Phytochemical Compounds Present in COVI-MXG Herbal Preparation Inhibits RNA-Dependent RNA Polymerase from SARS-CoV-2: Virtual Screening

Background: COVID-19 pandemic disease, caused by coronavirus 2 (SARS-CoV-2) is expressed as severe acute respiratory syndrome. There are currently no proven effective therapeutic agents or vaccines against the virus. However clinical management includes infection prevention, control measures, supportive care using drug therapy based on previous scientic experience, pathophysiology and pharmacology of the drug. Some of the therapeutic agents exploited include; antiviral and antimalarial agents (remdesivir, hydroxychloroquine, chloroquine, lopinavir, umifenovir, favipiravir, and oseltamivir), Zinc and selenium. There are claims of herbal preparations with palliative or therapeutic effects. The novel formulation of herbal preparation, COVI-MXG for the management of Covid-19 contains a unique combination of ve (5) plants. In silico studies was carried out using robust methods and software to evaluate the plant constituents to determine its possible antiviral activity, safety and pharmacokinetic prole. Results: Pharmacokinetic predictions showed phytochemicals with varying degrees of gastrointestinal absorption rates and blood brain barrier permeability. Toxicity class fall between 3 and 5 with high LD 50 values. Conclusions: When compared with the listed therapeutic agents, the phytochemical compounds present in COVI-MXG showed varying degrees of binding anities for SARS-CoV-2 (7BV2.pdb) better than the drugs currently in use at the target sites. The preparation contains high concentration of Zinc and other micronutrients. hepatotoxicity, immunotoxicity, mutagenicity and cytotoxicity


Background
Coronavirus disease 2019 , caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rst originated in Central China and has now spread to almost all countries of the world.
The disease is responsible for extensive mortality with serious public health implications and there are no clinically approved vaccines or speci c therapeutic drugs available at the moment [1,2] . SARS-CoV-2 requires the viral RNA-dependent RNA polymerase (RdRp) for replication which is a target for antiviral drugs such as Remdesivir and Favipiravir [3] . As a result of this challenge, it is thought that herbal medicines and/or puri ed natural products may be helpful in guiding development of novel and effective drugs against the virus. It was reported that between 85 to 92% of patients in some cities in China resort to herbal drugs in addition to other remedies [4,5] . Earlier reports show that even though the herbal medications are effective relatively good safety margin, the mechanism of activity is poorly understood. It however thought that the activity might be through viral inhibition [6] .
From the forgoing and given the impact of the pandemic, there is the urgent search for effective treatment using innovative strategies. One of such techniques involves computational approaches [7,8] .
This approach is advantageous, as it drastically cuts down on the time-consuming animal and in vitro assay laboratory work [9] . In addition it reduces costs associated with the attempt to physically screen large banks of compounds or of plant extracts for biological activity [10] . The strategy is particularly helpful in the attempt to tame the rampaging COVID-19 pandemic as it can highlight compounds which can then be evaluated for in vitro effectiveness and toxicity using cell-based assays before being subjected to other investigations such as animal and clinical trials [4] .
The herbal formulation COVI-MXG contains Monodora myristica, Xylopia aethiopica, Gongronema latifolium, Viscum album and Garcinia kola. There is no report in the literature to our knowledge to the effect that this combination of plants and in the proportions has ever been documented for use in the management of Covid 19.
The study used virtual screening to evaluate inhibitory effect of the phytochemical compounds present in COVI-MXG on SARS-CoV-2 RdRp.

Methods
All methods used in this study were entirely In Silico.

Hardware, Ligand Library and Target Preparation
All the computational analysis/ screening were done using x64-based PC, windows 10 Pro, with 32-Bit operating system.
A compound library of 21 phytochemical constituent (table 1) contained in COVI-MXG were downloaded from PubChem [11] and optimized in discovery studio 4.5 visualizer [12] . Also included in the ligand library were 9 therapeutic compounds currently in use for the treatment of COVID-19; Lopinavir, Remdesivir, Ritonavir, Umifenovir, Favipiravir, Oseltamivir, Quinine, Chloroquine and Hydroxychloroquine. The crystal structure of the SARS-CoV-2 target with pdb code 7BV2.pdb was downloaded from the Protein Data Bank (pdb) (https://www.rcsb.org/). The pdb le was opened in WordPad, water molecules and its original ligands (remdesivir) eliminated and le re-saved.

Virtual Screening
The library of ligands were docked into the protein target and the binding a nities determined in Autodock Tools using PyRx 0.8 package [13] and Autodock Vina [14] . between protein target and ligands were analysed using PyMol [15] and discovery studio 4.5 visualizer [12] .
In Silico ADMETox Analysis SwissADME [16] was used to study the absorption and distribution of the phytochemicals while accessing; bioavailability score, Blood-brain barrier permeability, and gastrointestinal tract absorption.
ProTox-II [17] was used to study the rodent oral toxicity of the phytochemicals. Parameters such as carcinogenicity, hepatotoxicity, immunotoxicity, mutagenicity and cytotoxicity were assessed.
Ethics approval and consent to participate Not applicable

Results
Phytochemicals present in COVI-MXG showed varying degrees of binding a nities for SARS-CoV-2 (7BV2.pdb) as shown by the change in binding energy (Kcal/mol) in Table 2. The molecules were seen to exhibit different types of interactions including hydrogen-bonding, covalent bonding, alkyl and van der waal's force interactions (Figure 1). Major phytochemical in Garcinia kola showed the highest binding a nity followed closely by phytochemicals in Viscum album. These molecules were seen to have been better than current therapies. Three (3) pytochemicals in Xylopia aethiopica had a better binding a nity than Favipiravir but the same with umifenovir while two (2) in Monodora myristica had the same binding a nity as Favipiravir (Table 1 & 2). The molecules were observed to have docked properly within the target without global changes ( Figure 2) Toxicity predictions (Table 3) showed phytochemicals with varying degrees of gastrointestinal absorption rates, blood brain barrier permeability and bioavailability. Toxicity class fell between 3-5 with high LD 50 except for P-cymene with a predicted toxicity class of 1 and a predicted rodent LD 50 of 3 mg/Kg.   of Kaempferol [18] , Limonene [19] , and myricetin [20] against coronavirus, and herpes simplex virus. Furthermore, Monoterpenes such as γ-terpinene, α-pinene, p-cymene, and 1, 8-cineole have been shown to have a better antiviral activity and low toxicity when in a mixture than as single entities [21] .
Structure activity relationship could be said to have an effect on position of binding among the phytochemicals as can be seen by remdesivir and kolaviron (Figure 2a & b). Most of the other smaller phytochemical compounds were seen to share the same binding pocket with hydroxchloroquine ( Figure  2c and d).
In silico ADMETox pro ling of the phytochemicals showed a very promising low toxicity pro le for COVI-MXG. This is because most of the molecules were of low toxicity except p-cymene which had a toxicity class 1 prediction and an LD 50 of 3 mg/kg. The toxicity of p-cymene should not pose a problem seeing as it is only 7.3% of Xylopia aethiopica fruit oil.
Phytochemicals such as α-and β-Pinene which exhibited considerable binding a nities with the molecular target but much lower than the current drug management of COVID-19 (Table 2), have been reported to possess antiviral and antibacterial activities [19,22] .
The formulation of COVI-MXG would afford a synergistic effect, seeing as it contains Gongronema latifolium which has an appreciable amount of Zinc [23] and the zinc ionophore activity of quercetin [24] which showed a higher binding a nity ( Table 2). Zinc has been shown to play an important role in the inhibition of coronavirus RNA polymerase [25] .

Conclusion
Pharmacokinetic predictions showed phytochemicals with varying degrees of gastrointestinal absorption rates and blood brain barrier permeability. Toxicity class ranged from 3-5 with a high LD 50 .
Phytochemicals exhibited considerable binding a nities with the molecular target. The formulation of COVI-MXG would afford a synergistic effect, since Gongronema latifolium contains high level of Zinc and the zinc ionophore activity of quercetin, which showed a very high binding a nity. Also Zinc has been shown to play an important role in the inhibition of coronavirus RNA polymerase. This formulation is safe, and has shown good activity against the coronavirus 2 (SARS-CoV-2 Availability of data and materials: These have been reported and cited in the methodology part of the manuscript Con ict of interest: The authors declare no con ict of competing interests Funding: No funding received Authors' Contributions: FKD and KJA did the conception and design of the work. PCO did the Acquisition, analysis and interpretation of the data. FKD drafted the manuscript. All authors substantively revised the manuscript, read and approved the nal copy of the manuscript for submission. Figure 1 2D binding interactions of Remdesivir (a), Kolaviron (b), kaempferol (c), α-phellandrene (d) and Transpinocarveol (e) to the active site residues of SARS-CoV-2 (7BV2.pdb). Ligands are shown in stick forms while amino acid residues are shown in disc forms. Hydrogen-bond interaction with amino acid main chain are indicated by green discontinuous lines, green colored discs shows van der waal's interaction, pink discs shows alkyl interactions while purple discs shows pi-sigma interactions. Discs and lines shown in red colour represent unfavourable bumps and interactions.