In Silico Study of Natural Compound on "Wedang Uwuh" as a COVID-19 Therapeutic Agent

Abstract

In 2019, the coronavirus disease (COVID-19), which started in Wuhan, China, spread throughout the world. COVID-19 is a disease that infects the human respiratory system. It has developed more widely and is reported to have mutated into several variants, one of which is Omicron. Until now, there is no specific antiviral drug compound used to cure COVID-19. Therefore, several drugs were used to treat COVID-19 (such as interferon, antiviral, or antibiotics) without evidence of efficacy in humans. Therefore, several drugs are used to treat COVID-19, such as antiviral drugs, antibiotics, and interferons, without evidence of efficacy in humans. However, the use of this drug is limited due to side effects. Based on that fact, the use of natural products as therapeutic agents found in nature can be explored for the treatment of COVID-19. Formulating various herbs with the ingredient rosella (Hibiscus sabdariffa), sappan wood (Caesalpinia sappan), ginger (Zingiber officinale), and honey is one traditional drink from Indonesia. These ingredients contain a lot of natural products that are potent to be drug compounds, such as rosmarinic acid (1) from rosella, brazilin (2) in sappan wood, 10-gingerol (3) in ginger, and kaempferol (4) in honey has been explored their potency to be COVID-19 therapeutic agent. The docking method has been validated by redocking the native ligand to Mpro of COVID-19, omicron variant, (PDB ID 7TOB) as a receptor protein. Compound 4 (ΔG =-8.2 kcal mol-1) has more negative binding energy than native ligan (ΔG =-8.1 kcal mol-1) of 7TOB. Instead, the binding energy of compounds 1, 2, and 3 are-7.6 kcal mol-1, 7.4 kcal mol-1, and-5.5 kcal mol-1. Based on the analysis of binding energy and binding similarity of active sites, the compound in traditional drinks from Indonesia is expected to be a potential alternative to a therapeutic agent for COVID-19.

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RESEARCH ARTICLE | FE BR UA RY 06 2024
In silico study of natural compound on “Wedang Uwuh” as a
COVID-19 therapeutic agent 
Mokhamat Ariefin ; Rizki Rachmad Saputra; Indah Nur Pramesti
AIP Conf. Proc. 3055, 040001 (2024)
https://doi.org/10.1063/5.0193646
08 February 2024 01:16:25

In Silico Study of Natural Compound on “Wedang Uwuh”
as a COVID-19 Therapeutic Agent
Mokhamat Ariefin1,a), Rizki Rachmad Saputra1,b) and Indah Nur Pramesti2,c)
1Department of Chemistry, Faculty of Mathematic and Natural Sciences, University of Palangka Raya, Palangka
Raya, Central Kalimantan, Indonesia
2Department of Chemistry, Faculty of Mathematic and Natural Sciences, State Univesity of Malang, Malang,
Indonesia
a) Corresponding author: mokhamatariefin@mipa.upr.ac.id
b)rizkirachmads@mipa.upr.ac.id
c)indahnur.pramesti.fmipa@um.ac.id
Abstract. In 2019, the coronavirus disease (COVID-19), which started in Wuhan, China, spread throughout the world.
COVID-19 is a disease that infects the human respiratory system. It has developed more widely and is reported to have
mutated into several variants, one of which is Omicron. Until now, there is no specific antiviral drug compound used to
cure COVID-19. Therefore, several drugs were used to treat COVID-19 (such as interferon, antiviral, or antibiotics) without
evidence of efficacy in humans. Therefore, several drugs are used to treat COVID-19, such as antiviral drugs, antibiotics,
and interferons, without evidence of efficacy in humans. However, the use of this drug is limited due to side effects. Based
on that fact, the use of natural products as therapeutic agents found in nature can be explored for the treatment of COVID-
19. Formulating various herbs with the ingredient rosella (Hibiscus sabdariffa), sappan wood (Caesalpinia sappan), ginger
(Zingiber officinale), and honey is one traditional drink from Indonesia. These ingredients contain a lot of natural products
that are potent to be drug compounds, such as rosmarinic acid (1) from rosella, brazilin (2) in sappan wood, 10-gingerol
(3) in ginger, and kaempferol (4) in honey has been explored their potency to be COVID-19 therapeutic agent. The docking
method has been validated by redocking the native ligand to Mpro of COVID-19, omicron variant, (PDB ID 7TOB) as a
receptor protein. Compound 4 (ΔG = -8.2 kcal mol-1) has more negative binding energy than native ligan (ΔG = -8.1 kcal
mol-1) of 7TOB. Instead, the binding energy of compounds 1, 2, and 3 are -7.6 kcal mol-1, 7.4 kcal mol-1, and -5.5 kcal
mol-1. Based on the analysis of binding energy and binding similarity of active sites, the compound in traditional drinks
from Indonesia is expected to be a potential alternative to a therapeutic agent for COVID-19.
INTRODUCTION
Coronaviruses are viruses belonging to Coronaviridae virus that are capable to infect the respiratory system of
humans and other mammals. In 2019, a novel corona virus was named 2019 novel Corona Virus Disease (COVID-
19), which was originally identified in Wuhan, China [1]. A novel disease class, COVID-19, has never been identified
in humans. The virus, named Sars-CoV-2, already spreads quickly and easily infect human. Early in 2020, the World
Health Organization (WHO) announced COVID-19 as a worldwide pandemic epidemic [2]. All nations in the world—
including Indonesia—have been affected by the Covid-19 virus. Based on the WHO Coronavirus Disease (COVID-
19) Dashboard, in Indonesia, the number of infection cases recorded was 6.446.143 cases with around 158.219 death
from January 2020 until October 2022. (WHO Corona Viruses Disease (COVID-19) Dashboard).
COVID-19 is an enveloped RNA virus, positive-sense single-stranded, that is classified as orthocoronavirinae
family. COVID-19 genome analysis disclosed that the structure of the COVID-19 virus consists of a lipid bilayer
composed of three types of protein, i.e., spike protein, envelope protein, membrane protein, and nucleocapsid (N)
protein. In addition, its analysis shows several non-structural proteins of COVID-19 such as Papain Like Protease
International Conference on Organic and Applied Chemistry (ICOAC) 2022
AIP Conf. Proc. 3055, 040001-1–040001-8; https://doi.org/10.1063/5.0193646
Published by AIP Publishing. 978-0-7354-4843-8/$30.00
040001-1
08 February 2024 01:16:25

(PLpro), 3-chymotrypsin-like (3CLpro) or main protease (Mpro), helicase and RNA Polymerase [3, 4]. Over time,
COVID-19 has evolved into several reported variants, such as the alpha, beta, gamma, and delta variants. The latest
variant detected is omicron which was first reported to WHO in November 2021 and detected in South Africa [5]. In
the omicron variant, several mutations were detected, both in structural proteins and Non-Structural Proteins (NSP).
However, 64% of proteins that undergo mutations are spike proteins. Because of that omicron can bind more easily to
the ACE-2 receptor than another variant [6]. The study of the structural protein of the Mpro micron variant showed
that the mutation is not close to the active site [7].
There has been a lot of development in handling COVID-19 such as vaccine development or drug development.
Main protease (Mpro) is an enzyme that plays a role in viral replication. Inhibition of proteases is one of the alternative
targets in treating COVID-19 because protease enzymes have a key role in the early life cycle of the virus. This is
supported by the fact that the antiviral component of PAXLOVIDTM, has been introduced as a therapeutic agent for
inhibition of Mpro COVID-19 [8]. Atmar group [9], also reported the usage of molnupiravir and nirmatlevir/ritonavir
can target polymerase and cause lethal mutase during the replication process of the virus. Nirmatlevir targets COVID-
19 Mpro to inhibit the virus replication combine with ritonavir to delay the metabolism of the virus.
Up to the current moment, few antiviral drugs have been accepted for the treatment of COVID-19, such as
nirmatrelvir, ritonavir, and malnupiravir. However, the use of this antiviral drug allows some users to have side effects,
such as diarrhea, increased blood pressure, or muscle ache [9]. To overcome this situation, it needs an alternative
therapy that utilizes herbal medicine available in nature. Indonesia is a country that has natural wealth in the form of
abundant medicinal plants such as rosella (Hibiscus sabdariffa), cinnamon (Cinnamons verum), cloves (Syzygium
aromaticum), bajakah (Spatholobus littoralis), ginger (Zingiber officinale), etc [10–13]. Therefore, a natural product
from Indonesian herbal medicine in Indonesia for COVID-therapeutic needs to be explored.
Traditional herbal medicines have long been consumed, but not everyone enjoys them. This includes spices with
high antioxidant content. Formulating various herbs with the ingredient’s rosella (Hibiscus sabdariffa), sappanwood
(Caesalpinia sappan), cinnamon (Cinnamomum burmannii), cloves (Syzygium aromaticum) and honey is one way to
enhance the intake of high-antioxidant functional drinks. The selection of this material is anticipated to contribute to
an improvement in sensory features in addition to being an effort at diversification. Obtaining the ideal wedang uwuh
recipe [14]. On the other components in wedang uwuh, the active compounds found in the leaves of Moringa oleifera
contain some compounds such as tannins, flavonoids, and phenol. These compounds can serve as antioxidants and
reduce hyperglycemia and hyperlipidemia [15].
In the previous study, simulation molecular docking was done to analyze the natural product and their derivatives
for their ability to inhibit Mpro of COVID-19. Sumaryada’s Group has conducted docking simulations for some
natural compounds contained in black seed, such as nigellidine. Nigellidine has more negative binding energy than
drug Mpinhibitortor like darunavir and lopinavir [16]. Yuanita, et.al., has been reported xanthone and it's derivative
as an antiviral drug through in silico study for COVID-19. They reported hydroxyxanthone with chloro and sulfonate
as a good alternative to a therapeutic agent for COVID-19 because of their good binding energy [17]. Ferdian, et. al.
[18], also reported the usage of a compound consisting in honey for Mpro COVID-19 inhibitor. They conduct a
docking simulation to Mpro COVID-19 (PDB ID: 6LU7) first variant. In that study, kaempferol gets binding affinity
-7.6 kcal/mol using Autodock Vina. Al-Karmalawy groups have conducted salvianolic acid, rosmaric acid, and
glychyrretinic acid as Mpro (PDB ID: 6LU7) inhibitor and spike protein (PDB ID: 6VW1) inhibitors [19]. However,
the study of kaempferol and rosmarinic acid toward the Mpro COVID-19 Omicron variant is still not available.
Furthermore, wedang uwuh ingredient also consists of sappan wood that contains rich of brazilin and ginger rich of
10-gingerol. Therefore, a study of the interaction of a compound consisting of wedang uwuh with Mpro of COVID-
19 is required to identify the potency of wedang uwuh. In this study, we use 10-gingerol (1) from ginger, brazilin (2)
from sappan wood, rosmarinic acid (3) from rosmary, and kaempferol (4) from honey as ligand test. All the structures
of ligand tests are available in Fig. 1.
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08 February 2024 01:16:25

Figure 1. Strucure of ligand test. (1) 10-gingerol, (2) brazlin, (3) rosmarinic acid, (4) kaempferol.
EXPERIMENTAL SECTION
Hardware and software
ASUS Laptop with processor Intel® Core i5-7200, CPU @2.50 GHz with 8 Gb RAM, windows 10 64-bit, Graphic
processing unit (GPU) an Intel® HD Graphic 620 was used to conduct molecular docking and analysis. The software
was used is AutodockTools 1.5.6 (The Scripps Research Institute), Autodock Vina [20, 21], BIOVIA Discovery
Studio, and PyMol.
Ligand and Macromolecule Preparation
The macromolecule (Mpro) of COVID-19 Omicron Type was downloaded from Protein Data Bank (PDB)
(https://www.rcsb.org/) with PDB ID: 7TOB. This protein already binded with native ligand (GC376) with acceptable
resolution (2.05 Å). The preparation was done using Autodock Tools 1.5.6. to delete native ligand and water molecule
from 7TOB. Polar hydrogen and Kollman Charge were added to protein, then converted to pdbqt format.
Ligand (natural product) 3D structure was downloaded frum PubChem (https://pubchem.ncbi.nlm.nih.gov/). The
ligand is 10-gingerol (CID: 168115), brazilin (CID: 73384), rosmarinic acid (CID: 5281792), and kaempferol (CID:
5280863). Format file of ligand structure was converted into. mol2 using OpenBabel GUI. After that, the ligand was
optiized using Autodock Tools 1.5.6 by adding Gasteiger charge to ligand, set the number of rotatable bonds, and
convert the ligand format file to pdbqt file.
Molecular Docking Methods
The docking performed in this simulation used semiflexible docking which means ligand act as flexible molecules,
but the protein remains a rigid structure. The conformation of the ligand will be changed to get minimum binding
energy with protein [22]. The method used in this study was using specific docking. This docking was simulated in
the area whereas the previous compound bound, in this case, the native ligand GC376. Specific docking is more
accurate than blind docking which uses a whole area of protein as a docking area.
To find the specific area where GC376 bound and for the validated method, the redocking of native ligand was
conducted. The Redocking method is a method to docking GC376 back into its original position in 7TOB. For the
center of the ligand, we used coordinates x = -18.370, y = 0.250, and z = 13.918 with grid box size
40 ×40 ×40
Å.
After that, the root means square deviation (RMSD) was calculated using PyMol. The docking method was valid if
the RMSD value of redocking was below 2 Å [18]. After validating the method, docking simulation was continued to
the ligand.
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08 February 2024 01:16:25

RESULT AND DISCUSSION
Validation results
Based on the validation method, the RMSD value of the redocking is 0.406 Å. The docking method was valid if
the RMSD value of redocking native ligand was below 2 Å. The binding cavity and binding pose of native ligand are
available in Fig. 2. In terms to increase accuracy of methods, the interaction between GC376 and 7TOB was analyzed.
Interaction formed between GC376 got from docking simulation and crystallography is compared. The similarity of
interaction between docking simulation and crystallography indicates that the docking parameter was valid. The
interaction of GC376 and 7TOB is shown in Fig. 3. According to Fig. 3, GC376 can make hydrogen bonding with
Phe140, Gly143, His163, Glu166, and Gln189. Meanwhile, the hydrogen bonding interaction between GC376 and 7TOB
from crystallographers is able to generate hydrogen bonding with Phe140, Cys 145, Glu166, and His164. In this study, this
method is unable to create hydrogen bonding with Cys145 and His164. However, the interaction with Cys145 and His164
was detected as a hydrophobic interaction. The interaction of GC376 by docking simulation and crystallography is
slightly different due to the rigid structure of the protein in this simulation. In the real condition, both ligand and
protein are flexible compounds that the pose of the structure can flexibly adapt to the structure of the ligand.
Figure 2. Binding poses and binding cavity of GC376 after redocking and original position. Yellow structure is original structure
of GC376 and light blue structure is redocking pose.
Figure 3. Binding inteaction of native ligand, GC376. (a) interaction from crystallography study and (b) interaction result from
redocking native ligand.
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08 February 2024 01:16:25

Table 1. Binding interaction GC376 from crystallography and redocking
Compound
Interaction
Hydrogen bond
Hydrophobic bond
Crystallography
Phe140, Cys145, His164,
Glu166
His41, Met49, Tyr54, Leu141,
Asn142, Gly143, Ser144, Cys145,
His163, Met165, His172, Asp187,
Arg188, Gln189.
Redocking
Phe140, Gly146, His163,
Glu166, Gln189
His41, Met49, Tyr54, Leu141,
Asn142, Ser144, Cys145, His164,
Met165, Leu167, Pro168, Asp187,
Arg188, Thr190, Ala191.
Binding Energy Analysis of Docking Simulation
The docking result in this study is available in Table 2. The docking score of all ligands is negative. It indicates
that all ligands can bind with 7TOB, and more negative binding energy mean the ligand can bind protein more stable.
Brazillin and rosmarinic acid have slightly different binding energy than GC376 with values -7.4 kcal mol-1 and 7.2
kcal mol-1 but 10-gingerol has lower binding energy than GC376. These results indicate that brazilin and rosmarinic
acid may have similar stability and ability to bind with 7TOB compared to GC376, but 10-gingerol has the worst
stability and ability to bind with 7TOB compared to another ligand test. Meanwhile, kaempferol binding energy is
slightly higher than the native ligand binding energy. It may indicate that kaempferol can bind with 7TOB more stable
than GC376.
Interaction analysis in this study only focused on hydrogen bond and hydrophobic interaction. Hydrogen bonding
is an important interaction that can influence the energy binding score [23]. The interaction between ligand and 7TOB
is categorized as a hydrogen bonding interaction if it has 2.2 – 2.5 Å. Meanwhile, hydrophobic interaction also has an
important role in the interaction of ligands and protein [24].
The pose of all ligands in binding cavity is available in Fig.4 and the 2-dimensional interaction of all ligands and
7TOB is shown in Fig. 5. Even though they are in the same binding cavity, each ligand will have a binding pose that
is very different from one another, so it will cause a difference in the binding site. According to Fig. 5, GC376 formed
hydrogen bonding with Phe140, Gly146, His163, Glu166, Gln189. However, all ligand tests, only formed hydrogen bonding
with some of that protein residue. 10-gingerol only formed hydrogen bonding with Thr190. This result explains the
reason 10-gingerol has less negative binding energy. Brazilin and rosmarinic acid have slightly less negative than
GC376. Brazillin formed hydrogen bond with Phe140 and Glu166, the same protein residue compared to GC376. It may
reason the energy binding of brazillin has slightly lower than GC376. Rosmarinic acid has energy binding -7.8 kcal
mol-1, slightly lower than the native ligand. It formed hydrogen bond with residue Thr190 and Gln189. Compared to
GC376, Thr190 was detected as a hydrophobic interaction, but rosmarinic acid has a higher similarity than another test
ligand. This result explained the reason rosmarinic acid has slightly lower binding energy compared to GC376.
Kaempferol has most negative than another ligand test and GC376, but this compound only formed two hydrogen
bonds with Leu141 and His163. The reason for this result is it generated stronger hydrogen bond compared to the native
ligand GC376.
Furthermore, the selected compound conducted in this study can exhibits interaction with at least one catalytic
dyad of COVID-19, His41 and Cys145 [25], except brazilin. 10 gingerol can form interaction with His41, rosmarinic
acid with His41, and kaempferol with His41 and Cys145, but brazilin can not form interaction with both of catalytic dyad
residue. The catalytic dyad is important residue to facilitate the clevage of COVID-19 polyprotein [25]. Jin, et. al. in
their report informs the interaction between Mpro inhibitor with some residue in Domain I (Thr24, Thr25, Met49) and
domain II (Phe140, Asn142, His163, Met165, Asp187, and Gln189) can help to lock the inhibitor to the binding cavity [26].
In domain I, 10-gingerol, brazilin, rosmarinic acid, and kaempferol can form binding with Met49. In domain II, the
interaction of ligand test more variatif. 10-gingerol can form binding with Phe140, Asn142, His163, Met165, and Gln189.
Brazilin and rosmarinic acid can form binding with Phe140, Asn142, His163, Met165, Asp187, and Gln189. Kaemferol can
form binding with Phe140, His163, Met165, Asp187, and Gln189. Based on that result compared to Jin, et. al. result of
binding site of Mpro inhibitor, the ligand test may can make good binding with the binding cavity of Mpro COVID-
19, so that compound can inhibit the activity of Mpro COVID-19.
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08 February 2024 01:16:25

Figure 4. binding pose of (a) 10-gingerol, (b) brazlin, (c) rosmarinic acid, (d) kaempferol
Table 2. Binding affinity, binding interacton, and binding similarity of native ligand (GC376) and ligand test
Compound
Binding
affinity
(Kcal mol-1)
Interaction
Binding similarity
Hydrogen bond
Hydrophobic bond
GC376
-8.1
Phe140, Gly146, His163,
Glu166, Gln189
His41, Met49, Tyr54, Leu141,
Asn142, Ser144, Cys145, His164,
Met165, Leu167, Pro168, Asp187,
Arg188, Thr190, Ala191.
100%
10-gingerol
-5.5
Thr190
His41, Met49, Phe140, Leu141,
Asn142, His163, His164, Met165,
Glu166, Pro168, Arg188, Gln189,
Ala191, Gln192
65%
Brazilin
-7.4
Phe140, Glu166
Met49, Leu141, Asn142, Cys145,
Ser144, His164, Met165, His163,
His172, Gln189
60%
Rosmarinic
acid
-7.6
Thr190, Gln189
His41, Met49, Phe140, Leu141,
Asn142, Ser144, His163, His164,
Met165, Glu166, His172, Asp187,
Arg188, Ala191
80%
Kaempferol
-8.2
Leu141, His163
His41, Met49, Pro52, Phe140, Ser144,
Cys145, His164, Met165, Glu166,
Gln189
60%
The binding similarty exhibits the probability of a compound has same inhibitor mechanism with native ligan or
reference ligand. In addition, rosmarinic acid has higher binding similarity than another ligand test. The binding
similarity of compound 1, 2, 3, and 4 is 65%, 60%, 80%, and 60%.
040001-6
08 February 2024 01:16:25

Figure 5. Binding interaction of all ligand test with 7TOB. (a) 10-gingerol, (b) brazilin, (c) rosmarinic acid, and (d) kaempferol
CONCLUSION
Based on the docking simulation, energy binding and bonding interaction, the four natural product from Indonesian
traditional medicine, namely 10-gingerol, rosmarinic acid, and kaempferol are potential to inhibit the replication of
COVID-19, with docking score 7.2 kcal mol-1, 7.5 kcal mol-1, and 8.2 kcal mol-1. Kaempferol has binding energy mre
negative than native ligand GC376. This result indicate kaempferol has most potencial natural product that can act as
alternative drug for COVID-19 therapeutic agent.
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