Silver Nanoparticles of Artemisia sieberi Extracts: Chemical Composition and Antimicrobial Activities

Background: Artemisia sieberi (mugwort) is a member of the daisy family Asteraceae and is widely propagated in Saudi Arabia. A. sieberi has historical medical importance in traditional societies. The current study aimed to assess the antibacterial and antifungal characteristics of the aqueous and ethanolic extracts of A. sieberi. In addition, the study investigated the effect of silver nanoparticles (AgNPs) synthesized from the A. sieberi extract. Methods: The ethanolic and aqueous extracts and AgNPs were prepared from the shoots of A. sieberi. The characteristics of AgNPs were assessed by UV–visible spectroscopy, transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), and dynamic light scattering (DLS). The antibacterial experiments were performed against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa. The fungal species used were Candida parapsilosis, Candida krusei, Candida famata, Candida rhodotorula, and Candida albicans. The antibacterial and antifungal characteristics were evaluated by measuring the diameter of growing organisms in Petri dishes treated with different concentrations of either extracts or AgNPs compared to the untreated controls. Furthermore, TEM imaging was used to investigate any ultrastructure changes in the microbes treated with crude extracts and AgNO3. Results: The ethanolic and aqueous extracts significantly decreased the growth of E. coli, S. aureus, and B. subtilis (p < 0.001), while P. aeruginosa was not affected. Unlike crude extracts, AgNPs had more substantial antibacterial effects against all species. In addition, the mycelial growth of C. famata was reduced by the treatment of both extracts. C. krusei mycelial growth was decreased by the aqueous extract, while the growth of C. parapsilosis was affected by the ethanolic extract and AgNPs (p < 0.001). None of the treatments affected the growth of C. albicans or C. rhodotorula. TEM analysis showed cellular ultrastructure changes in the treated S. aureus and C. famata compared to the control. Conclusion: The biosynthesized AgNPs and extracts of A. sieberi have a potential antimicrobial characteristic against pathogenic bacterial and fungal strains and nullified resistance behavior.


Introduction
Artemisia spp. is an important imperishable shrubby medicinal herb among the 500 different species related to the Asteraceae family. It is an imperishable plant native to Asia, the Middle East, Europe, and North Africa [1]. Artemisia spp. has several conversational names including green gusto, absinthe, absinthium, and mugwort. The flower heads are nanotechnology-based drug delivery systems, such as polymeric nanoparticles, solid lipid nanoparticles, liquid crystal systems, and precursor systems for liquid crystals, liposomes, and microemulsions, is an intriguing approach to improving the most desirable properties of a formulation [21].
The current study aimed to estimate the use of silver nanoparticles (AgNPs) synthesized from the aerial parts (shoot) of the A. sieberi strain growing in the northern region of Saudi Arabia as an antimicrobial agent against different bacterial and fungal species. To our knowledge, this is the first research to investigate the antimicrobial activities of ethanolic and aqueous extracts of the shoots of A. sieberi against specific pathogens, as most of the previous studies reported these activities from essential oils only.

Chemical Composition of A. sieberi
The aqueous extract and the biosynthesized AgNPs of A. sieberi were analyzed by FTIR to identify the important functional groups. As shown in Figures 1 and 2, the FTIR spectrum confirmed the presence of various functional groups.

Biosynthesis and Characterization of A. sieberi AgNPs
AgNPs were biosynthesized from the aqueous extract of A. sieberi. The synthesis process of AgNPs was interrupted by the change in color of AgNO 3 (2 µM) mixed with the extract of A. sieberi (20%) from colorless to brown ( Figure 3A). The transmission electron microscopy (TEM) images confirmed the biosynthesis of AgNPs, which appeared in a spherical shape with a diameter size of 27.5 ± 3.5 nm ( Figure 3B) compared to the 13 ± 2.42 nm of the unconjugated crude extract particles ( Figure 3C).
The purified AgNPs were analyzed by UV-visible spectra ( Figure 4A), which showed a surface plasmon resonance (SPR) peak at 445 nm. The results were compared to the spectrum obtained by the aqueous extract of A. sieberi, which revealed a comparable lower spectrum than that of the biosynthesized AgNPs. Furthermore, to measure the stability of the colloidal nanoparticles, we measured their effective surface charge, or zeta potential, which reflected their surface energy. The dynamic light scattering (DLS) method calculated the polydispersity index (PDI) (0.212) and the Z-average (101.2 d.nm) of AgNPs ( Figure 4B).

Biosynthesis and Characterization of A. sieberi AgNPs
AgNPs were biosynthesized from the aqueous extract of A. sieberi. The synthesis process of AgNPs was interrupted by the change in color of AgNO3 (2 µM) mixed with the extract of A. sieberi (20%) from colorless to brown ( Figure 3A). The transmission electron microscopy (TEM) images confirmed the biosynthesis of AgNPs, which appeared in a spherical shape with a diameter size of 27.5 ± 3.5 nm ( Figure 3B) compared to the 13 ± 2.42 nm of the unconjugated crude extract particles ( Figure 3C).

Biosynthesis and Characterization of A. sieberi AgNPs
AgNPs were biosynthesized from the aqueous extract of A. sieberi. The synthesis process of AgNPs was interrupted by the change in color of AgNO3 (2 µM) mixed with the extract of A. sieberi (20%) from colorless to brown ( Figure 3A). The transmission electron microscopy (TEM) images confirmed the biosynthesis of AgNPs, which appeared in a spherical shape with a diameter size of 27.5 ± 3.5 nm ( Figure 3B) compared to the 13 ± 2.42 nm of the unconjugated crude extract particles ( Figure 3C).

Biosynthesis and Characterization of A. sieberi AgNPs
AgNPs were biosynthesized from the aqueous extract of A. sieberi. The synthesis process of AgNPs was interrupted by the change in color of AgNO3 (2 µM) mixed with the extract of A. sieberi (20%) from colorless to brown ( Figure 3A). The transmission electron microscopy (TEM) images confirmed the biosynthesis of AgNPs, which appeared in a spherical shape with a diameter size of 27.5 ± 3.5 nm ( Figure 3B) compared to the 13 ± 2.42 nm of the unconjugated crude extract particles ( Figure 3C).

Biosynthesis and Characterization of A. sieberi AgNPs
AgNPs were biosynthesized from the aqueous extract of A. sieberi. The synthesis process of AgNPs was interrupted by the change in color of AgNO3 (2 µM) mixed with the extract of A. sieberi (20%) from colorless to brown ( Figure 3A). The transmission electron microscopy (TEM) images confirmed the biosynthesis of AgNPs, which appeared in a spherical shape with a diameter size of 27.5 ± 3.5 nm ( Figure 3B) compared to the 13 ± 2.42 nm of the unconjugated crude extract particles ( Figure 3C).

Biosynthesis and Characterization of A. sieberi AgNPs
AgNPs were biosynthesized from the aqueous extract of A. sieberi. The synthesis process of AgNPs was interrupted by the change in color of AgNO3 (2 µM) mixed with the extract of A. sieberi (20%) from colorless to brown ( Figure 3A). The transmission electron microscopy (TEM) images confirmed the biosynthesis of AgNPs, which appeared in a spherical shape with a diameter size of 27.5 ± 3.5 nm ( Figure 3B) compared to the 13 ± 2.42 nm of the unconjugated crude extract particles ( Figure 3C).

Biosynthesis and Characterization of A. sieberi AgNPs
AgNPs were biosynthesized from the aqueous extract of A. sieberi. The synthesis process of AgNPs was interrupted by the change in color of AgNO3 (2 µM) mixed with the extract of A. sieberi (20%) from colorless to brown ( Figure 3A). The transmission electron microscopy (TEM) images confirmed the biosynthesis of AgNPs, which appeared in a spherical shape with a diameter size of 27.5 ± 3.5 nm ( Figure 3B) compared to the 13 ± 2.42 nm of the unconjugated crude extract particles ( Figure 3C).

Biosynthesis and Characterization of A. sieberi AgNPs
AgNPs were biosynthesized from the aqueous extract of A. sieberi. The synthesis process of AgNPs was interrupted by the change in color of AgNO3 (2 µM) mixed with the extract of A. sieberi (20%) from colorless to brown ( Figure 3A). The transmission electron microscopy (TEM) images confirmed the biosynthesis of AgNPs, which appeared in a spherical shape with a diameter size of 27.5 ± 3.5 nm ( Figure 3B) compared to the 13 ± 2.42 nm of the unconjugated crude extract particles ( Figure 3C).

Biosynthesis and Characterization of A. sieberi AgNPs
AgNPs were biosynthesized from the aqueous extract of A. sieberi. The synthesis process of AgNPs was interrupted by the change in color of AgNO3 (2 µM) mixed with the extract of A. sieberi (20%) from colorless to brown ( Figure 3A). The transmission electron microscopy (TEM) images confirmed the biosynthesis of AgNPs, which appeared in a spherical shape with a diameter size of 27.5 ± 3.5 nm ( Figure 3B) compared to the 13 ± 2.42 nm of the unconjugated crude extract particles ( Figure 3C).

Biosynthesis and Characterization of A. sieberi AgNPs
AgNPs were biosynthesized from the aqueous extract of A. sieberi. The synthesis process of AgNPs was interrupted by the change in color of AgNO3 (2 µM) mixed with the extract of A. sieberi (20%) from colorless to brown ( Figure 3A). The transmission electron microscopy (TEM) images confirmed the biosynthesis of AgNPs, which appeared in a spherical shape with a diameter size of 27.5 ± 3.5 nm ( Figure 3B) compared to the 13 ± 2.42 nm of the unconjugated crude extract particles ( Figure 3C).

Antibacterial Activities of A. sieberi
In the current study, three doses of both extracts of A. sieberi were used to assess their antibacterial activities on the growth of different species (S. aureus, P. aeruginosa, B. subtilis, and E. coli) and compare them to antibacterial effects of the cephalexin, a known broad-spectrum antibiotic, at a dose of 5 µg/mL. The zone of inhibition of each treatment was measured in mm and compared to the negative control of DMSO-treated species (1 µL/dish). At the doses of 20 µg/mL and 40 µg/mL of the aqueous extract of A. sieberi ( Figure 5), significant inhibition of the growth of S. aureus, B. subtilis, and E. coli (p < 0.001) was shown, while the growth of P. aeruginosa was not affected. As shown in Table 3 and Figure 5, the largest inhibition zones were for S. aureus (3, 16.7 ± 0.6 mm, MIC: <10 µg/mL), followed by B. subtilis (13.2 ± 1.3 mm, MIC: <10 µg/mL) and E. coli (11.7 ± 0.3 mm, MIC: 15 µg/mL) at the dose of 40 µg/mL. The box-plot analysis represents the medians (solid horizontal lines) and the means (the square box) of each zone of inhibition (ZOI) of each strain. As shown in Figure 5B, the box plots showed low median ranges and a standard deviation (SD) among all replicates, which indicated accurate and constant imbibitional effects. The purified AgNPs were analyzed by UV-visible spectra ( Figure 4A), which showed a surface plasmon resonance (SPR) peak at 445 nm. The results were compared to the spectrum obtained by the aqueous extract of A. sieberi, which revealed a comparable lower spectrum than that of the biosynthesized AgNPs. Furthermore, to measure the stability of the colloidal nanoparticles, we measured their effective surface charge, or zeta potential, which reflected their surface energy. The dynamic light scattering (DLS) method calculated the polydispersity index (PDI) (0.212) and the Z-average (101.2 d.nm) of AgNPs ( Figure 4B).

Antibacterial Activities of A. sieberi
In the current study, three doses of both extracts of A. sieberi were used to assess their antibacterial activities on the growth of different species (S. aureus, P. aeruginosa, B. subtilis, and E. coli) and compare them to antibacterial effects of the cephalexin, a known broad- Similarly, different doses of the ethanolic extract of A. sieberi ( Figure 6) showed significant inhibition of the growth of S. aureus, B. subtilis, and E. coli (p < 0.001), while the growth of P. aeruginosa was not affected. As shown in Table 3 and Figure 4, the largest inhibition zones were for S. aureus (18 ± 0.5 mm, MIC: <10 µg/mL), followed by B. subtilis (16.5 ± 0.5 mm, MIC: <10 µg/mL) and E. coli (10.2 ± 0.3 mm, MIC: 15 µg/mL) at the dose of 40 µg/mL. Comparing both results, it was obvious that the ethanolic extract had more inhibitory effects than the aqueous extract against S. aureus (median: 18 versus 17 mm) and B. subtilis (median: 16.5 versus 13 mm). In contrast, E. coli growth was more sensitive to the aqueous (median: 11.5 mm) than the ethanolic extract (median: 10 mm) ( Table 3). In addition, the box plots showed low median ranges and SD among all replicates, which indicated accurate and constant imbibitional effects.
12.5 (11.5-13) 14 (14) 16.5 (16)(17) p value 1 <0.001 * <0.001 * <0.001 * <0.001 * Similarly, different doses of the ethanolic extract of A. sieberi ( Figure 6) showed significant inhibition of the growth of S. aureus, B. subtilis, and E. coli (p < 0.001), while the growth of P. aeruginosa was not affected. As shown in Table 3 and Figure 4, the largest inhibition zones were for S. aureus (18 ± 0.5 mm, MIC: <10 µg/mL), followed by B. subtilis (16.5 ± 0.5 mm, MIC: <10 µg/mL) and E. coli (10.2 ± 0.3 mm, MIC: 15 µg/mL) at the dose of 40 µg/mL. Comparing both results, it was obvious that the ethanolic extract had more inhibitory effects than the aqueous extract against S. aureus (median: 18 versus 17 mm) and B. subtilis (median: 16.5 versus 13 mm). In contrast, E. coli growth was more sensitive to the aqueous (median: 11.5 mm) than the ethanolic extract (median: 10 mm) ( Table 3). In addition, the box plots showed low median ranges and SD among all replicates, which indicated accurate and constant imbibitional effects. The biosynthesized AgNPs induced significant inhibitory effects against all strains at all concentrations (p < 0.001), as shown in Table 3 and Figure 7. All calculated MIC results were < 10 µg/mL for AgNPs, which indicated stronger effects. The largest inhibition zones were for S. aureus (19 ± 1 mm), followed by P. aeruginosa (17 ± 0.1 mm), E. coli (14.8 ± 0.3 mm), and B. subtilis (14 ± 0.5 mm) at the highest dose. Compared to the crude extract, AgNPs had the strongest inhibitory effects at all doses. The calculated ZOI of AgNO3 (2 mM) showed some inhibitory effect on the growth of all species, which was lower than AgNPs at all concentrations. In addition, the box plots showed low median ranges and SD among all replicates, which indicated accurate and constant inhibition effects. As shown from the above data, S. aureus was the most sensitive species to different treatments of A. sieberi among the other tested species. The comparative data analysis revealed that AgNPs induced higher zones of inhibition than the crude extracts. However, AgNPs induced significant growth inhibition of B. subtilis versus the ethanolic extract (p < 0.05) and P. aeruginosa versus both extracts (p < 0.001). The biosynthesized AgNPs induced significant inhibitory effects against all strains at all concentrations (p < 0.001), as shown in Table 3 and Figure 7. All calculated MIC results were <10 µg/mL for AgNPs, which indicated stronger effects. The largest inhibition zones were for S. aureus (19 ± 1 mm), followed by P. aeruginosa (17 ± 0.1 mm), E. coli (14.8 ± 0.3 mm), and B. subtilis (14 ± 0.5 mm) at the highest dose. Compared to the crude extract, AgNPs had the strongest inhibitory effects at all doses. The calculated ZOI of AgNO 3 (2 mM) showed some inhibitory effect on the growth of all species, which was lower than AgNPs at all concentrations. In addition, the box plots showed low median ranges and SD among all replicates, which indicated accurate and constant inhibition effects. As shown from the above data, S. aureus was the most sensitive species to different treatments of A. sieberi among the other tested species. The comparative data analysis revealed that AgNPs induced higher zones of inhibition than the crude extracts. However, AgNPs induced significant growth inhibition of B. subtilis versus the ethanolic extract (p < 0.05) and P. aeruginosa versus both extracts (p < 0.001).

Antifungal Activities of A. sieberi
In the current study, three doses of both extracts of A. sieberi were used to assess their antifungal activities on the growth of different species (C. rhodotorula, C. parapsilosis, C. krusei, C. famata, and C. albicans). The results of both extracts were compared to a positive control of a known antifungal medicine (Terbinafine) at a dose of 5 µg/mL.
As shown in Figure 8 and Table 4, the aqueous extract of A. sieberi affected the mycelial growth of C. krusei and C. famata at all concentrations (p < 0.001). It was obvious that the greatest zone inhibition diameters were for C. krusei at the three doses. The mycelial growth of C. rhodotorula, C. parapsilosis, or C. albicans was not affected by any doses of the aqueous extract of A. sieberi (Figure 8). Despite variable effects, the terbinafine antifungal activity proved that all species were not restricted to any source of antifungal resistance. The box plot analysis revealed low SD and median values, which revealed the constant effect at all replicates.

Antifungal Activities of A. sieberi
In the current study, three doses of both extracts of A. sieberi were used to assess their antifungal activities on the growth of different species (C. rhodotorula, C. parapsilosis, C. krusei, C. famata, and C. albicans). The results of both extracts were compared to a positive control of a known antifungal medicine (Terbinafine) at a dose of 5 µg/mL.
As shown in Figure 8 and Table 4, the aqueous extract of A. sieberi affected the mycelial growth of C. krusei and C. famata at all concentrations (p < 0.001). It was obvious that the greatest zone inhibition diameters were for C. krusei at the three doses. The mycelial growth of C. rhodotorula, C. parapsilosis, or C. albicans was not affected by any doses of the aqueous extract of A. sieberi (Figure 8). Despite variable effects, the terbinafine antifungal activity proved that all species were not restricted to any source of antifungal resistance. The box plot analysis revealed low SD and median values, which revealed the constant effect at all replicates. Plants 2023, 12, x FOR PEER REVIEW 13 of 25 For the ethanolic extract of A. sieberi, there was significant inhibition of the mycelial growth of C. famata and C. parapsilosis at all the concentrations (p < 0.001). Unlike the aqueous extract, the mycelial growth of C. famata treated with the ethanolic extract was the most affected ( Figure 9). The mycelial growth of C. rhodotorula, C. krusei, or C. albicans was not affected by any doses (Figure 9). The box plot analysis revealed low SD and median values, which revealed the constant effect at all replicates. The highest inhibitory effect against C. famata was induced by the ethanolic extract (35.8 ± 0.3 mm) compared to the aqueous extract (14.8 ± 0.3 mm) at a dose of 40%. We noticed that the inhibitory effects of both plant extracts were higher than those of the AgNPs at different doses. However, AgNPs could inhibit the growth of all the tested Candida species (Table 4).   For the ethanolic extract of A. sieberi, there was significant inhibition of the mycelial growth of C. famata and C. parapsilosis at all the concentrations (p < 0.001). Unlike the aqueous extract, the mycelial growth of C. famata treated with the ethanolic extract was the most affected ( Figure 9). The mycelial growth of C. rhodotorula, C. krusei, or C. albicans was not affected by any doses (Figure 9). The box plot analysis revealed low SD and median values, which revealed the constant effect at all replicates. The highest inhibitory effect against C. famata was induced by the ethanolic extract (35.8 ± 0.3 mm) compared to the aqueous extract (14.8 ± 0.3 mm) at a dose of 40%. We noticed that the inhibitory effects of both plant extracts were higher than those of the AgNPs at different doses. However, AgNPs could inhibit the growth of all the tested Candida species (Table 4). The effect of the biosynthesized AgNPs was stronger than that of both extracts of A. sieberi at all concentrations. Furthermore, AgNPs affected the mycelial growth of all the studied species ( Figure 10). As shown in Table 4, the highest antifungal activity of AgNPs was against C. famata (26.2 ± 0.8 mm), followed by C. krusei (14.7 ± 0.8 mm), C. albicans (14.7 ± 0.8 mm), and C. rhodotorula (10.3 ± 0.6 mm), where the least sensitive species was C. parapsilosis (8.3 ± 0.6 mm) ( Table 4). AgNO3 (2 mM) showed some inhibitory effect on the growth of all species, which was lower than that of AgNPs at all concentrations. In addition, the box plots showed low median ranges and SD among all replicates, which indicated accurate and constant inhibition effects. As shown from the above data, C. famata was the most sensitive species to different treatments of A. sieberi among the other tested species. The comparative data analysis revealed that the ethanolic extract induced more significant growth inhibition than the aqueous extract against C. famata and C. parapsilosis (p < 0.001). AgNPs induced higher zones of inhibition than the aqueous extract (p < 0.001). However, the ethanolic extract induced higher significant growth inhibition of C. famata and C. parapsilosis than AgNPs (p < 0.001). The effect of the biosynthesized AgNPs was stronger than that of both extracts of A. sieberi at all concentrations. Furthermore, AgNPs affected the mycelial growth of all the studied species ( Figure 10). As shown in Table 4, the highest antifungal activity of AgNPs was against C. famata (26.2 ± 0.8 mm), followed by C. krusei (14.7 ± 0.8 mm), C. albicans (14.7 ± 0.8 mm), and C. rhodotorula (10.3 ± 0.6 mm), where the least sensitive species was C. parapsilosis (8.3 ± 0.6 mm) ( Table 4). AgNO 3 (2 mM) showed some inhibitory effect on the growth of all species, which was lower than that of AgNPs at all concentrations. In addition, the box plots showed low median ranges and SD among all replicates, which indicated accurate and constant inhibition effects. As shown from the above data, C. famata was the most sensitive species to different treatments of A. sieberi among the other tested species. The comparative data analysis revealed that the ethanolic extract induced more significant growth inhibition than the aqueous extract against C. famata and C. parapsilosis (p < 0.001). AgNPs induced higher zones of inhibition than the aqueous extract (p < 0.001). However, the ethanolic extract induced higher significant growth inhibition of C. famata and C. parapsilosis than AgNPs (p < 0.001).

Ultrastructural Characterizations by TEM of Selected Species Treated with Different Preparations of A. sieberi
As shown from the above data, S. aureus and C. famata were the most sensitive species to different A. sieberi treatments. The morphological and ultrastructural changes and their effect on the growth of the sensitive microbes were detected by TEM. As shown in Figure  11, the extracts of A. sieberi induced some morphological changes on the cells of S. aureus that looked more granulated and less proliferated compared to the control. In the AgNPs settings, more extensive ultrastructural changes occurred, where larger vacuoles were formed and the cellular division was terminated.

Ultrastructural Characterizations by TEM of Selected Species Treated with Different Preparations of A. sieberi
As shown from the above data, S. aureus and C. famata were the most sensitive species to different A. sieberi treatments. The morphological and ultrastructural changes and their effect on the growth of the sensitive microbes were detected by TEM. As shown in Figure 11, the extracts of A. sieberi induced some morphological changes on the cells of S. aureus that looked more granulated and less proliferated compared to the control. In the AgNPs settings, more extensive ultrastructural changes occurred, where larger vacuoles were formed and the cellular division was terminated.
Similarly, the mycelial growth of C. famata by different extracts of A. sieberi had serious morphological changes in the cellular structure. The aqueous extract induced the formation of large vacuoles, while the ethanolic extract induced extreme damage, as indicated by the rapture of the fungal cells, which further affected the mycelial growth ( Figure 12 Similarly, the mycelial growth of C. famata by different extracts of A. sieberi had serious morphological changes in the cellular structure. The aqueous extract induced the formation of large vacuoles, while the ethanolic extract induced extreme damage, as indicated by the rapture of the fungal cells, which further affected the mycelial growth ( Figure 12). Larger vacuoles and undivided cells were induced by AgNPs.

Discussion
Medicinal plants are traditionally used worldwide as remedies for various diseases, including asthma, gastrointestinal symptoms, skin disorders, respiratory and urinary problems, and hepatic and cardiovascular diseases [22]. The current study aimed to investigate the antimicrobial activity of A. sieberi extracts and the biosynthesized AgNPs against selected strains of human pathogenic bacteria and fungi.  Similarly, the mycelial growth of C. famata by different extracts of A. sieberi had serious morphological changes in the cellular structure. The aqueous extract induced the formation of large vacuoles, while the ethanolic extract induced extreme damage, as indicated by the rapture of the fungal cells, which further affected the mycelial growth ( Figure 12). Larger vacuoles and undivided cells were induced by AgNPs.

Discussion
Medicinal plants are traditionally used worldwide as remedies for various diseases, including asthma, gastrointestinal symptoms, skin disorders, respiratory and urinary problems, and hepatic and cardiovascular diseases [22]. The current study aimed to investigate the antimicrobial activity of A. sieberi extracts and the biosynthesized AgNPs against selected strains of human pathogenic bacteria and fungi.

Discussion
Medicinal plants are traditionally used worldwide as remedies for various diseases, including asthma, gastrointestinal symptoms, skin disorders, respiratory and urinary problems, and hepatic and cardiovascular diseases [22]. The current study aimed to investigate the antimicrobial activity of A. sieberi extracts and the biosynthesized AgNPs against selected strains of human pathogenic bacteria and fungi.
The phytochemical analysis of A. sieberi revealed that it was rich in amines, carboxylic groups (such as alcohols and phenols), and alkenes. In agreement with these findings, a previous study showed that the FTIR analysis of the leaf extract of A. sieberi had the highest peaks for hydroxyl groups, alkanes, alkenes, amides, and amines [23,24]. In addition, the study conducted by Alotibi and Rizwana (2019) revealed that the methanolic extract of A. sieberi was rich in the functional groups of OH, CH stretching, C=O stretching, and aromatic skeletal stretches, which agreed with the current results as well [25]. Other species of the Asteraceae family had a similar composition of alkyl halides, alkanes, alkenes, aldehydes, and amide groups, such as Matricaria chamomilla [26], Centaurea cyanus [27], Artemisia maritima [28], Artemisia indica, and Artemisia vestita [29].
In the current study, AgNPs produced a SPR peak of 445 nm compared to the crude extract, which did not reveal any absorption peaks. The appearance of an absorption peak at a wavelength of 400-500 nm is an indication that AgNPs were formed [30]. Similar studies reported the UV-visible spectrum of AgNPs biosynthesized from the extracts of A. sieberi at 482 nm [31], 407 nm [32], and 410 nm [33].
In the current study, we compared the antimicrobial activities of the crude aqueous and ethanolic extracts of A. sieberi against Gram-positive and Gram-negative bacteria and Candida fungal strains. In addition, we studied the antimicrobial properties of the biosynthesized AgNPs of A. sieberi. Significant activities were reported against the growth of Gram-positive bacteria B. subtilis and S. aureus. The Gram-positive bacteria showed a larger ZOI than the Gram-negative bacteria due to the variation in cell wall composition. Additionally, in human pathogenic fungal strains treated with AgNPs nanoparticles of the extracts of A. sieberi at different concentrations (10, 20, and 40 µg/mL), significant variability in the inhibition rates was reported against different fungal strains such as C. albicans, C. parapsilosis, C. famata, C. krusei, and C. rhodotorula.
Previous studies showed that the significant arability and the selective activity of plant extracts towards the bacteria strains might be related to the presence of an impermeable barrier of lipopolysaccharide on the outer membrane of Gram-negative bacteria that inhibit diffusion of active compounds [39]. Moreover, Gram-positive bacteria freely allow the direct contact of active constituents with the phospholipid bilayer of the cell membrane, leading to enhanced ion permeability [40]. In agreement with our findings, the previous study of Alotibi and Rizwana (2019) [25] revealed that the methanol and dichloromethane extracts from A. sieberi had significant inhibitory effects against the growth of Alternaria alternata, Fusarium moniliforme, F. solani, F. oxysporum, and Macrophomina phaseolina. Another study showed that 8R-dihydroxygermacr-4(15),9(10)-dien-6S,7S,11RH,12,6-olide, 1R, 8Sdihydroxy-11R,13-dihydrobalchanin, 11-epiartapshin, and 3 -hydroxygenkwanin isolated from A. sieberi had moderate antifungal activities against C. albicans, C. tropicalis, Aspergillus niger, F. solani, and A. alternata [41]. The study conducted by Ghasemi et al. (2021) suggested that the strong antifungal activity of A. sieberi against Botrytis cinerea occurs due to the monoterpene compounds in the essential oils of A. sieberi [42]. Another study revealed that the essential oils extracted from Saudi-native species of Artemisia absinthium, Artemisia scoparia, and A. sieberi showed significant inhibitory activities against the growth of Grampositive (S. aureus, Bacillus licheniformis, and Micrococcus luteus) and Gram-negative bacteria (Enterobacter xiangfangensis, Escherichia fergusonii, and P. aeruginosa) and yeast strains (C. albicans, C. parapsilosis, and Aspergillus parasiticus) with MIC values of 0.1-3 µg/mL [3]. In addition, in the study conducted by Mahboubi et al. (2015), the essential oils of A. sieberi had significant antimicrobial activities against S. aureus (MIC: 6.25-9.37 µL/mL), C. albicans, P. aeruginosa, and E. coli (MIC: 12-19 µL/mL) [43].
The antimicrobial activity of synthesized AgNPs depends mainly on the physical and chemical characteristics of the green synthesized particles, and plants use spherical shapes and small sizes of nanoparticles, which are more effective than the triangular ones [44,45]. However, the exact mechanism of how shape affects the antibacterial activity is still unclear. In addition, the antibacterial activity increases with AgNPs concentration and exposure time to microbes such as Gram-negative and Gram-positive microorganisms [46,47]. Similarly, our findings demonstrated that antibacterial and antifungal activity against some drug-resistant pathogenic strains was maximal at the highest concentrations of AgNPs. Additionally, more studies explained the mechanism of AgNPs impact on microbial cells via intracellular physicochemical processes, particularly the oxidation of protoplasm and its destruction by oxygen; AgNPs, in turn, play the role of catalysts [48]. In addition, other studies have proposed that the effect of AgNPs on membrane permeability and membraneassociated enzymes, including the proton FOF1-ATPase, can be the main mechanism of AgNPs [49]. Moreover, the complexation of cellular nucleic acid with heavy metals in Ag-NPs particles leads to DNA impairment and bacterial viability [50]. Other studies revealed the significant antimicrobial activities of AgNPs biosynthesized from culture supernatant of bacterial strains Pseudoduganella eburnea against P. aeruginosa (MIC: 6.25 µg/mL) and S. aureus (MIC: 100 µg/mL) [51], Paenarthrobacter nicotinovorans against P. aeruginosa (MIC: 25 µg/mL) and Bacillus cereus (MIC: 12.5 µg/mL) [52], and Sphingobium sp. against E. coli (MIC: 6.25 µg/mL) and S. aureus (MIC: 50 µg/mL) [53], which are drug-resistant pathogenic microbes. In general, it was proposed that AgNPs might be adsorbed on the cell membrane, therefore obstructing the growth of bacteria and having a protective function [33,54].
Recently, it was reported that the green synthesis of AgNPs from Artemisia extracts has a versatile range of biological applications and can be used as an eco-friendly material without harmful effects on the environment [33]. In the current study, the results suggested that the biosynthesized AgNPs using A. sieberi extract had a higher antimicrobial activity which significantly increased with higher concentrations of biogenic AgNPs. Thus, it could be used as an excellent source against the tested microbes. However, the microbicidal effect of biosynthesized AgNPs could be different depending on the organism tested.
The results revealed more reliable antimicrobial activity of AgNPs than that of the crude extracts of A. sieberi against all bacterial and Candida species. However, the inhibitory effects induced by the plant extracts were higher than those of the AgNPs at different doses. The high biocidal activity of silver nanoparticles is explained by their large surface area, which provides better contact with microorganisms. Moreover, silver nanoparticles act as reservoirs for the AgNPs bactericidal agent. Previously, it was reported that a combination of silver nanoparticles and herbal extracts, their metabolites, and antibiotics exhibited a synergistic effect [55].
TEM microscopic analysis showed that different concentrations of aqueous and ethanolic extracts of A. sieberi significantly bind with cellular organelles and compartments of S. aureus cell membranes. S. aureus cultured in a control medium displayed some conserved morphological characteristics as intact cells. The treatment with the extract at different concentrations facilitated the penetration of the cell membranes of S. aureus, which led to the disruption of the cell wall and an increase in vacuole size with dilatation of the cell wall and lysis with increasing the time of exposure. The treatment with AgNPs at different concentrations induced the disintegration of bacterial cells, disruption of the cell walls and cell membranes, separation of the cytoplasmic membrane from the cell wall, and cytoplasmic dissolution, which significantly increased at higher concentrations of AgNPs.
Similarly, the ultrastructural changes in C. famata were identified by TEM analysis. It revealed a typical cellular structure with a conserved and intact layered cell wall and distinctive cytoplasmic membranes in the untreated C. famata. In contrast, fungal cells exposed to crude extracts of A. sieberi lost their cell wall permeability. In cells treated with AgNPs, disruption of the outer cell walls causes, subsequently, more permeabilization, which allows smaller AgNPs get inside the yeast. In consequence, that led to cellular vacuolation, destruction, and more accumulation of nanoparticles, which increased disruption, cellular vacuoles, aggregation, and dilatation of the cell walls, which more precisely affected the survival of both the yeast and the filamentous forms of the fungus compared to that which occurs in cells treated with pure aqueous and ethanolic extracts. The effect of AgNPs significantly occurred in a dose-dependent manner, which consequently increased with high doses.
TEM images revealed that AgNPs formed or were deposited on the cell membrane of bacterial or fungal species, whereas various magnifications of AgNPs-treated microbes (bacteria and fungi) showed several black spots on the cellular membrane and cytoplasm. Based on ultrastructural analyses, it was proposed that the antimicrobial activity of the synthesized AgNPs of A. sieberi depends mainly on the physical interactions between the AgNPs and the microbial cell surface. That depends on the superior surface area of the particles of the Artemisia extracts which increase the long-term stability of AgNPs and affect cell wall permeability [56]. The antibacterial efficacy of AgNPs from A. sieberi against Gram-positive bacteria was better than that of Gram-negative bacteria due to the difference in biological activity and thickness of the cell walls of individual bacterial species.
These results proved that AgNPs were mostly synthesized in the outer membrane of the cells of targeted microbes [57]. Previous studies on chitosan alone or as AgNPs proved antibacterial activity against S. aureus and E. coli. TEM analysis showed that chitosan nanoparticles induced cellular ultrastructure changes such as collapsed cell walls, condensed chromatin, and an increase in intracellular structures, such as vacuoles and cell debris, minimizing the growth and increasing the mortality score of the bacteria [58]. In agreement with our findings, the TEM results of the study conducted by Al-Otaibi and Rizwana (2019) [25] showed that damage to the fungal cells treated with an ethanolic extract of A. sieberi was represented by heavy vacuolation, proliferation of lipid bodies, septal damage, and undeveloped conidia.
In general, past observations supported the finding that that AgNPs nanoparticles alone or fabricated with plant extracts proved that nanoparticles were highly toxic and caused changes in the morphology and dimensions of the target organisms studied [59,60]. It was reported previously that increasing the concentration of AgNPs leads to a reduction in cell growth and more inhibition, which might reach 98.7% at higher concentrations. In addition, images of SEM and TEM analysis reflected the effect of nanoparticles via a shrinking and damaging of cell walls, indicating the toxicity of silver nanoparticles toward microorganisms such as cyanobacteria [60]. Similarly, previous studies proved that nanoparticles could affect bacterial replication, protein leakage, and cell death. That might be a potentially effective agent against the propagation and overgrowth of chronic microbial infections [57].

Plant Material and Preparation of Extracts
A. sieberi plant material was collected in March 2019 from the northern region (Rafha region), Kingdom of Saudi Arabia. The aerial parts (shoot) from each plant were washed, dried in shade, finely powdered, and stored at 4 • C for further use.
For the preparation of extracts, different solvents (ethanol and water) were used. A total of 30 g of the fresh, dry plant material was socked in 300 mL of either distilled water (aqueous extract) or absolute ethanol (ethanolic extract) for 24 h at room temperature. The mixture was transferred to a rotary shaker for another night, then filtered through cotton and tissue papers (Sartolab ® RF Vacuum Filtration Units 180C8, Goettingen, Germany). Finally, the remaining filtrates were left to dry in cleaned metallic trays, collected in clean glass containers, and stored at 4 • C. All plant extract (ethanol, water) concentrations were prepared as grams per milliliter. We used <1% concentrations combined with the extracts in culture media to increase their solubility. DMSO is widely used to solubilize different therapeutic applications, and studies indicated that a 10% v/v concentration did not modify culture viability [61]. Each stock-sterile-filtered extract was mixed with either the Mueller-Hinton agar medium for bacteria or the Sabouraud agar (Sigma-Aldrich, St. Louis. MO, USA) for Candida sp. to obtain different concentrations (0, 10, 20, and 40 µg/mL).

Biosynthesis and Characterization of Silver Nanoparticles
The prepared extract of A. sieberi (2 g) was soaked in 10 mL of sterile water at 50 • C for 20 min, also filtered. The biosynthesis of silver nanoparticles was carried out by mixing 2 mL of A. sieberi extract with 100 mL of AgNO 3 (2 mM). The set admixture was left unperturbed under the sun till the color of the response admixture changed to sanguinebrown. The color change is the primary suggestion of the conflation of the AgNPs, as shown by Kumar et al. (2015) with variations [62].
TEM imaging was used to confirm the liquid structure of the synthesized nanoparticles. The medication and processing of slides were performed according to the manufacturer's instructions, as described preliminarily [63]. The samples were fixed by overnight buffering with glutaraldehyde at 2.5%, then washed and dehydrated by immersion in serial concentrations of ethanol. These samples were embedded by resin mixture and cut at thicknesses of 70-80 nm by a UC6 ultramicrotome (Leica, Wetzlar, Germany). After loading on the cupper grid, the slides were stained by uranyl acetate and lead citrate and analyzed by the JEM-1400 transmission electron microscope (JEOL Ltd. Inc., Tokyo, Japan).
The synthesized nanoparticles were purified by the PD-10 desalting column (Sigma-Aldrich, St. Louis, MI, USA) according to the manufacturer's instructions [63].
A UV-visible spectrophotometer (Shimadzu, Tokyo, Japan) was used for the characterization of the purified biosynthesized AgNPs at 200-800 nm, according to the manufacturer's instructions [64,65].
To measure the stability of the colloidal nanoparticles and detect the particle size distribution, we used the DLS technique, which calculated the PDI and the Z-average. The Zetasizer Pro (Malvern Panalytical, Malvern, UK) was used according to the manufacturer's instructions.

Gas Chromatography-Mass Spectrometry (GC-MS)
GC-MS was performed on the ethanol extract of A. sieberi. Helium gas with an inflow of 1 mL/min served as the carrier gas. The instrument was GC coupled with MS (7890A, 5975C (Agilent Technologies, Santa Clara, CA, USA). A phenyl-methyl siloxane column was used (30 m × 250 µm × 0.25 µm). The ensuing conditions were maintained during the GC run time (90 min): volume (1 µL), temperature (280 • C; 250 • C), ion source, and resolve rate (201). Further, the temperature was held for 5 min at 40 • C; to begin with, it was also increased to 280 • C at 10 • C/min and at this temperature, it was maintained for another 5 min. An electron impact (70 eV) was generated for mass spectroscopy with a checkup range of 35 to 780 m/z. The mass digital library of the National Institute of Standards and Technology (NIST) was used for identification [66].

Fourier-Transfigure Infrared Spectroscopy (FTIR)
FTIR analysis was used to identify the important functional groups present in the ethanol extract. The spectrometer (Nicolet-6700, Thermo Fisher Scientific Inc., Waltham, MS, USA) enjoying a ray splitter and a sensor (DTGS) equipped with OMNIC software was used to collect and dissect the diapason in the checkup range of 500-4000 cm −1 . The obtained IR spectra were used to interpret the functional groups present according to the guidelines of LibreTexts libraries https://chem.libretexts.org/ (Accessed on 31 December 2022) [67].

Growth Inhibition Assay
Petri dishes of the Mueller-Hinton agar bacterial medium were prepared, and the different bacterial strains were plotted. With a metal cork, three discs were made and filled with one of the concentrations of the prepared treatments. Two dishes were prepared for the negative control (DMSO, <1%/dish) and positive control (cephalexin, 5 µg/mL). The cultures were incubated for 24 h at 37 • C. After incubation, all areas of inhibition were measured in millimeters. All experiments were prepared in triplicate.
Petri dishes of Sabouraud agar were prepared, and the different fungal strains were plotted. With a metal cork, three discs were made and filled with one of the concentrations of the prepared treatments. Another dish was prepared for the positive control (terbinafine, 5 µg/mL). The cultures were incubated for 72 h at 37 • C. After incubation, all areas of inhibition were measured in millimeters. All experiments were prepared in triplicate. Minimum inhibitory concentrations (MIC) and the evaluation of the susceptibility/resistance of the tested strains to various treatments were detected according to the guidelines of Clinical and Laboratory Standards Institute (CLSI) available from https://clsi.org/standards/products/microbiology/documents/m100/ (Accessed on 2 February 2022).

Determination of the Effect of Plant Extracts on the Morphology and Ultrastructure of the Organisms under Study
To prepare the bacterial and fungal samples for scanning electron microscopy (SEM), the selected bacteria and fungus were grown in the presence of a certain concentration of aqueous and ethanolic plant extracts, where both the sterilized extracts-the Mueller-Hinton agar and the Sabouraud agar medium-were mixed separately immediately before pouring into the sterilized Petri dishes. Control samples (extracts untreated) were prepared in the Mueller-Hinton agar and the Sabouraud agar medium plates only (plant extract-free). Then, the samples were inoculated with 10 µL of fresh subculture (old) of the selected bacterial and fungal strains and incubated at 28 • C for 24 h for bacterial isolation and for 48 h for Candida isolation. These treated and untreated plates were sent to the electron microscopy lab in the Central Laboratory at the Women Students' Medical Studies and Sciences Sections, King Saud University. All the sample preparations for the electron microscopy photography were performed according to their procedure.

Statistical Analysis
The results were analyzed using the STATISTIX 10 software, one-way analysis of variance (ANOVA), and Sidak's multiple comparisons tests. Mean values were separated on the basis of the least significant difference (LSD). The statistical significance was set at p < 0.05.

Conclusions
Artemisia's active metabolites may represent one of many examples of power hidden in natural sources; they might become a promising new antimicrobial drug, hopefully developed and introduced to clinical use shortly. Thus, more clinical trials are needed to increase and support the use of these extracts in clinical-based trials. The current study has several implications for scientific research. A. sieberi extracts produced by nanotechnology, in addition to being used in the inhibition programs of antimicrobials, might be used by food inspectors and food safety organizations. The results suggested that the AgNPs biosynthesized using A. sieberi extracts had potential antimicrobial activity against pathogenic bacterial and fungal strains and nullified resistance behavior. The biogenic AgNPs activity linked with the particles of A. sieberi extracts led to an increase in antibacterial activity and could be used as an excellent source against tested microbes. Despite the proposed high antimicrobial activity of the A. sieberi extracts and related AgNPs, further studies of their biocidal effect on animal models are needed before the use of synthesized AgNPs as antimicrobial agents.