Molecular Docking Terpinen-4-ol From Zingiber cassumunar Roxb.

as Anti-Inflammatory
in Atherosclerosis in Silico
Widya Safitri

Faculty of Mathematics and Natural Sciences, Departement of Biology, Hasanuddin University

Perintis Kemerdekaan KM. 10 street, Tamalanrea Indah, Tamalanrea, Makassar, Sulawesi Selatan, Indonesia
(safitriw716@gmail.com)

ABSTRACT

Atherosclerosis is a chronic inflammatory disease that begins with endothelial dysfunction, it caused fat
accumulation and plaque growth in the inner arteries walls. Endothelial dysfunction will activate the Mitogen
Activated Protein Kinase (MAPK) pathway involving ERK1, ERK2, JNK1, JNK2, and p38MAPK proteins, as
well as the Nuclear Factor Kappa B (NF-kB) pathway involving IKK proteins. Terpinen-4-ol is a constituent
found in the rhizome of bangle (Zingiber cassumunar Roxb.) which is known to have anti-inflammatory activity.
The purpose of this study was to determine the affinity and mechanism of terpinen-4-ol for MAPK10 protein as
an anti-inflammatory in atherosclerosis using molecular docking method. The research was carried out in an
exploratory manner with the stages of preparing the 3D structure of terpinen-4-ol, preparation of the 3D structure
of Mitogen-activated protein kinase 10, validation of the molecular docking method, and docking of terpinen-4-
ol on the protein. The results showed that the binding affinity of Mitogen-activated protein kinase 10 with the
active compound Terpinen-4-ol was -5.4 kcal/mol while the binding affinity of Atorvastatin as the control
compound with Mitogen-activated protein kinase 10 was -6.9 kcal/mole. By looking at the binding affinity, it
can be concluded that Atorvastatin, which is a chemical compound used in the treatment of atherosclerotic
inflammation, is 15% better than the active compound Terpinen-4-ol from (Zingiber cassumunar Roxb.). Based
on the Drug-likeness Test, the compound Terpinen-4-ol complies with Lipinski's rules. In addition, based on the
prediction results of ADMET, Terpinen-4-ol was found to be non-toxic and could be used as a medicinal
ingredient. Therefore, it can be concluded that Terpinen-4-ol from the rhizome of bangle (Zingiber cassumunar
Roxb.) has the potential as an alternative treatment for atherosclerosis inflammatory therapy.

Keywords : atherosclerosis, terpinen-4-ol, molecular docking, in silico

INTRODUCTION molecules include intercellular adhesion

Atherosclerosis is a disease caused by a
chronic inflammatory response in the
endothelium, which can lead to the formation
and thickening of plaques on the walls of blood
vessels (Warboys, 2011). Endothelial
dysfunction is a major factor involved in
plaque growth and atherosclerosis.
Dyslipidemia is one of the causes of
endothelial dysfunction that causes increased
permeability of endothelial cells, allowing the
accumulation of low-density lipoprotein (LDL)
in blood vessel walls (Douglas et al., 2010).
The longer accumulation of LDL in the blood
vessel wall tissue facilitates the modification of
LDL which results in the formation of ox-LDL.
LDL oxidation is caused by the presence
of free radicals that react with LDL and high
LDL levels in the blood. The emergence of
LDL-oxidation stimulates vascular
dysfunction, leading to the expression of
monocyte chemoathractant protein-1 (MCP-1),
macrophage colony-stimulating factor,
interferon-γ, interleukin (IL)-1, IL-6, and
tumor necrosis factor (TNF). -α) and adhesion
molecule (ICAM-1), vascular adhesion molecule
(VCAM-1), E-selectin, and P-selectin which
cause adhesion and infiltration of monocytes
and T lymphocytes to the intima area. Cytokines
will trigger monocyte differentiation into
macrophages and phagocytize LDL-oxidation so
as to form foam cells that contribute to the
occurrence of atherosclerotic plaques (Francis
and Pierce, 2011; Warboys, 2011). MAPKs are
central messenger molecules including
Extracellular Signal-Regulated Kinase (ERK
1/2), c-jun N-terminal kinase (JNK), and
p38MAPK. MAPKs pathway plays a role in
mediating intracellular signals related to cellular
activities including apoptosis, proliferation,
differentiation, and inflammation. Activation of
the MAPKs pathway is caused by the
phosphorylation of various downstream
substrates including transcription factors,
enzymes, and other kinases.
Treatment using natural ingredients is one
alternative for the treatment of atherosclerosis.
Bangle (Zingiber cassumunar Roxb.) is a plant
that contains phenylbutanoids, curcumin,
flavonoids, alkaloids, saponins, tannins, steroids,
 and terpenoids (Rachmadenawanti et al.,
2016). Terpinen-4-ol is one of the terpenoid
groups which is the main constituent contained
in the bangle rhizome (Zingiber cassumunar)
with a content reaching 30-35% of the total
compounds contained in the bangle rhizome
(Bhuiyan, 2018). In vitro test results showed
that Zingiber cassumunar ethanol extract could
inhibit COX and matrix metalloproteinase
MMP-2 production by blocking
proinflammatory signaling pathways involving
ERK 1/2, JNK, and p38 in the MAPK pathway
(Koontongkaew et al., 2013).
To determine the activity of terpinen-4-ol
as an anti-inflammatory in atherosclerosis, it
is necessary to conduct a preliminary test
using in silico molecular docking method. In
silico molecular docking technique can
predict the interaction between a protein and a
ligand molecularly, so that the activity of
bioactive compounds is known. This method
can increase efficiency and effectiveness in
new drug discovery research. Therefore, it is
important to conduct this study to determine
the anti-atherosclerotic activity of terpinen-4-
ol from the rhizome of bangle (Zingiber
cassumunar) in silico.
MATERIAL AND METHOD
Material
The material used is the MAPK 10
protein structure (PDB ID: 3OY1)
downloaded from the protein data bank
website (http://www.rcsb.org/pdb) and the 3-
dimensional (3D) structure of terpinen-4-ol
and Atorvastatin (as comparison ligands)
taken from the PubChem compound database
(https://pubchem.ncbi.nlm.nih.gov/) prepared
using the PyMOL v1.7.4.5 program.
Tools
The tool used in this research is a set of
computers with Windows 7 specifications (64
bit) equipped with PyRx 0.8 and PyMOL
v1.7.4 programs.
METHOD
Preparation of Terpinene-4-ol
The chemical structure terpinen-4-ol of
identified from Zingiber cassumunar Roxb. were
retrieved from PubChem compound database
(https://pubchem.ncbi.nlm.nih.gov/).The targeted
compounds were downloaded and saved in sdf
format. The ligand molecule was added to the
PyMOL v1.7.4.5 software (O’Boyle et al., 2011)
and converted to pdb format.
Target Protein Preparation
Potential target protein candidate for
molecular docking was validated using 3 data
banks, Pharmmapper (http://lilab.ecust.edu.cn),
SuperPred (http://prediction.charite.de), and also
STP (www.swisstargetprediction.ch) The target
compound was then searched using Uniprot
(https://www.uniprot.org) and then PDB 3OY1
was obtained. The code obtained was downloaded
on the Protein Data Bank website
(https://www.rcsb.org/) and then it was visualized
using the PyMOL v1.7.4.5 application, then saved
in pdb format.
Molecular Docking
To see the target protein docking with
terpinen-4-ol and atorvastatin, it can be done by
molecular docking using PyRx 0.8 software. The
reverse docking process is carried out using the
Vina Wizard feature integrated in PyRx 0.8.
Through this feature, the results of the docking of
the test compound show the binding affinity of the
active compound and target protein.
Data Analysis
The data were analyzed based on the binding
affinity from molecular docking. That is indicates
the strength of the affinity between the test
compound and target protein. The interaction
between MAPK10 target protein with Terpinen-4-
ol (active compound ligand) and atorvastatin
(control compound) was visualized in 3D and
analyzed using PyMOL v1.7.4.5 software.
Drug-likeness and ADME/TOX Test
Drug-likeness test of a compound is carried
out at SwissADME which is accessed at
(http://www.swissadme.ch.) Lipinski's rule can
determine the physicochemical properties of
ligandsto determine the hydrophobic/hydrophilic
character of a compound to pass through cell
membranes. Meanwhile, evaluation of the
properties of ADME/TOX was carried out to
determine which compounds could be used as
drug candidates by looking at carcinogenic
properties. ADME/TOX tests can be performed
by accessing the AdmetSAR website at
(http://lmdd.ecust.edu.cn/admetsar1.)
RESULT AND DISCUSSION
Preparation of Terpinene-4-ol
Terpinen-4-ol which is one of the anti-
inflammatory compounds was used and
atorvastatin as a control compound. Terpinen-4-ol
structure was drawn and optimized using PyMOL
v1.7.4.5program

(a) (b)
(c) (d)
Figure 1. Visualization 3D structure of compounds
Information:
a) Terpinen-4-ol 3D structure from Pubchem
b) Atorvastatin 3D structure from Pubhem
c) Terpinen-4-ol structure from PyMOL
d) Atorvastatin 3D structure from PyMOL
Target Protein Preparation
Potential target protein candidate for
molecular docking was validated using 3 data
banks, there are pharmmapper, SuperPred,and
Swiss Target Prediction, The results of the 3 data
banks are:
Table 1. Target Prediction Result
Pharmmapper SuP STP
MAPK10 - MAPK10
- - Adrenergic
receptor
alpha-2
Growth factor - -
receptorbound
protein 2
Based on data, the target protein is Mitogen
activated protein kinase 10 (MAPK 10) with PDB
code 3OY1. MAPK10 protein was then prepared
by separating the native ligand from the protein
structure using PyMOL v1.7.4.5 software to
produce a protein structure without its native
ligand. The separation of the native ligand from the
protein structure aims to provide a pocket that will
be used as a space where terpinen-4-ol binds to the
target protein.
In the protein preparation process, the
removal of water molecules (H2O) around the
protein structure is also carried out. It is intended
that water molecules do not interfere with the
docking process, so that it can be ascertained that
only ligands interact with proteins (Tjahyono and
Hamzah, 2013). The prepared native ligands and
proteins are then saved in pbd file format.
(a)
(b)
Figure 2. 3D Visualization structure of target
protein
Information:
a) MAPK10 structure (with water molecule)
b) MAPK10 structure (without water molecule
Molecular Docking
molecular docking is identified by redocking
the native ligand on the target protein using the
PyRx 0.8 software program. In the validation of the
molecular docking method, a grid box arrangement
is made which will become a space for the native
ligand to form a conformation when docked with the
target protein. The grid box is a place where the
ligand will interact with the amino acid residues on
the target protein. Determination of the grid box is
done to determine the coordinates of the binding
site. of a protein. The grid box settings are setting
the coordinates of the grid center and setting the
grid size (Rachmania et al., 2016).
The validation parameter in molecular
docking is the Root Mean Square Deviation
(RMSD) value. RMSD shows the comparison of the
native ligand conformation from docking with the
native ligand conformation from crystallographic
measurements (Saputri et al., 2016). The limit of the
acceptable RMSD value is 3Ǻ (Jain and Nicholls,
2008). Based on the RMSD value, the method used
can be said to be valid so that the terpinen-4-ol
docking process can be carried out.
 In addition, a similar process was also carried activated protein kinase 10 (MAPK10) according to
out for the control compound, namely Atorvastatin, the in silico results although it is 15% less effective
to compare its binding affinity. Binding affinity is compared to synthetic drugs on the market,
an important aspect that must be considered in the Atorvastatin.
interaction of ligands and receptors. A lower
binding affinity indicates that a compound requires (a)
less energy to bind or interact with the receptor. In
other words, lower binding affinity values have a
greater potential to interact with target proteins
(Muttaqin, 2019).

(b)

Figure 5. 3D Visualization of MAPK10 docking

results with compounds using PyMOL v1.7.4.5

Figure 3. Docking Results

Information:
a) MAPK10 docking with Terpinen-4-ol compounds
b) MAPK10 docking with Atorvastatin compounds
Table 2. Molecular Docking Result
origin Compound Binding Drug-likeness and ADME/TOX Test
Affinity Drug-Likeness Test is an initial test of drug
(kkal/mol) candidates by looking at the characteristics of drug
Zingiber Terpinen-4-ol -5,4 chemicals. This test uses Lipinski's 5 rules as a
cassumunar basis for looking at the safe range of drugs to select
drug candidates (Jia et al., 2020). Lipinski's rule can
Roxb.
determine the physicochemical properties of
synthetic Atorvastatin -6,9 ligandsto determine the hydrophobic /hydrophilic
character of a compound to pass through cell
membranes.
Data Analysis
Table 3. Drug-Likeness Test with Lipinski’s Rules
Binding affinity analysis was carried out to
determine the spontaneous reaction of the
compound and the stability of the ligand-receptor
interaction. The stability of the interaction is
proportional to the potential of the compound to
bind chemically strongly (Adelina, 2016). The
results of the docking of the test compound as
ADME/TOX is a test to see the nature of
showed in Table 2. The test ligand had ΔG = -5,4,
absorption, distribution, metabolism, elimination or
tended to be larger than the control ligand, which
excretion, and toxicity of drug candidates to
was -6,9. The result aslo visualized in Figure 4 for
determine which compounds could be used as drug
3D Visualization. Based on the molecular docking
using PyRx, Terpinen-4-ol compounds have an candidates by looking at carcinogenic properties.
effectiveness as an inhibitor of the Mitogen
 Table 3. Drug-Likeness Test with Lipinski’s Rules
Table 4. Drug-Likeness Test with Lipinski’s Rules
Based on Table 3 and Table 4, Terpinen-4-ol
is known to be non-toxic, safe for consumption by
the body, and effective/suitable as a consumption
drug. therefore, Terpinen-4-ol still has enormous
potential to be a candidate for a new Mitogen-
activated protein kinase 10 inhibitor drug in the
treatment of atherosclerotic inflammation.
CONCLUSIONS
Based on Molecular Docking carried out with
PyRx software and visualization with Pymol
software, the binding affinity of Mitogen-activated
protein kinase 10 with the active compound
Terpinen-4-ol was -5.4 kcal/mol while the binding
affinity of Atorvastatin as a control compound
with Mitogen-activated protein kinase 10 was -6.9
kcal/mol. Therefore, it can be concluded that
Terpinen-4-ol has potential as an anti-
inflammatory in atherosclerosis molecularly
because it has affinity with MAPK10 protein.
however, Terpinen-4-ol was still 15% less good
than Atorvastatin in inhibiting this target protein.
In long-term use, of course the use of the
active compound Terpinen-4-ol from Zingiber
cassumunar is more friendly (less side effects) than
the use of the chemical compound Atorvastatin
which if consumed continuously can have serious
side effects so that Zingiber cassumunar Roxb.
Very good as an alternative in overcoming the
problem of inflammation in atherosclerosis.
Testing Terpinen-4-ol to be used as a therapy
for atherosclerosis inflammation, Drug-likeness
Test and Prediction of ADME/TOX were carried
out. Based on the results obtained, the test ligand
Terpinen-4-ol is known to have no toxicity, is safe
for consumption by the body, and is
effective/suitable for use as a drug consumption.
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