Predictive Assessment of the Antiviral Properties of Imperata cylindrica against SARS-CoV-2

The omicron variant and its sublineages are highly contagious, and they still constitute a global source of concern despite vaccinations. Hospitalizations and mortality rates resulting from infections by these variants of concern are still common. The existing therapeutic alternatives have presented various setbacks such as low potency, poor pharmacokinetic profiles, and drug resistance. The need for alternative therapeutic options cannot be overemphasized. Plants and their phytochemicals present interesting characteristics that make them suitable candidates for the development of antiviral therapeutic agents. This study aimed to investigate the antiviral potential of Imperata cylindrica (I. cylindrica). Specifically, the objective of this study was to identify I. cylindrica phytochemicals that display inhibitory effects against SARS-CoV-2 main protease (Mpro), a highly conserved protein among coronaviruses. Molecular docking and in silico pharmacokinetic assays were used to assess 72 phytocompounds that are found in I. cylindrica as ligands and Mpro (6LU7) as the target. Only eight phytochemicals (bifendate, cylindrene, tabanone, siderin, 5-hydroxy-2-[2-(2-hydroxyphenyl)ethyl]-4H-1-benzopyran-4-one, maritimin, 5-methoxyflavone, and flavone) displayed high binding affinities with Mpro with docking scores ranging from −5.6 kcal/mol to −9.1 kcal/mol. The in silico pharmacokinetic and toxicological assays revealed that tabanone was the best and safest phytochemical for the development of an inhibitory agent against coronavirus main protease. Thus, the study served as a baseline for further in vitro and in vivo assessment of this phytochemical against Mpro of SARS-CoV-2 variants of concern to validate these in silico findings.


Introduction
Commonly known as "speargrass," "cogongrass," "alangalang" or "kunai grass," Imperata cylindrica is a member of the Poaceae family [1].It is a perennial rhizomatous plant that can grow on dry soils or soils with high moisture content to reach 0.6-3 m height.Te roots of the plant are fbrous and can thrive 1 to 1.2 m deep in the soil.Te leaves are stif, measuring up to 120 cm long and 2 cm wide at the base with a narrowing to sharp point at the top and a prominent white midrib.Te fowers are white fufy spikelike heads of 5-20 cm long and 2.5 cm diameter.Te plant produces brown, oblong seeds that are 1-1.5 mm long, with a ring of silky white hairs that are 10 mm long around the base (Figure 1) [2].It is native and widely found in tropical and subtropical Asia, Oceania, Australia, Southern Europe, and Eastern Africa [3].It was inadvertently introduced in Latin America, the Caribbean, and the south-eastern United States through contaminated shipping packages and intentionally as an ornamental plant and as forage grass for erosion control [4].As a result of its pyrophytic nature, it has been associated with wildfres in areas that it colonizes and is often regarded as an invasive weed which naturalises aggressively through self-seeding [5].In such cases, it forms a monoculture that displaces native species, leading to low crop yield, low crop quality, reduction of farm size, increased labour requirement, and increased susceptibility to crop pathogens [5,6].
Despite these negative aspects, I. cylindrica extracts have numerous benefcial attributes that justify its usage as a traditional medicine to treat various ailments [1,3,7,8].For example, in the traditional system of the "Shen Nong's herbal Classic" of the Han Dynasty (China), the plant's medicinal benefts are attributed to the lung, stomach, and bladder meridian and it is also acclaimed for its antidiuretic efects and fever-relieving properties [1].Moreover, it is indicated in the replenishment of the spleen, the treatment of polydipsia, blood stasis, lung heat, difculty in micturition, irregular menses, edema, and jaundice [8].About 72 phytochemicals have been isolated from I. cylindrica and characterized extensively.Te main types of phytochemicals that have been characterized belong to the classes of favonoids, saponins, coumarins, glycosides, and phenols.A few compounds of each class of phytochemicals are shown in Figure 1 [7,[9][10][11][12][13].Other unclassifed compounds such as sesquiterpenoid, cylindrene, tabanone, palmitic acid, and phytol were also isolated from this plant.It has shown that I. cylindrica extracts which contain these compounds display various biological activities.For instance, phytol, palmitic acid, and coumarin were proven to exhibit antibacterial activities [1,14], while imperanene (a phenol) and chromone (a favonoid) exhibit platelet aggregation activity and neuroprotective properties, respectively [15,16].Other biological activities of I. cylindrica extracts are hepatoprotective activities [17], vasodilative properties [18], antioxidant properties [19], anti-cancer activities [19][20][21], and anti-infammatory activities [1].Moreover, the rhizome of this plant can be used alone or in combination with other medicinal plants such as Panax quinquefolius, Rehmannia glutinosa, and Wolfporia cocos to treat hematuresis [1].In addition, it is also used in combination with Artemisia capillaris Tunb and Gardenia jasminoides Ellis to reduce dampness and heat in the management of jaundice.Furthermore, I. cylindrica is used together with Morus alba L. and Puerariae lobatae Radix for the treatment of asthma and high fever, respectively [1].
SARS-CoV-2 emerged as a life-threatening virus in 2019 and claimed millions of life worldwide.With the continued emergence of variants of concern, coupled with the inefcacy of the initial drugs and a rise in the number of hospitalizations and deaths, the call for an efective vaccine became urgent [22].Precisely, the omicron variant of SARS-CoV-2 (B.1.1.529),classifed as a variant of concern (VOC), has swiftly propagated across the globe, posing a signifcant new threat to public health.Tis variant is particularly notable due to the extensive number of mutations it has Glycosides: • Siderin • 7-O-Glucosyloxy-4-methoxy- • 7-Hydroxy-4-methoxy-5methylcoumarin Coumarins:
2 Advances in Virology accumulated [23].Te most dominant strain currently is now the FLiRT variant, which is derived from the omicron lineage.FLiRT variants, including subvariants such as KP.3, KP.2, and KP.1.1,account for a signifcant portion of new cases [24,25].Tese variants are particularly notable for their enhanced ability to evade immunity and diagnostic detection, whether from previous infections or vaccinations as a result of specifc mutations in the spike protein, that increase their transmissibility and ability to bypass immune defences [24,25].Furthermore, the symptoms of FLiRT infections remain similar to earlier variants, including sore throat, body aches, cough, runny nose, and shortness of breath, particularly in unvaccinated individuals or those whose immunity has waned over time [24].Te emergence of these variants could potentially lead to a surge in cases, as vaccines are inefective against these variants [24].
In response, research institutions and pharmaceutical companies rapidly developed drugs and vaccines that could efectively target SARS-CoV-2 variants [26].Tus, drugs such as oseltamivir, lopinavir, ritonavir, remdesivir, favipiravir, ribavirin, and chloroquine demonstrated some potency with the frst cases of COVID-19 but they were proven to be inefective in immunocompromised patients and those with comorbidities [27].Up to this point, only a few therapeutic agents have been verifed to exhibit certain levels of efcacy against SARS-CoV-2 strains of concern with the majority targeting one of the three key proteins associated with coronaviral pathogenicity [28].While a handful have received approval, others are in the pipeline for ofcial authorization, and some are still undergoing thorough investigation [28][29][30][31][32]. Tese agents encompass both synthetic and natural compounds [33], as well as newly developed [34,35] or repurposed ones [36][37][38].Te most signifcant compounds, including engineered antibodies with an anticipated nonspecifc efcacy against all emerging strains and potential future coronavirus species, are lengthily outlined in a scientifc report [28].Several vaccines received approval from the US Food and Drug Administration (US FDA), and even though they have been rolled out globally, they became less efective as new variants continued to emerge [39][40][41][42].
Natural products from medicinal plants, such as Lycoris radiata, Rheum ofcinale, Curcuma longa, and Polygonum multiforum (just to name a few), have demonstrated some potency against various coronavirus strains [33,[43][44][45].I. cylindrica also displays interesting characteristics that highlight its potential in the development of therapeutic compounds against pathogens such as SARS-CoV-2.One method that is mostly used to combat COVID-19 is by designing compounds that can hinder viral attachment to the host cells.SARS-CoV-2 is a positive-sense singlestranded (++SS) RNA virus with a genome of 14 open reading frames (ORFs) of 30kb [46].Coronaviruses have 2 long polyproteins (pp1a and pp1ab), nine accessory proteins, and four major structural proteins, namely, the spike (S), the membrane (M), the envelope (E), and the nucleocapsid (N) that are encoded by these ORFs.Te structural proteins are potent virulence factors involved in the replication, transmission, and viral host-cell interaction processes [46].Te S glycoprotein promotes viral entry into the host cell through binding with the host angiotensinconverting enzyme 2 (ACE-2) receptor [46].Tis highly conserved protein is the main target of anti-SARS-CoV-2 drugs that are currently developed and investigated.Similarly, the main protease (M pro or 3CL pro ) and the papain-like protease (PL pro ) are located within the ORF1a that are highly conserved among the variants of SARS-CoV, and they constitute ideal targets of the current drug candidates since human proteases do not have similar cleavage patterns [46][47][48].Tese are cysteine proteases that are involved in the cleavage and the maturation of the polyproteins pp1a and pp1ab into nonstructural proteins (NSPs), including RNAdependent RNA polymerase (RdRp), helicase, exoribonucleases, 2′-O-methyltransferase, and uridine-specifc endoribonuclease [47].Tus far, SARS-CoV-2 M pro inhibitors (Ensitrelvir, Simnotrelvir, and Nirmatrelvir) [49][50][51] and RdRp inhibitors (Remdesivir, Molnupiravir, Renmindevir, and Azvudine) [51][52][53][54][55] have been approved by the US FDA and the China Medical Products Administration but they present setbacks such as suboptimal potency, poor oral bioavailability, low oral drug exposure, moderate stability in human hepatic microsomes [53][54][55], and emergence of variants that are resistant to these drugs [56][57][58][59][60][61][62].
Te aim of this study is to use molecular docking tools to screen potential I. cylindrica phytochemicals that display antiviral activities against SARS-CoV-2 through molecular docking assays.

Protein Preparation.
As highlighted in the literature, the main protease (M pro ) is an essential protein in the replication of SARS-CoV-2 and it is highly conserved among the diferent variants [47].It is also one of the main targets of the current antivirals that are developed against SARS-CoV-2 [48].Tus, the three-dimensional X-ray crystal structure of the SARS-CoV-2 main protease with the identifer PDB ID: 6LU7 (resolution: 2.16 Å) was retrieved from the Research Collaboratory Structural Bioinformatics-Protein Data Bank (RCSB-PDB) and was used for in silico studies.It was frst visualised on BIOVIA Discovery Studio ® version 4.0 modelling environment (Dassault Systèmes, San Diego, 2021) [63].Water molecules and heteroatoms were then removed, and polar hydrogen was added to all noncarbon atoms to fll valencies.Finally, the restrained energy of the structure was minimised as previously described [64].

Ligand Preparation.
Seventy-two I. cylindrica phytochemicals were selected for this assay.Te N3 inhibitor (Supplementary Figure 1) was used to validate the procedure used in this study.Te library was prepared manually by downloading structure data fle (SDF) formats of favonoids, saponins, phenols, coumarins, glycosides, and other unclassifed phytochemicals of I. cylindrica from PubChem database (https://pubchem.ncbi.nlm.nih.gov/) as well as the PDB format of the N3 inhibitor.Teir drug likeness was assessed as per Lipinski's rule of fve [65].Te ligands were Advances in Virology energy-minimised by inducing the universal force feld (UFF) and converted into .pdbqtformat with OpenBabel ® (version 3.1.1x64)[66].Hydroxychloroquine was used for comparative purposes in this assay, and its SDF fle was retrieved from DrugBank database (accession number: DB01611) (https://go.drugbank.com/drugs/DB01611).

Binding Sites' Prediction and Receptor Grid Generation.
Te virtual screening and the molecular docking of the phytochemicals were carried out using Autodock Vina [67].Tis turnkey software stands out as one of the swiftest and extensively employed open-source docking engines which relies on a straightforward scoring function and swift gradient-optimization conformational search.BIOVIA Discovery Studio ® (version 4.0) was used to generate the receptor-binding grid.Te grid box was large enough to cover the binding site of the protein structure (x, y, and z coordinates were −10.729204, 12.417653, and 68.816122, respectively).An exhaustiveness value of 9 was applied to maximise the probability of detecting global minimum scoring function in Autodock Vina.Te interaction of the hit phytochemicals was assessed for their 2D and 3D interactions.Phytochemicals with the highest binding afnities were assessed with Discovery Studio Visualiser (version 4.0).

Pharmacokinetic Properties' Predictions.
Te Swis-sADME web tool (https://www.swissadme.ch/)[68] was used to predict the absorption, distribution, metabolism, and excretion (ADME) profles of the phytochemicals that were druggable (as a result of a suitable molecular weight and a demonstrated binding energy).To predict the toxicity and the toxicological efects of the phytochemicals, ProTox-II web tool was used [69].

Molecular Dynamics Simulation.
Molecular dynamics (MD) simulations of the most promising compound docked to M pro was performed with the Desmond package.Te complex was solvated in an explicit water box of size 10 Å using the TIP3P water model with periodic boundary conditions (PBC).Te OPLS3e force feld was employed to model the protein, ligand, and Na+/Cl− ions were assigned to neutralise the system's total charge.Te system underwent energy minimisation for 2000 steps before a 60 ns production run.After minimization, the complex was subjected to a production run in the NPT ensemble.Te system was gradually heated to maintain a temperature of 300 K and pressure using the Nose-Hoover thermostat algorithm and the Martina-Tobias-Klein method.Long-range electrostatic interactions were calculated using the particle mesh Ewald (PME) method with a grid spacing of 0.8 Å. Final trajectories were analyzed using UCSF Chimera [70].Te ligand-binding energy using MM-PBSA was calculated for the analyses.Te molecular mechanics potential energy and the free energy of solvation for each complex were analyzed using the equation ΔG binding � E gas + G sol − TΔS considering all frames from the MD simulation trajectories [71].
As illustrated in the fgures below, the best docking poses and the molecular interactions between the various docked phytochemicals and SARS-CoV-2 main protease (M pro ) were recorded.Hydroxychloroquine and N3 inhibitor molecular interactions with SARS-CoV-2 main protease were also illustrated (Figures 2(a) and 3(a)).Hydroxychloroquine interacted with SARS-CoV-2 main proteasebinding pocket by forming stable hydrogen bonds at amino acid residues Gly143, Leu141, Ser144, and Cys145.Alkyl bonds with M pro were formed at amino acid residues Pro168 and Met165 (Figure 2(b)).Te N3 inhibitor bonded with the SARS-CoV-2 M pro binding pocket through the formation of stable hydrogen bonds at amino acid residues Gly143, Cys145, and Ser144.In addition, an alkyl bond and a Pi sulphur bond were also formed at the amino acids residues His163 and Cys145, respectively (Figure 3(b)).
Bifendate interacted with M pro through cationic bonds (Figure 4(a)) at amino acid residues His41 and Glu166 as well as van der Waals bonds at amino acid residues His41 and Asn142 (Figure 4(b)).
Furthermore, cylindrene interacted with SARS-CoV-2 M pro through hydrogen bonds (Figure 5(a)) at amino residues Cys145 and Ser144, and it also formed a Pi-alkyl bond at amino acid residue His163 (Figure 5(b)).
Tabanone and siderin best binding poses highlighted an interaction between these ligands and SARS-CoV-2 M pro through three hydrogen bonds at amino acid residues Cys145, Ser144, and Gly143 (Figures 6(b  Advances in Virology 5-methoxyfavone are the only CYP3A4 inhibitors (Table 3).Te only phytochemical that was not permeable through the blood brain barrier was bifendate but all the phytochemicals displayed a high intestinal absorption (Table 3 and Figure 12).
Te prediction of permeability coefcient (Kp) for the transport of compounds through mammalian epidermis is based on the linear model by Potts and Guy and indicates the ability of a compound to pass through the mammalian skin.Te more negative the log Kp value of a molecule, the less  3).All the investigated phytocompounds displayed a high gastrointestinal absorption index and a good bioavailability score but only bifendate was not permeant to the blood brain barrier (BBB) (Table 3).P-glycoprotein, an ABC transporter that has undergone extensive research, acts as a biological barrier by expelling toxins and xenobiotics from cells.Both laboratory experiments and studies involving living organisms have shown that P-glycoprotein signifcantly infuences the absorption and distribution of drugs [73].In theory, Pgp-inducers (or Pgp substrates) can cause a reduction in the drug  bioavailability, an increased renal clearance of the drug and a reduced distribution in the peripheral tissues [74].Terefore, an ideal drug candidate should not be a Pgp substrate.All the phytocompounds investigated in this report are not Pgp substrate (Table 3).

Molecular Dynamics Simulation.
Te conformational fexibilities of the tabanone-docked compound with the M pro enzyme (PDB: 6LU7) were examined via MD simulations to obtain reliable drug-receptor-binding afnities.Figure 13 displays its kinetic energy plot (A), its stability on a temperature versus time plot (B), and its atomic potential energy function (C).As shown in Figure 13(a), the complex kinetic energy remains stable until around 500 ps.Its remains stable at temperatures varying from 260 to 350 K until around 500 ps (Figure 13(b)).To assess the dynamic stability of the 6LU7/tabanone complex, its time-dependent potential energy was calculated throughout the MD trajectory and its potential energy stabilises at around 500 ps (Figure 13(c)).14 Advances in Virology

Discussion
SARS-CoV-2 variants of concern (VOCs) such as omicron and its sublineages are highly transmissible and still constitute a source of concern [40].Despite vaccinations, breakthrough infections, hospitalizations, and mortality rates resulting from infections by these VOCs are still high and existing therapeutic alternatives have presented various setbacks such as low potency, poor pharmacokinetic profle, and drug resistance [26,58,60,61].Even though there are promising therapeutic options that could alleviate the burden of symptoms resulting from infection by SARS-CoV-2 VOCs [28,29,32,[34][35][36][37][38][76][77][78][79], there is still a need to identify compounds that could be efective against these variants, especially in this context of emerging resistant strains.Phytochemicals are still poorly investigated, they display interesting properties that make them ideal substitutes in the design and the development of novel therapeutic compounds, and in silico modelling of phytocompounds has yield interesting results that could be highly relevant in drug design and development [80][81][82].
Natural compounds had proven to be efective on SARS-CoV 2 by previous studies [33,[43][44][45]79].In this study, we used molecular docking tools to assess the antiviral properties of Imperata cylindrica phytochemicals against SARS-CoV-2 and their overall safety.Of the 72 compounds that are commonly found in this plant, eight displayed interesting docking results against SARS-CoV-2 main protease (M pro ), a highly conserved protein among all the coronaviruses [47].M pro is a homodimer which is vital in viral replication and the maturation of coronavirus nonstructural proteins, and it is an ideal drug target as humans do not possess its homologue.It is structured by three domains which confgure it into two substrate-binding sites: an oxyanion hole (which is made up of Gly143, Ser144, and Cys145) and a substratebinding pocket [83].Te substrate-binding pocket includes su-sites s1 (F140, L141, N142, H163, and E166), s2 (M49, Y54, H164, D187, and R188), s4 which is hydrophobic (M165, L167, Q189, T190, and Q192), and hydrophilic subsites such as s3 (E166), s5 (including T190, A191, and Q192), and s1′ (H41, G143, S144, and C145) [84][85][86].Te s1′ subsite is a catalytic dyad with residues Cys145-His41 where cysteine acts as a nucleophile while histidine plays the role of a proton acceptor.M pro inhibitors act through various modes of action.One of the mechanisms is by forming covalent bonds with Cys145-His41 of the catalytic dyad, leading to M pro inhibition.Another mode of action is by embedding in the oxyanion hole of M pro [86].
Bifendate displayed the highest binding afnity (−9.1 kcal/mol) to M pro and would be safe for human consumption based on the toxicological assessment data.It may be efective in inhibiting the main protease because it bound to the protein by forming van der Waal bonds with amino acid residues Asn142 and His41, and Pi-cationic bonds with amino acid residues Glu166 and His41.However, it would be a poor antiviral drug candidate due to the following: (i) no hydrogen bonds were formed with the M pro binding sites, (ii) it is not permeant to the blood brain barrier, and (iii) it inhibits CYP1A2, CYP2C19, CYP2C9, and CYP3A4.Te binding afnity of cylindrene was adequate (−6.5 kcal/mol) and it interacted with SARS-CoV-2 M pro by forming hydrogen bonds at amino residues Cys145 and Ser144 of the oxyanion hole, and a Pi-alkyl bond at amino acid residue His163.Tese parameters coupled with its acceptable pharmacokinetic profle and safety (based on toxicological assessment) suggest that cylindrene would be one of the best drug candidates to inhibit M pro .Tabanone and siderin also formed stable hydrogen bonds with M pro oxyanion hole at amino acid residues Cys145, Ser144, and Gly143.In addition, tabanone formed two alkyl bonds at amino acid residues His163 and Cys145, while siderin formed van der Waals bonds at amino acid residues His163 and Gln189.Tese two phytochemicals can therefore inhibit M pro .However, siderin would be a poor drug candidate despite its acceptable pharmacokinetic properties.As revealed in this study, siderin is carcinogenic and mutagenic when compared to tabanone which is safer with a predicted LD50 of 10,000 mg/kg (meaning that it would be lethal only at high doses).Interestingly, 5-hydroxy-2-[2-(2-hydroxyphenyl)ethyl]-4H-1-benzopyran-4-one formed six covalent hydrogen bonds in the M pro oxyanion hole by binding to Ser144, Cys145, His163, Gly143, and Leu141 amino acid residues, respectively.Besides the fact that it inhibits CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4, its other pharmacokinetic parameters are relatively acceptable and it is safe for human consumption.Contrastingly, even though maritimin, 5-methoxyfavone, and favone formed stable hydrogen bonds with amino acid residues of the M pro oxyanion-binding site, they are unsafe.In particular, maritimin is carcinogenic and highly mutagenic with an intriguing lethal dose of 100 mg/kg.Flavone is carcinogenic and highly cytotoxic while 5-methoxyfavone may be carcinogenic, mutagenic, and cytotoxic.Tese features indicate that these phytochemicals are not acceptable drug candidates.Te fndings on these I. cylindrica favonoids (5methoxyfavone and favone) are contrasting with another study which demonstrated the inhibitory properties of 31 polyphenolic and favonoid bioactive compounds on SARS-CoV-2 main protease and papain-like protease (PLpro) enzymes [87].Tis indicates that the binding of bioactive compounds can undergo structural adjustments in such a way that they interfere with the enzyme functions and this can be achieved through molecular dynamics simulation [87].
Conclusively, tabanone seems to be the best candidate for the development of an M pro inhibitor, among all the I. cylindrica phytochemicals tested in this study.Tis was justifed by the following observations: (i) tabanone will be lethal to human beings only at very high doses (predicted LD50 � 10000 mg/kg) (Table 4 and Supplementary Table 2); (ii) it is neither hepatotoxic nor cytotoxic (Table 4); (iii) it is neither carcinogenic nor immunotoxic (Table 4); (iv) it is not mutagenic (Table 4) and it displayed a good skin permeability index (−5.61cm/S) (Table 3); (v) its bioavailability score, its gastrointestinal absorption index, and its intestinal absorption index are high (Table 3); (vi) it displayed a high ability to pass through the blood brain barrier; (vii) it is not a P-glycoprotein substrate and it does not inhibit any of the Advances in Virology cytochromes (Table 3 and Supplementary Table 2); (viii) it neither violated any of Lipinski's rule of fve nor that of the other scientists (Veber, Muegge, Ghose, and Egan) (Table 3 and Supplementary Table 2).As compared to hydroxychloroquine (binding afnity � −5.4) and N3 inhibitor (binding afnity � −3.7), tabanone displayed a better binding afnity to SARS-CoV-2 M pro (binding afnity � −5.6) (Table 1).Tey all interacted with the active site of the main protease-binding pocket by forming covalent and stable hydrogen bonds at amino acid residues Gly143, Ser144, and Cys145 (Figures 2(b), 3(b), and 6(b)).However, only N3 inhibitor and tabanone were able to form an alkyl bond with M pro at amino acid residue His163.Furthermore, the MD simulations indicate that tabanone is stable when forming a complex with the M pro enzyme (Figures 13(a), 13(b), and 13(c)).
Tis study is the frst to assess I. cylindrica phytochemicals against a viral protein using molecular docking assays.However, a similar study investigated the inhibitory properties of phytocompounds from four medicinal plants (Salvia ofcinalis L, Anacardium occidentale, Crinum jagus, and Andrographis paniculata) against SARS-CoV nsp16 protein (a conserved protein that is involved in coronavirus RNA methylation) [88].Of the 100 phytocompounds that were screened, 59 passed the drug likeness assays and only six compounds (oxoproline, andrographolide, deacetylbowdensine, 12dimethyl sageone, sageone, and quercetin) showed good binding afnities after the docking process, with docking scores that ranged from −7.9 to −8.4 kcal/mol.Te toxicological profles of all the six compounds were acceptable, making the compounds ideal candidates for the development of nsp16 inhibitors.Furthermore, another study investigated the inhibitory properties of Andrographis paniculata (a plant that is used as an antiinfammatory and an antiviral therapy) phytochemicals and the nitrobenzoxadiazole-conjugated andrographolide (andro-NBD) suppressed SARS-CoV M pro activities [89].In addition, Zanthoxylum piperitum, Withania somnifera, Calophyllum inophyllum, and Centella asiatica phytochemicals were found to be efective against SARS-CoV-2 after in silico screening (with molecular docking), in vitro and in vivo assays [90].Another study docked the bioactive phytocompounds of tea against SARS-CoV-2 nonstructural protein 15 (nsp15) [91].Tree molecules from the tea extract (barrigenol, kaempferol, and myricetin) displayed adequate docking scores.Similarly, another interesting study investigated the inhibitory potential of an ayurvedic herbal formulation (Triphala) on SARS-Cov-2 main protease, and it was demonstrated that some of its phytocompounds (terfavin A, chebulagic acid, chebulinic acid, and corilagin) where potential inhibitors of M pro .In summary, several other studies have investigated the inhibitory efects of phytocompounds on SARS-CoV-2 main protease and other vital proteins (such as PLpro and RdRp) [92][93][94][95], and they reported acceptable docking scores showing that medicinal plants constitute potential alternatives in the development of therapeutic drugs against SARS-CoV VOCs [96][97][98][99][100][101][102][103][104][105].
Further in vitro and in vivo assessment of these phytochemicals against SARS-CoV-2 proteins should be considered to validate the fndings of this study.

Conclusion
Te current antiviral agents that are destined to cure COVID-19 through the inhibition of coronavirus main protease, a highly conserved protein among coronaviruses, have demonstrated limitations such as the rise of resistant strains to these drugs, poor pharmacokinetic profles, and a suboptimal potency, rendering them inefective against emerging strains of SARS-CoV-2.Tis calls for the development of new therapeutic options that could be efective against such variants of concern.Phytochemicals of medicinal plants present as unexplored potentials which makes them ideal candidates for the development of efective therapeutic alternatives.Imperata cylindrica phytochemicals were screened with molecular docking tools and in silico pharmacokinetic assays in this study.Of the 79 phytocompounds that were investigated, bifendate, cylindrene, tabanone, siderin, 5-hydroxy-2-[2-(2-hydroxyphenyl)ethyl]-4H-1-benzopyran-4-one, maritimin, 5-methoxyfavone, and favone displayed the best docking scores.Tabanone was revealed as the safest compound after toxicological assessment and the best drug candidate based on its pharmacokinetic profle.Tabanone could be used in the development of an efcient M pro inhibitor, provided that adequate in vitro and in vivo assays are conducted to support the fndings of this study.

Figure 6 :Figure 7 :
Figure 6: (a) Tabanone best binding pose and binding site with M pro .(b) Tabanone amino acid residues within the M pro binding pocket.

Table 3 :
Pharmacokinetic properties of I. cylindrica phytochemicals with the best docking scores.

Table 4 :
Toxicity assessment of I. cylindrica phytochemicals with the best docking scores.