Article

Thymus vulgaris L. Essential Oil Solid Formulation:

Chemical Profile and Spasmolytic
and Antimicrobial Effects
Matteo Micucci 1,†, Michele Protti 2,†, Rita Aldini 1, Maria Frosini 3, Ivan Corazza 4,
Carla Marzetti 5, Laura Beatrice Mattioli 1, Gabriella Tocci 5, Alberto Chiarini 1,6,
Laura Mercolini 2,* and Roberta Budriesi 1,*
1 Department of Pharmacy and Biotechnology, Nutraceutical Lab, Alma Mater Studiorum—University of
Bologna, 40126 Bologna, Italy; [email protected] (M.M.); [email protected] (R.A.);
[email protected] (L.B.M.); [email protected] (A.C.)
2 Department of Pharmacy and Biotechnology, Research group of Pharmaco‐Toxicological Analysis

(PTA Lab), Alma Mater Studiorum—University of Bologna, 40126 Bologna, Italy; [email protected]
3 Department of Life Sciences, Vita, University of Siena, 53100 Siena, Italy; [email protected]

4 Department of Experimental, Diagnostic and Specialty Medicine—DIMES,

Alma Mater Studiorum—University of Bologna, 40138 Bologna, Italy; [email protected]

5 Valsambro S.r.l., 40121 Bologna, Italy; [email protected] (C.M.);

[email protected] (G.T.)
6 GVM Care & Research, 48022 Lugo, Ravenna, Italy

* Correspondence: [email protected] (L.M.); [email protected] (R.B.);

Tel.: +39‐051‐20‐9‐9726 (L.M.); +39‐051‐20‐9‐9737 (R.B.)
† The two Authors contributed equally to the work.

Received: 24 April 2020; Accepted: 29 May 2020; Published: 4 June 2020

Abstract: A new Thymus vulgaris L. solid essential oil (SEO) formulation composed of liquid EO
linked to solid excipients has been chemically analysed and evaluated for its intestinal spasmolytic
and antispastic effects in ex vivo ileum and colon of guinea pig and compared with liquid EO and
excipients. Liquid EO and solid linked EO were analysed by original capillary
electrochromatography coupled to diode array detection (CEC‐DAD) and liquid chromatography‐
tandem mass spectrometry (LC‐MS/MS) methodologies. The main bioactive constituents are thymol
and carvacrol, with minor constituents for a total of 12 selected analysed compounds. Liquid EO
was the most effective in decreasing basal contractility in ileum and colon; excipients addiction
permitted normal contractility pattern in solid linked EO SEO. In ileum and colon, the Thymus
vulgaris L. solid formulation exerted the relaxant activity on K+‐depolarized intestinal smooth
muscle as well as liquid EO. The solid essential oil exhibits antimicrobial activity against different
strains (Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa, Escherichia coli,
Salmonella Thyphimurium, Candida albicans) similarly to liquid oil, with activity against pathogen, but
not commensal strains (Bifidobacterium Breve, Lactobacillus Fermentum) in intestinal homeostasis.
Therefore, Thymus vulgaris L. solid essential oil formulation can be proposed as a possible
spasmolytic and antispastic tool in medicine.

Keywords: capillary electrochromatography; diarrhoea; intestinal contractility; L‐type Calcium

channels; LC‐MS/MS; solid based formulation

1. Introduction
In traditional medicine, humans have greatly benefited from plants and their secondary
metabolites. Plants have been used not only for their properties occasionally attributed to their
Biomolecules 2020, 10, 860; doi:10.3390/biom10060860 www.mdpi.com/journal/biomolecules
Biomolecules 2020, 10, 860 2 of 20

secondary metabolites, but interest is also due to essential oils (EOs) diffused in or obtained from the
surface of plant organs, particularly their aerial parts—flowers and leaves. EOs are complex mixtures
of chemical compounds that have long been known and used as natural food preservatives, aromatic
additives [1], in personal care products, and in aromatherapy [2]. The interest of the scientific
community in EOs has been increasing, as their therapeutic properties have been gradually
confirmed [1]. Among all the EOs used in the therapeutic field, there is a growing commercial interest
in the EO of Thymus vulgaris L. (common thyme), now one of the 10 most widely traded EOs in the
world. It is an aromatic and medicinal plant. So far, 928 species of the Thymus genus have been
identified in Europe, North Africa, Asia, South America, and Australia [3]. Thymus EOs are mainly
composed of terpenes, terpene alcohols, esters, and phenolic derivatives. Among them, thymol and
carvacrol present the highest interest. The traditional and the therapeutic use, supported by in vitro
studies, has shown that Thymus and its derivatives present a wide biological spectrum—antioxidant
[4,5], antibacterial [5,6], antifungal [7], and muscle antispasmodic activities [8].
Thymus EOs have beneficial effects on neurodegenerative, cardiovascular, cancer, and
inflammatory diseases [9]; are traditionally used to treat respiratory tract problems [8]; and relieve
gastrointestinal spasm and digestion [3]. Therefore, Thymus and its EO are functional and promising
in medicine [3].
In this paper, we have studied a peculiar formulation of Thymus vulgaris L. EO absorbed onto a
solid matrix of excipients (solid essential oil—SEO) inserted into a capsule (operculum) for a potential
use in intestinal pathologies. The operculum was opened, and the solid liquid oil was obtained. The
rationale was to obtain information about the effect of this solid form of EO on intestinal contractility.
EO solid formulation, in fact, possessed many advantages, such as the ability to overcome patient
taste and smell distaste; to modulate EO release; to increase the stability of the active essential oil;
and to reduce volatility, toxicity, and interactions with the intestinal substances, thus improving
patient compliance and convenience. In addition, an original analytical methodology based on
capillary electrochromatography coupled to diode array detection (CEC‐DAD) was developed for
the separation and determination of twelve selected constituents of Thymus vulgaris L. EO as a
representative set of bioactive compounds that can be correlated with the biological activities object
of this study. The target analytes were thymol, carvacrol, p‐cymene, α‐terpinene, γ‐terpinene,
linalool, borneol, β‐cariophyllene, β‐myrcene, α‐terpineol, β‐pinene, and limonene. After method
validation with good results in terms of linearity, precision and accuracy, this original strategy was
applied for the analysis of Thymus vulgaris L. EO and a derived formulation. In addition, thymol and
carvacrol being among the most abundant and characteristic bioactive constituents of Thymus EOs,
an original method based on liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) was
set up and validated in order to accurately confirm thymol and carvacrol levels in the analysed
samples and to demonstrate the effectiveness of CEC‐DAD analysis. Since the current focus on
natural products is to develop their formulation to improve bioavailability, pharmacokinetics, and to
reduce adverse effects for the treatment of various human diseases [10], the main components of the
formulation (SEO, EO, and excipients) were then evaluated for a potential use in intestinal
pathologies, by assessing their effects on spontaneous and induced contractility of guinea pig smooth
muscle isolated gallbladder, gastric fundus, ileum, and colon. Otilonium bromide (OB), an
antispasmodic drug, has been considered as a positive control. At the same time, potential
antimicrobial activity of Thymus EO alone, SEO, and excipients has been tested against some bacteria
and fungi most commonly involved in the onset and progression of gastrointestinal diseases.

2. Materials and Methods

The Thymus vulgaris L. solid essential oil (SEO) formulation named Aromatoil® (manifactured
by Coima, Bastia (RA), Italy) used in this study was supplied by BIO‐LOGICA, Via della Zecca 1,
40100, Bologna, Italy. The used essential oil has been obtained by steam distillation of the summit
flowers. The formulation was made by Thymus vulgaris L. EO absorbed to a solid matrix of excipients
(SEO) inserted into a capsule (operculum). The operculum was discharged, and the solid liquid oil
SEO was obtained. Each operculum, contains summit flowers Thymus vulgaris L. essential oil (0.6 mg)
Biomolecules 2020, 10, 860 3 of 20

and 340.4 mg of excipients consisting of pregelatinized corn starch, soy lecithin, ascorbic acid, calcium
carbonate, levilite, vegetable magnesium stearate (Invention Patent Application N:
102018000007395—(I0174439)).

2.1. Instrumental Analysis: CEC‐DAD and LC‐MS/MS

Chemicals and solutions. Analytical‐grade standards of thymol [5‐methyl‐2‐(propan‐2‐
yl)phenol], carvacrol [2‐methyl‐(propan‐2‐yl)phenol], p‐cymene [1‐methyl‐4‐(propan‐2‐yl)benzene],
α‐terpinene [4‐methyl‐1‐(1‐methylethyl)‐1,3‐cyclohexadiene], γ‐terpinene [4‐methyl‐1‐(1‐
methylethyl)‐1,4‐cyclohexadiene], linalool (3,7‐dimethyl‐1,6‐octadien‐3‐ol), borneol (endo‐1,7,7‐
trimethyl‐ bicycle [2 .2.1]heptan‐2‐ol), β‐cariophyllene {(1R,4E,9S)‐4,11,11‐Trimethyl‐8‐
methylidenebicyclo[7.2.0]undec‐4‐ene}, β‐myrcene (7‐methyl‐3‐methylene‐octa‐1,6‐diene), α‐
terpineol [2‐(4‐Methylcyclohex‐3‐en‐1‐yl)propan‐2‐ol], β‐pinene (6,6‐dimethyl‐2‐
methylidenebicyclo[3.1.1]heptane), limonene [1‐methyl‐4‐(prop‐1‐en‐2‐yl)cyclohex‐1‐ene] and
xylene (dimethylbenzene), used as the internal standard (IS) for CEC‐DAD, were obtained from
Sigma Aldrich (St. Louis, MO, USA). MS‐grade acetonitrile (ACN) and methanol (MeOH), analytical‐
grade formic acid, and ammonium acetate were also purchased from Sigma Aldrich, while ultrapure
water (18.2 MΩ∙cm) was obtained by means of a Milli‐Q system from Millipore (Burlington, MA,
USA). Analyte and IS stock solutions (1 mg/mL) were prepared in MeOH and stored at −20 °C,
working solutions were prepared daily by dilution in the mobile phase of each system and stored
protected from light in amber glass vials.

2.1.1. Analytical Conditions

CEC‐DAD analyses were carried out on a 3DCE capillary electrophoresis apparatus from Agilent
Technologies, equipped with a DAD operating at 210 nm. Fused silica capillary (32 cm total length x
100 μm ID, 375 OD) was from Polymicro Technologies (Phoenix, AZ, US) and packed with
LiChrospher 100 RP‐18 endcapped particles (5 μm particle size, 100 Å pore size) from Merck‐
Millipore. The optimised mobile phase was a mixture of 50 mM, pH 5.5 ammonium acetate solution
and ACN (15/85, V/V), while the capillary temperature was kept constant at 25 °C. Analyses were
carried out applying a 30 kV voltage and 8 bar pressure at both ends of the capillary and samples
were injected at the anodic end of the capillary by applying a pressure of 5 bar for 30 s. LC‐MS/MS
analysis was exploited in order to confirm quali‐quantitative results obtained by CEC‐DAD as
regards major components thymol and carvacrol. The LC‐MS/MS analytical conditions developed
ad‐hoc for this research work are described in Supporting Material (S1).

2.1.2. Sample Analysis

In order to be applied for the analysis of EO and formulations, both CEC‐DAD and LC‐MS/MS
methods were fully validated on analyte standard solutions, according to the main international
guidelines [11] in terms of linearity (including limit of detection, LOD and limit of quantitation, LOQ),
precision and accuracy. As regards EO analysis, a 100‐μL aliquot was suitably diluted in the mobile
phase of both the instrumental systems, filtered through 0.2 μm pore diameter nylon syringe filters
and injected in the two analytical systems described above. As regards formulations containing
Thymus EO, three capsules were individually weighed, their content was mixed, and an aliquot of
100 mg was extracted with 10 mL of mobile phase by vortex agitation for 30 s and ultrasonic bath for
15 min. The suspension was then centrifuged at 4500 RPM for 10 min, the supernatant was transferred
in autosampler vials and analysed by both CEC‐DAD and LC‐MS/MS. Compound quantitation was
obtained by integrating peak areas obtained from sample analysis and interpolation on the linearity
curve of each analyte. All analyses were carried out in triplicate by both CEC‐DAD and LC‐MS/MS
on a single batch of both EO and formulations. Quantitative results were then expressed as μg of
analyte for 100 μL of EO (% m/V) for Tymus vulgaris L. EO samples and as μg/cps for EO‐based
formulations.
Biomolecules 2020, 10, 860 4 of 20

2.2. Ex Vivo Muscle Contractibility Evaluations

Male Guinea‐pig (200–400 g) obtained from Charles River (Calco, Como, Italy) were used. The
animals were housed according to the ECC Council Directive regarding the protection of animals
used for experimental and other scientific purposes. The work was conducted according to the
guidelines set forth to EU Directive 2010/63/EU and to ARRIVE guidelines [12]. The protocol was
approved by the Institutional Ethics Committee of the University of Bologna (Protocol PR 21.79.14)
and transmitted to the Ministry of Health. Humane end points were followed (https://www.humane‐
endpoints.info/en).
The animals were sacrificed by cervical dislocation. The organs studied were stomach, ileum,
proximal colon and gallbladder. Briefly, the organs were set up rapidly under a suitable resting
tension in 15 mL organ bath, containing appropriate physiological salt solution (PSS) consistently
warmed and buffered to pH 7.4 by saturation with 95% O2—5% CO2 gas and used as previously
described [13].
For detailed information about gastric fundus, ileum, proximal colon, and gallbladder, see
Supporting Material (S2).

2.2.1. Contractility Spontaneous

The tracing graphs of Spontaneous Contractions (SC) (g/min) of ileum, colon, gallbladder and
gastric fundus were continuously recorded with the LabChart Software (version 5.04, GraphPad
Software Inc., San Diego, CA, USA). After the equilibration period (about 30 min to 45 min according
to each tissue) cumulative‐concentration curves (0.1, 0.5, 1, 5, 10 mg/mL) to samples were constructed.
At the end of each single dose, a 5 min stationary period was selected and, for each interval, the
following parameters were evaluated: mean contraction amplitude (MCA), calculated as the mean
force value (g); the force contractions standard deviations, considered as an index of the spontaneous
contraction variability (SCV); and basal spontaneous contraction activity (BSCA), calculated as the
percentage (%) variation of each mean force value (g) with respect the control. For details about
spontaneous contractions rates through a standard FFT analysis, see Supplementary Material S2.
In order to avoid errors due to the presence of artefacts, the period of analysis was chosen by a
skilled operator.

2.2.2. Contractility
The spasmolytic activity via action on L‐type calcium channel was studied using ileum, colon,
and gallbladder contracted by high K+‐concentration. Tension changes in smooth muscle relaxation
were recorded isometrically as previously described [14].

2.3. Antibacterial Activity

The antibacterial activity was performed against Gram+: Staphylococcus aureus (ATCC 25923
KS2), Streptococcus pyogenes (ATCC 19615), Bifidobacterium Breve (ATCC 15700), Lactobacillus
Fermentum (ATCC 9338); Gram: Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC
700728), Salmonella Thyphimurium (ATCC 14028); and fungus: Candida albicans (ATCC 14053). For
detailed information, see Supporting Material (S3).

2.4. Statistical Analysis

For in vitro studies on isolated organs, data are presented as described below. Spontaneous
contractility: the samples were added in a cumulative manner. Variation higher than 10% percent
variations of each range were considered statistically significant. On spontaneous contractility
experiments, data from concentration‐response curves were analysed by GraphPad Prism® version
5.04, GraphPad Software Inc., San Diego, CA, USA [15,16]. Induced contractility: the spasmolytic
activity of samples was expressed as the percent inhibition of calcium‐induced contraction on K+‐
depolarized ileum, colon and gallbladder strips (smooth muscle activity) and presented as mean ±
Biomolecules 2020, 10, 860 5 of 20

S.E.M. The potency of all samples defined as IC50 was evaluated from log concentration–response
curves (Probit analysis by Litchfield and Wilcoxon, n = 6–8) in the appropriate pharmacological
preparations [15–17]. Antibacterial activity: the minimal inhibitory concentrations (MICs) values
were determined by the microdilution method [18]. Data were evaluated using the IBM SPSS
software program (version 19; IBM SPSS Inc., IL, USA). All tested samples and control groups were
compared at the 95% confidence interval.

3. Results

3.1. Analytical Characterization

In order to effectively analyse the content of representative compounds in Thymus EO and EO‐
based formulations, original CEC‐DAD and LC‐MS/MS methodologies were optimised and fully
validated. CEC‐DAD was exploited to perform a qualitative and quantitative evaluation of 12
compounds, while LC‐MS/MS was used to accurately confirm the quantitative levels of thymol and
carvacrol in samples.
Both CEC‐DAD and LC‐MS/MS methods were fully validated in terms of linearity, precision
and accuracy. Method development and complete validation data are reported in Supplementary
Material S4. Briefly, method sensitivity was between 2 μg/mL and 5 μg/mL in terms of limit of
quantitation (LOQ) while linearity was deemed good (r2 ≥ 0.9991) over the 5–200 μg/mL range for all
the analytes. Method precision was also satisfactory, being the percentage relative standard deviation
(RSD%) always < 5.7%, while accuracy was ≥ 85%.
Qualitative and quantitative results obtained from the analysis of Thymus EOs and derived
formulation, by applying CEC‐DAD methodologies are reported in Table 1.

Table 1. Chemical composition of Thymus vulgaris L. essential oil and derived formulation.

Thymus vulgaris L. EO EO‐based Formulation

Class Compound
(μg/100 μL) (μg/cps)
p‐Cymene 9.4 ± 0.6 41.8 ± 6.5
α‐Terpinene 0.7 ± 0.2 4.4 ± 0.5
Monoterpenes γ‐Terpinene 4.0 ± 0.7 19.3 ± 1.0
β‐Myrcene 2.1 ± 0.4 12.1 ± 0.8
Limonene 0.4 ± 0.1 1.8 ± 0.3
Bicyclic monoterpenes β‐Pinene 1.1 ± 0.2 4.3 ± 0.6
Thymol 43.3 ± 1.4 210.2 ± 5.6
Carvacrol 20.7 ± 2.3 99.7 ± 7.4
Monoterpenols
Linalool 0.7 ± 0.2 3.4 ± 0.6
α‐Terpineol 0.2 ± 0.1 0.9 ± 0.2
Bicyclic monoterpenols Borneol 1.3 ± 0.4 6.4 ± 0.7
Sesquiterpene lactones β‐Cariophyllene 3.1 ± 0.5 13.9 ± 1.0

As can be seen, all 12 compounds included in the CEC‐DAD method were successfully identified
and quantified in both samples. As regards confirmatory analysis performed by LC‐MS/MS for
thymol and carvacrol, these provided quantitative results in good agreement with those obtained by
CEC‐DAD, namely 43.5 ± 0.3 μg/100 μL and 21.0 ± 1.1 μg/100 μL for thymol and carvacrol in EO
samples respectively, and 213.3 ± 3.4 μg/cps and 102.2 ± 4.8 respectively for thymol and carvacrol in
EO‐based formulations, thus proving the effectiveness of CEC‐DAD analysis. The
electrochromatogram obtained from the analysis of a Thymus vulgaris L. essential oil sample under
the optimised conditions is shown in Figure 1, while the LC‐MS/MS chromatogram obtained from
the analysis of a Thymus vulgaris L. essential oil–based formulation sample is shown in Figure 2.
Biomolecules 2020, 10, 860 6 of 20

Figure 1. Capillary electrochromatography coupled to diode array detection (CEC‐DAD)

electrochromatogram obtained from the analysis of a Thymus vulgaris L. essential oil sample under
the optimised conditions: 1, borneol; 2, linalool; 3, α‐terpineol; 4, thymol; 5, carvacrol; 6, p‐cymene; 7,
β‐pinene; 8, α‐terpinene; 9, β‐myrcene; 10, β‐Cariophyllene; 11, γ‐terpinene; 12, limonene.

Figure 2. Liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) chromatogram obtained

from the analysis of a Thymus vulgaris L. essential oil–based formulation sample under the optimised
conditions: 1, thymol; 2, carvacrol.

3.2. Ex Vivo Muscle Contractibility Evaluations

Liquid EO and SEO effects were studied on gastric fundus, ileum, colon, and gallbladder
contractility, on both spontaneous and induced contractility (K+ 80 mM). In addition, the excipients
(Table 1) were also tested. The results were compared with antispasmodic and spasmolytic activity
of OB, taken as a positive reference drug [19]. A quantitative comparison between EO, SEO, and OB
is not possible, since OB is a single molecule, EO and SEO are a mixture of chemical compounds.
Only a qualitative comparison is possible, most important for a possible therapeutic use.

3.2.1. Spontaneous Contractility

The variation of spontaneous contraction for isolated stomach, ileum, colon, and gallbladder
tissues was evaluated through concentration‐response curves. For all tissues, the changes of basal
Biomolecules 2020, 10, 860 7 of 20

activity induced by these chemical compounds were evaluated and were expressed as BSMA, SC and
following the modification of frequency bands of interest in each tissue. For all tissues, the original
record tract was shown.

Ileum
Liquid EO has the highest effect and induces a decrease (by 40%) in ileal tone as early as at 0.1
mg/mL concentration, up to a 44% at 10 mg/mL; SEO presents an effect concentration dependent,
with maximal activity of 48% at 10 mg/mL. Excipients have minimal effect: up to 15% at 10mg/mL
(Figure 3). At the maximal concentration, (10 mg/mL) EO and SEO effects are similar, although the
EO effective concentration is 500 times lower in SEO than in EO. The OB IC50 (5x10‐7 M) on basal
spontaneous contraction activity (BSCA) is close to the effects elicited by EO and SEO at the maximal
concentration.

A B
EO SEO Excipients OB
20 20
Basal Spontaneously Contraction Activity (%)

Basal Spontaneously Contraction Activity (%)

0.01 0.1 1 10 100 0.1 1 10 100 1000
0 0
(mg/mL) (ng/mL)
(0.001)

-20 (0.002) -20

(0.01)

-40 -40

(0.02)

-60 -60

-80 -80

Figure 3. Ileum: basal spontaneous contraction activity. Zero represents the basal tone and each point
is the percent variation from the baseline after cumulative addition of each dose. (A) Essential oil
(EO), solid essential oil (SEO), and excipients (mg/mL); numbers in brackets represent the effective
EO concentration (mg/mL) in SEO; (B) otilonium bromide (OB) (ng/mL), an antispasmodic drug, used
as a positive control. Each value (expressed as percent variation) is the mean ± SEM; when the error
bar is not shown, it is covered by the point.

Spontaneous contraction greatly decreases for EO at the minimal concentration, it decreases for
SEO at the maximal concentration (10mg/mL), consistently for the lower dose of essential oil in SEO;
spontaneous contraction decreases slightly and minimally for excipients and OB, respectively.
Therefore, the excipients effect is compliant with essential oil.
Contractility decreases at the lowest concentration (0.1 mg/mL) for EO, while it remains almost
constant for SEO and drops with the maximum concentration (10 mg/mL), and variability decreases
at 10 mg/mL. MCA and variability progressively decrease up to the maximal concentration; mean
amplitude and variability progressively decrease for OB (Figure 4).
Therefore, liquid essential oil has the highest power of decreasing the ileal contractility, since the
solid formulation presents 0.18% of essential oil rather than the liquid form. Excipients do not get in
the way of essential oil, but they act synergistically. Moreover, spontaneous contraction rates (FFT)
analysis showed that variability drops from the control to the first concentration and then remains
unchanged (on all frequencies) with a minimum for 10 mM concentrations (Supplementary Material
S5). OB maintains the ileal contraction, but in presence of a modest decrease of contraction amplitude
and low frequencies waves. Therefore, SEO profile seems similar to OB profile, since the decrease in
ileal tone seems associated to modest reduction in waves morphology (Supplementary Material S5).
Biomolecules 2020, 10, 860 8 of 20

Figure 4. Focus on experimental original recording of the concentration‐response curve of EO and

SEO, excipients, and otilonium bromide (OB) on spontaneous ileum basal contractility. Spontaneous
contraction (SC); spontaneous contraction variability (SCV); mean contraction amplitude (MCA).
Biomolecules 2020, 10, 860 9 of 20

Colon
Liquid EO reduces by 44% the ileal tone, independently on the dose; SEO decreases the tone in
a concentration dependent manner up to 50% at the highest dose. Excipients effect is much less
significant, as it reached the maximum of 20% at the highest concentration of 10 mg/mL. OB decreases
the tone, dose dependently up to 30% at 5x10‐5 M (Figure 5).

A B
EO SEO Excipient
20 20 OB
Basal Spontaneously Contraction Activity (%)

Basal Spontaneously Contraction Activity (%)

0.01 0.1 1 10 100 0.01 0.1 1 10 100
0 0
(mg/mL) (µg/mL)
(0.002)
-20 -20

-40 -40
(0.01)
(0.02)
-60 -60

-80 -80

Figure 5. Basal spontaneous contraction activity (BSCA) in the colon. Zero represents the basal tone
and each point is the percent variation from the baseline after cumulative addition of each dose. (A)
EO, SEO and excipients (mg/mL); numbers in brackets represent the effective EO concentration
(mg/mL) in SEO. (B) Otilonium bromide (OB) (μg/mL), an antispasmodic drug, used as a positive
control. Each value (expressed as percent variation) is the mean ± SEM; when the error bar is not
shown, it is covered by the point.

Spontaneous contraction is decreased severely by EO, less by SEO, even less by excipients, and
not by OB. Mean contraction amplitude is severely reduced by EO, with minimal variability; it is
progressively reduced by SEO, with reduced variability; and by excipients, reduced by OB only at
the highest concentration but without variability (Figure 6). In addition, the size of the bars in the FFT
are very high for the control and smaller but similar to each other for the different concentrations.
(Supplementary Material S6).
Excluding the control (for which the variability is very different), the contractility for SEO and
OB is similar.
The effect of SEO is similar in the two organs, liquid EO is the most effective in both the organs;
excipients decrease the effect of EO to the values of SEO. The SEO decrease in intestinal tone is 50%
both in the ileum and in the colon and is similar to OB in ileum.
Ileal and colonic contraction is maintained in presence of a reduction of the tone, although the
SCV is maintained more in the ileum than in colon, suggestive of a stronger effect on the colon.
Biomolecules 2020, 10, 860 10 of 20

Figure 6. Focus on experimental original recording of the concentration‐response curve of liquid EO

and SEO, excipients, and OB on spontaneous colon basal contractility. Spontaneous Contraction (SC);
spontaneous contraction variability (SCV); mean contraction amplitude (MCA). Absolute band
powers of control and after addition of each concentration observed in the same experiment.

Gallbladder
Liquid EO and SEO decrease the BSCA independently on concentration at minimal value (about
5%). Surprisingly, a dose‐dependent effect is obtained by excipients, but without influence on SEO
values, that are similar to liquid EO (Figure 7).
Biomolecules 2020, 10, 860 11 of 20

EO SEO Excipients
20

Basal Spontaneously Contraction Activity (%)

0.01 0.1 1 10 100
0
(mg/mL)

-20

-40

-60

-80

Figure 7. Basal spontaneous contraction activity elicited by free EO, SEO, and excipients in the
gallbladder. Zero represents the basal tone and each point is the percent variation from the baseline
after cumulative addition of each dose. Each value (expressed as percent variation) is the mean ± SEM;
when the error bar is not shown, it is covered by the point.

Spontaneous contraction is constant, contractility slightly modified without variability of

spontaneous contractions, both for EO and for SEO. Excipients decrease contraction but not
variability of contractions; therefore, the effect of essential oil on gallbladder are very small (Figure
8). In addition, the variability on FFT is observed only at higher concentrations (Supplementary
Material S7).
Biomolecules 2020, 10, 860 12 of 20

Figure 8. Focus on experimental original recording of the concentration‐response curve of liquid EO,
SEO, and excipients on spontaneous gallbladder basal contractility. Spontaneous contraction (SC);
spontaneous contraction variability (SCV); mean contraction amplitude (MCA).

Gastric Fundus
Liquid EO and excipients induce a modest decrease in the gastric fundus tone. SEO
progressively decreases the gastric tone up to 70% at the highest studied dose (Figure 9).

EO SEO Excipients
20

Basal Spontaneously Contraction Activity (%)

0.01 0.1 1 10 100
0
(mg/mL)

(0.0002)
-20

(0.001)
-40

(0.002)

-60
(0.01)

(0.02)
-80

Figure 9. Basal spontaneous contraction activity elicited by free EO, SEO, and excipients in the gastric
fundus. Zero represents the basal tone and each point is the percent variation from the baseline after
cumulative addition of each dose. Numbers in brackets represent the effective EO concentration
(mg/mL) in SEO. Each value (expressed as percent variation) is the mean ± SEM; when the error bar
is not shown, it is covered by the point.

Liquid EO, SEO, and excipients do not influence spontaneous contractility pattern in a
concentration dependent manner (Figure 10). The pattern of the contractions is regularly maintained.
EO tone remains constant and increases by 1 mg/ml concentration and then decreases. Compared to
the control, the variability drops but shows a slight increase to 10 mg/mL concentration compared to
the smaller ones (Figure 10). SEO tone gradually decreases; the variability increases for
concentrations greater than 1 mg/mL (Figure 10). Excipients tone drops (Figure 10); the variability is
constant up to 1 mg/ml, and then it increases to 10 mg/mL (Figure 10). In all case regarding the FFT,
low frequency prevails (Supplementary Materials S8).
Biomolecules 2020, 10, 860 13 of 20

Figure 10. Focus on experimental original recording of the concentration‐response curve of liquid EO,
SEO and excipients on spontaneous gastric fundus basal contractility. Spontaneous contraction (SC);
spontaneous contraction variability (SCV); mean contraction amplitude (MCA).

3.2.2. Induced Contractility

Liquid EO, SEO, and excipients have been studied on intestinal segments depolarized by high
K (80 mM) to evaluate its spasmolytic effects by affecting calcium movements through L‐type
+

calcium channels.

Ileum and Colon

Liquid EO inhibition is independent on dose while on the contrary, SEO and excipients act in
dose‐dependent manner and present overlapping curves. EO inhibited the activity by 77.4 ± 0.2%
already at the lowest concentration tested of 0.1 mg/mL. The same inhibition was attained by SEO
and excipients at 50‐fold and 100‐fold higher concentration, respectively. (Figure 11A). The
excipients, separately studied, have similar values of intrinsic activity: 72 ± 1.9 but at five times greater
concentrations (Figure 11). The same trend is repeated in the colon (Figure 11C), with the difference
that the excipients, separately taken, have a maximum intrinsic activity at 5 mg/mL with less potency
(Table 2). OB taken as a positive control has higher spasmolytic potency on colon than ileum (IC50
ileum 8.3 μM, colon 4.8 μM). Although it is not possible to make quantitative comparisons, since
otilonium is a single molecule and EO is a complex mixture of compounds, the activity profile of is
very similar, suggesting that they probably act on the same targets.
Percent inhibition is independent on concentration for liquid EO and similar in the ileum and
colon. Excipients present a similar concentration‐dependent decrease, overlapping SEO in both
organs, as the effect of excipients seems the same.
Biomolecules 2020, 10, 860 14 of 20

A B
120 120
EO SEO Excipients OB

100 (0.02) 100

IC50=8.3 µM (28.2 µg)

80 (0.01) 80

% OF INHIBITION

% OF INHIBITION
60 (0.002) 60
(5.64 µg)

40 40
(2.8 µg)
(0.56 µg)
20 20
(0.001) ILEUM (56.3 ng) (0.28 µg) ILEUM
0 0

0.01 0.1 1 10 100 -8 -7 -6 -5 -4 -3

(mg/mL) Log [M]

C D
120 120
EO SEO Excipients OB

100 100

IC50=4.8 µM (28.2 µg)

80 80
% OF INHIBITION

% OF INHIBITION

60 (0.02) 60 (5.64 µg)

(0.01)
(2.8 µg)
40 (0.002) 40

(0.56 µg)
20 20
(56.3 ng)
(0.28 µg)
(0.001)
COLON COLON
0 0 (28.2 ng)

0.01 0.1 1 10 100 -8 -7 -6 -5 -4 -3

(mg/mL) Log [M]

Figure 11. Cumulative concentration‐response curves of spasmolytic activity of Thymus vulgaris L.

free EO, SEO, and excipients and otilonium bromide (OB) against potassium chloride‐ (80 mM)
induced contraction on guinea pig ileum (panels A and B) and colon (panels C and D). Each point is
the mean ± SEM of four‐six experiments. Where error bars are not shown these are covered by the
point itself. For a better comparison of the effects, numbers in brackets represent the effective EO
concentration (mg/mL) in SEO (panels A and C) or the amount of OB present in 1 mL (panels B and
D).

Gallbladder
The same study done on the gallbladder showed free EO and SEO action. The intrinsic activity
is 84 ± 2.4 (0.1 mg/mL) and 77 ± 1.6 (10 mg/mL), respectively. As for potency, SEO is 30 times less
potent than liquid EO. The excipients have no intrinsic activity worthy of note like the reference
compound (Table 2). Interestingly, intrinsic spasmolytic activity of OB on gallbladder is negligible.

Table 2. Relaxant activity of tested samples on K+‐depolarized guinea pig intestinal smooth muscle.

Activitya Potencyb
Tissue Comp. M ± SEM IC50 95% conf lim
Liquid EO 87 ± 1.6 (1)
SEO 100 ± 1.3 (5) 1.12 1.02−1.47
Ileum
Excip 72 ± 1.9 (5) 1.64 1.36−1.98
OB 90 ± 3 (0.005) 0.0048 0.0040−0.0057
Colon Liquid EO 85 ± 1.9 (0.5) 0.031 0.009−0.043
Biomolecules 2020, 10, 860 15 of 20

SEO 61 ± 2.4 (1) 0.70 0.57−0.85

Excip 95 ± 1.7 (5) 1.33 0.99−1.48
OB 90 ± 2.3 (0.02) 0.0019 0.0015−0.0025
Liquid EO 84 ± 2.4 (0.1) 0.048 0.035−0.055
SEO 77 ± 1.6 (10) 1.44 1.06−2.03
Gallbladder
Excip 38 ± 2.6 (1)
OB 10 ± 0.7 (0.02)
aPercent inhibition of calcium‐induced contraction on K+‐depolarized (80 mM) guinea pig ileum,
colon, and gallbladder. In parenthesis the indicated concentrations that give the maximum effect
expressed as mg/mL. bIC50, expressed as mg/mL, represent the concentration that inhibited 50% of the
maximum contraction induced by K+ 80 mM and was calculated from concentration‐response curves
(probit analysis by Litchfield and Wilcoxon [17] with n = 6–7).

3.3. Antimicrobial Activity

In order to confirm the antibacterial activity of SEO, we have evaluated effects on some lines of
bacteria and fungi. Table 3 shows minimal inhibitory concentration (MIC) values. As can be seen, the
solid version maintains the bactericidal action against some pathogens taken as a reference while the
excipients are devoid of effects as already documented in literature [20].
Unlike cyprofloaxacin, taken as positive control, SEO and free EO did not show any effect on
Bifido and Lactobacillus. In addition, in negative control no growth inhibition was observed.

Table 3. Antimicrobial activity of tested samples.

Microorganism strain MIC (mg/mL)

Liquid EO SEO Excipient Cyprofloaxacin
Gram bacteria
+

Staphylococcus aureus 0.28 >50 Inactive 0.005

Streptococcus pyogenes 0.004 2 Inactive 0.002
Bifidobacterium Breve Inactive Inactive Inactive 0.005
Lactobacillus Fermentum Inactive Inactive Inactive 0.005
Gram bacteria
–

Pseudomonas aeruginosa 0.0002 0.1 Inactive 0.0004

Escherichia coli 0.4 >50 Inactive 0.005
Salmonella Thyphimurium 0.33 >50 Inactive 0.005
Fungus
Candida albicans 0.0018 9 Inactive 0.005
10% DMSO Inactive Inactive Inactive Inactive
a Minimal inhibition concentration (MIC) values.

4. Discussion
The three pharmacologic agents currently indicated in the USA for treatment of irritable bowel
syndrome with diarrhea (IBS‐D) are non‐systemic antibiotic rifaximin, the mixed μ‐ and κ‐opioid
receptor agonist/δ‐opioid antagonist eluxadoline, and the selective serotonin 5‐HT3 antagonist
alosetron [21]. An acceptable initial therapy, especially for patients with mild disease, is lifestyle
modification and education. In this context, the antispasmodic and spasmolytic action of a drug is
used to treat excessive painful muscle contractility of the intestine [22]. However, loperamide, which
inhibits peristalsis and increases colonic transit time, is not helpful with abdominal pain [23], and
often more than a drug is necessary. The association of antispasmodic and antinociceptive activity
should be important. Moreover, the available synthetic antispasmodic and/or spasmolytic molecules
Biomolecules 2020, 10, 860 16 of 20

often present severe side effects, limiting treatment efficiency and patient compliance. Therefore, the
pharmaceutical industry is now searching for developing new drug candidates from plants rich in
essential oils [24]. EOs antioxidant, anti‐inflammatory, and antitumoral effects are widely known,
together with their antinociceptive activities; they act on the digestive system [25] and improve the
digestion process by stimulating the olfactory nerve endings [26,27]. Their antispasmodic effect has
been less investigated, mainly for their difficult oral administration and their local aggression, though
they are used in worldwide medicine. Peppermint oil administered orally in an enteric coated form
[28,29], was efficacious in reducing global symptoms and pain in IBS [30].
With this view, we have evaluated the effect of a solid formulation of Thymus vulgaris L. EO, that
presents spasmolytic and nociceptive effects [31] on the modulation of guinea pig ileum and colon
basal and induced contractility, in order to have experimental evidences of its antispasmodic and
spasmolytic intestinal effect. The study of the contractility of gallbladder and gastric fundus has been
done to rule out the possibility of side effects.
In Thymus EO, mainly phenolic compounds containing hydrogen, carbon, and oxygen are
present. By applying the original CEC‐DAD and LC‐MS/MS methodologies developed ad hoc for this
study to Thymus EO and to an EO‐based formulation, it was observed how the main bioactive
constituents of both considered samples are represented by thymol and carvacrol together with other
minor constituents for a total of 12 selected analysed compounds, among monoterpenes, bicyclic
monoterpenes, monoterpenols, bicyclic monoterpenols ,and sesquiterpene lactones. Such results are
consistent with the literature on the subject [32,33], also considering that the type of cultivar, the
geographical area, and seasonality significantly influence the content of bioactive compounds in
Thymus essential oil composition and thus its chemotype [34,35]. Based on these results, the most
represented phytochemicals thymol and carvacrol are responsible for the modulation of contractility
of EO and SEO. However, the interactions of different phytochemicals present in the phytocomplex
could produce synergistic antispastic and spasmolytic effects observed in ex vivo experiments [36].
In the basal conditions, SEO, consisting in excipients associated to liquid EO, is less effective
than liquid EO in reducing the muscular tone in both the ileum and colon; its effects are dose‐
dependent and comparable to OB. However, in the ileum, the association of the excipients to liquid
EO permits the maintenance of a normal pattern of waves that are abolished by liquid EO,
consistently with SCV results. Therefore, the solid based formulation can functionally be proposed
as ileal antispastic. In the colon, EO and SEO decrease the basal tone, and their effect is twice that of
OB. The contractions are almost abolished by EO and SEO, differently from OB, which maintains a
normal contractility route. The fact that EO and SEO possess comparable effects is surprising as it
should be considered that in SEO, which is given to humans, EO represents the 0.18 % w/w (i.e., 1
mg SEO contains 0.002 mg EO). This suggest that EO, when administered with excipients, exert
consistent effects also in the micromolar range of concentration. Consistently, it has been reported
[37] that a thyme extract possesses spasmolytic activity both on trachea and intestinal smooth muscle
due to thymol and carvacrol, as shown by experiments with each molecule were tested separately. In
that study, the activity was not directly proportional to concentration, since the lower doses were the
most active. This observation is in agreement with the present data in which very low EO
concentration are very active, especially when considering that the thymol and carvacrol
concentration in the solid form are comparable to those of the above‐described reported data [37].
Another possibility is that the phytocomplex and excipients cooperate. As excipients per se are poorly
active, we can speculate that their components strongly potentiate the effects of EO; in the presence
of excipients, in fact, a 500‐fold lower EO amount elicits effects comparable to EO per se.
The effect of essential oil seems stronger in the colon than in the ileum: this fact is important
because, in the ileum, the contractile activity is maintained, and the low frequency waves are present
also if the intestinal tone is reduced. The presence of the basal contraction rate pattern allows the
mixing of the internal luminal content that is specific of the small intestine and not typical of the
colon. The areas of alternate contraction and stretching present segmentation that may be particularly
important in securing mixing: a recent paper speculates that the timing of segmentation contractions
is largely, if not entirely, the result of slow wave activity in the intestinal smooth muscle coat [38]. In
Biomolecules 2020, 10, 860 17 of 20

the colon, the low frequency bands and the contractility pattern are really diminished leading to a
low contractile activity that associated to the decreased tone, may be helpful in colonic diarrheal
syndrome.
Regarding the L‐type calcium channels effects, in the ileum, liquid EO presents high activity and
high potency, SEO lower activity, and much lower potency than EO, probably due to excipients; in
the colon, liquid EO shows the same activity as in the colon, but SEO shows less activity than in the
ileum, and EO and SEO show half their respective potency than in the ileum. OB has the highest
activity and potency in the colon with respect to the ileum. The ability of SEO to block calcium‐
mediated events in gastrointestinal smooth muscle would lead to a local reduction in ileal and
intestinal muscle tone.
A possible direct modulation of the formulation on the L‐type calcium channels on the self‐
excitable cells of the ileum and colon opens an interesting set of potential targets for its activity. We
can speculate, in fact, that thyme oil affects indirectly the mechanisms which drive motility trough
membrane receptors, the activation of which is linked to the entry of calcium into the cell. Moreover,
the possibility that the phytocomplex could directly bind other receptors involved in gut motility, as
already demonstrated for the cholinergic receptor [36], cannot be ruled out. To the SEO, spasmolytic
action also contributes to monoterpenes, for which antispasmodic activity has already been shown
[39]. In particular, in SEO, there is an interesting amount of p‐cymene (Table 1), to which the literature
attributes antispasmodic action through interaction with receptors directly involved in the control of
motility such as cholinergic ones [40].
In addition, we have studied gallbladder and gastric fundus contractility as off target districts.
EO and SEO do not modify the spontaneous basal contraction of gallbladder; on calcium induced
contractility, EO exerts a relaxant activity on gallbladder, but the solid formulation, although
maintaining a relaxant activity on ileum and colon, reduces the potency by four and two times on
gallbladder with respect to the ileum and colon (Table 2).
The antibacterial activity of essential oils has long been known [41] and seems to be linked to the
prevalent chemical chemotype, with phenols being the most active compounds. The scientific
community is in agreement that the actions of these natural phytocomplexes depend not only on the
compounds present in greater quantities but on the chemotype. Indeed, it is possible to find very
powerful actions [42,43] but also phytocomplexes with much lower antimicrobial action [44,45]. The
Thymus oil used contains predominantly phenolic monoterpenes (Table 1) and maintains its
antibacterial action even in the solid form by selectively acting on pathogenic bacteria (Table 3). It is
interesting to underline how the formulation maintains action in line with liquid oil even if with
lower power. Both are without effects on commensal bacteria. This data is particularly interesting for
the importance of the microbiota in intestinal homeostasis [46] and particularly for the strong action
on Streptococcus pyogenes (gram+) and Pseudomonas aeruginosa (Gram‐). The same can be said for
Candida albicans (Table 3). Carvacrol and thymol, being hydrophobic, can interfere with the lipid
bilayer of cytoplasmic membranes of bacteria, bringing loss of integrity and increasing its fluidity
and permeability and leakage of cellular material such as ions [47]. Biologically active molecules
probably maintain the ability to pass the bacterial wall, enter the cytoplasm and perform their
bactericidal action compromising the vital functions of the bacterium itself. This action is also
described in the literature for thymol which represents the prevalent compound [48] and for p‐
cymene, which has been proven to possess interesting in vitro antimicrobial activity [40].
In conclusion, our work focuses mainly on spontaneous contractility by highlighting an
interesting activity profile of SEO. This is probably due to the direct action of the formulation on the
L‐type calcium channels on the self‐excitable cells of the ileum and colon. The possible modulation
of L‐type calcium channels opens up an interesting set of potential targets for its activity. In
conclusion, this formulation probably modulates various nodes of the target network connected to
diarrhoea owing to spasmolytic and antispasmodic action on ileum and colon. The solid form allows
systemic applications and makes it possible for use in systemic diseases. In addition, the anti‐tumour
action of essential oil demonstrated for some cell lines [49] can be an interesting added value.
Biomolecules 2020, 10, 860 18 of 20

Supplementary Materials: The following are available online at www.mdpi.com/2218‐273X/10/6/860/s1, File S1.
LC‐MS/MS conditions; File S2. Details for in vitro studies; File S3. Details for antibacterial activity; File S4.
Analytical method development and validation; Table S1. CEC‐DAD and LC‐MS/MS method validation results;
Figure S5. Effects of EO, SEO, excipients, and OB on ileum spontaneous contraction rates (FFT). Absolute band
powers of control and after addition of each concentration observed in the same experiment (FFT); Figure S6.
Effects of EO, SEO, excipients, and OB on colon spontaneous contraction rates (FFT). Absolute band powers of
control and after addition of each concentration observed in the same experiment (FFT); Figure S7. Effects of EO,
SEO, excipients, and OB on gallbladder spontaneous contraction rates (FFT). Absolute band powers of control
and after addition of each concentration observed in the same experiment (FFT); Figure S8. Effects of EO, SEO,
excipients, and OB on gastric fundus spontaneous contraction rates (FFT). Absolute band powers of control and
after addition of each concentration observed in the same experiment (FFT); File S9. Supplementary references.

Author Contributions: Conceptualization: R.B., L.M., M.M., and M.P.; methodology: M.M. and M.P.; validation,
formal analysis, and investigation: M.M,, M.P., L.B.M., G.T., and C.M.; resources: R.B. and L.M.; data curation:
M.M., M.P., I.C., and M.F.; writing—original draft preparation: R.B., M.M., L.M., and M.P. ; writing—review and
editing: R.A., R.B., and L.M.; visualization: M.F.; supervision: A.C.; project administration: R.B.; funding
acquisition: R.B. and L.M. All authors have read and agreed to the published version of the manuscript.

Funding: This research was funded by University of Bologna, ‘Ricerca Fondamentale Orientata’ (RFO), 2018.

Conflicts of Interest: The authors declare no conflict of interest.

References
1. Sharifi‐Rad, J.; Sureda, A.; Tenore, G.C.; Daglia, M.; Sharifi‐Rad, M.; Valussi, M.; Tundis, R.; Sharifi‐Rad,
M.; Loizzo, M.R.; Ademiluyi, A.O.; et al. Biological Activities of Essential Oils: From Plant Chemoecology
to Traditional Healing Systems. Molecules 2017, 22, pii: E70.
2. Wei, A.; Shibamoto, T. Medicinal Activities of Essential Oils: Role in Disease Prevention. In Bioactive Foods
in Promoting Health Fruit and Vegetables; Elsevier Inc: Cambridge, MA, USA, 2010; pp. 59–70.
3. Li, X.; He, T.; Wang, X.; Shen, M.; Yan, X.; Fan, S.; Wang, L.; Wang, X.; Xu, X.; Sui, H.; et al. Traditional Uses,
Chemical Constituents and Biological Activities of Plants from the Genus Thymus. Chem. Biodivers. 2019,
16, e1900254.
4. Orłowska, M.; Kowalska, T.; Sajewicz, M.; Pytlakowska, K.; Bartoszek, M.; Polak, J.; Waksmundzka‐Hajnos,
M. Antioxidant Activity of Selected Thyme (Thymus L.) Species and Study of the Equivalence of Different
Measuring Methodologies. J. AOAC Int. 2015, 98, 876–882.
5. El‐Nekeety, A.A.; Mohamed, S.R.; Hathout, A.S.; Hassan, N.S.; Aly, S.E.; Abdel‐Wahhab, M.A. Antioxidant
properties of Thymus vulgaris oil against aflatoxin‐induce oxidative stress in male rats. Toxicon 2011, 57,
984–991.
6. Cosentino, S.; Tuberoso, C.I.; Pisano, B.; Satta, M.; Mascia, V.; Arzedi, E.; Palmas, F. In vitro antimicrobial
activity and chemical composition of Sardinian Thymus essential oils. Lett. Appl. Microbiol. 1999, 29, 130–
135.
7. de Lira Mota, K.S.; de Oliveira Pereira, F.; de Oliveira, W.A.; Lima, I.O.; de Oliveira Lima, E. Antifungal
activity of Thymus vulgaris L. essential oil and its constituent phytochemicals against Rhizopus oryzae:
Interaction with ergosterol. Molecules 2012, 17, 14418–14433.
8. Alarcón, R.; Pardo‐de‐Santayana, M.; Priestley, C.; Morales, R.; Heinrich, M. Medicinal and local food
plants in the south of Alava (Basque Country, Spain). J. Ethnopharmacol. 2015, 176, 207–224.
9. Pereira, O.; Peres, A.; Silva, A.; Domingues, M.; Cardoso, S. Simultaneous characterization, and
quantification of phenolic compounds in Thymus x citriodorus using a validated HPLC–UV and ESI‐MS
combined method. Food Res. Int. 2013, 54, 1773–1780.
10. Nagoor Meeran, M.F.; Javed, H.; Al Taee, H.; Azimullah, S.; Ojha, S.K. Pharmacological Properties and
Molecular Mechanisms of Thymol: Prospects for Its Therapeutic Potential and Pharmaceutical
Development. Front. Pharmacol. 2017, 8, 380.
11. ICH Harmonised Tripartite Guideline. Validation of Analytical Procedures: Text and Methodology Q2(R1);
International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals
for Human Use: Geneva, Switzerland, 2005; pp. 1–13.
12. McGrath, J.C.; Drummond, G.B.; McLachlan, E.M.; Kilkenny, C.; Wainwright, C.L. Guidelines for reporting
experiments involving animals: The ARRIVE guidelines. Br. J. Pharmacol. 2010, 160, 1573–1576.
Biomolecules 2020, 10, 860 19 of 20

13. Micucci, M.; Angeletti, A.; Cont, M.; Corazza, I.; Aldini, R.; Donadio, E.; Chiarini, A.; Budriesi, R. Hibiscus
flowers and olive leaves extracts based formulation for hypertension care: In vitro efficacy and toxicological
profile. J. Med. Food 2016, 19, 504–512.
14. Micucci, M.; Ioan, P.; Aldini, R.; Cevenini, M.; Alvisi, V.; Ruffilli, C.; Chiarini, A.; Budriesi, R. Castanea
sativa mill. extract induces gallbladder contraction and sphincter of Oddi relaxation in guinea pig: A
natural approach to biliary tract motility disorders. J. Med. Food 2014, 17, 795–803.
15. Motulsky, H.; Christopoulos, A. Fitting Models to Biological Data Using Linear and Non Linear Regression.
2003. Available online: www.graphpad.com (accessed on 13 April 2020).
16. Motulsky, H.J. Prism 5 Statistics Guide; GraphPad Software Inc.: San Diego, CA, USA, 2007. Available
online: www.graphpad.com (accessed on 13 April 2020).
17. Tallarida, R.J.; Murray, R.B. Manual of Pharmacologic Calculations with Computer Programs, 2nd ed.; Springer‐
Verlag: New York, NY, USA, 1987; pp 31–35.
18. Swenson, J.M.; Killgore, G.E.; Tenover, F.C. Antimicrobial Susceptibility Testing of Acinetobacter spp. by
NCCLS Broth Microdilution and Disk Diffusion Methods. J. Clin. Microbiol. 2004, 42, 5102–5108.
19. Evangelista, S. Otilonium bromide: A selective spasmolytic for the gastrointestinal tract. J. Int. Med. Res.
1999, 27, 207–222.
20. Natavi, S.M.; Marchese, A.; Izadi, M.; Curti, V.; Daglia, M.; Natavi, S.F. Plants belonging to the genus
Thymus as antibacterial agents: From farm to pharmacy. Food Chem. 2015, 173, 339–347.
21. Brenner, D.M.; Sayuk, G.S. Current US Food and Drug Administration‐Approved Pharmacologic
Therapies for the Treatment of Irritable Bowel Syndrome with Diarrhea. Adv. Ther. 2020, 37, 83–96.
22. Hicks, G.A. Irritable Bowel Syndrome. In Comprehensive Medicinal Chemistry; Taylor, J.B., Triggle, D.J., Eds.;
Elsevier Science: London, UK, 2007; pp. 643–670.
23. Lembo, A.J.; Lacy, B.E.; Zuckerman, M.J.; Schey, R.; Dove, L.S.; Andrae, D.A.; Davenport, J.M.; McIntyre,
G.; Lopez, R.; Turner, L.; et al. Eluxadoline for irritable bowel syndrome with diarrhea. N. Engl. J. Med.
2016, 374, 242–253.
24. Heghes, S.C.; Vostinaru, O.; Rus, L.M.; Mogosan, C.; Iuga, C.A.; Filip, L. Antispasmodic Effect of Essential
Oils and Their Constituents: A Review. Molecules 2019, 24, pii: E1675.
25. Zou, Y.; Xiang, Q.; Wang, J.; Peng, J.; Wei, H. Oregano Essential Oil Improves Intestinal Morphology and
Expression of Tight Junction Proteins Associated with Modulation of Selected Intestinal Bacteria and
Immune Status in a Pig Model. Biomed. Res. Int. 2016, 2016, 5436738.
26. Sowndhararajan, K.; Kim, S. Influence of Fragrances on Human Psychophysiological Activity: With Special
Reference to Human Electroencephalographic Response. Sci. Pharm. 2016, 84, 724–751.
27. Kako, H.; Fukumoto, S.; Kobayashi, Y.; Yokogoshi, H. Effects of direct exposure of green odor components
on dopamine release from rat brain striatal slices and PC12 cells. Brain Res. Bull. 2008, 75, 706–712.
28. Grigoleit, H.G.; Grigoleit, P. Peppermint oil in irritable bowel syndrome. Phytomedicine 2005, 12, 601–606.
29. Merat, S.; Khalili, S.; Mostajabi, P.; Ghorbani, A.; Ansari, R.; Malekzadeh, R. The effect of enteric‐coated,
delayed‐release peppermint oil on irritable bowel syndrome. Dig. Dis. Sci. 2010, 55, 1385–1390.
30. Hills, J.M.; Aaronson, P.I. The mechanism of action of peppermint oil on gastrointestinal smooth muscle.
An analysis using patch clamp electrophysiology and isolated tissue pharmacology in rabbit and guinea
pig. Gastroenterology 1991, 101, 55–65.
31. de Cássia da Silveira, E.; Sá, R.; Lima, T.C.; da Nóbrega, F.R.; de Brito, A.E.M.; de Sousa, D.P. Analgesic‐
Like Activity of Essential Oil Constituents: An Update. Int. J. Mol. Sci. 2017, 18, pii: E2392.
32. Tohidi, B.; Rahimmalek, M.; Arzani, A. Essential oil composition, total phenolic, flavonoid contents, and
antioxidant activity of Thymus species collected from different regions of Iran. Food Chem. 2017, 220, 153–
161.
33. Nikolić, M.; Glamočlija, J.; Ferreira, I.C.F.R.; Calhelha, R.C.; Fernandes, Â.; Marković, T.; Marković, D.;
Giwelie, A.; Soković, M. Chemical composition, antimicrobial, antioxidant and antitumor activity of
Thymus serpyllum L., Thymus algeriensis Boiss. and Reut and Thymus vulgaris L. essential oils. Ind. Crops Prod.
2014, 52, 183–190.
34. Schmidt, E.; Wanner, J.; Höferl, M.; Jirovetz, L.; Buchbauer, G.; Gochev, V.; Girova, T.; Stoyanova, A.;
Geissler, M. Chemical composition, olfactory analysis and antibacterial activity of Thymus vulgaris
chemotypes geraniol, 4‐thujanol/terpinen‐4‐ol, thymol and linalool cultivated in southern France. Nat. Prod.
Commun. 2012, 7, 1095–1098.
Biomolecules 2020, 10, 860 20 of 20

35. Vaičiulytė, V.; Butkienė, R.; Ložienė, K. Effects of meteorological conditions and plant growth stage on the
accumulation of carvacrol and its precursors in Thymus pulegioides. Phytochemistry 2016, 128, 20–26.
36. Schönknecht, K.; Krauss, H.; Jambor, J.; Fal, A.M. Treatment of cough in respiratory tract infections‐the
effect of combining the natural active compounds with thymol. Wiad. Lek. 2016, 69, 791–798.
37. Begrow, F.; Engelbertz, J.; Feistel, B.; Lehnfeld, R.; Bauer, K.; Verspohl, E.J. Impact of thymol in thyme
extracts on their antispasmodic action and ciliary clearance. Planta Med. 2010, 76, 311–318.
38. Thuneberg, L.; Peters, S. Toward a concept of stretch‐coupling in smooth muscle. I. Anatomy of intestinal
segmentation and sleeve contractions. Anat. Rec. 2001, 262, 110–124.
39. Astudillo, A.; Hong, E.; Bye, R.; Navarrete, A. Antispasmodic activity of extracts and compounds of
Acalypha phleoides Cav. Phytother. Res. 2004, 18, 102–106.
40. Rivero‐Cruz, I.; Duarte, G.; Navarrete, A.; Bye, R.; Linares, E.; Mata, R. Chemical Composition and
Antimicrobial and Spasmolytic Properties of Poliomintha longiflora and Lippia graveolens Essential Oils. J.
Food Sci. 2011, 76, C309–C317.
41. Man, A.; Santacroce, L.; Jacob, R.; Mare, A.; Man, L. Antimicrobial activity of six essential oils against a
group of human pathogens: A comparative study. Pathogens 2019, 8, pii: E15.
42. Borugă, O.; Jianu, C.; Mişcă, C.; Goleţ, I.; Gruia, A.T.; Horhat, F.G. Thymus vulgaris essential oil: Chemical
composition and antimicrobial activity. J. Med. Life 2014, 7, 56–60.
43. Gedikoğlu, A.; Sökmen, M.; Çivit, A. Evaluation of Thymus vulgaris and Thymbra spicata essential oils and
plant extracts for chemical composition, antioxidant, and antimicrobial properties. Food Sci. Nutr. 2019, 7,
1704–1714.
44. Cáceres, M.; Hidalgo, W.; Stashenko, E.; Torres, R.; Ortiz, C. Essential Oils of Aromatic Plants with
Antibacterial, Anti‐Biofilm and Anti‐Quorum Sensing Activities against Pathogenic Bacteria. Antibiotics
2020, 9, pii: E147.
45. Kerekes, E.B.; Vidács, A.; Takó, M.; Petkovits, T.; Vágvölgyi, C.; Horváth, G.; Balázs, V.L.; Krisch, J. Anti‐
Biofilm Effect of Selected Essential Oils and Main Components on Mono‐ and Polymicrobic Bacterial
Cultures. Microorganisms 2019, 7, pii: E345.
46. Tojo, R.; Suárez, A.; Clemente, M.G.; de los Reyes‐Gavilán, C.G.; Margolles, A.; Gueimonde, M.; Ruas‐
Madiedo, P. Intestinal microbiota in health and disease: Role of bifidobacteria in gut homeostasis World J.
Gastroenterol. 2014, 20, 15163–15176.
47. Lambert, R.J.; Skandamis, P.N.; Coote, P.J.; Nychas, G.J. A study of the minimum inhibitory concentration
and mode of action of oreg‐ano essential oil, thymol and carvacrol. J. Appl. Microbiol. 2001, 91, 453–462.
48. Marchese, A.; Orhan, I.E.; Daglia, M.; Barbieri, R.; Di Lorenzo, A.; Nabavi, S.F.; Gortzi, O.; Izadi, M.; Natavi,
S.M. Antibacterial and antifungal activity of thymol: A brief review of the literature. Food Chem. 2016, 210,
402–414.
49. Sertel, S.; Eichhorn, T.; Plinkert, P.K.; Efferth, T. Cytotoxicity of Thymus vulgaris essential oil towards
human oral cavity squamous cell carcinoma. Anticancer Res. 2011, 31, 81–87.

© 2020 by the authors. 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 (http://creativecommons.org/licenses/by/4.0/).