Agathis robusta Bark Essential Oil Effectiveness against COVID-19: Chemical Composition, In Silico and In Vitro Approaches

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2), the causative agent of Coronavirus Disease 2019 (COVID-19), has seriously threatened global health. Alongside the approved vaccines, the discovery of prospective anti-COVID-19 drugs has been progressively targeted. Essential oils (EOs) provide a rich source of compounds with valuable antiviral activities that may contribute as effective agents against COVID-19. In this study, the EO of Agathus robusta bark was investigated for its chemical composition and its antiviral activity against SARS-CoV2. Overall, 26 constituents were identified by gas chromatography-mass spectrometry (GC-MS) analysis. α-Pinene, tricyclene, α-terpineol, limonene, d-camphene, trans-pinocarveol, α-phellandren-8-ol, L-β-pinene and borneol were the major components. In silico docking of these constituents against viral key enzymes, spike receptor-binding domain (RBD), main protease (Mpro) and RNA-dependent RNA polymerase (RdRp), using Molecular Operating Environment (MOE) software revealed good binding affinities of the components to the active site of the selected targets, especially, the RBD. In Vitro antiviral MTT and cytopathic effect inhibition assays demonstrated a promising anti SARS-CoV2 for A. robusta bark EO, with a significant selectivity index of 17.5. The results suggested using this EO or its individual components for the protection against or treatment of COVID-19.


Introduction
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) is the latest emerged contagious respiratory pathogen associated with the global outbreak of atypical pneumonia pandemic . It was discovered for the first time in December 2019 in Wuhan (China), sequenced by January 2020 and was announced as a pandemic disease by the World Health Organization (WHO) on 11 March 2020 [1,2]. Once exposed to SARS-CoV2, clinical symptoms may appear including fever, headache, runny nose, cough, sore throat and trouble breathing. However, most of the patients with SARS-CoV-2 showed normal and mild symptoms, and the mortality rate is lower than in its relatives: SARS-CoV and MERS-CoV [3]. SARS-CoV2 has been recognized as a positive-sense enveloped, single-stranded RNA virus from the Coronaviridae family [4]. Morphologically, the virion is spherical, having a central nucleus and jutting surface proteins through which the virus is entrenched in the lipid membranes from the host cells. One of these surface proteins was identified as a spike [S] protein that gives a characteristic solar-crown-like appearance to the viruses, thus the name coronaviruses (CoVs) [4]. this century, Baker and Smith studied the steam volatile oils of Australian araucaroids, including A. robusta leaves, where d-α-pinene was noticed as the major constituent of the oil [31,32]. Again, the leaf EO of A. robusta growing in Australia was analyzed at the end of 20th century, and it consisted of mainly spathulenol (36.7%) and rimuene (5.6%) [33]. Recently, A. robusta growing in India was investigated for the chemical compositions of resin and leaf EOs, where 34 constituents were identified in the resin EO comprising isobornyl acetate (37.9%) and limonene (12.3%) as major constituents. On the other hand, the leaf oil composition was quite different, with a total of 43 identified constituents, including β-selinene (18.1%) and rimuene (14.2%) as major constituents [34].
The literature survey indicated that there is no attempt has been made until now to investigate the chemical composition and possible biological activities of the bark EO of A. robusta. Therefore, the present work aimed to explore of the chemical constituents of A. robusta bark EO growing in Egypt and to investigate the oil's ability to inhibit SARS-CoV2 using in silico (on different virus key enzymes) and In Vitro approaches.

Volatile Constituents in A. robusta Bark EO
The hydrodistilled EO obtained from A. robusta bark offered an average yield of 1.6 ± 0.07% v/dried weight (from three independent experiments). A total of 26 components, comprising 88.34% of the total composition, were identified and quantified using gas chromatography-mass spectrometry (GC-MS) analysis. The area percentages of identified compounds and their chemical names are illustrated in Table 1. The different separated essential oil structures as classified according to structure class, oxygenation and cyclization in Table 2. Figure 1 shows the GC/FID chromatogram of the A. robusta bark in a whole chromatogram view ( Figure 1a) and a focused view (Figure 1b). The focused chromatogram view demonstrates the major identified compounds, and the structures of those compounds are revealed in Figure 1c. In elution order from RTX-5MS ® column. a Rt: retention time in minutes. b RI = identification based on retention index relative to standard n-alkanes. c All reported RI are from [35]. d The mean of three independent plant samples from the same area. Standard error of mean was removed to simplify reading and it did not exceed 10% of the mean value.    Three proteins of the COVID-19 virus, the main protease (Mpro), the RNA-dependent RNA polymerase (RdRp) and the virus spike receptor-binding domain (RBD), were implemented to provide insight on binding efficiencies of the A. robusta bark EO with the active sites of the targeted COVID-19 key targets.
Docking studies of tricyclene, α-pinene, d-camphene, limonene, trans-pinocarveol, borneol, α-phellandren-8-ol and α-terpineol on COVID-19 RNA-dependent RNA polymerase (RdRp) revealed that all the compounds reached the catalytic site of the enzyme. In comparison to the native ligand (H3U: 8-(3-(3-aminobenzamido)-4-methylbenzamido) naphthalene-1,3,5-trisulfonic acid), the docked components showed a fair binding affinity and lower RSMD resulting in more stable binding with lower rotations into the active site of RdRp. It was observed that the hydroxyl groups of trans-pinocarveol, borneol, α-phellandren-8-ol and α-terpineol formed hydrogen bond interactions with one of the key amino acid residues in the RdRp active site: Thr591, The hydroxyl group of trans-pinocarveol formed H-bond interactions with the Val367 of RBD, and hydroxyl groups of borneol, α-phellandren-8-ol and α-terpineol interacted with Asn343 through hydrogen bonds, Figure 2. Additionally, minor components also exhibited interactions with the key amino acid residues of the RBD active site, where camphor, cis-verbenol and myrtenal formed H-bonded interactions with Gly339, Ser371 and Leu 368, respectively. Another interesting finding is the H-π interactions between m-cymene, trans-pinocamphone, (-)-carvone and bornyl acetate and Phe338, Trp436, Phe342 and Trp436 amino acid residues, respectively, (   The energy scores for binding of the major components with the three targeted COVID-19 enzymes are represented in Table 3.

Flexible Alignments
The major components were examined for their conformational similarity with the cocrystallized ligand using the flexible alignment tool in Molecular Operating Environment (MOE). The best results of components are demonstrated in Table 4. As indicated from the 3D flexible alignment results, trans-pinocarveol, borneol, α-phellandren-8-ol and αterpineol have similar conformations to the co-crystallized ligand.
The cyclic moiety in the components overlaps with the 2-deoxy-beta-D-glucopyranose moiety of the co-crystallized ligand. This elucidates that trans-pinocarveol, borneol, αphellandren-8-ol and α-terpineol would share the same active site interactions. This was approved by the docking process as they exhibited hydrogen bond interactions with the key amino residues present in the active site. Table 4.

S kcal/mol Flexible Alignment Component
The cyclic moiety in the components overlaps with the 2-deoxy-beta-D-glucopy nose moiety of the co-crystallized ligand. This elucidates that trans-pinocarveol, borne α-phellandren-8-ol and α-terpineol would share the same active site interactions. This w approved by the docking process as they exhibited hydrogen bond interactions with key amino residues present in the active site.

S kcal/mol Flexible Alignment Component
The cyclic moiety in the components overlaps with the 2-deoxy-beta-D-glucopy nose moiety of the co-crystallized ligand. This elucidates that trans-pinocarveol, borne α-phellandren-8-ol and α-terpineol would share the same active site interactions. This w approved by the docking process as they exhibited hydrogen bond interactions with key amino residues present in the active site.

S kcal/mol Flexible Alignment Component
The cyclic moiety in the components overlaps with the 2-deoxy-beta-D-glucopy nose moiety of the co-crystallized ligand. This elucidates that trans-pinocarveol, borne α-phellandren-8-ol and α-terpineol would share the same active site interactions. This w approved by the docking process as they exhibited hydrogen bond interactions with t key amino residues present in the active site.

S kcal/mol Flexible Alignment Component
The cyclic moiety in the components overlaps with the 2-deoxy-beta-D-glucop nose moiety of the co-crystallized ligand. This elucidates that trans-pinocarveol, born α-phellandren-8-ol and α-terpineol would share the same active site interactions. This approved by the docking process as they exhibited hydrogen bond interactions with key amino residues present in the active site.
Log P o/w (MLOGP) for the compound lipophilicity should be lower than 4.15 so as not to violate the Lipinski's rule. Limonene, trans-pinocarveol, borneol, α-phellandren-8-ol and α-terpineol do not violate Lipinski's rule, as the MLOGP ranged from 2.20 to 3.27. However, tricyclene, α-pinene and d-camphene slightly violate the rule, as the MLOGP was > 4.15, ranging from 4.29 to 4.43. The pink region of the bioavailability radar ( Figure 3) indicted a favorable properties range, Table 5.
Overall, the components relatively fulfil the criteria of drug-likeness, suggesting that they are potential drug candidates.

Cytotoxicity and Antiviral Activity of A. robusta Bark EO
To evaluate the antiviral activity of A. robusta bark EO, the half maximal cytotoxic concentration (CC 50 ) and the 50% inhibitory concentration (IC 50 ) were calculated (Figure 4). The oil showed CC 50 = 5.675 µL/mL and IC 50 = 0.324 µL/mL. The overall therapeutic activity was determined by calculating the selectivity index (SI). The results revealed that A. robusta bark EO exhibited a promising In Vitro activity against NRC-03-nhCoV with a significant selectivity index (17.5) for antiviral activity relative to cellular toxicity.

Discussion
The current crisis of COVID-19 demonstrates the need for potential antiviral drug therapies. Scientists from several medical fields have a serious interest in the invention of new bio-active antiviral molecules from the EOs of aromatic medicinal plants as promising sources to prevent viral replication and infection.
Several EOs and their isolated components have been described in In Vitro and in vivo models as effective antiviral agents against DNA and RNA viruses in different host cell lines by obstructing different steps of the viral life cycle [38]. These impacts are mainly based on the oil composition of mono-and sesquiterpene hydrocarbons together with phenolic, alcoholic, and other oxygenated other components [38,39]. Several EOs have been recognized for their antiviral activities against a diversity of viruses, including human immunodeficiency virus (HIV), human herpes virus (HSV), avian influenza A virus (H5N1), influenza A virus (H1N1) and Zika virus [23]. Additionally, modern studies disclosed that some EOs have exerted a huge antiviral activity against SARS-CoV2. For example, Eucalyptus oil and its active constituent (eucalyptol) exhibited a promising therapeutic potential in the prevention and treatment of COVID-19 [40]. Furthermore, garlic EOs and their isolated constituents, particularly diallyl sulphide and allyl trisulphide, lemon and geranium EOs and their derivative compounds exemplify potential natural antiviral agents, which hinder coronavirus entry into the human body [41]. Various plants have been proposed as potential sources for anti-SARS-CoV-2 EOs, including Eugenia brasiliensis, Melissa officinalis, Cedrus libani, Zingiber zerumbet, Zataria multiflora and Vetiveria zizanoides [42].
Isolated monoterpenes such as α-pinene and α-terpineol exerted significant antiviral activity against HSV-1, with elevated selectivity index, demonstrated by the inhibition of HSV-1 plaque formation [43]. α-Terpineol owns activity against IFV-A by delaying the early steps in the viral replication cycle [38]. α-Pinene and β-pinene inhibit the binding between RNA and the IBV N-protein, thereby possessing anti-IBV activity [44]. Moreover, in silico studies emphasized their activity against SARS-CoV-2 by binding to the active site of Mpro, human serine protease TMPRSS2 and spike (S) glycoprotein [38,43].
Virtual screening of these constituents was carried out using Molecular Operating Environment (MOE) software to explore their possibilities of creating effective docking with the spike receptor-binding domain (RBD), in addition to the viral vital enzymes, main protease (Mpro) and RNA-dependent RNA polymerase (RdRp). Moreover, 3D crystal structures of the possible ligands for these COVID-19 targets (PDB: 6LU7, 7D4F and 7BZ5) were utilized in molecular modelling design to elucidate the inhibitory activity of A. robusta bark EO against the virus. The molecular docking of major components of A. robusta bark essential oil with COVID-19 Mpro (PDB: 6LU7) demonstrated good binding affinity revealed by the docking scores ranging from −4.1916 to −5.0752 kcal/mol. Moreover, visual inspection of the docking poses showed that the major components could fit within the main cleft of Mpro active site through hydrogen bond formation with Met49, His164 and Met165 in addition to the H-pi bond interaction with His41. The docking results with COVID-19 RdRp show that major components of A. robusta bark essential oil bind to the active site of the targeted enzyme, so they could interfere with the viral RNA synthesis. Each component of A. robusta bark essential oil spike RBD show favorable binding affinities obtained from docking study in an energy score ranging of 3.8556 to −4.7246 kcal/mol in comparison with the native ligand (−4.5304 kcal/mol). More negative expected binding affinity suggested a stronger favorable protein-ligand complex. Binding of the components of A. robusta bark essential oil with the active pocket of the spike RBD of COVID-19 could interfere in the refolding of the COVID-19 spike, preventing COVID-19 spikes from binding with human ACE2 and inhibiting the process of viral infection. Moreover, ADME assessment revealed that the components relatively fulfill the criteria of drug-likeness, indicating the studied components of the oil as potential drug candidates. These results recommend using each component in this EO, especially the major ones or the whole EO in the treatment or protection of COVID-19.
The in silico study encouraged investigation into the antiviral activity of the A. robusta bark EO, which was determined by calculating the half-maximal cytotoxic concentration (CC 50 ) and the 50% inhibitory concentration (IC 50 ) that required reducing the virus-induced cytopathic effect (CPE) by 50%. The overall therapeutic activity was determined in terms of the selectivity index (SI), which is the ratio of the CC 50 to IC 50 . High SI values (>10) suggest the drug is a good candidate as antiviral drug and that the drug effect is more directed toward viral inhibition with less cytotoxic activity toward the host cell [50,51]. The results declared a promising In Vitro activity for A. robusta bark EO exhibited against SARS-CoV2 with a significant selectivity index (17.5) for antiviral activity relative to cellular toxicity.
In a summary, A. robusta bark EO could be an auspicious drug candidate in treatment of COVID-19 infection. The EO could be used either as a prophylactic or on the inception of SARS-CoV2 infection to act as anti SARS-CoV2 through the interaction with the RBD resulting in the subsequent inhibition of the viral envelope fusion with host cellular membranes or through the inhibition of viral replication by targeting the viral Mpro and/or RdRp. Clinical trials in humans are necessary to assist realizing the role of A. robusta bark EO intake during COVID-19 infection.

Plant Material
The fresh bark of the cultivated tree of A. robusta (50 years old in age) was collected at the cone maturation stage from El-Orman Botanical Garden, Giza, Egypt (July 2021). Eng. Therese Labib, Consultant of Plant Taxonomy at Ministry of Agriculture and the Former Director of Orman Botanical Garden, Giza, Egypt, kindly confirmed the identity of the plant. A voucher specimen (ZU-Ph-Cog-0100) was kept at the Herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Zagazig University.

Isolation of Bark EO
The shade-dried bark of A. robusta (300 g) was pulverized into a course powder and then subjected to hydrodistillation by the Clevenger-type apparatus according to Perveen, et al. [52]. Hydrodistillation was performed under atmospheric pressure at about 100 • C for 4 h. The recovered volatile fraction was dried (anhydrous sodium sulphate), and the obtained EO samples were retained in brown vials (to protect photosensitive compounds) in the refrigerator (4 • C) until used.

GC-FID and GC-MS Analyses
The isolated bark EO was subjected to GC-MS analysis according to Perveen, et al. [52]. Gas chromatography/flame ionization (GC/FID) analysis was carried out using GC-2010 Plus (Shimadzu Corporation, Kyoto, Japan) gas chromatograph supplied with FID-2010 Plus detector and a split/splitless injector. The column was RTX-5MS ® fused silica capillary (0.25 µm film thickness and 30 m × 0.25 mm i.d.); the oven temperature was initially kept at 45 • C for 4 min and then increased to 270 • C at 4 • C/min and then held for 15 min. The carrier gas was helium with a flow rate of 2.0 mL/min; the temperatures of detector and injector were 300 and 250 • C, respectively, the split ratio was 1:20 and the injection volume is 1 µL. Quantification of EO components was accomplished by relative parentage area calculations. The relative percentage of the EO constituents was estimated on basis of the FID responses from the total peak area using percentage area normalization [53][54][55]. Gas chromatography/mass spectrometry (GC/MS) data were recorded on GCMS-QP2010 Plus (Shimadzu corporation, Kyoto, Japan). For the mass spectrometer, the ionization energy was 70 eV. The other conditions were identical to those mentioned for GC/FID; however, EI mass spectra were collected over the range of m/z 40-700 in full scan mode. Kovat's retention indices (RI) were determined with respect to a set of co-injected standard hydrocarbons (C10-C28, Sigma Aldrich, Darmstadt, Germany). Identification of the compounds was carried out by comparing their spectral data and retention indices with Wiley Registry of Mass Spectral Data 10th edition (April 2013), NIST 11 Mass Spectral Library (NIST11/2011/EPA/NIH) and literature data [35]. The identified compounds and their percentages are listed in Table 1.  [56]. The docking placement methodology is triangle matcher, using London dG as the initial scoring function and GBVI/WSAdG as the final scoring function. The receptor was kept rigid, and the ligands were allowed to be flexible during the refinement process. The docking energy scores of the best-fitted poses of the ligands with the active site at the protein sites were recorded.
The crystal structures of the selected SARS-COVID-19 enzymes were retrieved from the RCSB Protein Data Bank website (http://www.rcsb.org (accessed on 28 October 2021)) [16] (PDB codes: 6LU7, 7D4F and 7BZ5) [18,57,58] , Table S4. The crystalized water molecules were ignored from the enzyme complexes. Then, the protein structures were prepared individually using the MOE QuickPrep protocol for adding hydrogen atoms and partial charges through the Amber10: EHT forcefield.

Ligand Preparation
The major components of A. robusta bark EO (tricyclene, α-pinene, d-camphene, limonene, trans-pinocarveol, borneol, α-phellandren-8-ol and α-terpineol) were drawn utilizing the Chemdraw ® program, then transferred to the MOE using smiles format. The energy of the components was minimized with root mean square (RMS) gradient 0.0001 kcal/mol and finally preparing a database file. The co-crystallized ligands were re-docked into the active site of the corresponding enzyme following the same set of the parameters as mentioned above for active site validation. Root mean square deviation (RMSD), docking energy score (S) and visual inspection of 2D and 3D planes of the componenttargeted enzyme interactions were utilized for the analysis of the docking results.

Flexible Alignments
Flexible alignment studies were performed on trans-pinocarveol, borneol, α-phellandren-8-ol and α-terpineol and the co-crystallized ligand of COVID-19 virus spike receptorbinding domain (RBD) using MOE 2019 software [56]. Flexible alignment mode and the resulting conformations were assessed according to the alignment score of the configuration (S). The latter is the sum of the similarity measure of configuration (F), and the average strain energy of the molecules in the alignment in kcal/mol (U). The lower S value indicates better alignment.

ADME Assessment of the Major Components of A. robusta Bark EO
The major components of A. robusta bark EO (tricyclene, α-pinene, d-camphene, limonene, trans-pinocarveol, borneol, α-phellandren-8-ol and α-terpineol) were subjected to ADME prediction using the SwissADME server using compounds in SMILES format. Swiss ADME [59], supported by SIB Swiss Institute of Bioinformatics, gives free access to several factors and predictive models to compute the physicochemical properties and estimate the pharmacokinetics and drug likeness of various small molecules.

MTT Cytotoxicity Assay
The half-maximal cytotoxic concentration (CC 50 ) was assessed according to Mosmann [60]. The pure separated EO sample was serially diluted (v/v) to make 10 working solutions with Dulbecco's Modified Eagle's medium (DMEM). The 3-(4, 5-dimethylthiazol -2-yl)-2, 5-diphenyltetrazolium bromide (MTT) method, with minor modifications, was used to evaluate the cytotoxic activity of the A. robusta bark EO in VERO-E6 cells. Briefly, the cells were seeded in 96 well-plates (100 µL/well at a density of 3 × 10 5 cells/mL) and then incubated for 24 h at 37 • C in 5% CO 2 . After 24 h, cells were treated with various concentrations of the tested sample in triplicates. After 24 h, the supernatant was discarded, cell monolayers were washed with sterile 1x phosphate buffer saline (PBS) 3 times and MTT solution (20 µL of 5 mg/mL stock solution) was added to each well and incubated at 37 • C for 4 h followed by medium aspiration. In each well, the formed formazan crystals were dissolved with 200 µL of acidified isopropanol (0.04 M HCl in absolute isopropanol = 0.073 mL HCL in 50 mL isopropanol). Absorbance of formazan solutions was measured at λ max 540 nm with 620 nm as a reference wavelength using a multi-well plate reader. The CC 50 values were calculated using nonlinear regression analysis of GraphPad Prism software (version 5.01) by plotting log inhibitor versus normalized response (variable slope).

Inhibitory Concentration 50 (IC 50 ) Determination
Determination of the inhibitory concentration 50 (IC 50 ) was carried out as previously described by Mostafa, et al. [61]. In 96-well tissue culture plates, 2.4 × 10 4 Vero-E6 cells were allocated in each well and incubated overnight at a humidified 37 • C incubator under 5% CO 2 condition. The cell monolayers were then washed once with 1× PBS and exposed to virus adsorption (hCoV-19/Egypt/NRC-03/2020 (Accession Number on GSAID: EPI_ISL_430820)) for 1 h at room temperature (RT). The cell monolayers were further overlaid with 100 µL of DMEM containing different serial concentrations of A. robusta bark EO as specified above in working solution. After incubation at 37 • C in 5% CO 2 incubator for 72 h, the cells were fixed with 100 µL of 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet in distilled water for 15 min at RT. The crystal violet dye was then dissolved using 100 µL absolute methanol per well and the optical density of the color is measured at 570 nm using Anthos Zenyth 200rt plate reader (Anthos Labtec Instruments, Heerhugowaard, The Netherlands). The IC 50 of the sample is that required to reduce the virus-induced cytopathic effect (CPE) by 50%, relative to the virus control.

Calculation of Selectivity Index
The selectivity index (SI) is a ratio the cytotoxic concentration of a drug to its effective bioactive concentration. The more elevated the SI ratio, the theoretically more efficient and safer a drug is. The SI value was calculated using the following equation: SI = CC 50 /IC 50 .
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/plants11050663/s1, Table S1: 3D-Binding mode of the major components of A. robusta bark EO inside the Mpro active site (PDB code: 6LU7); Table S2: 3D-Binding mode of the major components of A. robusta bark EO inside COVID-19 RNA-dependent RNA polymerase (PDB code: 7D4F); Table S3: Molecular docking details of A. robusta bark EO with RBD, Table S4: The target enzymes involved for docking of the major components of A. robusta bark EO.