NPs was determined by Scanning Electron Microscopy (SEM-ZEISS-SUPRA 40/gemini column) equipped with Electron Backscatter Diffraction (EBSD) detector and Energy Dispersive X-ray (EDX) detector -SEM-EDX analysis. Moreover, the size and shape of Ag NPs were evaluated by transmission electron microscopy (LEOL JEM-1400 TEM). The total Ag content was determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES, Horiba JobinYvonUltima 2).

Antibacterial Activity of Biosynthesized Ag NPs
The antibacterial activity of biosynthesized Ag NPs was evaluated against E. coli ATCC 25922, and kanamycin-resistant E. coli pARG-25 (Scientific-Production Center "Arm-Biotechnology", NAS, Yerevan, Armenia) strains by a disk-diffusion method employing disks with 6 mm in diameter, as described by Hambardzumyan et al., 2020 [7,48]. The Mueller-Hinton agar was exploited for the growth of bacteria. The antibacterial activity of Ag NPs was recorded in terms of Minimum Inhibitory Concentration (MIC). MIC values were recorded after 18 h of incubation at 37 • C. Kanamycin and ampicillin (50 µg/mL) were used as positive controls. Results were compared to the antibacterial activity of colloidal Ag, commercially available under the "Silverton" trademark ("Tonus-Les" Lab, Yerevan, Armenia), and produced by an electrochemical process [48,56].

Growth Kinetics of E. coli ATCC 25922 and E. coli pAPG-25 Strains under the Influence of Biosynthesized Ag NPs
The growth kinetics of E. coli ATCC 25922 and E. coli pARG-25 strains were monitored in the presence of R. nigrum leaf extract. Fresh bacterial strains were isolated from Mueller-Hinton agar plates and transferred to LB broth (pH 7.5) followed by incubation for 18 h at 37 • C. The antibacterial effect of the extract was monitored at the same concentration employed to produce Ag NPs i.e., 0.5 mg/mL. Bacterial growth curves were determined by measuring the turbidity of samples containing bacteria at 565 ± 15 nm every 60 min, exploiting a densitometer (DEN-1B McFarland, Biosan, Latvia) [57].
The specific growth rate (µ) of bacteria was calculated according to µ = (lnOD t − lnOD 0 )/t, where OD 0 represents the initial value of optical density (OD); OD t represents the value of OD after 6 h; µ is expressed as h −1 [48].

Determination of H + -fluxes
The H + -fluxes through the membrane were determined in whole bacterial cells by employing an appropriate selective electrode (HJ1131B, HANNA Instruments, Portugal. Bacterial cells were transferred into the assay medium containing 150 mmol/L Tris-phosphate buffer (pH 7.5), 0.4 mmol/L MgSO 4 , 1 mmol/L KCl and 1 mmol/L NaCl followed by the addition of 11 mmol/L of glucose. The H + -fluxes were expressed in mmol H + per min per 10 10 cells [48,58]. N, N'-dicyclohexylcarbodiimide (DCCD) served as an inhibitor of the F O F 1 -ATPase and the bacterial cultures were incubated with 0.2 mmol/L of DCCD for 10 min [58].
In order to understand the possible mechanisms underlying the antibacterial activity of Ag NPs, the changes in energy-dependent H + -fluxes through the bacterial membrane of E. coli (both ATCC 25922 and kanamycin-resistant E. coli pARG-25 strains) in the presence of Ag NPs and R. nigrum leaf extract were investigated.

Determination of F O F 1 -ATPase Activity in Membrane Vesicles in the Presence of Ag NPs and R. nigrum Leaf Extract
The impact of Ag NPs at the H + -translocating F O F 1 -ATPase activity was investigated in E. coli ATCC 25922 and E. coli pARG-25 strains to identify possible targets. ATPase activity assay was performed in bacterial membrane vesicles, which were obtained by the Kaback method [48,56]. Bacterial cultures were grown in the presence of R. nigrum leaf extract and Ag NPs (10 µg/mL). F O F 1 -ATPase activity was evaluated by quantifying the amount of inorganic phosphate liberated after adding ATP to membrane vesicles of bacteria. Inorganic phosphate was detected by Tausski and Shorr method. The membrane vesicles of bacteria were incubated with 0.2 mmol/L DCCD for 10 min [48,56]. F O F 1 -ATPase activity is expressed in nmol Pi µg −1 protein min −1 .

Conclusions
The leaves of R. nigrum are widely used in European traditional medicine due to their high content of biologically active compounds. The metabolomic characterization of phenolic components of R. nigrum leaf extract affirms that it can serve as an excellent reducing agent due to the presence of large amounts of flavan-3-ols, hydroxycinnamates, flavonols, quercetin and quercetin derivatives.
Ag NPs synthesized with the use of R. nigrum extract have exhibited excellent antibacterial activity possibly due to the relatively small size and round shapes of nanoparticles. The identified substances can also serve as stabilizers for the synthesized nanoparticles and enhance their shelf life. We suggest that these biogenic Ag NPs can exhibit enhanced antibacterial properties through changes in membrane permeability as a result of the impact of these particles on the H + -translocating ATPase activity, energy-dependent H + -fluxes and formate hydrogenlyase (FHL) proton-potassium transporter. This phenomenon not only explains the antibacterial potential of R. nigrum extract-mediated Ag NPs but also suggests their potential application in biomedicine. Data Availability Statement: The datasets and materials used and/or analyzed during the current study are available from the author upon reasonable request.