Surfaces and Interfaces 27 (2021) 101456

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Surfaces and Interfaces

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Antibacterial and antifungal activities of encapsulated Au and Ag

nanoparticles synthesized using Argemone mexicana L extract, against
antibiotic-resistant bacteria and Candida albicans
Diana Guadalupe Téllez-de-Jesús a, N.S. Flores-Lopez b, J.A. Cervantes-Chávez a, *, A.
R. Hernández-Martínez b, *
a
Facultad de Ciencias Naturales, Unidad de Microbiología Básica y Aplicada, Universidad Autónoma de Querétaro, anillo vial Fray Junipero Serra km 8s/n cp 76000,
Carretera a Chichimequillas, Santiago de Qro, México
b
Centro de Física Aplicada y Tecnología Avanzada (CFATA), Universidad Nacional Autónoma de México (UNAM), Blvd. Juriquilla 3000, Querétaro, México

A R T I C L E I N F O A B S T R A C T

Keywords: Recent studies showed that plant extracts enhance the antibacterial activity of conventional antibiotics. Those
Gold nanoparticles extracts could be used to synthesize nanoparticles for producing antibiotics with better results against microbial
Silver nanoparticles infections. We selected the medicinal plant Argemone mexicana L. and evaluated its antibacterial activity. An
Biosynthesis
extract of this plant was used for synthesizing gold and silver nanoparticles. The antibacterial activity of
Gram-negative bacteria
nanoparticles was tested against pathogenic bacteria by the Kirby-Bauer protocol. Strains bacteria were obtained
Gram-positive bacteria
Antibiotic resistant bacteria directly from clinical isolates from a Hospital in Mexico. SEM analysis indicates that A. mexicana L. extract allows
Candida albicans the synthesis of Au and Ag nanoparticles. Results indicate that the organic molecules from the extract and the Ag
nanoparticles act synergically to increase inhibition effect in the growth of resistant bacteria, wild type bacteria
and Candida albicans. In conclusion, the Ag nanoparticles obtained by A. mexicana L. extract have an excellent
performance against antibiotic-resistant bacteria from a hospital environment, and in consequence, they have the
potential to be used for producing more efficient antibiotics.

1. Introduction
Since the introduction of antibiotics in 1940, the number of bacterial
strains resistant to several antibiotics has been increasing, mainly due to
its misuse. Resistance is the ability shown by bacterial strains to survive
at a concentration of antimicrobial compounds that inhibit/kill other
strains of the same species. Due to the short generation time of bacteria,
they can easily develop mechanisms to escape the toxic effect exerted by
antibiotics. For example, mechanisms related to the horizontal gene
transfer between strains such as overexpression of efflux pumps, biofilm
formation, inactivation of the antibiotic molecule or change of the
antibiotic target [1–4].
Those mechanisms produce emergent strains known as multidrug-
resistant bacteria, that by the end of 2050, according to some studies,
will be the main cause of death associated with nosocomial infections.
The occurrence of resistant strains to new antibiotics is increasing, such
as the case of extended spectrum β-lactamases-producing microorgan­
isms, or carbapenem and meropenem resistant strains. Regarding fungi,
in the particular case of Candida albicans, some resistant strains emerge
in patients under medical treatment [5]; therefore, it is important to
develop new antimicrobial strategies to counter-attack the infections
produced by multidrug-resistant strains [3–7].
On the path of searching for new strategies, plant extracts have been
used to enhance the antibacterial activity of conventional antibiotics,
and some studies propose synergistic treatments using those new com­
pounds [8]. Moreover, the use of synergistic treatments incorporating
plant extracts or purified compounds from plants has become an
emerging area of great interest in the medical and scientific community.
In our research, we selected A. mexicana L (Mexican Prickly Poppy)
for being a known medicinal plant to obtain an extract and evaluate its
potential. A. mexicana L belongs to the Papaveraceae family, in Mexico is
commonly known as “chicalote” or “cardosanto”. This allelopathic plant
is widely distributed in tropical and subtropical countries, in Mexico is
abundant, it usually grows on the edge of the roads; it blooms
throughout the year, its stems and leaves are surrounded by thorns, and
it can grow in nutrient-poor environments. In Mexico, this plant is used

* Corresponding authors.
E-mail addresses: jose.antonio.cervantes@uaq.mx (J.A. Cervantes-Chávez), arhm@fata.unam.mx (A.R. Hernández-Martínez).

https://doi.org/10.1016/j.surfin.2021.101456
Received 29 August 2021; Accepted 3 September 2021
Available online 15 September 2021
2468-0230/© 2021 Elsevier B.V. All rights reserved.
D.G. Téllez-de-Jesús et al.
in traditional medicine as a treatment of gastrointestinal and skin in­
fections. Some compounds from the plant have been recently identified
for having anti-inflammatory and antimicrobial properties, for instance:
protopine, barberin, and sanguinarine. Also, it has been identified some
secondary metabolites against HIV, malaria and plant pathogen micro­
organisms [9–12].
The plant extract itself has demonstrated antimicrobial activity as a
result of the high content of phenolic compounds, in particular flavo­
noids and tannins, it is effective against diverse strains of Bacillus subtilis,
Escherichia coli, and Pseudomonas aeruginosa antibiotic-resistant [9,
13-16]. Also, it has been demonstrated its effectiveness against fungi like
Aspergillus niger, Aspergillus flavus, Penicillum notatum, Mucor indicus
[17], A. mexicana L is also effective against Trichomonas vaginales, a
sexually transmitted protozoan which produced trichomoniasis in
humans [18].
On the other hand, nanotechnology has altered the perspectives to
counter-attack the infections produced by multidrug-resistant strains.
The nanomedicine has also shown impressive potential in tackling
almost every aspect of microbial infection; where nanoparticle-based
technologies have the greatest potential. Physicochemical characteris­
tics of nanoparticles have a critical role in antimicrobial properties of the
materials, which are rarely expressed in materials’ bulk form. Some
nanoparticle-based nanotechnologies can affect antibiotic resistance
mechanisms of bacteria.
Nanomaterials can produce more efficient immune responses against
microbial infection [19]. Recently, materials with nanoparticles have
been used for medical purposes, obtaining excellent results. Silver and
gold nanoparticles often are obtained by chemical synthesis, but the
current trend is using green synthesis as an environmental-friendly
method that also allows to produce materials with less cytotoxicity
[20–22].
Therefore, we used green synthesis to obtain silver and gold nano­
particles using an A. mexicana L extract and we performed a systematic
analysis to evaluate the antibacterial and fungicide activity of the ma­
terials obtained with the purpose to set the bases for finding a new
strategy to counter-attack the infections produced by multidrug-
resistant strains.
2. Material and methods
2.1. Materials
All reactants were analytical grade, supplied by Sigma Aldrich and
used as received unless otherwise specified. These reactants are listed
below:
2-Propanol (C3H8O; CAS 67–63–0; PubChem CID: 3776), ethanol
(C2H6O; 99.8%, CAS 64–17–5, PubChem CID: 702); methanol (CH4O;
99.8%, CAS 67–56–1, PubChem CID: 887); 2–2-Diphenyl-1-picrylhydrazyl
(DPPH; C18H12N5O6; > 99%, CAS 1898–66–4, PubChem CID: 74,358);
CuCl2×2H2O (99%), neocuproine (> 98%), ammonium acetate (98.0%),
trolox (C14H18O4; CAS 53,188–07–1; PubChem CID: 40,634; 97%), Folin-
Ciocalteu’ phenol reagent (3H2OxP2O5×14WO3×4MoO3×10H2O; Prod­
uct Number at Sigma Aldrich F9252); AgNO3, NaOH, and HCl.
Millipore water purity (H2O) from Millipore Elix 3 UV Ultraviolet
Water Purification
System with two filters Millipak 0.22 μm and 15 MΩcm@25 ◦ C.
Argemone mexicana L plant was collected from Queretaro in Mexico
(20.6481033, − 100.3321360), in February 2018. The collection was
carried out following the description in a previous report [10], the
material was powdered using a grinding machine and finally, it was
stored and protected from light for later use.gallic acid (C₆H₂(OH)₃­
COOH, CAS 149-91-7).
2.2. Methods, characterization and techniques
The plant extract was obtained using 10 gs of the powder of
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Surfaces and Interfaces 27 (2021) 101456
A. mexicana L and 150 mL of 2-propanol (isopropyl alcohol) in a Soxhlet
extractor apparatus. The liquid fraction obtained was transferred into
rotary evaporation equipment (BUCHI R-100®) at 40 ◦ C in order to
remove the alcohol and the powder obtained was stored at 4 ◦ C to be
saved in the dark for further tests.
The obtained extract powders were weighed and redissolved in iso­
propanol into 10 mL volumetric flasks to prepare a saturated solution
(stock solution; at 200 ppm) of known concentration, from which
various solutions (from 30 to 200 ppm) were prepared to generate a
calibration curve to determine the concentration (ppm) of any other
future extraction. The lower limit of the concentration was fixed at 30
ppm because a factor of 10 times the lower reproducible limit of the
spectrometer was taken, which is 0.3 ppm; while the upper limit of 200
ppm corresponds to the saturated solution. In order to obtain the cali­
bration curve, UV–vis spectra were recorded using Thermo Evolution
Array UV–Visible spectrophotometer VWR-1600 PC and the GraphPad
Prism 7 program, in the range from 200 to 800 nm at room temperature.
The antioxidant content of alcoholic extract from A. mexicana L was
determined by DPPH assay, following the reported methodology [23].
Briefly, 200 μL of 150 mM DPPH in methanol was added to 20 µL of
A. mexicana L stock solution. The reaction was left to rest, in dark con­
ditions for 60 min and absorbance was measured at 520 nm using a
UV–Visible spectrophotometer VWR-1600 PC. The antioxidant activity
was determined by comparison with a standard Trolox curve and the
results were reported as mg Trolox equivalents per gram of sample (mg
Trolox EQ / g of sample).
Total phenolic content was determined using Folin-Ciocalteu re­
agent. An aliquot of 1 mL of crude extract of A. mexicana L was mixed
with 4 mL of 7% sodium carbonate (Na2CO3). Then 5 mL of 10% Folin-
Ciocalteu reagent was added to the mixture; and kept at room temper­
ature for 60 min, then absorbance was recorded at 765 nm using UV–Vis
spectrophotometer (Thermo Multiskan GO®). The total phenolic con­
tent was calculated by a standard curve of Gallic acid prepared with
distilled water in the range 10–50 µg/mL (R2=0.9734).
For the preparation of silver nanoparticles (AgNPs), 3 mL of 1 mM
AgNO3 (aqueous solution) was mixed with 3 mL of extract previously
adjusted to 100 ppm with distilled water. This mixture was stirred at
25 ◦ C (as the lower setpoint) and 80 ◦ C (as the upper setpoint) for 30 min
with a reflux system was used at both temperatures to avoid solvent loss
due to evaporation. The reaction product was monitored by UV–Vis
spectroscopy photometer (Thermo Multiskan GO®). Golden nano­
particles were prepared with the same procedure used for silver nano­
particles but with the precursor of 1 mM gold (III) chloride.
The antibacterial activity of the prepared nanoparticles with
A. mexicana L extract was tested against pathogenic bacteria by the
Kirby-Bauer protocol on Mueller Hinton agar (MCD LAB®) Fig. 1 [24].
We selected representatives of Gram positive and Gram negative bac­
teria. Bacteria were grown on nutritive agar (MCD LAB®) for 24 h at
37 ◦ C with constant shaking (150 RPM). Due to the current problematic
of antimicrobial resistance, clinical bacteria isolates resistant to antibi­
otics (obtained from a Hospital in Morelia, Michoacan, Mexico) were
included in the assays; P. aeruginosa resistant to imipenem and mer­
openem IMP/MEM (R), Klebsiella pneumoniae and E. coli extended
spectrum beta lactamases BLEE (+) and E. coli ertapenem were resistant
to ETP (R) Then, each strain was inoculated in nutritive broth (5 mL),
and cultures were incubated at 37 ◦ C/18 h with constant shaking.
The Optical Density at 600 nm (DO600) of the cultures was measured
using the spectrophotometer (Genesys 10S UV–vis®), and adjusted to
1.00 units. Then, 200µL were spreaded on Muller Hinton agar and 60 µL
of the nanoparticles were deposited on a paper filter disk (Whatman 1).
These assays were conducted by triplicate; data was analyzed by two
way Anova. The fungicide activity was assessed as above mentioned, but
the yeast C. albicans was grown in liquid YPD (1% yeast extract, 2%
peptone, 2% glucose) and cultures were incubated at 28 ◦ C for 24 h.
Kirby Bauer protocol was used for evaluating antibacterial activity of
the samples (Fig. 1). Wild-type strains of Staphylococcus aureus, P.
D.G. Téllez-de-Jesús et al. Surfaces and Interfaces 27 (2021) 101456

Fig 1. Schematic representation of the arrangement for the Kirby Bauer protocol.

aeruginosa and E. coli were selected as representatives of Gram positive
and Gram negative bacteria regarding their differences in the cell wall
composition, resulting in different interaction with the nanoparticles. In
the hospital environment, the Gram negative bacteria are the most
abundant, being especially important the presence of P. aeruginosa
because it is able to resist several antibiotics due to its genetics plasticity,
contributing to longer hospital stays [25].
3. Results and discussion
3.1. Isopropanolic A. mexicana L. extract characterization
UV–vis absorption spectra of the A. mexicana L. extract is presented
in Fig. 2 for concentrations from 30 to 200 ppm. In this region, the
UV–vis absorption spectra presented two absorption bands inside
wavelength ranges of 200–400 nm (ultraviolet spectroscopy), around

Fig 2. Absorption spectra of A. mexicana L. extracts (solvent isopropyl alcohol) at different concentrations (ppm) with distilled water. The A. mexicana L. image is
shown in the insert (left). The concentration versus absorbance up to the maximums at 411 nm is shown.

3
D.G. Téllez-de-Jesús et al.
226 and 269 nm; the latter is not distinguishable from spectrum noise for
30 ppm solutions. Similarly, five absorption bands inside wavelength
ranges of 400–800 nm (visible spectroscopy) of 411, 470, 537, 608, and
667 nm were observed.
The absorption bands at wavelength ranges between 200 and 400 nm
are related to electronic transitions in unsaturated molecules. Therefore,
the extract has at least two regions of molecular conjugation; they can be
two unsaturated rings in the same molecule as is the case of some
organic amine as b-Carboline alkaloids [26], or the case of some
phenolic compounds as the flavonoids. The latter compounds have a
C6-C3-C6 skeleton with two benzene rings and a pyrone or heterocyclic
pyran ring. Flavonoids subclasses are anthocyanins, flavanols, flavones,
flavanones, and isoflavones. Characteristic absorption maximum of
flavonoids is around 240–290 nm, which is characteristic of the conju­
gation of ring A and its substitution pattern [25–29]. The absorption
bands at 608 nm and 667 nm could be associated with the presence of
small concentrations in the extract of chlorophyll type a and b.
Normally, UV–vis spectroscopy is used to determine molecular
concentrations quantitatively in a solution according to Beer-Lambert
law. However, this is not feasible in a complex mixture such as the
A. mexicana L. extract; in which its chemical compounds were not pre­
viously separated and purified. However, in this case, the UV–vis ab­
sorption spectrum was useful as a control test between different
extraction and collection of A. mexicana L. Any variation in the UV–vis
absorption spectrum profile will indicate that the extract does not
contain the same mixture of chemical compounds. Similarly, the in­
tensity of the bands can be associated to an approximation of the com­
pound mixture concentration in each extract, because there is a
relationship between the concentration (in ppm) with the intensities of
absorption bands at 411 nm as observed in Fig. 2 (right insert). The
above was used to guarantee the reproducibility of the extraction
method and the proper comparison between results.
As mentioned in the experimental section, total phenolic, phenolic
compounds, and antioxidant activity of the isopropanolic A. mexicana L.
extract were determined, and the results are shown in Table 1.
As it is seen in Table 1, isopropanolic A. mexicana L. extract is rich in
phenolic compounds (20.031±0.0698 mgGAE/g). Total flavonoids and
tannins compounds are expressed in “Catechin Equivalent”, and they
were found at 202±0.078 mg/g and 398.753±0.031 mg/g, respectively.
Each sample had three replicates and data were shown as mean ±
standard deviation (SD). Similarly, an antioxidant activity close to 15%/
g of extract was determined. These properties of the isopropanolic
extract are decisive to carry out the biosynthesis of metallic nano­
particles by a chemical oxidation/reduction mechanism.
3.2. AgNPs and aunps synthesis and characterization
The synthesis of the nanoparticles was carried out from solution to
colloidal suspension, and the change in color was the main indicator to
determine the completion of each synthesis. In this way, the reaction
mixture changed from green to brown (indicating the presence of silver
nanoparticles) and from pink to purple in the case of the gold
Table 1
Total phenolic, phenolic compounds, and antioxidant activity of the iso­
propanolic Argemone mexicana L. extract.
Plant TPC [mg GAE/ TFC [mg TTC [mg QE/g] Antioxidant
sample g]** QE/g]** ** activity
[%/g]**
A. mexicana 20.031±0.0698 202±0.078 398.753±0.031 15
L.
TPC: Total phenol content, TFC: total flavonoid content; TTC: total tannis con­
tent, GAE: gallic acid equivalents.
*Each simple had three replicates and data were shown as mean ± standard
deviation.
**g dry extract.
4
Surfaces and Interfaces 27 (2021) 101456
nanoparticles after 30 min of reaction. When temperature was
increased, color change occurred in less time. UV–vis spectroscopy was
used for monitoring nanoparticle formation (of gold or silver). Fig. 3
shows the optical absorption of spectra corresponding to biosynthesis of
silver (Fig. 3a) and gold nanoparticles (Fig. 3b) in a colloidal suspension
at different temperatures, 25 ◦ C and 80 ◦ C in both cases.
The spectra showed an increase in wavelength absorption at 420 nm
for AgNPs (Fig. 3a) and at 520 nm for AuNPs (Fig. 3b). In both cases, the
increase in the reaction temperature (from 25 ◦ C to 80 ◦ C) intensified
UV–vis absorption, as well as a slight shift towards red; the increase in
intensity is related to a greater amount of nanoparticles formed, while
the red shift is related to a greater average size; as previously reported
[30–32]. Therefore, it was expected that the synthesis at 80 ◦ C will
present, under the microscope, greater dispersion in particle size.
X-ray diffraction (XRD) analysis was used to determine the crystal­
line nature of the particles. As shown in Fig. 4ab, biosynthesized AgNPs
also showed diffraction peaks as follows: 37.9◦ (111), 45.1◦ (200), 64.9◦
(220) and 78.1◦ (311). It was confirmed that both nanoparticles have a
face-centered cubic structure from their XRD patterns. And finally, in
both cases, each XRD pattern suggests that the nanoparticles were
essentially crystalline. However, in the background of each XRD pattern
also, some evidence of amorphous material is observed, related to the
presence of biomolecules from A. mexicana L. extract.
Fig. 4b shows the XRD pattern of the biosynthesized gold nano­
particles after the complete reduction of Au3+ to Au0. In which
diffraction peaks at 2θ values of 38.3◦ , 44.5◦ , 64.7◦ and 77.5◦ were
observed, corresponding to diffraction planes (111), (200), (220), (311)
from the International Center of Diffraction Data card (ICDD, formerly
JCPDS) data of gold (No. 00–004–0784) for the standard gold metal
(Au0).
Average crystallite sizes were calculated using the Debye-Scherrer
equation to AgNPs and AuNPs, which were 23.53 nm and 18.56 nm
respectively.
Nanoparticles were also characterized by electron microscopy to
confirm the presence of biomolecules from A. mexicana L. extract and to
study their structural and morphological features. Fig. 5 shows the
Scanning Electron Microscopy (SEM) images of nanoparticles at syn­
thesis temperatures (25 ◦ C and 80 ◦ C). In all cases, coarse-shaped
nanoparticles with an apparently “organic envelope” were observed.
The presence of the “organic envelope” explains the amorphous nature
observed in the background of the XRD patterns.
As shown in Fig. 5, the encapsulation of the nanoparticles within the
organic material occurred in the four synthesis cases; i.e. in AgNPs
synthesized at 25 ◦ C and at 80 ◦ C, as well as in AuNPs synthesized at
25 ◦ C and at 80 ◦ C; this means that the temperature (at least below
80 ◦ C) does not affect the organic encapsulation of the nanoparticles.
However, the temperature did affect the final particle size distribution as
evidenced by histograms.
Particle sizes (histograms, Fig. 5) were found for AgNPs synthesized
at 25 ◦ C in the order from 3 to 28 nm, with the highest concentration
around 12 nm; AgNPs synthesized at 80 ◦ C in the range from 2 to 68 nm,
with the highest concentration around 5 nm; AuNPs synthesized at 25 ◦ C
in the range from 8 to 70 nm, with the two modes of highest concen­
tration, the first around 14 nm and the second around 36 nm; and,
AuNPs synthesized at 80 ◦ C have sizes from 4 to 18 nm, with the highest
concentration around 11 nm.
Those results show a clear influence of the temperature of synthesis
on distribution and average size of the nanoparticles. Apparently, the
representative size decreases at higher synthesis temperature. However,
it is not possible to conclude a similar trend with respect to the disper­
sion of the particle size, but the dispersion is affected by temperature.
Transmission electron microscopy (TEM) of AgNPs synthesized at
25 ◦ C was obtained (Fig. 6). TEM showed a higher concentration of
particles between 8 and 14 nm. Nevertheless, the information found by
TEM and SEM studies for AgNPs synthesized at 25 ◦ C is similar; showing
the same order in particle size distribution (8 nm to 26 nm). Therefore,
D.G. Téllez-de-Jesús et al.
Fig 3. Optical absorption spectra of biosynthesis of: a) Silver nanoparticles (AgNPs) at 25 ◦ C and 80 ◦ C, b) Gold nanoparticles (AuNPs) at 25 ◦ C and 80 ◦ C.
A. mexicana L. extract adjusted at 100 ppm was included as a control.
Fig 4. XRD patterns of a) Silver nanoparticles (AgNPs) and b) Gold nanoparticles (AuNPs), both nanoparticles synthesized by A. mexicana L. extract.
TEM for the other samples was not obtained.
Thermogravimetric analysis (TGA) of the A. mexicana L. extract and
the metallic NPs was also performed to determine the percentage of
organic mass around the NPs. TGA curves of the extract and metallic NPs
are shown in Fig. 7. Each TGA curve was recorded over a wide tem­
perature range (20 ◦ C – 850 ◦ C). A. mexicana L. extract presented a
significant weight loss (close to 78%) and 22 wt% of residual mass,
which can be residual inorganic materials and cinder (Fig. 7a). The
weight loss was carried out in four stages that are distinguished from
each other by the change in slope in the thermogravimetric curve. Each
stage is highlighted with a colored rectangle and labelled with its tem­
perature range and the accumulated wt%. The thermogravimetric pro­
file of Fig. 7a was useful to analyze the thermograms of each metallic NP
and determine the percentage of organic material.
Comparing the thermogravimetric profiles of AgNPs synthesized at
25 ◦ C (Fig. 7b), at 80 ◦ C (Fig. 7c); as well as AuNPs synthesized at 25 ◦ C
(Fig. 7d), and at 80 ◦ C (Fig. 7e), with the A. mexicana L. extract ther­
mogravimetric profile of the extract, it was possible to determine that;
(i) the organic material degraded around (600–620) ◦ C; (ii) the constant
weight that occurs after (600–620) ◦ C corresponds to the weight of the
metallic NPs plus 22 wt% of the original amount of organic matter; and
(iii) the temperature of synthesis did not affect the percentage of organic
matter that surrounds the metallic NPs. From the thermograms in Fig. 7,
it can be estimated that approximately 51 wt% of the encapsulated
metallic Nps is organic material from A. mexicana L. biomolecules, i.e.,
5
Surfaces and Interfaces 27 (2021) 101456
in 1 g of the encapsulated metallic nanoparticles, there are 0.51 g of
organic material from A. mexicana L. biomolecules.
3.3. Antibacterial activity
The effect of the A. mexicana L. extract diluted at 100 ppm was tested
against E. coli, P. aeruginosa and S. aureus strains, since this was the
concentration used to carry out the synthesis of the AgNPs and AuNPs.
That effect was compared with the result produced by the concentrated
extract. In the wild type strains only the concentrated extract was
effective, the higher inhibition was observed for E. coli, next
P. aeruginosa and S. aureus, obtaining inhibition halos of 25 mm, 22 mm
and 20 mm respectively, in contrast the diluted extract did not produced
any effect, these data could be related with the amount of secondary
metabolites presented in the two concentrations tested (Fig. 8).
A clear effect is shown in the growth of the three bacteria with the
AgNPs synthesized using the A. mexicana L. extract (100 ppm), since
inhibition halos of 18 mm for P. aeruginosa for both AgNPs synthesized
at 25 ◦ C and 80 ◦ C were formed, E. coli 15 mm (AgNPs at 25 ◦ C) and 18
mm (AgNPs at 80 ◦ C) and the least effect was recorded for S. aureus,
obtaining inhibition halos of 14 mm (AgNPs at 25 ◦ C) and 13 mm
(AgNPs at 80 ◦ C) (Fig. 8). According to the statistics analysis (Fig. 9), in
general the best effect was achieved by AgNPs synthesized at 25 ◦ C.
Considering that during the synthesis not all the extract initially (100
ppm) added to the reaction is integrated into the AgNPs obtained, and
D.G. Téllez-de-Jesús et al. Surfaces and Interfaces 27 (2021) 101456

Fig 5. From A to C, SEM observation of AgNPs biosynthesized at 25 ◦ C; from D to E, SEM observation of AgNPs biosynthesized at 80 ◦ C; from G to I, SEM observation
of AuNPs biosynthesized at 25 ◦ C; and from J to L, SEM observation of AuNPs biosynthesized at 80 ◦ C. At the end of each series in a row, the particle size histogram
is displayed.

the structure of the secondary metabolites presented in the extract could
be diminished or change during the experimental conditions, the results
observed could indicate a synergistic effect between the extract and the
AgNPs, because these silver nanoparticles are able to interact with a
wide bacterial surface, and also enter into the cell and damage DNA
synthesis and the respiratory chain, disrupt the formation of biofilm
[33].
As a control experiment, the sensitivity to the antibiotic gentamicin
for S. aureus and P. aeruginosa, and ampicillin for E. coli was tested. In the
three cases these bacteria were more susceptible to the effect of these
commercial drugs as compared with the effect produced with the AgNPs
prepared with A. mexicana L. extract, nevertheless this fact is important
considering the emergence of bacterial resistance and the difficulties
associated with the development for new antibiotics [34]. Besides, it is
possible to couple different molecules to the AgNPs in order to improve
their activity, since they are good carriers of antibiotics [35].
On the other hand, the AgNPs produced by chemical synthesis were
barely effective against P. aeruginosa and E. coli, both Gram negative
bacteria, but null effect was obtained in the case of the Gram positive
S. aureus; previous reports showed that Gram positive bacteria are more
affected by AgNPs due to the general negative charge on the cell wall of
Gram positive bacteria [36].

6
D.G. Téllez-de-Jesús et al.
Fig 6. Silver nanoparticles (AgNPs) at 25 ◦ C. TEM image, low magnification (left), Histogram of the particle size in nm (right).
In a similar way, the growth of these three bacteria was not affected
by the AuNPs produced either by chemical or green synthesis. In other
words, results show that gold nanoparticles, either the biosynthesis and
chemical synthesis, have no effect in the inhibition of the bacteria
growth. The bactericidal effect of AgNPs synthesized by A. mexicana L.
extract and the bactericidal effect of extract itself not diluted is better
than biosynthesis (Fig. 10a). However, this can be explained for the
accumulation of secondary metabolites in extract. When the extract was
diluted at 100 ppm and tested, it had no effect; but in the biosynthesis of
AgNPs, it was used at the same concentration of the extract (100 ppm)
and the AgNPs showed an effect against antibiotic resistant bacterial
strains (Fig. 10b). Finally, two commercial antibiotics (ampicillin and
kanamycin) were used to confirm their effect.
Similar to the results obtained in wildtype E. coli, S. aureus, and
P. aeruginosa strains, the chemical synthesis of AgNPs only affected the
growth in E. coli (R) y E. coli ETP (R) even the inhibition halos obtained
are very small showing 9 mm y 7 mm respectively (Fig. 9). For the rest of
bacteria, no effect was recorded. In contrast, with the AgNPs produced
with the A. mexicana L. extract, a clear effect on the growth of these
bacteria was obtained, being the most effective the particles synthesized
at 25 ◦ C against P. aeruginosa IMP/MEM (R) showing a 20.3 mm inhi­
bition halo, followed by E. coli BLEE (+)(18 mm), and the least
K. pneumoniae BLEE (+)(13 mm). On the other hand, different response
was observed with the AgNPs synthesized at 80 ◦ C; in this case the
bacteria more susceptible was E. coli BLEE (+), 16 mm, very similar
result was observed in P. aeruginosa IMP/MEM (R) and E. coli (R) 14.6
mm and 14.3 mm respectively, and 10.6 mm for K. pneumoniae BLEE
(+).
In general, the inhibition halos obtained with the concentrated
A. mexicana L. extract were bigger, as those obtained with the AgNPs,
but as aforementioned it is necessary to consider the amount of the
extract used. This is an important fact for future experiments, where the
use of more concentrated extract would produce AgNPs more effective
to control the growth of bacteria.
Previously, the antibacterial effect of the A. mexicana L. extract was
reported against strains of Pseudomonas antibiotic resistant isolated from
clinical samples, nevertheless the solvents used in that report were
methanol, acetone and ethanol [37]. In that case, the bigger effect was
observed for methanol extract (15 mm y 17 mm), and none of the
Pseudomonas strains were resistant to imipenem or meropenem [37]. On
the other hand, our experiments reveal a different behavior in bacteria
7
Surfaces and Interfaces 27 (2021) 101456
growth, that is to say, a 19.3 mm inhibition halo in the case of
P. aeruginosa IMP/MEM®, a large quantity compared with those previ­
ous studies of meropenem and imipenem (both are wide spectrum car­
bapenem antibiotics widely used to eradicate multiresistant bacteria)
[38,39]. This behavior suggests that the extract obtained with iso­
propanol has different bio-molecules that result in a different activity.
Other reports analyze the antimicrobial activity of
chemically-synthesized AgNPs against clinical isolates of P. aeruginosa
resistant to antibiotics [40], but the results in our report show feasible to
implement the green synthesis of AgNPs using A. mexicana L. extract
prepared with some other solvents in order to improve the effectiveness
of those AgNPs. Silver nanoparticles (AgNPs) had been recently shown
the low cytotoxic effect produced against human cell lines of AgNPs
[40].
3.4. Fungicide activity of biosynthesis of silver nanoparticles
C. albicans was selected considering that yeasts belonging to the
genus Candida are the most frequent opportunistic fungal pathogen
isolate in humans samples, such as Candida tropicalis, Candida glabrata,
Candida parapsilosis and Candida krusei [41]. Hence this is the fungus
with the highest incidence in nosocomial infections. It can be present in
different anatomical places in the human body, being a commensal and
possess the ability to be pathogenic if the host conditions allow it
[42–44].
The results showed that the concentrated extract itself has the better
response in the inhibition growth, but the extract at 100 ppm has not the
same effect; these can be explained by the dilution of secondary me­
tabolites obtained from the plant. In the biosynthesis of silver nano­
particles, it was used the extract at 100 ppm, and the inhibition zone was
15 mm for the biosynthesis at 25 ◦ C and for the biosynthesis at 80 ◦ C the
results show an inhibition of 17 mm. In this case, C. albicans was more
affected by the presence of the biosynthesis of silver nanoparticles
(AgNPs) in comparison with the effect obtained by Hygromycin B since
halos of inhibition around 10 mm were obtained (Fig. 11). In our ex­
periments the AgNPs produced by the chemical method were not
effective to inhibit the growth of C. albicans. This result contrasts with a
previous report showing effectiveness of this type of AgNPs against this
yeast [45–47], suggesting that differences in the methodology followed
to prepare the AgNPs are important to obtain products with some spe­
cific characteristics related with the capacity to regulate the growth of
D.G. Téllez-de-Jesús et al.
Fig 7. Thermogravimetric Analysis (TGA) of: (a) A. mexicana L. extract silver, Silver nanoparticles at (b) 25 ◦ C and (c) 80 ◦ C, Gold nanoparticles at (d) 25 ◦ C and
(e) 80 ◦ C.
some specific microorganism.
Even though AuNPs were prepared with the same A. mexicana L.
extract, these particles were not effective against C. albicans. This result
must be related with the fact that gold in the nano-scale is not able to
trigger the cell death process by means of the production of oxygen
reactive species, unless the particles’ size are 10 nm or less since the
antibiotic activity of gold nanoparticles is related with the shape and size
[47].
As mentioned before, the bactericidal effect of A. mexicana L. extract
has been previously reported [48], but there are very few reports on
fungicide capacity of A. mexicana L. against C. albicans, considering that
it is one of the most important opportunistic pathogens able to produce
local as well as systemic infections [45–49], and it is resistant to echi­
nocandins, azoles and polioles [5,50]. Recently, it was demonstrated the
capacity of C. albicans to specifically enhance the tolerance to mer­
openem in P. aeruginosa strains, only when both pathogens are presented
in the biofilm, this data is important because around the 80% of the
human infections are related with this association between microor­
ganisms, where the biofilm constitutes a source of persister pathogen
cells [51]. Even though for some Pseudomonas strains different
8
Surfaces and Interfaces 27 (2021) 101456
impairment on their growth exerted by the AgNPs studied was observed
[34], recently, evidence about the antibacterial activity against
P. aeruginosa multidrug resistant strains was reported [52]. Hence the
evidence here presented, using the A. Mexicana L: extract and the AgNPs
would be a promising alternative mainly to treat infections produced by
microorganism resistant to antibiotics, nevertheless more studies are
required to elucidate the mechanism of the antibacterial and antifungal
activity display by this composite and carry out the corresponding trials
to be approved by Federal Drug Administration.
4. Conclusion
Characterization results suggest that the isopropanolic extract of
A. mexicana L. is a mixture of flavonoids and is rich in phenolic com­
pounds. Chemical properties of this extract allow the green synthesis of
metallic nanoparticles. Gold and silver nanoparticles formation was
monitored through UV–vis spectroscopy. The structure of the nano­
particles was determined as crystalline by XRD patterns, but surrounded
by organic molecules from the A. mexicana L. as an additional amor­
phous material. SEM analysis confirmed those characteristics in metallic
D.G. Téllez-de-Jesús et al. Surfaces and Interfaces 27 (2021) 101456

Fig 8. Antibacterial activity of Arg-AgNPs and Arg-AuNPs against S. aureus, P. aeruginosa, and E. coli. Inhibition halos are shown.

Fig 9. Antibacterial activity of silver nanoparticles (AgNPs) against sensitive bacterial strains. As a negative control (NC) distilled water was used. Additionally, 5 μg,
10 μg, 15 μg of gentamicin and 10 μg, 20 μg, 30 μg of ampicillin were included. The growth of each bacteria was inhibited by 60μL of AgNPs (chemical and biological
synthesis) and 60μL of A. mexicana L. extract. Different letters indicate significant differences. Each experiment was conducted by triplicate. ANOVA P=<0.05.

nanoparticles structure. Temperature of nanoparticles synthesis has not
any effect on the organic encapsulation of the nanoparticles, but it is
related with their particle size distribution. Results indicate that the
organic molecules from the extract and the silver nanoparticles act
synergically to increase inhibition effect in the growth of resistant bac­
teria, wild type bacteria and fungi. Statistics analysis showed that the
AgNPs obtained by green synthesis at 25 ◦ C had the best performance in
the inhibition of bacteria. The growth of C. albicans was also controlled
by the addition of AgNPs produced with A. mexicana L extract. Our
systematic analysis on antibacterial and antifungal activity of the ma­
terial obtained from the A. mexicana L. extract and the AgNPs, shows a
promising alternative to treat infections produced by microorganism

9
D.G. Téllez-de-Jesús et al. Surfaces and Interfaces 27 (2021) 101456

Fig 10. Antibacterial activity of silver and gold nanoparticles (AgNPs, AuNPs) against antibiotic resistant bacterial strains. a) The growth of each bacterium was
inhibited by 60μL of AgNPs and 60μL extract of A. mexicana L. Disk diffusion test of antibacterial activity of A. mexicana L. extract and AgNPs (chemical and
biological synthesis). As a negative control water was used. b) size of the zone of inhibition formed around each dis. c) Additionally 10 μg, 20 μg and 30 μg of
Ampicillin and 5 μg, 10 μg, 15 μg of kanamycin were included against multidrug resistant bacteria as a control water was used. Kp BLEE: K. penumoniae Beta
lactamases extended spectrum positive, EC (R): E. coli resistant, Ec-ETP: E. coli Enteropathogenic resistant, Pa IMP/MEM: P. aeruginosa imipenem and meropenem
resistant, Ec BLEE: E. coli Beta lactamases extended spectrum positive. Each experiment was conducted by triplicate. ANOVA p=<0.05.

10
D.G. Téllez-de-Jesús et al.
Fig 11. Fungicide activity of silver and gold nanoparticles (AgNPs, AuNPs) against Candida albicans. disk diffusion test of fungicide activity of biosynthesis of silver
nanoparticles 60μL (AgNPs) and A. mexicana L. extract (60μL), Additionally 50 μg, 100 μg, 150 μg of Hygromycin B were included. Distilled water was used as a
negative control. Each experiment was conducted by triplicate. ANOVA p=<0.05.
resistant to antibiotics, and provides the possibility to conduct further
studies of the Argemone/AgNPs material for the future development of
novel medical treatments.
CRediT authorship contribution statement
Diana Guadalupe Téllez-de-Jesús: Investigation, Methodology,
Writing – original draft, Data curation. N.S. Flores-Lopez: Investiga­
tion, Validation, Visualization, Writing – original draft, Data curation. J.
A. Cervantes-Chávez: Funding acquisition, Formal analysis, Project
administration, Supervision, Writing – review & editing. A.R.
Hernández-Martínez: Funding acquisition, Formal analysis, Supervi­
sion, Project administration, Validation, Writing – review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
Authors are grateful to Dra. Ana Angélica Feregrino Pérez and Silvia
Pineda for their help to determine phenolic compounds, and to the
“Laboratorio Nacional de Caracterización de Materiales” (LaNCaM).
Authors are also grateful to A. L. Ramos-Jacques for her valuable
contribution in language editing and proofreading the final manuscript.
Part of this research was supported by the “Dirección General de Asuntos
del Personal Académico’’ of “Universidad Nacional Autónoma de
México’’ through its Program to Support Research Projects and Tech­
nology Innovation (UNAM’s DGAPA program PAPIIT; Project No.
IN209517). Another part of the research was supported by the Project
FOFIUAQ2018,-FNB201819.
11
Surfaces and Interfaces 27 (2021) 101456
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