Potential anti-COVID-19 activity of Egyptian propolis using computational modeling

Aim: To investigate the prospective anti COVID-19 activity of Egyptian propolis. Material & methods: Propolis samples were collected from different Egyptian geographical areas and characterized using standardized methods, scanning electron microscope and gas chromatography/mass spectrometry along with computational modeling to predict the anti-COVID-19 activity. Results & conclusion: Gas chromatography/mass spectrometry analysis of Menoufia propolis proved the presence of Octatriacontyl pentafluoropropionate (4.2%). Docking analyses declared that Octatriacontyl pentafluoropropionate is well oriented inside the enzyme pockets, in addition to excellent binding manner with the active site of the target macromolecules (RNA-dependent RNA polymerase, Spike protein S1 and main protease) in relation to some broad-spectrum antiviral agents. Menoufia propolis could be a promising candidate in the combat against the pandemic COVID-19.

anti-inflammatory, antihepatotoxic and antitumor [7]. Sforcin [12] reported the safety profile of the propolis as a nontoxic compound to humans or mammals with DL50 ranged from 2 to 7.3 g/kg in mice; however for humans DL50 reached 1.4 mg/kg (∼70 mg/day).
Novelty impeded in the present work is that propolis samples from different Egyptian geographical areas were collected and comparative chemical composition was conducted through analytical and chromatographic methods with concomitant to the anti-COVID-19 activity.

Propolis analysis Scanning electron microscopic examination of propolis samples
Propolis samples were examined using scanning electron microscope JEOL JSM-6390LV (USA). Each sample was oven dried (105˚C/45 min), then coated with metallic gold ions and stored in plastic boxes, which were sealed with parafilm (PARAFILM 1M) to prevent moisture absorption and left at room temperature for 24 h. The samples were then analyzed at different magnifications (Voltage 12 kV, Working Distance 12 mm, Spot size 44, Vacuum Mode HV) [13].

Preparation of the propolis extract
Propolis samples were chopped into small pieces and extracted (20% w/v) by maceration with ethanol (99.9%) for 7 days in the dark with continuous stirring followed by sonification for one hour at 70 • C [14] then filtered through a Whatman filter paper No. 1 and the extracts were stored in a dark jar at 4 • C for further analysis.

Chemical analysis
Qualitative method Standard procedures were used to determine the presence or absence of different secondary metabolites of propolis ethanolic extracts such as phenolic compounds, terpenoids, steroids, flavonoids, tannins and glycosides [15].

Quantitative method
Gas chromatography/mass spectrometry analysis of different propolis samples About 2 mg of the propolis ethanolic extract was dissolved in 20 μl dry pyridine, then 30 μl N,0bis(trimethylsilyl)trifluoroacetamide (BSTFA) were added. The mixture was heated at 80 • C for 20 min and diluted with 100 μl pyridine then each sample was analyzed using gas chromatography/mass spectrometry (GC/MS). Compounds identification was done using computer search user-generated reference libraries, incorporating mass spectra (Wiley 138 and Nist 98 libraries) [16].

Molecular modeling
In the current study, molecular docking was performed to predict the binding affinity of the test compounds toward the target enzymes RNA-dependent RNA polymerase (RdRp), Spike protein S1 and main protease (3CLpro, or 3-chymotrypsin-like protease). This study could describe mode of binding of screened compounds to the active site of target macromolecules and type of interactions of different conformations. The scoring function (S) could compute the binding energy to select orientations with the lowest energy state. It also could be used as indicator of relative strength of interactions with the active site [17].
Molecular Operating Environment (MOE) software was utilized in all the docking experiments, with the x-ray crystal structures of the docking targets; optimized SARS-CoV-2 RdRp (PDB ID: 7BTF), SARS-CoV-2 Spike protein S1 (PDB ID: 6VW1) and SARS-CoV-2 3CLpro (PDB ID: 6Y2G) models were obtained from the Protein Data Bank (PDB) website.
Molecular docking results are often validated using a training set of experimental ligand-protein complexes, and the accuracy of these docking programs is mainly dependent on the used training set [18]. Due to the lack of known ligands, it is important to ensure that the used software is able to replicate the binding mode of a known experimental  inhibitor for the studied enzymes. Although neither an effective antiviral drug nor a vaccine against COVID-19 is currently available, several reports have indicated that RdRp inhibitors (remdesivir), influenza glycoproteins inhibitors (umifenovir), in addition to HIV-1 protease inhibitors (lopinavir) have the potential for designing active SARS-CoV-2 inhibitors [19,20]. In the attempt to have reference values (positive control), remdesivir, umifenovir and lopinavir antiviral drugs were considered as comparative standards for the molecular docking.

Propolis analysis
Propolis used in the present study was collected from different geographic areas namely: Alexandria, Tanta and Menoufia. Alexandria and Menoufia propolis were dark brown while Tanta propolis was light brown. Propolis classification showed variations according to the botanical origin due to the difference in the dominant pollen grains [21]. Pollen grain analysis was investigated according to Temizer et al. [21], which revealed the relatedness of Alexandria propolis to the family Cyperaceae (that was occasionally found in Tanta propolis) while that of Tanta and Menoufia were related to the family Asteraceae.

Scanning electron microscopic examination of propolis samples
Scanning electron microscopic analysis of the propolis samples under test ( Figure 1A-C) showed 3D structures and morphological features of the propolis samples. It was revealed that no foreign materials were detected and wrinkled surfaces covered with wax layers were observed. Table 1. Qualitative chemical analysis of the propolis samples.

Chemical analysis of the propolis Qualitative analysis
The qualitative chemical analysis for the tested propolis samples showed variations among the samples that were geographically different. Steroids were not detected in all the tested samples. In the present study a detailed investigation on the composition of the Egyptian propolis collected from specific areas was analyzed (Table 1).

GC/MS analysis
On referring the results of the present work to the data base with the corresponding acquisition time (Figures 2A-C), 26 compounds have been identified. It was revealed that the common detected components in the tested extracts were heptacosane, octacosanol and pinocembrin that were in average concentrations except for that of Menoufia and Alexandria propolis, respectively ( Table 2). The presence of unusual substances such as sterol precursors and some alcohols may be due to the different geographic areas of propolis in Egypt.
Anti-COVID-19 activity using molecular docking In the current study, molecular docking was performed to predict the binding affinity of the test compounds toward the target enzymes RNA-dependent RNA polymerase, spike protein S1 and main protease ( Table 3). Results of the docking studies showed that most compounds possessed promising binding scores, in addition to excellent binding manner with the active site of target macromolecules in comparison to the reference antiviral drugs remdesivir, lopinavir and umifenovir. The binding interactions of test compounds were investigated to study their modes of binding and orientations related to their antiviral activity.
The interaction of 3CLpro with the reference drug KALETRA R (lopinavir) displayed hydrogen bond interaction with Glu166 and hydrophobic interaction with Thr26 and Asn142 ( Figure 5A). Docking results for octatriacontyl  pentafluoropropionate revealed hydrogen bond interaction with Met49 ( Figure 5B).

Discussion
In the present study, Egyptian propolis samples were analyzed and investigated for their anti-COVID-19 activity using molecular docking. Pollen grain analysis was investigated according to Temizer et al. [22] which revealed that the family Cyperaceae was dominant in Alexandria propolis while family Asteraceae was dominant in Tanta and Menoufia propolis samples. Scanning electron microscope analysis of propolis samples revealed that all the propolis samples have a putative gel like morphology. Rahim et al. [23] stated that the propolis samples with a homogenized flat topography (with intervals of flaking out and crumpled-like surface topography) may act as building blocks of the hive. Further chemical analysis was applied on the propolis samples, which revealed that the presence of tannins, phenols and steroids varies among the samples which prove that the propolis composition differs according to the correspondence area and the environmental conditions including flora [24]. GC/MS analysis revealed that acids were the main component in the propolis samples. Hegazi et al. [24] reported that the characteristic groups Table 3. The binding scores of test compounds toward three protein targets (RNA-dependent RNA polymerase, spike protein S1 and main protease) from 2019-nCoV.    of poplar propolis were aliphatic, aromatic acids, aromatic acid esters and flavonoids and it was characterized by the presence of some triterpenoids. Ibrahim et al. [25] found that 11 phenolic compounds in propolis samples that were collected from Abees Apiculture Unit, Agricultural Research Farm, University of Alexandria, Alexandria.
In the current study, molecular docking was performed to predict the binding affinity [26] of the propolis compounds toward RNA-dependent RNA polymerase, spike protein S1 and main protease ( Table 3). Most of the tested fractions showed promising binding scores in relation to the reference antiviral drugs namely: remdesivir, lopinavir and umifenovir. In a trial to investigate the binding interactions of the tested fractions; the modes of binding and orientations were investigated in relation to their antiviral activity. Octatriacontyl pentafluoropropionate showed hydrogen bond interaction with Lys798, Asp420 and Met49, in addition to H-arene interaction with Tyr505 inside RNA-dependent RNA polymerase, spike protein S1 and main protease enzymes pockets, respectively.

Conclusion
Data from the present study concluded that Egyptian propolis samples proved to have variable chemical composition according to the botanical origin and geographical distribution. Menoufia propolis reported high anti-COVID-19 activity in relation to the tested BSAA and it is recommended as a promising candidate in the combat against the pandemic COVID-19.

Summary points
• The present study aimed to investigate the prospective anti-COVID-19 activity of Egyptian propolis.
• Propolis samples were collected from different Egyptian geographic areas.
• Qualitative and quantitative propolis analyses were evaluated. Gas chromatography/mass spectrometry proved the presence of octatriacontyl pentafluoropropionate (4.2%). • Computational modeling of propolis fractions was evaluated in relation to some known antiviral drugs namely: remdesivir, lopinavir and umifenovir. • Most of the tested compounds showed a promising binding score.
• Docking analyses declared that octatriacontyl pentafluoropropionate is well oriented inside the enzyme pockets and showed hydrogen bond interaction with Lys798, in addition to excellent binding manner with the active site of the target macromolecules (RNA-dependent RNA polymerase, Spike protein S1 and main protease) in relation to some broad-spectrum antiviral agents. • Menoufia propolis could be recommended as a promising candidate in the combat against the pandemic COVID-19.
Financial & competing interests disclosure