Heliyon 7 (2021) e05896

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Heliyon
journal homepage: www.cell.com/heliyon

Research article

Nanoemulsion of Minthostachys verticillata essential oil. In-vitro evaluation

of its antibacterial activity
M.E. Cecchini b, c, C. Paoloni a, N. Campra b, c, N. Picco a, M.C. Grosso a, M.L. Soriano Perez d,
F. Alustiza d, N. Cariddi b, c, R. Bellingeri a, e, *
a
Universidad Nacional de Río Cuarto, Facultad de Agronomía y Veterinaria, Departamento de Anatomía Animal, Laboratorio de Biotecnología Animal, Ruta 36 Km 601,
Río Cuarto, C
ordoba, 5800, Argentina
b
Universidad Nacional de Río Cuarto, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Departamento de Microbiología e Inmunología, Laboratorio de
Inmunología, Ruta 36 Km 601, Río Cuarto, C
ordoba, 5800, Argentina
c
Instituto de Biotecnología Ambiental y Salud (INBIAS) CONICET, Universidad Nacional de Río Cuarto, Ruta 36 Km 601, Río Cuarto, C
ordoba, 5800, Argentina
d
Instituto Nacional de Tecnología Agropecuaria (INTA), Estaci
on Experimental Marcos Ju
arez, Marcos Ju

arez, X2580, C
ordoba, Argentina
e
Instituto de Investigaciones en Tecnologías Energ
eticas y Materiales Avanzados (IITEMA) CONICET, Universidad Nacional de Río Cuarto, Ruta 36 Km 601, Río Cuarto,
C
ordoba, 5800, Argentina

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

Keywords: Infectious diseases constitute a problem of great importance for animal and human health, as well as the
Minthostachys verticillata increasing bacterial resistance to antibiotics. In this context, medicinal plants emerge as an effective alternative to
Essential oil replace the use antibiotics. The essential oil (EO) of Minthostachys verticillata (Griseb.) Epling (Lamiaceae) has
Nanoemulsion
demonstrated a strong antimicrobial activity. However, its instability and hydrophobicity under normal storage
Antibacterial activity
conditions are limitations to its use. Nanoemulsion technology is an excellent way to solubilize, microencapsulate,
and protect this compound. This study aimed to obtain a nanoemulsion based on M. verticillata EO and evaluate its
antibacterial activity against Staphylococcus aureus. The EO was obtained by steam distillation. Identification and
quantification of their components were determined by GC-MS revealing that the dominated chemical group was
oxygenated monoterpenes. Nanoemulsions (NE) were characterized by measuring pH, transmittance, separation
percentage, release profile, and morphology. The effect of NE on the growth of S. aureus and cyto-compatibility
was also evaluated. The results showed that NE containing a higher percentage of tween 20 exhibited higher
stability with an approximated droplet size of 10 nm. The effect of encapsulation process was evaluated by GC-MS
revealing that the volatile components in EO were no affected. After 24 h, 74.24
0.75% of EO was released from
NE and the antibacterial activity of EO was enhanced considerably by its encapsulation. The incubation of
S. aureus with the NE and pure EO, show a bacterial growth inhibition of 58.87%
0.99 and 46.72%
3.32 (p <
0.05), respectively. In addition, nanoemulsi
on did not cause toxicity to porcine and equine red blood cells. The
results obtained showed that NE could be a potential vehicle for M. verticillata EO with promissory properties to
emerge as a tool for developing advanced therapies to control and combat infections.

1. Introduction 2020a, 2020b, 2020c, 2020d; Sharifi-Rad et al., 2018; Adorjan and
Buchbauer, 2010). Minthostachys verticillata (Griseb.) Epling, also known
The irrational use of antibiotics has led to a considerable decrease in as “peperina”, is a species of the Lamiaceae family. M. verticillata essential
their effectiveness generating resistance in certain microorganisms and a oil (EO) is used to alleviate respiratory and digestive disorders in folkloric
significant impact on human and animal health (Foster, 2017). Natural medicine and as a food preservative and flavoring (Ojeda et al., 2001;
products of plant origin could be a viable alternative to the use of anti- Schmidt-Lebuhn, 2008).
biotics (Lillehoj et al., 2018; Salehi et al., 2018). It has been reported that The main components of EOs are complex mixtures of hydrocarbon
they possess significant antiseptic, antibacterial, antiviral, antioxidant, terpenes and terpenoids (El Asbahani et al., 2015). The majority of the
anti-parasitic, antifungal, and insecticidal effects (Salehi et al., 2019, first group consists of monoterpenes and sesquiterpenes, and the

* Corresponding author.
E-mail address: [email protected] (R. Bellingeri).

https://doi.org/10.1016/j.heliyon.2021.e05896
Received 28 May 2020; Received in revised form 28 July 2020; Accepted 30 December 2020
2405-8440/© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
M.E. Cecchini et al. Heliyon 7 (2021) e05896

second group consists of oxygenated derivatives of hydrocarbon ter- 2.2. Identification and quantification of essential oil pure compounds
penes. Studies about volatile constituents of M. verticillata from various
phytogeographic areas of Argentina revealed that there is a high ge- The essential oil recovered was analyzed by gas chromatography
netic diversity in this species, which appears to be divided into three coupled to mass spectrometry (GC-MS) using a gas chromatograph Clarus
chemotypes: carvone, thymol-carvacrol and pulegone-menthone (Zyg- 600 (Perkin Elmer) equipped with DB5 column. Gas chromatographic
adlo et al., 1996). Testing the EO chemical composition is fundamental (GC) conditions were as follows: injector temperature, 260  C; flame
because several biological activities are attributed to the EO com- ionization detection temperature, 240  C; carrier gas, Helium; flow rate,
pounds (Le
on-M
endez et al., 2019). Recent studies on M. verticillata EO 1 mL/min; and split injection with split ratio 1:40. The oven temperature
(pulegone-menthone chemotype) have revealed in vitro antimicrobial was initially 60  C and was increased to 240  C at a rate of 5ºC/min. One
and anti-biofilm properties on Streptococcus uberis, Escherichia coli, microliter of each sample, dissolved in hexane (1:100 mg/μL), was
Enterococcus faecium and Bacillus pumilus strains isolated from cows injected into a DB-5 column (0.25 mm I. D, 60 m in length, and 0.25 μm
with mastitis (Montironi et al., 2016; Cerioli et al., 2018). In addition, film thickness). The chromatogram was obtained in “scan” mode, from
M. verticillata EO has also demonstrated immunomodulatory effects by m/z ¼ 30 am/z ¼ 450 (scan time: 0.2 s, inter-scan time: 0.1s), solvent
decreasing pro-inflammatory mediators as histamine and β-hexosa- delay: 5 min. Data were acquired using TurboMass 5.4.2 software. The
minidase or pro-inflammatory cytokines (TNF-α, IL-1β) and increasing identification of the peaks was made by comparison with the mass
anti-inflammatory cytokines (IL-10) both in vitro and in experimental spectra of the components found with that of the NIST MS Search 2.0
mouse models (Cariddi et al., 2011; Montironi et al., 2019). However, program libraries. To confirm the identity of the compounds, pure
the limitations in the use and application of EOs are mainly attributed compounds (standard grade) were injected under the same chromato-
to their photosensitive, volatile, and hydrophobic nature, which has graphic conditions. Retention rates were calculated by analyzing a
stimulated the search for new conservation systems to exploit their C8–C20 alkane standard (Fluka cod 101385968) under the same condi-
great potential. For this purpose, a large number of encapsulation tions as the sample (Montironi et al., 2016).
methods have been developed in recent years. These methods include
film hydration (Asprea et al., 2017), coacervation (Gonçalves et al.,
2.3. Nanoemulsion preparation
2017), spray drying (Herculano et al., 2015), ionic gelation (Noppa-
kundilograt et al., 2015), complexation (Da Rocha Neto et al., 2018;
Nanoemulsions (NE) consisted of M. verticillata EO, Tween 20 as a
Shrestha et al., 2017), liposomes (Cui et al., 2020; Lin et al., 2019),
surfactant and deionized water were synthesized as Rodrigues et al.
chitosan nanoparticles embedded gelatin nanofibers (Cui et al., 2018)
(2014) with some modifications. The essential oil and Tween 20 were
and nanoemulsions (Lou et al., 2017; Chang et al., 2013). The encap-
stirred at 800 rpm using a magnetic stirrer for 30 min. Then, water was
sulation of EO in a nanoemulsion (NE) is a promising strategy to
added dropwise at a flow rate of 2 mL/min. The mixture was stirred at
facilitate its applicability and enhance its properties. These formula-
800 rpm for 30 min. Final homogenization was achieved using a Hei-
tions offer numerous advantages including a controlled small droplet
dolph DIAX 900 homogenizer (Germany) equipped with a 10 G disperser
size which are kinetically stabilized against aggregation and gravita-
for 5 min (8000 rpm). Formulation variable was oil/surfactant ratio (1:1,
tional separation (Donsí and Ferrari, 2016; Qian and McClements,
1:2, 1:3 and 1:4). EO percentage was 6% for all formulations.
2011). Additionally, NEs have a larger surface area of active in-
gredients compared to conventional emulsions and, therefore, the
functionality is enhanced when they interact with biological systems, 2.4. Physic-chemical characterization
improving the diffusion and biological efficacy of dispersed bioactive
agents (Salvia-trujillo et al., 2013; Bajerski et al., 2016). NEs have been 2.4.1. Percentage transmittance
extensively exploited in many fields, including the pharmaceutical The turbidity of all the formulated NE was analyzed by measuring the
field, due to their medicinal properties. Several EO formulations were transmittance of undiluted emulsions at a wavelength of 600 nm ultra-
tested against Gram-positive and Gram-negative bacteria (Donsí and violet using Shimadzu UV/VIS spectrophotometer (Jasco, Mod. V-630
Ferrari, 2016). However, to the best of our knowledge there are no Bio).
reports about M. verticillata EO based NE and their antibacterial effect.
The present study aimed to characterize M. verticillata EO nano- 2.4.2. pH measurement
emulsions and evaluate its antibacterial activity against a Staphylo- The pH value of the NE was measured by immersing the electrode
coccus aureus strain (ATCC, 209213) when compared with that of the directly into the NE using a calibrated pH meter (Cole-Parmer pH/mV/ºC
pure EO. meter), at 25  C
1  C. The measurement was carried out in triplicates.

2.4.3. Stability study

2. Materials and methods Formulated NE were centrifuged at 3,500 rpm for 30 min to prove
stability and observe phase separation. Separation percentage was
2.1. Plant material and essential oil extraction calculated using the following equation:

The plant material was obtained in a commercial herb store in Separation percentage (%) ¼ (separated aqueous phase weight/total weigh of
February 2017. To obtain the EO, 60 g of ground leaves from the nanoemulsion) * 100 (1)
collected specimens were subjected to a steam distillation process. For
this, the dry leaves of M. verticillata were placed in an extraction col-
umn. A 1 L capacity balloon, containing distilled water, was heated
until boiling; the water steam, flowing through the plant material in 2.5. Morphological analysis by transmission electron microscopy
the column, carrying the volatile components, which condensed in the
glass condenser and then by decantation, separated into two phases, The morphology of the NE was visualized by transmission electron
one oily and one aqueous. The emulsified water inside the oil was microscopy (TEM) (Siemens, Elmiskop 101). Samples (50 mL) were
separated from it, by freezing. The EO fraction was stored at -20  C and placed on 200 mesh formvar coated copper TEM grids, and then nega-
protected from light until use (Cariddi et al., 2011; Montironi et al., tively stained with 50 mL of 1.5% (weight/volume) phosphotungstic acid
2016). for 10 min at room temperature. Excess liquid was blotted with Whatman

2
M.E. Cecchini et al. Heliyon 7 (2021) e05896

filter paper. The measurements of droplets were performed using Axio- The total number of bacteria was determined by plating serial di-
Vision software (Carl Zeiss; Oberkochen, Germany). lutions (1:104 to 1:108) of the content of each well on Petri dishes con-
taining Trypticase soy agar (TSA); the dishes were incubated at 37  C for
24 h, and colonies counted. The number of bacteria was calculated, based
2.6. Effect of nanoemulsification on M. verticillata EO compounds
on the dilution factor. To normalize the data, a positive control (0.2 %
Triton x100) and negative control (PBS) were used.
The effects of nanoencapsulation process on the EO volatile constit-
uents was evaluated using GC-MS as previously was described. A sample
2.8.2. Hemolytic activity
of pure EO obtained from dry leaves of M. verticillata (collected in
Bovine and equine peripheral blood samples were freshly collected
February 2020) and NE were analyzed. As Tween 20 and water are not
and put into a test tube containing an anticoagulant (EDTA-Na2 10%).
compatible with the GC system, EO was separated using the non-polar
For the quantification of hemolytic activity, same concentrations of NE
solvent, n-hexane. The hexane layer was evaporated using a vacuum
used in the previous assay were tested (50, 150, 250, 350 and 450 mg/
rotary evaporator. The residual oil was dried over anhydrous Na2SO4 and
mL). Aliquots of 50 μL of blood were mixed with the different solutions of
kept in a refrigerator (4  C) until its use. The relative percentage of the
NE; the tubes were homogenized and incubated for 60 min at room
identified constituents was calculated from the GC peak areas.
temperature. The samples were centrifuged at 2000 rpm for 5 min, and
the supernatant was removed to measure the absorbance (540 nm) cor-
2.7. In vitro release of M. verticillata EO from nanoemulsion rected with the blank (buffered solution containing red blood cells). The
results were compared with a positive control in which the cells were
The in vitro release experiments of the EO were carried out using the completely lysed by distilled water. Experiments were carried out in
dialysis technique (Rodrigues et al., 2018). An aliquot of nanoemulsion triplicate. The hemolytic activity of each sample was calculated by the
was placed inside a dialysis bag (cellulose membrane, molecular weight following equation:
cut-off 14,000 Da, Roth®), sealed, and immersed in a vessel containing
Hemolysis (%) ¼ (DO sample - DO control (-)) / (DO control (þ) - DO control
150 mL of 10 mM phosphate buffer solution (PBS, pH 7.4). The releasing
(-))  100 (2)
system was maintained at 37
1  C, under magnetic stirring (100 rpm).
Samples (1 mL) were taken out of the dissolution medium at different
time intervals (1, 3, 6, 9 and 24 h). This volume was replaced with fresh
PBS, and the sample was analyzed by UV spectrophotometry at 260 nm.
In order to establish the linearity of the proposed method, three cali- 2.9. Statistical analysis
bration curves were constructed at eight concentrations levels within the
range of 2–10 μg/mL solubilizing a known amount of EO in ethanol. The Statistical analysis was performed using Infostat software (Di Rienzo
release profile of M. verticillata EO was expressed as cumulative amount et al., 2011). Data were represented as means standard deviation (SD).
of released EO (mean
SD) and plotted versus time. The experiments Differences were considered significant at the level of p < 0.05. Levels of
were carried out in triplicate. significance were evaluated by Kruskal-Wallis test.

2.8. In vitro evaluation of nanoemulsi
on 3. Results

2.8.1. Antimicrobial activity 3.1. Main compounds detected in M. verticillata EO

The essential oil obtained from fresh leaves showed a yield of 4.6%
2.8.1.1. Bacterial culture. Staphylococcus aureus ATCC 29213 (wound
(w/v). Oil density was 0.95 g/mL and pH was equal to 5.0. In total, 13
isolated) was cultured in Trypticase Soy Broth (TSB, Britania Laboratory,
compounds were identified with the predominance of monoterpenes and
Ref. B0410361, Argentina) and grown overnight at 37  C. The concen-
sesquiterpenes (98%). Chromatography showed that the most abundant
tration of the inoculum was estimated by spectrophotometric turbidity
chemical group of M. verticillata essential oil was oxygenated mono-
measurement at 600 nm on a microplate reader (Biolatin, Venezuela) and
terpenes, a monoterpenes group attached to an oxygen atom and fol-
adjusted to an optical density (OD) of 0.07 which corresponds to a 0.5
lowed by monoterpenes (C10H16). Among these, the main oxygenated
McFarland standard (1  108 CFU/mL).
monoterpenes reported were monocyclic ketone such as pulegone
(76.96%) and menthone (20.38%), and limonene (1.54%). Sesquiter-
2.8.1.2. Antibacterial assay. In order to compare antibacterial activity, it pene group (C15H24) was represented by γ-elemene and spathulenol
was important to make sure that the concentrations of EO used both pure (Table 1).
and encapsulated in the NE, were the same. Five concentrations of EO
were chosen to be tested: 3, 9, 15, 21, and 27 mg/mL. Then, dilutions of
the NE that would contain equal concentrations of EO mentioned above 3.2. Nanoemulsion characterization
were calculated based on the density of the EO (950 mg/mL) and its ratio
in the NE formulation (6%). The concentrations obtained were 50, 150, 3.2.1. Physicochemical properties
250, 350 and 450 mg/mL of NE, which corresponded to 3, 9, 15, 21 and A continuous increase in pH was observed when surfactant concen-
27 mg/mL of EO encapsulated in the NE, respectively. In all cases, stock tration increased from 1:1 to 1:4 ratio by maintaining the oil concen-
solutions of each concentration of EO and NE were prepared immediately tration constant. The turbidity of NE decreased when the surfactant
before been used, diluted in phosphate buffered saline (PBS). concentration increased, while its stability increased (Table 2). This in-
Antibacterial activity assay was conducted in 96-wells plates. Briefly, dicates that varying o/s ratio has a high impact on the nature of colloidal
10 μL of the initial inoculum standardized at 1  108 CFU/mL and 90 μL surfaces. To carry out the electron microscopy and the antibacterial
of the stock solutions of EO and NE (corresponding to the different properties test, it was decided to use nanoemulsion D, due to its higher
concentrations) were added to each well. Then, all the wells were filled stability.
with 100 μL of fresh TSB for a final volume of 200 μL per well with a
bacteria concentration of 1  106 CFU/mL in the wells. Growth control 3.2.2. Morphology study
wells contained 10 μL of inoculum and 190 μL of TSB. Plates were To study the NE morphology and distribution of the droplets size,
incubated at 37  C for 24 h. transmission electron microscopy photomicrographs were taken, as

3
M.E. Cecchini et al. Heliyon 7 (2021) e05896

Table 1. Chemical composition of M. verticillata EO.

Identified compounds Molecular formula Relative percentage (%)

δ-carene C10H16 0.10
β-pinene C10H16 0.20
Limonene C10H16 1.54
Menthone C10H18O 20.38
ρ-menthone C10H18O 0.10
Isopulegone C10H16O 0.15
2-Isopropyl-2, C11H20O 0.14
5-dimethylcyclohexanone
Pulegone C10H16O 76.96
Piperitone C10H16O 0.15
bicyclo,heptan-2-ol, C10H16O 0.10 Figure 1. TEM image of nanoemulsion morphology (scale bar ¼ 50nm;
2-allyl-1,7,7-trimet 300,000x Frame).
Piperitenone C10H14O 0.06
γ-elemene C15H24 0.07
3.5. In vitro evaluation of nanoemulsi
on
Spathulenol C15H24O 0.05
Total 100 3.5.1. Antibacterial activity
The antibacterial activity after the incubation for 24 h of S. aureus
shown in Figure 1. TEM showed discrete droplets of spherical shape, with with the different concentration of EO and NE was evaluated. The
an approximated diameter of 10 nm and without any aggregation. average percentage of bacterial growth inhibition of the highest con-
centration tested of both the EO (27 mg/mL) and the NE (450 mg/mL ¼
27 mg/mL of EO) was 46.72%
3.32 and 58.87%
0.99, respectively. A
3.3. Effect of nanoemulsification on EO compounds dose-dependent effect was not observed between all the concentrations
of NE evaluated; however, a clear statistical significant difference was
The volatiles composition of the NE was found to be similar from that observed between the highest and lowest (50 mg/mL ¼ 3 mg/mL of EO)
of the EO. GC-MS analysis showed that the use of ultrahomogenization to NE concentrations. All the NE concentrations tested showed a stronger
prepare nanoemulsions led to no significant quantitative differences in growth inhibition with statistical significant difference when compared
the predominant compounds so the pulegone/menthone chemotype was to the same concentration of pure EO. No dose-dependent effect was
maintained. Sample components of less than 1%are not reported observed between the concentrations of EO (Table 4).
(Table 3, Figure 2). The effect of incubation with NE and EO on bacterial growth is
showed in Figure 4. A significant reduction in bacterial growth was
observed after 3 h (about 1.28 log UFC/mL). The greatest effect was
3.4. In vitro release of M. verticillata EO from nanoemulsion
found after 24 h of incubation with NE (450 mg/mL, equivalent to 27
mg/mL of EO) that produced a 5 log reduction approximately.
Release profile assay was carried out using in vitro dialysis experi-
ment. As can be seen in Figure 3, after 3 h, the release of EO was 29.48

3.5.2. Hemolytic activity
0.96%. However, a sustained release of EO from the NE was observed
The results of the hemolysis test are presented in Table 5. The
during the first 6 h, in which more than 55% of EO was found to be
absorbance of the erythrocytes suspension supernatant at 450 nm after
released (58.75
1.75). At the end of 24 h of experiment, 74.24
0.75%
incubation with different concentrations of nanoemulsion was studied.
of EO was released from NE.

Table 2. Physicochemical parameters of nanoemulsions.

Formulation (oil:surfactant) pH Transmittance percentage (%) Separation percentage (%)

A (1:1) 3.02
0.01 0.12
0.02 20.35
0.12
B (1:2) 3.31
0.03 0.37
0.04 18.55
0.11
C (1:3) 3.41
0.01 0.65
0.01 18.98
0.20
D (1:4) 3.64
0.02 1.04
0.02* 12.71
0.14*

Data presented as mean (n ¼ 3)
standard deviation (SD). * Indicate statistical significance (p < 0.05). Kruskal Wallis test, Infostat, 2011.

Table 3. Comparison between relative percentage of chemical compounds of pure M. verticillata EO and nanoemulsified (NE).

Identified compounds Retention index (min) Molecular formula Relative percentage (%) in EO Relative percentage (%) in NE
α-pinene 6.79 C10H16 1.17 1.66
β-pinene 8.27 C10H16 1.38 1.74
Limonene 10.04 C10H16 3.49 3.83
Menthone 14.17 C10H18O 21.22 20.86
Isopulegone 14.78 C10H16O 2.15 2.75
Pulegone 16.67 C10H16O 67.96 65.49
γ-elemene 23.59 C15H24 1.23 1.78
Spathulenol 25.55 C15H24O 1.40 1.89
Total 100 100

4
M.E. Cecchini et al. Heliyon 7 (2021) e05896

chromatographic analysis carried out in the present work reported that

the main components identified in the EO were pulegone and menthone,
in concordance with the chemotype reported by Zygadlo et al. (1996) for
M. verticillata specimens from the central region of Argentina. Several
studies focused on same species from this area have reported the same
chemotype (Cariddi et al., 2011). It is important to highlight that
although the results are coincident, there may be variations in the
chemical composition of the EO. The percentage of every chemical
compound not only depends on the aromatic species in question but also
on its stage of development and the environmental conditions to which
the plant is exposed at the time of collection (climate, soil, season)
(Bakkali et al., 2005; Zygadlo et al., 1996).
Essential oils are known for their numerous biological activities
(Conti et al., 2010), and the interest in their use has been renewed due to
the need for innovative agents, capable of fighting bacterial resistance
(Czaplewski et al., 2016; Franklyne et al., 2016; Thormar, 2010). How-
ever, its use is usually limited by its high volatility and tendency to
readily degrade by oxidation and isomerization, which could be a
drawback for its application as an antibacterial agent (Ghosh et al., 2013;
Turek and Stintzing, 2013). In this context, nanoemulsions (NE) consti-
tute a valid strategy to overcome this problem since they have a wide
variety of advantages over pure essential oils, including better stability of
the volatile compounds, and protect them against environmental factors
that may cause chemical degradation (de Matos et al., 2019).
In the present study, NE was synthesized by ultrasonic emulsification,
a high-energy method that rapidly and efficiently produces NEs with
Figure 2. Chromatograms for EO (green) and NE (red) GC/MS measurement. small droplet diameters (Manchun et al., 2014; Ghosh et al., 2013). The
physical-chemical characterization of NE revealed that the different
The hemolysis percentage values of all samples were less than 5%, sug-
formulations presented similar pH, but the transmittance percentage and
gesting no hemolytic activity by NE.
stability increased significantly with the enhancements of surfactant
content. Scanning electron microscopy allowed establishing the average
4. Discussion
diameter of droplets, in around 10 nm, leading to the formation of a more
homogeneous structure. Ghosh et al. (2014), obtained similar results
Antimicrobial resistance has become a major medical and public
with eugenol-loaded nanoemulsions using a non-ionic surfactant (tween
health problem. New strategies to treat the infections caused by
80). Previous studies revealed that nanoemulsions that minimize droplet
antibiotic-resistant pathogens are needed. In the present study, the
size have a more homogeneous structure that leads to higher stability (de
development of antibacterial nanoemulsi
on based in natural products,


Oca-Avalos et al., 2017). According to Bruxel et al. (2012), the use of
was studied as an alternative method for controlling these diseases.
nonionic surfactants from the group of poloxamer and polyoxyethylene
The extraction of M. verticillata essential oil (EO) by steam distillation
sorbitans (tweens) has shown a favorable association with phospholipids,
of dry leaves resulted in a slightly yellow liquid with and estimated yield
as they lead to the formation of compact mixed films, conferring greater
of 4.6% (w/v) based on dry weight. In a previous work, Cariddi et al.
stability to the formulation.
(2011), obtained a similar yield percentage to EO with M. verticillata
The application of encapsulated essential oils also supports their
species from central region of Argentina. Studies of wild peperina shows
controlled and sustained release, which enhances their bioavailability
higher contents of EO in plants of the central area of Argentina than in
and efficacy against multidrug-resistant pathogens (Prakash et al., 2018).
those from northwest region, this differences were accompanied by
Owing to its greater stability, the formulation with 1:4 rate (oil:-
changes in the number and size of the glandular trichomes, structures
surfactant) was selected for studying the antibacterial activity against
where the EO is synthesized and stored (Arteaga et al., 2016). The

Figure 3. In vitro release profile of EO from the NE through dialysis membrane. Illustrated values correspond to the mean
standard deviation (n ¼ 3). Infostat, 2011.

5
M.E. Cecchini et al. Heliyon 7 (2021) e05896

Table 4. Antibacterial effect after 24 h.

EO Concentration (mg/mL) Growth inhibition percentage (%) Log CFU/mL

EO NE EO NE
3 43.67
1.83 53.50
1.36* 5.66
0.26a 4.67
0.18b
9 44.52
4.10 55.76
1.89* 5.57
0.49a 4.44
0.24ab
15 42.37
0.45 54.46
2.01* 5.79
0.13a 4.57
0.26ab
a
21 45.48
4.07 56.55
0.94* 5.47
0.35 4.36
0.15ab
27 46.72
3.32 58.87
0.99* 5.35
0.41a 4.13
0.11a

Data represents the mean (n ¼ 3)
standard deviation (SD). * Indicate statistical significance between essential oil (EO) and nanoemulsi
on (NE) at same concentrations
(p < 0.05). Different letters indicate statistical significance between concentrations of EO or NE (p < 0.05). Kruskal Wallis test, Infostat, 2011. Log CFU/mL in control
group at 24 h ¼ 10.04
0.15.

Figure 4. Staphylococcus aureus growth (Log

CFU/mL) after 24 h incubation with essential oil
(EO, 27 mg/mL) and nanoemulsi
on (NE, 450 mg/
mL equivalent to 27 mg/mL of EO). The illus-
trated values represent the mean
standard de-
viation (n ¼ 3). Different letters indicate
significant differences between groups at same
time (p < 0.05). Infostat, 2011. Initial log CFU/
mL values at 0 h: Control (8.16
0.04), EO (8.17

0.03) and NE (8.20
0.02). Final log CFU/mL
values at 24 h: Control (10.04
0.15), EO (5.35

0.41) and NE (4.13
0.11).

S. aureus. This is a common opportunistic microorganism that is found in conducted in simple model solutions, while the information regarding
skin abrasions and open wounds and the most commonly isolated con- possible effects of the encapsulation process on the EO chemical com-
tagious organism from mastitis and dermatitis in cattle, sheep, goats, ponents is scarce. For this reason, we evaluate the effect of encapsulation
pigs, and horses (Peton and Le Loir, 2014; Boss et al., 2011). process on EO volatile compounds. GC-MS analysis showed no significant
Factors determining the activity of the EO are the composition differences between EO and NE profile. Only few studies have evaluated
functional groups present in the active components (Dorman and Deans, changes on essential oil constituents after encapsulation. Donsì et al.
2000) and the encapsulation method. The main compounds identified in (2012) reported that high-pressure homogenization (HPH) as well as
M. verticillata EO were oxygenated terpenes. Previous studies state that high shear homogenization (HSH), results in decomposition of active
oxygenated terpenes exhibit better antimicrobial activity than hydro- constituents of EO particularly p-cymene, terpinenes, carveol, carvacrol
carbons (Guimar~ aes et al., 2019) maybe through the inhibition of and others. During emulsification by HPH there is an intrinsic increase in
essential processes to microbial survival and alterations in the lipid the temperature of the sample, which must be taken into account,
membrane function (Mahizan et al., 2019; Cano et al., 2008). especially when it comes to the production of nanoemulsions incorpo-
An important requirement in the synthesis of essential oil based rating heat-sensitive active compounds (Salvia-Trujillo et al., 2017).
nanoemulsion is that the encapsulation system protects the active in- Shukat and Relkin (2011) observed a substantial degradation of
gredients from chemical degradation and that the encapsulation process α-tocopherol in palm oil nanoemulsions produced with HPH. Ali et al.
does not alter the bioactivity of the components. Although EOs are (2020) studied the effect of nanoencapsulation on volatile constituents of
complex mixtures, their bioactivity is mainly dependent on a few mole- Algerian Origanum glandulosum Desf. EO that was encapsulated via HSH
cules present at high concentrations. However, the compounds contained at 18000 rpm and via HPH. The results reported a significant decrease for
at low percentages are useful to enhance the effectiveness. Most of the carvacrol as the process intensity increased from HSH to HPH and
actually studies that evaluate the functionality of nanoemulsions are showed that the volatiles profile of oil nanocapsules generated by HSH is
still similar to that of the oil and corresponds to the thymol and/or
carvacrol chemotype. In the present work, we used a lower homogeni-
Table 5. Percentage of hemolysis of NE in blood samples. zation speed than the previous reported works (approximately 8000
rpm for 5 min), only a slight percentage (~2.5%) of pulegone was
Nanoemulsion Porcine blood Equine blood affected.
concentration (mg/mL)
A killing kinetics assay was performed to establish changes in the
50 1.36
0.58 0.68
0,.20
viability of S. aureus upon interaction with pure EO or NE over 24 h. A
150 1.04
0.20 0.85
0.03 reduction of 1.28 log CFU/mL was observed after 3 h interaction of NE
250 0.81
0.56 1.27
0.45 with the bacteria. The greatest effect was found after 24 h with NE (450
350 1.07
0.60 1.04
0.60 mg/mL, equivalent to 27 mg/mL of EO) reaching a reduction of
450 0.71
0.10 0.54
0.30 approximately 5 Log CFU/mL. Sugumar et al. (2014) studied the anti-
Data represents mean
standard deviation (SD) (n ¼ 3). Infostat, 2011. bacterial activity of eucalyptus oil nanoemulsion (undiluted) against

6
M.E. Cecchini et al. Heliyon 7 (2021) e05896

S. aureus and obtained a complete loss of viability within 15 min of Funding statement
interaction. The antibacterial activity results founded in the present work
clearly shows that the NE was more effective at inactivating the bacteria This work was supported by grants from Agencia Nacional de
than pure EO. These results were in agreement with other studies that Promoci
on Científica y Tecnol

ogica (ANPCyT) and Secretaría de Ciencia
showed that the conversion of an EO (T. daenensis) into a NE greatly y T
ecnica de la Universidad Nacional de Río Cuarto (SECYT-UNRC).
enhanced its antibacterial activity against an important food-borne
pathogenic bacterium (E. coli) (Moghimi et al., 2016; Bhargava et al., Data availability statement
2015). The superior antimicrobial effect of NE can be explained by an
increase in surface area that improves the efficacy of EO as it ensures Data included in article/supplementary material/referenced in
larger contact with bacteria cells. Donsì et al. (2011) have shown that article.
nanoemulsion-based delivery systems of d-limonene and a terpenes
mixture significantly increased the antimicrobial activity of encapsulated Declaration of interests statement
compounds in comparison to non-encapsulated ones. This is probably
because the small lipid particles inside the NE are capable of transport the The authors declare no conflict of interest.
EO towards the surface of the cell membranes, suggesting that the pure
oil (which has little solubility) could not easily interact with the mem- Additional information
branes (Bilia et al., 2017).
Safety is an important concern in the use of novel products such as No additional information is available for this paper.
NE. In vitro erythrocyte-induced hemolysis can be considered a simple
and reliable measure for estimating the membrane damage caused in vivo Acknowledgements
(Pape and Hoppe, 1990). The behavior of M. verticillata EO based NE was
studied by investigating the degree of hemolysis in vitro. No hemolytic R. Bellingeri, L.N. Cariddi are permanent researchers of CONICET.
activity on both porcine and equine blood was detected by any of NE M.E. Cecchini, N. Campra and ML Soriano thanks CONICET for a grad-
concentrations evaluated. The NE toxicity is highly dependent on the uate fellowship.
chemical composition of the used oil (Mendanha et al., 2013). Safety
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