Phytomedicine Plus 3 (2023) 100498

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Phytomedicine Plus
journal homepage: www.sciencedirect.com/journal/phytomedicine-plus

Chemical composition, in vitro and in silico approaches of the relaxant effect

of the jejunum using Thymus algeriensis Boiss. and Reut essential oil
Leila Beyi a, b, Mohamed Marghich a, c, *, Ouafa Amrani a, Ahmed Karim a, Tarik Harit d,
Mohammed Aziz a
a
Laboratory of Bioresources, Biotechnology, Ethnopharmacology and Health, Faculty of Sciences, Mohammed First University, 60000, Oujda, Morocco
b
Regional Center for the Professions of Education and Training, Oriental Region, 60000, Oujda, Morocco
c
Nutritional Physiopathology, Neurosciences and Toxicology Team, Laboratory of Anthropogenetic, Biotechnology and Health, Faculty of Sciences, Chouaib Doukkali
University, 24000, El Jadida, Morocco
d
Laboratory of Applied Chemistry and Environment–ECOMP, Faculty of Sciences, Mohammed First University, 60000, Oujda, Morocco

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

Keywords: Background: Gastrointestinal problems are among the most common diseases in Morocco people, who frequently
Thymus algeriensis use aromatic and medicinal plants to eliminate these problems. Thymus algeriensis is one of these plants widely
Essential oil used to treat gastrointestinal problems. This research aimed to identify the phytochemical compounds present in
Antispasmodic
Thymus algeriensis essential oil (TaEO), assess its acute toxicity, and investigate its potential myorelaxant and
Cholinergic receptor
Voltage-dependent calcium channel
antispasmodic effects.
Thujone Methods: In vitro experiments were conducted on rat and rabbit jejunum using an isotonic transducer and in silico
docking calculations were conducted on L-type voltage-gated Ca2+and muscarinic receptor active sites.
Results: The GC–MS analysis of TaEO revealed the presence of 11 compounds, with 89.77 % of the compounds
identified being oxygenated monoterpenes, among which Thujone dominates the composition with 32.89 %. The
administration of TaEO did not produce any observable signs of toxicity or mortality at 1 g/Kg.bw. TaEO exhibit
a dose-dependent decrease in the basal contractions of the rabbit jejunum with an IC50 value of 5.66 ± 1.88 µg/
mL. In addition, TaEO induced an antispasmodic effect on KCl and CCh-induced contractions in the rat jejunum.
The inhibitory effect demonstrated are similar to those produced by a non-competitive antagonist of voltage-
dependent calcium channel and cholinergic receptors. These results are boosted by the docking study when
we showed that Thujone could bind to the active site of muscarinic receptor and, L-type voltage-gated Ca2+ with
a binding energy equal to -59.401 and, -47.441 Kcal/mol respectively.
Conclusions: These results provide strong confirmation for the traditional use of Thymus algeriensis in Moroccan as
an effective antispasmodic remedy and give a new lead to find a phytomedicament against gastrointestinal
problems.

IP3 Inositol 145-trisphosphate

KCl Potassium Chloride
Abbreviations KHB Krebs-Henseleit buffer solution
Ach Acetylcholine SEM Standard error of the mean
Cav1.1 L-type voltage-gated Ca2+ channels TaEO Thymus algeriensis essential oil
CCh Carbachol VDCCs Voltage-dependent calcium channels
DMSO Dimethyl sulfoxide
EDTA Ethylenediaminetetraacetic acid
GC–MS Gas Chromatography-Mass Spectrometry analysis
IC50 The half-maximal inhibitory concentration

* Corresponding author at: Nutritional Physiopathology, Neurosciences and Toxicology Team, Laboratory of Anthropogenetic, Biotechnology and Health, Faculty
of Sciences, Chouaib Doukkali University, 24000, El Jadida, Morocco.
E-mail address: m.marghich@ump.ac.ma (M. Marghich).

https://doi.org/10.1016/j.phyplu.2023.100498

Available online 2 November 2023

2667-0313/© 2023 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
L. Beyi et al.
1. Introduction
Morocco and its neighboring countries frequently use aromatic and
medicinal plants due to their availability, affordability, and efficacy in
treating various ailments, with gastrointestinal issues being the most
commonly treated (Fakchich and Elachouri, 2014). Thymus algeriensis is
one of the commonly used plants in Moroccan pharmacopeia. It is uti­
lized in the form of an infusion or decoction of the aerial part to address
digestive and respiratory problems (Bellakhdar, 1997). Various phar­
macological activities have been demonstrated in this plant such as
antiacetylcholinesterase (Jaouadi et al., 2023), antimicrobial (Souiy
et al., 2023), anti-inflammatory and antioxidant activities (Righi et al.,
2023). These potential pharmacological activities attributed to the
diverse range of phytochemicals present in it, including flavonoids,
phenolic acids, and essential oils (Guesmi et al., 2019). In our prior
research, we demonstrate that Thymus algeriensis aqueous extract exhibit
relaxant and antispasmodic activities (Beyi et al., 2021). The current
study aims to continue the research on this plant by determining the
phytochemical compounds present in its essential oil, evaluating its
acute toxicity, as well as assess its myorelaxant and antispasmodic
effects.
2. Material and methods
2.1. Chemicals
All chemicals employed were acquired from Sigma Chemicals, pos­
sessing analytical grade, and were dissolved in distilled water.
2.2. Plant material
In the Oujda region, Morocco, Thymus algeriensis samples were ob­
tained during the flowering period (April and May 2019). Professor
Benyounes Haloui, a botanist from the Department of Biology, Faculty of
Sciences Oujda (Morocco), conducted the botanical identification of the
plant. A reference specimen was deposited in the plant section of the
herbarium of the Faculty of Sciences, Oujda (No. HUMPOM 425). The
name of the plant has been checked with http://www.theplantlist.org
2.3. Essential oil preparation
To extract the Thymus algeriensis essential oil (TaEO), the Dean–Stark
apparatus was used for hydrodistillation. Firstly, 200 g of the plant’s
aerial parts are placed in 1200 mL of distilled water, and then the
hydrodistillation system was initiated (3 h). The oil was then stored at a
temperature of -20 ◦ C until needed. The extraction resulted in a yield of
0.24 %. Before use, TaEO was dissolved in DMSO.
2.4. GC–MS analysis
Gas chromatography coupled with mass spectrometry GC–MS (Shi­
madzu QP2010 model, Kyoto, Japan) was used to characterize TaEO.
The carrier gas helium was used at a flow rate of 3 mL/min in a BPX 25
capillary column (30 m × 0.25 mm, film thickness 0.25 m). The column
temperature program started at 50 ◦ C for 5 min, followed by an increase
of 10 ◦ C per minute up to 250 ◦ C, which was maintained for 10 min. The
injector temperature was 225 ◦ C, and the detector temperature was
maintained at 250 ◦ C. Electron impact mass spectra were collected at an
ionization voltage of 70 eV and a scan rate of five scans/s. The ionization
source temperature was 200 ◦ C, and the interface temperature was
250 ◦ C. The NIST 147 database (National Institute for Standard Tech­
nology–NIST-147, 198 compounds LabSolutions version 2.5. USA) was
used to identify the compounds by comparing their mass spectra. The
quantity of each compound identified was estimated based on the per­
centage of its peak area relative to the total of other peaks obtained.
2
Phytomedicine Plus 3 (2023) 100498
2.5. Animals
Male and female Wistar rats weighing between 200 and 300 g, Al­
bino mice weighing between 25 and 30 g, and New Zealand rabbits
weighing between 1.5–2 kg were used for the experiments. The animals
were housed in a climate-controlled room with regulated lighting,
adhering to a 12 h light-darkness cycle at the animal house of the faculty
of sciences, Oujda, Morocco. The animals were subjected to an 18 h food
withdrawal period before the experiment. All procedures involving the
animals were conducted according to ethical guidelines as outlined by
the National Research Council’s for the care and use of laboratory ani­
mals (NRC, 2011).
2.6. Acute toxicity study
Following the guidelines outlined by the Organization for Economic
Co-operation and Development number 425 (OECD, 2008), this study
evaluated the acute toxicity of TaEO. Twenty-four Albino mice were
randomly divided into four groups, each consisting of six mice (3 males
and 3 females).
Group 1: receives DMSO (1 %) by gavage.
Group 2: receives TaEO (0.2 g/Kg) by gavage.
Group 3: receives TaEO (0.5 g/Kg) by gavage.
Group 4: receives TaEO (1.0 g/Kg) by gavage.
The mice were under close observation for 14 days to detect any
indications of toxicity, changes in behavior, or death.
2.7. Isolated jejunum experiments
To remove the jejunum we followed the protocol described in detail
by our team (Marghich et al., 2021).
2.8. Myorelaxant activity
After stabilization of spontaneous contractions of rabbit jejunum,
cumulative doses of the TaEO (3, 10, and 30 µg/mL) or verapamil hy­
drochloride (0.1, 0.3, and 1 µM) a standard calcium channel blocker,
used as a positive control were added to the isolated organ chamber.
The dose of TaEO (30 µg/mL) that gave the maximum myorelaxant
was tested in the presence of three adrenergic receptor antagonists:
prazosin (α1), propranolol (β1), and yohimbine (α2) at a concentration of
5.10− 5 M for each.
2.9. Antispasmodic effect of TaEO on pre-contracted rat jejunum
To evaluate the ability of TaEO to prevent spasms in rat jejunum, we
pre-contracted jejunum smooth muscle with KCl (75 mM) or CCh (10− 6
M), and then cumulative doses of TaEO have been added in the isolated
organ tank for final concentrations of 3, 10 and 30 µg/mL.
2.10. Antispasmodic effect of TaEO on concentration-response curves of
CaCl2 and CCh
The effect of TaEO on the contractile response to CCh was evaluated
using the following method: In a single experiment, concentration-
dependent tests were conducted with CCh (ranging from 10− 8 M to
10− 5 M) both before and after the administration of atropine (10− 6 M) or
TaEO at concentrations of 3, 10, and 30 µg/mL.
Similar procedures were employed in additional tests, wherein the
jejunum segments were contracted using CaCl2 (0.1, 0.3, 1, 3, and 10
mM), except in this experiment we used verapamil (10− 6 M) as a positive
control used, and the KHB rich in potassium and without calcium (NaCl
48, KCl 75, CaCl2 0, MgSO4 1.2, NaHCO3 25, KH2PO4, 1.2 and glucose
10 mM)
L. Beyi et al. Phytomedicine Plus 3 (2023) 100498

2.11. Docking study finally, camphor represents 9.15 % (Fig. 1) (Table 1).

The “iGemdock program” known as a free available software was
used to perform the docking calculations (Yang and Chen, 2004). The 3D
structure of Thujone (4098408) was extracted from ZINC20 database, a
free database of commercially available compounds (https://zinc.doc
king.org/). iGemdock predicts the muscarinic-Thujone or Rabbit
Cav1.1-Thujone interaction profiles according to the method described
in detail by our last published article (Marghich et al., 2023).
3.2. Acute toxicity study
Mice that were given the essential oil of Thymus algeriensis suffered
no deaths and no toxic symptoms even when exposed to doses of up to 1
g per kg of body weight. The mortality rate observed during the
experiment was 0 % (Table 2).

3.3. Myorelaxant activity

2.12. Statistical analysis
The TaEO, with an IC50 value of 5.66 ± 1.88 µg/mL (Table 3),
Pharmacological responses are presented as mean values ± SE induced a dose-dependent decrease in the basal contractions of the
(standard error) for separate experiments using n tissues. Graphs of rabbit jejunum. The most significant inhibition was observed at 30 µg/
concentration – responses curves were determined using nonlinear mL (p ˂ 0.001) (Fig. 2A). The effect was found to be reversible as the
regression were fitted to the Hill equation by an iterative least-squares spontaneous activity of the tissue was restored after being washed twice.
method (GraphPad Prism 8.0 Software, San Diego, CA, USA) to pro­ Verapamil, a calcium channel antagonist, used as a positive control, also
vide estimates of the maximum effective concentration IC50 (the nega­ caused a dose-dependent decrease in basal contractions of rabbit
tive logarithm of the agonist concentration producing 50% of maximum jejunum (Fig. 2B) with an IC50 value of 0.092 ± 0.001 µM (Table 3).
inhibition). Antagonist affinity of TaEO was expressed as an apparent Even in the presence of α and β adrenergic receptor blockers such as
pA2 value. pA2 was calculated from a single concentration of antagonist
using the following equation: pA2 = - log [B] + log (r-1), where [B] is the Table 1
concentration of the antagonist and r the ratio of agonist EC50, deter­ Chemical composition of the essential oil of Thymus algeriensis.
mined in the presence or absence of the antagonist. For the comparison
Pic N∘ Retention time Chemical compounds Abundance (%)
of the different effects against the control, a one-way analysis of variance (min)
(ANOVA) was performed followed by Bonferroni’s multiple comparison
1 4.999 α-pinene 0.35
tests considering p ˂ 0.05 as statistically significant. 2 5.233 Camphene 1.08
3 5.600 Thujene 3.90
3. Results 4 6.508 Cineole 11.90
5 7.850 Thujone 32.89
6 8.308 Camphor 9.15
3.1. GC–MS analysis 7 8.767 Terpinene-4-ol 1.16
8 10.067 (+)-3-Carene, 2 14.76
By using GC–MS, the TaEO was analyzed, and the results revealed 11 (acetylmethyl)-
compounds, with over half of them being monoterpenes. Specifically, 9 4.550 Santolinatriene 9.95
10 8.245 Santolinatriene 4.60
89.77 % of the compounds identified in TaEO are oxygenated mono­ Total monoterpenes 89.77
terpenes, among which Thujone dominates the composition with 32.89 11 6.458 1,2-Diisopropenyl cyclobutane 10.22
%, followed by 2-acetylmethyl 3-carene with 14.76 %. Moreover, san­ Total 99.99
tolinatriene accounts for 14.55 %, cineole represents 11.90 %, and

Fig. 1. Gas chromatogram of the essential oil of Thymus algeriensis.

3
L. Beyi et al. Phytomedicine Plus 3 (2023) 100498

Table 2 inhibitors (Fig. 3).

Mortality and toxic symptoms of acute toxicity study.
Samples Death/Total Toxic symptoms 3.4. Antispasmodic effect of TaEO on pre-contracted rat jejunum
DMSO (1%) 0/6 None
TaEO (0.2 g/Kg) 0/6 None TaEO exhibits a dose-dependent antispasmodic effect on KCl-
TaEO (0.5 g/Kg) 0/6 None induced contractions and CCh-induced contractions in the rat jejunal
TaEO (1.0 g/Kg) 0/6 None
tissue (Fig. 4), which is statistically significant (p ˂ 0.001) across a range
of 3 to 30 μg/mL. The IC50 value for this inhibition was 10.35 ± 2.15 and
8.76 ± 1.63 μg/mL respectively (Table 4).
Table 3
IC50 value of the myorelaxant effect of Thymus algeriensis essential and
3.5. Antispasmodic effect of TaEO on concentration-response curves of
Verapamil.
CaCl2 and CCh
TaEO (µg/mL) Verapamil (µM)

IC50 5.66 ± 1.88 0.092 ± 0.001
C50 (Inhibitory concentration 50): The concentration of TaEO or Verapamil
that inhibit the basic contraction by half.
Propranolol, Prazosin, and Yohimbine, TaEO (30 µg/mL) was able to
produce its relaxing effect. The level of inhibition was similar to that
observed when TaEO (30 µg/mL) was administered without any
The outcomes of our study reveal that various doses of TaEO (3, 10,
and 30 μg/mL) were capable of inhibiting the increasing tonus caused by
cumulative doses of CaCl2 (Fig. 5) and CCh (Fig. 6A). This inhibition was
observed by the concentration-response curves shifting downwards and
to the right. The dose of 30 μg/mL was found to be particularly effective,
and its impact was comparable to that of Verapamil (10− 6 M) (Fig. 5)
and Atropine (10− 6 M) (Fig. 6A) was used as a positive control for the
two tests respectively.

Fig. 2. Relaxation effect of Thymus algeriensis essential oil (A) and Verapamil (B) on the amplitude of basal contractions of the rabbit jejunum. (Mean ± SEM, n = 6).

Fig. 3. Original plot showing the effect of essential oil of Thymus algeriensis (TaEO) on the basal contractions of rabbit jejunum in the presence of different adrenergic
inhibitors (Adr inhibitors): Propranolol, Prazosin, and Yohimbine at 5.10− 5 M for each.

4
L. Beyi et al.
Fig. 4. Antispasmodic effect of essential oil of Thymus algeriensis (TaEO) on
contractions of rat jejunum pre-contracted by KCl (75 mM) or CCh (10− 6 M).
(Mean ± SEM, n = 6).
Table 4
IC50 value of antispasmodic effect of essential oil of Thymus algeriensis (TaEO) on
rat jejunum pre-contracted with KCl (75 mM) or CCh (10− 6 M).
KCl CCh
IC50 (µg/mL) 10.35 ± 2.15 8.76 ± 1.63
IC50 (Inhibitory concentration 50): The concentration of TaEO that inhibit the
contraction induced by KCl or CCh by half.
Fig. 5. CaCl2 concentration-response curves in the presence and absence of
different doses of Thymus algeriensis essential oil or Verapamil. (Mean ± SEM, n
= 6).
In the absence of TaEO, the EC50 value for CaCl2 was measured at
2.14 ± 1.11 mM. However, when 30 μg/mL of TaEO was introduced, the
EC50 value increased to 12.59 ± 1.60 mM. This indicates that the
presence of TaEO the concentration required to induce a 50 %
contraction was higher (Table 5).
The EC50 value for CCh in the control was 2.06 × 10− 7 M. However,
in the presence of 30 μg/mL of TaEO, the EC50 value increased to 1.97 ×
10− 5 M. This suggests that the concentration needed to induce a 50 %
contraction by CCh was higher in the presence of TaEO (Table 6). In
addition, we demonstrate that the concentration of the TaEO that
5
Phytomedicine Plus 3 (2023) 100498
reduces the effect of the CCh by half was 10 μg/mL (Fig. 6B).
3.6. Doking study
In this study, we performed the docking of the major component
“Thujone” on the active sites of L-type voltage-gated Ca2+ (Cav1.1)
channels in complexing form with verapamil and M1 muscarinic
acetylcholine receptor in complexing form with the atropine. The vali­
dation of the docking protocol, which will be used to conduct the cal­
culations, was reported in our recent work (Marghich et al., 2023). The
3D and 2D binding configurations of this compound are displayed in
Fig. 7.
In the case of an L-type voltage-gated Ca2+ channel, the Thujone is
associated with its active site via a conventional hydrogen bond between
the oxygen atom and the residue PHE656. The formed complex is also
stabilized by two Van der Walls interactions LEU 563 and ASN654 as
well as alkyl interaction between LEU566 and LEU 652 and the iso­
propyl moiety. For the M1 muscarinic receptor toxin complex, the oxy­
gen atom of the carbonyl group also forms a conventional hydrogen
bond with TYR 408. We note the presence of several Van der Wall in­
teractions, besides two π-alkyl interactions with TYR381 and CYS407.
On the other hand, the binding energy of Thujone with M1 muscarinic
receptor toxin complex was found lower than that of Thujone with L-
type voltage-gated Ca2+ channel with a binding energy equal to -59.401
and -47.441 Kcal/mol respectively (Table 7).
4. Discussion
In our study, we identified 11 compounds, with over half of them
being monoterpenes. Specifically, 89.77 % of the compounds identified
in TaEO are oxygenated monoterpenes, among which Thujone domi­
nates the composition with 32.89 %, followed by 2-acetylmethyl 3-
carene with 14.76 %. Moreover, santolinatriene accounted for 14.55 %,
cineole 11.90 %, and finally, camphor 9.15 %. Another study performed
on the essential oil of Thymus algeriensis from the oriental region of
Morocco found more than forty compounds (Ouariachi et al., 2014). The
proportion of monoterpenes among these compounds is over 50%. The
most significant compounds identified are borneol (18.3 %), camphene
(11.8 %), camphor (10.0 %), myrcene (8.6 %), and geranyl acetate (6.9
%). These five compounds alone make up over half of the essential oil’s
composition, totaling 55.3 % out of over forty compounds. Salhi et al.
(2016) conducted a study in the Al Hoceima region of Morocco, where
they identified geranyl acetate as the predominant chemical type in the
essential oil of Thymus algeriensis, comprising 80.8 % of the composition.
Therefore, we observe a specific diversity of compounds and the lack of a
clearly identified chemotype in Moroccan Thymus algeriensis but it is
characterized by the dominance of oxygen-type monoterpene com­
pounds. Toxicity tests have shown that the consumption of 1 g/kg.bw of
TaEO did not cause any risk or sign of toxicity in mice. However, Righi
et al. (2023) observed reversible signs of toxicity when they increased
the dose of TaEO to 2 g/kg.bw, which disappeared after 4 h. The mice
remained stable and healthy 24 h after the treatment. Nonetheless,
microscopic analysis of liver and kidney tissues revealed preserved
general architecture, and there were no signs of vascular congestion,
inflammatory infiltrates, or necrosis.
Cajal’s interstitial cells are responsible for triggering spontaneous
contractions in the intestinal smooth muscle (Farre et al., 1991). How­
ever, the addition of TaEO inhibit these contractions in the isolated
rabbit jejunum with an IC50 equal to 5.66 ± 1.88 µg/mL. The relaxation
effect showed in a dose-dependent and reversible manner, as the tone
and amplitude of basic contractions recover after washing with a
physiological medium. Compared to other essential oil plants such as
Anthemis mauritiana (Karim et al., 2010), Origanum majorana (Makrane
et al., 2019), and Warionia saharae oils (Amrani et al., 2022), which
respectively inhibited rabbit jejunum contractions with IC50 of 32.26 ±
1.48, 64.08 ± 2.42, and 16.80 ± 0.63 µg/mL, the relaxing effect
L. Beyi et al. Phytomedicine Plus 3 (2023) 100498

Fig. 6. Carbachol concentration-response curves in the presence and absence of different doses of Thymus algeriensis essential oil or Atropine (A) and the PA2; the
concentration of the antagonist (TaEO) that reduced the effect of the agonist (CCh) by half (B). (Mean ± SEM, n = 6).

demonstrated (30 µg/mL) produced a clear relaxing effect in the pres­

Table 5
ence of these three inhibitors. This suggests that the effect of TaEO is not
EC50 value obtained from CaCl2 concentration-response curves in the presence
mediated by adrenergic receptors, as is the case with other essential oils
and absence of different doses of Thymus algeriensis essential oil (TaEO) or
Verapamil.
derived from plants such as Artemisia herba-alba (Aziz et al., 2012),
Anthemis mauritiana (Karim et al., 2010) and, Artemisia campestris
Control TaEO TaEO TaEO Verapamil
(Marghich et al., 2023).
(3 µg/mL) (10 µg/mL) (30 µg/mL) (10− 6 M)
Medicinal plants have a spasmolytic effect by either opening K+
EC50 (mM) 2.14 3.01 6.55 12.59 2772.60
channels or blocking Ca2+channels (Makrane et al., 2019; Gilani et al.,
SEM 1.11 1.17 0.93 1.60 1.08
2006). Substances that inhibit contraction caused by KCl must somehow
EC50 (Effective concentration 50): The concentration of the agonist that gives prevent the entry of Ca2+ions (Godfraind et al., 1986). In this study,
half-maximal contraction. TaEO is tested under conditions where contraction is induced by KCl 75
mM. This high concentration results in a biphasic contraction: a phasic
contraction and a sustained tonic contraction separated by a brief
Table 6
relaxation period. This type of response has also been observed in the
EC50 value obtained from Carbachol concentration-response curves in the
presence and absence of different doses of Thymus algeriensis essential oil (TaEO) colon and, the ileum (Dai et al., 2003).
or Atropine. TaEO showed a dose-dependent inhibitory effect on the contractions
of the rat jejunum induced by a KCl, with an IC50 value of 10.35 ± 2.15
Control TaEO TaEO TaEO Atropine
µg/mL. This suggests that the reduction in rat jejunum contraction may
(3 µg/mL) (10 µg/mL) (30 µg/mL) (10− 6 M) be attributed to the blocking of Ca2+input. To strengthen this hypoth­
EC50 2.06 × 2.67 × 4.14 × 1.97 × 0.054
esis, we tested the effect of increasing doses of CaCl2 both in the presence
(M) 10− 7 10− 7 10− 7 10− 5
and in the absence of TaEO. This later caused a significant inhibition of
EC50 (Effective concentration 50): The concentration of the agonist (CCh) that the maximal response to CaCl2 and shifted the dose-response curves
gives half-maximal contraction. downwards and to the right. This suggests that TaEO has a non-
competitive antagonistic effect on VDCCs. Additionally, verapamil also
achieved by our plant is stronger. Artemesia herba-alba essential oil, reduced the maximal response in the CaCl2-induced curves. That is in
which inhibited contractions with an IC50 of 97.33 ± 2.59 ng/mL (Aziz agreement with our previous study when obtained that the aqueous
et al., 2012), are more potent than TaEO. Verapamil, a standard channel extract of Thymus algeriensis can act also on the voltage-dependent cal­
blocker that inhibits voltage-dependent type L (Fleckenstein, 1977), was cium channels (Beyi et al., 2021).
utilized as a positive control, and it completely suppressed the Carbachol (CCh) was used to test the pathway involving cholinergic
contraction of smooth muscle fibers at a concentration of 1 μM. This receptors. In the gut, the response is mediated by activation of two
outcome has also been demonstrated in numerous previous studies specific muscarinic receptor types (M2 and M3) (Ehlert, 2003). TaEO
(Marghich et al., 2023; Karim et al., 2010). exhibited a dose-dependent inhibitory effect on the tonic contraction of
The activity of the gastrointestinal tract’s smooth muscles is modu­ rat jejunum induced by carbachol, with an IC50 value of 8.76 ± 1.63
lated via the enteric nervous system. This modulation has an inhibitory μg/mL. TaEO demonstrated a significant inhibitory effect on the
effect on the motility of the tract by the action of Adrenaline (Hansen, maximum contraction induced by carbachol while simultaneously
2003). Our objective was to determine whether our extracts acted via shifting the concentration-response curves downward and to the right.
the adrenergic pathway. To accomplish this, we blocked α1 adrenergic This effect was similar to that observed with the use of Atropine as a
receptors with Prazosin, α2 receptors with Yohimbine, and β1 and β2 positive control. Based on these findings, it can be postulated that one or
receptors with Propranolol. We observed that Adrenaline had no impact more constituents of the TaEO may act as a non-competitive antagonist
on the spontaneous contractions of the rabbit jejunum in the presence of through one of the two contraction pathways linked to muscarinic re­
these inhibitors, while TaEO, at the concentration previously ceptors. These results are comparable to those obtained previously of a

6
L. Beyi et al. Phytomedicine Plus 3 (2023) 100498

Fig. 7. 3D and 2D binding modes of Thujone with L-type voltage-gated Ca2+ (Cav1.1) channel active site (A and B) and the active site of M1 muscarinic receptor toxin
complex (C and D).

Thujone which is abundantly present in TaEO also has a probable

Table 7
antispasmodic effect, since it exists in various plants known for their
Binding energy of the Thujone with the active sites of Cav1.1 channel
strong antispasmodic effect (Perfumi et al., 1995; Heghes et al., 2019).
and M1 muscarinic receptor toxin complex.
In this context and to correlate with the experimental results, we verify
Receptor Binding energy (Kcal/mol) the ability of this molecule to inhibit Cav1.1 channel and muscarinic
Cav1.1 channel -47.441 receptor active sites via an in silico study as possible targets. The ob­
M1 muscarinic -59.4014 tained results showed that Thujone could bind to their active site with a
preference to that of muscarinic receptor. Such results suggest that the
IC50 value of TaEO for the inhibition of muscarinic receptors is less than
medicinal plant that exhibit non-competitive antagonistic activity
that in the case of Cav1.1, which is in good agreement with the experi­
against CCh in the smooth muscles of ileum (Câmara et al., 2003).
mental finding. Therefore, these pathways are probably involved in the
The antispasmodic properties of TaEO may be attributed to their
mechanism of action of TaEO on the spasmolytic effect. These various
high content of compounds known for their antispasmodic effects, such
constituents may exert their effects independently or in combination.
as camphor and terpinene (Astudillo et al., 2004). α- and β-pinene can
relax the duodenum by their action on acetylcholine-induced contrac­
5. Conclusion
tion (Matthews Jucá et al., 2011; Câmara et al., 2003). Furthermore, a
recent in silico investigation by Marghich et al., al.(2023) demonstrate
Thymus algeriensis essential oil-induced myorelaxant and antispas­
that the isomers α-pinene and β-pinene interacted with L-type
modic effects mediated by the antagonist of muscarinic receptors and an
voltage-gated Ca2+channel through various van der Waals interactions
L-type voltage Ca2+channels. These findings explain the traditional use
involving the residues PHE608, ILE1049, ILE1050, and ALA1053. In
of Thymus algeriensis in gastrointestinal problems and we give a new lead
addition, demonstrate that these two molecules interact with the active
to find a phyto-medication against digestive tract problems.
site of muscarinic receptors. On the rat ileum, β-pinene has a spasmolytic
effect via the 5-HT3 receptor channel system (Riyazi et al., 2007).

7
L. Beyi et al.
Funding source
This work was funded by the budget allocated to research at
Mohammed First University by the Ministry of Higher Education, Sci­
entific Research, and Innovation (Morocco).
CRediT authorship contribution statement
Leila Beyi: Methodology, Formal analysis, Writing – review &
editing. Mohamed Marghich: Software, Formal analysis, Validation,
Writing – original draft, Writing – review & editing. Ouafa Amrani:
Formal analysis, Validation, Writing – review & editing. Ahmed Karim:
Conceptualization, Validation, Writing – review & editing. Tarik Harit:
Software, Validation, Writing – original draft. Mohammed Aziz:
Conceptualization, Resources, Funding acquisition, Validation, Writing
– review & editing, Supervision, Project administration.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
The chemistry department of the Faculty of Sciences at Mohammed
First University is acknowledged for conducting the GC–MS analysis of
the sample, with gratitude towards Pr. Abdelmonaem Talhaoui head of
the department. Mustapha Badraoui, Karim Ramdaoui, and A. Joudar
provided technical support and animal breeding. Additionally, Professor
Benyounes Haloui played a key role in identifying the plant.
References
Amrani, O., Marghich, M., Addi, M., Hano, C., Chen, J.T., Makrane, H., Alem, C.,
Karim, A., Aziz, M., 2022. The antispasmodic effect of Warionia saharae essential oil
in experimental models and its mechanism of action. Front. Biosci. Sch. 14, 10.
https://doi.org/10.31083/j.fbs1402010.
Astudillo, A., Hong, E., Bye, R., Navarrete, A., 2004. Antispasmodic activity of extracts
and compounds of Acalypha phleoides Cav. Phyther. Res. 18, 102–106. https://doi.
org/10.1002/ptr.1414.
Aziz, M., Karim, A., El-Ouariachi, E.M., Bouyanzer, A., Amrani, S., Mekhfi, H., Ziyyat, A.,
Melhaoui, A., Bnouham, M., Legssyer, A., 2012. Relaxant effect of essential oil of
Artemisia herba-alba Asso. on rodent jejunum contractions. Sci. Pharm. 80, 457–467.
https://doi.org/10.3797/scipharm.1106-13.
Bellakhdar, J., 1997. La Pharmacopée Marocaine traditionnelle, Fung Kwan Tam ed. Ibis
Press, Paris France.
Beyi, L., Zrouri, H., Makrane, H., Mekhfi, H., Ziyyat, A., Bnouham, M., Aziz, M., 2021.
Relaxant and antispasmodic activities of aqueous extract from Thymus algeriensis
boiss. And reut. J. Nat. Remedies. 21, 165–171. https://doi.org/10.18311/jnr/
2021/24752.
Câmara, C.C., Nascimento, N.R.F., Macêdo-Filho, C.L., Almeida, F.B.S., Fonteles, M.C.,
2003. Antispasmodic effect of the essential oil of Plectranthus barbatus and some
major constituents on the guinea-pig ileum. Planta Med. 69, 1080–1085. https://doi.
org/10.1055/s-2003-45186.
Dai, Y., Liu, J.X., Li, J.X., Xu, Y.F., 2003. Effect of pinaverium bromide on stress-induced
colonic smooth muscle contractility disorder in rats. World J. Gastroenterol. 9,
557–561. https://doi.org/10.3748/wjg.v9.i3.557.
Ehlert, F.J., 2003. Contractile role of M2 and M3 muscarinic receptors in gastrointestinal,
airway and urinary bladder smooth muscle. Life Sci. 74, 355–366. https://doi.org/
10.1016/j.lfs.2003.09.023.
Fakchich, J., Elachouri, M., 2014. Ethnobotanical survey of medicinal plants used by
people in Oriental Morocco to manage various ailments. J. Ethnopharmacol. 154,
76–87. https://doi.org/10.1016/j.jep.2014.03.016.
Farre, A.J., Colombo, M., Fort, M., Gutierrez, B., 1991. Differential effects of various Ca2
+
antagonists. Gen. Pharmac. 22, 177–181. https://doi.org/10.1016/0306-3623(91)
90331-y.
Phytomedicine Plus 3 (2023) 100498
Fleckenstein, A., 1977. Specific pharmacology of calcium in myocardium, cardiac
pacemakers, and vascular smooth muscle. Annu. Rev. Pharmacol. Toxicol. 17,
149–166. https://doi.org/10.1146/annurev.pa.17.040177.001053.
Gilani, A.H., Khan, A.U., Ghayur, M.N., Ali, S.F., Herzig, J.W., 2006. Antispasmodic
effects of Rooibos tea (Aspalathus linearis) is mediated predominantly through K+-
channel activation. Basic Clin. Pharmacol. Toxicol. 99, 365–373. https://doi.org/
10.1111/j.1742-7843.2006.pto_507.x.
Godfraind, T., Miller, R., Wibo, M., 1986. Calcium antagonism and calcium entry
blockade. Pharmacol. Rev. 38, 321–416.
Guesmi, F., Saidi, I., Bouzenna, H., Hfaiedh, N., Landoulsi, A., 2019. Phytocompound
variability, antioxidant and antibacterial activities, anatomical features of glandular
and aglandular hairs of Thymus hirtus Willd. Ssp. algeriensis Boiss. and Reut. over
developmental stages. S. Afr. J. Bot 127, 234–243. https://doi.org/10.1016/j.
sajb.2019.09.004.
Hansen, M.B., 2003. The enteric nervous system II: gastrointestinal functions. Pharmacol.
Toxicol 92, 249–257. https://doi.org/10.1034/j.1600-0773.2003.920601.x.
Heghes, S.C., Vostinaru, O., Rus, L.M., Mogosan, C., Iuga, C.A., Filip, L., 2019.
Antispasmodic effect of essential oils and their constituents: a review. Molecules. 24,
1475. https://doi.org/10.3390/molecules24091675.
Jaouadi, R., Boussaid, M., Zaouali, Y., 2023. Variation in essential oil composition within
and among Tunisian Thymus algeriensis Boiss et Reut. (Lamiaceae) populations: effect
of ecological factors and incidence on antiacetylcholinesterase and antioxidant
activities. Biochem. Syst. Ecol 106, 104543. https://doi.org/10.1016/j.
bse.2022.104543.
Karim, A., Berrabah, M., Mekhfi, H., Ziyyat, A., Legssyer, A., Bouali, A., Haloui, B.,
Amrani, S., Aziz, M., 2010. Effect of essential oil of Anthemis mauritiana Maire &
Sennen flowers on intestinal smooth muscle contractility. J. Smooth Muscle Res. 46,
65–75. https://doi.org/10.1540/jsmr.46.65.
F.K. Makrane, H., Aziz, M., Berrabah, M., Mekhfi, H., Ziyyat, A., Bnouham, M.,
Legssyer, A., Elombo, F.K., Gressier, B., Eto, B., 2019. Myorelaxant Activity of
essential oil from Origanum majorana L on rat and rabbit J. Ethnopharmacol. 228,
40–49. https://doi.org/10.1016/j.jep.2018.08.036.
Marghich, M., Amrani, O., Mekhfi, H., Ziyyat, A., Bnouham, M., Aziz, M., 2021.
Myorelaxant and antispasmodic effect of an aqueous extract of Artemisia campestris L.
via calcium channel blocking and anticholinergic pathways. J. Smooth. Muscle. Res.
57, 35–48. https://doi.org/10.1540/jsmr.57.35.
Marghich, M., Amrani, O., Karim, A., Harit, T., Beyi, L., Mekhfi, H., Bnouham, M.,
Aziz, M., 2023. Myorelaxant and antispasmodic effects of the essential oil of
Artemisia campestris L., and the molecular docking of its major constituents with the
muscarinic receptor and the L-type voltage-gated Ca2+ channel. J. Ethnopharmacol.
311, 116456 https://doi.org/10.1016/j.jep.2023.116456.
Matthews Jucá, D., Da Silva, M.T.B., Palheta Junior, R.C., De Lima, F.J.B., Okoba, W.,
Lahlou, S., De Oliveira, R.B., Dos Santos, A.A., Magalhães, P.J.C., 2011. The essential
oil of Eucalyptus tereticornis and its constituents, α-and β-pinene, show accelerative
properties on rat gastrointestinal transit. Planta Med. 77, 57–59. https://doi.org/
10.1055/s-0030-1250116.
NRC (National Research Council\’s), 2011. Guide For the Care and Use of Laboratory
animals, Eight Edition. Institute for Laboratory Animal ResearchThe National
Academeis Press, Washighnton, United States of America.
OECD (The Organization of Economic Co-operation and Development Guidelines), 2008.
Guidelines for the testing of chemicals. acute oral toxicity – up-and-down-procedure.
No 425.
Ouariachi, E.M., Hamdani, I., Bouyanzer, A., Hammouti, B., Majidi, L., Costa, J.,
Paolini, J., Chetouani, A., 2014. Chemical composition and antioxidant activity of
essential oils of Thymus broussonetii Boiss. and Thymus algeriensis Boiss. from
Morocco. Asian Pacific J. Trop. Dis. 4, 281–286. https://doi.org/10.1016/S2222-
1808(14)60573-9.
Perfumi, M., Paparelli, F., Cingolani, M.L., 1995. Spasmolytic activity of essential oil of
Artemisia thuscula Cav. From the Canary Islands. J. Essent. Oil Res. 7, 387–392.
https://doi.org/10.1080/10412905.1995.9698545.
Righi, N., Deghima, A., Ismail, D., Fernandes, P.A.R., Baali, F., Boumerfeg, S.,
Baghiani, A., Coimbra, M.A., Coelho, E., 2023. Chemical composition and in vivo/in
silico anti-inflammatory activity of an antioxidant, non-toxic essential oil from
Thymus algeriensis Boiss & Reut. S. Afr. J. Bot 157, 64–74. https://doi.org/10.1016/j.
sajb.2023.03.050.
Riyazi, A., Hensel, A., Bauer, K., Geißler, N., Schaaf, S., Verspohl, E.J., 2007. The effect of
the volatile oil from ginger rhizomes (Zingiber officinale), its fractions and isolated
compounds on the 5-HT3 receptor complex and the serotoninergic system of the rat
ileum. Planta Med. 73, 355–362. https://doi.org/10.1055/s-2007-967171.
Salhi, A., Bouyanzer, A., El Mounsi, I., Bendaha, H., Hamdani, I., El Ouariachi, E.,
Chetouani, A., Chahboun, N., Hammouti, B., Desjobert, J.M., Costa, J., 2016.
Chemical composition, antioxidant and anticorrosive activities of Thymus Algeriensis.
J. Mater. Environ. Sci 7, 3949–3960.
Souiy, Z., Al-Shaikh, T.M., Alghamdi, O.A., Krifi, B., 2023. Optimization of extraction
yield, chemical composition, antioxidant, and antimicrobial activities of Thymus
algeriensis Boiss. & Reut essential oils. Biocatal. Agric. Biotechnol. 47, 102593
https://doi.org/10.1016/j.bcab.2022.102593.
Yang, J.M., Chen, C.C., 2004. GEMDOCK: a generic evolutionary method for molecular
docking. Proteins 55, 288–304. https://doi.org/10.1002/prot.20035.