Molecular Docking Study of Lemon (Citrus limon (Linn) Burm. f)

Flavonoid Derivatives Compound in Receptor Cyclooxygenase-1

(COX-1) as Antiplatelet in Ischaemic Stroke Disease

Rizky Arcinthya Rachmania*, Hariyanti, Nurul Rochmah

Faculty of Pharmacy and Science, Universitas Muhammadiyah Prof. DR. HAMKA (UHAMKA)

Islamic Center, Jl. Delima II/IV Perumnas Klender, Jakarta Timur, 13460, Telp. (021) 8611070

Keywords: Citrus limon, COX-1, flavonoid derivatives, antiplatelet, docking.

Abstract: Lemon (Citrus limon (Linn) Burm. f) is a plant that has efficacy as antiplatelet. Flavonoids in lemon

potentially obstruct COX-1 (Cyclooxygenase-1) receptor which has an important role in increasing

thromboxane A2 in the process of ischaemic stroke. This research aims at looking for flavonoids activity

from citrus lemon which is expected to be the antiplatelet drug candidates. The method used in this research

was the molecular docking method using Autodock Vina and Pymol software programs. The results showed

that the value of ΔG binding affinity Aspirin as the standard ligand was -6,5 kcal/mol and lemon flavonoid

derivatives that have the lowest ΔG binding affinity value was on Neohesperidin -15,4 kcal/mol and Rutin -

15,3 kcal/mol. This research shows that Neohesperidin and Rutin in lemon can be used as drug candidate of

antiplatelet in ischaemic stroke disease.

1 INTRODUCTION

Stroke is the third most common disease after heart

disease and cancer. According to the World Health

Organization (WHO), the definition of stroke is a

rapidly growing clinical sign of focal (or global)

brain dysfunction, with symptoms that last for 24

hours or more, can cause death, with no cause other

than vascular. Stroke is characterized by a sudden

loss of blood circulation to the brain area resulting in

a neurological deficit (Gund et al., 2013). In general,

strokes are classified as ischaemic and hemorrhagic.

Ischaemic stroke is an ischaemic brain tissue arising

from a blockage in the cervical vascular blood

vessels or brain tissue hyperfusion by various factors

such as atherothrombosis, embolism, or

hemodynamic instability (Chung and Caplan, 2007).

Stroke is the third leading cause of death in the

United States and Britain after heart disease and

cancer, and the main cause of adult disability. In the

United States, more than 160,000 adult Americans

die of strokes every year. In Europe, around 650,000

people died from strokes. In the United States,

people who report strokes over 65 years (Gund et

al., 2013). According to the latest data from Riset

Kesehatan Dasar in 2013, the number of stroke

patients in Indonesia in 2013 based on the diagnosis

of Health Workers is estimated to be 1,236,825

people (7.0 ‰), whereas based on diagnosis

symptoms are estimated as many as 2,137,941

people (12.1 ‰) (Balitbang, 2013).

Antiplatelet therapy is an important long-term

treatment for all patients at risk of atherothrombosis

such as ischaemic stroke. A comparison of some

antiplatelet drugs statistically indicated a significant

difference in outcomes (Shinohara et al., 2010).

Strong platelet function inhibitors have been

developed in recent years with different drug-action

mechanisms, because when combined the effects are

additive or even synergistic (Ringleb et al., 2011).

These antiplatelet drugs are classified into several

groups based on their mechanism of action, namely

inhibition of prostaglandin synthesis (aspirin),

inhibition of ADP-induced platelet aggregation

(clopidogrel, prasugrel, ticlopidine), and blockade of

the receptor glycoprotein IIb/IIIa in platelets

(abciximab, tirofiban, and eptifibatide) (Ringleb et

al., 2011). However, these drugs can cause serious

side effects for users such as gastrointestinal

bleeding, leukopenia, and thrombocytopenia

(Ringleb et al., 2011). Because of the side effects of

Rachmania, R., Hariyanti, . and Rochmah, N.

Molecular Docking Study of Lemon (Citrus limon (Linn) Burm. f) Flavonoid Derivatives Compound in Receptor Cyclooxygenase-1 (COX-1) as Antiplatelet in Ischaemic Stroke Disease.

DOI: 10.5220/0008238700190025

In Proceedings of the 1st Muhammadiyah International Conference on Health and Pharmaceutical Development (MICH-PhD 2018), pages 19-25

ISBN: 978-989-758-349-0

these drugs, herbal medicine is also an option for

patients.

Indonesia has a wealth of herbs, one of which

is lemon (Citrus limon (Linn) Burm F.). One of the

compounds in lemon that is thought to be potential

as an antiplatelet is flavonoids. Based on studies that

have been conducted, lemon plants are known to

have activity as an anticoagulant and antiplatelet in

vitro/in vivo (Riaz et al., 2014). Flavonoids are one

type of antioxidant that can inhibit adhesion,

aggregation and platelet secretion (Retnaningsih et

al. 2007). The ability of flavonoids to inhibit platelet

aggregation is caused by the flavonoid inhibiting the

metabolism of arachidonic acid by cyclooxygenase

enzyme, thus reducing the amount of thromboxane

A2 (TXA2) production and platelet aggregate

production causing blood vessel blockage

(Middleton et al., 2000).

Cyclooxygenase (COX) is a functional enzyme

bound to the membrane acting to catalyze two

important stages in the formation of prostanoid,

cyclooxygenation, and peroxidation reaction. The

cyclooxygenation reaction stage is the stage at which

COX conducts a cyclization process and the addition

of two oxygen molecules to arachidonic acid to form

prostaglandin G2 (PGG2). The peroxidation stage is

the reduction stage of PGG2 into an unstable

endoperoxide compound called prostaglandin H2

(PGH2). There are two main isoforms of the

cyclooxygenase enzyme, cyclooxygenase-1 (COX-

1) and cyclooxygenase-2 (COX-2). COX-1 is

expressed continuously and has a function as a

regulator of homeostasis in the function of

protecting the gastric mucosa, maintaining platelet

integrity, and maintaining the function of renal

perfusion. COX-2 plays a role in pathologies such as

inflammation, pain, and cancer (Claria, 2003).

Molecular docking is a device that can be used to

study the interactions that occur from a molecular

complex. Molecular docking helps in studying drug

or ligand interactions with receptors or proteins.

Molecular docking is conducted by identifying the

corresponding active site of the receptor/protein,

obtaining the best geometry of the receptor ligand

and calculating the interaction energy of each

different ligand for designing a more effective

ligand. To perform molecular docking, the first thing

required is a three-dimensional structure of ligand

and receptor. Virtual screening is a computational

technique in the design of new computer-based

drugs (in silico) to identify the structures most likely

to bind to a targeted drug, usually a protein or

enzyme receptor (Mukesh and Rakesh, 2011).

2 MATERIALS AND METHODS

2.1 Materials

The tool used in this research was hardware and

software. Hard performances were equipped with

AMD E1-2100 APU with Radeon ™ HD Graphics

CPU GHz processor, 2GB RAM, and Microsoft

Windows 7 Ultimate 64-bit operating system, 24-

inch Hp® Monitor, and Bolt® modem for internet

access. The software programs were equipped with

the MGL Tools 1.5.6 Package consisting of

Autodock Vina, Autodock Tools, Pymol (DeLano

Scientific LLC.), Discovery Studio 4.5 Client, CLC

Drug Discovery Workbench 2.5, Chem office 2010,

Protein Data Bank (http://www.rcsb.org/pdb).

The material used was the 3D structure of the

platelet receptor that was downloaded from Protein

Data Bank which has formatted .pdb,i.e.

prostaglandin H2 synthase-1 (PDB ID: 1CQE) and

3D structure used was flavonoid derived compound

among others were eriocitrin, hesperidin,

neohesperidin, diosmin, rutin, luteolin, nobiletin,

sinensetin, and tangeritin (Molina et al., 2010).

2.2 Methods

Preparation of Prostaglandin H2 Synthase-1 (COX-

1) structure was conducted by downloading the

COX-1 receptor macromolecule from the Protein

Data Bank from http://www.rcsb.org/pdb formatted

from .pdb website to .pdb. Cavity must be

determined to find the residues in the receptor. The

cavity determination was performed using the

offline CLC Drug Discovery Workbench 2.5

software that was downloaded from

http://www.clcbio.com/products/clc-drug-discovery-

workbench/. Receptor macromolecules were

separated from solvents and ligands or non-standard

residues. The separation of macromolecules from

unnecessary molecules was done using the

Discovery Studio 4.0 program. The result of the

separation was saved in .pdb format. The design of

the ligand structure of the flavonoid derived

compound consists of eriocitrin, hesperidin,

neohesperidin, diosmin, rutin, luteolin, nobiletin,

sinensetin, and tangeritin were downloaded from the

PubChem site (http://pubchem.ncbi.nlm.nih.gov./).

The docking file preparation was conducted by

using Autodock Tools that was optimized by setting

the number of action torsion and converting the

format to .pdbqt. While the receptor preparation was

being conducted by adding hydrogen polar, the grid

MICH-PhD 2018 - 1st Muhammadiyah International Conference on Health and Pharmaceutical Development

box was set to know the position of the binding site

and the format was changed to .pdbqt. This file was

saved in a single folder in the C: drive on the

computer. Molecular Docking Process was

conducted using Autodock Vina. Ligands and

receptor that were already in drive C: copied and

converted in the form of notepad were saved with a

conf.txt name, Autodock Vina was executed with

command prompt program.

Molecular docking analysis was done by looking

at the free energy value of binding docking results,

viewed at the output in log.txt format. The selected

ligand-receptor complex was the complex which has

the lowest free binding energy value for further

analysis. The interaction between receptor and

ligand can be observed in Pymol software

3 RESULTS AND DISCUSSION

The macromolecule that was used as the docking

target was the Cyclooxygenase-1 enzyme (COX-1).

COX-1 was downloaded from the Protein Data

Bank. The PDB ID of COX-1 used was 1CQE

which has been used as a reference in predicting

antiplatelet activity based on previous research (Wu

et al., 2007). 1CQE consisted of 580 amino acid

residues. The 1CQE structure was downloaded from

the RCSB site with the format .pdb.

Cyclooxygenase-1 (COX-1) downloaded from the

RCSB must be cleaned using the offline Discovery

Studio 4.5 software because the receptor on RCSB is

holoprotein, which contains many ligands in the

receptor. The reason for the cleansing was to remove

the original disturbing ligands and water attached to

the receptor to speed up the docking process.

Molecular docking is carried out on the specific

region of the target protein, which is to be a binding

site. The location of this site is based on the ligand

or cofactor position co-crystallized with the structure

of the target protein, or the position of the amino

acids known for the binding position.

To get the cyclooxygenase-1 receptor inhibitory

effect, it must first recognize the residues that form

cavity and pocket in the target (receptor). The cavity

is a substance that is owned by the receptor. The

pocket is a space inside the cavity as access to the

bond between the ligand and the receptor, resulting

in the expected effect. Cavity search was carried out

using the CLC Drug Discovery Workbench 2.5

software located at the site

http://www.clcbio.com/products/clc-drug-discovery-

workbench/ (Glaab 2015). CLC Drug Discovery

Workbench 2.5 is a software managed by CLC Bio

A QIAGEN Company. This software can detect

bindings on receptors through the programs

provided.

Usually, more than one cavity is found in the

target receptor. Therefore, it was necessary to

evaluate the cavity to saw the possibility of the

cavity being a binding site that actually used the

CLC Drug Discovery Workbench 2.5 software. In

the search for COX-1 cavity receptors, many

cavities were detected in the COX-1 receptor regions

(Figure 1). It was necessary to do cavity evaluation

by setting a binding site on the Drug Discovery

Figure1: Results of COX-1 receptor site binding detection (CLC Drug Discovery Software). Description: Areas marked

with green spots are cavity areas. The area in the white circle is the actual cavity as a binding site on the COX-1 receptor.

Molecular Docking Study of Lemon (Citrus limon (Linn) Burm. f) Flavonoid Derivatives Compound in Receptor Cyclooxygenase-1

(COX-1) as Antiplatelet in Ischaemic Stroke Disease

Workbench 2.5 CLC work program. After cavity

evaluation, the COX-1 receptor area that had been

detected by cavity showed a more specific area that

described the actual cavity as a binding site (Figure

1). This area is the place where interactions between

amino acid residues and receptors and ligands will

be used as the grid box area. The selection of cavity

binding pocket by CLC drug design based on the

largest pocket size (Li et al., 2008) and more open

and compact pockets have good properties for drug

binding (Cheng et al., 2007). Ligands, receptor, and

Autodock Vina software was saved in a folder

located on drive C: windows in the vina folder. The

destination was saved in one folder so that the

docking process can be carried out through the

command prompt. The command prompt is a

command line interface based program with written

work orders. At the receptor, the grid box must be

determined according to the results of the cavity

binding pocket from CLC Drug Design which was

set in the offline software of Autodock Tools. The

grid box used for 1CQE in the oriented docking

process was at the center_x coordinate = 28,453;

center_y = 7.271; center_z = 195.072, grid size 56 x

40 x 122Å with a spacing of 0.375Å (Figure 2).

Analysis of results in molecular docking includes

values of ΔG binding affinity and Root Mean Square

Deviation (RMSD). Molecular docking was

conducted to see the complex conformation of the

receptor-the docked ligand with Autodock Vina.

Determination of ligand conformation can be seen

from the results that come out in the command

prompt program which will be selected one of the

best out of nine conformations out of the docking

results by using Autodock Vina. The docking result

was the value of ΔG binding affinity (kcal/mol) for

one ligand. Affinity binding is a docking parameter

using Autodock Vina. The smaller the value of the

ΔG binding affinity, the affinity between the

receptor - the ligand will be higher and

otherwise, the

greater the value of ΔG binding affinity, the affinity

between receptor-ligand complex will be lower

(Rachmania et al., 2015).

Table 1: Results of standard ligand docking (Aspirin),

lemon plant ligand with COX-1 receptor (1CQE) using

Autodock Vina Software.

Ligands

ΔG Binding Affinity

(kcal/mol)

RMSD

(Å)

Aspirin -6.5 0

Neohesperidin -15.4 0

Rutin -15.3 0

Eriocitrin -14.9 0

Hes

eridin -14.7 0

Diosmin -14.1 0

Luteolin -10.0 0

Nobiletin -9.5 0

Sinensetin -9.5 0

Tangeritin -9.5 0

Figure 2: Results of COX-1 receptor site binding detection (CLC Drug Discovery Software And Autodock Tools).

Description: The area in the blue box is the actual cavity as a binding site on the COX-1 receptor.

MICH-PhD 2018 - 1st Muhammadiyah International Conference on Health and Pharmaceutical Development

Based on table 1, it can be seen that of the ten

ligands that were analyzed, the lowest values of ΔG

binding affinity in lemon are neohesperidin -15.4

kcal/mol and rutin -15.3 kcal/mol. The ΔG binding

value of affinity aspirin as a standard ligand is -6.5

kcal/mol. These values suggest that neohesperidin

and rutin ligands have better affinity than aspirin and

have antiplatelet potency. RMSD is the value used to

determine whether the prediction of the bond mode

is successful and important for validating the

docking program with a default value of ≤ 2Å. With

increasing deviations, the greater the error of

predicting the ligand interaction with receptors

(Brooijmans, 2009). The RMSD value obtained

from the docking of each ligand in the best

conformation is 0. This is caused by Vina compared

the value of each conformation with its best

conformation value.

The interaction between the receptor and the

ligand resulted in the distance between the bonds

Table 2: The distance of amino acid bond and residue, ligand functional group between Aspirin, Neohesperidin, and Rutin

with COX-1 Receptor (1CQE) using PyMol Software.

Ligands

The Distance of hydrogen

ond (Å)

Amino Acid Residue

Binding

Functional groups binding

Aspirin 2,8 Cys

-O

3,4 Cys

-O

Neohes

eridin 3,

469

-OH

3,1 Ar

469

-OH

3,5 Gln

-O

3,3 Cys

-OH

3,4 Cys

-OH

3,2 L

468

-OH

Rutin 3,4 L

468

-OH

2,3 Glu

465

-OH

3,0 Glu

465

-OH

2,8 Asp

135

-OH

3,2 Asp

135

-OH

3,2 Gl

-OH

3,4 Gl

-OH

2,6 C

-O

3,2 His

-OH

Figure 3. Residue contact between Neohesperidin and 1CQE receptor (Software Pymol)

Molecular Docking Study of Lemon (Citrus limon (Linn) Burm. f) Flavonoid Derivatives Compound in Receptor Cyclooxygenase-1

(COX-1) as Antiplatelet in Ischaemic Stroke Disease

and the bonded amino acid residues. Hydrogen

bonding is a bond that can occur by involving the

interaction of hydrogen atoms bonded covalently

with electronegative atoms such as Flour (F),

Nitrogen (N), Oxygen (O) (Glowacki et al., 2013).

Table 2 shows distances, amino acids and binding

groups that illustrate the interaction between

ligands/drugs with COX-1 receptors. The interaction

between aspirin as a standard drug against the COX-

1 receptor shows the presence of hydrogen bonds

from the -O group at Cys41 residues. For ligands

having the lowest ΔG binding affinity values,

neohesperidin and rutin, show that hydrogen bonds

are formed from the -O and -OH groups to the

Arg469, Gln44, Cys41, and Lys468 residues in

neohesperidin (Figure 3). The hydrogen bonds are

formed from the -O and -OH groups to the residues

Glu465, Cys41, Gly45, Asp135, Lys468, and His43

on the rutin (Figure 4). Hydrogen bonds may occur

between intermolecular and intramolecular, a good

range of hydrogen bonds at 2.5-3.5 Å (Syahputra et

al., 2014). The hydrogen bond distance that occurs

between amino acids residues in the COX-1 receptor

and neohesperidin and the rutin is a good hydrogen

bond because it is in the range 2.5-3.5 Å. So from

the results of the in silico analysis using molecular

docking method, it can be concluded that

neohesperidin and rutin compounds in lemon are

predicted to have potential as antiplatelet ischaemic

in stroke.

4 CONCLUSIONS

The flavonoid derived compounds found in lemon

(Citrus limon (Linn) Burm f) ie neohesperidin and

rutin result values of ΔG binding affinity (kcal/mol),

i.e. -15.4 kcal/mol and -15.3 kcal/mol. These

numbers are lower compared with aspirin

comparative drugs that has a value of ΔG binding

affinity -6.5 kcal/mol. neohesperidin and rutin

compounds have a better affinity than aspirin so that

it can be used as an antiplatelet candidate in

ischaemic stroke disease.

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Molecular Docking Study of Lemon (Citrus limon (Linn) Burm. f) Flavonoid Derivatives Compound in Receptor Cyclooxygenase-1

(COX-1) as Antiplatelet in Ischaemic Stroke Disease