Antimicrobial and cytotoxic activity of green synthesis silver nanoparticles targeting skin and soft tissue infectious agents

Combining traditional medicine with nanotechnology therefore opens the door to innovative strategies for treating skin and soft tissue infections (SSTIs) and also contributes to the fight against the rise of antimicrobial resistance. Acanthospermum australe (Loefl.) Kuntze is a medicinal plant used by indigenous peoples in northeastern Argentina to treat SSTIs. Spherical and stable silver nanoparticles (AgNPs) of 14 ± 2 nm were synthesized from the aqueous extract of A. australe and silver nitrate. The antimicrobial activity against main species causing SSTIs and cytotoxicity on peripheral blood mononuclear cells of AgNP solution and its synthesis components were evaluated. Compared to its synthesis components, AgNP solution showed greater antimicrobial activity and lower cytotoxicity. The antimicrobial activity of AgNPs was due to the silver and not to the metabolites of the aqueous extract present on the surface of the nanoparticles. The plant extract played an important role in the formation of stable AgNPs and acted as a modulator of cytotoxic and immune responses.

Preparation of the aqueous extract of A. australe (TAE). Exactly 50 g of the comminuted plant material was boiled with 1000 mL of sterile deionized water for 5 min and allowed to stand for 1 h at room temperature. It was then filtered using Whatman filter paper No. 1 followed by a Millipore filter (0.22 µm). The obtained aqueous extract was frozen at -70 °C, lyophilized and stored at 4 °C in the dark under sterile conditions for later use. The percentage yield of extraction (Y) was determined using the following equation: Total phenolic content. The total phenolic content of TAE was analyzed spectrophotometrically using the Folin-Ciocalteu method 18 , with some modifications (Supplementary file). The results are expressed as the mean gallic acid equivalent mass in mg per g of lyophilized extract (mg GAE/g LE) ± standard deviation (SD).
Total flavonoid content. It was determined for TAE according to Dewanto et al. 19 , with modifications (Supplementary file). Total flavonoid content was expressed as the mean quercetin equivalent mass in mg per g of lyophilized extract (mg QE/g LE) ± SD.
Green synthesis of silver nanoparticles. In order to obtain small and stable nanoparticles, the green synthesis was carried out according to the results obtained by other authors 12,20,21 , with some modifications (Supplementary file).
Characterization of silver nanoparticles. The synthesized AgNPs were characterized using the following techniques: (A) UV-visible spectrophotometer (Multiskan Go, Thermo Fisher, Finland) was used to measure absorbance and record optical density at wavelengths from 300 to 700 nm. (B) The shape and size of AgNPs were determined by transmission electron microscopy (TEM; JEOL, Tokyo, Japan; JEM-2100) at an accelerating voltage of 200 kV. Samples were prepared by drop coating 10 µL of AgNP solution onto carbon coated copper grids followed by air drying, forming a thin layer on the surface. (C) Energy dispersive X-ray spectroscopy (EDX) was Ferric reducing antioxidant power (FRAP) assay. The antioxidant activity of TAE, AgNO 3 and AgNPs was determined using the FRAP assay according to Quiroz-Reyes et al. 22 (Supplementary file). The results are expressed as the mean Trolox equivalent mass in μmol per g of lyophilized extract (μmol TE/g LE) ± SD.
DPPH radical scavenging capacity. The antiradical capacity was determined using the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay according to the methodology proposed by Molyneux 23 , with some modifications (Supplementary file). The effective concentration 50% (EC 50 ), defined as the amount of antioxidant required to reduce the initial concentration of DPPH radical by 50%, was calculated. In addition, the percentage of DPPH reduction was calculated using the following equation: Cytotoxicity assay. The cytotoxicity of TAE, AgNO 3 , and AgNPs was determined against peripheral blood mononuclear cells (PBMCs) as previously described 24  In vitro inhibitory activity. Minimum inhibitory concentration (MIC). To evaluate the inhibitory activity of TAE, AgNO 3 and AgNPs, MICs were determined using the broth microdilution method in accordance with the CLSI M27 document for yeasts 26,27 , CLSI M38 for filamentous fungi 28 and CLSI M07 for bacteria 29 . Dimethyl sulfoxide was used as the solvent (final concentration ≤ 1%).
MIC was determined by visual reading of growth inhibition at three endpoints using the following numerical scale: MIC-0 as the lowest concentration capable of inhibiting 100% of microbial growth; MIC-1 as the lowest concentration capable of inhibiting ≥ 80% of microbial growth compared to the growth control; and MIC-2 as the lowest concentration capable of inhibiting ≥ 50% of microbial growth compared to the growth control. The criteria for antimicrobial activity defined in a previous work were followed 17 . These criteria specify that plant extracts are considered active when MIC-2 ≤ 1024 μg/mL and inactive at higher values, while pure compounds are considered active when MIC-2 ≤ 256 μg/mL and inactive at higher values. In this work, AgNP and AgNO 3 solutions were considered as pure compounds and TAE as plant extract. The inhibitory controls used were: itraconazole (ITZ) (Sigma-Aldrich) for fungi, penicillin (PEN) (Sigma-Aldrich) for S. pyogenes, and gentamicin (GEN) (Sigma-Aldrich) for S. aureus and P. aeruginosa. For the control drugs, the endpoint values reported in the CLSI documents were used.
Minimum fungicidal concentration (MFC) and minimum bactericidal concentration (MBC). The MFC and MBC of TAE, AgNO 3 and AgNPs were determined according to the procedures established by other authors [30][31][32][33] , with modifications (Supplementary file). The MBC and MFC were defined as the lowest TAE, AgNO 3 or AgNP concentrations at which no colonies were observed.
MFC:MIC and MBC:MIC ratios were calculated for each isolate using the MIC-0 value. By extrapolating the conventional definition used for bacterial assays, in this work a compound was considered bactericidal or fungicidal if the ratio was ≤ 4, and bacteriostatic or fungistatic if the ratio was > 4 33,34 .
All assays were performed in duplicates. Range and mode values were determined. All MIC, MFC, and MBC data were expressed as μg LE/mL for TAE and μg silver/mL for AgNO 3 and AgNPs.

Results
Yield and phytochemical analysis. The process used to obtain the TAE showed a yield of 12%. On the other hand, the total phenolic and flavonoid contents of the extract were 155.9 ± 3.9 mg GAE/g LE and 20.97 ± 0.32 mg QE/g LE, respectively. from pale yellow to dark brown within a few min, indicating the formation of AgNP. The main features of the synthesized nanoparticles are shown in Fig. 1. The UV-visible spectrum showed a maximum absorption band at 410 nm, which corresponds to the local surface plasmon resonance absorption typical of nanoparticles 35 .
The TEM images showed non-agglomerated spherical nanoparticles. The sizes of at least 100 particles were measured using ImageJ software. The data were transferred to Origin software for statistical analysis, which revealed a diameter of 14 ± 2 nm. DLS analysis revealed strongly anionic nanoparticles with a PDI of 0.269, a hydrodynamic diameter of 36 ± 13 nm, and a zeta potential of -37 ± 6 mV. The FTIR spectrum of AgNPs showed peaks at about 1636, 2100 and 3300 cm -1 . Finally, EDX analysis revealed a strong signal of elemental silver with an optical absorption band peak in the range of 3 to 4 keV, which is typical for the absorption of metallic silver nanocrystallites 35 . In addition, other elemental signals were recorded in smaller amounts.
Antioxidant activity. The results of FRAP and DPPH assays of TAE, AgNO 3 and AgNPs are shown in Table 1.  Table 2.
The values obtained with the control drugs were similar to those obtained by other authors 27,[36][37][38][39] , which allowed us to confirm that we were working under standardized conditions.

Discussion
In recent years, the World Health Organization (WHO) has emphasized the importance of research and development of new antimicrobial drugs as morbidity and healthcare costs have increased due to the rise of resistant strains 40 . In response, nanoparticles and medicinal plants have emerged as promising alternatives for the treatment of skin infections 3,8,10,11,16,17,41 . In addition, the development of more efficient green synthesis methods has become an important focus of researchers. Biological synthesis of silver nanoparticles using plants has proven to be cost-efficient and environmentally friendly and is a valuable alternative for large-scale production 10,13,41 . Ayurveda, the traditional Indian medicine, was perhaps the first to use metallic herbal preparations. These preparations, often called bhasmas, are used to treat many diseases. Silver bhasma is a medicine known since the seventh century BC, obtained after exposure of metallic silver through a series of physico-chemical processes in the presence of medicinal herbs. Today, bhasmas are claimed to be biologically produced nanoparticles 42 .
Aqueous extract of A. australe (TAE). The selection of plant material for this study was based on the ancestral knowledge of indigenous people in northeastern Argentina about medicinal plants for the treatment of skin infections 14,43 . TAE was used as both reducing and capping agent for AgNP synthesis. The obtained yield of 12% from TAE was lower than the values of 17.1%, 22.8% and 28.5% obtained by other authors [44][45][46] . Several factors influence this performance, including the plant material used and the extraction methodology, but also the place and time of collection, the chemical nature of the metabolites present in the plant, and their affinity for the solvent 47 . It has been shown that growth conditions lead to changes in plant composition, which translates into changes in extraction yield 17 .
On the other hand, chemical analysis of TAE revealed the presence of phenolic compounds, mainly flavonoids. Similar to other reports, the presence of these compounds was also detected in tapekué extracts, which are associated with antimicrobial properties and are responsible for the reducing property of the extract [48][49][50] . Likewise, it is known that the flavonoids contained in the aqueous plant extracts are mainly responsible for the reduction of silver ions and the formation of stable nanoparticles 13,21,41 . Silver nanoparticles (AgNPs). In this work, the ionized chemical groups present in the extract allowed the rapid formation of AgNPs when TAE was mixed with AgNO 3 solution at pH 9.5. The color change indicated the presence of AgNPs in the solution.
TEM images were used to determine the size of the metallic core and DLS to determine the hydrodynamic size, i.e., the size of the nanoparticle in solution including the metallic core, the coating phytochemicals, and the solvent layer. TEM images confirmed the presence of AgNPs with sizes and shapes similar to those obtained by other authors with other plants 41 . DLS analysis revealed a monomodal population. Moreover, absolute values of zeta potential higher than 30 mV indicate stable nanoparticles, which could be related to the electrostatic repulsion of the anions present in the colloidal suspension 51 . These results explain the good stability of the synthesized AgNPs, which remained stable as colloids for more than 6 months.
EDX analysis verified the purity of the AgNPs and the presence of traces of other elements that could have originated from the biomolecules of TAE that were bound to the surface of the nanoparticles. FTIR analysis allowed us to identify the functional groups responsible for the reduction of silver ions. It revealed peaks at 1636, 2100 and 3300 cm -1 corresponding to the C = O functional groups of the amide (1640 cm -1 ), the C≡C stretch (2100 cm -1 ) and the N-H/O-H vibrational stretch of the amine (3370 cm -1 ), which are similar to the functional groups of AgNPs synthesized with quercetin 21 . In addition, other authors have shown that quercetin is present in tapekué extracts 48,52 . The results obtained in this work suggest that quercetin could be the main metabolite responsible for the reduction of silver ions to AgNPs, according to Jain et al. 21  www.nature.com/scientificreports/ negative zeta potential could be due to the hydroxyl groups of quercetin present on the surface of the nanoparticles, generating a negative charge on these particles. Antioxidant activity. In this work, TAE showed significant antioxidant activity. In contrast, AgNO 3 solution showed no activity, while AgNPs showed lower antioxidant activity (55%) than the TAE in both assays (DPPH and FRAP). This resulted in the inability to determine the EC 50 for AgNP and AgNO 3 solutions. The activity shown by AgNPs could be due to the phenolic compounds of TAE present on the surface of the nanoparticles, which act as antioxidants and capping agents.
The antioxidant property of a plant is given mainly by the composition and concentration of phenolic compounds and to a lesser extent by other components. Phenolic compounds are involved in the stabilization of free radicals and metals by acting as electron donors. The antioxidant activity of many polyphenols results essentially from the ease with which the hydroxyl group is donated to a free radical and the ability of the aromatic structure to carry an unpaired electron 54 . The results show that TAE is rich in phenolic compounds, which justifies its higher antioxidant activity. Therefore, TAE polyphenols played an important role both as silver ion reducing agents and AgNP capping agents.

Cytotoxic activity. The cytotoxicity of TAE, AgNO 3 and AgNP solutions was studied on PBMCs. PBMCs are cells of the immune system consisting of lymphocytes (T cells, B cells, NK cells) and monocytes. Cytotoxicity
on PBMCs represents the potential for immune responses and immunotoxicity and allows prediction of immune effects during preclinical safety evaluation. It also helps to understand the interactions between nanomaterials and cells of the immune system 55 .
Cytotoxicity assays of the components used for the synthesis of the nanoparticles showed that AgNO 3 was cytotoxic, while TAE was not cytotoxic to PBMCs at the concentrations evaluated. Moreover, the plant extract induced low cell proliferation compared to untreated cells. It has been reported that metabolites present in plants, mainly phenolic compounds, affect the nonspecific immune response by mainly increasing phagocytosis and proliferation of macrophages and neutrophils 56 . The non-toxicity property shown by TAE is of utmost importance in the context of AgNP production for medicinal applications, especially as a potential topical treatment for skin infections.
On the other hand, AgNP solution showed variations in cell viability as a function of dose and time. Cell viability reached values below 70% only at AgNP concentrations ≥ 16 µg/mL after 24 h of incubation, but was above 70% after 72 h of incubation, indicating the non-cytotoxicity of the synthesized nanoparticles at the tested concentrations 25 .
Since AgNO 3 was cytotoxic, while AgNPs and TAE were found to be non-cytotoxic and even TAE showed a slight proliferative effect, we can conclude that the reduction in cell viability shown by the synthesized AgNPs is related to the silver content, but TAE metabolites present on the surface of the nanoparticles reduce the cytotoxic effect and modulate the immune response. Therefore, the use of silver in humans may have toxic effects, but these effects can be attenuated when silver is incorporated as coated nanoparticles. In other words, a certain amount of silver in the form of silver ions is more toxic than the same amount in the form of nanoparticles coated with phytocompounds.
Our results are in agreement with those of Orta-García et al. 57 who showed that uncoated AgNPs induce significant cytotoxic effects on PBMCs at relatively low concentrations (< 5 μg/mL) and short exposure times (3 to 12 h), suggesting that the coating reduces the area of the active surface of AgNPs and thus the sites of interaction with cellular components. Antimicrobial activity. The antimicrobial activity of AgNPs, AgNO 3 and TAE, was tested against 298 fungi and bacteria causing SSTIs. Three endpoint values (MIC-0, MIC-1, and MIC-2) were used along with the determination of CFM, as there is still no consensus on which is the best predictor of the in vivo behavior of these compounds.
Although tapekué is used in traditional medicine to treat skin infections, TAE was inactive against the microorganisms tested in this work. However, considering the possibility of poor water solubility of the active compound, we cannot claim that the plant lacks this well-known traditional property. This position is in agreement with the results of Portillo et al. 45 , who showed that tapekué extracts obtained with organic solvents had higher antimicrobial activity than the aqueous extract. www.nature.com/scientificreports/ AgNO 3 showed antimicrobial activity but with higher MIC and CFM values than those of the synthesized nanoparticles, and its toxicity precludes it as an antimicrobial agent. Therefore, AgNP solution was a better antimicrobial agent than the components used for its synthesis (Fig. 3).
Variations in MIC and CFM values were observed between isolates of the same species and between species of the same genus. These variations highlight the importance of testing multiple isolates in the evaluation of antimicrobial activity.
According to the MFC values, the tested S. pyogenes strains and Malassezia species were most inhibited by AgNPs. M. globosa, the main causative agent of pityriasis versicolor and seborrheic dermatitis 5 , was the most susceptible microorganism to the synthesized nanoparticles. Moreover, methicillin-resistant S. aureus strains were inhibited by AgNPs. This indicates the potential use of AgNPs as antimicrobial agents against fungi and bacteria.
The values of MFC:MIC ratio obtained for AgNPs against the tested fungi were consistent with fungicidal activity. However, a different behavior was obtained against bacteria. According to the mode values obtained for MBC:MIC ratios, AgNPs showed bactericidal activity, but some strains of S. aureus and P. aeruginosa showed values ≥ 4 associated with bacteriostatic activity.
MIC-0 and MFC values greater than 1024 μg/mL were obtained for AgNO 3 against bacteria; therefore, the MFC:MIC ratio could not be determined. However, the MIC-2 values were determined. In addition, sustained growth was observed at AgNO 3 concentrations above the MIC-2 (trailing effect). This effect occurs with drugs that have bacteriostatic activity and is not normally observed with bactericidal drugs. Although AgNO 3 may have a bacteriostatic effect, we did not consider AgNO 3 itself as an antimicrobial agent because of its toxicity.
The use of less stringent endpoint readings, such as MIC-2 and MIC-1, is associated with drugs with fungistatic/bacteriostatic activity, whereas MIC-0 is generally associated with drugs with biocidal activity 28,29 .  www.nature.com/scientificreports/ Consequently, MIC-0 could be considered as the best endpoint reading for AgNPs against the tested fungi and bacteria.

Species (N) Compound
To confirm fungicidal or bactericidal activity, further studies, such as time-kill assays, are needed to correlate these results. In addition, it is important to note that the choice of endpoint reading for a clinical drug is based on the distribution of their MIC, pharmacokinetic and pharmacodynamic parameters, animal models, and therapeutic outcomes 58 .
The reasons for the excellent antimicrobial activity of AgNPs are not yet clear. However, it is known that the mode of action of AgNPs is the result of several simultaneous processes by which AgNPs can circumvent most microbiological resistance mechanisms that occur with classical antimicrobial agents. Therefore, new strategies against multidrug-resistant microorganisms are increasingly based on the search for synergistic effects between antimicrobial agents of clinical use and metallic nanoparticles 16,59 .

Conclusion
In summary, green synthesis using TAE proved to be a simple, environmentally friendly and economical method for the preparation of AgNPs with improved properties compared to their synthesis components.
AgNPs were not cytotoxic at the concentrations required to inhibit microbial growth, whereas AgNO 3 was cytotoxic. AgNPs were fungicidal and bactericidal, while AgNO 3 was fungicidal but bacteriostatic. TAE was inactive against the main microorganisms causing SSTIs and not cytotoxic to PBMCs.
The antimicrobial activity of AgNPs is due to the silver and not to the phytocompounds present on the surface of the nanoparticles. TAE plays a more important role in the formation of stable AgNPs and possibly as a modulator of cytotoxic and immune response.
The combination of traditional medicine with nanotechnology gives us a great opportunity to develop new antimicrobial agents. This work enabled us to rediscover a technology formerly known as bhasmas for the treatment and prevention of SSTIs.