Study in Silico Red Betel (Piper crocatum) as Candidate of Anti-Inflammatory Drug on Mice

(Mus musculus) Rheumatoid Arthritis Model

Siti Imroatul Maslikah1, Sri Rahayu Lestari1, Nursasi Handayani1, Alif Rofiqotun Nafiah2
1
Lecturer Department of Biology, Faculty of Mathematics and Natural Sciences, State University of
Malang,
2
Student Departement of Biology, State University of Malang,

email: siti.imroatul.fmipa@um.ac.id

Abstract
Rheumatoid Arthritis is a systemic autoimmune disease that causes inflammation of the
synovial tissue and ends in joint damage. RA is due to the production of proinflammatory
cytokines in the expansion of NF-kB (Nuclear factor-kappa B) expression. Apigenin,
Luteolin, Hydroxychavicol, Eugenol, Chavibetol, are active compounds contained in red
betel leaves (Piper crocatum). The compound can be predicted as a therapeutic drug in
mouse RA model by inhibiting NF-kB expression. The 3D structure of active compound of
red betel leaves is obtained from PubChem web server, while NF-kB is obtained from
Uniprot. Prediction of virtual screening through molecular docking method and compared
with control drugs, namely aspirin. Of the five active compounds of red betel, Apigenin
and Luteolin have smaller bond affinity values, -6.3 kcal / mol and -6 kcal / mol than with
aspirin control medications. Red betel active compound can be used as alternative
candidate of Rheumatoid Arthritis therapy drug.

Keywords: in silico, red betel, apigenin, rhematoid arthritis, active compound betel red

Background
Rheatoid Arthritis (RA) is a chronic disease characterized by inflammation of the synovial tissue and
can cause bone and cartilage tissue damage (Tsubaki et al., 2015). Several studies have shown the
cause of RA remains uncertain, but genetic abnormalities and environmental factors may trigger
inflammatory processes (Okada et al., 2014). The prevalence of RA in Southeast Asia is 0.4%, the
Eastern Mediterranean is 0.37%, the European area is 0.62%, the American area is 1.25%, and 0.42%
in the Western Pacific, exposed to RA (Rudan et al., 2015). Families with positive RA have a risk of RA
up to 40-60% (Smolen et al., 2016). The inflammation caused by RA is due to the expression of
nuclear factor-kB (7). NF-kB acts as an inflammatory response regulator, in addition it can induce
some proinflammatory genes in innate immune cells (Oeckinghaus and Ghosh, 2009). NF-kB also
regulates on activation, differentiation, and effector function in inflammatory T cells. This underlies
the inflammatory disease as a result of NF-kB activation. Therefore, treatment of inflammatory
diseases should have a target to suppress NF-kB activation (Liu et al., 2017). Alternative treatments
that many people use to suppress NF-kB expression include red betel (Piper crocatum) (Salminen et
al., 2018).
Red betel is a tropical plant that is widely grown in Indonesia and widely used as a traditional
medicine. Methanol extract 96% red betel leaf contains flavonoids, saponins, triterpenoids, and
tannins. The flavonoid species in red betel are flavonone, isoflavone, auron, catechin, anthocyanidin,
and kalkone (Lister et, al., 2014). Red betel contains flavonoids also contain biofenolics such as
hydroxychavicol, eugenol, khavibetol, and piperol, some of them as antimutagenic (Chang et al.,
2002; Gundala and Aneja et al., 2014). Epigenin compound is the main compound of red betel leaf
ethanol extract (Maslikah et al., 2016). In this study aims to predict the active compound in red betel
to suppress the expression of NF-kB in mouse RA model.
Method
a. Ligand and Preparation Data
The three-dimensional structure of the red betel active compound, Apigenin (CID 5280443), Luteolin
(CID 5280445), Hydroxychavicol (CID 70775), Eugenol (CID 3314), Chavibetol (CID 596375), and
Aspirin (CID 2244) obtained from PubChem database in .sdf format.
b. Protein Data and Preparation
The complex three-dimensional structure with NF-kappaB-DNA dimer DNA is taken from Protein
Data Bank (GDP Code: 1NFK) with a selection of mice organisms. The water molecule is removed and
the addition of hydrogen atoms to proteins using PyMOL software.
c. Docking Simulation
The identification of apigenin compounds potentially as anti-inflammatory drug candidates in RA
mice was performed using the Autodock Vina program in the PyRx 0.8 software. The grid on the
coordinate box is set on the active side of NF-kB (x = -1,195 Å; y = 9,0149 Å; z = 19,759 Å) which has
hydrogen bond residues in Agr54, Agr56, Tyr57, Cys59, Lys241, Gln306 and Thr143 of hydrogen bond
with DNA. The results obtained based on the docking process of the affinity value of binding of
compounds or ligands with NF-kB protein. Docking process in addition to using red betel active
compounds used Aspirin as a control drug. Binding affinity values are obtained from ligand-protein
complex values at the lowest free-bond energy (ΔG).

Results and Discussion

Predicted inhibition with active compound of red betel and didoking on NF-kB complex. The results
of the dosage of free energy values are shown in Table 1. Apigenin binds to 1NFK with ΔG of -6.3 kcal
/ mol and has a hydrogen bond with a side chain Lys-144, Tyr-57, and Arg-54 (Fig. 1A) . Chavibetol
binds to 1NFK with ΔG of -4.7 kcal / mol and has a hydrogen bond with a Glu-60 and Arg-54 side
chain (Fig. 1B). Eugenol binds to 1NFK with ΔG of -4.7 kcal / mol and has a hydrogen bond with a Glu-
60 and Arg-54 side chain (Fig. 1C). Hydroxychavicol binds to 1NFK with ΔG of -4.8 kcal / mol and has
a hydrogen bond with the Arg-54 and His-141 side chains (Fig. 1D). Luteolin binds to 1NFK with ΔG of
-4.7 kcal / mol and has a hydrogen bond with Asp-239, His-141, Arg-54, and Glu-60 side chains
(Figure 1 E), while aspirin (control) binds to 1NFK with ΔG of -4.7 kcal / mol and has a hydrogen bond
with a Thr-143, Cys-59, and Val-58 side chain (Fig. 1F).
The red betel active compound forms most of the polar bonds with Lys-144, Tyr-57, Arg-54, Thr-143,
Cys-59, Val-58, Glu-60, Lys-241, His-141, Asp-239, which form a hydrogen bond with the DNA
cosensus sequence on the kB side of NF-kB. The red betel active compound may interfere with the
NF-kB bond on the kB side with Lys-144 and Lys-241 on the NF-kB complex (Jutooru, et al, 2010).
Among the five active compounds of red betel (Apigenin, Chavibetol, eugenol, Hydroxychavicol, and
Luteolin) and are predicted to show inhibition after didoking with NF-kB which shows the best bond
with the lowest free-bonded energy values, Apigenin, Luteolin, Hydroxychavicol, Chavibeto, and
Eugenol.
The physicochemical properties of red betel active compounds are predicted using the
Molinspiration Property Calculator webserver shown in Table 2. The physicochemical prediction
aims to estimate the resemblance to the drug in terms of inhibition. According to Kumar and Bora
(2012) there are some things to note in order to estimate the resemblance of the drug, ie the LogP
of the compound should be less than 5, the hydrogen bond donor must be less than 5, the H bond
acceptor less than 10 and the molecular weight should be less than 500. Membrane permeability
cells in a molecule must have a topological polar surface area (TPSA) of less than 140 Å2. The
inhibitor molecules of red betel all have values less than the maximum value, it can be predicted that
they have good cell membrane permeability. The three-dimensional visualization of the NF-kB
complex (1NFK) with the red betel active compound attached to the Hydrogen bond is described in
Figure 1 below.
 (A) (B) (C)

(D) (E) (F)

Figure 1. Three-dimensional visualization of NF-κB complex (1NFK) with red betel active compound
bonded with hydrogen bond. (A) Apigenin with Lys-144, Tyr-57, Arg-54, (B) Chavibetol with Glu-60
and Arg-54, (C) Eugenol with Lys-241, (D) Hydroxychavicol with Arg-54 and His- 141, (E) Luteolin with
Asp-239, His-141, Arg-54, and Glu-60, (F) Aspirin with Thr-143, Cys-59, and Val-58.

Table 1. Free Energy Association ΔG in red betel active compounds after tethered with NF-kB.
No Compound ΔG (kcal / mol)
1 Apigenin -6,3
2 Eugenol -4,1
3 Chavibetol -4,7
4 Hydroxychavicol -4,8
5 Luteolin -6
6 Aspirin (Kontrol) -4,7

Table 2. Prediction of Physicalochemical Properties of Red Better Compound Compound

Inhibitor LogP TPSA Molecular Aceptor H Donor H Violation
(Å2 —) weight bond bond (Lipinski
rule)
Apigenin 2.46 90.89 270.24 5 3 0
Eugenol 2.10 29.46 164.20 2 1 0
Chavibetol 2.10 29.46 164.20 2 1 0
Hydroxychavicol 1.79 40.46 150.18 2 2 0
Luteolin 1.97 111.12 286.24 6 4 0
Aspirin (Kontrol) 1.43 63.60 180.16 4 1 0
Conclusion
Today's in-silico research provides much insight into the inhibition of NF-kB by red betel active
compounds. Residues Lys-144, Asp-239, and Lys-241 play an important role in the binding of red
betel to NF-kB. Red betel active compounds (Apigenin, Chavibetol, eugenol, Hydroxychavicol, and
Luteolin) can be predicted to be NF-kB inhibitors for Rhematoid Arthritis treatment therapy. This can
be supported by physicochemical properties that show good results for drug similarities.

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