News Reader.info

ABSTRACT The development of methods for obtaining new materials with antimicrobial properties, based on green chemistry principles has been a target of research over the past few years. The

present paper describes the phyto-mediated synthesis of metallic nano-architectures (gold and silver) _via_ an ethanolic extract of _Melissa officinalis_ L. (obtained by accelerated solvent

extraction). Different analytic methods were applied for the evaluation of the extract composition, as well as for the characterization of the phyto-synthesized materials. The

cytogenotoxicity of the synthesized materials was evaluated by _Allium cepa_ assay, while the antimicrobial activity was examined by applying both qualitative and quantitative methods. The

results demonstrate the synthesis of silver nanoparticles (average diameter 13 nm) and gold nanoparticles (diameter of ca. 10 nm); the bi-metallic nanoparticles proved to have a core-shell

flower-like structure, composed of smaller particles (ca. 8 nm). The Ag nanoparticles were found not active on nuclear DNA damage. The Au nanoparticles appeared nucleoprotective, but were

aggressive in generating clastogenic aberrations in _A. cepa_ root meristematic cells. Results of the antimicrobial assays show that silver nanoparticles were active against most of the

tested strains, as the lowest MIC value being obtained against _B. cereus_ (approx. 0.0015 mM). SIMILAR CONTENT BEING VIEWED BY OTHERS SUSTAINABLE PHYTO-FABRICATION OF SILVER NANOPARTICLES

USING _GMELINA ARBOREA_ EXHIBIT ANTIMICROBIAL AND BIOFILM INHIBITION ACTIVITY Article Open access 07 January 2022 GREEN SYNTHESIS OF _BRASSICA CARINATA_ MICROGREEN SILVER NANOPARTICLES,

CHARACTERIZATION, SAFETY ASSESSMENT, AND ANTIMICROBIAL ACTIVITIES Article Open access 26 November 2024 BIOENGINEERED SYNTHESIS OF PHYTOCHEMICAL-ADORNED GREEN SILVER OXIDE (AG2O)

NANOPARTICLES VIA _MENTHA PULEGIUM_ AND _FICUS CARICA_ EXTRACTS WITH HIGH ANTIOXIDANT, ANTIBACTERIAL, AND ANTIFUNGAL ACTIVITIES Article Open access 13 December 2022 INTRODUCTION The

conventional chemical methods for obtaining metallic nanoparticles with organic solvents, toxic chemicals and non-biodegradable stabilizing agents are no longer a trend for the future

research1. In order to obtain materials with controlled size and morphology, as well as with certain properties, research efforts are nowadays focused on the border areas of science,

combining knowledge from chemistry, biology and nanotechnology2,3,4,5. In addition, because both the number of severe infection diseases and the increase in resistance of most pathogens to

the available drugs are steadily increasing nowadays, another challenge in this area is the discovery of new materials that can be used as drugs3. Due to their abilities to damage the

bacterial DNA, cell membrane and critical enzymes, noble metal nanoparticles are considered to be promising antibacterial agents6,7. In a very close correlation with their content in

bioactive molecules, plant extracts have been successfully used to generate noble metal naoparticles8,9,10,11,12. Among the noble metals, silver is the most common and widely applied as an

antibacterial agent13. Gold nanoparticles can also be found in a wide range of applications, from imaging to drug delivery thermotherapy14. Regarding their cytotoxicity, silver nanoparticles

were found to have a relatively high toxicity15, gold nanoparticles are inert16, while the gold/silver bi-metallic nanoparticles have an intermediary biologic action17,18. _Melissa

officinalis_ L., commonly known as lemon balm, is a well-known medicinal plant, belonging to the Lamiaceae family, with a strong specific scent; it is commonly used in folk medicine of many

countries. Since ancient times, when plants were used as teas, decoctions or they were used as such, _Melissa officinalis_ L. was used for the treatment of mental diseases, cardiovascular

and respiratory problems, as memory enhancer, antidepressant, sleeping aid and antidote19,20. Recent studies have shown its antihyperlipidemic, anti-inflammatory and antioxidant

properties21,22,23. Besides the pharmaceutical use, other industrial applications of _M. officinalis_ are related to its use in apiculture (lemon balm being also known as _bee balm_ or

_honey balm_ 24, in food and liquors industries, in cosmetics or as ornaments20. Also, it has been revealed that the _M. officinalis_ leaf extract possesses antiviral properties due to the

content of phenolic acids, while its essential oil has antibacterial, antifungal, and antihistaminic activities because of constituents such as geranial, neral, (E)-anethole,

(E)-caryophyllene, and citronellal25,26,27. Literature data20 shows a high content of phytochemicals (known to be involved in the phytosynthesis of metallic nanoparticles, including terpenes

and phenolic compounds) in the _M. officinalis_ extracts; this, in turn, suggests a very high potential for the synthesis of metallic nanoparticles, an application of _M. officinalis_ not

sufficiently explored in the published literature data. The present manuscript describes the phyto-mediated synthesis of metallic nano-architectures (gold, silver and gold/silver bimetallic

nanoparticles) _via_ the ethanolic extract of _M. officinalis_ (obtained by accelerated solvent extraction). Analytic methods (XRD, XRF, UV-Vis, electron microscopy, chromatographic

techniques – GC-MS, HPLC) were applied for the evaluation of the extract composition, as well as for the characterization of the phyto-synthesized materials. The cytotoxicity of the

synthesized materials was evaluated in _Allium cepa_ assay, while the antimicrobial activity was evaluated by using both qualitative and quantitative methods. Qualitative screening was

performed by diffusion method (adapted from CLSI standard methods) and quantitative analysis was performed by binary serial micro-dilution method in liquid medium, in order to determine the

Minimal Inhibitory Concentration values. Microbial strains included in the present study belong to different genera and species (molds, yeast and bacteria strains). MATERIALS AND METHODS

VEGETAL MATERIAL, EXTRACTION PROCEDURE AND METAL NANOPARTICLE PHYTOSYNTHESIS Leaves of certified _M. officinalis_ L. were harvested at full matureness stage from the ecological crops of S.C.

HOFIGAL Export Import S.A (Bucharest, Romania). The plants were shade dried and grinded to powder. The extract was obtained using an accelerated solvent extractor ASE 350 (Dionex, Thermo

Scientific), equipped with a solvent controller unit. The dried plant samples (5 g) were mixed with silica beads and packed into 34 mL cells using ethanol as an extraction solvent. Prior to

solvent extraction an initial heat-up step was applied. Static extractions were performed and the cell was rinsed with the selected solvent; the solvent was purged from the cell using N2

gas. The extraction conditions were as follows: temperature: 100 °C; pressure: 1500 psi; static time: 5 min; static cycles: 1; flush volume: 60%; purge time: 120 s. The extraction procedure

was selected considering our previous results regarding nanoparticle synthesis that showed that more efficient extraction procedure leads to smaller and more active nanoparticles4. The

phytosynthesis of the metallic nanoparticles was accomplished using a source of metallic ions (AgNO3 and HAuCl4 × 3H2O; Merck KGaA, Germany) and ethanolic extract of _M. officinalis_ as

reducing agent. The general recipe involves the drop-wise addition of 10 mL extract in 40 mL 10−3M metal containing solution, under vigorous stirring. For the synthesis of bi-metallic

nanoparticles, the metals (Ag and Au) were in equal molar concentration (0.5 × 10−3 M). ANALYTICAL CHARACTERIZATION METHODS The obtained extract was characterized by means of GC-MS and HPLC,

in order to evaluate the extract phytochemical composition. The GC-MS analysis was carried out using a 7010 GC-MS triple Quad system (Agilent, USA). The system is equipped with a HP-5MS

Inert column with dimensions of 30 mm × 0.25 mm ID × 0.25 μm. The carrier gas used is helium with a flow of 1.0 mL min−1. The injector was operated at 250 °C and the oven temperature was

programmed as follows: 50 °C for 2 min, then gradually increased to 290 °C at 5 °C min−1. The identification of compounds was based on Flavor2 and NIST14 libraries as well as comparison of

their retention indexes. The HPLC analyses were performed on a Varian system (solvent delivery pumps Prostar 410, DAD detector Prostar 335 and autosampler Prostar 410); obtained data were

analyzed using Varian Workstation 6.3 software. The mobile phase consisted of two different solutions, solution A (water, acidified with 1% CH3COOH) and solution B (acetonitrile, acidified

with 1% CH3COOH). The flow rate was 1 mL min−1 with an injection volume of 10 μL. Calibration curves were constructed for each of the analyzed compounds (R2 > 0.999), using commercial

available standards: phenolic acids (chlorogenic acid, ferulic acid, gallic acid and rosmarinic acid), flavonoids (rutin, quercetin and apigenin) and phenylpropenes (_trans_-anethole; all

supplied by Merck KGaA, Germany). Both formation and stability of the nanoparticles were monitored by UV-VIS spectrometry, using a SPECORD 210 Plus UV-VIS Spectrometer, in the wavelength

range of 370-450 nm for silver, 500-600 nm for gold and 370-600 nm for gold/silver, respectively. The measurements were performed every four hours, for a period of six days. X-Ray

diffraction measurements were performed using a Rigaku SmartLab equipment, operating at 45 kV and 200 mA, Cu Kα radiation (1.54059 Å), parallel beam configuration (2θ/θ scan mode), from 25

to 90 2θ degrees; the components were identified using the Rigaku Data Analysis Software PDXL 2, database provided by ICDD. The X-Ray fluorescence measurements were performed in order to

evaluate the synthesis of nanoparticles, using an energy-dispersive spectrometer, EDXRF PW4025, type MiniPal 2 (PANalytical, B.V., The Netherlands), with a Si-PIN detector, at 20 kV and

automatic current intensity, measurement time 300 seconds, in Helium atmosphere. Aluminum and molybdenum filters were used for the analysis of silver and silver/gold nanoparticles,

respectively gold nanoparticles, in order to remove the Rh lines (from the X-ray tube) and other light elements. The Transmission Electron Microscopy images were recorded using a JEOL JEM

1400 electron microscope (80 kV), using copper grids. CYTOGENOTOXICITY TEST Bulbs of common onion _Allium cepa_ L. were purchased from a local market and used as indicator plant, being

carefully cleaned without destroying the primordial roots. Bulbs were selected for their phytosanitary status. Three bulbs were used in each experimental group depending on the types and

concentration of extracts, distilled water being used as a negative control. The toxicity assay was performed as a 96-h static exposure at 10% and 20% concentrations of ethanolic extract of

_M. officinalis_ (R), ethanolic extract of _M. officinalis_ with Ag nanoparticles, (S1), ethanolic extract of _M. officinalis_ with Au nanoparticles (S2), and ethanolic extract of _M.

officinalis_ with Ag and Au nanoparticles (S3). The onion bulbs were grown in distilled water for the first 48 h and then transferred for another 48 hours in containers containing test

extract and their nanoparticles, respectively. Root tips at a length of 10 mm were cut off and placed for 24 hours in 3:1 ratio glacial acetic acid: ethanol. 5 root tips for each sample were

heated 15 min at 60 °C in hydrochloric acid solution 1 N. Fixed and macerated root tips were then stained with aceto-orcein solution 1% for 15 min at 60 °C. The roots were placed on a slide

in a drop of acetic-water (glacial acetic acid 45%) and the terminal root tips were used to made slides via squash technique. The coded slides were sealed with a few coats of nail varnish,

and refrigerated. All slides were examined using Olympus CX-31 microscope, at 400× magnification. The microscopic analysis was focused on scoring of mitosis phases, chromosome aberrations

and nuclear abnormalities, per 3000 scored cells for each sample. The mitotic index was determined as number of mitotic cells among the total amount of scored cells being shown as a

percentage28. The number of cells at different mitotic stages (prophase, metaphase, anaphase and telophase) was calculated as percentage of the number of dividing cells. The frequency of

chromosome aberrations and nuclear abnormalities were calculated as percentage of the number of dividing cells in the appropriate mitotic stage and cell cycle phase, respectively. The data

were analyzed for statistical significance using analysis of variance (one-way ANOVA), and Duncan test was used to determine significant differences among means. Significant differences were

set at P = 0.05. Results are presented as the Mean ± standard error of more independent experiments. ANTIMICROBIAL TESTS The antimicrobial assay was performed using the following standard

strains: _Staphylococcus aureus_ (ATCC 25923), _Bacillus cereus_ (B1079), _Pseudomonas aeruginosa_ (ATCC 27853), _Escherichia coli_ (ATCC 25922), _Candida albicans_ (ATCC 10231), _C.

parapsilosis_ (ATCC 22109), _C. glabrata_ (ATCC 64677), _C. krusei_ (ATCC 14243), _Aspergillus niger_ (strain isolated from soil), _Trichoderma viride_ (strain isolated from soil). An

adapted spot diffusion method was followed for the qualitative screening29. For the experiments, yeast suspensions of 1.5 × 108 CFU mL−1 (0.5 McFarland density) were obtained from cultures

developed on yeast extract peptone dextrose solid media. For the filamentous fungi experiments, the density of the spore suspension in phosphate buffer saline (PBS), supplemented with 1 µl 

mL−1 Tween 20 was 0.4–5 × 104 CFU mL−1. Petri dishes with YPG (for yeasts) or PDA (potato dextrose agar) (for filamentous fungi) were seeded with fungal inoculums, and an amount of 10 mL

solution of each compound of 10 mg mL−1 concentration was spotted after seeding. DMSO was used as negative control. After inoculation, the plates were initially kept at room temperature (in

order to ensure the equal diffusion of the compound in the medium) and then incubated at 37 °C (for clinical strains) or room temperature (for reference strains) for 24–48 hrs (yeasts) or

5–7 days (for filamentous fungi). The positive results were read as the occurrence of an inhibition zone of fungal growth around the spot. For the quantitative screening, a binary serial

microdilution technique using 96-well microtitre plates was used to obtain the MIC values of the tested compounds against the various microorganisms30. The quantitative assay was performed

in YPG broth medium for yeasts, Muller Hinton broth for bacteria, and RPMI for filamentous fungi, according with “Performance Standard for Antimicrobial Susceptibility Testing”31 and

“Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi; Approved Standard”32. For establishing the MIC (minimum inhibitory concentration) values, a

microdilution method performed in nutritive broth was used. The sterile broth was added in sterile 96 well plates and binary dilutions of each tested compound were performed in a final

volume of 150 μL. After preparing the binary dilutions, 15 μL of microbial suspension adjusted to an optical density of 0.5 McFarland (1,5 × 108 CFU mL−1) were added in each well. The MIC

values were established by visual analysis and spectrophotometric measurement (absorbance reading at 600 nm). Each experiment was performed in triplicate and repeated on at least three

separate occasions. For the assessment of adherence on the inert substratum, 96-multi well plastic plates containing binary dilutions of the tested compounds in a final volume of 200 µL were

inoculated with 50 µL microbial suspensions of 107 CFU mL−1 prepared in sterile saline and inoculated and incubated for 24 h at 37 °C. After incubation, the wells were discarded, washed

three times by PBS and the bacterial cells adhered to the plastic walls were stained by 1% violet crystal solution for 15 min. The dyed biofilm was thereafter fixed by cold methanol for 5 

minutes and re-suspended by 33% acetic acid solution. The absorbance at 490 nm of the blue suspension was measured using BIOTEK SYNERGY-HTX ELISA multi-mode reader, the obtained values being

proportional with the number of the adhered microbial cells. RESULTS Several chromatographic techniques (Table 1 – results of the GC-MS analysis, chromatogram presented in Supplementary

Figure S1; Table 2 – results of HPLC analysis, HPLC chromatogram presented in Supplementary Figure S2) were applied in order to analyze the phytochemical composition of the obtained extract.

In order to evaluate the synthesis and the stability of the mono and bi-metallic architectures, the UV-Vis spectra were recorded from the reaction medium as a function of a time (every four

hours, over six days period, in order to evaluate their stability) (Fig. 1). The analysis focused in the wavelength range of 370–450 nm for silver, 500–600 nm for gold and 370–600 nm for

gold/silver. Peaks were recorded at 417 nm for silver, 537 nm for gold and two peaks (at 413 nm and 541 nm) for the bimetallic nanoparticles, respectively. For the XRD and XRF analyses, the

samples were centrifuged for 60 minutes at 6000 rpm and the sediments containing nanoparticles were dried (for the X-ray diffraction directly on the sample plates and for X-ray fluorescence

on a cellulosic support). The XRF analysis identified the dried sediments as having as major constituent silver, gold, and silver and gold, respectively (Fig. 2). The XRD results (Fig. 3)

confirm the synthesis of the nanoparticles. In the diffractograms could be discerned the specific peaks of: silver (S, PDF card no. 01-071-4613) at 38.13, 44.31 and 77.13 degrees,

corresponding to (111), (200) and, respectively, (311) planes; gold (G – 03-065-2870) at 38.16, 44.38, 64.53 and 77.75 degrees, corresponding to (111), (200), (220) and, respectively, (311)

planes; for the bi-metallic nanoparticles, the peaks corresponding to the nanoparticles are found at 38.31, 44.45, 64.75, 77.01, 77.68 and 81.71 degrees. In the silver and gold/silver

nanoparticles, peaks specific to silver oxides (Ag2O – 01-078-5867 and Ag3O4 – 03-065-9750) can also be observed, due to the oxidation of silver nanoparticles during sample preparation.

Transmission electron microscopy was used to visualize the size and shape of obtained nano-architectures (Fig. 4). The dimensions of the nanoparticles (determined from over 250 measurements)

had an average of 13 nm for silver nanoparticles, 10 nm for gold nanoparticles and 100 nm for the bi-metallic nano-architectures, respectively. Considering the mutagenicity assay, all

tested samples decreased the mitotic index when compared to the control (Fig. 5). The results demonstrated a nearly lethal effect of the extract (encoded as R). The decrease effect of R

indicates a correlation with the extract concentration, since the MI of R 20% was almost null. The phytosynthesized nanoparticles inhibited the mitodepressive effect of the _M. officinalis_

extract. The activity of nanoparticles/nanoarchitectures showed a dose-response correlation, the highest concentration (20%) being more efficient to prevent the mitodepressive effect of R.

The most effective in improving de _A. cepa_ MI was S3 (bi-metallic nanoparticles) 20%, for which the MI was not significantly different compared to control. Distribution of various mitotic

phases showed significant dose-dependent differences from control and _R_ regulated by the type of nanoparticles/nanoarchitectures. It has been noticed therefore that S2 (gold nanoparticles)

20% and S3 10% induced a significant accumulation of cells in the prophase compared to other samples (Supplementary Figure S3). This increased proportion of cells blocked in prophase may be

attributed to inhibition of the formation or functioning of the spindle fibers or extension of cell cycle G2 stage where DNA repair occurs. Table 3 indicates the results of mutagenicity

test obtained by the _A. cepa_ bioassay. Various kinds of aberrations induced by the extracts/nanoparticles are shown in Supplementary Figure S7. The most frequent clastogenic aberrations

were C-mitosis, ana-telophase bridges, alongside with a low frequency of micronuclei, vagrants and laggards. Multipolarity and stickiness were very rarely noticed. Bridges and stickiness are

due to chromatin dysfunction, and micronuclei, C-mitosis and laggards are due to spindle failure33. It worth being noticed here that different samples described by ethanolic extract _M.

officinalis_ with or without nanoparticles and/or nanoarchitectures induced specific chromosomal aberrations, with a characteristic and significant preponderance. The results of the

antimicrobial qualitative screening (Table 4, images presented in Supplementary Figures S8 to S13) revealed that silver nanoparticles are active against most of the tested strains (except

for _Candida glabrata, Aspergillus niger, Trichoderma viride_). For _Bacillus cereus_, not only the silver nanoparticles showed a strong effect, but also the extract itself. Gold

nanoparticles exhibited activity against a limited number of strains (_B. cereus –_ most probably due to the extract effect_, Pseudomonas aeruginosa, C. krusei_). The gold/silver

nanoparticles usually exhibit an intermediary effect (except for _Staphylococcus aureus_), similar with the values obtained for silver nanoparticles. It can also be observed that the

combination between silver and gold (in the form of core-shell architectures) decreased the antimicrobial effect of silver nanoparticles, expressed by smaller inhibition zone diameters and

higher MIC values (Tables 4 and 5). The results of the quantitative assay (determination of the minimum inhibitory concentration) are presented in Table 5, while their graphic representation

is presented in Supplementary Figures S14 to S21. The results of the evaluation of biofilm eradication properties are presented in Table 6, while their graphic representations are shown in

Supplementary Figures S22 to S28. DISCUSSIONS The synthesis of noble metal nanoparticles using either microorganisms or plant extracts has emerged in the last decades as an alternative

approach to the classical (either physical or chemical) methods, due to their several advantages: they are simple (usually one-pot), cost-effective, give high yields, yet non-toxic and

therefore environmentally friendly34. Although it has the advantages of green chemistry approach, the use of microorganisms (such as bacteria, yeasts, actinomycetes or fungi) has certain

limitations, such as the difficulty of implementing on a large scale and the need for maintaining cell cultures. Thus, the use of plant extracts is distinguished by other methods in that the

method is simple and scalable35. As a first step of the analytic protocol developed for the current study, the extract was subjected to chromatographic analyses. By means of the GC-MS

analysis performed, 67 peaks were identified using the databases, from a total of 75 peaks found to be present in the extract. The area of the 8 unidentified peaks totalizes ca. 3.33%.

Following the identification of the volatile components, the HPLC analyses were performed in order to quantify different compounds as markers of _M. officinalis_ extract36,37,38. The

obtained HPLC results are consistent with the literature data regarding _M. officinalis_ extracts. For example, Arceusz and Wesolowski36, in a study covering 19 lemon balm samples from 12

Polish manufacturers, achieved values ranging from 0.15 to 42.3 mg/g for rosmarinic acid, from 0.006 to 1.59 mg/g for ferulic acid, from 0.01 to 0.33 mg/g for chlorogenic acid and from under

the detection limit to 0.067 mg/g for gallic acid; Arceusz _et al_.39 identified rutin (from under the detection limit to 2.34 mg/g) and quercetin (0.17–27 mg/g) in different types of

extract obtained from lemon balm. Upon the addition of the reducing agent (extract) a color change can be rapidly observed. The change of solution color is an indicator of the nanoparticles

formation: a yellowish color is specific for small size silver nanoparticles; a ruby-red color is specific to gold nanoparticles, while the bi-metallic nanoparticles are characterized by a

dark brownish color40. The UV-Vis spectra present characteristic absorption peak for silver nanoparticles (Fig. 1A) at 417 nm (suggesting particle dimensions under 30 nm) and gold

nanoparticles (Fig. 1B) at 537 nm (suggesting particle dimensions around 50 nm). The spectrum in Fig. 1C presents a decrease of the specific peak of gold nanoparticles (accompanied by a

slight shift, to 541 nm) and an increase of the silver peak (with a slight shift to 413 nm). The peaks variation obtained for silver and gold plasmon bands suggest core-shell architecture,

with a coating of silver around the gold nanoparticles37. The exact pathway involved in the reduction of metallic ions to metallic nanoparticles is still under debate, several mechanisms

being proposed by different authors focusing on different active compounds, including polyphenols41, flavonoids42 and other bio-active compounds43. Figure 6 presents both the proposed

mechanism and the involved chemical reactions for metal reduction using plant extracts. In our opinion, the assignment of a particular active substance as the main reducing agent is

hazardous, as, most probably, the combination of various biomolecules found in natural extracts acts both as a reducing and stabilizing agent, in a synergic manner43. Moreover, we consider

that the most probable biomolecules involved in the reduction of metallic salts to nanoparticles are the phenolic compounds, as several authors proposed35,41. Different studies stressed out

that plant metabolites like polyphenols are natural reducing agents41 and capping agent with contribution to metallic nanoparticles stabilization. This effect was most emphasized when

different solvent extraction was used and the quantities of polyphenols had significant variation, hence the effectiveness in the formation and stabilization of nanoparticles also varied44.

In present study, the HPLC analysis was carried out in order to link the formation of NPs to some of the metabolites found in _Melissa officinalis_ L. extract. Besides usual metabolites

(gallic acid, quercetin, rutin, etc.) which can be responsible for NPs synthesis45 it was found that rosmarinic acid is present in large amount in studied extract. This compound is very

effective in NPs synthesis according to literature data46. It was found that the rosmarinic acid molecules are able to generate and stabilize the gold and silver nanoparticles. Rutin was

also found in large quantities in plant extract and therefore can act as a reducing and capping agent in the process. The GC-MS analyses, complementary to the HPLC determinations, identified

some metabolites (numbers 13, 15, 38, 44, 48, 57, 62, 65, 66 and 75 from Table 1) in the _Melissa officinalis_ L. extract, known to present antifouling capacity, which could contribute to

stabilization of the nanoparticles47. The XRD analysis, besides confirmation of the phytosynthesis of the nanoparticles, also provides information regarding the particle dimensions: the

crystallite size, determined by the Debye-Scherrer equation, showed silver nanoparticles with an average dimension of 14 nm and gold nanoparticles with an average dimension of 10 nm. Similar

dimensions were obtained for the bi-metallic nanoparticles (ca. 8.5 nm). The investigation of the samples using transmission electron microscopy (as presented in Fig. 4) confirmed the

findings of XRD. Uniform silver nanoparticles could be observed, with an average dimension around 13 nm (determined from over 250 measurements), in a relatively narrow range of dimensions

(Fig. 4A). The analysis of gold nanoparticles revealed smaller nanoparticles (around 10 nm, determined from over 250 measurements), but very inhomogeneous in shape (Fig. 4B). Besides

spherical nanoparticles different shaped particles, such as triangular, hexagonal, rhombic, etc. could be observed. A very interesting case is observed for the bi-metallic nanoparticles: at

a first glance (Fig. 4C), it appears that very big particles were formed, with dimensions around 100 nm, with some dispersed smaller particles. A closer look (Fig. 4D) reveals that the large

particles are actually clusters of very small nanoparticles (around 8 nm), that tend to aggregate in a flower-like core-shell configuration, as other authors previously reported48,49. The

_Allium cepa_ test system is widely used as an ideal bioindicator for the first screening of genotoxicity, as a suitable indicator for the evaluation of clastogenic and/or aneugenic effects

of a potential genotoxic agent50, providing similar results with _in vitro_ animal test systems51. The reduction of the mitotic index was usually associated with a significant increase in

the frequency of cells in metaphase, when comparing with control (Supplementary Figure S4). Samples defined by the biogenic Ag and Au nanoparticles at a concentration of 10% proved to be

valuable statmokinetic agent. Significant incidence of metaphase in the root tips of _A. cepa_ L. incubated with S3 10%, suggested a lower mitostimulatory effect of these nanoarchitectures.

An increased number of anaphase associated to a reduced number of telophase was observed for the slides defined by ethanolic extract of _M. officinalis_, irrespective of the tested

concentration. However, statistical analysis revealed no significant differences between the samples S1, S2, S3 for their effect on the rate of anaphase and telophase (Supplementary Figures 

S5 and S6). Formation of micronuclei in a significant low frequency was characteristic for those meristematic root cells of _A. cepa_ incubated in R and S1, suggesting a minor toxicity of R

along with a lack of chemoprotective activity of Ag nanoparticles. According to Carvalho _et al_.52 ethanolic and aqueous extracts of _M. officinalis_ induced a low frequency of

micronucleated cells in CF-1 male mice, but not significant enough to indicate mutagenic and genotoxic activities. Moreover, the study of Kamdem _et al_.53 did not reveal either the

cytotoxic nor genotoxic effects of _M. officinalis_ extract to human leukocytes. In this study, the formation of micronuclei might be due to either the 48 hours contact of meristematic root

tips with ethanol used as solvent for extract preparation or to inappropriate concentrations. Microscopic analysis revealed a protective effect of _M. officinalis_ extracts containing Au

nanoparticles or Ag/Au nano-architectures on nuclear DNA damage, expressed in absence of micronuclei. Meristematic root cells of _A. cepa_ exposed to S2 and S3 for 48 h were affected by

turbagenic changes, such as C-mitosis and bridges. Significant high frequency of C-mitosis observed in root tip cells incubated in particular with S3 10%, recommend Ag/Au nano-architecture

as a potential antitumor agent. From the presented antimicrobial assays, very small MIC values are noticed for silver nanoparticles solution obtained for most of the tested strains (except

_Aspergillus niger_ strain). _A. niger_ is known to be the most resistant pathogen among fungi. The resistance of this strain to AgNPs may result either from the activity of some chemical

components which are produced by _A. niger_ and inhibits the bioactivity mechanisms of the silver ions or from the structure particularity of the cell wall54. Nevertheless, details about the

specific resistance mechanism need to be further investigated. The antimicrobial activity of silver nanoparticles has been reported against fungus, yeast, Gram-negative and Gram-positive

bacteria55,56. Silver nanoparticles (AgNP) are known to possess antimicrobial properties, but the mechanisms regarding their microbial toxicity are not fully known. One of the hypotheses is

that silver nanoparticles can cause cell lysis or inhibit cell growth, thereby causing structural changes in the cell membrane like the permeability of the cell membrane and consequently

death of the cell56,57. The results of Hsueh _et al_.58 confirmed that AgNP toxicity is likely mediated by the released Ag+ ions from AgNPs, which penetrate bacterial cells and are

subsequently intracellularly oxidized to Ag2O. These findings provide conclusive evidence for the role of Ag+ ions in AgNP microbial toxicity58. When comparing our results with literature

data, it should be noticed that the MIC values are better even than the ones obtained for silver nanoparticles obtained _via_ chemical route: Kim59 _et al_. obtained MIC values of 0.033 mM

and 0.0033 mM against _S. aureus_ and, respectively, _E. coli_, while Guzman _et al_.60 obtained MIC values ranging from 14.38 to 259 mg/L (for _S. aureus_ and _E. coli_) and from 6.74 to

215.74 mg/L for _P. aeruginosa_, using silver nanoparticles synthesized _via_ citrate method. However, when comparing results regarding antimicrobial properties of phytosynthesized

nanomaterials with the properties of nanoparticles obtained using classical chemical or physical methods, we must consider that “green” nanoparticles have the advantage of a functionalized

surface due to presence of bioactive molecules, such as organic ligands, proteins, polysaccharides, and polyatomic alcohols61. Regarding the assessment of adherence on the inert substratum

assay, it is worth to notice that the silver nanoparticles appeared active against all studied strains, while the gold nanoparticles showed no effect on the adherence on inert substratum.

The bi-metallic nanoparticles are active against all studied strains, with MCBE (minimal concentration for biofilm eradication) values higher than the ones obtained for silver nanoparticles.

In natural sites, microorganisms can adhere to biotic or abiotic surfaces, generating biofilms62. Because of the medical and industrial implication, this phenomenon has been studied in many

different environments, generating new control strategies that follow the use of different anti-biofilm strategies like bio-solutions (enzymes, antimicrobial peptides, quorum sensing

molecules, plant extracts) or nanoparticles63,64. In their research, Kalishwaralal _et al_.65 treated _Pseudomonas aeruginosa_ and _Staphylococcus epidermidis_ with silver nanoparticles over

24 h, obtaining more than 95% inhibition in biofilm formation. Also, another recent study showed that AgNPs are also effective against _Mycobacterium spp_. biofilms66. Silver nanoparticles

express synergistic activity with ampicillin, kanamycin, streptomycin or vancomycin against _E. coli_ and _P. Aeruginosa_ 67. Other studies have revealed synergy of AgNPs with compounds

other than antibiotics. As an example, Ammons _et al_.68 showed that a silver wound dressing combined with the immune molecule lactoferrin and the rare sugar-alcohol xylitol, reduced biofilm

viability more effectively than standard silver hydrogel. Our results are in agreement with previous studies, the phytosynthesized nanoparticles inhibiting the adherence capacity of the

majority of tested strains, the MCBE values being very low (under 0.05 mM). CONCLUSIONS By utilizing an eco-friendly route, three types of nanoparticles were obtained, _i.e_. silver and gold

nanoparticles, and silver/gold nano-architectures. The analytical results proved the successful phytosynthesis, offering information regarding their size and morphology. The obtained

nanomaterials were tested for their mutagenicity and antimicrobial properties. Mitosis was inhibited by ethanolic extract of _M. officinalis_. The Ag nanoparticles were not active on nuclear

DNA damage. The Au nanoparticles appeared nucleoprotective, but were aggressive in generating clastogenic aberrations in _A. cepa_ root meristematic cells. The evaluation of the

antimicrobial properties of the phytosynthesized nanoparticles showed that silver nanoparticles are active against most of the tested strains (especially for adherence on inert substratum).

From both assays, it can be concluded that the bi-metallic nanoparticles seem to be the most promising materials for further applications. REFERENCES * Nadagouda, M. N., Hoag, G., Collins,

J. & Varma, R. S. Green synthesis of Au nanostructures at room temperature using biodegradable plant surfactants. _Cryst. Growth Des._ 9, 4979–4983 (2009). Article  CAS  Google Scholar 

* Kharisov, B. I. _et al_. Iron-containing nanomaterials: synthesis, properties, and environmental applications. _RSC Adv._ 2, 9325–9358 (2012). Article  CAS  Google Scholar  * Tahir, K. _et

al_. Facile and green synthesis of phytochemicals capped platinum nanoparticles and _in vitro_ their superior antibacterial activity. _J. Photochem. Photobiol._ 166, 246–251 (2017). Article

  CAS  Google Scholar  * Ortan, A. _et al_. Innovative phytosynthesized silver nanoarchitectures with enhanced antifungal and antioxidant properties. _Appl. Surf. Sci._ 358, 540–548 (2015).

Article  ADS  CAS  Google Scholar  * Sutan, N. A., Fierascu, I., Fierascu, R. C., Manolescu, D. S. & Soare, L. C. Comparative analytical characterization and _in vitro_ cytogenotoxic

activity evaluation of _Asplenium scolopendrium_ L. leaves and rhizome extracts prior to and after Ag nanoparticles phytosynthesis. _Ind. Crop. Prod._ 83, 379–386 (2016). Article  CAS 

Google Scholar  * Chamakura, K., Perez-Ballestero, R., Luo, Z., Bashir, S. & Liu, J. Comparison of bactericidal activities of silver nanoparticles with common chemical disinfectants.

_Colloids Surf. B Biointerfaces_ 84, 88–96 (2011). Article  CAS  PubMed  Google Scholar  * Morones, J. R. _et al_. The bactericidal effect of silver nanoparticles. _Nanotechnol._ 16,

2346–2353 (2005). Article  ADS  CAS  Google Scholar  * Ferraris, S. _et al_. _In situ_ reduction of antibacterial silver ions to metallic silver nanoparticles on bioactive glasses

functionalized with polyphenols. _Appl. Surf. Sci._ 396, 461–470 (2017). Article  ADS  CAS  Google Scholar  * Panigrahi, S., Kundu, S., Ghosh, S. K., Nath, S. & Pal, T. General method of

synthesis for metal nanoparticles. _J. Nanopart. Res._ 6, 411–414 (2004). Article  ADS  CAS  Google Scholar  * Sun, Q. _et al_. Green synthesis of silver nanoparticles using tea leaf

extract and evaluation of their stability and antibacterial activity. _Colloids Surf. A_ 444, 226–231 (2014). Article  CAS  Google Scholar  * Sen, I. K., Maity, K. & Islam, S. S. Green

synthesis of gold nanoparticles using a glucan of an edible mushroom and study of catalytic activity. _Carbohydr. Polym._ 91, 518–528 (2013). Article  CAS  PubMed  Google Scholar  *

Goudarzi, M., Mir, N., Mousavi-Kamazani, M., Bagheri, S. & Salavati-Niasar, M. Biosynthesis and characterization of silver nanoparticles prepared from two novel natural precursors by

facile thermal decomposition methods. _Sci. Rep._ 6, 32539 (2016). Article  ADS  CAS  PubMed  PubMed Central  Google Scholar  * Wijnhoven, S. W. P. _et al_. Nano-silver - a review of

available data and knowledge gaps in human and environmental risk assessment. _Nanotoxicol._ 3, 109–138 (2009). Article  CAS  Google Scholar  * Barcikowski, S. & Mafune, F. Trends and

current topics in the field of laser ablation and nanoparticle generation in liquids. _J. Phys. Chem. C_ 115, 4985–4985 (2011). Article  CAS  Google Scholar  * Kittler, S. _et al_. The

influence of proteins on the dispersability and cell-biological activity of silver nanoparticles. _J. Mater. Chem._ 20, 512–518 (2010). Article  CAS  Google Scholar  * Mahl, D., Greulich,

C., Meyer-Zaika, W., Köller, M. & Epple, M. Gold nanoparticles: dispersability in biological media and cell-biological effect. _J. Mater. Chem._ 20, 6176–6181 (2010). Article  CAS 

Google Scholar  * Li, T. _et al_. Comparative toxicity study of Ag, Au, and Ag-Au bimetallic nanoparticles on _Daphnia magna_. _Anal. Bioanal. Chem._ 398, 689–700 (2010). Article  ADS  CAS 

PubMed  Google Scholar  * Mahl, D. _et al_. Silver, gold, and alloyed silver–gold nanoparticles: characterization and comparative cell-biologic action. _J. Nanopart. Res._ 14, 1153 (2012).

Article  ADS  Google Scholar  * Dastmalchi, K. _et al_. Chemical composition and _in vitro_ antioxidative activity of a lemon balm (_Melissa officinalis L_.) extract. _LWT – Food Sci.

Technol._ 41, 391–400 (2008). Article  CAS  Google Scholar  * Shakeri, A., Sahebkar, A. & Javadi, B. Melissa officinalis L. – A review of its traditional uses, phytochemistry and

pharmacology. _J. Ethnopharmacol._ 188, 204–228 (2016). Article  CAS  PubMed  Google Scholar  * Weidner, C. _et al_. Lemon balm extract causes potent antihyperglycemic and antihyperlipidemic

effects in insulin-resistant obese mice. _Mol. Nutr. Food Res._ 58, 903–907 (2014). Article  CAS  PubMed  Google Scholar  * Birdane, Y., Buyukokuroglu, M., Birdane, F., Cemek, M. &

Yavuz, H. Anti-inflammatory and antinociceptive effects of _Melissa officinalis L_. in rodents. _Rev. Med. Vet._ 158, 75–81 (2007). Google Scholar  * Mimica-Dukic, N., Bozin, B., Sokovic, M.

& Simin, N. Antimicrobial and antioxidant activities of _Melissa officinalis_ L. (_Lamiaceae_) essential oil. _J. Agric. Food Chem._ 52, 2485–2489 (2004). Article  CAS  PubMed  Google

Scholar  * Yilmaz, O. Traditional honey beekeeping in Turkey. _J. Vet. Adv._ 6, 1317–1324 (2016). Article  Google Scholar  * Stanojevic, D., Comic, L. J., Stefanovic, O. & Solujic

Sukdolak, S. _In vitro_ synergistic antibacterial activity of _Melissa officinalis_ L. and some preservatives. _Span. J. Agric. Res._ 8, 109–115 (2010). Article  Google Scholar  * Sharopov,

F. S. _et al_. Composition and bioactivity of the essential oil of _Melissa officinalis_ L. growing wild in Tajikistan. _Int. J. Trad. Nat. Med._ 2, 86–96 (2013). CAS  Google Scholar  *

Rabbani, M., Etemadifar, Z., Karamifard, F. & Borhani, M. S. Assessment of the antimicrobial activity of _Melissa officinalis_ and _Lawsonia inermis_ extracts against some bacterial

pathogens. _Comp. Clin. Path._ 25, 59–65 (2016). Article  Google Scholar  * Tedesco, S. B. & Laughinghouse, H. D. Bioindicator of genotoxicity: the _Allium cepa_ test, In: _Environmental

Contamination_, (ed. Srivastava, J. K.), 137–156 (InTech Publisher, 2012). * Badiceanu, C. D. _et al_. Antimicrobial activity of some new 2-thiophene carboxylic acid thioureides. _Rom.

Biotechnol. Lett._ 15, 5545–5551 (2010). CAS  Google Scholar  * Ditu, L. M. _et al_. _In vitro_ assessment of the antimicrobial activity of new N-acyl-thiourea derivatives. _Roum. Arch.

Microbiol. Immunol._ 69, 41–47 (2010). CAS  PubMed  Google Scholar  * CLSI, _Performance Standards for Antimicrobial Susceptibility Testing_, 27th edition, CLSI supplement M100. Wayne P.A.:

Clinical & Laboratory Standards Institute, USA, 2017. * CLSI. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi; Approved Standard- Second

edition. CLSI document M38-A2. Wayne P.A.: Clinical & Laboratory Standards Institute, USA, 2008. * Mesi, A. & Kopliku, D. Cytotoxic and genotoxic potency screening of two peticides

on _Allium cepa_ L. _Procedia Technol._ 8, 19–26 (2013). Article  Google Scholar  * Zhang, Y., Cheng, X., Zhang, Y., Xue, X. & Fu, Y. Biosynthesis of silver nanoparticles at room

temperature using aqueous aloe leaf extract and antibacterial properties. _Colloids Surf. A: Physicochem. Eng. Aspects_ 423, 63–38 (2013). Article  CAS  Google Scholar  * AbdelHamid, A. A.,

Al-Ghobashy, M. A., Fawzy, M., Mohamed, M. B. & Abdel-Mottaleb, M. M. Phytosynthesis of Au, Ag and Au-Ag bimetallic nanoparticles using aqueous extract of sago pondweed (_Potamogeton

pectinatus_ L.). _ACS Sustainable Chem. Eng._ 1(12), 1520–1529 (2013). Article  CAS  Google Scholar  * Arceusz, A. & Wesolowski, M. Quality consistency evaluation of _Melissa

officinalis_ L. commercial herbs by HPLC fingerprint and quantitation of selected phenolic acids. _J. Pharm. Biomed. Anal._ 83, 215–220 (2013). Article  CAS  PubMed  Google Scholar  *

Hanganu, D. _et al_. The study of some polyphenolic compounds from _Melissa officinalis_ L. (_Lamiaceae_). Rev. _Med. Chir. Soc. Med. Nat. Iasi_ 112, 525–529 (2008). Google Scholar  *

Sharopov, F. S., Zhang, H., Wink, M. & Setzer, W. N. Aromatic Medicinal Plants from Tajikistan (CentralAsia). _Medicines_ 2, 28–46 (2015). Article  CAS  PubMed  PubMed Central  Google

Scholar  * Arceusz, A., Wesolowski, M. & Ulewicz-Magulska, B. Flavonoids and phenolic acids in methanolic extracts, infusions and tinctures from commercial samples of lemon balm. _Nat.

Prod. Commun._ 10, 977–981 (2015). PubMed  Google Scholar  * Ahmed, S. _et al_. Evaluation of Gold, Silver and Silver–Gold (bimetallic) nanoparticles as radiosensitizers for radiation

therapy in cancer treatment. _Cancer Oncol. Res._ 4, 42–51 (2016). Google Scholar  * Barua, S. _et al_. One step preparation of biocompatible, antimicrobial reduced graphene oxide-silver

nanohybrid as a topical antimicrobial agent. _RSC Adv._ 4, 9777–9783 (2014). Article  CAS  Google Scholar  * Terenteva, E. A., Apyari, V. V., Dmitrienko, S. G. & Zolotov, Yu. A.

Formation of plasmonic silver nanoparticles by flavonoid reduction: A comparative study and application for determination of these substances. _Spectrochim. Acta A: Mol. Biomol. Spectrosc_

151, 89–95 (2015). Article  CAS  Google Scholar  * Ahmed, S., Ahmad, M., Swami, B. L. & Ikram, S. Plants extract mediated synthesis of silver nanoparticles for antimicrobial

applications: a green expertise. _J. Adv. Res._ 7, 17–28 (2016). Article  CAS  PubMed  Google Scholar  * Kumar, V., Yadav, S. C. & Yadav, S. K. Syzygium cumini leaf and seed extract

mediated biosynthesis of silver nanoparticles and their characterization. _J. Chem. Technol. Biotechnol._ 85, 1301–1309 (2010). Article  CAS  Google Scholar  * Kumar, H. A. K. _et al_.

Antimicrobial and antioxidant activities of _Mimusops elengi_ seed extract mediated isotropic silver nanoparticles. _Spectrochim. Acta Mol. Biomol. Spectrosc._ 130, 13–18 (2014). Article 

ADS  Google Scholar  * Sarkar, A., Shukla, S. P. & Adhikari, S. Synthesis, stabilisation and surface modification of gold and silver nanoparticles by rosmarinic acid and its analogues.

_Int. J. Nanotechnol._ 7, 1027–1037 (2010). Article  ADS  CAS  Google Scholar  * Thangaraju, N., Venkatalakshmi, R. P., Chinnasamy, A. & Kannaiyan, P. Synthesis of silver nanoparticles

and the antibacterial and anticancer activities of the crude extract of _Sargassum polycystum_ C. _Agardh. Nano Biomed. Eng._ 4, 89–94 (2012). Google Scholar  * Lu, L., Burkey, G.,

Halaciuga, I. & Goia, D. V. Core–shell gold/silver nanoparticles: Synthesis and optical properties. _J. Colloid Interface Sci._ 392, 90–95 (2013). Article  ADS  CAS  PubMed  Google

Scholar  * Zhang, G., Li, J., Shen, A. & Hua, J. Synthesis of size-tunable chitosan encapsulated gold–silver nanoflowers and their application in SERS imaging of living cells. _Phys.

Chem. Chem. Phys._ 17, 21261–21267 (2015). Article  CAS  PubMed  Google Scholar  * Firbas, P. & Amon, T. Chromosome damage studies in the onion plant Allium cepa L. _Caryologia_ 67,

25–35 (2014). Article  Google Scholar  * Teixeira, R. O., Camparoto, M. L., Mantovani, M. S. & Vicentini, V. E. P. Assessment of two medicinal plants _Psidium guajava_ L. and _Achillea

millefolium_ L., in _in vitro_ and in _vivo assays_. _Genet. Mol. Biol._ 26, 551–555 (2003). Article  Google Scholar  * Carvalho, N. C. _et al_. Evaluation of the genotoxic and antigenotoxic

potential of _Melissa officinalis_ in mice. _Genet. Mol. Biol._ 34, 290–297 (2013). Article  Google Scholar  * Kamden, J. P. _et al_. Antioxidant activity, genotoxicity and cytotoxicity

evaluation of lemon balm (_Melissa officinalis_ L.) ethanolic extract: Its potential role in neuroprotection. _Ind. Crop. Prod._ 51, 26–34 (2013). Article  Google Scholar  * Rawashdeh, R.

& Haik, Y. Antibacterial mechanisms of metallic nanoparticles: a review. _Dyn. Biochem. Process. Biotechnol. Mol. Biol._ 3, 12–20 (2009). Google Scholar  * Kim, J. S. _et al_.

Antimicrobial effects of silver nanoparticles. _Nanomedicine_ 3, 95–101 (2007). Article  CAS  PubMed  Google Scholar  * Sondi, I. & Salopek-Sondi, B. Silver nanoparticles as

antimicrobial agent: a case study on E. coli as a model for Gram-negative bacteria. _J. Colloid Interface Sci._ 275, 177–182 (2004). Article  ADS  CAS  PubMed  Google Scholar  * Prabhu, S.

& Poulose, E. K. Silver nanoparticles: mechanism of antimicrobial action, synthesis, medical applications, and toxicity effects. _Int. Nano Lett._ 2, 1–10 (2012). Article  Google Scholar

  * Hsueh, Y. H. _et al_. The antimicrobial properties of silver nanoparticles in _Bacillus subtilis_ are mediated by released Ag+ ions. _PLoS One_ 10, doi:10.1371/journal.pone.0144306

(2015). * Kim, J. S. _et al_. Antimicrobial effects of silver nanoparticles. _Nanomed. Nanotech. Biol. Med._ 3, 95–101 (2007). Article  CAS  Google Scholar  * Guzman, M. G., Dille, J. &

Godet, S. Synthesis of silver nanoparticles by chemical reduction method and their antibacterial activity. _Int. J. Chem. Biomol. Eng._ 2, 104–111 (2009). CAS  Google Scholar  * Bjarnsholt,

T. The role of bacterial biofilms in chronic infections. _APMIS Suppl._ 136, 1–51 (2013). Article  Google Scholar  * Makarov, V. V. _et al_. “Green” nanotechnologies: synthesis of metal

nanoparticles using plants. _Acta Naturae_ 6, 35–44 (2014). CAS  PubMed  PubMed Central  Google Scholar  * Simoes, M., Simoes, L. & Vieira, M. J. A review of current and emergent biofilm

control strategies. _Food Sci. Technol._ 43, 573–583 (2010). CAS  Google Scholar  * de Faria, A. F. _et al_. Anti-adhesion and antibacterial activity of silver nanoparticles supported on

graphene oxide sheets. _Colloids Surf. B: Biointerfaces_ 113, 115–124 (2014). Article  PubMed  Google Scholar  * Kalishwaralal, K., BarathManiKanth, S., Pandian, S. R. K., Deepak, V. &

Gurunathan, S. Silver nanoparticles impede the biofilm formation by _Pseudomonas aeruginosa_ and _Staphylococcus epidermidis_. _Colloids Surf. B: Biointerfaces_ 79, 340–344 (2010). Article 

CAS  PubMed  Google Scholar  * Islam, M. S., Larimer, C., Ojha, A. & Nettleship, I. Antimycobacterial efficacy of silver nanoparticles as deposited on porous membrane filters. _Mater.

Sci. Eng. C_ 33, 4575–4581 (2013). Article  CAS  Google Scholar  * Wolska, K. I., Grzes, K. & Kurek, A. Synergy between novel antimicrobials and conventional antibiotics or bacteriocins.

_Pol. J. Microbiol._ 61, 95–104 (2012). CAS  PubMed  Google Scholar  * Ammons, M. C. B., Ward, L. S. & James, G. A. Anti-biofilm efficacy of a lactoferrin/xylitol wound hydrogel used in

combination with silver wound dressings. _Int. Wound J._ 8, 268–273 (2011). Article  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS The authors gratefully acknowledge the

support obtained through the project SusMAPWaste, SMIS 104323, Contract No. 89/09.09.2016, from the Operational Program Competitiveness 2014-2020, project co-financed from the European

Regional Development Fund. AUTHOR INFORMATION Author notes * Irina Fierascu, Milen I. Georgiev, Alina Ortan and Radu Claudiu Fierascu contributed equally to this work. AUTHORS AND

AFFILIATIONS * University of Agronomic Science and Veterinary Medicine, 59 Marasti Blvd, 011464, Bucharest, Romania Irina Fierascu, Milen I. Georgiev, Alina Ortan, Radu Claudiu Fierascu, 

Sorin Marius Avramescu & Daniela Ionescu * The National Institute for Research & Development in Chemistry and Petrochemistry-ICECHIM, 202 Spl. Independentei, 060021, Bucharest,

Romania Irina Fierascu & Radu Claudiu Fierascu * Laboratory of Applied Biotechnologies, Institute of Microbiology, Bulgarian Academy of Sciences, 139 Ruski Boulevard, 4000, Plovdiv,

Bulgaria Milen I. Georgiev * University of Bucharest, Faculty of Chemistry, PROTMED Research center, 36-46M. Kogalniceanu Blvd., 050107, Bucharest, Romania Sorin Marius Avramescu * S.C.

HOFIGAL EXPORT IMPORT S.A., 2 Intrarea Serelor, 042124, sector 4, Bucharest, Romania Daniela Ionescu * University of Pitesti, Faculty of Science, 1 Targu din Vale Str, 110040, Pitesti,

Romania Anca Sutan * Romanian Academy, Institute of Biology – Bucharest, 296 Spl. Independentei, 060031, Bucharest, Romania Alexandru Brinzan * University of Bucharest, Microbiology

Department, 1-3 Aleea Portocalelor, 060101, Bucharest, Romania Lia Mara Ditu Authors * Irina Fierascu View author publications You can also search for this author inPubMed Google Scholar *

Milen I. Georgiev View author publications You can also search for this author inPubMed Google Scholar * Alina Ortan View author publications You can also search for this author inPubMed 

Google Scholar * Radu Claudiu Fierascu View author publications You can also search for this author inPubMed Google Scholar * Sorin Marius Avramescu View author publications You can also

search for this author inPubMed Google Scholar * Daniela Ionescu View author publications You can also search for this author inPubMed Google Scholar * Anca Sutan View author publications

You can also search for this author inPubMed Google Scholar * Alexandru Brinzan View author publications You can also search for this author inPubMed Google Scholar * Lia Mara Ditu View

author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS I.F., M.I.G., A.O. and R.C.F designed and performed the research. A.O. and S.M.A performed the

extraction. D.I. provided the vegetal material and data performed the study regarding _M. officinalis_. S.M.A. performed the chromatographic analyses. I.F. and R.C.F. performed the XRD and

EDXRF analyses. A.S. performed the cytotoxicity assay. A.B. performed the TEM analyses. L.M.D. performed the antimicrobial assays. I.F., M.I.G., A.O. and R.C.F. analyzed data. I.F., M.I.G.,

A.O. and R.C.F. wrote the manuscript. All authors reviewed the manuscript. CORRESPONDING AUTHORS Correspondence to Alina Ortan or Radu Claudiu Fierascu. ETHICS DECLARATIONS COMPETING

INTERESTS The authors declare that they have no competing interests. ADDITIONAL INFORMATION PUBLISHER'S NOTE: Springer Nature remains neutral with regard to jurisdictional claims in

published maps and institutional affiliations. ELECTRONIC SUPPLEMENTARY MATERIAL SUPPLEMENTARY MATERIAL RIGHTS AND PERMISSIONS OPEN ACCESS This article is licensed under a Creative Commons

Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original

author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the

article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use

is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit

http://creativecommons.org/licenses/by/4.0/. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Fierascu, I., Georgiev, M.I., Ortan, A. _et al._ Phyto-mediated metallic

nano-architectures _via Melissa officinalis_ L._:_ synthesis, characterization and biological properties. _Sci Rep_ 7, 12428 (2017). https://doi.org/10.1038/s41598-017-12804-7 Download

citation * Received: 23 June 2017 * Accepted: 14 September 2017 * Published: 29 September 2017 * DOI: https://doi.org/10.1038/s41598-017-12804-7 SHARE THIS ARTICLE Anyone you share the

following link with will be able to read this content: Get shareable link Sorry, a shareable link is not currently available for this article. Copy to clipboard Provided by the Springer

Nature SharedIt content-sharing initiative