foods

Article
In Silico and In Vitro Evaluation of the Antimicrobial and
Antioxidant Potential of Mentha × smithiana R. GRAHAM
Essential Oil from Western Romania
Călin Jianu 1 , Daniela Stoin 1 , Ileana Cocan 1 , Ioan David 1, *, Georgeta Pop 2 ,
Alexandra Teodora Lukinich-Gruia 3 , Marius Mioc 4 , Alexandra Mioc 4 , Codrut, a S, oica 4 , Delia Muntean 5,6 ,
Laura-Cristina Rusu 7,8 , Ionut, Golet, 9 and Delia Ioana Horhat 5

1 Faculty of Food Engineering, Banat’s University of Agricultural Sciences and Veterinary Medicine “King
Michael I of Romania” from Timisoara, Calea Aradului 119, RO-300645 Timis, oara, Romania;
[email protected] (C.J.); [email protected] (D.S.); [email protected] (I.C.)
2 Faculty of Agriculture, Banat’s University of Agricultural Sciences and Veterinary Medicine “King Michael I
of Romania” from Timisoara, Calea Aradului 119, RO-300645 Timis, oara, Romania; [email protected]
3 OncoGen Centre, County Hospital “Pius Branzeu”, Blvd. Liviu Rebreanu 156, RO-300736 Timisoara,
Romania; [email protected]
4 Faculty of Pharmacy, “Victor Babes” University of Medicine and Pharmacy, 2nd Eftimie Murgu Square,
RO-300041 Timisoara, Romania; [email protected] (M.M.); [email protected] (A.M.);
[email protected] (C.S, .)
5 Faculty of Medicine, “Victor Babes” University of Medicine and Pharmacy, 2nd Eftimie Murgu Square,





 RO-300041 Timisoara, Romania; [email protected] (D.M.); [email protected] (D.I.H.)
6 Multidisciplinary Research Center on Antimicrobial Resistance, “Victor Babes” University of Medicine and
Citation: Jianu, C.; Stoin, D.; Cocan,
Pharmacy, 2nd Eftimie Murgu Square, RO-300041 Timisoara, Romania
I.; David, I.; Pop, G.; Lukinich-Gruia, 7 Faculty of Dental Medicine, “Victor Babes” University of Medicine and Pharmacy, 2nd Eftimie Murgu Square,
A.T.; Mioc, M.; Mioc, A.; S, oica, C.; RO-300041 Timisoara, Romania; [email protected]
Muntean, D.; et al. In Silico and In 8 Multidisciplinary Center for Research, Evaluation, Diagnosis and Therapies in Oral Medicine, “Victor Babes”
Vitro Evaluation of the Antimicrobial University of Medicine and Pharmacy, Spl. Tudor Vladimirescu 14A, RO-300173 Timisoara, Romania
9 Faculty of Economics and Business Administration, West University of Timis, oara, 300233 Timisoara,
and Antioxidant Potential of Mentha
× smithiana R. GRAHAM Essential Romania; [email protected]
Oil from Western Romania. Foods * Correspondence: [email protected]
2021, 10, 815. https://doi.org/
10.3390/foods10040815 Abstract: This study was conducted to identify the volatile compounds of Mentha × smithiana essen-
tial oil (MSEO) and evaluate its antioxidant and antibacterial potential. The essential oil (EO) content
Academic Editors: Severino Zara and was assessed by gas chromatography–mass spectrometry (GC-MS). Carvone (55.71%), limonene
Giacomo Zara (18.83%), trans-carveol (3.54%), cis-carveol (2.72%), beta-bourbonene (1.94%), and caryophyllene
oxide (1.59%) were the main identified compounds. The MSEO displayed broad-spectrum an-
Received: 21 March 2021
tibacterial effects and was also found to be the most effective antifungal agent against Candida
Accepted: 7 April 2021
albicans and Candida parapsilosis. The antioxidant activity of MSEO was tested against cold-pressed
Published: 9 April 2021
sunflower oil by peroxide, thiobarbituric acid, 1,1-diphenyl-2-picrylhydrazyl radical (DPPH), and
β-carotene/linoleic acid bleaching methods. The EO showed strong antioxidant effects as reflected
Publisher’s Note: MDPI stays neutral
by IC50 values of 0.83 ± 0.01 mg/mL and relative antioxidative activity of 87.32 ± 0.03% in DPPH
with regard to jurisdictional claims in
and β-carotene/linoleic acid bleaching assays, respectively. Moreover, in the first 8 days of the incu-
published maps and institutional affil-
iations.
bation period, the inhibition of primary and secondary oxidation compounds induced by the MSEO
(0.3 mg/mL) was significantly stronger (p < 0.05) than that of butylated hydroxyanisole. In silico
molecular docking studies were conducted to highlight the underlying antimicrobial mechanism as
well as the in vitro antioxidant potential. Recorded data showed that the antimicrobial activity of
MSEO compounds could be exerted through the D-Alanine-d-alanine ligase (DDl) inhibition and may
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland. be attributed to a cumulative effect. The most active compounds are minor components of the MSEO.
This article is an open access article Docking results also revealed that several mint EO components could exert their in vitro antioxidant
distributed under the terms and activity by employing xanthine oxidase inhibition. Consequently, MSEO could be a new natural
conditions of the Creative Commons source of antioxidants and antiseptics, with potential applications in the food and pharmaceutical
Attribution (CC BY) license (https:// industries as an alternative to the utilization of synthetic additives.
creativecommons.org/licenses/by/
4.0/).

Foods 2021, 10, 815. https://doi.org/10.3390/foods10040815 https://www.mdpi.com/journal/foods

Foods 2021, 10, 815 2 of 17

Keywords: essential oil; Mentha × smithiana R. GRAHAM; antioxidant activity; antimicrobial activity;
molecular docking

1. Introduction
The consumption of minimally processed and additive-free foods has increased in
recent decades, demanding the replacement of the traditional preservation methods by the
food industry [1]. Different emerging technologies (e.g., high-pressure processing, pulsed
electric field, modified atmosphere packaging) have been studied in order to prolong the
shelf life of foodstuff [2], including the use of natural extracts and essential oils (EOs). EOs
are aromatic, volatile, and complex liquids extracted from different plant parts (flowers,
leaves, seeds, fruits, bark, roots) [3]. These are secondary metabolites mainly involved
in plants’ defensive mechanisms and usually contain monoterpenes, sesquiterpene, and
phenolic compounds, as well as oxygenated or non-oxygenated derivatives [4]. Aside
from their multiple applications in the cosmetic, pharmaceutical, and food industry, these
are also recognized for their biological properties (antimicrobial, antioxidant, carminative,
antiviral, anti-inflammation, analgesic, antispasmodic, etc.) [5–8].
Mentha species (Lamiaceae), which includes 42 species and hundreds of subspecies,
are spread worldwide, mainly in Asia, Africa, Australia, North America, and Europe [9–11].
Romanian flora includes about 25 species and several varieties and subspecies from the
genus Mentha [12]. The aerial parts (e.g., leaves, flowers, and stems) of Mentha species
have been applied for centuries in folk medicine to treat multiple dysfunctions of the gas-
trointestinal tract or cholecystopathies [12–14]. Several Mentha species, such as spearmint
(Mentha spicata), peppermint (M. × piperita), and corn mint (M. canadensis) are extensively
utilized as industrial crops for the purpose of EO production [11]. These oils have many
applications as flavoring agents in chewing gums, beverages, bakery products, cosmetics,
oral hygiene products, and pharmaceuticals [11,15]. The plants belonging to the Mentha
species are mentioned as promising free radical scavengers, as well as primary antioxi-
dants that can react with free radicals and reduce the attack of reactive oxygen species on
biological and food systems [16–18]. Additionally, multiple investigations reported the
antimicrobial and antifungal properties of the Mentha species EOs and/or extracts against
pathogenic bacteria and fungi [3,19–22]. Nevertheless, some members of the Mentha genus
remain partly explored, such as Mentha × smithiana R. GRAHAM, an accepted hybrid of
M. aquatica × M. arvensis × M. spicata [23]. To the best of our knowledge, no data have
been reported yet on the antioxidant properties of Mentha × smithiana essential oil (MSEO).
Therefore, this research investigated: (i) the chemical composition; (ii) the antimicro-
bial and antioxidant activities of MSEO; and (iii) the mechanisms of interaction between
MSEO chemical compounds and target proteins associated with antibacterial effects and
intracellular antioxidant mechanisms, thus aiming for its possible recommendation in food
and pharmaceutic industries as a green preservative.

2. Materials and Methods

2.1. Chemicals
Anhydrous sodium sulphate, C8 –C20 alkane standard mixture, hexane, chloroform,
ethanol, methanol, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), thio-
barbituric acid (TBA), 1,1-diphenyl-2-picrylhydrazyl radical (DPPH), and β-carotene were
purchased from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany). All substances
were used as received.

2.2. Essential Oil Extraction

The aerial parts (flowers, leaves, and stems) of M. smithiana, at the full flowering
phenological stage, were manually harvested from the experimental fields of Banat’s Uni-
versity of Agricultural Sciences and Veterinary Medicine “King Michael I of Romania” from
Foods 2021, 10, 815 3 of 17

Timisoara (BUASVM Timisoara) in June 2019. A voucher specimen (VSNH.BUASTM-90/1)

was collected, identified, and deposited in the BUASVM Timisoara Herbarium. MSEO
was obtained by steam distillation, using a modified Clevenger apparatus (Corning Life
Sciences, Kennebunk, ME, USA) [24], with a cooled oil collector to prevent the generation
of artefacts during isolation [25]. A slightly yellow oil with a characteristic odor and sharp
taste was obtained. After decantation, the water traces were removed by using anhydrous
sodium sulphate, and the product was deposited at −18 ◦ C until use.

2.3. Gas Chromatography–Mass Spectrometry

The MSEO was diluted 1:1000 in hexane and vortexed before injection. In total, 1 µL
of the prepared sample was injected in splitless mode in a HP6890 Gas-Chromatograph
coupled with a HP5973 Mass Spectrometer (Agilent Technologies, Santa Clara, CA, USA).
The injected sample was run into a Bruker Br-5MS capillary column (30 m × 0.25 mm
id × 0.25 µm, Bruker, Billerica, MA, USA) with helium flow of 1 mL/minute. The oven
temperature ranged from 50 ◦ C to 300 ◦ C with 6 ◦ C/minute rate and a final hold for
5 min; the solvent delay was 3 min. The mass spectrometer had the source set at 230 ◦ C,
the MS Quad at 150 ◦ C, and ionization energy at 70 eV. The compounds’ mass values
were scanned between 50 and 550 amu. The identification of the MSEO components
was based on retention indices (RIs), calculated by means of a C8 –C20 alkane standard
mixture calibration curve and subsequently compared with Adams indices [26], computer
matching with the NIST0.2 mass spectra library (USA National Institute of Science and
Technology software, NIST, Gaithersburg, MD, USA), and by co-injection with reference
samples (limonene and carvone).

2.4. Antioxidant Activity

In order to evaluate the antioxidant activity of the MSEO, cold-pressed sunflower oil
purchased from the local market was used (1.92 meq·kg−1 initial peroxide value). This oil
is frequently used in the South-East European countries’ cuisines [27] and is somewhat
unstable due to the rich content in fatty acids [28]. The MSEO antioxidant activity was
studied by peroxide, thiobarbituric acid, 1,1-diphenyl-2-picrylhydrazyl radical (DPPH),
and β-carotene/linoleic acid bleaching tests, respectively.

2.4.1. Sample Preparation

Samples were prepared by adding 0.1 mg/mL and 0.2 mg/mL, 0.3 mg/mL, respec-
tively, of MSEO to 10 mL volume of cold-pressed sunflower oil. Additionally, 0.2 mg/mL of
BHA and BHT, the maxim amount of these synthetic additives in fats and oils according to
the European Union (EU) food legislation [29], were added in 10 mL cold-pressed sunflower
oil. A control sample without any additive was prepared under similar conditions.

2.4.2. Peroxide Value

The peroxide value (PV) of the above-prepared samples, expressed in meq of oxygen
·kg−1 , was measured at 0, 4, 8, 12, 16, 20, and 24 days, according to ISO 27107: 2008, “Animal
and vegetable fats and oils. Determination of peroxide value” [30]. All measurements were
performed in triplicate.

2.4.3. Thiobarbituric Acid Value

The thiobarbituric acid (TBA) test, as described by Jianu et al. [31], was adopted; briefly,
2 g of each sample, benzene (5 mL), and 0.67% aqueous thiobarbituric acid solution (4 mL)
were mixed and continuously homogenized for 1 h with a mechanical shaker. Subsequently,
the supernatant was maintained on a hot water bath for 45 min. After cooling, the solutions
were spectrophotometrically assessed at 540 nm (Specord 210 spectrophotometer, Analytik
Jena, Jena, Germany). The TBA value (µg malondialdehyde g−1 ) was measured every
4 days, and all the measurements were performed in triplicate.
Foods 2021, 10, 815 4 of 17

2.4.4. Scavenging Effect on 1,1-Diphenyl-2-picrylhydrazyl Radical (DPPH)

The scavenging effect of the MSEO on the DPPH radical was analyzed by using
the adapted Brand-Williams method [32–34]. In total, 10 µL methanolic DPPH solution
(1 mg/mL) was mixed with each analyzed sample (100 µL) at different serial concentrations
(1.5 to 0.093 mg/mL). The absorbances were measured at 515 nm (Tecan i-control, 1.10.4.0
infinite 200Pro) after 30 min of incubation (in the dark at room temperature). BHT and
BHA served as positive controls, while methanol was used as a negative control. The
inhibition of free DPPH radical (I%) was calculated according to the following equation:
I% = (Amethanol − Asample /Amethanol ) × 100, where: Amethanol is the absorbance of the
methanol and Asample is the absorbance of the tested oil. BioDataFit 1.02 program (Chang
Broscience Inc., Fremont, CA, USA) was used to calculate the IC50 . All measurements were
performed in triplicate.

2.4.5. β-Carotene Bleaching Assay

The analysis was conducted using the method described by Oke et al. [35], with
some modifications [31]. Briefly, a β-carotene (0.5 mg) stock solution was prepared in a
mixture of chloroform (1 mL), Tween 40 (200 mg), and linoleic acid (25 µL). The chloroform
was removed under vacuum at 40 ◦ C for 5 min by using a rotary evaporator (Heidolph,
Schwabach, Germany). The residue was treated with 3% hydrogen peroxydeaqueous
solution (100 mL) and stirred vigorously (2–3 min) to obtain an emulsion. Aliquots of the
emulsion (2.5 mL) were added in the test tubes containing MSEO (350 µL); BHT was used
as a positive control. All tubes were kept for 48 h at room temperature before measuring
the absorbances at 490 nm. All measurements were performed in triplicate.

2.5. Determination of Antimicrobial Activity

2.5.1. Bacterial Strains
The MSEO antibacterial and antifungal activity was tested against six Gram-positive
and Gram-negative bacteria: Salmonella enterica serotype Typhimurium (ATCC 14028), Es-
cherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Shigella flexneri serotype
2b (ATCC 12022), Staphylococcus aureus (ATCC 25923), and Streptococcus pyogenes (ATCC
19615) and two fungus strain Candida albicans (ATCC 10231) and Candida parapsilosis (ATCC
22019) (Microbiologics, France). According to the EFSA One Health 2019 Zoonoses Re-
port, the selected microbial strains are the most frequently reported causes of foodborne
outbreaks across the EU and other reporting countries [36].

2.5.2. Antibacterial Activity Assay

The MSEO antimicrobial activity was assessed according to the Clinical and Labora-
tory Standards Institute standard [37], with some modifications [3]. A bacterial suspension
of each tested strain was prepared to a concentration of 0.5 MacFarland, approximatively
1–2 × 108 colony forming units (CFU)/mL for bacterial strains and 1–5 × 106 CFU/mL
for Candida strains. The Mueller-Hinton (MH) agar or Mueller-Hinton agar-fastidious
organisms’ agar (MHF) supplemented with defibrinated horse blood and β-nicotinamide
adenine dinucleotide (bioMérieux, Marcy-l’Étoile, France) was inoculated with 0.1 mL
standardized suspension. In total, 10 µL MSEO was added to a 6 mm diameter blank
paper disk (BioMaxima, Lublin, Poland) and deposited on the MH or MHF plates’ surface,
respectively, previously inoculated with microbial strains. The plates were incubated for
24 h at 35–37 ◦ C (for bacterial strains) and for 48 h at 28 ◦ C (for yeasts), after which the
diameters of the inhibitory halos (in millimeters) formed around the paper disk were
measured. Gentamycin (10 µg/disk) and fluconazole (25 µg/disk) disks were used as a
positive control for bacteria and yeast (BioMaxima, Lublin, Poland), respectively. Dimethyl-
sulphoxide (DMSO) was used as the negative control. All analyses were carried out in
triplicate for each microbial strain.
Foods 2021, 10, 815 5 of 17

2.5.3. Minimum Inhibitory Concentration (MIC)

MSEO strongly inhibits the growth of all tested bacterial and yeast strains; minimum
inhibitory concentration (MIC) values were determined by applying microdilution in broth
assay according to the Clinical and Laboratory Standards Institute (CLSI) guidelines M07-
A10 for bacteria [38] and European Committee on Antimicrobial Susceptibility Testing
(EUCAST) definitive document E.Def 7.2 for yeasts [39]. Standardized inoculum (0.5 Mc-
Farland meaning 108 CFU/mL for bacterial strains and 2 × 106 CFU/mL for fungal strains)
was prepared by dilution, resulting in 1–5 × 105 CFU/mL microbial suspension. Successive
dilutions of MSEO in DMSO were prepared in order to achieve various concentrations
(400, 200, 100, 50, 25, 12.5 mg/mL). In total, 0.1 mL of each MSEO dilution was treated
with 0.4 mL MH or MHF broth and 0.5 mL microbial suspension, with a final microbial
inoculum of approximately 0.5 × 105 CFU/mL. After 24 h of incubation at 35–37 ◦ C (for
bacteria) and 28 ◦ C (for yeasts), the MIC (the lowest concentration without visible growth)
was assessed. As a control, 0.1 mL DMSO was added in a tube with 0.5 mL microbial
suspension and 0.4 mL MH or MHF broth for bacteria and fungi, respectively. All analyses
were carried out in triplicate for each microbial strain.

2.5.4. Minimum Bactericidal Concentration (MBC) and Minimum Fungicidal

Concentration (MFC)
The minimum bactericidal concentration (MBC) and minimum fungicidal concentra-
tion (MFC) were determined according to the method described by Danciu et al. and Jianu
et al. [40,41], with some modifications. In total, 1 µL culture from each test tube at MIC
(with no visible growth) was inoculated using a loop (NuovaAptaca SRL, Canelli, Italy) on
Columbia agar supplemented with 5% blood and maintained for 24 h at 37 ◦ C for bacteria
or on Sabouraud supplemented with chloramphenicol medium and maintained for 48 h at
28 ◦ C for the yeast. The MBC/MFC was established as the lowest EO concentration, which
killed 99.5% of the inoculated microorganisms [42,43]. All analyses were carried out in
triplicate for each microbial strain.

2.6. In Silico Molecular Docking

Corresponding 3D structures of the protein targets (Table 1) were obtained from the
RCSB Protein Data Bank [44]. The protein structures were prepared as suitable targets,
using the Autodock Tools (version 1.5.6, The Scripps Research Institute, La Jolla, CA, USA).
Water molecules, undesired protein chains, metal atoms, and the co-crystalized ligands
were removed from the protein structure, after which polar hydrogen atoms and Gesteiger
charges were added. The target was saved as the required file format (pdbqt). Ligand
molecules corresponding to the 39 MSEO components were drawn as mol files using Biovia
Draw (Dassault Systems Biovia, San Diego, CA, USA) and were subsequently converted
into 3D structures using PyRx’s Open Babel module. Structure geometry optimization was
also achieved with Open Babel, using the ghemical force field. Compound docking was
performed with the GUI software PyRx, using Autodock Vina’s scoring function [45]. In
order to validate the docking method, the co-crystalized ligands were previously removed
from their respective proteins, prepared as suitable pdbqt files and re-docked in their
original binding sites. The predicted docking pose was compared with the experimental co-
crystallized binding pose. This docking validation was performed for all protein structures.
The grid box, which delimitates the search space, was defined in terms of coordinates and
size (Table 1) to best fit the active binding site. Data results for docked molecules were
recorded as free binding energy values (∆G, kcal/mol). Ligand-protein binding features
were analyzed using Accelerys Discovery Studio 4.1 (Dassault Systems Biovia).
Foods 2021, 10, 815 6 of 17

Table 1. Molecular docking parameters and protein targets.

Conformers
Protein PDB ID Grid Box Centre Coordonates Grid Box Size
Generated per Ligand
center_x = −26.7358277569 size_x = 19.8110325702
Isoleucyl-tRNA
1JZQ center_y = 6.92671107775 size_y = 19.2750015157 10
synthetase (IARS)
center_z = −27.8259282538 size_z = 15.5426417959
center_x = 19.4639026798 size_x = 13.8319651582
DNA gyrase 1KZN center_y = 31.387371307 size_y = 20.5700336941 10
center_z = 36.3586907625 size_z = 21.360339073
center_x = 31.8624471237 size_x = 13.8319651582
Dihydropteroate
2VEG center_y = 49.6265167401 size_y = 14.65456219 10
synthase (DHPS)
center_z = 1.88555734697 size_z = 14.9994242074
center_x = 48.3562458265 size_x = 18.0967836094
D-alanine: D-alanine
2ZDQ center_y = 18.8505150195 size_y = 8.89317511714 10
ligase (Ddl1)
center_z = −1.46703160733 size_z = 9.35800790526
center_x = −34.0399241986 size_x = 18.0967836094
Type IV topoisomerase 3RAE center_y = 68.8943424518 size_y = 14.65456219 10
center_z = −24.2150819768 size_z = 18.0726700658
center_x = −5.43716183713 size_x = 14.8382078869
Dihydrofolate
3SRW center_y = −31.0341681565 size_y = 12.9264417759 10
reductase (DHFR)
center_z = 5.38290214414 size_z = 11.0341937468
center_x = 15.5996662331 size_x = 16.9958735218
DNA gyrase subunit B 3TTZ center_y = −18.1561399124 size_y = 14.685112087 10
center_z = 7.09296891151 size_z = 12.2001752611
center_x = 34.9424942577 size_x = 25.0
Penicillin binding
3UDI center_y = 1.47896841514 size_y = 12.4533701393 10
protein 1a (PBP1a)
center_z = 9.89373816917 size_z = 21.6899971175
center_x = 22.362960394 size_x = 12.3991873959
Lipoxygenase 1N8Q center_y = 1.27287112362 size_y = 10.6627584168 10
center_z = 20.265022301 size_z = 12.0420500164
center_x = −19.8236696285 size_x = 12.397236391
CYP2C9 1OG5 center_y = 86.6979336918 size_y = 11.6533632259 10
center_z = 38.2757994523 size_z = 11.6533632259
center_x = 18.9974990948 size_x = 13.9673646775
NADPH-oxidase 2CDU center_y = −5.67040299733 size_y = 15.0103503874 10
center_z = −1.71861856213 size_z = 18.8052690382
center_x = 37.4736743805 size_x = 7.33311695257
Xanthine oxidase 3NRZ center_y = 19.3078554887 size_y = 10.3360607773 10
center_z = 18.1521505909 size_z = 9.12399788674

2.7. Statistical Analysis

Statistical analysis was carried out using the SPSSv25 software (SPSS Inc., Chicago, IL,
USA). In the preliminary analysis, the Shapiro–Wilk test was performed to test the normality
of data for each incubation period (day 0, day 4, day 8, day 12, day 16, day 20, and day 24).
Due to the non-normally found in our data, nonparametric tests were further applied. As
an overall omnibus test, Kruskal–Wallis was employed to find significant differences in
the case of independent samples, BHA, BHT, and three concentrations of MSEO (0.1, 0.2,
and 0.3 mg/mL, respectively). The Mann–Whitney test was performed to make multiple
pairwise comparisons to detect statistically significant differences between specific samples.
All the above-mentioned tests were applied for each level of the incubation period. The
Friedman test for dependent samples and repeated measurements was performed to
analyze the time evolution over the incubation period levels. Each statistical testing
procedure was applied distinctively for peroxide and TBA values. The Tukey test assessed
Foods 2021, 10, 815 7 of 17

differences among values obtained from three replicates performed in the antimicrobial
analysis. For all the testing mentioned above, differences were considered significant when
p < 0.05.

3. Results and Discussion

3.1. MSEO Chemical Composition
The steam distillation of fresh aerial parts of M. smithiana gave a slightly yellow
EO, with an average calculated yield of 1.24 ± 0.03% (expressed as mean ± SD). The
chemical composition is shown in Table 2, according to their elution order on a Br-5MS
capillary column.

Table 2. Components of essential oil (EO) from M. smithiana growing in Western Romania.

No Compound % RI a Identification b
1 alpha-Thujene tr. 912 MS, RI
2 alpha-Pinene 0.97 918 MS, RI
3 Camphene 0.37 933 MS, RI
4 alpha-Phellandrene 0.45 954 MS, RI
5 beta-Pinene 0.87 959 MS, RI
6 beta-Myrcene 0.59 970 MS, RI
7 3-Octanol 0.31 976 MS, RI
8 p-Mentha-1 (7),8-diene 0.09 985 MS, RI
9 p-Cymene 0.23 1006 MS, RI
10 Limonene 18.83 1013 MS, RI, co-GC
11 Eucalyptol 0.96 1015 MS, RI
12 Terpineol, cis-beta 0.12 1054 MS, RI
13 Linalool 0.33 1087 MS, RI
14 Nonanal 0.06 1092 MS, RI
15 3-Octanol, acetate 0.07 1109 MS, RI
16 trans-p-Mentha-2,8-dien-1-ol 0.25 1111 MS, RI
17 cis-Limonene oxide 0.12 1124 MS, RI
18 cis-p-Mentha-2,8-dien-1-ol 0.47 1128 MS, RI
19 Isopinocarveol 0.08 1133 MS, RI
20 cis-Verbenol 0.06 1139 MS, RI
21 Menthone 0.48 1149 MS, RI
22 Borneol 0.76 1168 MS, RI
23 p-Menthan-1-ol 1.05 1175 MS, RI
24 cis-Dihydro carvone 0.84 1197 MS, RI
25 cis-Carveol 2.72 1222 MS, RI
26 trans-Carveol 3.54 1226 MS, RI
27 Carvone 55.71 1256 MS, RI, co-GC
28 cis-Carvone oxide 0.60 1284 MS, RI
29 (1R,4R)-p-Mentha-2,8-diene, 1-hydroperoxide 0.35 1332 MS, RI
30 Limonene-diol 1.07 1359 MS, RI
31 Carveol acetate 0.57 1374 MS, RI
32 Lavamenthe 0.89 1388 MS, RI
33 8-Oxabicyclo [5.1.0]oct-2-en-4-one, 3,6,6-trimethyl 0.34 1396 MS, RI
34 beta-Bourbonene 1.94 1402 MS, RI
35 cis-Jasmone 0.38 1409 MS, RI
36 beta-Cubebene 0.26 1450 MS, RI
37 (−)-Calamenene 0.28 1540 MS, RI
38 (−)-Spathulenol 0.37 1595 MS, RI
39 Caryophyllene oxide 1.59 1601 MS, RI
Total 98.97%
a b
The retention index (RI) was calculated using a homologous series of n-alkanes C8 –C20 ; co-GC: Co-injection with an authentic sample; tr.
(trace): <0.05.

Thirty-nine compounds, accounting for 98.97% of total oil compositions, were iden-
tified. The major components were carvone (55.71%), limonene (18.83%), trans-carveol
Foods 2021, 10, 815 8 of 17

(3.54%), cis-carveol (2.72%), beta-bourbonene (1.94%), and caryophyllene oxide (1.59%),

comprising 84.33% of the EO. A previous study, conducted in Romania, also reported
carvone (72.72%) and limonene (14.66%) as the major compounds of MSEO [46]. On the
other hand, Lawrence reported carvone (22.1–38.4%) together with dihydrocarvyl acetate
(16.8%), beta-pinene (17.1%), 1,8-cineole (10.9%), and beta-caryophyllene (10.4%) as the
main components of MSEO [23]. The yield and compositional variation of the EOs may
be due to the harvesting time at different stage, storage, and extraction methods [47]
and also depends on the physiological and environmental conditions (e.g., seasonal and
geographical location, soil composition, climate) [11,48].

3.2. Assessment of Antioxidant Activity

The assessment of the MSEO antioxidant activity has been conducted on cold-pressed
sunflower oil. Figure 1A shows the changes in PVs of the investigated cold-pressed sun-
flower oil samples. The PV measures the total peroxide and hydroperoxide oxygen content
of the edible oil system [49]. The Shapiro–Wilk test was applied to test the distribution
of experimental data. The analysis showed that most of our data did not have a normal
distribution (p < 0.05), except for the initial day (p = 0.619) and the fourth day (p = 0.36),
which concluded the use of non-parametric tests. For each storage period time, we applied
the Kruskal–Wallis test to see if we have differences between the PVs of all six tested
samples. In all cases, we obtained extremely significant differences (p < 0.001), tested the
variance of the samples using the ANOVA test, and obtained the same statistical conclusion
(p < 0.001).
Further on, we applied the Mann–Whitney test to compare two samples at the time.
The tests revealed that the PVs of the samples treated with MSEO were significantly
different from those treated with BHA and BHT. One can notice that, for the initial day, the
MSEO (0.2 mg/mL and 0.3 mg/mL, respectively) had significantly lower PVs compared
to BHA (p = 0.007; p = 0.02). For day four, we obtained even better results for MSEO
(0.2 mg/mL and 0.3 mg/mL, respectively) compared to BHT (p < 0.001). The same results
were obtained when the MSEO (0.1, 0.2, and 0.3 mg/mL, respectively) was compared with
the BHA (p < 0.001). On day eight, the MSEO (0.1, 0.2, and 0.3 mg/mL) had registered
the same tendency versus BHA (p < 0.001). Moreover, we wanted to compare the treated
samples’ PV variance between the storage days by using the Friedman test. The test
revealed extremely significant differences (p < 0.001) between the time points included in
the PV daily evolution study.
Peroxides are products of the primary lipid oxidation, which subsequently decom-
posed into carbonylic and other compounds. The peroxide decomposition products may
catalyze further oxidation. TBA values of the cold-pressed sunflower oil samples were also
recorded during the storage period to measure such secondary oxidation products.
The TBA value quantifies the malondialdehyde produced from unsaturated fatty acids
that result from the oxidation of a lipid system [50]. Figure 1B shows the changes in the TBA
values of the studied samples. The Shapiro–Wilk normality test revealed that, besides the
initial day, where we had a normal distribution (p = 0.48), the other data were not normally
distributed (p < 0.05). Extremely significant differences (p < 0.001) between the TBA values
of the tested MSEO samples (0.1, 0.2, and 0.3 mg/mL, respectively) were obtained after
running the Kruskal–Wallis test for each testing period. To strengthen this conclusion,
we ran a variance analysis between the samples by applying an ANOVA test, which led
to the same statistical conclusion (p < 0.001). In order to emphasize the most important
differences related to this study, we tested two samples at the time by applying the Mann–
Whitney test. We also analyzed the possible differences that can be measured between the
TBA values of MSEO (0.1, 0.2, and 0.3 mg/mL), BHA, and BHT, respectively. On the first
day, significantly lower TBA values were obtained in the case of MSEO (0.2 mg/mL and
0.3 mg/mL) compared to BHT (p = 0.014; p = 0.009) and MSEO (0.2 mg/mL and 0.3 mg/mL)
compared to BHA (p = 0.03; p < 0.001). On day four, we observed extremely significantly
lower TBA values in the case of MSEO (0.3 mg/mL) versus BHT and BHA (p < 0.001). The
 3.2. Assessment of Antioxidant Activity
The assessment of the MSEO antioxidant activity has been conducted on cold-
pressed sunflower oil. Figure 1A shows the changes in PVs of the investigated cold-
pressed sunflower oil samples. The PV measures the total peroxide and hydroperoxide
Foods 2021, 10, 815 9 of 17
oxygen content of the edible oil system [49]. The Shapiro–Wilk test was applied to test the
distribution of experimental data. The analysis showed that most of our data did not have
a normal distribution (p < 0.05), except for the initial day (p = 0.619) and the fourth day (p
= 0.36),
samewhich concluded
tendency was notedthe use of non-parametric
for day tests. For
eight with extremely each storage
significantly period
lower TBAtime,
valueswe in
applied theofKruskal–Wallis
the case MSEO (0.3 mg/mL) test to see if wetohave
compared BHTdifferences
(p < 0.001). between the PVs
Additionally, of alltreated
for each six
tested samples.
sample, In allthecases,
we tested dailywe obtained
evolution by extremely significant
applying the Friedmandifferences (p <extremely
test, revealing 0.001),
tested the variance
significant of the samples
differences using the ANOVA test, and obtained the same statistical
(p < 0.001).
conclusion (p < 0.001).
a
16 a a
A
a a
Peroxide value (meq/kg)

a a
12 a Control sample
a a a a
a
a a a BHT
a a BHA
a a a
8 a a MSEO 0.1 mg/mL
ab b a MSEO 0.2 mg/mL
a a a
ab MSEO 0.3 mg/mL
4 abc bc c ab a a
ab ab b ab a a

0
0 4 8 12 16 20 24 Storage time (days)
a
B
TBA value (μg malondialdehyde · g–1)

25
a

a a Control sample
20
BHT
a a
a a a BHA
15 a
a a MSEO 0.1 mg/mL
a a
a MSEO 0.2 mg/mL
ab a MSEO 0.3 mg/mL
10 a a
ab ab a
abc a a
a a a
abc ab a
5 abc abc b
b c a a abc b c a

0
0 4 8 12 16 20 24 Storage time (days)

Figure 1. The
Figure effect
1. The of Mentha
effect × smithiana
of Mentha essential
× smithiana oil (MSEO),
essential butylated
oil (MSEO), hydroxyanisole
butylated hydroxyanisole(BHA), andand
(BHA), butylated hydroxy-
butylated hydroxy-
toluene (BHT) on peroxide values (PVs) (A) and thiobarbituric acid (TBA) values, (B) of cold-pressed sunflower oil
toluene (BHT) on peroxide values (PVs) (A) and thiobarbituric acid (TBA) values, (B) of cold-pressed sunflower oil during
during
24 days of the storage period. Values are the mean, and the error bars indicate standard deviations. Letters indicate ho-
24 days of the storage period. Values are the mean, and the error bars indicate standard deviations. Letters indicate
mogenous groups within incubation periods (p > 0.05).
homogenous groups within incubation periods (p > 0.05).

DPPH-radical scavenging models are frequently employed in antioxidant experiments

due to their suitable stability [51,52]. DPPH radicals can be scavenged based on the ability
of EOs to donate hydrogen radicals to DPPH free radicals, thus reducing them to DPPH-H
(2,2-diphenyl-1-picrihydrazine) [53]. The color of DPPH-H turns from purple to yellow,
a process that can be quantified by measuring the absorbance at 517 nm [51]. The MSEO
antioxidant activity, evaluated by the DPPH radical scavenging assay and expressed
as 50% inhibition (IC50 ), is shown in Table 3. Despite the fact that the IC50 values of
the MSEO (IC50 : 0.83 ± 0.01 mg/mL) and BHA (IC50 : 0.76 ± 0.01 mg/mL) are rather
close, when we tested the statistical significance of their scavenging activity, extremely
significant differences (p < 0.001) were revealed. In contrast, BHT exhibits an extremely
significantly (p < 0.001) stronger antioxidant activity (IC50 : 0.43 ± 0.08 mg/mL) than
MSEO (Table 3). No previous investigations were reported in the literature concerning
Foods 2021, 10, 815 10 of 17

the DPPH radical scavenging capacity of MSEO. However, de Sousa Barros et al. [54]
reported a similar IC50 value (0.86 ± 0.01 mg/mL) for the EO of M. longifolia (Himalayan
silver mint) grown in Brasil. Compared to results reported on M. × rotundifolia from
Tunisia (IC50 : 3.77 mg/mL) [55], M. pulegium from Iran (IC50 : 14.736 ± 0.156 mg/mL) [56],
M. × piperita (IC50 : 5.72 ± 0.06 mg/mL), M. aquatica (IC50 : 6.75 ± 0.23 mg/mL), and M.
arvensis (IC50 : 57.72 ± 0.11 mg/mL) from Brasil [54], the MSEO exhibited higher activity. In
contrast, EOs isolated from M. spicata from Tunisia (IC50 : 10 ± 0.24 µg/mL) [19], Iran (IC50 :
13.3 ± 0.6 µg/mL) [57], and Cyprus (IC50 : 7.74 ± 0.20 µg/mL) [58] were more effective in
scavenging DPPH free radical.

Table 3. Antioxidant activity of the essential oil of M. smithiana growing in western Romania.

Parameter MSEO BHA a BHT b

DPPH, IC50 (mg/mL) 0.83 ± 0.01 0.76 ± 0.01 0.43 ± 0.08
β-carotene bleaching (RAA c ) (%) 87.32 ± 0.03 Nd d 100
a Butylated hydroxyanisole (BHA); b butylated hydroxytoluene (BHT); c relative antioxidative activity (RAA);
d not detected (Nd).

In the β-carotene and linoleic acid test, in the absence of an antioxidant, β-carotene
undergoes rapid bleaching due to oxidation, which leads to the formation of free radicals.
The new radicals generated by the loss of a hydrogen atom from the diallylic methylene
groups attack the unsaturated β-carotene molecules, which they oxidize and partially
decompose [59]. The rate of β-carotene discoloration can be decreased in the presence
of antioxidants [31,60]. The relative antioxidant activity (RAA) of MSEO was calculated
according to the following equation: RAA = AMSEO /ABHT , where ABHT is the absorption
of BHT (positive control used) and AMSEO is the absorption of MSEO. Higher RAA values
translated to higher antioxidant capacity. Tested through the linoleic acid system, MSEO
exhibited strong antioxidant activity (87.32 ± 0.03%); however, RAA% was lower than the
activity calculated for BHT (100%) (Table 3). No previous reports concerning the β-carotene
and linoleic acid test expressed as RAA were available on the Mentha species’ antioxidant
activity to compare the results directly.

3.3. Assessment of Antimicrobial Activity

The antibacterial activity was tested against eight bacterial and fungal strains (Table 4).
The diameters of the inhibition halos induced by MSEO against the tested microbial strains
varied between 17.66 ± 0.57 mm and 32.33 ± 2.51 mm, suggesting that the oil exerts
broad-spectrum antimicrobial effects.

Table 4. Antimicrobial of the essential oil of M. smithiana growing in Western Romania by disk diffusion, minimum inhibitory
concentration (MIC), minimum bactericidal concentration (MBC), and minimum fungicidal concentration (MFC) 1 .

Disk Diffusion MIC Value MBC Value MFC Value

Bacterial and Yeast Strains
(mm) (mg/mL) (mg/mL) (mg/mL)
Streptococcus pyogenes (ATCC 19615) 29.33 ± 0.57 5 10 N.T.
Staphylococcus aureus (ATCC 25923) 27.66 ± 0.57 10 10 N.T.
Escherichia coli (ATCC 25922) 19.66 ± 0.57 20 20 N.T.
Salmonella typhimurium (ATCC 14028) 17.66 ± 0.57 20 20 N.T.
Shigella flexneri (ATCC 12022) 18.33 ± 0.57 20 20 N.T.
Pseudomonas aeruginosa (ATCC 27853) 19.33 ± 2.08 20 20 N.T.
Candida albicans (ATCC 10231) 32.33 ± 2.51 2.5 N.T. 2.5
Candida parapsilosis (ATCC 22019) 31.33 ± 1.52 2.5 N.T. 2.5
1The diameters of the inhibition halos are presented as the mean (n = 9) ± standard deviation, and the mean value for MIC, MBC, and
MFC; N.T. not tested; no significant difference (p > 0.05) was observed by applying the Tukey test.
Foods 2021, 10, 815 11 of 17

The results revealed that C. albicans and C. parapsilosis were the most susceptible tested
strains to the MSEO action, followed by S. pyogenes > S. aureus > E. coli > P. aeruginosa >
S. flexneri > S. typhimurium. Our results are in agreement with Jianu et al. [46], which re-
ported that MSEO exerted significant antimicrobial activity against S. aureus, S. typhimurium,
P. aeruginosa, and C. albicans. Furthermore, the oils extracted from other members of the
mint genus (e.g., M. longifolia, M. spicata, M. viridis, M. suaveolens) significantly inhibited the
growth of P. aeruginosa, L. monocytogenes, C. albicans, S. typhimurium, and S. aureus [19–22].
The recorded MICs of the tested strains were 2.5, 5, 10, and 20 mg/mL, respectively. The
MBCs and MFCs were 2.5, 10, and 20 mg/mL, respectively. The MICs and MBCs were con-
sistent with those of the inhibition zones; that is, the larger the inhibition zones’ diameter
was, the smaller the MICs and MBCs would be (Table 4). Overall, the Gram-positive strains
were more susceptible to the oil’s action than the Gram-negative strains, in agreement with
previous investigations [61]. These susceptibility differences were presumably due to the
presence of phospholipids and lipopolysaccharides in the composition of Gram-negative
bacteria membrane, which provides protection against the external environment [20,62].

3.4. In Silico Prediction of Mechanism by Molecular Docking Analysis

Molecular docking is a powerful tool, often used to gain valuable insight into the
possible molecular mechanisms of pharmacologically active substances. Herein molecular
docking was employed to identify a possible mechanism of action correlated with the
recorded antimicrobial effect of MSEO components. This process was used to evaluate
the binding affinity of the 39 compounds from the essential oil to target proteins, usually
correlated with bactericidal/bacteriostatic effects, such as DHPS, DHFR, Ddl, penicillin
binding protein 1a PBP1a, DNA gyrase, type IV topoisomerase, and isoleucyl-tRNA syn-
thetase IARS. Molecular docking was also used to assess the studied compounds’ potential
to act as protein inhibitors of targets involved in intracellular antioxidant mechanisms. For
this purpose, lipoxygenase, CYP2C9, NADPH-oxidase, and xanthine oxidase was used as
protein targets.
The molecular docking results are shown as free binding energies (kcal/mol), the
lowest values indicating a high affinity for the target protein (Table 5). The 39 compounds
identified by GC/MS analysis on MSEO represent monoterpene or derived monoterpene
structures, most of them having structurally similar scaffolds. Therefore, docking scores
were presented as a heat map-type table, using a red-yellow color scheme, ranging from
the lowest energy values, highlighted in red (in most cases corresponding to the docking
score of the native ligand) to the highest, highlighted as yellow (Table 5), to easily identify
a tendency of a set of compounds to act as potential inhibitor compounds with the lowest
values close to that of the cocrystalized ligand) for a certain protein.
Monoterpenes are secondary plant metabolites with known antioxidant effects exerted
mostly due to their conjugated double bond system within their structure. Some derivatives
exhibit this effect by the additional presence of easily oxidizable groups, such as phenolic
or alcoholic hydroxyls [63]. The docking results for the second subset of target proteins
showed a tendency for several compounds to behave as potential inhibitors for xanthine
oxidase (3NRZ). Xanthine oxidoreductase is the enzyme responsible for the catalyzed
oxidation of hypoxanthine to xanthine followed by xanthine’s transformation to uric acid.
In addition to this function, mammalian xanthine oxidase is a physiological source of
reactive oxygen species (ROS), such as superoxide ion and hydrogen peroxide, which can
function as second messengers in activating various pathways [64].
Foods 2021, 10, 815 12 of 17

Table 5. Heat map of recorded docking scores (binding free energy—kcal/mol) of the essential oil of M. smithiana
components 1 .
Protein PBD ID 1JZQ 1KZN 2VEG 2ZDQ 3RAE 3SRW 3TTZ 3UDI 1N8Q 1OG5 2CDU 3NRZ
Ligand Binding Free Energy ∆G (kcal/mol)
Native co-crystalized ligand −8.3 −9.4 −6.9 −6.2 −5.6 −10.0 −8.5 −7.4 −5.8 −9.8 −9.3 −6.7
(1R,4R)-4-Isopropenyl-1-methyl-2-
cyclohexen-1-yl −5.7 −6.6 −5.1 −6.6 −3.9 −6.1 −6.2 −5.6 −4.6 −6.4 −6.1 −7.4
hydroperoxide
3,6,6-Trimethyl-8-oxabicyclo[5.1.0]oct-2-
−5.6 −5.7 −4.7 −6.5 −4.3 −6.1 −5.7 −5.2 −3.3 −5.8 −6.0 −3.1
en-4-one
3-octanol −4.5 −4.6 −4.0 −4.8 −3.0 −4.7 −4.7 −3.8 −5.1 −4.8 −4.4 −5.6
3-octanyl acetate −5.3 −5.3 −4.3 −5.4 −3.5 −5.2 −5.3 −4.4 −5.0 −5.1 −5.1 −5.3
Alpha-phellandrene −5.8 −6.2 −4.6 −6.0 −3.6 −5.7 −5.9 −4.9 −6.0 −6.7 −6.0 −7.2
Alpha-pinene −5.1 −5.8 −4.2 −5.0 −3.3 −5.8 −5.5 −4.6 −5.6 −6.0 −5.9 −3.3
Alpha-tujene −5.0 −5.5 −3.8 −5.2 −3.4 −5.7 −5.3 −4.7 −6.5 −5.7 −5.6 −5.2
Beta-myrcene −6.9 −6.4 −5.0 −3.5 −3.9 −7.6 −6.6 −5.7 −0.6 −7.3 −6.8 −1.2
Beta-pinene −5.5 −5.0 −4.2 −5.3 −3.4 −5.3 −5.3 −4.3 −5.3 −5.5 −4.9 −6.2
Beta-bourbonene −5.2 −6.0 −4.0 −5.4 −3.5 −5.7 −5.5 −4.6 −5.1 −5.9 −5.5 −4.5
Beta-cubebene −6.7 −6.4 −5.1 −4.7 −4.1 −7.8 −6.8 −5.5 −4.4 −7.4 −6.8 2.1
Borneol −5.1 −4.3 −4.3 −4.2 −3.3 −5.5 −4.7 −4.9 −2.8 −5.7 −5.3 2.0
Calamenene −6.7 −6.2 −5.3 −6.3 −3.9 −7.6 −7.7 −6.1 −3.8 −7.6 −7.3 0.9
Camphene −5.2 −4.6 −3.9 −4.7 −3,0 −5.4 −4.8 −4.5 −3.9 −5.7 −5.6 1.0
Cariophillene oxyde −7.1 −6.7 −5.4 −6.4 −4.3 −8.0 −6.9 −6.1 −0.4 −7.7 −7.2 1.5
Carveol −5.7 −6.0 −4.7 −6.0 −3.8 −5.8 −6.1 −5.1 −5.8 −6.3 −6.1 −6.9
Carvone oxide −5.4 −5.3 −4.5 −6.0 −3.9 −5.9 −5.6 −5.6 −4.0 −5.9 −5.9 −4.6
Carvone −5.9 −6.0 −4.8 −6.1 −3.8 −5.9 −6.0 −5.1 −5.3 −6.5 −6.2 −7.3
Carvyl acetate −6.1 −6.8 −5.2 −6.6 −4.2 −6.5 −6.9 −5.6 −3.5 −6.7 −6.4 −5.6
Cis-Dihydrocarvone −5.8 −6.0 −4.8 −6.1 −3.7 −5.9 −6.0 −5.1 −4.9 −6.4 −6.1 −7.2
Cis-Limonene oxide −5.8 −6.1 −4.8 −6.0 −3.6 −5.9 −6.0 −5.0 −5.7 −6.1 −6.0 −6.7
Cis-jasmone −5.6 −5.8 −4.5 −6.0 −3.5 −5.7 −5.9 −5.1 −4.4 −6.4 −5.8 −7.1
Cis-p-Mentha-2,8-dien-1-ol −5.6 −6.3 −4.8 −6.1 −3.9 −6.0 −5.9 −5.1 −5.3 −6.3 −5.9 −7.8
Cis-verbenol −5.3 −5.1 −4.3 −5.5 −3.8 −6.1 −5.4 −5.1 −4.7 −5.8 −5.7 0.3
Eucalyptol −5.5 −4.6 −3.9 −4.9 −3.8 −5.8 −5.0 −4.8 −3.3 −5.5 −5.9 2.9
Isopinocarveol −5.4 −4.9 −4.3 −5.4 −3.7 −6.1 −5.4 −5.3 −4.4 −5.6 −5.7 −1.3
Lavamenthe −6.2 −6.5 −4.9 −6.7 −4.2 −6.3 −6.3 −5.8 −3.2 −6.8 −6.0 −7.1
Limonene diol −5.6 −6.1 −5.1 −6.2 −4.1 −5.9 −6.1 −5.8 −5.2 −6.0 −6.3 −6.3
Limonene −5.4 −5.8 −4.3 −5.6 −3.5 −5.6 −5.8 −4.6 −5.6 −6.3 −5.7 −6.8
Linalool −5.4 −5.4 −4.3 −5.7 −3.4 −5.7 −5.9 −4.6 −4.8 −5.5 −5.0 −5.0
Menthone −5.2 −5.7 −4.3 −5.8 −3.9 −5.8 −5.8 −5.1 −4.6 −6.3 −5.6 −7.0
Nonanal −4.4 −4.8 −4.0 −4.5 −3.0 −4.7 −4.9 −3.9 −4.9 −4.9 −4.7 −5.7
P-cymene −5.5 −5.8 −4.5 −5.7 −3.5 −5.6 −5.7 −4.7 −6.0 −6.2 −5.7 −6.9
P-Mentha-1(7),8-diene −5.4 −5.8 −4.4 −5.6 −3.5 −5.6 −5.8 −4.6 −5.5 −6.3 −5.7 −6.8
P-menthan-1-ol −5.5 −6.2 −4.7 −5.9 −3.7 −5.7 −5.8 −5.1 −4.0 −6.1 −5.8 −7.2
Spathulenol −6.8 −6.4 −5.4 −5.5 −4.3 −8.0 −7.1 −6.0 −1.2 −7.9 −7.1 4.5
Terpineol, cis-beta −5.6 −6.2 −4.7 −6.0 −3.8 −5.7 −5.8 −5.1 −4.1 −6.2 −5.8 −7.4
Trans-p-Mentha-2,8-dien-1-ol −5.7 −6.2 −4.7 −6.0 −3.8 −5.7 −5.8 −5.1 −4.4 −6.3 −5.8 −7.6
1 Color scale varies from red to yellow (lowest recorded binding free energy to highest). First subset (left) corresponds to targets involved
in antimicrobial activity, while the second subset (right) corresponds to proteins involved in antioxidant activity.

According to the obtained docking scores, 15 of the analyzed compounds show

superior affinity compared to the native cocrystallized ligand (hypoxanthine), registering
free binding energies lower than the value calculated for hypoxanthine (−6.7 kcal/mol).
Our predictions are in line with the findings of a previously published study, which
highlighted the antioxidant potential of rich monoterpene EOs obtained from the genus
Oscimum, assessed by HPLC-based hypoxanthine/xanthine oxidase assay [65]. Therefore,
we can assume that the MSEO can exert an in vitro antioxidant effect through xanthine
oxidase inhibition. The best scoring structures were the two isomers (cis-trans) of p-mentha-
2,8-dien-1-ol, with the cis isomer recording the lowest binding energy (−7.8 kcal/mol).
Binding analysis showed that the structure was well accommodated in the xanthine oxidase
binding pocket through 3 HB formed with Glu802, Glu1261, and Ala1079, by means of the
hydroxyl group, and was well stabilized by several additional hydrophobic interactions
(Figure 2).
 can assume that the MSEO can exert an in vitro antioxidant effect through xanthine oxi-
dase inhibition. The best scoring structures were the two isomers (cis-trans) of p-men-
tha−2,8-dien−1-ol, with the cis isomer recording the lowest binding energy (−7.8 kcal/mol).
Binding analysis showed that the structure was well accommodated in the xanthine oxi-
Foods 2021, 10, 815 dase binding pocket through 3 HB formed with Glu802, Glu1261, and Ala1079, by means 13 of 17
of the hydroxyl group, and was well stabilized by several additional hydrophobic inter-
actions (Figure 2).

Figure2.2.Structure
Figure Structureof
ofxanthine
xanthineoxidase
oxidase(3NRZ)
(3NRZ)in incomplex
complexwith
withcis-p-Mentha-2,8-dien-1-ol;
cis-p-Mentha−2,8-dien−1-ol;hydrogen
hy-
drogen bond interactions are depicted as green dotted lines, hydrophobic interactions as purple
bond interactions are depicted as green dotted lines, hydrophobic interactions as purple dotted lines,
dotted lines, and electrostatic interactions in orange; interacting amino acids are shown as green
and electrostatic interactions in orange; interacting amino acids are shown as green sticks.
sticks.
Regarding the set of target protein structures involved in antimicrobial activity, the
Regarding the set of target protein structures involved in antimicrobial activity, the
results indicate an increased affinity of most docked structures towards the D-Alanine-
results indicate an increased affinity of most docked structures towards the D-Alanine-d-
d-alanine ligase (DDI) (2ZDQ). DDl is the enzyme that catalyzes the condensation of
alanine ligase (DDI) (2ZDQ). DDl is the enzyme that catalyzes the condensation of two D-
two D-Ala molecules using ATP to produce d-Ala-d-Ala, which is the terminal pep-
Ala molecules using ATP to produce d-Ala-d-Ala, which is the terminal peptide of a pep-
tide of a peptidoglycan monomer. The cell-wall peptidoglycan polymer is produced
tidoglycan monomer. The cell-wall peptidoglycan polymer is produced through cross-
through cross-linking of peptidoglycan monomer units [66]. Of the docked compounds,
linking of peptidoglycan monomer units [66]. Of the docked compounds, 17 showed sim-
17 showed similar affinity for DDl compared to that of the cocrystallized ligand (alanine,
ilar affinity for DDl compared to that of the cocrystallized ligand (alanine, −6.2 kcal/mol)
−6.2 kcal/mol) and 6 of them ((1R,4R)-4-Isopropenyl-1-methyl-2-cyclohexen-1-yl hydroper-
and 6 of them ((1R,4R)−4-Isopropenyl−1-methyl−2-cyclohexen−1-yl hydroperoxide, −6.6
oxide, −6.6 kcal/mol; 3,6,6-Trimethyl-8-oxabicyclo [5.1.0] oct-2-en-4-one, −6.5 kcal/mol;
kcal/mol; 3,6,6-Trimethyl−8-oxabicyclo [5.1.0] oct−2-en−4-one, −6,5 kcal/mol; Calamenene
Calamenene −6.3 kcal/mol; Cariophillene oxyde, −6.4 kcal/mol; Carvyl acetate,
−6.3 kcal/mol; Cariophillene oxyde, −6.4 kcal/mol; Carvyl acetate, −6.6 kcal/mol; Lava-
−6.6 kcal/mol; Lavamenthe, −6.7 kcal/mol) showed lower binding energies than those
menthe, −6.7 kcal/mol) showed lower binding energies than those recorded for the cocrys-
recorded for the cocrystallized ligand, indicating a possible higher affinity for the target
tallized ligand, indicating a possible higher affinity for the target protein. Other studies
protein. Other studies have shown that monoterpenes, such as menthol, thymol, and
have shown that monoterpenes, such as menthol, thymol, and linalyl acetate, also exert
linalyl acetate, also exert their bactericidal effect by bacterial cell wall denaturation, causing
their bactericidal effect by bacterial cell wall denaturation, causing leakage of essential
leakage of essential nutrients [67]. Therefore, the assumption that one of the antibacterial
nutrients [67]. Therefore, the assumption that one of the antibacterial mechanisms of the
mechanisms of the EO’s monoterpene components is the inhibition of bacterial wall syn-
thesis, by inhibiting the DDl enzyme, is highly plausible. These results also support the
fact that the antibacterial effect of the MSEO is mainly attributed to the lower concentration
components. According to our results, while carvone (first major component) showed good
binding affinity (−6.1 kcal/mol), slightly lower than the value calculated for Ala, limonene
(second major component) is not ranked between the 17 active compounds mentioned
above. In fact, the 6 compounds with the highest predicted affinity make up a little over
4% of the total oil composition.
Lavamenthe was recorded as the compound with the best docking score. Binding
analysis showed a good accommodation of the structure within the protein binding site
(Figure 3A). The oxo group is very well oriented, forming three hydrogen bonds (HBs) with
Arg268, Gly288, and Ser293. The structure also interacts with ATP through an electrostatic
interaction and is stabilized in the binding pocket through several hydrophobic interactions.
On the other hand, compounds, such as 3,6,6-Trimethyl-8-oxabicyclo [5.1.0] oct-2-en-4-one,
bearing a different scaffold than the usual monoterpene structure also recorded very good
 oil composition.
Lavamenthe was recorded as the compound with the best docking score. Binding
analysis showed a good accommodation of the structure within the protein binding site
(Figure 3A). The oxo group is very well oriented, forming three hydrogen bonds (HBs)
Foods 2021, 10, 815
with Arg268, Gly288, and Ser293. The structure also interacts with ATP through an elec- 14 of 17
trostatic interaction and is stabilized in the binding pocket through several hydrophobic
interactions. On the other hand, compounds, such as 3,6,6-Trimethyl−8-oxabicyclo [5.1.0]
oct−2-en−4-one, bearing a different scaffold than the usual monoterpene structure also
docking
recorded very good scores.
docking Binding
scores. analysis
Binding showed
analysis a somewhat
showed different
a somewhat interaction
different inter- in the protein
binding
action in the protein pocketpocket
binding (Figure 3B). The
(Figure compound
3B). formedformed
The compound two HBs onHBs
two opposite
on op-sides of the core
ring,
posite sides of the with
core Ser159
ring, withand Ser293,
Ser159 andand interacted
Ser293, with His82
and interacted through
with His82 athrough
hydrophobic
a interaction
but did not interact with the ATP structure.
hydrophobic interaction but did not interact with the ATP structure.

Figure 3.ofStructure
Figure 3. Structure of DDl
DDl (2ZDQ) (2ZDQ) in
in complex complex
with with lavamenthe
lavamenthe (A) and 3,6,6-Trimethyl−8-oxabi-
(A) and 3,6,6-Trimethyl-8-oxabicyclo [5.1.0] oct-2-en-4-one
cyclo [5.1.0] oct−2-en−4-one (B); hydrogen bond interactions are depicted as green dotted lines,
(B); hydrogen bond interactions are depicted as green dotted lines, hydrophobic interactions as purple dotted lines, and
hydrophobic
electrostatic interactions interactions as purple dotted
in orange; interacting lines,are
aminoacids andshown
electrostatic
as greeninteractions
sticks. in orange; interact-
ing aminoacids are shown as green sticks.
4. Conclusions
4. Conclusions Our findings revealed that MSEO is rich in monoterpene or monoterpene derivatives,
Our findings revealed
mainly that MSEO
carvone, is rich
limonene, in monoterpene
trans-carveol, or monoterpene
cis-carveol, derivatives,
beta-bourbonene, and caryophyllene
mainly carvone,oxide.
limonene,
MSEOtrans-carveol,
in different cis-carveol, beta-bourbonene,
concentrations (0.1, 0.2, and and caryophyllene
0.3 mg/mL, respectively) and
oxide. MSEO instorage
different concentrations
period time points (0.1, 0.2, and
inhibits the0.3 mg/mL, respectively)
formation of primary and andsecondary
stor- oxidation
age period timeproducts.
points inhibits the formation
Moreover, the results ofof primary and −
1,1-diphenyl secondary oxidation
2-picrylhydrazyl andprod-
β-carotene/linoleic
ucts. Moreover,acidthe bleaching assays indicated that the MSEOand
results of 1,1-diphenyl−2-picrylhydrazyl exhibits a strong antioxidant
β-carotene/linoleic acid activity. The
bleaching assays oilindicated
also exerted
thatbroad-spectrum
the MSEO exhibits antibacterial and antifungal
a strong antioxidant effects.
activity. TheMolecular
oil docking
studies show antibacterial
also exerted broad-spectrum that the MSEO andcompounds’ antimicrobial
antifungal effects. Molecular activity could
docking be exerted due to
stud-
ies show that thetheMSEO
DDl enzyme
compounds’inhibition and may activity
antimicrobial be attributed
could tobe aexerted
cumulative
due to effect;
the however, the
most active
DDl enzyme inhibition andcompounds are minor
may be attributed to components
a cumulative ofeffect;
the oil.however,
In addition,
the the
mostresults suggested
active compounds are minor components of the oil. In addition, the results suggested that inhibition. In
that the in vitro antioxidant activity presumably employs xanthine oxidase
light of these
the in vitro antioxidant findings,
activity MSEOemploys
presumably might represent
xanthineaoxidase
new source of natural
inhibition. preservatives with
In light
potential
of these findings, MSEO application in theafood
might represent newand pharmaceutical
source industries. with po-
of natural preservatives
tential application in the food and pharmaceutical industries.
Author Contributions: Conceptualization and methodology, C.J., C.S, ., and D.S.; investigation,
Author Contributions: Conceptualization
D.M., G.P., and
A.T.L.-G., L.-C.R., methodology,
A.M., M.M., and, C.J., C.Ș., and analysis,
I.C.; statistical D.S.; investigation,
I.G.; writing—original draft
D.M., G.P., A.T.L.-G., L.-C.R., C.J.,
preparation, A.M., M.M.,and
D.I.H., and, I.C.;
I.D.; statistical analysis,
writing—review and I.G.; C.J.,—
writing
editing, original
D.I.H., anddraft
I.D. All authors have
read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Acknowledgments: The authors would like to thank the entire team of the Interdisciplinary Research
Platform belonging to Banat University of Agricultural Sciences and Veterinary Medicine “King
Michael I of Romania” from Timisoara for their support during our study. This paper is published
from the own funds of the Banat University of Agricultural Sciences and Veterinary Medicine “King
Michael I of Romania” from Timisoara.
Conflicts of Interest: The authors declare no conflict of interest.

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