Naturally Available Flavonoid Aglycones as Potential Antiviral Drug Candidates against SARS-CoV-2

Flavonoids are important secondary plant metabolites that have been studied for a long time for their therapeutic potential in inflammatory diseases because of their cytokine-modulatory effects. Five flavonoid aglycones were isolated and identified from the hydrolyzed aqueous methanol extracts of Anastatica hierochuntica L., Citrus reticulata Blanco, and Kickxia aegyptiaca (L.) Nabelek. They were identified as taxifolin (1), pectolinarigenin (2), tangeretin (3), gardenin B (4), and hispidulin (5). These structures were elucidated based on chromatographic and spectral analysis. In this study, molecular docking studies were carried out for the isolated and identified compounds against SARS-CoV-2 main protease (Mpro) compared to the co-crystallized inhibitor of SARS-CoV-2 Mpro (α-ketoamide inhibitor (KI), IC50 = 66.72 µg/mL) as a reference standard. Moreover, in vitro screening against SARS-CoV-2 was evaluated. Compounds 2 and 3 showed the highest virus inhibition with IC50 12.4 and 2.5 µg/mL, respectively. Our findings recommend further advanced in vitro and in vivo studies of the examined isolated flavonoids, especially pectolinarigenin (2), tangeretin (3), and gardenin B (4), either alone or in combination with each other to identify a promising lead to target SARS-CoV-2 effectively. This is the first report of the activity of these compounds against SARS-CoV-2.


Introduction
Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) represents an evolving global threat worldwide; its infection is characterized by acute respiratory symptoms, such as fever, dry cough, and shortness of breath with an incubation period of about 5 days (average 2-14 days) [1]. By 19 October 2021, 194 vaccines are in the pre-clinical phase and 127 candidate vaccines are in clinical progress (WHO, Vaccine tracker, and landscape) [2]. Currently, different types of vaccines are approved for use in many countries, such as Sanofi-GSK, BioNTech-Pfizer, Curevac, AstraZeneca (The University of Oxford), Moderna, and Johnson & Johnson [3]. However, vaccines are a prophylactic approach and cannot be implemented for treatment, especially in pandemic situations [4]. Furthermore, the search for new therapeutic drugs from safe natural sources is crucial during pandemics [2,[5][6][7][8][9][10][11][12][13]. Several naturally existing bioactive compounds were reported to behave as antiviral agents [14][15][16]. Flavonoids demonstrated antiviral and immunomodulatory activities against coronaviruses [17]. Therefore, the antiviral properties of flavonoids might also be applicable in the current COVID-19 pandemic. The antiviral activity of some flavonoids against coronaviruses (CoVs) is recognized by inhibiting 3C-like protease (3CLpro), which is capable of blocking the enzymatic activity of SARS-CoV 3CLpro [18].
The SARS-CoV-2 main protease (Mpro) enzyme plays an important role in the synthesis of viral functional proteins from its basic polypeptides [19][20][21]. Therefore, it seems to be responsible for both viral transcription and replication [22,23]. Based on the given facts, it is recommended to target the SARS-CoV-2 Mpro enzyme to obtain a fast and promising lead to solve the COVID-19 pandemic situation as soon as possible [24][25][26].
One of the most important methods for drug discovery processes nowadays is computational drug design [27,28]. Molecular docking studies assist scientists greatly to discover new drugs in a fast-track manner [29][30][31][32]. Moreover, molecular dynamic simulations confirm the results of molecular docking, especially in absence of in vitro studies [6,20]. Previous computational studies have revealed that taxifolin could be a potential inhibitor against the SARS-CoV-2 Mpro enzyme [33]. Moreover, tangeretin showed potential for the treatment and prevention of COVID-19 [34], while, hispidulin showed a better binding affinity to Mpro of SARS-CoV-2 and ACE2 receptor than hydroxychloroquine and could be used as a therapeutic candidate against COVID-19 [35]. No studies, either computational or in vitro, were reported for the compounds pectolinarigenin and gardenin B regarding their effects on SARS-CoV-2. Therefore, we take the responsibility for their investigations.
It is evident that the Glu166 amino acid seems to be very crucial for SARS-CoV-2 Mpro pocket binding and inhibition.
From Tables 1 and 2 it can be observed that the docking results of the isolated and identified five flavonoids from the aerial parts of A. hierochuntica and K. aegyptiaca and the citrus peel of C. reticulata fruits, namely taxifolin (1), pectolinarigenin (2), tangeretin (3), gardenin B (4), and hispidulin (5), examined against SARS-CoV-2 Mpro and compared to the docked KI, give us a clear promising idea towards their binding affinities, which indicates, subsequently, their expected intrinsic activities as well their importance to combat the SARS-CoV-2 pandemic.

Hispidulin (5)
The red dash represents H-bonds and the black dash represents H-pi interactions.

In Vitro Validation
Based on the in silico studies, pectolinarigenin, tangeretin, and gardenin B showed the best evidence of the studied drugs to be selected for further in vitro validation against SARS-CoV-2. Hence, the in vitro study was conducted on the five compounds and the results were effective with pectolinarigenin, tangeretin, and gardenin B. To identify the proper concentrations to define the antiviral activity of pectolinarigenin, tangeretin, and gardenin B, the half-maximal cytotoxic concentration "CC 50 " was calculated by a crystal violet assay ( Figure 2). All compounds showed a wide range of safety within the tested concentrations (10 ng/mL-100 mg/mL). The antiviral screening revealed that pectolinarigenin (2) and tangeretin (3) exhibited a promising cytotoxic inhibitory activity against NRC-03-nhCoV with IC 50 = 12.4 and 2.5 µg/mL, respectively (Figure 2b,c). Both natural compounds exerted their anti-SARS-CoV-2 activities with high selectivity indices (CC 50 /IC 50 > 1000). In previous reports that mentioned the biological activities of pectolinarigenin and gardenin B; pectolinarigenin showed potent inhibitory activities on melanogenesis [40] and exhibited powerful in vitro anti-diabetic, hepatoprotective, and anticancer activities [41][42][43]. On the same line, gardenin B, which is a methoxylated flavonoid derived from a tangeretin, showed slight anti-SARS-CoV-2 activity (IC 50 = 128 µg/mL). Interestingly, gardenin B, motioned previously for its induction of cell death in human leukemia cells, involves multiple caspases [44] and also shows in vitro antiviral activity against the Encepehalomyocarditis virus (EMV) [45].

Plant Material
Three plant species were collected and identified as belonging to three different families: A. hierochuntica L. (Brassicaceae), C. reticulata Blanco (Rutaceae), and K. aegyptiaca (Plantaginaceae). The aerial parts of the first and last species were collected from the northern coast of El Dabaa road, in March 2019, while the fresh matured fruits of C. reticulata were obtained from the traditional market, Giza, Egypt.

Extraction, Isolation and Structure Elucidation
The aerial parts of A. hierochuntica and K. aegyptiaca as well as the peel of C. reticulata fruits were air-dried and ground. Each obtained powder was extracted with MeOH:H 2 O (7:3) 3 times at room temperature. All extract were evaporated under reduced pressure and temperature to obtain residues. Each residue was subjected to an acid hydrolysis process (2N HCl, 100 • C, 2 h) [46]. The acidic solutions were extracted with ethyl acetate several times, affording aglycones extracts upon evaporation. Each extract was subjected to a Sephadex LH-20 column; using MeOH:H 2 O (1:1) afforded fractions. Each fraction was subjected to PPC using BAW and 50% AcOH several times to isolate the flavonoid aglycones. All compounds were finally purified with a Sephadex LH-20 column, using 100% MeOH as eluent to reach pure aglycones. Compound (1) was obtained from A. hierochuntica, compound (2) from K. aegyptiaca, while compounds (3)(4)(5) were obtained from C. reticulata (tangerine). The structures of the isolated flavonoids were elucidated by extensive chromatographic, chemical, and spectroscopic methods (HRESI-MS, UV, and NMR) as well as Co-PC with reference samples. Their spectroscopic data were compared with previously reported values [38][39][40][41]. HRESI-MS and NMR chromatograms are provided as supplementary files (Supplementary Material Figures S1-S10).
Moreover, they were compared to the co-crystallized inhibitor of SARS-CoV-2 Mpro (KI) as a reference standard.

Preparation of the Isolated and Identified Five Flavonoids (1-5)
The 2D chemical structures of the isolated five flavonoids-taxifolin (1), pectolinarigenin (2), tangeretin (3), gardenin B (4), and hispidulin (5)-were sketched using Chem-Draw Professional. Each chemical structure was introduced separately into the MOE window, converted to the 3D orientation, adjusted for partial charges, and energy minimized to be prepared for docking according to the default preparation steps described earlier [53][54][55][56]. After saving each prepared compound separately using the (.moe) extension, the co-crystallized native inhibitor of SARS-CoV-2 Mpro (KI) was extracted and saved in a separate MOE file as well. Furthermore, all of the aforementioned prepared compounds (1)(2)(3)(4)(5) were imported in the same database file and saved as (.mdb) extension to be uploaded during the docking step.

Target Mpro of SARS-CoV-2 Preparation
The target Mpro enzyme (as a dimer) of SARS-CoV-2 was extracted from the Protein Data Bank (PDB code: 6Y2G) [57]. Moreover, it was subjected to the detailed preparation steps described before [58][59][60][61] to be ready for the docking process.

Docking of the Database Compounds (1-5) to the Dimer Mpro of SARS-CoV-2
The previously discussed database, containing the KI in addition to the five isolated and identified flavonoids (1)(2)(3)(4)(5), was uploaded in place of the ligand during a general docking process. The binding site of the co-crystallized α-ketoamide inhibitor was identified as the docking site. Moreover, the program specifications were adjusted as follows: triangle matcher for the placement methodology, London dG for the first scoring methodology, GBVI/WSA dG for the final scoring methodology to select the best 10 poses from 30 different poses for each docked compound, and rigid receptor for the refinement methodology [61][62][63][64]. Finally, the best pose for each tested compound, based on the score and RMSD values, was selected for further studies.
Furthermore, a MOE program validation process was carried out before applying the previously described docking process by redocking the co-crystallized KI alone at its binding site of Mpro. The obtained low RMSD values (<2) between the native co-crystallized and the redocked α-ketoamide inhibitor confirmed the valid performance [65][66][67].

Cytotoxicity (CC 50 ) Determination
To assess the half-maximal cytotoxic concentration (CC 50 ), stock solutions of the compounds were prepared in 10% DMSO in ddH 2 O and diluted further to the working solutions with DMEM. The cytotoxic activity of the extracts was tested in VERO-E6 cells by using a crystal violet assay, as previously described [68] with minor modifications. Briefly, the cells were seeded in 96 well-plates (100 µL/well at a density of 3 × 10 5 cells/mL) and incubated for 24 h at 37 • C in 5% CO 2 . Control cells were treated with 1% DMSO in DMEM (the concentration of DMSO in the highest concentration of the tested samples). After 24 h, the cells were treated with various concentrations of the compounds in triplicates. After 72 h, the supernatant was discarded, and the cell monolayers were fixed with 10% formaldehyde for 1 h at room temperature (RT). The fixed monolayers were, then, dried and stained with 50 µL of 0.1% crystal violet for 20 min on a bench rocker at RT. The monolayers were, then, washed and dried, and the crystal violet dye in each well was dissolved with 200 µL methanol for 20 min on a bench rocker at RT. The absorbance of the crystal violet solutions was measured at λmax 570 nm as a reference wavelength using a multi-well plate reader. The cytotoxicity of the various concentrations, compared to the untreated cells and the blank background, was determined using nonlinear regression analysis by plotting the log inhibitor versus the normalized response.

Inhibitory Concentration 50 (IC 50 ) Determination
The IC 50 values for the compounds were determined as previously described [69], with minor modifications. Briefly, in 96 well tissue culture plates, 2.4 × 10 4 Vero-E6 cells were distributed in each well and incubated overnight in a humidified 37 • C incubator under 5% CO 2 conditions. The cell monolayers were then washed once with 1× PBS. An aliquot of the SARS-CoV-2 "NRC-03-nhCoV" virus [70] containing 100 TCID 50 was incubated with serially diluted concentrations of the tested compound and kept at 37 • C for 1 h. The Vero-E6 cells were treated with a virus/compound mix and co-incubated at 37 • C in a total volume of 200 µL per well. Untreated cells infected with the virus represented virus control; however, cells that were not treated and not infected were cell control. Following incubation at 37 • C in a 5% CO 2 incubator for 72 h, the cells were fixed with 100 µL of 10% paraformaldehyde for 20 min and stained with 0.5% 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 was measured at 570 nm using an Anthos Zenyth 200 rt plate reader (Anthos Labtec Instruments, Heerhugowaard, Netherlands). The IC 50 is the concentration of the compound required to reduce the virus-induced cytopathic effect (CPE) by 50%, relative to the virus control.

Statistical Analyses
All experiments were performed in three biological repeats. Statistical tests and graphical data presentation were carried out using GraphPad Prism 5.01 software. Data are presented as the average of the means. The IC 50 and CC 50 curves represent the nonlinear fit of "normalize" of "transform" of the obtained data; their values were calculated using GraphPad prism as "best fit value".