Investigating the Potential Anti-SARS-CoV-2 and Anti-MERS-CoV Activities of Yellow Necklacepod among Three Selected Medicinal Plants: Extraction, Isolation, Identification, In Vitro, Modes of Action, and Molecular Docking Studies

The anti-MERS-CoV activities of three medicinal plants (Azadirachta indica, Artemisia judaica, and Sophora tomentosa) were evaluated. The highest viral inhibition percentage (96%) was recorded for S. tomentosa. Moreover, the mode of action for both S. tomentosa and A. judaica showed 99.5% and 92% inhibition, respectively, with virucidal as the main mode of action. Furthermore, the anti-MERS-CoV and anti-SARS-CoV-2 activities of S. tomentosa were measured. Notably, the anti-SARS-CoV-2 activity of S. tomentosa was very high (100%) and anti-MERS-CoV inhibition was slightly lower (96%). Therefore, the phytochemical investigation of the very promising S. tomentosa L. led to the isolation and structural identification of nine compounds (1–9). Then, both the CC50 and IC50 values for the isolated compounds against SARS-CoV-2 were measured. Compound 4 (genistein 4’-methyl ether) achieved superior anti-SARS-CoV-2 activity with an IC50 value of 2.13 µm. Interestingly, the mode of action of S. tomentosa against SARS-CoV-2 showed that both virucidal and adsorption mechanisms were very effective. Additionally, the IC50 values of S. tomentosa against SARS-CoV-2 and MERS-CoV were found to be 1.01 and 3.11 µg/mL, respectively. In addition, all the isolated compounds were subjected to two separate molecular docking studies against the spike (S) and main protease (Mpr°) receptors of SARS-CoV-2.


Introduction
Coronavirus disease 2019 (COVID-19) is defined as an illness and the outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) started in Wuhan, China, spread to several other countries, and now is in its exponential phase of spread [1,2]. The most recent outbreaks of SARS-CoV and the Middle East respiratory syndrome-related coronavirus (MERS-CoV) happened in China and Saudi Arabia, respectively [3,4]. The ongoing coronavirus disease 2019 (COVID- 19) pandemic has created an alarming situation were reported as the best solvents to be used to obtain an extract from many medicinal plants with potent antiviral activities [18,30]. The highest antiviral activity of several medicinal plants was associated with the crude ethyl acetate and/or dichloromethane extract, indicating the possibility of synergism among the antiviral constituents of the extract which may act by a different mode of action [32]. So, the ethyl acetate/dichloromethane (1:1, v/v) extract of each medicinal plant was used in the current study.
There is no doubt that the in silico studies in the process of drug discovery constitute very important and crucial pathways for the rapid introduction of new drugs [33,34]. Computational methods help scientists to save effort and time [7,35,36]. Notably, molecular docking is one of the most widely used computational methods to predict and/or explain the mechanism of action for a certain ligand against a specific receptor [37][38][39].
Herein, a comparative screening of the selected plants to evaluate their potential anticoronavirus activities against MERS-CoV propagation was carried out. Their cytotoxic activities were tested in Vero-E6 cells and the plot of % cytotoxicity versus sample concentration was used to calculate the concentration which exhibited 50% cytotoxicity (CC 50 ). A plaque reduction assay was employed using the safe dose of each extract to evaluate its effect on virus propagation. Furthermore, the work was extended to study the possible mode of action of virus inhibition at three different levels: viral replication, viral adsorption, and virucidal activity with different safe concentrations. Additionally, isolation and structural identification of the compounds of S. tomentosa which were proposed to cause the antiviral activity against MERS and SARS were carried out. Finally, the anti-SARS-CoV-2 activities of the nine isolated and identified compounds from S. tomentosa ( Figure 1) were recorded as well.   (7), 6-methoxy-7-O-β-D-glucoside apigenin (8), and daucosterol (9).
It is worth mentioning that compounds 1, 6, and 8 were isolated for the first time from the Sophora genus. All compounds were isolated for the first time from this species except compounds 3 and 5, while compound 9 was recently isolated from S. mollis (Royle) Graham Ex Baker [47].

Antiviral Activity for Three Medicinal Plants against MERS-CoV by Plaque Reduction Assay
In the present study, in searching for new anti-MERS-CoV agents, the study started with measuring the cytotoxic activity of each extract of the three selected plants in Vero-E6 cells using an MTT assay with some modification [48]. The concentration which exhibited 50% cytotoxic concentration (CC 50 ) was calculated and was found to be equal to 22.52, 31.60, and 20.86 µg/mL for A. indica (Neem), A. judaica, and S. tomentosa, respectively, Figure 2. According to the results of the cytotoxicity assay ( Figure 2) to determine CC50, different safe concentrations were selected to start plaque reduction assays for each extract against MERS-CoV virus propagation [50] (Table 1).   According to the results of the cytotoxicity assay ( Figure 2) to determine CC 50 , different safe concentrations were selected to start plaque reduction assays for each extract against MERS-CoV virus propagation [49] (Table 1).  Table 1 represents the antiviral effects of the three plants' extracts after being measured using a plaque reduction assay. The antiviral effects were from 96 to 88% for Sophora with concentrations of 12.50 to 3.13 µg/mL. On the other hand, they were from 92 to 85% for Artemisia with concentrations of 12.50 to 3.13 µg/µL. However, the lowest effect was for neem with 54% inhibition at the highest concentration (3.13 µg/mL).
Investigation of the chemical composition of the extract of S. tomentosa revealed the presence of four isoflavonoids (3)(4)(5)(6). The isoflavonoids are an important polyphenolic subclass of flavonoids with a skeleton based on a 3-phenylchroman structure and their antiviral powers have been proven in several scientific reports before. Some isoflavonoids and flavonoids have been reported as potential candidates against viral infection [50]. The current results demonstrated that necklacepod plant extract showed 96% inhibition against the MERSrelated coronavirus. This is in agreement with the previous results that showed that the flavones (luteolin, quercetin, and apigenin) isolated from S. tomentosa and Artemisia judaica L. were reported to inhibit severe acute RS-CoV 3CL pr• activity with IC 50 values of 20.2, 23.8, and 280.8 µM, respectively [51]. Polyphenols (flavonoids) attack viral proteins present in the viral membrane or inside the virus particle. Phenolics are active against free viral particles but not-or to a lesser degree-after a virus has entered a host cell [52]. Limited data associated with the antiviral activities of neem may be due to its phytoconstituents not having a promising effect of as antivirals.

Mode of Action against MERS-CoV
The possible mode of action for herbal products may be investigated at three different levels: inhibition of viral replication, viral adsorption, and virucidal activity. Herein, the mode of action for the most promising medicinal plants with different safe concentrations was illustrated for both S. tomentosa and A. judaica ( Figure 3).
The results showed that they achieved 99.5% and 92% inhibition effects at 1.56 µg/mL for S. tomentosa and A. judaica, respectively, with virucidal as the main mode of action. This may be attributed to their direct effect on the virus which lost the ability of infectivity. So, the two plants have a virucidal effect more than the effect on viral adsorption to the cells and have less effect on viral replication. 2)). The virucidal effect was the main mode of action for the two extracts but has less effect on viral adsorption and a very low effect on viral replication.

Comparison between the Antiviral Activity of S. tomentosa against MERS-CoV and SARS-CoV-2
The antiviral activity of S. tomentosa (necklacepod) was illustrated by plaque reduction assay against MERS-CoV isolate compared with SARS-CoV-2 isolate as depicted in Table 2. The results showed that antiviral activity against SARS-CoV-2 was very high (100%) and the extract succeeded in achieving full inhibition of viral propagation at different concentrations (12.50 and 6.25 µ g/mL). On the other hand, it showed a slightly lower inhibition against MERS-CoV (96%) at the highest concentration (12.50 µ g/mL). Table 2. Comparison between the antiviral activity of S. tomentosa (necklacepod) using plaque reduction assay against MERS-CoV isolate (NRCE-HKU270 (Accession Number: KJ477103.2)) and SARS-CoV-2 isolate (hCoV-19/Egypt/NRC-3/2020).  2)). The virucidal effect was the main mode of action for the two extracts but has less effect on viral adsorption and a very low effect on viral replication.

Comparison between the Antiviral Activity of S. tomentosa against MERS-CoV and SARS-CoV-2
The antiviral activity of S. tomentosa (necklacepod) was illustrated by plaque reduction assay against MERS-CoV isolate compared with SARS-CoV-2 isolate as depicted in Table 2. The results showed that antiviral activity against SARS-CoV-2 was very high (100%) and the extract succeeded in achieving full inhibition of viral propagation at different concentrations (12.50 and 6.25 µg/mL). On the other hand, it showed a slightly lower inhibition against MERS-CoV (96%) at the highest concentration (12.50 µg/mL). Table 2. Comparison between the antiviral activity of S. tomentosa (necklacepod) using plaque reduction assay against MERS-CoV isolate (NRCE-HKU270 (Accession Number: KJ477103.2)) and SARS-CoV-2 isolate (hCoV-19/Egypt/NRC-3/2020). The crystal violet assay to determine both the CC 50 and IC 50 of the isolated compounds from S. tomentosa L. against SARS-CoV-2 was applied ( Figure 4).

MERS-CoV
The crystal violet assay to determine both the CC50 and IC50 of the isolated compounds from S. tomentosa L. against SARS-CoV-2 was applied ( Figure 4).
Generally, the highest anti-SARS-CoV-2 activity of S. tomentosa was associated with the crude ethanolic extract, indicating the possibility of synergism between the antiviral phytochemicals 1-9 of the extract.
Herein, compound 4 (genistein 4'-methyl ether) was found to achieve superior anti-SARS-CoV-2 activity with an IC 50  It was important to test the mode of action of S. tomentosa against the SARS-CoV-2 isolate ( Figure 5). The results showed that two mechanisms of action (virucidal and adsorption) were effective at 12.50 and 6.25 µg/mL with an inhibition percent of more than 99%. On the other hand, the extract's efficacy by adsorption decreased when decreasing the concentration but was still high with a virucidal mechanism of action (>99%) at 3.12 and 1.56 µg/mL. It was important to test the mode of action of S. tomentosa against the SARS-CoV-2 isolate ( Figure 5). The results showed that two mechanisms of action (virucidal and adsorption) were effective at 12.50 and 6.25 µ g/mL with an inhibition percent of more than 99%. On the other hand, the extract's efficacy by adsorption decreased when decreasing the concentration but was still high with a virucidal mechanism of action (>99%) at 3.12 and 1.56 µ g/mL. Both the 50% cytotoxic concentration (CC50) and the 50% inhibitory concentration (IC50) were measured in the same conditions for S. tomentosa ( Figure 6).

Sophora tomentosa
The CC50 of S. tomentosa was recorded to be 21.57 µ g/mL. Furthermore, the IC50 values against SARS-CoV-2 and MERS-CoV were found to be 1.01 and 3.11 µ g/mL, respectively. The therapeutic indexes for S. tomentosa against SARS-CoV-2 and MERS-CoV were 21.18 and 6.92, respectively, as well.
Based on the above, we can conclude that S. tomentosa is more active against SARS-CoV-2 and it may be considered a promising anti-SARS-CoV-2 therapy after more advanced preclinical and clinical studies. The IC50 of each test was calculated using nonlinear regression analysis in triplicate for each concentration used. The best-fitting line was drawn between log concentrations and viral inhibition % using GraphPad Prism software.

Docking Studies
The X-ray structures of the S and M pr° receptors of SARS-CoV-2 were visualized and studied carefully based on the data introduced in the PDB and literature. It was clear that Asp80 is one of the most crucial amino acids in the S binding pocket of SARS-CoV-2 [54]. However, Glu166 is the most crucial amino acid for the inhibition of the dimeric M pr° pocket of SARS-CoV-2 [55].
Herein, the two most biologically active isolates (4 and 8) were selected for a further The CC 50 of S. tomentosa was recorded to be 21.57 µg/mL. Furthermore, the IC 50 values against SARS-CoV-2 and MERS-CoV were found to be 1.01 and 3.11 µg/mL, respectively. The therapeutic indexes for S. tomentosa against SARS-CoV-2 and MERS-CoV were 21.18 and 6.92, respectively, as well.
Based on the above, we can conclude that S. tomentosa is more active against SARS-CoV-2 and it may be considered a promising anti-SARS-CoV-2 therapy after more advanced preclinical and clinical studies.

Docking Studies
The X-ray structures of the S and M pr• receptors of SARS-CoV-2 were visualized and studied carefully based on the data introduced in the PDB and literature. It was clear that Asp80 is one of the most crucial amino acids in the S binding pocket of SARS-CoV-2 [53]. However, Glu166 is the most crucial amino acid for the inhibition of the dimeric M pr• pocket of SARS-CoV-2 [54].
Herein, the two most biologically active isolates (4 and 8) were selected for a further deep investigation to propose their expected mechanism of action and try to explain their inhibitory activity as well. Their scores, RMSD, 3D binding interactions, and 3D positioning inside the binding pockets of the S and M pr• pockets of SARS-CoV-2, besides the co-crystallized O6K inhibitor of M pr• , are represented in Table 3.
Regarding the docking process within the S binding pocket of SARS-CoV-2, we can observe that: and Asn137 amino acids. Its binding score was found to be −7.03 kcal/mol. However, concerning the docking process towards the M pr• receptor of SARS-CoV-2, we can show that: (a) The docked O6K inhibitor of the dimeric M pr• binding pocket formed three Hbonds with Glu166, Asn142, and Ser1 amino acids. Moreover, it achieved a score of −8.98 kcal/mol. (b) Notably, compound 4 bound the crucial Glu166 amino acid with one H-bond which was enough to stabilize itself and produce its inhibitory effect with a binding score of −6.44 kcal/mol. (c) Furthermore, compound 8 formed three H-bonds with Glu166, Asn142, and Gln192 with a binding score of −7.36 kcal/mol. Based on the above, we can conclude that the most biologically active compounds (4 and 8) formed H-bonds with the crucial amino acids which are important for the inhibition of the S and M pr• receptors of SARS-CoV-2 (Asp80 and Glu166, respectively). This greatly recommends the proposed mechanisms of action for the studied isolates as SARS-CoV-2 inhibitors targeting both the S and M pr• receptors. Notably, the docking results showed near matching with the previously discussed in vitro results as well.

Plant Materials
The pubescent leaves of shih-Balady (Artemisia judaica L., family Asteraceae) were purchased from an Egyptian market. A collection of the leaves of neem (Azadirachta indica A. juss., family: Meliaceae) was made at the Ministry of Agriculture, Giza, Egypt. The necklacepod (Sophora tomentosa L., family: Fabaceae) was collected from El Qanater, Qalyubia Governorate. Authentication was performed by Treas Labib, consultant of plant taxonomy at the Ministry of Agriculture and ex-director of El-Orman Garden (Giza, Egypt), and by Sherif S. El-Khanagry, Department of Flora and Phytotaxonomy Research Unit of the Agricultural Museum, Ministry of Agriculture of Giza, Egypt. Authentic reference material was available at the Department of Chemistry of Natural Compounds.

Preparation of Extracts for Antiviral Assays
The fresh leaves of each plant were dried in the shade in an air draft at room temperature. Each plant material (100 g) was separately refluxed in a dichloromethane-ethyl acetate (DCM/EA, 1:1, v/v) mixture (three extractions, each for 8 h with 1.25 L). The collected solution was filtered and dried in a vacuum to yield brown, greenish-brown, and dark brown amorphous residue of the DCM/EA extract of A. judaica (DCM/EA-Ar), A. indica (DCM/EA-Az), and S. tomentosa (DCM/EA-S), respectively. The percentage yields were 28.6, 34.2, and 11.6%, respectively. Part of each extract was re-dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), and the stocks (100 µg/mL) were stored at −20 • C until subsequent use.

General
The NMR spectra were recorded at 300, 400, 500 ( 1 H), and 75, 100, 125 ( 13 C) MHz on a Varian Mercury 300, Bruker High-Performance Digital FT-NMR 400 Avance III, and JOEl ECA 500 MHz spectrometer, respectively, using a convenient solvent. The chemical shifts (δ) are reported in parts per million (ppm) and coupling constants (J) in Hz. Herein, Gallenkamp electrothermal melting point apparatus and electrothermal digital apparatus were used. EI-MS spectra were taken on HP; MS-5988. The UV analyses of the pure samples were recorded, separately, as MeOH solutions and with different diagnostic UV shift reagents on a Shimadzu UV 240 (P/N 240-5800) UV-visible spectrophotometer.

Extraction and Isolation
The procedure depends on the isolation of compounds from DCM/EA (1:1, v/v) after the ethanol precipitation process for purifying sugar substances from the crude aqueous alcohol extract. Briefly, the air-dried and powdered leaves of S. tomentosa (1900 g) were exhaustively extracted with ethanol (80%). The ethanolic extract was evaporated to dryness under reduced pressure to afford a greenish-gray gummy residue. The residue was treated with the addition of excess ethanol. A yellowish-white precipitate was produced (F. I). The precipitate was purified using Sephadex LH-20 (MeOH as eluent) to give compound 1. Compound 1 was obtained as white crystals (29 mg) soluble in water and is observed as a colorless spot in visible light and a purple spot under UV light. The remaining solution, once the precipitate has been filtered out, is known as the filtrate and was dried in vacuo to give 89.3 g. The dried filtrate was fractionated with DCM/EA (1:1, v/v). A part of the total DCM/EA-S (40 g) was column chromatographed on a Si column and was eluted with n-hexane containing an increasing amount of ethyl acetate (100:0 → 0:100). A number of fractions (Fr. II-Fr. VI) were afforded which were combined based on TLC monitoring. Compound 2 was obtained from Fr. II eluted with n-hexane/ethyl acetate (7.5:2.5) and it was purified by crystallization (methanol as eluent) and obtained as a white powder (19 mg). Fr. III was re-chromatographed on the Si column by elution with EtOAc: MeOH (10:0-7:3) and divided into five subfractions (Fr. 02-Fr. 06). Fr. 02 and Fr. 03 were, separately, subjected to repeated CC on Si with n-hexane/acetone to give two semi-pure compounds. Each compound was crystallized with methanol to give compounds 3 and 4 as a yellow amorphous powder (24 mg) and yellowish-white crystals (28 mg), respectively. Compound 6 (20 mg) was obtained from Fr. 04 after final purification was achieved through Sephadex LH-20 (methanol as eluent) as a yellow amorphous powder (14 mg). The subfraction Fr. 05 was subjected to preparative TLC on Si CC with CHCl 3 -Me 2 CO (8:1) to give compounds 5 and 7 as yellow amorphous powder (22 mg) and light-yellow crystals (16 mg), respectively. On the other hand, fractions Fr. IV and Fr. V were combined and subjected to Si CC eluting with a solvent system of CHCl 3 -MeOH-H 2 O (9:1:0.1) to give compound 8 (15 mg). Fr. VI led to the isolation of compound 9. This fraction was subjected to repeat CC on Si with CHCl 3 /EtOAc to give a white crystalline solid (33 mg) of 9.
The purity of compounds 1-9 was checked by TLC using convenient solvent systems S 1 -S 6 . Spray reagents R 1 and R 2 were used for compounds 2 and 9, respectively. R 3 was used for compounds 3-8. All compounds were characterized mainly by spectroscopic methods, UV, 1 H, and 13 C NMR, and a comparison of the melting points with authentic samples or those in the literature was carried out.

Virus and Cells
The cell type used in the study was Vero-E6 cells from the National Research Centre (NRC). The MERS-CoV isolates (NRCE-HKU270 (Accession Number: KJ477103.2)) were a virus that is infecting humans. Moreover, SARS-CoV-2 isolates (hCoV-19/Egypt/NRC-03/2020 (Accession Number on GSAID: EPI_ISL_430820)) were used. The isolates were approved by the ethics committee of the NRC (Giza, Egypt).

Determination Titers of Viruses by Plaque Titration Assay
Plaque titration assay [55] was used for the determination of titers of MERS-CoV and SARS-CoV-2 to be used in other assays as the mode of action and plaque reduction assay. The full methodology is depicted in the Supplementary Material (SI 3).

MTT Cytotoxicity Assay (CC 50 )
The cytotoxic activity of the extracts was tested in Vero-E6 cells by using the MTT method with minor modification [56]. The applied full methodology is described in the Supplementary Material (SI 4).

Plaque Reduction Assay
The assay was performed according to the Hayden et al. method as previously described [57]. The full methodology is represented in the Supplementary Material (SI 5). The viral replication assay was applied according to Kuo et al. [58] as described in the Supplementary Material (SI 6.1).

Viral Adsorption
The viral adsorption assay using the Zhang et al. method [59] was performed as represented in the Supplementary Material (SI 6.2).

Virucidal
The virucidal assay was carried out [60] as depicted in the Supplementary Material (SI 6.3).

Inhibitory Concentration 50 (IC 50 ) Calculation
The inhibitory concentration 50 (IC 50 ) of the examined extracts was tested in Vero-E6 cells according to the reported methodology [61] described in detail in the Supplementary Material (SI 7).

Docking Studies
The nine isolated compounds from S. tomentosa (1-9) were inserted in two separate docking processes against both the S and M pr• receptors of SARS-CoV-2 using the MOE 2019.012 suite [62,63]. This was carried out to propose their expected mechanism of action as anti-SARS-CoV-2 agents targeting the S and/or M pr• receptors. Additionally, the co-crystallized inhibitor of the M pr• receptor pocket (O6K, 10) was used in the M pr• docking process as a reference standard.

Validation of the MOE Program
This was carried out to confirm the validity of the docking program to be able to consider the presented docking results [64,65]. Therefore, the co-crystallized inhibitor of the M pr• receptor (O6K) was redocked within its binding pocket, and both its binding mode and root mean square deviation (RMSD) were studied. The MOE validation was concluded based on obtaining approximately the same binding mode of the redocked O6K (green) compared to its native one (red) as depicted in Figure 7, and the low value of RMSD (1.41).
This was carried out to confirm the validity of the docking program to be able to consider the presented docking results [65,66]. Therefore, the co-crystallized inhibitor of the M pr° receptor (O6K) was redocked within its binding pocket, and both its binding mode and root mean square deviation (RMSD) were studied. The MOE validation was concluded based on obtaining approximately the same binding mode of the redocked O6K (green) compared to its native one (red) as depicted in Figure 7, and the low value of RMSD (1.41). Figure 7. Superimposition of the redocked O6K inhibitor (green) over its native one (red).

Preparation of the S. tomentosa Isolated Compounds
The nine isolated compounds (1-9) from S. tomentosa were sketched using the ChemDraw program. Each compound was introduced individually into the MOE program window and prepared for docking as discussed before [67,68]. Then, the nine prepared isolates (1-9) were imported into two different databases in order to perform two separate docking processes.

Preparation of the S and M pr° Receptors of SARS-CoV-2
The X-ray structures of both the S and M pr° receptors of SARS-CoV-2 (IDs: 7FCD [54] and 6Y2G [55], respectively) were downloaded from the Protein Data Bank (PDB). Each protein was prepared as described earlier in detail [69,70] Each database was uploaded in a separate general docking process according to the previously discussed methodology [71,72]. Moreover, the best pose for each tested compound was selected according to the binding mode, score, and RMSD as well [73,74].

Conclusions
Three selected medicinal plants (A. indica (neem), A. judaica, and S. tomentosa) were screened against MERS-CoV using a plaque reduction assay. The highest viral inhibition percentage (96%) was recorded for S. tomentosa (known as yellow necklacepod) with CC50 of 20.86 µ g/mL. Then, the mode of action for both S. tomentosa and A. judaica showed that they achieved ..99% and 92% inhibition effects, respectively, at a concentration of 1.56 µ g/mL, with virucidal as the main mode of action. Moreover, the antiviral activity of S. tomentosa against both MERS-CoV and SARS-CoV-2 using a plaque reduction assay was measured. It showed that the antiviral activity of S. tomentosa against SARS-CoV-2 was very high (100%) and the extract succeeded in achieving full inhibition for viral propagation at different concentrations (12.50 and 6.25 µ g/mL). In addition, it showed a slightly

Preparation of the S. tomentosa Isolated Compounds
The nine isolated compounds (1-9) from S. tomentosa were sketched using the Chem-Draw program. Each compound was introduced individually into the MOE program window and prepared for docking as discussed before [66,67]. Then, the nine prepared isolates (1-9) were imported into two different databases in order to perform two separate docking processes.

Preparation of the S and M pr• Receptors of SARS-CoV-2
The X-ray structures of both the S and M pr• receptors of SARS-CoV-2 (IDs: 7FCD [53] and 6Y2G [54], respectively) were downloaded from the Protein Data Bank (PDB). Each protein was prepared as described earlier in detail [68,69] Each database was uploaded in a separate general docking process according to the previously discussed methodology [70,71]. Moreover, the best pose for each tested compound was selected according to the binding mode, score, and RMSD as well [72,73].

Conclusions
Three selected medicinal plants (A. indica (neem), A. judaica, and S. tomentosa) were screened against MERS-CoV using a plaque reduction assay. The highest viral inhibition percentage (96%) was recorded for S. tomentosa (known as yellow necklacepod) with CC 50 of 20.86 µg/mL. Then, the mode of action for both S. tomentosa and A. judaica showed that they achieved 99.5% and 92% inhibition effects, respectively, at a concentration of 1.56 µg/mL, with virucidal as the main mode of action. Moreover, the antiviral activity of S. tomentosa against both MERS-CoV and SARS-CoV-2 using a plaque reduction assay was measured. It showed that the antiviral activity of S. tomentosa against SARS-CoV-2 was very high (100%) and the extract succeeded in achieving full inhibition for viral propagation at different concentrations (12.50 and 6.25 µg/mL). In addition, it showed a slightly lower inhibition against MERS-CoV (96%) at the highest concentration (12.50 µg/mL). Furthermore, the phytochemical investigation of the very promising S. tomentosa L. led to the isolation and structure determination of nine compounds (1-9) using different techniques. Notably, compound 4 (genistein 4'-methyl ether) was found to achieve superior anti-SARS-CoV-2 activity among other isolates with an IC 50 value of 2.13 µm. Interestingly, it was important to test the mode of action of S. tomentosa against SARS-CoV-2. The results showed that two mechanisms of action (virucidal and adsorption) were effective at 12.50 and 6.25 µg/mL with an inhibition of more than 99%. On the other hand, the CC 50 of S. tomentosa was recorded to be 21.57 µg/mL. Additionally, the IC 50 values against SARS-CoV-2 and MERS-CoV were found to be 1.01 and 3.11 µg/mL, respectively. The therapeutic indexes for S. tomentosa against SARS-CoV-2 and MERS-CoV were 21.18 and 6.92, respectively, as well. Obviously, we can conclude that S. tomentosa is more active against SARS-CoV-2 and it may be considered a promising anti-SARS-CoV-2 therapy after more advanced preclinical and clinical studies. Finally, molecular docking studies clarified that the most biologically active compounds (4 and 8) showed the formation of H-bonds with the crucial amino acids which are important for the inhibition of the S and M pr• receptors of SARS-CoV-2 (Asp80 and Glu166, respectively). This greatly recommends the proposed mechanisms of action for the studied isolates as SARS-CoV-2 inhibitors targeting both the S and M pr• receptors.