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Article title: An investigation of ethanolic propolis extracts: Their potential inhibitor properties against ACE-II
receptors for COVID-19 treatment by Molecular Docking Study
Authors: Halil Ibrahim Güler[1], Gizem Tatar[2], Oktay Yildiz[3], Ali Osman Belduz[4], Sevgi Kolayli[5]
Affiliations: aradeniz Technical University, Faculty of Science, Department of Molecular Biology and Genetics[1],
Karadeniz Technical University, Faculty of Medicine, Department of Biostatistics and Medical Informatics[2], Karadeniz
Technical University, Faculty of Pharmacy, Basic Pharmaceutical Sciences, Department of Biochemistry[3], Karadeniz
Technical University, Faculty of Science, Department Biology[4], Karadeniz Technical University, Faculty of Science,
Department of Chemisty[5]
Orcid ids: 0000-0002-7261-6790[1]
Contact e-mail: hiboguler@gmail.com
License information: This work has been published open access under Creative Commons Attribution License
http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited. Conditions, terms of use and publishing policy can be found at
https://www.scienceopen.com/.
Preprint statement: This article is a preprint and has not been peer-reviewed, under consideration and submitted to
ScienceOpen Preprints for open peer review.
DOI: 10.14293/S2199-1006.1.SOR-.PP5BWN4.v1
Preprint first posted online: 22 April 2020
Keywords: Coronavirus, Covid-19, propolis, Flavonoids, ACE-II, Molecular docking
An investigation of ethanolic propolis extracts: Their potential inhibitor
properties against ACE-II receptors for COVID-19 treatment by Molecular
Docking Study
Halil Ibrahim GULER1*, Gizem TATAR2, Oktay YILDIZ3, Ali Osman BELDUZ4, Sevgi
KOLAYLI5
1Karadeniz Technical University, Faculty of Science, Department of Molecular Biology and Genetics,
61080 Trabzon, TURKEY
2Karadeniz Technical University, Faculty of Medicine, Department of Biostatistics and Medical
Informatics, 61080 Trabzon, TURKEY
3Karadeniz Technical University, Faculty of Pharmacy, Basic Pharmaceutical Sciences, Department
of Biochemistry, 61080 Trabzon, TURKEY
4Karadeniz Technical University, Faculty of Science, Department Biology, 61080 Trabzon, TURKEY
5Karadeniz Technical University, Faculty of Science, Department of Chemisty, 61080 Trabzon,
TURKEY
*Corresponding author
E-mail: hiboguler@gmail.com
Phone: +90462 377 3553
Present adress: Karadeniz Technical University, Faculty of Science, Department of Molecular
Biology and Genetics, 61080 Trabzon, Turkey
ORCID: https://orcid.org/0000-0002-7261-6790
Conflict of interest
No conflict of interest is declared.
Abstract
The angiotensin-converting enzyme (ACE)-related carboxypeptidase, ACE-II, is a type
I integral membrane protein of 805 amino acids that contains one HEXXH-E zinc binding
consensus sequence. ACE-II has been implicated in the regulation of heart function and also
as a functional receptor for the coronavirus that causes the severe acute respiratory syndrome
(SARS). In this study, the potential of some flavonoids present in propolis to bind to ACE II
receptors was calculated in silico.
Binding constants of ten flavonoids, caffeic acid, caffeic acid phenethyl ester, chrysin,
galangin, myricetin, rutin, hesperetin, pinocembrin, luteolin and quercetin were measured
using the AutoDock 4.2 molecular docking program. And also, these binding constants were
compared to reference ligand of MLN-4760.
The results are shown that rutin has the best inhibition potentials among the studied
molecules with high binding energy -8,97 kcal/mol and Ki 0,261 M, and it is followed by
myricetin, caffeic acid phenethyl ester, hesperetin and pinocembrin. However, the reference
molecule has binding energy of -7,28 kcal/mol and 4,65 M. In conclusion, the high potential
of flavonoids in ethanolic propolis extracts to bind to ACE II receptors indicates that this
natural bee product has high potential for Covid- 19 treatment, but this needs to be supported
by experimental studies.
Keywords: Coronavirus, Covid-19, propolis, flavonoids, ACE-II, molecular docking
Introduction
Propolis is a natural mixture that honey bees collect from nature in order to protect their
hives. Honey bees use propolis for insulating hives, mummification of dead insects and bees
and as an antibacterial, antiviral, antioxidant and anti-inflammatory agent for many biological
activities. Crude propolis is a highly viscous, slightly soluble mixture in water and best
dissolved in 60-80% ethanol. Propolis has been an indispensable component of apitherapy for
centuries and has recently been used as a food additive, or supplementary, under the name of
traditional and complementary medicine [1, 2].
Its composition varies according to the flora of the region where it is collected, but the
majority of active ingredients of propolis comprise the family of polyphenols. Phenolic acids,
flavonoids (flavanones, flavones, flavonols etc.), stilbenes, tannins are the active polyphenols
of propolis [1, 3]. Propolis are not consumed as raw, but their ethanolic and aqueous extracts
are widely consumed in different formulations.
It has been reported that polyphenolic agents such as gallic acid, caffeic acid,
protocatechuic acid, chrysin, quercetin, rutin, galangin, kaempferol, hesperetin, pinocembrin,
pinobanksin, apigenin, luteolin, daidzein, caffeic acid phenyl ester (CAPE) are the most active
polyphenols of propolis and these secondary metabolites vary depending on the propolis
source [1, 4, 5].
Studies show that propolis extracts have high immunomodulatory effect and inhibition
potential for some clinically important enzymes, such as urease, xanthine oxidase (XO),
acetylcholinesterase (AChE), -amylase, -glucosidase [6, 7]. In addition, in vivo and in vitro
studies show that flavonoids, one of the active ingredients of propolis, have high potential for
Angiotensin-Converting Enzyme (ACE) Inhibition [8, 9, 10].
The newly discovered SARS-CoV-2 was characterized as a beta-coronavirus and
recognized as the seventh discrete coronavirus species capable of causing human disease. The
disease caused by the virus is officially named Coronavirus Disease 2019 (Covid-19) by
Word Health Organization (WHO). The emerged global epidemic spread rapidly with
2.246.291 confirmed cases and 152.707 deaths across 213 countries, areas and territories
(COVID-19 situation Report WHO, 20 April 2020). Subsequent studies have shown that
SARS-CoV-2 has been suggested to recognize human ACE II more strongly than SARS-
CoV, thereby increasing the ability to be transmitted from person to person [11]. Therefore,
ACE II enzyme inhibition is important for treatments against these virus infections caused by
SARS-CoV-2.
The aim of this study was to calculate the inhibition constants of some flavonoids, one
of the active ingredients of Anatolian propolis, to the ACE II enzyme by molecular modeling
as a positive control (S, S) -2- {1-Carboxy-2- [3- (3,5-Dichloro-Benzyl) -3h-Imidazole-4-Yl] -
Ethylamino}-4-Methyl-Pentanoic Acid (MLN-4760). We analyzed therapeutic potential
compounds of Turkish propolis extracts tested against ACE-II in experimental studies with in
silico methods.
Up to now, there are very limited studies about Covid-19 and most researchers focused
primarily on clinical cases. However, to the authors' knowledge, no study has been made on
inhibition of ACE-II known to be associated with Covid-19. Therefore, the paper will
encourage further researches about Covid-19 and candidate drug compounds.
Materials and Methods
Materials
Raw propolis samples obtained experienced beekeepers in 2018 from Black Sea Region,
Turkey.
Chemicals
All phenolic standard for HPLC analyses of gallic acid, protocathequic acid, p-OH
benzoic acid, catechin, caffeic acid, syringic acid, epicatechin, p-coumaric acid, ferulic acid,
rutin, myricetin, resveratrol, daidzein, luteolin, t-cinnamic acid, hesperetin, chrysin,
pinocembrin, caffeic acid phenethyl ester (CAPE) were purchased from Sigma Chemical
Co.(St Louis, MO, USA). All solvent for using mobile phases were analytical grade.
Preparation of propolis extracts
The raw propolis samples were frozen at -20 °C and then grinded to powder. The
following method was used to prepare the ethanolic propolis extract: 10 g powdered crude
propolis was placed with 100 mL 70% ethanol in a glass flask and stirred with shaker
(Heidolph Promax 2020, Schwabach, Germany) at room temperature for 24 h and then
filtered with Whatman paper.
Determination of phenolic profiles
In this study, nineteen phenolic standards were used to high-performance liquid
chromatography (HPLC) (Elite LaChrom Hitachi, Japan) with a UV detector. Separation was
performed on a column with a reverse phase C18 column (150 mmx4.6 mm, 5μm; Fortis), in
gradient solvent systems A (2% acetic acid in water) and solvent B (70:30, acetonitrile/water),
which was sonicated before stirring and continuously degassed by the built-in HPLC system
[12, 13]. The flow rate was kept constant at 1 mL/min using gradient programming, starting
the flow of the mobile phase as B (5%) to 3 minutes, gradually increasing (up to 15, 20, 25,
40 and 80% at 8, 10, 18, 25 and 35 minutes, respectively) and decreasing to 5% at 40 minutes,
before being left for 10 minutes to equilibrate in the column. The standard phenolic
substances chromatogram is given in Figure 1.
Molecular Docking
In the study, some flavonoids detected in the ethanolic propolis extracts and they used
as ligand (see in Supplementary File) for ACE II receptors. The crystal structure of a ACE II
protein was downloaded from protein data bank web site (http://www.rcsb.org/pdb) (PDB ID:
1R4L: Resolution 3.00 Å). This crystal structure contains the inhibitory bound state of the
extracellular metallopeptidase domain of ACE II with MLN-4760 compound. Small
compounds of flavonoids used in docking studies were obtained from PubChem as SDF form
and were drawn in the Hyperchem software [14] then subjected to conformational search with
geometric optimization. Possible docking modes between compounds and the ACE II enzyme
were studied using the Autodock 4.2 [15] and Lamarckian genetic algorithm was employed
for docking simulations. The selected cavity is the binding site of reference inhibitor MLN-
4760 ((S,S)-2-{1-Carboxy-2-[3-(3,5-dichloro-benzyl)-3H-imidazol-4-YL]-ethylamino}-4-
methyl-pentanoic acid). A grid box dimensions of 52, 34, and 47 points in x, y, and z
directions was set with a grid spacing of 0.375 Å. The program was run for a total number of
100 Genetic algorithm runs. The default settings were applied for all other parameters. Results
of the molecular docking described the affinity represented by docking score and binding
interaction of each ligand on the interested protein target. The visualization of results was
performed with the help of the BIOVIA Discovery Studio 2018 [16].
Results and Discussion
Phenolic composition of propolis extract
In this study, a standard HPLC-UV chromatogram prepared with nineteen phenolic
standards including some phenolic acids and flavonoids is given in Fig 1. The analysis data
of the ethanolic propolis extract carried out according to this chromatogram are summarized
in Table 1. Although the hydroxybenzoic acids and catechin derivatives of the propolis
sample were found below determination limits, it was found to be rich in hydroxycinnamic
acids and flavanoids. Among the hydroxycinnamic acid derivatives, the caffeic acid phenyl
ester is the highest amount of phenolic component in the sample and followed it caffeic acid
and cinnamic acid. Ferulic acid could not be detected in the sample. Among the flavonoids
subclass of flavonoids, the highest amount of myrisetin was detected and rutin followed it.
Among these three flavanons studied, chrysin is the most abundant compounds
pinosebrin and hesperetin followed. A smaller amount of flavone derivative of luteolin, was
detected, while daidzein is not detected. Of all the studied compounds, chyrisin and
pinosembrin were detected as major flavonoids in the propolis sample.
Although composition of propolis varies according to the flora of the region where it is
produced, these flavonoids were also reported in propolis samples of different countries [17,
18, 19]. There are many scientific studies showing that propolis, a natural bee product, is a
very rich mixture of flavonoids and is an important agent of apitherapy. Polyphenolic profile
of propolis varies according to the flora of the region where it is collected, caffeic acid,
CAPE, rutin, quercetin, polyphenols such as myricetin, kampherol, hesperetin, galangin are
the active substances of Anatolian propolis [3, 4, 5, 20, 21]. Barbarić et al. (2011) studied
chemical composition of the ethanolic propolis extracts and determined ferulic acid, p-
coumaric acid, caffeic acid, tectochrysin, galangin, pinocembrin, chrysin, apigenin,
kaempferol, quercetin as phenolic compound in Croatia, Bosnia and Hercegovina and
Macedonia propolis [22]. Major compounds of red propolis samples from Brasilia were found
as luteolin (1.75 mg/g), naringenin (0.96 mg/g), kaempferol (0.59 mg/g), pinocembrin (0.41
mg/g) and biochanin A (0.39 mg/g) [23]. There are some differences between the findings
because the chemical composition of propolis varies according to the geographical region,
climate, environmental conditions and collection seasons [23, 24, 25]. The findings show that
propolis are a good source of phenolic substances. The literature states that propolis samples
from different geographical origins have a good antioxidant antimicrobial, antifungal and
antiviral (Avian influenza virus) activity [26, 27, 28, 29, 30].
Binding affinity analysis for proteins and ligands with molecular docking
We focus here on the Anatolian propolis compounds used by people to treat infections
against ACE II with molecular docking methods. For this purpose, we made docking analysis
with the compounds and found that quercetin, rutin, myrisetin and hesperetin have a better
affinity against ACE II enzyme than natural inhibitor MLN-4760 with low µM Ki values
among the evaluated compounds (Table 2).
Furthermore, these compounds interacted with Arg273, Thr371, His345, Pro346,
Tyr515, Glu402 and Glu375 in ACE II binding site. Especially, our in silico study showed
that, rutin has the best binding affinity to the ACE II enzyme (Binding energy: -8.98 kcal/mol,
Ki 0.261 M). This compound was observed to bind to the residues Asn149, His345, Asp269,
Glu375, Glu406, Thr371, Tyr127 and Asp368 of ACE II protein with the stronger hydrogen
bond (Figure 2). It can be suggested that these residues can contribute to the enhancement of
ligand affinity for ACE II enzyme. In addition, this compound has the pi-cation interaction
with Arg273, pi-pi T shape interaction with His374, alkyl interaction with Cys344 and pi-
alkyl interaction with Tyr 127 residues (Figure 2).
Therefore, in this study, in silico effects of Anatolian propolis on ACE II enzyme
inhibition was investigated with the ten flavonoids as major substances. The results of this
study showed that quercetin, rutin, myricetin and hesperetin compounds effectively inhibit the
ACE II enzyme. These compounds can be clinically tested and used for the treatment disease
role of ACE II. Furthermore, Arg273, Thr371, His345, Pro346, Tyr515, Glu402 and Glu375
are potential inhibitor targeting sites for the ACE II enzyme. Based on this information, we
propose guidelines to develop novel and specific inhibitors that target ACE II enzyme.
Guerrero et al. (2012) experimentally demonstrated that some flavonoids have a
relatively high inhibition potential for ACE-I [31]. With the molecular docking studies, we
have shown that some of these flavonoids inhibit ACE-II. ACE-I and ACE-II enzymes are
metalloproteases, both of which contain similar zinc fingers (HEXXH) in their active sites.
Molecular docking studies indicated that there are bond interactions between rutin and zinc
finger residues of ACE II. Because of similar active sites of ACE I and II, rutin may
functionally bind both ACE I and II similar way.
It is revealed that Covid-19 binds to human angiotensin-converting enzyme 2 (ACE2) to
enter the host cells. Rutin may compete with Covid-19 for ACE II and may prevent or delay
the entry of Covid-19 into the cell.
In recent years, flavonoids have gained a great amount of interest with regards to their
potential for cardiovascular protection. In fact, many epidemiological studies associate an
increased consumption of foods and beverages rich in flavonoids with a reduced risk of CVD
death [32, 33, 34]. Additionally, several of these flavonoids or their derivatives (i.e., diosmin,
rutin and quercetin) are widely used as pharmaceutical agents for their vasoprotective
properties (i.e., Daflon 500, cantaining flavonoid derivatives hesperedin and diosmin) [35].
Therefore, rutin and other flavonoids used in this study can be used for prophylactic purposes
as ACE II inhibitors and competitor [36, 37].
In conclusion, in silico study is shown that the high binding constants for the ACE II
receptors of flavanones in the ethanolic propolis extract make it a good competitive inhibitor
and protective natural agents for the treatment of Covid-19. However, this study should be
supported with further in vivo studies.
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Figure 1. Chromatogram of nineteen phenolic compounds of HPLC-UV; (1): Gallic acid,
(2): Protocathequic acid, (3): p-OH benzoic acid, (4):catechin, (5): caffeic acid, (6): syringic
acid, (7): epicatechin, (8): p-coumaric acid, (9): ferulic acid, (10): rutin, (11): myricetin, (12):
resveratrol, (13).daidzein, (14); luteolin, (15): t-cinnamic acid, (16): hesperetin, (17): chrysin,
(18):pinocembrin, (19): Caffeic acid phenethyl ester (CAPE).
Figure 2. The two-dimension (2D) and three-dimension (3D) interaction analysis of
ACE II with rutin
Table 1. Phenolic composition of propolis
Group name
Compound name
mg/100g
Hydroxybenzoic acids
Gallic acid
n.d.
Protocateuic acid
n.d.
p-OH Benzoic acid
n.d.
Syringic acid
n.d.
Catechin
Catechin
n.d.
Epicatechin
n.d.
Hydroxycinnamic acids
Caffeic acid
254.501
p-Coumaric acid
63.871
Ferulic acid
n.d.
t-Cinnamic acid
145.455
CAPE
541.213
Flavanons
Rutin
770.970
Myricetin
1567.750
Flavones
Hesperetin
258.010
Chrysin
4678.423
Pinocembrin
1467.260
Isoflavons
Luteolin
684.752
Stilbands and Lignans
Daidzein
n.d
Resveratrol
2.372
n.d: not detected
Table 2: Summary of reference and flavonoids compounds aganist ACE II with binding energy,
Ki and interacted residues in the ACE II binding site.
Ligand Name
Binding
Energy
(kcal/mol)
Ki
(uM)
ACE II residues interacting with ligands
MLN-4760 ((S,S)-2-{1-Carboxy-2-[3-
(3,5-Dıchloro-Benzyl)-3h-Imıdazol-4-
Yl]-Ethylamıno}-4-Methyl-Pentanoic
Acid)*
-7.28
4.65
Tyr127, Arg273, Phe274, Trp271, Arg273,
Phe274, His345, Pro346,Cys361, Thr371,
His374, Glu375, His378,Glu406, Phe504,
His505, Tyr515, Arg518
Caffeic acid (3,4-dihyroxycinnamic
acid)
-5.53
89.08
Arg273, His345, Pro346, Thr347, Ala348,
His374, Glu375, His378, Phe504, His505,
Tyr515
Caffeic acid phenethyl ester (Caffeic
acid 2-phenylethyl ester; β-Phenylethyl
caffeate) (2-Phenylethyl (2E)-3-(3,4-
dihydroxyphenyl)acrylate)
-7.76
2.04
Tyr127, Ser128, Leu144, Glu145, Asn149,
Trp271, Val343, Cys344, His345, Pro346,
Met360, Cys361, Thr362, Lys363, Asp368,
Phe504
Chrysin (5,7-Dihydroxyflavone)
-7.08
6.41
Tyr127, Ser128, Glu145, Asn149, Cys344,
His345, Pro346, Met360, Cys361, Thr362,
Lys363, Asp368, Phe504
Galangin ( 3,5,7-Trihydroxyflavone)
-7.18
5.41
Arg273, Phe274, His345, Pro346, Thr347,
Ala348,Thr371, His374, Glu375, His378,
Glu406, Phe504, His505, Tyr515, Arg518
Myricetin (3,3′,4′,5,5′,7-
Hexahydroxyflavone)
-7.70
2.28
Arg273, Phe274, His345, Pro346, Thr347,
Ala348, Thr371, His374, Glu375, His378,
Glu406, Phe504, His505, Tyr515, Arg518,
Rutin (Quercetin-3-rutinoside hydrate)
-8.98
0.261
Tyr127, Ser128, Leu144, Glu145, Asn149,
Asp269, Met270, Trp271, Arg273, Phe274,
Val343, Cys344, His345, Pro346, Met360,
Cys361, Lys363, Asp367, Asp368, Thr371,
His374, Glu375, Glu406, Phe504,Arg518
Hesperetin (3',5,7-Trihydroxy-4'-
methoxyflavanone)
-7.40
3.79
Arg273, His345, Pro346, Thr347, Ala348,
Trp349, Thr371, His374, Glu375, His378,
Glu406, Phe504, His505, Tyr510, Tyr515,
Arg518
Pinocembrin (5,7-Dihydroxy-2-phenyl-
2,3-dihydro-4H-chromen-4-one)
-7.46
3.38
Tyr127, Ser128, Leu144, Glu145, Asn149,
Trp271, Val343, Cys344, His345, Pro346,
Met360, Cys361, Thr362, Lys363, Asp368
Luteolin (2-(3,4-Dihydroxyphenyl)- 5,7-
dihydroxy-4-chromenone)
-6.93
8.36
Arg273, Phe274, His345, Pro346, Thr347,
Ala348, Trp349, His374, Glu375, His378,
Asp382, Glu402, Phe504, His505, Tyr510,
Tyr515, Arg518
Ouercetin
-7.62
2.62
Arg273, Phe274, His345, Pro346, Thr347,
Ala348, Thr371, Glu375, His374, His378,
Glu406, Phe504, His505, Tyr515, Arg518
*Reference compund
Supplementary Table: Chemical compounds used for the molecular docking screening
Chemical compound
2D-Structure
Caffeic acid (3,4-
dihyroxycinnamic acid)
Caffeic acid phenethyl ester
(Caffeic acid 2-phenylethyl ester;
β-Phenylethyl caffeate) (2-
Phenylethyl (2E)-3-(3,4-
dihydroxyphenyl)acrylate)
Chrysin (5,7-Dihydroxyflavone)
Galangin ( 3,5,7-
Trihydroxyflavone)
Myricetin (3,3′,4′,5,5′,7-
Hexahydroxyflavone)
Rutin (Quercetin-3-rutinoside
hydrate)
Hesperetin (3',5,7-Trihydroxy-4'-
methoxyflavanone)
Pinocembrin (5,7-Dihydroxy-2-
phenyl-2,3-dihydro-4H-chromen-
4-one)
Luteolin (2-(3,4-
Dihydroxyphenyl)- 5,7-
dihydroxy-4-chromenone)
