New broth macrodilution volatilization method for antibacterial susceptibility testing of volatile agents and evaluation of their toxicity using modified mtt assay in vitro

Article abstract of DOI:10.3390/molecules26144179

In this study, a new broth macrodilution volatilization method for the simple and rapid determination of the antibacterial effect of volatile agents simultaneously in the liquid and vapor phase was designed with the aim to assess their therapeutic potenti

Full text of DOI:10.3390/molecules26144179

molecules
Article
New Broth Macrodilution Volatilization Method for
Antibacterial Susceptibility Testing of Volatile Agents and
Evaluation of Their Toxicity Using Modified MTT Assay
In Vitro
Marketa Houdkova 1, Aishwarya Chaure 1 , Ivo Doskocil 2 , Jaroslav Havlik 3 and Ladislav Kokoska 1,
*
1
Department of Crop Sciences and Agroforestry, Faculty of Tropical AgriSciences, Czech University of Life
Sciences Prague, 16500 Prague, Czech Republic; houdkovam@ftz.czu.cz (M.H.); chaure@ftz.czu.cz (A.C.)
Department of Microbiology, Nutrition and Dietetics, Faculty of Agrobiology, Food and Natural Resources,
Czech University of Life Sciences Prague, 16500 Prague, Czech Republic; doskocil@af.czu.cz
Department of Food Science, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life
Sciences Prague, 16500 Prague, Czech Republic; havlik@af.czu.cz
2
3
*
Correspondence: kokoska@ftz.czu.cz; Tel.: +420-224382180
Abstract: In this study, a new broth macrodilution volatilization method for the simple and rapid
determination of the antibacterial effect of volatile agents simultaneously in the liquid and vapor
phase was designed with the aim to assess their therapeutic potential for the development of new in-
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
halation preparations. The antibacterial activity of plant volatiles (β-thujaplicin, thymohydroquinone,
Citation: Houdkova, M.; Chaure, A.;
Doskocil, I.; Havlik, J.; Kokoska, L.
New Broth Macrodilution
thymoquinone) was evaluated against bacteria associated with respiratory infections (Haemophilus in-
fluenzae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes) and their cytotoxicity
was determined using a modified thiazolyl blue tetrazolium bromide assay against normal lung
fibroblasts. Thymohydroquinone and thymoquinone possessed the highest antibacterial activity
Volatilization Method for
Antibacterial Susceptibility Testing of
Volatile Agents and Evaluation of
Their Toxicity Using Modified MTT
Assay In Vitro. Molecules 2021, 26,
4179. https://doi.org/10.3390/
molecules26144179
against H. influenzae, with minimum inhibitory concentrations of 4 and 8 µg/mL in the liquid and
vapor phases, respectively. Although all compounds exhibited cytotoxic effects on lung cells, thera-
peutic indices (TIs) suggested their potential use in the treatment of respiratory infections, which was
especially evident for thymohydroquinone (TI > 34.13). The results demonstrate the applicability of
the broth macrodilution volatilization assay, which combines the principles of broth microdilution
volatilization and standard broth macrodilution methods. This assay enables rapid, simple, cost- and
labor-effective screening of volatile compounds and overcomes the limitations of assays currently
used for screening of antimicrobial activity in the vapor phase.
Academic Editor: Domenico
Montesano
Received: 28 May 2021
Accepted: 5 July 2021
Published: 9 July 2021
Keywords: antimicrobial; cytotoxicity; macrodilution method; respiratory infections; β-thujaplicin;
thymohydroquinone; thymoquinone; vapor phase; volatile compound
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
Bacterial infections of the lower respiratory tract, such as pneumonia and bronchi-
tis, are some of the leading global causes of death, especially among children under
five years of age and elderly people [
infections include Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae,
and Streptococcus pyogenes [ ]. Moreover, co-infection with viruses (e.g., severe acute res-
piratory syndrome coronavirus) can significantly increase the morbidity and mortality
rates [ ]. Inhalation therapy represents the appropriate way to treat respiratory disor-
ders. If a medication is inhaled, it is directly delivered into the airways to the site of
infection. This gives a relatively faster onset of action, while using a lower dose of active
agent, which consequently causes fewer side effects in districts where its action is not
1,2]. Typical causative bacterial species of respiratory
3
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Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
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4.0/).
4
needed [
5,6]. Although antibiotic treatment is generally considered as effective against
most infective strains, it is increasingly failing due to some limitations including allergies,
Molecules 2021, 26, 4179. https://doi.org/10.3390/molecules26144179
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 4179
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bacterial resistance, inadequate penetration in lung tissues, and undesirable adverse ef-
fects [7]. Several types of devices for the delivery of inhaled medications to the lungs have
been invented, such as nebulizers, pressurized metered-dose and dry-powder inhalers;
however, they all face various limitations, including a short-life because of pulmonary
clearance, enzymatic degradation, fast systemic absorption, and poor bioavailability of
bioactive agents at the target site [8]. Moreover, the efficiency of the inhalable therapies
may be affected by the deposition of the aerolized particles in the oropharyngeal region
and upper airways while the deposition of drugs in the lungs can be reduced due to the
inappropriate size of its droplets or due to specific respiratory tract anatomy, and proper
operation, especially in children and elderly patients [9,10].
Since plant-derived antimicrobial agents have been traditionally used in the pre-
vention and treatment of respiratory infections, they are considered promising sources
of novel chemical scaffolds for the development of new drugs against bacteria causing
respiratory diseases [11]. For this reason, the biological potential of natural substances, in-
cluding volatile plant-derived compounds has been intensively studied in recent years with
the aim to find potential alternatives to conventional synthetic antimicrobial agents [12
,13].
For example, -thujaplicin, a monoterpenoid isolated from the wood of the Cupressaceae
β
species, and thymohydroquinone, a monoterpenoid phenol occurring in the Ranunculaceae
and Lamiaceae families, have been reported to exhibit antimicrobial properties, includ-
ing effects against bacteria causing respiratory infections [14–17]. Due to the high vapor
pressure of volatile plant-derived products at the ambient temperature, they have the bene-
fit of bioactivity in the vapor phase [18], and thus, plant volatiles have great potential for
the development of novel preparations for inhalation [19,20]. For example, thymoquinone,
a benzoquinone occurring in the seeds of Nigella sativa (Ranunculaceae), has been recorded
to possess a relatively strong antibacterial effect in the vapor phase against pathogens
that cause pneumonia [21]. Although plant-derived products are generally considered
as relatively safe, the toxicological evaluation of volatile agents is necessary to confirm
their non-toxicity for their practical application in inhalation therapy [22]. Despite the
availability of several lung cell in vitro models, there is an urgent need for the development
of more appropriate non-animal methods of inhalation toxicity, particularly for predicting
effects in humans [23].
In vitro screening is typically the first step in the process of discovery of new antimi-
crobial drugs, including those derived from plant volatiles. However, the susceptibility
testing of microorganisms to volatile agents using standard methods, such as broth dilution
and disk diffusion assays, is a challenging task because of their specific physico-chemical
properties, including high volatility, hydrophobicity, and viscosity [24]. The main problem
is that their hydrophobic nature worsens the solubility of these compounds in water-based
media (e.g., agar, broth) and their volatility increases the risk of loss of active substances
via evaporation during sample handling, experiment preparation, and incubation. In ad-
dition, the transition of the vapors can affect the microplate assay results, as described in
our previous studies [25
vapors testing. In contrast to well-established methods for antimicrobial susceptibility
testing on solid (agar disc diffusion) and liquid (broth dilution) media [27 32], there are no
,26]. This is even more complicated in the case of antimicrobial
–
standardized methods for the determination of microbial sensitivity to volatile compounds
in the vapor phase. In recent years, several methods for the testing of the antimicro-
bial effects of volatile plant-derived products in the vapor phase have been developed.
However, most of them have some specific limitations, such not being designed for high-
throughput screening, and some of them need special equipment that is not commonly
available [33]. Recently, we proposed a broth microdilution volatilization assay [21] based
on the principles of broth microdilution and disc volatilization methods, which is suitable
for high-throughput screening of volatile compounds simultaneously in the liquid and
vapor phase. This method can also be easily used for determination of the antibacterial ef-
fects of essential oil vapors [34
,35]. Although the broth microdilution volatilization method
is fast, simple and labor-effective, it has several weaknesses. For example, clamps and

Molecules 2021, 26, 4179
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wooden pads are required for a better sealing and fixing the microtiter plate and its lid
together. Moreover, the limited volume of agar that is applied on the lid can affect the
growth of the microorganisms tested.
With the aim to overcome the above mentioned drawbacks of previously developed
methods used for testing of volatile antimicrobial agents in the vapor phase, we designed a
novel macrodilution volatilization assay that combines the principles of broth microdilu-
tion volatilization [21] and standard broth macrodilution [27] methods. The validity of the
method for susceptibility testing of bacterial pathogens causing respiratory infections was
evaluated using three antimicrobial phytochemicals, namely β-thujaplicin, thymohydro-
quinone, and thymoquinone (Figure 1). In addition, the cytotoxicity of these compounds
was analyzed using a modified thiazolyl blue tetrazolium bromide (MTT) cytotoxicity
assay to assess their safety for use in the treatment of respiratory infections.
Figure 1. Chemical structures of plant volatile compounds tested.
2. Results and Discussion
2.1. Antimicrobial Activity
The results of the antibacterial effect of plant-derived volatiles against respiratory
pathogens in the liquid and vapor phases assessed using the broth macrodilution volatiliza-
tion method are listed in Table 1. All the compounds tested exhibited a certain degree
of growth-inhibitory effect in both the liquid and vapor phase and their effectiveness
varied in the ranges 4–64 µg/mL and 8–1024 µg/mL in the broth and agar media, respec-
tively. To the best of our knowledge, this is the first report on the antibacterial activity
of thymohydroquinone in the vapor phase. Moreover, the antimicrobial susceptibility of
H. influenzae to β-thujaplicin and thymohydroquinone, S. pyogenes to thymohydroquinone
and thymoquinone, and S. pneumoniae to thymohydroquinone was also described for the
first time in this study.
In the liquid phase, the lowest minimal inhibitory concentration (MIC) values were
observed for thymohydroquinone and thymoquinone against H. influenzae (4
followed by the growth-inhibitory effects against S. aureus, S. pneumoniae, and S. pyogenes
with respective MIC values of 8, 16, and 32 g/mL. -thujaplicin exhibited the same
level of antibacterial activity against all bacterial strains tested with a MIC of 64 g/mL.
µg/mL),
µ
β
µ
As well as in the broth, thymohydroquinone and thymoquinone were found to be the
most active antibacterial agents against H. influenzae in the vapor phase with a MIC of
8
µ
g/mL. In addition, both compounds effectively inhibited the growth of S. aureus and
S. pyogenes on agar medium at the same concentrations, 16 and 32 g/mL, respectively.
However, their activity differed against S. pneumoniae, where a lower MIC value was
detected for thymoquinone (16 g/mL) than for thymohydroquinone (32 g/mL). In the
case of -thujaplicin, a moderate antibacterial efficacy was recorded against H. influenzae
µ
µ
µ
β
and S. aureus (512
S. pyogenes.
µg/mL) and a low antibacterial efficacy against S. pneumoniae and

Molecules 2021, 26, 4179
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Table 1. Antibacterial activity of plant volatile compounds in the liquid and vapor phases against respiratory pathogens.
Bacterium/Minimal Inhibitory Concentration
Haemophilus influenzae
Broth
Agar
Staphylococcus aureus
Streptococcus pneumoniae
Streptococcus pyogenes
Broth
Agar
Plant Volatile Compound
x-MIC
Broth
Agar
Broth
Agar
(µg/cm3)
(µg/cm3)
(µg/cm3)
(µg/cm3)
(µg/mL)
(µg/mL)
(µg/mL)
(µg/mL)
(µg/mL)
(µg/mL)
(µg/mL)
(µg/mL)
β-thujaplicin
thymohydroquinone
thymoquinone
64
4
512
8
320
5
64
8
512
16
320
10
64
16
1024
32
640
20
64
32
32
1024
32
640
20
64
15
15
-
4
8
5
8
16
10
16
16
10
32
20
0.5 b
0.25 d
positive antibiotic control
1 a
n.d.
n.d.
n.d.
n.d.
0.25 c
n.d.
n.d.
n.d.
n.d.
a
x-MIC: mean value of minimal inhibitory concentrations in broth medium, positive antibiotic control: ampicillin, b oxacillin, c amoxicillin, d tetracycline, n.d.: not detected.

Molecules 2021, 26, 4179
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In general, the results of antibacterial potential of plant-derived volatiles obtained
by our novel broth macrodilution volatilization assay correspond with those observed
by other authors using standard broth and agar dilution methods. The MICs previously
determined by Domon et al., Morita et al., and Inoue et al. [17
against various strains of S. aureus in the range 12.5–160 g/mL were close to the MIC
detected in our study. However, a much higher antimicrobial efficacy of this compound
was recorded against S. pneumoniae and S. pyogenes with MICs of 0.3–1 g/mL in previ-
ously published studies [17]. Other authors [25 38] observed the growth-inhibitory effect
of thymoquinone against S. aureus ATCC 29213 and ATCC 25923 with the MIC value of
g/mL, which is the same as that in our study. In the case of thymohydroquinone, a fifty-
times-higher MIC value of 400 g/mL was detected against S. aureus ATCC 25923 [39].
,36,37] for β-thujaplicin
µ
µ
,
8
µ
µ
As it has previously been observed, various bacterial strains with different susceptibilities
to antibacterial agents [40], as well as different antimicrobial assays used for the testing of
volatiles [41], may be responsible for the variability of the results obtained by other authors.
In contrast to a number of published papers on the antibacterial potential of plant-derived
volatile compounds in the liquid phase, literature on the activity of their vapors is limited.
Wang et al. [42] recorded a certain level of growth-inhibitory effect of β-thujaplicin against
oral microorganisms in the gaseous phase and Inouye et al. [43] described a very potent
vapor activity of thymoquinone against Trichophyton mentagrophytes; both by using the
disc volatilization method. Our previous results on the antimicrobial activity of thymo-
quinone against respiratory pathogens H. influenzae, S. aureus, and S. pneumoniae obtained
by using the broth microdilution volatilization assay with respective MIC values of 8, 16,
and 16 µg/mL in the liquid phase and 8, 16, and 32 µg/mL in the vapor phase [21] corre-
spond to this current study and demonstrate the validity of our newly developed method.
In general, the compounds tested in this study showed higher or equal antibacterial effects
in the liquid phase than in the vapor phase. The largest differences were observed in the
case of β-thujaplicin when sixteen times lower MIC values were detected in broth than on
the agar against S. pneumonieae and S. pyogenes.
The above-described results demonstrate the validity of our novel broth macrodilution
volatilization assay, which combines the principles of broth microdilution volatilization [21
]
and standard macrodilution methods [27] and overcomes some of their drawbacks. In com-
parison with previously established liquid matrix volatilization techniques, the broth
macrodilution volatilization method is performed in commercially available microtubes,
which can be tightly closed with a snap cap, and therefore, it does not require specialized
equipment, such as wooden pads and clamps to prevent the losses of the active agents
by evaporation. A variable number of sample concentrations tested in one experiment is
another benefit of this method, while the design is not limited by the quantity of wells,
as in the case of microplate-based assays. Whereas a higher amount of appropriate me-
dia can be applied in microtubes and their caps, which are suitable for the cultivation of
slower growing microorganisms (e.g., fungi) that require longer incubation time to produce
enough growth for MIC determination [44]. Despite the obvious benefits of our novel
antimicrobial susceptibility test based on microtubes, it has some specific limits, such as a
lower potential for the automation (e.g., use of automated pipetting platform and reader)
and the need for an extra step for the preparation of an appropriate amount of serially
diluted concentrations of samples in the test tubes. Due to the transition of antimicrobial
compounds between the liquid and vapor phases and their possible losses during the exper-
iment preparation, the final concentration should be considered as indicative only. For that
reason, the concentrations of the samples tested in the vapor phase were expressed as the
weight of the volatile agent per volume unit of a microtube, that is, 640, 320, 160, 80, 40, 20,
10, and 5 µ µg/mL, respectively. Nevertheless,
g/cm3 for 1024, 512, 128. 64, 32, 16, 8, and 4
the real quantity of volatile agents evaporated from the broth should be determined, e.g.,
using a combination of solid-phase microextraction and gas chromatography analysis [45].

Molecules 2021, 26, 4179
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2.2. Cytotoxicity
The results of the modified MTT assay performed on lung fibroblast cells are sum-
marized in Table 2. The cytotoxic effect of twelve two-fold serially diluted concentrations
of compounds tested are displayed in Figure 2.
exhibited moderate toxicity to the lung cells with respective half maximal inhibitory concen-
tration (IC50) values of 4.15 and 2.64 g/mL. Thymoquinone was evaluated as toxic with
an IC50 value of 1.21 g/mL. In the case of an 80% inhibitory concentration of proliferation
(IC80) determination, the lowest cytotoxic effect was observed for thymohydroquinone
followed by -thujaplicin with IC80 values >12.00 and 214.85 g/mL, respectively. Simi-
lar to IC50, the lowest IC80 value was recorded for thymoquinone (IC80 = 15.00 g/mL).
β-Thujaplicin and thymohydroquinone
µ
µ
β
µ
µ
Although according to the WHO [46], all the compounds were classified as toxic and
moderately toxic, the therapeutic index (TI) calculated to compare their antibacterial and
cytotoxic effects indicate that thymohydroquinone (TI > 34.13) can be a safe and effective
antibacterial agent for inhalation therapy.
Table 2. Cytotoxicity of plant volatile compounds to the normal lung fibroblast cells MRC-5.
Samples
IC50 ± SD (µg/mL)
IC80 ± SD (µg/mL)
TI
Plant volatile compound
β-thujaplicin
thymohydroquinone
thymoquinone
Positive control
vinorelbin
4.15 ± 0.45
2.64 ± 0.33
1.21 ± 0.24
214.85 ± 9.71
>512.00
15.00 ± 4.46
3.36
>34.13
1.00
0.54 ± 0.26
>10
-
IC50: half maximal inhibitory concentration of proliferation in µg/mL, IC80: 80% inhibitory concentration of
proliferation in µg/mL, SD: standard deviation, TI: therapeutic index (TI = IC80/x-MIC).
Figure 2. Cytotoxic activity of twelve two-fold serially diluted concentrations (0.25–512 µg/mL) of
plant volatile compounds to lung fibroblast cells tested by using MTT assay performed in microtiter
plates sealed with vapor barrier EVA Capmat.
Series of assays examining the toxic potential of thymoquinone have been performed
including our previous study [21], which observed that this compound has a very similar
cytotoxic effect on healthy human lung MRC-5 cell lines with a IC50 value of 1.70
while Gurung et al. [47] found that thymoquinone did not alter the viability of normal IMR-
90 lung fibroblasts at a concentration of 32.84 g/mL. In contrast to the well-documented
µg/mL,
µ

Molecules 2021, 26, 4179
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toxicity of thymoquinone, there are only few data available on the safety of other com-
pounds tested in normal human lung fibroblasts. In a study of Ivankovic et al. [48],
thymohydroquinone exhibited a certain level of cell growth inhibition against mouse fi-
broblasts at concentrations of 10 and 100
of -thujaplicin was detected against various types of human lung cancer tissues, with IC50
values of 0.26–12.32 g/mL [49 51]. Regarding the relatively high toxicity of compounds
µg/mL (21% and 63%, respectively). A toxic effect
β
µ
–
tested in lung cell cultures, their practical application for inhalation therapy to treat respi-
ratory infection seems to be limited. However, their specific structural and technological
modifications reducing toxicity are possible. For example, liposomal encapsulation of
thymoquinone was found to be effective in decreasing its toxic effects [52].
3. Materials and Methods
3.1. Chemicals and Reagents
The following plant-derived volatile antibacterial agents were assayed: β-thujaplicin
(99%, CAS 499-44-5), thymohydroquinone (98%, CAS 2217-60-9), and thymoquinone
(99%, CAS 490-91-5). Amoxicillin (90%, CAS 26787-78-0), ampicillin (84.5%, CAS 69-52-3),
oxacillin (86.3%, CAS 7240-38-2), and tetracycline (98–102%, CAS 60-54-8) were used as
positive antibiotic controls. The chemicals used for antimicrobial susceptibility testing were
as follows: dimethyl sulfoxide (DMSO, CAS 67-68-5), thiazolyl blue tetrazolium bromide
dye (MTT, CAS 298-93-1), and Tween 20 (CAS 9005-64-5). With the exception of thymo-
hydroquinone prepared by reduction of thymoquinone according to a method described
further, all other chemicals were obtained from Sigma-Aldrich (Prague, Czech Republic).
The following chemicals were used for thymohydroquinone preparation and char-
acterization: acetic acid (CAS 64-19-7) purchased from Merck (Darmstadt, Germany),
then deuterium oxide containing 3-(trimethylsilyl) propionic-2,2,3,3-d4 acid sodium salt
(CAS 7789-20-0), vanillin (CAS 121-33-5) and zinc (CAS 7440-66-6) purchased from Sigma-
Aldrich (Praha, Czech Republic), and others obtained from Penta (Praha, Czech Republic),
namely chloroform (CAS 67-66-3), ethanol (96%, CAS 64-17-5), hexane (CAS 110-54-3),
hydrochloric acid (35%, CAS 7647-01-0), and sulfuric acid (CAS 7664-93-9).
3.2. Thymohydroquinone Preparation and Characterization
Thymohydroquinone was prepared by the reduction of thymoquinone using acetic
acid in the presence of zinc powder as a catalyst according to the method previously
described by Tesarova et al. [53]. Briefly, 50 mg of thymoquinone (Sigma Aldrich, Praha,
Czech Republic) was dissolved in 3 mL of concentrated acetic acid (99%) and 5 g of zinc
powder was subsequently added. The reaction mixture was stirred for 4 h, monitored by
thin layer chromatography (TLC) under laboratory conditions, and then filtered. The TLC
was performed on TLC Silica gel 60 F254 (Merck, Darmstadt, Germany) plates with chloro-
form and ethanol in a ratio of 9:1 as the mobile phase. Vanillin dissolved in sulfuric acid in
ethanol (1%) was used as a visualizing agent. The liquid fraction was evaporated under
vacuum in a rotary evaporator (Rotavapor R-210, Buchi, Flawil, Switzerland). The solid
residue was dissolved in water, acidified by the addition of hydrochloric acid (to 5% v/v)
and yellow residues of thymoquinone were removed by repeated extraction with hexane.
Thymohydroquinone was then extracted three times with distilled diethyl ether and evap-
orated under a stream of N2. For further purification, sublimation at 168 ◦C was used and
colorless needle crystals of thymohydroquinone were stored in the dark.
The identity and purity of obtained thymohydroquinone were confirmed using gas
chromatography/mass spectrometry analysis (GC/MS) and a nuclear magnetic resonance
(NMR). The mass spectra of thymohydroquinone were recorded by Agilent GC-7890B and
MSD-5977B (Agilent Technologies, Santa Clara, CA, USA) equipped with a fused-silica
HP-5MS column (30 m
×
0.25 mm, film thickness 0.25 µm, Agilent 19091s-433) and a
flame ionization detector coupled with single quadrupole mass selective detector Agilent
MSD-5977B (Agilent Technologies, Santa Clara, CA, USA). Operational parameters were:
helium as a carrier gas at 1 mL/min, injector temperature 250 ◦C. The oven temperature

Molecules 2021, 26, 4179
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was raised from 50 to 280 ◦C. Thymohydroquinone was diluted in n-hexane for GC/MS
at a concentration of 20 µg/mL. One microliter of solution was injected in a split mode
(split ratio 1:50). The mass detector was set to the following conditions: ionization energy
70 eV, ion source temperature 230 ◦C, scan time 1 s, mass range 40–600 m/z. Identification
of the sample was based on the comparison of its retention index (RI), retention time (RT)
and mass spectra with the National Institute of Standards and Technology Library ver. 2.0.f
(National Institute of Standards and Technology, USA) [54].
The 1H-NMR spectra of thymohydroquinone standard (2 mg/mL) were recorded on a
Bruker Avance III HD BBFO (Bruker BioSpin GmbH, Rheinstetten, Germany), operating at
◦
500 MHz for 1H-NMR and 126 MHz for 13C-NMR using noesypr1d pulse sequence, at 25 C.
The sample was dissolved in H2O containing 10% deuterium oxide, at pH 7.4, and includ-
ing 3-(trimethylsilyl) propionic-2,2,3,3-d4 acid sodium salt (99%) as an internal standard.
Supplementary evidence was given by 13C-NMR, HSQC, HMBC and COSY experiments.
1
The experimental chemical shifts in H and 13C-NMR spectra of thymohydroquinone
closely matched the theoretically predicted chemical shifts obtained using www.nmrdb.org
(accessed on 21 June 2021) [55,56].
The signals were confirmed and assigned after inspection of the 1D and 2D spectra
and GC/MS data and were as follows: Thymohydroquinone. White crystals; RI: 1520, MS,
m/z (rel. int.): 166 (M+, 43%), 151 (100), 152 (14), 77 (8), 123 (7), 95 (7); 1H-NMR (H2O/D2O
9:1, 500 MHz):
δ
6.79 (s, 1H, 2-H), 6.73 (s, 1H, 5-H), 3.14 (sept, 1H, J = 6.9 Hz, 8-H), 2.13 (s, 3H,
147.5 (C-6),
7-H), 1.17 (d, 6H, J = 6.9 Hz, 9,10-H); 13C-NMR (H2O/D2O 9:1, 126 MHz):
δ
145.8 (C-3), 134.4 (C-4), 123.3 (C-1), 118.3 (C-2), 113.2 (C-5), 26.0 (C-8), 22.2 (C-9), 22.2 (C-10),
14.9 (C-7).
3.3. Bacterial Strains and Culture Media
The following four bacterial standard strains from the American Type Culture Collec-
tion (ATCC, Manassas, VA, USA) were used: Haemophilus influenzae ATCC 49247, Staphylo-
coccus aureus ATCC 29213, Streptococcus pneumoniae ATCC 49619, and Streptococcus pyogenes
ATCC 19615. Cultivation and assay media (broth/agar) were Mueller–Hinton (MH) com-
plemented by Haemophilus Tested Medium (H. influenzae), MH (S. aureus), and Brain
Heart Infusion (S. pneumoniae and S. pyogenes). The pH of the broths was equilibrated to a
final value of 7.6 using Trizma base (Sigma-Aldrich, Praha, Czech Republic). All microbial
strains and cultivation media were purchased from Oxoid (Basingstoke, UK).
◦
Stock cultures of bacterial strains were cultivated in broth medium at 37 C for
24 h prior to testing. For the preparation of inoculum, the turbidity of the bacterial
suspension was adjusted to 0.5 McFarland standard using a Densi-La-Meter II (Lachema,
Brno, Czech Republic) to obtain a final concentration of 108 CFU/mL.
3.4. Cell Cultures
Lung fibroblast cell line MRC-5, obtained from ATCC, was propagated in Eagle’s Min-
imum Essential Medium (EMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM
glutamine, 10 µL/mL non-essential amino acids, and 1% penicillin–streptomycin solution
(10,000 units/mL of penicillin and 10 mg/mL of streptomycin); all these components were
purchased from Sigma-Aldrich. The cells were pre-incubated in 96-well microtiter plates
at a density of 2.5
atmosphere of 5% CO2 in air.
×
103 cells per well for 24 h at 37 ◦C in a humidified incubator in an
3.5. Antimicrobial Assay
The antibacterial potential of volatile plant-derived compounds in the liquid and
vapor phases was determined using a newly developed broth macrodilution volatilization
method performed in standard 2 mL microtubes with snap caps (Eppendorf, Hamburg,
Germany). Initially, each sample of compound was dissolved in DMSO at maximum
concentration of 1% and diluted in the appropriate broth medium. With the aim to prepare
a sufficient amount of stock solutions of the compounds assayed, six two-fold serially

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diluted concentrations of samples were prepared in 15 mL test tubes closed with plugs
to avoid the losses of active compounds by evaporation (Gama Group, Ceske Budejovice,
Czech Republic). The concentration of
128 g/mL for thymohydroquinone and thymoquninone. In the second step, 90
melted agar was pipetted into rims on the caps (Figure 3a) and inoculated with 5
β-thujaplicin started from 1024 µg/mL and from
µ
µ
L of
L of
µ
bacterial suspension after agar solidification. Subsequently, the appropriate concentrations
of each sample previously prepared in test tubes were pipetted into microtubes in a final
volume of 1500 µL. Then, the microtubes were inoculated with 10 µL of bacterial suspension
and closed properly (Figure 3b). Microtubes containing inoculated and non-inoculated
media were prepared as growth and purity controls simultaneously. After incubation at
37 ◦C for 24 h, the MICs were evaluated by the visual assessment of bacterial growth after
coloring metabolically active bacterial colonies with MTT dye. The respective volumes of
30 and 375 µL of MTT at a concentration of 600 µg/mL were pipetted into the caps and
in the microtubes when the interface of color change from yellow to purple (relative to
that of colors in control wells) was recorded in broth and agar (Figure 3c). A black and
white scheme of a cross-sectional view of a microtube filled with broth and agar shows the
effective flow of sample vapors in the closed testing system (Figure 4). The MIC values
were determined as the lowest concentrations that inhibited bacterial growth compared
with the compound-free control and are expressed in
phase, the concentration was also expressed in
g/cm3 as the weight of the volatile agent
per volume unit of a microtube. DMSO, assayed as the negative control, did not inhibit
any of the strains at the tested concentration ( 1%). The respective susceptibilities of
µg/mL. In the case of the vapor
µ
≤
H. influenzae, S. aureus, S. pneumoniae, and S. pyogenes to ampicillin, oxacillin, amoxicillin,
and tetracycline were checked as positive antibiotic controls [57]. All tests were performed
as three independent experiments, each carried out in triplicate, and the results were
presented as median/modal values. According to the widely accepted norm in MIC testing,
the mode and median were used for the final value calculation when triplicate endpoints
were within the two- and three-dilution ranges, respectively.
3.6. Cytotoxicity Assay
A modified method based on the metabolization of MTT to blue formazan by mito-
chondrial dehydrogenases in living lung cells previously described by Mosmann [58] was
used. The lung fibroblast cells were treated for 72 h with the tested compounds dissolved
in DMSO at a maximum concentration of 1% and diluted in the EMEM medium supple-
mented with 10% FBS. Twelve two-fold serially diluted concentrations of these agents
ranging from 512 to 0.25 µg/mL were prepared. The microtiter plates were covered with
EVA capmatsTM at 37 ◦C in a humidified atmosphere of 5% CO2 in air and cultivated for
72 h. Thereafter, MTT reagent (1 mg/mL) in EMEM solution was added to each well and
the plates were incubated for an additional 2 h at 37 ◦C in a humidified atmosphere of
5% CO2 in air. The media were removed, and the intracellular formazan product was
dissolved in 100
µL of DMSO. The solvent used did not affect the viability of the lung cells
at the tested concentration (
≤
1%). The absorbance was measured at 555 nm using a Tecan
Infinite M200 spectrometer (Tecan Group, Mannedorf, Switzerland), and the viability was
calculated in comparison to that of the untreated control. Three independent experiments
(two replicates each) were performed for each test. The results of the cytotoxicity effect
were calculated using GraphPad Prism software (GraphPad Software, La Jolla, CA, USA)
and expressed as average IC50 with standard deviation in
effects were classified according to the Special Programme for Research and Training in
Tropical Diseases (WHO–Tropical Diseases) [46] as cytotoxic (IC50 < 2 g/mL), moderately
cytotoxic (IC50 2–89 g/mL), and non-toxic (IC50 > 90 g/mL). Furthermore, IC80 was
calculated as equivalent to the MIC endpoint [59] for comparison of microbiological and
toxicological data. Therapeutic indices (TIs) were defined as the ratio of -IC80 and -MIC
µg/mL. The levels of cytotoxic
µ
µ
µ
x
x
values with the aim of determining the amount of effective antibacterial agents with the
quantity causing toxicity [60].

Molecules 2021, 26, 4179
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Figure 3. Broth macrodilution volatilization method (
of agar is pipetted into rim of every cap; ( ) incubation: after inoculation, microtubes containing liquid
medium with serially diluted samples of tested volatiles and their caps containing solid medium are
properly closed together to prevent the losses of active compounds; ( ) MIC determination: the results
a) pipetting of agar in the microtube caps: 90 µL
b
c
are evaluated visually after coloring of living bacterial colonies with MTT dye.
Figure 4. Detail of the cross-sectional view of the closed microtube with snap cap containing broth
and agar media.

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4. Conclusions
As a result of this study, a new broth macrodilution volatilization method was devel-
oped for the simultaneous determination of the antimicrobial effects of volatile agents in the
liquid and the vapor phases at variable concentrations. This rapid, simple, cost- and labor-
effective technique, which combines the principles of broth microdilution volatilization and
standard broth macrodilution methods, is performed in commercially available microtubes
and, therefore, does not require specialized equipment. It can also be a suitable option
for the testing of slower growing organisms (e.g., fungi) that require longer incubation
time to produce enough growth for MIC determination. Nevertheless, further research
focusing on the optimization of the novel broth macrodilution volatilization method for
susceptibility testing of a broader spectrum of microorganisms will be necessary to confirm
this assumption. In addition, the validity of the method for the susceptibility testing of
bacterial pathogens causing respiratory infection was evaluated using three antimicrobial
phytochemicals (β-thujaplicin, thymohydroquinone, and thymoquinone). As a result of
this research, thymohydroquinone was found to be a promising antibacterial agent for
application in inhalation therapy that is safe to human lung cell lines. However, in vivo
experiments are required to verify the therapeutic potential of this compound.
Author Contributions: Coordination and performance of the antimicrobial activity and cytotoxicity
testing and its related data analysis, the manuscript drafting, M.H.; performance of the antimicrobial
activity, A.C.; performance of the cytotoxicity testing, I.D.; performance of the NMR analysis, J.H.;
conceptualization and coordination of the whole study and finalization of the manuscript, L.K.
All authors have read and agreed to the published version of the manuscript.
Funding: This research was financially supported by the Czech University of Life Sciences Prague
[project IGA 20213109] and METROFOOD-CZ Research Infrastructure Project [MEYS Grant No:
LM2018100].
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
Brooks, W.A. Bacterial pneumonia. In Hunter’s Tropical Medicine and Emerging Infectious Diseases, 10th ed.; Ryan, E.T., Hill, D.R.,
Solomon, T., Aronson, N.E., Endy, T.P., Eds.; Elsevier: Amsterdam, The Netherlands, 2020; pp. 446–453.
The Top 10 Causes of Death. Available online: https://www.who.int/news-room/fact-sheets/detail/the-top-10-causes-of-death
(accessed on 19 March 2021).
Zafar, A.; Hasan, R.; Nizamuddin, S.; Mahmood, N.; Mukhtar, S.; Ali, F.; Morrissey, I.; Barker, K.; Torumkuney, D. Antibiotic sus-
ceptibility in Streptococcus pneumoniae, Haemophilus influenzae and Streptococcus pyogenes in Pakistan: A review of results from the
Survey of Antibiotic Resistance (SOAR) 2002-15. J. Antimicrob. Chemother. 2016, 71 (Suppl. 1), 103–109. [CrossRef] [PubMed]
Manohar, P.; Loh, B.; Nachimuthu, R.; Hua, X.T.; Welburn, S.C.; Leptihn, S. Secondary bacterial infections in patients with viral
pneumonia. Front. Med. 2020, 7, 720. [CrossRef] [PubMed]
4.
5.
6.
7.
8.
9.
Borghardt, J.M.; Kloft, C.; Sharma, A. Inhaled therapy in respiratory disease: The complex interplay of pulmonary kinetic
processes. Can. Respir. J. 2018, 2018, 2732017. [CrossRef] [PubMed]
Sorino, C.; Negri, S.; Spanevello, A.; Visca, D.; Scichilone, N. Inhalation therapy devices for the treatment of obstructive lung
diseases: The history of inhalers towards the ideal inhaler. Eur. J. Intern. Med. 2020, 75, 15–18. [CrossRef]
Cock, I.E.; van Vuuren, S.F. The traditional use of southern African medicinal plants for the treatment of bacterial respiratory
diseases: A review of the ethnobotany and scientific evaluations. J. Ethnopharmacol. 2020, 263, 113204. [CrossRef]
Jain, H.; Bairagi, A.; Srivastava, S.; Singh, S.B.; Mehra, N.K. Recent advances in the development of microparticles for pulmonary
administration. Drug Discov. Today 2020, 25, 1865–1872. [CrossRef]
Ibrahim, M.; Verma, R.; Garcia-Contreras, L. Inhalation drug delivery devices: Technology update. Med. Devices 2015, 8, 131–139. [CrossRef]
10. Klepser, M.E. Role of nebulized infections antibiotics for the treatment of respiratory. Curr. Opin. Infect. Dis. 2004, 17, 109–112. [CrossRef]
11. Kokoska, L.; Kloucek, P.; Leuner, O.; Novy, P. Plant-derived products as antibacterial and antifungal agents in human health care.
Curr. Med. Chem. 2019, 26, 1–38. [CrossRef]
12. Cos, P.; Vlietinck, A.J.; Vanden Berghe, D.; Maes, L. Anti-infective potential of natural products: How to develop a stronger
in vitro ‘proof-of-concept’. J. Ethnopharmacol. 2006, 106, 290–302. [CrossRef] [PubMed]
13. Mutlu-Ingok, A.; Devecioglu, D.; Dikmetas, D.N.; Karbancioglu-Guler, F.; Capanoglu, E. Antibacterial, antifungal, antimycotoxi-
genic, and antioxidant activities of essential oils: An updated review. Molecules 2020, 25, 4711. [CrossRef]
14. Taborsky, J.; Kunt, M.; Kloucek, P.; Lachman, J.; Zeleny, V.; Kokoska, L. Identification of potential sources of thymoquinone and related
compounds in Asteraceae, Cupressaceae, Lamiaceae, and Ranunculaceae families. Cent. Eur. J. Chem. 2012, 10, 1899–1906. [CrossRef]

Molecules 2021, 26, 4179
12 of 13
15. Fotopoulou, T.; Ciric, A.; Kritsi, E.; Calhelha, R.; Ferreira, I.C.F.R.; Sokovic, M.; Zoumpoulakis, P.; Koufaki, M. Antimicro-
bial/antibiofilm activity and cytotoxic studies of beta-thujaplicin derivatives. Arch. Pharm. 2016, 349, 698–709. [CrossRef]
16. Abdelazeem, A.H.; Mohamed, Y.M.A.; Gouda, A.M.; Omar, H.A.; Al Robaian, M.M. Novel thymohydroquinone derivatives as
potential anticancer agents: Design, synthesis, and biological screening. Aust. J. Chem. 2016, 69, 1277–1284. [CrossRef]
17. Domon, H.; Hiyoshi, T.; Maekawa, T.; Yonezawa, D.; Tamura, H.; Kawabata, S.; Yanagihara, K.; Kimura, O.; Kunitomo, E.;
Terao, Y. Antibacterial activity of hinokitiol against both antibiotic-resistant and -susceptible pathogenic bacteria that predominate
in the oral cavity and upper airways. Microbiol. Immunol. 2019, 63, 213–222. [CrossRef] [PubMed]
18. Reyes-Jurado, F.; Navarro-Cruz, A.R.; Ochoa-Velasco, C.E.; Palou, E.; Lopez-Malo, A.; Avila-Sosa, R. Essential oils in vapor phase
as alternative antimicrobials: A review. Crit. Rev. Food Sci. Nutr. 2020, 60, 1641–1650. [CrossRef] [PubMed]
19. Leigh-de Rapper, S.; van Vuuren, S.F. Odoriferous therapy: A review identifying essential oils against pathogens of the respiratory
tract. Chem. Biodivers. 2020, 17, e2000062. [CrossRef] [PubMed]
20. Jaradat, N.A.; Al Zabadi, H.; Rahhal, B.; Hussein, A.M.; Mahmoud, J.S.; Mansour, B.; Khasati, A.I.; Issa, A. The effect of inhalation
of Citrus sinensis flowers and Mentha spicata leave essential oils on lung function and exercise performance: A quasi-experimental
uncontrolled before-and-after study. J. Int. Soc. Sports Nutr. 2016, 13, 36. [CrossRef] [PubMed]
21. Houdkova, M.; Rondevaldova, J.; Doskocil, I.; Kokoska, L. Evaluation of antibacterial potential and toxicity of plant volatile compounds
using new broth microdilution volatilization method and modified MTT assay. Fitoterapia 2017, 118, 56–62. [CrossRef] [PubMed]
22. Kiani, S.; Minaei, S.; Ghasemi-Varnamkhasti, M. Application of electronic nose systems for assessing quality of medicinal and
aromatic plant products: A review. J. Appl. Res. Med. Aromat. Plants 2016, 3, 1–9. [CrossRef]
23. BeruBe, K.; Aufderheide, M.; Breheny, D.; Clothier, R.; Combes, R.; Duffin, R.; Forbes, B.; Gaca, M.; Gray, A.; Hall, I.; et al. In vitro
models of inhalation toxicity and disease. Altern. Lab. Anim. 2009, 37, 89–141.
24. Reyes-Jurado, F.; Franco-Vega, A.; Ramirez-Corona, N.; Palou, E.; Lopez-Malo, A. Essential oils: Antimicrobial activities,
extraction methods and their modeling. Food Eng. Rev. 2015, 7, 275–297. [CrossRef]
25. Novy, P.; Kloucek, P.; Rondevaldova, J.; Havlik, J.; Kourimska, L.; Kokoska, L. Thymoquinone vapor significantly affects the results
of Staphylococcus aureus sensitivity tests using the standard broth microdilution method. Fitoterapia 2014, 94, 102–107. [CrossRef]
26. Houdkova, M.; Albarico, G.; Doskocil, I.; Tauchen, J.; Urbanova, K.; Tulin, E.E.; Kokoska, L. Vapors of volatile plant-derived
products significantly affect the results of antimicrobial, antioxidative and cytotoxicity microplate-based assays. Molecules 2020,
25, 6004. [CrossRef] [PubMed]
27. Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Disk Susceptibility Tests, 11th ed.;
Approved Standard, CLSI Document M02-A11; CLSI: Wayne, PA, USA, 2012; p. 32, ISBN 1-56238-782-0.
28. European Committee on Antimicrobial Susceptibility Testing. Antimicrobial Susceptibility Testing EUCAST Disk Diffu-
sion Method, Version 9. Available online: https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Disk_test_
documents/2021_manuals/Manual_v_9.0_EUCAST_Disk_Test_2021.pdf (accessed on 22 May 2021).
29. Clinical and Laboratory Standards Institute (CLSI). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow
Aerobically; Approves Standard, 10th ed.; CLSI document M07-A10; CLSI: Wayne, PA, USA, 2015; p. 35, ISBN 1-56238-988-2.
30. National Committee for Clinical Laboratory Standards (NCCLS). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria
That Growth Aerobically; Approved Standard, 6th ed.; NCCLS document M7-A6; NCCLS: Wayne, PA, USA, 2003.
31. Food and Drug Administration (FDA). Guidance for industry and FDA. Class II Special Controls Guidance Document: Antimicrobial
Susceptibility Test (AST) Systems; Center for Devices and Radiological Health, FDA: Rockville, MD, USA, 2009.
32. International Organization for Standardization (ISO). Susceptibility Testing of Infectious Agents and Evaluation of Performance of
Antimicrobial Susceptibility Devices, Part 1. Broth Micro-Dilution Reference Method for Testing the In Vitro Activity of Antimicrobial Agents
against Rapidly Growing Aerobic Bacteria Involved in Infectious Diseases, 2nd ed.; ISO/DIS 20776-1; ISO: Geneva, Switzerland, 2019.
33. Houdkova, M.; Kokoska, L. Volatile antimicrobial agents and in vitro methods for evaluating their activity in the vapour phase:
A review. Planta Med. 2020, 86, 822–857. [CrossRef]
34. Houdkova, M.; Urbanova, K.; Doskocil, I.; Rondevaldova, J.; Novy, P.; Nguon, S.; Chrun, R.; Kokoska, L. In vitro growth-inhibitory
effect of Cambodian essential oils against pneumonia causing bacteria in liquid and vapour phase and their toxicity to lung
fibroblasts. S. Afr. J. Bot. 2018, 118, 85–97. [CrossRef]
35. Houdkova, M.; Doskocil, I.; Urbanova, K.; Tulin, E.K.C.B.; Rondevaldova, J.; Tulin, A.B.; Kudera, T.; Tulin, E.E.; Zeleny, V.;
Kokoska, L. Evaluation of antipneumonic effect of Philippine essential oils using broth microdilution volatilization method and
their lung fibroblasts toxicity. Nat. Prod. Commun. 2018, 13, 1059–1066. [CrossRef]
36. Morita, Y.; Matsumura, E.; Tsujibo, H.; Yasuda, M.; Sakagami, Y.; Okabe, T.; Ishida, N.; Inamori, Y. Biological activity of alpha-
thujaplicin, the minor component of Thujopsis dolabrata Sieb. et Zucc. var. hondai Makino. Biol. Pharm. Bull. 2001, 24, 607–611.
[CrossRef] [PubMed]
37. Inoue, Y.; Suzuki, R.; Murata, I.; Nomura, H.; Isshiki, Y.; Kanamoto, I. Evaluation of antibacterial activity expression of the
hinokitiol/cyclodextrin complex against bacteria. Acs Omega 2020, 5, 27180–27187. [CrossRef] [PubMed]
38. Chaieb, K.; Kouidhi, B.; Jrah, H.; Mahdouani, K.; Bakhrouf, A. Antibacterial activity of thymoquinone, an active principle of
Nigella sativa and its potency to prevent bacterial biofilm formation. BMC Complement Altern Med. 2011, 11, 29. [CrossRef]
39. Halawani, E. Antibacterial activity of thymoquinone and thymohydroquinone of Nigella sativa L. and their interaction with some
antibiotics. Adv. Biol. Res. 2009, 3, 148–152.

Molecules 2021, 26, 4179
13 of 13
40. Muthaiyan, A.; Biswas, D.; Crandall, P.G.; Wilkinson, B.J.; Ricke, S.C. Application of orange essential oil as an antistaphylococcal
agent in a dressing model. BMC Complement. Altern. Med. 2012, 12, 125. [CrossRef]
41. Valgas, C.; de Souza, S.M.; Smania, E.F.A.; Smania, A. Screening methods to determine antibacterial activity of natural products.
Braz. J. Microbiol. 2007, 38, 369–380. [CrossRef]
42. Wang, T.H.; Hsia, S.M.; Wu, C.H.; Ko, S.Y.; Chen, M.Y.; Shih, Y.H.; Shieh, T.M.; Chuang, L.C.; Wu, C.Y. Evaluation of the
antibacterial potential of liquid and vapor phase phenolic essential oil compounds against oral microorganisms. PLoS ONE 2016
11, e0163147. [CrossRef] [PubMed]
,
43. Inouye, S.; Uchida, K.; Takizawa, T.; Yamaguchi, H.; Abe, S. Evaluation of the effect of terpenoid quinones on Trichophyton
mentagrophytes by solution and vapor contact. J. Infect. Chemother. 2006, 12, 100–104. [CrossRef]
44. Espinel-Ingroff, A.; Canton, E. Antifungal susceptibility testing of yeasts. In Antimicrobial Susceptibility Testing Protocols; Schwalbe,
R., Steele-Moore, L., Goodwin, A.C., Eds.; CRC Press: Boca Raton, FL, USA, 2007; pp. 173–208.
45. Vihanova, K.; Houdkova, M.; Promgool, T.; Urbanova, K.; Kanokmedhakul, S.; Kokoska, L. In vitro growth-inhibitory effect of
essential oils and supercritical carbon dioxide extracts from Cinnamomum spp. barks and fruits against food bacterial pathogens
in liquid and vapor phase. J. Food Saf. 2021, e12900. [CrossRef]
46. Special Programme for Research and Training in Tropical Diseases. Available online: http://www.who.int/tdr/grants/
workplans/en/cytotoxicity_invitro.pdf (accessed on 11 March 2021).
47. Gurung, R.L.; Lim, S.N.; Khaw, A.K.; Soon, J.F.F.; Shenoy, K.; Ali, S.M.; Jayapal, M.; Sethu, S.; Baskar, R.; Hande, M.P. Thymoquinone
induces telomere shortening, DNA damage and apoptosis in human glioblastoma cells. PLoS ONE 2010, 5, e12124. [CrossRef]
48. Ivankovic, S.; Stojkovic, R.; Jukic, M.; Milos, M.; Milos, M.; Jurin, M. The antitumor activity of thymoquinone and thymohydro-
quinone in vitro and in vivo. Exp. Oncol. 2006, 28, 220–224.
49. Li, L.H.; Wu, P.; Lee, J.Y.; Li, P.R.; Hsieh, W.Y.; Ho, C.C.; Ho, C.L.; Chen, W.J.; Wang, C.C.; Yen, M.Y.; et al. Hinokitiol induces DNA
damage and autophagy followed by cell cycle arrest and senescence in gefitinib-resistant lung adenocarcinoma cells. PLoS ONE
2014, 9, e104203. [CrossRef] [PubMed]
50. Lee, T.B.; Seo, E.J.; Lee, J.Y.; Jun, J.H. Synergistic anticancer effects of curcumin and hinokitiol on gefitinib resistant non-small cell
lung cancer cells. Nat. Prod. Commun. 2018, 13, 1667–1671. [CrossRef]
51. Lee, T.B.; Jun, J.H. Can hinokitiol kill cancer cells? Alternative therapeutic anticancer agent via autophagy and apoptosis. Korean
J. Clin. Lab. Sci. 2019, 51, 221–234. [CrossRef]
52. Allemailem, K.S.; Alnuqaydan, A.M.; Almatroudi, A.; Alrumaihi, F.; Aljaghwani, A.; Khalilullah, H.; Younus, H.; Khan, A.
;
Khan, M.A. Safety and therapeutic efficacy of thymoquinone-loaded liposomes against drug-sensitive and drug-resistant Acineto-
bacter baumannii. Pharmaceutics 2021, 13, 677. [CrossRef]
53. Tesarova, H.; Svobodova, B.; Kokoska, L.; Marsik, P.; Pribylova, M.; Landa, P.; Vadlejch, J. Determination of oxygen radical absorbance
capacity of black cumin (Nigella sativa) seed quinone compounds. Nat. Prod. Commun. 2011, 6, 213–216. [CrossRef] [PubMed]
54. NIST WebBook Chemie. NIST Standard Reference Database Number 69. 2017. Available online: http://webbook.nist.gov/
chemistry/ (accessed on 29 September 2020).
55. Castillo, A.M.; Patiny, L.; Wist, J. Fast and accurate algorithm for the simulation of NMR spectra of large spin systems. J. Magn.
Reson. 2011, 209, 123–130. [CrossRef]
56. Steinbeck, C.; Krause, S.; Kuhn, S. NMRShiftDB-Constructing a free chemical information system with open-source components.
J. Chem. Inform. Comput. Sci. 2003, 43, 1733–1739. [CrossRef]
57. Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing; 25th Informational
Supplement M100-S25; CLSI: Wayne, PA, USA, 2015.
58. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays.
J. Immunol. Methods 1983, 65, 56–63. [CrossRef]
59. Kokjohn, K.; Bradley, M.; Griffiths, B.; Ghannoum, M. Evaluation of in vitro activity of ciclopirox olamine, butenafine HCl and
econazole nitrate against dermatophytes, yeasts and bacteria. Int. J. Dermatol. 2003, 42, 11–17. [CrossRef] [PubMed]
60. Trevor, A.J.; Katzung, B.G.; Kruidering-Hall, M. Katzung and Trevor’s Pharmacology Examination and Board Review, 11th ed.;
McGraw-Hill Education: New York, NY, USA, 2015; p. 20, ISBN 978-0-07-182639-6.