Synergistic Hypnotic Effects of Sesamol and Thymol Possibly through GABAergic Interaction Pathway: In Vivo and In Silico Studies

Abstract

Background: Sleep is essential to human homeostasis and affects the immune system. Sleep disorders are a group of conditions that disturb normal sleep patterns and can affect overall health, safety, and quality of life. Poor or insufficient sleep has been associated with various dysfunctions in most body systems, such as endocrine, metabolic, higher cortical function, and neurological disorders. This study aims to evaluate the effects of the natural plant derivatives Sesamol (SES) and Thymol (THY) on sodium pentobarbital-induced sleeping mice. Methodology: The animals were given Sesamol (SES) (25, 50 mg/kg), Thymol (THY) (30 mg/kg), Diazepam (DZP) (3 mg/kg), and Caffeine (CAF) (10 mg/kg) orally (p.o.) in the respective groups individually and in combination. After 30 minutes, the treated mice were given sodium thiopental (10 mg/kg) intraperitoneally (i.p.) to induce sleep, and latency of sleeping time and duration were observed. Additionally, an in silico study was undertaken to predict the involvement of gamma-aminobutyric acid (GABA) receptors in the sleep mechanism. Results: In the current study, we observed that SES and THY increased the duration of sleeping time and decreased the latency of sleep induction. When SES, DZP, and THY were administered together, they demonstrated the greatest hypnotic activity. SES and THY exhibited strong binding affinity with different GABA receptor subtypes in in silico studies. The pharmacokinetic analysis of SES and THY using SwissADME indicated good absorption, distribution, metabolism, and excretion properties. Conclusion: SES and THY produced a hypnotic-like effect in the mice model, possibly through the GABAA and GABAB receptor interaction pathways.

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Article J. Biol. Regul. Homeost. Agents. 2023; 37(11): 6419–6435
https://doi.org/10.23812/j.biol.regul.homeost.agents.20233711.609
Copyright: © 2023 The Author(s). Published by Biolife Sas. This is an open access article under the CC BY 4.0 license.
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Synergistic Hypnotic Effects of Sesamol and Thymol
Possibly through GABAergic Interaction Pathway: In
Vivo and In Silico Studies
Md. Showkoth Akbor1, Mehedi Hasan Bappi1, Abdullah Al Shamsh Prottay1,
Mst. Farjanamul Haque1, Md. Sakib Al Hasan1, Shoyaeb Ahammed1,
Rokibul Islam Chowdhury1, Md. Sabbir Hosain1, Clara Mariana Gonçalves Lima2,
Sheikh F. Ahmad3, Sabry M. Attia3, Talha Bin Emran4,5,6,
Henrique Douglas Melo Coutinho7,*, Micheline Azevedo Lima8,
Muhammad Torequl Islam1,*
1Department of Pharmacy, Bangabandhu Sheikh Mujibur Rahman Science and Technology University, 8100 Gopalganj, Bangladesh
2Department of Food Science, Federal University of Lavras, CEP 37200-900 Lavras, Minas Gerais, Brazil
3Department of Pharmacology and Toxicology, College of Pharmacy, King Saud University, 11451 Riyadh, Saudi Arabia
4Department of Pathology and Laboratory Medicine, Warren Alpert Medical School, Brown University, Providence, RI 02912, USA
5Legorreta Cancer Center, Brown University, Providence, RI 02912, USA
6Department of Pharmacy, Faculty of Allied Health Sciences, Daffodil International University, Dhaka 1207, Bangladesh
7Laboratory of Microbiology and Molecular Biology, Department of Biological Chemistry, Regional University of Cariri, CEP 63105-000 Crato, Ceará,
Brazil
8Department of Molecular Biology, Federal University of Paraiba – UFPB, 58297-000 João Pessoa, PB, Brazil
*Correspondence: hdmcoutinho@gmail.com (Henrique Douglas Melo Coutinho); dmt.islam@bsmrstu.edu.bd (Muhammad Torequl Islam)
Published: 20 November 2023
Background: Sleep is essential to human homeostasis and affects the immune system. Sleep disorders are a group of conditions
that disturb normal sleep patterns and can affect overall health, safety, and quality of life. Poor or insufficient sleep has been as-
sociated with various dysfunctions in most body systems, such as endocrine, metabolic, higher cortical function, and neurological
disorders. This study aims to evaluate the effects of the natural plant derivatives Sesamol (SES) and Thymol (THY) on sodium
pentobarbital-induced sleeping mice.
Methodology: The animals were given Sesamol (SES) (25, 50 mg/kg), Thymol (THY) (30 mg/kg), Diazepam (DZP) (3 mg/kg), and
Caffeine (CAF) (10 mg/kg) orally (p.o.) in the respective groups individually and in combination. After 30 minutes, the treated
mice were given sodium thiopental (10 mg/kg) intraperitoneally (i.p.) to induce sleep, and latency of sleeping time and duration
were observed. Additionally, an in silico study was undertaken to predict the involvement of gamma-aminobutyric acid (GABA)
receptors in the sleep mechanism.
Results: In the current study, we observed that SES and THY increased the duration of sleeping time and decreased the latency
of sleep induction. When SES, DZP, and THY were administered together, they demonstrated the greatest hypnotic activity.
SES and THY exhibited strong binding affinity with different GABA receptor subtypes in in silico studies. The pharmacokinetic
analysis of SES and THY using SwissADME indicated good absorption, distribution, metabolism, and excretion properties.
Conclusions: SES and THY produced a hypnotic-like effect in the mice model, possibly through the GABAAand GABABrecep-
tor interaction pathways.
Keywords: sleep disorders; Sesamol; Thymol; GABAergic interaction
Introduction
Sleep disorders are characterized by abnormal sleep
patterns that impair one’s capacity to function physically,
mentally, and emotionally [1]. Sleep disturbances are
prevalent among the general population and can have se-
rious health consequences. 30% of people report having
sleep problems that last for at least a few nights each month
[2]. Sleep problems affect sleep duration, quality, and
quantity, resulting in daily pain and functional impairment
[3,4]. Sleep-wake disorders commonly coexist with phys-
ical illnesses or other mental health conditions, including
anxiety, depression, or cognitive deficits [5]. The Inter-
national Classification of Sleep Disorders, second edition
(ICSD-2) classified more than 80 distinct sleep disorders

6420
into eight categories, including parasomnias, central hyper-
somnia, insomnia, and sleep-related movement disorders
[6]. Sleep problems are commonly connected to neurode-
generative diseases, including those caused by Alzheimer’s
disease, schizophrenia, bipolar disorder, and major depres-
sive disorder [7,8].
Numerous factors, both known and unknown, can
cause sleep disorders, such as genetic mutation, psychiatric
disorders, stressful life, neurological disorders, pregnancy,
ageing, cancer, allergy, nocturia, migraine, heavy use of
alcohol and Caffeine, physical ailment, and heavy use of
medication [9–13]. Healthy sleep and waking states are
controlled by a complex neuronal network in the brain reg-
ulated by homeostatic and circadian processes [14]. Rapid
eye movement (REM) and non-REM (NREM) sleep are
the basic sleep phases. When REM sleep includes phasic
episodes such as REM and bursts of muscular activity re-
sembling waking, it is often referred to as paradoxical sleep
[15]. Synchronized electroencephalographic (EEG) activ-
ity, muscular relaxation, drop-in heart rate, blood pressure,
and tidal volume are all characteristics of NREM sleep [16].
Among several neurotransmitters, gamma-
aminobutyric acid (GABA) plays a vital role in sedation
[17] and is the most prevalent inhibitory neurotransmitter
in the central nervous system [18]. GABA receptors
have three subtypes: GABAA, GABAB, and GABAC
[19]. GABABreceptors are a form of G-protein coupled
heterodimer receptor with a muscle relaxant site, while
GABAAreceptors (containing α1,α2,α3, or α5subunits)
are a ligand-gated ion channel [20]. The inward flow of
ions, primarily chloride ions, is made possible by activating
the GABAAreceptor and opening the ligand-gated ion
channel, causing hyperpolarization and inhibiting signal
transmission [21]. On the other hand, GABABreceptors
are trans-membrane GABA receptors connected to potas-
sium channels through G-proteins [22]. The presynaptic
cell becomes hyperpolarized due to the subsequent influx
of potassium, also causing a reduction in both the inflow of
calcium ions (Ca2+) and the release of neurotransmitters
[23,24].
Benzodiazepines, barbiturates, barbiturates-related
agents, GABA receptor agents, chloral hydrate, and an-
tihistamines are among the most commonly prescribed
drugs that are used to treat various sleeping problems
[25]. The widely used sleep aids based on benzodi-
azepines and non-benzodiazepine hypnotic medications are
frequently linked to adverse side effects including daytime
sleepiness, respiratory arrest, disorientation, dependence,
headache, depression, insomnia caused by hypnotic with-
drawal, and even increased mortality [26,27]. Long-term
usage of benzodiazepines can cause cognitive impairment
[28]. Phenobarbitol-group drugs have several adverse ef-
fects, including drowsiness, withdrawal symptoms such as
convulsions and muscular relaxation, anterograde amne-
sia, and an elevated risk of accidents [29,30]. Prolonged
use of these medicines can result in dependency, tolerance,
paradoxical responses, teratologic risk, and psychomotor
effects. GABA receptor medication is utilized for treat-
ing both short-term and long-term insomnia and erratic
daytime sleeping [31]. Daytime sedation and disorienta-
tion are caused by diphenhydramine, the antihistamine H1
blocker [32]. The development of hypnotic and depressive
medicines is the GABA receptor [33]. However, given the
great care taken in using side effects and traditional drugs,
this interest has risen and there has been a rise in interest
in finding new pharmacological research to develop safer,
more focused drugs with hypnotic activity with no/minimal
side effects.
Natural plants are the mainstay of medical treatment
worldwide, with up to 38–80% (depending on the nation of
origin) of the population using photo-derived therapeutic
chemicals directly or indirectly to meet a range of health-
care demands [34–36]. Therefore, natural products made
up of various components, including flavonoids, glyco-
sides, alkaloids, and terpenoids, are one of the promis-
ing and alternative routes in the context of drug discov-
ery and development. They make up a sizable portion of
the approved chemotherapeutic agents utilized in the man-
agement of anxiety and depression-like illnesses [37–40].
According to research, plant-derived flavonoids have dif-
ferent medicinal properties that can be utilized for treating
a wide range of illnesses and disorders, including micro-
bial infection, inflammation, pro-oxidant-related diseases,
malaria, neurodegenerative diseases, mutagenic toxicity,
cancer, and infertility [41].
Sesamol (SES) is a phenolic derivative containing
a methylenedioxy group found in sesame seed oil [42].
SES has different pharmacological effects, including an-
tioxidant, anticancer, neuroprotective, cardio-protective,
anti-inflammatory, hypolipidemic, radio-protective, anti-
aging, anti-ulcer, anti-dementia, antiplatelet, anticonvul-
sant, wound healing, cosmetic (skin whitening), anti-
microbial, matrix metalloproteinase (MMP) inhibition,
and hepatoprotective activity [43,44]. The anticonvul-
sant potential of SES (30 mg/kg) is assessed against
pentylenetetrazol-induced seizures, cognitive decline, and
oxidative stress [45]. Thymol (THY) is a naturally occur-
ring phenolic monoterpene chemical that has been the sub-
ject of several studies examining its possible therapeutic
benefits. As a result, this molecule currently has a wide
range of functional possibilities in the medical area. THY
possesses antioxidant, analgesic, anti-carcinogenesis, an-
timicrobial, anti-inflammatory, antiseptic, antifungal, an-
tiviral, carminative, diaphoretic, and even anti-cancer, anti-
hyperlipidemic, and anti-hyperglycemic activity [46,47].
THY exhibits sedation at 25 mg/L dose in the muscle and
brain of silver catfish in previous experiments [48]. Recent
studies have demonstrated that Thymol has anxiolytic ac-
tivity in albino mice [49]. For this reason, SES and THY
may have a hypnotic effect.

6421
The current study aims to assess the effects of SES
and THY on sodium pentobarbital-induced sleeping mice
[50]. Furthermore, the possible sedative or hypnotic mech-
anisms of these bioactive compounds have been evaluated
using a conventional technique to determine the possible
interactions of these drug candidates with target receptors
responsible for the sedative or hypnotic effects in experi-
mental animals.
Fig. 1. Chemical structure of Sesamol, Thymol, Diazepam,
and Caffeine.
Materials and Method
In Vivo Study
Chemicals and Reagents
Caffeine, Diazepam, and Thiopental sodium were
kindly provided by ACME Laboratories Ltd., Square Phar-
maceuticals Ltd., and Gonoshasthaya Pharmaceuticals Ltd.
(Dhaka, Bangladesh), respectively. Sesamol (SES) 98%
(Catalog No. S3003) and Thymol (THY) 98.5% (Cat-
alog No. T0501) were purchased from Sigma-Aldrich
(Taufkirchen, Germany), while Tween 80 (Catalog No.
822187) was purchased from Merck (Mumbai, India).
Experimental Animals
For the current investigation, healthy Swiss albino
mice (Mus musculus) of either sex were housed at the Phar-
macology Lab of Bangabandhu Sheikh Mujibur Rahman
Science and Technology University (BSMRSTU), Gopal-
ganj, Bangladesh. The mice were obtained from Jahangir-
nagar University (JU), Bangladesh. They were six weeks
old and weighed between 22 and 28 g. The animals had
free access to regular supplies of water and food at all
times. These supplies were stored at 27 ±1 °C with a 12
h dark/light cycle under monitored illumination before the
test started. The current study was conducted from 8:00
a.m. to 3:00 p.m., and the mice were watched for 17 hours
to check for any potential post-study death. Experimental
strategy and processes were accepted by the Human and
Animal Ethics Committee of BSMRSTU (#2023-33).
Selection of Test Doses for Sesamol and Thymol
In different studies, SES demonstrated different phar-
macological activities at 25–50 mg/kg doses. SES exerts
significant anxiolytic activity in adult Swiss albino mice at
25, 50, and 100 mg/kg [51]. In another study, SES demon-
strated its cardio-protective activity at 50 mg/kg oral dose
on isoproterenol- (ISO-) induced myocardial infarction in
adult male albino Wistar rats [52]. On the other hand, the
anti-anxiety activity of THY was observed at doses of 5, 10,
and 20 mg/kg intraperitoneally (i.p.) in Swiss albino mice
[49]. Researchers also indicated that THY exerted cardio-
protective effects against adrenaline-induced rats at 15, 30,
and 60 mg/kg [53] and gastro-protective actions at 10, 30,
and 100 mg/kg [54] oral (p.o.) doses. Therefore, we se-
lected the significant doses for SES at 25 and 50 mg/kg and
THY at 30 mg/kg oral doses for the current study. When
testing hypnotic, sedative, anxiolytic, and other effects on
mice and rats, Diazepam (DZP) (1 to 3 mg/kg) is frequently
utilized as a standard drug [33].
Study Design (Sodium Thiopental-Induced Sleeping Test
in Mice)
The mice were randomly divided into several groups
after three days of acclimatization, as presented in Table 1.
The test sample, the reference medication (Diazepam: 3
mg/kg, p.o.), and the vehicle (control) were each delivered
in turn. After 0.5 hours of the treatments, each animal re-
ceived sodium thiopental (10 mg/kg, i.p.) to induce sleep
before being put in an observation chamber (e.g., plastic
cage). After administering sodium thiopental, the righting
reflex was lost, and the latent time was noted. The duration
of sleep, or the time that had elapsed between the loss and
recovery of the reflex, was also noted. The percentages of
occurrence of sleep and modulation (increase/decrease) of
latency or sleep duration were calculated using the follow-
ing equations:
% Incidence of sleep = ( Number of slept mice ÷
Total mice in the group )×100
% Decrease in latency = [( Latency of test group −
Latency of control group )÷Latency of test group ]×100
% Increase in sleep duration = [( Sleeping time of control group −
Sleeping time of test group )÷Sleeping time of control group ]×100

6422
Table 1. Name of treatments, their dose, and target receptor.
Treatment group Composition Dose Target receptor
First squad (individual treatments)
Gr-I Vehicle (0.5% Tween 80 dis-
solved in normal saline)
10 mL/kg -
Gr-II Sesamol (SES) 25 mg/kg Under investigation
Gr-III 50 mg/kg
Gr-IV Thymol (THY) 30 mg/kg Under investigation
Gr-V Diazepam (DZP) 3 mg/kg GABAA5
Gr-VI Caffeine (CAF) 10 mg/kg A2A, GABAA
Second squad (combined treatments)
Gr-VII SES-50+THY-30 50 mg/kg + 30 mg/kg Under investigation
Gr-VIII SES-50+DZP-3 50 mg/kg + 3 mg/kg Under investigation
Gr-IX SES-50+CAF-10 50 mg/kg + 10 mg/kg Under investigation
Gr-X SES-50+DZP-3+THY-30 50 mg/kg + 3 mg/kg + 30 mg/kg Under investigation
Gr-XI SES-50+DZP-3+CAF-10 50 mg/kg + 3 mg/kg + 10 mg/kg Under investigation
Gr-XII SES-50+CAF-10+THY-30 50 mg/kg + 10 mg/kg + 30 mg/kg Under investigation
All treatments are given at 10 mL/kg via oral gavage (p.o.) (n = 6). GABA, gamma-aminobutyric acid.
Statistical Analysis
The results are presented as Mean ±S.E.M. (stan-
dard error of the mean) or percentage values. The data
are analyzed using one-way analysis of variance (ANOVA)
followed by t-Student–Newman–Keuls test as a post hoc
test with multiple comparisons using the statistical software
GraphPad Prism v9.5.0.73 (GraphPad Software LLC, In-
sightful Science, La Jolla, CA, USA). The experimental
groups are compared to the vehicle (control) group. The
levels of statistical significance were p<0.0001 at 95%
confidence intervals.
Molecular Docking (In Silico) Study
GABA Homology Model and Preparation of
Macromolecule
The Swiss model was utilized for developing ho-
mology modelling of human gamma-aminobutyric acid
(GABA) [55]. The sequence was obtained from UniProt
before modelling [56], followed by BLAST analysis us-
ing the NCBI BLAST program [57] to choose the tem-
plate. The amino acid sequences of GABA subtype (A2,
A4, A5, and B1) collected by UniProt (UniProt acces-
sion ID: P47869, P48169, P31644, and Q9, UBS5, respec-
tively) were subjected to the NCBI Blast Program for se-
lecting the closest homologous template homology model
of GABA (A2, A4, A5, and B1: 6wiv.1.B, 7t0w.1.B,
7qne.1, and 7c7s.1.A) generated by modelling the SWISS-
MODEL online server (https://swissmodel.expasy.org/inte
ractive). Macromolecules were prepared using PyMOL
(version 2.5.5, Schrodinger Inc., New York, NY, USA).
Optimization of the GABA molecules was achieved us-
ing the Swiss-PDB Viewer (version 4.1.0, Swiss Institute
of Bioinformatics (SIB), Basel, Switzerland). On the con-
trary, these GABA homology models were validated using
the Ramachandran plot performed by PROCHECK [58].
Ligand Preparation
The chemical structure of SES (PubChem ID: 68289)
and THY (PubChem ID: 6989) (Fig. 1) was obtained from
the PubChem repository sample in the ‘pdf’ file format.
With the Chem3D Pro21.0 software (PerkinElmer Infor-
matics, Inc., Boston, MA, USA) packages, the internal en-
ergies of all ligands were optimized [59,60].
Docking Protocol
For the development of new drugs, Molecular dock-
ing has grown in importance [61]. Molecular docking is
a type of computer modelling that makes it easier to an-
ticipate the preferred binding orientation of a molecule
(such as a ligand) to a different one (such as a receptor)
when they interact to create a stable complex [62] using
the PyRx-virtual screening tool (ver. 0.8, Laboratory of
Computational Biology at the National Heart, Lung, and
Blood Institute (NHLBI), part of the National Institutes of
Health, Bethesda, MD, USA) [63]. After preparing the lig-
ands and macromolecule (protein), Molecular docking was
performed with ligands and proteins using the PyRx (ver-
sion 0.8.0.0, The Scripps Research Institute, La Jolla, CA,
USA). Using docking date from the PyRx-virtual screening
tool, the degree of ligand interaction with the targeted pro-
tein’s active site was determined, and two-dimensional (2D)
and three-dimensional (3D) ligand-protein interaction im-
ages were taken using visualizer software, Pymol v1.7.4.5
Edu and BIOVIA Discovery Studio (version 21.1.0, Das-
sault, Systemes, France) [64,65].
ADME Prediction
The SwissADME online platform (http://www.swissa
dme.ch/) was utilized for analyzing the physicochemical
properties of effective candidates, including aqueous sol-
ubility, lipid affinity, and pharmacokinetics [66,67]. Ad-

6423
sorption, distribution, metabolism, and excretion (ADME)
properties of SES and THY were predicted using the Swis-
sADME online tool.
Results
In Vivo Study
In this in vivo test, minimum latency of sleeping was
noticed in the SES-50 group among the Negative control
(NC), DZP, SES-25, THY-30, and CAF groups. Inter-
estingly, almost similar latency was observed between the
DZP and THY groups. However, the CAF group showed
the highest latency. Dose-dependent changes were ob-
served in the SES-50 and SES-25 groups. However, no
significant difference was observed in the sleep latency for
those given NC, SES-50, SES-25, DZP-3, and THY-30 after
intraperitoneally injected sleep latency (mg/kg). However,
for those given CAF-10, the sleep latency increased signifi-
cantly (p<0.0001). The lowest latency was observed when
SES-50 was administered with DZP, and when THY SES-
50 was administered with DZP and THY, the lowest latency
was observed. CAF increased the latency of SES-50, DZP,
and THY when used with them in combination (Fig. 2).
There was an eye-catching alteration in the duration of
sleeping time among the tested groups. For SES-50, the du-
ration of sleeping time increased incredibly in comparison
with the NC groups. In the case of the CAF group, a smaller
duration of sleeping time was observed. Despite the high-
est duration of sleeping time in the SES-50 groups, there
was no significant (p<0.05) difference among the SES-50,
SES-35, and DZP groups. Those were administered with
DZP, SES-50, SES-25, and DZP; the sleeping time was
changing significantly in comparison with the NC group.
The duration of sleeping time increased in combined groups
more than in any other single drug-administered group. In
the case of combined drug administration, when SES-50
was administered with DZP, the sleeping time of DZP was
the maximum observed when THY was administered with
the combination of DZP+SES time. It was observed that
sleeping time increased significantly (p<0.0001) in this
group in comparison with SES-50+CAF and in the SES-
50+DZP+THY groups in comparison with SES-50+CAF.
Moreover, we observed that CAF decreased the sleeping
time of SES-50, DZP, and THY when used with them in
combination (Fig. 2).
Furthermore, it was shown that the SES-50 and THY-
30 groups slept longer and with less latency than the NC
group and more than the DZP group. There were increases
in sleeping time of 74.6% and 127% and decreases in la-
tency of 16.2% and 4.2% in the SES-50+DZP and SES-
50+DZP+THY groups, respectively (Table 2).
Table 2. Percentage of modulation of latency and sleep
duration by the tests or standards or their combined groups
compared to the control (vehicle) group.
Treatment group Latency decrease (%) Sleeping time increase (%)
Gr-II 3.57 54.61
Gr-III 42.86 63.76
Gr-IV 17.86 40.82
Gr-V 17.86 59.70
Gr-VI - -
Gr-VII 50.00 68.25
Gr-VIII 50.00 69.59
Gr-IX - 49.87
Gr-X 25.00 70.55
Gr-XI 50.00 67.31
Gr-XII 28.57 63.69
Values are percentage increase/decrease in comparison to the con-
trol (Gr-I: vehicle) group; Gr-II: Sesamol (SES) 25 mg/kg; Gr-
III: Sesamol (SES) 50 mg/kg; Gr-IV: Thymol (THY) 30 mg/kg;
Gr-V: Diazepam (DZP) 3 mg/kg; Gr-VI: Caffeine (CAF) 10
mg/kg; Gr-VII: SES-50+THY-30; Gr-VIII: SES-50+DZP-3; Gr-
IX: SES-50+CAF-10; Gr-X: SES-50+DZP-3+THY-30; Gr-XI:
SES-50+DZP-3+CAF-10; Gr-XII: SES-50+CAF-10+THY-30; “-
” means no percentage modulation compared to Negative control
(NC).
In Silico Studies
GABA Homology Model
A comparative or homology modelling process em-
ploys the basic principle that proteins with similar se-
quences have similar structures to predict the structure of
proteins. The drug discovery process may be rationalized,
improved, accelerated, and made more cost-effective using
high throughput docking (HTD), lead compound optimiza-
tion, and hit identification [68]. The homology modelling
method is improved and standardized using fully automated
frameworks and databases, allowing even people without a
specialized computational background to build correct pro-
tein maps and have a quick and clear reference to modelling
findings, representation, and assessment [69,70]. Fig. 3dis-
plays the 3D homology model of the GABA receptor sub-
types. Validation of these subtypes of GABA receptor ho-
mology models was acquired using a Ramachandran plot
performed by PROCHECK, illustrated in Fig. 4.
The Ramachandran plot is a method that makes it sim-
ple to see how many torsion angles are present in a protein
structure [71]. It evaluates the integrity of protein three-
dimensional structures and provides an overview of the ac-
ceptable and forbidden torsion angle value ranges. All of
the phi-psi torsion angles of the structure’s residues are
shown on the Ramachandran map (except those at the chain
termini). Since glycine residues do not just occur in the plot
areas assigned for one of the other side chain types, trian-
gles indicate glycine residues. The shade and hue of the
plot depict the numerous locations mentioned. The areas

6424
Fig. 2. Latency of sleeping observed in test samples, control and combinations [values are Mean ±S.E.M. (n = 6)]. (A)
a-cdef, c-def, def, e-fns; a-b, b-cdef p<0.0001; aNC (vehicle); bCAF; cDZP; dSES-50; eSES-25; fTHY-30. (B) a-cdef, c-def, def, e-fns; a-b, b-cdefp<
0.0001; aSES-50+DZP; bSES-50+CAF; cSES-50+THY; dSES-50+DZP+CAF; eSES-50+DZP+THY; fSES-50+CAF+THY. (C) c-dens;
e-f, d–ep<0.05; c-f p<0.001; a-cdef, b-cdefp<0.0001; aNC (vehicle); bCAF; cDZP; dSES-50; eSES-25; fTHY-30. (D) c-def, def, a-de ns; e-f, b-f, a-cp
<0.05; a-f, b-cp<0.01; a-b, b-de p<0.0001; aSES-50+DZP; bSES-50+CAF; cSES-50+THY; dSES-50+DZP+CAF; eSES-50+DZP+THY;
fSES-50+CAF+THY. Values are Mean ±S.E.M. (n = 5); (one-way ANOVA and t-Student-Neuman-Keuls post hoc test with multiple
comparisons). Mean ±S.E.M., standard error of the mean. ANOVA, one-way analysis of variance. ns, not significant.
with the deepest hues (shown in red) are the “core” regions,
corresponding to the best phi-psi value combinations. In
an ideal world, almost 90% of the leftovers would have
been present in these “core” areas. One of the most signif-
icant ways to assess the stereochemical integrity of a sam-
ple is the percentage of residues in the “core” portions [72]
(Fig. 4). According to Ramachandran plot data, the most fa-
vorable residues for GABA (A2), (A4), (A5), and (B1) are
96.40%, 96.44%, 92.08%, and 95.71%, respectively.
Interaction of Sesamol with GABA Receptor. SES
demonstrated significant binding affinities with GABA re-
ceptor subunits such as (A2), (A5), and (B1). The bind-
ing affinities were –5.5, –5.6, and –6.1 kcal/mol, respec-
tively. SES is bound to the GABA (A2) subunit through
four H-bonds with ARG556 and GLU646, PHE537, and
PHE537. Furthermore, SES displayed a binding affinity
with GABA (A5) through seven H-bonds and two alkyl
bonds with ARG283 and ILE422. SES exhibited a bind-
ing interaction with GABA (B1) through one H-bond with
ILE841 and two alkyl bonds with CRYS637 and ALA640
(Table 3). The 2D and 3D structures of non-bond interac-
tions of SES with GABA receptor subunits are displayed in
Fig. 5.

6425
Fig. 3. Homology model of GABA receptors. (A) GABA (A2), (B) GABA (A4), (C) GABA (A5), and (D) GABA (B1) through the
Swiss model.
Interaction of Thymol with GABA Receptors. The bind-
ing affinities of THY were –6.7, –6.2, and –6.4 kcal/mol
with GABA (A4), (A5), and (B1), respectively (Table 4).
Thymol bound with GABA (A4) subunit through two
H-bonds with SER238 and SER238, two pi-pi-stacked
bonds with TYR193 and TYR243, one alkyl bond with
ILE236, and four pi-alkyl bonds. Furthermore, Thymol
showed a binding affinity with GABA (A5) through two H-
bonds with GLU284 and ASN420, one pi-anion bond with
GLU284, two alkyl bonds, and two pi-alkyl bonds. For
GABA (B1), THY displayed binding interaction through
two H-bonds with ILE841 and SER845, one pi-stacked
bond with GLU636, three alkyl bonds, and three pi-alkyl
bonds. Compared with SES, THY exhibited higher binding
affinities with GABA receptor subunits, including GABA
(A5) and GABA (B1), respectively. The 2D and 3D struc-
tures of non-bond interactions of THY with GABA receptor
subunits are illustrated in Fig. 6.
Pharmacokinetics and Drug-Likeness Properties
According to the in silico analysis, the SwissADME
online tool was utilized for examining the absorption, dis-
tribution, metabolism, and excretion (ADME) profile and
drug-likeness features of SES and THY, as illustrated in
Fig. 7and summarized in Table 5. This section lists the
basic physicochemical and molecular properties such as
Molecular refractivity (MR), Molecular weight (MW), the
sum of exact atom types, polar surface area (PSA), number
of heavy atoms, aromatic heavy atoms, H-bond acceptors
(HBAs), and H-bond donors (HBDs) [67].
The partition coefficient between n-octanol and water
(log Po/w) is a typical lipophilicity descriptor for pharma-
cokinetic drug design and discovery [73]. SwissADME of-
fers five lipophilicity prediction models, one of which is an
atomistic method with a knowledge-based repository and
correction factors [74]. Water solubility is a unique prop-
erty in drug development affecting absorption and provid-
ing an appropriate quantity of active ingredients in a few
therapeutic dosages [75]. The Log S (ESOL) model is a
water solubility prediction tool for use in SwissADME. Re-
garding bioavailability, the drug-likeness property is crucial
in predicting the chance of a molecule producing an oral
treatment.
SwissADME provides the Ghose method (a rule-
based filter) with various criteria classifying the molecule
as drug-like [67]. The results meet all the desired criteria for
forceful hits’ physicochemical, pharmacokinetic, and drug-
like properties for oral administration. The results in Fig. 8
and Table 5indicated that SES and THY were water-soluble
and met Lipinski’s requirements. Their equivalent to the
norm and high gastrointestinal absorption (GIA) demon-
strated that they were well absorbed orally.
Discussion
The term “sleep disorders” describes a broad range of
mental health conditions encompassing all types of sleep
dysfunctions, including parasomnias, trouble falling asleep
at night, slightly earlier awakenings, poor sleep quality,
sleep-related movement disorders, circadian rhythm dis-

6426
Fig. 4. The optimized model of human GABA receptors. (A) A2, (B) A4, (C) A5, and (D) B1using PROCHECK.
eases, and sleep-related breathing disorders (SBDs) [76].
Nowadays, nearly 15% of adults complain of excessive
sleepiness, and about 40% complain of insomnia [77]. The
inability to do everyday tasks, including memory, learning,
logical thinking, and mathematical operations, is reported
by those who have sleep problems [78]. Several physiolog-
ical systems, including endocrine, higher cortical function,
metabolic, and neurological disorders, obesity and weight
gain, hypertension, vulnerability to the common cold, sig-
nificant depression, and overall mortality [79–81], are all at
risk for sleep disturbances. Humans who have sleep de-
privation have altered levels of alertness and psychomo-
tor performance, moral reasoning mood, emotion modula-
tion, consolidation of memory, metabolism, regulation of
hunger, and immunological function [82].
Barbiturates were the first family of hypnotic drugs to
be developed in the early 20th century [83]. They func-
tion by directly activating the GABAAreceptor, which has
a distinct binding site from GABA [21]. Barbiturates do
not prefer certain GABAAreceptor compositions. By act-
ing on GABAAreceptor subunits, phenobarbital prolongs
the time that chloride channels are open, which reduces the
activity of the central nervous system [84]. The chloride
ion gates open when phenobarbital interacts with these re-
ceptors and remain open, allowing these ions to continu-
ously flow into neuronal cells [85]. Consequently, the cell
membrane becomes hyperpolarized, raising the action po-
tential threshold. This medication efficiently treats seizures
due to its enhanced action potential [84]. Across a range
of receptor compositions, the GABAAallosteric modula-
tors, sometimes called second-generation sedatives (benzo-
diazepines), bind at the interface between the subunits [86].
The receptor responsible for the effects of benzodiazepine
is GABAA, which is one of the three GABA receptor sub-
types A, B, and C [87]. The ligand-gated, chloride-selective
GABAAreceptor has five subunits: 2α, 2β, and γ. Benzo-

6427
Table 3. The best three results of a Molecular docking study of SES with GABA receptors.
Protein (receptor) Binding affinity (Kcal/mol) H-bond Other bonds
Residues Types Bond length (Å) Residue Types Bond length (Å)
GABA (A2) –5.5
ARG556 Conventional 2.36
-
GLU646 Conventional 1.95
PHE537 Carbon 2.61
PHE537 Carbon 3.01
GABA (A5) –5.6
ARG430 Conventional 2.69
ARG283 Carbon 2.75
ILE422 Carbon 2.65 ARG283 Alkyl 5.18
LEU282 Carbon 2.63 ILE422 Alkyl 4.32
ASP426 Carbon 2.81
ASP426 Carbon 3.03
GLU284 Carbon 2.71
GABA (B1) –6.1 ILE841 Carbon 2.95 CYS637 Alkyl 4.85
ALA640 Alkyl 4.46
Fig. 5. Two-dimensional (2D) and three-dimensional (3D) structures of molecular docking contacts between. (A) GABA (A2) and
SES, (B) GABA (A5) and SES, and (C) GABA (B1) and SES.
diazepines function as positive allosteric modulators when
they bind to a pocket formed by the alpha and gamma sub-
units. The GABAAreceptor has a single benzodiazepine
binding site in comparison with two GABA molecules that
may bind to the receptor [88]. The central nervous sys-
tem is inhibited by benzodiazepines when they bind with
the GABAAreceptor and modify the conformation of the
chloride channel [89] (Fig. 8). Moreover, GABAArecep-
tors may be divided into subtypes according to their alpha
subunits, including subtypes α1,α2,α3,α4, and α5, while
α1receptors are in charge of sedative effects [20].
The usefulness of animal models for drug research was
proven by evaluating mice models [90]. Since it is impos-
sible to perform proper human clinical trials on several new

6428
Table 4. The best three results of a Molecular docking study of THY with GABA receptors.
Protein (receptor) Binding affinity (Kcal/mol) H-bond Other bonds
Residues Types Bond length (Å) Residue Types Bond length (Å)
GABA (A4)
TYR193 Pi-Pi 4.75
TYR243 Pi-Pi 4.04
SER238 Conventional 2.13 ILE236 Alkyl 5.00
–6.7 PHE133 Pi-Alkyl 5.18
SER238 Carbon 2.76 TYR193 Pi-Alkyl 3.74
TYR243 Pi-Alkyl 4.66
TYR243 Pi-Alkyl 5.27
GABA (A5)
GLU284 Pi-Anion 4.30
GLU284 Conventional 2.47 ARG283 Alkyl 3.98
–6.2 ASN420 ILE422 Alkyl 4.20
Conventional 2.27 ARG283 Pi-Alkyl 5.14
ILE422 Pi-Alkyl 4.46
GABA (B1)
GLU636 Amide-Pi 4.67
ALA640 Alkyl 3.77
ILE841 Conventional - ILE841 Alkyl 4.18
–6.4 SER845 CYS637 Alkyl 4.02
Conventional - PHE674 Pi-Alkyl 5.43
CYS637 Pi-Alkyl 4.83
ALA640 Pi-Alkyl 4.48
Fig. 6. Two-dimensional (2D) and three-dimensional (3D) structures of molecular docking contacts between (A) GABA (A4) and
THY, (B) GABA (A5) and THY, and (C) GABA (B1) and THY.

6429
Fig. 7. ADME properties analysis. ADME properties of (A) SES and (B) THY generated by SwissADME. [The colored zone of both
(A and B) are the suitable physicochemical space for oral bioavailability. In both case (A and B), LIPO (lipophilicity): –0.7 <XLOGP3
<+5.0; SIZE: 100 g/mol <Molecular weight (MW) <500 g/mol; POLAR (polarity): 20 Å2<Topological polar surface area (TPSA)
<130 Å2; INSOLU (insolubility): –6 <Log S (ESOL) <0; INSATU (insaturation): 0.25 <Fraction Csp 3 <1; FLEX (flexibility): 0
<Number of. rotatable bonds <9.] ADME, absorption, distribution, metabolism, and excretion.
Fig. 8. Sedative effect caused by GABA, benzodiazepine, and phenobarbital sodium. Cl−, chlorine ion.
drugs, early pharmacological research typically depends on
mice models [91]. In research using mice, targeted ther-
apy may also be helpful in modifying treatment responses
to the genetic profile of the individuals in the target group
[92]. Similar to how pharmacological action is studied in
only one human, the effectiveness of therapeutic action has
traditionally only been investigated in one mouse genotype
[90]. This usually results in false judgments about the ef-
fectiveness and drawbacks of new medications. Since using
mouse models, creative medication delivery systems and
treatment plans have also been developed [93].

6430
Table 5. Pharmacokinetic properties and drug-likeness properties of SES and THY.
Properties Factors SES THY
Physicochemical properties
Formula C7H6O3C10H14 O
Molecular weight (MW) (g mol−1) 138.12 150.22
Heavy atoms 10 11
Arom. heavy atoms 6 6
H-bond acceptors (HBAs) 3 1
H-bond donors (HBDs) 1 1
Molar refractivity 38.69 48.01
Lipophilicity log Po/w (XLOGP3) 1.23 2.97
Water solubility Log S (ESOL) Very Soluble Soluble
Pharmacokinetics GI absorption High High
Drug likeness Lipinski Yes Yes
Bioavailability score 0.55 0.55
Medicinal chemistry Synthetic accessibility 2.27 1.00
Over 80% of the world’s population relies solely on
medicinal plants for their medical needs, and traditional
medicine is still acknowledged as the preferred primary
healthcare system in numerous communities [94]. This is
due to several factors, including affordability, accessibil-
ity, and low cost [95]. The majority of herbal treatments
for anxiety and insomnia are quite safe, frequently having
ten times fewer adverse effects than prescription medica-
tions [96]. Several herbal remedies have been suggested
to enhance GABAergic signaling, many of which inter-
act with the GABAAreceptor [97]. For ages, a variety of
sleep problems have been treated with herbal therapy, us-
ing plants or chemicals derived from plants for medicinal
purposes. Notable examples of such plants and chemicals
are valerian (Valeriana officinalis L.), lemon balm (Melissa
officinalis L.), passionflower (Passiflora incarnata L.), and
Californian poppy (Eschscholzia californica Cham.) [85].
An enormous decline in quality of life is linked to insom-
nia, a common illness that is frequently chronic and affects
5–15% of the general population [98]. The GABAAre-
ceptor is the primary mechanism of action for most herbal
remedies [85]. Alkanes and alkaloids, flavones, flavonoids
and iso-flavonoids, phenols, terpenes, coumarins, and oth-
ers, are compounds operating as natural modulators of the
GABAAreceptor. They were selected because they ad-
dressed the unique pharmacological aspects of their interac-
tions with the receptor [99]. Numerous naturally occurring
substances that target GABAergic signaling include medic-
inal herbs with well-known sleep-inducing effects [100].
GABAergic neurotransmission is responsible for anx-
iolytic and sedative remedies [100]. GABAA, GABAB,
and GABACare three distinct GABA receptors controlling
alertness and sleep [101]. In particular, allosteric regulation
of the benzodiazepine site is one mechanism through which
the most widely utilized hypnotics affect GABA systems
[102]. The fast-acting ionotropic GABAAreceptors were
the first to be identified and have been the focus of three
generations of hypnotics and anxiolytics. GABAArecep-
tors are pentameric, ligand-gated chlorine ion (Cl−) chan-
nels. The classical synaptic subtypes are formed of two
α, two β, and one γor δsubunit, the α1,β2,γ2recep-
tor being the most abundant [103]. Binding to the GABAB
receptor is responsible for sleep-promoting. The strength
and duration of slow-wave sleep are improved by activat-
ing GABABreceptors on hypocretin/orexin neurons [104].
In adult Swiss albino mice, the effects of SES at
dosages of 25, 50, and 100 mg/kg on anxiety were ex-
amined. SES has a dose-dependent anxiolytic effect [47].
SES exerts ameliorative effects in the experimental model
of epilepsy [105]. SES (30 mg/kg) significantly delayed
the development of kindling and prevented seizure-induced
cognitive impairment and oxidative stress [106]. THY
acts as a GABABagonist [49]. THY inhibits the calcium
channel and lowers the amount of cytosolic calcium [107].
Anxiety and depression are consistently correlated with
GABAergic activity. Both anxiety and depression can be
treated using GABAAand GABABreceptors [108]. SES
and THY may bind with GABAAor GABABreceptors and
prolong the Cl−influx or K+efflux, causing hyperpolariza-
tion, ultimately resulting in a hypnotic effect. The possible
mechanism is displayed in Fig. 9.
In the current study, we observed that SES and THY
increased the duration of sleeping time and reduced the
sleep-induced latency. SES at 50 mg/kg showed the maxi-
mum duration of sleeping time in comparison with the NC
and DZP groups. THY at 30 mg/kg also exhibited greater
hypnotic activity than NC and DZP. When SES, DZP, and
THY were administered together, they demonstrated the
greatest hypnotic activity and increased sleep by 70.05%
in comparison with NC. On the contrary, when stimulating
agents like CAF were combined, sleep time duration de-
creased and the latency of SES, THY, and DZP increased.
Computer-aided Molecular docking has become a re-
liable, affordable, and speedy method for identifying lead
compounds. It is a crucial tool for understanding the bind-
ing interactions between a ligand and its target receptor

6431
Fig. 9. Possible sedation modulatory mechanism of Sesamol (SES) and Thymol (THY).
[109]. In this in silico study, the amino acid sequences
of GABA (A2, A4, A5, and B1) were collected using
Uniprot and subjected to the NCBI Blast Program for se-
lecting the closest homologous template homology by the
Swiss model. It was observed that SES and THY have a
strong binding affinity with different GABA receptor sub-
units. SES showed binding affinity of –5.5, –5.6, and –
6.1 kcal/mol through the GABA (A2), (A5), and (B1), re-
spectively. SES exhibited maximum H-bond through the
GABA (A5) subunit. THY showed greater affinity of 6.7,
–6.2, and –6.4 kcal/mol through GABA (A4), (A5), and
(B1), respectively. The pharmacokinetic investigation of
SES and THY by SwissADME identified no violations of
Lipinski rules, indicating good absorption qualities. The
current study revealed that SES and THY exhibited a hyp-
notic effect on the mice model.
Conclusions
In the present study, the hypnotic effects of SES
and THY were observed individually in mice models. In
sodium thiopental-induced sleeping mice, both increased
the sleeping duration and decreased the sleeping-induced
time. They also showed greater efficacy than conventional
DZP. Further in silico studies have demonstrated that SES
and THY exhibited good pharmacokinetic properties and

6432
highly interacted with the GABAAand GABABreceptors
to generate hypnotic-like action, most likely at receptor sub-
types including GABA (A1), GABA (A4), GABA (A5), and
GABA (B1). SES and THY may exhibit their hypnotic ef-
fect through the GABAAor GABABreceptor interaction
pathway.
Availability of Data and Materials
This published paper comprises the data acquired or
researched during this project. Information will be supplied
upon proper request.
Author Contributions
All authors contributed to the study conception and
design. MSA, MHB, MFH and MTI designed the study.
MHB, MFH, AASP, MAL and MSAH help in data curation.
SA, RIC, MSH, CMGL and SFA help in formal analysis.
SFA and SMA help in funding acquisition. HDMC, MTI
and TBE provide the resources/software. MSA and MHB
perform the initial drafting. SFA, HDMC, MTI and TBE
help in review and editing. All authors contributed to edi-
torial changes in the manuscript. All authors read and ap-
proved the final manuscript. All authors have participated
sufficiently in the work and agreed to be accountable for all
aspects of the work.
Ethics Approval and Consent to Participate
The current project was approved by the Human and
Animal Ethics Committee at BSMRSTU (#2023-33).
Acknowledgment
The authors are grateful to the Department of Phar-
macy at BSMRSTU for approving this project and pro-
viding laboratory facilities. The authors acknowledge and
extend their appreciation to the Researchers Supporting
Project Number (RSPD2023R748), King Saud University,
Riyadh, Saudi Arabia, for funding the current study.
Funding
The Research Center of BSMRSTU (Approval No.
2023-33) funded the present project. This research was
funded by King Saud University, Riyadh, Saudi Arabia,
Project Number (RSPD2023R748).
Conflict of Interest
The authors declare no conflict of interest. Talha Bin
Emran is serving as one of the Guest editors of this journal.
We declare that Talha Bin Emran had no involvement in the
peer review of this article and has no access to information
regarding its peer review.
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