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Hypericum perforatum and Echinacea purpurea have been reported to have antiviral activity against various viral infections. In this study, H. perforatum (Hypericum perforatum) and Echinacea purpurea were tested in vitro for their antiviral activity against the newly identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using Vero E6 cells during its infectious cycle. 0 to 48 hours after infection. Hypericin from H. perforatum and various parts (roots, seeds, aerial parts) of two species of Echinacea purpurea (Echinacea purpurea and Echinacea angustifolia) were tested for their antiviral activity using real-time viral load quantification. Polymerase chain reaction (qRT) inhibition . -PCR) Cell culture analysis. Interestingly, a mixture of H. perforatum-Echinacea (1:1 ratio) of H. perforatum and Echinacea was also tested for SARS-CoV-2 and showed key antiviral activity comparable to the antiviral therapeutic effects of H. perforatum and Echinacea. The results of Hypericum perforatum and Echinacea species used in this study showed significant antiviral and virucidal effects in the following order of effectiveness: Hypericum perforatum, Hypericum perforatum-Echinacea mixture and Echinacea purpurea against the SARS-CoV-2 infectious cycle. In addition, molecular analysis of compounds with the main proteins of SARS-CoV-2 (Mpro and RdRp) revealed the most potent biologically active compounds, such as echinacein, echinacein, anthocyanin, anthocyanin 3-(6”-alonylglucoside, quercetin-3-O- glucuronide, proanthocyanidins, rutin, kaempferol-3-O-rutinoside, and quercetin-3-O-xyloside, therefore, based on the results of this study, it is necessary to establish specific treatment regimens in clinical trials.
Human coronaviruses (HCoV) continue to pose a global threat to human health, first with the spread of the severe acute respiratory syndrome coronavirus (SARS-CoV)1 and then with the emergence of the Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012. and finally due to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic in Wuhan, China. The pandemic caused the global COVID-193 pandemic. As the disease progresses, pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6, IFN-γ, CXCL10, MCP-1 are produced in large amounts, leading to vasculitis, a hypercoagulable state, and multiple organ damage, eventually leading to to death 4. A major protease (Mpro), known as 3-chymotrypsin-like cysteine protease (3CLpro), is considered to be an important functional target in the life cycle of the virus and therefore a candidate for antiviral drugs against the target of SARS-CoV-256. due to its role in the release of functional polypeptides encoded by all HCoVs7,8. Recent reports on the treatment of COVID-19 disease have targeted its structure, pathology, and mechanisms for optimal management of the infection9. For example, many effective treatments for SARS-CoV-2 have been proposed, such as: neuraminidase inhibitors, remdesivir, peptide (EK1), arbidol, RNA synthesis inhibitors (TDF and 3TC), anti-inflammatory drugs (hormones and other molecules). and traditional Chinese medicine. In addition, there are several studies on synergism between antimalarial drugs such as chloroquine-hydroxychloroquine, remdesivir-favipiravir for the treatment of COVID-1911,12,13.
Evaluation of herbs and plant extracts that have demonstrated antiviral activity against other coronaviruses may offer an alternative approach to developing treatments for COVID-19. Several studies have examined antiviral activity against other coronaviruses with high activity and low cytotoxicity. For example, glycyrrhizin, a glycyrrhizinate root extract, has been shown to completely block viral replication15,16.
H. perforatum (Hypericum) or St. John’s wort (SJW) has long been known as a potent medicinal plant used to treat a range of infectious and non-infectious diseases such as depression, bacterial and viral infections, dermatological wounds, and inflammation17,18. . The metabolites extracted from each part of the plant (root, seed and aerial part) of H. perforatum vary and are chemically defined as naphthodianthrones (hypericin), phloroglucinol (hyperforin), flavonoid glycosides (hyperoside), rutin, flavonoids quercetin and myricetin 19,20. As an antiviral agent, H. perforatum was evaluated in vitro and in vivo for its activity against infectious bronchitis virus (IBV), hepatitis C virus, HIV, and coronaviruses other than SARS-CoV-221,22. Moreover, both hyperforin alone and other extracts of H. perforatum inhibit the action of cytokines in β-cell lines and isolated rat and human islets 20, 23 . In addition, ethyl acetate-extracted sections of St. John’s wort (HPE) showed significantly lower relative viral titers of IBV in vitro and in vivo, as well as reduced levels of IL-6, TNF-α, and NF-kB17 mRNA expression. Based on the ability of St. John’s wort extract and its main polyphenolic component hypericin to counteract the pro-inflammatory effects of various cytokines, its use to prevent cytokine storm in patients with COVID-1924 was reviewed and proposed.
Another herb that is used in many traditional and common remedies for cold and flu symptoms and to boost the immune system is echinacea. Echinacea is known as nine of several plants in the Echinacea genus, however, only three of them are used as herbal supplements: E. angustifolia, E. purpurea, and E. pallida. Echinacea contains compounds responsible for its medicinal properties, such as: phenols, including derivatives of caffeic acid and echinacea, polysaccharides, flavonoids, ketones, and lipophilic alkylamides25,26. Previous in vitro and in vivo studies have shown that echinacea affects cytokine production27,28, increases CD6929 expression, acts on natural killer cells30 and reduces disease severity. In vivo studies have shown an anti-inflammatory therapeutic effect on human THP-1 monocyte cells31. In addition, alkylamides and ketones of Echinacea purpurea extracts have been reported to have anti-inflammatory effects32,33,34,35. A 2009 study on the H5N1 HPAIV strain showed that extracts of E. purpurea prevent the entry of the virus into cells by blocking the virus’ receptor-binding activity36. Another study examined the in vitro virucidal and antiviral potential of Echinacea purpurea (Echinaforce®) herb and root extracts against human coronaviruses, including SARS-CoV-237. This study reports the inactivation of MERS-CoV, SARS-CoV-1 and SARS-CoV-2 using Echinacea purpurea extracts.
Following our previous studies evaluating the antiviral properties of natural antiviral agents against certain human coronaviruses 16, 38, 39, in this study we report that Hypericum perforatum species (aerial parts) and Echinacea species (roots, seeds, aerial parts) against SARS-CoV – 2. We also report molecular modeling studies of registered biologically active compounds in selected plants against powerful therapeutic targets, viz. Basic protein (Mpro) and RNA-dependent polymerase (RdRp) of SARS-CoV-2.
Vero E6 cells (ATCC® CRL-1586™) and HEK 293 cells (ATCC® CRL-1573™) were incubated in 75 cm2 culture flasks containing Dulbecco’s modified Eagle medium. Medium (DMEM) was maintained at 37°C in 5% CO2 atmosphere. For testing purposes, 96-well plates were seeded with Vero E6 cells at a density of 3 x 104 cells/well and incubated for 24 hours at 37°C and 5% CO2 until a confluent monolayer was obtained.
The SARS-CoV-2 isolate used in this study was isolated from a well-characterized clinical specimen (SARS-CoV-2/human/SAU/85791C/2020, GenBank accession number: MT630432) in the BSL-3 laboratory. Released earlier at 41. Briefly, isolation was performed in 75 cm2 cell culture flasks containing Vero E6 cells in Minimal Basic Medium (Gibco, Thermo Fisher)2 containing 4% fetal bovine serum and 1% aminoamide glutamate. Cytopathic effects were monitored daily. Approximately 72 hours later, almost complete cell lysis was observed, resulting in a TCID50 of 3.16 x 106 infectious particles per ml. Virus supernatants were used for inoculation in subsequent experiments. All experiments with live SARS-CoV-2 viruses were conducted in a biosafety level 3 room at the Special Infectious Pathogens Division of King Abdulaziz University.
H. perforatum and Echinacea products were purchased from gaia HERBS® (Brevard, NC, USA) in gelatin capsule and liquid form and sold as nutritional and herbal supplements, respectively. According to the supplier, the entire area of the plant is treated in a biosecurity zone and the plant components are extracted with water and ethanol, avoiding the use of oral extraction solvents. After extraction and filtration, the extract is concentrated at low temperature and pressure to slowly remove the solvent and preserve the delicate plant components. Finally, an HPLC analysis is performed to ensure that the extract is concentrated to the desired level of activity.
Plant material at a concentration of 100 mg/mL42 was prepared in a light protected BSL-2 laboratory and dissolved in a minimum volume of dimethyl sulfoxide (DMSO, ≥ 99.5%, plant cell culture test, SIGMA) and diluted to working concentration. Use the medium and filter through a 0.22 µm43 filter. All extracts are filtered to remove any plant fibers. A mixture of Hypericum perforatum and Echinacea purpurea was prepared by mixing each plant material at a concentration of 100 mg/mL.
Cytotoxicity assays were performed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Roche, Germany) protocol with minor modifications as previously reported44. A monolayer of Vero E6 cells at a concentration of 4×103 cells/ml was seeded on a 96-well culture plate and washed 3 times with phosphate-buffered saline (PBS) 1×, pH 7.4. Dispense 100 µl of the prepared working solution of St. John’s wort, Echinacea perforatum, and a mixture of St. John’s wort and Echinacea perforatum, diluted in serum-free DMEM (two-fold dilutions from 0.039 to 5 µg/mL), into the wells of a 96-well plate and add an additional 100 µl of maintenance medium (3 wells per dilution in two independent experiments). Control cells were incubated with appropriate concentrations of DMSO solution as solvent control and unspiked wells as negative control. The cells were incubated at 37°C in an atmosphere of 5% CO2 for 48 h, the supernatant was removed, the cells were washed 3 times with PBS, 20 μL of MTT solution were added to each well, and incubated for 4 h at 37°C. Add solubilizing solution and incubate overnight at 37°C. The plates were read using an ELISA reader (Synergy 2 Microplate, BIOTIK, Korea) with a reference wavelength of 570 nm (OD570). Cytotoxicity was calculated from the mean value according to the following equation: (1 – (OD570 drug/OD570 control) × 100). The Graphpad 9 prism (version 9.0.0) then plotted percent cytotoxicity versus log10 drug concentration, plotted cytotoxicity, and calculated CC50 using a non-linear curve fitted with a variable slope, where the equation for the fitted curve is: Y = 100/ (1 + 10^((LogCC50-X)*HillSlope))), where Y is the percentage of cytotoxicity and X is the concentration.
Plant extracts have been described previously45 with some modifications. Briefly, 96-well plates were prepared with Vero E6 cells as described above. Cells were then washed twice with PBS and 2-fold serial dilutions of plant material (H. perforatum, Echinacea and H. perforatum-Echinacea mixture) (0.316–5 μg/mL) in culture were treated with various products according to the procedure described in the Antiviral Activity Assay section. , 1 SARS-CoV-2 isolate was infected and incubated at 37°C and 5% CO2 for three days. The results were quantified as described previously. Dose-response curves were used to express percent inhibition compared to control viruses. Graphpad Prism 9 (version 9.0.0) was then used to generate graphs by plotting percent inhibition versus log10 drug concentration, and IC50 values were calculated using non-linear curve fitting with variable slope, where the fitted curve equation is represented by the equation . (1), where Y is the percentage of inhibition and X is the concentration.
The effects of medicinal plants were tested in the BSL-3 Biosecurity Laboratory using an antiviral assay (shown in Figure 1). Prepared Vero E6 96-well plates were treated with plant extracts in triplicate in two independent experiments:
Overview of antiviral tests. Vero E6 cells were cultured in 96-well culture plates and used in three workflows: post-treatment of virus-infected cells (A), pre-treatment of cells before viral infection (B), and virucidal treatment (C).
To determine the effect of Hypericum perforatum, Echinacea perforatum, and Hypericum perforatum-Echinacea mixture on SARS CoV-2 infected cells, Vero E6 cells cultured in 96-well plates were infected with viral isolates at MFI = 1 for 2 h at 37°C. . C as previously described 17. They were then treated with H. perforatum, echinacea, and H. perforatum-Echinacea at 37°C for 48 hours, respectively. Collect supernatants from cell samples at 12, 16, 24, 36, and 48 hours (3 wells each)46 and pool wells at each time point to obtain sufficient material for RNA extraction and use for quantitative RT-PCR41. Percent inhibition of SARS-CoV-2 was evaluated against a positive viral control (SARS-CoV-2 on Vero E6 cells to which no compound was added).
To study the effect of medicinal plants on blocking cell receptors, Vero E6 cells cultured in 96-well plates were treated with a solution of Hypericum perforatum, Echinacea perforatum and a mixture of Hypericum perforatum-Echinacea at 37°C for 2 h, and then treated with PBS. The solution was washed 3 times17. The cells were then inoculated with SARS-CoV-2 (MOI = 1) and incubated at 37°C for 48 hours. Supernatants were collected at 12, 16, 24, 36 and 48 hours (3 wells at time), pooled and used for quantitative PCR. Percent inhibition of SARS-CoV-2 was assessed against a positive viral control (SARS-CoV-2 on Vero E6 cells to which no compound was added)41.
To analyze the direct effect of medicinal plants on SARS-CoV-2 infection, SARS-CoV-2 (MOI = 1) was incubated with Hypericum perforatum, Echinacea perforatum, and a mixture of St. Then SARS-CoV-2 treated with three extracts was used to infect Vero E6 cells at 37°C for 48 hours. Assessment of SARS-CoV-2 in cell supernatants at 12, 16, 24, 36 and 48 hours using qRT-PCR41 compared to a positive viral control (SARS-CoV-2 on Vero E6 cells, no compound added) % inhibition Sample 46 . During the post-infection cycle, plates were analyzed by visualizing virus-induced CPE using light microscopy (Nikon-ECLIPSE-Ti, Japan) and stained with crystal violet (Sigma-Aldrich, USA) as previously described.
Viral RNA was extracted from all samples collected directly in 96-well plates for antiviral assays using the QIAmp Viral RNA mini kit (Qiagen, Germany) according to the manufacturer’s instructions as described previously41. Relative quantification of SARS-CoV-2 viral load was performed using one-step, dual-target real-time RT-PCR (RealStar SARS-CoV-2 RT-PCR Kit 1.0, Altona Diagnostics, Germany) using 7500 Fast Real-time PCR according to the manufacturer’s instructions systems (Applied Biosystems, USA). PCR detection of betacoronavirus-specific target (E gene), SARS-CoV-2 specific target (RdRP gene), and internal control. Viral load reduction was expressed by comparing the cycle threshold for each sample with the Ct value of the pretreated inoculated sample47. SARS-CoV-2 titers are expressed as PFU per milliliter (PEq/mL) using a standard curve constructed from serial dilutions of stock qRT-PCR test virus samples under the same testing conditions as for test samples. Each run included a positive control of the virus template and a negative control without a template. Each sample was tested in duplicate and the mean values are presented as PEq/mL. Y = 100/(1 + 10^((LogIC50-X)*HillSlope)) where Y is the percent inhibition and X is the concentration of 4.9.
Data were analyzed using one-way or two-way analysis of variance, and Tukey’s test was used for multiple comparisons. P<0.05 and <0.005 were considered statistically significant. All analyzes were performed using GraphPad Prism version 8.
In silico studies have been performed to predict the synergistic effects of phytochemicals from Eucalyptus angustifolia, Eucalyptus angustifolia, and Hypericum perforatum on SARS-CoV-2. Briefly, the published literature was searched for information on phytochemicals present in selected plants and their 3D structures were extracted from the PubChem database in SDF format48,49. In addition, (b) the crystal structures of SARS-CoV-2 major protease (Mpro) and RNA-dependent RNA polymerase (RdRp) proteins were collected from the RCSB Protein Database (http://www.rcsb.org/) and PDB . ID: 6W6350 and PDB ID: 7B3C51 respectively. Both target structures were prepared using Chimera’s built-in Dockprep tool and all compounds were prepared in PyRx software prior to screening. The active sites of the two drug targets were predicted using the Cavity Plus52 tool. Predicted pockets with natural ligand binding residues were considered for meshing in PyRx. Initially, structure-based virtual screening (SBVS) of phytochemicals collected from each selected plant was performed using the PyRx53 software as described previously54. After the completion of six independent SBVS experiments, i.e. Mpro-E. Eleopsis, Mpro-E. Purple, Mpro-H. perforatum, RdRp-E.angustifolia, RdRp-E. Purpura and RdRp-H. perforatum, the most potential connections with the highest docking rates in SBVS were additionally selected for redocking analysis in the settings of the Chimera-AutoDock Vina plugin55,56. Before redocking, the selected ligand and target protein constructs were prepared using the Dockprep tool built into the Chimera program. For Mpro, the grid (30 Å x 30 Å x 30 Å) is set at three central coordinates (119.44 x -4.95 x 16.78), covering all key target protein residues for ligand binding. RdRp is gridded (30 Å × 30 Å × 40 Å) over three central coordinates (97.36 × 97.76 × 116.69). A similar approach was used to dock two targets with control ligands [X-77 (Mpro), Remdesivir (RdRp)]. Later, in Free Academic Schrödinger-Maestro v12.7 (Schrödinger reprint 2021-1: Maestro, Schrödinger, LLC, New York, NY, 2021).
Selected redocking complexes from each set of dockings were studied in a 100 ns molecular dynamics simulation to understand the activity of phytochemicals on selected viral proteins (e.g., SARS-CoV-2 Mpro and SARS-CoV-2 RdRp). Binding stability in the pocket as described above57. Briefly, the selected complex interoperability poses were preprogrammed with default parameters using the protein preparation tool from the free Maestro-Desmond academic package (Maestro-Desmond Interoperability Tools, Schrödinger, New York, NY, 2018)58. Thereafter, the complexes were placed in an orthorhombic water bath (10 × 10 × 10 Å) supplemented with aqueous TIP4P solvent, and the entire simulated system was neutralized with counterions using a system design to place ions at a distance of 20 Å from the ligand location instrument. In addition, in order to mimic the in vitro physicochemical environment, 0.15 M salts were also modified in the simulated system using the system build tool. The entire system was then minimized using the Desmond minimization tool with default parameters, followed by a 100 ns MD simulation at 300 K and 1.01325 bar using the molecular dynamics simulation tool with default parameters. Each docked complex was subjected to MD modeling with the same parameters. Subsequently, the MD simulation trajectories were analyzed using the simulation interaction graph tool in the Maestro-Desmond package.
Cytotoxicity of St. John’s wort, Echinacea, and a mixture of St. John’s wort with Echinacea was assessed by MTT assay after 48 h of treatment of Vero E6 cells. The results showed that the CC50s of St. John’s wort and St. John’s wort/Echinacea mixture were: 66.78 and 141.1 µg/mL, respectively, while Echinacea showed the highest cytotoxicity with concentration-dependent cytotoxicity (Fig. S1).
Further evaluation of the cytotoxicity of plant extracts was performed on HEK293 cells as a human cell line. The cytotoxicity of the extracts was independent of concentration, with echinacea extract showing the highest cytotoxicity (Fig. S1).
The antiviral activity of medicinal plants was assessed against control viruses using qRT-PCR assay, as shown in Figure S2, which shows the dose-response curves of the tested plants. The transient response of the test plants was assessed using the following assays:
Analysis of virus-infected cells after treatment. The results of this analysis indicated that St. John’s wort was the most effective (Fig. 2A) with a minimum inhibitory concentration (MIC) of 1.56 μg/mL, while a mixture of St. John’s wort and Echinacea showed an MIC of 6.25 μg/mL, the worst effect. has echinacea, which is 6.25 mcg / ml. Viral load over time (12, 18, 24, 36, 48 hours) (Figure 3A) showed a significant decrease in H. perforatum viral load after the mixture was added up to 36 hours of addition (p = 0.0047). up to 36 hours with the addition of a mixture of H. perforatum-Echinacea (p = 0.0048) and up to 24 hours with the addition of echinacea (p = 0.0060).
Cell pretreatment prior to viral infection assay: The results of this assay showed an MIC of 1.56 μg/mL for St. John’s wort and 6.25 μg/mL for Echinacea and a mixture of St. John’s wort and Echinacea. Figures 2B and 3B (shown earlier) show viral suppression (%) and viral load reduction over time (12–48 hours) for Hypericum perforatum, Hypericum perforatum-Echinacea complex, and Echinacea purpurea, respectively.
Virucidal activity analysis (virus pretreatment before infection): the results of this analysis showed that Hypericum perforatum had the highest effect (Fig. 2C and 5c) at 1.56 µg/mL, followed by Hypericum perforatum-Echinacea mixture, then Echinacea purpurea 6.25 mcg. /ml The time of addition of the drug (during the infectious cycle) showed a significant decrease in the viral load of H. perforatum within 48 hours, as did the mixture of H. perforatum-Echinacea, however, echinacea showed virucidal activity only within 36 hours.
The inhibitory effect of Hypericum perforatum, Echinacea perforatum, and a mixture of St. John’s wort and Echinacea purpurea on SARS-CoV-2 RNA levels in Vero E6 cells was assessed by qRT-PCR. Three experiments were designed: post-treatment of virus-infected cells (A), pre-treatment of cells prior to viral infection (B), and virucidal assay (C). In (A–C), the percent inhibition of H. perforatum virus was 18%, 30%, and 36% up to 36 hours, while the percent inhibition of H. perforatum-Echinacea was 7%, 24%, and 32%. %, respectively, and showed that the inhibition rate of Echinacea purpurea can be as low as 24 hours. Inhibition is representative of two independent experiments performed in triplicate. Statistical analysis showed significant differences, p < 0.05 and p < 0.005 (one-way and two-way ANOVA).
Effect of Hypericum perforatum, Echinacea perforatum and Hypericum perforatum-Echinacea perforatum mixture on viral load of SARS-CoV-2 infection in Vero E6 cells assessed by qRT-PCR. Three experimental designs: post-treatment of virus-infected cells (A), pre-treatment of cells before virus infection (B), and virucidal assay (C). H. perforatum viral load was less than 25,000 PFU/mL for up to 48 hours in (A) compared to control virus, almost zero in (B) and (C), and for the H. perforatum-Echinacea mixture, which showed up to 36 hours of echinacea viral load. Viral load is representative of two independent experiments performed in triplicate. Statistical analysis shows a significant difference, p < 0.005 (one-way and two-way ANOVA), PC represents a positive viral control without treatment.
The antiviral activity of H. perforatum, echinacea, and the H. perforatum-Echinacea mixture (Figs. 2 and 3) was evaluated at maximum non-toxic concentrations (1.56, 6.25, and 6.25). 2 was higher than the mixture of Hypericum perforatum-Echinacea purpurea and Echinacea purpurea. In addition, Echinacea has been shown to be a weaker inhibitor than St. John’s wort and St. John’s wort/Echinacea mixture, but has a stronger virucidal effect for up to 24 hours. In addition, the effect of plant material on SARS-CoV-2 infection was assessed by staining cells with viral CPE with crystal violet (Fig. 4). On fig. 4 shows the evaluation of the effect of addition of extracts on antiviral activity in three different antiviral assays (panels A, B and C) followed by observation for 48 hours after addition. PC is a positive virus control without further processing, while NC is a negative cell control without added virus and additional processing.
Antiviral activity of Hypericum perforatum, Echinacea perforatum and Hypericum perforatum-Echinacea mixture on SARS-CoV-2 infected Vero E6 cells (A, B, C). Photographic images of cell viability and CPE during the SARS-CoV-2 infection cycle visualized by crystal violet analysis (blue circles) 24–48 hours post-infection. Capture images when CPE starts 24 hours later against a positive control. The results are representative of two independent experiments carried out in triplicate.
In order to decipher the potential compounds present in the respective extracts of the selected plants, the phytochemicals indicated in each plant were collected from the literature and putative inhibitory mechanisms were investigated using computational methods. Such assistance includes molecular docking, molecular contact formation, and molecular dynamics modeling.
SBVS of a total of 394 phytochemicals from E. angustifolia, E. purpurea and H. perforatum for SARS-CoV-2 Mpro and SARS-CoV-2 RdRp predicted their binding affinity to be between -11 and -1 kcal/mol . (Tables S1-S6) The following are the top 10 connections for each SBVS from Mpro-E. Eleopsis, Mpro-E. Purple, Mpro-H. Perforated grass, RdRp-E. Eleopsis, RdRp-E. Purpura and RdRp-H. Select a perforation for re-docking analysis. The binding energies of the re-docked molecules of each plant to two drug targets are listed in Tables S7-S12. In case of repeated docking of SARS-CoV-2 Mpro, echinacein (-9.0 kcal/mol) and echinacein (-8.6 kcal/mol), anthocyanins (-9.6 kcal/mol) and anthocyanin 3-(6” – malonyl glucoside from E. purpurea) (-9.0 kcal/mol) and quercetin-3-O-glucuronide (-9.6 kcal/mol) from H. perforatum and proanthocyanidins (-9.1 kcal/mol) show significant binding energy to viral proteases. , much better than the natural ligand X77 (-8.4 kcal/mol).
Whereas, in the case of SARS-CoV-2 RdRp redocking, echinacea (-9.0 kcal/mol) and rutin (-8.0 kcal/mol) from E. angustifolia, kaempferol-3-O-rutin ( -9.3 kcal/mol) /mol) mol) and echinacein (-9.2 kcal/mol) from E. purpurea, as well as rutin (-9.2 kcal/mol) and quercetin-3-O-wood from H. perforatum Glycosides (-9.0 kcal/mol) showed significant binding energies to the target protein. These observed binding energies of the aforementioned compounds were much better than those of the control ligand remdesivir (-7.6 kcal/mol) and some previously registered FDA-approved drugs that were repurposed for SARS-CoV-260,61. This suggests that all of the compounds described above could be potential inhibitors of these two SARS-CoV-2 drug targets. Molecular interaction analysis results showed good molecular contacts with catalytic residues and other substrate-binding residues (Tables 1 and 2).
The intermolecular interactions of the corresponding SARS-CoV-2 Mpro docking complexes were also evaluated (Table 1). Notably, analysis of the Mpro-Echinacea SARS-CoV-2 complex showed that Mpro Echinacea and SARS-CoV-2 residues Phe140, His163, Glu166, and Gln192 form four hydrogen bonds, while Thr190, Arg188, Asn142, Glu166, Gly143, Thr26 SARS-CoV-2 Mpro Thr25, His41, and Cys44 residues form ten hydrogen bonds with echinaline in Echinacea angustifolia (Fig. 5). Anthocyanins from E. purpurea form six hydrogen bonds with Tyr54, Thr190, Cys44, Glu166, Hie163, and Asn142, and cyanidin 3-(6′-malonylglucoside) binds to Glu166, Gln189 of SARS-CoV-2, Asp187, and Cys44 residues. five hydrogen bonds to Mpro (Fig. 5). H. perforatum quercetin-3-O-glucuronide forms six hydrogen bonds with His163, Asn142, Glu166, Met49, Cys44, and Thr190, while proanthocyanidins form six hydrogen bonds with Arg188, Thr190, Glu166, Asn142, and the Ser144 residue forms five hydrogen bonds: 2 Mpro (Fig. 5). The observed interactions for each of the above molecules are much better than those described in the SARS-CoV-2 Mpro crystal structure for binding to the potent broad spectrum non-covalent inhibitor X77, as only three hydrogen bonds (to Gly143, His163 and Glu166) were observed in this particular complex structure, as well as in the re-docked SARS-CoV-2 Mpro-X77 complex (Fig. S5)50.
Two-dimensional molecular contact analysis of SARS-CoV-2 Mpro redocking with selected phytochemicals in selected plants, viz. (a) SARS-CoV-2 Mpro-Echinacin, (b) SARS-CoV-2 Mpro-Echinacin, (c) SARS-CoV-2 Mpro-cyanin, (d) SARS-CoV-2 Mpro-Cyanidin 3-( 6′-alonylglucoside), (e) SARS-CoV-2 Mpro-quercetin-3-O-glucuronide, and (f) SARS-CoV-2 Mproanthocyanidins. These postures demonstrate hydrogen bonding (pink arrows), π–π (green line), hydrophobic 55, polar (blue), negative (red), positive (purple), and glycine (grey) molecular interactions for the respective docking complexes. .
Similarly, the interaction of selected SARS-CoV-2 RdRp docking positions with selected phytochemicals was investigated (Table 2). Interestingly, however, SARS-CoV-2 RdRp-echinacoside showed 10 hydrogen bond formation at Asp452, Thr687, Asp760, Asp618, Lys551, Arg553, Ala554, and Asp623, while SARS Asp760, Asp452, Arg555, Arg553 -CoV-2 RdRp forms eight hydrogen bonds and three π-cationic contacts (Arg555 and Lys551) with rutin from E. angustifolia at Lys551 and Asp618 (Fig. 6). In this study, kaempferol-3-O-rutinoside from E. purpurea formed nine hydrogen bonds with Asp452, Lys551, Asp623, Lys621, Tyr619, Asp618, and Asp760 RdRp SARS-CoV-2, while quercetin-3-O- SARS-CoV-2 RdRp xyloside from H. perforatum forms six hydrogen bonds (with Asn497, Arg569, Gly683, Asn543) and one pi-cation interaction (Lys500) (Fig. 6). Notably, the important role of receptor-ligand hydrogen bond formation in complex stability has been discussed and established in the field of drug discovery (cit., Patil R., Das S., Stanley A., Yadav L., Sudhakar A., Varma AK Optimized hydrophobicity Interactions and hydrogen bonding at the target-ligand interface open opportunities for drug development SARS-CoV-2 RdRp compared to Remdesivir, a potential inhibitor of SARS-CoV-2 RdRp, reported The latter has a docking fraction of – 7.6 kcal/mol and interacts with similar active residues in RdRp of SARS-CoV-258,60.,61.
Two-dimensional molecular contact analysis of SARS-CoV-2 Rdrp redocking with selected phytochemicals from selected plants, viz. (a) SARS-CoV-2 RdRp-echinealin, (b) SARS-CoV-2 RdRp-rutin, (c) SARS-CoV-2 Rdrp-kaempferol-3-O-rutin, and (d) SARS-CoV-2 Rdrp-Quercetin-3-o-xyloside. These postures demonstrate hydrogen bonding (pink arrows), π–π (green line), hydrophobic 55, polar (blue), negative (red), positive (purple), and glycine (grey) molecular interactions for the respective docking complexes. .
In the field of drug discovery, molecular dynamics simulations are performed on proven molecular docking complexes to understand the stability of the respective complexes and intermolecular interactions over time. In this study, the MD simulation intervals of selected docking complexes were examined using standard deviation (RMSD), root mean square fluctuation (RMSF), and protein-ligand contact mapping versus 100 ns simulation intervals.
An analysis of the standard deviation along the MD trajectories of the docking facility helps to understand the convergence of the facility in time. Initially, proteins (Cα) and ligands suitable for proteins, phytochemicals selected from three plants were collected from their respective MD trajectories (Fig. 7). Notably, the calculated RMSD values for the alpha carbons (Cα) of SARS-CoV-2 Mpro and SARS-CoV-2 RdRp showed an acceptable deviation of <3 Å throughout the simulation. These observations showed that there were no significant structural changes in the docked viral proteins during the 100 ns interval (Fig. 7). Similarly, all selected phytochemicals showed < 5 Å stability as protein ligands with their respective viral proteins, with the exception of SARS-CoV-2 M-pro-quercetin-3-O-glucuronide (< 6.5 Å), M-proanthocyanidin SARS-CoV-2 ( < 11.3 Å) and SARS-CoV-2 RdRp-rutin (< 7.5 Å) showed high deviations at the end of the 100 ns simulation interval, indicating a high stability of the docking complex (Fig. 7) . In addition, these observations are also supported by the allowable fluctuations of the RMSF of the protein (Mpro SARS-CoV-2 less than 3 Å and RdRp SARS-CoV-2 less than 6 Å) and the RMSF of the ligand suitable for the protein (both proteins less than 3 Å). values confirm the respective docking complexes, except that the SARS-CoV-2 RdRp has a higher bias <6 Å at 300–325 residues of the SARS-CoV-2 RdRp-echinacoside and the C-terminal region of the viral RdRp (Fig. S3-S4 ). Overall, RMSD and RMSF analyzes of the docking complex confirmed the stability of the docking complex in the active pockets of the viral protease and RdRp during 100 ns of MD simulation.
Calculated RMSD values of alpha carbons (Cα) (blue curve) of SARS-CoV-2 protein and docking ligand (red curve), viz. (a) SARS-CoV-2 Mpro-Echinacin, (b) SARS-CoV-2 Mpro-Echinacin, (c) SARS-CoV-2 Mpro-cyanin, (d) SARS-CoV-2 Mpro-Cyanidin 3-( 6′-alonylglucoside), (e) SARS-CoV-2 M-procyanidin-3-O-glucuronide, (f) SARS-CoV-2 M-procyanidin, (g) SARS-CoV-2 RdRp-echinacoside, (h) SARS-CoV -2 RdRp-rutin, (i) SARS-CoV-2 RdRp-kaempferol-3-O-rutinoside, and (j) SARS-CoV-2 RdRp-quercetin-3-O-xyloside with modeling interval of about 100 ns.
To further assess the stability of the docking complex, protein-ligand interaction profiles were extracted from the corresponding MD simulation trajectories, including hydrogen bonds, hydrophobic interactions, ionic interactions, and water bridge formation (Fig. 8). Notably, the simulated complex showed significant formation of molecular contacts with basic residues in the active pocket of the viral protein over a simulated 100 ns interval. Interestingly, interacting residues were also noted in the initial docking positions of the corresponding complexes (Tables 1, 2). It is noteworthy that in SARS-CoV-2 Mpro, individual phytochemicals make a significant contribution to the formation of hydrogen bonds and the assimilation of water bridges, while in SARS-CoV-2, in addition to the formation of hydrogen bridges and water bridges, a significant contribution of ionic interactions was also noted. for RdRp docking with selected phytochemicals during the simulation interval (Fig. 8). Thus, these protein-ligand contact maps suggest significant stabilization of the docking ligand through the formation of intermolecular contacts with active residues of viral proteins. Thus, the analysis of MD modeling showed that the selected phytochemicals, namely echinacein, echinacein, anthocyanin, anthocyanin 3-(6′-alonylglucoside), quercetin-3-O-glucuronide, proanthocyanidin, echinacea, rutin, kaempferol-3-O Rutin and quercetin-3-O-xyloside as key components in some plant extracts were responsible for the observed SARS-CoV-2 inhibition effects.
Mapping of protein-ligand interaction of SARS-CoV-2 proteins with attached ligands, namely (a) SARS-CoV-2 Mpro-echinacin, (b) SARS-CoV-2 Mpro-echinacin, (c) SARS-CoV-2 Mpro -Echinacin, (c) SARS-CoV-2 Mpro-Cyanin, (d) SARS-CoV-2 Mpro-Cyanidin 3-(6′-alonylglucoside), (e) SARS-CoV-2 Mpro-Quercetin- 3-O -glucuronide and (f) SARS-CoV-2 Mproanthocyanidins, (g) SARS-CoV-2 RdRp-Echinakin, (h) SARS-CoV-2 RdRp-rutin, (i) SARS-CoV-2 RdRp-kaempferol-3 -O-rutinoside and (j) SARS-CoV-2 RdRp-quercetin-3-O-xyloside extracted from their respective 100-ns molecular dynamics simulation trajectories.
The COVID-19 pandemic is creating global challenges for the global economy, society and health systems. The pandemic has resulted in more than 5 million confirmed cases and 288 million deaths worldwide64. Although some vaccines have been developed and are urgently used due to the pandemic, their effectiveness remains questionable, especially with the emergence of worrying new variants in the genome structure63,64,65,66. Several non-specific treatment options have been evaluated and are in clinical trials, including repurposing known treatments for other diseases3. Therefore, the improvement and research of effective antiviral therapies is an urgent need for the treatment of SARS-CoV-2 infection.
Our study evaluated the anti-SARS-CoV-2 effects of the medicinal plants Hypericum perforatum, Echinacea purpurea and their combinations. Medicinal plants are purchased from commercial sources to ensure ingredient consistency and are tested individually or in combination with each other as a single treatment. Their mechanism of action was evaluated using three methods: post-treatment of virus-infected cells, pre-treatment of cells before viral infection, and virucidal methods. The cytotoxic effect of the test medicinal plants was assessed by the MTT assay and the results were expressed as percent cytotoxicity relative to the cell control (cells to which the test medicinal plants were not added). The CC50s of H. perforatum and H. perforatum-Echinacea mixtures were 66.78 and 141.1 µg/mL, respectively, while Echinacea was highly toxic.
It has been previously reported that the antiviral activity of H. perforatum extract and hyperforin reduces IL-6 and TNF-α mRNA expression levels and is effective in preventing the pro-inflammatory effects of various cytokines20,23,63. When exposed to light, hypericin exhibits various modes of antiviral activity, such as inhibition of the budding of new virions64, cross-linking of the capsid to prevent viral shedding65, and inhibition of protein kinase activity required for the replication of many viruses66,67. In addition, it binds to cell membrane phospholipids such as as phosphatidylcholine and binds to retroviral particles possibly associated with membrane lipid envelopes68. Gibbon et al. Many species of St. John’s wort have been noted to contain the biologically active acylphloroglucin69,70. In addition, the polycyclic quinones of hypericin have been reported to have potent light-induced antiviral activity against many enveloped viruses, including HIV-164, 65, 71, 72, 73. In the presence of light, the molecular field of action of hypericin is increased by more than 100-fold64,65,71, 72.73 as it is a photosensitive compound74. During light exposure, singlet oxygen is efficiently produced with a quantum yield of 0.7375, which is thought to be a causal factor in the antiviral activity of hypericin64,65,72 or a complex mechanism involving the superoxide anion and hypericin76. Other studies have shown64,65 that hypericin induces significant changes in the HIV p24 capsid protein upon exposure to light and can inhibit reverse transcriptase activity.
Our results showed that Hypericum perforatum containing 0.9 mg/grain of hypericin significantly reduced the viral load of SARS-CoV-2 compared to the positive control during the period of viral infection from 0 to 48 hours (Table S13, Figures 2, 3). This decline has been demonstrated. in the three mechanisms investigated in this study. Notably, the results showed that the highest antiviral effect was through the virucidal mechanism (Figure 3). Compared to the other two mechanisms, the highest inhibitory effect was observed 36 hours after infection, as shown in Figure 3. 2. While St. John’s wort had the strongest inhibitory effect (35.77%) and reduction in viral load up to 48 hours, echinacea and H. perforatum-echinacea mixture had 3.30 and 31.36%, respectively, up to 36 hours . The inhibitory effect of the H.perforatum-Echinacea mixture was lower than that of H.perforatum alone, which was expected because the active compound of H.perforatum was diluted with the addition of echinacea.
Hypericin has been reported to be the active antiviral compound in St. John’s wort extract against various viral infections including IBV17, bovine diarrhea virus (BVDV)77 and hepatitis C virus (HCV)47, where hypericin has been identified as the active ingredient. Other studies have shown that St. John’s wort extract has antiviral activity against influenza A virus and HIV78,79,80. In addition to the antiviral effect of the H. perforatum extract, H. perforatum ethyl acetate (HPE) extract significantly reduced the concentration of IL-6 and TNF-α in the lung tissue of mice infected with influenza A 80 virus and the content of hypericin. in the trachea and kidneys of IBV-infected chickens is mainly derived from HPE17.
Post time: Mar-27-2023