Chemical profile of lippia thymoides, evaluation of the acetylcholinesterase inhibitory activity of its essential oil, and molecular docking and molecular dynamics simulations

Article abstract of DOI:10.1371/journal.pone.0213393

The essential oils of the fresh and dry flowers, leaves, branches, and roots of Lippia thymoides were obtained by hydrodistillation and analyzed using gas chromatography (GC) and GC-mass spectrometry (MS). The acetylcholinesterase inhibitory activity of the essential oil of fresh leaves was investigated on silica gel plates. The interactions of the key compounds with acetylcholinesterase were simulated by molecular docking and molecular dynamics studies. In total, 75 compounds were identified, and oxygenated monoterpenes were the dominant components of all the plant parts, ranging from 19.48% to 84.99%. In the roots, the main compounds were saturated and unsaturated fatty acids, having contents varying from 39.5% to 32.17%, respectively. In the evaluation of the anticholinesterase activity, the essential oils (detection limit (DL) = 0.1 ng/spot) were found to be about ten times less active than that of physostigmine (DL = 0.01ng/spot), whereas thymol and thymol acetate presented DL values each of 0.01 ng/spot, equivalent to that of the positive control. Based on the docking and molecular dynamics studies, thymol and thymol acetate interact with the catalytic residues Ser203 and His447 of the active site of acetylcholinesterase. The binding free energies (ΔGbind) for these ligands were -18.49 and -26.88 kcal/mol, demonstrating that the ligands are able to interact with the protein and inhibit their catalytic activity.

Full text of DOI:10.1371/journal.pone.0213393

RESEARCH ARTICLE
Chemical profile of Lippia thymoides,
evaluation of the acetylcholinesterase
inhibitory activity of its essential oil, and
molecular docking and molecular dynamics
simulations
1
1
2
´
Sebastião Gomes Silva , Renato Araujo da Costa , Mozaniel Santana de Oliveira
,
ID
Jorddy Neves da Cruz 3, Pablo Luis B. Figueiredo 1,4, Davi do Socorro Barros Brasil1,
a1111111111
a1111111111
a1111111111
a1111111111
a1111111111
ID
ID
Lidiane Diniz Nascimento5,6, Antonio Maia de Jesus Chaves Neto 3,5, Raul Nunes de
ˆ
ID
Carvalho Junior 2,5*, Eloisa Helena de Aguiar Andrade1,6
ID
´
1 Program of Post-Graduation in Chemistry, Federal University of Para, Belem, PA, Brazil, 2 LABEX/FEA
(Faculty of Food Engineering), Program of Post-Graduation in Food Science and Technology, Federal
´
´
University of Para, Belem, PA, Brazil, 3 Laboratory of Preparation and Computation of Nanomaterials,
Federal University of Para, Belem, PA, Brazil, 4 Department of Natural Sciences, State University of Para,
´
´
´
´
Belem, PA, Brazil, 5 Program of Post-Graduation in Engineering of Natural Resources of Amazon, Federal
University of Para, Belem, PA, Brazil, 6 Adolpho Ducke Laboratory, Botany Coordinating, Museu Paraense
´
´
´
´
Emılio Goeldi, Belem, PA, Brazil
OPEN ACCESS
Citation: Silva SG, da Costa RA, de Oliveira MS, da
Cruz JN, Figueiredo PLB, Brasil DdSB, et al. (2019)
Chemical profile of Lippia thymoides, evaluation of
the acetylcholinesterase inhibitory activity of its
essential oil, and molecular docking and molecular
dynamics simulations. PLoS ONE 14(3):
e0213393. https://doi.org/10.1371/journal.
pone.0213393
* raulncj@ufpa.br
Abstract
The essential oils of the fresh and dry flowers, leaves, branches, and roots of Lippia thy-
moides were obtained by hydrodistillation and analyzed using gas chromatography (GC)
and GC–mass spectrometry (MS). The acetylcholinesterase inhibitory activity of the essen-
tial oil of fresh leaves was investigated on silica gel plates. The interactions of the key com-
pounds with acetylcholinesterase were simulated by molecular docking and molecular
dynamics studies. In total, 75 compounds were identified, and oxygenated monoterpenes
were the dominant components of all the plant parts, ranging from 19.48% to 84.99%. In the
roots, the main compounds were saturated and unsaturated fatty acids, having contents
varying from 39.5% to 32.17%, respectively. In the evaluation of the anticholinesterase
activity, the essential oils (detection limit (DL) = 0.1 ng/spot) were found to be about ten
times less active than that of physostigmine (DL = 0.01ng/spot), whereas thymol and thymol
acetate presented DL values each of 0.01 ng/spot, equivalent to that of the positive control.
Based on the docking and molecular dynamics studies, thymol and thymol acetate interact
with the catalytic residues Ser203 and His447 of the active site of acetylcholinesterase.
The binding free energies (ΔGbind) for these ligands were -18.49 and -26.88 kcal/mol, dem-
onstrating that the ligands are able to interact with the protein and inhibit their catalytic
activity.
Editor: Marco Bonizzoni, The University of
Alabama, UNITED STATES
Received: October 22, 2018
Accepted: February 19, 2019
Published: March 8, 2019
Copyright: © 2019 Silva et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Information files.
Funding: This work was supported by scholarships
awarded to SGS. SGS thanks Secretaria Estadual
´
de Educac¸ão do Para.; Oliveira M. S (Process
Number: 1662230) and Costa W. A. (Process
Number: 1427204), thanks CAPES for the
doctorate scholarship. The funder had no role in
PLOS ONE | https://doi.org/10.1371/journal.pone.0213393 March 8, 2019
1 / 17

Chemical profile of Lippia thymoides
study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
Introduction
Alzheimer’s disease is considered one of the major public health problems worldwide and one
of the main complications of this pathology is the activity deficit of cholinergic neurons. This
fact can be reversed and/or attenuated by elevating the levels of the neurotransmitter acetyl-
choline in the neuronal synaptic area. The use of cholinesterase inhibitors is an effective thera-
peutic approach [1]. The inhibitors increase the availability of neurotransmitters by inhibiting
their main catalytic enzymes, acetyl- and butyrylcholinesterase, thus diminishing the choliner-
gic deficit and relieving the symptoms of Alzheimer’s patients [2]. The oldest inhibitor of these
enzymes is physostigmine, an alkaloid of the shrub Physostigma venenosum Balf [3]. Synthetic
and semisynthetic inhibitors, such as galantamine, donepezil, tacrine, and rivastigmine, can
also be used, but these drugs have disadvantages such as short half-lives and adverse side effects
including hepatotoxicity and gastrointestinal irritation [1,4,5]. This has encouraged a search
for new inhibitors from natural sources, and some examples are the secondary metabolites
present in essential oils [1,4,6–8].
Competing interests: The authors have declared
that no competing interests exist.
Lippia thymoides is a native and endemic Brazilian species with distribution in the states of
Bahia and Minas Gerais in the Caatinga and Cerrado types of vegetation [9]. Folk medicine
makes use of this plant for the treatment of wounds, and the leaves are used as an antipyretic
and digestive, as well as in the treatment of bronchitis and rheumatism [10,11].
The objective of this study was to obtain and analyze the chemical composition of the essen-
tial oils from different parts of L. thymoides and to evaluate the anticholinesterase potential of
the oil of the fresh leaves and its main constituents(thymol and thymol acetate), as well as to
evaluate the interactions of acetylcholinesterase (AChE) with thymol and thymol acetate by
molecular docking and molecular dynamics simulations and free energy calculations using the
molecular mechanics generalized Born surface area (MM/GBSA) method.
Materials and methods
Plant material and essential oil extraction
The authors declare that no specific permissions were required for these locations/activities;
and we confirm that the field studies did not involve endangered or protected species.
L. thymoides was collected in the Municipality of Abaetetuba, Eastern Amazon, State of
´
Para, Brazil. The botanical identification was made by comparison with authentic samples and
incorporated into the "João Murc¸a Pires" herbarium of the Museu Paraense Emilio Goeldi
´
´
(Belem, Para, Brazil) under catalog number MG 213373.
The essential oils were obtained from fresh and dried parts of L. thymoides.The drying of
the plant material was conducted in a forced air convection oven for two days at 34˚C. After
that the material was ground and submitted to hydrodistillation.
Hydrodistillation
The extraction of the essential oils of fresh flowers (FFL, 10g), leaves (FLE, 40g), branches (FB,
40g), and roots (FR, 40g), and dried flowers (DFL, 10g), leaves (DLE, 40g), branches (DB, 40g),
and roots (DR, 40g) was carried out by hydrodistillation in a Clevenger apparatus for 3 h. Sub-
sequently, the oils were submitted to centrifugation, dehydrated with anhydrous Na2SO4 and
stored at 8˚C in a freezer. The moisture content was determined using a moisture analyzer
(ID50, Marte) at the moment of extraction. The yield of essential oil (%) was calculated and is
expressed as milliliters / 100-g of dried material [12].
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Chemical profile of Lippia thymoides
Chemical constituent identification
The chemical composition was determined according to the approach described by Da Silva
et al. [13], where the qualitative analyses of essential oils was carried using a gas chromatogra-
phy–mass spectrometry (GC-MS) Thermo Focus DSQ-II system under the following the oper-
ating conditions: DB-5MS silica capillary column (30 m × 0.25 mm; 0.25 μm film thickness),
temperature program: 60–250˚C with a gradient of 3˚C/min); injector temperature: 240˚C,
helium carrier gas (linear velocity of 32 cm/s, measured at 100˚C), injection type: split less
(0.1 μL of a 2:1000 n-hexane solution),and ion-source temperature and other parts: 200˚C.
The ionization was achieved by electron impact at 70 eV. Quantitative sample data were
obtained using a GC with a flame ionization detector in a Focus GC-FID, which was operated
under the same conditions as the GC-MS, except for the carrier gas, which was nitrogen. The
identification of volatile components was based on the linear retention index (IR), which was
calculated in relation to the retention times of a homologous series of n-alkanes and the frag-
mentation pattern observed in the mass spectra by comparison with authentic samples from
the libraries of the data system and the literature [14,15].
Acetylcholinesterase assay
The AChE assay was conducted according to the method reported by Marston et al. [16] The
enzyme AChE (500 U), from Electrophorus electric us (electric eel, Sigma Aldrich, Missouri,
EUA, E.C. 3.1.1.7), was dissolved in tris-hydrochloric acid buffer (pH 7.8) and stabilized by the
addition of bovine serum albumin fraction V (0.1%). Thymol, thymol acetate, and the essential
oil of L. thymoides fresh leaves were applied to thin-layer chromatography (TLC) plates to
obtain spots with concentrations from 0.01 to 1000 ng/spot. Physostigmine was used as a posi-
tive control. The plates were sprayed with the AChE solution (3.33 U/mL), dried, and incu-
bated at 37˚C for 20 min. The enzyme activity was detected by spraying with a solution of
0.25% of 1-naphtyl acetate in ethanol and a 0.25% aqueous solution of Fast Blue B salt (20 mL).
Potential acetylcholinesterase inhibitors appeared as clear zones on a purple background.
Semisynthesis of thymol acetate
To obtain thymol acetate, thymol (Sigma–Aldrich), acetic anhydride, and pyridine, which acts
as a catalyst, were used. Thymol (4g) was acetylated with acetic anhydride in the presence of
pyridine for 24 h at 25˚C. The excess acetic anhydride was removed by storage of the sample in
a desiccator for 12 h. The reaction mixture was partitioned with dichloromethane and water
for the removal of acetic acid. The semi synthesis and identification of the pure compound was
monitored by GC and GC-MS analysis.
Molecular docking
To analyze the interactions between the ligands (thymol and thymol acetate) and AChE,
molecular docking simulations were carried out using the Molegro Virtual Docker (MVD) 5.5
program [17]. The crystallographic structure of AChE was obtained from the Protein Data
Bank (https://www.rcsb.org/) (PDB code: 1C2B) [18]. Both the structures of the enzyme and
the ligands were prepared using the MVD module. Before the docking simulation, for each
complex, hydrogen atoms and partial atomic charges were added. The active site of AChE was
positioned in a spherical grid of 10-Å diameter, and all residues of AChE binding site were
included using the following spatial coordinates of the central cavity: x = 26.40, y = 79.03, and
z = 20.20. A grid resolution equal to 0.3 Å was used. Molecular docking was performed using
the standard MolDock algorithm in MVD.
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Chemical profile of Lippia thymoides
Molecular dynamics (MD) simulations
Molecular dynamics (MD) simulation for all ligand–protein systems obtained after docking
were performed to evaluate possible conformational changes in the protein structure. First, the
partial atomic charges were calculated using Gaussian09 [19], using the restrained electrostatic
potential (RESP) protocol at the HF/6-31G� level of theory [20,21]. The antechamber [22]
module of Amber 16 package was used to parameterize the ligand [23,24], which was described
by the General Amber Force Field (GAFF) [25].
The protonation state of all ionizable residues was determined by the PDB2PQR server
[26,27], and the protein structure was treated with the ff14SB force-field [28] in all MD simula-
tions. The protein system was solvated in octahedral periodic box with the TIP3P explicit sol-
vation model [29]. Counterions were added to neutralize the system charges. The system
contained approximately 16,200 water molecules and 55,500 atoms. A cut-off distance of 12 Å
and the particle mesh Ewald method [30] was used for electrostatic calculations, and the
SHAKE algorithm was used to keep all the hydrogen bonds at their pre-defined equilibrium
distances during minimization [31]. Before the MD simulation, production systems under-
went simulations for energy minimization, heating, and equilibration that were performed
with the Sander module and pmemd. CUDA [32].
Energy minimization was performed in two steps. In each step, 1,000 steepest descent and
conjugate gradient algorithm cycles were applied. In these steps, bad contacts and possible ste-
ric conflicts were removed, and the protein system acquired the most energetically favorable
conformational state. First, the protein structure was restrained with a harmonic force con-
stant of 100 kcal/mol Å−2, and the water molecules and counterions were not treated with har-
monic restraints. Then, the harmonic constraint was removed to perform the MD run of the
protein system (protein, water, and counterions). This system was then heated to 300 K in five
steps for 500 ps. In the first four steps, we applied a harmonic force constant of 25 kcal/mol
Å−2 to restrain all heavy atoms. In the last heating step, the harmonic force constraint was
removed. The Langevin thermostat [33] within a collision frequency of 2 ps−1was used to
maintain the temperature. Thus, 5 ns of MD simulation without harmonic restraints at 300 K
was carried out to obtain system equilibrium. Finally, a 100-ns MD simulation was performed
for all protein systems.
Binding free energy calculations using the MM-GBSA approach
The molecular mechanics–generalized Born surface area (MM-GBSA) method [34,35] was
used to measure the binding free energies of the ligands, thymol and thymol acetate. The bind-
ing free energies (ΔGbind) were calculated according to the following equations:
DGbind ¼ DGcomplex À DGreceptor À DGligand
ð1Þ
Each free energy state is calculated by the following equations:
DGbind ¼ DH À TDS � DEMM þ DGsolv À TDS
ð2Þ
ð3Þ
ð4Þ
DEMM ¼ DEinternal þ DEele þ DEvdW
DGsolv ¼ DGGB þ DGSA
The ΔGbind values correspond to the sum of interaction energies in the gas phase between
the protein and ligand (ΔEMM), the desolvation free energy (ΔGsolv), and the system entropy
(−TΔS). Here, ΔEMM is the sum of internal energy (ΔEinternal), sum of bond length, angle, and
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Chemical profile of Lippia thymoides
dihedral energies), electrostatic contributions (ΔEelectrostatic), and van der Waals terms
(ΔEvdW). ΔGsolv is the sum of the polar contributions (ΔGGB) and non-polar contributions
(ΔGSA). The external dielectric constant of the solute was defined as 80, while the internal
dielectric constant was defined as 1. ΔGSA was determined from the solvent accessible surface
area (SASA) estimated using the LCPO algorithm. For the free energy calculations, 1,000 MD
frames were used, corresponding to the last 5 ns of the simulations.
Per-residue binding free energy decomposition
To analyze the free energy contributions of each amino acid residue of protein pocket for
ligand interaction, the binding free energy was decomposed into the van der Waals (ΔEvdW
)
and electrostatic (ΔEelectrostatic) contributions in the gas phase, as well as the polar solvation
(ΔGpol) and nonpolar solvation (ΔGnonpol) contributions [36], using the following equation:
DGinhibitorÀ residue ¼ DEvdW þ DEele þ DGpol þ DGnonpol
ð5Þ
Results and discussion
Yield and composition of essential oil
The yield of L. thymoides essential oil varied in quantity in the different plant parts and with
treatment (fresh and dried). The flowers produced the highest essential oil yields, both fresh
and dried (FFL 5.8%; DFL 7.3%). DB presented an oil yield equal to 0.14%, whereas FB, FR,
and DR contained only traces of essential oil (Table 1).
Generally, the essential oil yield of a plant varies depending on the part, seasonality, and
geographical distribution, among other factors. For example, samples of L. citriodora produced
different oil yields depending on the part extracted, and it was verified that the highest yields
were obtained from the flowers [37]. Low oil yields from branches and roots (0.1% for both)
were also registered in a species of domesticated L. muliflora [38]. The yield of dried leaves of
L. thymoides from state of Bahia (Brazil) showed seasonal variation, and its values varied
between 2.14% and 2.93% [39].
Seventy-five components were identified in the oil, comprising (95.53–99.54%) of the total
composition (Table 1). Oxygenated monoterpenes were identified in all plant organs, and the
percentage contents of this group varied from 19.48% (FR) to 84.99% (FFL)
The monoterpene and sesquiterpene hydrocarbons varied respectively from 0.26% (FFL) to
28.78% (DFL) and 2.41% (FB) to 12.95% (FFL), but these were absent in the essential oils of
the roots. Saturated fatty acids were also identified, whose content varied from 0.06% (DFL) to
41.62% (FR), but these were absent in the leaves; unsaturated fatty acids were detected only in
the branches and roots, ranging from 6.26% (FB) to 36.37% (FR).
Thymol was the only constituent present in all the organs of L. thymoides and was the main
component of the oils of the flowers, leaves, and branches. This oxygenated monoterpenoid
varied among all the plant organs from 19.34% (FR) to 66.33% (FLE). In the three parts where
thymol was the main component. In addition, for the fourth plant sample, quantitative varia-
tions in p-cymene (0.07% FFL and 8.36% DLE), β-caryophyllene (1.29% FB and 9.55% FFL),
γ-terpinene (0.15% FFL and 15.06% DFL), and thymol acetate (5.07% FB and 33.81% FFL)
were observed.
The essential oils from the roots were characterized, and hexadecanoic (palmitic) acid was
found to be the main component (38.02% DR; 40.92% FR), followed by (9Z)-octadecenoic
(oleic) acid (27.4% DR; 28.21% FR), thymol (19.34% FR; 22.18% DR), (9Z,12Z)-9,12-octadeca-
dienoic (linoleic) acid (4.49% FR), and (11Z)-11-hexadecenoic acid (2.73% DR; 3.67% FR). It
is important to note that hexadecanoic (palmitic) acid was also present in the oils obtained
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Chemical profile of Lippia thymoides
Table 1. Essential oil composition of different organs of Lippia thymoides.
Oil Yield
FFL
5.80
DFL
7.30
FLE
1.87
DLE
1.25
FB
tr
DB
FR
tr
DR
tr
0.14
RI
Constituents
Composition %
904
1-ethylbutyl hydroperoxide
1-methylpentyl hydroperoxide
α-thujene
0.28
0.32
0.21
0.20
914
923
1.46
0.20
0.11
0.21
0.05
1.67
0.30
1.99
7.18
0.28
0.30
0.13
15.06
0.12
0.10
0.04
0.07
0.04
0.10
0.05
0.03
0.23
0.37
0.02
2.00
37.86
21.44
0.24
0.81
0.05
0.24
0.26
933
α-pinene
947
Camphene
0.02
0.04
0.04
0.68
0.10
0.55
5.30
0.15
0.39
0.10
7.58
0.03
0.08
0.10
0.43
0.07
0.57
3.27
0.16
0.24
0.06
3.39
969
Sabinense
0.04
0.12
0.60
0.08
0.77
3.35
0.14
0.30
0.10
4.84
974
β-pinene
0.18
1.34
0.13
1.48
8.36
0.23
0.48
0.11
9.36
989
Myrcene
1003
1016
1022
1026
1028
1045
1057
1068
1089
1092
1097
1100
1143
1147
1166
1169
1175
1188
1233
1293
1352
1358
1372
1373
1386
1401
1419
1432
1433
1441
1454
1479
1486
1495
1496
1499
1510
α-phellandrene
α-terpinene
0.02
0.07
p-cymene
Limonene
1,8-cineole
0.19
(E)-β-ocimene
γ-terpinene
0.15
0.14
cis-sabinene hydrate
Terpinolene
0.06
0.03
0.14
0.09
0.04
0.16
0.06
0.15
0.08
para-cymenene
Linalool
0.02
0.08
0.03
0.10
0.06
trans-sabinene hydrate
Camphor
0.10
0.02
0.06
0.02
0.29
0.03
1.01
66.33
7.49
0.09
0.12
0.02
0.05
0.03
0.52
0.06
3-methyl-3-butenyl-3-methylbutanoate
Borneol
Umbellulone
terpinen-4-ol
α-terpineol
0.44
0.32
0.32
0.41
0.33
0.56
0.03
1.39
66.20
5.96
0.07
0.02
ether methyl thymol
Thymol
1.82
1.27
58.9
8.10
0.08
0.03
1.47
63.59
5.07
0.07
48.04
33.81
19.34
22.18
thymol acetate
Eugenol
0.12
0.49
α-copaene
0.04
0.03
0.06
carvacrol acetate
β-bourbonene
Methyleugenol
β-caryophyllene
trans-α-bergamotene
γ-elemene
0.05
0.04
0.09
0.03
4.16
0.13
9.55
0.15
0.06
5.93
0.11
0.03
5.32
0.16
4.53
0.10
1.29
0.07
Aromadendrene
α- humulene
γ-muurolene
germacrene-D
γ-amorphene
α-selinene
0.04
0.73
0.13
0.26
0.05
0.04
0.05
0.03
0.61
0.12
0.65
0.05
0.05
0.71
0.15
0.61
0.07
0.03
0.08
0.06
1.35
0.16
1.22
0.04
0.69
0.05
0.70
0.26
0.09
0.42
0.02
0.04
0.03
α-muurolene
δ-amorphene
0.03
0.04
0.02
(Continued)
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Chemical profile of Lippia thymoides
Table 1. (Continued)
Oil Yield
FFL
5.80
DFL
7.30
FLE
1.87
DLE
1.25
FB
tr
DB
FR
tr
DR
tr
0.14
RI
Constituents
Composition %
1514
1521
1521
1535
1539
1562
1579
1583
1609
1637
1641
1645
1653
1663
1668
1862
1900
1920
1955
1961
2080
2158
γ-cadinene
δ-cadinene
0.12
0.23
0.05
0.10
0.10
0.20
0.03
0.02
0.02
0.04
0.09
0.16
0.02
0.08
0.02
0.13
0.24
0.05
0.02
0.02
0.05
trans-calamenene
trans-cadina-1,4-diene
α-cadinene
0.02
0.03
germacrene B
0.02
0.02
0.66
0.07
Spathulenol
caryophyllene oxide
humulene epoxide II
epi-α-cadinol
0.61
0.05
0.02
0.29
0.03
0.02
0.40
0.04
0.05
0.33
0.03
0.42
0.04
0.09
epi-α-muurolol
0.02
0.03
0.14
α-muurolol
0.03
0.16
α-cadinol
0.07
0.03
0.04
0.07
0.03
0.05
0.03
tetradecanoic acid
14-hydroxi-9-epi-(E)-caryophyllene
pentadecanoic acid
Nonadecane
0.35
0.33
0.09
0.03
0.35
0.37
1.15
0.72
2.04
2.73
38.02
2-ethylhexyl-3-(4-methoxyphenyl)-2-propenoate
(11Z)-11-hexadecenoic acid
hexadecanoic acid
3.67
40.92
4.49
0.16
0.06
6.11
2.77
´
(9Z,12Z)-9,12-octadecadienoic acid
(9Z)-octadecenoic acid
monoterpene hydrocarbons
oxygenated monoterpenes
sesquiterpene hydrocarbons
saturated fatty acids
unsaturated fatty acids
Others
6.26
8.40
1.58
10.36
75.00
6.71
28.21
27.40
22.18
0.26
84.99
12.95
0.16
28.78
62.58
10.69
0.06
14.89
75.86
7.19
22.18
69.63
6.48
71.21
2.41
19.48
6.11
2.77
41.62
36.37
1.46
39.50
32.17
1.13
6.26
1.58
0.84
0.43
0.7
0.54
1.15
0.80
Total
99.20
99.54
98.64
98.90
95.53
97.20
98.93
96.05
FFL: fresh flower; DFL: dried flower; FLE: fresh leaves; DLE: dried leaves; FB: fresh branche; DB: dried branche; FR: fresh root; DR: dried root. tr: traces
https://doi.org/10.1371/journal.pone.0213393.t001
from the branches (2.77% DB; 6.11% FB) and flowers (0.06% DFL; 0.16% FFL), and(9Z)-octa-
decenoic (oleic) acid was also identified in the oils of the branches (1.58% DB; 6.26% FB).
Please refer to S1 and S2 Figs with the chromatogram ions for the chemical composition of the
fractions of essential oils.
Silva et al. [39]reported that the essential oil from the leaves of a specimen of L. thymoides-
contains sesquiterpeneβ-caryophyllene (17.22–26.27%) as the main component, followed by
borneol (4.45–7.36%), camphor (3.22–8.61%), camphene (2.64–5.66%), and germacrene-D
(4.72–6.18%). This chemical profile is different from described in the present study. This is
possible related to biotic and abiotic factors of the collection site of these two specimens;
according to Ribeiro et al. [40], these factors qualitatively and quantitatively affect the yield
and composition of secondary metabolites.
Studies with species of the same genus, such as L. multiflora Moldenke, showed that the
essential oils obtained from the leaves, flowers, branches, and roots do notvary in chemical
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Chemical profile of Lippia thymoides
Fig 1. Synthesis reaction of thymol acetate.
https://doi.org/10.1371/journal.pone.0213393.g001
profile, and the composition was essentially dominated by monoterpenes [38]. Similarly, the
volatiles of the vegetative parts (leaves and branches, flowers, and fruit) of Lippia citriodora
were characterized as having the same constituents in all parts of the plant: geranial (30.67–
36.87%), neral (21.71–28.33%), and limonene (6.07–7.27%) [37].
Species of other genera, such as Hertia cheirifolia, showed significant variation in the com-
position of the essential oil between the different parts of the plant, where the flower buds and
flowers were characterized by a higher content of drimane-type sesquiterpenes (34.08% and
32.55%, respectively), while the leaves and fruits predominantly contained α-pinene (35.63%
and 33.17%, respectively) [41]. In addition, the essential oils of the roots, leaves, branches,
inflorescences, and fruits of Kelussia odoratissima Mozaff. allcontained the same major com-
pounds: (Z)-ligustilide (54.0–86.0%) and (2e)-decen-1-ol (2.0–12.3%). However, among the
samples of these parts, a complex mixture of up to thirty-two different chemical compounds
was identified [42].
Obtaining thymol acetate
Thymol acetate was obtained by the acetylation of thymol (Fig 1) in 99.38% purity. The chro-
matogram and mass spectrum of thymol acetate are shown in Figs 2 and 3.
Anticholinesterase activity
The aim of this study is to contribute to the search for new inhibitors of AChE from natural
sources. Thus, the anticholinesterase activity of the essential oil from the leaves of L. thymoides
was investigated. In addition, we determined the activity of two of the main constituents of the
oil, thymol and thymol acetate (99.38% purity).
Fig 2. Thymol acetate chromatogram.
https://doi.org/10.1371/journal.pone.0213393.g002
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Chemical profile of Lippia thymoides
The essential oil of the leaves of L. thymoides (thymol: 66.33%) showed a detection limit
(DL) at a concentration of 0.1 ng/spot, about ten times less active than physostigmine
(DL = 0.01 ng/spot). Because thymol and thymol acetate presented a DL values of 0.01 ng/
spot, equivalent to that of physostigmine, the alkaloid was used as a positive control. The
higher detection concentration of thymol and thymol acetate in the oil may be because these
components have a lower total content in the sample mixture compared to the isolate. Thus,
the anticholinesterase action of the essential oil of L. thymoides may be tied to these two com-
ponents. These results are in agreement with data from the literature, which indicates that the
essential oil of Origanum ehrenbergii (thymol: 19.6%) shows strong inhibitory activity against
AChE, having a 50% inhibitory concentration (IC50) of 0.3 0.02 μg/mL when physostigmine
was used as positive control and exhibited an IC50equal to 0.1 0.003 μg/mL [43]. The essen-
tial oil of Thymus vulgaris L. (thymol: 12%) showed an IC50 of 0.216 0.011 mg/mL [44]. O.
vulgare subsp. vulgare essential oil (thymol: 58.31%) was found to have a concentration
of1.64 0.002 mg galantamine equivalents per gram (GALAEs/g) [1].
Thymol and carvacrol have anticholinesterase activity reported by several authors, with dif-
ferent values of IC50: 0.74 mg/mL (thymol) and IC50 equivalent to 0.063 mg/mL (carvacrol)
[45]; 0.212 0.011 mg/mL (thymol) and IC50 0.091 0.011 mg/mL (carvacrol) [44];
47.5 1.08 mg/mL (thymol) and IC50 182 1.32 mg/mL (carvacrol) [46]. These studies have
shown that the anticholinesterase activity of thymol is promising. Thus, the specimen of L. thy-
moides described in this work can be a natural source with potential for the development of
new phytotherapics for the treatment of Alzheimer’s disease or even be included in the diet of
people with Alzheimer’s. It is important to emphasize that in our previous work [47] it was
shown that this specimen presents thymol as the majority constituent of essential oil through-
out its seasonal cycle, which reinforces its viability as a source of obtaining this component.
Interactions observed by molecular docking
Previously, molecular docking has been used to elucidate the interactions that occur between
the ligands with the active site residues of AChE [4,48,49]. In the present study, molecular
docking simulations were used to evaluate the complementarity and interactions between thy-
mol and thymol acetate with the AChE binding site.
The binding energies, expressed by the MolDock scores obtained from molecular docking
simulations, are shown in Table 2. The differences in energies can be explained by the differ-
ence in the molecular volume between thymol and thymol acetate.
Thymol and thymol acetate form hydrogen bonding (H-bond) interactions with the Ser203
catalytic residue in the AChE binding pocket (Fig 4).
The O1 atom of the hydroxyl group, which belongs to thymol, forms a H-bond interaction
with Ser203 at a distance of 3.14 Å. In contrast, thymol acetate forms two H-bond interactions
Fig 3. Thymol acetate mass spectrum.
https://doi.org/10.1371/journal.pone.0213393.g003
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Chemical profile of Lippia thymoides
Table 2. Results of docking energies obtained by the MolDock score.
Compound
Thymol
MolDock score (kcal/mol)
-72.82
-84.49
Thymol acetate
https://doi.org/10.1371/journal.pone.0213393.t002
with the Ser203 residue. The interactions with oxygen atom O2 of the Ser203 residue have dis-
tances of 2.66 and 2.98 Å.
Considering the catalytic mechanism of AChE, residues Ser203 and His447 are directly
involved in covalent bond formation and breakage. Residue Ser203 is a nucleophilic site,
whereas the His447 imidazole group acts as a catalytic base, accepting a proton transferred
from Ser203 [50,51]. Thus, because molecular docking calculations have shown that thymol
and thymol acetate interact with the same catalytic site residues of the enzyme, these inhibitors
may show similar anti-AChE activity.
Structural dynamics of the AChE systems
The structural dynamics of AChE complexed with the ligands thymol and thymol acetate dur-
ing MD simulations were analyzed using the root mean square deviation (RMSD). The RMSD
plots of the AChE structure were analyzed based on the Cα backbone, whereas the ligand
structure was analyzed using the heavy atoms alone.
The RMSD plot of AChE complexed to thymol and thymol acetate reaches a plateau (Fig
5), indicating that AChE structure is stable, showing few conformational changes over the
100-ns MD simulation. The ligands showed high stability at the AChE binding site. Over the
100-ns MD run, the ligands remained bound to AChE and did not undergo drastic conforma-
tional changes that altered their interactions with the catalytic residues.
Binding free energy decomposition
Binding energy calculations show that thymol and thymol interact with high affinity in the
AChE binding pocket, having ΔGbind values of -18.49 and -26.88 kcal/mol, respectively. The
binding free energy decomposition reveals that the van der Waals (ΔEvdW) contribution repre-
sents a large proportion of the interactions of the ligands in AChE binding pocket. The electro-
static (ΔEelectrostatic) and non-polar (ΔGSA) contributions were also favorable for the formation
of the complexes. The ΔG values and their components are listed in Table 3.
Intermolecular interactions analyses
To explore the relative energy contribution to the overall ligand binding energy for each resi-
due in the AChE binding pocket, the per-residue binding free energy decomposition was ana-
lyzed in Amber16. The results obtained are shown in Fig 6.
The AChE structure contains different binding sites [52]. The catalytic residues Ser203,
Glu337, and His447 are located in the same cavity of the enzyme, which also contains other
important residues for enzymatic activity, for example, Gly121, Gly122, and Ala204 (oxyanion
hole), Trp86, Tyr133, Tyr337, and Phe338 (anionic subsite), and Phe295 and Phe297 (acyl
pocket) [53].
Thymol acetate (ΔGbind = -26.88 kcal/mol) has a better binding energy than that ofthymol
(ΔGbind = -18.49 kcal/mol); that is, the binding energy of thymol acetate is greater than that of
thymol, and both the van der Waals and electrostatic energies are important for ligand binding
in AChE cavity.
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Chemical profile of Lippia thymoides
Fig 4. The result of molecular docking conformation obtained. (A) Molecular interactions for Thymol and (b) Thymol acetate in AChE
binding pocket.
https://doi.org/10.1371/journal.pone.0213393.g004
Thymol formed intermolecular interactions with different residues of the AChE binding
pocket, in particular, with residue His447, which belongs to the catalytic triad (His 447,
Tyr337, and Trp86) located on the anionic subsite. Residues His447 and Gly122 belong to the
oxyanion hole. In contrast, thymol acetate formed different intermolecular interactions with
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Chemical profile of Lippia thymoides
Fig 5. RMSD values for 100 ns of MD for AChE systems. The protein backbone is colored in black, thymol in blue
and thymol acetate in green. (a) RMSD sistem plot Thymol- AChE e (b) RMSD sistem plot Thymol acetate-AChE.
https://doi.org/10.1371/journal.pone.0213393.g005
Table 3. Free energy variation (ΔGbind) and its components. ΔEvdW represents the Van de Waals energy contribution, ΔEele represents the electrostatic energy, ΔGGB
polar contribution and ΔGSA non-polar contribution. All values are expressed in kcal/mol.
Compound
Thymol
ΔEvdW
-23.89
-33.22
ΔEele
-7.97
ΔGGB
16.47
24.52
ΔGSA
-3.10
-4.05
ΔGbind
-18.49
-26.88
Thymol acetate
-14.11
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Chemical profile of Lippia thymoides
Fig 6. Binding free energy decomposition for each residue that interact with (A) thymol and (B) thymol acetate. The bars
represent the energy contribution values: van der Waals contributions (blank bars), electrostatic contributions (striped bars) and total
energy contribution for each residue (black bars). Residues highlighted in red are from catalytic site, blue are from anionic subsite and
green are from oxyanion hole.
https://doi.org/10.1371/journal.pone.0213393.g006
AChE binding pocket and the electrostatic and van der Waals energy contributions were
found to be significant in the interactions with residues Ser203 and His447 (catalytic triad),
thus stabilizing ligand binding. Thymol acetate also formed important interactions with resi-
dues from two other sites, Trp86 and Tyr133 (anionic subsite) and Gly121 (oxyanion hole).
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Chemical profile of Lippia thymoides
Conclusions
Thymol is the main constituent in the essential oil from the flowers (37.86% DFL; 48.04%
FFL), leaves (58.9% DLE; 66.33% FLE), and branches (63.59% FB; 66.2% DB) of L. thymoides,
and the essential oil of the fresh leaves had the highest content of this oxygenated monoter-
pene. In the roots, hexadecanoic acid (palmitic acid) was the main constituent (38.02% DR;
40.92% DF) of the oil. The essential oil of the fresh leaves (DL = 0.1 ng/spot) and two of its
mains components, thymol (DL = 0.01 ng/spot) and thymol acetate (DL = 0.01 ng/spot),
showed inhibitory activity against acetylcholinesterase on TLC layers.
The MolDock scores obtained for thymol and thymol acetate were favorable. The poses
obtained from molecular docking simulations showed that the ligands forms H-bond interac-
tions with the Ser203 residue, which belongs to the catalytic triad. The RMSD values obtained
over 100 ns of MD simulation showed that the ligands are stable in the AChE binding pocket.
The binding free energies for thymol (ΔGbind = -18.49 kcal/mol) and thymol acetate (ΔGbind
-26.88 kcal/mol) indicate the ligands are stable and bind with affinity to AChE. The per-resi-
due binding free energy decomposition revealed that the ligands form interactions with resi-
dues that are important for catalytic activity. Some residues belonging to the catalytic triad
(Ser203 and His447), anionic subsite (Trp86, Tyr333, and Tyr337), and oxyanion hole (Gly121
and Gly122) form intermolecular interactions that stabilize ligand binding.
Supporting information
S1 Fig. ion chromatogram relative to the essential oil composition of leaves and flowers of
Lippia thymoides.
(DOCX)
S2 Fig. ion chromatogram relative to the essential oil composition of branches and roots of
Lippia thymoides.
(DOCX)
Author Contributions
´
Conceptualization: Sebastião Gomes Silva, Renato Araujo da Costa, Davi do Socorro Barros
Brasil, Raul Nunes de Carvalho Junior, Eloisa Helena de Aguiar Andrade.
´
Formal analysis: Renato Araujo da Costa.
Funding acquisition: Raul Nunes de Carvalho Junior, Eloisa Helena de Aguiar Andrade.
Investigation: Mozaniel Santana de Oliveira, Pablo Luis B. Figueiredo, Lidiane Diniz Nasci-
mento, Eloisa Helena de Aguiar Andrade.
Methodology: Pablo Luis B. Figueiredo, Raul Nunes de Carvalho Junior, Eloisa Helena de
Aguiar Andrade.
Project administration: Raul Nunes de Carvalho Junior, Eloisa Helena de Aguiar Andrade.
ˆ
Resources: Antonio Maia de Jesus Chaves Neto.
ˆ
Software: Antonio Maia de Jesus Chaves Neto.
Validation: Pablo Luis B. Figueiredo.
Visualization: Mozaniel Santana de Oliveira, Jorddy Neves da Cruz, Pablo Luis B. Figueiredo,
Raul Nunes de Carvalho Junior.
Writing – original draft: Lidiane Diniz Nascimento.
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Chemical profile of Lippia thymoides
Writing – review & editing: Raul Nunes de Carvalho Junior, Eloisa Helena de Aguiar
Andrade.
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