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Anti-bacterial/fungal and anti-cancer performance
of green synthesized Ag nanoparticles using summer
savory extract
Shahram Ahmadi
a
, Mohammad Fazilati
a
, Seyyed Mojtaba Mousavi
b
and
Habibollah Nazem
a
a
Department of Science, Payame Noor University (PNU), Tehran, Iran;
b
Department of Chemical
Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan
ABSTRACT
Nowadays, the expansion of nanoparticles as antimicrobial and
anticancer agents is one of the most exciting methods for cancer
therapy and microbial diseases that can vanquish the problems of
drug resistance. In this research, the biosynthesis of silver nano-
particles (AgNPs) was stated using Summer Savory extract.
Moreover, these AgNPs by UV-Vis spectroscopy, FTIR, XRD, FESEM,
EDX, PSA, and zeta potential analysis were characterized, and by
antimicrobial and anti-cancerous performance was evaluated. The
maximum absorption peak at 472 nm by UV-Visible spectra con-
firmed the formation of Ag nanoparticles. The results obtained by
FT-IR determined the presence of organic compounds such as
amines, alkenes, alcohols, phenols, which performed significant
roles in the fixating of these nanoparticles. The results obtained
from FESEM, EDX, PSA, and Zeta Potential Analysis revealed that
synthesized AgNPs had a hexagonal structure with a size ranging
from 10 to 100 nm. The antimicrobial properties of the synthesized
AgNPs were examined by Minimum Inhibitory Concentration,
Minimum Bactericidal Concentration, and Minimum Fungal
Concentration against five microbial strains included Klebsiella
pneumonia,Escherichia coli,Enterococcus faecalis,Staphylococcus
aureus,andSaccharomyces cerevisiae. The results demonstrated
that biosynthesized AgNPs had the most antimicrobial activity,
especially against E. Coli. The anti-cancerous results of these nano-
particles through MTT assay demonstrated exceptional anticancer
effects against K-562 and MCF-7 human cancer cell lines with IC50
values 50 and 200 mg/ml, respectively.
ARTICLE HISTORY
Received 29 April 2020
Accepted 14 July 2020
KEYWORDS
Summer savory; Ag
nanoparticle; green
synthesis; anti-cancer;
anti-bacterial/fungal
1. Introduction
Nanotechnology is the manipulation of material on an atomic, molecular, and supra-
molecular measurement, and it also is a field of applied sciences and technology that
CONTACT Shahram Ahmadi sh.ahmadi@pnu.ac.ir Department of Science, Payame Noor University (PNU),
Tehran 19569, Iran.
ß2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
JOURNAL OF EXPERIMENTAL NANOSCIENCE
2020, VOL. 15, NO. 1, 363–380
https://doi.org/10.1080/17458080.2020.1799981
covers a wide range of fields. The main issue is the inhibition of matter or devices less
than one micrometer in diameter, usually about 1 to 1000 nanometers [1–3]. There are
different ways to produce nanoparticles, such as chemical, physical, and green synthesis
methods. The green bioassays are more useful than other methods because of their non-
toxicity, higher stability, and more valuable produced nanoparticles [3–5]. Recent amazing
progress in Nanotechnology and Nanoscience changed the methods of diagnosing, treat-
ing, and preventing various illnesses of humans [6]. AgNPs is one of the most biotic and
fascinating nanomaterials among metallic NPs. AgNPs are involved in many medical and
therapeutic applications. AgNPs, due to their unique properties, have been remarkably
effective in various fields of science, including biology, biomedicine, chemical engineering,
environment, and many other fields of science. It also plays a significant role in nanotech-
nology and nanoscience, particularly in nanomedicine [7–9]. Silver NPs have antimicro-
bial, antitumor, antioxidant, and anti-inflammatory properties, which have provided
extensive open possibilities for the usage of these nanoparticles in biomedicine. [7–10]. A
schematic diagram representing various applications of AgNPs has been provided in
Figure 1.
The capability of microorganisms and organic compounds to reduce metal ions and
stabilize them into nanoparticles forms the foundation of green synthesis of these nano-
particles. The green synthesis of different types of nanoparticles using a diverse array of
plant extracts has been reported [10–12]. One of the most essential herbs in Iran, which
is native to different regions of the country, is Satureja sp [13–16]. Satureja is a plant that
is widely used in various food products. This herb is a one-year-old herbaceous plant that
belongs to the family of mint (Figure 2).
This plant is resistant to various weather conditions and has many medicinal proper-
ties, such as antioxidant, antifungal, antibacterial, and antiseptic properties. Moreover, it
Figure 1. Various usages of Ag NPs.
364 S. AHMADI ET AL.
is an LDL cholesterol-lowering agent rich in oily and protein substances [16–19].
Interestingly, from the 305 articles searched in the Scopus database between the years
1989 to 2018, 209 papers had focused on the summer savory effects [19]. In this research,
considering the mentioned therapeutic properties regarding the Summer Savory plant and
the role and importance of AgNPs, the aqueous extract of Summer Savory was applied
for the generation of AgNPs. The morphological, chemical and optical properties of syn-
thesized AgNPs were investigated and characterized using a UV-Vis, FT-IR, EDX, XRD,
FE-SEM, PSA, and Zeta Potential assays. Also, the Anti-Microbial performance of the
green synthesized AgNPs was evaluated by MIC, MBC, and MFC methods against
selected bacterial. The anticancerous performance of the synthesized AgNPs was analyzed
through MTT colorimetric assay with exposing nanoparticles against K562 and MCF-7
human cancer cell lines.
Figure 2. Summer Savory (Satureja hortensis) plant.
JOURNAL OF EXPERIMENTAL NANOSCIENCE 365
2. Experimental section
2.1. Collection and investigation of summer savory plant
In this regard, Summer Savory plant was collected from Shiraz and Gachsaran
(Southwestern, Iran). The genus of the collected plant was identified and confirmed by
comparing it with herbarium specimen existing in the Department of Agriculture, Shiraz
University (Iran). Plant samples were placed in sterile plastic bags, and they were brought
to the laboratory for further processes.
2.2. Extraction of aqueous extract of SS and fabrication of Ag NP
Some chemical materials, such as the silver nitrate (AgNO
3
), were purchased from Merck
Co (Germany). After washing the aerial parts of SS with deionized water, they were dried
at room temperature for two weeks. Afterward, 20 grams of the dried plant of Summer
Savory were powdered and poured into a glass beaker. Then, 100 mL of deionized water
was added to a pure powdered plant and boiled for 20 min at 70 C. In the following, the
compound was cooled, and the aqueous extract using Whatman filter paper No.1 was fil-
tered and stored at 4 C in a dark bottle. Herein, for the biosynthesis of AgNPs, 5 mL of
the obtained aqueous extract by sterile pipette was poured into a glass jar. Then, 95 ml of
an aqueous solution of 1 mM AgNO
3
was added. Finally, for future studies, this solution
was placed in the room for 24 h at 37 C.
2.3. Characterization
The formation, morphological and optical structure, particle size, constituent elements
and functional group of biosynthesized silver nanoparticles, the nature of synthetic nano-
particles were investigated. The characterization of AgNPs was used using routine analysis
such as Uv-Vis spectroscopy (øambda35, PERKIN Elmer, Germany), FTIR spectrometer
(Bruker model Tensor II, Germany), X-ray diffractometer (XRD-7000, Shimadzu, Japan),
FESEM (TESCAN-MIRA3, Czech Republic’s), EDX (Tescan model S Max detector
MIRA3), PSA (Malvern model MS1002, England) and zeta potential Analyzer (Horiba
model SZ- 100, Japan).
2.4. Antibacterial activity
2.4.1. Reference strains and preparation of test samples
In this research, five microbial strains, including four bacterial strains (two g negative and
two g positive) and one fungi spice, were used for antimicrobial performance. Microbial
Strains were purchase from the Iranian Biological Resources Center (IBRC). five microbial
strains included Klebsiella pneumonia ATCC 7881, Escherichia coli ATCC 33876,
Enterococcus faecalis ATCC 6057, Staphylococcus aureus ATCC 6538, and Saccharomyces
cerevisiae ATCC 9763. For the antimicrobial experiment of synthesized nanoparticles, the
stock solution of silver nanoparticles had first to be prepared, and then the required con-
centrations were prepared. Silver nanoparticles were first dissolved in 5% Dimethyl
Sulfoxide (DMSO) solution; thus the stock solution with a concentration of 20 mg L
1
was produced. Finally, different concentrations (7.8 ،15.62 ،31.25 ،62.5 ،125 ،250،500 ،
1000 mgmL
1
) of this stock solution were provided for antimicrobial testing.
366 S. AHMADI ET AL.
2.4.2. Appraisement of antimicrobial of Ag NPs and summer savory extract
In this section, the antimicrobial activity of Ag NPs and Summer Savory extract was com-
pared and determined against five selected microbial strains using Minimum Inhibitory
Concentration (MIC), Minimum Bactericidal Concentration (MBC) and Minimum Fungal
Concentration (MFC) methods. Herein, the related experiments were done corresponding
to the dilution Instructions presented by the Clinical and Laboratory Standard Institute
(CLSI) [20]. The lowermost concentration of antimicrobial agents that prevented about 90
percent of the bacteria and fungi growth, respectively, after 24 h incubation was compared
with the negative control of dimethyl sulfoxide (DMSO), and were defined as MIC, MBC,
and MFC. To experiment antimicrobial of Summer Savory extract, sequential dilutions
(7.8 ،15.62 ،31.25 ،62.5 ،125 ،250،500 ،1000 (mg/ml)) of Summer Savory extract were
made ready and poured into a sterile 96-well microplate containing 90 microliters of
M€
uller Hinton Broth (MHB) medium. In the following, the chosen microbial strains in
the MHB attaining the turbidity of nearly 5 108 CFU mL
1
were cultured. In the next
part, the microbial suspensions were accumulated for inoculation via diluting them to
yield 5 106 CFU/mL. Then, 10 mL of each prepared microbial suspension was insemi-
nated into microplates. Finally, after 24 h incubation, the optical density of microplates
was measured using a microplate reader (Hyperion, model MPR4 Plus) at a wavelength
of 600 nm. However, to investigate the effects of synthesized silver nanoparticles, first ser-
ial dilutions of the AgNPs (Similar to Summer Savory Extract) and control groups
(Normal saline was used as negative control, and neomycin was considered positive con-
trol) were provided in 96-well microplates containing BHI medium. In the following, the
bacteria and fungi suspension with the turbidity standard of 0.5 McFarland was added to
microplates. Then, these microplates were incubated for 24 h at 37 C. Finally, the optical
density of plates, as stated above, was measured by a microplate reader (Hyperion, model
MPR4 Plus) at 600 nm. For both synthesized compounds, this method was repeated three
times. For the measurement of MBC and MFC (the lowermost concentration from the
composition that leads off to no growth of bacteria and fungi, as MBC and MFC were
considered, respectively.) experiment, each five selected microbial strains were cultured
24 h in BHI medium, afterward for each one of microorganisms a stock with a concentra-
tion of 10
5
–10
6
CFU/mL was gathered. Then, 90 lL of diluted concentrations of composi-
tions (7.8 to 1000 lg/mL) was added to a microplate containing 90 lg/ml BHI medium.
Afterward, 10 lg/mL of any micro-suspension was added to each cell, and then
Microplates were incubated within 24 h at a temperature of 37 C. In the following, 10 lL
of each prepared microbial suspension was added to a recently prepared BHI medium. To
assess the microbial activity of each compound, microplates were incubated for 24 h
at 37 C.
2.5. Anticancer performance of silver nanoparticles and summer savory extract
The human cancer cell lines of K562 and MCF-7 were brought from the cancer center of
Shiraz medical university, Iran. Various concentrations of green synthesized AgNPs, and
Summer Savory extract were applied to study the anticancer performance on these human
cell lines via exploring 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide
(MTT) method. 10
6
cells of K562 and MCF-7 cell lines were grown adequately in
RPMI1640 on 96-well microplate and incubated with 2% carbon dioxide for 24 h at 37 C.
The microplates studied the cytotoxic performance of provided materials on the cell lines
and were incubated with various concentrations (1, 10, 50, 100, 200, and 500 mg/mL) of
AgNPs and Summer Savory extract. After completing the incubation time, micro-plates
JOURNAL OF EXPERIMENTAL NANOSCIENCE 367
were entirely washed with phosphate buffer saline (PBS). In the following, micro-plate
with MTT solution was treated and again, as in the previous step, it was incubated. Then,
to dissolve the crystals formed via MTT solution, the micro-plates were treated with
100 ml of Dimethyl sulfoxide and were put on the rotary shaker so that the color of dis-
solved crystals could be expanded. The intensity of the color reaction was measured using
a microplate reader at a wavelength of 570 nm to study the percentage of survivability of
cell lines by the formula presented below.
Cells survivability %
ðÞ¼AbsorbanceofAgNPstreatedcell
absorbance of Untreated cells 100
The mentioned formula was used to determine the IC50 value of cells. In this test,
Dimethyl Sulfoxide and was applied as control samples.
2.6. Statistical analysis
In this study, data analysis was performed using SPSS IBM computer software. To evalu-
ate the results of biological experiments, the data were displayed in each group as mean-
± standard deviation, and the significant difference between the mean groups was
analyzed by One-Way Analysis of Variance (ANOVA), and then Tukey test was applied.
In all tests, an average of 3 measurements was used for each group. The significant level
in all statistical tests was considered less than 0.05 (p<0.05). Excel software (2010) was
also used to prepare the graphs.
3. Results and discussion
3.1. Characterization of synthesized nanoparticles
3.1.1. Visual observation and UV-Vis analysis
Metal ions such as Silver, Iron, Copper, and Gold due to the property of Surface Plasmon
Resonance (SPR) of the optical properties, are unique [21]. In this section, the formation
of AgNPs was evaluated by a color change and UV-Vis assay. The change in reaction
color from light yellow to dark brown after the addition of AgNO3 to the plant extract
indicated the formation of AgNPs (Figure 3). The generation of these nanoparticles hap-
pened due to the presence of active molecules in the Summer Savory plant extract, which
reduced the Ag metal ions present in the reaction from Ag
þ
to Ag
0
. Several articles have
reported this color change due to SPR stimulation [15,22,23]. A characteristic peak for
AgNPs using UV-Visible Spectrophotometric analysis was obtained at around k472 nm.
The obtained spectrum, according to numerous papers and lectures, was related to the
formation of AgNPs. According to these documents, the formation of the spectrum of
AgNPs has been reported in the wavelength range between 400 to 500 nm [15,24].
3.1.2. Fourier transform infrared (FT-IR) spectroscopy
The purpose of the FT-IR experiment is the recognition of the possible functional groups
of bio-molecules that are involved in capping, effective stabilization, and reduction of
recently synthesized AgNPs [15,25]. FT-IR analysis of AgNPs was shown in Table 1 and
Figure 4. In this figure, several distinct absorption peaks can be seen in the3405.88,
2427.61, 2359.46, 2100.99, 1788.81, 1635.08, 1338.55, 1048.96, and 832.54 cm
1
areas. The
peaks at 3405.88 cm
1
in the synthesized samples were related to the O-H stretching of
phenols/alcohols [15,25] and N-H group of amide A [15,26]. The peak at 2427.61 cm
1
368 S. AHMADI ET AL.
was related to the C-H group of Alkanes [15,25,26]. The existing peaks at 2359.46 cm
1
to 2100.99 cm
1
were related to CN, and CC stretches in Aromatic/Aliphatic com-
pounds [25–28]. The peaks at 1788.81 cm
1
regions of 1635.08 cm
1
were related to the
C¼C stretch of Aromatic compositions [29]. Another document has determined that
these peaks are related to Amid I (C ¼O stretch) of proteins [27]. The existing peak at
1338.55 cm
1
indicated the CH3 group of Sugars (carbohydrates) [30]. The existing peak
at 1048.96 cm
1
regions was related to the CO group of in-plane bending of carboxylic
acids, alcohols, esters, ethers, and alkanes [15]. Eventually, the existing peak at
832.54 cm
1
area was related to ¼CH stretch of Aromatic bi-cyclic monoterpenes, accord-
ing to previous reports [15,31]. FT-IR Spectroscopy results have demonstrated the pres-
ence of various biomolecules that play a major role in the stabilizing of Ag NPs.
3.1.3. X-Ray Diffraction (XRD) spectrometer
The crystalline structure of synthesized silver nanoparticles was confirmed using the XRD
method. The results obtained from XRD assay for these nanoparticles were shown in
Figure 5 and Table 2. Obtained results were demonstrated by the Hexagonal structure of
AgNPs, showing that Ag was facing centered cubic (fcc). The X-ray patterns revealed that
Table 1. FT-IR analysis of synthesized AgNPs.
The peak for synthesized
Ag NPs (cm
1
) Functional Group Compositions present
3405.88 O-H stretch
N-H stretch
Phenols, alcohols
amide A
2427.61 C-H stretch Alkanes
2359.46 to 2100.99 CN stretch
CC stretch
Aromatic or Aliphatic
1788.81 to 1635.08 C ¼C stretch
C¼O stretch
Aromatic
proteins
1338.55 CH3 group carbohydrates
1048.96 CO group Carboxylic acids, alcohols, esters,
ethers, and alkanes
832.54 ¼CH stretch Aromatic bi-cyclic monoterpenes
Figure 3. UV-Vis spectra analysis of synthesized AgNPs using Summer Savory extract.
JOURNAL OF EXPERIMENTAL NANOSCIENCE 369
silver nanoparticles had different peaks at 2hof about 29.448, 35.679, 38.395, 48.702,
55.649, 60.231, and 61.105 that could be attributed to the 221, 042, 111, 243, 003,030 and
442 orientations, respectively. These orientations were matched to the fcc phase of Ag
0
ions. The formation of these peaks was due to the organic compounds present in the
summer savory plant extract, which were responsible for reducing Ag ions and fixation of
synthesized nanoparticles [32]. These results were compared with synthesized AgNPs
using various plant extracts, reported by other researchers reported a similar crystalline
structure for AgNPs [10,33,34].
The average crystalline size of the AgNPs was measured by Equation (1) (Debye-
Scherrer equation):
D¼Ƙk
bcosh(1)
In this Equation, D is the average crystalline size of synthesized nanoparticles, k is a
geometric factor (0.9), kis the wavelength of the X-Ray radiation source and bis the
angular FWHM (full-width at half maximum) of the XRD peak at the diffraction angle h
[35]. In this research, the average crystallite size of the biosynthesized silver nanoparticles
was 69.2 nm using Summer Savory extract.
3.1.4. Field emission scanning electron microscope (FESEM)
The microstructural and morphological analysis of synthesized silver nanoparticles was carried
out by FE-SEM experiments. FE-SEM image from Ag nanoparticles is shown in Figure 6.As
observed from the figure, all particles were shown hexagonal and spherical crystal structure,
and the diameter of AgNPs was in the range of 10 100 nm. The aggregation of Ag NPs was
observed in FE-SEM Images, which could be due to the evaporation and removal of solvent
used during sample preparation. Solvent removal causes electrostatic forces to bring Ag NPs
Figure 4. FT-IR spectra Analysis for synthesized AgNPs and Summer Savory Extract.
370 S. AHMADI ET AL.
closer together and aggregated [36]. This factor has probably led to variations in the size of
synthesized silver nanoparticles. According to the results obtained from the past researches,
the silver nanoparticles were derived from different plant extracts, depending on the chemical
composition of the plant extract and the concentration, and pH of the media can have vari-
ous forms such as hexagonal, spherical and triangular [15,36–38].
3.1.5. Particle size analyzer and zeta potential (PSA and Zeta)
The amount of size and the potential size of the synthesized silver nanoparticles by
Summer savory extract were calculated using the PSA and Zeta method (Figure 7).
According to the obtained diagrams, the size of silver nanoparticles using PSA assay was
in the range of 70 nm, and the zeta potential of these particles was about 15.8 mV. The
negative potential value supports properties including good colloidal nature, long term
stability, and high dispersity of green synthesized Ag nanoparticles [39–41].
3.1.6. EDX spectroscopy
The purpose of the EDX experiment was to identify quantitive and qualitative elements in
the reaction that may be involved in the synthesis of AgNPs. The EDX spectra from Ag
NPs (Figure 8) also revealed and confirmed the establishment of Ag NPs. According to
Figure 5. XRD Analysis of synthesized Ag NPs by Summer Savory extract for determination of Ag crystals.
Table 2. Output data of XRD assay from synthesized AgNPs using summer savory.
No
Peak
Pos.
[2Th.]
Pos. [2Th.]
of Ref.
plane
(hkl)
crystalline
structure
chemical
formula
Crystallite
Size only [Å]
MicroStrain
only [%] Ref.
1 29.5721 29.448 2 2 1 Hexagonal Mg73.44Ag18.09 695.6756 0.217112 96-810-3420
2 35.6104 35.679 0 4 2 Hexagonal Mg73.44Ag18.09 705.902 0.17858 96-810-3420
3 38.7394 38.395 1 1 1 Hexagonal O2.00Ag6.00 60.33804 1.926199 96-710-9279
4 48.5404 48.702 2 4 3 Hexagonal Mg73.44Ag18.09 340.8293 0.275149 96-810-3420
5 55.7198 55.649 0 0 3 Hexagonal O2.00Ag6.00 172.5947 0.47792 96-710-9279
6 60.0606 60.231 0 3 0 Hexagonal O2.00Ag6.00 436.1658 0.176591 96-710-9279
7 61.1347 61.105 4 4 2 Hexagonal Mg73.44Ag18.09 560.5623 0.135217 96-810-3420
JOURNAL OF EXPERIMENTAL NANOSCIENCE 371
Figure 6. FE-SEM image of green synthesized AgNPs using summer Savory extract.
Figure 7. (a) Zeta potential graph of Synthesized AgNPs, (b) Particle size analysis diagram of Green Synthesized Silver
nanoparticles by Summer savory extract.
372 S. AHMADI ET AL.
the results obtained from past researches, AgNPs due to their SRP has shown a typic
optical absorption peak approximately at 3 keV [21,42]. EDX quantification of synthe-
sized AgNPs (Table 3) provided atomic percentages of 18.93% C, 42.42% O, 21.64% N,
7.70% Ag, 8.60% Na, 0.04% Cl, 0.11% Ca, 0.42% Mg and 0.14% K. According to the table,
it can be stated that the presence of Silver and Oxygen elements in the composition indi-
cated the formation of AgNPs, and also, the synthesized nanoparticles were in the form
of silver oxide, Ag2O [43,44]. Also, the presence of carbon and oxygen elements in the
composition can be related to the presence of polyphenolic compounds or any carbon or
oxygenated composition found in the Summer Savory extract [45]. The presence of nitro-
gen in the compound can be explained how that this ion most likely results from the
decomposition of excess AgNO
3
in a composition released during the reaction and syn-
thesis of AgNPs. Other elements found in the composition, such as sodium, potassium,
magnesium, and chlorine, are elements of the Summer Savory extract that play a critical
role in enzymatic activities of reaction such as neutralizing the organic anions in the plant
extract and stabilizing the pH between 7 and 8. In the meantime, the role of potassium
and sodium is more important than other elements [46]. Therefore, this is one of the
major benefits of using plant extract to synthesized nanoparticles than using chemicals.
3.2. Antimicrobial performance of synthesized compounds
Silver nanoparticles, due to their high antimicrobial properties, are one of the most
important metal nanoparticles in the treatment of microbial diseases. Today, the use of
Figure 8. EDX spectrum of green synthesized Ag NPs.
Table 3. Outcome of EDAX analysis for Ag NPs with Summer savory.
Compound Element Intensity Weight % Atomic %
Ag NPs / Summer savory C 79.6 13.89 18.93
N 64.9 18.59 21.64
O 328.0 42.20 42.42
Na 475.2 12.30 8.60
Ag 173.3 11.69 7.70
Cl 7.1 0.08 0.04
K 27.3 0.35 0.14
Ca 19.1 0.26 0.11
Mg 32.4 0.63 0.42
JOURNAL OF EXPERIMENTAL NANOSCIENCE 373
this nanoparticle has received much attention. It has a wide range of nanotechnology
researches, especially in the field of medical sciences and against various microbial species.
In this section, the antimicrobial performance of Summer Savory extract and bio-synthe-
sized silver nanoparticles were evaluated against selected pathogenic microorganisms,
including two G
-
(Escherichia coli and Klebsiella pneumonia) and two G
þ
(Staphylococcus
aureus and Enterococcus faecalis) bacteria and one fungus spices (Saccharomyces cerevi-
siae). In Figure 9 and Table 4, the outcome of MIC, MBC and MFC evaluations for an
aquatic extract of Summer Savory and green synthesized Ag NPs against diverse kinds of
bacteria and fungi specie can be seen. In this regard, at high concentrations (500 to 1000
lgmL
1
), all of the desired compounds had significant antimicrobial effects against
selected microbial and fungi strains. Based on the obtained data, the aquatic extract of
Figure 9. Antibacterial and Antifungal performance of an aqueous extract of Summer Savory and green synthesized
AgNPs derived from Summer Savory against (a) E.Coli, (b) Klebsiella pneumonia, (c) Enterococcus faecalis,(d)
Staphylococcus aureus and (e) Saccharomyces cerevisiae, at diverse concentrations. Data are statistically meaningful at
the p<0.05 level.
374 S. AHMADI ET AL.
Summer Savory showed approximately the same MIC against all tested samples at a con-
centration of 250 lgmL
1
. However, the MBC and MFC varied in the range of 250 to
500 lgmL
1
. On the other, in the case of synthesized Ag NPs, different outcomes were
obtained, where lower MIC, MBC, and MFC values were achieved compared with the
aquatic extract of summer savory. Developed Ag NPs showed the highest MIC/MBC at a
concentration of 62.5 mgmL
1
for gram-negative E.Coli bacterium. Concerning the mech-
anism of the antimicrobial effect of nanoparticles, although there is no comprehensive
theory yet, different hypotheses have been proposed by researchers. One theory is that
these nanoparticles attached to the outer surface of the microbial membrane, disrupt the
permeability and cellular respiration of the microbial membrane and ultimately cause bac-
terial cell death [3,21,40,47]. Another theory mentioned that the size of NPs is effective
in bacterial death. Proponents of this theory believed that the smaller size of the synthe-
sized nanoparticles would cause more significant antimicrobial activity [3,21,40,48,49].
Generally, it was observed that fabricated AgNPs through green protocol had a more
potent inhibitory and lethal effect on the studied microorganisms than the aqueous
extract of Summer Savory, which can be an effective antibiotic against pathogenic
microorganisms.
3.3. Cytotoxic effect of Ag NPs
Widespread cancer is one of the main reasons for mortality worldwide, which requires
practical tumor-targeting strategies to minimize the death rate. Nowadays, the use of sil-
ver nanoparticles in the diagnosis and treatment of cancer-related diseases has attracted
physicians, pharmacists, and researchers in these fields [50,51]. However, several articles
have evaluated the anticancer effects of Satureja Species extract [52–55]. Nevertheless,
there was no article or report on the effect of synthesized silver nanoparticles by Summer
Savory on selected cancer cell lines. In this research, the anticancer performance of
Summer Savory extract and AgNPs was evaluated with the MCF-7 and K-562 human can-
cer cell lines by MTT experiment. As shown in Figure 10, at high concentrations (200
and 500 lgmL
1
), both compounds had a high cytotoxic effect against the target cell
groups compared to the control sample. However, with decreasing concentration, the
Anti-cancer effect of the substances has also decreased. Thus, at concentrations below 200
lgmL
1
, the anticancer properties of Summer Savory extract on both cells have been sig-
nificantly reduced. However, for synthesized silver nanoparticles, the antioxidant levels or
cell survivability (IC
50
) were 50 and 100 lgmL
1
, respectively, for the MCF-7 and K-562
human cancer cells. So, it can be deduced that synthesized silver nanoparticles had an
anticancer effect on evaluated cell lines, and this cytotoxic effect on MCF-7 cancer cell
was very significant, which highlighted the potential of developed nanoparticles as capable
anticancer drugs. The results of this research were compared with previous researches on
Satureja family plants. The results showed a potential anticancer effect of Summer Savory
Table 4. Calculated MIC and MBC/MFC values of Summer Savory extract and green synthesized AgNPs.
Microorganisms
SS extract (lgmL
1
) AgNPs (lgmL
1
)
MIC MBC MIC MBC
Staphylococcus aureus 250 500 125 250
Enterococcus faecalis 250 500 250 <250
Klebsiella pneumonia 250 <250 125 <125
Escherichia coli 250 <250 62.5 <62.5
Saccharomyces cerevisiae 250 500 125 250
JOURNAL OF EXPERIMENTAL NANOSCIENCE 375
compared to other plants [56,57]. Many efforts have been made to identify the mechanism
of the effect of AgNPs on different cancer cell lines. One of the most influential theories
widely accepted has attributed the death of cancer cells, the potential production of free
radicals by AgNPs, which prevents cell aggregation and, ultimately, cell death [58–63]. The
result of a study by Rahman et al. (2009) [64] showed that the interaction between AgNPs
and cancer cells could generate electrons that can lead to the production of ROS and,
ultimately, cell death. Also, modification of AgNPs via green protocol could promote anti-
cancer features of nanoparticles through a culmination of harmful radicals, which can
attract protein and cause oxidative stress. These oxidative stress wills have partial or per-
manent damages on the functionality of proteins [58,65]. In another theory, the effects of
nanoparticles on cellular protein function have been considered stating that AgNPs regulate
the activity of DNA-dependent kinase enzymes involved in cellular damage [65].
4. Conclusion
In this study, we tried to introduce a simple method, and in the shortest possible time to
produce silver nanoparticles use the particular plant of summer Savory, which has
received less attention. Concerns about the synthesis of AgNPs using chemical and phys-
ical methods such as the use of precursor chemicals and toxic solvents and generation of
toxic using products have led to a new alternative approach, green synthesis. This eco-
friendly technique incorporates the use of biological agents, plants or microbial agents as
reducing and capping agents. Ag NPs synthesized using green chemistry suggest a novel
and potential alternative to chemically and physically synthesized nanoparticles. Silver
nanoparticles have antimicrobial, antitumor, antioxidant, and anti-inflammatory proper-
ties, which have provided extensive open possibilities for the usage of these nanoparticles
in biomedicine. In previous studies, only one or two bacteria have been work, but in this
presentation we have increased the range of antimicrobial functions and evaluated both
bacterial and fungal. Eventually, its anticancer function has been reported to be complete.
In this research, the green biosynthesis of AgNPs was investigated using Summer Savory
extract. The synthesized AgNPs demonstrated a hexagonal structure with a size ranging
from 10 to 100 nm, as observed by FESEM, PSA, Zeta Potential, and XRD assay. The
Figure 10. MTT assay for confirming the cytotoxic effect of Summer Savory extract and Green Synthesized Ag NPs to
the K-562 and MCF-7 cancer cells for 48h. (a) Cytotoxic effects on K-562, (b) Cytotoxic effects on MCF. Data are statis-
tically meaningful at the p<0.05 level.
376 S. AHMADI ET AL.
possible functional groups from bio-molecules of Summer Savory extract that involved in
capping, active stabilization, and bio-reduction of AgNPs, were detected using the FT-IR
method. The EDX analysis determined the Ag, C, O, Na, and N elements as the main
compositions of the present synthesized nanoparticles. The synthesized silver nanopar-
ticles have shown antibacterial, antifungal, and anticancer activities against selected micro-
organisms, and two cell lines established their application in biomedicine. Therefore, it
can be concluded that the green synthesis of AgNPs by Summer Savory extract was a rela-
tively simple, completely practical, and also bio-environmental method. These nanopar-
ticles not only could eliminate the disadvantages of chemical drugs, but also could be
commercialized for mass production if their antimicrobial and anticancer effects on ani-
mal samples are examined.
Acknowledgments
This research was conducted in Fars Science and Technology Park, Shiraz. The author appreciates all the
executives of this collection, especially the researchers of Borjobaro Fars Company. A sincere thanks to
Dr. Alireza Keikhosravi and Dr. Ayoob Khosravi for their proofreading of this paper.
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