Green Synthesis of Silver Nanoparticles Using Extract of Artemisia absinthium L., Humulus lupulus L. and Thymus vulgaris L., Physico-Chemical Characterization, Antimicrobial and Antioxidant Activity

: The novelty of this study is twofold: AgNPs were obtained and characterized using Artemisia absinthium ( A. absinthium ), Humulus lupulus ( H. lupulus ), and Thymus vulgaris ( T. vulgaris ) plants extracts; moreover, a green and environmentally friendly approach for the synthesis of silver nanoparticles (AgNPs) using aqueous extracts was developed. This paper discusses new approaches about the synthesis of AgNPs. T. vulgaris , H. lupulus, and A. absinthium, which are renewable and common plants, perfect as reducing, stabilizing, and capping agent for green synthesis of silver nanoparticles (AgNPs). The extracts and synthesized AgNPs were characterized by various physico-chemical, phytochemical, morphological scanning electron microscope (SEM/EDS) and transmission electron microscope scanning (TEM), and antibacterial activity. The antioxidant activity of extracts and AgNPs were also assessed by 2,2 (cid:48) -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS • + ), 2,2-diphenyl-1-picrylhydrazyl (DPPH • ), cupric reducing antioxidant capacity (CUPRAC), ferric reducing antioxidant power (FRAP), and triﬂuoperazine dihydrochloride (TFPH • ) scavenging assays. Extracts/AgNPs showed signiﬁcant antioxidant activity in all cases. A. absinthium /AgNPs, H. lupulus /AgNPs, and T. vulgaris /AgNPs displayed activities against DPPH • (0.14 ± 0.00; 0.11 ± 0.00 and 0.14 ± 0.00 mmol/g), ABTS • + (0.55 ± 0.05; 0.86 ± 0.05 and 0.55 ± 0.05 mmol/g), respectively. TEM analysis conﬁrmed the average particle size, it estimated t A. absinthium /AgNPs–46 nm, H. lupulus /AgNPs size of synthesized particles was 42 nm and T. vulgaris /AgNPs–48 nm. SEM analysis revealed that T. vulgaris /AgNPs showed in solitary cases as snowﬂake-like, branched, but in a general spheric shape, H. lupulus /AgNPs were wedge-shaped, and A. absinthium /AgNPs were the spherical shape of the synthesized AgNPs. EDS analysis conﬁrmed the purity of the synthesized AgNPs with a strong signal at 3.2 keV. A. absinthium /AgNPs, H. lupulus /AgNPs, and T. vulgaris /AgNPs exhibited higher antibacterial activity against all tested bacterial strains compared to their respective pure extracts. It is concluded that AgNPs synthesized in extracts have a broad range of biological applications, which can be used as an eco-friendly material without having negative effects on the environment.

Processes 2021, 9, x FOR PEER REVIEW 3 of 16 metric studies helped to determine the total phenolic, flavonoid, proanthocyanidin content, and hydroxycinnamic acid derivatives. The antimicrobial activity was investigated against Gram-negative/positive bacteria cultures by the agar disk diffusion test method.

Plant Material
Finely cut A. absinthium, H. lupulus, and T. vulgaris (manufacturer UAB "Švenčionių vaistažolės") material were purchased from a public pharmacy, operating in Kaunas, Lithuania. Plant material was ground to a powder using a mill (IKA ® A11 basic "Staufen", Germany). Loss on drying before the analysis was determined by drying powdered raw plant material in a laboratory drying oven to complete evaporation of water and volatile compounds (temperature: 105°C; the difference in weight between measurements: up to 0.01 g) and by calculating the difference in raw material weight before and after drying. The data were recalculated for dry weight.

Extraction
Fifty grams of raw material was weighed (exact mass of ground material) and soaked for 2 h in 70 (v/v) ethanol. The soaked raw material was transferred to a percolator, covered with extractant-70 (v/v) ethanol, and left to macerate for 24 h. An ethanolic phase of liquid extract was evaporated using a rotary evaporator. The obtained plant extracts were used as green reductants and capping agents for the biosynthesis of AgNPs.

Preparation of Silver Nanoparticles
Silver nitrate was purchased in Merck, Germany. An amount of 0.03 g silver nitrate was weighed and dissolved in 5 mL distilled well-mixed water. Next, 30 mL of each of the aqueous extracts-A. absinthium, H. lupulus, and T. vulgaris-was added to a silver nitrate aqueous solution under vigorous stirring (speed 500 rpm) at 25°C temperature for 2 h, then a 24-h incubation at room temperature was completed. During synthesis, gradual change in color from mild yellow to dark brown in the reaction mixture indicated the presence of silver nanoparticles.

Determination of Total Phenolic Content
All spectrophotometric measurements were carried out using a M550 (Spectronic CamSpec, Garforth, England, United Kingdom). The total phenolic content in the extracts was determined using the Folin-Ciocalteu method [22]. Gallic acid concentration range

Plant Material
Finely cut A. absinthium, H. lupulus, and T. vulgaris (manufacturer UAB "Švenčionių vaistažolės") material were purchased from a public pharmacy, operating in Kaunas, Lithuania. Plant material was ground to a powder using a mill (IKA ® A11 basic "Staufen", Germany). Loss on drying before the analysis was determined by drying powdered raw plant material in a laboratory drying oven to complete evaporation of water and volatile compounds (temperature: 105 • C; the difference in weight between measurements: up to 0.01 g) and by calculating the difference in raw material weight before and after drying. The data were recalculated for dry weight.

Extraction
Fifty grams of raw material was weighed (exact mass of ground material) and soaked for 2 h in 70 (v/v) ethanol. The soaked raw material was transferred to a percolator, covered with extractant-70 (v/v) ethanol, and left to macerate for 24 h. An ethanolic phase of liquid extract was evaporated using a rotary evaporator. The obtained plant extracts were used as green reductants and capping agents for the biosynthesis of AgNPs.

Preparation of Silver Nanoparticles
Silver nitrate was purchased in Merck, Germany. An amount of 0.03 g silver nitrate was weighed and dissolved in 5 mL distilled well-mixed water. Next, 30 mL of each of the aqueous extracts-A. absinthium, H. lupulus, and T. vulgaris-was added to a silver nitrate aqueous solution under vigorous stirring (speed 500 rpm) at 25 • C temperature for 2 h, then a 24-h incubation at room temperature was completed. During synthesis, gradual change in color from mild yellow to dark brown in the reaction mixture indicated the presence of silver nanoparticles.

Determination of Total Phenolic Content
All spectrophotometric measurements were carried out using a M550 (Spectronic CamSpec, Garforth, England, United Kingdom). The total phenolic content in the extracts was determined using the Folin-Ciocalteu method [22]. Gallic acid concentration range was 0.25-4 mg/mL. The total phenolic content was expressed as mg/g gallic acid equivalent (GAE) per gram of dry weight (DW) (mg GAE/g DW).

Determination of Total Flavonoid Content
The total amount of flavonoids was determined using the described methodology by Urbonaviciute et al. (2006). Rutin concentration range was 0-0.85 mg/mL, while the total Processes 2021, 9, 1304 4 of 15 amount of flavonoids was expressed as mg/g rutin equivalent (RE) per gram of dry weight (mg RE/g DW).

Determination of Total Content of Hydroxycinnamic Acid Derivatives
The total content of hydroxycinnamic acid derivatives was determined using the described methodology [9]. Sample preparation: 2 mL 0.5 M HCl, 2 mL Arnow reagent, and 2 mL NaOH attenuate solution was added to 1 mL of extract and diluted with distilled water up to 10 mL. Blank solution: 2 mL 0.5 M HCl, 2 mL NaOH attenuate solution was added to 1 mL of extract and diluted with distilled water up to 10 mL. Total content of hydroxycinnamic acid derivatives in the extract was expressed as mg/mL of chlorogenic acid equivalents (ChAE) using chlorogenic acid calibration curve, while chlorogenic acid concentration range was 0.0625-1 mg/mL. In dry material, total content of hydroxycinnamic acid derivatives was expressed as mg ChAE/g DW.

ABTS •+ Radical Cation Scavenging Assay
Parental ABTS •+ radical solution is prepared by oxidizing ABTS with potassium persulfate. An amount of 2 mM of ABTS was dissolved in purified water and 0.7 mM of potassium persulfate was added. Then, prepared solution was stored for 16-17 h in the dark at a room temperature, until ABTS and potassium persulfate fully reacted. Meanwhile, a stable ABTS •+ radical cation formed during this reaction. Parental ABTS •+ solution, when protected from light, at room temperature, remained stable for more than 2 days.
Working ABTS •+ solution was prepared by diluting a parent ABTS •+ solution with distilled water to 0.8 ± 0.03 absorption units at wavelength of 734 nm [24]. Ten microliters of extract was mixed with 3 mL of working ABTS •+ solution in a quartz cuvette. After 30 min, a decrease in sample light absorbance was determined using a spectrophotometer. Antiradical activity was expressed as TE based on Trolox calibration curve; Trolox concentration range-8000-24,000 µM.

DPPH • Free Radical Scavenging Assay
The DPPH • free radical scavenging activity was determined using the method proposed by Brand-Williams et al. [25]. Ten microliters of the extract sample was mixed with the DPPH • solution in 96.3 v/v ethanol (3 mL, 6 × 10 −5 M) in a quartz cuvette. Using a spectrophotometer, a decrease in light absorbance was determined at a wavelength of 517 nm, until absorption equilibrium was achieved (approximately after 30 min.). Antiradical activity when applying DPPH • method was expressed as TE based on Trolox calibration curve: Trolox concentration range-400-20000 µM.

CUPRAC Assay
In the meantime, CUPRAC solution included copper (II) chloride dihydrate (0.01 M in water), ammonium acetate buffer solution (0.001 M, pH = 7), and neocuproine (0.0075 M in ethanol) (ratio 1:1:1). During the evaluation, 3 mL of CUPRAC reagent was mixed with 10 µL of extracts. An increase in absorbance was recorded at λ = 450 nm. Reduction activity when applying FRAP method was expressed as TE based on Trolox calibration curve; Trolox concentration range-2000-48,000 µM. Contents of FRAP reagent included sodium acetate buffer solution 300 mM (pH = 3.6), 10 mM TPTZ solution in 40 mM hydrochloric acid, iron chloride hexahydrate 20 mM. The working FRAP reagent solution was prepared by mixing sodium acetate buffer solution, TPTZ solution in hydrochloric acid and iron chloride hexahydrate solution in a ratio of 10:1:1. The working solution was prepared right before the use 10 µL of extract was mixed with 3 mL of working FRAP reagent solution in a quartz cuvette [6]. After 30 min, an increase in light absorbance was determined at a wavelength of 593 nm using a spectrophotometer. Reduction activity when applying FRAP method was expressed as TE based on Trolox calibration curve; Trolox concentration range-400-24,000 µM.

TFPH •+ Radical Cation Scavenging Assay
Preparation of parental solution was accomplished as following. Parental TFPH solution was prepared by dissolving 0.480 g (exact mass) of TFPH in 10 mL of purified water, which remained stable for 2 months when stored in the dark at 4 • C temperature.
Preparation of working solution: 0.5 mL of parental TFPH solution was mixed with 0.1 mL of 100 mM K 2 S 2 O 8 and 70 mL of 4 M sulfuric acid in a 100 mL volumetric flask. Then 4 M sulfuric acid was added to a total volume of 100 mL, while prepared solution was stored in the dark for 15-20 min. Measured light absorption should be equal to 0.70 ± 0.1 absorption units at wavelength of 502 nm. Working solution remained stable for 2 h when stored in the dark at a room temperature [26].
Ten microliters of extract was mixed with 3 mL of working TFPH solution in a quartz cuvette. After 30 min, a decrease in sample light absorbance was determined using a spectrophotometer. Antiradical activity when applying TFPH method was expressed as TEAC based on Trolox calibration curve; Trolox concentration range-2000-16,000 µM.

Calculation of Antioxidant Activity of the Extracts
The antioxidant activity of the extracts was calculated from the Trolox calibration curve and was expressed as mmol of the Trolox equivalent (TE) per one gram of dry weight (DW). TE was calculated according to the following formula: where, c: the concentration of Trolox established from the calibration curve (in µM); V: the volume of the extract (in mL); m: the weight (exact) of the herbal raw material (in grams).
2.6. Microscopy 2.6.1. Scanning Electron Microscopy (SEM) The particle size and structures were studied from the images obtained by SEM FEI Quanta 200 FEG (FEI Company, Hillsboro, OR, USA) manufacturer, city, state abbreviation, country). The samples were examined in a low vacuum mode operating at 20.0 kV using an LDF detector. The content of AgNPs and chemical analysis is of nanocomposites were performed by the energy dispersive spectroscopy SEM/EDS technique with a BruckerXFlash 4030 detector (accelerating voltage 10 kV, distance between the bottom of the objective lens and the object 10 mm).

Transmission Electron Microscopy (TEM)
Tecnai G2 F20 X-TWIN (FEI, USA) examined the size of the synthesized nanoparticles under a transparent electron microscope (TEM). For the TEM experiment, the diluted samples were deposited drop-wise onto carbon-coated copper TEM grids. The Schottky emission electron source was used, the accelerating voltage was 20-200 kV. The resolution of the microscope ranged from 0.8 to 1.0 nm. EDAX spectrometer with r-TEM detector and 11 MPix ORIUS SC1000B (Gatan Inc., Pleaston, California, USA) manufacturer, city, state abbreviation, country) CCD camera were used. The spot/linear resolution was 0.25/0.102 nm.

Antimicrobial Activity
The antimicrobial activity was investigated against Gram-negative and Gram-positive bacteria cultures. The antimicrobial activity of extracts was tested via an agar well diffusion assay [27]. For this purpose, a 0.5 McFarland Unit density suspension (~108 CFU/mL) of each pathogenic bacterial strain was inoculated onto the cooled Mueller Hinton Agar (Oxoid, Basingstoke, UK), using sterile cotton swabs. Wells of 6 mm in diameter were punched in the agar and filled with 50 µL of extracts. The experiments were repeated three times, and the average size of the inhibition zones was calculated. The antimicrobial activities against the tested bacteria were determined by measuring the inhibition zones' diameter (mm). The antimicrobial activity of extracts were determined against Staphylococcus aureus (S. aureus), Staphylococcus haemolyticus (S. haemolyticus), Enterococcus durans (E. durans), Bacillus pseudomycoides (B. pseudomycoides), Salmonella enterica (S. enterica), Aeromonas hydrophila (A. hydrophila), Aeromonas veronii (A. veronii), Acinetobacter baumannii (A. baumannii), Acinetobacter johnsonii (A. johnsonii), Enterobacter cloacea (E. cloacea), Cronobacter sakazakii (C. sakazakii), Kluyvera cryocrescens (K. cryocrescens), Klebsiella pneumoniae (K. pneumoniae), Escherichia coli (E. coli), and Pseudomonas aeruginosa (P. aeruginosa), which were obtained from the collection of the Lithuanian University of Health Sciences (Kaunas, Lithuania).

Statistical Analysis
The statistical analysis was performed using SPSS 20 software (SPSS Inc., Armonk, NY, USA). All the experiments were implemented in triplicate and the results are conferred as mean ± standard error mean (SEM) of all calculated values.

Content of Phytochemicals
In the present work, aqueous extracts of A. absinthium, H. lupulus, and T. vulgaris were used for the synthesis of AgNPs. As shown in Figure 2, the biomolecules present in the extracts are responsible for the reduction of Ag + ions to Ag 0 in a single step. emission electron source was used, the accelerating voltage was 20-200 kV. The resolution of the microscope ranged from 0.8 to 1.0 nm. EDAX spectrometer with r-TEM detector and 11 MPix ORIUS SC1000B (Gatan Inc., Pleaston, California, USA) manufacturer, city, state abbreviation, country) CCD camera were used. The spot/linear resolution was 0.25/0.102 nm.

Antimicrobial Activity
The antimicrobial activity was investigated against Gram-negative and Gram-positive bacteria cultures. The antimicrobial activity of extracts was tested via an agar well diffusion assay [27]. For this purpose, a 0.5 McFarland Unit density suspension (~108 CFU/mL) of each pathogenic bacterial strain was inoculated onto the cooled Mueller Hinton Agar (Oxoid, Basingstoke, UK), using sterile cotton swabs. Wells of 6 mm in diameter were punched in the agar and filled with 50 μL of extracts. The experiments were repeated three times, and the average size of the inhibition zones was calculated. The antimicrobial activities against the tested bacteria were determined by measuring the inhibition zones' diameter (mm). The antimicrobial activity of extracts were determined against

Statistical Analysis
The statistical analysis was performed using SPSS 20 software (SPSS Inc., Armonk, New York, USA). All the experiments were implemented in triplicate and the results are conferred as mean ± standard error mean (SEM) of all calculated values.

Content of Phytochemicals
In the present work, aqueous extracts of A. absinthium, H. lupulus, and T. vulgaris were used for the synthesis of AgNPs. As shown in Figure 2, the biomolecules present in the extracts are responsible for the reduction of Ag + ions to Ag 0 in a single step.  The phenolic compounds and reducing sugars are responsible for the bioreduction of Ag + ions to AgNPs. Moreover, flavonoids and proanthocyanidins may also cause reduction of Ag + ions to AgNPs and act as a capping agent to prevent agglomeration. Exceeding, flavonoids can also directly scavenge molecular species of active oxygen. All the three tested extracts contained flavonoids, consisting of hydroxyl groups with a stronger ability to bind with silver ions and acted as a reducing agent.
The following substances were studied based on the reviewed literature: phenols, hydroxycinnamic acid, flavanoids, and proanthocyanidin. A great deal of scientific literature have analyzed and discussed the bactericidal properties of plant extracts and their possible application. One of the most important biological effects of plant extracts is high antioxidant activity, which is closely related to anticancer antibacterial, anti-inflammatory, antiallergic, antiviral, hepatoprotective, antithrombogenic, and many other effects [28].
The extracts that were obtained from A. absinthium, H. lupulus, and T. vulgaris material and their phytochemical composition, i.e., phenolic compounds, flavonoids, proanthocyanidins, and hydroxycinnamic acid, was investigated and summarized in Table 1. It was found that H. lupulus extract contained at most proanthocyanidins from all the tested extracts was at 6.31 ± 0.26 mg EE/g, and similarly to phenolic compounds, this extract contained 10.79 ± 1.44 mg GAE/g. It was reported that methanol extract of hop cones contained 7.4 µg and 7.12 ±0.09 mg GAE/g total polyphenol [15,29]. Further on, T. vulgaris extract contained at most phenolic and flavonoid compounds 55.83 ± 2.55 GAE/g DW and 24.77 ± 0.37 RE/g DW, respectively. Other researchers obtained similar results with phenolic and flavonoid compounds 44.16 ± 1.46 GAE/g DW and 17.89 ± 0.71 RE/g DW, respectively [30]. The results showed that the total amount of proanthocyanidins, hydroxycinnamic acid derivatives, phenolic content, and flavonoids decrease after synthesized silver nanoparticles in all extracts (Table 1).
Notably, A. absinthium crude extract and A. absinthium/AgNPs total phenolic content in all tested cases decrease about one time. The more significant change of H. lupulus was observed, where the total amount of proanthocyanidins and flavonoids decreased 1.66 times. These changes in phenolic compounds may be associated with the synthesis of AgNPs particles. Therefore these secondary metabolites, specifically phenols, and flavanoids, may interact with AgNPS as capping agents and increase the surface area due to efficient scavenging properties [27].

Determination of Antioxidant Properties
Epidemiological studies had demonstrated the ability of an antioxidant to reduce or stop the progression of many chronic diseases [31,32]. The antiradical and reductive activity of selected plant sample extracts in vitro is suitable for prospective antioxidant studies. The results, obtained during the studies, will provide a consumer with products rich in antioxidants, valid for the assessment and standardization of the quality of plant raw materials and their products. Plant extracts are natural multicomponent matrices containing various biologically active compounds, the antioxidant activity of which is manifested by different mechanisms. Therefore, plant extracts antioxidant activity cannot be properly evaluated using only one methodology [33]. For these reasons, to fully assess plant extracts antioxidant activity, it is recommended to apply at least two methods to determine antioxidant activity [34]. ABTS, CUPRAC, TFPH, FRAP, and DPPH UV-VIS spectrophotometry methods to the in vitro studies of selected plant extracts' antioxidant activity were applied. They are often used in antioxidant tests, mostly for natural extracts. The results are summarized in Table 2. Examination of selected plant extracts using five different in vitro spectrophotometric methods showed that the strongest in vitro antiradical activity, assessed by ABTS and TFPH methods, and in vitro reducing activity, determined by FRAP method, were characterized by extracts of T. vulgaris samples. Extracts of H. lupulus samples showed the strongest antiradical activity in vitro as measured by the DPPH methodology and in vitro reduction activity by the CUPRAC methodology ( Table 2). The total amount of phenolic compounds (TPC) and flavonoid content (TFC) of T. vulgaris/AgNPs was significantly higher than A. absinthium/AgNPs and H. lupulus/AgNPs. In comparison with the other studies these are secondary metabolites. These results may indicate the impact of the synthesis of nanoparticles in different plant extract. Similar results were also obtained by other researchers with grapefruit pomace extract/AgNPs and Thymus citriodorus and Thymus vulgaris [27,35].

SEM Analysis
SEM technique was employed to visualize the morphology, size, and shape of the influence on the reaction medium of AgNPs. Antibacterial activity of AgNPs depends on the nanoparticle morphology: size and shape [3]. The size of synthesized silver nanoparticles might increase their surface area, promoted bacterial cell wall interactions and membrane permeability, and enhance their antimicrobial activity by leakage of bacterial cellular contents [36]. Figure 3 presents typical SEM images of the biosynthesized nanomaterials. Since secondary plant metabolites act as reducing agents and as capping agents for AgNPs, they differ in the phytochemical composition. Different extract biomolecules cap the silver nanoparticles. Microscopic imagines indicate these morphological differences. For A. absinthium/AgNPs were spherical-shaped with a size of~50 nm (Figure 3a). The silver nanoparticles assisted by H. lupulus/AgNPs were also spherical and size of~40 nm. (Figure 3b). The AgNPs assisted by T. Vulgaris was spherical-shaped with 45 nm (Figure 3c).

T. vulgaris/AgNPs
0.55 ± 0.05 0.14 ± 0.00 0.10 ± 0.00 0.068 ± 0.01 0.26 ± 0.01 Examination of selected plant extracts using five different in vitro spectrophotometric methods showed that the strongest in vitro antiradical activity, assessed by ABTS and TFPH methods, and in vitro reducing activity, determined by FRAP method, were characterized by extracts of T. vulgaris samples. Extracts of H. lupulus samples showed the strongest antiradical activity in vitro as measured by the DPPH methodology and in vitro reduction activity by the CUPRAC methodology ( Table 2). The total amount of phenolic compounds (TPC) and flavonoid content (TFC) of T. vulgaris/AgNPs was significantly higher than A. absinthium/AgNPs and H. lupulus/AgNPs. In comparison with the other studies these are secondary metabolites. These results may indicate the impact of the synthesis of nanoparticles in different plant extract. Similar results were also obtained by other researchers with grapefruit pomace extract/AgNPs and Thymus citriodorus and Thymus vulgaris [27,35].

SEM Analysis
SEM technique was employed to visualize the morphology, size, and shape of the influence on the reaction medium of AgNPs. Antibacterial activity of AgNPs depends on the nanoparticle morphology: size and shape [3]. The size of synthesized silver nanoparticles might increase their surface area, promoted bacterial cell wall interactions and membrane permeability, and enhance their antimicrobial activity by leakage of bacterial cellular contents [36]. Figure 3 presents typical SEM images of the biosynthesized nanomaterials. Since secondary plant metabolites act as reducing agents and as capping agents for AgNPs, they differ in the phytochemical composition. Different extract biomolecules cap the silver nanoparticles. Microscopic imagines indicate these morphological differences. For A. absinthium/AgNPs were spherical-shaped with a size of ~50 nm (Figure 3a). The silver nanoparticles assisted by H. lupulus/AgNPs were also spherical and size of ~ 40 nm. (Figure 3b). The AgNPs assisted by T. Vulgaris was spherical-shaped with 45 nm ( Figure  3c).  The assessment of the elemental composition on the surface of the biosynthesized nanomaterials was done using EDS spectroscopy. In general, the optical absorption peak of silver appears at around 3 keV [4,37]. Similar patterns of EDS spectra were reported Processes 2021, 9, 1304 9 of 15 earlier for the AgNPs with an absorption range of 2-4 keV, which may arise because of the possibility of X-ray energy range in different levels [38].
As shown in Figure 3, EDS analysis revealed silver in the nanoparticles formulated for A. absinthium, H. lupulus, and T. vulgaris. Furthermore, the prepared nanoparticles did not exhibit any nitrogen. It indicated the absence of noticeable traces of ions from the AgNO 3 precursor used, which confirms the presence of metallic silver in the sample and evidence of successful reduction of silver ions. Figure 4 indicates carbon and oxygen atoms in all nanoparticle samples. Once again this confirms the existence of phytochemicals on the surface of the biosynthesized AgNPs since secondary plant metabolites act not only as reducing agents but also as capping agents for AgNPs [36].
Moreover, the presence of magnesium (Mg) in A. absinthium/AgNPs and T. vulgaris/ AgNPs extracts samples were indicated. This mineral is a main component of chlorophyll molecule found in green plants. Comparable, in H. lupulus/AgNPs magnesium, was not detected. For A. absinthium and T. vulgaris extracts stem, and leave morphological parts were used. Meanwhile, H. lupulus extract was made with only dried fins.
Processes 2021, 9, x FOR PEER REVIEW 10 of 16 The assessment of the elemental composition on the surface of the biosynthesized nanomaterials was done using EDS spectroscopy. In general, the optical absorption peak of silver appears at around 3 keV [4,37]. Similar patterns of EDS spectra were reported earlier for the AgNPs with an absorption range of 2-4 keV, which may arise because of the possibility of X-ray energy range in different levels [38].
As shown in Figure 3, EDS analysis revealed silver in the nanoparticles formulated for A. absinthium, H. lupulus, and T. vulgaris. Furthermore, the prepared nanoparticles did not exhibit any nitrogen. It indicated the absence of noticeable traces of ions from the AgNO3 precursor used, which confirms the presence of metallic silver in the sample and evidence of successful reduction of silver ions. Figure 4 indicates carbon and oxygen atoms in all nanoparticle samples. Once again this confirms the existence of phytochemicals on the surface of the biosynthesized AgNPs since secondary plant metabolites act not only as reducing agents but also as capping agents for AgNPs [36].
Moreover, the presence of magnesium (Mg) in A. absinthium/AgNPs and T. vulgaris/AgNPs extracts samples were indicated. This mineral is a main component of chlorophyll molecule found in green plants. Comparable, in H. lupulus/AgNPs magnesium, was not detected. For A. absinthium and T. vulgaris extracts stem, and leave morphological parts were used. Meanwhile, H. lupulus extract was made with only dried fins.    Figure 5 presents typical TEM images of the biosynthesized nanomaterials. These micrographs show the presence of individual nanoparticles with almost spherical shape and roughly, which is characteristic of AgNPs [36]. The particle size and size distribution of the synthesized AgNPs using plant extracts were estimated under TEM micrographs using ImagineJ software. A. absinthium/AgNPs size were found to be about 46 nm, and polydispersity of 38.    Figure 5 presents typical TEM images of the biosynthesized nanomaterials. These micrographs show the presence of individual nanoparticles with almost spherical shape and roughly, which is characteristic of AgNPs [36]. The particle size and size distribution of the synthesized AgNPs using plant extracts were estimated under TEM micrographs using ImagineJ software. A. absinthium/AgNPs size were found to be about 46 nm, and polydispersity of 38.2, H. lupulus/AgNPs size of synthesized particles were 42 nm with a polydispersity of 41.0. Consistently, T. vulgaris/AgNPs picture the size of particles 48 nm and the polydispersity of 46.1.

TEM
Results indicate the nanoparticulate nature of the synthesized materials regarding the successful formation of colloidal particles from silver ions in the presence of aqueous plant extracts. In addition, all the ImageJ graphs described a Gaussian distribution peak at around the average sizes estimated for each nanoparticle formulation ( Figure 5), which showed decent particle size distribution as observed with polydispersity values below 50%.

Antimicrobial Activity
Three plant extracts and AgNPs synthesized in these extracts were investigated to evaluate their antibacterial activity against 15 different pathogenic and opportunistic bacteria strains using the disc diffusion method. Evaluation of antibacterial activity of these plant extracts and AgNPs was recorded in Table 3. The results revealed that all plant extracts were potentially effective in suppressing microbial growth bacteria with variable potency. Results showed that significant antibacterial activity was observed against S. aureus with a zone of inhibition to 13.3-25.6 mm of AgNPs synthesized in tested extracts. Other researchers obtained similar results with AgNPs against S. aureus where the zone of inhibition was 23-27 mm [39]. A. absinthium/AgNPs inhibit all tested bacteria strains-growth with the inhibition zone size of 9 to 19.3 mm, but not all bacteria tested were insensitive to pure A. absinthium extract.  activities against DPPH • (0.14 ± 0.00; 0.11 ± 0.00 and 0.14 ± 0.00 mmol/g), ABTS •+ (0.55 ± 0.05; 0.86 ± 0.05 and 0.55 ± 0.05 mmol/g), respectively. SEM characterization techniques confirmed the successful formation of spherical AgNPs. T. vulgaris, A. absinthium, and H. lupulus plants serve well as reducing, stabilizing, and capping agents to synthesize silver nanoparticles. Besides this, synthesized silver nanoparticles demonstrated good bactericidal properties with inhibition zones in the range 9.0 ± 0.1 to 20.4 ± 0.3 mm against all tested pathogenic and opportunistic bacteria strains. These microorganisms are commonly involved in infections; therefore, the extracts/AgNPs may be beneficial for the future application.

Conflicts of Interest:
The authors declare no conflict of interest.