An investigation of ethanolic propolis extracts: Their potential inhibitor properties against ACE-II receptors for COVID-19 treatment

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 Covid19 treatment, but this needs to be supported by experimental studies.


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.
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]. 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 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
Raw propolis samples obtained experienced beekeepers in 2018 from Black Sea Region, Turkey.

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:

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, pcoumaric 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 K i 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, K i 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 pialkyl 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. 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.