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Investigation into SARS-CoV‑2 Resistance of Compounds in Garlic
Essential Oil
Bui Thi Phuong Thuy, Tran Thi Ai My, Nguyen Thi Thanh Hai, Le Trung Hieu, Tran Thai Hoa,
Huynh Thi Phuong Loan, Nguyen Thanh Triet, Tran Thi Van Anh, Phan Tu Quy, Pham Van Tat,
Nguyen Van Hue, Duong Tuan Quang,*Nguyen Tien Trung, Vo Thanh Tung, Lam K. Huynh,
and Nguyen Thi Ai Nhung*
Cite This: https://dx.doi.org/10.1021/acsomega.0c00772
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sıSupporting Information
ABSTRACT: Eighteen active substances, including 17 organosulfur
compounds found in garlic essential oil (T), were identified by GC−MS
analysis. For the first time, using the molecular docking technique, we report
the inhibitory effect of the considered compounds on the host receptor
angiotensin-converting enzyme 2 (ACE2) protein in the human body that
leads to a crucial foundation about coronavirus resistance of individual
compounds on the main protease (PDB6LU7) protein of SARS-CoV-2. The
results show that the 17 organosulfur compounds, accounting for 99.4%
contents of the garlic essential oil, have strong interactions with the amino
acids of the ACE2 protein and the main protease PDB6LU7 of SARS-CoV-
2. The strongest anticoronavirus activity is expressed in allyl disulfide and
allyl trisulfide, which account for the highest content in the garlic essential
oil (51.3%). Interestingly, docking results indicate the synergistic interactions of the 17 substances, which exhibit good inhibition of
the ACE2 and PDB6LU7 proteins. The results suggest that the garlic essential oil is a valuable natural antivirus source, which
contributes to preventing the invasion of coronavirus into the human body.
1. INTRODUCTION
Garlic (Allium sativum L.) (cf. Figure 1) is considered as an
important herb thanks to its variety of uses, including either a
common spice for family meals or a popular component in
folk-medicine prescriptions.
1,2
For thousands of years, garlic
has been used as a medication for common colds, influenza,
and other kinds of infections.
3,4
Recent pharmacological
studies indicate that essential oil of garlic is an exceptional
source of organosulfur compounds, possessing strong anti-
oxidant, antibacterial, antifungal, anticancer, and antimicrobial
properties. The oil is also proven to be conducive to
hypoglycemia, hypotension, antithrombotic, immunomodula-
tory, and prebiotic therapy. Besides, allicin is a typical reactive
sulfur species found in the essential oil.
5
Recently, many people have been infected with a novel
coronavirus (SARS-CoV-2), and the death toll has reached
thousands and been increasing day by day, which is a major
problem in the world.
6
Therefore, the demand to seek for
natural and safe medicines to prevent coronavirus is of great
concern for all scientists around the world. With abundant
medicinal resources in Vietnam and the specific healing
properties of garlic,
7
we herein recommend a solution for the
Received: February 21, 2020
Accepted: March 20, 2020
Figure 1. Picture of garlic (A. sativum L.).
Article
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https://dx.doi.org/10.1021/acsomega.0c00772
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prevention and treatment of the Coronavirus disease 2019
(COVID-19) using the garlic essential oil.
The fact is that coronaviruses belong to a large family of
viruses that often cause the common cold in humans. Middle
East respiratory syndrome (MERS), severe acute respiratory
syndrome (SARS), and lately SARS-CoV-2 are the more severe
symptoms caused by the coronavirus family.
6,8
SARS-CoV-2 is
a new strain that has been unprecedentedly found in humans.
Angiotensin-converting enzyme 2 (ACE2) is an integral
membrane glycoprotein that is known for the highest
expression in most tissues such as kidneys, endothelium,
lungs, and heart.
9,10
The ACE2 protein is the same functional
host-cell receptor shared by SARS-CoV-2 and SARS.
8,11,12
The
structural database of ACE2 can be referenced from
UniProtKB.
13
Therefore, besides inhibiting SARS-CoV-2, the
inhibition of the ACE2 protein is absolutely necessary to
reduce the operability of the host receptor of SARS-CoV-2. If
the ACE2 protein is inhibited, it suggests that coronavirus is
prevented and treated.
12
As mentioned above, the high organosulfur compounds in
garlic essential oil are expected to have strong interactions with
the amino acids of the ACE2 protein. In this study, the idea is
to determine the inhibition capacity of ligands in garlic
essential oil not only to the ACE2 protein (host receptor for
SARS-CoV-2) but also directly to the PDB6LU7 protein (main
protease of SARS-CoV-2).
14
The structure of the PDB6LU7
protein of SARS-CoV-2 has been determined recently by
Worldwide Protein Data Bank.
14
It shows that the two proteins
selected from Uniprot and Worldwide Protein Data Bank have
three-dimensional structures, as shown in Scheme 1 with the
DOI: 10.2210/pdbACE2/pdb presented for the ACE2 protein
and the DOI: 10.2210/pdb6LU7/pdb shown for the
PDB6LU7 protein in SARS-CoV-2.
11−14
In this study, garlic essential oil was extracted from
commercial garlic and collected in Hue, Vietnam, by steam
distillation. The composition of the essential oil was identified
by GC−MS. The fact is that the study of HIV-1 resistance to
reverse transcriptase inhibitors was reported by Tarasova et
al.
15
The inhibition abilities of the garlic essential oil toward
the ACE2 and PDB6LU7 proteins were determined using the
molecular docking technique to investigate the interactions of
ligands in the garlic essential oil with the ACE2 and PDB6LU7
proteins. The docking results (i.e., docking score energy (DS),
root-mean-square deviation (RMSD), distances, and types of
interactions) indicate the inhibitory effects of the compounds
in the garlic essential oil on the two proteins. To the best of
our knowledge, this is the first report on the inhibitory effects
of the considered garlic compounds on the ACE2 protein,
which is a crucial foundation about SARS-CoV-2 resistance of
those compounds on the main protease (PDB6LU7) protein
of SARS-CoV-2 using docking simulations. The results of this
study show that natural garlic essential oil is considered as a
valuable resource recommended for preventing SARS-CoV-2
invasion into the human body.
2. RESULTS AND DISCUSSION
2.1. Composition of Garlic Essential Oil. The density
and refractive index of the garlic essential oil (A. sativum L.)
are 1.019 g·mL−1and 1.467, respectively. The results of the
analysis of physicochemical properties in this study agree well
with the results (1.025−1.029 g·mL−1; 1.464−1.469) reported
by Boukeria et al.
16
The qualitative and quantitative composition of the garlic
essential oil was determined by GC−MS analysis, and the data
are recorded in Figure 2 and Table 1, where the compounds
are listed in order of their elution. A total of 18 compounds
were identified in the garlic essential oil, covering more than
96.6% of the GC−MS profiles. The main constituents in the
garlic essential oil were allyl disulfide (28.4%), allyl trisulfide
(22.8%), allyl (E)-1-propenyl disulfide (8.2%), allyl methyl
trisulfide (6.7%), and diallyl tetrasulfide (6.5%). These results
had differences in concentrations of chemical composition
compared to those in the previous studies.
3,17−19
Li et al.
indicated that major essential oil components of garlic from
Suzhou City in China were 3-vinyl-4H-1,2-dithiin (31.9%),
diallyl trisulfide (13.3%), diallyl sulfide (2.2%), diallyl disulfide
(6.9%), propyl allyl disulfide (13.9%), and dimethyl disulfide
(7.1%).
17
On the contrary, diallyl trisulfide (37.3−45.9%),
diallyl disulfide (17.5−35.6%), and methyl allyl trisulfide (7.7−
10.4%) were the major components of the garlic essential oil
from the state of Ariana (Tunisia).
19
The main essential oil
components obtained from the east of Algeria were diallyl
disulfide (33.4−38.4%) and diallyl trisulfide (23.35−30.0%).
20
It has been known that the difference in the chemical
composition of essential oils can be attributed to the
importance of geo-ecological factors in the production of
metabolites of plants.
3,17−19
Although the bioactivities of garlic essential oil have not
been investigated in this study, they were documented in
previous studies.
21,22
According to Rattanachaikunsopon
Pongsak and Parichat Phumkhachorn, allyl disulfide (T1)
was also able to inhibit Escherichia coli O157:H7 in a food
model.
25
Moreover, Wodek et al. indicated that allyl disulfide
can constitute a source of sulfane sulfur for liver; therefore, it
can be used for cyanide detoxification in mouse tissues.
22
Methyl allyl disulfide and diallyl trisulfide were inhibitory
agents against Sitophilus zeamais Motschulsky and Tribolium
castaneum (Herbst) for contact toxicity, fumigant toxicity, and
antifeedant activity.
23
Besides, according to Seki et al., the
anticancer effect of diallyl trisulfide on cancer cells HCT-15
and DLD-1 was reported.
24
The biological activities of the
essential oil depend on the number of sulfur atoms, and the
higher the number of sulfur atoms, the stronger the biological
activities.
19
In this study, the high amount of sulfur (95.63%)
in the garlic essential oil suggests a further application of this
essential oil in the medicine and pharmaceutical industry.
2.2. Docking Simulation. To the best of our knowledge,
there are no results related to SARS-CoV-2 resistance through
Scheme 1. (A) Angiotensin-Converting Enzyme 2 (ACE2)
in the Human Body and (B) PDB6LU7 Protein in SARS-
CoV-2 Main Protease
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theoretical and experimental studies of compounds in essential
oils.
2.2.1. Docking Simulation Results of Compounds in
Garlic Essential Oil into ACE2 Protein. This is our first
successful demonstration of the model describing the docking
molecules of 17 out of 18 compounds in the garlic essential oil
into the complex structures of the ACE2 protein. Docking
simulations of the interactions between compounds T1−T17
and the ACE2 protein in the human body are presented as T1-
ACE2 to T17-ACE2 in the Supporting Information. We found
that T18-ACE2 has the lowest content in the essential oil, and
due to its bulky structure, the interaction with the ACE2
protein is not easy; hence, we do not demonstrate its
simulation in this study. The results of DS energy and
RMSD between compounds in garlic essential oil and proteins
with various interactions such as hydrogen bonds, cation−π
bonds, π−πbonds, and ionic interactions, as well as the
interaction distance between amino acids and the active sites of
compounds are presented in Table 2,Figures 3, and S1
(Supporting Information). There are also van der Waals
interactions between compounds with other amino acids of the
ACE2 protein, but since they are weak interactions, they are
not identified in this study.
The model describing the docking molecules indicated that
all of the 17 compounds in the garlic essential oil from T1-
ACE2 to T17-ACE2 have the binding ability toward the areas
affected by the ACE2 protein. The five presented representa-
tives T5-ACE2,T11-ACE2,T1-ACE2,T2-ACE2, and T4-
ACE2 have good DS energies of −14.06, −14.01, −12.84,
−12.76, and −12.5 kcal·mol−1, respectively (cf. Table 2 and
Figure 3). It can be seen that T5-ACE2 interacts best with
regions of the ACE2 protein, and the same trend is found for
T11-ACE2. The priority order of the interactions of
compounds in the garlic essential oil was as follows: T5 =
T11 > T1 = T2 > T4 > T8 > T9 > T12 > T13 > T14 > T15 >
T3 > T7 > T10 > T16 > T17 > T6.
We found that the sulfur compounds had strong interactions
with amino acids in the ACE2 protein. It is revealed from the
results of the model describing the docking molecules that the
relation in terms of structure and inhibition ability of the 17
substances in the garlic essential oil toward the ACE2 protein
was as follows: the compounds had very strong interactions
with the amino acids Pro 565, Trp 566, Ala 396, Gln 102, Gln
101, Glu 208, Gly 205, Gln 98, Asn 210, Lys 94, Lys 562, Val
209, and Ser 563. The interactions mainly took place with the
molecule groups containing sulfur in their compounds.
Interestingly, T10-ACE2 and T13-ACE2 mainly interacted
with oxygen in the compounds. The total content of the 17
compounds in the garlic essential oil resistant to the ACE2
protein was 99.4%. This proves that the entire essential oil is
appropriate for the prevention and treatment of pneumonia
caused by SARS-CoV-2. Thus, finding active sites of
compounds in the essential oil to inhibit the ACE2 protein
is of great significance to the orientation of using the garlic
essential oil in the prevention and treatment of SARS-CoV-2 in
specific and other viruses causing flu or pneumonia in general.
2.2.2. Docking Results of Compounds in Garlic Essential
Oil into the PDB6LU7 Protein of SARS-CoV-2. The model
describing the docking molecules of 18 substances in the garlic
essential oil into the complex structure of the PDB6LU7
protein was constructed using MOE 2015.10 software. The
docking was successful in 17 compounds of T1-SARS-CoV-2
to T17-SARS-CoV-2. The DS energy and RMSD values
between ligands and proteins shown through hydrogen bonds,
cation−πbonds, π−πbonds, and ionic interactions, as well as
the interaction distance between amino acids and the active
sites in the title molecules are presented in Table 3 and Figures
4and S2 (Supporting Information).
The anti-SARS-CoV-2 activity of the garlic essential oil
composition into the PDB6LU7 protein of SARS-CoV-2 was
found in the following order: T2 = T1 > T5 > T4 > T11 >
T15 > T8 > T16 > T9 > T12 > T13 > T3 > T6 > T7 > T10 >
T14 > T17. The compounds T1 and T2 indicated the highest
content in the garlic essential oil (51.3%) and gave the best
anti-SARS-CoV-2 activity, followed by the three compounds
T5,T4, and T11. These five substances account for 65.83%
composition of the garlic essential oil (Figure 4). The results
demonstrated that these 17 compounds have a stronger
inhibition on PDB6LU7 of SARS-CoV-2 than that on the
ACE2 protein in the human body. The DS energy was
negative, in the range of −15.32 to −11.68 kcal·mol−1.
Interactions with amino acid included Pro 565, Gln 102, Glu
208, Asn 210, Gly 205, Gln 98, Trp 566, Lys 94, Val 209, Gln
101, Asp 206, Asn 103, Ser 563, Ala 396, and Lys 562. The
total content of 17 out of 18 compounds in the garlic essential
oil exhibiting inhibition ability toward the PDB6LU7 protein
of SARS-CoV-2 is 99.4%. The docking simulation results are
excellent evidence of the anti-SARS-CoV-2 activity of the garlic
essential oil.
Figure 2. GC−MS chromatogram of garlic essential oil.
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The simultaneous interaction of 17 out of 18 compounds in
the garlic essential oil with the ACE2 protein and the
PDB6LU7 protein is described in Figure 5. Hydrogen bonds,
cation−πbonds, π−πbonds, and ionic interactions of 17
compounds with the amino acids of the ACE2 protein
included Trp 566, Asn 103, Gly 205, Lys 94, Asp 206, Glu
208, Val 209, Pro 565, Asn 210, Ser 563, Gln 98, Gln 102, Gln
101, Ala 396, Lys 562, and Ala 396, and those with the amino
acids of the PDB6LU7 protein included Gly 143, Glu 166, Asn
142, Leu 141, Cys 145, Ser 144, His 163, and Met 165. Based
on the percentage of each compound in the garlic essential oil
(shown by GC−MS) and docking score (DS) energy of each
compound, the average DS energies of these 17 compounds
with the ACE2 protein and the PDB6LU7 protein were
calculated as −11 311 and −13 871 kcal·mol−1, respectively.
This shows an excellent result to confirm the effects of the
garlic essential oil on the virus host receptor (ACE2) inhibition
and SARS-CoV-2 resistance. Therefore, it is possible to use
each compound in garlic essential oil or the whole essential oil
system to act simultaneously on the ACE2 protein and the
PDB6LU7 protein. This result opens the direction of research
and application of the essential oils in general, garlic essential
Table 1. Identification of the Bioactive Compounds in Garlic Essential Oil
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oil in particular, in the prevention and treatment of SARS-
CoV-2.
3. CONCLUSIONS
This study proposes a potential approach to the use of natural
essential oils, in general, and garlic essential oil, in particular, to
tackle the current pandemic SARS-CoV-2. The compounds in
the garlic essential oil inhibit the ACE2 protein, leading the
virus to lose the host receptor and attacking the PDB6LU7
proteinthe main protease of SARS-CoV-2at the same
time. This prevents protein maturation of the virus and the
spread of infection. Docking simulation suggests the active
binding site of most active compounds in garlic essential oil
with the ACE2 protein and the PDB6LU7 protein. From the
analysis of the docking data, it is revealed that 17 (T1−T17)
out of 18 compounds of the garlic essential oil are capable of
inhibiting ACE2 and resisting SARS-CoV-2 and that the total
content of these 17 compounds accounts for 99.4%
Table 2. Docking Simulation Results with Docking Score Energy (DS) and Root-Mean-Square Deviation (RMSD) between
Compounds in Garlic Essential Oil and the ACE2 Protein
T5 = T11 > T1 = T2 > T4 > T8 > T9 > T12 > T13 > T14 > T15 > T3 > T7 > T10 > T16 > T17 > T6
compound symbol (compound-
protein) DS (
kcal·mol−1)RMSD
(Å) interaction with amino acid
allyl disulfide T1-ACE2 −12.84 1.46 Pro 565 (2.85 Å), Trp 566 (2.87 Å), Ala 396 (3.14 Å)
allyl trisulfide T2-ACE2 −12.76 1.47 Gln 102 (2.89 Å; 3.11 Å), Asn 103 (3.45 Å)
allyl (E)-1-propenyl disulfide T3-ACE2 −9.07 0.66 Glu 208 (3.06 Å), Gly 205 (3.70 Å)
allyl methyl trisulfide T4-ACE2 −12.50 1.56 Asn 210 (3.13 Å; 3.05 Å), Lys 94 (3.22 Å)
diallyl tetrasulfide T5-ACE2 −14.06 1.23 Gly 205 (3.43 Å), Asp 206 (2.95 Å), Lys 562 (2.92 Å), Ala 396 (3.82 Å)
1,2-dithiole T6-ACE2 −7.89 3.09 Asn 210 (3.29 Å)
allyl (Z)-1-propenyl disulfide T7-ACE2 −9.04 1.58 Gln 98 (4.14 Å), Glu 208 (3.24 Å)
2-vinyl-4H-1,3-dithiine T8-ACE2 −11.83 0.62 Gln 98 (3.10 Å), Val 209 (3.56 Å), Asn 210 (3.93 Å)
3-vinyl-1,2-dithiacyclohex-4-ene T9-ACE2 −10.57 1.19 Trp 566 (2.78 Å), Pro 565 (3.95 Å), Glu 208 (3.12 Å)
carvone T10-ACE2 −8.58 1.53 Lys 94 (2.13 Å), Gln 98 (1.72 Å)
trisulfide, 2-propenyl propyl T11-ACE2 −14.01 1.85 Trp 566 (3.51 Å), Glu 208 (3.66 Å), (3.05 Å), Val 209 (3.25 Å), Gln 98
(3.52 Å)
methyl allyl disulfide T12-ACE2 −10.32 1.16 Val 209 (2.31 Å), Gln 98 (3.45 Å), Lys 94 (3.15 Å)
diacetonalcohol T13-ACE2 −9.71 0.85 Gln 101 (2.13 Å), Asn 210 (1.97 Å; 2.05 Å)
trisulfide, (1E)-1-propenyl 2-
propenyl T14-ACE2 −9.57 0.68 Asn 210 (3.20 Å; 1.72 Å), Ser 563 (3.73 Å)
allyl sulfide T15-ACE2 −9.38 1.48 Asn 210 (2.31 Å), Lys 94 (3.15 Å), Gln 98 (3.45 Å)
1-propenyl methyl disulfide T16-ACE2 −8.06 0.88 Asp 206 (2.81 Å), Trp 566 (3.46 Å)
trisulfide, (1Z)-1-propenyl 2-
propenyl T17-ACE2 −8.06 2.35 Glu 208 (3.29 Å), Gln 98 (3.64 Å)
Figure 3. (A) Native human angiotensin-converting enzyme 2 (ACE2) crystal structure. Docking simulation of the interaction between
compounds T5,T11,T1,T2,T4, and the ACE2 protein in the human body: (B) T5-ACE2, (C) T11-ACE2, (D) T1-ACE2, (E) T2-ACE2, and
(F) T4-ACE2. The inhibitory effects of the compounds on the ACE2 protein are of the order: T5 ≈T11 > T1 ≈T2 > T4.
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composition of the garlic essential oil. The docking score (DS)
energy of compounds for the ACE2 protein ranges from
−14.06 to −7.89 kcal·mol−1, and the DS energy of compounds
for the PDB6LU7 protein of SARS-CoV-2 ranges from −15.32
to −11.68 kcal·mol−1. The order of active compounds
inhibiting the ACE2 protein is as follows: T5 = T11 > T1 =
T2 > T4 > T8 > T9 > T12 > T13 > T14 > T15 > T3 > T7 >
T10 > T16 > T17 > T6. Meanwhile, the order of active
Table 3. Docking Simulation Results with Docking Score Energy (DS) and Root-Mean-Square Deviation (RMSD) between the
Title Compounds and PDB6LU7 Protein of SARS-CoV-2
T2 = T1 > T5 > T4 > T11 > T15 > T8 > T16 > T9 > T12 > T13 > T3 > T6 > T7 > T10 > T14 > T17
compound symbol (compound-
protein) DS (
kcal·mol−1)RMSD
(Å) interaction with amino acid
allyl disulfide T1-SARS-CoV-2 −15.32 1.35 Gly 143 (2.59 Å), Asn 142 (2.92 Å), Leu 141 (2.94 Å), Cys 145 (2.98 Å), Ser 144
(3.15 Å), His 163 (2.95 Å)
allyl trisulfide T2-SARS-CoV-2 −15.02 0.66 Gly 143 (3.93 Å; 2.79 Å), Asn 142 (2.87 Å; 2.67 Å), Cys 145 (3.25 Å; 3.63 Å), Ser
144 (3.06 Å)
allyl (E)-1-propenyl disulfide T3-SARS-CoV-2 −13.25 1.49 Leu 141 (2.98 Å), Ser 144 (3.07 Å)
allyl methyl trisulfide T4-SARS-CoV-2 −14.36 1.37 Gly 143 (3.69 Å), Asn 142 (2.81 Å), Cys 145 (3.61 Å; 3.12 Å), Ser 144 (2.96 Å)
diallyl tetrasulfide T5-SARS-CoV-2 −14.47 0.94 Gly 143 (2.88 Å; 2.98 Å), Asn 142 (2.10 Å), Cys 145 (3.94 Å; 3.56 Å), Ser 144
(3.14 Å)
1,2-dithiole T6-SARS-CoV-2 −13.21 2.30 Asn 142 (3.75 Å), Cys 145 (3.75 Å)
allyl (Z)-1-propenyl disulfide T7-SARS-CoV-2 −12.60 1.72 Gly 143 (3.32 Å), Leu 141 (3.62 Å)
2-vinyl-4H-1,3-dithiine T8-SARS-CoV-2 −14.04 4.35 Gly 143 (2.73 Å), Asn 142 (3.82 Å), Cys 145 (3.11 Å), Ser 144 (3.02 Å)
3-vinyl-1,2-dithiacyclohex-4-
ene T9-SARS-CoV-2 −13.83 1.05 Glu 166 (2.86 Å), Met 165 (2.88 Å), Cis 145 (2.13 Å)
carvone T10-SARS-CoV-2 −12.36 1.62 His 163 (2.15 Å)
trisulfide, 2-propenyl propyl T11-SARS-CoV-2 −14.36 1.37 Gly 143 (2.62 Å; 2.81 Å), Asn 142 (2.85 Å), Cys 145 (3.15 Å; 3.50 Å), Ser 144
(2.87 Å), His 163 (2.97 Å)
methyl allyl disulfide T12-SARS-CoV-2 −13.56 1.51 Cys 145 (2.43 Å), Ser 144 (2.82 Å), His 163 (2.65 Å)
diacetonalcohol T13-SARS-CoV-2 −13.26 1.41 Cys 145 (2.93 Å), Ser 144 (2.26 Å)
trisulfide, (1E)-1-propenyl 2-
propenyl T14-SARS-CoV-2 −12.00 2.12 Leu 141 (3.32 Å), Ser 144 (2.39 Å)
allyl sulfide T15-SARS-CoV-2 −14.24 1.20 Gly 143 (2.75 Å), Asn 142 (2.75 Å), Leu 141 (3.56 Å), Cys 145 (3.47 Å), Ser 144
(3.32 Å)
1-propenyl methyl disulfide T16-SARS-CoV-2 −13.84 0.91 Gly 143 (2.89 Å), Asn 142 (3.88 Å), Cys 145 (2.83 Å), Ser 144 (2.80 Å)
trisulfide, (1Z)-1-propenyl
2-propenyl T17-SARS-CoV-2 −11.68 3.60 Leu 141 (3.38 Å)
Figure 4. (A) Crystal structure of the virus main protease in the complex (PDB6LU7). Docking simulation of the interaction between compounds
T1,T2,T4,T5, and T11, and the PDB6LU7 protein of SARS-CoV-2: (B) T1-SARS-CoV-2, (C) T2-SARS-CoV-2, (D) T4-SARS-CoV-2, (E)
T5-SARS-CoV-2, and (F) T11-SARS-CoV-2. The inhibitory effects of the compounds on the PDB6LU7 protein of SARS-CoV-2 were of the
order: T1 ≈T2 > T5 > T4 > T11.
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compounds resisting SARS-CoV-2 is T2 = T1 > T5 > T4 >
T11 > T15 > T8 > T16 > T9 > T12 > T13 > T3 > T6 > T7 >
T10>T14>T17. The synergistic interactions of 17
substances of the garlic essential oil exhibited good inhibition
on the ACE2 protein (host receptor of the virus) and the
PDB6LU7 protein of the virus. This study opens the door
toward the use of the garlic essential oil in discovering and
treating SARS-CoV-2 to prevent the current pandemic.
4. MATERIALS AND METHODS
4.1. Experiment. 4.1.1. Sample Collection. Garlic (A.
sativum L.) was purchased from a local market (Thua Thien
Hue province in Central Vietnam). Then, it was botanically
identified, and a voucher specimen was deposited at the
Department of Biology, University of Sciences, Hue University.
During the experiment, all of the samples were maintained in
our lab at appropriate conditions (e.g., 25 °C in the dark).
4.1.2. Chemicals and Equipment. All analytical-grade
chemicals obtained from Sigma-Aldrich Co. were used without
any further purification. The major equipment includes a
hydrodistillation apparatus (Merck Specialities Pvt. Ltd.,
India), a polarimeter (Reichert Cat #14003000), a Jasco V-
630 spectrophotometer (Japan), and a gas chromatography−
mass spectrometry (GC−MS) device (Aligent GC 7890B-MS
5975C).
4.1.3. Isolation of Essential Oil. A hydrodistillation
Clevenger apparatus was used to extract the garlic essential
oil. Peeled garlic (250 g) was minced and then put into a 1 L
flask, in which 400 mL of distilled water was added. The
essential oil was obtained by water distillation at 100 °C for 90
min, according to the Vietnamese Pharmacopoeia.
25
A
sterilized glass vial was used to collect the essential oil. The
essential oil was dried by anhydrous Na2SO4before being
stored at 4 °C for further analysis. The extraction process was
repeated three times to check the repeatability of the analytical
procedure.
25
4.1.4. Refractive Index and Density of the Essential Oil.
Refractive index and density are two common physical
parameters for checking essential oil composition and purity.
The refractive index of essential oils was determined using a
polarimeter, and the density of the essential oil was determined
using a pycnometer flask according to the Vietnamese
Pharmacopoeia
25
and Boukeria et al.
16
4.1.5. Gas Chromatography−Mass Spectrometry (GC−
MS) Analysis. An Agilent GC 7890B-MS 5975C instrument
combined with an HP-5MS column (30 m ×250 μm×0.25
μm) was used to identify the chemical composition of the
garlic essential oil. The utilized carrier gas was helium at
constant pressure (13 psi). The essential oil (1 μL) was
injected into the GC with a split ratio of 20:1. The injection
temperature was 250 °C. The column temperature program
was applied as follows: start at 70 °C and then increased at the
rate of 10 °C every minute until it reached 280 °C. After the
analytes were separated on a capillary column, they passed
through the ionization zone in the MS source (ionization
energy: 70 eV; interface temperature: 280 °C; MS temper-
ature: 230 °C; quadrupole temperature: 150 °C), and the
neutral molecules were ionized yielding specific mass/charge
(m/z) ratios. C8−C30 Alkanes Calibration Standard (Sigma-
Aldrich) was applied to identify unknown compounds through
their retention indices by comparing their mass spectra to
those contained in the NIST02 database. The concentration of
each analyte will be calculated based on its peak area in the
chromatogram.
4.2. Molecular Docking Simulation. The molecular
docking technique is used to predict and describe the
interaction (including the binding energy and structural
parameters) of compounds resistant to the ACE2 protein in
the human body and the PDB6LU7 protein in SARS-CoV-2.
The docking results reveal that there are potential compounds
in the essential oil to prevent and treat SARS-CoV-2. The
molecular docking modeling consists of five essential
steps
15,26−28
as follows:
Step 1: Selection of proteins
•Selection of proteins in PDB: The biological targets are
the ACE2 protein and the PDB6LU7 protein presented
at Uniprot
13
and Worldwide Protein Data Bank.
14
•Determination of bonding position: The protein action
areas were determined based on the ligand positions
within a radius of 4.5 Å and the presence of important
amino acids. Water molecules were removed, and the
structures of amino acids were checked before
reestablishing the enzyme action areas.
Step 2: Preparation of proteins
•Creating 3D structures: The 2D chemical structure (flat
structure) of the compounds was automatically con-
verted to the 3D chemical structure (three-dimensional
structure) by ChemBioOffice 2018 software.
•Lowest-energy state: The 3D molecular structures of the
compounds were then optimized using SYBYL-X 1.1
software with the purpose to correct the mismatch
values of bond lengths, bond angles, bending angles, and
unusual nonbonding interactions due to the atoms in
Figure 5. 3D-docking results with simultaneous interaction of all 17 substances (17T = T1−T17) in the garlic essential oil with (A) ACE2 protein
and (B) PDB6LU7 protein of SARS-CoV-2.
ACS Omega http://pubs.acs.org/journal/acsodf Article
https://dx.doi.org/10.1021/acsomega.0c00772
ACS Omega XXXX, XXX, XXX−XXX
G
different parts of the compounds occupying the same
space. The energy minimization program used the Conj
Grad method (conjugate gradient) to choose the stop
point where the energy change is less than the cutoff
value of 0.001 kcal·mol−1with partial charges of
Gasteiger−Huckel and the maximum number of
iterations needed was 10 000. This method accumulates
the information about the energy function from each
iteration, which is suitable for small and large molecules.
Molecular dynamics conduction was used to obtain the
structures with the lowest energy via the principle of
simulated annealing in Sybyl-X 1.1 software. The
molecular structure was heated at a high temperature
(700 K) for a certain time (1000 ps) so that the
molecule rearranges its current state and then cooled to
200 K for another 1000 ps to a steady state for
computing the final configuration. The program
automatically iterated five times to find the various
configurations needed, and then the minimized energy
was observed again to determine steric energy. In this
way, the structural parameters of compounds with
different energies were found, which were more durable
than the original structures.
Step 3: Redocking
Redocking of protein-compound cocrystal structures: the
redocking of protein−ligand complex cocrystal structures aims
to assess the suitability of docking parameters. The process was
carried out with three structures of compounds as follows:
(1) Separation of compounds from homogenized complexes
in proteins.
(2) Separation of compounds from homogeneous complexes
and repreparation using Sybyl-X 1.1 software.
(3) Preparation of new compounds (structure drawing,
structural optimization parameters on minimal energy,
molecular dynamics calculations).
Root-mean-square deviation (RMSD) values reflect the
deviation of compounds’structures after docking compared to
available structures in the crystal structure and comparing the
interactions of compounds in the crystal structure after
docking. The docking results are considered reliable when
the RMSD value is <1.5 Å, and the interactions between
compounds and the initial enzyme are not significantly
different.
Step 4: Molecules docking into protein: docking inves-
tigations
•Docking of compounds into prepared proteins and
compounds via conducting the docking process with
MOE 2015.10 software with the following options: the
method of placing compound fragments into the triangle
matching; the maximum number of results for each
iteration is 1000; the maximum number of results for
each compound fragmentation is 200; retain the five best
configurations of each compound in the bonding
complex for further analysis. The best configuration is
the one with the lowest docking score (DS) energy
(kcal·mol−1). This score is the total energy consumed for
the formation of bonding interactions between the
compounds and the two proteins selected.
Step 5: Docking results analysis
•Evaluation of docking score (DS): Analysis of the
interactions between the compounds and targeted
proteins, and performance of interaction on 2D and
3D planes using MOE 2015.10 software. Various
interactions, such as van der Waals interactions,
hydrogen bonds, cation−πbonds, π−πbonds, and
ionic interactions, and the interaction distance between
amino acids and the active sites of compounds are
plotted. Van der Waals interactions are detected by
contact with hydrophilic and hydrophobic surfaces
between the compounds and the bonding point.
■ASSOCIATED CONTENT
*
sıSupporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsomega.0c00772.
Docking simulation of the interaction between com-
pounds and the ACE2 protein in human body;
inhibitory effects of compounds on the ACE2 protein:
T8 > T9 > T12 > T13 > T14 > T15 > T3 > T7 > T10
>T16>T17>T6; docking simulation of the
interaction between compounds and the PDB6LU7
protein of SARS-CoV-2; and inhibitory effects of
compounds on the PDB6LU7 protein: T15 > T8 >
T16 > T9 > T12 > T13 > T3 > T6 > T7 > T10 > T14
> T17 (PDF)
■AUTHOR INFORMATION
Corresponding Authors
Duong Tuan Quang −Department of Chemistry, University of
Education, Hue University, Hue City 530000, Vietnam;
orcid.org/0000-0002-4926-0271; Email: dtquang@
hueuni.edu.vn
Nguyen Thi Ai Nhung −Department of Chemistry, University
of Sciences, Hue University, Hue City 530000, Vietnam;
orcid.org/0000-0002-5828-7898; Email: ntanhung@
hueuni.edu.vn
Authors
Bui Thi Phuong Thuy −Faculty of Fundamental Science, Van
Lang University, Ho Chi Minh City 700000, Viet Nam
Tran Thi Ai My −Department of Chemistry, University of
Sciences, Hue University, Hue City 530000, Vietnam
Nguyen Thi Thanh Hai −Department of Chemistry, University
of Sciences, Hue University, Hue City 530000, Vietnam
Le Trung Hieu −Department of Chemistry, University of
Sciences, Hue University, Hue City 530000, Vietnam
Tran Thai Hoa −Department of Chemistry, University of
Sciences, Hue University, Hue City 530000, Vietnam
Huynh Thi Phuong Loan −Department of Chemistry,
University of Sciences, Hue University, Hue City 530000,
Vietnam; orcid.org/0000-0002-3659-4571
Nguyen Thanh Triet −Faculty of Traditional Medicine,
University of Medicine and Pharmacy at Ho Chi Minh City, Ho
Chi Minh City 700000, Vietnam
Tran Thi Van Anh −Faculty of Pharmacy, University of
Medicine and Pharmacy at Ho Chi Minh City, Ho Chi Minh
City 700000, Vietnam
Phan Tu Quy −Department of Natural Sciences &Technology,
Tay Nguyen University, Buon Ma Thuot City 630000, Vietnam
Pham Van Tat −Department of Environmental Engineering,
Hoa Sen University, Ho Chi Minh City 700000, Vietnam
ACS Omega http://pubs.acs.org/journal/acsodf Article
https://dx.doi.org/10.1021/acsomega.0c00772
ACS Omega XXXX, XXX, XXX−XXX
H
Nguyen Van Hue −Faculty of Engineering and Food
Technology, University of Agriculture and Forestry, Hue
University, Hue City 530000, Vietnam
Nguyen Tien Trung −Laboratory of Computational Chemistry
and Modeling, Department of Chemistry, Quy Nhon University,
Quy Nhon City 590000, Vietnam; orcid.org/0000-0001-
5710-5776
Vo Thanh Tung −University of Sciences, Hue University, Hue
City 530000, Vietnam
Lam K. Huynh −Department of Chemical Engineering,
International University, Ho Chi Minh City 700000, Vietnam;
Vietnam National University, Ho Chi Minh City 700000,
Vietnam; orcid.org/0000-0002-3683-2787
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsomega.0c00772
Notes
The authors declare no competing financial interest.
■ACKNOWLEDGMENTS
This research was supported by the Vietnam National
Foundation for Science and Technology Development
(NAFOSTED). The authors would like to thank Prof. Jong
Seung Kim (Korea University) for his valuable comments and
discussion. Also, they highly appreciate Dr Mingle Li (Korea
University) and Khue Lai (IU) for helping with figure
preparation.
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